Effects of growth phase and nitrogen starvation on expression of fatty acid desaturases and fatty acid composition of Isochrysis aff. galbana (TISO)

Very long-chain polyunsaturated fatty acids (VLC-PUFAs) are important dietary requirements for maintaining human health. Many marine microalgae are naturally high in ω − 3 VLC-PUFAs, however, the molecular mechanisms underpinning fatty acid (FA) desaturation and elongation in algae are poorly understood. An advanced molecular understanding would facilitate improvements of this nascent industry. We aimed to investigate expression responses of four front-end fatty acid desaturase genes and downstream effects on FA profiles to nitrogen limitation and cultivation growth stage in Isochrysis aff. galbana (TISO). Cultures were grown in nitrogen-replete and -deplete medium; samples were harvested during logarithmic, late logarithmic and stationary growth phases to analyse FA content/composition and gene expression of ?⁶-, ?⁸-, ?⁵- and ?⁴-desaturases (d6FAD (putative), d8FAD, d5FAD and d4FAD, respectively). d6FAD (putative) exhibited no differential expression, while d8FAD, d5FAD and d4FAD were significantly upregulated during logarithmic growth of nutrient-replete cultures, coinciding with rapid cell division. In conclusion, it is demonstrated that expression of some FADs in I. aff. galbana varies with culture age and nitrogen status which has downstream consequences on FA desaturation levels. This has implications for the commercial production of VLC-PUFAs where a trade-off between total lipid yield and VLC-PUFAs has to be made.     

Analysis of the role of Ser1/Ser2/Thr9 phosphorylation on myosin II assembly and function in live cells

Background

The therapeutic efficacy of human mesenchymal stem cells (hMSCs) for the treatment of hypoxic-ischemic diseases is closely related to level of hypoxia in the damaged tissues. To elucidate the potential therapeutic applications and limitations of hMSCs derived from human umbilical cords, the effects of hypoxia on the morphology and proliferation of hMSCs were analyzed.

Results

After treatment with DFO and CoCl₂, hMSCs were elongated, and adjacent cells were no longer in close contact. In addition, vacuole-like structures were observed within the cytoplasm; the rough endoplasmic reticulum expanded, and expanded ridges were observed in mitochondria. In addition, DFO and CoCl₂ treatments for 48 h significantly inhibited hMSCs proliferation in a concentration-dependent manner (P < 0.05). This treatment also increased the number of cells in G0/G1 phase and decreased those in G2/S/M phase.

Conclusions

The hypoxia-mimetic agents, DFO and CoCl₂, alter umbilical cord-derived hMSCs morphology and inhibit their proliferation through influencing the cell cycle.     

Hypoxia-mimetic agents desferrioxamine and cobalt chloride induce leukemic cell apoptosis through different hypoxia-inducible factor-1α independent mechanisms

Hypoxia presents pro-apoptotic and anti-apoptotic biphasic effects that appear to be dependent upon cell types and conditions around cells. The substantial reports demonstrated that commonly used hypoxia-mimetic agents cobalt chloride (CoCl₂) and desferrioxamine (DFO) could also induce apoptosis in many different kinds of cells, but the mechanism was poorly understood. In this work, we compare the apoptosis-inducing effects of these two hypoxia-mimetic agents with acute myeloid leukemic cell lines NB4 and U937 as in vitro models. The results show that both of them induce these leukemic cells to undergo apoptosis with a loss of mitochondrial transmembrane potentials (ΔΨ m), the activation of caspase-3/8 and the cleavage of anti-apoptotic protein Mcl-1, together with the accumulation of hypoxia-inducible factor-1 alpha (HIF-1α) protein, a critical regulator for the cellular response to hypoxia. Metavanadate and sodium nitroprusside significantly abrogate DFO rather than CoCl₂-induced mitochondrial Δ Ψ m collapse, caspase-3/8 activation, Mcl-1 cleavage and apoptosis, but they fail to influence DFO and CoCl₂-induced HIF-1α protein accumulation. Moreover, inducible expression of HIF-1α gene dose not alter DFO and CoCl₂-induced apoptosis in U937 cells. In conclusion, these results propose that although both DFO and CoCl₂-induced leukemic cell apoptosis by mitochondrial pathway-dependent and HIF-1α-independent mechanisms, DFO and CoCl₂-induced apoptosis involves different initiating signal pathways that remain to be investigated.     

