High-resolution numerical modelling of flow-vegetation interactions

In this paper, we present and apply a new three-dimensional model for the prediction of canopy-flow and turbulence dynamics in open-channel flow. The approach uses a dynamic immersed boundary technique that is coupled in a sequentially staggered manner to a large eddy simulation. Two different biomechanical models are developed depending on whether the vegetation is dominated by bending or tensile forces. For bending plants, a model structured on the Euler-Bernoulli beam equation has been developed, whilst for tensile plants, an N-pendula model has been developed. Validation against flume data shows good agreement and demonstrates that for a given stem density, the models are able to simulate the extraction of energy from the mean flow at the stem-scale which leads to the drag discontinuity and associated mixing layer.

Cell surface expression of the epithelial Na channel and a mutant causing Liddle syndrome: a quantitative approach.

The epithelial amiloride-sensitive sodium channel (ENaC) controls transepithelial Na+ movement in Na(+)-transporting epithelia and is associated with Liddle syndrome, an autosomal dominant form of salt-sensitive hypertension. Detailed analysis of ENaC channel properties and the functional consequences of mutations causing Liddle syndrome has been, so far, limited by lack of a method allowing specific and quantitative detection of cell-surface-expressed ENaC. We have developed a quantitative assay based on the binding of 125I-labeled M2 anti-FLAG monoclonal antibody (M2Ab*) directed against a FLAG reporter epitope introduced in the extracellular loop of each of the alpha, beta, and gamma ENaC subunits. Insertion of the FLAG epitope into ENaC sequences did not change its functional and pharmacological properties. The binding specificity and affinity (Kd = 3 nM) allowed us to correlate in individual Xenopus oocytes the macroscopic amiloride-sensitive sodium current (INa) with the number of ENaC wild-type and mutant subunits expressed at the cell surface. These experiments demonstrate that: (i) only heteromultimeric channels made of alpha, beta, and gamma ENaC subunits are maximally and efficiently expressed at the cell surface; (ii) the overall ENaC open probability is one order of magnitude lower than previously observed in single-channel recordings; (iii) the mutation causing Liddle syndrome (beta R564stop) enhances channel activity by two mechanisms, i.e., by increasing ENaC cell surface expression and by changing channel open probability. This quantitative approach provides new insights on the molecular mechanisms underlying one form of salt-sensitive hypertension.

Trypsin cleaves acid-sensing ion channel 1a in a domain that is critical for channel gating.

Acid-sensing ion channels (ASICs) are neuronal Na(+) channels that are members of the epithelial Na(+) channel/degenerin family and are transiently activated by extracellular acidification. ASICs in the central nervous system have a modulatory role in synaptic transmission and are involved in cell injury induced by acidosis. We have recently demonstrated that ASIC function is regulated by serine proteases. We provide here evidence that this regulation of ASIC function is tightly linked to channel cleavage. Trypsin cleaves ASIC1a with a similar time course as it changes ASIC1a function, whereas ASIC1b, whose function is not modified by trypsin, is not cleaved. Trypsin cleaves ASIC1a at Arg-145, in the N-terminal part of the extracellular loop, between a highly conserved sequence and a sequence that is critical for ASIC1a inhibition by the venom of the tarantula Psalmopoeus cambridgei. This channel domain controls the inactivation kinetics and co-determines the pH dependence of ASIC gating. It undergoes a conformational change during inactivation, which renders the cleavage site inaccessible to trypsin in inactivated channels.

The epithelial sodium channel and the control of sodium balance.

