Mammalian sialidase Neu3 overexpression in Cos-7 cells : effects on Newcastle disease virus-cell interaction

Newcastle Disease Virus (NDV) is an avian single-stranded RNA enveloped virus that belongs to the Paramyxoviridae family. The interaction of enveloped viruses with cell surface receptors is the first step in the viral cycle and an important determinant of viral host range. Although it is estabilished that NDV, such as the most of Paramyxovirus, infects cells binding sialic acid-containing glycoconjugates, the exact nature of these receptors is not well known. It is thought that both sialoglycoproteins and gangliosides may serve as viral receptors. Infection by NDV requires the combined action of its two glycoproteins: the hemagglutinin-neuraminidase (HN) protein and the fusion (F) protein. HN protein has three main biological activities: hemagglutinin, sialidase and fusion promotion. The hemagglutinin activity allows the binding of the virus to the host cell surface by recognizing specific cellular receptors. The sialidase activity facilitates the viral spread of newly born virions, while the fusion promotion activity induces F protein to begin the fusion between the virus and host membranes. After fusion, viral nucleocapsid is delivered into the cytoplasm and here the replication cycle takes place. After synthesis, once correctly folded and assembled, HN and F are transported to the host plasma membrane. F protein is synthetized as a precursor, Fo, and it becomes fusogenic only after cleavage into disulfide-linked F1 and F2 polypeptide before catching up the host membrane (Iwata et al., 1994). The NDV clone used in our experiments is \u201cClone 30\u201d that belongs to the lentogenic group. The lentogenic viruses\u2019 F protein can be cleaved only by tripsin-like enzyme. In this work we investigated the effects caused by mammalian sialidase Neu3 overexpression in COS7 cells in terms of NDV infection, NDV-cell interaction and its entry. First, we studied if the overexpression of Neu3 sialidase could interfere with the virus entry. To this end, we performed immunofluorescence assays in collaboration with the Department of Biochemistry and Molecular Biology of the University of Salamanca, and we observed that NDV infection was reduced (19%) in Neu3 overexpressing COS7 cells compared to controls. Because Neu3 sialidase is mainly located in regions of the membrane named \u201clipid rafts\u201d (Simons et al., 1997), we studied the possible involvement of these domains in the infection process by Western Blot analysis and HN activity assays. Mock and Neu3 overexpressing COS7 infected with 1 moi of NDV at 4\ub0C were collected after infection. Cell lysates were separated on an optiprep gradient. The 8 collected fractions were analyzed for sialidase activity toward Mu-NeuAc substrate and by western blot with antibodies a-caveolin 1 and a-NDV. We observed that NDV\u2019s neuraminidase is concentrated in the fraction corresponding to that of lipid rafts. Gangliosides have been identified as key receptors for some of caveolae-dependent viruses, such as for the polyomaviruses SV40 and Py (Tsai et al., 2003). Since it has been reported that NDV interacts with several gangliosides bearing both the Neu5 Ac and N-glycolylneuraminic sialic acid forms (Suzuky et al., 1985) and in vitro with different monosialogangliosides, disialogangliosides and trisialogangliosides, carrying sialic acid residues attached to a terminal or internal galactose (Ferreira et al.,2004), we tried to delineate if one or more gangliosides may act as receptors. We made a metabolic labelling with [1-3H]Sphingosine with a 2h pulse followed by a 48h chase; at 24h of chase cells were infected with NDV at 1 or 10 moi at 37\ub0C and collected after infection. Total lipids from lyophilized cells were extracted. [3H]Sphingolipids from aqueous and organic phases were separated by HPTLC. No differences could be detected between infected cells and controls in both aqueous and organic phases. It seems that NDV probably only hydrolize a very small number of sialo-glycosphingolipid to infect COS7 cells. Subsequently, we defined the glycosphingolipid pattern of COS7 cells at 24h post-infection, and we could observe a 50% decrease of GD1a accompained by a 46% increase of GM1 in infected cells compared to controls. This suggests that the newly syntetized HN specifically hydrolizes GD1a present on the host\u2019s surface to allow the virus spread by budding. To be sure that newly syntetized HN hydrolized only GD1a present on the host cell surface, we labelled COS7 with [1-3H]Sphingosine with a 2h pulse followed by a 48h chase: at 24h of chase, we made a co-colture for 24h of the labeled cells with COS7 cells infected by NDV. Analyzing the lipids from aqueous and organic phases, we could observe that GD1a of neighboring cells wasn\u2019t hydrolized by HN present on the infected cell\u2019s surface. The present study demonstrates that: \uf0d8 The overexpression of Neu3 sialidase leads to a decrease in the ability of NDV to infect host cells. \uf0d8 NDV\u2019s neuraminidase (HN) co-localizes with lipid rafts. \uf0d8 NDV\u2019s neuraminidase acts preferentially on ganglioside GD1a of host cells. GD1a of neighboring cells is not hydrolized by HN present on the infected cell\u2019s surface. Iwata et al.,(1994), J Virol. 68, 3200-3206 Simons et al., (1997) Nature 387, 569-572 Suzuky et al., (1985) JB 97,1189-1199 Ferreira et al., (2004) ICBCB 36, 2344-235

