Delocalization of the multifunctional RNA splicing factor TLS/FUS in hippocampal neurones: exclusion from the nucleus and accumulation in dendritic granules and spine heads.

International audienceLong-term synaptic change in the cortex and the hippocampus is believed to require the highly localized delivery and translation of mRNAs in the dendritic shafts and spines. The molecular interactions that underlie local signalling between synapses and mRNAs are still largely undefined. After purification from total brain extracts, the NMDA receptor is known to be associated with numerous proteins, including the multifunctional RNA-binding factor TLS (also called FUS). In non-neural tissue, TLS is a vital nuclear protein with roles in DNA repair, homologous recombination, transcriptional regulation and pre-mRNA processing. We have examined the distribution of TLS in hippocampal neurones, both in the adult brain and in mature primary cultures, using subcellular fractionation and immunofluorescence techniques. TLS immunoreactivity is largely excluded from the neuronal nucleus and is found in the cytosol and in somatodendritic particles. In some of these particles, TLS colocalizes with Sam68, a nuclear RNA-binding protein that we previously showed is incorporated into dendritic RNA granules. Some of the TLS clusters also colocalize with NMDA receptor clusters. Finally, TLS clusters are occasionally seen within spine heads. The apparent removal of TLS from the nucleus might result in specific patterns of mRNA transcription or splicing in hippocampal neurones. TLS may also contribute to steering, anchoring or regulating mRNAs at synaptic sites

Depolarization-induced translocation of the RNA-binding protein Sam68 to the dendrites of hippocampal neurons.

International audienceThe traffic and expression of mRNAs in neurons are modulated by changes in neuronal activity. The regulation of neuronal RNA-binding proteins is therefore currently receiving attention. Sam68 is a ubiquitous nuclear RNA-binding protein implicated in post-transcriptional processes such as signal-dependent splice site selection. We show that Sam68 undergoes activity-responsive translocation to the soma and dendrites of hippocampal neurons in primary culture. In unstimulated neurons transiently expressing a GFP-Sam68 fusion protein, 90% of the cells accumulated the protein exclusively in the nucleus, and 4% showed extension of GFP-Sam68 to the dendrites. This nuclear expression pattern required the integrity of the Sam68 N-terminus. When present, the dendritic GFP-Sam68 formed granules, 26% of which were colocalized with ethidium bromide-stained RNA clusters. Most of the GFP-Sam68 granules were completely stationary, but a few moved in either a retrograde or anterograde direction. Following depolarization by 25 mM KCl, 50% of neurons displayed dendritic GFP-Sam68. GFP-Sam68 invaded the dendrites after 2 hours with high KCl, and returned to the nucleus within 3 hours after termination of the KCl treatment. A control GFP fusion derived from the SC-35 splicing factor remained fully nuclear during depolarization. No significant change was observed in the phosphorylation of Sam68 after depolarization. Translocation of Sam68 to the distal dendrites was microtubule dependent. Blockade of calcium channels with nimodipine abolished the translocation. Furthermore, inhibition of CRM-1-mediated nuclear export by leptomycin B partially prevented the depolarization-induced nuclear efflux of GFP-Sam68. These results support the possible involvement of Sam68 in the activity-dependent regulation of dendritic mRNAs

SU(5) monopoles and non-abelian black holes

We construct spherically and axially symmetric monopoles in SU(5) Yang-Mills-Higgs theory both in flat and curved space as well as spherical and axial non-abelian, ''hairy'' black holes. We find that in analogy to the SU(2) case, the flat space monopoles are either non-interacting (in the BPS limit) or repelling. In curved space, however, gravity is able to overcome the repulsion for suitable choices of the Higgs coupling constants and the gravitational coupling. In addition, we confirm that indeed all qualitative features of (gravitating) SU(2) monopoles are found as well in the SU(5) case. For the non-abelian black holes, we compare the behaviour of the solutions in the BPS limit with that for non-vanishing Higgs self-coupling constants.Comment: 14 Revtex pages, 9 PS-figure

Tubulin tyrosination regulates synaptic function and is disrupted in Alzheimer's disease