Studies aiming at the elucidation of the genetic basis of rare monogenic forms of hypertension have identified mutations in genes coding for the epithelial sodium channel ENaC, for the mineralocorticoid receptor, or for enzymes crucial for the synthesis of aldosterone. These genetic studies clearly demonstrate the importance of the regulation of Na(+) absorption in the aldosterone-sensitive distal nephron (ASDN), for the maintenance of the extracellular fluid volume and blood pressure. Recent studies aiming at a better understanding of the cellular and molecular basis of ENaC-mediated Na(+) absorption in the distal part of nephron, have essentially focused on the regulation ENaC activity and on the aldosterone-signaling cascade. ENaC is a constitutively open channel, and factors controlling the number of active channels at the cell surface are likely to have profound effects on Na(+) absorption in the ASDN, and in the amount of Na(+) that is excreted in the final urine. A number of membrane-bound proteases, kinases, have recently been identified that increase ENaC activity at the cell surface in heterologous expressions systems. Ubiquitylation is a general process that regulates the stability of a variety of target proteins that include ENaC. Recently, deubiquitylating enzymes have been shown to increase ENaC activity in heterologous expressions systems. These regulatory mechanisms are likely to be nephron specific, since in vivo studies indicate that the adaptation of the renal excretion of Na(+) in response to Na(+) diet occurs predominantly in the early part (the connecting tubule) of the ASDN. An important work is presently done to determine in vivo the physiological relevance of these cellular and molecular mechanisms in regulation of ENaC activity. The contribution of the protease-dependent ENaC regulation in mediating Na(+) absorption in the ASDN is still not clearly understood. The signaling pathway that involves ubiquitylation of ENaC does not seem to be absolutely required for the aldosterone-mediated control of ENaC. These in vivo physiological studies presently constitute a major challenge for our understanding of the regulation of ENaC to maintain the Na(+) balance.

TECTONICS OF THE HELVETIC NAPPE SYSTEM (W SWITZERLAND): CONTROL OF STRAIN LOCALIZATION AND BASEMENT-COVER DEFORMATION. Insights from models based on continuum mechanics

