Cerebral cortex activation mapping upon electrical muscle stimulation by 32-channel time-domain functional near-infrared spectroscopy

The application of different EMS current thresholds on muscle activates not only the muscle but also peripheral sensory axons that send proprioceptive and pain signals to the cerebral cortex. A 32-channel time-domain fNIRS instrument was employed to map regional cortical activities under varied EMS current intensities applied on the right wrist extensor muscle. Eight healthy volunteers underwent four EMS at different current thresholds based on their individual maximal tolerated intensity (MTI), i.e., 10 % < 50 % < 100 % < over 100 % MTI. Time courses of the absolute oxygenated and deoxygenated hemoglobin concentrations primarily over the bilateral sensorimotor cortical (SMC) regions were extrapolated, and cortical activation maps were determined by general linear model using the NIRS-SPM software. The stimulation-induced wrist extension paradigm significantly increased activation of the contralateral SMC region according to the EMS intensities, while the ipsilateral SMC region showed no significant changes. This could be due in part to a nociceptive response to the higher EMS current intensities and result also from increased sensorimotor integration in these cortical regions.

Impact of extracellular matrix derived from osteoarthritis subchondral bone osteoblasts on osteocytes : role of integrinβ1 and focal adhesion kinase signaling cues

INTRODUCTION: Our recent study indicated that subchondral bone pathogenesis in osteoarthritis (OA) is associated with osteocyte morphology and phenotypic abnormalities. However, the mechanism underlying this abnormality needs to be identified. In this study we investigated the effect of extracellular matrix (ECM) produced from normal and OA bone on osteocytic cells function. METHODS: De-cellularized matrices, resembling the bone provisional ECM secreted from primary human subchondral bone osteoblasts (SBOs) of normal and OA patients were used as a model to study the effect on osteocytic cells. Osteocytic cells (MLOY4 osteocyte cell line) cultured on normal and OA derived ECMs were analyzed by confocal microscopy, scanning electron microscopy (SEM), cell attachment assays, zymography, apoptosis assays, qRT-PCR and western blotting. The role of integrinβ1 and focal adhesion kinase (FAK) signaling pathways during these interactions were monitored using appropriate blocking antibodies. RESULTS: The ECM produced by OA SBOs contained less mineral content, showed altered organization of matrix proteins and matrix structure compared with the matrices produced by normal SBOs. Culture of osteocytic cells on these defective OA ECM resulted in a decrease of integrinβ1 expression and the de-activation of FAK cell signaling pathway, which subsequently affected the initial osteocytic cell's attachment and functions including morphological abnormalities of cytoskeletal structures, focal adhesions, increased apoptosis, altered osteocyte specific gene expression and increased Matrix metalloproteinases (MMP-2) and -9 expression. CONCLUSION: This study provides new insights in understanding how altered OA bone matrix can lead to the abnormal osteocyte phenotypic changes, which is typical in OA pathogenesis.

mTOR function in skeletal muscle : a focal point for overnutrition and exercise

The mammalian target of rapamycin (mTOR) is a highly conserved atypical serine-threonine kinase that controls numerous functions essential for cell homeostasis and adaptation in mammalian cells via 2 distinct protein complex formations. Moreover, mTOR is a key regulatory protein in the insulin signalling cascade and has also been characterized as an insulin-independent nutrient sensor that may represent a critical mediator in obesity-related impairments of insulin action in skeletal muscle. Exercise characterizes a remedial modality that enhances mTOR activity and subsequently promotes beneficial metabolic adaptation in skeletal muscle. Thus, the metabolic effects of nutrients and exercise have the capacity to converge at the mTOR protein complexes and subsequently modify mTOR function. Accordingly, the aim of the present review is to highlight the role of mTOR in the regulation of insulin action in response to overnutrition and the capacity for exercise to enhance mTOR activity in skeletal muscle.

