Synthesis of Indolizine and Pyrrolo[1,2- a ]azepine Derivatives via a Gold(I)-Catalyzed Three-Step Cascade

International audienceLinear N-alkenyl or alkynyl N-sulfonyl 1-aminobut-3-yn-2-ones are converted into bicyclic indolizines and pyrrolo[1,2-a]azepine-type alkaloids upon gold(I) catalysis (17 examples, 10-85%). The reaction cascade allowed to form C-N, O-S and CC bonds via a cycloisomerization/sulfonyl migration/cyclization process using 10 mol % of [(2-biphenyl)di-tert-butylphosphine]gold(I) triflimide complex in dichloromethane

Shape Self-Regulation in Early Lung Morphogenesis

The arborescent architecture of mammalian conductive airways results from the repeated branching of lung endoderm into surrounding mesoderm. Subsequent lung’s striking geometrical features have long raised the question of developmental mechanisms involved in morphogenesis. Many molecular actors have been identified, and several studies demonstrated the central role of Fgf10 and Shh in growth and branching. However, the actual branching mechanism and the way branching events are organized at the organ scale to achieve a self-avoiding tree remain to be understood through a model compatible with evidenced signaling. In this paper we show that the mere diffusion of FGF10 from distal mesenchyme involves differential epithelial proliferation that spontaneously leads to branching. Modeling FGF10 diffusion from sub-mesothelial mesenchyme where Fgf10 is known to be expressed and computing epithelial and mesenchymal growth in a coupled manner, we found that the resulting laplacian dynamics precisely accounts for the patterning of FGF10-induced genes, and that it spontaneously involves differential proliferation leading to a self-avoiding and space-filling tree, through mechanisms that we detail. The tree’s fine morphological features depend on the epithelial growth response to FGF10, underlain by the lung’s complex regulatory network. Notably, our results suggest that no branching information has to be encoded and that no master routine is required to organize branching events at the organ scale. Despite its simplicity, this model identifies key mechanisms of lung development, from branching to organ-scale organization, and could prove relevant to the development of other branched organs relying on similar pathways

Mu opioid receptors on primary afferent nav1.8 neurons contribute to opiate-induced analgesia: insight from conditional knockout mice.

Opiates are powerful drugs to treat severe pain, and act via mu opioid receptors distributed throughout the nervous system. Their clinical use is hampered by centrally-mediated adverse effects, including nausea or respiratory depression. Here we used a genetic approach to investigate the potential of peripheral mu opioid receptors as targets for pain treatment. We generated conditional knockout (cKO) mice in which mu opioid receptors are deleted specifically in primary afferent Nav1.8-positive neurons. Mutant animals were compared to controls for acute nociception, inflammatory pain, opiate-induced analgesia and constipation. There was a 76% decrease of mu receptor-positive neurons and a 60% reduction of mu-receptor mRNA in dorsal root ganglia of cKO mice. Mutant mice showed normal responses to heat, mechanical, visceral and chemical stimuli, as well as unchanged morphine antinociception and tolerance to antinociception in models of acute pain. Inflammatory pain developed similarly in cKO and controls mice after Complete Freund's Adjuvant. In the inflammation model, however, opiate-induced (morphine, fentanyl and loperamide) analgesia was reduced in mutant mice as compared to controls, and abolished at low doses. Morphine-induced constipation remained intact in cKO mice. We therefore genetically demonstrate for the first time that mu opioid receptors partly mediate opiate analgesia at the level of Nav1.8-positive sensory neurons. In our study, this mechanism operates under conditions of inflammatory pain, but not nociception. Previous pharmacology suggests that peripheral opiates may be clinically useful, and our data further demonstrate that Nav1.8 neuron-associated mu opioid receptors are feasible targets to alleviate some forms of persistent pain

Germline loss-of-function mutations in EPHB4 cause a second form of capillary malformation-arteriovenous malformation (CM-AVM2) deregulating RAS-MAPK signaling

