Non-Gaussian water diffusion in aging white matter

Age-associated white matter degeneration has been well documented and is likely an important mechanism contributing to cognitive decline in older adults. Recent work has explored a range of noninvasive neuroimaging procedures to differentially highlight alterations in the tissue microenvironment. Diffusional kurtosis imaging (DKI) is an extension of diffusion tensor imaging (DTI) that accounts for non-Gaussian water diffusion and can reflect alterations in the distribution and diffusion properties of tissue compartments. We used DKI to produce whole-brain voxel-based maps of mean, axial, and radial diffusional kurtoses, quantitative indices of the tissue microstructure's diffusional heterogeneity, in 111 participants ranging from the age of 33 to 91 years. As suggested from prior DTI studies, greater age was associated with alterations in white-matter tissue microstructure, which was reflected by a reduction in all 3 DKI metrics. Prominent effects were found in prefrontal and association white matter compared with relatively preserved primary motor and visual areas. Although DKI metrics co-varied with DTI metrics on a global level, DKI provided unique regional sensitivity to the effects of age not available with DTI. DKI metrics were additionally useful in combination with DTI metrics for the classification of regions according to their multivariate “diffusion footprint”, or pattern of relative age effect sizes. It is possible that the specific multivariate patterns of age-associated changes measured are representative of different types of microstructural pathology. These results suggest that DKI provides important complementary indices of brain microstructure for the study of brain aging and neurologic disease.     

Freeze-drying of itraconazole-loaded nanosphere suspensions: a feasibility study

The present study concerns the stabilization of the association of the new hydrophobic triazole derivative itraconazole within poly-ϵ-caprolactone-nanospheres by means of freeze-drying. We have investigated the freeze-drying of nanospheres, and especially the cryopreservation conditions, with the help of differential scanning calorimetry and zeta potential measurements. Five commonly used cryoprotective agents were evaluated (glucose, sucrose, trehalose, dextran, mannitol at 0, 5, 10, 20, and 30% [w/v]) after freeze-thawing and freeze-drying. The addition of carbohydrates led to a partial protection of the colloidal suspension, with leakage of 30% of itraconazole under the best cryopreservation conditions (10% of glucose or sucrose). Zeta potential measurements revealed that the main destabilization mechanism during freeze-drying was surface modifications of the nanospheres, and particularly drug desorption. Therefore, the hydrophilic surfactant adsorbed at the surface of the nanospheres played an important role in the cryopreservation. Replacing the commonly used non ionic surfactant PLURONIC®PE F68 by the anionic surfactant sodium deoxycholate resulted in a complete stabilization of itraconazole-loaded nanospheres after freeze-drying, with no drug desorption, in the presence of 10% sucrose, but not in the presence of glucose. As shown by thermal analysis, PLURONIC®PE F68 may crystallize during freezing, which could lead to surface modifications and drug desorption, whereas sodium deoxycholate may not. Moreover, the Tg′ of glucose-containing suspensions is 10°C lower than Tg′ of sucrose-containing suspensions, which may explain the shrinkage of the cake observed in the case of glucose and the homogeneous appearance of the dried product in the case of sucrose. © 1996 Wiley-Liss, Inc.     

Ghrelin receptor conformational dynamics regulate the transition from a preassembled to an active receptor:Gq complex

