Age-regulated cycling metabolites are relevant for behavior

Circadian cycles of sleep:wake and gene expression change with age in all organisms examined. Metabolism is also under robust circadian regulation, but little is known about how metabolic cycles change with age and whether these contribute to the regulation of behavioral cycles. To address this gap, we compared cycling of metabolites in young and old Drosophila and found major age-related variations. A significant model separated the young metabolic profiles by circadian timepoint, but could not be defined for the old metabolic profiles due to the greater variation in this dataset. Of the 159 metabolites measured in fly heads, we found 17 that cycle by JTK analysis in young flies and 17 in aged. Only four metabolites overlapped in the two groups, suggesting that cycling metabolites are distinct in young and old animals. Among our top cyclers exclusive to young flies were components of the pentose phosphate pathway (PPP). As the PPP is important for buffering reactive oxygen species, and overexpression of glucose-6-phosphate dehydrogenase (G6PD), a key component of the PPP, was previously shown to extend lifespan in Drosophila, we asked if this manipulation also affects sleep:wake cycles. We found that overexpression in circadian clock neurons decreases sleep in association with an increase in cellular calcium and mitochondrial oxidation, suggesting that altering PPP activity affects neuronal activity. Our findings elucidate the importance of metabolic regulation in maintaining patterns of neural activity, and thereby sleep:wake cycles.     

Development of a primary microglia screening assay and its use to characterize inhibition of system xc- by erastin and its analogs

The inflammatory response in the central nervous system involves activated microglia. Under normal conditions they remove damaged neurons by phagocytosis. On the other hand, neurodegenerative diseases are thought to involve chronic microglia activation resulting in release of excess glutamate, proinflammatory cytokines and reactive oxygen species, leading to neuronal death. System x_C^- cystine/glutamate antiporter (SXC), a sodium independent heterodimeric transporter found in microglia and astrocytes in the CNS, imports cystine into the cell and exports glutamate. SXC has been shown to be upregulated in neurodegenerative diseases including multiple sclerosis, ALS, neuroAIDS Parkinson's disease and Alzheimer's disease. Consequently, SXC inhibitors could be of use in the treatment of diseases characterized by neuroinflammation and glutamate excitotoxicity. We report on the optimization of a primary microglia-based assay to screen for SXC inhibitors. Rat primary microglia were activated using lipopolysaccharides (LPS) and glutamate release and cystine uptake were monitored by fluorescence and radioactivity respectively. LPS-induced glutamate release increased with increasing cell density, time of incubation and LPS concentration. Conditions to screen for SXC inhibitors were optimized in 96-well format and subsequently used to evaluate SXC inhibitors. Known SXC inhibitors sulfasalazine, S-4CPG and erastin blocked glutamate release and cystine uptake while R-4CPG, the inactive enantiomer of S-4CPG, failed to inhibit glutamate release or cystine transport. In addition, several erastin analogs were evaluated using primary microglia and found to have EC₅₀ values in agreement with previous studies using established cell lines.     

Cu(I)-glutathione complex: a potential source of superoxide radicals generation

Cu(2+) ions and GSH molecules interact swiftly to form the complex Cu(I)-glutathione. We investigated the potential capacity of such complex to reduce molecular oxygen. The addition of SOD to a solution containing Cu(I)-glutathione led to a sustained decline of the basal oxygen level. Such effect was partially reverted by the addition of catalase. The complex was able to induce the reduction of cytochrome c and the oxidation of dyhydroethidium into 2-hydroxyethidium. Both effects were totally blocked by SOD. The ability of the complex to generate superoxide radicals was confirmed by EPR spin-trapping. Cu(I)-glutathione induces no oxidation of fluorescein, a hydroxyl radical-sensitive probe. We conclude that in solutions containing the complex, oxygen is continually reduced into superoxide, and that-in absence of interceptors-the latter radicals are quantitatively re-oxidized into molecular oxygen. We suggest that, by functioning as a continuous source of superoxide, the complex could potentially affect a broad range of susceptible biological targets.     

Chelicerae as male grasping organs in scorpions: sexual dimorphism and associated behaviour

Specialised structures that enable males to grasp females during sexual interactions are highly susceptible to selection and thus diverge relatively rapidly over evolutionary time. These structures are often used to test hypotheses regarding sexual selection such as sexually antagonistic co-evolution and sexual selection by female choice. In the present study, we determine whether there is a relationship between a novel record of scorpion sexual dimorphism, the sexual dimorphism of chelicerae (CSD), and the presence of the mating behaviour termed “cheliceral grip” (CG). The presence of both traits in the order Scorpiones is also reviewed from a phylogenetic perspective. The results confirm a strong relationship between CSD and the presence of CG. The morphological and behavioural patterns associated with “CSD–CG” are opposed to the predictions postulated by the hypothesis of sexually antagonistic co-evolution. However, if the female shows resistance after the deposition of the spermatophore, the possibility that the male exerts pressure as a “cryptic form” of coercion to prevent the interruption of mating cannot be ruled out completely. Female choice by “mechanical fit” could be another explanation for some aspects of the CG's contact zone. The possibility that the “CG–CSD” complex has evolved under natural selection in order to ensure sperm transfer is also considered.     

Crystal Structure of a Yeast Aquaporin at 1.15 ? Reveals a Novel Gating Mechanism

Aquaporins are transmembrane proteins that facilitate the flow of water through cellular membranes. An unusual characteristic of yeast aquaporins is that they frequently contain an extended N terminus of unknown function. Here we present the X-ray structure of the yeast aquaporin Aqy1 from Pichia pastoris at 1.15 Å resolution. Our crystal structure reveals that the water channel is closed by the N terminus, which arranges as a tightly wound helical bundle, with Tyr31 forming H-bond interactions to a water molecule within the pore and thereby occluding the channel entrance. Nevertheless, functional assays show that Aqy1 has appreciable water transport activity that aids survival during rapid freezing of P. pastoris. These findings establish that Aqy1 is a gated water channel. Mutational studies in combination with molecular dynamics simulations imply that gating may be regulated by a combination of phosphorylation and mechanosensitivity.     

A Complex Population Structure of the Cassava Pathogen Xanthomonas axonopodis pv. manihotis in Recent Years in the Caribbean Region of Colombia

Cassava bacterial blight, caused by Xanthomonas axonopodis pv. manihotis (Xam), is the most important bacterial disease affecting this crop. A continuous surveillance of the pathogen population dynamics is required to develop an efficient disease management program. During the 1990s, Xam populations showed high levels of genetic variation and relevant migratory processes that were important determinants of the distribution of the pathogen diversity in Colombia. Aiming to characterize the current population structure of the pathogen and the evolutionary forces that shape these populations, sampling collections were carried out from September 2008 until November 2010 in the Colombian Caribbean Region. One hundred and sixty bacterial isolates were characterized using amplified fragment length polymorphism (AFLP) markers. Additionally, a subset of effector genes were sequenced in some isolates to determine their usefulness in Xam population studies and to provide additional information to that obtained with AFLPs. Virulence patterns of ten isolates were determined in nine cassava accessions. Our results show a complex architecture of population and confirm migratory process previously reported in the Caribbean Region. Chinú, one of the locations sampled, presented remarkable features in population dynamics such as longer genetic distances, higher diversity indices, and a genetically differentiated population when it was compared with other locations. Virulence tests showed that MCOL2215, one of the most cultivated cassava varieties in the Caribbean coast, was susceptible to the majority of Xam isolates tested. This study shows the current condition of populations of Xam in the Caribbean Region of Colombia, and it contributes to improve the existing bacterial blight control practices.