text:
Cortical γ-aminobutyric acid (GABA)ergic interneurons are characterized by extraordinary neurochemical and functional diversity. Although recent studies have uncovered some of the molecular components underlying interneuron development, including the cellular and molecular mechanisms guiding their migration to the cortex, the intracellular components involved are still unknown. Rac1, a member of the Rac subfamily of Rho-GTPases, has been implicated in various cellular processes such as cell cycle dynamics, axonogenesis, and migration. In this study, we have addressed the specific role of Rac1 in interneuron progenitors originating in the medial ganglionic eminence, via Cre/loxP technology. We show that ablation of Rac1 from Nkx2.1-positive progenitors, results in a migratory impairment. As a consequence, only half of GABAergic interneurons are found in the postnatal cortex. The rest remain aggregated in the ventral telencephalon and show morphological defects in their growing processes in vitro. Ablation of Rac1 from postmitotic progenitors does not result in similar defects, thus underlying a novel cell autonomous and stage-specific requirement for Rac1 activity, within proliferating progenitors of cortical interneurons. Rac1 is necessary for their transition from G1 to S phase, at least in part by regulating cyclin D levels and retinoblastoma protein phosphorylation.
γ-Aminobutyric acid (GABA)ergic interneurons of the cerebral cortex, comprising ∼20% of cortical neurons, play important roles in cortical function and have been implicated in severe disorders such as schizophrenia and epilepsy. These cells are characterized by a remarkable morphological, molecular, and functional diversity, and recent studies have uncovered some of the molecular components underlying the generation of this diversity (Marin and Rubenstein 2003; Metin et al. 2006). All cortical interneurons originate from the ganglionic eminences, which are well-defined domains of the subpallial ventricular area, and migrate tangentially to populate the developing cortex (Marin and Rubenstein 2001; Marin and Rubenstein 2003). The cellular and molecular mechanisms guiding inteneurons from their subpallial origins to the cortex have only recently started to be elucidated. The long list of cues involved includes long-range attractive and repulsive factors, surface-bound permissive and instructive molecules, and motogenic factors (Marin and Rubenstein 2003; Metin et al. 2006; Butt et al. 2007). In contrast to the multiplicity of extracellular signals, the intracellular proteins that mediate the response to these cues are unknown (Martini et al. 2009).
Proteins of the Rac subfamily of Rho-GTPases are involved in many cellular functions, such as regulation of actin dynamics, cell cycle progression, establishment of polarity, and axonogenesis (Jaffe and Hall 2005; Watabe-Uchida et al. 2006; Koh 2007). This subfamily consists of 3 members: Rac1, a ubiquitously expressed protein; Rac2, expressed mostly in the hematopoietic system; and Rac3, which is highly enriched in the nervous system (Malosio et al. 1997).
Specifically, within the context of neuronal migration, experimental studies have shown that the migration defect in Lis1-deficient neurons correlates with disregulated Rho-GTPases and actin cytoskeleton (Kholmanskikh et al. 2003) and that Rac1 is involved in the interkinetic nuclear migration of cortical progenitor cells (Minobe et al. 2009). Specific inactivation of Rac1 from the ventricular zone (VZ) of the telencephalon (Rac1fl/fl;Foxg1Tg(Cre)) shows a wide spectrum of defects that range from severe defects in axonogenesis to perturbation in radial migration of cortical neurons as well as increased cell death of nascent neurons (Chen et al. 2007, 2009). In addition, interneurons derived from the medial ganglionic eminence (MGE) are absent from the cortex (Chen et al. 2007). In this mouse line, however, it is hard to distinguish cell autonomous from non–cell autonomous defects. An increase in cell cycle exit in the lateral ganglionic eminence (LGE) in Rac1fl/fl;Foxg1Tg(Cre) embryos has also been observed (Chen et al. 2009). Therefore, Rac1 appears to play a crucial role in different cell biological processes in distinct neuronal populations and, possibly, distinct developmental time windows. This is not surprising, given the fact that small Rho-GTPases play various roles in the development of different cell types and tissues (Jaffe and Hall 2005).
The precise role of Rac1 in the development of cortical interneurons remains unclear. To restrict ablation of Rac1 in the MGE and examine the specific role of Rac1 in cortical interneurons originating in this region, we combined an Nkx2.1-Cre transgenic mouse (Fogarty et al. 2007) with a conditional allele of Rac1 (Walmsley et al. 2003), thus deleting the Rac1 gene in the MGE. Our analysis indicates that 50% of Rac1-deficient GABAergic interneurons originating in the MGE fail to migrate toward the cortex due to an intrinsic defect. This is likely due to their inability to progress through the restriction point within the G1 phase of the cell cycle. Specific deletion of Rac1 in postmitotic interneurons resulted in no discernible phenotype, confirming the specific requirement for this protein in Nkx2.1-positive progenitors rather than postmitotic migratory MGE-derived neurons. These findings suggest a novel cell autonomous and stage-specific requirement of Rac1 activity within proliferating progenitors of cortical interneurons.
Materials and Methods
Generation of Nkx2.1-Cre- and Lhx6-Cre-Directed Conditional Rac1-Deficient Mice
Animals carrying a floxed allele of Rac1 (Rac1fl/fl) (the fourth and fifth exon of the Rac1 gene are flanked with loxP sites, Walmsley et al. 2003) were crossed to Nkx2.1Tg(Cre) mice (Nkx2.1 transgenic Cre, Fogarty et al. 2007), in order to generate the Rac1fl/fl;Nkx2.1Tg(Cre) genotype, and to Lhx6Tg(Cre) mice (Fogarty et al. 2007), in order to generate the Rac1fl/fl;Lhx6Tg(Cre) genotype. The ROSA26fl-STOP-fl-YFP allele was also inserted as an independent marker (Srinivas et al. 2001) to allow visualization of the Rac1-mutant neurons, via yellow fluorescent protein (YFP) expression. Rac1fl/fl;Nkx2.1Tg(Cre);R26R-YFP+/− and Rac1+/fl;Nkx2.1Tg(Cre);R26R-YFP+/− animals will be referred to as Rac1fl/fl;Nkx2.1+/Cre and Rac1+/fl;Nkx2.1+/Cre, respectively, in Materials and Methods, and Figures and similarly, Rac1fl/fl;Lhx6Tg(Cre);R26R-YFP+/− and Rac1+/fl;Lhx6Tg(Cre);R26R-YFP+/−animals will be written as Rac1+/fl;Lhx6+/Cre and Rac1fl/fl;Lhx6+/Cre, respectively, in Materials and Methods, and Figures. The genotyping was performed by polymerase chain reaction (shown in Supplementary Fig. 1E), using specific primers for:
Additional details for the genotyping of animals are available on request.
For timed pregnancies, the day of the vaginal plug was designated as embryonic day E0.5 and the day of birth was considered as P0.
For immunohistochemistry, embryonic brains were dissected in phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde (PFA) overnight, whereas adult animals were first perfused with 4% PFA and then the brains were dissected and immersed in the same fixative for 3 h. Following fixation, the tissues were cryoprotected by infiltration in 30% sucrose in PBS and 10 μm cryostat sections were collected on superfrost slides. For immunostaining, the slides were washed in 0.1%Triton X-100 in PBS (PBT), then blocked in 1% fetal bovine serum and 0.1% BSA in PBT for 1 h at room temperature (RT), and subsequently incubated with primary antibodies, diluted in blocking solution, for 18 h at 4 °C. Following this, the slides were washed in PBS (3 washes, 10 min each) and incubated with the secondary antibody, diluted in blocking solution, for 2 h at RT. After another 3 washes (10 min each), the slides were mounted, using MOWIOL (Calbiochem, Darmstadt, Germany). Primary antibodies used: mouse anti-Rac1 (1:20; Cytoskeleton, Denver, CO), mouse monoclonal anti-GFP (1:500; Invitrogen, San Diego, CA), rabbit polyclonal anti-GFP (1:5000; Minotech biotechnology, Heraklion, Greece), rat monoclonal anti-GFP (1:500; Nacalai Tesque, Kyoto, Japan), rabbit polyclonal anti-Lhx6 (1:200; Lavdas et al. 1999), rabbit polyclonal anti-GABA (1:1000; Sigma, Saint Louis, MI), mouse monoclonal anti-parvalbumin (PV) (1:1000; Chemicon, Temecula, CA), rabbit polyclonal anti-calretinin (CR) (1:1000; Swant, Bellinzona, Switzerland), rat monoclonal anti-NPY (Sigma, 1:500), rabbit polyclonal anti-somatostatin (Som) (1:500; Abcam, Cambridge, MA), rat polyclonal anti-BrdU (1:1000; Oxford Biotech, Oxford, UK), rabbit polyclonal anti-Ki67 (1:1000; Vector Laboratories, Burlingame, CA), rabbit anti-cleaved caspase 3 (1:200; Cell signaling, Beverly, MA), rabbit anti-PH3 (1:200; Upstate, Temecula, CA), mouse anti-Tuj1 (1:500; Covance, Berkeley, CA), rabbit anti-neurofilament (1:500; NF-M, Covance), mouse monoclonal anti-cortactin (1:200; Upstate, NY). Secondary antibodies used: goat anti-mouse-Alexa Fluor-488, -555, or -633, goat anti-rabbitAlexa Fluor-488, -555, or -633 and goat anti-rat-Alexa Fluor-488, -555, or -633 (all from Molecular Probes, Eugene, OR, all 1:800). Alexa 555-phalloidin was used for F-actin staining (Invitrogen).
BrdU Incorporation and Staining
Pregnant females of designated embryonic stages were injected intraperitoneally with BrdU (50 μg/animal gr) and sacrificed at appropriate stages to collect the embryos. The brains were processed for immunohistochemistry, with GFP, Ki67, or PH3 antibody, and then, they were treated with 2 N HCl to expose the BrdU antigen, so that BrdU antibody staining could follow.
Western Blot
MGE and LGE from E13.5 forebrain tissue was isolated and lysed in 85 mM Tris pH7.5, 30 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 120 mM glucose, 1% Triton X-100, 60 mM octyl β-d glucopyranoside (Sigma), and 1 mM phenylmethylsulfonyl fluoride. Lysates were run on a 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred into nitrocellulose membranes (Whatman GmbH, Dassel, Germany). Membranes were subsequently blocked with 5% milk in PBS, 0.1% Tween-20, and immunoblotted with mouse anti-Rac1 (1:500, Cytoskeleton), rabbit anti-Phospho-Rb (1:1000, Cell Signaling), rabbit anti-cyclin D2 (1:500, Santa-Cruz Biotechnology, Santa-Cruz, CA, recognizing also cyclin D1), and mouse anti-actin (1:4000, Chemicon) diluted in blocking solution. Secondary antibodies used: anti-mouse-IgG-Horseradish Peroxidase and anti-rabbit-Horseradish Peroxidase (1:4000; GE Healthcare, Buckinghamshire, UK).
RNA In Situ Hybridization
Nonradioactive in situ hybridization on fixed cryostat sections was performed as described previously (Denaxa et al. 2001; Kyriakopoulou et al. 2002). Riboprobes used were specific for Gad67 (kindly provided by Dr F. Guillemot, National Institute for Medical Research, Medical Research Council, Mill Hill) Lhx6 (Grigoriou et al. 1998), NPY, and Somatostatin (Liodis et al. 2007).
MGE Matrigel Explants
MGE explants were prepared from forebrains derived from E13.5 mouse embryos. Small tissue fragments corresponding to the subventricular zone (SVZ) of the MGE were incubated for 1 h in L15 + 10% fetal calf serum at 37 °C in 5% CO2. Subsequently, explants were placed in a 3D Matrigel gel matrix (BD Biosciences, San Jose, CA) and cultured for 24 or 48 h in Neurobasal medium in 4-well plates (Nunc, Naperville, IL) as described previously (Metin et al. 1997). Explants were then fixed in 4% PFA and analyzed using an epifluorescence microscope (Axiophot/Zeiss, Oberkochen, Germany).
MGE Dissociated Cell Culture
MGEs derived from forebrains of E13.5 mouse embryos were treated with trypsin (0.2%, Gibco) 10 min at 37 °C in 5% CO2. Subsequently, the cells were plated on coverslips coated with collagen (5 mg/mL) (Gibco) or Matrigel gel matrix using Neurobasal medium for 48 h.
Quantification and Statistical Analysis
For quantification of GABAergic interneurons at P0 or for assessing the different interneuronal subpopulations in P15 brains, at least 3 pairs of littermate animals were used (Rac1+/fl;Nkx2.1+/Cre vs. Rac1fl/fl;Nkx2.1+/Cre). For each pair, 4 sections corresponding to distinct bregmata along the rostrocaudal axis (−1.94 and 2) were selected, all including the barrel cortex field. Using appropriate molecular markers (PV, CR, somatostatin, NPY, YFP, or GABA), images were obtained with a confocal microscope (Leica TCS SP2, Leica, Nussloch, Germany), cells in the entire barrel field were counted and an average rostrocaudal number was calculated for the interneuron subpopulations of Rac1+/fl;Nkx2.1+/Cre and Rac1fl/fl;Nkx2.1+/Cre animals. In all cases, data are presented as mean ± standard error of mean (SEM). The effect of the genotype on each subpopulation was assessed using ANOVA for repeated measurements and Student's t-test.
For quantification on embryonic sections, at least 3 pairs of littermates were used in each case (Rac1+/fl;Nkx2.1+/Cre vs. Rac1fl/fl;Nkx2.1+/Cre). For each pair, 4 sections were used for confocal microscopy, all containing the region corresponding to the MGE, from rostral to caudal levels. From these 4 sections, an average number of cells was calculated for comparison of the 2 genotypes. The effect was assessed using ANOVA for repeated measurements, and Student's t-test, and all data are presented as mean ± SEM. The ImageJ program was used for the measurements of the leading process length and angles in vivo. Thirty cells were randomly picked on each of 4 consecutive sections per animal, and the statistical analysis was performed using ANOVA for repeated measurements and Student's t-test.
In order to quantify the Rac1 defect on MGE matrigel explants, 4 pairs of E13.5 littermate embryos were used (Rac1+/fl;Nkx2.1+/Cre and Rac1fl/fl;Nkx2.1+/Cre). From each embryo, the MGE was isolated and 5 explants were cultured from each MGE. After 48 h in culture, the 10 further away positioned neurons were selected from each explant, and their distance from the explant perimeter was measured. An average migrating distance was obtained for each MGE, and the effect of the genotype was assessed using ANOVA for repeated measurements and Student's t-test. The data represent mean ± SEM.
Quantification of axon lengths in vitro, on matrigel or collagen, was performed using the ImageJ program, and statistical significance was assessed using Student's t-test. At least 100 cells were measured of each genotype in 3 different experiments.
Conditional Ablation of Rac1 from MGE-Derived Interneurons by Nkx2.1-Cre: Defects during Embryogenesis
To examine the role of Rac1 in the development of the subpopulation of cortical interneurons originating in the MGE, we generated mice homozygous for a floxed allele of Rac1 (Rac1fl) (Walmsley et al. 2003) in the presence of an Nkx2.1-Cre transgene (Nkx2.1Tg(Cre)) (Kessaris et al. 2006; Fogarty et al. 2007). Expression of this transgene recapitulates the pattern of expression of the endogenous Nkx2.1 gene and has been previously used to lineally mark MGE-derived cortical interneurons, which can be identified by the expression of calbindin (CB), PV, and somatostatin (Som) (Sussel et al. 1999; Fogarty et al. 2007; Butt et al. 2008; Wonders et al. 2008). To be able to identify and follow the cells in which Rac1 has been deleted, we also introduced the reporter Rosa26StopYFP which expresses YFP in a Cre-dependent manner (Srinivas et al. 2001). Rac1+/fl;Nkx2.1Tg(Cre);R26R-YFP+/− animals were used as controls, while Rac1fl/fl;Nkx2.1Tg(Cre);R26R-YFP+/− were used as Rac1 mutants. Ablation of the Rac1 protein from the MGE of Rac1fl/fl;Nkx2.1Tg(Cre);R26R-YFP+/− animals occurs at E12 (Supplementary Fig. 1A,B) and by E13.5 (Supplementary Fig. 1c1, c2, C, d1, d2, D), no YFP+ MGE-derived cells coexpress Rac1 and YFP, as assessed by immunohistochemistry. In addition, by western blotting, we do not recognize the Rac1-specific signal in E13.5 lysates from the MGE (Fig. 5C and Supplementary Fig. 1F).
