In vitro and in vivo studies on oxygen free radical and DNA adduct formation in rat lung and liver during benzo[a]pyrene metabolism

Reactive oxygen species (ROS), possibly produced during the metabolic conversion of benzo(a)pyrene (B[a]P), could be involved in B[a]P-induced genotoxicity and, eventually, carcinogenicity. Therefore, ROS formation by rat lung and liver microsomes was studied in vitro by electron spin resonance (ESR/EPR) spectrometry. B[a]P-mediated generation of ROS was detected in incubations with rat lung, but not with liver microsomes. Inhibition of cytochrome P450 (CYP450) by the non isoform-specific inhibitor SKF-525A resulted in a complete inhibition of B[a]P-dependent ROS formation, whereas ROS formation was not affected by inhibition of prostaglandin H synthase by indomethacin. Subsequently, bulky DNA adduct formation and 8-oxo-dG levels after a single oral dose of B[a]P were examined in vivo in rat lung and liver, in combination with urinary excretion of 8-oxodG. B[a]P exposure resulted in increased urinary 8-oxo-dG levels. On the contrary, 8-oxo-dG levels decreased in liver and lung after B[a]P exposure. Bulky DNA adducts reached higher levels and were more persistent in rat lung than in liver. These results indicate that ROS are generated during the CYP450 dependent metabolism of B[a]P, particularly in the rat lung, but this does not necessarily result in increased levels of oxidative DNA damage in vivo, possibly by induction of DNA repair mechanisms.     

Dominance of Geobacteraceae in BTX-degrading enrichments from an iron-reducing aquifer

Microbial community structure was linked to degradation potential in benzene-, toluene- or xylene- (BTX) degrading, iron-reducing enrichments derived from an iron-reducing aquifer polluted with landfill leachate. Enrichments were characterized using 16S rRNA gene-based analysis, targeting of the benzylsuccinate synthase-encoding bssA gene and phospholipid fatty acid (PLFA) profiling in combination with tracking of labelled substrate. 16S rRNA gene analysis indicated the dominance of Geobacteraceae, and one phylotype in particular, in all enrichments inoculated with polluted aquifer material. Upon cultivation, progressively higher degradation rates with a concomitant decrease in species richness occurred in all primary incubations and successive enrichments. Yet, the same Geobacteraceae phylotype remained common and dominant, indicating its involvement in BTX degradation. However, the bssA gene sequences in BTX degrading enrichments differed considerably from those of Geobacter isolates, suggesting that the first steps of toluene, but also benzene and xylene oxidation, are carried out by another member of the enrichments. Therefore, BTX would be synthrophically degraded by a bacterial consortium in which Geobacteraceae utilized intermediate metabolites. PLFA analysis in combination with (13)C-toluene indicated that the enriched Geobacteraceae were assimilating carbon originally present in toluene. Combined with previous studies, this research suggests that Geobacteraceae play a key role in the natural attenuation of each BTX compound in situ.     

Long‐term recovery of the functional community assembly and carbon pools in an African tropical forest succession

On the African continent, the population is expected to expand fourfold in the next century, which will increasingly impact the global carbon cycle and biodiversity conservation. Therefore, it is of vital importance to understand how carbon stocks and community assembly recover after slash‐and‐burn events in tropical second growth forests. We inventoried a chronosequence of 15 1‐ha plots in lowland tropical forest of the central Congo Basin and evaluated changes in aboveground and soil organic carbon stocks and in tree species diversity, functional composition, and community‐weighted functional traits with succession. We aimed to track long‐term recovery trajectories of species and carbon stocks in secondary forests, comparing 5 to 200 + year old secondary forest with reference primary forest. Along the successional gradient, the functional composition followed a trajectory from resource acquisition to resource conservation, except for nitrogen‐related leaf traits. Despite a fast, initial recovery of species diversity and functional composition, there were still important structural and carbon stock differences between old growth secondary and pristine forest, which suggests that a full recovery of secondary forests might take much longer than currently shown. As such, the aboveground carbon stocks of 200 + year old forest were only 57% of those in the pristine reference forest, which suggests a slow recovery of aboveground carbon stocks, although more research is needed to confirm this observation. The results of this study highlight the need for more in‐depth studies on forest recovery in Central Africa, to gain insight into the processes that control biodiversity and carbon stock recovery.     

Aphid populations are frequently infected with facultative endosymbionts

The occurrence of facultative endosymbionts has been studied in many commercially important crop pest aphids, but their occurrence and effects in non-commercial aphid species in natural populations have received less attention. We screened 437 aphid samples belonging to 106 aphid species for the eight most common facultative aphid endosymbionts. We found one or more facultative endosymbionts in 53% (56 of 106) of the species investigated. This likely underestimates the situation in the field because facultative endosymbionts are often present in only some colonies of an aphid species. Oligophagous aphid species carried facultative endosymbionts significantly more often than monophagous species. We did not find a significant correlation between ant tending and facultative endosymbiont presence. In conclusion, we found that facultative endosymbionts are common among aphid populations. This study is, to our knowledge, the first of its kind in the Netherlands and provides a basis for future research in this field. For instance, it is still unknown in what way many of these endosymbionts affect their hosts, which is important for determining the importance of facultative endosymbionts to community dynamics.     

Measuring endothelial glycocalyx dimensions in humans: a potential novel tool to monitor vascular vulnerability.

