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14 Sep 2012: Repetto E, Briata P, Kuziner N, Harfe BD, McManus MT, et al. (2012) Correction: Let-7b/c Enhance the Stability of a Tissue-Specific mRNA during Mammalian Organogenesis as Part of a Feedback Loop Involving KSRP. doi: info:doi/10.1371/annotation/c93e4b67-d237-48fb-aec7-0db398407bb4 View correction
Gene silencing mediated by either microRNAs (miRNAs) or Adenylate/uridylate-rich elements Mediated mRNA Degradation (AMD) is a powerful way to post-transcriptionally modulate gene expression. We and others have reported that the RNA–binding protein KSRP favors the biogenesis of select miRNAs (including let-7 family) and activates AMD promoting the decay of inherently labile mRNAs. Different layers of interplay between miRNA– and AMD–mediated gene silencing have been proposed in cultured cells, but the relationship between the two pathways in living organisms is still elusive. We conditionally deleted Dicer in mouse pituitary from embryonic day (E) 9.5 through Cre-mediated recombination. In situ hybridization, immunohistochemistry, and quantitative reverse transcriptase–PCR revealed that Dicer is essential for pituitary morphogenesis and correct expression of hormones. Strikingly, αGSU (alpha glycoprotein subunit, common to three pituitary hormones) was absent in Dicer-deleted pituitaries. αGSU mRNA is unstable and its half-life increases during pituitary development. A transcriptome-wide analysis of microdissected E12.5 pituitaries revealed a significant increment of KSRP expression in conditional Dicer-deleted mice. We found that KSRP directly binds to αGSU mRNA, promoting its rapid decay; and, during pituitary development, αGSU expression displays an inverse temporal relationship to KSRP. Further, let-7b/c downregulated KSRP expression, promoting the degradation of its mRNA by directly binding to the 3′UTR. Therefore, we propose a model in which let-7b/c and KSRP operate within a negative feedback loop. Starting from E12.5, KSRP induces the maturation of let-7b/c that, in turn, post-transcriptionally downregulates the expression of KSRP itself. This event leads to stabilization of αGSU mRNA, which ultimately enhances the steady-state expression levels. We have identified a post-transcriptional regulatory network active during mouse pituitary development in which the expression of the hormone αGSU is increased by let7b/c through downregulation of KSRP. Our study unveils a functional crosstalk between miRNA– and AMD–dependent gene regulation during mammalian organogenesis events.
Author Summary
Pituitary gland development has served as a powerful model system by providing insights into the molecular strategies that underlie emergence of distinct cell types from a common primordium during organogenesis, permitting the discovery of novel regulatory mechanisms. Adenylate/uridylate–rich elements Mediated Degradation (AMD) is a central post-transcriptional mechanism to control the half-life of labile transcripts that contributes to their temporal and spatial expression regulation during tissue differentiation and development. Here, we report that the targeted deletion of Dicer in developing mouse pituitary reveals a role of miRNAs in regulating mRNA stability networks through a direct expression control of the AU-rich element mediated degradation regulator, KSRP. KSRP in turn directly regulates the stability of alpha glycoprotein subunit mRNA, common to three pituitary hormones, ultimately leading to an upregulation of its steady-state level expression. These findings define a hierarchical mechanism by which the upregulation of specific miRNAs controls the expression of KSRP and the subsequence decay rate of its target labile mRNAs during mammalian organogenesis.
Citation: Repetto E, Briata P, Kuziner N, Harfe BD, McManus MT, Gherzi R, et al. (2012) Let-7b/c Enhance the Stability of a Tissue-Specific mRNA during Mammalian Organogenesis as Part of a Feedback Loop Involving KSRP. PLoS Genet 8(7): e1002823. doi:10.1371/journal.pgen.1002823
Editor: T. Rajendra Kumar, The University of Kansas Medical Center, United States of America
Received: February 24, 2012; Accepted: May 22, 2012; Published: July 26, 2012
Copyright: © 2012 Repetto et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work has been partly supported by grants from AVENIR/INSERM to MT and NIH grants NS34934 and DK018477 to MGR. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
MicroRNAs (miRNAs) are small regulatory RNAs that mediate post-transcriptional silencing of specific target messenger RNAs (mRNAs) [1]. miRNAs control a wide range of cellular activities, including development, immune function and neuronal plasticity [1]. The importance of miRNAs is underscored by mounting evidence that misregulation of specific miRNA pathways is associated with complicated health afflictions such as cancer [2]. Mature miRNAs are formed by two sequential processing reactions: primary transcripts of miRNA genes (pri-miRNAs) are first processed into hairpin-containing intermediate precursors (pre-miRNAs) by the Drosha microprocessor complex, and pre-miRNAs are then cleaved into mature miRNAs by Dicer [3]. Finally, mature miRNAs are loaded in the RNA-induced silencing complex (RISC) to mediate degradation and/or block translation of specific target mRNAs via Watson-Crick base pairing partial sequence complementarity [1].
The decay rate of mRNAs varies considerably from one species to another and plays an important role in modulating gene expression [4]. mRNA decay rate is increased by the presence of specific AU-rich sequences called ARE (AU-rich element) within the mRNA molecule. AREs can be present in the 3′UTRs of short-lived transcripts and numerous RNA-binding proteins have been described to bind them (ARE-binding protein (ARE-BP)) [5]. ARE-BPs regulate the recruitment of mRNA decay enzymes to target mRNAs . Some ARE–BPs, such as tristetraprolin (TTP), butyrate response factor 1 and 2 (BRF1 and BRF2), T-cell intracellular antigen 1 (TIA-1), KH-type splicing regulatory protein (KSRP) promote decay of ARE-containing RNAs, while others, such as Hu antigen R (HuR), stabilize them. AU-rich element RNA-binding protein 1 (AUF1) either stabilizes or destabilizes ARE-containing mRNAs depending on the experimental systems used [5]. Different layers of interplay between miRNA- and AMD (Adenylate/uridylate–rich elements Mediated mRNA Degradation)- mediated gene silencing have been proposed in cultured cells but the relationship between the two pathways in living organisms is still elusive [6], [7], [8], [9].
KSRP is a multifunctional RNA binding protein that modulates many steps of RNA life including pre-mRNA splicing, ARE-mediated mRNAs decay, and maturation of select miRNAs from precursors [10]. Studies in cells and animal models revealed that KSRP is essential for the control of cell proliferation and differentiation as well as for the regulation of the innate immune response against viral infections, and the response to DNA damage [11], [12].
In this report we have investigated a post-transcriptional regulatory network active during mouse pituitary development that integrates miRNA-dependent and AMD-mediated gene silencing. Temporal and spatial regulation of gene expression is essential for cell fate determination in pituitary where distinct hormone-producing cell types arise from a common ectodermal primordium [13], [14]. The mature anterior pituitary gland contains distinct hormone-producing cell types, including corticotropes secreting adrenocorticotrophic hormone (ACTH), a proteolytic product of proopiomelanocortin (POMC); thyrotropes secreting thyroid-stimulating hormone (TSH); somatotropes, secreting growth hormone (GH); lactotropes, secreting prolactin (PRL); gonadotropes, secreting luteinizing hormone (LH) and follicle-stimulating hormone (FSH). TSH, LH, and FSH are heterodimeric glycoproteins containing a common α-subunit (αGSU) and a hormone-specific β-subunit (TSHβ, LHβ, and FSHβ).
Here, we demonstrated that Dicer is essential for mouse pituitary morphogenesis and correct expression of hormones with αGSU being absent in Dicer-deleted pituitaries. We found a negative feedback regulatory mechanism centered on KSRP that promotes let7b/c maturation from precursors and is, in turn, downregulated by let7b/c themselves. KSRP downregulation proved to be essential for the temporarily and spatially correct expression of αGSU through stabilization of its mRNA. Our studies unveil a functional crosstalk between miRNA- and AMD- dependent gene regulation during cell lineage determination in an animal model.
Dicer-null mouse embryos die at E7.5 [15], before pituitary development starts. Therefore, in order to investigate miRNAs relevance in modulating pituitary organogenesis, we generated a pituitary-specific Dicer gene deletion by crossing Dicerflox/flox [16] and Pitx1 CRE+ mouse lines (Figure 1A) [17]. Pitx1 CRE+ mice selectively express Cre recombinase in the pituitary primordium and have been shown to execute effective recombination of the ROSA26 locus in nearly all pituitary cells by E9–E9.5 [17]. Reverse transcription followed by PCR (RT-PCR) of mRNAs from microdissected E12.5 pituitary glands revealed a complete loss of the Dicer second RNaseIII domain in Dicerflox/flox Pitx1 CRE+ (Figure S1A), which results in miRNA processing blockade in comparison with control Dicerwt/flox Pitx1 CRE+ and Dicerwt/wt Pitx1 CRE+, as examined by Northern blot using a representative miRNA antisense probe (let-7c, Figure S1B). Dicerflox/flox Pitx1 CRE+ mice died shortly after birth due to cleft palate (data not shown). Dicer-deleted pituitaries appeared hypoplastic with an enlarged lumen (Figure 1B) probably as a result of cell death (Figure 1C and Figure S1C) indicating that Dicer is required for cell survival during pituitary development. Similarly, an increased apoptosis has been observed in the other conditional Dicer knockout mice, such as in limb [16], lung [18], skeletal muscle [19], and brain [20]. Cell proliferation, assessed by BrdU incorporation and Ki-67 expression, was unaffected in mutant mice (Figure 1D and Figure S1D). Importantly, at E17.5, expression of TSHβ, LHβ and αGSU (also known as Cga) was undetectable by immunostaining in Dicer-deleted pituitaries (Figure 2). Expression of GH and POMC, markers for somatotrope and the corticotropes/melanotrope cell types, respectively, was also remarkably decreased in Dicer-delete pituitaries. Therefore, Dicer is essential for the pituitary morphogenesis and the correct expression of all pituitary hormones.