BACKGROUND: Most arteriovenous malformations (AVMs) are localized and occur sporadically. However, they also can be multifocal in autosomal-dominant disorders, such as hereditary hemorrhagic telangiectasia and capillary malformation (CM)-AVM. Previously, we identified RASA1 mutations in 50% of patients with CM-AVM. Herein we studied non-RASA1 patients to further elucidate the pathogenicity of CMs and AVMs. METHODS: We conducted a genome-wide linkage study on a CM-AVM family. Whole-exome sequencing was also performed on 9 unrelated CM-AVM families. We identified a candidate gene and screened it in a large series of patients. The influence of several missense variants on protein function was also studied in vitro. RESULTS: We found evidence for linkage in 2 loci. Whole-exome sequencing data unraveled 4 distinct damaging variants in EPHB4 in 5 families that cosegregated with CM-AVM. Overall, screening of EPHB4 detected 47 distinct mutations in 54 index patients: 27 led to a premature stop codon or splice-site alteration, suggesting loss of function. The other 20 are nonsynonymous variants that result in amino acid substitutions. In vitro expression of several mutations confirmed loss of function of EPHB4. The clinical features included multifocal CMs, telangiectasias, and AVMs. CONCLUSIONS: We found EPHB4 mutations in patients with multifocal CMs associated with AVMs. The phenotype, CM-AVM2, mimics RASA1-related CM-AVM1 and also hereditary hemorrhagic telangiectasia. RASA1-encoded p120RASGAP is a direct effector of EPHB4. Our data highlight the pathogenetic importance of this interaction and indicts EPHB4-RAS-ERK signaling pathway as a major cause for AVMs

The conditional deletion of mu receptor in Nav1.8 primary neurons does not abrogate tolerance to morphine-induced antinociception.

<p>Morphine dose-dependent antinociception was measured following repeated 4-day i.p. injections of morphine or saline in mu^fl controls, mu-cKO and mu-KO animals. The shift to right for both mu^fl and mu-cKO chronic-morphine animals indicates the development of a comparable tolerance to analgesia. Total mu-KO animals show no antinociception. n=6-7/genotype/treatment, two-way ANOVA (genotype x treatment F(1,30) = 19.919, <i>P</i> <0.001 for treatment; F(2,30) = 97.039, <i>P</i> <0.001 for genotype); post-hoc Fisher test for individual morphine doses, ✰ <i>P</i> <0.05, ✰✰ <i>P</i><0.01, chronic morphine (tolerance) <i>vs</i> chronic saline in mu^fl mice; ★ <i>P</i><0.05, ★★ <i>P</i><0.00 chronic morphine (tolerance) <i>vs</i> chronic saline in mu-cKO mice.</p

Conditional mu-cKO and control mice show comparable systemic morphine analgesia in nociceptive assays.

<p>Top. Morphine induced dose-dependent antinociception in both mu-cKO and mu^fl mice in the three heat assays, (<b>A</b>) tail immersion, (<b>B</b>) tail flick and (<b>C</b>) hot plate. Morphine-induced analgesia was abolished in conventional mu-KO animals (tail immersion, n=10/genotype; tail flick, n=10-14/genotype; hot plate, n=6-17/ genotype), confirming the selective effect of morphine on mu receptor. Bottom. Mu-cKO and mu^fl control mice show similar systemic morphine analgesia in the tail pressure (n=9-13/genotype) and acetic acid-induced visceral nociceptive (n=13-14/genotype) assays. Two-way ANOVA, post-hoc Fisher test for individual time points, ✰ <i>P</i> <0.05, ✰<b>✰ <i>P</i><0.01</b>, ✰✰✰ <i>P</i><0.001, morphine <i>vs</i> saline.</p

Inflammation increased the number of small/medium <i>Oprm1</i>-positive neurons in DRGs of mu^fl but not of mu-cKO mice.

<p>Inflammation was induced by intra-paw CFA as in previous figures. The cell size distribution of <i>Oprm1</i>-positive neurons in DRGs was evaluated by <i>In </i><i>Situ</i> Hybridization. The % of <i>Oprm1</i>-positive neurons in naïve mu^fl and mu-cKO DRGs are shown in white and black, respectively. The % of <i>Oprm1</i>-positive neurons in ipsilateral DRGs of CFA mu^fl and mu-cKO DRGs are shown in dotted white and black bars. ✰✰ <i>P</i> <0.01, ✰✰✰ <i>P</i> <0.001, CFA <i>vs</i> naïve; ★★★ <i>P</i><0.001 mu-cKO <i>vs</i> mu^fl, Student t-test.</p

Spontaneous guarding pain behavior after paw-CFA and CFA-inflammatory pain at day 9 in conditional mu-cKO mice.

