Direct regulation of tRNA and 5S rRNA gene transcription by Polo-like kinase 1.

Polo-like kinase Plk1 controls numerous aspects of cell-cycle progression. We show that it associates with tRNA and 5S rRNA genes and regulates their transcription by RNA polymerase III (pol III) through direct binding and phosphorylation of transcription factor Brf1. During interphase, Plk1 promotes tRNA and 5S rRNA expression by phosphorylating Brf1 directly on serine 450. However, this stimulatory modification is overridden at mitosis, when elevated Plk1 activity causes Brf1 phosphorylation on threonine 270 (T270), which prevents pol III recruitment. Thus, although Plk1 enhances net tRNA and 5S rRNA production, consistent with its proliferation-stimulating function, it also suppresses untimely transcription when cells divide. Genomic instability is apparent in cells with Brf1 T270 mutated to alanine to resist Plk1-directed inactivation, suggesting that chromosome segregation is vulnerable to inappropriate pol III activity.

A proteomic approach to identify endosomal cargoes controlling cancer invasiveness.

We have previously shown that Rab17, a small GTPase associated with epithelial polarity, is specifically suppressed by ERK2 (also known as MAPK1) signalling to promote an invasive phenotype. However, the mechanisms through which Rab17 loss permits invasiveness, and the endosomal cargoes that are responsible for mediating this, are unknown. Using quantitative mass spectrometry-based proteomics, we have found that knockdown of Rab17 leads to a highly selective reduction in the cellular levels of a v-SNARE (Vamp8). Moreover, proteomics and immunofluorescence indicate that Vamp8 is associated with Rab17 at late endosomes. Reduced levels of Vamp8 promote transition between ductal carcinoma in situ (DCIS) and a more invasive phenotype. We developed an unbiased proteomic approach to elucidate the complement of receptors that redistributes between endosomes and the plasma membrane, and have pin-pointed neuropilin-2 (NRP2) as a key pro-invasive cargo of Rab17- and Vamp8-regulated trafficking. Indeed, reduced Rab17 or Vamp8 levels lead to increased mobilisation of NRP2-containing late endosomes and upregulated cell surface expression of NRP2. Finally, we show that NRP2 is required for the basement membrane disruption that accompanies the transition between DCIS and a more invasive phenotype.

CLIC3 controls recycling of late endosomal MT1-MMP and dictates invasion and metastasis in breast cancer.

Chloride intracellular channel 3 (CLIC3) drives invasiveness of pancreatic and ovarian cancer by acting in concert with Rab25 to regulate the recycling of α5ß1 integrin from late endosomes to the plasma membrane. Here, we show that in two estrogen receptor (ER)-negative breast cancer cell lines, CLIC3 has little influence on integrin recycling, but controls trafficking of the pro-invasive matrix metalloproteinase MT1-MMP (also known as MMP14). In MDA-MB-231 cells, MT1-MMP and CLIC3 are localized primarily to late endosomal/lysosomal compartments located above the plane of adhesion and near the nucleus. MT1-MMP is transferred from these late endosomes to sites of cell-matrix adhesion in a CLIC3-dependent fashion. Correspondingly, CLIC3-knockdown opposes MT1-MMP-dependent invasive processes. These include the disruption of the basement membrane as acini formed from MCF10DCIS.com cells acquire invasive characteristics in 3D culture, and the invasion of MDA-MB-231 cells into Matrigel or organotypic plugs of type I collagen. Consistent with this, expression of CLIC3 predicts poor prognosis in ER-negative breast cancer. The identification of MT1-MMP as a cargo of a CLIC3-regulated pathway that drives invasion highlights the importance of late endosomal sorting and trafficking in breast cancer.

A randomized controlled trial of web-based training to increase activity in children with cerebral palsy.