(A) Schematic representation of the Dicer conditional targeting construct. (B) Hematoxylin and eosin staining of control versus Dicer-deleted pituitaries; representative sagittal sections of E13.5 and 15.5 pituitaries are shown. (C) Apoptosis is observed by TUNEL in control or Dicer-deleted pituitaries at E12.5; a representative sagittal section of pituitary gland is shown. (D) BrdU-positive cells in control or Dicer-deleted pituitaries at E12.5 by immunohistochemistry; a representative sagittal section of pituitary gland is shown.
Protein expression of TSHβ, GH, POMC (expression was detected using anti-ACTH immunoglobulin G), LHβ, and αGSU in control or Dicer-deleted pituitaries at E17.5 by immunohistochemistry; a representative sagittal section of pituitary gland is shown.
αGSU expression, which is expressed in pituitary from E12.5 [21] was also undetectable at early embryonic stages of Dicer-deleted pituitaries (Figure 3A, 3B). αGSU expression is known to be differentially regulated in gonadotropes and thyrotropes during development [22], while mice harboring a knockout of αGSU display hypertrophy and hyperplasia of the anterior pituitary [23]. In addition, deregulation of αGSU expression was observed in different pituitary adenomas [24], suggesting that αGSU expression is finely regulated in development and pathological conditions. Because the expression of transcription factors that control αGSU promoter activity, such as Gata binding protein 2 (Gata2), Pituitary homeobox 1 and 2 (Pitx1/2), LIM-homeodomain transcription factors (Lhx3 and Lhx4), and Upstream stimulatory factor 1 (USF1) [14], were either unchanged or slightly decreased in Dicer-deleted pituitaries at E12.5 (Figure S2A, S2B and data not shown) we tested the hypothesis that αGSU may be post-transcriptionally regulated. In favor of this hypothesis, αGSU mRNA is a labile transcript that contains a canonical ARE (AUUUA) in its 3′UTR (Figure S2C) and displays a half-life that increases during pituitary development [25]. Interestingly, mRNA profiling analysis and quantitative RT-PCR from microdissected pituitaries during development revealed that KSRP mRNA was significantly upregulated in Dicer-deleted pituitaries compared to control (Figure 3B and Table S1), indicating that KSRP expression is also under regulatory control of Dicer during pituitary development. KSRP, in turn, may control the decay rate of αGSU mRNA. Indeed, KSRP expression displayed an inverse temporal relationship to αGSU mRNA expression during pituitary development. In particular, between E12.5 and E15.5, KSRP mRNA levels significantly declined while αGSU mRNA level increased nearly fivefold (Figure 3B). In contrast, between E15.5 and E17.5 KSRP mRNA levels did not change while αGSU mRNA level slightly increased nearly 1.4 fold, which may indicate a transcriptional control of αGSU expression and/or an accumulation of the stabilized αGSU transcript in the later phases of pituitary development. Moreover, KSRP expression, evaluated by immunofluorescence, is upregulated in Dicer-deleted pituitaries, including in the αGSU expression area (Figure 3A). We then utilized two pituitary-derived cell lines expressing endogenous αGSU, the gonadotrope αT3-1 cell line and the thyrotrope TαT-1 cell line (Figure 3C and Figure S2D) to measure the expression levels of αGSU and KSRP. Strikingly, αT3-1 cells express significantly higher levels of KSRP than T αT1 cells (Figure 3C), consistent with the lower expression and the shorter half-life of endogenous αGSU mRNA in αT3-1 cells (Figure 3C, 3D and Figure S2D). Indeed, quantitative RT-PCR analyses demonstrated that αGSU mRNA was more unstable in αT3-1 than in T αT-1 cells displaying a half-life upon actinomycin D treatment of approximately 30 and 75 minutes (min), respectively (Figure 3D). Therefore, this data suggest that KSRP regulates αGSU expression in pituitary development.
(A) In situ hybridization (upper panels) and immunohistochemical analyses of αGSU (middle panels) and KSRP (lower panels) in control or Dicer-deleted pituitaries at E12.5; a representative sagittal section of pituitary gland is shown. (B) Reverse transcription (RT) followed by quantitative PCR to analyze αGSU (upper panel) and KSRP (lower panel) mRNA expression in control or Dicer-deleted pituitaries at E12.5, E15.5, and E17.5. The data were normalized by β2-MG mRNA. a.u., arbitrary units compared to the value of Dicerwt/flox at E12.5. (C) immunoblot analysis of αGSU, KSRP and β-tubulin from several pituitary cell lines as indicated (GC-somatotrope, ATT20-corticotrope, αT3-1-gonadotrope, MMQ-lactotrope, and TαT1-thyrotrope). (D) Quantitative RT-PCR analysis of αGSU transcript in αT3-1 and TαT-1 cells. Total RNA was isolated at the indicated times after addition of actinomycin D. The data were normalized by β2-MG mRNA. All data are presented as mean and s.d. (n = 4).
To assess whether αGSU transcript is directly regulated by KSRP, we first performed an RNA immunoprecipitation experiment (RIP) from αT3-1 cell extract and found that anti-KSRP antibody immunoprecipitated αGSU mRNA but not the control GAPDH mRNA (Figure 4A). We then demonstrated that recombinant KSRP directly interacted with αGSU 3′UTR in a concentration-dependent fashion and that, as expected [26], KH3–4 domains accounted for the highest affinity binding to αGSU 3′UTR (Figure 4B and Figure S3A). To determine whether KSRP controls αGSU mRNA half-life, we constructed reporter plasmids in which αGSU 3′UTR was cloned downstream a luciferase open reading frame (ORF) with or without a mutation that deletes the ARE sequence motif. Then, we cotransfected the luciferase reporter with either a vector overexpressing KSRP or a siRNA to silence it (si-KSRP) into either human HeLa or mouse NIH-3T3 cells (Figure S3B). KSRP significantly promoted the downregulation of both luciferase mRNA and activity of the construct containing wt αGSU 3′UTR, but not the mutant, while cotransfection with either empty plasmid or siRNA control had no effect (Figure 4C and Figure S3C, S3D). We then investigated whether KSRP controls the half-life of endogenous αGSU mRNA in stably transfected KSRP knockdown αT3-1 cells (αT3-1–shKSRP) [27]. Indeed, αGSU mRNA was more stable in αT3-1–shKSRP compared to the mock-transfected cells (Figure 4D). In vitro degradation experiments using αT3-1 cell extracts from mock, overexpressing KSRP or sh-KSRP stable cells demonstrated that ARE is essential for KSRP-dependent control of αGSU mRNA decay. As presented in Figure 4E, KSRP promoted rapid decay rate of αGSU 3′UTR, but not the mutant substrate. Finally, KSRP knockdown in αT3-1 cells led to a more than sevenfold increase of the steady-state level of αGSU mRNA as compared to control cells (Figure 4F). These data indicate that in pituitary development Dicer modulates the turnover rate of αGSU mRNA through regulation of KSRP expression, resulting in a significant increase of the steady-state level of αGSU mRNA-protein.
(A) Anti-KSRP antibody immunoprecipitates αGSU mRNA in αT3-1 cell extracts. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (B) UV-crosslink assay to analyze the interaction of the recombinant KSRP (40–200 nM) as well as the indicated KSRP deletion mutants (150 nM) with radiolabeled αGSU 3′UTR or pitx2 3′UTR stable region (E3) as a negative control [38]. (C) Luciferase reporter plasmid bearing either wt or Δ-ARE αGSU 3′UTR sequences were cotransfected into HeLa cells with either a vector overexpressing KSRP or siRNA knocking it down. Quantitative RT-PCR analysis of luciferase transcript was normalized using Renilla mRNA. siCTL, control siRNA (D) Quantitative RT-PCR analysis of αGSU transcript in either sh-KSRP or empty pSUPER vector stably transfected into αT3-1 cells. Total RNA was isolated at the indicated times after addition of actinomycin D. The data were normalized by β2-MG mRNA. (E) In vitro RNA degradation assays were performed by incubating S100s from mock αT3-1, αT3-1–shKSRP, or -overexpressing KSRP cells with either wt or Δ-ARE mutant αGSU 3′UTR internally 32P-labeled and capped RNA substrates. The decay was monitored at the indicated times. (F) RT-PCR analysis of both αGSU and β2-MG transcripts from either mock αT3-1 or αT3-1–shKSRP cells. Student's t-test: *P<0.05. All data are presented as mean and s.d. (n = 4).