Bafilomycin A1 activates HIF-dependent signalling in human colon cancer cells via mitochondrial uncoupling

Mitochondrial uncoupling is implicated in many patho(physiological) states. Using confocal live cell imaging and an optical O₂ sensing technique, we show that moderate uncoupling of the mitochondria with plecomacrolide Baf (bafilomycin A1) causes partial depolarization of the mitochondria and deep sustained deoxygenation of human colon cancer HCT116 cells subjected to 6% atmospheric O₂. A decrease in iO₂ (intracellular O₂) to 0–10 μM, induced by Baf, is sufficient for stabilization of HIFs (hypoxia inducible factors) HIF-1α and HIF-2α, coupled with an increased expression of target genes including GLUT1 (glucose transporter 1), HIF PHD2 (prolyl hydroxylase domain 2) and CAIX (carbonic anhydrase IX). Under the same hypoxic conditions, treatment with Baf causes neither decrease in iO₂ nor HIF-α stabilization in the low-respiring HCT116 cells deficient in COX (cytochrome c-oxidase). Both cell types display equal capacities for HIF-α stabilization by hypoxia mimetics DMOG (dimethyloxalylglycine) and CoCl₂, thus suggesting that the effect of Baf under hypoxia is driven mainly by mitochondrial respiration. Altogether, by activating HIF signalling under moderate hypoxia, mitochondrial uncoupling can play an important regulatory role in colon cancer metabolism and modulate adaptation of cancer cells to natural hypoxic environments.     

Atypical Biosynthetic Properties of a Δ12/ν+3 Desaturase from the Model Basidiomycete Phanerochaete chrysosporium

The model white-rot basidiomycete Phanerochaete chrysosporium contains a single integral membrane Δ¹²-desaturase FAD2 related to the endoplasmic reticular plant FAD2 enzymes. The fungal fad2-like gene was cloned and distinguished itself from plant homologs by the presence of four introns and a significantly larger coding region. The coding sequence exhibits ca. 35% sequence identity to plant homologs, with the highest sequence conservation found in the putative catalytic and major structural domains. In vivo activity of the heterologously expressed enzyme favors C₁₈ substrates with ν+3 regioselectivity, where the site of desaturation is three carbons carboxy-distal to the reference position of a preexisting double bond (ν). Linoleate accumulated to levels in excess of 12% of the total fatty acids upon heterologous expression of P. chrysosporium FAD2 in Saccharomyces cerevisiae. In contrast to the behavior of the plant FAD2 enzymes, this oleate desaturase does not 12-hydroxylate lipids and is the first example whose activity increases at higher temperatures (30°C versus 15°C). Thus, while maintaining the hallmark activity of the fatty acyl Δ¹²-desaturase family, the basidiomycete fad2 genes appear to have evolved substantially from an ancestral desaturase.     

A Four-Way Comparison of Cardiac Function with Normobaric Normoxia,Normobaric Hypoxia,Hypobaric Hypoxia and Genuine High Altitude

