6E11, a highly selective inhibitor of Receptor-Interacting Protein Kinase 1, protects cells against cold hypoxia-reoxygenation injury.

Necroptosis is a programmed cell death pathway that has been shown to be of central pathophysiological relevance in multiple disorders (hepatitis, brain and cardiac ischemia, pancreatitis, viral infection and inflammatory diseases). Necroptosis is driven by two serine threonine kinases, RIPK1 (Receptor Interacting Protein Kinase 1) and RIPK3, and a pseudo-kinase MLKL (Mixed Lineage Kinase domain-Like) associated in a multi-protein complex called necrosome. In order to find new inhibitors for use in human therapy, a chemical library containing highly diverse chemical structures was screened using a cell-based assay. The compound 6E11, a natural product derivative, was characterized as a positive hit. Interestingly, this flavanone compound: inhibits necroptosis induced by death receptors ligands TNF-α (Tumor Necrosis Factor) or TRAIL (TNF-Related Apoptosis-Inducing Ligand); is an extremely selective inhibitor, among kinases, of human RIPK1 enzymatic activity with a nM Kd; has a non-ATP competitive mode of action and a novel putative binding site; is weakly cytotoxic towards human primary blood leukocytes or retinal pigment epithelial cells at effective concentrations; protects human aortic endothelial cells (HAEC) from cold hypoxia/reoxygenation injury more effectively than necrostatin-1 (Nec-1) and Nec-1s. Altogether, these data demonstrate that 6E11 is a novel potent small molecular inhibitor of RIPK1-driven necroptosis.

Phenotype-driven molecular autopsy for sudden cardiac death.

A phenotype-driven approach to molecular autopsy based in a multidisciplinary team comprising clinical and laboratory genetics, forensic medicine and cardiology is described. Over a 13 year period, molecular autopsy was undertaken in 96 sudden cardiac death cases. A total of 46 cases aged 1-40 years had normal hearts and suspected arrhythmic death. Seven (15%) had likely pathogenic variants in ion channelopathy genes [KCNQ1 (1), KCNH2 (4), SCN5A (1), RyR2(1)]. Fifty cases aged between 2 and 67 had a cardiomyopathy. Twenty-five had arrhythmogenic right ventricular cardiomyopathy (ARVC), 10 dilated cardiomyopathy (DCM) and 15 hypertrophic cardiomyopathy (HCM). Likely pathogenic variants were found in three ARVC cases (12%) in PKP2, DSC2 or DSP, two DCM cases (20%) in MYH7, and four HCM cases (27%) in MYBPC3 (3) or MYH7 (1). Uptake of cascade screening in relatives was higher when a molecular diagnosis was made at autopsy. In three families, variants previously published as pathogenic were detected, but clinical investigation revealed no abnormalities in carrier relatives. With a conservative approach to defining pathogenicity of sequence variants incorporating family phenotype information and population genomic data, a molecular diagnosis was made in 15% of sudden arrhythmic deaths and 18% of cardiomyopathy deaths.

GM1 monosialoganglioside pretreatment protects against soman-induced seizure-related brain damage.

The effects of GM1 monosialoganglioside pretreatment on brain damage resulting from soman-induced seizure activity were examined in this study. Male Sprague-Dawley rats were infused with GM1 via an osmotic minipump connected through a permanent cannula implanted intracerebroventricularly and challenged with soman (83 micrograms/kg, i.e., 1.25 x LD50) 4 d after initiation of GM1 infusion. Electrocorticographic recordings were monitored via indwelling cortical electrodes. Twenty-seven hours after soman administration, anesthetized rats were euthanized via transcardial perfusion with buffered paraformaldehyde. Brains were processed for hematoxylin and eosin (H&E), cresyl violet (CV), and acetylcholinesterase (AChE) histochemistry, and glial fibrillary acidic protein (GFAP) and microtubule-associated protein 2 (MAP2) immunohistochemistry. All soman-challenged rats not infused with GM1 (n = 14) developed status epilepticus (SE).

Making team working work--a real life experience.

Southern Derbyshire Speech and Language Therapy Department has a team of six specialist speech and language therapists who support children with Specific Language Impairment in their mainstream schools via joint commissioning with education. As the team expanded in October 1995 this coincided with health trust and education changes and produced great instability and insecurity within the team. This paper outlines the implementation of a formal team working package using a facilitator provided by the trust's quality team and shows how it is possible to problem solve and develop within a framework of group rules, such as honesty and openness. Following its success its principles have allowed us to co-operate more effectively with both school staff and the community team and to support colleagues through a re-organisation of their service.

Microtubule-associated protein 2 (MAP-2): a sensitive marker of seizure-related brain damage.

We have assessed the efficacy of MAP-2 immunohistochemistry as a marker of seizure-related brain damage and its suitability for quantitation of the damage using densitometric and morphometric image analysis. Seizures were produced in rats by administration of 1.5 LD50 soman, an irreversible AChE inhibitor. Our results demonstrate that neuronal damage, assessed using hematoxylin and eosin, and cresyl violet staining, was colocalized on adjacent serial sections with clearly demarcated reductions in MAP-2 staining. The most severely damaged brain regions were devoid of MAP-2 staining. Reductions in MAP-2 immunostaining were found to be exceptionally well suited for quantitation using densitometric and morphometric image analysis. This study represents the first demonstration of seizure-induced excitotoxic alterations in MAP-2.

Anticonvulsant effects of memantine and MK-801 in guinea pig hippocampal slices.

The anticonvulsant properties of memantine (Mem) were compared to those of MK-801. Extracellular field recordings were obtained from area CA1 of guinea pig hippocampal slices in a total submersion chamber at 32 degrees C in normal oxygenated artificial cerebrospinal fluid (ACSF). Evoked responses were elicited by 0.07 Hz stimulation of the Schaffer collateral and commissural fibers. Bath perfusion of slices with Mg(2+)-free ACSF and N-methyl-D-aspartate (NMDA)-containing ACSF induced epileptiform afterdischarges following evoked responses. Pretreatment of slices by bath application of 100 microM Mem for 18-20 min prevented epileptiform afterdischarges under both convulsant conditions. Perfusion with 100 microM Mem alone for up to 50 min had no discernible effect on evoked responses. MK-801 was as effective at < or = 10 microM and required application for over 15 min to suppress afterdischarges completely. Both Mem and MK-801 suppressed epileptiform activity when applied after such activity was induced by NMDA or MG(2+)-free ACSF. The EC50 of Mem was 16.6 microM and that of MK-801 was 0.19 microM for blocking NMDA-induced evoked response suppression. Thus, in the guinea pig hippocampal slice preparation, Mem appeared to have anticonvulsant properties qualitatively similar to those of MK-801, but was 10-100 fold less potent.