Suplementação de vacas da raça holandesa pastejando aveia e azevém e a variação no escore de condição corporal nos primeiros 100 dias de lactação.

O escore de condição corporal (ECC) é uma medida subjetiva das reservas corporais, baseado em avaliações visuais e táteis de pontos específicos do corpo, importante para monitorar os animais no início da lactação. O objetivo deste trabalho foi avaliar o ECC de vacas Holandesas em lactação mantidas em pastagem de aveia e azevém e suplementadas com níveis de 0, 4 e 8 kg/animal/dia de concentrado. O experimento foi realizado na EMBRAPA Pecuária Sul, de maio a dezembro de 2010, totalizando seis períodos de avaliações e 220 observações de 37 animais. Foi utilizado uma escala de 1 a 5, com subunidades de 0,25 pontos. Os resultados foram submetidos à análise de variância e as médias comparadas pelo teste de Dunnett a 5%, utilizando-se o SPSS 11.0. O ECC médio foi de 2,87 ± 0,56. Houve efeito (P0,05). O nível de concentrado influenciou negativamente o ECC de vacas Holandesas em lactação, em pastagem de aveia e azevém

Effet de la pression interstitielle sur la réponse sismique des sols : modélisation numérique 1D-3 Composantes

International audienceDuring strong quakes, the propagation of seismic waves in soil layers involves nonlinearities changing with the excitation level. A nonlinear hysteretic law is necessary to describe the variations of the stiffness and the energy dissipation during the seismic shaking. Furthermore, the influence of the pore pressure (cyclic mobility and liquefaction) cannot be neglected for saturated soils under strong quakes. Starting from a FEM formulation describing 1D propagation and three-dimensional loading ("1D-3 components approach"), the influence of the water is accounted for through a relation between the pore pressure and the work of the shear stress initially proposed by Iai. This model describes the variations of the pore pressure from the three-dimensional stress state of the soil. It has been validated through comparisons to laboratory tests (cyclic triaxial tests on saturated sands) and an analysis under three-dimensional excitations (seismic loading polarized along the 3 directions of space). The results involving 3 simultaneous excitation components and a single component in 3 separated analyses show the influence of the loading path on the seismic response and the pore pressure build-up

Efeito de diferentes níveis de concentrado nos componentes do leite de vacas holandesas em sistema a pasto.

O objetivo deste trabalho foi avaliar o efeito de três níveis de concentrado sobre os componentes do leite de vacas da raça Holandesa. Foram analisadas 453 amostras de leite provenientes de vacas da raça Holandesa recebendo diariamente três níveis de concentrado comercial (0Kg, 2Kg e 4Kg) e mantidas em pastagens cultivadas temperadas e nativas melhoradas durante o período de primavera/verão na oferta de 6 kg por dia de matéria seca para cada 100 kg de peso vivo. Os resultados foram submetidos à análise de variância e as médias submetidas ao teste de Dunnett T3 a 5%. Os valores médios e seus respectivos desvios-padrões para a porcentagem de gordura (%GORD), proteína (%PROT) e lactose (%LACT) foram de 3,15 ± 0,77%, 3,14 ± 0,34%; e 5,30 ± 20,28%. Os valores médios para a %GORD, %PROT e %LACT foram de 3,36 ± 0,83%, 3,20 ± 0,36%, e 4,29 ± 0,25%; de 3,24 ± 0,80%, 3,18 ± 0,37%, e 4,36 ± 0,25%; e de 2,86 ± 0,60%, 3,05 ± 0,24%, e 7,22 ± 34,89%, para o níveis 0Kg, 2Kg e 4Kg de concentrado comercial, respectivamente. Houve diferença (P=0,000) dos níveis de concentrado para a %GORD e %PROT, todavia, não houve diferença (P=0,356) para a %LACT. As vacas que receberam 4Kg de concentrado apresentaram a menor %GORD e %PROT. O nível de concentrado influenciou a porcentagem de gordura e de proteína no leite de vacas Holandesas em sistema a pasto

