7 research outputs found

    Integrated Microfluidic Chip and Online SCX Separation Allows Untargeted Nanoscale Metabolomic and Peptidomic Profiling

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    Metabolomics and peptidomics are systems biology approaches in which broad populations of molecular species produced in a cell or tissue sample are identified and quantified. These two molecular populations, metabolites and peptides, can be extracted from tissues in a similar fashion, and we therefore have here developed an integrated platform for their extraction and characterization. This was accomplished by liquid–liquid extraction of peptides and metabolites from tissue samples and online strong cation exchange (SCX) separation to allow characterization of each population individually. The platform was validated both by a mixed set of purified standards and by an analysis of splenic tissue from SIV-infected macaques, showing both good reproducibility in chromatography, with relative standard deviation (RSD) of hold time less than 0.4%, and clear separation of charge state, with ∼95% of molecular features in SCX separated runs at charge states of +1 or +2. Finally, we used this platform to analyze the physiological response to infection in the spleen, showing that the spleen contains an abundance of hemoglobin-derived peptides, which do not appear to change in response to infection, and that there appears to be a large and variable metabolic response to infection. We therefore present a method for peptidomic and metabolomic profiling which is simple, robust, and easy to implement

    Single Chain Variable Fragment Displaying M13 Phage Library Functionalized Magnetic Microsphere-Based Protein Equalizer for Human Serum Protein Analysis

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    Single chain variable fragment (scFv) displaying the M13 phage library was covalently immobilized on magnetic microspheres and used as a protein equalizer for the treatment of human serum. First, scFv displaying M13 phage library functionalized magnetic microspheres (scFv@M13@MM) was incubated with a human serum sample. Second, captured proteins on scFv@M13@MM were eluted with 2 M NaCl, 50 mM glycine-hydrochloric acid (Gly-HCl), and 20% (v/v) acetonitrile with 0.5% (v/v) trifluoroacetic acid in sequence. Finally, the tightly bonded proteins were released by the treatment with thrombin. The eluates were first analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining. Results indicated that the difference of protein concentration was reduced obviously in NaCl and Gly-HCl fractions compared with untreated human serum sample. The eluates were also digested with trypsin, followed by online 2D-strong cation exchange (SCX)-RPLC–ESI-MS/MS analysis. Results demonstrated that the number of proteins identified from an scFv@M13@MM treated human serum sample was improved 100% compared with that from the untreated sample. In addition, the spectral count of 10 high abundance proteins (serum albumin, serotransferrin, α-2-macroglobulin, α-1-antitrypsin, apolipoprotein B-100, Ig γ-2 chain C region, haptoglobin, hemopexin, α-1-acid glycoprotein 1, and α-2-HS-glycoprotein) decreased evidently after scFv@M13@MM treatment. All these results demonstrate that scFv@M13@MM could efficiently remove high-abundance proteins, reduce the protein concentration difference of human serum, and result in more protein identification

    VS1 exhibits malaria transmission-blocking activity in an <i>in vivo</i> system.

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    <p>Panels (<b>A–C</b>) show midgut oocyst intensities from a representative experiment in which <i>An. stephensi</i> mosquitoes were fed on mice infected with <i>Plasmodium berghei</i> pre- and post-injection with VS1-3,000. Each panel represents a different treatment group (A, PBS; B, PVP; C, VS1-3,000) and the X-axis of each is arranged by parasitized mouse (labeled 1–5) pre- and post-injection with VS1.</p

    VS1 exhibits marked transmission-blocking activity against <i>P. falciparum</i> in two divergent anopheline species.

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    <p>(<b>A</b>) Structure of compounds designed to interfere with ionic interactions between <i>Plasmodium</i> ookinetes and midgut apical-surface associated glycosaminoglycans (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003757#ppat.1003757.s001" target="_blank">Figure S1A</a>). The table indicates the average molecular weight (MW) and polydispersity index (PDI) for each species of compound tested in the study. (<b>B–C</b>) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003757#s3" target="_blank">Results</a> from representative replicate SMFAs showing <i>P. falciparum</i> midgut-oocyst intensities in <i>An. gambiae</i> plotted by treatment group. Below each graph, experimental details are provided, where N is the number of mosquito midguts dissected and scored for oocysts, prev is the infection prevalence among mosquitoes in a given treatment group, median is the median oocyst number, mean is the mean oocyst number, and % inhibition is calculated as (median<sub>control</sub> – median<sub>treatment</sub>)/median<sub>control</sub>. (<b>D–E</b>) Same as panels B–C except that <i>An. stephensi</i> were used. The level of statistical significance is denoted by asterisks following Bonferroni correction of <i>z-</i>scores, * = p&lt;0.05, ** = p&lt;0.01, *** = p&lt;0.001.</p

