85 research outputs found
Neuronal Intermediate Filaments in Amyotrophic Lateral Sclerosis
Neuronal intermediate filaments (NIFs) are the most abundant cytoskeletal element in mature neurons. They are composed of different protein subunits encoded by separate genes such as neurofilament light chain (NFL), neurofilament medium chain (NFM), neurofilament heavy chain (NFH), ɑ‐internexin and peripherin. NIFs are dynamic structures playing important functions in cell architecture and differentiation, interactions between proteins or subcellular organelles, and in axonal calibre determination and myelination. Consequently, their presence modulates electrophysiological properties of axons. NIFs have long been assigned a role in the pathogenesis of amyotrophic lateral sclerosis (ALS). Indeed, accumulation and abnormal phosphorylation of NIF subunits in motor neuron are one of the major pathological features in both sporadic and familial forms of the disease. Moreover, mutations in the NFH and peripherin genes and elevated cerebrospinal fluid NIF levels reported in ALS cases, associated with studies in transgenic mice, provided the evidence that primary defects in NIFs could be causative for motor neuron disease. However, the processes leading to the NIF abnormalities and the links to the pathogenesis of ALS remain unclear, leaving a challenging open field for further investigations in this highly disabilitating disease. Here, we review the main characteristics of these NIFs and their involvement in the pathomechanisms of ALS
Diagnostic value of minor salivary glands biopsy for the detection of Lewy pathology
The recent demonstration of the presence of Lewy pathology in the submandibular glands of Parkinson\u27s disease (PD) patients prompted us to evaluate the diagnostic performance of minor salivary gland biopsy for PD. Minor salivary glands were examined for Lewy pathology using phosphorylated alpha-synuclein antibody in 16 patients with clinically diagnosed PD and 11 control subjects with other neurological disorders. Abnormal accumulation of alpha-synuclein was found in 3 out of 16 PD patients. Two control subjects exhibited weak phosphorylated alpha-synuclein immunoreactivity. Our results do not support the use of minor salivary glands biopsy for the detection of Lewy pathology in living subjects
High throughput screening for identification of mycolactone targets : Relations between M. ulcerans and nervous system
Buruli ulcer is an infectious disease transmitted by arthropod vectors harboring Mycobacterium ulcerans, a mycobacterium which belong to the same family of bacteria causing tuberculosis and leprosy. The infection causes painless swelling and severe skin lesions. One key feature of M. ulcerans bacterium is its ability to secrete a necrotic toxin, the mycolactone within small lipophilic vesicles, which are critical for the bacterial induced cytotoxicity. The biological knowledge as well as the preventive and therapeutic means for this invalidating disease is still very limited.
Our first approach was to investigate whether the mycolactone toxin could be involved in the neutralization of pain by acting directly on the peripheral nervous system without causing destruction of nervous fibers. By use of live time fluorescence microscopy and appropriate markers, we showed that the addition of toxin at sub-toxic dose provokes modification of ionic currents of neuron cells. Based on this ability of the toxin, a molecular high throughput methodology was developed for the screening of a genome wide siRNA library and small molecules inhibitors to enable the search of the cellular targets for the toxin. The cell-based assay relies on automated confocal microscopy on macrophages coupled with dedicated image analysis. These two screening allowed us to identify a putative toxin target, and a toxin inhibitor. A binding assay confirmed that the putative target is a receptor of the toxin. Together these results allowed us to build a potential signaling pathway activated by the mycolactone and implicated in ionic channel activities.
The second approach was to confirm this model in the mouse model of M. ulcerans infection and its role in the hypoesthesia of the lesions. Toxin inhibitor, daily administered to mice, which were experimentally infected by M. ulcerans, conducted to the absence of the hypoesthesia of the lesions. Furthermore, a histological study of neuronal fibers did not show a destruction of neuronal cells. Moreover, in vitro studies have showed that M. ulcerans are able to colonize neuronal cells. Then, these results suggested that the hypoesthesia of the M. ulcerans lesions could be caused by a non-destructive process of nervous cells.
Is M. ulcerans able to colonize neuronal cells?
