Ischemic and traumatic brain injury is associated with increased risk for death and disability. The inhibition of penumbral tissue damage has been recognized as a target for therapeutic intervention, ...because cellular injury evolves progressively upon ATP-depletion and loss of ion homeostasis. In patients, thiopental is used to treat refractory intracranial hypertension by reducing intracranial pressure and cerebral metabolic demands; however, therapeutic benefits of thiopental-treatment are controversially discussed. In the present study we identified fundamental neuroprotective molecular mechanisms mediated by thiopental. Here we show that thiopental inhibits global protein synthesis, which preserves the intracellular energy metabolite content in oxygen-deprived human neuronal SK-N-SH cells or primary mouse cortical neurons and thus ameliorates hypoxic cell damage. Sensitivity to hypoxic damage was restored by pharmacologic repression of eukaryotic elongation factor 2 kinase. Translational inhibition was mediated by calcium influx, activation of the AMP-activated protein kinase, and inhibitory phosphorylation of eukaryotic elongation factor 2. Our results explain the reduction of cerebral metabolic demands during thiopental treatment. Cycloheximide also protected neurons from hypoxic cell death, indicating that translational inhibitors may generally reduce secondary brain injury. In conclusion our study demonstrates that therapeutic inhibition of global protein synthesis protects neurons from hypoxic damage by preserving energy balance in oxygen-deprived cells. Molecular evidence for thiopental-mediated neuroprotection favours a positive clinical evaluation of barbiturate treatment. The chemical structure of thiopental could represent a pharmacologically relevant scaffold for the development of new organ-protective compounds to ameliorate tissue damage when oxygen availability is limited.
Oxygen deprivation during ischemic or hemorrhagic stroke results in ATP depletion, loss of ion homeostasis, membrane depolarization, and excitotoxicity. Pharmacologic restoration of cellular energy ...supply may offer a promising concept to reduce hypoxic cell injury. In this study, we investigated whether carbimazole, a thionamide used to treat hyperthyroidism, reduces neuronal cell damage in oxygen-deprived human SK-N-SH cells or primary cortical neurons. Our results revealed that carbimazole induces an inhibitory phosphorylation of eukaryotic elongation factor 2 (eEF2) that was associated with a marked inhibition of global protein synthesis. Translational inhibition resulted in significant bioenergetic savings, preserving intracellular ATP content in oxygen-deprived neuronal cells and diminishing hypoxic cellular damage. Phosphorylation of eEF2 was mediated by AMP-activated protein kinase and eEF2 kinase. Carbimazole also induced a moderate calcium influx and a transient cAMP increase. To test whether translational inhibition generally diminishes hypoxic cell damage when ATP availability is limiting, the translational repressors cycloheximide and anisomycin were used. Cycloheximide and anisomycin also preserved ATP content in hypoxic SK-N-SH cells and significantly reduced hypoxic neuronal cell damage. Taken together, these data support a causal relation between the pharmacologic inhibition of global protein synthesis and efficient protection of neurons from ischemic damage by preservation of high-energy metabolites in oxygen-deprived cells. Furthermore, our results indicate that carbimazole or other translational inhibitors may be interesting candidates for the development of new organ-protective compounds. Their chemical structure may be used for computer-assisted drug design or screening of compounds to find new agents with the potential to diminish neuronal damage under ATP-limited conditions.
Autoimmune diseases, such as psoriasis and arthritis, show a patchy distribution of inflammation despite systemic dysregulation of adaptive immunity. Thus, additional tissue-derived signals, such as ...danger-associated molecular patterns (DAMPs), are indispensable for manifestation of local inflammation. S100A8/S100A9 complexes are the most abundant DAMPs in many autoimmune diseases. However, regulatory mechanisms locally restricting DAMP activities are barely understood. We now unravel for the first time, to our knowledge, a mechanism of autoinhibition in mice and humans restricting S100-DAMP activity to local sites of inflammation. Combining protease degradation, pull-down assays, mass spectrometry, and targeted mutations, we identified specific peptide sequences within the second calcium-binding EF-hands triggering TLR4/MD2-dependent inflammation. These binding sites are free when S100A8/S100A9 heterodimers are released at sites of inflammation. Subsequently, S100A8/S100A9 activities are locally restricted by calcium-induced (S100A8/S100A9)2 tetramer formation hiding the TLR4/MD2-binding site within the tetramer interphase, thus preventing undesirable systemic effects. Loss of this autoinhibitory mechanism in vivo results in TNF-α-driven fatal inflammation, as shown by lack of tetramer formation in crossing S100A9-/- mice with 2 independent TNF-α-transgene mouse strains. Since S100A8/S100A9 is the most abundant DAMP in many inflammatory diseases, specifically blocking the TLR4-binding site of active S100 dimers may represent a promising approach for local suppression of inflammatory diseases, avoiding systemic side effects.