Using narrow beam profiles to quantify focal spot size, for accurate Monte Carlo simulations of SRS/SRT systems

This study investigates the variation of photon field penumbra shape with initial electron beam diameter, for very narrow beams. A Varian Millenium MLC (Varian Medical Systems, Palo Alto, USA) and a Brainlab m3 microMLC (Brainlab AB. Feldkirchen, Germany) were used, with one Varian iX linear accelerator, to produce fields that were (nominally) 0.20 cm across. Dose profiles for these fields were measured using radiochromic film and compared with the results of simulations completed using BEAMnrc and DOSXYZnrc, where the initial electron beam was set to FWHM = 0.02, 0.10, 0.12, 0.15, 0.20 and 0.50 cm. Increasing the electron-beam FWHM produced increasing occlusion of the photon source by the closely spaced collimator leaves and resulted in blurring of the simulated profile widths from 0.26 to 0.64 cm, for the MLC, from 0.12 to 0.43 cm, for the microMLC. Comparison with measurement data suggested that the electron spot size in the clinical linear accelerator was between FWHM = 0.10 and 0.15 cm, encompassing the result of our previous output-factor based work, which identified a FWHM of 0.12. Investigation of narrow-beam penumbra variation has been found to be a useful procedure, with results varying noticeably with linear accelerator spot size and allowing FWHM estimates obtained using other methods to be verified.

Staurosporine augments EGF-mediated EMT in PMC42-LA cells through actin depolymerisation, focal contact size reduction and Snail1 induction : a model for cross-modulation

Background A feature of epithelial to mesenchymal transition (EMT) relevant to tumour dissemination is the reorganization of actin cytoskeleton/focal contacts, influencing cellular ECM adherence and motility. This is coupled with the transcriptional repression of E-cadherin, often mediated by Snail1, Snail2 and Zeb1/δEF1. These genes, overexpressed in breast carcinomas, are known targets of growth factor-initiated pathways, however it is less clear how alterations in ECM attachment cross-modulate to regulate these pathways. EGF induces EMT in the breast cancer cell line PMC42-LA and the kinase inhibitor staurosporine (ST) induces EMT in embryonic neural epithelial cells, with F-actin de-bundling and disruption of cell-cell adhesion, via inhibition of aPKC. Methods PMC42-LA cells were treated for 72 h with 10 ng/ml EGF, 40 nM ST, or both, and assessed for expression of E-cadherin repressor genes (Snail1, Snail2, Zeb1/δEF1) and EMT-related genes by QRT-PCR, multiplex tandem PCR (MT-PCR) and immunofluorescence +/- cycloheximide. Actin and focal contacts (paxillin) were visualized by confocal microscopy. A public database of human breast cancers was assessed for expression of Snail1 and Snail2 in relation to outcome. Results When PMC42-LA were treated with EGF, Snail2 was the principal E-cadherin repressor induced. With ST or ST+EGF this shifted to Snail1, with more extreme EMT and Zeb1/δEF1 induction seen with ST+EGF. ST reduced stress fibres and focal contact size rapidly and independently of gene transcription. Gene expression analysis by MT-PCR indicated that ST repressed many genes which were induced by EGF (EGFR, CAV1, CTGF, CYR61, CD44, S100A4) and induced genes which alter the actin cytoskeleton (NLF1, NLF2, EPHB4). Examination of the public database of breast cancers revealed tumours exhibiting higher Snail1 expression have an increased risk of disease-recurrence. This was not seen for Snail2, and Zeb1/δEF1 showed a reverse correlation with lower expression values being predictive of increased risk. Conclusion ST in combination with EGF directed a greater EMT via actin depolymerisation and focal contact size reduction, resulting in a loosening of cell-ECM attachment along with Snail1-Zeb1/δEF1 induction. This appeared fundamentally different to the EGF-induced EMT, highlighting the multiple pathways which can regulate EMT. Our findings add support for a functional role for Snail1 in invasive breast cancer.

