Modulation of BMP signaling in the wing results in <i>wg</i>-like phenotypes.

<p>(A) Wildtype adult wing. (B) Inhibition of Wg signaling by expression of <i>vg>dTcfΔN</i> caused extensive wing notching. Ectopic expression of <i>omb>Mad</i> (C), <i>vg>Medea</i> (D), <i>vg>Mad</i> (E) and the positive regulator of Dpp, Sara, in <i>vg>Sara</i> (G) resulted in wing notching. Co-expression of Mad and dTcf with <i>vg-Gal4</i> suppressed the wing notches (F). Heterozygosity for <i>dTcf³</i> enhanced the <i>vg>Sara</i> notching (H) while heterozygosity for <i>sgg^M1-1</i> suppressed the notching (I). (J) The wildtype distal portion of the third longitudinal vein (L3). Loss of function transheterozygous alleles of <i>dpp</i>, <i>dpp^d5/dpp^hr56</i> show ectopic bristles (K) that phenocopy ectopic Wg signaling seen in <i>T93>Arm^s10</i> (L). (M) An enlarged view of a wildtype proximal anterior wing margin, showing the normal pattern of bristles. (N) Wing from a <i>dpp^d5</i> adult that lacks most veins, and displays ectopic bristles (arrow). (O) A higher magnification of the patch of extra bristles (arrow) seen in (N).</p

IBMX and db-cAMP upregulate <i>Nurr1</i> expression in a redundant manner and RA and IBMX synergistically regulate <i>NF-L</i> expression.

<p>(A) RT-PCR analysis showing mRNA expression of <i>Nurr1</i> and <i>NF-L</i> following different treatments. Note that IBMX and db-cAMP are sufficient for upregulating <i>Nurr1</i> expression. IBMX and db-cAMP have redundant roles as reflected by the reduction of <i>Nurr1</i> expression only in the absence of both components from the medium. RA and IBMX act synergistically on <i>NF-L</i> expression. (B) Quantification of RT-PCR results of (A). Experiments were performed in duplicates and data is expressed as mean ± SEM.</p

BMP signaling can inhibit Wg-target gene expression <i>in vivo.</i>

<p>(A–R) Dpp signaling was activated ectopically in 3^rd instar wing discs and the expression of Wg target genes was examined. <i>nmo-lacZ</i> (as detected by anti-βgal antibody, red in B, C) is suppressed in <i>UAS-Tkv^QD</i> flip out clones (A–C; clone is marked by GFP in A, B; arrow in C) and <i>UAS-mad</i> flip out clones (D–F; arrow in F). The arrowhead in C points to the <i>nmo</i> expression in vein primordia (15). (G–I) Dll protein expression is suppressed (arrow in I) by <i>dpp-Gal4>UAS-Tkv^QD, UAS-GFP</i> and in <i>UAS-mad</i> flip out clones (J–L). (M–O) Sens expression is suppressed (arrow in O) in <i>UAS-Tkv^QD</i> clones. (P–R) Ac expression is suppressed in <i>UAS-Tkv^QD</i> flip out clones (arrow in R). (S, T) Adult wing phenotypes derived from larvae in which flip-out <i>UAS-Tkv^QD</i> clones were induced show inhibition of Wg signaling and Wg target gene expression.</p

MSC^hUCB1 express neural specific markers suggesting neural differentiation.

<p>(A) Protein expression profile. Expression of general neural markers (Tau, NSE and GFAP) and dopaminergic markers (TH and P-TH) are upregulated. The four P-TH isoforms are of sizes between 50.7 kDa and 90 kDa. Note that the different P- TH isoforms (isoforms 1, 3 and isoforms 2, 3) are differentially regulated (day 3 and day 5 post-induction, respectively). A small molecular weight band (∼28.6 kDa), possibly representing an unknown P-TH isoform is marked by ##. The MAPK pathway is down-regulated as a consequence of differentiation as shown by decreasing levels of P-ERK1/2 correlated to increasing length of differentiation. (B) The small molecular weight band (∼28.6 kDa) is strongly elevated in the dopaminergic SY5Y neuroblastoma cells in response to the differentiation medium. β-actin is used as loading controls (A, B). (C) Summary of the FACS analysis data showing the average geometric mean fluorescence intensity (GMFI) of CD133 expression of the experimental and the respective isotype control. CD133 is expressed more strongly in a small population of undifferentiated MSCs and weakly in the rest of the population. However, CD133 is completely down-regulated within 24 h of differentiation. Analysis was done in triplicates and data is expressed as mean ± SEM. (D–G) Immunocytochemical analysis of controls (D, F) versus differentiated MSC^hUCB1 (E, G), showing cells stained with α-synaptophysin (Syp) (D and E) and α-P-TH (F and G). Cells were fixed after 3 days of incubation in differentiation medium. Note that Syp is strongly expressed at the projections (arrowheads in E). Although P-TH expression is already present at basal levels in the control, it becomes upregulated during differentiation. Arrowheads in G point to some cells with neurite-like extensions.</p

Domains of Wg-target gene expression in vivo.