In E13.5 control embryos, YFP-expressing interneurons are migrating tangentially forming the characteristic 2 cellular streams in the marginal (MZ) and the intermediate (IZ)/SVZ (Marin and Rubenstein 2003; Metin et al. 2006). In contrast, in Rac1fl/fl;Nkx2.1Tg(Cre);R26R-YFP+/− embryos, the migration of MGE-derived interneurons is disrupted since YFP+ cells are found only ventrally with respect to the pallial–subpallial boundary (cf. Fig. 1A,D). Consistently, analysis of the expression of MGE-specific interneuronal markers, such as Lhx6 (Lavdas et al. 1999; Cobos et al. 2005, 2006; Liodis et al. 2007) and Som, showed absence of positive cells within the developing neocortex of E13.5 embryos (Fig. 1A–D). Similarly, the expression of gad67 mRNA reveals fewer migrating cells in the cortex of E14.5 Rac1fl/fl;Nkx2.1Tg(Cre);R26R-YFP+/− embryos (Fig. 1H) compared with the cortex of control littermates (Fig. 1G).
At E16.5 (Fig. 1I–P), we observe that YFP+ interneurons manage to reach the developing cortex of Rac1fl/fl;Nkx2.1Tg(Cre);R26R-YFP+/− embryos, but their number is significantly reduced compared with their control littermates. However, relative to control embryos, the vast majority of YFP+ cells in the forebrain of Rac1fl/fl;Nkx2.1Tg(Cre);R26R-YFP+/− embryos aggregate ventrally in their place of birth (asterisks in Fig. 1I,M and big white arrows in Fig. 1J,K,N,O). The ventral aggregation of YFP+ cells in the brains of Rac1fl/fl;Nkx2.1Tg(Cre);R26R-YFP+/− animals can be seen even at P0 (Supplementary Fig. 2), resulting in a significant reduction of GABA+YFP+ interneurons that are present in the mutant cortex at P0 (arrows in Fig. 1S,T), compared with the controls (arrows in Fig. 1Q,R). Almost half of the MGE-derived cells are absent from the mutant brains (Fig. 1U). Taken together, these developmental studies suggest that the specific deletion of Rac1 in the VZ of the MGE results in a dramatic reduction in the number of interneurons that enter the pallium.
Nkx2.1+ MGE-resident progenitors contribute to other cell types in addition to the cortical GABAergic interneurons, such as oligodendrocytes and striatal interneurons (Kessaris et al. 2006). We investigated whether ablation of Rac1 affects these cells, as well. Indeed, using immunohistochemistry against YFP, we observed that the total number of YFP cells in the striatum of Rac1fl/fl;Nkx2.1Tg(Cre);R26R-YFP+/− animals were significantly reduced compared with control brains (P15, Supplementary Fig. 3A′, A, a). Nevertheless and in agreement with our data on the cortical subpopulations (see below, Fig. 2 and Supplementary Fig. 5), the percentage of ChAT+;YFP+ cells to the total YFP+ population in the striatum remained unaffected (Supplementary Fig. 3B,B′, b).
Similar analysis for the expression of Olig2 and YFP showed that the number of Olig2+ MGE-derived oligodendrocytes (P0, Supplementary Fig. 3C, C′, c; E14.5 and 16.5, data not shown) was significantly reduced in the cortex of Rac1fl/fl;Nkx2.1Tg(Cre);R26R-YFP+/− animals, while its ratio to the total YFP+ cells remained unaffected. Therefore, Rac1 ablation from the MGE affects all cell populations originating there.
Delayed Onset of Migration of Rac1-Deficient MGE-Derived Interneurons
To further examine the migratory behavior of Rac1-deficient MGE-derived interneurons, we placed fragments of MGE dissected from E13.5 control and Rac1fl/fl;Nkx2.1Tg(Cre);R26R-YFP+/− embryos in explant cultures for 24 or 48 h in a 3D Matrigel matrix (Supplementary Fig. 4A,B). At the end of the culture period, we measured the longest distance traveled by interneurons exiting the explant. At 24 h, a relatively large number of cells had emigrated from control explants (Supplementary Fig. 4A), but no cells had emerged from Rac1fl/fl;Nkx2.1Tg(Cre);R26R-YFP+/− MGE explants (Supplementary Fig. 4B). At 48 h, cells invading the surrounding matrigel matrix were observed in both the control and Rac1fl/fl;Nkx2.1Tg(Cre);R26R-YFP+/− explants. However, the longest distance traveled by cells in mutant explants after 48 h was approximately half to that observed in control explants (Supplementary Fig. 4E). Nevertheless, the distance traveled by the mutant cells during the second 24 h in culture was not significantly different from the distance traveled by control cells during the same period (Supplementary Fig 4F), suggesting that the speed of the migrating cells is not altered when Rac1 is ablated.
These findings indicate that the interneuron defect observed in Rac1fl/fl;Nkx2.1Tg(Cre);R26R-YFP+/− animals is intrinsic to the MGE-derived cells and not due to a secondary defect in the cellular environment. In addition, they suggest that upon deletion of Rac1, the initiation of migration of MGE cells occurs later than in control cases. Importantly, these ex vivo observations correlate with the observed in vivo phenotype, where mutant cells start their migration later than the control ones.
Rac1 Ablation from MGE-Derived Interneurons by Nkx2.1-Cre: Defects in Postnatal Brains
To examine the long-term consequences of Rac1 ablation in the development of GABAergic neurons in the cortex, we also analyzed the mice postnatally. Since the vast majority of these animals die at around weaning time, our analysis was carried out at P15. At this stage, we observed no differences in the overall structure of the brain between control and mutant animals, as assessed by histological staining with cresyl violet (data not shown). The number and distribution of interneurons in the neocortex were examined either by expression of the lineage marker YFP (Fig 2A and Supplementary Fig. 5A,A′) and the general marker GABA (at P0, Fig. 1Q,R) or markers that are specifically expressed in MGE-derived interneuronal subpopulations such as Lhx6, PV, and Som (Fig. 2B,B′,C,C′,D,D′, respectively, and Supplementary Fig. 5B,B′,C,C′D,D′, respectively). We have also examined calretinin (CR) and NPY (Fig. 2E,E′,F, respectively, and Supplementary Fig. 5E,E′F,F′, respectively), which are primarily expressed by cortical interneurons derived from the caudal ganglionic eminence (CGE) (Fogarty et al. 2007; Butt et al. 2008). Although many areas of the cortex were examined, we focused primarily in the barrel cortex (Fig. 2G) from where images were obtained and counts were performed (see Materials and Methods). A significant reduction of the number of YFP cells in Rac1fl/fl;Nkx2.1Tg(Cre);R26R-YFP+/− cortices is shown in Figure 2A. A similar reduction was observed in the number of cells expressing markers appropriate for MGE-derived interneuron subpopulations, namely Lhx6, PV, and Som (Fig. 2B,C,D, respectively). However, the percentage of YFP+ neurons that co-express Lhx6, PV, and Som was not significantly different between control and Rac1fl/fl;Nkx2.1Tg(Cre);R26R-YFP+/−(Fig. 2B′,C′,D′, respectively), indicating that the ability of Rac1-deficient precursors to differentiate into different mature interneuron subtypes is not compromised, despite their reduced numbers. CGE-derived interneurons, expressing calretinin and NPY, show no significant change in their numbers or distribution in the adult cortex (Fig. 2E,E′,F and Supplementary Fig. 5E,E′F,F′). Together, our results show that the neocortex of Rac1fl/fl;Nkx2.1Tg(Cre);R26R-YFP+/− P15 animals has a dramatic reduction in the number of MGE-derived interneurons.
Rac1 Ablation in Postmitotic Interneurons Does Not Influence Their Development
In order to verify the necessity of Rac1 in the Nkx2.1-positive progenitors of the MGE and to exclude an additional essential role for Rac1 in postmitotic cells, we deleted Rac1 specifically in postmitotic MGE-derived interneurons expressing Lhx6 using Lhx6-Cre transgenic mice (Fogarty et al. 2007). Ablation was confirmed by western blot analysis using protein extracts from E13.5 MGE tissue (Supplementary Fig. 1F). Rac1fl/fl;Lhx6Tg(Cre);R26R-YFP+/− mice die soon after birth, rendering analysis of the adult brains impossible. This could be a result of defects in other systems where Lhx6 is expressed (i.e., oral ectomesenchyme, Grigoriou et al. 1998; Denaxa et al. 2009). Therefore, we restricted our experiments to embryonic and early postnatal stages. Control and Rac1fl/fl;Lhx6Tg(Cre);R26R-YFP+/− cortices from E13.5 embryos (Fig. 3A, a, D, d) and newborn mice (Fig. 3B,C,E,F) were analyzed using a similar set of markers employed for the analysis of Rac1fl/fl;Nkx2.1Tg(Cre);R26R-YFP+/− animals, such as Lhx6 and somatostatin. As expected, approximately 97% of YFP cells were also expressing Lhx6 (Fig. 3H). No difference is observed in cortical interneurons between the 2 genotypes, either in their number or distribution, as indicated by immunohistochemistry for YFP and Lhx6 at E13.5 (Fig. 3G,H) or in situ hybridization for Lhx6 and Som mRNA at P0 (Fig. 3I). These findings contrast those obtained from Rac1fl/fl;Nkx2.1Tg(Cre);R26R-YFP+/− mice and indicate that the requirement of Rac1 activity for the development of cortical interneurons is restricted to the progenitors that reside in the VZ of the MGE.
Rac1 Ablation Does Not Affect the Proliferation or Survival of MGE-Derived Progenitors
In order to study whether the reduction in the number of cortical interneurons is due to perturbed proliferation in the VZ, we assessed the pool size of proliferating progenitors and their mitotic potential in the MGE of control and Rac1fl/fl;Nkx2.1Tg(Cre);R26R-YFP+/− embryos. For this, we pulse-labeled neural progenitors with BrdU at E12.5 and E13.5, collected the embryos half an hour later, and immunostained for BrdU and phospho-histone-3 (PH3; Fig. 4A,B). This analysis reveals no differences in the number and distribution of mitotic cells in Rac1fl/fl;Nkx2.1Tg(Cre);R26R-YFP+/− embryos compared with their control littermates (Fig. 4C). Thus, the reduced number of GABAergic cells in the cortex of mutant mice is not due to defective proliferation or aberrant mitotic activity of the Rac1-deficient cells.
To further explore the cellular mechanisms leading to the observed phenotype of Rac1fl/fl;Nkx2.1Tg(Cre);R26R-YFP+/− mice, we examined apoptotic cell death at several embryonic stages of these animals. Immunostaining of coronal sections at E12.5, E13.5, E14.5, and E16.5 control and Rac1fl/fl;Nkx2.1Tg(Cre);R26R-YFP+/− embryos for activated caspase 3 reveals no difference in apoptotic cell death between the 2 groups (Supplementary Fig. 6 and data not shown). Therefore, it is unlikely that apoptotic cell death can account for the reduced number of cortical interneurons present in the Rac1fl/fl;Nkx2.1Tg(Cre);R26R-YFP+/− brains by the end of embryogenesis.
Inactivation of Rac1 Perturbs G1 Phase Progression and Reduces Cell Cycle Exit of MGE-Derived Progenitors
We show that Rac1-deficient cortices are missing half of their MGE-derived interneurons at P15. Our in vivo and explant culture studies thus far are consistent with the idea that deletion of Rac1 in the MGE results in a defect in the initiation of migration by interneuron precursors. To further investigate the cause of this defect, we compared the cell cycle dynamics of MGE progenitors between control and Rac1fl/fl;Nkx2.1Tg(Cre);R26R-YFP+/−embryos. For this, we pulse-labeled neural progenitors with BrdU at E12.5 and collected the embryos 24 h later for anti-BrdU and anti-Ki67 double-labeling (Supplementary Fig. 7) Cells expressing Ki67 are considered to be in the cell cycle, whereas Ki67-negative cells have exited the cell cycle. The fraction of BrdU+ cells that do not express Ki67 (Ki67−) was estimated in the MGE of control and mutant embryos, as well as in the LGE which served as an internal control, as the Nkx2.1-Cre transgene is not expressed in this region (Supplementary Fig. 7). This analysis revealed a specific decrease of BrdU+; Ki67− progenitors from the MGE in mutant embryos, indicating that fewer progenitors had exited the cell cycle in the absence of Rac1. The respective percentage of LGE-derived progenitors is not altered between mutant and control littermates (Fig. 5A). Similar analysis was performed on E14, E15, and E16 brains, 24 h after BrdU injection, in order to examine whether Rac1-deficient cells of the MGE exhibit a delayed cell cycle exit even at later developmental stages (Fig. 5B). We observed a similar reduction in the cell cycle exit index of mutants versus control animals confirming that even at later developmental stages fewer progenitors exit the cell cycle in the absence of Rac1.
Our data so far suggest a defect in the cell cycle of Rac1-deficient MGE-derived progenitors. However, PH3 and BrdU detection indicate normal G2-M and S phases of the cell cycle, respectively. To further characterize the observed defect, we performed western blot analysis on MGE tissue from E13.5 embryos, in order to detect the protein levels of phosphorylated retinoblastoma tumor suppressor protein (Rb), as a marker for G1 phase of the cell cycle. We found that Rac1fl/fl;Nkx2.1Tg(Cre);R26R-YFP+/− embryos have greatly diminished amounts of phosphorylated Rb in their MGE cells, compared with control littermates (Fig. 5C). We additionally analyzed the levels of cyclin D proteins since cyclins are crucial for phosphorylation of Rb and progression of the cell cycle through the G1 phase. We find a 2-fold reduction in the levels of cyclin D proteins in the MGE of Rac1fl/fl;Nkx2.1Tg(Cre);R26R-YFP+/− embryos compared with control littermates (Fig. 5C). The defect is specific to MGE-derived cells since similar analysis, using tissue from the LGE at E13.5, revealed no differences in the levels of cyclin D proteins. Altogether, these data indicate a specific role for Rac1 in the progression of the MGE-derived cells through the cell cycle, possibly during the G1 phase.
The Majority of Mature Interneurons in Rac1fl/fl;Nkx2.1Tg(Cre);R26R-YFP+/− Brains Derive from Early Born MGE-Derived Progenitors
Given the fact that ablation of Rac1 occurs at E12 and that cells are found in dramatically reduced numbers in the mature brain, we wanted to examine the birth date of the cells present in the mutant cortices at P0. For this, we pulse-labeled pregnant females with BrdU at 2 developmental stages, E11.5, when Rac1 is still present in the progenitors, and E14.5, when Rac1 is completely absent from MGE-derived cells. P0 brains were processed for BrdU and YFP immunolabeling (Fig. 6). The percentage of early-born cells (BrdU at E11.5, white arrows in Fig. 6A,B) to the total YFP+ population is not significantly altered in the mutant P0 brains (Fig. 6B,E) compared with P0 control brains (Fig. 6A,E). In contrast, the percentage of late born cells (BrdU at E14.5, white arrows in Fig. 6C,D), present in the mutant P0 brains (Fig. 6D,E), is significantly reduced compared with the control brains (Fig. 6C,E). These results indicate that the majority of MGE-derived interneurons, found in the postnatal cortex, descend from early progenitors, whereby Rac1 is still active and fewer numbers derive from late-born progenitors.
