Validation of the novel fibrosis markers by Western blotting indicates that they are promising compared to the markers in ELF test, FibroTest, Hepascore and FIBROSpect.

<p>The novel markers of fibrosis were validated alongside the markers for the ELF test, FibroTest, Hepascore and FIBROSpect using plasma samples from individuals in each of the seven Ishak stages of hepatic scarring as indicated at the top of the figure. (A) Western blots of our novel markers of fibrosis: LTIP, complement C3d, apolipoprotein L1 (ApoL1), apolipoprotein J (ApoJ), corticosteroid-binding globulin (CBG); (B) ELF test, FibroTest, Hepascore and FIBROSpect markers. Western blots of TIMP-1, PIIIP, apolipoprotein A1 (Apo A1), alpha 2 macroglobulin (a2M) and haptoglobin, ELISA data for hyaluronic acid (HA) and levels of bilirubin and gamma glutamyltranspeptidase. For hyaluronic acid, normal individuals are recognised to have hyaluronic acid below 120 ng/ml and cirrhotic patients above 250 ng/ml as indicated with the dashed lines. The two letter codes indicate if the marker is used in ELF test (EL), FibroTest (FT), Hepascore (HS) or FIBROSpect (FS).</p

Details of the 20 plasma samples used for 2-DE and Western blotting.

<p>Sample name for 2-DE (in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039603#pone.0039603.s001" target="_blank">Figure S1</a>) and lane number for Western blotting (in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039603#pone-0039603-g004" target="_blank">Figure 4</a>) are shown.</p><p>na  =  not analysed.</p><p>Other clinical details for these samples and the other 30 plasma samples studied are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039603#pone.0039603.s007" target="_blank">Table S1</a>.</p

Magnified regions of the gels showing changes for selected potential novel fibrosis biomarkers.

<p>The relative position of the identified protein is circled. (A) LTIP is present in normal plasma but decreased in plasma from cirrhotic patients; (B) Zinc-alpha-2-glycoprotein is present in normal plasma and decreased in plasma from cirrhotic patients; (C) Decreased feature of beta haptoglobin at pH 5.46–5.49. The top panel shows evenly spaced array of beta haptoglobin spots showing no significant difference between normal plasma and plasma from cirrhotic patients. The bottom panel shows zoomed image of the beta haptoglobin spot observed at approximately pH 5.46–5.49 which is present in normal plasma and decreased in plasma from cirrhotic patients; (D) Complement C3dg is absent in normal plasma but present in plasma from cirrhotic patients.</p

DataSheet1_Crystal polymorphism in fragment-based lead discovery of ligands of the catalytic domain of UGGT, the glycoprotein folding quality control checkpoint.PDF

None of the current data processing pipelines for X-ray crystallography fragment-based lead discovery (FBLD) consults all the information available when deciding on the lattice and symmetry (i.e., the polymorph) of each soaked crystal. Often, X-ray crystallography FBLD pipelines either choose the polymorph based on cell volume and point-group symmetry of the X-ray diffraction data or leave polymorph attribution to manual intervention on the part of the user. Thus, when the FBLD crystals belong to more than one crystal polymorph, the discovery pipeline can be plagued by space group ambiguity, especially if the polymorphs at hand are variations of the same lattice and, therefore, difficult to tell apart from their morphology and/or their apparent crystal lattices and point groups. In the course of a fragment-based lead discovery effort aimed at finding ligands of the catalytic domain of UDP–glucose glycoprotein glucosyltransferase (UGGT), we encountered a mixture of trigonal crystals and pseudotrigonal triclinic crystals—with the two lattices closely related. In order to resolve that polymorphism ambiguity, we have written and described here a series of Unix shell scripts called CoALLA (crystal polymorph and ligand likelihood-based assignment). The CoALLA scripts are written in Unix shell and use autoPROC for data processing, CCP4-Dimple/REFMAC5 and BUSTER for refinement, and RHOFIT for ligand docking. The choice of the polymorph is effected by carrying out (in each of the known polymorphs) the tasks of diffraction data indexing, integration, scaling, and structural refinement. The most likely polymorph is then chosen as the one with the best structure refinement Rfree statistic. The CoALLA scripts further implement a likelihood-based ligand assignment strategy, starting with macromolecular refinement and automated water addition, followed by removal of the water molecules that appear to be fitting ligand density, and a final round of refinement after random perturbation of the refined macromolecular model, in order to obtain unbiased difference density maps for automated ligand placement. We illustrate the use of CoALLA to discriminate between H3 and P1 crystals used for an FBLD effort to find fragments binding to the catalytic domain of Chaetomium thermophilum UGGT.</p

Synthetic 2-DE image representing all protein spots present in plasma samples in the comparison between normal healthy controls and cirrhosis patients.