The Helvetic nappe system in Western Switzerland is a stack of fold nappes and thrust sheets em-placed at low grade metamorphism. Fold nappes and thrust sheets are also some of the most common features in orogens. Fold nappes are kilometer scaled recumbent folds which feature a weakly deformed normal limb and an intensely deformed overturned limb. Thrust sheets on the other hand are characterized by the absence of overturned limb and can be defined as almost rigid blocks of crust that are displaced sub-horizontally over up to several tens of kilometers. The Morcles and Doldenhom nappe are classic examples of fold nappes and constitute the so-called infra-Helvetic complex in Western and Central Switzerland, respectively. This complex is overridden by thrust sheets such as the Diablerets and Wildhörn nappes in Western Switzerland. One of the most famous example of thrust sheets worldwide is the Glariis thrust sheet in Central Switzerland which features over 35 kilometers of thrusting which are accommodated by a ~1 m thick shear zone. Since the works of the early Alpine geologist such as Heim and Lugeon, the knowledge of these nappes has been steadily refined and today the geometry and kinematics of the Helvetic nappe system is generally agreed upon. However, despite the extensive knowledge we have today of the kinematics of fold nappes and thrust sheets, the mechanical process leading to the emplacement of these nappe is still poorly understood. For a long time geologist were facing the so-called 'mechanical paradox' which arises from the fact that a block of rock several kilometers high and tens of kilometers long (i.e. nappe) would break internally rather than start moving on a low angle plane. Several solutions were proposed to solve this apparent paradox. Certainly the most successful is the theory of critical wedges (e.g. Chappie 1978; Dahlen, 1984). In this theory the orogen is considered as a whole and this change of scale allows thrust sheet like structures to form while being consistent with mechanics. However this theoiy is intricately linked to brittle rheology and fold nappes, which are inherently ductile structures, cannot be created in these models. When considering the problem of nappe emplacement from the perspective of ductile rheology the problem of strain localization arises. The aim of this thesis was to develop and apply models based on continuum mechanics and integrating heat transfer to understand the emplacement of nappes. Models were solved either analytically or numerically. In the first two papers of this thesis we derived a simple model which describes channel flow in a homogeneous material with temperature dependent viscosity. We applied this model to the Morcles fold nappe and to several kilometer-scale shear zones worldwide. In the last paper we zoomed out and studied the tectonics of (i) ductile and (ii) visco-elasto-plastic and temperature dependent wedges. In this last paper we focused on the relationship between basement and cover deformation. We demonstrated that during the compression of a ductile passive margin both fold nappes and thrust sheets can develop and that these apparently different structures constitute two end-members of a single structure (i.e. nappe). The transition from fold nappe to thrust sheet is to first order controlled by the deformation of the basement. -- Le système des nappes helvétiques en Suisse occidentale est un empilement de nappes de plis et de nappes de charriage qui se sont mis en place à faible grade métamorphique. Les nappes de plis et les nappes de charriage sont parmi les objets géologiques les plus communs dans les orogènes. Les nappes de plis sont des plis couchés d'échelle kilométrique caractérisés par un flanc normal faiblement défor-mé, au contraire de leur flanc inverse, intensément déformé. Les nappes de charriage, à l'inverse se caractérisent par l'absence d'un flanc inverse bien défini. Elles peuvent être définies comme des blocs de croûte terrestre qui se déplacent de manière presque rigide qui sont déplacés sub-horizontalement jusqu'à plusieurs dizaines de kilomètres. La nappe de Mordes et la nappe du Doldenhorn sont des exemples classiques de nappes de plis et constitue le complexe infra-helvétique en Suisse occidentale et centrale, respectivement. Ce complexe repose sous des nappes de charriages telles les nappes des Diablerets et du Widlhörn en Suisse occidentale. La nappe du Glariis en Suisse centrale se distingue par un déplacement de plus de 35 kilomètres qui s'est effectué à la faveur d'une zone de cisaillement basale épaisse de seulement 1 mètre. Aujourd'hui la géométrie et la cinématique des nappes alpines fait l'objet d'un consensus général. Malgré cela, les processus mécaniques par lesquels ces nappes se sont mises en place restent mal compris. Pendant toute la première moitié du vingtième siècle les géologues les géologues ont été confrontés au «paradoxe mécanique». Celui-ci survient du fait qu'un bloc de roche haut de plusieurs kilomètres et long de plusieurs dizaines de kilomètres (i.e., une nappe) se fracturera de l'intérieur plutôt que de se déplacer sur une surface frictionnelle. Plusieurs solutions ont été proposées pour contourner cet apparent paradoxe. La solution la plus populaire est la théorie des prismes d'accrétion critiques (par exemple Chappie, 1978 ; Dahlen, 1984). Dans le cadre de cette théorie l'orogène est considéré dans son ensemble et ce simple changement d'échelle solutionne le paradoxe mécanique (la fracturation interne de l'orogène correspond aux nappes). Cette théorie est étroitement lié à la rhéologie cassante et par conséquent des nappes de plis ne peuvent pas créer au sein d'un prisme critique. Le but de cette thèse était de développer et d'appliquer des modèles basés sur la théorie de la méca-nique des milieux continus et sur les transferts de chaleur pour comprendre l'emplacement des nappes. Ces modèles ont été solutionnés de manière analytique ou numérique. Dans les deux premiers articles présentés dans ce mémoire nous avons dérivé un modèle d'écoulement dans un chenal d'un matériel homogène dont la viscosité dépend de la température. Nous avons appliqué ce modèle à la nappe de Mordes et à plusieurs zone de cisaillement d'échelle kilométrique provenant de différents orogènes a travers le monde. Dans le dernier article nous avons considéré le problème à l'échelle de l'orogène et avons étudié la tectonique de prismes (i) ductiles, et (ii) visco-élasto-plastiques en considérant les transferts de chaleur. Nous avons démontré que durant la compression d'une marge passive ductile, a la fois des nappes de plis et des nappes de charriages peuvent se développer. Nous avons aussi démontré que nappes de plis et de charriages sont deux cas extrêmes d'une même structure (i.e. nappe) La transition entre le développement d'une nappe de pli ou d'une nappe de charriage est contrôlé au premier ordre par la déformation du socle. -- Le système des nappes helvétiques en Suisse occidentale est un emblement de nappes de plis et de nappes de chaînage qui se sont mis en place à faible grade métamoiphique. Les nappes de plis et les nappes de charriage sont parmi les objets géologiques les plus communs dans les orogènes. Les nappes de plis sont des plis couchés d'échelle kilométrique caractérisés par un flanc normal faiblement déformé, au contraire de leur flanc inverse, intensément déformé. Les nappes de charriage, à l'inverse se caractérisent par l'absence d'un flanc inverse bien défini. Elles peuvent être définies comme des blocs de croûte terrestre qui se déplacent de manière presque rigide qui sont déplacés sub-horizontalement jusqu'à plusieurs dizaines de kilomètres. La nappe de Morcles and la nappe du Doldenhorn sont des exemples classiques de nappes de plis et constitue le complexe infra-helvétique en Suisse occidentale et centrale, respectivement. Ce complexe repose sous des nappes de charriages telles les nappes des Diablerets et du Widlhörn en Suisse occidentale. La nappe du Glarüs en Suisse centrale est certainement l'exemple de nappe de charriage le plus célèbre au monde. Elle se distingue par un déplacement de plus de 35 kilomètres qui s'est effectué à la faveur d'une zone de cisaillement basale épaisse de seulement 1 mètre. La géométrie et la cinématique des nappes alpines fait l'objet d'un consensus général parmi les géologues. Au contraire les processus physiques par lesquels ces nappes sont mises en place reste mal compris. Les sédiments qui forment les nappes alpines se sont déposés à l'ère secondaire et à l'ère tertiaire sur le socle de la marge européenne qui a été étiré durant l'ouverture de l'océan Téthys. Lors de la fermeture de la Téthys, qui donnera naissance aux Alpes, le socle et les sédiments de la marge européenne ont été déformés pour former les nappes alpines. Le but de cette thèse était de développer et d'appliquer des modèles basés sur la théorie de la mécanique des milieux continus et sur les transferts de chaleur pour comprendre l'emplacement des nappes. Ces modèles ont été solutionnés de manière analytique ou numérique. Dans les deux premiers articles présentés dans ce mémoire nous nous sommes intéressés à la localisation de la déformation à l'échelle d'une nappe. Nous avons appliqué le modèle développé à la nappe de Morcles et à plusieurs zones de cisaillement provenant de différents orogènes à travers le monde. Dans le dernier article nous avons étudié la relation entre la déformation du socle et la défonnation des sédiments. Nous avons démontré que nappe de plis et nappes de charriages constituent les cas extrêmes d'un continuum. La transition entre nappe de pli et nappe de charriage est intrinsèquement lié à la déformation du socle sur lequel les sédiments reposent.

