Proposed reductive metabolism of artemisinin by glutathione transferases in vitro

Artemisinin is a sesquiterpene lactone containing an endoperoxide bridge. It is a promising new antimalarial and is particularly useful against the drug resistant strains of Plasmodium falciparum. It has unique antimalarial properties since it acts through the generation of free radicals that alkylate parasite proteins. Since the antimalarial action of the drug is antagonised by glutathione and ascorbate and has unusual pharmacokinetic properties in humans, we have investigated if the drug is broken down by a typical reductive reaction in the presence of glutathione transferases. Cytosolic glutathione transferases (GSTs) detoxify electrophilic xenobiotics by catalysing the formation of glutathione (GSH) conjugates and exhibit glutathione peroxidase activity towards hydroperoxides. Artemisinin was incubated with glutathione, NADPH and glutathione reductase and GSTs in a coupled assay system analogous to the standard assay scheme with cumene hydroperoxide as a substrate of GSTs. Artemisinin was shown to stimulate NADPH oxidation in cytosols from rat liver, kidney, intestines and in affinity purified preparations of GSTs from rat liver. Using human recombinant GSTs hetelorogously expressed in Escherichia coli, artemisinin was similarly shown to stimulate NADPH oxidation with the highest activity observed with GST M1-1. Using recombinant GSTs the activity of GSTs with artemisinin was at least two fold higher than the reaction with CDNB. Considering these results, it is possible that GSTs may contribute to the metabolism of artemisinin in the presence of NADPH and GSSG-reductase We propose a model, based on the known reactions of GSTs and sesquiterpenes, in which (1) artemisinin reacts with GSH resulting in oxidised glutathione; (2) the oxidised glutathione is then converted to reduced glutathione via glutathione reductase; and (3) the latter reaction may then result in the depletion of NADPH via GSSG-reductase. The ability of artemisinin to react with GSH in the presence of GST may be responsible for the NADPH utilisation observed in vitro and suggests that cytosolic GSTs are likely to be contributing to metabolism of artemisinin and related drugs in vivo.     

Freshwater biota and rising pCO2?

Rising atmospheric carbon dioxide (CO₂) has caused a suite of environmental issues, however, little is known about how the partial pressure of CO₂ (pCO₂) in freshwater will be affected by climate change. Freshwater pCO₂ varies across systems and is controlled by a diverse array of factors, making it difficult to make predictions about future levels of pCO₂. Recent evidence suggests that increasing levels of atmospheric CO₂ may directly increase freshwater pCO₂ levels in lakes, but rising atmospheric CO₂ may also indirectly impact freshwater pCO₂ levels in a variety of systems by affecting other contributing factors such as soil respiration, terrestrial productivity and climate regimes. Although future freshwater pCO₂ levels remain uncertain, studies have considered the potential impacts of changes to pCO₂ levels on freshwater biota. Studies to date have focused on impacts of elevated pCO₂ on plankton and macrophytes, and have shown that phytoplankton nutritional quality is reduced, plankton community structure is altered, photosynthesis rates increase and macrophyte distribution shifts with increasing pCO₂. However, a number of key knowledge gaps remain and gaining a better understanding of how freshwater pCO₂ levels are regulated and how these levels may impact biota, will be important for predicting future responses to climate change.     

Nutrient modulation of intestinal gas dynamics in healthy humans: dependence on caloric content and meal consistency

The actions of nutrients on gut transit of liquids and solids have been extensively studied, but the effects of meal ingestion on intestinal gas flow are unexplored. We hypothesized that meals of varying caloric content and consistency modulate gas transit to different degrees. Nine healthy volunteers underwent jejunal perfusion of physiological gas mixtures at 12 ml.min(-1).3 h, with ingestion of nothing (control), water (240 ml), 240-kcal liquid meals, and 240-kcal solid meals at the end of the second hour in separate studies. Gas was quantified from an intrarectal catheter. After an initial lag phase, gas evacuation approached steady state by the end of the fasting period. Solid and liquid caloric meals increased total gas volumes evacuated from 5-40 min after ingestion vs. control studies (P < 0.05). These increases resulted from increased numbers of bolus gas evacuations (P < 0.05), whereas bolus volumes, pressures, and flow rates were similar for all test conditions. Solid and liquid caloric meals elicited similar effects on bolus gas dynamic parameters, whereas water did not affect these measures vs. control (NS, not significant). Both caloric meals and the noncaloric liquid meal increased continuous gas flow, which represented <2% of total gas expulsion. In conclusion, caloric meals promote bolus gas transit in healthy humans, whereas noncaloric liquids have no effect. Solids stimulate early postprandial gas dynamics to the same extent as liquid meals of similar caloric content. Thus modulatory effects of meals on intestinal gas transit depend on their caloric content but not their consistency.     