<p>Gels were run using pH 3–5.6 nonlinear immobilized pH gradient DryStrips with 9–16% (w/v) SDS-PAGE gradient gels and were stained using the fluorescent dye OGT 1238. The synthetic image shown was created using accurate spot matching as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039603#pone.0039603-Garcia1" target="_blank">[19]</a>. Differentially expressed features are indicated by arrows and the Swiss-Prot entry names are shown in parentheses. The names of selected proteins are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039603#pone-0039603-t002" target="_blank">Table 2</a> and a full list of all proteins shown on this image can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039603#pone.0039603.s008" target="_blank">Table S2</a>. N, feature present only in gels of plasma from normal healthy controls; C, feature present only in gels of plasma from cirrhosis patients; *, features present in gels of plasma from both normal healthy controls and cirrhosis patients but expressed to a higher extent in the group indicated. For complete gel figures, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039603#pone.0039603.s001" target="_blank">Figure S1</a>.</p

Uncleaved C3dg is elevated in hepatic cirrhosis.

<p>Using pH 3–5.6 gels, complement C3 was identified in a feature at approximately pH 4.9, MWt 38 kDa, only in gels for cirrhotic plasma. The full length sequence of complement C3 is shown with the alpha chain underlined, beta chain in italics, C3dg in bold and identified peptides highlighted in grey. Highlighted in black is the thioester site which is known to be cleaved by the fibrinolytic enzyme plasmin.</p

Western blot band densitometry.

<p>The five plots on the left show densitometry data for our five markers; from top to bottom: LTIP, complement C3d, apolipoprotein L1, apolipoprotein J and corticosteroid-binding globulin. The five plots on the right show densitometry data for all the markers that were blotted for in the ELF test (TIMP1 and PIIIP), FibroTest (apolipoprotein A1, alpha 2 macroglobulin and haptoglobin), Hepascore (alpha 2 macroglobulin) and FIBROSpect (TIMP-1, and alpha 2 macroglobulin). Each point represents the average band intensity for four patient samples. Error bars show +/− standard error.</p

Efficacy of <i>N</i>B-DNJ in guinea pigs challenged with EBOV.

<p>Female guinea pigs were infected with 10³ pfu of EBOV (Zaire strain) via IV cannula. Animals were untreated (n = 3), treated IV TID (8 hourly) with 1850 mg/kg/day <i>N</i>B-DNJ (n = 6) or placebo (n = 3) for 14 days. (A) Percentage of surviving animals. (B) Signs of illness. (C) Body temperature, compared to their temperature on day 0 (baseline) and (D) weight change as a percentage of body weight on day 0 (baseline). Mean for each group +/- standard deviation is plotted. Note: Temperatures in panel C show means of only 5 out of the 6 animals treated with <i>N</i>B-DNJ due to the temperature chip failing in one of the animals in this group.</p

Severity of histological lesions in samples of EBOV challenged animals after treatment (second efficacy study).

<p>Severity of histological lesions in samples of EBOV challenged animals after treatment (second efficacy study).</p

Effect of iminosugar treatment on guinea pig weight.

<p>Female guinea pigs were treated via IV cannula TID with 1850 mg/kg/day <i>N</i>B-DNJ (n = 6), 120 mg/kg/day M<i>O</i>N-DNJ (n = 4) or placebo (n = 4) for 16 days. Weight change as a percentage of body weight on day 0 (baseline) is shown for placebo (black circle), <i>N</i>B-DNJ (blue square, including euthanised animal) and M<i>O</i>N-DNJ (orange triangle) treatment groups. Means for each group +/- standard deviation are plotted.</p