AIM: To determine the efficacy of web-based training on activity capacity and performance in children with unilateral cerebral palsy (CP). METHOD: In a matched-pairs randomized waitlist controlled trial, independently ambulant children and adolescents with unilateral CP were allocated to receive 30 minutes of training (intervention) 6 days per week, or usual care (waitlist control) for 20 weeks. Activity capacity was assessed using maximal repetitions of functional strength tasks and 6-minute walk test (6MWT); performance using 4-day ActiGraph GT3X+ accelerometer records at baseline and 20 weeks. Data were analysed by intention to treat comparing between groups using hierarchical linear modelling. RESULTS: Participants were n=101, 52 males, mean age 11 years 3 months (SD 2y 4mo). Intervention participants completed a mean 32.4 hours (SD 17.2) of training, associated with significant improvements in functional strength (mean difference 19.3 repetitions; 95% confidence interval [CI] 10.8-27.7; p<0.001) and 6MWT distance (mean difference 38.9m; 95% CI 12.3-51.9; p<0.001) compared with the control group at 20 weeks, although not activity performance (p>0.05). INTERPRETATION: Training was effective at increasing functional strength and walking endurance in independently ambulant children with unilateral CP. This did not translate into improvements in activity performance.

Characteristics associated with physical activity among independently ambulant children and adolescents with unilateral cerebral palsy.

AIM: This study aimed to quantify the contribution of physical, personal and environmental characteristics to physical activity among independently ambulant children with unilateral cerebral palsy (CP). METHOD: One-hundred and two children with unilateral CP (52 males, 50 females; 52 right hemiplegia; mean age 11y 3mo, range 8-17y [SD 2y 4mo]) classified at Gross Motor Function Classification System (GMFCS) levels I = 44 and II = 58 participated. Physical activity was measured over 4 days using ActiGraph accelerometers recording as activity counts. GMFCS, functional strength, 6-minute walk test (6MWT), mobility limitations (MobQues28), age, sex, Assessment of Life-Habits recreation domain, Participation and Environment Measure for Children and Youth (PEM-CY) and environmental characteristics were considered for selection in a linear regression model. These served as independent variables which were determined using a backwards selection procedure. RESULTS: Younger age, male sex, increased performance on the 6MWT, and increased participation in the home and community measured using the PEM-CY were significantly associated with activity counts (p<0.001). However, the model fit was somewhat weak (R(2) =0.32), indicating that much of the variation was unexplained. Older age and reduced community participation were associated with high inactivity (p<0.001). INTERPRETATION: Physical activity interventions should primarily target adolescents and females. Walking endurance and participation in the home and community may represent modifiable characteristics to increase physical activity.

Epstein-Barr virus-encoded EBNA1 enhances RNA polymerase III-dependent EBER expression through induction of EBER-associated cellular transcription factors.

BACKGROUND: Epstein-Barr Virus (EBV)-encoded RNAs (EBERs) are non-polyadenylated RNA molecules transcribed from the EBV genome by RNA polymerase III (pol III). EBERs are the most abundant viral latent gene products, although the precise mechanisms by which EBV is able to achieve such high levels of EBER expression are not fully understood. Previously EBV has been demonstrated to induce transcription factors associated with EBER expression, including pol III transcription factors and ATF-2. We have recently demonstrated that EBV-encoded nuclear antigen-1 (EBNA1) induces cellular transcription factors, and given these findings, we investigated the role of EBNA1 in induction of EBER-associated transcription factors. RESULTS: Our data confirm that in epithelial cells EBNA1 can enhance cellular pol III transcription. Transient expression of EBNA1 in Ad/AH cells stably expressing the EBERs led to induction of both EBER1 and EBER2 and conversely, expression of a dominant negative EBNA1 led to reduced EBER expression in EBV-infected Ad/AH cells. EBNA1 can induce transcription factors used by EBER genes, including TFIIIC, ATF-2 and c-Myc. A variant chromatin precipitation procedure showed that EBNA1 is associated with the promoters of these genes but not with the promoters of pol III-transcribed genes, including the EBERs themselves. Using shRNA knock-down, we confirm the significance of both ATF-2 and c-Myc in EBER expression. Further, functional induction of a c-Myc fusion protein led to increased EBER expression, providing c-Myc binding sites upstream of EBER1 were intact. In vivo studies confirm elevated levels of the 102 kD subunit of TFIIIC in the tumour cells of EBV-positive nasopharyngeal carcinoma biopsies. CONCLUSIONS: Our findings reveal that EBNA1 is able to enhance EBER expression through induction of cellular transcription factors and add to the repertoire of EBNA1's transcription-regulatory properties.