Involvement of focal adhesion kinase in inhibition of motility of human breast cancer cells by sphingosine 1-phosphate

Sphingosine 1-phosphate (SPP), a bioactive sphingolipid metabolite, inhibits chemoinvasiveness of the aggressive, estrogen-independent MDA-MB-231 human breast cancer cell line. As in many other cell types, SPP stimulated proliferation of MDA-MB-231 cells, albeit to a lesser extent. Treatment of MDA-MB-231 cells with SPP had no significant effect on their adhesiveness to Matrigel, and only high concentrations of SPP partially inhibited matrix metalloproteinase-2 activation induced by Con A. However, SPP at a concentration that strongly inhibited invasiveness also markedly reduced chemotactic motility. To investigate the molecular mechanisms by which SPP interferes with cell motility, we examined tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin, which are important for organization of focal adhesions and cell motility. SPP rapidly increased tyrosine phosphorylation of FAK and paxillin and of the paxillin-associated protein Crk. Overexpression of FAK and kinase-defective FAK in MDA-MB-231 cells resulted in a slight increase in motility without affecting the inhibitory effect of SPP, whereas expression of FAK with a mutation of the major autophosphorylation site (F397) abolished the inhibitory effect of SPP on cell motility. In contrast, the phosphoinositide 3'-kinase inhibitor, wortmannin, inhibited chemotactic motility in both vector and FAK-F397- transfected cells. Our results suggest that autophosphorylation of FAK on Y397 may play an important role in SPP signaling leading to decreased cell motility.

Validity of accelerometry in ambulatory children and adolescents with cerebral palsy

To evaluate the validity of the ActiGraph accelerometer for the measurement of physical activity intensity in children and adolescents with cerebral palsy (CP) using oxygen uptake (VO 2) as the criterion measure. Thirty children and adolescents with CP (mean age 12.6 ± 2.0 years) wore an ActiGraph 7164 and a Cosmed K4b 2 portable indirect calorimeter during four activities; quiet sitting, comfortable paced walking, brisk paced walking and fast paced walking. VO 2 was converted to METs and activity energy expenditure and classiWed as sedentary, light or moderate-to-vigorous intensity according to the conventions for children. Mean ActiGraph counts min -1 were classiWed as sedentary, light or moderate-to-vigorous (MVPA) intensity using four diVerent sets of cut-points. VO 2 and counts min¡1 increased signiWcantly with increases in walking speed (P < 0.001). Receiver operating characteristic (ROC) curve analysis indicated that, of the four sets of cut-points evaluated, the Evenson et al. (J Sports Sci 26(14):1557-1565, 2008) cut-points had the highest classiWcation accuracy for sedentary (92%) and MVPA (91%), as well as the second highest classiWcation accuracy for light intensity physical activity (67%). A ROC curve analysis of data from our participants yielded a CP-speciWc cut-point for MVPA that was lower than the Evenson cut-point (2,012 vs. 2,296 counts min¡1), however, the diVerence in classiWcation accuracy was not statistically signiWcant 94% (95% CI = 88.2-97.7%) vs. 91% (95% CI = 83.5-96.5%). In conclusion, among children and adolescents with CP, the ActiGraph is able to diVerentiate between diVerent intensities of walking. The use of the Evenson cut-points will permit the estimation of time spent in MVPA and allows comparisons to be made between activity measured in typically developing adolescents and adolescents with CP. © 2011 Springer-Verlag.

Validation of accelerometry for physical activity measurement in ambulant adolescents with cerebral palsy

