Myristoylation exerts direct and allosteric effects on Gα conformation and dynamics in solution

Coupling of heterotrimeric G proteins to activated G protein-coupled receptors results in nucleotide exchange on the Gα subunit, which in turn decreases its affinity for both Gβγ and activated receptors. N-Terminal myristoylation of Gα subunits aids in membrane localization of inactive G proteins. Despite the presence of the covalently attached myristoyl group, Gα proteins are highly soluble after GTP binding. This study investigated factors facilitating the solubility of the activated, myristoylated protein. In doing so, we also identified myristoylation-dependent differences in regions of Gα known to play important roles in interactions with receptors, effectors, and nucleotide binding. Amide hydrogen-deuterium exchange and site-directed fluorescence of activated proteins revealed a solvent-protected amino terminus that was enhanced by myristoylation. Furthermore, fluorescence quenching confirmed that the myristoylated amino terminus is in proximity to the Switch II region in the activated protein. Myristoylation also stabilized the interaction between the guanine ring and the base of the α5 helix that contacts the bound nucleotide. The allosteric effects of myristoylation on protein structure, function, and localization indicate that the myristoylated amino terminus of Gα(i) functions as a myristoyl switch, with implications for myristoylation in the stabilization of nucleotide binding and in the spatial regulation of G protein signaling.     

Nrbf2 Protein Suppresses Autophagy by Modulating Atg14L Protein-containing Beclin 1-Vps34 Complex Architecture and Reducing Intracellular Phosphatidylinositol-3 Phosphate Levels

Autophagy is a tightly regulated lysosomal degradation pathway for maintaining cellular homeostasis and responding to stresses. Beclin 1 and its interacting proteins, including the class III phosphatidylinositol-3 kinase Vps34, play crucial roles in autophagy regulation in mammals. We identified nuclear receptor binding factor 2 (Nrbf2) as a Beclin 1-interacting protein from Becn1^−/−;Becn1-EGFP/+ mouse liver and brain. We also found that Nrbf2-Beclin 1 interaction required the N terminus of Nrbf2. We next used the human retinal pigment epithelial cell line RPE-1 as a model system and showed that transiently knocking down Nrbf2 by siRNA increased autophagic flux under both nutrient-rich and starvation conditions. To investigate the mechanism by which Nrbf2 regulates autophagy, we demonstrated that Nrbf2 interacted and colocalized with Atg14L, suggesting that Nrbf2 is a component of the Atg14L-containing Beclin 1-Vps34 complex. Moreover, ectopically expressed Nrbf2 formed cytosolic puncta that were positive for isolation membrane markers. These results suggest that Nrbf2 is involved in autophagosome biogenesis. Furthermore, we showed that Nrbf2 deficiency led to increased intracellular phosphatidylinositol-3 phosphate levels and diminished Atg14L-Vps34/Vps15 interactions, suggesting that Nrbf2-mediated Atg14L-Vps34/Vps15 interactions likely inhibit Vps34 activity. Therefore, we propose that Nrbf2 may interact with the Atg14L-containing Beclin 1-Vps34 protein complex to modulate protein-protein interactions within the complex, leading to suppression of Vps34 activity, autophagosome biogenesis, and autophagic flux. This work reveals a novel aspect of the intricate mechanism for the Beclin 1-Vps34 protein-protein interaction network to achieve precise control of autophagy.     

Sperm structure and ultrastructure of the Melittobia hawaiiensis, Perkins and M. australica, Girault (chalcidoidea: eulophidae)

Spermatozoa morphology has, for some years, been used to help answer some phylogenetic questions for Hymenoptera. This is the second study describing spermatozoa morphology of an Eulophidae species in which important characteristics were observed. Melittobia spermatozoa are spiralled and measure approximately 270mum in length. The head contains a small acrosome, apparently formed only by an acrosomal vesicle, which, together with the initial nuclear region, is surrounded by an extracellular sheath, from which innumerable filaments irradiate. The nucleus is helicoidal and completely filled with compact chromatin. A centriolar adjunct is observed at the nucleus-flagellum transition; it associates laterally with the nucleus and exhibits two small expansions, which reach around the centriole. In the flagellum there are two mitochondrial derivatives, which in cross-sections are asymmetric. In the derivative with the larger diameter, two distinct regions are observed, a small one, near the axoneme, with a clear "fissure" inside, and a larger region where the cristae occur. Both derivatives initiate at the nuclear base, but the larger diameter derivative finishes first, before the flagellum extremity. At the end of the axoneme, the accessory microtubules are the first to finish.     

