Characteristics of AE associated with Heparin & Dialysis 2000–2010.

<p>Characteristics of AE associated with Heparin & Dialysis 2000–2010.</p

Complement components C1r and C1s compete with FXIIa for C1inh.

<p>Kallikrein activity of (A) different doses of prekallikrein incubated with a fixed dose (1 ng) of FXIIa; (B) a fixed dose (0.5 µg) of prekallikrein incubated with different doses of FXIIa; (C) different doses of C1inh premixed with a fixed dose (1 ng) of FXIIa before combining with prekallikrein (1 µg). (D) Kallikrein activity of 0.5 µg of prekallikrein incubated with different combinations of FXIIa, C1inh and C1r/C1s. Experiments were repeated independently at least three times. Duplicate samples were used in panels C and D.</p

Factors that can influence bradykinin levels.

<p>The level of bradykinin (BK) is determined by the levels of kallikrein (KAL), high molecular weight kininogen (HMWK) and enzymes that degrade BK, such a angiotensin converting enzyme (ACE), aminopeptidase P (APP) and neutral carboxypeptidase (CPN). Activated factor XII (FXIIa) generates KAL from prekallikrein (PK). C1 inhibitor (C1inh) can inhibit both FXIIa and KAL. OSCS can facilitate FXIIa activity through enhancing its generation from FXII and/or stabilizing a complex of FXIIa, PK and HMWK. OSCS may also have a direct effect on C1inh. Other factors in the complement pathway and coagulation cascade can interact with C1inh and thus decrease C1inh availability for limiting BK production.</p

OSCS-induced kallikrein activation in patient plasma samples.

<p>(A) OSCS-induced kallikrein activation and (B) functional C1inh levels in plasma samples from normal individuals (n = 40) and from patients with septic shock (n = 32). (C) The relationship of OSCS-induced kallikrein activity and functional C1inh levels using the same samples. The error bars correspond to mean ± SD for each patient group. The correlation coefficient r = −0.413 indicates a negative association between functional C1inh levels and OSCS-induced kallikrein activation. Data shown here were representative of three independent experiments.</p

OSCS kallikrein activation kinetics and dose response.

<p>(A) Kinetics of kallikrein activation in normal plasma and C1inh-depleted plasma with the addition of 25 µg/ml of OSCS. Kallikrein activity was also determined in (B) normal human plasma and (C) C1inh-depleted plasma with different doses of OSCS. The OSCS dose response was also evaluated with plasma samples treated with antibody-coated bacteria. Experiments were repeated independently twice. Duplicate samples were used in panel A.</p

Bacteria incubation consumes C1 inhibitor and increases OSCS-induced kallikrein activity.

<p>(A) Natural antibody-coated <i>E. coli</i> BL21 bacteria were incubated with human plasma at 37°C, washed by PBS, and the level of C1 inhibitor deposited on the bacteria was determined by addition of horseradish peroxidase-conjugated anti-C1 inhibitor antibody and then developement with ABTS substrate as described in the Materials and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034978#s2" target="_blank">Methods</a>. (B) An ELISA assay showed decreased C1 inhibitor levels in plasma treated with antibody-coated <i>E. coli</i> BL21 bacteria. (C) OSCS and OSCS-contaminated heparin induced kallikrein activity were increased in plasma treated with antibody-coated bacteria as compared to untreated plasma. PBS, CSA or uncontaminated heparin did not induce kallikrein activity in either plasma. (D) Kallikrein activity in plasma treated with or without antibody-coated <i>E. coli</i> BL21 bacteria, C1 inhibitor (200 µg/ml), or OSCS (25 µg/ml) was measured. Data shown were representative of three independent experiments.</p

High-Throughput RNA FISH Analysis by Imaging Flow Cytometry Reveals That Pioneer Factor Foxa1 Reduces Transcriptional Stochasticity

