TLR-induced B-cell differentiation and immunoglobulin secretion of murine B-cell subsets.

<p>A, FACS sorted follicular B-cells (CD19⁺ B220⁺CD23⁺CD21⁻) were cultured in vitro with various TLR ligands as indicated for 5 days and antibody profile of culture supernatants measured by ELISA as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000863#s2" target="_blank">Materials and Methods</a>. B-D, FACS sorted marginal zone, B-1 and peyer's patch B-cells (CD19⁺ B220⁺CD23⁻CD21⁺ for marginal zone B, CD19⁺B220⁺CD23⁻for peritoneal B-1 B and CD19⁺B220⁺ for peyer's patch B cells) were cultured in vitro with various TLR ligands as indicated for 5 days and antibody secretion measured by ELISA as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000863#s2" target="_blank">Materials and Methods</a>.</p

A heat map summary of TLR expression and responsiveness to different TLR ligands by distinct murine B cell subsets.

<p>The values for TLR expression profile, proliferation and antibody secretion in response to various TLR ligands by different B-cell subsets shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000863#pone-0000863-g001" target="_blank">Figures 1</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000863#pone-0000863-g002" target="_blank"></a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000863#pone-0000863-g003" target="_blank">3</a> were plotted on a log scale, and represented as a heat map. For TLR expression, blue represents an 1 fold increase relative to β-actin, green represents 2 fold increase, yellow represents 3 fold increase, red represents 4 fold increase and dark brown represents ≥5 fold increase. For proliferation, blue represents an 1 fold increase relative to unstimulated medium controls, green represents 2 fold increase, yellow represents 3 fold increase, red represents 4 fold increase and dark brown represents 5 fold increase. For antibody secretion, blue represents an 1 fold increase relative to unstimulated medium controls, green represents 2 fold increase, yellow represents 3 fold increase, red represents 4 fold increase and dark brown represents 5 fold increase.</p

TLR expression profile of murine B-cell subsets.

<p>Real-time PCR profile of TLR expression in FACS sorted follicular B-cells (CD19⁺ B220⁺CD23⁺CD21⁻, panel A), marginal Zone B (CD19⁺ B220⁺CD23⁻CD21⁺, panel B), peritoneal B-1 (CD19⁺B220⁺CD23⁻, panel C) and peyer's patch B cells (CD19⁺B220⁺, panel D) as indicated with β-actin as loading control. CD11c+ dendritic cells were used as a positive control (panel E). Values represent the ratio of the TLR to β-actin.</p

Predicting Network Activity from High Throughput Metabolomics

<div><p>The functional interpretation of high throughput metabolomics by mass spectrometry is hindered by the identification of metabolites, a tedious and challenging task. We present a set of computational algorithms which, by leveraging the collective power of metabolic pathways and networks, predict functional activity directly from spectral feature tables without a priori identification of metabolites. The algorithms were experimentally validated on the activation of innate immune cells.</p></div

Application of mummichog to additional data sets.

<p>Metabolite prediction by <i>mummichog</i> is in good agreement with annotation in the original studies, 97% for the human urine data <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003123#pcbi.1003123-Roux1" target="_blank">[63]</a> and 86% for the yeast data <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003123#pcbi.1003123-Clasquin1" target="_blank">[64]</a>. The metabolites not in the original annotation (yellow) can not be compared. The “-z” option in <i>mummichog</i> enforces the presence of primary ion (M+H[+] for positive mode, M−H[−] for negative mode). This shifts the coverage in the huamn data set, but not much for the yeast data of limited annotation.</p

Modular organization of human metabolic network.

<p>A) Hierarchical clustering of the network by the steps between 4204 metabolic reactions, where the warmer color codes for fewer steps. Each red island represents a cluster of closely connected reactions. B) An insert by the while arrow in A. This demonstrates that network modules and pathways correlate with but not equate to each other. C) When measured by reaction steps between metabolites, most metabolites are connected in no more than four steps. This serves as a practical guide in searching subnetworks in the total metabolic network.</p

Metabolic activity network in dendritic cells stimulated by yellow fever virus.

<p>A) Prediction by <i>mummichog</i> directly from <i>m/z</i> feature tables (cell extracts after 6 hours of infection). Metabolites are colored according to log2 fold change. A high resolution copy is given in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003123#pcbi.1003123.s010" target="_blank">Figure S9</a>. B) Further investigation was focused on the subnetwork on the right. Glutamate was not significantly altered, but included for network connectivity.</p

How metabolomics data match metabolic models.

<p>The top section gives statistics of each of the three metabolic models. The bottom section gives the specific number of measured features and tentative metabolite matches in each study case. These tentative matches contain excessive ambiguity, which mummichog aims to resolve.</p

Mummichog redefines the work flow of untargeted metabolomics.

<p>A) In the work flow of untargeted metabolomics, the conventional approach requires the metabolites to be identified before pathway/network analysis, while mummichog (blue arrow) predicts functional activity bypassing metabolite identification. B) Each row of dots represent possible matches of metabolites from one <i>m/z</i> feature, red the true metabolite, gray the false matches. The conventional approach first requires the identification of metabolites before mapping them to the metabolic network. C) <i>mummichog</i> maps all possible metabolite matches to the network and looks for local enrichment, which reflects the true activity because the false matches will distribute randomly.</p

Gene expression confirms the activity network.

<p>A) Cytokines secreted after infection (ELISA) indicate the activation of innate immune programs. B) Down-regulation of transcripts of GCLC, GCLM (subunits of gamma-glutamylcysteine synthetase) and GSS (glutathione synthetase), the key enzymes for glutathione synthesis. C) Nitric oxide has feedback inhibition on the expression of eNOS and iNOS (nNOS was not detected). Gene expression was assayed by quantitative RT-PCR. Infected samples were compared to mocks by student's t-test (n = 3).</p