Effect of a temperature increase in the non-noxious range on proton-evoked ASIC and TRPV1 activity.

Acid-sensing ion channels (ASICs) are neuronal H(+)-gated cation channels, and the transient receptor potential vanilloid 1 channel (TRPV1) is a multimodal cation channel activated by low pH, noxious heat, capsaicin, and voltage. ASICs and TRPV1 are present in sensory neurons. It has been shown that raising the temperature increases TRPV1 and decreases ASIC H(+)-gated current amplitudes. To understand the underlying mechanisms, we have analyzed ASIC and TRPV1 function in a recombinant expression system and in dorsal root ganglion (DRG) neurons at room and physiological temperature. We show that temperature in the range studied does not affect the pH dependence of ASIC and TRPV1 activation. A temperature increase induces, however, a small alkaline shift of the pH dependence of steady-state inactivation of ASIC1a, ASIC1b, and ASIC2a. The decrease in ASIC peak current amplitudes at higher temperatures is likely in part due to the observed accelerated open channel inactivation kinetics and for some ASIC types to the changed pH dependence of steady-state inactivation. The increase in H(+)-activated TRPV1 current at the higher temperature is at least in part due to a hyperpolarizing shift in its voltage dependence. The contribution of TRPV1 relative to ASICs to H(+)-gated currents in DRG neurons increases with higher temperature and acidity. Still, ASICs remain the principal pH sensors of DRG neurons at 35°C in the pH range ≥6.

