8 research outputs found
Photocatalysis by 3,6-Disubstituted‑<i>s</i>‑Tetrazine: Visible-Light Driven Metal-Free Green Synthesis of 2‑Substituted Benzimidazole and Benzothiazole
<i>s</i>-Tetrazine based molecules were prepared for
visible-light-driven organic transformations. The 3,6-diÂ(pyridin-2-yl)-1,2,4,5-tetrazine
(<b>pytz</b>) derivative shows visible light absorption and
reversible one-electron reduction behavior. In the presence of <b>pytz</b> and aerial oxygen, aldehyde reacts with <i>o</i>-phenylenediamine or <i>o</i>-aminothiophenol under visible
light irradiation at ambient temperature to produce corresponding
2-substituted benzimidazoles and benzothiazoles, respectively. <b>Pytz</b> catalyst demonstrates excellent catalytic activity for
alkyl, aryl, organo-metallic substituted aldehydes and reducing sugar.
The reaction yield is high for both the electron-donating and electron
withdrawing substituents in aromatic aldehydes. The use of a metal-free
catalyst and visible light energy, along with the mild reaction conditions,
makes this reaction an environmentally benign and energy-saving chemical
process
Proteomic Screening of Human Targets of Viral microRNAs Reveals Functions Associated with Immune Evasion and Angiogenesis
<div><p>Kaposi's sarcoma (KS) is caused by infection with Kaposi's sarcoma-associated herpesvirus (KSHV). The virus expresses unique microRNAs (miRNAs), but the targets and functions of these miRNAs are not completely understood. In order to identify human targets of viral miRNAs, we measured protein expression changes caused by multiple KSHV miRNAs using pulsed stable labeling with amino acids in cell culture (pSILAC) in primary endothelial cells. This led to the identification of multiple human genes that are repressed at the protein level, but not at the miRNA level. Further analysis also identified that KSHV miRNAs can modulate activity or expression of upstream regulatory factors, resulting in suppressed activation of a protein involved in leukocyte recruitment (ICAM1) following lysophosphatidic acid treatment, as well as up-regulation of a pro-angiogenic protein (HIF1α), and up-regulation of a protein involved in stimulating angiogenesis (HMOX1). This study aids in our understanding of miRNA mechanisms of repression and miRNA contributions to viral pathogenesis.</p></div
KSHV miRNAs increase HIF1α and HMOX1 protein levels.
<p>(A) Western blot analysis of cells transfected with miRNAs and exposed to hypoxia (blots above, quantitation below). (B) Luciferase reporters containing hypoxia responsive elements (HRE-luc) or parental control (ctl-luc) in the promoter were transfected into 293 cells with miRNAs and exposed to hypoxia (1% oxygen for 16 hr.). Luciferase activity was normalized to an internal control reporter as well as the condition without hypoxia and transfected with the negative control miRNA. (C) Cells were treated as in (A) and HIF1α mRNA was measured using qPCR. (D) The same samples used in (A) were analyzed by Western blot analysis for proteins shown. Data were analyzed and presented as in (A). (E) Cells were transfected with KSHV miRNAs or controls and analyzed for BACH1 and HMOX1 protein levels using Western blot analysis. Average relative protein expression changes are shown with error bars representing S.D. from ≥3 biological replicates. Asterisks denote P<0.05 using a T-test.</p
Enriched classes of proteins repressed by KSHV miRNAs.
<p>A list of the 5% most repressed proteins (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003584#ppat-1003584-g001" target="_blank">Fig. 1C</a>) was analyzed for the most enriched networks of interacting gene products using Metacore (GeneGo). This is a similar analysis to gene ontology term enrichment analysis.</p
Proteomic screening for KSHV miRNA targets.
