C labeling experiments at metabolic nonstationary conditions: An exploratory study-4

Nts.<p><b>Copyright information:</b></p><p>Taken from "C labeling experiments at metabolic nonstationary conditions: An exploratory study"</p><p>http://www.biomedcentral.com/1471-2105/9/152</p><p>BMC Bioinformatics 2008;9():152-152.</p><p>Published online 18 Mar 2008</p><p>PMCID:PMC2373788.</p><p></p

C labeling experiments at metabolic nonstationary conditions: An exploratory study-2

D experiment S(right). Deep blue represents a correlation of 1, deep red represents -1.<p><b>Copyright information:</b></p><p>Taken from "C labeling experiments at metabolic nonstationary conditions: An exploratory study"</p><p>http://www.biomedcentral.com/1471-2105/9/152</p><p>BMC Bioinformatics 2008;9():152-152.</p><p>Published online 18 Mar 2008</p><p>PMCID:PMC2373788.</p><p></p

C labeling experiments at metabolic nonstationary conditions: An exploratory study-1

H a relative standard deviation of about 46% (first step of the black line). Only two parameters can be determined with less than 70% standard deviation. In contrast, using a labeled substrate, 10 parameters (about 40% of the parameters) can be determined with a standard deviation of less than 70%.<p><b>Copyright information:</b></p><p>Taken from "C labeling experiments at metabolic nonstationary conditions: An exploratory study"</p><p>http://www.biomedcentral.com/1471-2105/9/152</p><p>BMC Bioinformatics 2008;9():152-152.</p><p>Published online 18 Mar 2008</p><p>PMCID:PMC2373788.</p><p></p

C labeling experiments at metabolic nonstationary conditions: An exploratory study-0

And K) and v(K). Second to last row: Sensitivities of selected mass isotopomer concentrations to the same parameters.<p><b>Copyright information:</b></p><p>Taken from "C labeling experiments at metabolic nonstationary conditions: An exploratory study"</p><p>http://www.biomedcentral.com/1471-2105/9/152</p><p>BMC Bioinformatics 2008;9():152-152.</p><p>Published online 18 Mar 2008</p><p>PMCID:PMC2373788.</p><p></p

C labeling experiments at metabolic nonstationary conditions: An exploratory study-3

And K) and v(K). Second to last row: Sensitivities of selected mass isotopomer concentrations to the same parameters.<p><b>Copyright information:</b></p><p>Taken from "C labeling experiments at metabolic nonstationary conditions: An exploratory study"</p><p>http://www.biomedcentral.com/1471-2105/9/152</p><p>BMC Bioinformatics 2008;9():152-152.</p><p>Published online 18 Mar 2008</p><p>PMCID:PMC2373788.</p><p></p

Cell length over time graphs.

<p>Asterisks denote detected cell division events. <b>A</b> and <b>B</b> are derived from Case Study A, and show high quality tracks. Good tracks show the typical sawtooth curve of a cell repeatedly growing in length, then dividing. <b>C</b> and <b>D</b> are derived from Case Study B, and show a good track, as well as a bad track. The bad track is an example for a typical artifact, e.g. produced by continuously detected top or bottom channel structure fragments. As artifact tracks differ in structure from good tracks, filtering them is straigthforward.</p

Bacterial isolation and optimal sampling time point allow robust quantification of metabolites.

