Randomized controlled trial of N-acetylcysteine therapy for RYR1-related myopathies.

OBJECTIVE: To investigate the efficacy of N-acetylcysteine (NAC) for decreasing elevated oxidative stress and increasing physical endurance in individuals with ryanodine receptor 1-related myopathies (RYR1-RM). METHODS: In this 6-month natural history assessment (n = 37) followed by a randomized, double-blinded, placebo-controlled trial, 33 eligible participants were block-randomized (1:1) to receive NAC (n = 16) or placebo (n = 17), orally for 6 months (adult dose 2,700 mg/d; pediatric dose 30 mg/kg/d). The primary endpoint was urine 15-F2t isoprostane concentration and the clinically meaningful co-primary endpoint was 6-minute walk test (6MWT) distance. RESULTS: When compared to the general population, participants had elevated baseline 15-F2t isoprostane concentrations and most had a decreased 6MWT distance (mean ± SD 3.2 ± 1.5 vs 1.1 ± 1.7 ng/mg creatinine and 468 ± 134 vs 600 ± 58 m, respectively, both p < 0.001). 15-F2t isoprostane concentration and 6MWT distance did not change over the 6-month natural history assessment (p = 0.98 and p = 0.61, respectively). NAC treatment did not improve 15-F2t isoprostane concentration (least squares means difference 0.1 [95% confidence interval [CI] -1.4 to 1.6] ng/mg creatinine, p = 0.88) or 6MWT distance (least squares means difference 24 [95% CI -5.5 to 53.4] m, p = 0.11). NAC was safe and well-tolerated at the doses administered in this study. CONCLUSION: In ambulatory RYR1-RM-affected individuals, we observed stable disease course, and corroborated preclinical reports of elevated oxidative stress and decreased physical endurance. NAC treatment did not decrease elevated oxidative stress, as measured by 15-F2t isoprostane. CLASSIFICATION OF EVIDENCE: This study provides Class I evidence that, for people with RYR1-RM, treatment with oral NAC does not decrease oxidative stress as measured by 15-F2t isoprostane. CLINICALTRIALSGOV IDENTIFIER: NCT02362425.

Reliability and Validity of Self-Report Questionnaires as Indicators of Fatigue in RYR1-Related Disorders.

BACKGROUND: RYR1-related disorders (RYR1-RD), are a spectrum of genetic neuromuscular disorders. Affected individuals frequently experience fatigue yet appropriate tools to assess RYR1-RD-associated fatigue remain underdeveloped. OBJECTIVE: This study assessed the reliability and validity of two self-report questionnaires, the multidimensional fatigue inventory (MFI-20) and adult/pediatric functional assessment of chronic illness-fatigue (FACIT-F/Peds-FACIT-F) as potential fatigue measures in RYR1-RD affected individuals. METHODS: Participants (n = 37) were enrolled in an RYR1-RD combined natural history study and clinical trial. At baseline, participants completed fatigue questionnaires, six-minute walk test (6MWT), cardiopulmonary exercise test (CPET) and saliva collection for fatigue biomarker index (FBI) quantification. RESULTS: All questionnaires exhibited good test-retest reliability (n = 18, ICC > 0.80). MFI-20 (n = 37), and FACIT-F (n = 28) also showed good internal consistency (Cronbach's α> 0.80). All MFI-20 subscales, except mental fatigue, and FACIT-F demonstrated evidence of criterion validity when correlated against percent predicted 6MWT distance (MFI-20 n = 37; r = -0.34 to -0.47, all p < 0.05, mental fatigue, r = -0.16, p = 0.35; FACIT-F n = 28, r = 0.41, p = 0.03). This was not the case for percent predicted VO2 peak (all p > 0.05). FBI correlated with MFI-20 general fatigue dimension only (r = -0.35, p = 0.03). Comparison of standardized questionnaire scores revealed that RYR1-RD affected individuals experience significantly greater fatigue than the general population. CONCLUSIONS: MFI-20 and FACIT-F are valid and reliable tools for assessing RYR1-RD-associated fatigue, a symptom centrally implicated in this rare disorder.

Emergency Department Femoral Nerve Blocks and 1-Year Mortality in Fragility Hip Fractures.

