Sequestration of DROSHA and DGCR8 by Expanded CGG RNA Repeats Alters MicroRNA Processing in Fragile X-Associated Tremor/Ataxia Syndrome

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Highlights? DGCR8 binds to CGG RNA repeats, cause of the neurodegenerative FXTAS disease ? DGCR8 and its partner, DROSHA, are sequestered within CGG RNA aggregates ? DGCR8 rescues the neuronal cell death induced by expanded CGG RNA repeats ? MicroRNA processing is impaired in patients with FXTAS     

Reversibility of germinative and endocrine testicular function after long-term contraception with a GnRH-agonist implant in the tom—a follow-up study

A significantly reduced gonadotropin and testosterone secretion is a well-described result of long-term administration of GnRH agonists in the male dog and cat. To date, no data are available about the duration of efficacy and the reversibility of treatment-induced effects after long-term treatment with a 4.7 mg deslorelin implant. Seven healthy male European Shorthair cats (3.2 ± 0.5 kg, 1–6 years) were treated with a 4.7 mg deslorelin implant. Blood samples (testosterone, T), testicular volume, penile spines, and mating behavior were recorded once weekly. Considering T > 0.5 ng/mL as the biological endpoint, mean duration of efficacy was 78.8 ± 12.9 weeks (range: 61.7–100.7 weeks) with T concentrations increasing rapidly after the last T less than 0.1 ng/mL (basal) (P < 0.0001), and pretreatment T concentrations being reached after 3 weeks. Testicular volume rapidly increased after the first increase of T (P < 0.001) with pretreatment testicular volume being reached after 6.9 ± 3.4 weeks (5–11 weeks). “Normal” libido reoccurred 88.7 ± 12.4 weeks after treatment, and “normal” mating behavior was observed even later. Fertile matings occurred 7 to 42 weeks after the last T less than 0.1 ng/mL with a mean of 4.0 ± 0.0 kittens, and 13.6 to 47.6 weeks afterwards testicular histology revealed normal spermatogenesis. The present data confirm that the use of slow-release GnRH-agonist implants containing deslorelin in tomcats represents an effective and safe reversible alternative for long-term contraception; however, as number of animals is low, further fertility trials are recommended.     

Targeting DNA with novel diphenylcarbazoles

Double-stranded DNA is a therapeutic target for a variety of anticancer and antimicrobial drugs. Noncovalent interactions of small molecules with DNA usually occur via intercalation of planar compounds between adjacent base pairs or minor-groove recognition by extended crescent-shaped ligands. However, the dynamic and flexibility of the DNA platform provide a variety of conformations that can be targeted by structurally diverse compounds. Here, we propose a novel DNA-binding template for construction of new therapeutic candidates. Four bisphenylcarbazole derivatives, derived from the combined molecular architectures of known antitumor bisphenylbenzimidazoles and anti-infectious dicationic carbazoles, have been designed, and their interaction with DNA has been studied by a combination of biochemical and biophysical methods. The substitutions of the bisphenylcarbazole core with two terminal dimethylaminoalkoxy side chains strongly promote the interaction with DNA, to prevent the heat denaturation of the double helix. The deletion or the replacement of the dimethylamino-terminal groups with hydroxyl groups strongly decreased DNA interaction, and the addition of a third cationic side chain on the carbazole nitrogen reinforced the affinity of the compound for DNA. Although the bi- and tridentate molecules both derive from well-characterized DNA minor-groove binders, the analysis of their binding mode by means of circular and linear dichroism methods suggests that these compounds form intercalation complexes with DNA. Negative-reduced dichroism signals were recorded in the presence of natural DNA and synthetic AT and GC polynucleotides. The intercalation hypothesis was validated by unwinding experiments using topoisomerase I. Prominent gel shifts were observed with the di- and trisubstituted bisphenylcarbazoles but not with the uncharged analogues. These observations, together with the documented stacking properties of such molecules (components for liquid crystals), prompted us to investigate their binding to the human telomeric DNA sequence by means of biosensor surface plasmon resonance. Under conditions favorable to G4 formation, the title compounds showed only a modest interaction with the telomeric quadruplex sequence, comparable to that measured with a double-stranded oligonucleotide. Their sequence preference was explored by DNase I footprinting experiments from which we identified a composite set of binding sequences comprising short AT stretches and a few other mixed AT/GC blocks with no special AT character. The variety of the binding sequences possibly reflects the coexistence of distinct positioning of the chromophore in the intercalation sites. The bisphenylcarbazole unit represents an original pharmacophore for DNA recognition. Its branched structure, with two or three arms suitable to introduce a structural diversity, provides an interesting scaffold to built molecules susceptible to discriminate between the different conformations of nucleic acids.     

