Presentation_1_Tachykinin-3 Genes and Peptides Characterized in a Basal Teleost, the European Eel: Evolutionary Perspective and Pituitary Role.PDF

<p>In mammals, neurokinin B (NKB) is a short peptide encoded by the gene tac3. It is involved in the brain control of reproduction by stimulating gonadotropin-releasing hormone (GnRH) neurons, mainly via kisspeptin. We investigated tac3 genes and peptides in a basal teleost, the European eel, which shows an atypical blockade of the sexual maturation at a prepubertal stage. Two tac3 paralogous genes (tac3a and tac3b) were identified in the eel genome, each encoding two peptides (NKBa or b and NKB-related peptide NKB-RPa or b). Amino acid sequence of eel NKBa is identical to human NKB, and the three others are novel peptide sequences. The four eel peptides present the characteristic C-terminal tachykinin sequence, as well as a similar alpha helix 3D structure. Tac3 genes were identified in silico in 52 species of vertebrates, and a phylogeny analysis was performed on the predicted TAC3 pre-pro-peptide sequences. A synteny analysis was also done to further assess the evolutionary history of tac3 genes. Duplicated tac3 genes in teleosts likely result from the teleost-specific whole genome duplication (3R). Among teleosts, TAC3b precursor sequences are more divergent than TAC3a, and a loss of tac3b gene would have even occurred in some teleost lineages. NKB-RP peptide, encoded beside NKB by tac3 gene in actinopterygians and basal sarcopterygians, would have been lost in ancestral amniotes. Tissue distribution of eel tac3a and tac3b mRNAs showed major expression of both transcripts in the brain especially in the diencephalon, as analyzed by specific qPCRs. Human NKB has been tested in vitro on primary culture of eel pituitary cells. Human NKB dose-dependently inhibited the expression of lhβ, while having no effect on other glycoprotein hormone subunits (fshβ, tshβ, and gpα) nor on gh. Human NKB also dose-dependently inhibited the expression of GnRH receptor (gnrh-r2). The four eel peptides have been synthesized and also tested in vitro. They all inhibited the expression of both lhβ and of gnrh-r2. This reveals a potential dual inhibitory role of the four peptides encoded by the two tac3 genes in eel reproduction, exerted at the pituitary level on both luteinizing hormone and GnRH receptor.</p

Regulation of the expression of the two eel leptin receptors (LEPRs) in the anterior brain and ovary during experimental maturation.

<p>The mRNA levels of eel LEPRa and b were measured by qPCR and normalized to beta-actin mRNA. (Fig 8A) Experiment 1: Each bar represents the mean ± SD from 10 control female eels sacrificed at the beginning of the experiment (T0 controls) and 10 matured female eels sacrificed at the end of the experiment. Significant difference between control and matured groups were analyzed by Mann-Whitney <i>U</i> test; **<i>P</i><0.05 and *<i>P</i><0.05. (Fig 8B) Experiment 2: Each bar represents the mean ± SD from 6 control females sacrificed at the beginning of the experiment (T0 controls), 6 control females sacrificed at the end of the experiment (End controls), and 6 matured females. Significant differences between each group were analyzed by Kruskal-Wallis ANOVA.</p

Consensus phylogenetic tree of actinopterygian leptins.

<p>Phylogenetic analysis of 34 actinopterygian leptin amino acid sequences was performed using the Maximum Likelihood method, with 1,000 boostrap replicates (for the alignment and references of sequences see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126008#pone.0126008.s020" target="_blank">S3 Fig</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126008#pone.0126008.s018" target="_blank">S1 Table</a>). The number shown at each branch node indicates in percentage the boostrap value. Only values above 50% are indicated. The tree was rooted using mammalian (human and bull) leptin sequences as outgroup. Teleost duplicated leptin groups are indicated in yellow (“A” type) and in green (“B” type).</p

Consensus phylogenetic trees of vertebrate leptin receptors (LEPRs).

<p>Phylogenetic analyses of 21 vertebrate LEPR amino acid sequences (Fig 4A) and 22 partial vertebrate LEPR amino acid sequences (Fig 4B) were performed using the Maximum Likelihood method, with 1,000 boostrap replicates (for the alignments and references of sequences, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126008#pone.0126008.s009" target="_blank">S9</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126008#pone.0126008.s010" target="_blank">S10</a> Figs, and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126008#pone.0126008.s019" target="_blank">S2 Table</a>). The number shown at each branch node indicates, in percentage, the boostrap value. The trees were rooted using human and zebrafish GCSFR sequences as outgroups. Teleost LEPR group is indicated in yellow and sarcopterygian LEPR group is indicated in blue.</p

Conserved genomic synteny of vertebrate leptin receptors (LEPR).