Morphology of Rac1-Deficient Interneurons
Due to the known role of Rac1 in modulating cytoskeletal processes, we proceeded to assess whether there were any defects in the morphology and migrating processes of Rac1-deficient interneurons. Using YFP immunostaining on E13.5 brain sections, we examined the morphology of the cells in vivo in order to detect possible differences in the number of their processes, their length, and their branching during migration. We observe no morphological differences in the control and mutant YFP+ interneurons that have left the MGE and are found close to the pallial–subpallial boundary (cf. Fig. 7F,G). Consistently, the length of the leading processes (Fig. 7A), their number (Fig. 7B), and the number of primary branches (Fig. 7C) remain unaffected.
In addition, we examined the ability of Rac1-deficient cells to position themselves toward the expected direction during migration by measuring the percentage of cells with orientation opposite to the migration route and the angle formed between their leading process and the presumptive migration axis. Our measurements reveal no significant differences between the Rac1-deficient and control cells, regarding their angles during migration or the orientation of their leading process toward the cortex (Fig. 7D,E).
Although the morphology of migrating interneurons in vivo appears to be normal, we wanted to gain better information on the general morphology of Rac1-deficient cells, including those that do not move and remain aggregated ventrally. To achieve this, we dissociated cells from E13.5 MGE tissue and cultured them on collagen (Fig. 7I,J) or matrigel-coated coverslips (data not shown). After 48 h in culture, the cells were fixed and stained for YFP and cytoskeletal markers such as F-actin and cortactin (Fig. 7I,J). A statistically significant number of Rac1-deficient neurons appear to have defects with respect to the actin cytoskeleton of their growth cone (Fig. 7K). In addition, a large percentage of the Rac1-deficient cells cultured in vitro fail to exhibit normal growth cone morphology, while the length of their neurites is significantly shorter compared with control cells (Fig. 7K).
Taken together, these data suggest that despite their migratory impairment, Rac1-deficient cells that migrate toward the cortex do so in a similar fashion to control cells. However, a large percentage of Rac1-deficient neurons, most likely the population that remains aggregated in the ventral telencephalon, appear to have affected growth cones and abnormal axon extension, which is consistent with previously reported roles of Rac1 in cytoskeletal dynamics (Jaffe and Hall 2005; Watabe-Uchida et al. 2006; Koh 2007; Tahirovic et al. 2010).
In this manuscript, we show that the specific ablation of Rac1 function from Nkx2.1-expressing MGE-derived progenitors leads to a perturbation of their cell cycle exit accompanied by decreased levels of cyclin D proteins and reduced phosphorylation of Rb, a critical checkpoint of the cell cycle. Our results suggest that cells do not progress normally through cell cycle and a large number of them fail to migrate out of the MGE, thus aggregating in the ventral telencephalon. As a consequence, we observe decreased migration of cortical interneurons from the MGE toward the developing cortex, which results in a severely reduced number of all MGE-derived neurons in the adult brain. In contrast, ablation of Rac1 from Lhx6-positive interneurons that have already become postmitotic does not affect their development, further confirming the necessity of Rac1 in Nkx2.1-positive progenitors in order for them to complete their cell cycle.
Rac1 Deficiency Results in Reduced Numbers of MGE-Derived GABAergic Interneurons in the Cortex
Although many cues have been implicated in cortical interneuron development and migration, little information exists about the intracellular mechanisms involved. Since Rac1 is known to be downstream of several guidance cues, like ephrins/Eph, netrin1/DCC, Robo/Slit, and Semaphorins/Plexins (Jaffe and Hall 2005; Lowery and Van Vactor 2009), we reasoned that it may play a role in the signal transduction events that allow GABAergic interneurons to respond to guidance cues. However, we found that Rac1 activity is dispensable in postmitotic interneurons that are undergoing migration. Instead, its function is required in the mitotic progenitors of interneurons, still resident in the VZ of the MGE.
We have shown that interneurons populating the cortex are half in number and mainly derive from early-born MGE progenitors, before Rac1 ablation. These cells differentiate normally into the different subpopulations of interneurons as assessed by the expression of GABA and specific calcium-binding proteins and neuropeptides. In addition, the proportions of the different interneuron subpopulations relative to the total YFP-positive MGE-derived population are maintained. More specifically, like in control animals, ∼80% of Rac1-deficient cells in the cortex visualized by YFP, express Lhx6, which is known to regulate the PV+ and Som+ fate of MGE-derived interneurons of the Nkx2.1 lineage (Fogarty et al. 2007; Liodis et al. 2007), and the percentage of Lhx6-expressing and PV-expressing cells is not significantly altered in mutant compared with control cortices. As expected, subpopulations of interneurons expressing CR and NPY that derive primarily from the CGE, which is a territory negative for Nkx2.1 expression (Fogarty et al. 2007; Butt et al. 2008), are not affected in number or distribution in the Rac1fl/fl;Nkx2.1Tg(Cre);R26R-YFP+/− brains.
The 50% reduction we observe in Lhx6+ GABAergic interneurons is in disagreement with Chen et al. (2007), who showed a complete absence of Lhx6+ cells in the cortex at E16.5 and absence of GAD67+ cells at E18.5. This disagreement may originate in the fact that Chen et al. are using a different Cre line (namely, the Foxg1 Cre) to ablate Rac1 from the VZ of the entire telencephalon. Therefore, it is likely that defects outside the MGE may contribute to the reported phenotype.
Rac1-Deficient Interneurons: Morphology and Migration
Previous studies have demonstrated that Rac1 affects migration in several cellular populations. More particularly, Rac1 acting through WAVE and Arp2/3 regulates neuronal polarization (Tahirovic et al. 2010) and is required to promote actin polymerization at the front of migrating cells, thus pushing forward the leading edge membrane (Gardiner et al. 2002; Itoh et al. 2002). Rac1 is also required for microtubule assembly during cell movement, acting via IQGAP1/CLIP170 (Fukata et al. 2002). The dynamic interplay between microtubule and actin elements is crucial to the response of neuronal growth cones to guidance cues (Dent and Kalil 2001; Zhou and Cohan 2004). Therefore, one could hypothesize that in our study, Rac1-deficient MGE-derived interneurons might have defects in actin/microtubule dynamics, leading to their inability to migrate according to the appropriate spatiotemporal context. Our in vitro data from explant cultures of the mutant cells reveal a significant impairment in migration in agreement with our in vivo data.
Both our in vivo analysis and in vitro assays allowed us to follow the migrating cells. However, a large number of Rac1-deficient neurons remain in their place of origin. Dissociation of the ventral telencephalic tissue and in vitro cultures on collagen permitted us to visualize their morphology and cytoskeletal aspects. We observed that a significant number of these YFP+ cells fail to extend their axons beyond a certain length and their growth cones lack normal F-actin distribution. Additionally, cortactin, an important nucleation-promoting factor of Arp2/3 that has an essential role in many actin-remodeling processes such as migration and axon guidance (Ammer and Weed 2008), appears to be extensively reduced in the mutant neurons.
Taken together, our data suggest that Rac1 plays a role in the cytoskeletal reorganization of interneurons during the extension of their leading processes. Nevertheless, a large number of cells manage to migrate toward the cortex with normal morphology in terms of their leading processes, underlying a compensating mechanism, possibly involving other Rho-GTPases that share functional similarity with Rac1, like Cdc42, that could take over the role of Rac1 in migrating mutant cells (Fukata et al. 2002; Jaffe and Hall 2005). Such a possibility cannot be excluded and could hinder the elucidation of the unique role of Rac1. Future studies with combinatorial depletion of potentially compensating GTPases could provide more precise information on their functions.
Rac1 Regulates Progression of the Cell Cycle in MGE-Derived Progenitor Cells
It is known that the function of Rac1 may be different, depending on the specific cell type and tissue (Jaffe and Hall 2005). Previous studies have reported that Rac1 affects neuronal development in several aspects. It has been demonstrated that Rac1 along with Cdc42 play crucial roles in the proliferation of neural crest stem cells (Fuchs et al. 2009). In addition, ablating its function from the VZ of the entire telencephalon by the Foxg1 Cre line leads to severe axonal malformations (Chen et al. 2007) and results in a reduced pyramidal progenitor pool and microcephaly (Chen et al. 2009; Leone et al. 2010). The reduced size of the brain reported in Chen et al. (2009) is caused by accelerated cell cycle exit and increased apoptosis. Our results are consistent with the hypothesis that ablation of Rac1 in the MGE (using the Nkx2.1 Cre line) is causing progenitors to remain longer in the cell cycle with no accompanying effect on apoptosis.
In our study, we specifically ablated Rac1 from MGE-derived cells, either from the progenitor stage (with Nkx2.1-Cre) or postmitotically (with Lhx6-Cre). In the former case, in the absence of Rac1, reduced numbers of MGE-derived progenitors exit the cell cycle. Instead, they retain Ki67 expression, which characterizes cells actively cycling. It is known that Rho-GTPases influence the activity of cyclin-dependent kinases during G1 phase and the organization of microtubules and actin during M phase (Jaffe and Hall 2005). Our data suggest that Rac1-deficient cells are most likely not halted during the S, G2 or the M phase, as we do not observe any differences in the incorporation of BrdU (S phase) and the expression of PH3 (late G2-M phase), respectively. Therefore, the observed difference could be due to a percentage of cells that remain in the G1 phase and do not migrate toward the cortex.
Cell cycle progression is accomplished via tightly regulated expression of Rho, Rac1, and ERK, which exert an important role in the balanced expression of cyclin-dependent kinases (Welsh et al. 2001). Rac1-deficient MGE precursors display an imbalance in cyclin expression, as we observe reduced levels of cyclin D proteins. Additionally, we find that Rac1-deficient cells have barely detectable levels of phosphorylated Rb. This protein regulates the cell cycle by controlling progression through the restriction point within the G1 phase of the cell cycle (Sherr 1996). Cyclins of the D family and their downstream kinases are necessary for the phosphorylation of Rb (reviewed in Jaffe and Hall 2005). Once active CDK4/6–cyclin D complexes form, they phosphorylate Rb, and allow the progression of the cell cycle through the G1 checkpoint. In our study, the ablation of Rac1 in MGE-derived cells could result in the 2-fold decrease in cyclin D expression observed, through a yet unknown cascade. This could in turn lead to a severely reduced phosphorylation of Rb. Such an impediment could result in a decrease in the number of progenitors that exit the cell cycle to start migrating on time. The mutant cells remain aggregated ventrally, thus resulting in the phenotype observed in the mature brain of Rac1 mutant mice. In contrast to previous studies, whereby ablation of Rac1 from the entire forebrain caused decreased proliferation and premature cell cycle exit for pyramidal neurons (Chen et al. 2009; Leone et al. 2010), we show that ablation of the protein specifically from MGE-derived interneurons does not affect proliferation but leads to a decreased cell cycle exit. This finding underlies the differential role of Rac1 in distinct neuronal populations.
The specificity of the defect in the cell cycle is even more evident, when taking into account that no defect is observed in interneuron development if Rac1 is ablated postmitotically in vivo. The lack of phenotype in these Rac1fl/fl;Lhx6 Cre mice highlights the fact that Rac1 is indispensable for interneurons when they are still mitotic and nicely correlates with the cytoskeletal phenotype observed in the mutant neurons. By exiting the cell cycle late, it is possible that MGE-derived cells of Rac1fl/fl;Nkx2.1Tg(Cre);R26R-YFP+/− mice fail to receive crucial cues, acting at the subventricular zone in a strict spatiotemporal manner, which would allow the cells to extend their axons and respond to important guidance molecules that would direct them to the cortex. In support of this, progenitors born after the ablation of Rac1 at E12 are found in reduced numbers in the mature cortex compared with early-born cells (i.e., E11.5). It is also possible that a percentage of the aggregated cells found ventrally in the brain even postnatally are cells that failed to exit the cell cycle, thus remaining in their place of origin. Consequently, they do not migrate and interneurons are found in reduced numbers in the mature cortex.
Although several studies have been carried out to reveal the role of Rho-GTPases in multiple systems, the molecular details of their role in the development of the brain is largely unknown, mainly because of the differences, these proteins exhibit regarding their function, in different cellular populations. Our study is the first one to demonstrate a novel intrinsic role of Rac1 specifically in MGE-derived neuronal progenitors. Through potential regulation of cyclin D protein levels and Rb phosphorylation Rac1 could facilitate the transition from the G1 phase of an actively cycling condition to the postmitotic/migrating phase.
Supplementary Material
Supplementary material can be found at: http://www.cercor.oxfordjournals.org/.
European Union (CELLIMAGE Tok Grant #39528), the Royal Society of the UK, the IMBB and the graduate program of Molecular Biology-Biomedicine of the University of Crete (to D.K.); the Medical Research Council and the European Research Council (to N.K.); and the Medical Research Council Grant-in-Aid (to V.P.). M.V. has been the recipient of the Manasaki Fellowship of the University of Crete.
The authors would like to thank F. Stylianopoulou, S. Taraviras, and members of the Karagogeos and Pachnis laboratories for helpful discussions and comments. We would also like to thank P. Liodis, R. Lasrado, V. Sasseli, S. Bogni, K. Kourouniotis, and N. Vardouli for help with the animals and Drs A. Malliri, C. Tsatsanis, N. Sauvonnet, M. Averof, and E. Papakonstanti for kind supply of reagents. Conflict of Interest : None declared.