Structural, metamorphic and geochronological relations between the Zanskar Shear Zone and the Miyar Shear Zone (NW Indian Himalaya): Evidence for two distinct tectonic structures and implications for the evolution of the High Himalayan Crystalline of Zanskar

The geologic structures and metamorphic zonation of the northwestern Indian Himalaya contrast significantly with those in the central and eastern parts of the range, where the high-grade metamorphic rocks of the High Himalayan Crystalline (HHC) thrust southward over the weakly metamorphosed sediments of the Lesser Himalaya along the Main Central Thrust (MCT). Indeed, the hanging wall of the MCT in the NW Himalaya mainly consists of the greenschist facies metasediments of the Chamba zone, whereas HHC high-grade rocks are exposed more internally in the range as a large-scale dome called the Gianbul dome. This Gianbul dome is bounded by two oppositely directed shear zones, the NE-dipping Zanskar Shear Zone (ZSZ) on the northern flank and the SW-dipping Miyar Shear Zone (MSZ) on the southern limb. Current models for the emplacement of the HHC in NW India as a dome structure differ mainly in terms of the roles played by both the ZSZ and the MSZ during the tectonothermal evolution of the HHC. In both the channel flow model and wedge extrusion model, the ZSZ acts as a backstop normal fault along which the high-grade metamorphic rocks of the HHC of Zanskar are exhumed. In contrast, the recently proposed tectonic wedging model argues that the ZSZ and the MSZ correspond to one single detachment system that operates as a subhorizontal backthrust off of the MCT. Thus, the kinematic evolution of the two shear zones, the ZSZ and the MSZ, and their structural, metamorphic and chronological relations appear to be diagnostic features for discriminating the different models. In this paper, structural, metamorphic and geochronological data demonstrate that the MSZ and the ZSZ experienced two distinct kinematic evolutions. As such, the data presented in this paper rule out the hypothesis that the MSZ and the ZSZ constitute one single detachment system, as postulated by the tectonic wedging model. Structural, metamorphic and geochronological data are used to present an alternative tectonic model for the large-scale doming in the NW Indian Himalaya involving early NE-directed tectonics, weakness in the upper crust, reduced erosion at the orogenic front and rapid exhumation along both the ZSZ and the MSZ.

The role of tributary relative timing and sequencing in controlling large floods

Hydrograph convolution is a product of tributary inputs from across the watershed. The time-space distribution of precipitation, the biophysical processes that control the conversion of precipitation to runoff and channel flow conveyance processes, are heterogeneous and different areas respond to rainfall in different ways. We take a subwatershed approach to this and account for tributary flow magnitude, relative timing, and sequencing. We hypothesize that as the scale of the watershed increases so we may start to see systematic differences in subwatershed hydrological response. We test this hypothesis for a large flood (T >100 years) in a large watershed in northern England. We undertake a sensitivity analysis of the effects of changing subwatershed hydrological response using a hydraulic model. Delaying upstream tributary peak flow timing to make them asynchronous from downstream subwatersheds reduced flood magnitude. However, significant hydrograph adjustment in any one subwatershed was needed for meaningful reductions in stage downstream, although smaller adjustments in multiple tributaries resulted in comparable impacts. For larger hydrograph adjustments, the effect of changing the timing of two tributaries together was lower than the effect of changing each one separately. For smaller adjustments synergy between two subwatersheds meant the effect of changing them together could be greater than the sum of the parts. Thus, this work shows that while the effects of modifying biophysical catchment properties diminishes with scale due to dilution effects, their impact on relative timing of tributaries may, if applied in the right locations, be an important element of flood management.

An external site controls closing of the epithelial Na+ channel ENaC.

Members of the ENaC/degenerin family of ion channels include the epithelial sodium channel (ENaC), acid-sensing ion channels (ASICs) and the nematode Caenorhabditis elegans degenerins. These channels are activated by a variety of stimuli such as ligands (ASICs) and mechanical forces (degenerins), or otherwise are constitutively active (ENaC). Despite their functional heterogeneity, these channels might share common basic mechanisms for gating. Mutations of a conserved residue in the extracellular loop, namely the 'degenerin site' activate all members of the ENaC/degenerin family. Chemical modification of a cysteine introduced in the degenerin site of rat ENaC (betaS518C) by the sulfhydryl reagents MTSET or MTSEA, results in a approximately 3-fold increase in the open probability. This effect is due to an 8-fold shortening of channel closed times and an increase in the number of long openings. In contrast to the intracellular gating domain in the N-terminus which is critical for channel opening, the intact extracellular degenerin site is necessary for normal channel closing, as illustrated by our observation that modification of betaS518C destabilises the channel closed state. The modification by the sulfhydryl reagents is state- and size-dependent consistent with a conformational change of the degenerin site during channel opening and closing. We propose that the intracellular and extracellular modulatory sites act on a common channel gate and control the activity of ENaC at the cell surface.