In order to identify candidate miRNAs that may directly regulate KSRP mRNA during pituitary development, we used three different algorithms, namely Target Scan (http://www.targetscan.org/), miRanda (http://cbio.mskcc.org/mirnaviewer/), and PicTar (http://pictar.mdc-berlin.de/). Eight candidate miRNAs targeting KSRP 3′UTR were predicted by these programs (Figure S4A), but only let-7b, let-7c, miR-24, miR-27a, and miR-27b were expressed during pituitary development, as evaluated by a miRNA profiling array experiment (Table S2). Strikingly, quantitative RT-PCR data demonstrated that only let-7b/c expression display an inverse temporal relationship to KSRP mRNA during pituitary development (Figure 3B, Figure 5A, and Figure S4B, S4C, S4D). Furthermore, to determine which miRNAs can suppress endogenous KSRP mRNA expression, NIH-3T3 cells were singularly transfected with let-7b, let-7c, miR-24 or miR-27b mimic and the steady-state KSRP expression level was assayed by western blot and quantitative RT-PCR. KSRP expression levels were significantly decreased in presence of let-7b/c (Figure 5B and Figure S4E), while the expression of other ARE-BPs, including TTP and HuR, was not affected (Figure S4F, S4G). In addition, let7b/c knockdown significantly increased KSRP protein levels in NIH-3T3 cells (Figure S4H), overall indicating that let-7b/c may directly regulate KSRP expression. According to bioinformatic predictions, there is a single let-7b/c binding site in KSRP 3′UTR (Figure 5C and Figure S4I). To assess whether KSRP mRNA is directly targeted by let-7b/c, we constructed reporter plasmids in which KSRP 3′UTR fragments were cloned downstream to a luciferase ORF (Figure S5A). Overexpression of either pri-let-7b or pri-let-7c-1 significantly reduced the luciferase activity in 293T cells transfected with the construct containing KSRP 3′UTR segment A, which possesses the predicted let-7b/c binding site (Figure S5A–S5D). In addition, both pri-let-7b and pri-let-7c-1 reduced the expression of the luciferase mRNA encoded by the construct containing KSRP 3′UTR segment A (Figure S5E). Importantly, both pri-let-7b and pri-let-7c-1 overexpression in 293T cells significantly repressed luciferase activity in presence of a reporter construct containing the wild-type (wt) multimerized binding site for let-7b/c, but not the mutant reporter construct (Figure 5D, Figure S5F, Table S3). In vitro degradation experiments using cell extracts from NIH-3T3 cells transfected with let-7b/c mimics and controls confirmed that let-7b/c induce KSRP mRNA degradation (Figure S5G). These data suggest that let-7b/c directly regulate KSRP mRNA expression.
(A) Ontogeny of let-7c expression in pituitary development by quantitative RT-PCR. The data were normalized by U6 RNA. (B) Immunoblot analysis of KSRP and β-tubulin in NIH-3T3 cells singularly transfected with the indicated miRNA mimics. (C) RNA duplex expected to result from base pairing of KSRP mRNA with let-7c. (D) Relative luciferase activity of reporter constructs containing four either wt or mutant let-7b/c binding sites from KSRP 3′UTR sequence in 293T cells overexpressing pri-let-7c-1. The data were normalized using Renilla activity. (E) Ontogeny of pri-let-7b/c-2, and (F) Lin28A mRNA expression in pituitary development by quantitative RT-PCR. The data were normalized by β2-MG mRNA. Student's t-test: **P<0.01. All data are presented as mean and s.d. (n = 4).
Let-7c originates from two different transcripts, namely pri-let-7c-1 and pri-let-7c-2, while let-7b originates from a unique transcript, pri-let-7b, which forms a cluster with pri-let-7c-2. Interestingly, while mature let-7b/c was significantly upregulated during pituitary development, pri-let-7-c1 and pri-let-7b/c2 levels showed a poor increase, suggesting that a post-transcriptional control of let-7b/c expression occurs during pituitary development (Figure 5E and Figure S6). In accord with this hypothesis, we found that the expression of the mRNA encoding for Lin28A, which blocks let-7 biogenesis [28], [29], has an inverse temporal relationship to let-7b/c expression (Figure 5A, 5F and Figure S4B). This observation suggests that Lin28A may postranscriptionally regulate let-7 expression during pituitary development. Importantly, we and others have recently reported that, besides its activity promoting mRNA decay, KSRP is able to favor miRNA maturation [12], [30], [31], [32]. Among miRNA precursors the processing of which is regulated by KSRP, we found the let-7 family [30], [32]. KSRP recognizes an evolutionary conserved "G" triplet in the terminal loop sequence of let7 precursors to promote the processing. Therefore, we propose a model in which let-7b/c operate within a negative feedback loop in pituitary development. In concert with the reduction of Lin28A, KSRP specifically induces the biogenesis of let-7b/c, and, in turn, KSRP expression is post-transcriptionally downregulated by let-7b/c. This downregulation of KSRP expression ultimately affects the decay of KSRP-target labile mRNAs, as we demonstrated for αGSU mRNA (Figure 6). In conclusion, these data reveal the existence of an unsuspected KSRP-dependent molecular strategy for αGSU mRNA regulation during pituitary development, promoting on the one hand the biogenesis of microRNAs and on the other hand the degradation of unstable mRNAs, based on its versatile ability to recognize different classes of RNA motifs [26].
miRNA- and AMD-based gene silencing mechanisms have emerged as two key post-transcriptional pathways that function on a genome-wide scale to modulate tissue development and differentiation [1], [5]. The interplay between these two pathways has been demonstrated in cell culture models in which changes in AMD activity occur upon downregulation of HuR that is directly controlled by cell-specific miRNAs [6], [8]. Some ARE-BPs, including HuR and TTP, may contribute to the degree of miRNA-targeting complexity by associating with RISC and either promoting [9], [33] or inhibiting [7] the silencing of target mRNAs. Moreover, some miRNAs, including miR-369-3p and miR-466l, directly target ARE and compete with TTP for ARE binding to prevent mRNA degradation mediated by AMD [34], [35], uncovering additional layers of complexity for biochemical crosstalk between the two pathways. Here we identified in an animal model a post-transcriptional regulatory network operative during pituitary development in which mRNA stability of the pituitary hormone αGSU is significantly increased through let7b/c-mediated regulation of KSRP expression. Interestingly, some other ARE-BPs, including TTP and HuR, examined also demonstrated a Dicer-dependent expression regulation in pituitary development, indicating a large potential for linked programs of post-transcriptional regulation (Figure S7). Altogether, these findings define a unanticipated functional crosstalk between miRNA- and AMD- dependent gene regulation during mammalian organogenesis events.
Materials and Methods
Animals were kept in a pathogen-free barrier facility and maintained in accordance with Institutional Animal Care and Use Protocol of University of California at San Diego (UCSD) in accordance with appropriate national regulations concerning animal welfare. Dicerflox/flox, Pitx1 CRE+ mutant embryos were obtained by crossing Dicerflox/flox mice [16] with mice heterozygous for floxed Dicer and Pitx1 CRE+ [17]. Dicer knockout animals were genotyped by PCR using primers indicated in Table S3.
In situ hybridization and immunohistochemistry
In situ hybridization and immunofluorescence were carried out as previously described [36]. Mouse embryos from E12.5 to E17.5 were fixed in 10% neutral formalin, penetrated with 20% sucrose in PBS, and embedded in OCT compound. Serial 12-µm sections were hybridized with 35S-labeled antisense RNA probes. The probes used in this study were generated by RT-PCR from various tissues and verified by sequencing. For immunofluorescence staining, the sections were boiled for 10 min in 10 mM citrate buffer (pH 6.0) to retrieve antigens and stained with mouse Ki-67 (Pharmingen, 1∶50), BrdU (ICN Biomedicals, 1∶20), KSRP (1∶50), rabbit polyclonal antibodies against cleaved caspase-3 (Cell Signaling, 1∶200), GH (DAKO, 1∶200), TSHβ (National Hormone and Pituitary Program, National Institute of Diabetes and Digestive and Kidney Diseases, rabbit, 1∶400), ACTH (Sigma, 1∶100), αGSU (Novus Biologicals, 1∶200), and LHβ (1∶200). Secondary Alexa Fluor 488-, and Alexa Fluor 594-conjugated antibodies were from Jackson ImmunoResearch and Molecular Probes. Slides were coverslipped in Vectashield Mounting Medium with DAPI (Vector Laboratories). The results were analyzed on a Zeiss Axioplan2 microscope with a Hamamatsu camera.
Microarray analysis
For mRNA profiling analysis pituitaries were microdissected from 8 specimens of either Dicer knockout or wt at E12.5, the total RNA was isolated by using RNAeasy kit (Qiagen) and the Agilent whole genome array platform was used. The normalized data were analyzed with Biometric Research Branch (BRB) array tools 3.8.0 [37]. Signal intensity below 500 was discarded. Scatterplots of log2-transformed signal intensities were used to identify probes with at least 1.2 fold change.
MicroRNA profiling analysis was provided by LC Sciences (Houston, TX, USA). Total RNAs from 30 microdissected pituitaries at E12.5 and E17.5 were isolated using miRNAeasy kit (Qiagen) to interrogate a human/mouse/rat miRNA array (LC Sciences) comprising a total of 1256 unique mature miRNAs (837 human, 599 mouse, and 350 rat, based on Sanger miRBase 11.0; Sanger Institute, Hinxton, UK). Data were analyzed by first subtracting the background and then normalizing the signals using a LOWESS filter (locally weighted regression).
Cell culture and transfections
αT3-1, NIH-3T3, HeLa, MMQ, GC, TαT-1, ATT20, and HEK293T (293T) cells were cultured in DMEM containing 4500 mg/L glucose, 110 mg/L pyruvate, and 548 mg/L L-glutamine (Gibco) supplemented with 10% fetal bovine serum (Gibco), and 1% pen/strep antibiotics (Gibco). Cells were transiently transfected for 48 hours with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. miRNA mimics for control, let-7b, let-7c, miR-24, and miR-27b (Qiagene) as well as miRNA inhibitors for control, let-7b, let-7c, miR-24, and miR-27b (Qiagene) were singularly transfected at 100 nM final concentration. siRNA scramble sequence control, siRNAs against human and mouse KSRP were transfected at the final concentration of 24 nM.