Phosphorylation of Threonine 794 on Tie1 by Rac1/PAK1 Reveals a Novel Angiogenesis Regulatory Pathway

The endothelial receptor tyrosine kinase (RTK) Tie1 was discovered over 20 years ago, yet its precise function and mode of action remain enigmatic. To shed light on Tie1’s role in endothelial cell biology, we investigated a potential threonine phosphorylation site within the juxtamembrane domain of Tie1. Expression of a non-phosphorylatable mutant of this site (T794A) in zebrafish (Danio rerio) significantly disrupted vascular development, resulting in fish with stunted and poorly branched intersomitic vessels. Similarly, T794A-expressing human umbilical vein endothelial cells formed significantly shorter tubes with fewer branches in three-dimensional Matrigel cultures. However, mutation of T794 did not alter Tie1 or Tie2 tyrosine phosphorylation or downstream signaling in any detectable way, suggesting that T794 phosphorylation may regulate a Tie1 function independent of its RTK properties. Although T794 is within a consensus Akt phosphorylation site, we were unable to identify a physiological activator of Akt that could induce T794 phosphorylation, suggesting that Akt is not the physiological Tie1-T794 kinase. However, the small GTPase Ras-related C3 botulinum toxin substrate 1 (Rac1), which is required for angiogenesis and capillary morphogenesis, was found to associate with phospho-T794 but not the non-phosphorylatable T794A mutant. Pharmacological activation of Rac1 induced downstream activation of p21-activated kinase (PAK1) and T794 phosphorylation in vitro, and inhibition of PAK1 abrogated T794 phosphorylation. Our results provide the first demonstration of a signaling pathway mediated by Tie1 in endothelial cells, and they suggest that a novel feedback loop involving Rac1/PAK1 mediated phosphorylation of Tie1 on T794 is required for proper angiogenesis.     

Sense-overlapping lncRNA as a decoy of translational repressor protein for dimorphic gene expression

Long noncoding RNAs (lncRNAs) are vastly transcribed and extensively studied but lncRNAs overlapping with the sense orientation of mRNA have been poorly studied. We analyzed the lncRNA DAPALR overlapping with the 5´ UTR of the Doublesex1 (Dsx1), the male determining gene in Daphnia magna. By affinity purification, we identified an RNA binding protein, Shep as a DAPALR binding protein. Shep also binds to Dsx1 5´ UTR by recognizing the overlapping sequence and suppresses translation of the mRNA. In vitro and in vivo analyses indicated that DAPALR increased Dsx1 translation efficiency by sequestration of Shep. This regulation was impaired when the Shep binding site in DAPALR was deleted. These results suggest that Shep suppresses the unintentional translation of Dsx1 by setting a threshold; and when the sense lncRNA DAPALR is expressed, DAPALR cancels the suppression caused by Shep. This mechanism may be important to show dimorphic gene expressions such as sex determination and it may account for the binary expression in various developmental processes.     

