Functional characterization of different epilepsy associated SCN2A and CACNA1E mutations

Epilepsie ist eine der häufigsten neurologischen Erkrankungen weltweit. Epilepsie kann viele unterschiedliche Ätiologien haben. Monogetische Veränderungen machen einen kleinen Bruchteil hieran aus. In dieser Arbeit wurden Mutationen in zwei Genen untersucht, die bei Patienten mit genetischer Epilepsie gefunden wurden. Diese kodieren für einen Kalzium- oder für einen Natriumkanal Bei einem französischen Patienten mit fokaler Epilepsie und schwerer geistiger Retardierung wurde die c.632 G>A (p.G211D) Mutation in SCN2A, einem bereits bekannten Epilepsiegen, gefunden. SCN2A kodiert NaV1.2, einen der Hauptnatriumkanäle des menschlichen Gehirns während der Phasen der neuronalen Entwicklung. Die Mutation wurde in das Wildtyp Gen eingeführt und in tsA201 Zellen überexprimiert. Voltage-clamp Aufnahmen der transfizierten Zellen zeigten eine Verschiebung der Aktivierungskurve um -7mV und einen fraglichen kleineren Reststrom im Vergleich zum Wildtyp-Kanal. Wir gehen davon aus, dass der hauptsächliche Funktionsgewinn in diesem Kanal, welcher aus der Verschiebung der Aktivierungskurve resultiert, der Grund für die Erkrankung des Patienten ist. Der kleinere Reststrom könnte Hinweis auf einen zusätzlichen Funktionsverlust sein. In einer weiteren weltweiten Kollaboration wurden Mutationen im neuen Epilepsiegen CACNA1E bei 30 Kindern mit frühkindlicher Epilepsie und geistiger Retardierung identifiziert. Die meisten Patienten waren schwer betroffen und zeigten eine pharmakoresistente Epilepsie, sowie eine kongenitale Hypotonie und Kontrakturen. CACNA1E kodiert den im zentralen Nervensystem weithin exprimierten R-Typ Kalzium Kanal CaV2.3. Die drei Missense-Mutationen c.2093 T>C (p.F698S), c.2101 A>G (p.I701V) und c.2104 G>A (p.A702T) wurden in das Wildtyp- Gen eingeführt und heterolog in tsA201 Zellen exprimiert. Elektrophysiologische Messungen dieser transfizierten nicht-neuronalen Zellen mit der Patch-clamp Technik zeigten einen charakteristischen elektrophysiologischen Phänotyp, der aus einer Verschiebung der Aktivierungskurve in die negative Richtung, sowie einer verlängerten Inaktivierung im Vergleich zum Wildtyp bestand. Wir gehen davon aus, dass dieser sehr konsistente Funktionsgewinn in diesem Kanal der Grund für die Erkrankung der Patienten ist.Epilepsy is one of the most common neurological diseases worldwide. Even though many etiologies exist, only a small percentage of epilepsy cases can be traced back to monogenetic alterations. In this work, mutations in two genes, encoding a sodium or a calcium channel, that occur in patients with genetic epilepsy were studied. A patient in France with focal epilepsy and severe intellectual disability was diagnosed with the c.632 G>A (p.G211D) mutation in SCN2A, a well-known epilepsy gene. SCN2A encodes for NaV1.2, one of the main sodium channels in the human brain during early stages of development. The mutation was introduced into the wild type gene and overexpressed in tsA201 cells. Voltage clamp recordings of the transfected cells showed a -7 mV shift in the voltage dependence of activation and a debatable smaller persistent current compared to the wild type channel. We suspect the major gain of function in the channel, resulting out of the shift in the activation curve, to be the main cause of disease in this patient. Furthermore, the reduced persistent current might indicate an additional loss of function. In another worldwide collaborative study, mutations in the novel epilepsy gene CACNA1E were identified in 30 children with early infantile epilepsy and developmental delay. Most patients were severely affected and showed pharmacoresistant epilepsy and congenital hypotonia and contractures. CACNA1E encodes the R-Type calcium channel CaV2.3, which is widely expressed in the central nervous system. Three missense mutations c.2093 T>C (p.F698S), c.2101 A>G (p.I701V), and c.2104 G>A (p.A702T) were introduced into the wild type gene and expressed heterologously in tsA201 cells. Voltage clamp recordings of the transfected non-neuronal cells showed a distinctive electrophysiological phenotype with a shift of the voltage dependence of activation to more negative values and a prolonged inactivation in comparison to the wild type. We suspect that this very consistent gain of function in the channel is the cause of disease in these patients