Livers with Constitutive mTORC1 Activity Resist Steatosis Independent of Feedback Suppression of Akt

Insulin resistance is an important contributing factor in non-alcoholic fatty liver disease. AKT and mTORC1 are key components of the insulin pathway, and play a role in promoting de novo lipogenesis. However, mTORC1 hyperactivity per se does not induce steatosis in mouse livers, but instead, protects against high-fat diet induced steatosis. Here, we investigate the in vivo mechanism of steatosis-resistance secondary to mTORC1 activation, with emphasis on the role of S6K1-mediated feedback inhibition of AKT. Mice with single or double deletion of Tsc1 and/or S6k1 in a liver-specific or whole-body manner were generated to study glucose and hepatic lipid metabolism between the ages of 6–14 weeks. Following 8 weeks of high-fat diet, the Tsc1-/-;S6k1-/- mice had lower body weights but higher liver TG levels compared to that of the Tsc1-/- mice. However, the loss of S6k1 did not relieve feedback inhibition of Akt activity in the Tsc1-/- livers. To overcome Akt suppression, Pten was deleted in Tsc1-/- livers, and the resultant mice showed improved glucose tolerance compared with the Tsc1-/- mice. However, liver TG levels were significantly reduced in the Tsc1-/-;Pten-/- mice compared to the Pten-/- mice, which was restored with rapamycin. We found no correlation between liver TG and serum NEFA levels. Expression of lipogenic genes (Srebp1c, Fasn) were elevated in the Tsc1-/-;Pten-/- livers, but this was counter-balanced by an up-regulation of Cpt1a involved in fatty acid oxidation and the anti-oxidant protein, Nrf2. In summary, our in vivo models showed that mTORC1-induced resistance to steatosis was dependent on S6K1 activity, but not secondary to AKT suppression. These findings confirm that AKT and mTORC1 have opposing effects on hepatic lipid metabolism in vivo.     

Demographic Clusters Identified within the Northern Gulf of Mexico Common Bottlenose Dolphin (Tursiops truncates) Unusual Mortality Event: January 2010 - June 2013

A multi-year unusual mortality event (UME) involving primarily common bottlenose dolphins (Tursiops truncates) was declared in the northern Gulf of Mexico (GoM) with an initial start date of February 2010 and remains ongoing as of August 2014. To examine potential changing characteristics of the UME over time, we compared the number and demographics of dolphin strandings from January 2010 through June 2013 across the entire GoM as well as against baseline (1990-2009) GoM stranding patterns. Years 2010 and 2011 had the highest annual number of stranded dolphins since Louisiana’s record began, and 2011 was one of the years with the highest strandings for both Mississippi and Alabama. Statewide, annual numbers of stranded dolphins were not elevated for GoM coasts of Florida or Texas during the UME period. Demographic, spatial, and temporal clusters identified within this UME included increased strandings in northern coastal Louisiana and Mississippi (March-May 2010); Barataria Bay, Louisiana (August 2010-December 2011); Mississippi and Alabama (2011, including a high prevalence and number of stranded perinates); and multiple GoM states during early 2013. While the causes of the GoM UME have not been determined, the location and magnitude of dolphin strandings during and the year following the 2010 Deepwater Horizon oil spill, including the Barataria Bay cluster from August 2010 to December 2011, overlap in time and space with locations that received heavy and prolonged oiling. There are, however, multiple known causes of previous GoM dolphin UMEs, including brevetoxicosis and dolphin morbillivirus. Additionally, increased dolphin strandings occurred in northern Louisiana and Mississippi before the Deepwater Horizon oil spill. Identification of spatial, temporal, and demographic clusters within the UME suggest that this mortality event may involve different contributing factors varying by location, time, and bottlenose dolphin populations that will be better discerned by incorporating diagnostic information, including histopathology.     

