Edmond J. Breen

Analysis of in vitro secretion profiles from adipose-derived cell populations

BackgroundAdipose tissue is an attractive source of cells for therapeutic purposes because of the ease of harvest and the high frequency of mesenchymal stem cells (MSCs). Whilst it is clear that MSCs have significant therapeutic potential via their ability to secrete immuno-modulatory and trophic cytokines, the therapeutic use of mixed cell populations from the adipose stromal vascular fraction (SVF) is becoming increasingly common.MethodsIn this study we have measured a panel of 27 cytokines and growth factors secreted by various combinations of human adipose-derived cell populations. These were 1. co-culture of freshly isolated SVF with adipocytes, 2. freshly isolated SVF cultured alone, 3. freshly isolated adipocytes alone and 4. adherent adipose-derived mesenchymal stem cells (ADSCs) at passage 2. In addition, we produced an ‘in silico’ dataset by combining the individual secretion profiles obtained from culturing the SVF with that of the adipocytes. This was compared to the secretion profile of co-cultured SVF and adipocytes. Two-tailed t-tests were performed on the secretion profiles obtained from the SVF, adipocytes, ADSCs and the ‘in silico’ dataset and compared to the secretion profiles obtained from the co-culture of the SVF with adipocytes. A p-value of < 0.05 was considered statistically different. To assess the overall changes that may occur as a result of co-culture we compared the proteomes of SVF and SVF co-cultured with adipocytes using iTRAQ quantitative mass spectrometry.ResultsA co-culture of SVF and adipocytes results in a distinct secretion profile when compared to all other adipose-derived cell populations studied. This illustrates that cellular crosstalk during co-culture of the SVF with adipocytes modulates the production of cytokines by one or more cell types. No biologically relevant differences were detected in the proteomes of SVF cultured alone or co-cultured with adipocytes.ConclusionsThe use of mixed adipose cell populations does not appear to induce cellular stress and results in enhanced secretion profiles. Given the importance of secreted cytokines in cell therapy, the use of a mixed cell population such as the SVF with adipocytes may be considered as an alternative to MSCs or fresh SVF alone.

Automatic Poisson peak harvesting for high throughput protein identification

High throughput identification of proteins by peptide mass fingerprinting requires an efficient means of picking peaks from mass spectra. Here, we report the development of a peak harvester to automatically pick monoisotopic peaks from spectra generated on matrix‐assisted laser desorption/ionisation time of flight (MALDI‐TOF) mass spectrometers. The peak harvester uses advanced mathematical morphology and watershed algorithms to first process spectra to stick representations. Subsequently, Poisson modelling is applied to determine which peak in an isotopically resolved group represents the monoisotopic mass of a peptide. We illustrate the features of the peak harvester with mass spectra of standard peptides, digests of gel‐separated bovine serum albumin, and with Escherictia coli proteins prepared by two‐dimensional polyacrylamide gel electrophoresis. In all cases, the peak harvester proved effective in its ability to pick similar monoisotopic peaks as an experienced human operator, and also proved effective in the identification of monoisotopic masses in cases where isotopic distributions of peptides were overlapping. The peak harvester can be operated in an interactive mode, or can be completely automated and linked through to peptide mass fingerprinting protein identification tools to achieve high throughput automated protein identification.

High Throughput Peptide Mass Fingerprinting and Protein Macroarray Analysis Using Chemical Printing Strategies

We describe a chemical printer that uses piezoelectric pulsing for rapid, accurate, and non-contact microdispensing of fluid for proteomic analysis of immobilized protein macroarrays. We demonstrate protein digestion and peptide mass fingerprinting analysis of human plasma and platelet proteins direct from a membrane surface subsequent to defined microdispensing of trypsin and matrix solutions, hence bypassing multiple liquid-handling steps. Detection of low abundance, alkaline proteins from whole human platelet extracts has been highlighted. Membrane immobilization of protein permits archiving of samples pre-/post-analysis and provides a means for subanalysis using multiple chemistries. This study highlights the ability to increase sequence coverage for protein identification using multiple enzymes and to characterize N-glycosylation modifications using a combination of PNGase F and trypsin. We also demonstrate microdispensing of multiple serum samples in a quantitative microenzyme-linked immunosorbent assay format to rapidly screen protein macroarrays for pathogen-derived antigens. We anticipate the chemical printer will be a major component of proteomic platforms for high throughput protein identification and characterization with widespread applications in biomedical and diagnostic discovery.

Bead-based multiplex immuno-assays for cytokines, chemokines, growth factors and other analytes: median fluorescence intensities versus their derived absolute concentration values for statistical analysis.