<p>(A–D) Wildtype expression patterns of the four examined Wg targets: (A) <i>nmo</i>, (B) Dll, (C) Sens and (D) Ac. The expression of <i>nmo-lacZ</i> and Dll are weaker along the A/P boundary, as indicated by arrows in A and B. (E–G) Expression domain of Dpp in the early 3^rd instar larvae: In early 3^rd instar discs Dpp expression is continuous along the A/P boundary (E) and intersects with the domain of Wg expression (F–G). This localization provides an opportunity for Dpp to affect the expression of the early Wg targets such as <i>nmo</i> and Dll in areas of highest Dpp activity (arrows in A and B). The endogenous expression patterns of <i>nmo</i> and Dll along the A/P boundary are slightly suppressed relative to the rest of the expression domain, suggesting that <i>in vivo</i> endogenous Dpp plays a role in fine-tuning the expression of these Wg target genes (arrows in A and B). Expression domains of Dpp (as seen by expression of <i>UAS-Dpp-GFP</i> with <i>dpp-Gal4</i>) and Wg intersect in early third instar wing discs (E–G) while later wing discs show a discontinuity of Dpp (I–K, arrow in I). In late 3^rd instar discs, the expression of Dpp is suppressed at the D/V boundary (I–K) due to the action of the Notch pathway <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003893#pone.0003893-Newfeld1" target="_blank">[20]</a>, and thus fails to intersect the highest domain of Wg expression. The absence of an intersection between Wg and Dpp domains may explain the continuous expression of Sens in this region (C). In contrast to the leg disc, ectopic Dpp signalling within the wing pouch using <i>omb-Gal4>UAS-mad</i> does not repress endogenous Wg ligand (H).</p

In vivo competition between dTcf and Mad affects Wg target genes.

<p>Flip-out clones (positively marked with GFP) were generated to express Mad and/or dTcf. (A–C) Sens expression was suppressed in Mad misexpression clones (arrows in C). (D–F) No reduction of Sens was seen in double flip-out clones expressing Mad and dTcf (arrows in F). (G–I) Flip-out dTcf clones showed no reductions in Sens (arrows in I). (J–L) Flip-out Mad clones induced ectopic expression of the Dpp target Salm. (M–O) Double flip-out clones expressing Mad and dTcf did not show suppression of the ectopic induction of Salm.</p

MSC^hUCBs upregulate neural specific markers.

<p>(A) Band quantification readouts of RT-PCR results of the three MSC^hUCBs after incubation in differentiation medium for 24 h, normalized to β-actin and the respective controls. Note that <i>Nurr1</i> upregulation is statistically significant in all the three MSC^hUCBs. <i>NF-M</i> levels are also elevated, although not significantly. In contrast to MSC^hUCB3, MSC^hUCB1 and MSC^hUCB6 could considerably upregulate other neuronal specific marker gene expression such as <i>NF-H</i> and <i>NF-L</i>, with MSC^hUCB1 also showing upregulation of <i>TH</i> expression. Data are expressed as mean ± SEM (n = 3). (B) Gene expression profiling of MSC^hUCB1 with RT-PCR. Representative RT-PCR products for each marker are shown. Neural markers such as <i>NSE</i>, <i>NF-H</i>, <i>NF-M</i>, <i>NF-L</i>, <i>GFAP</i> as well as the dopaminergic markers <i>Nurr1</i> and <i>TH</i> are upregulated following differentiation. Experiments have been performed in triplicates and data are expressed as mean ± SEM. Note the down-regulation of <i>dopamine-β-hydroxylase</i> (<i>DβH</i>) which encodes for the key enzyme that converts dopamine into nor-adrenaline.</p

Analysis of neurite-like extensions of MSC^hUCB1 in different media composition.

<p>(A) Control. (B) Cytokine induction medium (CIM). (C, D, E, F and G) Substraction of IBMX, db-cAMP, RA, bFGF and NGF from CIM, respectively. (H, I, J, K and L) MSC^hUCB1 incubated with IBMX, db-cAMP, RA, bFGF and NGF, respectively. Note that IBMX and db-cAMP are necessary and sufficient for neurite-like outgrowth as their removal (C, D) resulted in less cells with neurite-like extensions and individually, (H, I) they induced neurite-like extensions. Removal of RA resulted in a higher frequency of cells with neurite-like extensions possibly due to a toxic effect of RA. If applied individually, RA resulted in small but statistically significant increase in cells with neurite-like extensions. bFGF and NGF did not have significant effects on neurite extension. (M) Quantification of cells with neurite-like outgrowth when individual components were removed from CIM. (N) Quantification of neurite-like extensions when MSC^hUCB1 were incubated with single components. Data in (M) and (N) were analysed using Student's t-test. Experiments were performed in triplicates and data is expressed as mean ± SEM.</p

Mad and dTcf form a complex that competes with Arm-dTcf binding and blocks dTcf-dependent gene expression.