BackgroundThere has been considerable debate as to whether different modalities of simulated hypoxia induce similar cardiac responses.ResultsAll 14 subjects completed the experiment at GHA, 11 at NN, 12 under NH, and 6 under HH. The four groups were similar in age, sex and baseline demographics. At baseline rest right ventricular (RV) systolic pressure (RVSP, p = 0.0002), pulmonary vascular resistance (p = 0.0002) and acute mountain sickness (AMS) scores were higher and the SpO₂ lower (p<0.0001) among all three hypoxic groups (GHA, NH and HH) compared with NN. At both 15 minutes and 120 minutes post exercise, AMS scores, Cardiac output, septal S’, lateral S’, tricuspid S’ and A’ velocities and RVSP were higher and SpO₂ lower with all forms of hypoxia compared with NN. On post-test analysis, among the three hypoxia groups, SpO₂ was lower at baseline and 15 minutes post exercise with GHA (89.3±3.4% and 89.3±2.2%) and HH (89.0±3.1 and (89.8±5.0) compared with NH (92.9±1.7 and 93.6±2.5%). The RV Myocardial Performance (Tei) Index and RVSP were significantly higher with HH than NH at 15 and 120 minutes post exercise respectively and tricuspid A’ was higher with GHA compared with NH at 15 minutes post exercise.ConclusionsGHA, NH and HH produce similar cardiac adaptations over short duration rest despite lower SpO₂ levels with GHA and HH compared with NH. Notable differences emerge following exercise in SpO_2, RVSP and RV cardiac function.     

Purification and characterization of active-site components of the putative p-cresol methylhydroxylase membrane complex from Geobacter metallireducens

p-Cresol methylhydroxylases (PCMH) from aerobic and facultatively anaerobic bacteria are soluble, periplasmic flavocytochromes that catalyze the first step in biological p-cresol degradation, the hydroxylation of the substrate with water. Recent results suggested that p-cresol degradation in the strictly anaerobic Geobacter metallireducens involves a tightly membrane-bound PCMH complex. In this work, the soluble components of this complex were purified and characterized. The data obtained suggest a molecular mass of 124 ± 15 kDa and a unique αα′β₂ subunit composition, with α and α′ representing isoforms of the flavin adenine dinucleotide (FAD)-containing subunit and β representing a c-type cytochrome. Fluorescence and mass spectrometric analysis suggested that one FAD was covalently linked to Tyr³⁹⁴ of the α subunit. In contrast, the α′ subunit did not contain any FAD cofactor and is therefore considered to be catalytically inactive. The UV/visible spectrum was typical for a flavocytochrome with two heme c cofactors and one FAD cofactor. p-Cresol reduced the FAD but only one of the two heme cofactors. PCMH catalyzed both the hydroxylation of p-cresol to p-hydroxybenzyl alcohol and the subsequent oxidation of the latter to p-hydroxybenzaldehyde in the presence of artificial electron acceptors. The very low K_m values (1.7 and 2.7 μM, respectively) suggest that the in vivo function of PCMH is to oxidize both p-cresol and p-hydroxybenzyl alcohol. The latter was a mixed inhibitor of p-cresol oxidation, with inhibition constants of a K_ic (competitive inhibition) value of 18 ± 9 μM and a K_iu (uncompetitive inhibition) value of 235 ± 20 μM. A putative functional model for an unusual PCMH enzyme is presented.     

Short-Term Effects of Particulate Matter on Stroke Attack: Meta-Regression and Meta-Analyses

Background and Purpose

Currently there are more and more studies on the association between short-term effects of exposure to particulate matter (PM) and the morbidity of stroke attack, but few have focused on stroke subtypes. The objective of this study is to assess the relationship between PM and stroke subtypes attack, which is uncertain now.

Methods

Meta-analyses, meta-regression and subgroup analyses were conducted to investigate the association between short-term effects of exposure to PM and the morbidity of different stroke subtypes from a number of epidemiologic studies (from 1997 to 2012).

Results

Nineteen articles were identified. Odds ratio (OR) of stroke attack associated with particular matter (“thoracic particles” [PM₁₀]<10 µm in aerodynamic diameter, “fine particles” [PM_2.5]<2.5 µm in aerodynamic diameter) increment of 10 µg/m³ was as effect size. PM₁₀ exposure was related to an increase in risk of stroke attack (OR per 10 µg/m³ = 1.004, 95%CI: 1.001∼1.008) and PM_2.5 exposure was not significantly associated with stroke attack (OR per 10 µg/m³ = 0.999, 95%CI: 0.994∼1.003). But when focused on stroke subtypes, PM_2.5 (OR per 10 µg/m³ = 1.025; 95%CI, 1.001∼1.049) and PM₁₀ (OR per 10 µg/m³ = 1.013; 95%CI, 1.001∼1.025) exposure were statistically significantly associated with an increased risk of ischemic stroke attack, while PM_2.5 (all the studies showed no significant association) and PM₁₀ (OR per 10 µg/m³ = 1.007; 95%CI, 0.992∼1.022) exposure were not associated with an increased risk of hemorrhagic stroke attack. Meta-regression found study design and area were two effective covariates.

