3D mapping of the SPRY2 domain of ryanodine receptor 1 by single-particle Cryo-EM

The type 1 skeletal muscle ryanodine receptor (RyR1) is principally responsible for Ca(2+) release from the sarcoplasmic reticulum and for the subsequent muscle contraction. The RyR1 contains three SPRY domains. SPRY domains are generally known to mediate protein-protein interactions, however the location of the three SPRY domains in the 3D structure of the RyR1 is not known. Combining immunolabeling and single-particle cryo-electron microscopy we have mapped the SPRY2 domain (S1085-V1208) in the 3D structure of RyR1 using three different antibodies against the SPRY2 domain. Two obstacles for the image processing procedure; limited amount of data and signal dilution introduced by the multiple orientations of the antibody bound in the tetrameric RyR1, were overcome by modifying the 3D reconstruction scheme. This approach enabled us to ascertain that the three antibodies bind to the same region, to obtain a 3D reconstruction of RyR1 with the antibody bound, and to map SPRY2 to the periphery of the cytoplasmic domain of RyR1. We report here the first 3D localization of a SPRY2 domain in any known RyR isoform.The authors want to thank the Brigham and Women’s Hospital Biomedical Research Institute (to MS), the Australian National Health and the Medical Research Council (471418 to AD, MC and PB), and the European Commission (Marie Curie Action PIOF-GA-2009-237120 to AP-M)

Le basi neurologiche del rapporto tra il consumatore e la marca. Il contributo del neuro-imaging alle ricerche di marketing

Consumer develop very tight relationships with brands. In many cases, consumers develop positive relationships with their preferred brands and goods. In some of these cases true “love” relationship may occur. Sometimes, also negative relationships arise, often as a reaction toward unsatisfactory experiences, bad practices, etc. Companies aim at developing strong and positive emotional relationships between their brands and their customers. When they succeed, the brand is immediately recognized, it elicits positive affective responses, it is more difficult to be substituted for by competitors. The aim of the present study is to measure behavioral and emotional brain responses to covert visual recognition of brands. Functional magnetic resonance imaging (fMRI) was used to measure brain activity in 15 healthy subjects (7 females, 23-33 years) that were exposed to four stimulus types: coloured scrambled pictures, coloured squares, brand logos, and IAPS pictures with positive and negative valence scores. Sixty-three popular brands were selected among 8 different product categories. Two specific patterns of activation emerged for like (amygdale) and dislike brands (anterior medial cingulate, left inferior frontal gyrus, left middle temporal gyrus, medial cuneus). Implications for interpreting the role of brands in consumer mental processes are given, with special reference to the asymmetry between positive and negative evaluations

PROGRESS IN DUCTILE ALUMINIUM HIGH PRESSURE DIE CASTING ALLOYS FOR THE AUTOMOTIVE INDUSTRY

Today the die casting process is used to cast parts with high quality requirements such as engine cradles, cross members and nodes for space frame construction. This has presented the challenge to design alloys with superior mechanical properties. For crash relevant parts requiring high ductility (elongation > 12 %) one option to meet these properties is by heat treating a low iron Al-Si alloy to a T4 or T7 temper. However heat treatment can lead to part distortion and blistering resulting in higher costs for the producer. The second option is Al-Mg alloys type which fulfill these requirements in the as-cast state but the alloys are not easy to cast. Research was started using the easy to cast Al-Si alloy system targeting a high elongation (> 12 %) and yield-strength (> 120 MPa) already in temper F and not showing any long term aging behavior. This paper will discuss the technical progress which began in the early 1990ies, gaining in importance with the first series space frame Aluminum car, continuing with structural parts applied in the as-cast state and approaching a future with an increasing amount of light weight components replacing steel and heat treated Aluminum designs

Studio di saldabilità (LBW,FSW, EBW) di leghe da pressocolata a base alluminio

Le leghe da pressocolata sono generalmente difficilmente saldabili con i processi ad arco tradizionali acausa dei cicli termici di saldatura blandi che possono provocare la precipitazione di fasi fragili in ZTA e altempo stesso la complicata geometria dei pezzi può di fatto rendere inapplicabili certe tecnologie(saldature in interstizi non raggiungibili con torce di saldatura tradizionali). Di qui l’esigenza di uno studiodi saldabilità con processi alternativi come quelli ad energia concentrata (fascio laser e fascio elettronico) oil caratteristico processo Friction Stir che permette di saldare facilmente materiali basso fondenti come leleghe di alluminio senza portarle a fusione (fattore che in questo caso può rivelarsi molto positivo). Lo studio inquestione ha previsto l’utilizzo delle tre tecnologie di saldatura sopracitate tramite la tecnica conosciuta come“beads on plate” che consiste nella realizzazione di cordoni di saldatura direttamente su materiale base senzaunire fisicamente due pezzi ma che di fatto permette ugualmente di stabilire l’applicabilità o meno di undeterminato processo. Come materiali per la sperimentazione è stato previsto l’utilizzo di lastrinepressofuse da 2 e 4 mm di spessore, di due differenti leghe Al/Si modificate allo Stronzio riconducibili ai gruppiAlSi9Mn ed AlSi9MgMn. La prima caratterizzata dall’assenza di Magnesio, la seconda con tenori dello stessocompresi fra lo 0.1 e lo 0.5% (quindi inquadrabile come lega indurente per precipitazione)