Within the scientific literature, analyses of data from bead based multiplex immunoassays are based on either median fluorescence intensities (MFI) or derived absolute concentration values (ACV) but no consideration of which set of data is the most appropriate for analysis has been published. Here we look at the variance of MFI versus their ACV from the expression of 14 analytes in plasma, using 6 commercially available kits, across 177 patients, recorded at two time points and the associated analyte standards. In total 60 micro titre plates were used resulting in 4965 MFI. In doing so we develop a new background subtraction procedure that reduced by 50% the number of out-of-range values observed in our data set. Using a linear mixed-effect model, which normalizes for assay-to-assay variation, MFI produced similar significant differences than that observed using absolute concentration values. We show that subtracting analyte blanks produces 15% negative MFI resulting in uncertainty of the data being analysed. We argue for analysis of protein expression values MFI are generally a better choice than absolute concentration values. It is argued that analyte standards are not required on each plate, or not at all, in multi-plate experiments, but knowledge of the concentration curve and the range of MFI values that fall within the limits of this curve for each analyte is required. The significance of using MFI over concentration values for the life scientist means higher statistical power and lower costs.

The Statistical Value of Raw Fluorescence Signal in Luminex xMAP Based Multiplex Immunoassays.

Tissue samples (plasma, saliva, serum or urine) from 169 patients classified as either normal or having one of seven possible diseases are analysed across three 96-well plates for the presences of 37 analytes using cytokine inflammation multiplexed immunoassay panels. Censoring for concentration data caused problems for analysis of the low abundant analytes. Using fluorescence analysis over concentration based analysis allowed analysis of these low abundant analytes. Mixed-effects analysis on the resulting fluorescence and concentration responses reveals a combination of censoring and mapping the fluorescence responses to concentration values, through a 5PL curve, changed observed analyte concentrations. Simulation verifies this, by showing a dependence on the mean florescence response and its distribution on the observed analyte concentration levels. Differences from normality, in the fluorescence responses, can lead to differences in concentration estimates and unreliable probabilities for treatment effects. It is seen that when fluorescence responses are normally distributed, probabilities of treatment effects for fluorescence based t-tests has greater statistical power than the same probabilities from concentration based t-tests. We add evidence that the fluorescence response, unlike concentration values, doesn’t require censoring and we show with respect to differential analysis on the fluorescence responses that background correction is not required.

Coverage and consistency: Bioinformatics aspects of the analysis of multirun iTRAQ experiments with wheat leaves

The hexaploid genome of bread wheat (Triticum aestivum) is large (17 Gb) and repetitive, and this has delayed full sequencing and annotation of the genome, which is a prerequisite for effective quantitative proteomics analysis. Aware of these constraints we investigated the most effective approaches for shotgun proteomic analyses of bread wheat that would support large-scale quantitative comparisons using iTRAQ reagents. We used a data set that was generated by two-dimensional LC-MS of iTRAQ labeled peptides from wheat leaves. The main items considered in this study were the choice of sequence database for matching LC-MS data, the consistency of identification when multiple LC-MS runs were acquired, and the options for downstream functional analysis to generate useful insight. For peptide identification we examined the extensive NCBInr plant database, a smaller composite cereals database, the Brachypodium distachyon model plant genome, the EST-based SuperWheat database, as well as the genome sequence from the recently sequenced D-genome progenitor Aegilops tauschii. While the most spectra were assigned by using the SuperWheat database, this extremely large database could not be readily manipulated for the robust protein grouping that is required for large-scale, multirun quantitative experiments. We demonstrated a pragmatic alternative of using the composite cereals database for peptide spectra matching. The stochastic aspect of protein grouping across LC-MS runs was investigated using the smaller composite cereals database where we found that attaching the Brachypodium best BLAST hit reduced this problem. Further, assigning quantitation to the best Brachypodium locus yielded promising results enabling integration with existing downstream data mining and functional analysis tools. Our study demonstrated viable approaches for quantitative proteomics analysis of bread wheat samples and shows how these approaches could be similarly adopted for analysis of other organisms with unsequenced or incompletely sequenced genomes.