Conclusion

PM_2.5 and PM₁₀ had different effects on different stroke subtypes. In the future, it''s worthwhile to study the effects of PM to ischemic stroke and hemorrhagic stroke, respectively.     

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

We investigated the changes in both performance and selected physiological parameters following a Live High-Train Low (LHTL) altitude camp in either normobaric hypoxia (NH) or hypobaric hypoxia (HH) replicating current “real” practices of endurance athletes. Well-trained triathletes were split into two groups (NH, n = 14 and HH, n = 13) and completed an 18-d LHTL camp during which they trained at 1100–1200 m and resided at an altitude of 2250 m (P_iO₂  = 121.7±1.2 vs. 121.4±0.9 mmHg) under either NH (hypoxic chamber; F_iO₂ 15.8±0.8%) or HH (real altitude; barometric pressure 580±23 mmHg) conditions. Oxygen saturations (S_pO₂) were recorded continuously daily overnight. P_iO₂ and training loads were matched daily. Before (Pre-) and 1 day after (Post-) LHTL, blood samples, VO_2max, and total haemoglobin mass (Hb_mass) were measured. A 3-km running test was performed near sea level twice before, and 1, 7, and 21 days following LHTL. During LHTL, hypoxic exposure was lower for the NH group than for the HH group (220 vs. 300 h; P<0.001). Night S_pO₂ was higher (92.1±0.3 vs. 90.9±0.3%, P<0.001), and breathing frequency was lower in the NH group compared with the HH group (13.9±2.1 vs. 15.5±1.5 breath.min⁻¹, P<0.05). Immediately following LHTL, similar increases in VO_2max (6.1±6.8 vs. 5.2±4.8%) and Hb_mass (2.6±1.9 vs. 3.4±2.1%) were observed in NH and HH groups, respectively, while 3-km performance was not improved. However, 21 days following the LHTL intervention, 3-km run time was significantly faster in the HH (3.3±3.6%; P<0.05) versus the NH (1.2±2.9%; ns) group. In conclusion, the greater degree of race performance enhancement by day 21 after an 18-d LHTL camp in the HH group was likely induced by a larger hypoxic dose. However, one cannot rule out other factors including differences in sleeping desaturations and breathing patterns, thus suggesting higher hypoxic stimuli in the HH group.     

NADPH–Cytochrome P450 Reductase Mediates the Fatty Acid Desaturation of ω3 and ω6 Desaturases from Mortierella alpina

Fatty acid desaturases play an important role in maintaining the appropriate structure and function of biological membranes. The biochemical characterization of integral membrane desaturases, particularly ω3 and ω6 desaturases, has been limited by technical difficulties relating to the acquisition of large quantities of purified proteins, and by the fact that functional activities of these proteins were only tested in an NADH-initiated reaction system. The main aim of this study was to reconstitute an NADPH-dependent reaction system in vitro and investigate the kinetic properties of Mortierella alpina ω3 and ω6 desaturases in this system. After expression and purification of the soluble catalytic domain of NADPH–cytochrome P450 reductase, the NADPH-dependent fatty acid desaturation was reconstituted for the first time in a system containing NADPH, NADPH–cytochrome P450 reductase, cytochrome b5, M. alpina ω3 and ω6 desaturase and detergent. In this system, the maximum activity of ω3 and ω6 desaturase was 213.4 ± 9.0 nmol min⁻¹ mg⁻¹ and 10.0 ± 0.5 nmol min⁻¹ mg⁻¹, respectively. The highest k_cat/K_m value of ω3 and ω6 desaturase was 0.41 µM⁻¹ min⁻¹ and 0.09 µM⁻¹ min⁻¹ when using linoleoyl CoA (18:2 ω6) and oleoyl CoA (18:1 ω9) as substrates, respectively. M. alpina ω3 and ω6 desaturases were capable of using NADPH as reductant when mediated by NADPH–cytochrome P450 reductase; although, their efficiency is distinguishable from NADH-dependent desaturation. These results provide insights into the mechanisms underlying ω3 and ω6 fatty acid desaturation and may facilitate the production of important fatty acids in M. alpina.     