Superovulatory responses of Holstein cows

Approximately 1000 registered cows and heifers were superovulated one to 10 times. Nonsurgical embryo recoveries were performed on all donors which exhibited estrus. Healthy donors produced more total ova and cleaving embryos and had a higher ovum recovery rate, fertilization rate and pregnancy rate from embryos transferred than did cows classified as infertile. While ovum number was not affected during 10 repeated superovulations, fertilization rate and embryo number decreased. The number of ova recovered from healthy cows was affected by season, and from infertile cows by the day of the estrous cycle on which FSH was started and by the number of days since calving. More ova were recovered from infertile cows synchronized with prostaglandins prior to superovulation than following a natural estrous cycle. The number of embryos recovered from infertile cows was affected by age and from healthy cows by daily milk production. Fertilization rates in both healthy and infertile cows were affected by age, time since calving, daily milk production, day of cycle FSH was injected and season. There was no effect of the day of recovery on the number of ova or embryos recovered from healthy or infertile cows.     

A new reconstruction of the Le Moustier 1 skull and investigation of internal structures using 3-D-μCT data

Using the non-destructive technique of 3-D micro computed tomography (3-D-μCT), we present a new, virtual reconstruction of the Le Moustier 1 Neandertal skull. This new reconstruction corrects defects found in earlier reconstruction attempts by repositioning misaligned cranial fragments, addressing the problem of asymmetry caused by pressure during the fossilization process, and placing the basioccipital in its proper anatomical position. Metric comparisons between Le Moustier 1 and juvenile and adult Neandertals demonstrate that facial height proceeded at a faster rate of growth than facial prognathism at the beginning of the adolescent period. They also confirm the anterior placement of the basioccipital. A compound painted to match the colour of the fossilized bone was used in previous reconstruction attempts and the aim of this analysis was to remove the false material to reveal to what extent the fossilized bone was preserved. The areas with the most artificial material and glue include the palate, areas around the mandibular teeth, the left frontal, and parts of the right parietal and temporal bones. The μCT data were also used to examine internal structures of the skull including the frontal sinus and the labyrinth of the inner ear. An investigation of the frontal sinus reveals morphology similar to that found in adult Neandertals, although the structure does not extend to mid-orbit. The dimension of the radius of curvature of the lateral semicircular canal falls within one standard deviation, and the anterior and posterior canals within two standard deviations, of the published Neandertal mean. As in other Neandertals, the posterior semicircular canal is in an inferior position relative to the plane of the lateral canal.     

Population dynamics of two antilisterial cheese surface consortia revealed by temporal temperature gradient gel electrophoresis

Background  

Surface contamination of smear cheese by Listeria spp. is of major concern for the industry. Complex smear ecosystems have been shown to harbor antilisterial potential but the microorganisms and mechanisms involved in the inhibition mostly remain unclear, and are likely related to complex interactions than to production of single antimicrobial compounds. Bacterial biodiversity and population dynamics of complex smear ecosystems exhibiting antilisterial properties in situ were investigated by Temporal temperature gradient gel electrophoresis (TTGE), a culture independent technique, for two microbial consortia isolated from commercial Raclette type cheeses inoculated with defined commercial ripening cultures (F) or produced with an old-young smearing process (M).     

Activated endothelial cells induce neutrophil extracellular traps and are susceptible to NETosis-mediated cell death

Neutrophil interaction with activated endothelial cells (EC) is required for transmigration. We examined consequences of this interaction on NETosis. Co-culture of activated EC with neutrophils induced neutrophil extracellular trap (NET) formation, which was partially dependent on production of IL-8 by activated EC. Extended neutophil/EC co-culture resulted in EC damage, which could be abrogated by inclusion of either diphenyleneiodonium to inhibit the NAPDH oxidase pathway required for NETosis, or DNAse to disrupt NETs. These findings offer new insight into mechanisms whereby NETs trigger damage to the endothelium in sepsis, small vessel vasculitis and possibly the villous trophoblast in preeclampsia.     