A Novel Ribosomopathy Caused by Dysfunction of RPL10 Disrupts Neurodevelopment and Causes X-Linked Microcephaly in Humans

Neurodevelopmental defects in humans represent a clinically heterogeneous group of disorders. Here, we report the genetic and functional dissection of a multigenerational pedigree with an X-linked syndromic disorder hallmarked by microcephaly, growth retardation, and seizures. Using an X-linked intellectual disability (XLID) next-generation sequencing diagnostic panel, we identified a novel missense mutation in the gene encoding 60S ribosomal protein L10 (RPL10), a locus associated previously with autism spectrum disorders (ASD); the p.K78E change segregated with disease under an X-linked recessive paradigm while, consistent with causality, carrier females exhibited skewed X inactivation. To examine the functional consequences of the p.K78E change, we modeled RPL10 dysfunction in zebrafish. We show that endogenous rpl10 expression is augmented in anterior structures, and that suppression decreases head size in developing morphant embryos, concomitant with reduced bulk translation and increased apoptosis in the brain. Subsequently, using in vivo complementation, we demonstrate that p.K78E is a loss-of-function variant. Together, our findings suggest that a mutation within the conserved N-terminal end of RPL10, a protein in close proximity to the peptidyl transferase active site of the 60S ribosomal subunit, causes severe defects in brain formation and function.     

Early predictors of cardiac decompensation in experimental volume overload

The adaptation to chronic hypoxia confers long-lasting cardiac protection against acute ischemia–reperfusion injury. Protein kinase C (PKC) appears to play a role in the cardioprotective mechanism but the involvement of individual PKC isoforms remains unclear. The aim of this study was to examine the effects of chronic intermittent hypoxia (CIH; 7,000 m, 8 h/day) and acute administration of PKC-δ inhibitor (rottlerin, 0.3 mg/kg) on the expression and subcellular distribution of PKC-δ and PKC-ε in the left ventricular myocardium of adult male Wistar rats by Western blot and quantitative immunofluorescence microscopy. CIH decreased the total level of PKC-ε in homogenate without affecting the level of phosphorylated PKC-ε (Ser729). In contrast, CIH up-regulated the total level of PKC-δ as well as the level of phosphorylated PKC-δ (Ser643) in homogenate. Rottlerin partially reversed the hypoxia-induced increase in PKC-δ in the mitochondrial fraction. Immunofluorescent staining of ventricular cryo-sections revealed increased co-localization of PKC-δ with mitochondrial and sarcolemmal membranes in CIH hearts that was suppressed by rottlerin. The formation of nitrotyrosine as a marker of oxidative stress was enhanced in CIH myocardium, particularly in mitochondria. The expression of total oxidative phosphorylation complexes was slightly decreased by CIH mainly due to complex II decline. In conclusion, up-regulated PKC-δ in CIH hearts is mainly localized to mitochondrial and sarcolemmal membranes. The inhibitory effects of rottlerin on PKC-δ subcellular redistribution and cardioprotection (as shown previously) support the view that this isoform plays a role in the mechanism of CIH-induced ischemic tolerance.     

Hair canopy of cricket sensory system tuned to predator signals

Filiform hairs located on the cerci of crickets are among the most sensitive sensors in the animal world and enable crickets to sense the faintest air movements generated by approaching predators. While the neurophysiological and biomechanical aspects of this sensory system have been studied independently for several decades, their integration into a coherent framework was wanting. In order to evaluate the hair canopy tuning to predator signals, we built a model of cercal population coding of oscillating air flows by the hundreds of hairs on the cerci of the sand cricket Gryllus bimaculatus (Insecta: Orthoptera). A complete survey of all hairs covering the cerci was done on intact cerci using scanning electronic microscopy. An additive population coding of sinusoid signals of varying frequencies and velocities taking into account hair directionality delivered the cercal canopy tuning curve. We show that the range of frequencies and velocities at which the cricket sensory system is best tuned corresponds to the values of signals produced by approaching predators. The relative frequencies of short (< 0.5 x 10(-3) m) and long hairs and their differing responses to oscillating air flows therefore enable crickets to detect predators in a time-frequency-intensity space both as far as possible and at close range.