CSF extracellular vesicle proteomics demonstrates altered protein homeostasis in amyotrophic lateral sclerosis.

BACKGROUND: Extracellular vesicles (EVs) released by neurons and glia reach the cerebrospinal fluid (CSF). Studying the proteome of CSF-derived EVs offers a novel perspective on the key intracellular processes associated with the pathogenesis of the neurodegenerative disease amyotrophic lateral sclerosis (ALS) and a potential source from which to develop biomarkers. METHODS: CSF EVs were extracted using ultrafiltration liquid chromatography from ALS patients and controls. EV size distribution and concentration was measured using nanoparticle tracking analysis and liquid chromatography-tandem mass spectrometry proteomic analysis performed. RESULTS: CSF EV concentration and size distribution did not differ between ALS and control groups, nor between a sub-group of ALS patients with or without an associated hexanucleotide repeat expansion (HRE) in C9orf72. Univariate proteomic analysis identified downregulation of the pentameric proteasome-like protein Bleomycin hydrolase in ALS patients, whilst Gene Ontology enrichment analysis demonstrated downregulation of proteasome core complex proteins (8/8 proteins, normalized enrichment ratio -1.77, FDR-adjusted p = 0.057) in the ALS group. The sub-group of ALS patients associated with the C9orf72 HRE showed upregulation in Ubiquitin-like modifying-activating protein 1 (UBA1) compared to non-C9orf72 cases. CONCLUSIONS: Proteomic analysis of CSF EVs in ALS detects intracellular alterations in protein homeostatic mechanisms, previously only identified in pathological tissues. This supports the wider use of CSF EVs as a source of novel biomarkers reflecting key and potentially druggable pathological intracellular pathway alterations in ALS.

Creating a Defined Process to Improve the Timeliness of Serious Safety Event Determination and Root Cause Analysis.

Serious Safety Events (SSEs) are defined as events in which there is a deviation from clinically accepted performance standards, causation, and significant patient harm or death. Given the nature of SSEs, it is important that the processes for declaration of SSEs, the performance of a root cause analysis (RCA), and action plan formation occur quickly, such that the window for potential recurrence of similar events is as small as possible. This manuscript describes a process put in place to improve the timeliness of SSE determination and RCA conduction and evaluates the effect of the process change on these parameters. METHODS: A causal analysis was performed of the baseline process to determine factors contributing to long process times. A new process was created and implemented both for the SSE determination process and the RCA completion process. We calculated the mean time for the pre-implementation phase (April 2016-December 2017) and the post-implementation phase (March 2018-January 2019) for both SSE determination and RCA completion. We evaluated differences with a two-sided t test assuming unequal variances. RESULTS: Comparing pre- versus post- implementation phases, the mean time for SSE determination for events that met the SSE criteria decreased from 38.4 to 4.8 days (P < 0.0001), determination for events that did not meet the SSE criteria decreased from 38.4 to 3.8 days (P < 0.0001), and RCA completion time dropped from 118.0 to 26.2 days (P < 0.0001). CONCLUSIONS: A targeted intervention can significantly reduce SSE determination and RCA conduction times.

A glial cell line-derived neurotrophic factor delivery system enhances nerve regeneration across acellular nerve allografts.

Acellular nerve allografts (ANAs) are used clinically to bridge nerve gaps but these grafts, lacking Schwann cells and therapeutic levels of neurotrophic factors, do not support regeneration to the same extent as autografts. Here we investigated a local drug delivery system (DDS) for glial cell line-derived neurotrophic factor (GDNF) controlled release to implanted ANAs in rats using drug-loaded polymeric microspheres (MSs) embedded in a fibrin gel. In a rat hindlimb nerve gap model, a 10mm ANA was used to bridge a 5mm common peroneal (CP) nerve gap. Experimental groups received DDS treatment at both suture sites of the allografts releasing GDNF for either 2 weeks or 4 weeks. In negative control groups, rats received no DDS treatment or empty DDS. Rats receiving nerve isografts served as the positive control group. The numbers of motor and sensory neurons that regenerated their axons in all the groups with GDNF MS and isograft treatment were indistinguishable and significantly higher as compared to the negative control groups. Nerve histology distal to the nerve graft demonstrated increased axon counts and a shift to larger fiber diameters due to GDNF MS treatment. The sustained delivery of GDNF to the implanted ANA achieved in this study demonstrates the promise of this DDS for the management of severe nerve injuries in which allografts are placed. STATEMENT OF SIGNIFICANCE: This work addresses the common clinical situation in which a nerve gap is bridged using acellular nerve allografts. However, these allografts are not as effective in supporting nerve regeneration as the gold standard method of autografting. The novel local drug delivery system used in this study provides sustained and controlled release of glial cell line-derived neurotrophic factor (GDNF), one of the most potent neurotrophic factors, which significantly improves nerve regeneration following severe nerve injuries. Results from this research will provide a mean of improving nerve allografts with locally delivered GDNF. This strategy may lead to a novel "off the shelf" alternative to the current management of severe nerve injuries.

