Metabolic insights from a GHSR-A203E mutant mouse model

Objective: Binding of ghrelin to its receptor, growth hormone secretagogue receptor (GHSR), stimulates GH release, induces eating, and increases blood glucose. These processes may also be influenced by constitutive (ghrelin-independent) GHSR activity, as suggested by findings in short people with naturally occurring GHSR-A204E mutations and reduced food intake and blood glucose in rodents administered GHSR inverse agonists, both of which impair constitutive GHSR activity. In this study, we aimed to more fully determine the physiologic relevance of constitutive GHSR activity. Methods: We generated mice with a GHSR mutation that replaces alanine at position 203 with glutamate (GHSR-A203E), which corresponds to the previously described human GHSR-A204E mutation, and used them to conduct ex vivo neuronal electrophysiology and in vivo metabolic assessments. We also measured signaling within COS-7 and HEK293T cells transfected with wild-type GHSR (GHSR-WT) or GHSR-A203E constructs. Results: In COS-7 cells, GHSR-A203E resulted in lower baseline IP3 accumulation than GHSR-WT; ghrelin-induced IP3 accumulation was observed in both constructs. In HEK293T cells co-transfected with voltage-gated CaV2.2 calcium channel complex, GHSR-A203E had no effect on basal CaV2.2 current density while GHSR-WT did; both GHSR-A203E and GHSR-WT inhibited CaV2.2 current in the presence of ghrelin. In cultured hypothalamic neurons from GHSR-A203E and GHSR-deficient mice, native calcium currents were greater than those in neurons from wild-type mice; ghrelin inhibited calcium currents in cultured hypothalamic neurons from both GHSR-A203E and wild-type mice. In brain slices, resting membrane potentials of arcuate NPY neurons from GHSR-A203E mice were hyperpolarized compared to those from wild-type mice; the same percentage of arcuate NPY neurons from GHSR-A203E and wild-type mice depolarized upon ghrelin exposure. The GHSR-A203E mutation did not significantly affect body weight, body length, or femur length in the first ∼6 months of life, yet these parameters were lower in GHSR-A203E mice after 1 year of age. During a 7-d 60% caloric restriction regimen, GHSR-A203E mice lacked the usual marked rise in plasma GH and demonstrated an exaggerated drop in blood glucose. Administered ghrelin also exhibited reduced orexigenic and GH secretagogue efficacies in GHSR-A203E mice. Conclusions: Our data suggest that the A203E mutation ablates constitutive GHSR activity and that constitutive GHSR activity contributes to the native depolarizing conductance of GHSR-expressing arcuate NPY neurons. Although the A203E mutation does not block ghrelin-evoked signaling as assessed using in vitro and ex vivo models, GHSR-A203E mice lack the usual acute food intake response to administered ghrelin in vivo. The GHSR-A203E mutation also blunts GH release, and in aged mice leads to reduced body length and femur length, which are consistent with the short stature of human carriers of the GHSR-A204E mutation.Fil: Torz, Lola J.. Universidad de Copenhagen; DinamarcaFil: Osborne Lawrence, Sherri. Ut Southwestern Medical Center; Estados UnidosFil: Rodriguez, Juan. Ut Southwestern Medical Center; Estados UnidosFil: He, Zhenyan. Ut Southwestern Medical Center; Estados UnidosFil: Cornejo, María Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; ArgentinaFil: Mustafá, Emilio Román. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; ArgentinaFil: Jin, Chunyu. Universidad de Copenhagen; DinamarcaFil: Petersen, Natalia. Universidad de Copenhagen; DinamarcaFil: Hedegaard, Morten A.. Universidad de Copenhagen; DinamarcaFil: Nybo, Maja. Universidad de Copenhagen; DinamarcaFil: Martínez Damonte, Valentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; ArgentinaFil: Metzger, Nathan P.. Ut Southwestern Medical Center; Estados UnidosFil: Mani, Bharath K.. Ut Southwestern Medical Center; Estados UnidosFil: Williams, Kevin W.. Ut Southwestern Medical Center; Estados UnidosFil: Raingo, Jesica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; ArgentinaFil: Perello, Mario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; ArgentinaFil: Holst, Birgitte. Universidad de Copenhagen; DinamarcaFil: Zigman, Jeffrey M.. Ut Southwestern Medical Center; Estados Unido

