P2Y1 receptor-evoked glutamate exocytosis from astrocytes: control by tumor necrosis factor-alpha and prostaglandins.

ATP, released by both neurons and glia, is an important mediator of brain intercellular communication. We find that selective activation of purinergic P2Y1 receptors (P2Y1R) in cultured astrocytes triggers glutamate release. By total internal fluorescence reflection imaging of fluorescence-labeled glutamatergic vesicles, we document that such release occurs by regulated exocytosis. The stimulus-secretion coupling mechanism involves Ca2+ release from internal stores and is controlled by additional transductive events mediated by tumor necrosis factor-alpha (TNFalpha) and prostaglandins (PG). P2Y1R activation induces release of both TNFalpha and PGE2 and blocking either one significantly reduces glutamate release. Accordingly, astrocytes from TNFalpha-deficient (TNF(-/-)) or TNF type 1 receptor-deficient (TNFR1(-/-)) mice display altered P2Y1R-dependent Ca2+ signaling and deficient glutamate release. In mixed hippocampal cultures, the P2Y1R-evoked process occurs in astrocytes but not in neurons or microglia. P2Y1R stimulation induces Ca2+ -dependent glutamate release also from acute hippocampal slices. The process in situ displays characteristics resembling those in cultured astrocytes and is distinctly different from synaptic glutamate release evoked by high K+ stimulation as follows: (a) it is sensitive to cyclooxygenase inhibitors; (b) it is deficient in preparations from TNF(-/-) and TNFR1(-/-) mice; and (c) it is inhibited by the exocytosis blocker bafilomycin A1 with a different time course. No glutamate release is evoked by P2Y1R-dependent stimulation of hippocampal synaptosomes. Taken together, our data identify the coupling of purinergic P2Y1R to glutamate exocytosis and its peculiar TNFalpha- and PG-dependent control, and we strongly suggest that this cascade operates selectively in astrocytes. The identified pathway may play physiological roles in glial-glial and glial-neuronal communication.

Effects of total fishing prohibition on the rocky fish assemblages of Medes Islands marine reserve (NW Mediterranean)

Visual scuba diving censuses were used to assess the effects of fishing prohibition on abundance and size structure of littoral fish populations by comparing the same benthic communities inside and outside the protected area of Medes Islands (NE Catalonia, Spain). The total number of species found was 43 in the reserve and 44 outside, but the mean value of species richness per sampling station was significantly higher in the protected area. However, diversity, heavily affected by the presence or absence of large schools of pelagic species, showed no significant differences between sites. The prohibition of fishing for 6 years is the first factor affecting the qualitative and quantitative structure of fish populations ('reserve effect'), and depth is the second factor. Thus, except in the cases of Serranus cabrilla and Mullus surmuletus, all other vulnerable species are highly sensitive to the protection measures. The size structure of all vulnerable species was found to be absolutely different at the reserve sites than in the unprotected zones, and the modal size classes of size frequency distributions were always higher in the reserve than outside. The reserve effect was significantly responsible of the differences observed in this change on size structure. Some highly vulnerable species, such as Epinephelus guaza and Sciaena umbra, have only been found in the protected area. Others, such as Sparus aurata, Diplodus cervinus and Dicentrarchus labrax, were much more frequent inside the reserve.

Imaging exocytosis and recycling of synaptic-like microvesicles in astrocytes.

Optical imaging techniques are well suited for following the dynamics of physiological processes in living cells. Total internal reflection fluorescence (TIRF) microscopy based on evanescent wave illumination (EWi) allows spectacular, real-time visualization of individual vesicle movements, fusions, and retrievals at the cell surface (i.e., within 100 nm of the plasma membrane). TIRF microscopy is an ideal approach for studying the properties of exocytosis and recycling in cultured astrocytes, particularly because these cells have a rather flat surface and contain secretory vesicles with sparse distribution. Among all populations of secretory vesicles, we focus here on synaptic-like microvesicles (SLMVs). We illustrate how TIRF microscopy using EWi is useful to study exocytosis and recycling of SLMVs at the single-vesicle level and, when combined with epifluorescence illumination (EPIi), can provide detailed information on the kinetics of exocytosis, endocytosis, and re-acidification at the whole-cell level.

