Viral genetic determinants of h5n1 influenza viruses that contribute to cytokine dysregulation

Human disease caused by highly pathogenic avian influenza (H5N1) is associated with fulminant viral pneumonia and mortality rates in excess of 60%. Cytokine dysregulation is thought to contribute to its pathogenesis. In comparison with human seasonal influenza (H1N1) viruses, clade 1, 2.1, and 2.2 H5N1 viruses induced higher levels of tumor necrosis factor-α in primary human macrophages. To understand viral genetic determinants responsible for this hyperinduction of cytokines, we constructed recombinant viruses containing different combinations of genes from high-cytokine (A/Vietnam/1203/04) and low-cytokine (A/WSN/33) phenotype HlNl viruses and tested their cytokine-inducing phenotype in human macrophages. Our results suggest that the H5N1 polymerase gene segments, and to a lesser extent the NS gene segment, contribute to cytokine hyperinduction in human macrophages and that a putative H5 pandemic virus that may arise through genetic reassortment between H5N1 and one of the current seasonal influenza viruses may have a markedly altered cytokine phenotype. © 2009 by the Infectious Diseases Society of America. All rights reserved.published_or_final_versio

LES-based Study of the Roughness Effects on the Wake of a Circular Cylinder from Subcritical to Transcritical Reynolds Numbers

This paper investigates the effects of surface roughness on the flow past a circular cylinder at subcritical to transcritical Reynolds numbers. Large eddy simulations of the flow for sand grain roughness of size k/D = 0.02 are performed (D is the cylinder diameter). Results show that surface roughness triggers the transition to turbulence in the boundary layer at all Reynolds numbers, thus leading to an early separation caused by the increased momentum deficit, especially at transcritical Reynolds numbers. Even at subcritical Reynolds numbers, boundary layer instabilities are triggered in the roughness sublayer and eventually lead to the transition to turbulence. The early separation at transcritical Reynolds numbers leads to a wake topology similar to that of the subcritical regime, resulting in an increased drag coefficient and lower Strouhal number. Turbulent statistics in the wake are also affected by roughness; the Reynolds stresses are larger due to the increased turbulent kinetic energy production in the boundary layer and separated shear layers close to the cylinder shoulders.We acknowledge “Red Española de Surpercomputación” (RES) for awarding us access to the MareNostrum III machine based in Barcelona, Spain (Ref. FI-2015-2-0026 and FI-2015-3-0011). We also acknowledge PRACE for awarding us access to Fermi and Marconi Supercomputers at Cineca, Italy (Ref. 2015133120). Oriol Lehmkuhl acknowledges a PDJ 2014 Grant by AGAUR (Generalitat de Catalunya). Ugo Piomelli acknowledges the support of the Natural Sciences and Engineering Research Council (NSERC) of Canada under the Discovery Grant Programme (Grant No. RGPIN-2016-04391). Ricard Borrell acknowledges a Juan de la Cierva postdoctoral grant (IJCI-2014-21034). Ivette Rodriguez, Oriol Lehmkuhl, Ricard Borrell and Assensi Oliva acknowledge Ministerio de Economía y Competitividad, Secretaría de Estado de Investigación, Desarrollo e Innovación, Spain (ref. ENE2014-60577-R).Peer ReviewedPostprint (author's final draft

Discovery of a new reproductive hormone in teleosts: Pituitary adenylate cyclase-activating polypeptide-related peptide (PRP)

Pituitary adenylate cyclase-activating polypeptide (PACAP)-related peptide (PRP) is a peptide encoded with PACAP in the same precursor protein. Non-mammalian PRPs were previously termed growth hormone-releasing hormone (GHRH)-like peptide, and was regarded as the mammalian GHRH homologue in non-mammalian vertebrates until the discovery of authentic GHRH genes in teleosts and amphibians. Although a highly specific receptor for PRP, which is lost in mammals, is present in non-mammals, a clear function of PRP in vertebrates remains unknown. Using goldfish as a model, here we show the expression of PRP and its cognate receptor in the brain-pituitary-gonadal (BPG) axis, thus suggesting a function of goldfish (gf) PRP in regulating reproduction. We found that gfPRP controls the expression of reproductive hormones in the brain, pituitary and ovary. Goldfish PRP exerts stimulatory effects on the expression of salmon gonadotropin-releasing hormone (sGnRH) in the brain, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in pituitary primary culture cells, but inhibits the expression of LH in the ovary. Using the same technique, we showed that gfPRP did not alter the mRNA level of growth hormone in the pituitary primary culture. In summary, we have discovered the first function of vertebrate PRP in regulating reproduction, which provides a new research direction in studying the neuroendocrine control of reproduction not only in teleosts, but also in other non-mammalian vertebrates. © 2011 Elsevier Inc.link_to_subscribed_fulltex