Nucleant layer effect on nanocolumnar ZnO films grown by electrodeposition

Different ZnO nanostructured films were electrochemically grown, using an aqueous solution based on ZnCl2, on three types of transparent conductive oxides grow on commercial ITO (In2O3:Sn)-covered glass substrates: (1) ZnO prepared by spin coating, (2) ZnO prepared by direct current magnetron sputtering, and (3) commercial ITO-covered glass substrates. Although thin, these primary oxide layers play an important role on the properties of the nanostructured films grown on top of them. Additionally, these primary oxide layers prevent direct hole combination when used in optoelectronic devices. Structural and optical characterizations were carried out by scanning electron microscopy, atomic force microscopy, and optical transmission spectroscopy. We show that the properties of the ZnO nanostructured films depend strongly on the type of primary oxide-covered substrate used. Previous studies on different electrodeposition methods for nucleation and growth are considered in the final discussion.We thank Prof. A. Segura of the Universitat de Valencia for the facilities with the sputtering equipment. This work was supported by the project PROMETEO/2009/074 from the Generalitat Valenciana.Reyes Tolosa, MD.; Damonte, LC.; Brine, H.; Bolink, HJ.; Hernández Fenollosa, MDLÁ. (2013). Nucleant layer effect on nanocolumnar ZnO films grown by electrodeposition. Nanoscale Research Letters. 8:135-144. https://doi.org/10.1186/1556-276X-8-135S1351448Franklin JB, Zou B, Petrov P, McComb DW, Ryanand MP, McLachlan MA,J: Optimised pulsed laser deposition of ZnO thin films on transparent conducting substrates. Mater Chem 2011, 21: 8178–8182. 10.1039/c1jm10658aJaroslav B, Andrej V, Marie N, Šuttab P, Miroslav M, František U: Cryogenic pulsed laser deposition of ZnO. Vacuum 2012, 86(6):684–688. 10.1016/j.vacuum.2011.07.033Jae Bin L, Hyeong Joon K, Soo Gil K, Cheol Seong H, Seong-Hyeon H, Young Hwa S, Neung Hun L: Deposition of ZnO thin films by magnetron sputtering for a film bulk acoustic resonator. Thin Solid Films 2003, 435: 179–185. 10.1016/S0040-6090(03)00347-XXionga DP, Tanga XG, Zhaoa WR, Liua QX, Wanga YH, Zhoub SL: Deposition of ZnO and MgZnO films by magnetron sputtering. Vacuum 2013, 89: 254–256.Reyes Tolosa MD, Orozco-Messana J, Lima ANC, Camaratta R, Pascual M, Hernandez-Fenollosa MA: Electrochemical deposition mechanism for ZnO nanorods: diffusion coefficient and growth models. J Electrochem Soc 2011, 158(11):E107-E110.Ming F, Ji Z: Mechanism of the electrodeposition of ZnO nanosheets below room temperature. J Electrochem Soc 2010, 157(8):D450-D453. 10.1149/1.3447738Pullini D, Pruna A, Zanin S, Busquets Mataix D: High-efficiency electrodeposition of large scale ZnO nanorod arrays for thin transparent electrodes. J Electrochem Soc 2012, 159: E45-E51. 10.1149/2.093202jesPruna A, Pullini D, Busquets Mataix D: Influence of deposition potential on structure of ZnO nanowires synthesized in track-etched membranes. J Electrochem Soc 2012, 159: E92-E98. 