Additional methods are provided in Text S1.
Supporting Information
Figure S1.
Dicer promotes cell survival during pituitary development. (A) RT-PCR analysis of Dicer mRNA in control or Dicer-deleted pituitaries at E12.5. (B) Northern blotting for let-7c and U6 from control or Dicer-deleted pituitaries at E17.5. (C,D) Immunohistochemical analysis of c-Casp-3 and Ki67 in control or Dicer-deleted pituitaries at E12.5; a representative sagittal section of pituitary gland is shown. The star indicates a non-specific immunostaining.
Figure S2.
Dicer regulates transcriptional regulatory programmes in pituitary development. (A) Expression of USF1, Lhx3, Lhx4 and Pitx1 mRNAs in control or Dicer-deleted pituitaries at E12.5 by quantitative RT-PCR. The data were normalized by β2-MG mRNA. (B) Expression of Gata2 and Pitx2 in control or Dicer-deleted pituitaries at E12.5 and E13.5 by in situ hybridization; a representative sagittal section of pituitary gland is shown. (C) αGSU 3′UTR with the AUUUA pentamer ARE sequence motif in red. (D) RT-PCR analysis of αGSU and β2-MG (beta 2-microglobulin, control transcript) expression from several pituitary cell lines as indicated (GC-somatotrope, ATT20-corticotrope, αT3-1-gonadotrope, MMQ-lactotrope, and TαT1-thyrotrope). Student's t-test: *P<0.05. All data are presented as mean and s.d. (n = 4).
Figure S3.
KSRP promotes the degradation of αGSU mRNA. (A) The UV-crosslinking reactions were subjected to immunoblot with either anti-KSRP (upper panel) or anti-GST (lower panel). (B) Immunoblot analysis of total extracts from either HeLa (upper-left) or NIH-3T3 (upper-right) cells transiently transfected with either scramble siRNA (siCtrl), or human or mouse KSRP siRNA (si-KSRP). In the lower panel HeLa cells were transfected with either pcDNA-3 or a pcDNA-3 overexpressing HA-KSRP. (C) KSRP knockdown in HeLa cells reduced the luciferase activity of a reporter construct bearing αGSU 3′UTR sequence. Control, transfection of pcDNA-3. The data were normalized using Renilla activity. (D) Quantitative RT-PCR analysis of luciferase transcript bearing either wt or Δ-ARE αGSU 3′UTR cotransfected into NIH-3T3 cells with either KSRP siRNA or control. The data were normalized using Renilla mRNA. Student's t-test: *P<0.05, **P<0.01. All data are presented as mean and s.d. (n = 4).
Figure S4.
Let-7b/c control KSRP expression. (A) Schematic representation of the KSRP 3′UTR with the bioinformatically predicted targeting miRNAs. (B) Ontogeny of mature let-7b, (C) miR-27b, and (D) miR-24 expression during pituitary development by quantitative RT-PCR. The data were normalized by U6 RNA. (E) Quantitative RT-PCR analysis of KSRP, (F) TTP or (G) HuR mRNAs in NIH-3T3 cells transfected with the indicated miRNA mimics. The data were normalized by β2-MG mRNA. (H) Immunoblot analysis of KSRP and β-tubulin in NIH-3T3 cells singularly transfected with the indicated miRNA inhibitors. (I) RNA duplex expected to result from base pairing of KSRP mRNA with let-7b. All data are presented as mean and s.d. (n = 4).
Figure S5.
KSRP mRNA is directly regulated by let-7b/c. (A) Relative luciferase activity of reporter constructs containing KSRP 3′UTR segments in 293T cells overexpressing pri-let-7c-1. Schematic representation of the KSRP 3′UTR and the location of A, B, and C is shown at the bottom. The data were normalized using Renilla activity. (B) Northern blot analysis of let-7c and let-7b in 293T cells overexpressing either pri-let-7c-1 or pri-let-7b. (C,D) Relative luciferase activity of reporter constructs bearing the indicated segments of KSRP 3′UTR sequences in 293T cells cotransfected with either pri-let-7b or an empty pcDNA-3 vector as mock. The data were normalized using Renilla activity. (E) Luciferase reporter plasmid bearing KSRP 3′UTR A was cotransfected into 293T cells with either pri-let-7c-1 or pri-let-7b. Quantitative RT-PCR analysis of luciferase transcript was normalized using Renilla mRNA. (F) Relative luciferase activity of reporter constructs containing four wt or mutant let-7b/c binding sites from KSRP 3′UTR sequence in 293T cells overexpressing pri-let-7b. The data were normalized using Renilla activity. (G) In vitro RNA degradation assays were performed by incubating S100s from let-7b/c overexpressing-NIH-3T3 cells and control cells with four wt or mutant let-7b/c binding sites from KSRP 3′UTR sequence internally 32P-labeled and capped RNA substrates. The decay was monitored at the indicated times. Student's t-test: *P<0.05; **P<0.01. All data are presented as mean and s.d. (n = 4).
Figure S6.
Ontogeny of pri-let-7c-1 expression in pituitary development. Reverse transcription (RT) followed by quantitative PCR to analyze by quantitative RT-PCR to analyze pri-let-7c-1 expression in pituitary at E12.5, E15.5 and E17.5. The data were normalized by β2-MG mRNA. All data are presented as mean and s.d. (n = 4).
Figure S7.
Dicer-dependent expression profile of different ARE-BPs during pituitary development. Reverse transcription (RT) followed by quantitative PCR to analyze TTP, HuR, TIA-1, and AUF1 mRNA expression in control or Dicer-deleted pituitaries at E12.5, E15.5 and E17.5. The data were normalized by β2-MG mRNA. a.u., arbitrary units compared to the value of Dicerwt/flox at E12.5. All data are presented as mean and s.d. (n = 4).
Table S1.
mRNA profiling microarray data from Dicer-deleted microdissected pituiries and control at E12.5. mRNAs whose expression levels changed of at least 1.2 fold in Dicer-deleted pituitaries compared to control at E12.5 are listed.
Table S2.
miRNAs profiling analysis in pituitaries at E12.5 and E17.5.
Table S3.
Mouse Oligonucleotides used for RT-PCR, cloning and Northern blot analysis of U6 RNA.
Text S1.
Supporting Methods
We are indebted to Dr C. Y. Chen for the generous gift of the mouse monoclonal anti-KSRP antibody; to Dr. P. Mellon for kindly providing us αT3-1 and TαT-1 cells; to Dr. K. Scully for helpful discussions and reagents; and to Dr X. de Mollerat du Jeu, Dr P. Kozbial, C. Nelson, K. Ohgi, H. Taylor, and Dr X. Zhu for technical assistance. MT is an investigator with the AVENIR program of INSERM. MGR is an investigator with the Howard Hughes Medical Institute.
Author Contributions
Conceived and designed the experiments: MT. Performed the experiments: ER PB NK RG MT. Analyzed the data: ER MT. Contributed reagents/materials/analysis tools: PB RG BDH MTM. Wrote the paper: MGR MT.
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The American singer-songwriter returns to Hop Farm alongside Billy Ocean, Peter Gabriel and Damien Rice
It's been a month of dramatic contrasts for Kate Winslet.
Independent Distributor PIAS declares: "We can get through this"
MIDDLESBROUGH were left to thank Portsmouth's poor finishing as they picked up another point in their battle to beat the drop.
A FRUSTRATED James DeGale is threatening to give up his British crown.
JAMES DeGALE'S trainer Jim McDonnell has spent the past two months booing his fighter to prepare him for Saturday's clash with Paul Smith.
DAVID HAYE is splashing the cash to ensure a successful second defence of his WBA world heavyweight title – and to guarantee the humiliation of challenger Audley Harrison.
Striding around in his sharp suits, he could have come straight off the set of TV's Mad Men - hard to believe this vision of sartorial elegance is Tony Blair.
A MAN made a 320-volt Taser-style stun gun out of a camera, screws and tape after watching how to do it on a YouTube video.
Middlesbrough & District Table Tennis League
Monday morning here at Mirror.
Maximo Park's Paul Smith, left, kicked off Glasto in typical camp style.
They are only seven years old but the youngsters of Bournville are already mini heroes.
The Arctic Monkeys, Radiohead and Kings of Leon will headline this year's Reading and Leeds Festivals, organisers confirmed today.
Robbie Blake inspired Burnley to put the wind up feeble Forest at gale-lashed Turf Moor.
Keith Fahey was compared to Lionel Messi as his outrageous goal lit up St Andrews.
Matteo Alberti must have thought he had carved out a spot of history for QPR.
Dave Jones praised his super-fit Bluebirds after Billy Davies's unbeaten run as Nottingham Forest boss came to a shuddering halt.
Billy Davies celebrated his first game in charge of Nottingham Forest with a stunning smash-andgrab victory as Charlton's run without a win was extended to a club record 17 league games.
The perfect storm is brewing around Colin Calderwood - just as Palace enjoyed the perfect end to an almost perfect week.
Plymouth boss Paul Sturrock was full of praise for match winner Rory Fallon as Argyle recorded their first home success.
Charlton manager Alan Pardew blasted referee Karl Evans for his failure to red card Forest skipper Kelvin Wilson.
Having a "fat" day? That means you'll probably stick to dark colours, wear an over-sized jacket - and avoid vertical stripes like the plague.
Mick McCarthy roared to the top of the Championship on the back of two-goal Michael Kightly - but was he bovvered?
Coca Cola FOOTBALL LEAGUE CHAMPIONSHIP
Portsmouth fans Paul Smith, 34, and wife Hayley, 25, have given new son Jacob the middle name Kanu after the Cup Final hero.
Nathan Tyson's 12th goal of the season was enough to keep Forest's lingering hopes of automatic promotion to the Championship alive.
Coca Cola FOOTBALL LEAGUE ONE Nottm Forest 1 Swindon 0
Coca Cola FOOTBALL LEAGUE ONE