<p>(A) Experimental design shows HUVECs transfected with control or KSHV miRNAs, then labeled with stable isotope-labeled amino acids (normal/light “L”, medium-heavy “M”, and heavy “H”), cells from both conditions were combined and LC-MS/MS was used to measure relative abundance of peptides corresponding to the labeled amino acids. Green proteins symbolize proteins that were translated before the amino acid labeling and/or do not contain stable isotope-labeled amino acids. (B) Argonaute2 (AGO2) was immunoprecipitated from HUVECs. Western blot shows unbound lysate (flow-through, “FT”) and immunoprecipitated material (IP) probed with AGO2 antibody. Graph shows RT-PCR miRNA data from AGO2-immunoprecipiated material from at least three immunoprecipitations per sample from either KSHV-infected HUVECs (black) or HUVECs transfected with miRNA mimics (gray) as in pSILAC assay. (C). Known miRNA targets (TWEAKR and BCLAF1) are repressed when 16 miRNA mimics are co-transfected. Shown is two-color quantitative Western blot analysis from three biological replicates. (D) Range of relative changes in protein expression of all proteins detected with at least two peptides per protein and found in two biological replicates. (E) Table shows the most repressed proteins in the KSHV miRNA samples. Protein levels were determined by pulsed SILAC and mRNA levels were determined by microarray.</p
Validation of predicted miRNA targets with Western blotting and <i>de novo</i> infection.
<p>(A) Primary HUVECs were transfected without miRNAs (no), a negative control miRNA (ctl), or KSHV miRNAs. Whole cells lysates were analyzed using Western blotting and normalized to actin (loading control) and the negative control miRNA (ctl). MirVana miRNAs mimics for miR-K8 in STAT3 assays are shown. Other mirVana miRNA mimic results are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003584#ppat.1003584.s002" target="_blank">Figure S2</a>. Average relative protein expression changes are shown with error bars showing S.D. from ≥3 biological replicates. (B) Primary HUVECs were <i>de novo</i> infected with KSHV (3 or 7 days post infection) and whole cell lysates were analyzed using Western blot analysis as in (A). Asterisks denote P<0.05, n≥3 using a T-test. (C) Plot showing average changes in protein expression on the horizontal axis from SILAC data and mRNA changes from microarray data (vertical axis) from the same transfections. Gray open circles are gene products found in both assays and black filled circles represent gene products from six validated targets (HMGCS1, STAT3, GRB2, ROCK2, AKAP9, TSPAN3). Multiple microarray probes are indicated for a subset of genes, yielding multiple vertically-aligned circles from multiple microarray probes, but one protein measurement (See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003584#ppat-1003584-g001" target="_blank">Figure 1E</a> for examples).</p
Analysis of miRNA seed-matching sites.
<p>(A) Proteins identified in screen were analyzed for KSHV miRNA seed-matching sites in their corresponding 3′UTRs using TargetScan. Histogram shows the distribution of the number of sites per 3′UTR. (B) Graph displays the fraction of proteins whose transcripts contain no or at least one seed-matching site in the transcripts of proteins with indicated repression levels in the presence of KSHV miRNAs. (C–D) Empirical cumulative distribution graph showing protein expression changes whose transcripts contain at least one miRNA seed-matching site (C) or classes of multiple sites (D).</p
KSHV miRNAs repress <i>intercellular adhesion molecule 1</i> (ICAM1).
<p>(A) HUVECs transfected with control (neg ctl) or KSHV miRNAs were stimulated with lysophosphatidic acid (LPA) to activate ICAM1 protein expression. Shown are results from Western blot analysis of ICAM1 and ROCK2 protein levels (relative to internal control GAPDH) from LPA-treated cells. (B) Western blot analysis of the phosphorylation of Tyr705 of STAT3 and total levels of STAT3. (C) Luciferase assays with the STAT3 3′UTR were performed as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003584#ppat-1003584-g003" target="_blank">Figure 3</a>. (D) HUVECs were infected with KSHV, and ICAM1 protein levels were measured by Western blot analysis. (E) HUVECs were transfected with miRNA inhibitors shown, infected with KSHV, and ICAM1 protein levels were determined by Western blot analysis. Average relative protein expression changes are shown with error bars showing S.D. from ≥3 biological replicates. Asterisks denote P<0.05 using a T-test.</p