Pre-Existing Hypoxia Is Associated with Greater EEG Suppression and Early Onset of Evolving Seizure Activity during Brief Repeated Asphyxia in Near-Term Fetal Sheep

Spontaneous antenatal hypoxia is associated with high risk of adverse outcomes, however, there is little information on neural adaptation to labor-like insults. Chronically instrumented near-term sheep fetuses (125 ± 3 days, mean ± SEM) with baseline PaO₂ < 17 mmHg (hypoxic group: n = 8) or > 17 mmHg (normoxic group: n = 8) received 1-minute umbilical cord occlusions repeated every 5 minutes for a total of 4 hours, or until mean arterial blood pressure (MAP) fell below 20 mmHg for two successive occlusions. 5/8 fetuses with pre-existing hypoxia were unable to complete the full series of occlusions (vs. 0/8 normoxic fetuses). Pre-existing hypoxia was associated with progressive metabolic acidosis (nadir: pH 7.08 ± 0.04 vs. 7.33 ± 0.02, p<0.01), hypotension during occlusions (nadir: 24.7 ± 1.8 vs. 51.4 ± 3.2 mmHg, p<0.01), lower carotid blood flow during occlusions (23.6 ± 6.1 vs. 63.0 ± 4.8 mL/min, p<0.01), greater suppression of EEG activity during, between, and after occlusions (p<0.01) and slower resolution of cortical impedance, an index of cytotoxic edema. No normoxic fetuses, but 4/8 hypoxic fetuses developed seizures 148 ± 45 minutes after the start of occlusions, with a seizure burden of 26 ± 6 sec during the inter-occlusion period, and 15.1 ± 3.4 min/h in the first 6 hours of recovery. In conclusion, in fetuses with pre-existing hypoxia, repeated brief asphyxia at a rate consistent with early labor is associated with hypotension, cephalic hypoperfusion, greater EEG suppression, inter-occlusion seizures, and more sustained cytotoxic edema, consistent with early onset of neural injury.     

Hypoxia-inducible Factor-1α (HIF-1α) Protein Diminishes Sodium Glucose Transport 1 (SGLT1) and SGLT2 Protein Expression in Renal Epithelial Tubular Cells (LLC-PK1) under Hypoxia

In this work, we demonstrated the regulation of glucose transporters by hypoxia inducible factor-1α (HIF-1α) activation in renal epithelial cells. LLC-PK₁ monolayers were incubated for 1, 3, 6, or 12 h with 0% or 5% O₂ or 300 μm cobalt (CoCl₂). We evaluated the effects of hypoxia on the mRNA and protein expression of HIF-1α and of the glucose transporters SGLT1, SGLT2, and GLUT1. The data showed an increase in HIF-1α mRNA and protein expression under the three evaluated conditions (p < 0.05 versus t = 0). An increase in GLUT1 mRNA (12 h) and protein expression (at 3, 6, and 12 h) was observed (p < 0.05 versus t = 0). SGLT1 and SGLT2 mRNA and protein expression decreased under the three evaluated conditions (p < 0.05 versus t = 0). In conclusion, our results suggest a clear decrease in the expression of the glucose transporters SGLT1 and SGLT2 under hypoxic conditions which implies a possible correlation with increased expression of HIF-1α.     