Molecular cloning and expression studies of a prolactin receptor in goldfish (Carassius auratus)

A full-length cDNA clone, of a size of 4.6 kb, for the goldfish prolactin receptor has been isolated. This cDNA clone encodes a protein of 600 amino acids homologous to prolactin receptors of other species. A Kyte- Doolittle hydropathy analysis of the receptor indicates that the translated protein consists of a signal peptide of 22 amino acids, an extracellular domain of 228 amino acids, a single transmembrane domain of 24 amino acids, and an intracellular domain of 346 amino acids. Several characteristic landmarks of prolactin receptor could be identified in this clone. These include the four conserved cysteine residues and the WS motif within the extracellular domain, and the box 1 and box 2 regions of the intracellular domain. Among all the prolactin receptor sequences known to date, this clone bears the closest resemblance to the tilapia prolactin receptor, although homology between these two fish prolactin receptors is rather low. There are only 57.4% of nucleotide and 48.3% of amino acid sequence identities between these two fish receptors. This receptor cDNA was transfected into CHO-K1 cells for functional analysis. RT-PCR analysis with a pair of gene specific primers indicate that the receptor was transcribed in the transfected cells. Using a cell proliferation assay based on the reduction of the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, the receptor transfected CHO-K1 cells can be stimulated to proliferate upon the addition of ovine prolactin in the culture medium. The tissue distribution of the prolactin receptor in goldfish was studied by RT-PCR/Southern analysis and by Northern analysis. The results indicated that the receptor is expressed mostly in the kidney, the gill and the intestine of goldfish, corroborating with the osmoregulatory role of prolactin in fish. In addition, an appreciable level of the receptor is also found in the brain and gonads of goldfish. Northern analysis showed that there are two transcript sizes, a major 4.6 kb and a minor 3.5 kb mRNAs, in the kidney, gill and intestine.link_to_subscribed_fulltex

Production of recombinant goldfish prolactin and its applications in radioreceptor binding assay and radioimmunoassay

Goldfish prolactin cDNA was subcloned into a pRSET A vector and expressed in Escherichia coli. Recombinant goldfish prolactin was expressed mainly as insoluble inclusion bodies in the form of N-terminal 6x His-tagged fusion protein. This fusion protein was purified, refolded and 125I-labeled to generate a radioligand for receptor binding and validation of a radioimmunoassay for goldfish prolactin. Using goldfish gill membrane as the substrate for prolactin receptor binding, both recombinant and native forms of goldfish prolactin were effective in displacing the specific binding of the radioligand in a similar dose range, suggesting that the fusion protein was refolded properly and could be recognized by goldfish prolactin receptors. To quantify prolactin contents in biological samples from the goldfish, a radioimmunoassay using the 125I-labeled recombinant prolactin as a tracer was established. This assay was shown to be selective for goldfish prolactin without cross-reactivity with mammalian prolactin and pituitary hormones from other fish species (e.g., growth hormone and gonadotropin II). This newly validated assay system was used to investigate neuroendocrine and signal transduction mechanisms regulating prolactin release in the goldfish. In this case, the Ca2+ ionophore A23187 and protein kinase C activator TPA were effective in elevating basal levels of prolactin secretion in perifused goldfish pituitary cells. In parallel studies using a static incubation approach, somatostatin and dopamine, but not vasoactive intestinal polypeptide, were inhibitory to basal prolactin release in goldfish pituitary cells. These results suggest that somatostatin and dopamine may serve as negative regulators of basal prolactin secretion and that extracellular Ca2+ influx and protein kinase C activation may be important signaling events mediating prolactin release in the goldfish. ©2002 Elsevier Science (USA).link_to_subscribed_fulltex

Molecular cloning of a teleost growth hormone receptor and its functional interaction with human growth hormone

This paper reports the first full-length cDNA sequence of a growth hormone receptor (GHR) from a teleost fish and its functional expression in cultured eukaryotic cells. The cDNA sequence, from Carassius auratus (goldfish), encodes a protein of 602 amino acids (aa) akin in architecture to the GHRs of other species. Despite the presence of motifs characteristic of GHR, the overall homology between the goldfish GHR and other GHRs is very low (≈ 40% aa identity). CHO cells transfected with this receptor cDNA can be stimulated to proliferate by human growth hormone (hGH). In addition, the transfected cells can transactivate a co-expressed mammalian serine protease inhibitor (Spi) 2.1 promoter upon stimulation by hGH, indicating the successful interaction of the fish receptor with the mammalian ligand to evoke the down-stream post-receptor events. Tissue distribution studies indicated that the receptor is mostly expressed in the liver and hypothalamus of goldfish. A single mRNA transcript of a size of about 4 kb was found in the goldfish liver. © 2001 Elsevier Science B.V.link_to_subscribed_fulltex