Estratigrafía de alta resolución de depósitos de flujos gravitacionales de la Formación Los Molles (Grupo Cuyo - Jurásico) en la región de La Jardinera, cuenca Neuquina

El Grupo Cuyo en el área del arroyo La Jardinera (Pliensbachiano a Caloviano Temprano) refleja una fisiografía de cuenca de plataforma y talud. Una discordancia de orden mayor define la culminación de una espesa sucesión de turbiditas y representa un límite de secuencia de 2do orden. La más antigua secuencia de 2do orden comprende el tramo basal de la Formación Los Molles e incluye un ciclo transgresivo-regresivo asignado a estratos de base de talud y fondo de cuenca. La secuencia más joven, que incluye el resto del Grupo Cuyo, consiste de una sucesión regresiva puntuada por varios desplazamientos abruptos de elementos fisiográficos (límites de secuencia de 3er orden). Las secuencias depositacionales de 3er orden incluyen depósitos de talud y de base de talud del Aaleniano de la Formación Los Molles, facies de plataforma del Bajociano Temprano en la transición entre las formaciones Los Molles y Lajas, y sedimentitas de plataforma interna a nearshore del Bajociano medio a tardío de la Formación Lajas, que es a su vez cubierta por depósitos del Bathoniano aluvial de la Formación Challacó. Ciclos de más alta frecuencia alternan en la sucesión y han controlado el origen, depositación y abandono de los elementos arquitecturales. Análisis de facies y de elementos arquitecturales indican que los depósitos gravitatorios densos de la Formación Los Molles encuadran en un modelo de rampa submarina. Los tres tipos de flujos gravitacionales (GF) han sido relacionados a caídas del nivel del mar de 4to orden. Los GF1 fueron asignados a flujos hiperpícnicos producidos durante un cortejo temprano de mar bajo de 4to orden, coevo con incisión fluvial en la plataforma, que evolucionaran a flujos turbidíticos en el talud, produciendo cargas de pulsos depositacionales cuya signatura composicional es fluvial. El sistema de GF1 incluye canales relativamente pequeños sobre el talud, lóbulos bien desarrollados en la base del talud y la planicie de fondo de cuenca proximales, así como lóbulos adventicios en la planicie del fondo de cuenca. Los estratos GF2 fueron asignados a flujos hiperpícnicos influenciados por un componente oscilatorio y relacionados a cortejos tardíos de mar bajo de 4to orden, que también incluyen relleno de canales incididos. Ellos están caracterizados en el talud por areniscas onduladas y laminadas ampliamente distribuidas y facies heterolíticas, que incluyen scours de bajo relieve en sus porciones centrales. Ellos representan flujos densos más diluidos y de grano más fino con respecto a los GF1. Los GF3, también asociados con cuñas de mar bajo de 4to orden, comprenden depósitos de flujos en masa y debris de talud y de base de talud, que representan clásicos surges de corta duración causados por debilidades en taludes relativamente abruptos. Los cortejos transgresivos y de mar alto de 4to orden llevaron al abandono de la etapa turbidítica y extensiva depositación de fango.The Pliensbachian to Early Callovian Cuyo Group in the Arroyo La Jardinera area reflects a shelf-slope-basin plain physiography. A major unconformity defines the onset of a thick turbidite succession and represents a 2nd order sequence boundary. The older 2nd order depositional sequence comprises the base of Los Molles Formation and includes a transgressive-regressive cycle ascribed to basin plain and slope rise strata. The younger one, which includes the rest of the Cuyo Group, consists of a regressive succession punctuated by several abrupt shifts of physiographic elements (3rd order sequence boundaries). The 3rd order depositional sequences include Aalenian slope rise and slope deposits in the Los Molles Formation, Early Bajocian shelfal facies at the transition of the Los Molles and Lajas formations, and Middle to Late Bajocian inner shelf to nearshore strata in the Lajas Formation that are overlain by Bathonian alluvial deposits of the Challacó Formation. Higher frequency cycles punctuate the succession and have controlled the origin, deposition and abandonment of the architectural elements. Facies and architectural elements analyses indicate that gravity flow deposits in the Los Molles Formation fit in a submarine ramp model. The three identified gravity flow (GF) types have been related to 4th order relative sea-level falls. The GF1 was ascribed to hyperpycnal flows produced during 4th order early lowstand, coeval to fluvial incision on the shelf, which were ignited in the slope and produced depletive depositional pulses with a fluvial compositional signature. The GF1 system includes relatively small channels in the slope, well-developed lobes in the slope rise and proximal basin plain, and lobe fringes in the basin plain. The GF2 strata were ascribed to hyperpycnal flows influenced by an oscillatory component and related to the 4th order late lowstand systems tract that also includes the incised channels fill. They are characterized in the slope by widespread, undulating and laminated sandstone and heterolithic facies, which include low relief scours at their central portions. They represent more diluted and finer-grained density flows relative to the GF1. The GF3, also associated with the 4th order lowstand wedge, comprises mass and debris flow deposits in the slope and slope rise that represent classical, short-lived surges caused by slope failures on relatively steep slopes. The 4th order transgressive and highstand systems tracts led to the abandonment of the turbidite stages and widespread mud deposition.Fil: Paim, Paulo S. G.. Universidad de Vale do Rio dos Sinos; BrasilFil: Silveira, Ariane. Universidad de Vale do Rio dos Sinos; BrasilFil: Lavina, Ernesto L. C.. Universidad de Vale do Rio dos Sinos; BrasilFil: Faccini, Ubiratan F.. Universidad de Vale do Rio dos Sinos; BrasilFil: Leanza, Hector Armando. Secretaría de Industria y Minería. Servicio Geológico Minero Argentino; Argentina. Universidad Nacional de La Plata; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Museo Argentino de Ciencias Naturales "Bernardino Rivadavia"; ArgentinaFil: Teixeira de Oliveira, J. M. M.. Universidad de Vale do Rio dos Sinos; BrasilFil: D'Avila, Roberto S. F.. Universidad de Vale do Rio dos Sinos; Brasil. Petrobras Argentina S.a; Argentin

