Lien entre les pathologies rachidiennes et l’intensité de signal IRM dans le disque intervertébral

RÉSUMÉ La scoliose et le spondylolisthésis sont des pathologies rachidiennes qui touchent respectivement 1,5-3% et 13.6% des personnes et possèdent un potentiel d’évolution. Ces pathologies tridimensionnelles sont principalement étudiées par des indices géométriques bidimensionnels ne reflétant qu’une partie des modifications morphologiques et biomécaniques/biochimiques du rachis. L’étude clinique de ces pathologies et de leurs évolutions sont réalisées à partir d’informations limitées sur les modifications engendrées au rachis. Le disque intervertébral (DIV) est le tissu mou permettant la mobilité entre les segments vertébraux. Il subit les effets dégénératifs des pathologies de manière précoce en son sein. Notre étude se base sur l’hypothèse que l’intensité de signal IRM au sein d’images cliniques pondérées en T2 est sensible à la pathologie rachidienne et à sa sévérité, permettant ainsi à cette modalité d’imagerie de fournir des informations sur les propriétés géométriques, biochimiques et mécaniques du DIV, et donc de réaliser des suivis in-vivo de l’évolution des pathologies rachidiennes. Ce projet vise à développer des techniques permettant l’étude tridimensionnelle de la distribution géométrique et gaussienne du signal IRM pondéré en T2 au sein d’images cliniques de patients présentant des pathologies rachidiennes, et ce dans le disque intervertébral (DIV) complet, dans l’annulus fibrosus (AF) et dans le nucleus pulposus (NP). Ces outils permettront de vérifier si les dégénérescences des DIV sont spécifiques aux pathologies les ayant engendrées ainsi qu’à leurs sévérités. Afin d’analyser le signal IRM tridimensionnel du DIV de manière automatisée, il est nécessaire de segmenter les images cliniques. Une méthode semi-automatisée a été utilisée dans ce projet et sa reproductibilité a été testée. Cette méthode permet un gain de temps par rapport aux méthodes manuelles utilisées dans la littérature. Des outils d’analyse de signal IRM au sein du DIV ont été développés afin de détecter la sensibilité de celui-ci aux pathologies rachidiennes et à leurs sévérités. Ces outils ont permis de refléter les variations de distribution du signal IRM de manière géométrique en 3D et de manière gaussienne. Dans ce but, une cohorte de 79 sujets (32 scolioses, 32 spondylolithésis, 15 contrôles) a été étudiée. Une normalisation de l’intensité de signal a été nécessaire à la comparaison des signaux----------ABSTRACT Scoliosis and spondylolisthesis are spine pathologies affecting 1.5-3% and 13.6% of the population, respectively. These diseases have the potential to further progress. These tridimensional pathologies are mainly studied using two-dimensional geometric indices, which reflect only a fraction of the morphological, biomechanical and biochemical variations of the spine. Clinical interpretations of these pathologies and of their evolution are based on the limited information of spine modifications. The intervertebral disc (IVD) is the soft tissue between adjacent vertebrae that allow the mobility of the spine between the rigid segments. Spine pathologies lead to premature degeneration of the IVD. In our study, we hypothesize that the MRI signal intensity within clinical T2-weighted images is sensitive to the spine pathology and to its severity. Thus this imaging technique could provide information on the geometrical, biochemical and mechanical properties of the IVD, and facilitate in-vivo follow-up of the evolution of these spine pathologies. This project aims to develop techniques to analyse the tridimensional geometry as well as the Gaussian distribution of the T2- weighted MRI signal within clinical images of the IVD, the annulus fibrosus (AF) and the nucleus pulposus (NP) of patients affected with various spine pathologies. These tools will assess whether or not a specific degeneration of the IVD is caused by the spine pathologies depending on their severity. In order to analyse automatically the tridimensional MRI signal within the IVD, it is necessary to segment clinical images. A semi-automated method was used in this project and its reproducibility was tested. This method is less time-consuming compared to the commonly used manual methods that are reported in the literature. MRI signal analysis tools were developed to detect its sensitivity within the IVD to spine pathologies and their severities. These tools allowed a Gaussian and geometric distribution analysis of the MRI signal intensity within the IVD. A cohort of 79 subjects (32 scoliosis, 32 spondylolisthesis, 15 controls) was studied. A normalization of the signal intensity was done in order to compare images from patients with variable parameters such as the acquisition gain. This study tested two normalizations of the intensity of the signal. The first one was based on the intensity within the cerebrospinal flui

Electrocorticographic recording of cerebral cortex areas manipulated using an adeno-associated virus targeting cofilin in mice

