Differentiation, Quantification and Identification of Abrin and Abrus precatorius Agglutinin

Abrin, the toxic lectin from the rosary pea plant Abrus precatorius, has gained considerable interest in the recent past due to its potential malevolent use. However, reliable and easy-to-use assays for the detection and discrimination of abrin from related plant proteins such as Abrus precatorius agglutinin or the homologous toxin ricin from Ricinus communis are sparse. To address this gap, a panel of highly specific monoclonal antibodies was generated against abrin and the related Abrus precatorius agglutinin. These antibodies were used to establish two sandwich ELISAs to preferentially detect abrin or A. precatorius agglutinin (limit of detection 22 pg/mL for abrin; 35 pg/mL for A. precatorius agglutinin). Furthermore, an abrin-specific lateral flow assay was developed for rapid on-site detection (limit of detection ~1 ng/mL abrin). Assays were validated for complex food, environmental and clinical matrices illustrating broad applicability in different threat scenarios. Additionally, the antibodies turned out to be suitable for immuno-enrichment strategies in combination with mass spectrometry-based approaches for unambiguous identification. Finally, we were able to demonstrate for the first time how the developed assays can be applied to detect, identify and quantify abrin from a clinical sample derived from an attempted suicide case involving A. precatorius.Peer Reviewe

Discovery of Novel Biomarker Candidates for Liver Fibrosis in Hepatitis C Patients: A Preliminary Study

Background: Liver biopsy is the reference standard for assessing liver fibrosis and no reliable non-invasive diagnostic approach is available to discriminate between the intermediate stages of fibrosis. Therefore suitable serological biomarkers of liver fibrosis are urgently needed. We used proteomics to identify novel fibrosis biomarkers in hepatitis C patients with different degrees of liver fibrosis.Methodology/Principal Findings: Proteins in plasma samples from healthy control individuals and patients with hepatitis C virus (HCV) induced cirrhosis were analysed using a proteomics technique: two dimensional gel electrophoresis (2-DE). This technique separated the proteins in plasma samples of control and cirrhotic patients and by visualizing the separated proteins we were able to identify proteins which were increasing or decreasing in hepatic cirrhosis. Identified markers were validated across all Ishak fibrosis stages and compared to the markers used in FibroTest, Enhanced Liver Fibrosis (ELF) test, Hepascore and FIBROSpect by Western blotting. Forty four candidate biomarkers for hepatic fibrosis were identified of which 20 were novel biomarkers of liver fibrosis. Western blot validation of all candidate markers using plasma samples from patients across all Ishak fibrosis scores showed that the markers which changed with increasing fibrosis most consistently included lipid transfer inhibitor protein, complement C3d, corticosteroid-binding globulin, apolipoprotein J and apolipoprotein L1. These five novel fibrosis markers which are secreted in blood showed a promising consistent change with increasing fibrosis stage when compared to the markers used for the FibroTest, ELF test, Hepascore and FIBROSpect. These markers will be further validated using a large clinical cohort.Conclusions/Significance: This study identifies 20 novel fibrosis biomarker candidates. The proteins identified may help to assess hepatic fibrosis and eliminate the need for invasive liver biopsies.</br

Secondary School Leaving Examinations: The Impact of Expectancies, Values, and Dimensional Comparisons on Male and Female Students’ Science-Oriented Choices

In Germany, secondary school students have to choose at least one STEM subject (mathematics, biology, chemistry, and physics) for their Secondary School Leaving Examinations. In a representative sample of students in grade 13 in one federal state in Germany, we explore male and female students’ subject choices in an expectancy-value as well as dimensional comparison framework by considering prior performance, ability self-concept, and values in the chosen subject. We extend previous research by including dimensional comparisons that students make between the varying subjects they have to choose from. We discriminate between two opposing groups. One group shows a science-avoidance choice pattern by selecting only one science subject: biology (n = 439). The other group shows a science-oriented choice pattern by selecting either physics or chemistry or two STEM subjects of which one is physics or chemistry (n = 248). We measured achievement test scores, relative and absolute midterm grades, ability self-concepts, as well as attainment and utility values in chosen and non-chosen subjects and calculated logistic regressions as well as multigroup models. Our results showed that science-oriented final exam choices depended on two mechanisms. Within the expectancy-value framework, a science-oriented choice pattern was predicted by ability self-concept in mathematics for male and female students. However, attainment and utility values appeared to be irrelevant for this specific choice. Within the dimensional comparison framework, the relative mathematics-English midterm grade was relevant, but only for male students. Our findings raise the question whether male and female students should be encouraged differently in order to stay in the STEM pipeline and how structural conditions may shape pathways into or out of this pipeline

New proteomics approaches for discovering biomarkers: Searching for liver fibrosis markers in hepatitis c patients: Conference abstract from the 2nd International Conference on Proteomics and Bioinformatics

Two-dimensional gel electrophoresis (2-DE) is often used to separate plasma or serum proteins in an attempt to identify novel biomarkers. A major difficulty with this approach is due to high abundant plasma/serum proteins which limits the detection of low abundance features. To overcome this problem a novel proteomics approach was developed and used to identify new fibrosis biomarkers in patients with different stages of liver fibrosis. Plasma samples from healthy individuals and patients with hepatitis C virus (HCV) induced cirrhosis were analysed using 2-DE over a narrow pH 3-5.6 range, a range outside the pH of highly abundant albumin, transferrin and immunoglobulins. Novel markers identified by this approach were validated across all fibrosis stages by Western blotting. 44 candidate biomarkers were revealed of which 20 were novel. Western blot analysis with newly identified biomarkers showed a consistent change with increasing fibrosis stage and were promising when compared to the markers used in established fibrosis tests. This is the first time the pH 3−5.6 range has been used to separate plasma by 2-DE. This pH range is useful for discovering novel biomarkers in all diseases. The novel fibrosis markers identified by this new proteomics approach may help to assess hepatic fibrosis and eliminate the need for invasive liver biopsies

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

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

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

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

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