Editorial: Explainable artificial intelligence models and methods in finance and healthcare

This article is a foreword to a special issue on "Explainable artificial intelligence models and methods in finance and healthcare" and introduces the main articles of the collection. The core topic of this special issue is explainability and trusting algorithmic output

Parallaxes of southern extremely cool objects III : 118 L and T dwarfs

We present new results from the Parallaxes of Southern Extremely Cool dwarfs program to measure parallaxes, proper motions and multiepoch photometry of L and early T dwarfs. The observations were made on 108 nights over the course of 8 yr using the Wide Field Imager on the ESO 2.2m telescope. We present 118 new parallaxes of L and T dwarfs of which 52 have no published values and 24 of the 66 published values are preliminary estimates from this program. The parallax precision varies from 1.0 to 15.5mas with a median of 3.8mas. We find evidence for two objects with long term photometric variation and 24 new moving group candidates. We cross-match our sample to published photometric catalogues and find standard magnitudes in up to 16 pass-bands from which we build spectral energy distributions and H-R diagrams. This allows us to confirm the theoretically anticipated minimum in radius between stars and brown dwarfs across the hydrogen burning minimum mass. We find the minimum occurs between L2 and L6 and verify the predicted steep dependence of radius in the hydrogen burning regime and the gentle rise into the degenerate brown dwarf regime. We find a relatively young age of ~2 Gyr from the kinematics of our sample.Peer reviewedFinal Accepted Versio