Structural basis for RNA-genome recognition during bacteriophage Qβ replication

Upon infection of Escherichia coli by bacteriophage Qβ, the virus-encoded β-subunit recruits host translation elongation factors EF-Tu and EF-Ts and ribosomal protein S1 to form the Qβ replicase holoenzyme complex, which is responsible for amplifying the Qβ (+)-RNA genome. Here, we use X-ray crystallography, NMR spectroscopy, as well as sequence conservation, surface electrostatic potential and mutational analyses to decipher the roles of the β-subunit and the first two oligonucleotide-oligosaccharide-binding domains of S1 (OB_1–2) in the recognition of Qβ (+)-RNA by the Qβ replicase complex. We show how three basic residues of the β subunit form a patch located adjacent to the OB₂ domain, and use NMR spectroscopy to demonstrate for the first time that OB₂ is able to interact with RNA. Neutralization of the basic residues by mutagenesis results in a loss of both the phage infectivity in vivo and the ability of Qβ replicase to amplify the genomic RNA in vitro. In contrast, replication of smaller replicable RNAs is not affected. Taken together, our data suggest that the β-subunit and protein S1 cooperatively bind the (+)-stranded Qβ genome during replication initiation and provide a foundation for understanding template discrimination during replication initiation.     

The Network of Knowledge approach: improving the science and society dialogue on biodiversity and ecosystem services in Europe

The absence of a good interface between scientific and other knowledge holders and decision-makers in the area of biodiversity and ecosystem services has been recognised for a long time. Despite recent advancements, e.g. with the Intergovernmental Platform on Biodiversity and Ecosystem Services (IPBES), challenges remain, particularly concerning the timely provision of consolidated views from different knowledge domains. To address this challenge, a strong and flexible networking approach is needed across knowledge domains and institutions. Here, we report on a broad consultation process across Europe to develop a Network of Knowledge on biodiversity and ecosystem services (NoK), an approach aiming at (1) organising institutions and knowledge holders in an adaptable and responsive framework and (2) informing decision-makers with timely and accurate biodiversity knowledge. The consultation provided a critical analysis of the needs that should be addressed by a NoK and how it could complement existing European initiatives and institutions at the interface between policy and science. Among other functions, the NoK provides consolidated scientific views on contested topics, identification of research gaps to support relevant policies, and horizon scanning activities to anticipate emerging issues. The NoK includes a capacity building component on interfacing activities and contains mechanisms to ensure its credibility, relevance and legitimacy. Such a network would need to ensure credibility, relevance and legitimacy of its work by maximizing transparency and flexibility of processes, quality of outputs, the link to data and knowledge provision, the motivation of experts for getting involved and sound communication and capacity building.     

Modeling Interventions in the Owned Cat Population to Decrease Numbers,Knox County,TN

To find management strategies for controlling the owned cat population in Knox County, TN, the authors formulated a mathematical model using biological properties of such nonhuman animals and spay actions on certain age classes. They constructed this discrete-time model to predict the future owned cat population in this county and to evaluate intervention strategies to surgically sterilize some proportion of the population. Using the predicted population size and the number of surgeries for specific scenarios, they showed that focusing on specific age classes can be an effective feature in spay programs.     

Symptoms in Response to Controlled Diesel Exhaust More Closely Reflect Exposure Perception Than True Exposure

Background

Diesel exhaust (DE) exposures are very common, yet exposure-related symptoms haven’t been rigorously examined.

Objective

Describe symptomatic responses to freshly generated and diluted DE and filtered air (FA) in a controlled human exposure setting; assess whether such responses are altered by perception of exposure.

Methods

43 subjects participated within three double-blind crossover experiments to order-randomized DE exposure levels (FA and DE calibrated at 100 and/or 200 micrograms/m³ particulate matter of diameter less than 2.5 microns), and completed questionnaires regarding symptoms and dose perception.

Results

For a given symptom cluster, the majority of those exposed to moderate concentrations of diesel exhaust do not report such symptoms. The most commonly reported symptom cluster was of the nose (29%). Blinding to exposure is generally effective. Perceived exposure, rather than true exposure, is the dominant modifier of symptom reporting.

Conclusion

Controlled human exposure to moderate-dose diesel exhaust is associated with a range of mild symptoms, though the majority of individuals will not experience any given symptom. Blinding to DE exposure is generally effective. Perceived DE exposure, rather than true DE exposure, is the dominant modifier of symptom reporting.