How G protein-coupled receptor conformational dynamics control G protein coupling to trigger signaling is a key but still open question. We addressed this question with a model system composed of the purified ghrelin receptor assembled into lipid discs. Combining receptor labeling through genetic incorporation of unnatural amino acids, lanthanide resonance energy transfer, and normal mode analyses, we directly demonstrate the occurrence of two distinct receptor:Gq assemblies with different geometries whose relative populations parallel the activation state of the receptor. The first of these assemblies is a preassembled complex with the receptor in its basal conformation. This complex is specific of Gq and is not observed with Gi. The second one is an active assembly in which the receptor in its active conformation triggers G protein activation. The active complex is present even in the absence of agonist, in a direct relationship with the high constitutive activity of the ghrelin receptor. These data provide direct evidence of a mechanism for ghrelin receptor-mediated Gq signaling in which transition of the receptor from an inactive to an active conformation is accompanied by a rearrangement of a preassembled receptor:G protein complex, ultimately leading to G protein activation and signaling.G protein-coupled receptors (GPCRs), one of the largest cell surface receptor families, are involved in many cellular signaling processes (1). Based on this property, as well as their importance as drug targets, the molecular aspects of GPCR functioning have been extensively investigated. In particular, coupling to heterotrimeric G proteins has been the focus of numerous studies. Indeed, delineating the molecular mechanisms responsible for receptor:G protein interaction is absolutely required to better understand how signaling is controlled. Recent years have seen spectacular advances that have culminated in elucidation of the 3D structure of the β₂-adrenergic receptor:Gs complex (2). Nevertheless, the need for further progress remains, in particular to fully understand the dynamics of this interaction. This is a crucial question, given that how the receptor interacts with its G protein partner governs signaling, and thus biological and pathophysiological responses.To date, two different models for GPCR:G protein interaction have been proposed: collision coupling and preassembly. Originally, it was proposed that receptors and G proteins couple by collision (3, 4). One of the main features of this model is that only activated receptors interact with G proteins. Since then, alternative models of signaling have been developed. One of these, the preassembly model, proposes that the receptor and the G protein make a complex even in the absence of agonist (5–8). Discriminating between the two models is crucial. Indeed, signaling outputs, such as the kinetics of G protein activation, will be significantly different depending on whether the ligand-free receptor is always in complex with its G protein or must first be activated by the agonist to recruit the G protein and trigger signaling. Moreover, it has been shown that GPCR conformational dynamics (9–11) and signaling in the absence of ligand are key features of GPCR functioning (12). How receptor constitutive activity and conformational dynamics relate to their coupling to the G protein remains an open question.Here we used the purified ghrelin receptor GHS-R1a to analyze the way in which this GPCR interacts with its G protein partners. Ghrelin is a neuroendocrine peptide hormone that acts through its cognate GPCR to control important biological processes, such as growth hormone secretion, food intake, and reward-seeking behaviors (13). Among the GPCRs, GHS-R1a has been shown to have one of the highest basal Gq activation levels both in vitro (10, 14) and in vivo (15, 16). The physiological relevance of GHS-R1a basal activity is substantiated by the occurrence of a natural human mutation in the GHS-R1a gene (A²⁰⁴E substitution in the second extracellular loop of the receptor) that dramatically decreases constitutive activity and is associated with a short-stature phenotype (17). Along with its importance in drug design, GHS-R1a is a prototype for peptide-activated class A GPCRs.To delineate the way in which the ghrelin receptor interacts with G proteins, we used monomeric GHS-R1a reconstituted in a membrane-mimicking environment, lipid discs, and a combination of innovative biochemical [labeling with unnatural amino acid (UAA)] and biophysical [lanthanide resonance energy transfer (LRET) and normal mode (NM) analyses] approaches. By doing so, we provide the first direct evidence that ghrelin-mediated signaling involves a complex dialogue between the conformational dynamics of the receptor and its ability to interact with the different G protein subtypes to which it is coupled.     

The effectiveness of webcast compared to live lectures as a teaching tool in medical school

Objective: The purpose of this study is to investigate whether webcast lectures are comparable to live lectures as a teaching tool in medical school.

Methods: Three Otolaryngology-Head&Neck Surgery (OTO-HNS) lectures were given to third year medical students through their regular academic curriculum with one group receiving lectures in a live lecture format and the other group in a webcast format. All lectures (live or webcast) were given by the same lecturer and contained identical material. Three outcome measures were used: a student satisfaction survey, performance on the OTO-HNS component of their written examination, and performance on an OTO-HNS OSCE station in the general end of year OSCE examination session.

Results: Students performance on the written examination was equal between the two groups. The webcast group outperformed the live lecture group in the OSCE station. The majority of students in the webcast group felt it was an effective learning tool for them. Most viewed the lectures more than once, and felt that this was beneficial to their learning.

Conclusion: Webcasts appear equally effective to live lectures as a teaching tool.     

Solitary fibrous tumors of the pleura: clinical characteristics, surgical treatment and outcome

Objective: The aim of this paper is to study clinical characteristics, surgical treatment and outcome of patients with solitary fibrous tumor of the pleura operated in our institutions in a 20-year period. Methods: Clinical records of all patients operated for solitary fibrous tumors of the pleura between 1981 and 2000 were reviewed retrospectively. Tumors were classified as malignant in the presence of at least one of the following criteria: (1) high mitotic activity; (2) high cellularity with crowding and overlapping of nuclei; (3) presence of necrosis; (4) pleomorphism; otherwise they were considered as benign. Results: Sixty patients (mean age 55 years) were operated in this period. None had asbestos exposure. Symptoms were present in 31 cases. Surgical approaches included thoracotomy (n=53), video-assisted thoracoscopy (n=6), and median sternotomy (n=1). Tumors originated from visceral pleura in 48 cases, from parietal, mediastinal or diaphragmatic pleura in seven, two and three cases, respectively; their mean diameter was 8.5 cm. Tumors could be resected with their implantation basis in 49 patients. In the remaining 11, extended resections were performed, including lung parenchyma (lobectomy, n=4, pneumonectomy, n=2), osteomuscular chest wall structures (n=2), diaphragm (n=2), and pericardium (n=1). Two postoperative deaths (due to myocardial infarction and pulmonary embolism, respectively) occurred. Tumors were pathologically benign in 38 cases and malignant in 22 cases. Mean follow-up was 88 months. Resection was complete in all the patients with benign tumors and no recurrence occurred. Resection was considered as complete in 21/22 malignant tumors. Local recurrence was observed in two cases. Both could be successfully managed by iterative exeresis (no extended resection had been initially performed). Metastatic disease (responsible for patient's death) was observed following the only incomplete resection. Actuarial 5- and 10-year survival rates were 97% for benign tumors and 89% for malignant ones. Conclusions: Surgical resection provided cure in all the patients with benign tumors. As insufficiency of exeresis is associated with all recurrences in malignant tumors, completeness of resection is in our experience the best prognostic factor in these forms.     

