Anion and cation permeabilities of the mouse TMEM16F/ANO6 calcium-activated channel

TMEM16F/ANO6 is widely expressed in different tissues where it plays important physiological roles, such as the regulation of blood coagulation, bone mineralization and apoptosis. TMEM16F plays two different biological roles in cells: it acts both as scramblase and as ion channel. Both of these functions are activated by the increase of intracellular Ca2+ concentration. In physiological conditions the distribution of cellular membrane lipids is polarized, meaning that the composition of the outer leaflet is different than that of the inner one. When the intracellular Ca2+ increases, the scramblases are activated. Scramblases are integral membrane proteins responsible for the translocation of phospholipids between the two leaflets of a lipid bilayer upon their electrochemical gradient, abolishing the asymmetry of the bilayer. This is a key process in many physiological and pathological contests because the exposure of particular lipids at the cell surface induces several transduction signalling cascades. Many studies investigated in details the molecular mechanisms of TMEM16F lipid scrambling and identified a specific domain of the protein which is essential for the lipid transport. The mechanisms of TMEM16F ion channel activity are less clear and, in addition, several studies reported conflicting results about the ionic selectivity of the TMEM16F-mediated current. These data were partially obtained using different methodologies and analysis making difficult to reconcile the discrepancies. In particular, studies reporting that TMEM16F is more permeable for anions were performed only using the whole-cell recordings, a technique that does not allow a direct comparison of the results with or without intracellular calcium. In contrast, results showing a higher permeability for cations derive from recordings from excised patches in the inside-out configuration. Here, we decided to investigate the ionic selectivity of TMEM16F using both configurations of the patch clamp technique, whole-cell and inside-out excised patch, using the same solutions, in order to directly compare the results. Moreover, we investigated the properties of the Q559K mutant that has been reported to alter the ionic selectivity of TMEM16F in inside-out patches. First of all, our study revealed a characteristic which is shared between the configurations. Indeed, TMEM16F heterologous expression in HEK 293T cells generates a large outward rectifying Ca2+-activated current both in whole-cell and inside-out configuration which is due to the voltage-dependent gating of the channel. On the other hand, we found consistent differences in terms of channel time-dependent activation and selectivity. In fact, although in inside-out experiments the Ca2+-dependent activation is fast and the TMEM16F-mediated current is activated in few ms, in whole-cell recordings a full activation requires up to 4 minutes. Similar results are obtained also with Q559K mutant. In inside-out patches the Ca2+ concentration for half-maximal current activation (K1/2) is 42 \ub5M at +60 mV, while the Hill coefficient is >2. Q559K mutant shows a significant reduction of Ca2+ sensitivity with a K1/2 almost 5-folds higher and a reduction of Hill coefficient to 1.4, indicating a possible alteration of the gating mechanism. To determine the channel selectivity, we decided to calculate the relative permeability between Na+ and Cl\uaf (PNa/PCl) using the dilution method both in whole-cell and inside-out configuration. We found that TMEM16F-mediated current is highly non-selective but there are differences depending on the configuration of the recordings. Indeed, in whole-cell both TMEM16F wild type and Q559K mutant have a PNa/PCl around 0.5 indicating a slight preference for Cl\uaf permeation. In contrast, in inside-out experiments the Q559K mutant retains a higher permeability for Cl\uaf, while TMEM16F wild type channel shows a higher permeability for Na+ with a PNa/PCl reaching 3.6. These results have two major implications: First, the discrepancies found between the recordings in whole-cell and inside-out, could suggest the presence of a cellular mechanism factor which controls TMEM16F selectivity. When the patch is excised this cellular mechanism and its controlling activity are lost. Second, the residue in position 559 could be responsible to control the selectivity among cations and anions when this cellular mechanism is lost

City networks in the Lombardy region: an analysis in terms of communication flows