Studio di saldabilit\ue0 (LBW,FSW, EBW)di leghe da pressocolata a base alluminio

Le leghe da pressocolata sono generalmente difficilmente saldabili con i processi ad arco tradizionali a causa dei cicli termici di saldatura blandi che possono provocare la precipitazione di fasi fragili in ZTA e al tempo stesso la complicata geometria dei pezzi pu\uf2 di fatto rendere inapplicabili certe tecnologie (saldature in interstizi non raggiungibili con torce di saldatura tradizionali). Di qui l\u2019esigenza di uno studio di saldabilit\ue0 con processi alternativi come quelli ad energia concentrata (fascio laser e fascio elettronico) o il caratteristico processo Friction Stir che permette di saldare facilmente materiali basso fondenti come le leghe di alluminio senza portarle a fusione (fattore che in questo caso pu\uf2 rivelarsi molto positivo). Lo studio in questione ha previsto l\u2019utilizzo delle tre tecnologie di saldatura sopracitate tramite la tecnica conosciuta come \u201cbeads on plate\u201d che consiste nella realizzazione di cordoni di saldatura direttamente su materiale base senza unire fisicamente due pezzi ma che di fatto permette ugualmente di stabilire l\u2019applicabilit\ue0 o meno di un determinato processo. Come materiali per la sperimentazione \ue8 stato previsto l\u2019utilizzo di lastrine pressofuse da 2 e 4 mm di spessore, di due differenti leghe Al/Si modificate allo Stronzio riconducibili ai gruppi AlSi9Mn ed AlSi9MgMn. La prima caratterizzata dall\u2019assenza di Magnesio, la seconda con tenori dello stesso compresi fra lo 0.1 e lo 0.5% (quindi inquadrabile come lega indurente per precipitazione

Dual mechanism of TRKB activation by anandamide through CB1 and TRPV1 receptors

Background. Administration of anandamide (AEA) or 2-arachidonoylglycerol (2AG) induces CB1 coupling and activation of TRKB receptors, regulating the neuronal migration and maturation in the developing cortex. However, at higher concentrations AEA also engages vanilloid receptor TRPV1, usually with opposed consequences on behavior. Methods and Results. Using primary cell cultures from the cortex of rat embryos (E18) we determined the effects of AEA on phosphorylated TRKB (pTRK). We observed that AEA (at 100 and 200 nM) induced a significant increase in pTRK levels. Such effect of AEA at 100 nM was blocked by pretreatment with the CBI antagonist AM251 (200 nM) and, at the higher concentration of 200 nM by the TRPV1 antagonist capsazepine (200 nM), but mildly attenuated by AM251. Interestingly, the effect of AEA or capsaicin (a TRPV1 agonist, also at 200 nM) on pTRK was blocked by TRKB.Fc (a soluble form of TRKB able to bind BDNF) or capsazepine, suggesting a mechanism dependent on BDNF release. Using the marble-burying test (MBT) in mice, we observed that the local administration of ACEA (a CBI agonist) into the prelimbic region of prefrontal cortex (PL-PFC) was sufficient to reduce the burying behavior, while capsaicin or BDNF exerted the opposite effect, increasing the number of buried marbles. In addition, both ACEA and capsaicin effects were blocked by previous administration of k252a (an antagonist of TRK receptors) into PL-PFC. The effect of systemically injected CB1 agonist WIN55,212-2 was blocked by previous administration of k252a. We also observed a partial colocalization of CBI /TRPV1 /TRKB in the PL-PFC, and the localization of TRPV1 in CaMK2+ cells. Conclusion. Taken together, our data indicate that anandamide engages a coordinated activation of TRKB, via CB1 and TRPV1. Thus, acting upon CBI. and TRPV1, AEA could regulate the TRKB-dependent plasticity in both pre- and postsynaptic compartments.Peer reviewe

Elastase-2 Knockout Mice Display Anxiogenic- and Antidepressant-Like Phenotype : Putative Role for BDNF Metabolism in Prefrontal Cortex

Several pieces of evidence indicate that elastase-2 (ELA2; chymotrypsin-like ELA2) is an alternative pathway to the generation of angiotensin II (ANGII). Elastase-2 knockout mice (ELA2KO) exhibit alterations in the arterial blood pressure and heart rate. However, there is no data on the behavioral consequences of ELA2 deletion. In this study, we addressed this question, submitting ELA2KO and wild-type (WT) mice to several models sensitive to anxiety- and depression-like, memory, and repetitive behaviors. Our data indicates a higher incidence of barbering behavior in ELA2KO compared to WT, as well as an anxiogenic phenotype, evaluated in the elevated plus maze (EPM). While a decrease in locomotor activity was observed in ELA2KO in EPM, this feature was not the main source of variation in the other parameters analyzed. The marble-burying test (MBT) indicated increase in repetitive behavior, observed by a higher number of buried marbles. The actimeter test indicated a decrease in total activity and confirmed the increase in repetitive behavior. The spatial memory was tested by repeated exposure to the actimeter in a 24-h interval. Both ELA2KO and WT exhibited decreased activity compared to the first exposure, without any distinction between the genotypes. However, when submitted to the cued fear conditioning, ELA2KO displayed lower levels of freezing behavior in the extinction session when compared to WT, but no difference was observed during the conditioning phase. Increased levels of BDNF were found in the prefrontal cortex but not in the hippocampus of ELA2KO mice compared to WT. Finally, in silico analysis indicates that ELA2 is putatively able to cleave BDNF, and incubation of the purified enzyme with BDNF led to the degradation of the latter. Our data suggested an anxiogenic- and antidepressant-like phenotype of ELA2KO, possibly associated with increased levels of BDNF in the prefrontal cortex.Peer reviewe