Schematic diagram of the roles of MIF in DV-infected patients’ circulation.

<p>DV can stimulate monocytes and endothelial cells to express MIF. MIF signals through binding to a functional receptor complex CD74/CD44 and G-protein-coupled chemokine receptors (CXCR2/CXCR4) individually <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055018#pone.0055018-Leng1" target="_blank">[20]</a>. Following signaling may activate PI3K/MEK/ERK and downstream JNK signaling pathways, resulting in TM and ICAM-1 expression and causing soluble TM levels to increase in plasma.</p

MIF, sTM and PC concentrations are increased in plasma of patients with DHF.

<p>Levels of (A) MIF, (B) soluble thrombomodulin (sTM) and (C) Protein C (PC) in patients infected with dengue virus and in control groups. Sera from healthy controls (N, n = 12), dengue fever (DF) patients (n = 12) and dengue hemorrhagic fever patients (DHF) sorted into grades I–II (n = 28) and grades III–IV (n = 9) were assayed for MIF, sTM and PC. The amounts of MIF, sTM and PC in dengue patients were measured using ELISA kits as described in the Materials and Methods. *p<0.1, **p<0.01 and ***p<0.001 compared with corresponding values from normal controls (N). Levels of MIF and sTM were higher than in normal controls, but PC was lower than in the control.</p

rMIF induces thrombomodulin (TM) expression in endothelial cells.

<p>(A) HMEC-1 (1×10⁶) cells were stimulated with rMIF (0.4 µg/mL) for 24 h. TM and nuclei were stained with FITC conjugated anti-TM antibody and DAPI, respectively, and observed using fluorescence microscopy at 400× magnification. The controls were treated with 0.9% saline (Mock). (B) The expression of TM in HMEC-1 cells with/without rMIF treatment was determined using flow cytometry analysis. Cells treated with rMIF showed increased TM expression compared to the control (without). The thrombin-treated cells were used as a positive control.</p

rMIF induces ICAM-1 expression in endothelial cells and monocytes.

<p>(A) HMEC-1 (1×10⁶) and (B) HUVEC (1×10⁶) endothelial cells were stimulated with rMIF (0.4 µg/mL) for 24 h. ICAM-1 and nuclei were stained with FITC conjugated anti-ICAM-1 antibody and DAPI, respectively, and observed using fluorescence microscopy at 400× magnification. Negative controls were treated with 0.9% saline (Mock). Thrombin-treated HMEC-1 cells were used as a positive control. THP-1 monocytic cells (2×10⁶) (C and D) were stimulated with rMIF (0.4 µg/mL) for 24 h. (C) RNA was extracted and ICAM-1 expression was analyzed using a semi-quantitative RT-PCR with specific primers for ICAM-1. The gene expression of GADPH was used as the internal control. (D) The expression of ICAM-1 was detected by flow cytometry analysis and compared to the control (without rMIF treatment). Flow cytometry analysis showed increased ICAM-1 expression in THP-1 monocytes following rMIF treatment.</p

rMIF induces thrombomodulin (TM) expression in monocytes.

<p>(A) THP-1 cells (2×10⁶) were stimulated with rMIF (0.4 µg/mL) for 24 h. TM and nuclei were stained with FITC conjugated anti-TM antibody and DAPI, respectively, and observed using fluorescence microscopy at 400× magnification. The controls were treated with 0.9% saline (Mock). (B) Flow cytometry analysis of TM expression in THP-1 cells with/without rMIF treatment. A neutralizing anti-MIF mAb (20 µg/mL) was used to abrogate the MIF-induced TM expression. (C) RNA was extracted and TM expression was analyzed through a semi-quantitative RT-PCR with specific primers for TM. The GADPH was used as the internal control. TM expression was enhanced with rMIF treatment in THP-1 cells, compared to the control (c; without rMIF treatment). (D) Flow cytometry analysis also showed increased TM expression in PBMCs following rMIF treatment.</p

Dengue virus induces MIF production in monocytes and human umbilical cord vein endothelial cells (HUVECs).

<p>THP-1 cells or primary HUVECs were infected with DV2 (MOI = 10). (A, B) The concentrations of MIF in culture media were assayed at 48 h post-infection using ELISA kits as described in the Materials and Methods. Controls were uninfected cells (Mock) and cells treated with UV-inactivated DV (UVDV2). (C) DV2 induced MIF mRNA expression in HUVECs. HUVECs were infected with DV2 (MOI = 1 or 10) and incubated for 6 hours. RNA was extracted and the expression of MIF was analyzed by semi-quantitative RT-PCR with specific primers for MIF. The gene expression of GADPH was used as the internal control. Controls were cells without virus (Mock) and cells treated with UV-inactivated DV (UV).</p

rMIF enhances ICAM-1 and TM expression via the Erk, JNK MAPK and the PI3K signaling pathways in THP-1 cells.

