Segmental duplications are hot spots of copy number variants affecting barley gene content

Copy number variants (CNVs) are pervasive in several animal and plant genomes and contribute to shaping genetic diversity. In barley, there is evidence that changes in gene copy number underlie important agronomic traits. The recently released reference sequence of barley represents a valuable genomic resource for unveiling the incidence of CNVs that affect gene content and for identifying sequence features associated with CNV formation. Using exome sequencing and read count data, we detected 16 605 deletions and duplications that affect barley gene content by surveying a diverse panel of 172 cultivars, 171 landraces, 22 wild relatives and other 32 uncategorized domesticated accessions. The quest for segmental duplications (SDs) in the reference sequence revealed many low‐copy repeats, most of which overlap predicted coding sequences. Statistical analyses revealed that the incidence of CNVs increases significantly in SD‐rich regions, indicating that these sequence elements act as hot spots for the formation of CNVs. The present study delivers a comprehensive genome‐wide study of CNVs affecting barley gene content and implicates SDs in the molecular mechanisms that lead to the formation of this class of CNVs.     

Rosiglitazone induces autophagy in H295R and cell cycle deregulation in SW13 adrenocortical cancer cells

Thiazolidinediones, specific peroxisome proliferator-activated receptor-?γ (PPAR-γ) ligands, used in type-2 diabetes therapy, show favourable effects in several cancer cells. In this study we demonstrate that the growth of H295R and SW13 adrenocortical cancer cells is inhibited by rosiglitazone, a thiazolidinediones member, even though the mechanisms underlying this effect appeared to be cell-specific. Treatment with GW9662, a selective PPAR-γ-inhibitor, showed that rosiglitazone acts through both PPAR-γ-dependent and -independent mechanisms in H295R, while in SW13 cells the effect seems to be independent of PPAR-γ. H295R cells treated with rosiglitazone undergo an autophagic process, leading to morphological changes detectable by electron microscopy and an increased expression of specific proteins such as AMPKα and beclin-1. The autophagy seems to be independent of PPAR-γ activation and could be related to an increase in oxidative stress mediated by reactive oxygen species production with the disruption of the mitochondrial membrane potential, triggered by rosiglitazone. In SW13 cells, flow cytometry analysis showed an arrest in the G0/G1 phase of the cell cycle with a decrease of cyclin E and cdk2 activity, following the administration of rosiglitazone. Our data show the potential role of rosiglitazone in the therapeutic approach to adrenocortical carcinoma and indicate the molecular mechanisms at the base of its antiproliferative effects, which appear to be manifold and cell-specific in adrenocortical cancer lines.     

A molecular dynamics study of a miRNA:mRNA interaction

In this paper we present a methodology to evaluate the binding free energy of a miRNA:mRNA complex through molecular dynamics (MD)–thermodynamic integration (TI) simulations. We applied our method to the Caenorhabditis elegans let-7 miRNA:lin-41 mRNA complex—a validated miRNA:mRNA interaction—in order to estimate the energetic stability of the structure. To make the miRNA:mRNA simulation possible and realistic, the methodology introduces specific solutions to overcome some of the general challenges of nucleic acid simulations and binding free energy computations that have been discussed widely in many previous research reports. The main features of the proposed methodology are: (1) positioning of the restraints imposed on the simulations in order to guarantee complex stability; (2) optimal sampling of the phase space to achieve satisfactory accuracy in the binding energy value; (3) determination of a suitable trade-off between computational costs and accuracy of binding free energy computation by the assessment of the scalability characteristics of the parallel simulations required for the TI. The experiments carried out demonstrate that MD simulations are a viable strategy for the study of miRNA binding characteristics, opening the way to the development of new computational target prediction methods based on three-dimensional structure information.     

Antinociceptive profile of LP1, a non-peptide multitarget opioid ligand

AimsOpioid drugs are the principal treatment option for moderate to severe pain and exert their biological effects through interactions with opioid receptors that are widely distributed throughout the CNS and peripheral tissues. Ligands capable of simultaneously targeting different receptors could be successful candidates for the treatment of chronic pain. Enhanced antinociception coupled with a low incidence of side effects has been demonstrated for ligands possessing mixed mu-opioid receptor (MOR) and delta-opioid receptor (DOR) activity. We previously reported that 3-[(2R,6R,11R)-8-hydroxy-6,11-dimethyl-1,4,5,6-tetrahydro-2,6-methano-3-benzazocin-3(2 H)-yl]-N-phenylpropanamide (LP1) acted as a MOR-DOR ligand in in vitro functional assays and moreover this drug produced a valid antinociception that was longer lasting than that of morphine. The aim of this work was to determine whether the antinociceptive effect produced by LP1 was central or peripheral and to assess which opioid receptor subtypes are involved in its effects.Main methodsWe explored the effects of naloxone methiodide (NX-M), a quaternary opioid antagonist, administered either intracerebroventricularly (i.c.v.) or subcutaneously (s.c.), on LP1-mediated antinociception in male Sprague–Dawley rats. In addition, we administered s.c. selective antagonists for MOR, DOR and kappa-opioid receptor (KOR) to investigate the effects of LP1. To characterise this drug's DOR profile better, we also investigated the effects of LP1 on DPDPE, a selective DOR agonist.Key findingsData obtained by tail flick test showed that LP1 induced predominantly MOR-mediated supraspinal antinociception and was able to counteract DPDPE analgesia.SignificanceLP1, a multitarget opioid ligand, is a supraspinal acting antinociceptive agent that is useful for the treatment of chronic pain.     

miRNAs expression analysis in paired fresh/frozen and dissected formalin fixed and paraffin embedded glioblastoma using real-time pCR

miRNAs are small molecules involved in gene regulation. Each tissue shows a characteristic miRNAs epression profile that could be altered during neoplastic transformation. Glioblastoma is the most aggressive brain tumour of the adult with a high rate of mortality. Recognizing a specific pattern of miRNAs for GBM could provide further boost for target therapy. The availability of fresh tissue for brain specimens is often limited and for this reason the possibility of starting from formalin fixed and paraffin embedded tissue (FFPE) could very helpful even in miRNAs expression analysis. We analysed a panel of 19 miRNAs in 30 paired samples starting both from FFPE and Fresh/Frozen material. Our data revealed that there is a good correlation in results obtained from FFPE in comparison with those obtained analysing miRNAs extracted from Fresh/Frozen specimen. In the few cases with a not good correlation value we noticed that the discrepancy could be due to dissection performed in FFPE samples. To the best of our knowledge this is the first paper demonstrating that the results obtained in miRNAs analysis using Real-Time PCR starting from FFPE specimens of glioblastoma are comparable with those obtained in Fresh/Frozen samples.     

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.