Xenogeneic, extracorporeal liver perfusion in primates improves the ratio of branched-chain amino acids to aromatic amino acids (Fischer's ratio)

In fulminant hepatic failure (FHF), the development of hepatic encephalopathy is associated with grossly abnormal concentrations of plasma amino acids (PAA). Normalization of the ratio of branched-chain amino acids to aromatic amino acids (Fischer's ratio) correlates with clinical improvement. This study evaluated changes in PAA metabolism during 4 h of isolated, normothermic extracorporeal liver perfusion using a newly designed system containing human blood and a rhesus monkey liver. Bile and urea production were within the physiological range. Release of the transaminases AST, ALT and LDH were minimal. The ratio of branched (valine, leucine, isoleucine) to aromatic (tyrosine, phenylalanine) amino acids increased significantly. These results indicate that a xenogeneic extracorporeal liver perfusion system is capable of significantly increasing Fischer's ratio and may play a role in treating and bridging patients in FHF in the future

A Simple, Highly Efficient Method for Heterologous Expression in Mammalian Primary Neurons Using Cationic Lipid-mediated mRNA Transfection

Expression of heterologous proteins in adult mammalian neurons is a valuable technique for the study of neuronal function. The post-mitotic nature of mature neurons prevents effective DNA transfection using simple, cationic lipid-based methods. Adequate heterologous protein expression is often only achievable using complex techniques that, in many cases, are associated with substantial toxicity. Here, a simple method for high efficiency transfection of mammalian primary neurons using in vitro transcribed mRNA and the cationic lipid transfection reagent Lipofectamine™ 2000 is described. Optimal transfection conditions were established in adult mouse dissociated dorsal root ganglion (DRG) neurons using a 96-well based luciferase activity assay. Using these conditions, a transfection efficiency of 25% was achieved in DRG neurons transfected with EGFP mRNA. High transfection efficiencies were also obtained in dissociated rat superior cervical ganglion (SCG) neurons and mouse cortical and hippocampal cultures. Endogenous Ca2+ currents in EGFP mRNA-transfected SCG neurons were not significantly different from untransfected neurons, which suggested that this technique is well suited for heterologous expression in patch clamp recording experiments. Functional expression of a cannabinoid receptor (CB1R), a G protein inwardly rectifying K+ channel (GIRK4) and a dominant-negative G protein α-subunit mutant (GoA G203T) indicate that the levels of heterologous protein expression attainable using mRNA transfection are suitable for most functional protein studies. This study demonstrates that mRNA transfection is a straightforward and effective method for heterologous expression in neurons and is likely to have many applications in neuroscience research

Exposición natural y envejecimiento acelerado de polietilenos para invernaderos, en diferentes condiciones climáticas

p.221-228Se evaluaron las propiedades mecánicas y ópticas de cuatro films de polietileno durante tres años de envejecimiento natural a la intemperie y mil horas de envejecimiento acelerado en laboratorio. Se expusieron en Yavi (Jujuy, Argentina) y se siguieron las normas A.S.T.M. Se correlacionaron los resultados obtenidos a la intemperie con los de envejecimiento acelerado para predecir la vida útil de un plástico, en condiciones climáticas similares, realizando sólo ensayos de laboratorio. Se observó que las propiedades mecánicas alcanzan más rápido las condiciones de envejecimiento y, por lo tanto, son mas relevantes para caracterizar el comportamiento de los mismos. Se analizaron las propiedades en función de la radiación solar y la precipitación acumulada, para independizarse de las variaciones climáticas. En Yavi la duración de los plásticos fue mayor que en Buenos Aires (Capital Federal, Argentina), en el mismo período. Se concluye que aunque la radiación en Yavi es elevada, el menor deterioro de los films probablemente sea por la baja precipitación y ausencia de contaminación

Rapid Modification of Proteins Using a Rapamycin-Inducible Tobacco Etch Virus Protease System

The ability to disrupt the function of a specific protein on a rapid time scale provides a powerful tool for biomedical research. Specific proteases provide a potential method to selectively cleave a chosen protein, but rapid control of protease activity is difficult.A heterologous expression system for rapid target-directed proteolysis in mammalian cells was developed. The system consists of an inducible NIa protease from the tobacco etch virus (TEVp) and a chosen protein into which a TEVp substrate recognition sequence (TRS) has been inserted. Inducible activity was conferred to the TEVp using rapamycin-controlled TEVp fragment complementation. TEVp activity was assayed using a FRET-based reporter construct. TEVp expression was well tolerated by mammalian cells and complete cleavage of the substrate was possible. Cleavage at 37 degrees C proceeded exponentially with a time constant of approximately 150 minutes. Attempts to improve cleavage efficiency were hampered by substantial background activity, which was attributed to inherent affinity between the TEVp fragments. A second TEVp assay, based on changes in inactivation of a modified K(V)3.4 channel, showed that functional properties of a channel can be using altered using an inducible TEVp system. Similar levels of background activity and variability were observed in both electrophysiological and FRET assays.The results suggested that an optimum level of TEVp expression leading to sufficient inducible activity (with minimal background activity) exists but the variability in expression levels between cells makes the present system rather impractical for single cell experiments. The system is likely to be more suitable for experiments in which the cell-to-cell variability is less of an issue; for example, in experiments involving large populations of cells