Optical flow analysis of the ventral cellular layer of the migrating Dictyostelium discoideum slug

A digital image analysis system for extracting motion information from time-varying digital light microscopy images is presented. This system is then used to map out the movement profile of the surface layer of cells in contact with the substratum through the extracellular matrix (ECM) of the migrating Dictyostelium discoideum slug. From digital high magnification light microscopy images, the morphology of moving cells within the tail region of a young migrating wild-type WS380B slug is described, and compared with the morphology of streaming D. discoideum cells. It is shown that: (i) when the migrating tip of the slug touches the agar substrate, cells in the anterior ventral surface layer of the tip region slow dramatically; (ii) overall cell movement in the ventral surface layer of the migrating D. discoideum slug is slower than the movement of the slug as a whole; and (iii) in less than 10% of cases a wave of movement (groups of cells synchronously slowing down and then accelerating forward) propagates down the slug axis at approx. 1.2 microns s-1. The time interval between waves may be related to the time interval between tip-to-substratum contact that is periodically re-established during normal WS380B slug migration after each aerial projection of the tip.

Linking structural features of protein complexes and biological function

Protein–protein interaction (PPI) establishes the central basis for complex cellular networks in a biological cell. Association of proteins with other proteins occurs at varying affinities, yet with a high degree of specificity. PPIs lead to diverse functionality such as catalysis, regulation, signaling, immunity, and inhibition, playing a crucial role in functional genomics. The molecular principle of such interactions is often elusive in nature. Therefore, a comprehensive analysis of known protein complexes from the Protein Data Bank (PDB) is essential for the characterization of structural interface features to determine structure–function relationship. Thus, we analyzed a nonredundant dataset of 278 heterodimer protein complexes, categorized into major functional classes, for distinguishing features. Interestingly, our analysis has identified five key features (interface area, interface polar residue abundance, hydrogen bonds, solvation free energy gain from interface formation, and binding energy) that are discriminatory among the functional classes using Kruskal‐Wallis rank sum test. Significant correlations between these PPI interface features amongst functional categories are also documented. Salt bridges correlate with interface area in regulator‐inhibitors (r = 0.75). These representative features have implications for the prediction of potential function of novel protein complexes. The results provide molecular insights for better understanding of PPIs and their relation to biological functions.

Identification of ovarian cancer associated genes using an integrated approach in a Boolean framework

BackgroundCancer is a complex disease where molecular mechanism remains elusive. A systems approach is needed to integrate diverse biological information for the prognosis and therapy risk assessment using mechanistic approach to understand gene interactions in pathways and networks and functional attributes to unravel the biological behaviour of tumors.ResultsWe weighted the functional attributes based on various functional properties observed between cancerous and non-cancerous genes reported from literature. This weighing schema was then encoded in a Boolean logic framework to rank differentially expressed genes. We have identified 17 genes to be differentially expressed from a total of 11,173 genes, where ten genes are reported to be down-regulated via epigenetic inactivation and seven genes are up-regulated. Here, we report that the overexpressed genes IRAK1, CHEK1 and BUB1 may play an important role in ovarian cancer. We also show that these 17 genes can be used to form an ovarian cancer signature, to distinguish normal from ovarian cancer subjects and that the set of three genes, CHEK1, AR, and LYN, can be used to classify good and poor prognostic tumors.ConclusionWe provided a workflow using a Boolean logic schema for the identification of differentially expressed genes by integrating diverse biological information. This integrated approach resulted in the identification of genes as potential biomarkers in ovarian cancer.

Priming Adipose-Derived Mesenchymal Stem Cells with Hyaluronan Alters Growth Kinetics and Increases Attachment to Articular Cartilage

Background. Biological therapeutics such as adipose-derived mesenchymal stem cell (MSC) therapy are gaining acceptance for knee-osteoarthritis (OA) treatment. Reports of OA-patients show reductions in cartilage defects and regeneration of hyaline-like-cartilage with MSC-therapy. Suspending MSCs in hyaluronan commonly occurs in animals and humans, usually without supporting data. Objective. To elucidate the effects of different concentrations of hyaluronan on MSC growth kinetics. Methods. Using a range of hyaluronan concentrations, we measured MSC adherence and proliferation on culture plastic surfaces and a novel cartilage-adhesion assay. We employed time-course and dispersion imaging to assess MSC binding to cartilage. Cytokine profiling was also conducted on the MSC-secretome. Results. Hyaluronan had dose-dependent effects on growth kinetics of MSCs at concentrations of entanglement point (1 mg/mL). At higher concentrations, viscosity effects outweighed benefits of additional hyaluronan. The cartilage-adhesion assay highlighted for the first time that hyaluronan-primed MSCs increased cell attachment to cartilage whilst the presence of hyaluronan did not. Our time-course suggested patients undergoing MSC-therapy for OA could benefit from joint-immobilisation for up to 8 hours. Hyaluronan also greatly affected dispersion of MSCs on cartilage. Conclusion. Our results should be considered in future trials with MSC-therapy using hyaluronan as a vehicle, for the treatment of OA.