Effect of metal ions on HIF-1α and Fe homeostasis in human A549 cells

Several metals are carcinogenic but little is known about the mechanisms by which they cause cancer. A pathway that may contribute to metal ion induced carcinogenesis is by hypoxia signaling, which involves a disruption of cellular iron homeostasis by competition with iron transporters or iron-regulated enzymes. To examine the involvement of iron in the hypoxia signaling activity of these metal ions we investigated HIF-1α protein stabilization, IRP-1 activity, and ferritin protein levels in human lung carcinoma A459 cells exposed to various agents in serum- and iron-free salt–glucose medium (SGM) or in normal complete medium. We also studied the effects of excess exogenous iron on these responses induced by nickel ion exposure. Our results show the following: (1) SGM enhanced metals-induced HIF-1α stabilization and IRP-1 activation (e.g., nickel and cobalt ions). (2) If SGM was reconstituted with a slight excess level (25 μM of FeSO₄) of iron, this enhancing ability was significantly decreased. (3) The effect of a high level of exogenous iron (500 μM of FeSO₄) on metal-induced hypoxia and iron metabolism was highly dependent on the order of addition. If treatment with the Fe and metal ions was simultaneous (co-treatment), the effects of nickel ion exposure were overwhelmed, since the added Fe reversed HIF-1α stabilization, decreased IRP-1 activity, and increased ferritin level. Pre-treatment with iron was not able to reverse the responses caused by nickel ion exposure. These results imply that it is important to consider the available iron concentration and suitable exposure design when studying metal-induced hypoxia or metal-induced disruption of Fe homeostasis.     

Selective Irreversible Inhibition of Neuronal and Inducible Nitric-oxide Synthase in the Combined Presence of Hydrogen Sulfide and Nitric Oxide

Citrulline formation by both human neuronal nitric-oxide synthase (nNOS) and mouse macrophage inducible NOS was inhibited by the hydrogen sulfide (H₂S) donor Na₂S with IC₅₀ values of ∼2.4·10⁻⁵ and ∼7.9·10⁻⁵ m, respectively, whereas human endothelial NOS was hardly affected at all. Inhibition of nNOS was not affected by the concentrations of l-arginine (Arg), NADPH, FAD, FMN, tetrahydrobiopterin (BH4), and calmodulin, indicating that H₂S does not interfere with substrate or cofactor binding. The IC₅₀ decreased to ∼1.5·10⁻⁵ m at pH 6.0 and increased to ∼8.3·10⁻⁵ m at pH 8.0. Preincubation of concentrated nNOS with H₂S under turnover conditions decreased activity after dilution by ∼70%, suggesting irreversible inhibition. However, when calmodulin was omitted during preincubation, activity was not affected, suggesting that irreversible inhibition requires both H₂S and NO. Likewise, NADPH oxidation was inhibited with an IC₅₀ of ∼1.9·10⁻⁵ m in the presence of Arg and BH4 but exhibited much higher IC₅₀ values (∼1.0–6.1·10⁻⁴ m) when Arg and/or BH4 was omitted. Moreover, the relatively weak inhibition of nNOS by Na₂S in the absence of Arg and/or BH4 was markedly potentiated by the NO donor 1-(hydroxy-NNO-azoxy)-l-proline, disodium salt (IC₅₀ ∼ 1.3–2.0·10⁻⁵ m). These results suggest that nNOS and inducible NOS but not endothelial NOS are irreversibly inhibited by H₂S/NO at modest concentrations of H₂S in a reaction that may allow feedback inhibition of NO production under conditions of excessive NO/H₂S formation.     

HIF-1α Inhibition Reduces Nasal Inflammation in a Murine Allergic Rhinitis Model

Background

Hypoxia-inducible factor 1α (HIF-1α) is an important regulator of immune and inflammatory responses. We hypothesized that nasal allergic inflammation is attenuated by HIF-1α inhibition and strengthened by HIF-1α stabilization.

Objective

To elucidate the role of HIF-1α in a murine model of allergic rhinitis (AR).

