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)

A Structural Basis for Cellular Uptake of GST-Fold Proteins

It has recently emerged that glutathione transferase enzymes (GSTs) and other structurally related molecules can be translocated from the external medium into many different cell types. In this study we aim to explore in detail, the structural features that govern cell translocation and by dissecting the human GST enzyme GSTM2-2 we quantatively demonstrate that the α-helical C-terminal domain (GST-C) is responsible for this property. Attempts to further examine the constituent helices within GST-C resulted in a reduction in cell translocation efficiency, indicating that the intrinsic GST-C domain structure is necessary for maximal cell translocation capacity. In particular, it was noted that the α-6 helix of GST-C plays a stabilising role in the fold of this domain. By destabilising the conformation of GST-C, an increase in cell translocation efficiency of up to ∼2-fold was observed. The structural stability profiles of these protein constructs have been investigated by circular dichroism and differential scanning fluorimetry measurements and found to impact upon their cell translocation efficiency. These experiments suggest that the globular, helical domain in the 'GST-fold' structural motif plays a role in influencing cellular uptake, and that changes that affect the conformational stability of GST-C can significantly influence cell translocation efficiency.This work was supported by Grant DP0558315 Australian Research Council (http://www.arc.gov.au/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Human Cortical Excitability Increases with Time Awake

Prolonged wakefulness is associated not only with obvious changes in the way we feel and perform but also with well-known clinical effects, such as increased susceptibility to seizures, to hallucinations, and relief of depressive symptoms. These clinical effects suggest that prolonged wakefulness may be associated with significant changes in the state of cortical circuits. While recent animal experiments have reported a progressive increase of cortical excitability with time awake, no conclusive evidence could be gathered in humans. In this study, we combine transcranial magnetic stimulation (TMS) and electroencephalography (EEG) to monitor cortical excitability in healthy individuals as a function of time awake. We observed that the excitability of the human frontal cortex, measured as the immediate (0-20 ms) EEG reaction to TMS, progressively increases with time awake, from morning to evening and after one night of total sleep deprivation, and that it decreases after recovery sleep. By continuously monitoring vigilance, we also found that this modulation in cortical responsiveness is tonic and not attributable to transient fluctuations of the level of arousal. The present results provide noninvasive electrophysiological evidence that wakefulness is associated with a steady increase in the excitability of human cortical circuits that is rebalanced during slee

Diversidade de minhocas e atributos químicos em sistemas de plantio direto e integração lavoura-pecuária do oeste catarinense.

Resumo também apresentado no CONGRESSO DE INICIAÇÃO CIENTÍFICA E PÓS-GRADUAÇÃO, 2., 2012, São Leopoldo. Mostra de iniciação científica da UNISINOS. São Leopoldo: Casa Leiria, 2012. e-book. II CICPG. Disposição dos autores: ORSO, R.; BARTZ, M. L. C.; BROWN, G. G.; KLAUBER FILHO, O.; ROSA, M. G. da; LOCATELLI, M.; ZORTÉA, T.; CASAROTTO, K.; DECÄENS, T.; BARETTA, D

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

EEG Responses to TMS Are Sensitive to Changes in the Perturbation Parameters and Repeatable over Time

BACKGROUND: High-density electroencephalography (hd-EEG) combined with transcranial magnetic stimulation (TMS) provides a direct and non-invasive measure of cortical excitability and connectivity in humans and may be employed to track over time pathological alterations, plastic changes and therapy-induced modifications in cortical circuits. However, the diagnostic/monitoring applications of this technique would be limited to the extent that TMS-evoked potentials are either stereotypical (non-sensitive) or random (non-repeatable) responses. Here, we used controlled changes in the stimulation parameters (site, intensity, and angle of stimulation) and repeated longitudinal measurements (same day and one week apart) to evaluate the sensitivity and repeatability of TMS/hd-EEG potentials. METHODOLOGY/PRINCIPAL FINDINGS: In 10 volunteers, we performed 92 single-subject comparisons to evaluate the similarities/differences between pairs of TMS-evoked potentials recorded in the same/different stimulation conditions. For each pairwise comparison, we used non-parametric statistics to calculate a Divergence Index (DI), i.e., the percentage of samples that differed significantly, considering all scalp locations and the entire post-stimulus period. A receiver operating characteristic analysis showed that it was possible to find an optimal DI threshold of 1.67%, yielding 96.7% overall accuracy of TMS/hd-EEG in detecting whether a change in the perturbation parameters occurred or not. CONCLUSIONS/SIGNIFICANCE: These results demonstrate that the EEG responses to TMS essentially reflect deterministic properties of the stimulated neuronal circuits as opposed to stereotypical responses or uncontrolled variability. To the extent that TMS-evoked potentials are sensitive to changes and repeatable over time, they may be employed to detect longitudinal changes in the state of cortical circuits