Insulin and TOR signal in parallel through FOXO and S6K to promote epithelial wound healing

The TOR and Insulin/IGF signalling (IIS) network controls growth, metabolism and ageing. Although reducing TOR or insulin signalling can be beneficial for ageing, it can be detrimental for wound healing, but the reasons for this difference are unknown. Here we show that IIS is activated in the cells surrounding an epidermal wound in Drosophila melanogaster larvae, resulting in PI3K activation and redistribution of the transcription factor FOXO. Insulin and TOR signalling are independently necessary for normal wound healing, with FOXO and S6K as their respective effectors. IIS is specifically required in cells surrounding the wound, and the effect is independent of glycogen metabolism. Insulin signalling is needed for the efficient assembly of an actomyosin cable around the wound, and constitutively active myosin II regulatory light chain suppresses the effects of reduced IIS. These findings may have implications for the role of insulin signalling and FOXO activation in diabetic wound healing

Complete atrial-specific knockout of sodium-calcium exchange eliminates sinoatrial node pacemaker activity.

The origin of sinoatrial node (SAN) pacemaker activity in the heart is controversial. The leading candidates are diastolic depolarization by "funny" current (If) through HCN4 channels (the "Membrane Clock" hypothesis), depolarization by cardiac Na-Ca exchange (NCX1) in response to intracellular Ca cycling (the "Calcium Clock" hypothesis), and a combination of the two ("Coupled Clock"). To address this controversy, we used Cre/loxP technology to generate atrial-specific NCX1 KO mice. NCX1 protein was undetectable in KO atrial tissue, including the SAN. Surface ECG and intracardiac electrograms showed no atrial depolarization and a slow junctional escape rhythm in KO that responded appropriately to β-adrenergic and muscarinic stimulation. Although KO atria were quiescent they could be stimulated by external pacing suggesting that electrical coupling between cells remained intact. Despite normal electrophysiological properties of If in isolated patch clamped KO SAN cells, pacemaker activity was absent. Recurring Ca sparks were present in all KO SAN cells, suggesting that Ca cycling persists but is uncoupled from the sarcolemma. We conclude that NCX1 is required for normal pacemaker activity in murine SAN

Mechanism of virus attenuation by codon pair deoptimization

Codon pair deoptimization is an efficient virus attenuation strategy, but the mechanism that leads to attenuation is unknown. The strategy involves synthetic recoding of viral genomes that alters the positions of synonymous codons, thereby increasing the number of suboptimal codon pairs and CpG dinucleotides in recoded genomes. Here we identify the molecular mechanism of codon pair deoptimization-based attenuation by studying recoded influenza A viruses. We show that suboptimal codon pairs cause attenuation, whereas the increase of CpG dinucleotides has no effect. Furthermore, we show that suboptimal codon pairs reduce both mRNA stability and translation efficiency of codon pair-deoptimized genes. Consequently, reduced protein production directly causes virus attenuation. Our study provides evidence that suboptimal codon pairs are major determinants of mRNA stability. Additionally, it demonstrates that codon pair bias can be used to increase mRNA stability and protein production of synthetic genes in many areas of biotechnology

The Putative AKH Receptor of the Tobacco Hornworm, Manduca sexta, and Its Expression

Adipokinetic hormones are peptide hormones that mobilize lipids and/or carbohydrates for flight in adult insects and activate glycogen Phosphorylase in larvae during starvation and during molt. We previously examined the functional roles of adipokinetic hormone in Manduca sexta L. (Lepidoptera: Sphingidae). Here we report the cloning of the full-length cDNA encoding the putative adipokinetic hormone receptor from the fat body of M. sexta. The sequence analysis shows that the deduced amino acid sequence shares common motifs of G protein-coupled receptors, by having seven hydrophobic transmembrane segments. We examined the mRNA expression pattern of the adipokinetic hormone receptor by quantitative Real-Time PCR in fat body during development and in different tissues and found the strongest expression in fat body of larvae two days after molt to the fifth instar. We discuss these results in relation to some of our earlier results. We also compare the M. sexta adipokinetic hormone receptor with the known adipokinetic hormone receptors of other insects and with gonadotropin releasing hormone-like receptors of invertebrates

Mutations of Mitochondrial DNA Are Not Major Contributors to Aging of Fruit Flies

Mammals develop age-associated clonal expansion of somatic mtDNA mutations resulting in severe respiratory chain deficiency in a subset of cells in a variety of tissues. Both mathematical modeling based on descriptive data from humans and experimental data from mtDNA mutator mice suggest that the somatic mutations are formed early in life and then undergo mitotic segregation during adult life to reach very high levels in certain cells. To address whether mtDNA mutations have a universal effect on aging metazoans, we investigated their role in physiology and aging of fruit flies. To this end, we utilized genetically engineered flies expressing mutant versions of the catalytic subunit of mitochondrial DNA polymerase (DmPOLγA) as a means to introduce mtDNA mutations. We report here that lifespan and health in fruit flies are remarkably tolerant to mtDNA mutations. Our results show that the short lifespan and wide genetic bottleneck of fruit flies are limiting the extent of clonal expansion of mtDNA mutations both in individuals and between generations. However, an increase of mtDNA mutations to very high levels caused sensitivity to mechanical and starvation stress, intestinal stem cell dysfunction, and reduced lifespan under standard conditions. In addition, the effects of dietary restriction, widely considered beneficial for organismal health, were attenuated in flies with very high levels of mtDNA mutations