<p>(<b>A</b>) The effect the conditional mutation on ongoing pain behavior was evaluated by quantifying the duration of guarding behavior over 6 min in mu^fl, mu-cKO and mu-KO mice before CFA-induced inflammation and at days 1 and 2 post-CFA. All mouse lines showed the same behavior (mu^fl, n=14; mu-cKO, n=6; mu-KO, n=12). Results are expressed as means ± sem. ★ <i>P</i><0.05, ★★ <i>P</i><0.01, ★★★ <i>P</i><0.001 post-CFA <i>vs</i> naïve. (B) Following CFA injection into tail, mu-cKO and mu^fl mice showed similar heat hyperalgesia at days 2, 6 and 9. The dashed line represents baseline (pre-CFA) sensitivity in the tail immersion tests at 48°C. Morphine (i.p.) produced anti-hyperalgesia in both genotypes, and that was reduced in mu-cKO mice as compared to controls. n=19/genotype., two-way ANOVA (genotype x treatment F(1,71) = 48.812, <i>P</i> <0.001 for treatment; F(1,71) = 5.999, <i>P</i> <0.05 for genotype. Post-hoc Bonferroni test, ✰✰✰ <i>P</i><0.001, morphine <i>vs</i> saline; ★ <i>P</i><0.05, cKO vs flox controls.</p

Conditional mu-cKO mice show decreased opiate-induced analgesia in the CFA-induced inflammatory pain model.

<p>(<b>A</b>) Two days after CFA, morphine (i.p.) dose-dependently reduced heat and mechanical hypersensitivities in mu^fl control mice. This analgesia was diminished in mu-cKO mice. Dashed lines represent baseline (pre-CFA) sensitivities. White bars, mu^fl mice; black bars, mu-cKO mice. For plantar test, n=13-20/genotype. two-way ANOVA (genotype x treatment F(1,90) = 75.336, <i>P</i> <0.001 for treatment; F(1,90) = 23.313, <i>P</i> <0.001 for genotype. Post-hoc Bonferroni test, ✰✰✰ <i>P</i><0.001, morphine <i>vs</i> saline; ★★ <i>P</i><0.01, ★★★ <i>P</i><0.001 cKO vs flox controls. For Von Frey filaments test, n=7-18/genotype. two-way ANOVA (genotype x treatment F(1,91) = 41.573, <i>P</i> <0.001 for treatment; F(1,91) = 27.378, <i>P</i> <0.001 for genotype. Post-hoc Bonferroni test, ✰✰ <i>P</i><0.01, ✰✰✰ <i>P</i><0.001 morphine <i>vs</i> saline; ★ <i>P</i><0.05, ★★ <i>P</i><0.01 mu-cKO vs mu^fl controls. (<b>B</b>) Fentanyl produced a dose-dependent analgesia in mu^fl control mice 2 days after CFA. Mu-cKO mice displayed a decreased analgesic response in the mechanical sensitivity test for the 0.03 mg/kg fentanyl dose. n=5-10/genotype. For plantar test, two-way ANOVA (genotype X treatment F(1,54) = 8.979, <i>P</i> <0.001 for treatment, <i>P</i>= 0.19 for genotype), post-hoc Bonferroni test; ✰✰ <i>P</i> <0.01, ✰✰✰ <i>P</i><0.001, fentanyl 0.1 mg/kg <i>vs</i> saline; for fentanyl 0.03 mg/kg, <i>P</i> = 0.0675 in mu^fl animals, <i>P</i> = 0.21 in mu-cKO animals. For Von Frey test, two-way ANOVA (genotype X treatment F(1,45) = 22.802, <i>P</i> <0.001 for treatment, F(1,45) = 9.316, <i>P</i> <0.01 for genotype). Post-hoc Bonferroni test for treatment, ✰✰✰ <i>P</i><0.001, fentanyl 0.1 mg/kg <i>vs</i> saline; for fentanyl 0.03 mg/kg, <i>P</i> = 0.0575 mu^fl animals, <i>P</i> = 0.28 mu-cKO animals. Post-hoc Fisher test for genotype, ★ <i>P</i><0.05, mu-cKO vs mu^fl mice. (<b>C</b>) Morphine-induced analgesia is reduced by systemic administration of the peripheral antagonist naloxone methiodide (NM). Morphine (5 mg/kg) induced an antihyperalgesic effect in mu^fl mice for both heat and mechanical responses. Systemic NM diminished morphine-induced analgesia. Heat hypersensitivity, one-way ANOVA for treatment F(3,42) = 29.778, P<0.001 ; post-hoc Bonferroni test, ✰✰ <i>P</i> <0.01, ✰✰✰ <i>P</i> <0.001 morphine <i>vs</i> saline; ★★ <i>P</i><0.01 ★★★ <i>P</i><0.001 NM + morphine <i>vs</i> morphine. Mechanical hypersensitivity, one-way ANOVA for treatment F(3,52) = 23.467, P<0.001 ; post-hoc Bonferroni test, ✰✰✰ <i>P</i> <0.001 morphine <i>vs</i> saline; ★★★ <i>P</i><0.001 NM + morphine <i>vs</i> morphine.</p