A MELAS phenotype and a paternal inherited inversion of chromosome 10 in a female patient

A MELAS phenotype and a paternal inherited inversion of chromosome 10 in a female patient: We describe a patient suffering from encephalomyopathy with overlapping symptoms, including MELAS and Kearn-Sayre syndrome features. Mutations in tRNA LEU (UUR) were not found in mtDNA of blood cells, suggesting a different genetic defect. Cytogenetic studies revealed a paternal inherited pericentric inversion of chromosome 10 (p13;q22) pat. Although the presence of the same inversion in the father and in the apparently asymptomatic sister does rather suggest that the concurrence of the mitochondrial disease in the patient was due to chance, some alternative explanations to associate both events might be proposed.     

Barley Mla and Rar mutants compromised in the hypersensitive cell death response against Blumeria graminis f.sp. hordei are modified in their ability to accumulate reactive oxygen intermediates at sites of fungal invasion

 The pathogenesis-related accumulation of superoxide radical anions (O^·− ₂) and hydrogen peroxide (H₂O₂) was comparatively analyzed in a barley line (Hordeum vulgare L. cv Sultan-5) carrying the powdery mildew (Blumeria graminis f.sp. hordei, Speer, Bgh) resistance gene Mla12, and in susceptible mutants defective in Mla12 or in genes “required for Mla12-specified disease resistance” (Rar1 and Rar2). In-situ localization of reactive oxygen intermediates was performed both by microscopic detection of azide-insensitive nitroblue tetrazolium (NBT) reduction or diaminobenzidine (DAB) polymerization, and by an NBT-DAB double-staining procedure. The Mla12-mediated hypersensitive cell death occurred either in attacked epidermal cells or adjacent mesophyll cells of wild-type plants. Whole-cell H₂O₂ accumulation was detected in dying cells, while O^·− ₂ emerged in adjacent cells. Importantly, all susceptible mutants lacked these reactions. An oxalate oxidase, which is known to generate H₂O₂ and has been implicated in barley resistance against the powdery mildew fungus, was not differentially expressed between the wild type and all mutants. The results demonstrate that the Rar1 and Rar2 gene products, which are control elements of R-gene-mediated programmed cell death, also control accumulation of reactive oxygen intermediates but not the pathogenesis-related expression of oxalate oxidase. Received: 7 January 2000 / Accepted: 2 June 2000     

Integrated Paleoenvironmental Reconstruction and Taphonomy of a Unique Upper Cretaceous Vertebrate-Bearing Locality (Velaux,Southeastern France)

The Velaux-La Bastide Neuve fossil-bearing site (Bouches-du-Rhône, France) has yielded a diverse vertebrate assemblage dominated by dinosaurs, including the titanosaur Atsinganosaurus velauciensis. We here provide a complete inventory of vertebrate fossils collected during two large-scale field campaigns. Numerous crocodilian teeth occur together with complete skulls. Pterosaur, hybodont shark and fish elements are also represented but uncommon. Magnetostratigraphic analyses associated with biostratigraphic data from dinosaur eggshell and charophytes suggest a Late Campanian age for the locality. Lithologic and taphonomic studies, associated with microfacies and palynofacies analyses, indicate a fluvial setting of moderate energy with broad floodplain. Palynomorphs are quite rare; only three taxa of pollen grains occur: a bisaccate taxon, a second form probably belonging to the Normapolles complex, and another tricolporate taxon. Despite the good state of preservation, these taxa are generally difficult to identify, since they are scarce and have a very minute size. Most of the vertebrate remains are well preserved and suggest transport of the carcasses over short distances before accumulation in channel and overbank facies, together with reworked Aptian grains of glauconite, followed by a rapid burial. The bones accumulated in three thin layers that differ by their depositional modes and their taphonomic histories. Numerous calcareous and iron oxides-rich paleosols developed on the floodplain, suggesting an alternating dry and humid climate in the region during the Late Campanian.     

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.First we describe how to generate subcutaneous tumors in mice, using antigen-negative cell lines as negative controls and antigen-positive cells as positive controls in the same mice for intraindividual comparison. We outline how to administer intravenously near-infrared fluorophore labelled (AlexaFluor680) antigen-specific nanobodies and conventional antibodies. In vivo imaging was performed with a small-animal NIRF-Imaging system. After the in vivo imaging experiments the mice were sacrificed. We then describe how to prepare the tumors for parallel ex vivo analyses by flow cytometry and fluorescence microscopy to validate in vivo imaging results.The use of the near-infrared fluorophore labelled nanobodies allows for non-invasive same day imaging in vivo. Our protocols describe the ex vivo quantification of the specific labeling efficiency of tumor cells by flow cytometry and analysis of the distribution of the antibody constructs within the tumors by fluorescence microscopy. Using near-infrared fluorophore labelled probes allows for non-invasive, economical in vivo imaging with the unique ability to exploit the same probe without further secondary labelling for ex vivo validation experiments using flow cytometry and fluorescence microscopy.     

