Novel aspects of blood platelet function and biology measured by fluorescent flow cytometry.

Fluorescent flow cytometry (FFC) is an accessible, high-throughput and sensitive technique that is well suited to analysis of cells suspended in complex heterogeneous media. Using a wide variety of live, fixed, extracellular, intracellular, dye/antibody/peptide based FFC assays, multiple and novel aspects of platelet biology including; activation, inflammation and mitochondrial function were examined. Through the examination of platelet activation with multiparameter and multidimensional analysis three platelet subsets in PAR1/GPVI activated whole blood samples were described. This same assay in the presence of PGI2 suggested a dichotomy in inhibition when comparing fibrinogen binding, phosphatidylserine (PS) exposure and CD62P. Using novel phosphoflow protocols the inhibition resistant expression of CD62P was shown to be independent of robust cAMP signalling. This CD62P expression was then shown to mediate platelet monocyte interactions – in the presence of inhibition. Caspase-1 cleavage as a marker of NLRP3 inflammasome activity was measured using fluorescent peptide dyes. Activation was induced by canonical activators of the complex, but it was observed that this was significantly potentiated by oxidised LDL (oxLDL). ROS and calcium were also shown to have a vital role in activation of platelet NLRP3 and scavenging or chelation of either induced a loss of signal. Furthermore, active caspase-1 signal also correlated with PS exposure. Finally, mitochondrial function in response to oxLDL in vitro and in the context of murine hyperlipidaemia ex vivo was measured using live cell dyes. In vitro stimulation with oxLDL drove an increase in both mitochondrial superoxide production and mitochondrial membrane potential. However, this was demonstrated to be important in vivo, as transgenic models of hyperlipidaemia showed a significant increase in basal mitochondrial superoxide compared to wild type control. This was recapitulated diet-induced obesity murine models and a trend towards increased mitochondrial superoxide and membrane potential was described

An integrated approach to enhancing functional annotation of sequences for data analysis of a transcriptome

Given the ever increasing quantity of sequence data, functional annotation of new gene sequences persists as being a significant challenge for bioinformatics. This is a particular problem for transcriptomics studies in crop plants where large genomes and evolutionarily distant model organisms, means that identifying the function of a given gene used on a microarray, is often a non-trivial task. Information pertinent to gene annotations is spread across technically and semantically heterogeneous biological databases. Combining and exploiting these data in a consistent way has the potential to improve our ability to assign functions to new or uncharacterised genes. Methods: The Ondex data integration framework was further developed to integrate databases pertinent to plant gene annotation, and provide data inference tools. The CoPSA annotation pipeline was created to provide automated annotation of novel plant genes using this knowledgebase. CoPSA was used to derive annotations for Affymetrix GeneChips available for plant species. A conjoint approach was used to align GeneChip sequences to orthologous proteins, and identify protein domain regions. These proteins and domains were used together with multiple evidences to predict functional annotations for sequences on the GeneChip. Quality was assessed with reference to other annotation pipelines. These improved gene annotations were used in the analysis of a time-series transcriptomics study of the differential responses of durum wheat varieties to water stress. Results and Conclusions: The integration of plant databases using the Ondex showed that it was possible to increase the overall quantity and quality of information available, and thereby improve the resulting annotation. Direct data aggregation benefits were observed, as well as new information derived from inference across databases. The CoPSA pipeline was shown to improve coverage of the wheat microarray compared to the NetAffx and BLAST2GO pipelines. Leverage of these annotations during the analysis of data from a transcriptomics study of the durum wheat water stress responses, yielded new biological insights into water stress and highlighted potential candidate genes that could be used by breeders to improve drought response

An integrated approach to enhancing functional annotation of sequences for data analysis of a transcriptome

