Comparing the Diagnostic Classification Accuracy of iTRAQ, Peak-Area, Spectral-Counting, and emPAI Methods for Relative Quantification in Expression Proteomics.

Diagnostic classification accuracy is critical in expression proteomics to ensure that as many true differences as possible are identified with acceptable false-positive rates. We present a comparison of the diagnostic accuracy of iTRAQ with three label-free methods, peak area, spectral counting, and emPAI, for relative quantification using a spiked proteome standard. We provide the first validation of emPAI for intersample relative quantification and find clear differences among the four quantification approaches that could be considered when designing an experiment. Spectral counting was observed to perform surprisingly well in all regards. Peak area performed best for smaller fold differences and was shown to be capable of discerning a 1.1-fold difference with acceptable specificity and sensitivity. The performance of iTRAQ was dramatically worse than the label-free methods with low abundance proteins. Using the iTRAQ data set for validation, we also demonstrate a novel iTRAQ analysis regime that avoids the use of ratios in significance testing and outperforms a common commercial alternative.

Multisite tyrosine phosphorylation of the N-terminus of Mint1/X11α by Src kinase regulates the trafficking of amyloid precursor protein.

Mint/X11 is one of the four neuronal trafficking adaptors that interact with amyloid precursor protein (APP) and are linked with its cleavage to generate ß-amyloid peptide, a key player in the pathology of Alzheimer's disease. How APP switches between adaptors at different stages of the secretory pathway is poorly understood. Here, we show that tyrosine phosphorylation of Mint1 regulates the destination of APP. A canonical SH2-binding motif ((202) YEEI) was identified in the N-terminus of Mint1 that is phosphorylated on tyrosine by C-Src and recruits the active kinase for sequential phosphorylation of further tyrosines (Y191 and Y187). A single Y202F mutation in the Mint1 N-terminus inhibits C-Src binding and tyrosine phosphorylation. Previous studies observed that co-expression of wild-type Mint1 and APP causes accumulation of APP in the trans-Golgi. Unphosphorylatable Mint1 (Y202F) or pharmacological inhibition of Src reduced the accumulation of APP in the trans-Golgi of heterologous cells. A similar result was observed in cultured rat hippocampal neurons where Mint1(Y202F) permitted the trafficking of APP to more distal neurites than the wild-type protein. These data underline the importance of the tyrosine phosphorylation of Mint1 as a critical switch for determining the destination of APP. The regulation of amyloid precursor protein (APP) trafficking is poorly understood. We have discovered that the APP adapter, Mint1, is phosphorylated by C-Src kinase. Mint1 causes APP accumulation in the trans-Golgi network, whereas inhibition of Src or mutation of Mint1-Y202 permits APP recycling. The phosphorylation status of Mint1 could impact on the pathological trafficking of APP in Alzheimer's disease.

The sodium channel-blocking antiepileptic drug phenytoin inhibits breast tumour growth and metastasis.

BACKGROUND: Voltage-gated Na(+) channels (VGSCs) are heteromeric protein complexes containing pore-forming α subunits and smaller, non-pore-forming ß subunits. VGSCs are classically expressed in electrically excitable cells, e.g. neurons. VGSCs are also expressed in tumour cells, including breast cancer (BCa) cells, where they enhance cellular migration and invasion. However, despite extensive work defining in detail the molecular mechanisms underlying the expression of VGSCs and their pro-invasive role in cancer cells, there has been a notable lack of clinically relevant in vivo data exploring their value as potential therapeutic targets. FINDINGS: We have previously reported that the VGSC-blocking antiepileptic drug phenytoin inhibits the migration and invasion of metastatic MDA-MB-231 cells in vitro. The purpose of the present study was to establish whether VGSCs might be viable therapeutic targets by testing the effect of phenytoin on tumour growth and metastasis in vivo. We found that expression of Nav1.5, previously detected in MDA-MB-231 cells in vitro, was retained on cells in orthotopic xenografts. Treatment with phenytoin, at a dose equivalent to that used to treat epilepsy (60 mg/kg; daily), significantly reduced tumour growth, without affecting animal weight. Phenytoin also reduced cancer cell proliferation in vivo and invasion into surrounding mammary tissue. Finally, phenytoin significantly reduced metastasis to the liver, lungs and spleen. CONCLUSIONS: This is the first study showing that phenytoin reduces breast tumour growth and metastasis in vivo. We propose that pharmacologically targeting VGSCs, by repurposing antiepileptic or antiarrhythmic drugs, should be further studied as a potentially novel anti-cancer therapy.

Filter-aided N-glycan separation (FANGS): a convenient sample preparation method for mass spectrometric N-glycan profiling.