OBJECTIVE: Femoral nerve blocks (FNBs) for fragility hip fractures have benefits in improving pain relief and early mobilization while decreasing opioid use and rates of pneumonia. However, no study has looked at 1-year mortality outcomes for this intervention. This study aims to provide insight into 1-year outcomes. METHODS: A single-site retrospective case-control study from 2007 to 2016 in primary fragility hip fractures compared 665 patients who received an emergency department FNB to 326 patients who did not receive an FNB. The primary outcome was 1-year mortality. Secondary outcomes included mortality, mobility, and residence at discharge, 6 months, and 1-year intervals. RESULTS: There were no significant differences in preoperative characteristics. Although there was no statistically significant difference in 1-year mortality, patients who did not receive an FNB were more likely to be nonambulant at 1 year (odds ratio 1.71, 95% confidence interval, 1.14-2.57, P = .005). There were no other significant differences in mobility, residence, or mortality. CONCLUSION: There was no statistically significant difference in 1-year mortality, although individuals who did not receive an FNB were more likely to be nonambulant at 1 year.

Salivary biomarkers of physical fatigue as markers of sleep deprivation.

STUDY OBJECTIVE: Determine whether a salivary biomarker of physical fatigue, referred to as the fatigue biomarker index (FBI), can discriminate a control group from a sleep deprived group when saliva is collected under controlled conditions. The study expands on previous work examining changes in the composition of saliva during periods of prolonged exercise. METHODS: Thirty (30) young adults (14 Control [CON]; 16 Sleep Deprived [SDEP]) were monitored for mood state (Profile of Mood States [POMS]), cognitive performance (Stroop Color-Conflict Tests), and salivary biomarkers of physical fatigue over a 48-h period with sampling at 3-h intervals. Trials lasted from 06:00 on day 1 (time = -3 h) to 09:00 on day 3 (time = 48 h). Levels of salivary biomarkers were calculated from liquid chromatography-mass spectrometry (LC-MS) data. Statistical comparisons were made using Wilcoxon rank sum tests with a Bonferroni correction to limit type 1 error. Receiver-operator characteristic (ROC) analysis was used to evaluate the ability of the various parameters to distinguish the SDEP population from the CON population. RESULTS: Longitudinal analysis demonstrated significant between-group differences in all three parameters. ROC analysis demonstrated that cognitive performance tests and salivary biomarkers of physical fatigue distinguish the SDEP population from the CON population. CONCLUSIONS: A previously identified salivary biomarker of physical fatigue may provide an alternative method for discriminating sleep deprived from rested individuals. The salivary biomarker of physical fatigue holds promise as an objective measure of sleep deprivation, perhaps eventually removing the reliance on self-reported sleep diaries and/or repeated polysomnographs for longitudinal tracking of sleep quality and/or diagnosis of sleep disorders.

Fatigue biomarker index: an objective salivary measure of fatigue level.

Fatigue changed the composition of the small-molecular weight (sMW) proteome of saliva during a 10h session of moderate (70% of maximum ventilatory threshold) physical exertion. Saliva samples were collected from nine recreationally trained cyclists participating in a cross-over study designed to simulate prolonged manual labor, a military operation or wildfire-suppression work. During each hour of the study, participants performed an exercise program that included upper and lower body exercises separated by short periods of recovery. Over the course of the study, fatigue level increased as suggested by a significant increase in the participants' relative perceived exertion. The composition of the sMW proteome was investigated using reversed-phase liquid chromatography with mass-spectrometric detection. Isotopes of acetic anhydride were used for mass-specific labeling of samples and subsequent identification of ions with significant changes in intensity. Cluster analysis was used to identify a pair of peptides with concentrations that changed in opposite directions with fatigue level, i.e. concentration of one peptide increased while concentration of the other decreased. The sequences of the two peptides were determined by high-resolution mass spectrometry. The ratio of the ion intensities of these two peptides, referred to as the fatigue biomarker index, was calculated for subjects throughout the study. The FBI values from the start of the study likely arose from a different distribution than the FBI values measured at the end of the study (Mann-Whitney test, P<.05). While this study is restricted to a small population of recreationally trained cyclists performing exercise under controlled conditions, it holds promise for the development of an objective salivary measurement of fatigue that is applicable to a much broader population performing in uncontrolled environments.