(A) Uninfected host RAW264.7 cells (denoted by “-”), and host cells infected with a replication-deficient mutant (ΔssaV), or the wt were separated at 20 hpi by SDS-PAGE alongside serially diluted STm grown in monoculture (dilutions denoted above blot). Immunoblot was performed using an antibody against the bacterial RecA protein (38 kDa) for the quantification of bacterial material among host material in the infection assay by densitometry (numbers (a.u.) on the blot) via a standard curve. The experiment was performed in biological duplicates (S1 Raw Image). (B) Enrichment/depletion after filtration, and washing/concentration applied to infected RAW264.7 cells. Fold protein levels relative to the level before the filtration are shown for bacteria (anti-RecA), cytosolic proteins (anti-GAPDH), mitochondria (anti-VDAC1), histones from nuclei (anti-H3), and the endoplasmic reticulum (ER, anti-Calreticulin). Mitochondria and nuclei were removed by the filtering step, and solubilized ER and cytosolic proteins by centrifuging and washing the bacterial-containing flow-through. Bars are the averages of triplicates. Quantification by densitometry from immunoblots (Methods). (C) Same as in (B) but with the bacterial enrichment protocol applied to infected HeLa cells. Using the same protocol, bacterial enrichment from HeLa cells is less strong compared to RAW264.7 cells, reflecting physiological differences between the 2 different cell lines. Bars are the averages from biological duplicates. (D) Same data as in Fig 2D, but ratios of peak areas of control over the sample are separately depicted here for each metabolite. The solid, dashed, and dotted lines indicate the height at which the control reaches 100%, 50%, or 20%, respectively, of the peak area of the sample. Data are representative of 2 independent experiments in infected host RAW264.7 cells. (E) Total fractional labeling enrichment (TFLE, n = 38) from bacterial monocultures fed with 50% labeled mannitol compared between the standard and the fast enrichment protocol. Green dots indicate metabolites with significantly different (adjusted p-value STm inside HeLa cells in a mtl-containing medium. Line is the average of biological triplicates. (I) Absolute metabolite concentrations in the cell culture medium during STm replication inside RAW264.7 cells and HeLa cells. Lines are the average of biological duplicates. The black line on the right side of each figure indicates the concentration range covered by the standard curve. (J) Fractional 13C labeling enrichment for 31 metabolites (their 244 isotopologues are compared) from bacteria isolated from RAW264.7 cells compared between 12 and 16 hpi (r is the Pearson correlation). Dots are averages from biological duplicates. The data underlying this figure can be found in S1 Data. (TIF)</p

Metabolic activity sensing of cells exposed to unviable antibiotic concentrations and metabolic activity changes of descendants after antibiotic stress.

<p>After an initial growth phase in complex medium BHI, cells were exposed to <b>(A)</b> 10 μg/mL ampicillin (cell wall inhibition, n = 5 colonies) and <b>(B)</b> 10 μg/mL chloramphenicol (inhibition of protein synthesis, n = 5 colonies), respectively, for one hour. The antibiotics were added to the perfusion medium during the exposure time and after an hour perfusion with antibiotic free BHI was continued. Calcein mean single cell fluorescence revealed how the antibiotics change the bacterial fitness during antibiotic exposure. (<b>C</b>) The apparent growth rate was determined for five microcolonies treated for one hour with AMP or CHL.</p

Metabolic activity sensing under iron limitation at single cell level.

<p><b>(A)</b> Mean single cell fluorescence traces of all descendants of a progenitor cell are shown under intermittent iron supply. A daughter cell with higher calcein fluorescence than its siblings (light blue arrow) generated growing descendants with increased mean single cell fluorescence traces (light blue lines) in comparison to other descendants of the initial progenitor cell (grey mean single cell fluorescence traces). A single spontaneously non-growing cell changed from a dividing state to a non-growing state loosing esterase activity and intracellular calcein due to lysis (indicated by black arrow, lysed cell shown in <b>(C)</b> and <b>(D)</b>, respectively). <b>(B)</b> Mean single cell fluorescence traces of spontaneously non-growing cells of a microcolony are shown. Lysing cells (red lines) lost fluorescence spontaneously after lysis (red arrow). However, they were still detectable as apparently intact cells (end of recognition marked with red asterisks). The mean single cell fluorescence traces of a spontaneously non-growing but metabolically active cell (blue line) are shown in comparison. Mean single cell fluorescence increases shortly after cell birth (blue arrow). <b>(C)</b> A lysed but apparently intact cell (marked with black dashed line) and a cell directly before performing lysis (marked with red dashed line) are depicted. <b>(D)</b> Lysed cells still appear to be intact cells (red dashed line and black dashed line, respectively) after lysis. These non-growing cells showed no calcein fluorescence and were considered to be metabolically non-active. <b>(E)</b> A non-growing but metabolically active cell after re-supply of iron with elevated mean single cell fluorescence (marked by blue dashed line).</p

Mixing plots in model space (varying in reaction bidirectionalities, all replicates).

To certify that the replicates did not get stuck in different regions of model space, all replicates are shown. Each subplot represents 1 independent MCMC run. The plots show that the sampler mixes well in the model space and that the mixing is very reproducible for all 10 replicate chains. (A) Low glucose concentration, (B) high glucose concentration. (TIF)</p