Efeito da germinação de soja CV. BR-13 e Paraná sobre ácido fítico, fósforo total e inibidores de tripsina

Este trabalho foi conduzido para avaliar o efeito da germinação sobre os constituintes antinutricionais da soja, ácido fítico, P total e inibidores de tripsina, que interferem na utilização de proteínas e outras substâncias. A diminuição no aproveitamento de nutrientes resulta em inibição de crescimento, hipoglicemia ou danos a tecidos animais. Para os ensaios foram utilizadas as cultivares de soja (Glycine max (L.) Merrill ) BR-13 e Paraná. As sementes foram incubadas em câmara de germinação a 25°C e 100% de umidade, de 0 a 72 horas, com coleta de amostras em intervalos eqüidistantes de seis horas. A cultivar Paraná apresentou teores superiores de ácido fítico, P total e inibidores de tripsina, em relação à cultivar BR-13. As análises de variância e comparações entre médias indicaram que houve aumentos significativos (p < 0,05) nos teores de P total (8,5% e 3,6% nas cultivares BR-13 e Paraná, respectivamente) e nos níveis de inibidores de tripsina (22,05% e 17,10% nas cultivares BR-13 e Paraná, respectivamente), e reduções nos teores de ácido fítico (15,96% e 9,46% nas cultivares BR-13 e Paraná, respectivamente), em 72 horas de germinação.

Geostatistical inversion of seismic and GPR reflection images: what can we actually resolve?

Estimation of the spatial statistics of subsurface velocity heterogeneity from surface-based geophysical reflection survey data is a problem of significant interest in seismic and ground-penetrating radar (GPR) research. A method to effectively address this problem has been recently presented, but our knowledge regarding the resolution of the estimated parameters is still inadequate. Here we examine this issue using an analytical approach that is based on the realistic assumption that the subsurface velocity structure can be characterized as a band-limited scale-invariant medium. Our work importantly confirms recent numerical findings that the inversion of seismic or GPR reflection data for the geostatistical properties of the probed subsurface region is sensitive to the aspect ratio of the velocity heterogeneity and to the decay of its power spectrum, but not to the individual values of the horizontal and vertical correlation lengths.

SDF 1-alpha (CXCL12) triggers glutamate exocytosis from astrocytes on a millisecond time scale: Imaging analysis at the single-vesicle level with TIRF microscopy

Chemokines are small chemotactic molecules widely expressed throughout the central nervous system. A number of papers, during the past few years, have suggested that they have physiological functions in addition to their roles in neuroinflammatory diseases. In this context, the best evidence concerns the CXC-chemokine stromal cell-derived factor (SDF-1alpha or CXCL12) and its receptor CXCR4, whose signalling cascade is also implicated in the glutamate release process from astrocytes. Recently, astrocytic synaptic like microvesicles (SLMVs) that express vesicular glutamate transporters (VGLUTs) and are able to release glutamate by Ca(2+)-dependent regulated exocytosis, have been described both in tissue and in cultured astrocytes. Here, in order to elucidate whether SDF-1alpha/CXCR4 system can participate to the brain fast communication systems, we investigated whether the activation of CXCR4 receptor triggers glutamate exocytosis in astrocytes. By using total internal reflection (TIRF) microscopy and the membrane-fluorescent styryl dye FM4-64, we adapted an imaging methodology recently developed to measure exocytosis and recycling in synaptic terminals, and monitored the CXCR4-mediated exocytosis of SLMVs in astrocytes. We analyzed the co-localization of VGLUT with the FM dye at single-vesicle level, and observed the kinetics of the FM dye release during single fusion events. We found that the activation of CXCR4 receptors triggered a burst of exocytosis on a millisecond time scale that involved the release of Ca(2+) from internal stores. These results support the idea that astrocytes can respond to external stimuli and communicate with the neighboring cells via fast release of glutamate.

