Correction: Comparison of “Live High-Train Low” in Normobaric versus Hypobaric Hypoxia

[This corrects the article DOI: 10.1371/journal.pone.0114418.]

Activation of transcription factor Nrf2 signalling by the sphingosine kinase inhibitor SKI-II is mediated by the formation of Keap1 dimers.

BackgroundAnti-oxidant capacity is crucial defence against environmental or endogenous oxidative stress. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a redox-sensitive transcription factor that plays a key defensive role against oxidative and cytotoxic stress and cellular senescence. However, Nrf2 signalling is impaired in several aging-related diseases, such as chronic pulmonary obstructive disease (COPD), cancer, and neurodegenerative diseases. Thus, novel therapeutics that enhance Nrf2 signalling are an attractive approach to treat these diseases.Methodology/principal findingsNrf2 was stabilized by SKI-II (2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole), which is a known sphingosine kinase inhibitor, in human bronchial epithelial cell line, BEAS2B, and in primary human bronchial epithelial cells, leading to enhancement of anti-oxidant proteins, such as HO-1, NQO1 and GCLM. The activation of Nrf2 was achieved by the generation of inactive dimerized form of Keap1, a negative regulator of Nrf2 expression, which was independent of sphingosine kinase inhibition. Using mice that were exposed to cigarette smoke, SKI-II induced Nrf2 expression together with HO-1 in their lungs. In addition, SKI-II reduced cigarette smoke mediated oxidative stress, macrophages and neutrophil infiltration and markers of inflammation in mice.Conclusions/significanceSKI-II appears to be a novel activator of Nrf2 signalling via the inactivation of Keap1

Development of soil health benchmarks for managed and semi-natural landscapes

Efforts to improve soil health require that target values of key soil properties are established. No agreed targets exist but providing population data as benchmarks is a useful step to standardise soil health comparison between landscapes. We exploited nationally representative topsoil (0–15 cm) measurements to derive soil health benchmarks for managed and semi-natural environments across Great Britain. In total, 4587 soil organic matter (SOM), 3860 pH, 2908 bulk density (BD), and 465 earthworm abundance (EA) datapoints were used. As soil properties are sensitive to site-specific characteristics, data were stratified by habitat, soil type, and mean annual precipitation, with benchmarks defined as the middle 80 % of values in each distribution – yielding 135 benchmarks. BD and pH decreased with land management intensity (agriculture > semi-natural grasslands > woodlands > heathlands > wetlands), and vice versa for SOM and EA. Normalising benchmark ranges by medians revealed soil health indicator benchmark widths increased in the order: pH < BD < SOM < EA, while width increased with decreasing land management intensity. Arable and horticulture and improved grassland exhibited narrow benchmarks for SOM, pH and BD, yet the widest EA benchmark, suggesting additional drivers impact EA patterns. Upland wetlands had the widest BD benchmarks, important when determining carbon stocks. East Anglia currently possesses the largest proportions of atypical soils, including below typical SOM (19.2 %), above typical BD (17.4 %) and pH (39.1 %), and the smallest proportions of above typical SOM (2.4 %), and below typical BD (5.8 %) and pH (2.3 %). This is found even after land use, soil type and rainfall have been considered, underscoring how urgently soil health should be addressed here. Our benchmarking framework allows landowners to compare where their measured soil health indicators fall within expected ranges and is applicable to other biomes, national and multinational contexts

Effect of knocking-down SK1 and SK2 on Nrf2 expression in BEAS2B cells.

<p><b>A, B, C and D.</b> Cells transfected with random oligonucleotide (RO) control, SK1 or SK2 siRNA were analysed by immunoblotting (IB) for Nrf2, SK1, SK2, and normalized using β-actin (fold change vs. NT). *** p<0.0001, ** p<0.001, * p<0.05. <b>E.</b> BEAS2B cells were stimulated with SKI-II (1 µM) for 2 h, pellets were spiked with C17 sphingosine, dihydrosphyngosine, S1P and dihydroS1P and extracted sphingolipids (C18) determined by LC-MS/MS. Intensity peaks for Sphingosine (Sph), dihydrosphingosine (dhSph) and dyhydrosphingosine 1 phosphate (dhS1P) are indicated in the graph whereas sphingosine 1 phophate levels (S1P) were below the detection range. <b>F.</b> Nuclear extracts from cells treated with either sphingosine (10 and 20 µM) or dihydrosphingosine (10 and 20 µM) for 2 h were analysed by immunoblotting for Nrf2 expression and normalized using TBP (fold change vs. NT). <b>G.</b> Nuclear extracts from BEAS2B cells treated with fumonisin B1 (FB1; 2 µM) for 1 hour and SKI-II (0.5 µM) for 2 h were analysed by immunoblotting for Nrf2 and TBP expression. Results are representative of two or more independent experiments and are means and S.E. of triplicates.</p

Effect of SKI-II on Keap1.