N-myristoylation is a common form of co-translational protein fatty acylation resulting from the attachment of myristate to a required N-terminal glycine residue. We show that aberrantly acquired ...N-myristoylation of SHOC2, a leucine-rich repeat-containing protein that positively modulates RAS-MAPK signal flow, underlies a clinically distinctive condition of the neuro-cardio-facial-cutaneous disorders family. Twenty-five subjects with a relatively consistent phenotype previously termed Noonan-like syndrome with loose anagen hair (MIM607721) shared the 4A>G missense change in SHOC2 (producing an S2G amino acid substitution) that introduces an N-myristoylation site, resulting in aberrant targeting of SHOC2 to the plasma membrane and impaired translocation to the nucleus upon growth factor stimulation. Expression of SHOC2S2G in vitro enhanced MAPK activation in a cell type-specific fashion. Induction of SHOC2S2G in Caenorhabditis elegans engendered protruding vulva, a neomorphic phenotype previously associated with aberrant signaling. These results document the first example of an acquired N-terminal lipid modification of a protein causing human disease.
RAS signaling plays a key role in controlling appropriate cell responses to extracellular stimuli and participates in early and late developmental processes. Although enhanced flow through this ...pathway has been established as a major contributor to oncogenesis, recent discoveries have revealed that aberrant RAS activation causes a group of clinically related developmental disorders characterized by facial dysmorphism, a wide spectrum of cardiac disease, reduced growth, variable cognitive deficits, ectodermal and musculoskeletal anomalies, and increased risk for certain malignancies. Here, we report that heterozygous germline mutations in
CBL, a tumor-suppressor gene that is mutated in myeloid malignancies and encodes a multivalent adaptor protein with E3 ubiquitin ligase activity, can underlie a phenotype with clinical features fitting or partially overlapping Noonan syndrome (NS), the most common condition of this disease family. Independent
CBL mutations were identified in two sporadic cases and two families from among 365 unrelated subjects who had NS or suggestive features and were negative for mutations in previously identified disease genes. Phenotypic heterogeneity and variable expressivity were documented. Mutations were missense changes altering evolutionarily conserved residues located in the RING finger domain or the linker connecting this domain to the
N-terminal tyrosine kinase binding domain, a known mutational hot spot in myeloid malignancies. Mutations were shown to affect CBL-mediated receptor ubiquitylation and dysregulate signal flow through RAS. These findings document that germline mutations in
CBL alter development to cause a clinically variable condition that resembles NS and that possibly predisposes to malignancies.
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•First presentation of the spatiotemporal distribution of crayfish plague cases in Switzerland.•54 confirmed crayfish plague cases between 1980 and 2020.•Earliest detection of A. ...astaci DNA through qPCR in a sample of 1991.•Re-evaluation of archived samples increased the positive rate by 16.3%.
The oomycete Aphanomyces astaci is the causative agent of crayfish plague, a disease threatening susceptible freshwater crayfish species in Europe.
To detect its spatiotemporal occurrence in Switzerland, we reviewed (1) the literature regarding occurrence of crayfish plague and North American crayfish carrier species and (2) the necropsy report archive of the Institute for Fish and Wildlife Health (FIWI) from 1968 to 2020. In the past, crayfish plague was diagnosed through several methods: conventional PCR, culture, and histology. When available, we re-evaluated archived Bouin’s or formalin-fixed, paraffin-embedded samples collected during necropsies (1991–2020) with a recently published quantitative PCR.
Literature research revealed putative reports of crayfish plague in Switzerland between the 1870s and 1910s and the first occurrence of three North American crayfish species between the late 1970s and 1990s.
Finally, 54 (28.1%) cases were classified as positive and 9 (4.7%) cases as suspicious. The total number of positive cases increased by 14 (14.7%) after re-evaluation of samples. The earliest diagnosis of crayfish plague was performed in 1980 and the earliest biomolecular confirmation of A. astaci DNA dated 1991. Between 1980–1990, 1991–2000 and 2001–2010 crayfish plague spread from one to two and finally three catchment basins, respectively.
Similar to other European countries, crayfish plague has occurred in Switzerland in two waves: the first at the end of the 19th and the second at the end of the 20th century in association with the first occurrence of North American crayfish species. The spread from one catchment basin to another suggests a human-mediated pathogen dispersal.
Exome sequencing has markedly enhanced the discovery of genes implicated in Mendelian disorders, particularly for individuals in whom a known clinical entity could not be assigned. This has led to ...the recognition that phenotypic heterogeneity resulting from allelic mutations occurs more commonly than previously appreciated. Here, we report that missense variants in CDC42, a gene encoding a small GTPase functioning as an intracellular signaling node, underlie a clinically heterogeneous group of phenotypes characterized by variable growth dysregulation, facial dysmorphism, and neurodevelopmental, immunological, and hematological anomalies, including a phenotype resembling Noonan syndrome, a developmental disorder caused by dysregulated RAS signaling. In silico, in vitro, and in vivo analyses demonstrate that mutations variably perturb CDC42 function by altering the switch between the active and inactive states of the GTPase and/or affecting CDC42 interaction with effectors, and differentially disturb cellular and developmental processes. These findings reveal the remarkably variable impact that dominantly acting CDC42 mutations have on cell function and development, creating challenges in syndrome definition, and exemplify the importance of functional profiling for syndrome recognition and delineation.