Buruli ulcer, or Mycobacterium ulcerans infection, is an emerging disease, principally diagnosed in humid tropical countries and inducing large skin ulcers. These lesions are painless, a distinct feature that suggests that the mycolactone toxin and/or M. ulcerans impedes the signal transmission by the nervous system. In this context, the aim of this work was to study the interaction between M. ulcerans and neuronal cells by using in vitro and in vivo models. We showed that a virulent strain of M. ulcerans is able to enter into neurons cultivated from neonatal rat hippocampus. On the contrary, this phenomenon was less observed with a mycolactone-deficient strain. To support these data, we analysed nerve fibres from mouse-infected tissues and few bacilli were found in close contact with nerve fibres. The invasion process established by M. ulcerans to colonize the nervous system remains uncharacterised, but we hypothesise that this ability could be involved in the painless of the M. ulcerans infection
Assessment of a percutaneous iliosacral screw insertion simulator.
International audienceBACKGROUND: Navigational simulator use for specialized training purposes is rather uncommon in orthopaedic and trauma surgery. However, it reveals providing a valuable tool to train orthopaedic surgeons and help them to plan complex surgical procedures. PURPOSE: This work's objective was to assess educational efficiency of a path simulator under fluoroscopic guidance applied to sacroiliac joint percutaneous screw fixation. MATERIALS AND METHODS: We evaluated 23 surgeons' accuracy inserting a guide-wire in a human cadaver experiment, following a pre-established procedure. These medical trainees were defined in three prospective respects: novice or skilled; with or without theoretical knowledge; with or without surgical procedure familiarity. Analysed criteria for each tested surgeon included the number of intraoperative X-rays taken in order to achieve the surgical procedure as well as an iatrogenic index reflecting the surgeon's ability to detect any hazardous trajectory at the time of performing said procedure. RESULTS: An average number of 13 X-rays was required for wire implantation by the G1 group. G2 group, assisted by the simulator use, required an average of 10 X-rays. A substantial difference was especially observed within the novice sub-group (N), with an average of 12.75 X-rays for the G1 category and an average of 8.5 X-rays for the G2 category. As far as the iatrogenic index is concerned, we were unable to observe any significant difference between the groups
Melanoma tumour vasculature heterogeneity: from mice models to human
Tumour angiogenesis is defined by an anarchic vasculature and irregularities in alignment of endothelial cells. These structural abnormalities could explain the variability in distribution of nanomedicines in various tumour models. Then, the main goal of this study was to compare and to characterize the tumour vascular structure in different mouse models of melanoma tumours (B16F10 and SK-Mel-28) and in human melanomas from different patients. Tumours were obtained by subcutaneous injection of 106 B16F10 and 3.106 SK-Mel-28 melanoma cells in C57BL/6 and nude mice, respectively. Tumour growth was evaluated weekly, while vasculature was analysed through fluorescent labelling via CD31 and desmin. Significant differences in tumour growth and mice survival were evidenced between the two melanoma models. A fast evolution of tumours was observed for B16F10 melanoma, reaching a tumour size of 100 mm3 in 7 days compared to SK-Mel-28 which needed 21 days to reach the same volumes. Important differences in vascularization were exposed between the melanoma models, characterized by a significant enhancement of vascular density and a significant lumen size for mice melanoma models compared to human. Immunostaining revealed irregularities in endothelium structure for both melanoma models, but structural differences of vasculature were observed, characterized by a stronger expression of desmin in SK-Mel-28 tumours. While human melanoma mainly develops capillaries, structural irregularities are also observed on the samples of this tumour model. Our study revealed an impact of cell type and tumour progression on the structural vasculature of melanoma, which could impact the distribution of drugs in the tumour environment
Ethambutol-induced optic neuropathy linked to OPA1 mutation and mitochondrial toxicity
Ethambutol (EMB), widely used in the treatment of tuberculosis, has been reported to cause Leber’s hereditary optic neuropathy in patients carrying mitochondrial DNA mutations. We study the effect of EMB on mitochondrial metabolism in fibroblasts from controls and from a man carrying an OPA1 mutation, in whom the drug induced the development of autosomal dominant optic atrophy (ADOA). EMB produced a mitochondrial coupling defect together with a 25% reduction in complex IV activity. EMB induced the formation of vacuoles associated with decreased mitochondrial membrane potential and increased fragmentation of the mitochondrial network. Mitochondrial genetic variations may therefore be predisposing factors in EMB-induced ocular injury