<p><b>A.</b> Nuclear fractions from BEAS2B cells treated with SKI-II (1 µM) at increasing times (10–120 min) were analysed for Keap1 and Nrf2 expression and normalized using TBP (nuclear). Keap1 bands at 140 kDa and 69 kDa were analysed as fold change over non-treatment of the 69 kDa band. *** p<0.0001, ** p<0.001, * p<0.05. <b>B.</b> Cells were treated with cycloheximide (CXM) and SKI-II (1 µM) at different time points (1 to 24 h) and whole cell extracts were analysed for Keap1 expression normalized against β-actin. Keap1 bands at 140 kDa and 69 kDa were analysed as fold change over non-treatment of the 69 kDa band. *** p<0.0001 when CXM vs. CXM with SKI-II were compared. <b>C.</b> Whole-cell extracts from BEAS2B cells that were treated with SKI-II (1 µM) for 2 and 4 h were immunoprecipitated (IP) using a Keap1 antibody. IP Keap1 was analysed by immunoblotting for ubiquitin modification, p62 and Keap1 expression. IgG: Mouse immunoglobulin control (no cell lysates). Ub: ubiquitin. Hmw: High molecular weight. <b>D.</b> Whole-cell extracts from BEAS2B cells that were pre-treated with arachidonic acid (AA; 10 µM) or biotynilated arachidonic acid (AA-Bio; 10 µM) for 30 min were treated with SKI-II (1 µM) for 2.5 h and immunoprecipitated using Neutravidin Agarose Resin (IP-NA). IP-NA was analysed by immunoblotting for Keap1 expression. <b>E.</b> Whole-cell extracts from BEAS2B cells treated with SKI-II (SK; 1 µM), sulforaphane (SF; 5 µM) or CDDO-Imidazolide (CD; 50 nM) for 4 h, 8 h and 24 h were analysed by immunoblotting for Nrf2, Keap1, NQO1, HO-1 and β-actin. Results are representative of 3 independent experiments and are means and S.E. of triplicates.</p

Effects of intranasal SKI-II on Nrf2, HO-1 and other inflammatory markers in cigarette smoke exposed mice.

<p> Nrf2 protein level normalized to β-actin in lung. Mice were exposed to smoke only once or 12 times (once daily). The lung was collected 2 h after cigarette smoke exposure. <b>B.</b> HO-1 protein in lung. The Western Blot image from pooled samples was shown. <b>C.</b> MDA in BALF, <b>D.</b> Alveolar macrophage in BALF, <b>E.</b> Neutrophils in BALF, <b>F.</b> CXCL1(KC) in BALF, <b>G.</b> MMP9 in BALF. Mice were treated with SKI-II (10 µM), intranasally before each cigarette smoke. Results are representative of 4–5 independent experiments and are means and S.E. of triplicates.</p

Effect of SK inhibition on Nrf2 in human airway epithelial cells (BEAS2B).

<p><b>A.</b> Nuclear extracts from cells treated with increasing concentrations of SK inhibitor SKI-II (0.03 to 3 µM) for 2 h were analysed by immunoblotting for Nrf2 expression and normalized using TBP (fold change vs. NT). *** p<0.0001. <b>B.</b> Expression of antioxidant genes NQO1 and GCLM was determined 24 h after and HO-1 after 8 h treatment with SKI-II. GNB2L1 was used as housekeeping gene. ** p<0.001, * p<0.05. <b>C.</b> Nuclear fractions from cells treated with SKI-II (1 µM) at increasing times (10–120 min) were analysed for Nrf2 expression and normalized using TBP (nuclear). *** p<0.0001,* p<0.05. <b>D.</b> Nuclear fractions from cells treated with SKI-II (1 µM) at increasing times (10–120 min) were analysed for Anti-oxidant Response Element (ARE) binding. *** p<0.0001, ** p<0.001. <b>E.</b> BEAS2B cells were treated with cycloheximide (CXM) and SKI-II (1 µM) at different time points (5 to 60 min) and nuclear extracts were analysed for Nrf2 expression normalized against TBP. Data are representative of 3 independent experiments and are means and S.E. of triplicates.</p