Background Promoting participation physical activity (PA) is an important means of promoting healthy growth and development in children with cerebral palsy (CP). The ActiGraph is a uniaxial accelerometer that provides a realtime measure of PA intensity, duration and frequency. Its small, light weight design makes it a promising measure of activity in children with CP. To date no study has validated the use of accelerometry as a measure of PA in ambulant adolescents with CP. Objectives To evaluate the validity of the ActiGraph accelerometer for measuring PA intensity in adolescents with CP, using oxygen consumption (VO2), measured using portable indirect calorimetry (Cosmed K4b2), as the criterion measure. Design Validation Study Participants/Setting: Ambulant adolescents with CP aged 10–16 years, GMFCS rating of I-III. The recruitment target is 30 (10 in each GMFCS level). Materials/Methods Participants wore the ActiGraph (counts/min) and a Cosmed K4b2 indirect calorimeter (mL/kg/min) during six activity trials: quiet sitting (QS), comfortable paced walking (CPW), brisk paced walking (BPW), fast paced walking (FPW), a ball-kicking protocol (KP) and a ball-throwing protocol (TP). MET levels (multiples of resting metabolism) for each activity were predicted from ActiGraph counts using the Freedson age-specific equation (Freedson et al. 2005) and compared with actual MET levels measured by the Cosmed. Predicted and measured METs for each activity trial were classified as light (> 1.5 METs and <4.6 METs) or moderate to vigorous intensity (≥ 4.6 METs). Results To date 36 bouts of activity have been completed (6 participants x 6 activities). Mean VO2 increased linearly as the intensity of the walking activity increased (CPW=9.47±2.16, BPW=14.06±4.38, FPW=19.21±5.68 ml/kg/min) and ActiGraph counts reflected this pattern (CPW=1099±574, BPW=2233±797 FPW=4707±1013 counts/min). The throwing protocol recording the lowest VO2 (TP=7.50±3.86 ml/kg/min) and lowest overall counts/min (TP=31±27 counts/min). When each of the 36 bouts were classified as either light or moderate to vigorous intensity using measured VO2 as the criterion measure, the Freedson equation correctly classified 28 from 36 bouts (78%). Conclusion/Clinical Implications These preliminary findings suggest that there is a relationship between the intensity of PA and direct measure of oxygen consumption and that therefore the ActiGraph may be a promising tool for accurately measuring free living PA in the community. Further data collection of the complete sample will enable secondary analysis of the relationship between PA and severity of CP (GMFCS level).

Sequential versus simultaneous viewing of an environment : effects of focal attention to individual object locations on visual spatial learning

We investigated memories of room-sized spatial layouts learned by sequentially or simultaneously viewing objects from a stationary position. In three experiments, sequential viewing (one or two objects at a time) yielded subsequent memory performance that was equivalent or superior to simultaneous viewing of all objects, even though sequential viewing lacked direct access to the entire layout. This finding was replicated by replacing sequential viewing with directed viewing in which all objects were presented simultaneously and participants’ attention was externally focused on each object sequentially, indicating that the advantage of sequential viewing over simultaneous viewing may have originated from focal attention to individual object locations. These results suggest that memory representation of object-to-object relations can be constructed efficiently by encoding each object location separately, when those locations are defined within a single spatial reference system. These findings highlight the importance of considering object presentation procedures when studying spatial learning mechanisms.

Measuring reliability and validity of the ActiGraph GT3X accelerometer for children with cerebral palsy : a feasibility study

PURPOSE The purposes of this study were to: 1) establish inter-instrument reliability between left and right hip accelerometer placement; 2) examine procedural reliability of a walking protocol used to measure physical activity (PA), and; 3) confirm concurrent validity of accelerometers in measuring PA intensity as compared to the gold standard of oxygen consumption measured by indirect calorimetry. METHODS Eight children (mean age: 11.9; SD: 3.2, 75% male) with CP (GMFCS levels I-III) wore ActiGraph GT3X accelerometers on each hip and the Cosmed K4b^{2} portable indirect calorimeter during two measurement sessions in which they performed the six minute walk test (6MWT) at three self-selected speeds (comfortable/slow, brisk, fast). Oxygen consumption (VO2) and accelerometer step and activity count data were recorded. RESULTS Inter-instrument reliability of ActiGraph GT3X accelerometers placed on left and right hips was excellent (ICC=0.96-0.99, CI_{95}: 0.81-0.99). Reproducibility of the protocol was good/excellent (ICC=0.75-0.95, CI_{95}: 0.75-0.98). Concurrent validity of accelerometer count data and VO2 was fair/good (rho=0.67, p< 0.001). The correlation between step count and VO2 was not significant (rho=0.29, p=0.2). CONCLUSION This preliminary research suggests that ActiGraph GT3X accelerometers are reliable and valid devices to monitor PA during walking in children with CP and may be appropriate in rehabilitation research and clinical practice. ActiGraph GTX3 step counts were not valid for this sample and further research is warranted.

