The fourth extracellular loop of the alpha subunit of Na,K-ATPase. Functional evidence for close proximity with the second extracellular loop.

Na,K-ATPase is the main active transport system that maintains the large gradients of Na(+) and K(+) across the plasma membrane of animal cells. The crystal structure of a K(+)-occluding conformation of this protein has been recently published, but the movements of its different domains allowing for the cation pumping mechanism are not yet known. The structure of many more conformations is known for the related calcium ATPase SERCA, but the reliability of homology modeling is poor for several domains with low sequence identity, in particular the extracellular loops. To better define the structure of the large fourth extracellular loop between the seventh and eighth transmembrane segments of the alpha subunit, we have studied the formation of a disulfide bond between pairs of cysteine residues introduced by site-directed mutagenesis in the second and the fourth extracellular loop. We found a specific pair of cysteine positions (Y308C and D884C) for which extracellular treatment with an oxidizing agent inhibited the Na,K pump function, which could be rapidly restored by a reducing agent. The formation of the disulfide bond occurred preferentially under the E2-P conformation of Na,K-ATPase, in the absence of extracellular cations. Using recently published crystal structure and a distance constraint reproducing the existence of disulfide bond, we performed an extensive conformational space search using simulated annealing and showed that the Tyr(308) and Asp(884) residues can be in close proximity, and simultaneously, the SYGQ motif of the fourth extracellular loop, known to interact with the extracellular domain of the beta subunit, can be exposed to the exterior of the protein and can easily interact with the beta subunit.

Functional sphere profiling reveals the complexity of neuroblastoma tumor-initiating cell model.

Neuroblastoma (NB) is a neural crest-derived childhood tumor characterized by a remarkable phenotypic diversity, ranging from spontaneous regression to fatal metastatic disease. Although the cancer stem cell (CSC) model provides a trail to characterize the cells responsible for tumor onset, the NB tumor-initiating cell (TIC) has not been identified. In this study, the relevance of the CSC model in NB was investigated by taking advantage of typical functional stem cell characteristics. A predictive association was established between self-renewal, as assessed by serial sphere formation, and clinical aggressiveness in primary tumors. Moreover, cell subsets gradually selected during serial sphere culture harbored increased in vivo tumorigenicity, only highlighted in an orthotopic microenvironment. A microarray time course analysis of serial spheres passages from metastatic cells allowed us to specifically "profile" the NB stem cell-like phenotype and to identify CD133, ABC transporter, and WNT and NOTCH genes as spheres markers. On the basis of combined sphere markers expression, at least two distinct tumorigenic cell subpopulations were identified, also shown to preexist in primary NB. However, sphere markers-mediated cell sorting of parental tumor failed to recapitulate the TIC phenotype in the orthotopic model, highlighting the complexity of the CSC model. Our data support the NB stem-like cells as a dynamic and heterogeneous cell population strongly dependent on microenvironmental signals and add novel candidate genes as potential therapeutic targets in the control of high-risk NB.

Functional multidisciplinary rehabilitation versus outpatient physiotherapy for non specific low back pain: randomized controlled trial.

INTRODUCTION: In recent decades the treatment of non-specific low back pain has turned to active modalities, some of which were based on cognitive-behavioural principles. Non-randomised studies clearly favour functional multidisciplinary rehabilitation over outpatient physiotherapy. However, systematic reviews and meta-analysis provide contradictory evidence regarding the effects on return to work and functional status. The aim of the present randomised study was to compare long-term functional and work status after 3-week functional multidisciplinary rehabilitation or 18 supervised outpatient physiotherapy sessions. METHODS: 109 patients with non-specific low back pain were randomised to either a 3-week functional multidisciplinary rehabilitation programme, including physical and ergonomic training, psychological pain management, back school and information, or 18 sessions of active outpatient physiotherapy over 9 weeks. Primary outcomes were functional disability (Oswestry) and work status. Secondary outcomes were lifting capacity (Spinal Function Sort and PILE test), lumbar range-of-motion (modified-modified Schöber and fingertip-to-floor tests), trunk muscle endurance (Shirado and Biering-Sörensen tests) and aerobic capacity (modified Bruce test). RESULTS: Oswestry disability index was improved to a significantly greater extent after functional multidisciplinary rehabilitation compared to outpatient physiotherapy at follow-up of 9 weeks (P = 0.012), 9 months (P = 0.023) and 12 months (P = 0.011). Work status was significantly improved after functional multidisciplinary rehabilitation only (P = 0.012), resulting in a significant difference compared to outpatient physiotherapy at 12 months' follow-up (P = 0.012). Secondary outcome results were more contrasted. CONCLUSIONS: Functional multidisciplinary rehabilitation was better than outpatient physiotherapy in improving functional and work status. From an economic point of view, these results should be backed up by a cost-effectiveness study.

Pre-existing astrocytes form functional perisynaptic processes on neurons generated in the adult hippocampus.

The adult dentate gyrus produces new neurons that morphologically and functionally integrate into the hippocampal network. In the adult brain, most excitatory synapses are ensheathed by astrocytic perisynaptic processes that regulate synaptic structure and function. However, these processes are formed during embryonic or early postnatal development and it is unknown whether astrocytes can also ensheathe synapses of neurons born during adulthood and, if so, whether they play a role in their synaptic transmission. Here, we used a combination of serial-section immuno-electron microscopy, confocal microscopy, and electrophysiology to examine the formation of perisynaptic processes on adult-born neurons. We found that the afferent and efferent synapses of newborn neurons are ensheathed by astrocytic processes, irrespective of the age of the neurons or the size of their synapses. The quantification of gliogenesis and the distribution of astrocytic processes on synapses formed by adult-born neurons suggest that the majority of these processes are recruited from pre-existing astrocytes. Furthermore, the inhibition of astrocytic glutamate re-uptake significantly reduced postsynaptic currents and increased paired-pulse facilitation in adult-born neurons, suggesting that perisynaptic processes modulate synaptic transmission on these cells. Finally, some processes were found intercalated between newly formed dendritic spines and potential presynaptic partners, suggesting that they may also play a structural role in the connectivity of new spines. Together, these results indicate that pre-existing astrocytes remodel their processes to ensheathe synapses of adult-born neurons and participate to the functional and structural integration of these cells into the hippocampal network.