A 3.7 kb fragment of the mouse Scn10a gene promoter directs neural crest but not placodal lineage EGFP expression in a transgenic animal.

Under physiological conditions, the voltage-gated sodium channel Nav1.8 is expressed almost exclusively in primary sensory neurons. The mechanism restricting Nav1.8 expression is not entirely clear, but we have previously described a 3.7 kb fragment of the Scn10a promoter capable of recapitulating the tissue-specific expression of Nav1.8 in transfected neurons and cell lines (Puhl and Ikeda, 2008). To validate these studies in vivo, a transgenic mouse encoding EGFP under the control of this putative sensory neuron specific promoter was generated and characterized in this study. Approximately 45% of dorsal root ganglion neurons of transgenic mice were EGFP-positive (mean diameter = 26.5 μm). The majority of EGFP-positive neurons bound isolectin B4, although a small percentage (∼10%) colabeled with markers of A-fiber neurons. EGFP expression correlated well with the presence of Nav1.8 transcript (95%), Nav1.8-immunoreactivity (70%), and TTX-R INa (100%), although not all Nav1.8-expressing neurons expressed EGFP. Several cranial sensory ganglia originating from neurogenic placodes, such as the nodose ganglion, failed to express EGFP, suggesting that additional regulatory elements dictate Scn10a expression in placodal-derived sensory neurons. EGFP was also detected in discrete brain regions of transgenic mice. Quantitative PCR and Nav1.8-immunoreactivity confirmed Nav1.8 expression in the amygdala, brainstem, globus pallidus, lateral and paraventricular hypothalamus, and olfactory tubercle. TTX-R INa recorded from EGFP-positive hypothalamic neurons demonstrate the usefulness of this transgenic line to study novel roles of Nav1.8 beyond sensory neurons. Overall, Scn10a-EGFP transgenic mice recapitulate the majority of the Nav1.8 expression pattern in neural crest-derived sensory neurons.This work was supported by the Intramural program at the National Institutes of Health, National Institute on Alcohol Abuse and Alcoholis

Processes and products of turbidity currents entering soft muddy substrates

New laboratory experiments reveal that cohesionless turbidity currents are able to enter cohesive soft muddy substrates without losing their shape. These intrabed currents are driven by bed shear stress exceeding bed cohesive strength, and by flow density exceeding bed den - sity. The flows produce unique turbidites with internal mud layers, mixed cohesive-nonco - hesive sediment layers, and flame and load structures. A depositional model for intrabed (I) turbidites is proposed, comprising, from base to top: I1�sand-bearing mud, with a scoured base, dispersed mud, and mud clasts; I2�muddy sand from the intrabed portion of the tur - bidity current; I3�sandy mud with a speckled appearance; and I4�mud-poor sand from the suprabed portion of the flow. Complete I1�I4 turbidites are inferred to dominate loca - tions in nature where the currents mix with the bed and deep erosional scours form, filled with deformed or chaotic sand-mud mixtures. Further downflow, base-missing I2�I4 and I4 sequences signify gradual deceleration, loss of erosivity, and termination of intrabed flo

Controls in the development of transgressive dune fields of the Holocene coastal barrier, northern coast of Rio Grande do Sul