Methods

Mice were pretreated with the HIF-1α inhibitor 2-methoxyestradiol (2ME2) or the HIF-1α inducer cobalt chloride (CoCl₂) in an established AR murine model using ovalbumin (OVA)-sensitized BALB/c mice. HIF-1α and vascular endothelial growth factor (VEGF) expression in nasal mucosa was measured and multiple parameters of allergic responses were evaluated.

Results

HIF-1α and VEGF levels were locally up-regulated in nasal mucosa during AR. Inflammatory responses to OVA challenge, including nasal symptoms, inflammatory cell infiltration, eosinophil recruitment, up-regulation of T-helper type 2 cytokines in nasal lavage fluid, and serum OVA-specific IgE levels were present in the OVA-challenged mice. 2ME2 effectively inhibited HIF-1α and VEGF expression and attenuated the inflammatory responses. Stabilization of HIF-1α by CoCl₂ facilitated nasal allergic inflammation. HIF-1α protein levels in nasal airways correlated with the severity of AR in mice.

Conclusions

HIF-1α is intimately involved in the pathogenesis of nasal allergies, and the inhibition of HIF-1α may be useful as a novel therapeutic approach for AR.     

Extreme Variations of pCO2 and pH in a Macrophyte Meadow of the Baltic Sea in Summer: Evidence of the Effect of Photosynthesis and Local Upwelling

The impact of ocean acidification on benthic habitats is a major preoccupation of the scientific community. However, the natural variability of pCO₂ and pH in those habitats remains understudied, especially in temperate areas. In this study we investigated temporal variations of the carbonate system in nearshore macrophyte meadows of the western Baltic Sea. These are key benthic ecosystems, providing spawning and nursery areas as well as food to numerous commercially important species. In situ pCO₂, pH (total scale), salinity and PAR irradiance were measured with a continuous recording sensor package dropped in a shallow macrophyte meadow (Eckernförde bay, western Baltic Sea) during three different weeks in July (pCO₂ and PAR only), August and September 2011.The mean (± SD) pCO₂ in July was 383±117 µatm. The mean (± SD) pCO₂ and pH_tot in August were 239±20 µatm and 8.22±0.1, respectively. The mean (± SD) pCO₂ and pH_tot in September were 1082±711 µatm and 7.83±0.40, respectively. Daily variations of pCO₂ due to photosynthesis and respiration (difference between daily maximum and minimum) were of the same order of magnitude: 281±88 µatm, 219±89 μatm and 1488±574 µatm in July, August and September respectively. The observed variations of pCO₂ were explained through a statistical model considering wind direction and speed together with PAR irradiance. At a time scale of days to weeks, local upwelling of elevated pCO₂ water masses with offshore winds drives the variation. Within days, primary production is responsible. The results demonstrate the high variability of the carbonate system in nearshore macrophyte meadows depending on meteorology and biological activities. We highlight the need to incorporate these variations in future pCO₂ scenarios and experimental designs for nearshore habitats.     

Statistical Optimization of Ultraviolet Irradiate Conditions for Vitamin D2 Synthesis in Oyster Mushrooms (Pleurotus ostreatus) Using Response Surface Methodology

Response surface methodology (RSM) was used to determine the optimum vitamin D₂ synthesis conditions in oyster mushrooms (Pleurotus ostreatus). Ultraviolet B (UV-B) was selected as the most efficient irradiation source for the preliminary experiment, in addition to the levels of three independent variables, which included ambient temperature (25–45°C), exposure time (40–120 min), and irradiation intensity (0.6–1.2 W/m²). The statistical analysis indicated that, for the range which was studied, irradiation intensity was the most critical factor that affected vitamin D₂ synthesis in oyster mushrooms. Under optimal conditions (ambient temperature of 28.16°C, UV-B intensity of 1.14 W/m², and exposure time of 94.28 min), the experimental vitamin D₂ content of 239.67 µg/g (dry weight) was in very good agreement with the predicted value of 245.49 µg/g, which verified the practicability of this strategy. Compared to fresh mushrooms, the lyophilized mushroom powder can synthesize remarkably higher level of vitamin D₂ (498.10 µg/g) within much shorter UV-B exposure time (10 min), and thus should receive attention from the food processing industry.     