: Microtubules play fundamental roles in the maintenance of neuronal processes and in synaptic function and plasticity. While dynamic microtubules are mainly composed of tyrosinated tubulin, long-lived microtubules contain detyrosinated tubulin, suggesting that the tubulin tyrosination/detyrosination cycle is a key player in the maintenance of microtubule dynamics and neuronal homeostasis, conditions which go awry in neurodegenerative diseases. In the tyrosination/detyrosination cycle, the C-terminal tyrosine of α-tubulin is removed by tubulin carboxypeptidases and re-added by tubulin tyrosine ligase. Here we show that tubulin tyrosine ligase hemizygous mice exhibit decreased tyrosinated microtubules, reduced dendritic spine density, and both synaptic plasticity and memory deficits. We further report decreased tubulin tyrosine ligase expression in sporadic and familial Alzheimer's disease, and reduced microtubule dynamics in human neurons harboring the familial APP-V717I mutation. Finally, we show that synapses visited by dynamic microtubules are more resistant to oligomeric amyloid β peptide toxicity and that expression of tubulin tyrosine ligase, by restoring microtubule entry into spines, suppresses the loss of synapses induced by amyloid β peptide. Together, our results demonstrate that a balanced tyrosination/detyrosination tubulin cycle is necessary for the maintenance of synaptic plasticity, is protective against amyloid β peptide-induced synaptic damage, and that this balance is lost in Alzheimer's disease, providing evidence that defective tubulin retyrosination may contribute to circuit dysfunction during neurodegeneration in Alzheimer's disease

Isolation, Characterization and Biological Evaluation of Jellyfish Collagen for Use in Biomedical Applications

Fibrillar collagens are the more abundant extracellular proteins. They form a metazoan-specific family, and are highly conserved from sponge to human. Their structural and physiological properties have been successfully used in the food, cosmetic, and pharmaceutical industries. On the other hand, the increase of jellyfish has led us to consider this marine animal as a natural product for food and medicine. Here, we have tested different Mediterranean jellyfish species in order to investigate the economic potential of their collagens. We have studied different methods of collagen purification (tissues and experimental procedures). The best collagen yield was obtained using Rhizostoma pulmo oral arms and the pepsin extraction method (2–10 mg collagen/g of wet tissue). Although a significant yield was obtained with Cotylorhiza tuberculata (0.45 mg/g), R. pulmo was used for further experiments, this jellyfish being considered as harmless to humans and being an abundant source of material. Then, we compared the biological properties of R. pulmo collagen with mammalian fibrillar collagens in cell cytotoxicity assays and cell adhesion. There was no statistical difference in cytotoxicity (p > 0.05) between R. pulmo collagen and rat type I collagen. However, since heparin inhibits cell adhesion to jellyfish-native collagen by 55%, the main difference is that heparan sulfate proteoglycans could be preferentially involved in fibroblast and osteoblast adhesion to jellyfish collagens. Our data confirm the broad harmlessness of jellyfish collagens, and their biological effect on human cells that are similar to that of mammalian type I collagen. Given the bioavailability of jellyfish collagen and its biological properties, this marine material is thus a good candidate for replacing bovine or human collagens in selected biomedical applications

Outpatient-basis Chemotherapy of Oxaliplatin, 5-fluorouracil, and Leucovorin as First-line Treatment for Patients with Metastatic or Recurrent Colorectal Cancer

The objectives of the present study were to evaluate the efficacy and safety of an outpatient-basis chemotherapy of oxaliplatin, 5-fluorouracil, and leucovorin as the first-line treatment for patients with advanced colorectal cancer. Forty-three histologically confirmed patients with metastatic or recurrent colorectal cancer were enrolled. The chemotherapy consisted of oxaliplatin 85 mg/m2 as a 2-hr infusion on day 1, plus leucovorin 30 mg/m2 over 10 min, followed by bolus 5-fluorouracil 400 mg/m2 and an 8-hr infusion of 5-fluorouracil 600 mg/m2 on days 1 and 2 (modified FOLFOX4), all of which were administered on an outpatient basis every 2 weeks. The median age was 58 yr (range 33-72 yr), and 25 (58.1%) patients had metastatic diseases. Eventually, 39 patients were assessable for efficacy and all assessable for toxicity. Four (9.3%) complete responses and 11 (25.6%) partial responses were confirmed, giving an overall response rate of 34.9% (95% CI; 20.0-49.7%). The median time to progression and median overall survival for all patients was 6.1 months and 17.4 months, respectively. Grade 3/4 neutropenia occurred in 2 patients (4.7%) and febrile neutropenia was observed in 1 patient (2.3%). Modified FOLFOX4, an outpatient-basis regimen, was found to be well-tolerated and effective as the first-line chemotherapy in patients with advanced colorectal cancer