Progesterone down-regulates the open probability of the amiloride-sensitive epithelial sodium channel via a Nedd4-2-dependent mechanism.

Activation of the mitogen-activated protein (MAP) kinase cascade by progesterone in Xenopus oocytes leads to a marked down-regulation of activity of the amiloride-sensitive epithelial sodium channel (ENaC). Here we have studied the signaling pathways involved in progesterone effect on ENaC activity. We demonstrate that: (i) the truncation of the C termini of the alphabetagammaENaC subunits results in the loss of the progesterone effect on ENaC; (ii) the effect of progesterone was also suppressed by mutating conserved tyrosine residues in the Pro-X-X-Tyr (PY) motif of the C termini of the beta and gamma ENaC subunits (beta(Y618A) and gamma(Y628A)); (iii) the down-regulation of ENaC activity by progesterone was also suppressed by co-expression ENaC subunits with a catalytically inactive mutant of Nedd4-2, a ubiquitin ligase that has been previously demonstrated to decrease ENaC cell-surface expression via a ubiquitin-dependent internalization/degradation mechanism; (iv) the effect of progesterone was significantly reduced by suppression of consensus sites (beta(T613A) and gamma(T623A)) for ENaC phosphorylation by the extracellular-regulated kinase (ERK), a MAP kinase previously shown to facilitate the binding of Nedd4 ubiquitin ligases to ENaC; (v) the quantification of cell-surface-expressed ENaC subunits revealed that progesterone decreases ENaC open probability (whole cell P(o), wcP(o)) and not its cell-surface expression. Collectively, these results demonstrate that the binding of active Nedd4-2 to ENaC is a crucial step in the mechanism of ENaC inhibition by progesterone. Upon activation of ERK, the effect of Nedd4-2 on ENaC open probability can become more important than its effect on ENaC cell-surface expression.

Evolution of a debris flow channel monitored using a 3D terrestrial laser scanner

Like numerous torrents in mountainous regions, the Illgraben creek (canton of Wallis, SW Switzerland) produces almost every year several debris flows. The total area of the active catchment is only 4.7 km², but large events ranging from 50'000 to 400'000 m³ are common (Zimmermann 2000). Consequently, the pathway of the main channel often changes suddenly. One single event can for instance fill the whole river bed and dig new several-meters-deep channels somewhere else (Bardou et al. 2003). The quantification of both, the rhythm and the magnitude of these changes, is very important to assess the variability of the bed's cross section and long profile. These parameters are indispensable for numerical modelling, as they should be considered as initial conditions. To monitor the channel evolution an Optech ILRIS 3D terrestrial laser scanner (LIDAR) was used. LIDAR permits to make a complete high precision 3D model of the channel and its surroundings by scanning it from different view points. The 3D data are treated and interpreted with the software Polyworks from Innovmetric Software Inc. Sequential 3D models allow for the determination of the variation in the bed's cross section and long profile. These data will afterwards be used to quantify the erosion and the deposition in the torrent reaches. To complete the chronological evolution of the landforms, precise digital terrain models, obtained by high resolution photogrammetry based on old aerial photographs, will be used. A 500 m long section of the Illgraben channel was scanned on 18th of August 2005 and on 7th of April 2006. These two data sets permit identifying the changes of the channel that occurred during the winter season. An upcoming scanning campaign in September 2006 will allow for the determination of the changes during this summer. Preliminary results show huge variations in the pathway of the Illgraben channel, as well as important vertical and lateral erosion of the river bed. Here we present the results of a river bank on the left (north-western) flank of the channel (Figure 1). For the August 2005 model the scans from 3 viewpoints were superposed, whereas the April 2006 3D image was obtained by combining 5 separate scans. The bank was eroded. The bank got eroded essentially on its left part (up to 6.3 m), where it is hit by the river and the debris flows (Figures 2 and 3). A debris cone has also formed (Figure 3), which suggests that a part of the bank erosion is due to shallow landslides. They probably occur when the river erosion creates an undercut slope. These geometrical data allow for the monitoring of the alluvial dynamics (i.e. aggradation and degradation) on different time scales and the influence of debris flows occurrence on these changes. Finally, the resistance against erosion of the bed's cross section and long profile will be analysed to assess the variability of these two key parameters. This information may then be used in debris flow simulation.