The use of electrocorticographic (ECoG) recordings in rodents is relevant to sleep research and to the study of a wide range of neurological conditions. Adeno-associated viruses (AAVs) are increasingly used to improve understanding of brain circuits and their functions. The AAV-mediated manipulation of specific cell populations and/or of precise molecular components has been tremendously useful to identify new sleep regulatory circuits/molecules and key proteins contributing to the adverse effects of sleep loss. For instance, inhibiting activity of the filamentous actin-severing protein cofilin using AAV prevents sleep deprivation-induced memory impairment. Here, a protocol is described that combines the manipulation of cofilin function in a cerebral cortex area with the recording of ECoG activity to examine whether cortical cofilin modulates the wakefulness and sleep ECoG signals. AAV injection is performed during the same surgical procedure as the implantation of ECoG and electromyographic (EMG) electrodes in adult male and female mice. Mice are anesthetized, and their heads are shaved. After skin cleaning and incision, stereotaxic coordinates of the motor cortex are determined, and the skull is pierced at this location. A cannula prefilled with an AAV expressing cofilinS3D, an inactive form of cofilin, is slowly positioned in the cortical tissue. After AAV infusion, gold-covered screws (ECoG electrodes) are screwed through the skull and cemented to the skull with gold wires inserted in the neck muscles (EMG electrodes). The animals are allowed three weeks to recover and to ensure sufficient expression of cofilinS3D. The infected area and cell type are verified using immunohistochemistry, and the ECoG is analyzed using visual identification of vigilance states and spectral analysis. In summary, this combined methodological approach allows the investigation of the precise contribution of molecular components regulating neuronal morphology and connectivity to the regulation of synchronized cerebral cortex activity during wakefulness and sleep

Étude de la relation entre les oligomères amyloïde-bêta et la protéine d'adhésion synaptique Neuroligine-1

Dans la maladie d'Alzheimer (MA), les pertes synaptiques et la neurodégénérescence, causées en partie par les oligomères amyloïdes-bêta (Aßo), sont les meilleurs prédicteurs des troubles de mémoire. Une priorité actuelle est d'identifier un biomarqueur affecté précocement par les Aßo. La protéine post-synaptique Neuroligine-1 (NL-1) pourrait être une candidate due à son implication dans la plasticité synaptique et la mémoire. La première hypothèse de cette étude est que les dysfonctions synaptiques engendrées par les Aβo1-42 pourraient affecter de façon précoce et progressive le niveau de NL-1. L'expression de NL-1 a été mesurée lors d'injections hippocampiques chroniques d'Aßo1-42, d'exposition de neurones primaires d'hippocampe aux Aßo1-42, ainsi que chez les souris 3xTg. En utilisant 5 ensembles d’amorces informant sur les transcrits de Nl-1, une augmentation des niveaux d'ARNm après 2 jours d'injections d'Aßo1-42 a été détectée pour 2 ensembles d’amorces, et une diminution d’expression de 3 ensemble d’amorces a été mesuré après 4 jours d'injections. Par contre, aucun changement du niveau protéique n'a été observé. In vitro, le niveau de NL-1 diminue après 72h d'exposition aux Aβo1-42, concordant avec une diminution de la viabilité des neurones. Finalement, les souris 3xTg femelles démontrent une diminution de NL-1 à 4 mois. Notre deuxième hypothèse stipulant que l’absence de NL-1 entraîne une toxicité accrue des Aßo1-42 a été confirmée. Les souris NL-1 KO injectées aux Aßo1-42 ont obtenus de plus faibles performances au test de la reconnaissance d'objet et à la piscine de Morris, ainsi qu’une perte neuronale plus importante que les autres groupes testés. Cette étude confirme la relation étroite entre NL-1 et les Aßo1-42, apportant possiblement une nouvelle cible thérapeutique ou biomarqueur pour le développement de nouvelles thérapies aux stades précoces de la MA.Synaptic loss and neurodegeneration, partly induced by amyloid-beta oligomers (Aßo), are the best predictors of memory deficits in Alzheimer’s disease (AD). An actual priority in the field is to identify a novel biomarker affected early by Aßo. The post-synaptic protein Neuroligin-1 (NL-1) could be a good candidate due to its roles in synaptic plasticity and memory. The first hypothesis of this study is that the synaptic dysfunctions caused by Aβo could rapidly and gradually affect the level of NL1. NL-1 expression was measured in chronic hippocampal Aβo1-42 injections, in primary hippocampal neuronal cultures exposed to Aβo1-42, and in 3xTg mice. Using 5 sets of probes assessing Nl-1 transcripts, an increase in mRNA levels after 2 days of Aβo1-42 injections was detected for 2 sets of probes, and a decrease in 3 sets of probes expression were measured after 4 days of injections. However, no change in protein level was observed. In vitro, the level of NL-1 decreases after 72 hours of exposure to Aβo1-42, following the decrease in neuron viability. Finally, female 3xTg mice show a decrease in NL-1 at 4 months. In addition, the second hypothesis that the absence of NL-1 will lead to increased toxicity has been confirmed. Aβo1-42 injected NL-1 KO mice performed poorly in spatial object recognition and in the Morris water maze, and had greater neuronal loss than all the other groups tested. This study therefore confirms the close relationship between NL-1 and Aβo1-42, possibly providing a new therapeutic target or biomarker for the development of therapies in the early stages of AD