Linear mixed-effects modeling of the relationship between heart rate variability and fatigue arising from sleep deprivation.

INTRODUCTION: Fatigue degrades cognitive performance, yet there is no universally accepted objective measure of fatigue. We tested whether fatigue arising from sleep deprivation can be quantified objectively using heart rate variability (HRV). METHODS: There were 35 male subjects (mean +/- SD; age = 21.4 +/- 2.6 yr) who were assigned to one of two experimental groups: (1) control (N = 16), or (2) 48-h sleep-deprived (N=19). Using 3-h sampling intervals, we simultaneously tracked fatigue level, cognitive performance, and HRV. Linear mixed-effects (LME) models were used to evaluate linear relationships between fatigue level and cognitive performance, as well as between fatigue level and HRV. RESULTS: Significant negative slopes were observed in LME models of cognitive performance and fatigue level. Of the several HRV parameters examined during standing and supine rest, the ratio of low-frequency to high-frequency R-R interval in the supine position had the clearest significant relationship when modeled against fatigue level. DISCUSSION: In summary, our results suggest that HRV tracks fatigue arising from sleep deprivation. This noninvasive, objective tool can quantify fatigue in real time.

Predicting success in the tactical air combat party training pipeline.

OBJECTIVES: To develop a statistical model that predicts the likelihood of success or failure of military training candidates using tests administered before initial skill training as inputs. METHODS: Data were acquired from candidates before the start of U.S. Air Force Tactical Air Control Party training, including (1) demographic, (2) psychological composition evaluated using Emotional Quotient Inventory, (3) physical performance capability, (4) a physical activity questionnaire, and (5) salivary fatigue biomarker index. A total of 126 candidates were tracked until they either passed or failed the training, and a total of 55 variables were used as inputs for creation of the model. RESULTS: Clustering analysis of the data revealed that only 4 of 55 variables were useful for predicting success or failure. The variables in the order of their importance are as follows: run time, number of miles run per week in the last year, level of salivary fatigue biomarker, and height. CONCLUSIONS: The results suggest that simple testing methods can identify candidates at high risk of failure.

A comparison of cognitive performance decreases during acute, progressive fatigue arising from different concurrent stressors.

Fatigue is known to impair cognitive performance, but it remains unclear whether concurrent common stressors affect cognitive performance similarly. We used the Stroop Color-Word Conflict Test to assess cognitive performance over 24 hours for four groups: control, sleep-deprived (SD), SD + energy deficit, and SD + energy deficit + fluid restricted. Fatigue levels were quantified using the Profile of Mood States (POMS) survey. Linear mixed-effects (LME) models allowed for testing of group-specific differences in cognitive performance while accounting for subject-level variation. Starting fatigue levels were similar among all groups, while 24-hour fatigue levels differed significantly. For each cognitive performance test, results were modeled separately. The simplest LME model contained a significant fixed-effects term for slope and intercept. Moreover, the simplest LME model used a single slope coefficient to fit data from all four groups, suggesting that loss in cognitive performance over a 24-hour duty cycle with respect to fatigue level is similar regardless of the cause.

Ezrin mediates tethering of the gamma-aminobutyric acid transporter GAT1 to actin filaments via a C-terminal PDZ-interacting domain.

A high density of neurotransmitter transporters on axons and presynaptic boutons is required for the efficient clearance of neurotransmitters from the synapse. Therefore, regulators of transporter trafficking (insertion, retrieval, and confinement) can play an important role in maintaining the transporter density necessary for effective function. We determined the interactions that confine GAT1 at the membrane by investigating the lateral mobility of GAT1-yellow fluorescent protein-8 (YFP8) expressed in neuroblastoma 2a cells. Through fluorescence recovery after photobleaching, we found that a significant fraction ( approximately 50%) of membrane-localized GAT1 is immobile on the time scale investigated ( approximately 150 s). The mobility of the transporter can be increased by depolymerizing actin or by interrupting the GAT1 postsynaptic density 95/Discs large/zona occludens 1 (PDZ)-interacting domain. Microtubule depolymerization, in contrast, does not affect GAT1 membrane mobility. We also identified ezrin as a major GAT1 adaptor to actin. Förster resonance energy transfer suggests that GAT1-YFP8 and cyan fluorescent (CFP) tagged ezrin (ezrin-CFP) exist within a complex that has a Förster resonance energy transfer efficiency of 19% +/- 2%. This interaction can be diminished by disrupting the actin cytoskeleton. In addition, the disruption of actin results in a >3-fold increase in gamma-aminobutyric acid uptake, apparently via a mechanism distinct from the PDZ-interacting protein. Our data reveal that actin confines GAT1 to the plasma membrane via ezrin, and this interaction is mediated through the PDZ-interacting domain of GAT1.