Given the ever increasing quantity of sequence data, functional annotation of new gene sequences persists as being a significant challenge for bioinformatics. This is a particular problem for transcriptomics studies in crop plants where large genomes and evolutionarily distant model organisms, means that identifying the function of a given gene used on a microarray, is often a non-trivial task. Information pertinent to gene annotations is spread across technically and semantically heterogeneous biological databases. Combining and exploiting these data in a consistent way has the potential to improve our ability to assign functions to new or uncharacterised genes. Methods: The Ondex data integration framework was further developed to integrate databases pertinent to plant gene annotation, and provide data inference tools. The CoPSA annotation pipeline was created to provide automated annotation of novel plant genes using this knowledgebase. CoPSA was used to derive annotations for Affymetrix GeneChips available for plant species. A conjoint approach was used to align GeneChip sequences to orthologous proteins, and identify protein domain regions. These proteins and domains were used together with multiple evidences to predict functional annotations for sequences on the GeneChip. Quality was assessed with reference to other annotation pipelines. These improved gene annotations were used in the analysis of a time-series transcriptomics study of the differential responses of durum wheat varieties to water stress. Results and Conclusions: The integration of plant databases using the Ondex showed that it was possible to increase the overall quantity and quality of information available, and thereby improve the resulting annotation. Direct data aggregation benefits were observed, as well as new information derived from inference across databases. The CoPSA pipeline was shown to improve coverage of the wheat microarray compared to the NetAffx and BLAST2GO pipelines. Leverage of these annotations during the analysis of data from a transcriptomics study of the durum wheat water stress responses, yielded new biological insights into water stress and highlighted potential candidate genes that could be used by breeders to improve drought response

TRANSFERABILITY OF A PREVIOUSLY VALIDATED IMU SYSTEM FOR LOWER EXTREMITY KINEMATICS

This study tested transferability and validity of an Inertial Measurement Unit (IMU) system for estimation of lower limb kinematics. Peak hip, knee, and plantarflexion angles and sagittal plane range of motion (ROM) were compared during body weight squats (BWSQ) and countermovement jumps (CMJ) in 16 participants using root mean square error (RMSE) and intraclass correlation coefficients (ICC). RMSE wa

The circadian clock gene circuit controls protein and phosphoprotein rhythms in Arabidopsis thaliana

Twenty-four-hour, circadian rhythms control many eukaryotic mRNA levels, whereas the levels of their more stable proteins are not expected to reflect the RNA rhythms, emphasizing the need to test the circadian regulation of protein abundance and modification. Here we present circadian proteomic and phosphoproteomic time series from Arabidopsis thaliana plants under constant light conditions, estimating that just 0.4% of quantified proteins but a much larger proportion of quantified phospho-sites were rhythmic. Approximately half of the rhythmic phospho-sites were most phosphorylated at subjective dawn, a pattern we term the “phospho-dawn.” Members of the SnRK/CDPK family of protein kinases are candidate regulators. A CCA1-overexpressing line that disables the clock gene circuit lacked most circadian protein phosphorylation. However, the few phospho-sites that fluctuated despite CCA1-overexpression still tended to peak in abundance close to subjective dawn, suggesting that the canonical clock mechanism is necessary for most but perhaps not all protein phosphorylation rhythms. To test the potential functional relevance of our datasets, we conducted phosphomimetic experiments using the bifunctional enzyme fructose-6-phosphate-2-kinase/phosphatase (F2KP), as an example. The rhythmic phosphorylation of diverse protein targets is controlled by the clock gene circuit, implicating posttranslational mechanisms in the transmission of circadian timing information in plants

qpMerge: Merging different peptide isoforms using a motif centric strategy

Accurate quantification and enumeration of peptide motifs is hampered by redundancy in peptide identification. A single phosphorylation motif may be split across charge states, alternative modifications (e.g. acetylation and oxidation), and multiple miss-cleavage sites which render the biological interpretation of MS data a challenge. In addition motif redundancy can affect quantitative and statistical analysis and prevent a realistic comparison of peptide numbers between datasets. In this study, we present a merging tool set developed for the Galaxy workflow environment to achieve a non-redundant set of quantifications for phospho-motifs. We present a Galaxy workflow to merge three exemplar dataset, and observe reduced phospho-motif redundancy and decreased replicate variation. The qpMerge tools provide a straightforward and reusable approach to facilitating phospho-motif analysis. The source-code and wiki documentation is publically available at http://sourceforge.net/projects/ppmerge. The galaxy pipeline used in the exemplar analysis can be found at http://www.myexperiment.org/workflows/4186