A new approach in the treatment of high flow native AV fistula: the open-pore external scaffolding prosthesis

Objective: The vascular access steal syndrome is a complication occurring in 1-6% after native arterio-venous (AV) fistulas, often due to huge diameter of the vein. This results in very high flow, which could also be responsible for cardiac overload. The aim of this study is to evaluate the efficiency of a new approach in the treatment of this pathology using open-pore external scaffolding prosthesis.Methods: This a retrospective review of all patients presenting symptomatic high flow after native AV fistula between January 2007 and December 2009 in 3 vascular centers. Pre-operative duplex exam confirmed the diagnosis of high flow. The operation consisted in preparation of the whole fistula, measurement of the flow and section on the venous side. The vein was wrapped with this 6 to 8 mm open-pore external scaffolding prosthesis (ProVena, BBraun, Germany) according to its diameter and to the flow and then sutured. Measurement of the flow was repeated. Patients were followed by duplex exam at 1 week and at 1, 3, 6 and 12 months. Procedural success was defined as complete implantation of the prosthesis and reduction of the flow. Primary outcomes were reduction of the flow and recovery of the symptoms and secondary endpoint was patency of the fistula.Results: During the study period, 14 patients, with a mean age of 65・8 years old, have been operated with this technique.There were 2 native forearmfistulas and 12 on the armwith a mean pre-operative flow of 2600 ml/min (1800-3800). The mode of presentation was pain in 6 patients, neurological disorders in 10 and necrosis in 4. Moreover, 3 patients had cardiac insufficiency due to high flow in the fistula. The procedure was technically successful in 100% of cases. Re-intervention was necessary in 2 patients due to hematoma. Recovery of the initial symptoms occurred in 13 patients (93%). The mean flow reduction was 1200 ml/min (600-2000). In 1 patient, a persistent steal syndrome despite flow reduction to 1400 ml/min resulted in fistula closure 2 months later. At a mean follow-up of 22 months (4-35), all remaining patients (13/14) presented a patent fistula without recurrence.Conclusion: This new approach seems to be safe and effective in the treatment of symptomatic high flow native AV fistulas by significantly reducing the flow and avoiding closure of the vascular access. Longer follow-up with more patients are necessary to evaluate the risk of recurrence.

Intracellular ubiquitylation of the epithelial Na+ channel controls extracellular proteolytic channel activation via conformational change.

The epithelial Na(+) channel ENaC is a key player in the maintenance of whole body Na(+) balance, and consequently of blood pressure. It is tightly regulated by numerous signaling pathways including ubiquitylation via the ubiquitin-protein ligase Nedd4-2. This mechanism is itself under the control of several kinases, which phosphorylate Nedd4-2, thereby interfering with ENaC/Nedd4-2 interaction, or by Usp2-45, which binds to and deubiquitylates ENaC. Another, different regulatory mechanism concerns the proteolytic activation of ENaC, during which the channel is cleaved on its luminal side by intracellular convertases such as furin, and further activated by extracellular proteases such as CAP-1. This process is regulated as well but the underlying mechanisms are not understood. Previously, evidence was provided that the ubiquitylation status of ENaC may affect the cleavage of the channel. When ubiquitylation of ENaC was reduced, either by co-expressing Usp2-45, or mutating either the ENaC PY-motifs (i.e. the binding sites for Nedd4-2) or intracellular lysines (i.e. ubiquitylation sites), the level of channel cleavage was increased. Here we demonstrate that lysine-mutated ENaC channels are not ubiquitylated at the cell surface, are preferentially cleaved, and Usp2-45 does not affect their cleavage efficiency. We further show by limited proteolysis that the intracellular ubiquitylation status of ENaC affects the extracellular conformation of αENaC, by demonstrating that non-ubiquitylated channels are more efficiently cleaved when treated with extracellularly added trypsin or chymotrypsin. These results present a new paradigm in which an intracellular, post-translational modification (e.g. ubiquitylation) of a transmembrane protein can affect its extracellular conformation.