<p>(A) Erk inhibitor U0126, (B) JNK inhibitor SP60125, or (C) PI3K inhibitor LY294002 at 20 µM in DMSO was added to the THP-1 cell culture 30 min before and throughout rMIF treatment. The cells (2×10⁶) were stimulated with rMIF (0.4 µg/mL) for 24 h. The expression of ICAM-1 (Left panel) and TM (Right panel) was assessed by flow cytometry analysis. The rMIF-treated cells were compared to the controls.</p

A Hormone Receptor-Based Transactivator Bridges Different Binary Systems to Precisely Control Spatial-Temporal Gene Expression in <em>Drosophila</em>

<div><p>The GAL4/<em>UAS</em> gene expression system is a precise means of targeted gene expression employed to study biological phenomena in <em>Drosophila</em>. A modified GAL4/<em>UAS</em> system can be conditionally regulated using a temporal and regional gene expression targeting (TARGET) system that responds to heat shock induction. However heat shock-related temperature shifts sometimes cause unexpected physiological responses that confound behavioral analyses. We describe here the construction of a drug-inducible version of this system that takes advantage of tissue-specific GAL4 driver lines to yield either RU486-activated LexA-progesterone receptor chimeras (LexPR) or β-estradiol-activated LexA-estrogen receptor chimeras (XVE). Upon induction, these chimeras bind to a LexA operator (<em>LexAop</em>) and activate transgene expression. Using GFP expression as a marker for induction in fly brain cells, both approaches are capable of tightly and precisely modulating transgene expression in a temporal and dosage-dependent manner. Additionally, tissue-specific GAL4 drivers resulted in target gene expression that was restricted to those specific tissues. Constitutive expression of the active PKA catalytic subunit using these systems altered the sleep pattern of flies, demonstrating that both systems can regulate transgene expression that precisely mimics regulation that was previously engineered using the GeneSwitch/<em>UAS</em> system. Unlike the limited number of GeneSwitch drivers, this approach allows for the usage of the multitudinous, tissue-specific GAL4 lines for studying temporal gene regulation and tissue-specific gene expression. Together, these new inducible systems provide additional, highly valuable tools available to study gene function in <em>Drosophila</em>.</p> </div

Time-course and Dose-response Analysis of the Inducible LexPR Bridge System in Response to RU486.

<p>(A) The trans-activation of LexPR was monitored in flies (<i>Or22a</i>-Gal4/<i>UAS</i>-LexPR-attP40; +/<i>LexAop</i>-mCD8::GFP-attP2) fed various concentrations of RU486 (0, 0.5, 1, 1.5, 2, and 3 mM) for 5 days. <i>LexAop</i>-mCD8::GFP expression was observed in one of the antennal lobes of adult brains. (B) <i>LexAop</i>-mCD8::GFP expression in flies fed 1.5 mM RU486 for 1–6 days (d1–d6). (C) The inducer was removed by replacing the food with fresh food for 2–24 days ((−) d2–d24). Using 3D projections, (D) the green fluorescence intensity of single glomeruli was analyzed in 5 samples from each group of induction by different concentrations of RU486 (0, 0.5, 1, 1.5, 2, and 3 mM) for 5 days, and (E) the green fluorescence intensity of single glomeruli was analyzed from 5 samples for each group of induction for 1–6 days. Each bar represents the mean, and the error bars represent the standard error (± s.e.). Data from each panel were analyzed using Student's <i>t</i> test, and any differences between various concentrations or treatment durations are indicated: n.s. indicates no significant difference; *** indicates p<0.001; ** indicates p<0.01; and * indicates p<0.05. Scale bar, 20 µm.</p

LexPR or XVE as Bridges for Spatial Targeting of Gene Expression.

<p>In a single animal, 3 separate <i>p</i>-element constructs were combined. Two were used for the GAL4/<i>UAS</i> binary gene expression system to specifically express either the LexPR or XVE chimeric proteins in a specific area and one carried the target gene of interest under the control of the <i>LexAop</i> operator sequences (<i>LexAop</i>). (A) A schematic diagram of LexPR under the control of the yeast upstream activating sequence (<i>UAS</i>) with the GAL4 driver. LexPR is a chimeric protein that includes the LexA-DNA binding domain (LexA-BD) fused to the human progesterone receptor ligand-binding domain and the p65 (NFκB) activation domain. Following treatment with RU486, LexPR is activated and binds to <i>LexAop</i> to drive the expression of Your Favorite Gene (<i>YFG</i>) downstream. In the absence of RU486, the target transgene remains silent. (B) A schematic diagram of XVE under the control of the yeast <i>UAS</i> with the GAL4 driver. XVE is a chimeric protein that includes the LexA-BD fused to the estrogen receptor ligand-binding domain and the p65 activation domain. Following treatment with β-estradiol, XVE is activated and binds to <i>LexAop</i> to drive the expression of <i>YFG</i> downstream. In the absence of β-estradiol, the target transgene remains silent.</p