In Archaea the two major modes of DNA packaging are wrapping by histone proteins or bending by architectural non-histone proteins. To supplement our knowledge about the binding mode of the different DNA-bending proteins observed across the three domains of life, we present here the first model of a complex in which the monomeric Methanogen Chromosomal protein 1 (MC1) from Euryarchaea binds to the concave side of a strongly bent DNA. In laboratory growth conditions MC1 is the most abundant architectural protein present in Methanosarcina thermophila CHTI55. Like most proteins that strongly bend DNA, MC1 is known to bind in the minor groove. Interaction areas for MC1 and DNA were mapped by Nuclear Magnetic Resonance (NMR) data. The polarity of protein binding was determined using paramagnetic probes attached to the DNA. The first structural model of the DNA-MC1 complex we propose here was obtained by two complementary docking approaches and is in good agreement with the experimental data previously provided by electron microscopy and biochemistry. Residues essential to DNA-binding and -bending were highlighted and confirmed by site-directed mutagenesis. It was found that the Arg25 side-chain was essential to neutralize the negative charge of two phosphates that come very close in response to a dramatic curvature of the DNA.
Presurgical language mapping in dominant hemisphere epilepsy to evaluate the risk of postoperative deficit is particularly difficult in children. Extraoperative invasive cortical stimulation can show some areas critical to language, but not all of them, due to scarce sampling, poor cooperation, cortical immaturity, or network reorganization, whereas functional magnetic resonance imaging (fMRI) displays entire networks involved in, but not necessarily critical to, language. In a homogeneous series of children with epilepsy, we compared the contributions of language fMRI and depth electrode stimulations to optimize language mapping.Eight children (7.5-15.5 years) with left frontal or temporal epilepsy underwent language fMRI and language stimulation with depth electrodes as part of their comprehensive presurgical workup. fMRI data collected during sentence generation were analyzed using statistical parametric mapping (SPM2) (false discovery rate [FDR] p < 0.05). Bipolar stimulations were performed during language production tasks. By coregistering fMRI and postimplantation computed tomography (CT) images, we were able to directly compare the cortical areas identified by both investigations.fMRI during sentence generation robustly showed activation in the whole perisylvian regions with little reorganization (left hemisphere dominant in 7). Of the 184 electrode contacts tested for language, only 8 were positive (language disruption) in three of the seven patients with periictal language impairment and left language dominance. All of the positive contacts colocalized with an fMRI activated cluster, that is, fMRI did not miss any region critical to language (sensitivity = 100%). However, 54 of the 176 negative contacts were within activated clusters (low specificity).In children with epilepsy, the sensitivity of fMRI during sentence generation allows for the detection of all critical regions displayed by cortical stimulation within the large perisylvian language network, but with a low specificity. It is, therefore, useful to optimize the placement of intracranial electrodes when language mapping is necessary. Systematic planning of the electrode placement according to language fMRI maps should increase the yield of extraoperative cortical stimulation, which appears rather low in children when compared to adults.
Dystrophin is a large skeletal muscle protein located at the internal face of the plasma membrane and interacting with membrane phospholipids and a number of cytosolic proteins. Binding of neuronal nitric oxide synthase (nNOS) to dystrophin appears to be crucial for exercise-induced increases in blood supply in muscle cells. By contrast, utrophin, the developmental homologous protein of dystrophin, does not display nNOS interaction. Recent in vitro and in vivo experiments showed that the dystrophin region involved in nNOS binding is located in spectrin-like repeats R16 and R17 of its filamentous central domain. Using homology modeling and atomistic molecular dynamics simulation, we compared the structural organization and surface potentials of dystrophin, utrophin, and chimeric fragments, thus revisiting the dystrophin–nNOS binding region. Our simulation results are in good agreement with experimental data. They provide a three-dimensional representation of the repeats and give insight into the molecular organization of the regions involved in dystrophin–nNOS interaction. This study also further elucidates the physical properties crucial for this interaction, particularly the presence of a large hydrophobic patch. These results will be helpful to improving our understanding of the phenotypic features of patients bearing mutations in the nNOS-binding region of dystrophin.