10.1149/2.003205jesMarotti RE, Giorgi P, Machado G, Dalchiele EA: Crystallite size dependence of band gap energy for electrodeposited ZnO grown at different temperatures. Solar Energy Materials and Solar Cells 2009, 90(15):2356–2361.Yeong Hwan K, Myung Sub K, Jae Su Y: Structural and optical properties of ZnO nanorods by electrochemical growth using multi-walled carbon nanotube-composed seed layers. Nanoscale Res Lett 2012, 7: 13. 10.1186/1556-276X-7-13Elias J, Tena-Zaera R, Lévy-Clément C: Electrodeposition of ZnO nanowires with controlled dimensions for photovoltaic applications: role of buffer layer. Thin Solid Films 2007, 515(24):8553–8557. 10.1016/j.tsf.2007.04.027Zhai Y, Zhai S, Chen G, Zhang K, Yue Q, Wang L, Liu J, Jia J: Effects of morphology of nanostructured ZnO on direct electrochemistry and biosensing properties of glucose oxidase. J Electroanal Chem 2011, 656: 198–205. 10.1016/j.jelechem.2010.11.020Reyes Tolosa MD, Orozco-Messana J, Damonte LC, Hernandez-Fenollosa MA: ZnO nanoestructured layers processing with morphology control by pulsed electrodeposition. J Electrochem Soc 2011, 158(7):D452-D455. 10.1149/1.3593004Gouxa A, Pauporté T, Chivot J, Lincot D: Temperature effects on ZnO electrodeposition. Electrochim Acta 2005, 50(11):2239–2248. 10.1016/j.electacta.2004.10.007Kwok WM, Djurisic , Aleksandra B, Leung , Yu H, Li D, Tam KH, Phillips DL, Chan WK: Influence of annealing on stimulated emission in ZnO nanorods. Appl Phys Lett 2006, 89(18):183112. 183112–3 183112–3 10.1063/1.2378560Donderis V, Hernández-Fenollosa MA, Damonte LC, Marí B, Cembrero J: Enhancement of surface morphology and optical properties of nanocolumnar ZnO films. Superlattices and Microstructures 2007, 42: 461–467. 10.1016/j.spmi.2007.04.068Ghayour H, Rezaie HR, Mirdamadi S, Nourbakhsh AA: The effect of seed layer thickness on alignment and morphology of ZnO nanorods. Vacuum 2011, 86: 101–105. 10.1016/j.vacuum.2011.04.025Michael B, Mohammad Bagher R, Sayyed-Hossein K, Wojtek W, Kourosh K-z: Aqueous synthesis of interconnected ZnO nanowires using spray pyrolysis deposited seed layers. Mater Lett 2010, 64: 291–294. 10.1016/j.matlet.2009.10.065Jang Bo S, Hyuk C, Sung-O K: Rapid hydrothermal synthesis of zinc oxide nanowires by annealing methods on seed layers. J Nanomater 2011, 2011: 6.Peiro AM, Punniamoorthy R, Kuveshni G, Boyle DS, Paul O’B, Donal DC, Bradley , Jenny N, Durrant JR: Hybrid polymer/metal oxide solar cells based on ZnO columnar structures. J Mater Chem 2006, 16(21):2088–2096. 10.1039/b602084dVallet-Regí M, Salinas AJ, Arcos D: From the bioactive glasses to the star gels. J Mater Sci Mater Med 2006, 17: 1011–1017.Peulon S, Lincot D: Mechanistic study of cathodic electrodeposition of zinc oxide and zinc hydroxychloride films from oxygenated aqueous zinc chloride solutions. J Electrochem Soc 1998, 145: 864. 10.1149/1.1838359Dalchiele EA, Giorgi P, Marotti RE, Martín F, Ramos-Barrado JR, Ayouci R, Leinen D: Electrodeposition of ZnO thin films on n-Si(100). Sol. Energy Mater. Sol. Cells 2001, 70: 245. 10.1016/S0927-0248(01)00065-4Courtney IA, Dahn JR: Electrochemical and in situ X‐ray diffraction studies of the reaction of lithium with tin oxide composites. J Electrochem Soc 1997, 144(6):2045–2052. 10.1149/1.183774