An engineered biocompatible drug delivery system enhances nerve regeneration after delayed repair.

Localized drug delivery strategies could greatly benefit patients with peripheral nerve injury and could be easy for surgeons to implement. We developed a local drug delivery system (DDS) using drug-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres (MS) embedded in a fibrin gel. In an in vitro study, we investigated the biocompatibility of this DDS by performing a toxicity assay in which we incubated PC-12 cells with the medium released from the DDS in vitro. In an in vivo study, this DDS was applied at the rat common peroneal (CP) nerve injury site to deliver exogenous glial cell line-derived neurotrophic factor (GDNF) to the regenerating axons after delayed nerve repair. In vitro, PC-12 cells incubated with released media samples from the DDS had similar viability to control cells cultured with normal media, demonstrating that the DDS was not toxic. In vivo, the numbers of motor and sensory neurons that regenerated their axons with empty MS treatment were the same as when there was no MS treatment. The DDS increased the numbers of regenerating motor- and sensory neurons to levels indistinguishable from those observed with immediate nerve repair. The DDS increased neuron regeneration to levels double those observed with negative control groups. This biocompatible, nontoxic, fibrin gel-based DDS enhances outcomes following severe peripheral nerve injuries.

Extracellular microRNAs in Membrane Vesicles and Non-vesicular Carriers.

Great excitement has surrounded the finding that small RNAs are stable in various biofluids and carry specific signatures reflecting physiological and pathological states. In this chapter, we briefly describe the impact of this revolutionary discovery and introduce different subclasses of circulating microRNAs based on their mode of transport. Subsequently, we review the current state-of-the art knowledge on microRNA selection for export, secretion and possible uptake mechanisms and their potential function in circulation. Furthermore, we give an overview on the possible use of cell-free microRNAs as biomarkers and as therapeutic targets. Overall, we aim to highlight open questions and address some of the pitfalls of current extracellular RNA research.

Dose effect of caffeine on testosterone and cortisol responses to resistance exercise.

INTRODUCTION: Interest in the use of caffeine as an ergogenic aid has increased since the International Olympic Committee lifted the partial ban on its use. Caffeine has beneficial effects on various aspects of athletic performance, but its effects on training have been neglected. PURPOSE: To investigate the acute effect of caffeine on the exercise-associated increases in testosterone and cortisol in a double-blind crossover study. METHODS: Twenty-four professional rugby-league players ingested caffeine doses of 0, 200, 400, and 800 mg in random order 1 hr before a resistance-exercise session. Saliva was sampled at the time of caffeine ingestion, at 15-min intervals throughout each session, and 15 and 30 min after the session. Data were log-transformed to estimate percent effects with mixed modeling, and effects were standardized to assess magnitudes. RESULTS: Testosterone concentration showed a small increase of 15% (90% confidence limits, +/- 19%) during exercise. Caffeine raised this concentration in a dose-dependent manner by a further small 21% (+/- 24%) at the highest dose. The 800-mg dose also produced a moderate 52% (+/- 44%) increase in cortisol. The effect of caffeine on the testosterone:cortisol ratio was a small decline (14%; +/- 21%). CONCLUSION: Caffeine has some potential to benefit training outcomes via the anabolic effects of the increase in testosterone concentration, but this benefit might be counteracted by the opposing catabolic effects of the increase in cortisol and resultant decline in the testosterone:cortisol ratio.