Positive Expiratory Pressure Improves Oxygenation in Healthy Subjects Exposed to Hypoxia

Introduction

Positive end-expiratory pressure (PEEP) is commonly used in critical care medicine to improve gas exchange. Altitude sickness is associated with exaggerated reduction in arterial oxygenation. We assessed the effect of PEEP and pursed lips breathing (PLB) on arterial and tissue oxygenation under normobaric and hypobaric hypoxic conditions.

Methods

Sixteen healthy volunteers were exposed to acute normobaric hypoxia (Laboratory study, FiO₂=0.12). The protocol consisted in 3-min phases with PEEPs of 0, 5 or 10 cmH₂O, PLB or similar ventilation than with PEEP-10, interspaced with 3-min phases of free breathing. Arterial (pulse oximetry) and quadriceps (near-infrared spectroscopy) oxygenation, ventilation, cardiac function, esophageal and gastric pressures and subjects’ subjective perceptions were recorded continuously. In addition, the effect of PEEP on arterial oxygenation was tested at 4,350 m of altitude in 9 volunteers breathing for 20 min with PEEP-10 (Field study).

Results

During the laboratory study, PEEP-10 increased arterial and quadriceps oxygenation (arterial oxygen saturation +5.6±5.0% and quadriceps oxyhemoglobin +58±73 µmol.cm compared to free breathing; p<0.05). Conversely, PLB did not increase oxygenation. Oxygenation improvement with PEEP-10 was accompanied by an increase in expiratory esophageal and gastric pressures (esophageal pressure swing +5.4±3.2 cmH₂O, p<0.05) but no change in minute ventilation, breathing pattern, end-tidal CO₂ or cardiac function (all p>0.05) compared to PEEP-0. During the field study, PEEP-10 increased arterial oxygen saturation by +6.7±6.0% after the 3^rd minute with PEEP-10 without further significant increase until the 20^th minute with PEEP-10. Subjects did not report any significant discomfort with PEEP.

Conclusions

These data indicate that 10-cmH₂O PEEP significantly improves arterial and muscle oxygenation under both normobaric and hypobaric hypoxic conditions in healthy subjects. PEEP-10 could be an attractive non-pharmacological tool to limit blood oxygen desaturation and possibly symptoms at altitude.     

In vitro and in silico studies of bis (indol-3-yl) methane derivatives as potential α-glucosidase and α-amylase inhibitors

In this paper, bis (indol-3-yl) methanes (BIMs) were synthesised and evaluated for their inhibitory activity against α-glucosidase and α-amylase. All synthesised compounds showed potential α-glucosidase and α-amylase inhibitory activities. Compounds 5 g (IC₅₀: 7.54 ± 1.10 μM), 5e (IC₅₀: 9.00 ± 0.97 μM), and 5 h (IC₅₀: 9.57 ± 0.62 μM) presented strongest inhibitory activities against α-glucosidase, that were ∼ 30 times stronger than acarbose. Compounds 5 g (IC₅₀: 32.18 ± 1.66 µM), 5 h (IC₅₀: 31.47 ± 1.42 µM), and 5 s (IC₅₀: 30.91 ± 0.86 µM) showed strongest inhibitory activities towards α-amylase, ∼ 2.5 times stronger than acarbose. The mechanisms and docking simulation of the compounds were also studied. Compounds 5 g and 5 h exhibited bifunctional inhibitory activity against these two enzymes. Furthermore, compounds showed no toxicity against 3T3-L1 cells and HepG2 cells.

Highlights

1. A series of bis (indol-3-yl) methanes (BIMs) were synthesised and evaluated inhibitory activities against α-glucosidase and α-amylase.
2. Compound 5g exhibited promising activity (IC₅₀ = 7.54 ± 1.10 μM) against α-glucosidase.
3. Compound 5s exhibited promising activity (IC₅₀ = 30.91 ± 0.86 μM) against α-amylase.
4. In silico studies were performed to confirm the binding interactions of synthetic compounds with the enzyme active site.