Summary: Purpose: To study separately the functional value of each cerebral hemisphere in hemimegalencephaly (HME). HME is a unique model of unilateral hemispheric lesion, but one suspects that the non‐HME hemisphere also could be functionally impaired because the postsurgery outcome is less favorable than expected. Methods: We performed simultaneous prolonged EEG and 133‐xenon SPECT (single‐photon emission computed tomography); we measured the absolute values of cerebral blood flow (CBF) in both hemispheres and compared them with the normal values previously acquired. Thirteen patients (aged 5–38 months) underwent 31 examinations, 20 before surgery (hemispherotomy) and 11 after. Results: In the HME hemisphere, we confirmed the presurgical mixture of increased and decreased CBF due to intermittent ictal discharges. After surgery, CBF was decreased in most cases. In the non‐HME hemisphere, presurgery CBF was abnormal in 60% of the patients, increased and related mostly to diffuse interictal spikes on the same side, whereas normal CBF cases had focal spikes. After surgery, CBF was normal in 82% of cases, corresponding to an EEG without diffuse spikes. In the six patients longitudinally studied, CBF dramatically decreased after surgery in the HME hemisphere, whereas in the non‐HME hemisphere, CBF was mostly normal very early (three fourths before 2 months), increased as soon as 3 months, and normalized only after hemispherotomy, the more rapidly the child was operated on, the earlier it was. Conclusions: This study shows that the function of the nonmalformed hemisphere is impaired as soon as the first months of the course of HME but can be restored after surgery. Our data support the recommendations to operate on the children as early as possible.
Dystrophin is a large intracellular protein that prevents sarcolemmal ruptures by providing a mechanical link between the intracellular actin cytoskeleton and the transmembrane dystroglycan complex. Dystrophin deficiency leads to the severe muscle wasting disease Duchenne Muscular Dystrophy and the milder allelic variant, Becker Muscular Dystrophy (DMD and BMD). Previous work has shown that concomitant interaction of the actin binding domain 2 (ABD2) comprising spectrin like repeats 11 to 15 (R11-15) of the central domain of dystrophin, with both actin and membrane lipids, can greatly increase membrane stiffness. Based on a combination of SAXS and SANS measurements, mass spectrometry analysis of cross-linked complexes and interactive low-resolution simulations, we explored in vitro the molecular properties of dystrophin that allow the formation of ABD2-F-actin and ABD2-membrane model complexes. In dystrophin we identified two subdomains interacting with F-actin, one located in R11 and a neighbouring region in R12 and another one in R15, while a single lipid binding domain was identified at the C-terminal end of R12. Relative orientations of the dystrophin central domain with F-actin and a membrane model were obtained from docking simulation under experimental constraints. SAXS-based models were then built for an extended central subdomain from R4 to R19, including ABD2. Overall results are compatible with a potential F-actin/dystrophin/membrane lipids ternary complex. Our description of this selected part of the dystrophin associated complex bridging muscle cell membrane and cytoskeleton opens the way to a better understanding of how cell muscle scaffolding is maintained through this essential protein.
In order to validate the ability of ictal single photon emission computed tomography (SPECT) to localize the epileptogenic zone (EZ) in children, we compared in 20 patients aged from 10 months to 17 years (mean 6.5 years) the topography of the area of increased ictal perfusion (IPA), determined on the basis of ictal minus interictal scan values, with that of the EZ determined by intracranial EEG recordings and assessed its relationship with the postsurgical outcome. Eighteen patients had symptomatic epilepsy and 10 had extratemporal epilepsy. All patients except one had an ictal injection (mean time lag from clinical seizure onset was 18 s). Ictal and interictal SPECT images were successively co‐registered, normalized, subtracted, smoothed and superimposed on MRI. All patients with ictal injection exhibited one or several IPAs. The topography of the ‘highest’ IPA, i.e. the maximal cerebral blood flow (CBF) change between ictal and interictal SPECT, significantly colocalized with the site of onset of the discharge, and that of the lower IPAs with that of the area of propagation (P < 0.0001). At a threshold of 30% of the maximal CBF change, the IPAs detected the onset of the discharge with a sensitivity of 0.80 and a specificity of 0.70. The highest IPA localized the EZ in 12 out of 15 patients. In the three others it missed the EZ and showed the area of propagation because of rapid seizure propagation or of infraclinical seizure onset. Among the patients with favourable surgery outcome, the highest IPA colocalized with the resected area in 70% of cases. Ictal SPECT could therefore plays an important role as a non‐invasive presurgical method of investigation by optimizing the placement of intracranial electrodes, thus improving the postsurgery outcome of paediatric partial epilepsy.