We have developed a simple method for the release and isolation of glycoprotein N-glycans from whole-cell lysates using less than a million cells, for subsequent implementation with mass spectrometric analysis. Cellular protein extracts prepared using SDS solubilization were sequentially treated in a membrane filter device to ultimately release glycans enzymatically using PNGase F in the volatile buffer ammonium bicarbonate. The released glycans are recovered in the filtrate following centrifugation and typically permethylated prior to mass spectrometric analysis. We call our method "filter-aided N-glycan separation" and have successfully applied it to investigate N-glycan profiles of wild-type and mutant Chinese hamster ovary cells. This method is readily multiplexed and, because of the small numbers of cells needed, is compatible with the analysis of replicate samples to assess the true nature of glycan variability in tissue culture samples.

The Effects of Exercise Training on Brachial Artery Flow-Mediated Dilation: A Meta-analysis.

PURPOSE: Flow-mediated dilation, a barometer of cardiovascular (CV) health, is reported to increase with exercise training (ET); however, the potential moderating factors of ET are not clear to date. The purpose of this study was to determine the effect of ET assessed by brachial artery flow-mediated dilation (BAFMD). METHODS: Authors searched PubMed between January 1999 and December 2013, bibliographies, and reviews to identify studies examining ET and BAFMD. Two independent reviewers extracted quality, descriptive, exercise, and outcome data of eligible studies. Data were presented as weighted effect sizes (ESs) and 95% confidence limits. RESULTS: Analysis included 66 studies reporting BAFMD data (1865 ET and 635 control subjects). Overall, ET had significant improvements in BAFMD compared with controls (P < .0001). Exercise training at higher ET intensities resulted in a greater increase in BAFMD (9.29; 95% CI, 5.09-13.47) than lower ET intensities (3.63; 95% CI, -0.56 to 7.83) or control (-0.42; 95% CI, -2.06 to 1.21). Subjects whose ET duration was ≥150 min/wk (11.33; 95% CI, 7.15-15.51) had a significant improvement in BAFMD compared with those with <150 min/wk (4.79; 95% CI, 3.08-6.51) or control (-0.30; 95% CI, -1.99 to 1.39). Age (P = .11) and baseline artery diameter (P = .31) did not modify the BAFMD response to ET. CONCLUSION: Exercise training contributes to a significant increase in BAFMD. These results provide indirect evidence that ET alters a well-known factor associated with the primary and secondary prevention of CV diseases. Exercise training interventions, including greater intensity and duration, may optimize the increase in BAFMD.

Developmental differences in children's ballistic aiming movements of the arm.

An experiment was conducted to examine the change in the relation between programming and "on-line" correction as a developmental explanation of children's arm movement performance. Each of 54 children in three age groups (5, 8, and 10 yr.) completed two types of rapid aiming arm movements in the longitudinal plane on the surface of a digitizer. Percent primary submovements and timing variability were dependent variables. Analysis suggested that the 5-yr.-olds used "on-line" monitoring during the arm movement and did not perform the movement sequence as a functional unit. Compared with 8- and 10-yr.-olds, the 5-yr.-olds planned a smaller portion of movements, executed the arm movements with more variability in time to peak velocity. The 8- and 10-yr.-olds appeared to plan their movements and execute the sequence as a unit. The developmental implications were discussed.

Improved Data Reporting in RQES: from volumes 49, 59, to 84.

This commentary provides a review of changes in data reporting in Research Quarterly for Exercise and Sport from Volumes 49 and 59 to 84. Improvements were noted in that all articles reported means, standard deviations, and sample sizes, while most (87%) articles reported an estimate of effect size (ES). Additional reporting recommendations were made about ES for mean differences, use of appropriate estimates of variability, when to use a table or figure, use of multivariate analyses, and power analyses.

Advancing kinesiology through improved peer review.

Peer review of scholarship is essential to journal quality, evidence, knowledge advancement, and application of that knowledge in any field. This commentary summarizes recent literature on issues related to peer-review quality and current review practice in kinesiology and provides recommendations to improve peer review in kinesiology journals. We reviewed the literature on the characteristics of peer review in scientific journals and describe the status of peer review in kinesiology journals. Although the majority of scholars and editors strongly support the peer-review process, systematic research in several disciplines has shown somewhat positive but mixed results for the efficacy of peer review in evaluating the quality of and improving research reports. Past recommendations for improvement have focused on agreement between reviewers, standards for evaluating quality, and clarification of the editorial team roles. Research on interventions, however, indicates that improving reviewer performance is difficult. The specific research on peer review in kinesiology is limited. Six recommendations to improve peer review are proposed: publishing clear evaluation standards, establishing collaborative evaluation procedures and editorial team roles, utilizing online submission data to help improve reviewer comments, creating author appeals procedures, protecting reviewer time commitments, and improving reviewer recognition. There is considerable variation in peer-review criteria and procedures in kinesiology, and implementing several reasonable improvements may advance knowledge development and the field of kinesiology.