Habitual physical activity of independently ambulant children and adolescents with cerebral palsy: are they doing enough?

BACKGROUND: Despite the health benefits of regular physical activity, children with cerebral palsy (CP) are thought to participate in reduced levels of physical activity. OBJECTIVE: The study objective was to assess physical activity and determine the proportion adhering to the recommended 60 minutes of moderate-to-vigorous physical activity (MVPA) daily in independently ambulant children and adolescents with unilateral CP. DESIGN: This was a cross-sectional study. METHOD: Children (N=102; 52 boys, 50 girls; mean age=11 years 3 months, SD=2 years 4 months) with spastic hemiplegia classified at Gross Motor Function Classification System (GMFCS) levels I (n=44) and II (n=58) recorded physical activity over 4 days using an accelerometer. Activity counts were converted to daily and hourly time spent inactive and in light physical activity or MVPA using uniaxial cutpoints (inactive: ≤100 vertical counts·min(-1), light: 101 to 2,295 vertical counts·min(-1), MVPA: ≥2,296 vertical counts·min(-1)) and recorded step counts. Differences between groups were examined using t tests. RESULTS: Of a potential 396 days, 341 days (86%) were recorded. The average wear time was 11:44 (SD=1:56) hours. On a typical day, participants recorded 438 (SD=234) counts·min(-1), took 7,541 (SD=3,894) steps, spent 8:36 (SD=1:09) hours inactive, spent 2:38 (SD=0:51) hours in light activity, and spent 0:44 (SD=0:26) hours in MVPA. Only 25% of participants met the recommended level of MVPA on at least one day. Physical activity was highest in boys (versus girls), in children (versus adolescents), and on weekdays (versus weekends). LIMITATIONS: Participants were limited to children with unilateral spasticity who were classified at GMFCS levels I and II. CONCLUSIONS: The majority of independently ambulant children with unilateral CP did not perform sufficient physical activity to meet public health recommendations.

Variability in measuring physical activity in children with cerebral palsy.

INTRODUCTION: This study aimed to establish the variability in the measurement of habitual physical activity using the ActiGraph® GT3X+ accelerometer in children with cerebral palsy (CP). METHOD: Repeated measures: Independently ambulant children with unilateral CP (n = 30; age, 11 yr 3 months (2 yr 4 months)) completed standardized tasks over two consecutive days, wearing an ActiGraph® GT3X+ accelerometer and HR monitor. Testing protocol comprised 5 min of seated rest (REST), walking at light, moderate, and vigorous pace, and rapid stepping on/off a step. Agreement was calculated between days using intraclass correlation coefficients (ICC) (two-factor mixed agreement model). Minimum detectable difference was calculated (minimum detectable difference = [SD√1 - ICC] × 1.96√2). Performance variability: Participants (n = 102) wore an ActiGraph® GT3X+ accelerometer for 4 d in the community. Activity counts were converted into activity intensity using uniaxial-derived cut points to classify the time spent in moderate-to-vigorous physical activity (MVPA). Between-day intraclass reliability coefficients (R) and Spearman-Brown prophecy formula ([ICCdesired/(1 - ICCdesired)][(1 - ICCestimated)]/ICCestimated]) were calculated. RESULTS: Agreement between repeated measures was strong for light physical activity and MVPA (ICC, 0.80). For MVPA, the minimum detectable difference was 1412 counts per minute. In the community, 345 d (87%) were recorded. Three days of monitoring produced acceptable variability estimates of MVPA (R = 0.63-0.73). Spearman-Brown prophecy analysis estimated that 3 d would achieve a reliability coefficient of 0.7 and 11 d would achieve 0.9. CONCLUSIONS: Measurement of habitual physical activity using the ActiGraph® GT3X+ accelerometer is reliable under controlled walking and stepping conditions as well as in a community environment in independently ambulant children and adolescents with CP.

A systematic review of the clinimetric properties of measures of habitual physical activity in primary school aged children with cerebral palsy.