text:
FSC and WPS lucked out on Sunday - for once, the league's flaws gave us a white-knuckle thrill ride. The only downside was that the home team lost. Another league or network would have stooped to showing horrified and shocked Gold Pride fans crying tears of disbelief, but WPS doesn't do negative yet. In any case, audible on-field celebrations from Freedom players was appropriate enough, as a contrast to the utter shocked silence in Buck Shaw. I haven't gone back and compared frame-by-frame, but I doubt Abby Wambach was happier to have won the gold medal in 2004.
Wambach's return to form nearly overshadowed the triumphant return of Tiffeny Milbrett. As Grantland Rice would have said, girl was KILLING fools out there.
You could say that these are quality players putting on career-defining performances. Or, you could say that WPS teams are wildly uneven. There's a serious talent gap between the best players and the not-so-best at this point. The Gold Pride-Freedom game was reminiscent of the Red Bulls-Galaxy Beckham 5-4 demolition derby of a couple years ago, and the Crew-Wiz 6-4 scoreboard breaker from MLS' inaugural season. (And that's not an obscure game, my friends - people who don't remember who ran for President that year remember that match. Had the late Boots Randolph not already written "Yakety Sax," he would have composed it for that match.)
Those who love the art and skill of defense saw those games with looks straight out of Iron Eyes Cody. But neutral fans, passersby, and others who should know better ate it up like delicious opium. I certainly did, anyway.
It was much more suspenseful than the Red Stars chewing up and spitting out Smith-less Boston. And [Mark Rogondino] I didn't see the game, [/Rogondino] so for all I know the Solo v. LeBlanc showdown for best keeper in the hemisphere that ended up 2-1 (in penalty kick saves) was the finest game of the week. A's fans focusing on the Chalupny PK miss lead me to conclude otherwise, though.
But all these games are indicative of the same things - wildly unbalanced teams trying to cover their weaknesses and play to their strengths. Boston and Los Angeles couldn't get any business going without their international stars - and neither of those teams are what you'd call terrible. The Red Stars sure look terrific, so maybe we can forget that Sky Blue shut them out in Bridgeview. I think the Gold Pride are extremely fortunate that Milbrett was found frozen in the tundra and thawed out, because they're certainly not keeping the other teams from scoring more or less at will. (Keep an eye or two out for Kandace Wilson, though. She looks like she'll be a household name before too much longer.) (Well, in households that care about women's soccer.)
For the sake of the league, we hope to see more 4-3 track meets while the teams adjust to each other, but there's no guarantee we won't see a bunch more 0-0 or 1-0 slogs.
Still - you'd rather watch the US roll over teams like Chinese Taipei once every two months, instead?
On paper, this week's FSC game is a mismatch. Washington of course is on a huge wave of exuberance, and Athletica hasn't scored yet. Popular opinion has Saint Louis unlucky, rather than sucky, so maybe they'll get well against the Freedom's alleged defense. Fortunately, there's a nice juicy sideshow to keep us occupied. Hope Solo visits her close, personal friend Abby Wambach for the first time since they went to China together in 2007. Imagine how much fun they'll have, catching up on old times. Why on earth would you miss this game?
In other news, Julie Foudy says Birgit Prinz and Hanna Ljunberg are overrated garbage, neither of whose entire careers worth what Marta has already accomplished. She also says China was nothing without Sun Wen, and can't think of a single Swedish or Canadian player worthy of paying attnetion to. And don't get her started on how much April Heinrichs stunk, both as coach and player.
Well, actually, all she did was pick an all-time women's international dream team. I'm just jumping to conclusions based on her omissions, because that's what you do when you see a list like this. (What, no Greg Ryan?)