Anuran responses to spatial patterns of agricultural landscapes in Argentina

Context: Amphibians are declining worldwide and land use change to agriculture is recognized as a leading cause. Argentina is undergoing an agriculturalization process with rapid changes in landscape structure. Objectives: We evaluated anuran response to landscape composition and configuration in two landscapes of east-central Argentina with different degrees of agriculturalization. We identified sensitive species and evaluated landscape influence on communities and individual species at two spatial scales. Methods: We compared anuran richness, frequency of occurrence, and activity between landscapes using call surveys data from 120 sampling points from 2007 to 2009. We evaluated anuran responses to landscape structure variables estimated within 250 and 500-m radius buffers using canonical correspondence analysis and multimodel inference from a set of candidate models. Results: Anuran richness was lower in the landscape with greater level of agriculturalization with reduced amount of forest cover and stream length. This pattern was driven by the lower occurrence and calling activity of seven out of the sixteen recorded species. Four species responded positively to the amount of forest cover and stream habitat. Three species responded positively to forest cohesion and negatively to rural housing. Two responded negatively to crop area and diversity of cover classes. Conclusions: Anurans within agricultural landscapes of east-central Argentina are responding to landscape structure. Responses varied depending on species and study scale. Life-history traits contribute to responses differences. Our study offers a better understanding of landscape effects on anurans and can be used for land management in other areas experiencing a similar agriculturalization process.Facultad de Ciencias ExactasCentro de Investigaciones del Medioambient

Carrageenans from Chilean samples of Stenogramme interrupta (Phyllophoraceae): structural analysis and biological activity

Carrageenans extracted from cystocarpic and tetrasporic Stenogramme interrupta were analysed by chemical and spectroscopic methods. The carrageenan from cystocarpic plants is composed predominantly of 0.5 M KCl-insoluble and 1 M KCl-soluble fractions. The insoluble fraction contained iota-carrageenan as the major component with alpha-carrageenan and pyruvated carrageenan as minor components. The soluble fraction is highly heterogeneous and did not contain the precursors mu- and nu-carrageenans. The polysaccharide from tetrasporic plants is composed of zeta- and lambda-carrageenans, and low sulfated galactans. It is soluble in KCl and partly cyclized by alkaline treatment. The antiviral and anticoagulant properties of the insoluble polysaccharide fraction from cystocarpic S. interrupta and the polysaccharide from tetrasporic S. interrupta are reported the results of which suggest promising antiherpetic activity.Fil: Cáceres, Pablo J.. Universidad de Santiago de Chile; ChileFil: Carlucci, Maria Josefina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Virología; ArgentinaFil: Damonte, Elsa Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Virología; ArgentinaFil: Matsuhiro, Betty. Universidad de Santiago de Chile; ChileFil: Zuñiga, Elisa A.. Universidad de Santiago de Chile; Chil

On novel hydrogels based on poly(2-hydroxyethyl acrylate) and polycaprolactone with improved mechanical properties prepared by frontal polymerization