<p>(A) Binding of Mad and dTcf. pCMV-T7-Mad and pCMV-Myc-dTcf were co-transfected into HEK293 cells. Cell lysates were immunoprecipitated (IP'd) with anti-Myc, anti-T7 or IgG (control). Immunoblotting (IB) was performed with anti-Myc and anti-T7 antibodies. (B) Mad and Arm do not associate directly. pCMV-T7-Mad and pCMV-HA-Arm were co-transfected into HEK293 cells. Cell lysates were IP'd with anti-HA, anti-T7 or IgG (control). IB was performed with anti-HA and anti-T7 antibodies. (C) A schematic map of the dTcf and Mad truncation constructs and indication of their ability to bind the other. (D) dTcf binds the MH2 domain of Mad. HEK293 cells were transfected with Myc-dTcf and the indicated T7-Mad constructs. Cell lysates were IP'd with anti-Myc or IgG (control). IB was performed with anti-Myc and anti-T7 antibodies. (E) Mad interacts with the HMG domain of dTcf. HEK293 cells were transfected with T7-Mad and Myc-dTcf constructs. Cell lysates were IP'd with anti-Myc or IgG (control). IB was performed with anti-Myc and anti-T7 antibodies. WCL, whole cell lysates. (F) Increasing concentrations of Arm can inhibit the Mad/dTcf complex. 1.5 mg of T7-Mad, 1.5 mg of Myc-dTcf and increasing amounts of HA-Arm were co-transfected into HEK293 cells. Cell lysates were IP'd with anti-Myc. IB was performed with anti-HA, anti-Myc and anti-T7 antibodies. (G) High concentrations of Mad can inhibit Arm/dTcf complex formation. 500 ng of HA-Arm, 800 ng of Myc-dTcf and increasing amounts of T7-Mad were co-transfected into HEK293 cells. Cell lysates were IP'd with anti-Myc. IB was performed with anti-HA, anti-Myc and anti-T7 antibodies. (H) Transfected cell lysates expressing HA-Arm, Myc-dTCF and T7-Mad were IP'd with anti-T7, followed by IB with anti-T7 and anti-HA, showing that Mad did not bind to Arm (I) Transfected cell lysates expressing HA-Arm, Myc-dTCF and T7-Mad were IP'd with anti-HA, followed by IB with anti-T7 and anti-Myc, showing that Arm did not pull down Mad. (J) Topflash assays in HEK293 cells showed inhibition of dTcf/Arm-dependent transcription by Mad. Topflash values are indicated on the left. These values are from the average of three independent transfection experiments. Vectors used for each experiment are as indicated in the figure. The negative control Fopflash values are given on the right in white columns. (K) Only Mad forms that can bind dTcf can inhibit Topflash expression, indicating the interaction must be direct.</p

Activity Descriptor Identification for Oxygen Reduction on Platinum-Based Bimetallic Nanoparticles: <i>In Situ</i> Observation of the Linear Composition–Strain–Activity Relationship

Despite recent progress in developing active and durable oxygen reduction catalysts with reduced Pt content, lack of elegant bottom-up synthesis procedures with knowledge over the control of atomic arrangement and morphology of the Pt–alloy catalysts still hinders fuel cell commercialization. To follow a less empirical synthesis path for improved Pt-based catalysts, it is essential to correlate catalytic performance to properties that can be easily controlled and measured experimentally. Herein, using Pt–Co alloy nanoparticles (NPs) with varying atomic composition as an example, we show that the atomic distribution of Pt-based bimetallic NPs under operating conditions is strongly dependent on the initial atomic ratio by employing microscopic and <i>in situ</i> spectroscopic techniques. The Pt_<i>x</i>Co/C NPs with high Co content possess a Co concentration gradient such that Co is concentrated in the core and gradually depletes in the near-surface region, whereas the Pt_<i>x</i>Co/C NPs with low Co content possess a relatively uniform distribution of Co with low Co population in the near-surface region. Despite their different atomic structure, the oxygen reduction reaction (ORR) activity of Pt_<i>x</i>Co/C and Pt/C NPs is linearly related to the bulk average Pt–Pt bond length (<i>R</i>_Pt–Pt). The <i>R</i>_Pt–Pt is further shown to contract linearly with the increase in Co/Pt composition. These linear correlations together demonstrate that (i) the improved ORR activity of Pt_<i>x</i>Co/C NPs over pure Pt NPs originates predominantly from the compressive strain and (ii) the <i>R</i>_Pt–Pt is a valid strain descriptor that bridges the activity and atomic composition of Pt-based bimetallic NPs