subset: commoncrawl


text:
Monroe - This Is My Only Hobby [HD 1080p] (2014/WowGirls.com/787.44 MB)
Here you will find fresh porn videos, movies for every taste. You can download their by clicking on the link site file-sharing service Nitroflare, Uploaded, Gigapeta, Keep2Share.cc and many other.
Release date: December 10, 2014
File: 56677dс1_full.mp4
Video: AVC, 1920x1080, HD , 29.970 fps, 6065 Kbps
Audio: AAC, 2 channels, 44.1 KHz, 152 Kbps
Duration: 00:17:41
Size: 787 MB
Download (56677dc1_full.mp4) from ex-load.com
Download (56677dc1_full.mp4) from gigapeta.com
56677dc1_full.rar - 2 x 394.22 MB
Download (56677dc1_full.part1.rar) from k2s.cc
Download (56677dc1_full.part2.rar) from k2s.cc
Users of Guests are not allowed to comment this publication.
|User info


text:
Forgot Password? | Not a User? Sign up
Your browser does not support iframes.
Shelby County's Jillian Jacobs, along with four classmates at Capital Day School in Frankfort, are in the running for national recognition for a phone app designed to help feed the hungry.
The team was recently selected as Best in State and Best in Region winners in the fifth annual Verizon Innovative Learning app challenge for a mobile app concept "Waste Free America." For the honor, the team received a $5,000 award from the Verizon Foundation for their school and tablets for each team member.
Some parents gearing up to send their little one to kindergarten in the upcoming school year might have to hold off on their school supply shopping for another year.
Starting this coming school year, children must be five years old on or before Aug. 1 to enter kindergarten. Previously, the cutoff was October 1.
In 2012, however, the General Assembly changed the law with the mindset that younger students may not be ready for kindergarten.
That law is set to go into effect for the 2017-18 school year.
Although prom season as we traditionally know it is still a few months away, thousands of young men and women dressed in their formal best will make their way to one of 375 churches around the globe for their very own Night to Shine, an unforgettable prom night experience for people with special needs, ages 14 and older.
Sponsored by the Tim Tebow Foundation, the worldwide movement will take place in all 50 states and 11 countries around the world at 6 p.m. on Feb. 17, and one of those locations includes Spencer Christian Church in Spencer County.
After an intensive search for more human remains in Waddy yielded more skeletal bones last week, investigators are now concentrating on a more daunting task –identification.
"Any technology that's out there that can help identify, as to male, female, age – we are trying to determine if we're going to have an avenue where we can identify this individual," said Shelby County Sheriff Mike Armstrong.
Though moving painfully Princess, the miniature horse recently stabbed after being attacked by a dog, is slowly recuperating.
William "Snake" Bruns said that he is keeping a close eye on the miniature horse he rescued 16 years ago, after she was first attacked Jan. 19 at his residence on Jail Hill Road by a pit bull and then again Jan. 23 by a person.
For the first time in nearly four years, Shelbyville will once again feature an art gallery on Main Street.
Dogwood Art Gallery and Shops at Blue Gables on Main Street opens today, said Howard Griffith, chair of the Shelby Regional Arts Council.
"The community has made a significant investment in making this possible because of the hope that the arts can bring about some renewal to the west end of Main Street," he said.
Bright, new furnishings, a spacious living room, modern kitchen and cozy bedrooms – even a lone tropical fish swimming serenely in his bowl in the bathroom.
In a reception at the newly remodeled Omega House Friday night, select visitors were treated to a tour of the women's shelter run by Operation Care and it's new features.
But the most important feature in the home is one that never changed.
Seated at nearly a dozen tables at the Blair Center last week, public officials and business representatives from across Shelby County joined together with students from Shelby County Public Schools' Big Picture Learning Academy for an informational luncheon on their potential collaboration.
Lisa Rogers with Shelter Insurance, who hosted a Big Picture student intern during the program's first phase, shared with attendees her experience and encouraged others to jump on board.
When the Shelbyville City Council met last month, it pondered the idea of a non-partisan election and that conversation will continue Thursday when the council convenes for its regularly scheduled meeting at 6:30 at city hall, 315 Washington Street.
During the previous meeting, several members quickly expressed support of establishing an ordinance to erase political party lines from the city council election– an idea that developed following a suggestion from council member Donna Eaton during an earlier meeting.
Nearly seven years of energy efficiency has meant a savings of more than $2 million for Shelby County Public Schools.
Jon Swindler, facilities engineer for the district, shared with the board Thursday the annual energy management report and said the district has accumulated $2.4 million in savings from cost avoidance since 2010.
Those savings were accrued through the reduction of Energy Use Intensity (EUI), which measures the energy use per square foot.
Copyright © 2017
The Sentinel-News is your source for local news, sports, events and information in Shelby County and Shelbyville, KY, and the surrounding area.