Regulation of Postsynaptic Function by the Dementia-Related ESCRT-III Subunit CHMP2B

The charged multivesicular body proteins (Chmp1–7) are an evolutionarily conserved family of cytosolic proteins that transiently assembles into helical polymers that change the curvature of cellular membrane domains. Mutations in human CHMP2B cause frontotemporal dementia, suggesting that this protein may normally control some neuron-specific process. Here, we examined the function, localization, and interactions of neuronal Chmp2b. The protein was highly expressed in mouse brain and could be readily detected in neuronal dendrites and spines. Depletion of endogenous Chmp2b reduced dendritic branching of cultured hippocampal neurons, decreased excitatory synapse density in vitro and in vivo, and abolished activity-induced spine enlargement and synaptic potentiation. To understand the synaptic effects of Chmp2b, we determined its ultrastructural distribution by quantitative immuno-electron microscopy and its biochemical interactions by coimmunoprecipitation and mass spectrometry. In the hippocampus in situ, a subset of neuronal Chmp2b was shown to concentrate beneath the perisynaptic membrane of dendritic spines. In synaptoneurosome lysates, Chmp2b was stably bound to a large complex containing other members of the Chmp family, as well as postsynaptic scaffolds. The supramolecular Chmp assembly detected here corresponds to a stable form of the endosomal sorting complex required for transport-III (ESCRT-III), a ubiquitous cytoplasmic protein complex known to play a central role in remodeling of lipid membranes. We conclude that Chmp2b-containing ESCRT-III complexes are also present at dendritic spines, where they regulate synaptic plasticity. We propose that synaptic ESCRT-III filaments may function as a novel element of the submembrane cytoskeleton of spines

Automatic colorimetric calibration of human wounds

Contains fulltext : 88431.pdf (publisher's version ) (Open Access)BACKGROUND: Recently, digital photography in medicine is considered an acceptable tool in many clinical domains, e.g. wound care. Although ever higher resolutions are available, reproducibility is still poor and visual comparison of images remains difficult. This is even more the case for measurements performed on such images (colour, area, etc.). This problem is often neglected and images are freely compared and exchanged without further thought. METHODS: The first experiment checked whether camera settings or lighting conditions could negatively affect the quality of colorimetric calibration. Digital images plus a calibration chart were exposed to a variety of conditions. Precision and accuracy of colours after calibration were quantitatively assessed with a probability distribution for perceptual colour differences (dE_ab). The second experiment was designed to assess the impact of the automatic calibration procedure (i.e. chart detection) on real-world measurements. 40 Different images of real wounds were acquired and a region of interest was selected in each image. 3 Rotated versions of each image were automatically calibrated and colour differences were calculated. RESULTS: 1st Experiment: Colour differences between the measurements and real spectrophotometric measurements reveal median dE_ab values respectively 6.40 for the proper patches of calibrated normal images and 17.75 for uncalibrated images demonstrating an important improvement in accuracy after calibration. The reproducibility, visualized by the probability distribution of the dE_ab errors between 2 measurements of the patches of the images has a median of 3.43 dE* for all calibrated images, 23.26 dE_ab for all uncalibrated images. If we restrict ourselves to the proper patches of normal calibrated images the median is only 2.58 dE_ab! Wilcoxon sum-rank testing (p < 0.05) between uncalibrated normal images and calibrated normal images with proper squares were equal to 0 demonstrating a highly significant improvement of reproducibility. In the second experiment, the reproducibility of the chart detection during automatic calibration is presented using a probability distribution of dE_ab errors between 2 measurements of the same ROI. CONCLUSION: The investigators proposed an automatic colour calibration algorithm that ensures reproducible colour content of digital images. Evidence was provided that images taken with commercially available digital cameras can be calibrated independently of any camera settings and illumination features

Pooled Safety and Efficacy Analysis Examining the Effect of Performance Status on Outcomes in Nine First-Line Treatment Trials Using Individual Data From Patients With Metastatic Colorectal Cancer

Performance status (PS) is a prognostic factor in patients with metastatic colorectal cancer. Clinical trials typically enroll less than 10% of patients with a PS of 2 (PS2); thus, the benefit of systemic chemotherapy in PS2 patients is uncertain