    Summary of the results from two replicate direct feeding assays (DFA) for each VS1 compound using <i>Anopheles stephensi</i> that fed on <i>Plasmodium berghei</i> ANKA 2.34 -infected mice pre- and post-injection with either PBS (carrier-only control), PVP

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    <p>For each treatment group, the data are summarized into four columns. In the first two columns, the means of the median number of oocysts per mosquito for 4–5 mice per group are given for pre- and post-injection feedings, respectively, with the standard error for each reported in parentheses below the mean. In the third column, the means of percent Inhibition, calculated as the average of (median<sub>pre</sub> – median<sub>post</sub>)/median<sub>pre</sub> for each mouse, are reported for each treatment group along with standard errors in parentheses. In the fourth column, <i>P-</i>value results of Mann-Whitney U tests are reported for each set of pre- and post-injection feedings. Only <i>P</i>-values that are significant at a Bonferroni-corrected alpha of 0.0028 are given. NS, non-significant.</p

    VS1 binds to critical <i>Plasmodium</i> micronemal proteins.

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    <p>Immunofluorescence microscopy images of VS1 staining patterns of permeabilized wild type ookinetes from (<b>A</b>) <i>P. falciparum</i> (WT<sub>Pf</sub>) and (<b>B</b>) <i>P. berghei</i> (WT<sub>Pb</sub>). Each row of images depicts brightfield, followed by staining with VS1 (green), P28 (red), and the merged image of VS1, P28, and DAPI nuclear staining (blue). Note that different antibodies were used for each <i>Plasmodium</i> species to stain orthologous surface markers, α-P28 for <i>P. falciparum</i> and α-Pbs21 for <i>P. berghei</i>. Size bar = 10 µm. (<b>C</b>) Binding assays with recombinant <i>P. vivax</i> CTRP and WARP (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003757#ppat.1003757.s005" target="_blank">Figure S5</a>) demonstrate that biotinylated VS1 is recognized by PvWARP and the first vWA domain of PvCTRP. Two representative experiments are shown with assays performed in triplicate. Black bars represent PvWARP (PvW) and gray bars represent PvCTRP (PvC). Each protein, at two concentrations (10 and 5 µg/ml), was allowed to bind with biotinylated VS1 immobilized to a microplate, followed by detection using an anti-HIS MAb (Sigma). In addition, competitive binding assays were performed by incubating the recombinant proteins with heparin (HEP) and chondroitin sulfate A (CS-A) prior to incubation with VS1 and detection of PvWARP and PvCTRP binding to the VS1-coated plate as above. A no-protein control was included in each ELISA assay and used as the background subtraction value. Error bars represent +/−1 standard deviation. (<b>D–F</b>) Immunofluorescence microscopy images demonstrating selective VS1 binding to ookinetes that were generated <i>in vitro</i> from <i>P. berghei</i> (<b>D</b>) CTRP<sub>KO</sub>, (<b>E</b>) ΔTS<sub>7</sub>, and (<b>F</b>) ΔA<sub>6</sub> transgenic lines. VS1 staining of ΔTS<sub>7</sub> but not CTRP<sub>KO</sub> or ΔA<sub>6</sub> ookinetes suggest that the vWA domain of CTRP is the primary binding ligand of VS1, and that staining is specific to the localized expression of CTRP in micronemes. Each row of images depicts brightfield, followed by staining with VS1 (green), P28 (red), and the merged image of VS1, P28, and DAPI nuclear staining (blue). Size bar = 10 µm.</p

    Discovery of Novel Small-Molecule Scaffolds for the Inhibition and Activation of WIP1 Phosphatase from a RapidFire Mass Spectrometry High-Throughput Screen

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    Wild-type P53-induced phosphatase 1 (WIP1), also known as PPM1D or PP2Cδ, is a serine/threonine protein phosphatase induced by P53 after genotoxic stress. WIP1 inhibition has been proposed as a therapeutic strategy for P53 wild-type cancers in which it is overexpressed, but this approach would be ineffective in P53-negative cancers. Furthermore, there are several cancers with mutated P53 where WIP1 acts as a tumor suppressor. Therefore, activating WIP1 phosphatase might also be a therapeutic strategy, depending on the P53 status. To date, no specific, potent WIP1 inhibitors with appropriate pharmacokinetic properties have been reported, nor have WIP1-specific activators. Here, we report the discovery of new WIP1 modulators from a high-throughput screen (HTS) using previously described orthogonal biochemical assays suitable for identifying both inhibitors and activators. The primary HTS was performed against a library of 102 277 compounds at a single concentration using a RapidFire mass spectrometry assay. Hits were further evaluated over a range of 11 concentrations with both the RapidFire MS assay and an orthogonal fluorescence-based assay. Further biophysical, biochemical, and cell-based studies of confirmed hits revealed a WIP1 activator and two inhibitors, one competitive and one uncompetitive. These new scaffolds are prime candidates for optimization which might enable inhibitors with improved pharmacokinetics and a first-in-class WIP1 activator
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