The endothelial glycocalyx is increasingly considered as an intravascular compartment that protects the vessel wall against pathogenic insults. The purpose of this study was to translate an established experimental method of estimating capillary glycocalyx dimension into a clinically useful tool and to assess its reproducibility in humans. We first evaluated by intravital microscopy the relation between the distance between the endothelium and erythrocytes, as a measure of glycocalyx thickness, and the transient widening of the erythrocyte column on glycocalyx compression by passing leukocytes in hamster cremaster muscle capillaries. We subsequently assessed sublingual microvascular glycocalyx thickness in 24 healthy men using orthogonal polarization spectral imaging. In parallel, systemic glycocalyx volume (using a previously published tracer dilution technique) as well as cardiovascular risk profiles were assessed. Estimates of microvascular glycocalyx dimension from the transient erythrocyte widening correlated well with the size of the erythrocyte-endothelium gap (r = 0.63). Measurements in humans were reproducible (0.58 +/- 0.16 and 0.53 +/- 0.15 microm, coefficient of variance 15 +/- 5%). In univariate analysis, microvascular glycocalyx thickness significantly correlated with systemic glycocalyx volume (r = 0.45), fasting plasma glucose (r = 0.43), and high-density lipoprotein-cholesterol (r = 0.40) and correlated negatively with low-density lipoprotein-cholesterol (r = -0.41) as well as body mass index (r = -0.45) (all P < 0.05). In conclusion, the dimension of the endothelial glycocalyx can be measured reproducibly in humans and is related to cardiovascular risk factors. It remains to be tested whether glycocalyx dimension can be used as an early marker of vascular damage and whether therapies aimed at glycocalyx repair can protect the vasculature against pathogenic challenges.     

The Gustatory Signaling Pathway and Bitter Taste Receptors Affect the Development of Obesity and Adipocyte Metabolism in Mice

Intestinal chemosensory signaling pathways involving the gustatory G-protein, gustducin, and bitter taste receptors (TAS2R) have been implicated in gut hormone release. Alterations in gut hormone profiles may contribute to the success of bariatric surgery. This study investigated the involvement of the gustatory signaling pathway in the development of diet-induced obesity and the therapeutic potential of targeting TAS2Rs to induce body weight loss. α-gustducin-deficient (α-gust^-/-) mice became less obese than wild type (WT) mice when fed a high-fat diet (HFD). White adipose tissue (WAT) mass was lower in α-gust^-/- mice due to increased heat production as a result of increases in brown adipose tissue (BAT) thermogenic activity, involving increased protein expression of uncoupling protein 1. Intra-gastric treatment of obese WT and α-gust^-/- mice with the bitter agonists denatonium benzoate (DB) or quinine (Q) during 4 weeks resulted in an α-gustducin-dependent decrease in body weight gain associated with a decrease in food intake (DB), but not involving major changes in gut peptide release. Both WAT and 3T3-F442A pre-adipocytes express TAS2Rs. Treatment of pre-adipocytes with DB or Q decreased differentiation into mature adipocytes. In conclusion, interfering with the gustatory signaling pathway protects against the development of HFD-induced obesity presumably through promoting BAT activity. Intra-gastric bitter treatment inhibits weight gain, possibly by directly affecting adipocyte metabolism.     

Glucocorticoid‐mediated modulation of morphological changes associated with aging in microglia

Microglia dynamically adapt their morphology and function during increasing age. However, the mechanisms behind these changes are to date poorly understood. Glucocorticoids (GCs) are long known and utilized for their immunomodulatory actions and endogenous GC levels are described to alter with advancing age. We here tested the hypothesis that age‐associated elevations in GC levels implicate microglia function and morphology. Our data indicate a decrease in microglial complexity and a concomitant increase in GC levels during aging. Interestingly, enhancing GC levels in young mice enhanced microglial ramifications, while the knockdown of the glucocorticoid receptor expression in old mice aggravated age‐associated microglial amoebification. These data suggest that GCs increase ramification of hippocampal microglia and may modulate age‐associated changes in microglial morphology.     

Activation of immune receptor Rx1 triggers distinct immune responses culminating in cell death after 4 hours

Intracellular nucleotide-binding leucine-rich repeat (NLR)-type immune receptors are a fundamental part of plant immune systems. As infection occurs at foci, activation of immune responses is typically non-uniform and non-synchronized, hampering the systematic dissection of their cellular effects and determining their phasing. We investigated the potato NLR Rx1 using the CESSNA (Controlled Expression of effectors for Synchronized and Systemic NLR Activation) platform. CESSNA-mediated Potato virus X coat protein (CP) expression allowed the monitoring of Rx1-mediated immune responses in a quantitative and reproducible manner. Rx1 was found to trigger a reactive oxygen species (ROS) burst and ion leakage within 1 h and a change in autofluorescence within 2 h after the induction of CP production. After 2 h, HIN1 expression was increased and single-stranded DNA (ssDNA) damage and loss of cellular integrity became apparent, followed by double-stranded DNA (dsDNA) damage after 3 h and increased PR-1a, LOX, ERF1 and AOX1B expression and cell death at 4 h. Nuclear exclusion of Rx1 resulted in increased basal levels of ROS and permitted Rx1 activation by an Rx1-breaking CP variant. In contrast, nuclear-targeted Rx1 showed diminished basal ROS levels, and only avirulent CP could trigger a compromised ROS production. Both nuclear-excluded and nuclear-targeted Rx1 triggered a delayed ion leakage compared with non-modified Rx1, suggesting that ion leakage and ROS production originate from distinct signalling pathways. This work offers novel insights into the influence of Rx1 localization on its activity, and the interplay between Rx1-triggered processes.