Molecular modeling and imaging of initial stages of cellulose fibril assembly: Evidence for a disordered intermediate stage

International audienceThe remarkable mechanical strength of cellulose reflects the arrangement of multiple β-1,4-linked glucan chains in a para-crystalline fibril. During plant cellulose biosynthesis, a multimeric cellulose synthesis complex (CSC) moves within the plane of the plasma membrane as many glucan chains are synthesized from the same end and in close proximity. Many questions remain about the mechanism of cellulose fibril assembly, for example must multiple catalytic subunits within one CSC polymerize cellulose at the same rate? How does the cellulose fibril bend to align horizontally with the cell wall? Here we used mathematical modeling to investigate the interactions between glucan chains immediately after extrusion on the plasma membrane surface. Molecular dynamics simulations on groups of six glucans, each originating from a position approximating its extrusion site, revealed initial formation of an uncrystallized aggregate of chains from which a protofibril arose spontaneously through a ratchet mechanism involving hydrogen bonds and van der Waals interactions between glucose monomers. Consistent with the predictions from the model, freeze-fracture transmission electron microscopy using improved methods revealed a hemispherical accumulation of material at points of origination of apparent cellulose fibrils on the external surface of the plasma membrane where rosette-type CSCs were also observed. Together the data support the possibility that a zone of uncrystallized chains on the plasma membrane surface buffers the predicted variable rates of cellulose polymerization from multiple catalytic subunits within the CSC and acts as a flexible hinge allowing the horizontal alignment of the crystalline cellulose fibrils relative to the cell wall

Neuroligin-1 Is Altered in the Hippocampus of Alzheimer\u27s Disease Patients and Mouse Models, and Modulates the Toxicity of Amyloid-Beta Oligomers

Synapse loss occurs early and correlates with cognitive decline in Alzheimer’s disease (AD). Synaptotoxicity is driven, at least in part, by amyloid-beta oligomers (Aβo), but the exact synaptic components targeted by Aβo remain to be identified. We here tested the hypotheses that the post-synaptic protein Neuroligin-1 (NLGN1) is affected early in the process of neurodegeneration in the hippocampus, and specifically by Aβo, and that it can modulate Aβo toxicity. We found that hippocampal NLGN1 was decreased in patients with AD in comparison to patients with mild cognitive impairment and control subjects. Female 3xTg-AD mice also showed a decreased NLGN1 level in the hippocampus at an early age (i.e., 4 months). We observed that chronic hippocampal Aβo injections initially increased the expression of one specific Nlgn1 transcript, which was followed by a clear decrease. Lastly, the absence of NLGN1 decreased neuronal counts in the dentate gyrus, which was not the case in wild-type animals, and worsens impairment in spatial learning following chronic hippocampal Aβo injections. Our findings support that NLGN1 is impacted early during neurodegenerative processes, and that Aβo contributes to this effect. Moreover, our results suggest that the presence of NLGN1 favors the cognitive prognosis during Aβo-driven neurodegeneration

MRI signal distribution within the intervertebral disc as a biomarker of adolescent idiopathic scoliosis and spondylolisthesis