subset: commoncrawl


text:
A partnership between PayPal (EBAY) and leading Chinese e-commerce platform DHGate.com has unraveled: DHGate, which claims to have over 3M buyers and 20M product listings, says it no longer accepts PayPal as a payment method, due to mutual disagreements. The deal was PayPal's biggest in China, whose online payments market is dominated by Alibaba (ALBCF.PK), Tencent (TCEHY.PK), and China UnionPay.
A partnership between PayPal (EBAY) and leading Chinese e-commerce platform DHGate.com has...
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Video at CNBC.com (Thu, 2:23PM)
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Comments (3)
So, who in their right mind, would get into bed with eBay/PayPal in the first place, let alone stay there for any length of time in such an abusive relationship?
The Autonomous Guy #23, contributorComments (205) | + Follow Following - UnfollowAh , there he is...My favorite comedian...Pill Conehead....
If amongst any of the attending eBay shills there are other than fools such as the above commenter, if you have anything meaningful you want to say to me, David Smith has my direct email address ... In the meantime, what is causing the anxiety amongst the eBay shills ...
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text:
Video Industry News Video Company News Technology Industry News Technology Company News Novelty Industry News Novelty Company News Gay Industry News Gay Company News Legal Industry News All Company News
Released: Jun 01st, 2004
Running Time: 99
Director: Craven Moorehead
Company: NorthStar Associates
Cast: Claudia Bella, Chanelle Chavez, Peter North, Jazz Duro, Samara (I), Monique (I), Marcela, Alberto Rey, Patricia (I), Joel Lawrence
Critical Rating: AAAA
Genre: All-Sex
Strong Latin choice.
Sure, there's a ton of Latin product on the market. But this simple title from producer/performer Peter North and director Craven Moorhead is an excellent entry into the overflowing genre.
Claudia Bella, the beautiful piece of ass on the box cover, may have never looked better than she does in the opener with North. Bella has a seemingly perfect suntan and an ass that was meant for porn. The Hall of Fame veteran decks her pussy doggie-style before a cowgirl exhibition that includes some remarkable hip gyrations by Bella. An anal encounter puts a bow on it. Pre-nom it for Best Couples Scene.
Newcomer Chanelle Chavez ends up in an initiation-type scene with Joel Lawrence and Jazz Duro. She may not have known what she was getting herself into as the guys basically rip her a new pussy and asshole and cause her to make a series of crazy faces while she's being fucked. Chavez turns gun shy during the pops, but she does manage a smile.
North's threeway with two blondes that look like sisters highlights the rest of the action.


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Editor's Note: The Steps to End Poverty in McPherson County organization has established a goal of eliminating poverty in the county. The multi-step approach started early this year and is now in the Getting Ahead process in which low-income families attend a series of 18 courses to learn how to overcome obstacles and move out of poverty. During this time, volunteers are training to become allies of Getting Ahead class members while others are being trained to learn how to help low-income families change their situation. During this time, The Sentinel, in partnership with the STEPMC organization, will be offering weekly profiles on STEP volunteers. The profiles will highlight individuals that are members of one of six committeess. If you would like more information on any part of this process contact Brenda Sales of the United Way, 241-5152 or Neisha Dahlstrom, 242-6781, or by E-mail at firstname.lastname@example.com.
Fred Bohnenblust
Place of Residence:
Which team are you a member of and what are your responsibilities with that group?
Guiding Coalition.
-Collaborative efforts with the community businesses and McPherson Area Habitat for Humanity
Why did you choose to get involved with STEPMC?
STEPMC is trying to do what we at Habitat for Humanity have been trying to do since our beginning in McPherson in 1992 – and that is giving a "hand-up" rather than a hand out. Helping and empowering people with low incomes to better themselves. At Habitat for Humanity we do it through helping people have home ownership. But STEPMC is taking a more "Wholistic" approach by helping a person see what barriers are preventing them from bettering themselves and then build a plan to improve themselves with the help of others around them.
Why do you believe this initiative is important for the people of McPherson County?
McPherson County is blessed with a better wealth and living environment than most communities. We (McPherson) will be better when "everyone" can have access to that wealth and environment.
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Location, ST | website.com


text:
- Miss Florida 2017 stripped of crown days after win - US Weekly
- Lindsay Lohan steps out amid relationship drama - TMZ
- Was Taylor Swift snubbed by the VMAs? - Gossip Cop
- Rachel Platten sings "Fight Song" with lots of stars - Just Jared Jr
- Farrah Abraham sizzles in a revealing bikini - Radar
- New pics from the Clooneys' Italian summer - Lainey Gossip
- Fox News snubs major DNC moment - The Hollywood Reporter
16 March 2013 at 8:54 PM
Taylor Swift: Trampoline Jumper at Photo Shoot!
About this photo set: Taylor Swift shows off several looks at a photo shoot on Saturday (March 16) in Los Angeles. Later in the day, the 23-year-old country singer was spotted rocking… Read More Here
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text:
Potion (Base Set 94)(Redirected from Potion (Lucario Quarter Deck 7))
Potion (Japanese: きずぐすり Salve) is an Item card. It was first released in the Base Set as a Trainer card.
Card text
e-Reader data
The Expedition Base Set and Theater Limited VS Pack prints include a Dot Code strip for use with the e-Reader, and have the card ID A-25-#. The short strip contains a brief card summary. There is no long strip for this card.
Release information
This card has been released numerous times throughout the course of the TCG. It originated in the Base Set as a Trainer card with artwork by Keiji Kinebuchi. It was included in the Trainer Deck A with a red border and "Trainer Deck A" printed on the back. This deck was given to prototype Pokémon Leagues before the TCG's market release in North America. In September 2002, it was reprinted in the Expedition Base Set with updated artwork by the same artist. This print was used until the release of EX Unseen Forces in August 2005, where this card was reprinted with new artwork by Shin-ichi Yoshikawa. It was later released as an Item card in the Black & White expansion with new artwork by Ayaka Yoshida, originating from the Japanese Beginning Set. It was released in the English Black & White Trainer Kit. This print was reprinted in the Boundaries Crossed expansion, originating from the National Beginning Set. It was again reprinted in the Everyone's Exciting Battle collection.
Potion was reprinted again during the XY Series in the English Kalos Starter Set, first originating from the Japanese XY Beginning Set, with new artwork by 5ban Graphics. This artwork was later reprinted in the Japanese Xerneas Half Deck. It was reprinted in both Half Decks of several Deck Kits, namely the XY Trainer Kit, XY Trainer Kit: Bisharp & Wigglytuff, XY Trainer Kit: Latias & Latios, the Japanese Emboar-EX vs Togekiss-EX Deck Kit (which led to a reprint in the English BREAKpoint expansion), and the XY Trainer Kit: Pikachu Libre & Suicune. In Japan, it was reprinted again in the BREAK Starter Pack.
Potion is a healing item in the Pokémon games that was introduced in Pokémon Red and Blue.
|This article is part of Project TCG, a Bulbapedia project that aims to report on every aspect of the Pokémon Trading Card Game.