Quantification of aortic flow by phase-contrast magnetic resonance in patients with bicuspid aortic valve.

AIMS: Bicuspid aortic valve (BAV) causes complex flow patterns in the ascending aorta (AAo), which may compromise the accuracy of flow measurement by phase-contrast magnetic resonance (PC-MR). Therefore, we aimed to assess and compare the accuracy of forward flow measurement in the AAo, where complex flow is more dominant in BAV patients, with flow quantification in the left ventricular outflow tract (LVOT) and the aortic valve orifice (AV), where complex flow is less important, in BAV patients and controls. METHODS AND RESULTS: Flow was measured by PC-MR in 22 BAV patients and 20 controls at the following positions: (i) LVOT, (ii) AV, and (iii) AAo, and compared with the left ventricular stroke volume (LVSV). The correlation between the LVSV and the forward flow in the LVOT, the AV, and the AAo was good in BAV patients (r = 0.97/0.96/0.93; P < 0.01) and controls (r = 0.96/0.93/0.93; P < 0.01). However, in relation with the LVSV, the forward flow in the AAo was mildly underestimated in controls and much more in BAV patients [median (inter-quartile range): 9% (4%/15%) vs. 22% (8%/30%); P < 0.01]. This was not the case in the LVOT and the AV. The severity of flow underestimation in the AAo was associated with flow eccentricity. CONCLUSION: Flow measurement in the AAo leads to an underestimation of the forward flow in BAV patients. Measurement in the LVOT or the AV, where complex flow is less prominent, is an alternative means for quantifying the systolic forward flow in BAV patients.

Heterogeneity of human monocytes: an optimized four-color flow cytometry protocol for analysis of monocyte subsets.

Monocytes are central mediators in the development of atherosclerotic plaques. They circulate in blood and eventually migrate into tissue including the vessel wall where they give rise to macrophages and dendritic cells. The existence of monocyte subsets with distinct roles in homeostasis and inflammation suggests specialization of function. These subsets are identified based on expression of the CD14 and CD16 markers. Routinely applicable protocols remain elusive, however. Here, we present an optimized four-color flow cytometry protocol for analysis of human blood monocyte subsets using a specific PE-Cy5-conjugated monoclonal antibody (mAb) to HLA-DR, a PE-Cy7-conjugated mAb to CD14, a FITC-conjugated mAb to CD16, and PE-conjugated mAbs to additional markers relevant to monocyte function. Classical CD14(+)CD16(-) monocytes (here termed "Mo1" subset) expressed high CCR2, CD36, CD64, and CD62L, but low CX(3)CR1, whereas "nonclassical" CD14(lo)CD16(+) monocytes (Mo3) essentially showed the inverse expression pattern. CD14(+)CD16(+) monocytes (Mo2) expressed high HLA-DR, CD36, and CD64. In patients with stable coronary artery disease (n = 13), classical monocytes were decreased, whereas "nonclassical" monocytes were increased 90% compared with healthy subjects with angiographically normal coronary arteries (n = 14). Classical monocytes from CAD patients expressed higher CX(3)CR1 and CCR2 than controls. Thus, stable CAD is associated with expansion of the nonclassical monocyte subset and increased expression of inflammatory markers on monocytes. Flow cytometric analysis of monocyte subsets and marker expression may provide valuable information on vascular inflammation. This may translate into the identification of monocyte subsets as selective therapeutic targets, thus avoiding adverse events associated with indiscriminate monocyte inhibition.