Calcium Microdomains Control Exo-Endocytosis of Synaptic-Like Microvesicles in Astrocytes

During the last decade, the discovery that astrocytes possess a nonelectrical form of excitability (Ca21-excitability) that leads to the release of chemical transmitters, an activity called ''gliotransmission'', indicates that these cells may have additional important roles in brain function. Elucidating the stimulus-secretion coupling leading to the exocytic release of chemical transmitters (such as glutamate, Bezzi et al., Nature Neurosci, 2004) may therefore clarify i) whether astrocytes represent in full a new class of secretory cells in the brain and ii) whether they can participate to the fast brain signaling in the brain. Here by using a recently developed approach for studying vesicle recycling dynamics at synapses (Voglmaier et al., Neuron, 2006; Balaji and Ryan, PNAS, 2007) combined with epifluorescence and total internal reflection fluorescence (TIRF) imaging, we investigated the spatiotemporal characteristics of stimulus-secretion coupling leading glutamate exocytosis of synaptic-like microvesicles (SLMVs) in astrocytes. We performed the analysis at both the whole-cell and single-vesicle levels providing the first system for comparing exo-endocytic processes in astrocytes with those in neurons. Both the time course and modalities of secretion in astrocytes present more similarities to neurons then previously expected. We found that 1. the G-protein-coupled receptor (GPCR)-evoked exocytosis reached the maximum on a ms time scale and that 2. ER tubuli formed sub-micrometer domains beneath the plasma membrane in close proximity to exocytic vesicles, where fusion events were spatiotemporally correlated with fast Ca21 events.

Smart instrumentation for determination of ligament stiffness and ligament balance in total knee arthroplasty.

Ligament balance is an important and subjective task performed during total knee arthroplasty (TKA) procedure. For this reason, it is desirable to develop instruments to quantitatively assess the soft-tissue balance since excessive imbalance can accelerate prosthesis wear and lead to early surgical revision. The instrumented distractor proposed in this study can assist surgeons on performing ligament balance by measuring the distraction gap and applied load. Also the device allows the determination of the ligament stiffness which can contribute a better understanding of the intrinsic mechanical behavior of the knee joint. Instrumentation of the device involved the use of hall-sensors for measuring the distractor displacement and strain gauges to transduce the force. The sensors were calibrated and tested to demonstrate their suitability for surgical use. Results show the distraction gap can be measured reliably with 0.1mm accuracy and the distractive loads could be assessed with an accuracy in the range of 4N. These characteristics are consistent with those have been proposed, in this work, for a device that could assist on performing ligament balance while permitting surgeons evaluation based on his experience. Preliminary results from in vitro tests were in accordance with expected stiffness values for medial collateral ligament (MCL) and lateral collateral ligament (LCL).

Radiographic total disc replacement angle measurement accuracy using the Oxford Cobbometer: precision and bias.

Total disc replacement (TDR) clinical success has been reported to be related to the residual motion of the operated level. Thus, accurate measurement of TDR range of motion (ROM) is of utmost importance. One commonly used tool in measuring ROM is the Oxford Cobbometer. Little is known however on its accuracy (precision and bias) in measuring TDR angles. The aim of this study was to assess the ability of the Cobbometer to accurately measure radiographic TDR angles. An anatomically accurate synthetic L4-L5 motion segment was instrumented with a CHARITE artificial disc. The TDR angle and anatomical position between L4 and L5 was fixed to prohibit motion while the motion segment was radiographically imaged in various degrees of rotation and elevation, representing a sample of possible patient placement positions. An experienced observer made ten readings of the TDR angle using the Cobbometer at each different position. The Cobbometer readings were analyzed to determine measurement accuracy at each position. Furthermore, analysis of variance was used to study rotation and elevation of the motion segment as treatment factors. Cobbometer TDR angle measurements were most accurate (highest precision and lowest bias) at the centered position (95.5%), which placed the TDR directly inline with the x-ray beam source without any rotation. In contrast, the lowest accuracy (75.2%) was observed in the most rotated and off-centered view. A difference as high as 4 degrees between readings at any individual position, and as high as 6 degrees between all the positions was observed. Furthermore, the Cobbometer was unable to detect the expected trend in TDR angle projection with changing position. Although the Cobbometer has been reported to be reliable in different clinical applications, it lacks the needed accuracy to measure TDR angles and ROM. More accurate ROM measurement methods need to be developed to help surgeons and researchers assess radiological success of TDRs.