Fluorescent cargo proteins in peptidergic endocrine cells: cell type determines secretion kinetics at exocytosis.

Fluorescent fusion proteins are an important tool for the study of vesicle trafficking and exocytosis, especially when combined with newer types of microscopy. We previously reported that the design of a vesicle-targeted fluorescent fusion construct strongly influences the kinetics of fluorescence change at exocytosis. In the present study we demonstrate that the cell in which a construct is expressed also affects the kinetics of fluorescence change at exocytosis. We fused enhanced green fluorescent protein to the carboxy terminus of the vesicular cargo protein rodent islet amyloid polypeptide. The two proteins were separated by a "linker" sequence of 18 amino acids. We then compared kinetics of fluorescence change at exocytosis for this fluorescent cargo protein expressed in three different types of peptidergic endocrine cell: pancreatic alpha cell, pancreatic beta cell, and adrenal chromaffin cell. In resting cells of all three types, fluorescent spots of similar size and membrane-proximal density appeared near the plasma membrane as expected if the probe is stored in large dense-core secretory vesicles. Upon stimulation, the fluorescent spots displayed sudden changes in fluorescence intensity that were consistent with exocytosis. In beta and alpha cells the fluorescent spots consistently brightened and persisted, whereas in chromaffin cells the fluorescent spots always dispersed rapidly. Thus, for fluorescent cargo proteins in peptidergic endocrine cells, cell type influences the kinetics of fluorescence change at exocytosis. Together with our previous findings, this observation strongly highlights the fact that the behavior of vesicle-targeted fluorescent cargo may be unrelated to that of native cargo, and it emphasizes the need for caution in interpreting fluorescence kinetics in terms of an exocytosis mechanism.

Human insulin vesicle dynamics during pulsatile secretion.

In healthy individuals, plasma insulin levels oscillate in both fasting and fed states. Numerous studies of isolated pancreata and pancreatic islets support the hypothesis that insulin oscillations arise because the underlying rate of insulin secretion also oscillates; yet, insulin secretion has never been observed to oscillate in individual pancreatic beta-cells. Using expressed fluorescent vesicle cargo proteins and total internal reflection fluorescence (TIRF) microscopy, we demonstrate that glucose stimulates human pancreatic beta-cells to secrete insulin vesicles in short, coordinated bursts of approximately 70 vesicles each. Randomization tests and spectral analysis confirmed that the temporal patterns of secretion were not random, instead exhibiting alternating periods of secretion and rest, recurring with statistically significant periods of 15-45 s. Although fluorescent vesicles arrived at the plasma membrane before, during, and after stimulation, their rate of arrival was significantly slower than their rate of secretion, so that their density near the plasma membrane dropped significantly during the cell's response. To study in greater detail the vesicle dynamics during cyclical bursts of secretion, we applied trains of depolarizations once a minute and performed simultaneous membrane capacitance measurements and TIRF imaging. Surprisingly, young fluorescent insulin vesicles contributed at least half of the vesicles secreted in response to a first train, even though young vesicles were vastly outnumbered by older, nonfluorescent vesicles. For subsequent trains, young insulin vesicles contributed progressively less to total secretion, whereas capacitance measurements revealed that total stimulated secretion did not decrease. These results suggest that in human pancreatic beta-cells, young vesicles are secreted first, and only then are older vesicles recruited for secretion.

Mechanisms of peptide hormone secretion.

According to the classical view, peptide hormones are stored in large dense-core vesicles that release all of their cargo rapidly and completely when they fuse with and flatten into the plasma membrane. However, recent imaging studies suggest that this view is too simple. Even after vesicles fuse with the plasma membrane, cells might control the rate of dispersal of vesicle cargo - either by modulating the properties of the fusion pore that connects the vesicle lumen to the extracellular solution or by storing cargo in states that disperse slowly in the extracellular space. Understanding these mechanisms is important, owing to the increasing prevalence of diseases, such as type 2 diabetes mellitus, which arise from insufficient secretion of peptide hormones.