Hyperthermophilic Archae- and Eubacteria occurring within Indonesian Hydrothermal Areas

From 85 samples taken during cruise 45B of the R/V SONNE within the Sunda Arc subduction zone and from solfatara fields in Java, thermophilic and hyperthermophilic archae- and eubacteria were isolated. The archaebacteria belong to the genera Methanobacterium, Methanolobus, Methanosarcina, Acidianus, Thermoproteus, Desulfurococcus, Thermoplasma and to two up to now unknown genera of hyperthermophilic marine heterotrophs and continental metal mobilizers. The eubacterial hyperthermophilic isolates are a new species of Thermotoga and a novel strictly anaerobe, thriving by H2/N03"-autotrophy

Rapid Diagnosis of Central Nervous System Scedosporiosis by Specific Quantitative Polymerase Chain Reaction Applied to Formalin-Fixed, Paraffin-Embedded Tissue

Scedosporium (S.) apiospermum is a typical mold causing cerebral abscesses, often after near-drowning. Infections are associated with high morbidity and mortality due to diagnostic challenges including the need for prolonged incubation of cultures. In addition, histopathological differentiation from other filamentous fungi, including Aspergillus fumigatus, may not be possible, excluding early specific diagnosis and targeted therapy. Polymerase chain reaction (PCR) on tissue samples can rapidly identify fungi, leading to an earlier adequate treatment. Due to an extensive spectrum of causative fungi, broad-range PCRs with amplicon sequencing have been endorsed as the best DNA amplification strategy. We herein describe a case with brain abscesses due to S. apiospermum in a 66-year-old immunocompromised female patient. While broad-range PCR failed to identify a fungal pathogen from a cerebral biopsy demonstrating hyaline mold hyphae, specific quantitative PCR (qPCR) identified Scedosporium and ruled out Aspergillus, the most prevalent agent of central nervous system mold infection. A panel of specific qPCR assays, guided by the morphology of fungal elements in tissue or as a multiplex assay, may be a successful molecular approach to identify fungal agents of brain abscesses. This also applies in the presence of negative broad-range fungal PCR, therefore providing diagnostic and therapeutic potential for early specific management and improvement of patient clinical outcome

Setting the agenda, influencing public opinion, and advocating for social change: determinants of journalistic interventionism in 21 countries

This study seeks to contribute to the systematic explanation of journalists’ professional role orientations. Focusing on three aspects of journalistic interventionism – the importance of setting the political agenda, influencing public opinion and advocating for social change – multilevel analyses found substantive variation in interventionism at the individual level of the journalist, the level of the media organizations, and the societal level. Based on interviews with 2100 journalists from 21 countries, findings affirm theories regarding a hierarchy of influences in news work. We found journalists to be more willing to intervene in society when they work in public media organizations and in countries with restricted political freedom. An important conclusion of our analysis is that journalists’ professional role orientations are also rooted within perceptions of cultural and social values. Journalists were more likely to embrace an interventionist role when they were more strongly motivated by the value types of power, achievement and tradition

Thermococcus chitonophagus sp. nov., a novel, chitin-degrading hyperthermophilic archaeum from a deep-sea hydrothermal vent environment

From a hydrothermal vent site off the Mexican west coast (20°50′N, 109°06′W) at a depth of 2,600 m, a novel, hyperthermophilic, anaerobic archaeum was isolated. Cells were round to slightly irregular cocci, 1.2–2.5 μm in diameter and were motile by means of a tuft of flagella. The new isolate grew between 60 and 93°C (optimum: 85°C), from pH 3.5 to 9 (optimum: pH 6.7), and from 0.8 to 8% NaCl (optimum: 2%). The isolate was an obligate organotroph, using chitin, yeast extract, meat extract, and peptone for growth. Chitin was fermented to H2, CO2, NH3, acetate, and formate. H2S was formed in the presence of sulfur. The chitinoclastic enzyme system was oxygen-stable, cell-associated, and inducible by chitin. The cell wall was composed of a surface layer of hexameric protein complexes arranged on a p6 lattice. The core lipids consisted of glycerol diphytanyl diethers and acyclic and cyclic glycerol diphytanyl tetraethers. The G+C content was 46.5 mol%. DNA/DNA hybridization and 16S rRNA sequencing indicated that the new isolate belongs to the genusThermococcus, representing a new species,Thermococcus chitonophagus. The type strain is isoalte GC74, DSM 10152