text: We're sorry, Oak Loop Napkins Rings is no longer available Why not try one of these instead? Set Of Four Nordic Heart Wooden Napkin Rings by Dibor £11$18.87€13.18$14.09 Pack Of 10 Rustic Wooden Heart Napkin Rings by Creative and Contemporary £11$18.87€13.18$14.09 Deer Napkin Ring Pair by Victoria & Abigail £15$25.74€17.97$19.22 Pair Of Bamboo Napkin Rings by Monty's Vintage Shop £14$24.02€16.77$17.93 Pinch to zoom Oak Loop Napkins Rings by Wood Paper Scissors £14.95$25.65€17.91$19.15 This item is no longer available Favourite Favourite View your favourites quantityquantity (required)Set of fourSet of six (+ £3.50)Set of eight (+ £6)quantity (required)Set of fourSet of six (+ $6)Set of eight (+ $10.29)quantity (required)Set of fourSet of six (+ €4.19)Set of eight (+ €7.19)quantity (required)Set of fourSet of six (+ $4.48)Set of eight (+ $7.68) we're sorry we're sorry, This item is no longer available buy a gift voucher related categories cards & wrap > occasional supplies > napkin rings weddings > styling your day > table decorations > napkins & napkin holders by Wood Paper Scissors Location: Cornwall, GB read seller's profile see seller's complete range more items from this seller Chunky Jigsaw Coasters And Trivet by Wood Paper Scissors £32.95$56.54€39.49$42.22 Solid Oak 'Hot Stuff' Trivet by Wood Paper Scissors £35$60.06€41.94$44.84 The Big Button Coaster by Wood Paper Scissors £11.75$20.16€14.08$15.05 Personalised Wooden Beard And Moustache Comb by Wood Paper Scissors £12.95$22.22€15.52$16.59 Set Of Two Personalised Cut Out Walnut Jig... by Wood Paper Scissors £15.95$27.37€19.11$20.43 Set Of Four Personalised Cut Out Walnut Ji... by Wood Paper Scissors £24.75$42.47€29.66$31.71 view all you may also like PRODUCT DETAILS CUSTOMER REVIEWS DELIVERY RETURNS Made in Britain exclusive eco-friendly ask seller a question save for later Add a natural touch to your dining setting with these solid oak, loop napkin rings. Each of these loop rings are a perfect ellipse and are ideal for every occasion such as weddings and special events. Made and designed in Britain from locally sourced kiln dried oak and hand finished with a natural oil. This product is available as a set of four, six or eight. Postage within the UK is free and napkin rings will arrive gift wrapped in tissue paper and boxed, ready to give or enjoy for yourself. Clean with soft damp cloth. Dimensions: length 7cm, width 5cm, depth 2.3cm. product code: 126519 2 customer reviews read about our product rating system ratings guide trusted independent customer feedbackforum excellent bad good poor This product is dispatched to you by Wood Paper Scissors. You have 28 days, from receipt of cancellable goods, to notify the seller if you wish to cancel your order or exchange an item. Please note: goods that are personalised, bespoke or made-to-order to your specific requirements, perishable products and personal items sold with a hygiene seal (cosmetics, underwear) in instances where the seal is broken are non-refundable, unless faulty. Read more about our returns policy.Should you choose to return or exchange your order you will need to deliver the item(s) to the UK, where this seller is based. Wood Paper Scissors's return policy Please see notonthehighstreet.com's returns policy.


text:
25405 Broadway Ave., Unit 4, Oakwood Village, OH 44146
Email: email@example.com
VOICE: Toll Free 866-914-1252
Toll Free
Mon-Fri 9am-5pm Sat 10am-2pm
Search by Part Number or Description
Click for Brands and Diagrams
45-0366 - AGRI-FAB EQUIPMENT PARTS Parts
Item# AF-45-0366
GT 48" Rock Rake
The GT Rock Rake is designed to be used on garden tractors fitted with a sleeve hitch. It is the ideal tool for removing debris such as brush, sticks, and rocks from project areas. It is also good for seedbed preparation and giving an area a groomed look. The tines can be angled left or right for use in a variety of applications.
Your price: $442.43
All pages © 1997-2017 M-and-D.com


text:
English, , 208kb
This paper focuses on the identification of potential conflict of interest situations through effective supervisory techniques, and examines possible regulatory gaps in existing laws. First, it provides an overview of a typical pension plan cycle, and compares the conflicts of interest rules and regulations of six countries–Chile, Costa Rica, Hungary, Mexico, Poland, and Slovenia—and examines how these would apply to typical conflict
English, , 257kb
These guidelines, approved in September 2003 by the Working Party on Private Pensions, set forth core rights and protections for plan members and beneficiaries that are generally intended to be implemented and monitored by pension regulators and supervisors.
English, , 678kb
This Comparative Overview surveys the corporate governance framework and practices in Eurasia and identifies important corporate governance reform priorities for the region.
English, , 38kb
Developed by the OECD Insurance Committee, these guidelines for good practice address every step of the insurance claim management process, particularly: adequate information and assistance to the policyholder for claim reporting; efficient claim filing methods; operational fraud detection and prevention measures; adequate, fair and transparent claim assessment and processing; expeditious claim settlement; effective complaint and
Paris, 22-23 November 2004. This conference addressed the issue of the management and compensation of large-scale disasters - be they natural catastrophes, accidental man-made disasters, or terrorist acts.
This meeting, which took place in Paris on 19 November 2004, explored the complementary roles of the MENA-OECD Investment Programme and the G-8 Investment Task Force in promoting key reforms to improve the investment climate and fostering private sector development in the MENA region.
Participants discussed and endorsed "Priorities for Implementing IFRS in the Russian Federation" and "Implementing Effective Disclosure of Related Party Transactions and Beneficial Ownership in Russia".
The Fourth Forum for Asian Insolvency Reform (FAIR) took place in New Delhi, India, on 3-5 November 2004. The Forum was organised by the Organisation for Economic Co-operation and Development (OECD), the Asian Development Bank, in partnership with the World Bank and the Japanese government.
English, , 248kb
This publication provides a comprehensive overview of the legal and institutional frameworks for combating corruption in 21 Asian and Pacific countries and territories.
English, , 1,826kb
Proceedings from the Third Forum for Asian Insolvency Reform - November 2003. This volume examines both the theory and the practice of Asian insolvency reform. It provides an extensive regional overview and assessment..


text:
Tommy Hilfiger
Boys Blue V-Neck Jersey T-Shirt
$18.20Only a few left! Only a few left!
Boys mid blue marl, ribbed V-neck t-shirt by Tommy Hilfiger. This practical, short sleeved top is made in a soft cotton jersey. The designer's logo is embroidered on the front.
- Product number 148737
- 100% cotton (soft & stretchy jersey)
- Machine wash (40*C)
- Designer colour name: Indigo Heather
View More


text:
Hotels in Coca, Ecuador
Search & Compare Coca Hotels
Get Secret Prices on select hotels
These prices aren't available to everyone.
Trending now in Coca
- Most bookedHotel Coca ImperialNo Guest Reviews yet
11 people looked at this hotel in the last hour
- GRAN HOTEL DE LAGO EL COCANo Guest Reviews yet
10 people looked at this hotel in the last hour
8 people looked at this hotel in the last hour
7 people looked at this hotel in the last hour
Most recent review
"I booked for our family vacation without knowing the details. The lodge is NOT accessible by car so do not trust your Google map! You need to get to a hotel called Oasis in Coca to take the motor boat to the lodge which isn't anywhere in the researvation. We arrived at 7 pm, ie too late to take t..."
A Traveller, Dec 2015, US
Best hotels in Coca
You can compare all of our Coca hotel rooms including the major Coca hotel chains using our genuine Hotels.com guest reviews as well as TripAdvisor Coca hotel reviews to help find the perfect hotel room when booking your accommodation in Coca. We also feature Expedia Coca hotels and reviews.
When you book your Coca stay with Hotels.com you may also earn free nights on participating hotels by joining the Hotels.com Welcome Rewards program. It's free to join and only takes 2 minutes to sign up and when you stay 10 nights you receive 1 night free*. So even a short weekend break in Coca can get you on your way to a free night.
Why use Hotels.com to book your Coca lodging & accommodation?
We can help you to find the perfect Coca hotel room for almost any occaision. With our fast and secure hotel search you may compare different Coca hotels by:
- - Price
- - Location within Coca
- - Neighborhoods in Coca
- - Nearby Coca attractions
- - Hotels in different areas of Ecuador
- - Genuine Hotels.com guest reviews for Coca
- - Star rating
- - Amenities
- - Hotel name & chain
- - Theme
For every hotel in Coca Ecuador we provide:
- - Up to the minute hotel rates & availability
- - In depth hotel information and photos
- - Detailed Coca maps showing the locations of your hotel in Coca and the nearest transport and landmarks
- - TripAdvisor Coca hotel review score
And when you make a hotel reservation in Coca with Hotels.com we'll send you email and text confirmations with the reservation details of your Coca hotel booking along with contact details, directions, information on nearby Coca attractions, restaurants and even the weather - and if you get stuck, we're only a phone call away.
To make the most of our service, download our mobile & tablet apps and sign up to our newsletter or follow us on Facebook, Google+ or Twitter to receive the latest deals and Hotels.com Coca discount codes.