Efficient total variation algorithm for fetal brain MRI reconstruction.

Fetal MRI reconstruction aims at finding a high-resolution image given a small set of low-resolution images. It is usually modeled as an inverse problem where the regularization term plays a central role in the reconstruction quality. Literature has considered several regularization terms s.a. Dirichlet/Laplacian energy, Total Variation (TV)- based energies and more recently non-local means. Although TV energies are quite attractive because of their ability in edge preservation, standard explicit steepest gradient techniques have been applied to optimize fetal-based TV energies. The main contribution of this work lies in the introduction of a well-posed TV algorithm from the point of view of convex optimization. Specifically, our proposed TV optimization algorithm or fetal reconstruction is optimal w.r.t. the asymptotic and iterative convergence speeds O(1/n2) and O(1/√ε), while existing techniques are in O(1/n2) and O(1/√ε). We apply our algorithm to (1) clinical newborn data, considered as ground truth, and (2) clinical fetal acquisitions. Our algorithm compares favorably with the literature in terms of speed and accuracy.

Importance of polyethylene thickness in total shoulder arthroplasty: a finite element analysis.

BACKGROUND: Articular surfaces reconstruction is essential in total shoulder arthroplasty. Because of the limited glenoid bone support, thin glenoid component could improve anatomical reconstruction, but adverse mechanical effects might appear. METHODS: With a numerical musculoskeletal shoulder model, we analysed and compared three values of thickness of a typical all-polyethylene glenoid component: 2, 4 (reference) and 6mm. A loaded movement of abduction in the scapular plane was simulated. We evaluated the humeral head translation, the muscle moment arms, the joint force, the articular contact pattern, and the polyethylene and cement stress. Findings Decreasing polyethylene thickness from 6 to 2mm slightly increased humeral head translation and muscle moment arms. This induced a small decreased of the joint reaction force, but important increase of stress within the polyethylene and the cement mantel. Interpretation The reference thickness of 4mm seems a good compromise to avoid stress concentration and joint stuffing.

População microbiana total e solubilizadora de fosfato em solo submetido a diferentes sistemas de cultivo

O objetivo deste trabalho foi avaliar o efeito de diferentes espécies de plantas, fontes de fósforo e calagem sobre a população microbiana total e solubilizadora de fosfato. Foram isolados fungos e bactérias capazes de solubilizar hidroxiapatita, proporcionando P solúvel. O experimento utilizado foi em blocos ao acaso com fatorial 3x3x2. E os fatores avaliados foram espécies de plantas (controle, braquiária e guandu), fertilizantes (controle, superfosfato simples e fosfato de rocha, ambos na dose de 400 kg ha-1 de P(2)0(5) ) e calagem (com e sem calcário). A população bacteriana cresceu pelo efeito da calagem, e a de fungos aumentou, independentemente da calagem, nas parcelas cultivadas com braquiária e fertilizadas com superfosfato. Foi constatado incremento de biomassa-P microbiana sobre o controle por influência da braquiária (23,9%), do superfosfato (30,9%) e da calagem (46,9%). O número de bactérias solubilizadoras foi favorecido pela calagem ou pelo plantio de guandu adubado com fosfato natural ou com braquiária sem adubação. Os fungos solubilizadores aumentaram na ausência de planta ou de adubação e na presença de guandu com fosfato natural. Finalmente, a calagem favoreceumais o crescimento dos fungos solubilizadores, em comparação com o controle, nos tratamentos fosfato natural, braquiária ou guandu.