Sonography detection threshold for knee effusion

The aim of this study was to determine the threshold for detecting knee effusion by ultrasound (US). Five knee specimens from embalmed cadavers were studied. Intra-articular injection of saline, blood and synovial fluid was performed under ultrasound control and methylene blue dye instillation. The smallest amount of fluid detectable by US in the knee was 7.4 ml for synovial fluid, 10.1 and 10.4 ml for saline solution and 9.7 for blood. The threshold for detecting knee joint effusion by US in cadaver specimens was 10 ml for saline and blood and 7 ml for synovial fluid.Abbreviations US Ultrasound     

CCR7-mediated physiological lymphocyte homing involves activation of a tyrosine kinase pathway

Homing of blood-borne lymphocytes to peripheral lymph nodes (PLNs) is a multistep process dependent on the sequential engagement of L-selectin, which mediates lymphocyte rolling along the luminal surface of high endothelial venules (HEVs), followed by activation of lymphocyte integrins and transmigration through HEVs. Within lymphoid tissue, B and T lymphocytes then migrate toward specific microenvironments such as B-cell follicles and the paracortex, respectively. The lymphocyte-expressed chemokine receptor CCR7 is playing an important role during this process, as its HEV-presented ligands CCL19 and CCL21 can trigger rapid integrin activation under flow in addition to inducing a chemotactic response, which may participate in transmigration and/or interstitial migration. Here, we report that Tyrphostin (Tyr) AG490, a pharmacological inhibitor of Janus family tyrosine kinases (Jaks), blocked the chemotactic response of primary mouse lymphocytes to CCL19 and CCL21 in a dose-dependent manner. Furthermore, Tyr AG490 inhibited rapid CCL21-mediated up-regulation of alpha4 and beta2 integrin adhesiveness in static adhesion assays and under physiological flow, whereas adhesion induced by phorbol myristate acetate remained unaltered. Using intravital microscopy of subiliac PLNs in mice, we found that adoptively transferred Tyr AG490-treated lymphocytes adhered significantly less in HEVs compared with control cells, although L-selectin-mediated rolling was similar in both samples. Finally, we observed rapid Jak2 phosphorylation in CCL21-stimulated primary mouse lymphocytes. Thus, our study suggests a role for Jak tyrosine kinases during CCR7-mediated lymphocyte recirculation.     

CD10 expression by melanoma cells is associated with aggressive behavior in vitro and predicts rapid metastatic progression in humans

BackgroundNo biological or molecular marker of primary melanoma tumor cells has been shown to predict clinical outcome in melanoma.ObjectiveTo determine whether CD10, CD133, nestin and CD20 may evaluate the prognosis of melanoma.MethodsThe differential expression of these molecules was assessed in pairs of cell lines. We evaluated, by both immunohistochemical staining and RT-qPCR, their expression in a cohort of 32 patients (68 samples) with a history of metastatic melanoma, divided into two groups according to their clinical outcome profile.ResultsCD10 over expression in cancer cell lines was associated with more aggressive behavior in vitro. A CD10-positive staining was more frequent in patients in the “rapidly progressive” group than those in the “long survivor” group (23/35 versus 2/18, p < 10^?4). CD10 expression was associated with a lower median overall survival (1.15 year – IQR: [0.50–2.58] versus 4.27 – IQR: [1.66–6.33]; p = 10^?4). The Odds Ratio of displaying a “rapidly progressive” melanoma when tumor cells expressed CD10 was 15 (95% confidence interval: [3–78]). After adjusting for confounding factors, CD10 expression in melanoma tumor cells remained associated with an increased risk of death and more rapid disease progression (p = 6 × 10^?4; HR = 3.71).ConclusionCD10 may predict clinical outcome in melanoma patients.