Zusammenfassung
Das Netzwerk Universitätsmedizin (NUM) besteht aus den 36 Standorten der Universitätsmedizin in Deutschland. Der Auftrag ist die Koordinierung der universitätsmedizinischen ...COVID-19-Forschung auf nationaler Ebene. Dazu werden u. a. gemeinsame Infrastrukturen für die Sammlung, Haltung und Nutzung medizinischer Forschungsdaten benötigt. Diese standen beim Start des NUM-Projekts im April 2020 nicht im erforderlichen Rahmen zur Verfügung. Medizinische Forschungsdaten sind extrem heterogen und gehen weit über „Real World Data“ (Daten aus dem Versorgungsalltag) hinaus. Eine universelle Lösung dafür gab es nicht, deshalb hat das NUM fünf Forschungsinfrastrukturen für unterschiedliche Datenarten, unterschiedliche Wege der Datengewinnung und unterschiedliche Datenentstehungssettings aufgebaut. Um die Bildung neuer Datensilos zu verhindern, arbeiten alle fünf Infrastrukturen auf Basis der FAIR-Prinzipien, nach denen Daten auffindbar (findable), zugänglich (accessible), interoperabel (interoperable) und wiederverwendbar (reusable) sein sollen. Zudem implementiert das NUM einen übergreifenden Steuerungsrahmen (Governance Framework), um die Weiterentwicklung dieser fünf Infrastrukturen zentral zu steuern. Der Artikel beschreibt den aktuellen Stand der Infrastrukturentwicklung im NUM und mögliche Perspektiven. Ein starker Fokus wird dabei auf die technisch-organisatorischen Grundlagen gerichtet.
The GermAn Laser Lead Extraction RegistrY: GALLERY is a retrospective, national multicentre registry, investigating the safety and efficacy of laser lead extraction procedures in Germany.
Twenty-four ...German centres that are performing laser lead extraction have participated in the registry. All patients, treated with a laser lead extraction procedure between January 2013 and March 2017, were consecutively enrolled. Safety and efficacy of laser lead extraction were investigated. A total number of 2524 consecutive patients with 6117 leads were included into the registry. 5499 leads with a median lead dwell time of 96 (62-141) months were treated. The mean number of treated leads per patient was 2.18 ± 1.02. The clinical procedural success rate was 97.86% and the complete lead removal was observed in 94.85%. Additional extraction tools were used in 6.65% of cases. The rate of procedural failure was 2.14% with lead age ≥10 years being its only predictor. The overall complication rate was 4.32%, including 2.06% major and 2.26% minor complications. Procedure-related mortality was 0.55%. Female sex and the presence of abandoned leads were predictors for procedure-related complications. The all-cause in-hospital mortality was 3.56% with systemic infection being the strongest predictor, followed by age ≥75 years and chronic kidney disease.
In the GALLERY, a high success- and low procedure-related complication rates have been demonstrated. In multivariate analysis, female sex and the presence of abandoned leads were predictors for procedure-related complications, while the presence of systemic infection, age ≥75 years, and chronic kidney disease were independent predictors for all-cause mortality.
Abstract
We previously molecularly and clinically characterized Mazzanti syndrome, a RASopathy related to Noonan syndrome that is mostly caused by a single recurrent missense variant (c.4A > G, ...p.Ser2Gly) in SHOC2, which encodes a leucine-rich repeat-containing protein facilitating signal flow through the RAS-mitogen-associated protein kinase (MAPK) pathway. We also documented that the pathogenic p.Ser2Gly substitution causes upregulation of MAPK signaling and constitutive targeting of SHOC2 to the plasma membrane due to the introduction of an N-myristoylation recognition motif. The almost invariant occurrence of the pathogenic c.4A > G missense change in SHOC2 is mirrored by a relatively homogeneous clinical phenotype of Mazzanti syndrome. Here, we provide new data on the clinical spectrum and molecular diversity of this disorder and functionally characterize new pathogenic variants. The clinical phenotype of six unrelated individuals carrying novel disease-causing SHOC2 variants is delineated, and public and newly collected clinical data are utilized to profile the disorder. In silico, in vitro and in vivo characterization of the newly identified variants provides evidence that the consequences of these missense changes on SHOC2 functional behavior differ from what had been observed for the canonical p.Ser2Gly change but converge toward an enhanced activation of the RAS-MAPK pathway. Our findings expand the molecular spectrum of pathogenic SHOC2 variants, provide a more accurate picture of the phenotypic expression associated with variants in this gene and definitively establish a gain-of-function behavior as the mechanism of disease.
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