<div><p>Genes are regulated at the single-cell level. Here, we performed RNA FISH of thousands of cells by flow cytometry (flow-RNA FISH) to gain insight into transcriptional variability between individual cells. These experiments utilized the murine adenocarcinoma 3134 cell line with 200 copies of the MMTV-Ras reporter integrated at a single genomic locus. The MMTV array contains approximately 800–1200 binding sites for the glucocorticoid receptor (GR) and 600 binding sites for the pioneer factor Foxa1. Hormone activation of endogenous GR by dexamethasone treatment resulted in highly variable changes in the RNA FISH intensity (25–300 pixel intensity units) and size (1.25–15 µm), indicative of probabilistic or stochastic mechanisms governing GR and cofactor activation of the MMTV promoter. Exogenous expression of the pioneer factor Foxa1 increased the FISH signal intensity and size as expected for a chromatin remodeler that enhances transcriptional competence through increased chromatin accessibility. In addition, specific analysis of Foxa1-enriched cell sub-populations showed that low and high Foxa1 levels substantially lowered the cell-to-cell variability in the FISH intensity as determined by a noise calculation termed the % coefficient of variation. These results suggest that an additional function of the pioneer factor Foxa1 may be to decrease transcriptional noise.</p></div

Automated detection of RNA FISH by imaging flow cytometry.

<p>(A) Organization of the MMTV-Ras transgene in the 3134 cell line. The 3134 cell line contains approximately 200 tandem repeats of MMTV-LTR driving viral Ras RNA on chromosome 4. (B) A Dig-labeled DNA probe was generated by nick translation of the pM18 plasmid containing the viral Ras coding sequence. Agarose gel electrophoresis revealed a DNA probe size within a 250–500 base pair range. (C) Imaging of cells following probe hybridization showed the presence of distinct nuclear FISH signals at a 40X magnification. The FISH signal was detected with anti-Dig secondary antibodies conjugated to a green-fluorescent dye. BF = Bright Field; DRAQ5 = nuclear stain.</p

Time course experiments of the MMTV-Ras transcriptional response.

<p>(A) Frequency distribution of the percentage of cells exhibiting 1 FISH signal following dexamethasone treatment for 5, 30, 60, 120 and 240 mins. (B, C) Box-and-whisker plots of the RNA FISH signal intensity (mean pixel intensity units) and size (µm) distributions at each time point. (D, E) Computations of the individual cell-to-cell variability in the FISH signal intensity and size using the % coefficient of variation (% CV). Sample sizes for each time point are as follows: 5 mins (n = 7777), 30 mins (n = 10178), 60 mins (n = 11058), 120 mins (n = 10887) and 240 mins (n = 227). (F) Scatter plot showing a positive linear relationship between the FISH intensity (mean pixel intensity units) and the total FISH intensity (total pixel intensity units) within each FISH foci using the 60 min time point data (n = 11058). (G) Scatter plot showing a positive linear relationship between the FISH size (µm) and the total RNA FISH intensity (total pixel intensity units) using the 60 min time point data (n = 11058). * in (B,C), indicates a significant difference from the 5 mins time point as determined by the Mann-Whitney U Test (p< 0.001).</p

Foxa1 expression in the 3134 cell line enhances MMTV-Ras transcription.

<p>(A) Schematic of full-length Foxa1 and the C-terminal CT1 truncation mutant. The central DBD region interacts with DNA. Two C-terminal regions (black vertical bars) are important for core histone interactions. (B, C) Box-and-whisker plots of the RNA FISH signal intensity (mean pixel intensity units) and size (µm) distributions within each cell group. Sample sizes for each condition: Veh+pcDNA (n = 899), Dex+pcDNA (n = 4584), Veh+Foxa1 (n = 1067), Dex+Foxa1 (n = 2195), Veh+CT1 (n = 225), Dex+CT1 (n = 1959). (D) Comparative analysis of Ras RNA expression by qRT-PCR. Ras RNA expression is normalized to mouse β-actin. Data are expressed relative to that of Dex+pcDNA which is set to 100% (n≥3). (E) Bright Detail Similarity of Foxa1 colocalization with the RNA FISH foci. (F) Quantification of the percentage of total Foxa1-transfected cells showing positive Foxa1 positive (+) immunofluorescence. *, indicates a significant difference from the Dex+pcDNA group in each graph as determined by the Mann-Whitney U Test (p< 0.001).</p