No litoral norte do Rio Grande do Sul, ao norte do balneário Xangri-lá, a barreira holocênica encontra-se parcialmente coberta pela fase moderna e relativamente ativa de um campo de dunas transgressivo, enquanto ao sul deste balneário, o campo de dunas cobre completamente a barreira. Este trabalho contribuiu para o conhecimento acerca desse comportamento diferenciado de desenvolvimento da fase moderna do campo de dunas transgressivo sobre a barreira holocênica. Foram utilizadas séries temporais de vento (rumo e velocidade) e precipitação de estações meteorológicas localizadas ao sul e ao norte de Xangri-lá, bem como foram realizadas medições simultâneas de vento, por meio de anemômetros portáteis, imediatamente ao norte e ao sul desse balneário. Potenciais de transporte de areia foram calculados para ambas as estações meteorológicas. Os registros históricos da estação de Tramandaí (mais ao sul) indicaram maiores velocidades de vento e menores de precipitação em relação à estação de Torres (mais ao norte). Devido à maior velocidade dos ventos em Tramandaí, o potencial de transporte de areia nessa localidade foi superior ao de Torres, indicando favorecimento da migração das dunas em suas proximidades. O comportamento desigual de desenvolvimento da fase moderna do campo de dunas transgressivo pode então estar relacionado à maior velocidade e frequência dos ventos NNE, NE, ENE e E em Tramandaí (ou seja, ao sul de Xangri-lá), além da menor precipitação, que também favorece um transporte de areia mais eficiente.In the north coast of Rio Grande do Sul, to the north of Xangri-lá beach, the Holocene barrier is partially covered by the modern and relatively active phase of a transgressive dune field, while to the south of this balneario the dune field completely covers the barrier. This work contributed to the knowledge about this differentiated development behavior of the modern phase of the transgressive dune field on the Holocene barrier. Time series of wind (direction and speed) and precipitation from meteorological stations located to the south and north of Xangri-lá were used, as well as simultaneous wind measurements using portable anemometers, immediately to the north and to the south of this balneario. Sand transport potentials were calculated for both meteorological stations. Historical records of the Tramandaí station (further south) indicated higher wind speeds and lower precipitation relative to the Torres station (further north). Due to the greater speed of the winds in Tramandaí, the potential of sand transport in this locality was higher than Torres, indicating the favor of the dunes migration in its proximities. The uneven developmental behavior of the modern phase of the transgressive dune field can then be related to the higher speed and frequency of the NNE, NE, ENE and E winds in Tramandaí (ie, south of Xangri-lá), as well as the lower precipitation also favors more efficient sand transportation

Alfvén waves in the near-PSBL lobe: Cluster observations

Electromagnetic low-frequency waves in the magnetotail lobe close to the PSBL (Plasma Sheet Boundary Layer) are studied using the Cluster spacecraft. The lobe waves show Alfvénic properties and transport their wave energy (Poynting flux) on average toward the Earth along magnetic field lines. Most of the wave events are rich with oxygen (O+) ion plasma. The rich O+ plasma can serve to enhance the magnetic field fluctuations, resulting in a greater likelihood of observation, but it does not appear to be necessary for the generation of the waves. Taking into account the fact that all events are associated with auroral electrojet enhancements, the source of the lobe waves might be a substorm-associated instability, i.e. some instability near the reconnection site, or an ion beam-related instability in the PSBL

Plasma convection in the magnetotail lobes: statistical results from Cluster EDI measurements

A major part of the plasma in the Earth's magnetotail is populated through transport of plasma from the solar wind via the magnetotail lobes. In this paper, we present a statistical study of plasma convection in the lobes for different directions of the interplanetary magnetic field and for different geomagnetic disturbance levels. The data set used in this study consists of roughly 340 000 one-minute vector measurements of the plasma convection from the Cluster Electron Drift Instrument (EDI) obtained during the period February 2001 to June 2007. The results show that both convection magnitude and direction are largely controlled by the interplanetary magnetic field (IMF). For a southward IMF, there is a strong convection towards the central plasma sheet with convection velocities around 10 km s<sup>−1</sup>. During periods of northward IMF, the lobe convection is almost stagnant. A <I>B<sub>y</sub></I> dominated IMF causes a rotation of the convection patterns in the tail with an oppositely directed dawn-dusk component of the convection for the northern and southern lobe. Our results also show that there is an overall persistent duskward component, which is most likely a result of conductivity gradients in the footpoints of the magnetic field lines in the ionosphere

Causal trajectories description of atom diffraction by surfaces

9 pages, 7 figures -- PACS numbers: 79.20.Rf, 03.65.Sq, 03.65.BzThe method of quantum trajectories proposed by de Broglie and Bohm is applied to the study of atom diffraction by surfaces. As an example, a realistic model for the scattering of He off corrugated Cu is considered. In this way, the final angular distribution of trajectories is obtained by box-counting, which is in excellent agreement with the results calculated by standard S-matrix methods of scattering theory. More interestingly, the accumulation of quantum trajectories at the different diffraction peaks is explained in terms of the corresponding quantum potential. This non-local potential "guides" the trajectories causing a transition from a distribution near the surface, which reproduces its shape, to the final diffraction pattern observed in the asymptotic region, far from the diffracting object. These two regimes are homologous to the Fresnel and Fraunhofer regions described in undulatory optics. Finally, the turning points of the quantum trajectories provide a better description of the surface electronic density than the corresponding classical ones, usually employed for this task.This work was supported by DGES (Spain) under contracts No PB95-71, PB95-425 and PB96-76. A.S. Sanz also acknowledges the Universidad Autónoma de Madrid for a doctoral grant.Peer reviewe