Adaptation to developmental diet influences the response to selection on age at reproduction in the fruit fly

Experimental evolution (EE) is a powerful tool for addressing how environmental factors influence life‐history evolution. While in nature different selection pressures experienced across the lifespan shape life histories, EE studies typically apply selection pressures one at a time. Here, we assess the consequences of adaptation to three different developmental diets in combination with classical selection for early or late reproduction in the fruit fly Drosophila melanogaster. We find that the response to each selection pressure is similar to that observed when they are applied independently, but the overall magnitude of the response depends on the selection regime experienced in the other life stage. For example, adaptation to increased age at reproduction increased lifespan across all diets; however, the extent of the increase was dependent on the dietary selection regime. Similarly, adaptation to a lower calorie developmental diet led to faster development and decreased adult weight, but the magnitude of the response was dependent on the age‐at‐reproduction selection regime. Given that multiple selection pressures are prevalent in nature, our findings suggest that trade‐offs should be considered not only among traits within an organism, but also among adaptive responses to different—sometimes conflicting—selection pressures, including across life stages.     

Two functionally distinct Axin-like proteins regulate canonical Wnt signaling in C. elegans

Axin is a central component of the canonical Wnt signaling pathway that interacts with the adenomatous polyposis coli protein APC and the kinase GSK3beta to downregulate the effector beta-catenin. In the nematode Caenorhabditis elegans, canonical Wnt signaling is negatively regulated by the highly divergent Axin ortholog PRY-1. Mutation of pry-1 leads to constitutive activation of BAR-1/beta-catenin-dependent Wnt signaling and results in a range of developmental defects. The pry-1 null phenotype is however not fully penetrant, indicating that additional factors may partially compensate for PRY-1 function. Here, we report the cloning and functional analysis of a second Axin-like protein, which we named AXL-1. We show that despite considerable sequence divergence with PRY-1 and other Axin family members, AXL-1 is a functional Axin ortholog. AXL-1 functions redundantly with PRY-1 in negatively regulating BAR-1/beta-catenin signaling in the developing vulva and the Q neuroblast lineage. In addition, AXL-1 functions independently of PRY-1 in negatively regulating canonical Wnt signaling during excretory cell development. In contrast to vertebrate Axin and the related protein Conductin, AXL-1 and PRY-1 are not functionally equivalent. We conclude that Axin function in C. elegans is divided over two different Axin orthologs that have specific functions in negatively regulating canonical Wnt signaling.     

SIRT2 regulates adipocyte differentiation through FoxO1 acetylation/deacetylation

The family of mammalian Sirtuin proteins comprises seven members homologous to yeast Sir2. Here we show that SIRT2, a cytoplasmic sirtuin, is the most abundant sirtuin in adipocytes. Sirt2 expression is downregulated during preadipocyte differentiation in 3T3-L1 cells. Overexpression of SIRT2 inhibits differentiation, whereas reducing SIRT2 expression promotes adipogenesis. Both effects are accompanied by corresponding changes in the expression of PPARgamma, C/EBPalpha, and genes marking terminal adipocyte differentiation, including Glut4, aP2, and fatty acid synthase. The mechanism underlying the effects of reduced SIRT2 in 3T3-L1 adipocytes includes increased acetylation of FOXO1, with direct interaction between SIRT2 and FOXO1. This interaction enhances insulin-stimulated phosphorylation of FOXO1, which in turn regulates FOXO1 nuclear and cytosolic localization. Thus, Sirt2 acts as an important regulator of adipocyte differentiation through modulation of FOXO1 acetylation/phosphorylation and activity and may play a role in controlling adipose tissue mass and function.     