Plasma membrane proteomes of differentially matured dendritic cells identified by LC-MS/MS combined with iTRAQ labelling.

Dendritic cells (DCs) play a pivotal role in polarising Th lymphocyte subsets but it is unclear what molecular events occur when DCs generate Th2-type responses. Here, we analysed plasma membrane-enriched fractions from immature, pro-Th1 and pro-Th2 DCs and used a combination of iTRAQ labelling and LC-MS/MS to quantify changes in the proteomes. Analysis was performed on triplicate biological samples and changes verified by flow cytometry. MHC class II molecules and CD29 were up-regulated in pro-Th1 DCs whilst CD18 and CD44 were up-regulated in pro-Th2 DCs. One of the most down-regulated molecules in pro-Th1 DCs was YM-1 whilst the greatest decrease in pro-Th2 DCs was NAP-22. Other molecules up-regulated in pro-Th2 DC compared to pro-Th1 DCs included some potentially involved in protein folding during antigen processing (clathrin and Rab-7), whilst other non-membrane proteins such as enzymes/transporters related to cell metabolism (malate dehydrogenase, pyruvate kinase, and ATPase Na(+)/K(+)) were also recorded. This suggests that pro-Th2 DCs are more metabolically active while pro-Th1 DCs have a mature 'end state'. Overall, although several molecules were preferentially expressed on pro-Th2 DCs, our proteomics data support the view of a 'limited maturation' of pro-Th2 DCs compared to pro-Th1 DCs.

Developmental gender differences for overhand throwing in Aboriginal Australian children.

In a review of 46 meta-analyses of gender differences, overhand throwing had the largest gender difference favoring boys (ES > 3.0). Expectations for gender-specific performances may be less pronounced in female Australian Aborigines, because historical accounts state they threw for defense and hunting. Overhand throwing velocities and kinematics were recorded in 30 female and male Aboriginal Australian children 6-10 years old. Results indicated the Aboriginal girls and boys were more similar in horizontal ball velocities than U.S. girls and boys. Throwing kinematics between girls and boys were also more similar in Australian Aborigines than U.S. children. Aboriginal girls threw with greater velocities than U.S., German, Japanese, and Thai girls, while the boys were similar across cultures.

NopM and NopD are rhizobial nodulation outer proteins: identification using LC-MALDI and LC-ESI with a monolithic capillary column.

We have explored the potential of commercial polystyrene-divinylbenzene monolithic capillary nanoLC-MS/MS for identifying Sinorhizobium fredii HH103 nodulation outer proteins. Monolithic nanoLC with off-line MALDI-TOF/TOF and on-line ESI-q-oTOF is fast and robust, generating complementary data and offering high-confidence protein identifications from gel bands too weak for successful analysis using traditional approaches. This has allowed identification of two proteins not previously described as being type III-secreted in rhizobia, NopM and NopD.

The 75th anniversary of Research Quarterly for Exercise and Sport. An analysis of status and contributions.

In celebration of the 75th anniversary of The Research Quarterly/Research Quarterly for Exercise and Sport (RQ/ RQES) an analysis was conducted comparing RQ/RQES to numerous other journals in the field with regard to impact factors and citation rates. A series of analyses was conducted from the first publication of RQ/RQES in 1930 through this 75th edition to identify total citations by decade, the top 10 cited papers, top cited papers by decade, the top three papers in 5-year intervals from the 50th anniversary issue in 1980 through 2001, and the outstanding research writing award papers since this award began 23 years ago.

Arm movement control: differences between children with and without attention deficit hyperactivity disorder.

The purpose of this study was to examine performance differences in arm movement control (programmingvs. "on-line" control) between children with and without attention deficit hyperactivity disorder (ADHD). Twenty children (10 with ADHD and 10 without ADHD) from the ages of 8 to 13 years participated in the study. On the surface of a digitizer, each participant completed three types of aiming arm movements (10 trials for each) and 10 baseline trials (without accuracy requirement). Multivariate analyses of variance with repeated measures were used to analyze the variables of reaction time, movement time, normalized jerk, intersegment-interval (ISI), and movement timing. Children with ADHD appeared to use "on-line " monitoring during the arm movement and did not perform the entire movement sequence as afunctional unit. They executed the arm movements more slowly, had greater variability in movement timing, and demonstrated longer ISIs than their counterparts. Children with ADHD had multiple peaks in the velocity profiles. Children withoutADHD, however, appeared to program their entire arm movements and execute the sequence as a unit. Their velocity profiles were symmetrical with a single peak, and the movement segments were temporally coordinated. Thesefindings suggested that cognitive functions are important resources for controlling rapid aiming arm movements. Children with ADHD might rely more on visual feedback during the movements, which resulted in slower and more variant movement outcomes than children who did not have ADHD.