text:
Posts Tagged 'respondent experience'
GRIT Sneak Peek: The Secret To Success In 30 Years Is…Tuesday, May 19, 2015, 10:31 am No Comments
A sneak peek of the upcoming GRIT report section "If market researchers want to be successful in 30 years, the one thing they should be doing right now is…"
Jeffrey Henning's #MRX Top 10 – From Wearable Tech to Implantable/Edible TechTuesday, October 28, 2014, 10:57 am 1 Comment
Of the 3,766 unique links shared on #MRX last week, here are 10 of the most retweeted...
#IIeX Focus Series – Technology & Market Research (2 of 5): Social MediaWednesday, February 19, 2014, 7:59 am 3 Comments
Ahead of the IIEX Europe conference in Amsterdam,Phil Rance examines where market research is in technology adoption. Today: social media.
#IIeX Focus Series – Technology & Market Research 2014 (1 of 5): MobileTuesday, February 18, 2014, 12:19 pm 3 Comments
Ahead of the IIEX Europe conference in Amsterdam, I've tried to pull together my thoughts on where we are in use of technology in Market Research.
Get Into Their Shoes Before Getting Into Their MindsFriday, September 21, 2012, 6:49 am 5 Comments
Before you put any questionnaire in the field, take the survey yourself as a respondent. You'll be surprised what you find.


text:
* required fields
Repairing cracked or spalled concrete in commercial buildings seems like an obvious solution to keep ownership costs down. But if the underlying damage is severe, it may be cheaper over the long term to tear everything down and rebuild.
ChemCo Systems suggests considering the lifecycle costs of a building when deciding whether to repair or replace and offers the six following tips:
Concrete vs. Steel
Bacteria Stitches Together Concrete Cracks
Concrete: Understanding When and Why Repairs are Necessary
Polyglass U.S.A., Inc. has been manufacturing premium roofing products in North America for 25 years. Learn about our CURE Technology and ADESO Technology, innovations that have transformed the roofing industry.
Utility Vehicle Alert! Don't Get Framed. Rust can eat right through most UTV frames, destroy your investment and disable your crews. But this simple step stops rust in its tracks.


text: http://www.cdc.gov/h1n1flu/ Noting the above link to the CDC is not reporting the same numbers as each state. The second death related to the Swine Flu/H1N1 has been reported. Very sad... She had just given birth recently. http://news.yahoo.com/s/ap/20090506/ap_on_re_us/us_swine_flu_texas_12 Although the discussion of swine flu may not be "cool" or considered blase, the facts remain and we are not out of the woods.


text:
Job Post
Verscend Technologies (formerly Verisk Health) drives better healthcare outcomes through data analytics. Our payment accuracy, revenue integrity, risk assessment and stratification, and quality improvement solutions help organizations utilize their data so they can efficiently and cost-effectively succeed in the new era of healthcare. Learn more at www.verscend.com.
Our ideal candidate is a quick study who has solid communications and project management skills. We're looking for a team player to help us build our IT security and compliance content database and improve the questionnaire process. Attention to detail and deadlines are a must.
Verscend works with some of the healthcare industry's biggest players, so an understanding of technical jargon and industry trends is a plus. As healthcare is constantly evolving, the desire to research our industry and stay up-to-date with relevant healthcare-related topics is also critical.
- Manage the completion of IT security and compliance questionnaires requested by current and prospective clients through both Verscend's client services' and proposal teams
- Develop and maintain our IT security and compliance content database, which will serve as a library for standard responses received from subject-matter experts across our lines of business
- Work across the organization to assimilate information, partnering with leaders from IT, compliance, data operations, etc.
- Proof responses from internal subject-matter experts to ensure final responses are both accurate and client-facing
- Adhere to internal and client deadlines
- Continually assess and update content in response to changes in services
- Support tracking tools, such as Salesforce and Box
- 1-2 years of project management experience
- 1-2 years of database experience
- Excellent written and oral communications skills
- Ability to interact with colleagues at all levels
- Strong analytic, research, and interpersonal skills
Verscend is proud to be an Equal Opportunity Employer.


text:
An astronomer friend writes from the Cavendish Laboratory:
I notice we seem to have an amorphophallus flowering in Cambridge next
week! Thought you might be interested. I haven't been along yet, but I
will try to see the flowering next week - they say Sept 1 or 2.
One bloomed recently of the two at Virginia Tech. I saw an
article in the newspaper on the evening of 4 August. It opened that
night at 10pm. The next morning I skipped the ALMA Board Telecon
(living dangerously) to drive the 3 hours down there. It was already
closing up and the famous odor barely detectable at 2pm but it was the
thrill of the month by far.
You can see me and the strange flower at:
note the little window carved into the base to access the pollen.
Clear skies,
Aroid-l mailing list


text:
The British Asian community generates around 10 per cent of the country's GDP despite making up only 2.5 per cent of the population, according to research published today.
British Asians contribute some £103 billion a year to the economy despite many having arrived in this country "penniless, homeless and unable to speak the language", the Institute Of Asian Professionals (IAP) said.
The figures coincide with the publication of the first Asian Power 100 List, which recognises the most influential and successful members of the British Asian community, many of whom will attend a ceremony at the the Hilton Hotel in


text:
Cobra Martial Arts MorleySports Martial Arts Academies
515 Walter Rd East, Unit 11, Morley, WA 6062
Best match results for: CATEGORY = Martial Arts Academies for Kids + LOCATION = the Northern Region
515 Walter Rd East, Unit 11, Morley, WA 6062
unit 7/1924 Beach Rd, Malaga, WA 6090
This business can travel to you.
Have trained with this club almost 16 years now, fantastic instructors who will inspire and motivate you at every session, couldn't love it any more!
I have trained at ATI Martial Arts for about 5 years. The instructors, team and atmosphere at ATI is extremely supportive. You are provided with a safe environment and numerous opportunities to learn and grow as an individual. I cannot thank the directors enough for the self confidence and respect I have gained through my training.
My son trains there and it has help his motor skills and he always looks forward to going to training.
I put my son into the ATI School in Malaga to help him come out of his shell and give him some confidence. It helped him so much, I then joined the same club! Was the best thing I did for myself and my son. The Instructors are friendly and helpful in all areas of Martial Arts, the school teaches their classes grouped into ages instead of a giant class were I see young kids get lost in other clubs. Great people ! Great place! Highly recommend for parents to contact them for their children! I can honestly say I have nothing bad to say about them!
my 3 girls train Taekwondo with ATI and it is the most valuable activity they do . Instructors are great role models, not just another Martial arts school- the BEST
Martial arts schools are everywhere these days and choosing the right one can be daunting and confusing.dfsvd Read more
Kiara Coillege Gym, 368 Benara Rd, Kiara, WA 6054
Fantastic set up to get the maximum out of your training by having a children's session and adults session. Chief Instructor Meade is an inspirational teacher and brings great leadership with his personal set of skills. Together with his blackbelts he has a Dojang where you must have trained at least ones. You'll go back for more. Thank you for letting me (and my children) train here.
I have always found the training at this centre to be of a really high standard and I get a great workout. The instructors and assisting black belts are dynamic, helpful and inspire me to improve by giving me useful tips on how to better my technique. As a lady member it is great to have a good balance of female and male instructors with such a wealth of experience behind them.
Excellent class. My children train here, the instructors are very good and training is superb.
This is an excellent martial arts school, with experienced and professional instructors. A traditional and welcoming training centre.
PO Box 478, Morley, WA 6062
My 2 sons have been training with this group for the past 15 years. The self discipline, confidence, fitness & self defense skills my sons possess now are from all the years training with these excellent instructors. They still love their Taekwondo training today and never miss a lesson under their Instructor or the Master. I highly recommend First Taekwondo to all my friends and family.
A supportive training environment with professional, patient and encouraging instructors.
Great classes, excellent instructors with many years of experience. Traditional non competitive martial art. great for your mind and body
197 Scarborough Beach Rd, Mount Hawthorn, WA 6016
Kin Taekwondo is the best combination of health, fun, and fitness that I've come across. Not only am I fitter and stronger than I've ever been, I have also learned valuable leadership skills which I will carry with me for life. The instructors create a constructive, supportive environment, which gives students confidence to push themselves and learn. Thank you, Kin.
A very progressive martial arts school and training centre in Perth, that teaches traditional and effective tae kwon do. The Instructors are very caring and understanding and always provide teaching and training that is fun, dynamic and a challenge. They are also all very skilled in their own way and provide a very diverse range of skills, not just fighting ability. This is a very family friendly place where kids, teens and adults are welcome. The self defence skills that is taught is realistic and practical and the combat techniques are very sharp and dynamic. I highly recommend Kin Tae Kwon Do to all that want to train hard, improve their fitness and strength or discipline and confidence and just have better health and wellbeing.
Kin Taekwondo is the perfect school for all ages and abilities. There is an equal focus on skill and fitness, and excellent balance between self-defence and the traditional martial art. The Instructors take the time to get to know each student and work with them one-on-one when needed, and everyone is pushed to the best of their ability. I'm 20 and have been heavily involved in sport all my life, but I have never been physically or mentally healthier than I am since starting at Kin.
My 3 kids (8, 10, 12) have been learning from Ricky, Paul & Jesse (brothers and all black belts) for several years and love it. Exercise, discipline, heirarchy and skills. Highly recommend.
I started Kin Tae Kwon Do with no experience in martial art. I have always been active, however I significantly noticed a change in my physical and mental stamina when I joined. The community, instructors and students respect, include and learn from each other, which is something that is hard to find. As a young adult, I would highly recommend this activity for any and all ages. Whether you want to increase your physical fitness, mental discipline or want to try something new, I strongly suggest Kin Tae Kwon Do.
7A 44 Hutton St, Osborne Park, WA 6017
Unit 4, 108 Wanneroo Rd, Tuart Hill, WA 6060
7/1924 Beach Rd Malaga, Behind Parrys Carpets, Malaga, WA 6090
Great school with dedicated instructors who challenge, inspire and build confidence across all ages.
52 Railway Pde, Bayswater, WA 6053
Unit 5/5 Wellington rd, Morley, WA 6062
10 Boag Road, Morley, WA 6062
Woodville Pavillion & Forster Park Hall, Cloverdale, WA 6105
240 Star St, Unit 3, Welshpool, WA 6106
Unit 3, 15 Dyer Rd, Bassendean, WA 6054
35/515 Walter Rd East, Morley, WA 6062
18 Wellman St, Northbridge, WA 6003
Carramar Community Centre, Morley, WA 6062
The Atrium, 168 St Georges Tce Cnr King St, Perth CBD, WA 6000
Freindly and helpful instructors and students very technical but a lot of fun. Very proffessional witha solid history in Perth
17 Townsend St, Unit 7, Malaga, WA 6090
36 Wickham St, East Perth, WA 6004
12/64-66 Kent St, Cannington, WA 6107
328 Albany Hwy, Victoria Park, WA 6100
Beatty Park Swimming Centre Vincent St, North Perth, WA 6006
Queens Park Recreation Centre, Queens Park, WA 6107
Perth CBD, WA 6000
454 Scarborough Beach Rd, Osborne Park, WA 6017
Unit 7, 44 Hutton St, Osborne Park, WA 6017