This work aimed to improve the mechanical properties of poly(2-hydroxyethyl acrylate) (PHEA)-based hydrogels by performing their polymerization in the presence of an ad hoc synthesized star-shaped polymer, whose arms are composed of a biopolymer, i.e., polycaprolactone (PCL) end-capped with acrylic units to act as a crosslinker for PHEA. Indeed, this system was designed to have (i) biocompatible, biodegradable, and mechanical strong arms as well as (ii) a star structure with acrylic functionalities to promote the crosslinking of the material.H- 1 NMR, IR and TGA measurements confirmed the star PCL functionalization, which was carried out starting from a hydroxyl-terminated polymer. Hydrogels were prepared by varying the concentration of the synthesized star polymer, and the properties of the materials obtained by frontal (FP) and bulk polymerization (BP) were compared. Moreover, to evaluate the specific effect of PCL-star-4-2 k-tetraacrylate (PCL-TA) on the crosslinking of the systems, samples were also synthesized using a commercial acrylate crosslinker. For what concerns the frontal polymerization process, fronts were found to be stable in the presence of PCL-TA. The thermal characterization results showed a significant decrease in the PHEA glass transition temperature with increasing PCL-TA content, which was particularly evident in the samples prepared by FP. This result can be attributed to the partial miscibility of the two polymers, which become compatible during the polymerization process by forming a copolymer system. This was confirmed by analyzing the thermal behavior of hydrogels polymerized in the presence of a hydroxyl terminated PCL, i.e., a polymer without active functional end groups. The samples synthesized by FP with the star polymer and the commercial crosslinker, as well as neat PHEA, were subjected to mechanical tests. The mechanical behavior of PCL-based hydrogels was outstanding, exhibiting three time higher modulus than that of crosslinked PHEA, and were structurally stable even under high compression loadings. This remarkable property, combined with the fast and efficient polymerization method and the environmentally friendly properties of PCL, make the developed hydrogels promising systems for practical applications in various fields

AR-12 Inhibits Chaperone Proteins Preventing Virus Replication and the Accumulation of Toxic Misfolded Proteins

Fil: Booth, Laurence. Virginia Commonwealth University; Estados UnidosFil: Roberts, Jane L. Virginia Commonwealth University; Estados UnidosFil: Ecroyd, Heath. University of Wollongong; AustraliaFil: Reid, St Patrick. United States Army Medical Research Institute of Infectious Diseases; Estados UnidosFil: Proniuk, Stefan. Arno Therapeutics; Estados UnidosFil: Zukiwski, Alexander. Arno Therapeutics; Estados UnidosFil: Jacob, Abraham. University of Arizona; Estados UnidosFil: Damonte, Elsa Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Tunon, Maria J. Universidad de León; EspañaFil: Dent, Paul. Virginia Commonwealth University; Estados Unido

Synthesis and biological evaluation of novel thiazole- VX-809 hybrid derivatives as F508del correctors by QSAR-based filtering tools

The most common CF mutation, F508del, impairs the processing and gating of CFTR protein. This deletion results in the improper folding of the protein and its degradation before it reaches the plasma membrane of epithelial cells. Present correctors, like VX809 only induce a partial rescue of the mutant protein. Our previous studies reported a class of compounds, called aminoarylthiazoles (AATs), featuring an interesting activity as correctors. Some of them show additive effect with VX809 indicating a different mechanism of action. In an attempt to construct more interesting molecules, it was thought to generate chemically hybrid compounds, blending a portion of VX809 merged to the thiazole scaffold. This approach was guided by the development of QSAR analyses, which were performed based on the F508del correctors so far disclosed in the literature. This strategy was aimed at exploring the key requirements turning in the corrector ability of the collected derivatives and allowed us to derive a predictive model guiding for the synthesis of novel hybrids as promising correctors. The new molecules were tested in functional and biochemical assays on bronchial CFBE41o-cells expressing F508del-CFTR showing a promising corrector activity

Effect of the redox state on HIV-1 tat protein multimerization and cell internalization and trafficking

The redox state of the cysteine-rich region of the HIV Tat protein is known to play a crucial role in Tat biological activity. In this article, we show that Tat displays two alternative functional states depending on the presence of either one or three reduced sulphydryl groups in the cysteine-rich region, respectively. Using different approaches, a disulfide pattern has been defined for the Tat protein and a specific DTT-dependent breaking order of disulfide bonds highlighted. The Tat redox state deeply influences macrophage protein uptake. Immunoistochemistry analysis shows that the oxidized protein does not enter cells, whereas partially reduced protein reaches the cytosol and, to a limited extent, the nucleus. Finally electrophoretic analysis shows Tat high-molecular weight multi-aggregation, resulting in the loss of biological activity. This is due to strong electrostatic and metal-binding interactions, whereas Tat dimerization involves metal-binding interactions as well as disulfide bond formation