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Long before the station was up and running, space shuttle missions broke new ground in many different fields of research, taking advantage of the microgravity environment to perform studies that couldn't be done on terra firma.
As the last-ever shuttle launch nears — NASA's STS-135 mission aboard Atlantis will blast off July 8 — here's an admittedly subjective countdown at six of the coolest experiments ever done aboard NASA's iconic space plane.
Researchers first noticed this characteristic in studies performed aboard Atlantis' STS-115 flight in 2006 and the STS-123 mission of Endeavour two years later. And it's not a subtle change; Salmonella becomes three to seven times more virulent in microgravity conditions, researchers have said.
Scientists believe that the bacteria get ramped up because spaceflight tricks them into behaving as if they're inside the human gut. The shuttle missions also identified dozens of genes that seem to be involved in the hyper-virulence, as well as a "master switch" protein that regulates many of these genes.
The biotech firm Astrogenetix worked with NASA to conduct and extend this research, and the company recently developed a Salmonella vaccine based on it. Astrogenetix is also performing space-based studies of other pathogens, such as dangerous methicillin-resistant Staphylococcus aureus (MRSA) bacteria, with the aim of finding better treatments down the road.
During STS-46, the tether unspooled just 840 feet (256 meters) from Atlantis before the reel jammed. Four years later, 12.2 miles (19.7 km) of cable were released before the 0.1-inch (0.25 centimeter) tether snapped, sending the probe shooting away into a higher orbit.
Though neither attempt was 100 percent successful, the TSS belongs on this list for its scale and ambition alone. And the 1996 experiment did return some interesting results. Before the tether snapped, the TSS had been generating 3,500 volts and up to 0.5 amps of current, according to NASA officials.
But there was science, and business, behind the move. The company International Flavors and Fragrances (IFF) wanted to see how microgravity altered the sweet and familiar scent of a rose — and if a new perfume component might come out of the experiment.
So IFF sent a miniature rose plant up, and shuttle astronauts sampled its volatile oils, which carry the essential odors of the flower. It turned out that the space rose produced fewer volalites than its counterparts did back on Earth. And, more importantly, its overall fragrance was entirely different.
IFF commercialized the space rose odor, which has since been incorporated into "Zen," a perfume put out by the company Shiseido Cosmetics. NASA flew another rose on a space shuttle in 2007, but that flower was destined for the annual New Year's Rose Parade in Pasadena, Calif., not a perfume.
Of the dozens of studies performed on that 10-day flight, one stands out nearly three decades later: STS-9 astronauts cultivated the first protein crystals ever grown in space.
Scientists found that crystals grown in space are larger and more neatly ordered — and thus easier to subject to X-ray structural analysis — than those grown here on Earth. Space-grown crystals therefore have great potential to help scientists understand how certain proteins work, perhaps leading to better and more targeted drugs in the future.
The WSF would take advantage of this vacuum to grow extremely thin and pure films that could find applications in many different fields, such as the manufacture of semiconductors.
The experiment went up on three shuttle flights: Discovery's STS-60 in 1994, Endeavour's STS-69 in 1995 and Columbia's STS-80 in 1996. Hardware issues kept the WSF from being fully deployed on the first mission, relegating it to a place at the end of Discovery's robotic arm. But it flew successfully in the shuttle's wake on STS-69 and STS-80.
The WSF experiments helped researchers learn how to craft better photocells and thin films of various materials, including ceramics that could be used to make artificial retinas.
The nematodes, housed in specially designed canisters, survived the tragedy and were recovered alive. While the worms' survival is but a tiny and peripheral part of the heartbreaking Columbia story, it taught scientists some lessons about the tenacity of life, and how it might spread from planet to planet.
"It's the first demonstration that animals can survive a re-entry event similar to what would be experienced inside a meteorite," Catharine Conley, then of NASA's Ames Research Center in Moffett Field, Calif., said in 2005. Conley was principal investigator of the Columbia nematode experiment. "It shows directly that even complex small creatures originating on one planet could survive landing on another without the protection of a spacecraft."
Descendents of the STS-107 nematodes have flown on subsequent shuttle flights, including Endeavour's final STS-134 mission in May 2011.


text:
Client has DNS at a telephone company which provide emails for his domain (sudamerica.cl). I need to switch DNS in order to get control over them so I can properly set the A records for the new website, and the intranet.sudamerica.cl for the Intranet website.
I have suceeded before by coping the MX record by doing nslookup with http://network-tools.com/nslook/, the pop records and so on... but many other times it has failed without explanaition.
Is there any other records beside MX records that must be migrated? What I can do to ensure DNS is correclty configured for email delivery?


text: Memory from CATS for Clarinet Quartet Look Inside Listen Memory from CATS for Clarinet Quartet Clarinet, Woodwind Quartet, Bass Clarinet, Clarinet Quartet - IntermediateBy Andrew Lloyd Webber. Arranged by Doug Clyde. Broadway, Musicals. Score, Set of Parts. 18 pages. Published by ALBEDO MUSIC (H0.115069-541120). Available Instantly More info Add to Cart { "catalogRefId":"20232682", "productId":"prod20232682", "quantity":"1" }


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Join us as we celebrate 33 Years
of Poetry, Music, and Friends
January 30 through February 4, 2017 in Elko, Nevada
The National Cowboy Poetry Gathering is a week-long celebration of life in the rural West, featuring the contemporary and traditional arts of western ranching culture. Poetry, music, stories, gear, film, photography and food - all contribute to the texture of an event that has become an annual ritual and a place of personal meaning for thousands of people.
All day during the Gathering's three busiest days, Thursday through Saturday, you will find:
- Ticketed evening performances and shows.
- Performances of poetry and music with the purchase of a Deluxe Pass or Single Day Pass (Thursday through Saturday, click here for information on Passes).
- Open mic poetry and music sessions - sign up daily to perform. (Thursday through Saturday)
- Workshops and special talks to attend.
- Exhibitions at the Western Folklife Center.
- Film Screenings.
- Socializing in the Pioneer Saloon.
- Other activities around town (this list is updated for our guests every January).
If you are signed up for our email news, we will notify you of interesting shows and other Gathering-related news. Sign up for our email news here.


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Video Industry News Video Company News Technology Industry News Technology Company News Novelty Industry News Novelty Company News Gay Industry News Gay Company News Legal Industry News All Company News
Released: Aug 01st, 2006
Running Time: 106
Director: Astrux Entertainment
Company: 1st Strike
Distribution Companies: Nectar
Cast: Courtney Simpson, Sandra Romain, Nadia Styles, Denise K, Taryn Thomas, TonyT, Richard Kline, T.J. Cummings
Critical Rating: AAA 1/2
Genre: Gonzo
Although four big cocks are, indeed, stuck in deep, not a single girl actually weeps. In fact, while the title and hard cover would lead one to believe that this is a slobber and rough sex release, it's really fairly tame, has a curiously tender and sensitive behind the scene section, and an appealing soundtrack.
What it lacks in weeping it makes up for in volume, with Nadia Styles honking like a goose, moaning and screaming until her voice cracks, and ultimately begging Alex Sanders in a high pitched voice to cum on her face. Denise K. becomes loudly inarticulate while fucking, while Taryn Thomas coughs and chokes on cock before screaming, shouting, and swearing like a sailor during her anal, which she assures the audience is filled with cock. Only Courtney Simpson makes noise that appears in direct proportion to the action.
Retailing: A lively sex release, even if it's not a tear-inducing one.


text:
University Fairs
College/University Fair
Are you planning to transfer to a four-year university? If so, come meet with representatives from a variety of universities to learn about their various majors, programs and resources, and financial aid opportunities. Hope to see you there!!
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23,000 Members Strong - The Largest and Oldest Mom Blogger Social Network
Hi All! I am new to this group and love the idea of getting 200 followers. I have seen some blogs that I follow explode from 30 or 40 followers to 300 almost overnight! I am dying to know the secretCome visit me at www.lettersfromcrazyville.blogspot.comI will be happy to return the follow just LMK you are from here :-)
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Autism Screening and Diagnosis Center - Bloomington, IN
- WebMD Physician Directory of Bloomington Doctors
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- Bloomington Emergency Contacts
- Nearby Bloomington Hospitals
- Find a Local Pharmacy on RxList - Bloomington
Bloomington Doctors and Specialists for Autism Screening and Diagnosis
Autism Screening and Diagnosis
Autism screening and diagnosis overview
Diagnosing autism spectrum disorders (ASDs) can be difficult, since there is no medical test, like a blood test, to diagnose the disorders. Doctors look at the child's behavior and development to m...
Other Related Autism Screening and Diagnosis Articles
Emergency Contact for Bloomington
- In case of Emergency, call 911
Nearby Bloomington Hospitals results
- 1IU Health Bloomington Hospital
601 W 2nd St
Bloomington, Indiana 47403
(812) 336-682
- 2Bloomington Meadows Hospital
3600 N Prow Rd
Bloomington, Indiana 47404
(812) 331-800
- 3Monroe Hospital
4011 S Monroe Medical Park Blvd
Bloomington, Indiana 47403
(812) 825-111
- 4Indiana Univ Health Morgan Hosp
2209 John R Wooden Dr
Bloomington, Indiana 46151
(765) 342-844
- 5IU Health Bedford Hospital
2900 16th St
Bloomington, Indiana 47421
(812) 275-120
- 6St Vincent Dunn Hospital
1600 23rd St
Bloomington, Indiana 47421
(812) 275-333
- 7Franciscan St Francis Hospital
1201 Hadley Rd
Bloomington, Indiana 46158
(317) 831-116
- 8Johnson Memorial Hospital
1125 W Jefferson St
Bloomington, Indiana 46131
(317) 736-330
- 9Greene County General Hospital
Bloomington, Indiana 47441
(812) 847-228
- 10Columbus Behavioral Center
2223 Poshard Dr
Bloomington, Indiana 47203
(812) 376-171
- 11Columbus Regional Hospital
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Bloomington, Indiana 47201
(812) 379-444
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Citic Securities to Buy Credit Agricole's CLSA for $1.25 Billion
Citic Securities Co., China's largest brokerage by market value, said it agreed to buy Credit Agricole SA's CLSA unit for $1.25 billion.
Citic, based in Beijing, today completed the previously agreed purchase of a 19.9 percent stake in CLSA for $310.3 million, according to a statement to the Shanghai stock exchange. Citic will buy the remaining 80.1 percent stake fo$941.7 million, subject to it meeting conditions, the company said.
— With assistance by Keith Campbell
Before it's here, it's on the Bloomberg Terminal. LEARN MORE