A Drosophila

Glucocerebrosidase (GBA1) mutations are associated with Gaucher disease (GD), an autosomal recessive disorder caused by functional deficiency of glucocerebrosidase (GBA), a lysosomal enzyme that hydrolyzes glucosylceramide to ceramide and glucose. Neuronopathic forms of GD can be associated with rapid neurological decline (Type II) or manifest as a chronic form (Type III) with a wide spectrum of neurological signs. Furthermore, there is now a well-established link between GBA1 mutations and Parkinson's disease (PD), with heterozygote mutations in GBA1 considered the commonest genetic defect in PD. Here we describe a novel Drosophila model of GD that lacks the two fly GBA1 orthologs. This knock-out model recapitulates the main features of GD at the cellular level with severe lysosomal defects and accumulation of glucosylceramide in the fly brain. We also demonstrate a block in autophagy flux in association with reduced lifespan, age-dependent locomotor deficits and accumulation of autophagy substrates in dGBA-deficient fly brains. Furthermore, mechanistic target of rapamycin (mTOR) signaling is downregulated in dGBA knock-out flies, with a concomitant upregulation of Mitf gene expression, the fly ortholog of mammalian TFEB, likely as a compensatory response to the autophagy block. Moreover, the mTOR inhibitor rapamycin is able to partially ameliorate the lifespan, locomotor, and oxidative stress phenotypes. Together, our results demonstrate that this dGBA1-deficient fly model is a useful platform for the further study of the role of lysosomal-autophagic impairment and the potential therapeutic benefits of rapamycin in neuronopathic GD. These results also have important implications for the role of autophagy and mTOR signaling in GBA1-associated PD

Sclerotiorin stabilizes the assembly of nonfibrillar Abeta42 oligomers with low toxicity, seeding activity, and beta-sheet content

The self-assembly of the 42-residue amyloid-β peptide, Aβ42, into fibrillar aggregates is associated with neuronal dysfunction and toxicity in Alzheimer's disease (AD) patient brains, suggesting that small molecules acting on this process might interfere with pathogenesis. Here, we present experimental evidence that the small molecule sclerotiorin (SCL), a natural product belonging to the group of azaphilones, potently delays both seeded and non-seeded Aβ42 polymerization in cell-free assays. Mechanistic biochemical studies revealed that the inhibitory effect of SCL on fibrillogenesis is caused by its ability to kinetically stabilize small Aβ42 oligomers. These structures exhibit low β-sheet content and do not possess seeding activity, indicating that SCL acts very early in the amyloid formation cascade before the assembly of seeding-competent, β-sheet-rich fibrillar aggregates. Investigations with NMR WaterLOGSY experiments confirmed the association of Aβ42 assemblies with SCL in solution. Furthermore, using ion mobility-mass spectrometry we observed that SCL directly interacts with a small fraction of Aβ42 monomers in the gas phase. In comparison to typical amyloid fibrils, small SCL-stabilized Aβ42 assemblies are inefficiently taken up into mammalian cells and have low toxicity in cell-based assays. Overall, these mechanistic studies support a pathological role of stable, β-sheet-rich Aβ42 fibrils in AD, while structures with low β-sheet content may be less relevant

Evidence for a Grooming Claw in a North American Adapiform Primate: Implications for Anthropoid Origins

Among fossil primates, the Eocene adapiforms have been suggested as the closest relatives of living anthropoids (monkeys, apes, and humans). Central to this argument is the form of the second pedal digit. Extant strepsirrhines and tarsiers possess a grooming claw on this digit, while most anthropoids have a nail. While controversial, the possible presence of a nail in certain European adapiforms has been considered evidence for anthropoid affinities. Skeletons preserved well enough to test this idea have been lacking for North American adapiforms. Here, we document and quantitatively analyze, for the first time, a dentally associated skeleton of Notharctus tenebrosus from the early Eocene of Wyoming that preserves the complete bones of digit II in semi-articulation. Utilizing twelve shape variables, we compare the distal phalanges of Notharctus tenebrosus to those of extant primates that bear nails (n = 21), tegulae (n = 4), and grooming claws (n = 10), and those of non-primates that bear claws (n = 7). Quantitative analyses demonstrate that Notharctus tenebrosus possessed a grooming claw with a surprisingly well-developed apical tuft on its second pedal digit. The presence of a wide apical tuft on the pedal digit II of Notharctus tenebrosus may reflect intermediate morphology between a typical grooming claw and a nail, which is consistent with the recent hypothesis that loss of a grooming claw occurred in a clade containing adapiforms (e.g. Darwinius masillae) and anthropoids. However, a cladistic analysis including newly documented morphologies and thorough representation of characters acknowledged to have states constituting strepsirrhine, haplorhine, and anthropoid synapomorphies groups Notharctus tenebrosus and Darwinius masillae with extant strepsirrhines rather than haplorhines suggesting that the form of pedal digit II reflects substantial homoplasy during the course of early primate evolution