Targeting and transport : how microtubules control focal adhesion dynamics

Directional cell migration requires force generation that relies on the coordinated remodeling of interactions with the extracellular matrix (ECM), which is mediated by integrin-based focal adhesions (FAs). Normal FA turnover requires dynamic microtubules, and three members of the diverse group of microtubule plus-end-tracking proteins are principally involved in mediating microtubule interactions with FAs. Microtubules also alter the assembly state of FAs by modulating Rho GTPase signaling, and recent evidence suggests that microtubule-mediated clathrin-dependent and -independent endocytosis regulates FA dynamics. In addition, FA-associated microtubules may provide a polarized microtubule track for localized secretion of matrix metalloproteases (MMPs). Thus, different aspects of the molecular mechanisms by which microtubules control FA turnover in migrating cells are beginning to emerge.

Cerebral regions associated with verbal response initiation, suppression and strategy use

Cerebral activation associated with performance on a novel task involving two conditions was investigated with functional magnetic resonance imaging (fMRI). In the response initiation condition, subjects nominated the general superordinate category to which each of a series of exemplars (concrete nouns) belonged. In the response suppression condition, subjects were required to nominate a general superordinate category to which each exemplar did not belong, with the instruction that they were not to nominate the same category response twice in a row. Both conditions produced distinct patterns of activation relative to an articulation control condition employing identical stimuli. When initiation and suppression conditions were directly compared, response suppression produced activation in the right frontal pole, orbital frontal cortex and anterior cingulate, left dorsolateral prefrontal cortex and posterior cingulate, and bilaterally in the precuneus, visual association cortex and cerebellum. Response latencies were significantly longer in the suppression condition. Two broadly-defined strategies associated with the correct production of words during the suppression condition were a self-ordered selection from among the superordinate categories identified during the first section of the task and the generation of novel category responses. The neuroanatomical correlates of response initiation, suppression and strategy use are discussed, as are the respective roles of response suppression and strategy generation.

Multi-site study of additive genetic effects on fractional anisotropy of cerebral white matter: Comparing meta and megaanalytical approaches for data pooling

Combining datasets across independent studies can boost statistical power by increasing the numbers of observations and can achieve more accurate estimates of effect sizes. This is especially important for genetic studies where a large number of observations are required to obtain sufficient power to detect and replicate genetic effects. There is a need to develop and evaluate methods for joint-analytical analyses of rich datasets collected in imaging genetics studies. The ENIGMA-DTI consortium is developing and evaluating approaches for obtaining pooled estimates of heritability through meta-and mega-genetic analytical approaches, to estimate the general additive genetic contributions to the intersubject variance in fractional anisotropy (FA) measured from diffusion tensor imaging (DTI). We used the ENIGMA-DTI data harmonization protocol for uniform processing of DTI data from multiple sites. We evaluated this protocol in five family-based cohorts providing data from a total of 2248 children and adults (ages: 9-85) collected with various imaging protocols. We used the imaging genetics analysis tool, SOLAR-Eclipse, to combine twin and family data from Dutch, Australian and Mexican-American cohorts into one large "mega-family". We showed that heritability estimates may vary from one cohort to another. We used two meta-analytical (the sample-size and standard-error weighted) approaches and a mega-genetic analysis to calculate heritability estimates across-population. We performed leave-one-out analysis of the joint estimates of heritability, removing a different cohort each time to understand the estimate variability. Overall, meta- and mega-genetic analyses of heritability produced robust estimates of heritability.