Regular participation in physical activity is an important determinant of health for children and adolescents with cerebral palsy (CP). However, there is little consensus on the most valid or reliable method to measure physical activity in this population. This study aimed to systematically review the psychometric properties of habitual physical activity (HPA) measures in primary school-aged children with CP. Databases were systematically searched for measures assessing physical activity over more than one day and had evidence of validity, reliability and/or clinical utility in children aged 6-12 years with CP. Ten measures met inclusion criteria and their quality was assessed in twelve studies. Quality of the included studies was appraised using the consensus-based standards for the selection of health measurement instruments (COSMIN) checklist. Measures were moderately to strongly correlated to criterion measures, with study quality rated as Fair (+) to Poor (0). Only four measures had evidence of reliability. Accelerometers provide a valid measure of HPA with good clinical utility; however they do not have documented reliability in this population. No one measure appears ideal to record HPA in primary school-age children with CP and further research is necessary to determine the psychometric properties of HPA measurement instruments in this population.

Move it to improve it (Mitii): study protocol of a randomised controlled trial of a novel web-based multimodal training program for children and adolescents with cerebral palsy.

INTRODUCTION: Persons with cerebral palsy require a lifetime of costly and resource intensive interventions which are often limited by equity of access. With increasing burden being placed on health systems, new methods to deliver intensive rehabilitation therapies are needed. Move it to improve it (Mitii) is an internet-based multimodal programme comprising upper-limb and cognitive training with physical activity. It can be accessed in the client's home at their convenience. The proposed study aims to test the efficacy of Mitii in improving upper-limb function and motor planning. Additionally, this study hopes to further our understanding of the central neurovascular mechanisms underlying the proposed changes and determine the cost effectiveness of Mitii. METHODS AND ANALYSIS: Children with congenital hemiplegia will be recruited to participate in this waitlist control, matched pairs, single-blind randomised trial. Children be matched at baseline and randomly allocated to receive 20 weeks of 30 min of daily Mitii training immediately, or waitlisted for 20 weeks before receiving the same Mitii training (potential total dose=70 h). Outcomes will be assessed at 20 weeks after the start of Mitii, and retention effects tested at 40 weeks. The primary outcomes will be the Assessment of Motor and Process Skills (AMPS), the Assisting Hand Assessment (AHA) and unimanual upper-limb capacity using the Jebsen-Taylor Test of Hand Function (JTTHF). Advanced brain imaging will assess use-dependant neuroplasticity. Measures of body structure and functions, activity, participation and quality of life will be used to assess Mitii efficacy across all domains of the International Classification of Functioning, Disability and Health framework. ETHICS AND DISSEMINATION: This project has received Ethics Approval from the Medical Ethics Committee of The University of Queensland (2011000608) and the Royal Children's Hospital Brisbane (HREC/11/QRCH/35). Findings will be disseminated widely through conference presentations, seminars and peer-reviewed scientific journals. TRIAL REGISTRATION: ACTRN12611001174976.

A systematic review of the clinimetric properties of habitual physical activity measures in young children with a motor disability.

Aim. To identify and systematically review the clinimetric properties of habitual physical activity (HPA) measures in young children with a motor disability. Method. Five databases were searched for measures of HPA including: children aged <6.0 years with a neuromuscular disorder, physical activity defined as "bodily movement produced by skeletal muscles causing caloric expenditure", reported HPA as duration, frequency, intensity, mode or energy expenditure, and evaluated clinimetric properties. The quality of papers was assessed using the COSMIN-checklist. A targeted search of identified measures found additional studies of typically developing young children (TDC). Results. Seven papers assessing four activity monitors met inclusion criteria. Four studies were of good methodological quality. The Minimod had good ability to measure continuous walking but the demonstrated poor ability to measure steps during free-living activities. The Intelligent Device for Energy Expenditure and Activity and Ambulatory Monitoring Pod showed poor ability to measure activity during both continuous walking and free-living activities. The StepWatch showed good ability to measure steps during continuous walking in TDC. Interpretation. Studies assessing the clinimetric properties of measures of HPA in this population are urgently needed to allow assessment of the relationship between HPA and health outcomes in this group.