In this paper, it is shown how the logic that shapes the city-system is more complicated than the simple "territorial" and hierarchical logic of the traditional central-place model. The control of the market of outputs, inputs and innovative assets is performed by the firm, not only in terms of management of a gravity area, but also and increasingly in terms of network relationships. The new behavioural logic of the firm parallels and partly determines the new organisational logic of the city system, where phenomena of specialisation and networking also appear. Probability of interaction among cities may go far beyond what is expected on the basis of their size and distance. High density of information flows on a proximity basis, as for those taking place within specialised industrial "districts"; cooperation and spatial division of labour among specialised centres; and synergy among similarly specialised centres linked through high- performance information networks, are all elements calling for selected non-hierarchical linkages among centres, which we call "city networks". In the empirical realm of the Lombardy region, network relations among centres were discovered, mainly showing up in two specific spatial contexts: within the metropolitan area of Milan, shaping its emerging policentric structure; and within sub-regional industrial districts. In the first case, the empirical evidence suggests the existence of "synergy networks," occurring among similar centres performing headquarter and advanced production-services functions; in the second case, mainly "complementarity networks" and "milieu" type of interactions are revealed.Dans cet article, on montre que la logique interne d'un système de villes est plus complexe que la simple logique "territoriale" et hiérarchique du modèle traditionnel de la place centrale. La firme exerce son contrôle sur le marché des produits, des facteurs de production et de l'innovation non plus seulement au sein d'une aire gravitaire mais également par des relations en réseaux. Cette nouvelle stratégie des firmes accompagne et détermine en partie le nouveau principe d'organisation des systèmes de villes, où l'on commence également à observer des phénomènes de spécialisation et de mise en réseau. Les interactions entre villes peuvent aller bien au-delà de ce que l'on pourrait attendre compte tenu de leurs tailles respectives et de la distance entre elles. D'importants flux d'informations au sein d'espaces restreints (comme on en observe à l'intérieur des "districts" industriels spécialisés), la coopération et la division du travail entre centres spécialisés, la synergie entre centres identiquement spécialisés (reliés entre eux par des réseaux de communication à haute performance) : voilà autant d'éléments plaidant en faveur d'un principe de relations non hiérarchiques, pour former ce que nous nommons des "réseaux de villes". Des études empiriques menées en Lombardie ont montré l'existence de telles relations en réseau à deux niveaux : - dans l'agglomération de Milan, où émerge une structure polycentrique; - au sein des districts industriels de la région. Dans le premier cas, les données empiriques suggèrent l'existence de "réseaux de synergie" entre centres remplissant les mêmes fonctions de sièges sociaux et de fournisseurs de service haut de gamme. Dans le deuxième cas, on met surtout en évidence des "réseaux de complémentarité" et l'existence d'un milieu aux interactions spécifiques.Camagni Roberto, Stabilini Stefano, Diappi Lidia. City networks in the Lombardy region: an analysis in terms of communication flows. In: Flux, n°15, 1994. pp. 37-50

City Networks in the Lombardy Region: an Analysis in terms of Communication Flows

none3CAMAGNI R.; L. DIAPPI; STABILINI S.Camagni, Roberto; Diappi, Lidia; Stabilini, Stefan

Rilievo cronografico dei sistemi urbani del Corridoio Padano

Il testo analizza i caratteri spazio-temporali dei sistemi urbani del LIMES padano, la metropoli lineare che si estende da Torino a Trieste, innervata da un importante corridoio infrastrutturale. Si propongono metodologie di analisi cronografica alla scala vasta e si analizzano con maggiore dettaglio alcuni sistemi urbani significativi dell'area lombarda

Space-time design of the public city

Time has become an increasingly important topic in urban studies and urban planning. The spatial-temporal interplay is not only of relevance for the theory of urban development and urban politics, but also for urban planning and governance. The space-time approach focuses on the human being with its various habits and routines in the city. Understanding and taking those habits into account in urban planning and public policies offers a new way to improve the quality of life in our cities. Adapting the supply and accessibility of public spaces and services to the inhabitants’ space-time needs calls for an integrated approach to the physical design of urban space and to the organization of cities. In the last two decades the body of practical and theoretical work on urban space-time topics has grown substantially. The book offers a state of the art overview of the theoretical reasoning, the development of new analytical tools, and practical experience of the space-time design of public cities in major European countries. The contributions were written by academics and practitioners from various fields exploring space-time research and planning

Minimally invasive approach to incisional hernia in elective and emergency surgery: a SICE (Italian Society of Endoscopic Surgery and new technologies) and ISHAWS (Italian Society of Hernia and Abdominal Wall Surgery) online survey

Minimally invasive abdominal wall surgery is growing worldwide, with a constant and fast improvement of surgical techniques and surgeons' confidence in treating both primary and incisional hernias (IH). The Italian Society of Endoscopic Surgery and new technologies (SICE) and the ISHAWS (Italian Society of Hernia and Abdominal Wall Surgery) worked together to investigate state of the art in IH treatment in elective and emergency settings in Italy. An online open survey was designed, and Italian surgeons interested in abdominal wall surgery were invited to fill out a 20-point questionnaire on IH surgical procedures performed in their departments. Surgeons were asked to express their points of view on specific questions about technical and clinical variables in IH treatment. Preferred approach in elective IH surgery was minimally invasive (59.7%). Open surgery was the preferred approach in 40.3% of the responses. In emergency settings, open surgery was the preferred approach (65.4%); however, 34.5% of the involved surgeons declare to prefer the laparoscopic/endoscopic approach. Most respondents opted for conversion to open surgery in case of relevant surgical field contamination, with a non-mesh repair of abdominal wall defects. Among those that used the laparoscopic approach in the emergent setting, the majority (74%) used the size of the defect of 5 cm as a decisional cut-off. The spread of minimally invasive approaches to IH repair in emergency surgery in Italy is gaining relevance. Code-sharing through scientific societies can improve clinical practice in different departments and promote a tailored approach to IH surgery