Pathomechanisms of ALS8: altered autophagy and defective RNA binding protein (RBP) homeostasis due to the VAPB P56S mutation

Mutations in RNA binding proteins (RBPs) and in genes regulating autophagy are frequent causes of familial amyotrophic lateral sclerosis (fALS). The P56S mutation in vesicle-associated membrane protein-associated protein B (VAPB) leads to fALS (ALS8) and spinal muscular atrophy (SMA). While VAPB is primarily involved in the unfolded protein response (UPR), vesicular trafficking and in initial steps of the autophagy pathway, the effect of mutant P56S-VAPB on autophagy regulation in connection with RBP homeostasis has not been explored yet. Examining the muscle biopsy of our index ALS8 patient of European origin revealed globular accumulations of VAPB aggregates co-localised with autophagy markers LC3 and p62 in partially atrophic and atrophic muscle fibres. In line with this skin fibroblasts obtained from the same patient showed accumulation of P56S-VAPB aggregates together with LC3 and p62. Detailed investigations of autophagic flux in cell culture models revealed that P56S-VAPB alters both initial and late steps of the autophagy pathway. Accordingly, electron microscopy complemented with live cell imaging highlighted the impaired fusion of accumulated autophagosomes with lysosomes in cells expressing P56S-VAPB. Consistent with these observations, neuropathological studies of brain and spinal cord of P56S-VAPB transgenic mice revealed signs of neurodegeneration associated with altered protein quality control and defective autophagy. Autophagy and RBP homeostasis are interdependent, as demonstrated by the cytoplasmic mis-localisation of several RBPs including pTDP-43, FUS, Matrin 3 which often sequestered with P56S-VAPB aggregates both in cell culture and in the muscle biopsy of the ALS8 patient. Further confirming the notion that aggregation of the RBPs proceeds through the stress granule (SG) pathway, we found persistent G3BP- and TIAR1-positive SGs in P56S-VAPB expressing cells as well as in the ALS8 patient muscle biopsy. We conclude that P56S-VAPB-ALS8 involves a cohesive pathomechanism of aberrant RBP homeostasis together with dysfunctional autophagy.Subject terms: Mechanisms of disease, Amyotrophic lateral sclerosis     

Assessing agreement with multiple raters on correlated kappa statistics

In clinical studies, it is often of interest to see the diagnostic agreement among clinicians on certain symptoms. Previous work has focused on the agreement between two clinicians under two different conditions or the agreement among multiple clinicians under one condition. Few have discussed the agreement study with a design where multiple clinicians examine the same group of patients under two different conditions. In this paper, we use the intraclass kappa statistic for assessing nominal scale agreement with such a design. We derive an explicit variance formula for the difference of correlated kappa statistics and conduct hypothesis testing for the equality of kappa statistics. Simulation studies show that the method performs well with realistic sample sizes and may be superior to a method that did not take into account the measurement dependence structure. The practical utility of the method is illustrated on data from an eosinophilic esophagitis (EoE) study.     

A critical review on antiviral peptides derived from viral glycoproteins and host receptors to decoy herpes simplex virus

The health of the human population has been continuously challenged by viral infections. Herpes simplex virus (HSV) is one of the common causes of illness and can lead to death in immunocompromised patients. Existing anti-HSV therapies are not completely successful in eliminating the infection due to anti-viral drug resistance, ineffectiveness against the latent virus and high toxicity over prolonged use. There is a need to update our knowledge of the current challenges faced in anti-HSV therapeutics and realize the necessity of developing alternative treatment approaches. Protein therapeutics are now being explored as a novel approach due to their high specificity and low toxicity. This review highlights the significance of HSV viral glycoproteins and host receptors in the pathogenesis of HSV infection. Proteins or peptides derived from HSV glycoproteins gC, gB, gD, gH and host cell receptors (HSPG, nectin and HVEM) that act as decoys to inhibit HSV attachment, entry, or fusion have been discussed. Few researchers have tried to improve the efficacy and stability of the identified peptides by modifying them using a peptidomimetic approach. With these efforts, we think developing an alternative treatment option for immunocompromised patients and drug-resistant organisms is not far off.     

Effects of various conditions on the alkaline degradation of d-fructose and d-glucose

Dilute solutions of d-fructose and d-glucose undergo alkaline degradation, and, at temperatures in the range of 30–70°, almost two moles of alkali are consumed per mole of the carbohydrate. The degradation is partly guided by the dielectric constant of the medium; such additives as acetone and urea have specific effects where the reactions are not essentially guided by the medium dielectric. Acetone and urea presumably form complexes with the carbohydrates; this is revealed for the former by the formation of a dark red solution having a spectral band at 320 nm, like that observed earlier in the presence of ethylenediamine.     

Subcellular location of enzymes involved in oxidation of n-alkane by Cladosporium resinae

More than 70% of n-hexadecane-grown cells of Cladosporium resinae ATCC 22711 were converted to spheroplasts when they were treated with chitinase and lytic enzyme from Trichoderma harziamum. The light mitochondrial fraction, containing microbodies, mitochondria and vacuoles, was isolated from spheroplasts. Vacuoles in cells were demonstrated by the inability of acridine orange to stain organelles previously treated with 2.5 μM Bafilomycin A₁, a vacuolar ATPase inhibitor. Microbodies, mitochondria and vacuoles were separated from the light mitochondrial fraction by self-generated density-gradient ultracentrifugation using iodixanol as gradient medium. NADH-dependent n-alkane monooxygenase activity and fatty alcohol oxidase activity were located in the cytoplasm and mitochondrial fractions respectively. Received: 21 September 1998 / Received revision: 21 January 1999 / Accepted: 31 January 1999