Facies characteristics and diversity in carbonate eolianites

Carbonate eolian dunes can form huge sand bodies along the coasts but are seldom described in the pre-Quaternary record. The study of more than 600 thin-sections collected in present-day, Holocene and Pleistocene dunes from Sardinia, Crete, Cyprus, Tunisia, Morocco, Australia and Baja California confirms that these deposits can be easily misinterpreted as shallow marine at core or thin-section scale. The classical eolian criteria (fine-grained and well-sorted sands) are exceptional in carbonate dunes because the diversity of shapes and densities of carbonate particles lowers the critical shear velocity of the sediment thus blurring the sedimentary structures. Wind carbonate deposits are mainly heterogeneous in size and often coarse-grained. The paucity of eolianites in the pre-Quaternary record could be due to misinterpretation of these deposits. The recognition should be based on converging sedimentological and stratigraphic elements at core scale, and diagenetic (vadose diagenesis, pedogenetic imprints) and petrographical (grain verticalization, scarcity of micritic envelopes, broken and/or reworked foraminifera) clues in thin-section. Bioclastic or oolitic grainstones showing evidence of vadose diagenesis or pedogenetic imprints, should always be suspected of having an eolian origin.     

Acute hypertonicity alters aquaporin-2 trafficking and induces a MAPK-dependent accumulation at the plasma membrane of renal epithelial cells

The unique phenotype of renal medullary cells allows them to survive and functionally adapt to changes of interstitial osmolality/tonicity. We investigated the effects of acute hypertonic challenge on AQP2 (aquaporin-2) water channel trafficking. In the absence of vasopressin, hypertonicity alone induced rapid (<10 min) plasma membrane accumulation of AQP2 in rat kidney collecting duct principal cells in situ, and in several kidney epithelial lines. Confocal microscopy revealed that AQP2 also accumulated in the trans-Golgi network (TGN) following hypertonic challenge. AQP2 mutants that mimic the Ser(256)-phosphorylated and -nonphosphorylated state accumulated at the cell surface and TGN, respectively. Hypertonicity did not induce a change in cytosolic cAMP concentration, but inhibition of either calmodulin or cAMP-dependent protein kinase A activity blunted the hypertonicity-induced increase of AQP2 cell surface expression. Hypertonicity increased p38, ERK1/2, and JNK MAPK activity. Inhibiting MAPK activity abolished hypertonicity-induced accumulation of AQP2 at the cell surface but did not affect either vasopressin-dependent AQP2 trafficking or hypertonicity-induced AQP2 accumulation in the TGN. Finally, increased AQP2 cell surface expression induced by hypertonicity largely resulted from a reduction in endocytosis but not from an increase in exocytosis. These data indicate that acute hypertonicity profoundly alters AQP2 trafficking and that hypertonicity-induced AQP2 accumulation at the cell surface depends on MAP kinase activity. This may have important implications on adaptational processes governing transcellular water flux and/or cell survival under extreme conditions of hypertonicity.     

‘Glocal’ Robustness Analysis and Model Discrimination for Circadian Oscillators

To characterize the behavior and robustness of cellular circuits with many unknown parameters is a major challenge for systems biology. Its difficulty rises exponentially with the number of circuit components. We here propose a novel analysis method to meet this challenge. Our method identifies the region of a high-dimensional parameter space where a circuit displays an experimentally observed behavior. It does so via a Monte Carlo approach guided by principal component analysis, in order to allow efficient sampling of this space. This ‘global’ analysis is then supplemented by a ‘local’ analysis, in which circuit robustness is determined for each of the thousands of parameter sets sampled in the global analysis. We apply this method to two prominent, recent models of the cyanobacterial circadian oscillator, an autocatalytic model, and a model centered on consecutive phosphorylation at two sites of the KaiC protein, a key circadian regulator. For these models, we find that the two-sites architecture is much more robust than the autocatalytic one, both globally and locally, based on five different quantifiers of robustness, including robustness to parameter perturbations and to molecular noise. Our ‘glocal’ combination of global and local analyses can also identify key causes of high or low robustness. In doing so, our approach helps to unravel the architectural origin of robust circuit behavior. Complementarily, identifying fragile aspects of system behavior can aid in designing perturbation experiments that may discriminate between competing mechanisms and different parameter sets.