text:
Cappuccino Bon Bons
|Fudge brownie mix
|10 1⁄2 Ounce (Use Duncan Hines, 1 Package Family Size)
|1⁄3 Cup (5.33 tbs)
|1⁄3 Cup (5.33 tbs) (Use Crisco Puritan)
|Instant coffee
|1 1⁄2 Tablespoon (Use Folgers)
|Ground cinnamon
|1 Teaspoon
|Whipped topping
|1 Tablespoon
|Ground cinnamon/Fresh fruit
|1 Tablespoon (For Garnish)
Preheat oven to 350°F.
Place 1 1/2 inch foil cupcake liners on cookie sheet.
Combine brownie mix, eggs, water, oil, instant coffee and 1 teaspoon cinnamon.
Stir with spoon until well blended, about 50 strokes.
Fill each cupcake liner with 1 measuring tablespoon batter.
Bake at 350°F for 12 to 15 minutes or until wooden toothpick inserted in centers comes out clean.
Cool completely.
Garnish each bon bon with whipped topping and a dash of cinnamon or piece of fruit.
Refrigerate until ready to serve.
Rate It
Nutrition Rank
Nutrition Facts
Serving size: Complete recipe
Calories 2176 Calories from Fat 956
% Daily Value*
Total Fat 107 g165.1%
Saturated Fat 20.6 g103.2%
Trans Fat 0 g
Cholesterol 423 mg141%
Sodium 1502.4 mg62.6%
Total Carbohydrates 280 g93.4%
Dietary Fiber 19.3 g77%
Sugars 167.5 g
Protein 27 g53.1%
Vitamin A 10.9% Vitamin C 1.3%
Calcium 30.4% Iron 82.4%
*Based on a 2000 Calorie diet


text:
BHEL Recruitment 2017 Through GATE (50 Engineer Trainee) Openings Apply
Bharat Heavy Electricals Limited has propagated BHEL Recruitment 2017 for filling 50 posts of Executive Trainees in different disciplines through GATE 2017.
Further details with respect to Notification like Particular Category Wise Openings, Name of the post, age perceptions, educational abilities, application cost, and selection procedures including how to apply are proffered below. BHEL Notification has been freed to recruit 50 Executive Trainees Jobs. Under the page, you can get clear information with respect to recruitment.
The Commission has revealed BHEL Recruitment 2017 for assigning most legitimate candidates in discharged 50 number of opening. Appellants more probably must have graduation degree. Candidates need to complete their education from any Indian Govt Recognized Institute or Board.
BHEL Recruitment 2017 Details:
· Hiring Organization – Bharat Heavy Electricals Limited
· Post Name- Executive Trainees
· Total No. of Vacancies – 50 posts
· Employment Type – Full time
· Official site: http://careers.bhel.in/
Read Also: Last Minute Tips For प्रतियोगी परीक्षा
Educational Qualification: candidates should have completed graduation degree with relative stream from recognized university & good score in GATE 2017
Age Limit: kindly check the official notification. Age relaxation will be applicable as per government rules.
Read Also: Government Exam Age Calculator
Selection Procedure: Selection of candidates will be based on GATE 2017 & Interview.
How to Apply:
· Candidates first visit the official page
· Click on appropriate link for you want to apply
· Read it thoroughly to get the requirements
· Click on apply online application form
· fill the application form with required details without any mistake
· Use applicants personal and professional information
· Upload scanned photograph and signature.
· At last, take a printout for the future use.
Important Dates:
Commencement of Online Form
Last Date to Apply
Official Notification
Note: For more information you can visit the official website.
Read Also: कैसे पाये मनपसंद सरकारी नौकरी


text:
Is Top Exec Management Bureau incorrect or missing key business information? Update your Hotfrog listing today.
Contact details
Are these details correct?
Hotfrog SouthAfrica provides information regarding Top Exec Management Bureau in Randburg Gauteng. Top Exec Management Bureau is located at 84 Sophia St, Randburg Gauteng 2030 and provides Financial services. Contact them on 011 537 2400.
Is Top Exec Management Bureau in 84 Sophia St, Randburg Gauteng 2030 your business? Claim your listing and attract more leads by adding more content, photos and other business details.
We have more Financial services in Randburg Gauteng available on Hotfrog SouthAfrica. You can update your search for Randburg Financial by location, keyword or service options.
249 Arbeid Street Strijdom Park Randburg Gauteng 1724
8 Serruna Street Ferndale Randburg Gauteng 2194
32 Highland Crescent Bromhof Randburg Gauteng 2154
1st Floor, JMJ House Executive City Randburg Gauteng 2194
Small Business Hub
Sharing tips, ideas and advice to help grow your small business.
Your profile without ads
Have you considered upgrading to Hotfrog AdVantage? No competitor advertising means exclusive focus on your business. You take centre stage.
Create your free Hotfrog business profile
Follow Hotfrog on:
Share on:
Hotfrog Small Business Hub


text:
Cattle King's Ranch Down Under to Be Carved Up for Overseas Saleby
Ranch larger than Israel split off to allay security concerns
S. Kidman & Co. attracts at least one new bid, MD says
One of Australia's most iconic cattle companies, with ranches that span an area more than twice the size of Switzerland, is being broken up to clear the way for a foreign takeover.
The government last month blocked the sale of S. Kidman & Co. to an overseas buyer, saying the proximity of one of its ranches to a weapons testing range could compromise national security. Now the 116-year-old company is preparing to carve off its biggest ranch, Anna Creek, and offer it separately to existing shareholders or an Australian buyer.
Founded in 1899 by so-called Cattle King Sidney Kidman, the company's ranches span 101,000 square kilometers (39,000 square miles), or about 1.3 percent of Australia's total land area, and carry about 185,000 cattle. Hiving off Anna Creek, about a quarter of the total property, weakens the company's ability to drought-proof its business through ranches in different rainfall zones.
Managing Director Greg Campbell said in a statement Friday the carve-up was "undesirable" though necessary to address the government's national security concerns.
Kidman had attracted bids from China's Shanghai Pengxin Group and Hong Kong-based investment firm Genius Link Asset Management, a person with knowledge of the matter has said. Pengxin offered about A$350 million ($254 million), the Australian Financial Review has reported.
At least one of the final bidders has submitted to the government a fresh offer that excludes Anna Creek, Campbell said in a telephone interview from his office in Adelaide. He declined to name any suitor or say how much was offered.
Stripping out Anna Creek -- the nearest station to Kidman's cattle-fattening operation in South Australia -- could shape how the company is run under new ownership. A new owner could mitigate the loss of Anna Creek, which is bigger than Israel, by focusing more on live cattle exports out of northern Australia, Campbell said.
The sale process is now likely to run into January at least, he said.


text:
Blast off! Lunar Harvest will cultivate your taste buds with exploding flavors like berries, rock candy and blue cotton candy.
Be the first to review Lunar Harvest!
Stay up to date on the latest product releases, special offers & ZampleBox news by signing up for our newsletter
All purchases are age verified before fulfillment
Must be of legal smoking age to use zamplebox.com
Give friends $5 off their boxes each month and get $5 off yours for every friend that joins.


text:
Property Services Officer (Residences - Office Tower)
- Information Center
- Monitoring & Controlling
- Handling Tenant / Resident complaint
- Report and Administration
- Maximum age 28 years old
- Minimum D3 Tourism/Hotel or equivalent
- Having experience in Apartement / Hotel will be advantage
- Strong command in English, written & oral is a must
- Strong personality & communication skill, good appearance and pleasant personality
- Computer literate (Ms. Office)
- Able to work on shift duty
Lowongan pekerjaan ini kemungkinan telah berakhir.
Deskripsi Perusahaan
Senayan City integrated trade area was first introduced its Soft Launching on June 23, 2006 and Grand launching on September 21, 2006. Senayan City Shopping Mall is a five-star shopping mall with its target market of upper-class society. Senayan City positioned itself as "The World Class Shopping Destination". Built with super block concept, Senayan City Shopping Mall is surrounded by three towers, consist of office tower used by SCTV Tower and Panin Tower, the other is Apartment Tower namely Senayan City Residence.
Located in Senayan area with total area of 48.000 m2, Senayan City also located near SCBD (Sudirman Central Business District), which is known for its business movement and a very strategic location that easily reaches by customers all over Jakarta. Also this location is not affected by the 3 in 1 car policy. Thus, there is no timing issue; you can visit our mall any time within the mall's business operational hour.
Review ( PT Manggala Gelora Perkasa (Senayan City) )
Current Employee
- Gaji & Benefit
- Work/Life Balance
- Senior Manajemen
- Nilai & Budaya
- Jenjang Karir
Gaji yang ditawarkan oleh perusahaan sudah diatas Upah Minimum Regional Jakarta, sehingga kebutuhan sehari-hari pun terjamin dan bekerja pun bisa dengan penuh semangat.
Sayangnya, perusahaan tidak menyediakan upah lembur. Sebagai gantinya memang diberikan tambahan pengganti hari libur, tapi hal ini menurut saya tidak seimbang dengan lelahnya pekerja saat lembur.


text:
- Publisher: Rizzoli
- Pages: 256
- Illustrations: Full color throughout
- Dimensions: 5 1/4'' x 6 7/16''
- Format: Paperback
- Author: Will Jones
You currently have level 1 (first party) cookies disabled in your browser. Please be aware that you will not be able to access full site functionality unless level 1 cookies are enabled. See instructions on enabling level 1 cookies.
Skip to content
Item# 80-015497
Essential reading for both native New Yorkers and tourists alike, How to Read New York unveils the boundless diversity of Gotham's architectural wonders. Covering every era of New York architecture�from what remains of the colonial days to the latest postmodern skyscraper�this unique guidebook illuminates the fascinating architectural and urban history of New York.
Organized chronologically and by architectural style, the book covers key highlights of the built environment from the Battery to Inwood. Many of the skyline�s most iconic buildings are included, along with many lesser-known buildings that are architecturally interesting. Charmingly illustrated with 430 line drawings and vintage engravings that bring old New York to life, the book concludes with a map section that suggests themed walking tours.
|Order Total per Address
|Standard US Shipping
|Up to $50.00
|Over $150.00
For more information on international and expedited shipping options, please see our Shipping & Handling page.
Your satisfaction is our goal. Please inspect your order upon delivery, and if for any reason you are not satisfied, you may return the merchandise within 180 days from the date of purchase. See details.
Copyright � 2017 The Metropolitan Museum of Art. All rights reserved. 1000 Fifth Avenue, New York, NY 10028. | Terms & Conditions | Accessibility Visit metmuseum.org