Background: Early stages of scoliosis and spondylolisthesis entail changes in the intervertebral disc (IVD) structure and biochemistry. The current clinical use of MR T2-weighted images is limited to visual inspection. Our hypothesis is that the distribution of the MRI signal intensity within the IVD in T2-weighted images depends on the spinal pathology and on its severity. Therefore, this study aims to develop the AMRSID (analysis of MR signal intensity distribution) method to analyze the 3D distribution of the MR signal intensity within the IVD and to evaluate their sensitivity to scoliosis and spondylolisthesis and their severities. Methods: This study was realized on 79 adolescents who underwent a MRI acquisition (sagittal T2-weighted images) before their orthopedic or surgical treatment. Five groups were considered: low severity scoliosis (Cobb angle 50 degrees), low severity spondylolisthesis (Meyerding grades I and II), high severity spondylolisthesis (Meyerding grades III, IV and V) and control. The distribution of the MRI signal intensity within the IVD was analyzed using the descriptive statistics of histograms normalized by either cerebrospinal fluid or bone signal intensity, weighted centers and volume ratios. Differences between pathology and severity groups were assessed using one-and two-way ANOVAs. Results: There were significant (p < 0.05) variations of indices between scoliosis, spondylolithesis and control groups and between low and high severity groups. The cerebrospinal fluid normalization was able to detect differences between healthy and pathologic IVDs whereas the bone normalization, which reflects both bone and IVD health, detected more differences between the severities of these pathologies. Conclusions: This study proves for the first time that changes in the intervertebral disc, non visible to the naked eye on sagittal T2-weighted MR images of the spine, can be detected from specific indices describing the distribution of the MR signal intensity. Moreover, these indices are able to discriminate between scoliosis and spondylolisthesis and their severities, and provide essential information on the composition and structure of the discs whatever the pathology considered. The AMRSID method may have the potential to complement the current diagnostic tools available in clinics to improve the diagnostic with earlier biomarkers, the prognosis of evolution and the treatment options of scoliosis and spondylolisthesis

Study and optimization of freeze-drying cycles of a model probiotic strain

[EN] This work is based on the experimental study of the freeze-drying process to understand the impact of numerous factors on the survival rates of a model probiotic strain of Lactobacillus casei type. With the aim to find out if cell density in the matrix and survival rates are linked, we have studied the location of the cells after freeze drying inside a porous matrix composed of a lactose basis with a polymer, the polyvinylpyrrolidone (PVP) in various amounts. The best survival rate were obtained at slow freezing rate for a formulation containing 5% (m/V) of lactose and 5% (m/V) of PVP.Verlhac, P.; Vessot-Crastes, S.; Degobert, G.; Cogné, C.; Andrieu, J.; Beney, L.; Gervais, P. (2018). Study and optimization of freeze-drying cycles of a model probiotic strain. En IDS 2018. 21st International Drying Symposium Proceedings. Editorial Universitat Politècnica de València. 635-642. https://doi.org/10.4995/IDS2018.2018.7400OCS63564

Structural insights into chaperone addiction of toxin-antitoxin systems

International audienceSecB chaperones assist protein export by binding both unfolded proteins and the SecA motor. Certain SecB homologs can also control toxin-antitoxin (TA) systems known to modulate bacterial growth in response to stress. In such TA-chaperone (TAC) systems, SecB assists the folding and prevents degradation of the antitoxin, thus facilitating toxin inhibition. Chaperone dependency is conferred by a C-terminal extension in the antitoxin known as chaperone addiction (ChAD) sequence, which makes the antitoxin aggregation-prone and prevents toxin inhibition. Using TAC of Mycobacterium tuberculosis, we present the structure of a SecB-like chaperone bound to its ChAD peptide. We find differences in the binding interfaces when compared to SecB–SecA or SecB-preprotein complexes, and show that the antitoxin can reach a functional form while bound to the chaperone. This work reveals how chaperones can use discrete surface binding regions to accommodate different clients or partners and thereby expand their substrate repertoire and functions

Activity of EGFR Tyrosine Kinase Inhibitors in NSCLC With Refractory Leptomeningeal Metastases

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Structural and functional characterization of interactions involving the Tfb1 subunit of TFIIH and the NER factor Rad2

The general transcription factor IIH (TFIIH) plays crucial roles in transcription as part of the pre-initiation complex (PIC) and in DNA repair as part of the nucleotide excision repair (NER) machinery. During NER, TFIIH recruits the 3′-endonuclease Rad2 to damaged DNA. In this manuscript, we functionally and structurally characterized the interaction between the Tfb1 subunit of TFIIH and Rad2. We show that deletion of either the PH domain of Tfb1 (Tfb1PH) or several segments of the Rad2 spacer region yield yeast with enhanced sensitivity to UV irradiation. Isothermal titration calorimetry studies demonstrate that two acidic segments of the Rad2 spacer bind to Tfb1PH with nanomolar affinity. Structure determination of a Rad2–Tfb1PH complex indicates that Rad2 binds to TFIIH using a similar motif as TFIIEα uses to bind TFIIH in the PIC. Together, these results provide a mechanistic bridge between the role of TFIIH in transcription and DNA repair