Unexpected Receptor Functional Mimicry Elucidates Activation of Coronavirus Fusion

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Phloem import and storage metabolism are highly coordinated by the low oxygen concentrations within developing wheat seeds

We studied the influence of the internal oxygen concentration in seeds of wheat (Triticum aestivum) on storage metabolism and its relation to phloem import of nutrients. Wheat seeds that were developing at ambient oxygen (21%) were found to be hypoxic (2.1%). Altering the oxygen supply by decreasing or increasing the external oxygen concentration induced parallel changes in the internal oxygen tension. However, the decrease in internal concentration was proportionally less than the reduction in external oxygen. This indicates that decreasing the oxygen supply induces short-term adaptive responses to reduce oxygen consumption of the seeds. When external oxygen was decreased to 8%, internal oxygen decreased to approximately 0.5% leading to a decrease in energy production via respiration. Conversely, increasing the external oxygen concentration above ambient levels increased the oxygen content as well as the energy status of the seeds, indicating that under normal conditions the oxygen supply is strongly limiting for energy metabolism in developing wheat seeds. The intermediate metabolites of seed storage metabolism were not substantially affected when oxygen was either increased or decreased. However, at subambient external oxygen concentrations (8%) the metabolic flux of carbon into starch and protein, measured by injecting (14)C-Suc into the seeds, was reduced by 17% and 32%, respectively, whereas no significant effect was observed at superambient (40%) oxygen. The observed decrease in biosynthetic fluxes to storage compounds is suggested to be part of an adaptive response to reduce energy consumption preventing excessive oxygen consumption when oxygen supply is limited. Phloem transport toward ears exposed to low (8%) oxygen was significantly reduced within 1 h, whereas exposing ears to elevated oxygen (40%) had no significant effect. This contrasts with the situation where the distribution of assimilates has been modified by removing the lower source leaves from the plant, resulting in less assimilates transported to the ear in favor of transport to the lower parts of the plant. Under these conditions, with two strongly competing sinks, elevated oxygen (40%) did lead to a strong increase in phloem transport to the ear. The results show that sink metabolism is affected by the prevailing low oxygen concentrations in developing wheat seeds, determining the import rate of assimilates via the phloem.     

Development and validation of a gas chromatography-mass spectrometry method for the simultaneous determination of buprenorphine, flunitrazepam and their metabolites in rat plasma: application to the pharmacokinetic study

Buprenorphine (BUP), a synthetic opioid analgesic, is frequently abused alone, and in association with benzodiazepines. Fatalities involving buprenorphine alone seem very unusual while its association with benzodiazepines, such as flunitrazepam (FNZ), has been reported to result in severe respiratory depression and death. The quantitative relationship between these drugs remain, however, uncertain. Our objective was to develop an analytical method that could be used as a means to study and explore, in animals, the toxicity and pharmacological interaction mechanisms between buprenorphine, flunitrazepam and their active metabolites. A procedure based on gas chromatography-mass spectrometry (GC-MS) is described for the simultaneous analysis of buprenorphine, norbuprenorphine (NBUP), flunitrazepam, N-desmethylflunitrazepam (N-DMFNZ) and 7-aminoflunitrazepam (7-AFNZ) in rat plasma. The method was set up and adapted for the analysis of small plasma samples taken from rats. Plasma samples were extracted by liquid-liquid extraction using Toxi-tubes A. Extracted compounds were derivatized with N,O-bis-(trimethylsilyl)trifluoroacetamide (BSTFA), using trimethylchlorosilane (TMCS) as a catalyst. They were then separated by GC on a crosslinked 5% phenyl-methylpolysiloxane analytical column and determined by a quadrupole mass spectrometer detector operated under selected ion monitoring mode. Excellent linearity was found between 0.125 and 25 ng/microl plasma for BUP, 0.125 and 12.5 ng/microl for NBUP and N-DMFNZ, 0.125 and 5 ng/microl for FNZ, and between 0.025 and 50 ng/microl for 7-AFNZ. The limit of quantification was 0.025 ng/microl plasma for 7-AFNZ and 0.125 ng/microl for the four other compounds. A good reproducibility (intra-assay CV=0.32-11.69%; inter-assay CV=0.63-9.55%) and accuracy (intra-assay error=2.58-12.73%; inter-assay error=0.83-11.07%) were attained. Recoveries were 71, 67 and 81%, for BUP, FNZ and N-DMFNZ, respectively, and 51% for NBUP and 7-AFNZ, with CV ranging from 5.4 to 13.9%, and were concentration-independent. The GC-MS method was successfully applied to the pharmacokinetic study of BUP, NBUP, FNZ, DMFNZ and 7-AFNZ in rats, after administration of BUP and FNZ.