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Ijan Hilaire
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OK, I looked at that GDC presentation and I think I have an idea of where you might see a difference here.
By default, the viewport covers the entire with and height of the render target as well as the entire range of Z values in the interval [0,1].
Try changing to a non-default viewport that covers only a portion of the depth range, say [0,0.5]. Then toggle the DepthClipEnable flag in the rasterizer state. Do you get different results for primitives that have depth values in the interval [0.5,1]?
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04/23/2013 | Changes to FCC Radiotelegraph Operator Certificates Coming May 20
The FCC's changes to its Radiotelegraph Operator Certificates go into effect next month.
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04/18/2013 | FCC Again Denies ARRL's Petition in BPL Proceeding
Though BPL is both a commercial failure and a proven interference source, the FCC still remains unwilling to adopt rules sufficient to protect licensed radio services.
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04/15/2013 | FCC Issues Two Forfeiture Notices for Cell Phone Jamming
On April 9, the FCC found two businesses -- The Supply Room in Oxford, Alabama and Taylor Oilfield Manufacturing in Broussard, Louisiana -- to have "apparently willfully and repeatedly violated" the Commission's rules by operating multiple cellular phone
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04/04/2013 | FCC Seeks to Reassess RF Exposure Limits
On March 27, the FCC released a First Report and Order, Further Notice of Proposed Rulemaking and a Notice of Inquiry (ET Docket Nos. 13-84 and 03-137). The documents seek to reassess the FCC's RF exposure limits and policies, as well as to propose change
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03/26/2013 | FCC Chairman Julius Genachowski, FCC Commissioner Robert McDowell to Step Down
FCC Commissioner Julius Genachowski and FCC Commissioner Robert McDowell both announced last week that they would be leaving the Commission.
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03/21/2013 | FCC Reduces Pennsylvania Ham's Fine
After affirming it was going to uphold a forfeiture in the amount of $4000 to Jose Torres, N3TX, of Philadelphia, Pennsylvania, the FCC decided to lower the fine to $3500. The FCC issued the fine in May 2011, saying that Torres' willful and repeated opera
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03/18/2013 | ARRL Executive Committee Focused on FCC and Regulatory Items at March Meeting
The ARRL Executive Committee (EC) held its first 2013 meeting Saturday, March 9 in Irving, Texas. The meeting's agenda included items concerning the FCC and regulatory items, FCC enforcement, legislative issues and organizational matters.
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03/05/2013 | Florida Ham Issued $25,000 Fine for Operating an Unlicensed Radio Transmitter and Interfering with Licensed Communications
On March 1, the FCC issued a Notice of Apparent Liability for Forfeiture (NAL) in the amount of $25,000 to Terry L. VanVolkenburg, KC5RF, of Cocoa, Florida.
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Project Mudslinger - School Of Hard KnocksWritten by on March 1, 2004 Contributors: Jimmy Dimsdale, Joey Helms Project Mudslinger Earns Its Degree In Black And Blue Ink
Project Mudslinger's maiden voyage onto the racetrack (seen in last month's issue) ended in mixed reviews. The bad news-we set the wrong toe-in, making the car skittish, and we lost a radiator hose in the feature, ending our night early in a cloud of steam. The good news-we didn't do any major damage to the little red race car.
Preparations for week two were fairly easy. We simply beat out a few dents in the sheetmetal, fixed the toe-in problem (from 1 inch of toe to 1/4 inch), and replaced both radiator hoses with new ones. From the start, Scott Helms and I agreed to rotate the driving privileges. This week, it's my turn.
It was comforting to have a better idea of what was going on at the track. The previous weekend, both the car and driver were brand-new. No one knew what to expect from either. After everyone had cleared out that first night, the management at Lancaster Motor Speedway was kind enough to allow me a few practice laps. Coming into my first race, I had an advantage over Scott in that I knew what the car would feel like when it turned left.
In the hot laps, it soon became obvious that fixing the toe-in made a world of difference in the car's manner. Before, it was as skittish as a teenage boy meeting his girlfriend's daddy for the first time. Now it felt much more stable and predictable. Later, as the South Carolina clay dried out in the heat and feature races, it only got better.
The racing itself was fairly uneventful. Since it was my first time in real competition, I started the heat and feature from the back of the pack, which meant I had to do a fair amount of dodging other cars that had spun or wrecked. I wasn't comfortable, or, let's face it, fast enough yet to pass anyone on the track. At one point, after a big tangle, I had Darrell Waltrip'd myself all the way up to fourth, simply by puttering past cars that had wrecked or spun.
Trouble came during a caution well into the feature. While cruising around the track holding my place in line, a puff of white smoke came out from around the hood. For a moment, the rear end felt loose. The water temperature was at 240, but started going back down. I was having too much fun and didn't want to admit my night should end early, so I stayed on the track. Two laps later, when the green flag flew again, the car simply wouldn't go. I pulled into the pits on the backstretch and immediately shut it off.
I had ignored the warning signs that we had again lost a radiator hose. Most of the water had dumped under the car, which is why the rear end felt loose for a moment. Everyone in the stands knew exactly what happened, but I had blindly kept motoring around the track. I had taken the temperature gauge dropping as a good sign, but that was only because there was no longer fluid in the cooling system. After half-heartedly watching the rest of the Mod 4 race finish, and trying to avoid direct eye contact with Scott, we packed up and left for home as soon as the track cleared.
Back home, we pulled the head off the four-cylinder and discovered the damage wasn't as bad as we had feared. The engine had overheated and seized a couple of exhaust valves, but that was about it. Johnson's Machine Shop, Project Mudslinger's engine builder, had the head back in fighting shape in no time, and we were ready to go racing again the next week. For the record, we now have plans to install a water pressure gauge in the Project Mudslinger cockpit at the next opportunity. There will be a big-fat-idiot light connected to it for yours truly.
The car was back together for a third assault. This time around, we went with NAPA flexible hoses and double-clamped both on each end. The rotation had Scott back in the car, which was good because I had a scheduling conflict and couldn't make it.
Not being there turned out to be a big mistake because I have to resort to secondhand information to detail what happened. Scott and I planned for this to be another "start from the back" weekend with the express intent of shaking down the car to see if we had finally licked the cooling problem. We wanted to last an entire race to see what the checkered flag looked like at the end. That, however, isn't what happened.
During the heat race, Scott had a formal meeting with the concrete structure found at racetracks, aka the wall. The wall imposed its will upon Project Mudslinger. I didn't see the car until Sunday morning, where it sat crippled on the trailer. In the true fashion of men everywhere, Scott and I didn't discuss what happened for days. It was just too much to handle.
Scott said the car was running like a dream. He was gaining confidence and speed when the car suddenly jumped to the right, in the center between turns one and two. It headed right for the wall. Thankfully, the ButlerBuilt seat and Rebco belts did their jobs, and Scott walked away with nothing more than a bruised ego. If I had been there, I suspect he would have avoided eye contact with me.
At first, we suspected a broken tie rod or that a tire had come off the bead, but neither was the case. A racing friend who was watching from the infield said it looked like the car caught a tire in a rut and simply became unbalanced. I guess our best explanation is, "It's just one of them racin' deals."
It was hard to tell exactly how much damage had been done until all the sheetmetal was removed from the car. We found out the hit was even harder than Scott suspected, and he was sitting in the car when it happened. The list of bent and cracked parts is pretty long and keeps growing. The tube frame structure in front of the front strut mounts is bent. We're still unsure if we can bend it back or if we'll have to cut it off and start over.
We also have a crack in the transmission case, a broken ear on the engine block where the starter bolts up, a bent strut, two bad rims, and other cuts and bruises. Another concern is that a number of the car's original structural welds holding the unibody together have broken loose. We aren't sure if the frame is still straight. We are definitely in for a lot more diagnostic work before we can determine the scale of repairs needed, or even where to start.
The only thing we know is we have our work cut out for us. Thankfully, we were only one race away from the end of the season. Now that the off-season is upon us, there is no great rush to put our Mustang back together.
Oh, before I forget, there is some good news to relay. In the philosophy that every cloud has a silver lining, the NAPA radiator hoses held up just fine.
More Photos From This Article View Slideshow
Overall, we knew we had damaged at least three exhaust valves because of the marks on the pistons where the valves had gone into them. Thank- fully, neither the pistons nor the head itself were cracked or showed any damage.The source of our troubles seems to be the flex hoses we are using to route water between the radiator and the block. The wire spring pulls up, leaving the hose as resistant to collapse as wet tissue paper.For help, we took the head back to our engine builder, Johnson's Machine Shop, where Larry Teal jumped right on it. It's nice to have an engine builder you depend on like Johnson's. I just wish we didn't need to depend on them quite so often.All four exhaust valves needed to be replaced. Two were damaged so badly that Larry had to remove both the valve and the guide together.As part of the inspection process, Larry checked every valvespring. They were OK and able to go back in the head.Larry installed two new guides, lightly honed the rest, and touched up the valve seats. After re-installing the valves and springs, he pressure-tested the valve/seat seal and everything checked out OK.Larry set the camshaft advance for us at the shop. It can be done with little more than a wrench and an angle finder on an overhead cam engine. Now all we had to do was be careful not to use the cam as a carrying handle before installing the head back on the short-block.
Notice how much area there is in the front of the rear wheelwell. Although the tire still held air, the right rear rim was bent and ruined. We are hoping the bent rim, rather than a tweaked frame, is the reason the wheel is no longer being centered in the wheelwell.Back in the shop, Scott pulled off body panels. The more he pulled off, the more pieces he found that were bent, cracked, or otherwise beat up. At first glance, it appears the steering wheel is the only thing that is not bent, but we think the car is still fixable.We know the car's sole purpose is to absorb the force of impact, getting bent in the process, but it was still hard to pull off all the bumpers and supports, knowing they were going to have to be fabricated all over again.This shot shows how much the tube frame structure in front of the strut towers has been shoved over. We are thinking about taking it to a body shop to see if someone can straighten it out, but we aren't sure if it would be better to cut it off and start over.Here's what's left of the right-front wheel. The rim has been flattened under the impact. That absorption of energy likely saved the chassis from even more damage.