Principles of two-photon excitation fluorescence microscopy and other nonlinear imaging approaches.

The aim of this article is to review the basic principles of two-photon excitation fluorescence (2PEF) microscopy and to compare the advantages and disadvantages of 2PEF imaging to other microscopy methodologies. 2PEF imaging is a nonlinear approach that generates images of optical sections and that is particularly well suited for deep-tissue and in vivo imaging of live animals. The nonlinear excitation used for 2PEF offers the advantage, too, of being able to generate contrast from second or third harmonic generation as well as coherent anti-Stokes Raman scattering. We also review the recent use of nonlinear excitation to provide image resolution beyond the diffraction limit and discuss the progress in non-scanning (planar) 2PEF microscopy, an approach that holds great potential for large-scale quantitative imaging and plate reading, e.g., in screening applications.

Pancreatic beta-cells secrete insulin in fast- and slow-release forms.

Insulin vesicles contain a chemically rich mixture of cargo that includes ions, small molecules, and proteins. At present, it is unclear if all components of this cargo escape from the vesicle at the same rate or to the same extent during exocytosis. Here, we demonstrate through real-time imaging that individual rat and human pancreatic beta-cells secrete insulin in heterogeneous forms that disperse either rapidly or slowly. In healthy pancreatic beta-cells maintained in culture, most vesicles discharge insulin in its fast-release form, a form that leaves individual vesicles in a few hundred milliseconds. The fast-release form of insulin leaves vesicles as rapidly as C-peptide leaves vesicles. Healthy beta-cells also secrete a slow-release form of insulin that leaves vesicles more slowly than C-peptide, over times ranging from seconds to minutes. Individual beta-cells make vesicles with both forms of insulin, though not all vesicles contain both forms of insulin. In addition, we confirm that insulin vesicles store their cargo in two functionally distinct compartments: an acidic solution, or halo, and a condensed core. Thus, our results suggest two important features of the condensed core: 1) It exists in different states among the vesicles undergoing exocytosis and 2) its dissolution determines the availability of insulin during exocytosis.

Fluorescent cargo proteins in pancreatic beta-cells: design determines secretion kinetics at exocytosis.

We compared secretion kinetics for four different fluorescent cargo proteins, each targeted to the lumen of insulin secretory vesicles. Upon stimulation, individual vesicles displayed one of four distinct patterns of fluorescence change: i), disappearance, ii), dimming, iii), transient brightening, or iv), persistent brightening. For each fusion protein, a different pattern of fluorescence change dominated. Furthermore, we demonstrated that the dominant pattern depends upon both i), the specific choice of fluorescent protein, and ii), the sequence of amino acids linking the cargo protein to the fluorescent protein. Thus, in beta-cells, experiments involving fluorescent cargo proteins for the study of exocytosis must be interpreted carefully, as design of a fluorescent cargo protein determines secretion kinetics at exocytosis.

Correlation of local changes in extracellular oxygen and pH that accompany dopaminergic terminal activity in the rat caudate-putamen.

Terminal activity causes an increase in local cerebral blood flow that can be quantified by measuring the accompanying increase in tissue oxygen. Alkaline pH changes can also follow neuronal activation. The purpose of these studies was to determine whether these changes in extracellular oxygen and pH correlate. Fast-scan cyclic voltammetry was used to detect changes in dopamine, pH and oxygen levels simultaneously in the caudate-putamen after electrical stimulation of the substantia nigra in anesthetized rats. The biphasic increases in oxygen and pH followed similar time courses, and were delayed a few seconds from the immediate release and uptake of dopamine. The changes following administration of neurotransmitter receptor antagonists as well as agents that modulate blood flow were identical for oxygen and pH. Two distinct mechanisms were identified that give rise to the oxygen and pH changes: blood vessel dilatation caused by nitric oxide synthesis after muscarinic receptor activation and adenosine receptor activation. We conclude that changes in blood flow accompanying terminal activity cause alkaline pH shifts by the rapid removal of carbon dioxide, a component of the extracellular brain buffering system.