New liver cell mutants defective in the endocytic pathway

AbstractTo isolate mutant liver cells defective in the endocytic pathway, a selection strategy using toxic ligands for two distinct membrane receptors was utilized. Rare survivors termed trafficking mutants (Trf2–Trf7) were stable and more resistant than the parental HuH-7 cells to both toxin conjugates. They differed from the previously isolated Trf1 HuH-7 mutant as they expressed casein kinase 2 α″ (CK2α″) which is missing from Trf1 cells and which corrects the Trf1 trafficking phenotype. Binding of 125I-asialoorosomucoid (ASOR) and cell surface expression of asialoglycoprotein receptor (ASGPR) were reduced approximately 20%–60% in Trf2–Trf7 cells compared to parental HuH-7, without a reduction in total cellular ASGPR. Based on 125I-transferrin binding, cell surface transferrin receptor activity was reduced between 13% and 88% in the various mutant cell lines. Distinctive phenotypic traits were identified in the differential resistance of Trf2–Trf7 to a panel of lectins and toxins and to UV light-induced cell death. By following the endocytic uptake and trafficking of Alexa488-ASOR, significant differences in endosomal fusion between parental HuH-7 and the Trf mutants became apparent. Unlike parental HuH-7 cells in which the fusion of endosomes into larger vesicles was evident as early as 20 min, ASOR endocytosed into the Trf mutants remained within small vesicles for up to 60 min. Identifying the biochemical and genetic mechanisms underlying these phenotypes should uncover novel and unpredicted protein–protein or protein–lipid interactions that orchestrate specific steps in membrane protein trafficking

An Autonomous Wearable System for Predicting and Detecting Localised Muscle Fatigue

Muscle fatigue is an established area of research and various types of muscle fatigue have been clinically investigated in order to fully understand the condition. This paper demonstrates a non-invasive technique used to automate the fatigue detection and prediction process. The system utilises the clinical aspects such as kinematics and surface electromyography (sEMG) of an athlete during isometric contractions. Various signal analysis methods are used illustrating their applicability in real-time settings. This demonstrated system can be used in sports scenarios to promote muscle growth/performance or prevent injury. To date, research on localised muscle fatigue focuses on the clinical side and lacks the implementation for detecting/predicting localised muscle fatigue using an autonomous system. Results show that automating the process of localised muscle fatigue detection/prediction is promising. The autonomous fatigue system was tested on five individuals showing 90.37% accuracy on average of correct classification and an error of 4.35% in predicting the time to when fatigue will onset

Frontal plane hip and ankle sensorimotor function, not age, predicts unipedal stance time

Introduction: Changes occur in muscles and nerves with aging. In this study we explore the relationship between unipedal stance time (UST) and frontal plane hip and ankle sensorimotor function in subjects with diabetic neuropathy. Methods: UST, quantitative measures of frontal plane ankle proprioceptive thresholds, and ankle and hip motor function were tested in 41 subjects with a spectrum of lower limb sensorimotor function ranging from healthy to moderately severe diabetic neuropathy. Results: Frontal plane hip and ankle sensorimotor function demonstrated significant relationships with UST. Multivariate analysis identified only composite hip strength, ankle proprioceptive threshold, and age to be significant predictors of UST ( R 2 = 0.73), explaining 46%, 24%, and 3% of the variance, respectively. Conclusions: Frontal plane hip strength was the single best predictor of UST and appeared to compensate for less precise ankle proprioceptive thresholds. This finding is clinically relevant given the possibility of strengthening the hip, even in patients with significant peripheral neuropathy. Muscle Nerve, 2012Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/90607/1/22325_ftp.pd

Neural Adaptations Associated with Interlimb Transfer in a Ballistic Wrist Flexion Task

Cross education is the process whereby training of one limb gives rise to increases in the subsequent performance of its opposite counterpart. The execution of many unilateral tasks is associated with increased excitability of corticospinal projections from primary motor cortex (M1) to the opposite limb. It has been proposed that these effects are causally related. Our aim was to establish whether changes in corticospinal excitability (CSE) arising from prior training of the opposite limb determine levels of interlimb transfer. We used three vision conditions shown previously to modulate the excitability of corticospinal projections to the inactive (right) limb during wrist flexion movements performed by the training (left) limb. These were: (1) mirrored visual feedback of the training limb; (2) no visual feedback of either limb; and (3) visual feedback of the inactive limb. Training comprised 300 discrete, ballistic wrist flexion movements executed as rapidly as possible. Performance of the right limb on the same task was assessed prior to, at the mid point of, and following left limb training. There was no evidence that variations in the excitability of corticospinal projections (assessed by transcranial magnetic stimulation (TMS)) to the inactive limb were associated with, or predictive of, the extent of interlimb transfer that was expressed. There were however associations between alterations in muscle activation dynamics observed for the untrained limb, and the degree of positive transfer that arose from training of the opposite limb. The results suggest that the acute adaptations that mediate the bilateral performance gains realized through unilateral practice of this ballistic wrist flexion task are mediated by neural elements other than those within M1 that are recruited at rest by single-pulse TMS

Human Liver Cell Trafficking Mutants: Characterization and Whole Exome Sequencing

<div><p>The HuH7 liver cell mutant <i>Trf1</i> is defective in membrane trafficking and is complemented by the casein kinase 2α subunit CK2α’’. Here we identify characteristic morphologies, trafficking and mutational changes in six additional HuH7 mutants <i>Trf2-Trf7. Trf1</i> cells were previously shown to be severely defective in gap junction functions. Using a Lucifer yellow transfer assay, remarkable attenuation of gap junction communication was revealed in each of the mutants <i>Trf2-Trf7</i>. Electron microscopy and light microscopy of thiamine pyrophosphatase showed that several mutants exhibited fragmented Golgi apparatus cisternae compared to parental HuH7 cells. Intracellular trafficking was investigated using assays of transferrin endocytosis and recycling and VSV G secretion. Surface binding of transferrin was reduced in all six <i>Trf2-Trf7</i> mutants, which generally correlated with the degree of reduced expression of the transferrin receptor at the cell surface. The mutants displayed the same transferrin influx rates as HuH7, and for efflux rate, only <i>Trf6</i> differed, having a slower transferrin efflux rate than HuH7. The kinetics of VSV G transport along the exocytic pathway were altered in <i>Trf2</i> and <i>Trf5</i> mutants. Genetic changes unique to particular <i>Trf</i> mutants were identified by exome sequencing, and one was investigated in depth. The novel mutation Ile34Phe in the GTPase RAB22A was identified in <i>Trf4</i>. RNA interference knockdown of RAB22A or overexpression of RAB22AI34F in HuH7 cells caused phenotypic changes characteristic of the <i>Trf4</i> mutant. In addition, the Ile34Phe mutation reduced both guanine nucleotide binding and hydrolysis activities of RAB22A. Thus, the RAB22A Ile34Phe mutation appears to contribute to the <i>Trf4</i> mutant phenotype.</p></div