cGMP-Phosphodiesterase Inhibition Enhances Photic Responses and Synchronization of the Biological Circadian Clock in Rodents

The master circadian clock in mammals is located in the hypothalamic suprachiasmatic nuclei (SCN) and is synchronized by several environmental stimuli, mainly the light-dark (LD) cycle. Light pulses in the late subjective night induce phase advances in locomotor circadian rhythms and the expression of clock genes (such as Per1-2). The mechanism responsible for light-induced phase advances involves the activation of guanylyl cyclase (GC), cGMP and its related protein kinase (PKG). Pharmacological manipulation of cGMP by phosphodiesterase (PDE) inhibition (e.g., sildenafil) increases low-intensity light-induced circadian responses, which could reflect the ability of the cGMP-dependent pathway to directly affect the photic sensitivity of the master circadian clock within the SCN. Indeed, sildenafil is also able to increase the phase-shifting effect of saturating (1200 lux) light pulses leading to phase advances of about 9 hours, as well as in C57 a mouse strain that shows reduced phase advances. In addition, sildenafil was effective in both male and female hamsters, as well as after oral administration. Other PDE inhibitors (such as vardenafil and tadalafil) also increased light-induced phase advances of locomotor activity rhythms and accelerated reentrainment after a phase advance in the LD cycle. Pharmacological inhibition of the main downstream target of cGMP, PKG, blocked light-induced expression of Per1. Our results indicate that the cGMP-dependent pathway can directly modulate the light-induced expression of clock-genes within the SCN and the magnitude of light-induced phase advances of overt rhythms, and provide promising tools to design treatments for human circadian disruptions

Complete vertebrate mitogenomes reveal widespread repeats and gene duplications

Abstract: Background: Modern sequencing technologies should make the assembly of the relatively small mitochondrial genomes an easy undertaking. However, few tools exist that address mitochondrial assembly directly. Results: As part of the Vertebrate Genomes Project (VGP) we develop mitoVGP, a fully automated pipeline for similarity-based identification of mitochondrial reads and de novo assembly of mitochondrial genomes that incorporates both long (> 10 kbp, PacBio or Nanopore) and short (100–300 bp, Illumina) reads. Our pipeline leads to successful complete mitogenome assemblies of 100 vertebrate species of the VGP. We observe that tissue type and library size selection have considerable impact on mitogenome sequencing and assembly. Comparing our assemblies to purportedly complete reference mitogenomes based on short-read sequencing, we identify errors, missing sequences, and incomplete genes in those references, particularly in repetitive regions. Our assemblies also identify novel gene region duplications. The presence of repeats and duplications in over half of the species herein assembled indicates that their occurrence is a principle of mitochondrial structure rather than an exception, shedding new light on mitochondrial genome evolution and organization. Conclusions: Our results indicate that even in the “simple” case of vertebrate mitogenomes the completeness of many currently available reference sequences can be further improved, and caution should be exercised before claiming the complete assembly of a mitogenome, particularly from short reads alone

Heterogeneity and complexity within the nuclease module of the Ccr4-Not complex

The shortening of the poly(A) tail of cytoplasmic mRNA (deadenylation) is a pivotal step in the regulation of gene expression in eukaryotic cells. Deadenylation impacts on both regulated mRNA decay as well as the rate of mRNA translation. An important enzyme complex involved in poly(A) shortening is the Ccr4-Not deadenylase. In addition to at least six non-catalytic subunits, it contains two distinct subunits with ribonuclease activity: a Caf1 subunit, characterized by a DEDD (Asp-Glu-Asp-Asp) domain, and a Ccr4 component containing an endonuclease-exonuclease-phosphatase (EEP) domain. In vertebrate cells, the complexity of the complex is further increased by the presence of paralogs of the Caf1 subunit (encoded by either CNOT7 or CNOT8) and the occurrence of two Ccr4 paralogs (encoded by CNOT6 or CNOT6L). In plants, there are also multiple Caf1 and Ccr4 paralogs. Thus, the composition of the Ccr4-Not complex is heterogeneous. The potential differences in the intrinsic enzymatic activities of the paralogs will be discussed. In addition, the potential redundancy, cooperation, and/or the extent of unique roles for the deadenylase subunits of the Ccr4-Not complex will be reviewed. Finally, novel approaches to study the catalytic roles of the Caf1 and Ccr4 subunits will be discussed

Hydraulic transients caused by air expulsion during rapid filling of undulating pipelines

One of the main issues arising during the rapid filling of a pipeline is the pressure transient which originates after the entrapped air has been expelled at the air release valve. Because of the difference in density between water and air, a pressure transient originates at the impact of the water column. Many authors have analyzed the problem, both from the theoretical and the experimental standpoint. Nevertheless, mainly vertical or horizontal pipelines have been analyzed, whereas in real field applications, the pipe profile is a sequence of ascending and descending pipes, with air release/vacuum valves at high points. To overcome lack of knowledge regarding this latter case, laboratory experiments were carried out to simulate the filling of an undulating pipeline, initially empty at atmospheric pressure. The pipe profile has a high point where an orifice is installed for air venting, so as to simulate the air release valve at intermediate high point of a supply pipeline. In the experiments, the diameter of the orifice and the opening degree of both upstream and downstream valves were varied, in order to analyze their effect on the pressure transient. The experiments were also carried out with a longer descending pipe, in order to assess the effects on the pressure surge of the air volume downstream of the orifice