Evaluation of Multiband EPI Acquisitions for Resting State fMRI

Functional magnetic resonance imaging (fMRI) and particularly resting state fMRI (rs-fMRI) is widely used to investigate resting state brain networks (RSNs) on the systems level. Echo planar imaging (EPI) is the state-of-the-art imaging technique for most fMRI studies. Therefore, improvements of EPI might lead to increased sensitivity for a large amount of studies performed every day. A number of developments to shorten acquisition time have been recently proposed and the multiband technique, allowing the simultaneous acquisition of multiple slices yielding an equivalent reduction of measurement time, is the most promising among them. While the prospect to significantly reduce acquisition time by means of high multiband acceleration factors (M) appears tempting, signal quality parameters and the sensitivity to detect common RSNs with increasing M-factor have only been partially investigated up to now. In this study, we therefore acquired rs-fMRI data from 20 healthy volunteers to systematically investigate signal characteristics and sensitivity for brain network activity in datasets with increasing M-factor, M = 2 − 4. Combined with an inplane, sensitivity encoding (SENSE), acceleration factor, S = 2, we applied a maximal acceleration factor of 8 (S2×M4). Our results suggest that an M-factor of 2 (total acceleration of 4) only causes negligible SNR decrease but reveals common RSN with increased sensitivity and stability. Further M-factor increase produced random artifacts as revealed by signal quality measures that may affect interpretation of RSNs under common scanning conditions. Given appropriate hardware, a mb-EPI sequence with a total acceleration of 4 significantly reduces overall scanning time and clearly increases sensitivity to detect common RSNs. Together, our results suggest mb-EPI at moderate acceleration factors as a novel standard for fMRI that might increase our understanding of network dynamics in healthy and diseased brains.     

A New Method to Quantify Ifosfamide Blood Levels Using Dried Blood Spots and UPLC-MS/MS in Paediatric Patients with Embryonic Solid Tumours

Ifosfamide blood concentrations are necessary to monitor its therapeutic response, avoiding any adverse effect. We developed and validated an analytical method by UPLC-MS/MS to quantify ifosfamide in dried blood spots (DBS). Blood samples were collected on Whatman 903® filter paper cards. Five 3 mm disks were punched out from each dried blood spot. Acetonitrile and ethyl acetate were used for drug extraction. Chromatographic separation was carried out in an Acquity UPLC equipment with a BEH-C18 column, 2.1 x 100 mm, 1.7 μm (Waters®). The mobile phase consisted in 5 mM ammonium formate and methanol:acetonitrile (40:48:12 v/v/v) at 0.2 mL/min. LC-MS/MS detection was done by ESI+ and multiple reaction mode monitoring, ionic transitions were m/z¹⁺ 260.99 > 91.63 for ifosfamide and 261.00 > 139.90 for cyclophosphamide (internal standard). This method was linear within a 100–10000 ng/mL range and it was accurate, precise and selective. Ifosfamide samples in DBS were stable for up to 52 days at -80°C. The procedure was tested in 14 patients, ages 1 month to 17 years (9 males and 5 females), with embryonic tumours treated with ifosfamide, alone or combined, at a public tertiary referral hospital. Ifosfamide blood levels ranged from 11.1 to 39.7 μmol/L at 12 hours after the last infusion, while 24-hour levels ranged from 0.7–19.7 μmol/L. The median at 12 hours was 19.5 μmol/L (Q₂₅ 14.4–Q₇₅ 29.0) and 3.8 μmol/L (Q₂₅ 1.5–Q₇₅ 9.9) at 24 hours, p<0.001. This method is feasible to determine ifosfamide plasma levels in paediatric patients.     

Mechanics of DNA bridging by bacterial condensin MukBEF in vitro and in singulo

Structural maintenance of chromosome (SMC) proteins comprise the core of several specialized complexes that stabilize the global architecture of the chromosomes by dynamically linking distant DNA fragments. This reaction however remains poorly understood giving rise to numerous proposed mechanisms of the proteins. Using two novel assays, we investigated real‐time formation of DNA bridges by bacterial condensin MukBEF. We report that MukBEF can efficiently bridge two DNAs and that this reaction involves multiple steps. The reaction begins with the formation of a stable MukB–DNA complex, which can further capture another protein‐free DNA fragment. The initial tether is unstable but is quickly strengthened by additional MukBs. DNA bridging is modulated but is not strictly dependent on ATP and MukEF. The reaction revealed high preference for right‐handed DNA crossings indicating that bridging involves physical association of MukB with both DNAs. Our data establish a comprehensive view of DNA bridging by MukBEF, which could explain how SMCs establish both intra‐ and interchromosomal links inside the cell and indicate that DNA binding and bridging could be separately regulated.     

SynCAM 1 adhesion dynamically regulates synapse number and impacts plasticity and learning

Synaptogenesis is required for wiring neuronal circuits in the developing brain and continues to remodel adult networks. However, the molecules organizing synapse development and maintenance in?vivo remain incompletely understood. We now demonstrate that the immunoglobulin adhesion molecule SynCAM 1 dynamically alters synapse number and plasticity. Overexpression of SynCAM 1 in transgenic mice promotes excitatory synapse number, while loss of SynCAM 1 results in fewer excitatory synapses. By turning off SynCAM 1 overexpression in transgenic brains, we show that it maintains the newly induced synapses. SynCAM 1 also functions at mature synapses to alter their plasticity by regulating long-term depression. Consistent with these effects on neuronal connectivity, SynCAM 1 expression affects spatial learning, with knock-out mice learning better. The reciprocal effects of increased SynCAM 1 expression and loss reveal that this adhesion molecule contributes to the regulation of synapse number and plasticity, and impacts how neuronal networks undergo activity-dependent changes.