<p>Genomic synteny map comparing LEPR and their neighbouring genes from human, other tetrapods, basal sarcoterygian (coelacanth), non-teleost actinopterygian (spotted gar), and teleost species including the two eel LEPR (LEPRa and LEPRb) genomic regions, are represented. The LEPR genomic region has been duplicated in teleost species, likely as a result of the teleost specific third round of genome duplication. The duplicated LEPRb has been conserved in the eel but not in the other teleosts. Genes are named after their human orthologs according to the Human Genome Naming Consortium (HGNC). Orthologs of each gene are represented in the same color and displayed in the same column. The genes reproduced in this figure are not necessarily presented in the same order as they appear on the chromosomes and scaffolds, except for human, and their positions are indicated in 10⁶ base pairs. The detailed genomic locations of the genes are given in Supporting <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126008#pone.0126008.s026" target="_blank">S9 Table</a>.</p

Conserved genomic synteny of vertebrate leptins.

<p>Genomic synteny maps comparing leptins and their neighbouring genes from human, non-teleost actinopterygian (spotted gar), and teleost species including the two eel leptins (leptin1 and leptin2) genomic regions, are represented. The leptin genomic region has been duplicated in teleost species, likely as a result of the teleost specific third round of genome duplication. The duplicated leptins have been conserved in most of the teleosts, including the eel. In the current status of the European and Japanese eel draft genomes, the identification of a “A” or a “B” type for eel leptins would be based only on the presence of a RBM28 on the same scaffold as eel leptin1. Therefore, we chose to keep a distinct nomenclature in the present study, and named the eel leptins, eel leptin1 and leptin2. Genes are named after their human orthologs according to the Human Genome Naming Consortium (HGNC), except for si:dkey-5i3.5 that does not exist in human. This gene was named after its spotted gar ortholog. Orthologs of each gene are represented in the same color and displayed in the same column. The genes reproduced in this figure are not necessarily presented in the same order as they appear on the chromosomes and scaffolds, except for spotted gar, and their positions are indicated in 10⁶ base pairs. The detailed genomic locations of the genes are given in Supporting <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126008#pone.0126008.s023" target="_blank">S6 Table</a>.</p

MUC5AC and a Glycosylated Variant of MUC5B Alter Mucin Composition in Children With Acute Asthma.

BACKGROUND: Diffuse airway mucus obstruction is an important feature of severe and fatal asthma. MUC5AC and MUC5B are the principal gel-forming mucins found in airway mucus. The mucin composition of airway mucus likely affects its functional properties. METHODS: We quantified the principal airway mucins MUC5AC and MUC5B in the sputum of age-matched children with acute and stable asthma and healthy control subjects by using Western blotting. RESULTS: Sputum samples from 38 children (13 with acute asthma, 15 with stable asthma, 10 control subjects) were obtained. Sputum MUC5AC concentrations were 7.6 μg/mL in control subjects, 22.4 μg/mL in those with stable asthma (P = .17), and 44.7 μg/mL in those with acute asthma (P < .05). MUC5B concentrations showed less variation, with 156.2, 222.3, and 254.8 μg/mL in control subjects, those with stable asthma, and those with acute asthma, respectively. The greater MUC5AC concentration in those with acute asthma resulted in a significantly altered MUC5B:MUC5AC ratio between control subjects and those with acute asthma (P < .05). Significant differences in MUC5B glycoforms were present between the groups, with the low-charge-only glycoform being found uniquely in those with acute asthma. CONCLUSIONS: Increased MUC5AC and the presence of a low-charge-only MUC5B glycoform significantly altered mucin composition in children with acute asthma. These changes may be important contributory factors to the airway mucus obstruction observed during acute asthma

Proposed evolutionary scenario of leptin and LEPR genes in osteichthyans.

<p>Osteichthyan ancestor possessed one leptin gene and one LEPR gene. These genes were duplicated in teleosts, as a result of the teleost-specific third whole genome duplication event (3R), and further duplicated in salmonids after the 4R, specific to this group. LEPRb would have been lost during teleost radiation, some time after the elopomorph emergence and before the clupeocephal emergence.</p

Comparison of leptin receptor (LEPR) gene structure, in human and actinopterygian species.

<p>LEPR genes from human, non-teleost actinopterygian spotted gar, and teleost species including the two eel LEPRs are represented. Exons are symbolized either by black squares (untranslated nucleotides) or white squares (translated nucleotides). The nucleotide length is indicated above each translated exon. The total lengths of exons containing untranslated nucleotides are indicating below. The numbers of the exons (from 1 to 18) composing the coding sequence (CDS) are indicated below. Chr: chromosome; LG: linkage group.</p

Regulation of the expression of the two eel leptins in the pituitary and liver during experimental maturation.

<p>The mRNA levels of eel leptin1 and 2 were measured by qPCR and normalized to beta-actin mRNA. (Fig 7A) Experiment1: Each bar represents the mean ± SD from 10 control female eels sacrificed at the beginning of the experiment (T0 controls) and 10 matured female eels sacrificed at the end of the experiment. Significant differences between the two eel groups were analyzed by Mann-Whitney <i>U</i> test; ***<i>P</i><0.001. (Fig 7B) Experiment2: Each bar represents the mean ± SD from 6 control females sacrificed at the beginning of the experiment (T0 controls), 6 control females sacrificed at the end of the experiment (End controls), and 6 matured females. Significant differences between the three eel groups were analyzed by Kruskal-Wallis ANOVA; significant differences are indicated by different letters. ND: Not detectable.</p