OPA1 mutations induce mitochondrial DNA instability and optic atrophy ‘plus’ phenotypes
Mutations in OPA1, a dynamin-related GTPase involved in mitochondrial fusion, cristae organization and control of apoptosis, have been linked to non-syndromic optic neuropathy transmitted as an autosomal-dominant trait (DOA). We here report on eight patients from six independent families showing that mutations in the OPA1 gene can also be responsible for a syndromic form of DOA associated with sensorineural deafness, ataxia, axonal sensory-motor polyneuropathy, chronic progressive external ophthalmoplegia and mitochondrial myopathy with cytochrome c oxidase negative and Ragged Red Fibres. Most remarkably, we demonstrate that these patients all harboured multiple deletions of mitochondrial DNA (mtDNA) in their skeletal muscle, thus revealing an unrecognized role of the OPA1 protein in mtDNA stability. The five OPA1 mutations associated with these DOA ‘plus’ phenotypes were all mis-sense point mutations affecting highly conserved amino acid positions and the nuclear genes previously known to induce mtDNA multiple deletions such as POLG1, PEO1 (Twinkle) and SLC25A4 (ANT1) were ruled out. Our results show that certain OPA1 mutations exert a dominant negative effect responsible for multi-systemic disease, closely related to classical mitochondrial cytopathies, by a mechanism involving mtDNA instability
Radiographic evaluation of calcaneal fractures: To measure or not to measure
Objective: The aim of this study was to correlate the functional outcome after treatment for displaced intra-articular calcaneal fracture with plain radiography. Design: The design was a prognostic study of a retrospective cohort with concurrent follow-up. Patients: A total of 33 patients with a unilateral calcaneal fracture and a minimum follow-up of 13 months participated. Patients filled in three disease-specific questionnaires, graded their satisfaction and the indication for an arthrodesis was noted. Standardised radiographs were made of the previously injured side and the normal (control) side. Different angles and distances were measured on these radiographs and compared with values described in the literature. The differences in values in angles and distances between the injured and uninjured (control) foot were correlated with the outcome of the questionnaires, and the indication for an arthrodesis. Results: None of the angles correlated with the disease-specific outcome scores. Of the angles only the tibiotalar angle correlated with the VAS (r=0.35, p=0.045) and only the absolute foot height correlated with the indication for an arthrodesis (odds=0.70, CI=0.50-0.99). Conclusion: In this study the radiographic evaluation correlated poorly with the final outcome. Measurements on plain radiographs seem not to be useful in determining outcome after intra-articular calcaneal fractures
Mycolactone Gene Expression Is Controlled by Strong SigA-Like Promoters with Utility in Studies of Mycobacterium ulcerans and Buruli Ulcer
Mycolactone A/B is a lipophilic macrocyclic polyketide that is the primary virulence factor produced by Mycobacterium ulcerans, a human pathogen and the causative agent of Buruli ulcer. In M. ulcerans strain Agy99 the mycolactone polyketide synthase (PKS) locus spans a 120 kb region of a 174 kb megaplasmid. Here we have identified promoter regions of this PKS locus using GFP reporter assays, in silico analysis, primer extension, and site-directed mutagenesis. Transcription of the large PKS genes mlsA1 (51 kb), mlsA2 (7 kb) and mlsB (42 kb) is driven by a novel and powerful SigA-like promoter sequence situated 533 bp upstream of both the mlsA1 and mlsB initiation codons, which is also functional in Escherichia coli, Mycobacterium smegmatis and Mycobacterium marinum. Promoter regions were also identified upstream of the putative mycolactone accessory genes mup045 and mup053. We transformed M. ulcerans with a GFP-reporter plasmid under the control of the mls promoter to produce a highly green-fluorescent bacterium. The strain remained virulent, producing both GFP and mycolactone and causing ulcerative disease in mice. Mosquitoes have been proposed as a potential vector of M. ulcerans so we utilized M. ulcerans-GFP in microcosm feeding experiments with captured mosquito larvae. M. ulcerans-GFP accumulated within the mouth and midgut of the insect over four instars, whereas the closely related, non-mycolactone-producing species M. marinum harbouring the same GFP reporter system did not. This is the first report to identify M. ulcerans toxin gene promoters, and we have used our findings to develop M. ulcerans-GFP, a strain in which fluorescence and toxin gene expression are linked, thus providing a tool for studying Buruli ulcer pathogenesis and potential transmission to humans
- …