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Roasted Greek Potatoes
Total Time
Prep 15 mins
Cook 1 hr
These potatoes are just like the ones at our favourite Greek restaurant...Paros Taverna. They are lemony with just the right balance of garlic and oregano. I never weigh the potatoes...and I always double the liquid mixture....as we like lots of the reduction. I have reduced the salt to 1 tsp as the lemon/garlic/herb takes care of most of the flavour. And most of us don't need that much salt. The roasting time is appoximate as to the size of the potatoes.
Ingredients Nutrition
- 4 lbs potatoes, medium, whole
- 1 cup water
- 1⁄2 cup lemon juice
- 1⁄3 cup extra virgin olive oil
- 3 garlic cloves, minced
- 1 teaspoon salt
- 2 teaspoons dried oregano
- 1 teaspoon pepper
- Preheat oven to 325 degrees.
- Combine water, lemon juice, oil, garlic, salt, oregano and pepper in bowl.
- Peel potatoes and leave whole.
- Place potatoes in baking dish and pour liquid over all.
- Bake for 30 minutes and turn potatoes over.
- Repeat this procedure until potatoes are cooked and the liquid has reduced.
- Serve potatoes with a spoonful of the reduction.
- Enjoy!
Most Helpful
These greek potatoes were wonderful, lemony and garlicy with a nice amount of herbs. I used the suggested amount of sea salt and fresh ground pepper. So tasty, the potatoes were tender and very flaovrful. I did cook the potatoes for about 1 1/2 hours, it took awhile for the liquids to reduce. Thank you so much for sharing your recipe.
These were very good, quite lemony. I reduced the amounts of both salt and pepper. These make a nice change from the usual. Thanks for posting!
This is now my favorite potato recipe. It's simply wonderful. I didn't peel my potatoes since the skins were nice and thin and I cut some of the potatoes so they'd be more uniform in size. Other than that, I followed the recipe as it's written. I wish I'd doubled the sauce as suggested in the intro. Oh yes, I also used a bit less salt since I'm trying to reduce the sodium in my diet. I just ground sea salt over the potatoes before roasting. Thanks for posting.


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Two suspects arrested after officer-involved shooting in Grand Chute
A Grand Chute police officer was injured after a suspect stole a squad car, but he was not shot.
Six injured after Bonduel crash involving Boy Scouts
Authorities say an SUV wandered off the roadway, over-corrected, and then lost control when the second vehicle struck the SUV.
"Last Dance" prom to give Jerika Bolen the time of her life
Before her life ends, 14-year-old Jerika Bolen wanted one last dance. The community gave it to her in style.
Waupaca County house explosion sends two men to hospital
The two men injured were severely burned.
9 killed in Munich attack; suspected lone gunman found dead
Munich police say it now appears the gunman in the shopping mall shooting acted alone, then took his own life.
How to Play the Tundraland Gridiron Giveaway
On this page we describe Tundraland's Gridiron Giveaway contest, provide quick links for entering the contest online, and provide solutions …
Car show a fundraiser for American Legion post
GRAND CHUTE, Wis. (WBAY) – An American Legion post is trying to raise money after it received an assessment from the town totaling more than…
Car stolen from Kiel car dealership
Police say the man stole a royal blue 2005 Chevrolet Trailblazer from Vogel's Chevrolet.
Green Bay home damaged in electrical fire
No one was inside the home at the time. There were no injuries.
West Allis man charged with buying gun used to shoot officer
The Milwaukee police officer was ambushed last weekend.
RV destroyed in Green Bay fire
Firefighters say a propane refrigerator ignition caused the camper to catch fire.
Sheriff: Florida man stole dead man's identity 22 years ago
Investigators say he used the name of a man who drowned in 1991.
Milwaukee police shoot, kill stabbing suspect
Police say the man was a convicted felon from Milwaukee and was on probation for strangulation.
EAA AirVenture's 'million little details'
The "World's Greatest Aviation Celebration" officially kicks off on Monday, but the EAA grounds are buzzing.
Former loan officer convicted in Van Den Heuvel bank fraud case
Paul Piikkilla pleaded guilty to conspiracy. Prosecutors say he helped De Pere businessman Ron Van Den Heuvel get loans that weren't approve…
Authorities identify victim in fatal Highway 23 crash
A man from Sheboygan Falls suffered critical injuries. A woman from Plymouth was also hurt.
Heat is a concern at Packers running events
The 1K course is slightly modified and extra water, ice and medical staff are in place for the 1K and 5K runs.
Gov. Scott Walker talks RNC, Trump and running for president again
"If anything I'm going to run for governor in 2018. I haven't made that official yet but that's really where my heart's intention is," Walke…
Wisconsin DOJ appeals judge's ruling on Voter ID
The judge says voters lacking a photo ID can vote if they sign an affidavit explaining why they couldn't get identification.
Shawano Lake Treatment Hailed a Success
For those who live along the lake, the difference they've seen over the past two months is still hard to fathom.
Boy airlifted to hospital after ATV crash
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Hillary Clinton names Tim Kaine her VP running mate
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Thompson sees Trump as winner, Cruz as loser at convention
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Police: Man climbs rope to burglarize Manitowoc theater
The suspect climbed up a 25-foot downspout to reach the business' roof.
De Pere Police seek registered sex offender
There are several warrants out for Jason Mann's arrest.
Oshkosh man sentenced to life in prison for murder of girlfriend
It comes after a jury found 31-year-old Samson Gomoll guilty of first-degree intentional homicide.
Democrat Hillary Clinton could soon choose vice presidential candidate
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Former NFL coach Dennis Green dead at 67
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Trump, speaking to supporters in Cleveland the morning after accepting the Republican nomination for president, said Cruz "may have ruined h…


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Bix Youth Band
2015 Bix Youth Band Schedule
Blossoms At Butterworth
Sunday, June 21, 1 - 4 PM
Catfish Jazz Society
Sunday, July 26, 3:00 pm
Bix Beiderbecke Memorial Jazz Festival
July 30 - August 1
2015 Bix Beiderbecke Memorial Youth Jazz Band
Nicholas Alt, Edison Jr. High
Sabrina Alt, Rock Island High School
Alexandra Axup, Rock Island High School
Ansel Bare, North Scott High School
Reid Barlow, Pleasant Valley High School
Lucas Boley, United Township High School
Cierra Brabant, North Scott High School
Nathan Brackevelt, United Township High School
Karlie Bunn, North Scott North Scott High School
Alexis Ernst, North Scott High School
Haylee Ernst, North Scott High School
Katy Fier, Clinton High School
David Fudge, Moline High School
Matthew Gray, United Township High School
Dale Huntley, Jr., Alleman High School
Andrey Lyamin, Edison Jr. High
Jacob Maranda, Rock Island High School
Koby Peterson, Rock Island High School
Joel Whitmore, Calamus-Wheatland High School
Directed By Jim Buennig, Western Illinois University
Bix Youth Band
The Bix Youth Band is sponsored by the Bix Memorial Society of Davenport, IA. An auditioned group, the band seeks to spread the music of Bix Beiderbecke and other early 20th century artists and further jazz education in the Quad Cities. The Youth Band is made up of middle and high school students from throughout the Quad Cities who express an interest in early jazz and complete a successful audition. Come see us this summer!
More Bix 2011


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Doctors Put Foster Children at Risk With Mind-Altering Drugs
Florida foster child killed self after being prescribed a powerful mix of meds.
Govt. Fails to Oversee Foster Kids' Drug Treatment
GAO report released today caps off year-long investigation by ABC News
Foster Kids and Meds: Finding Solutions
Part 2: Congress holds hearing on foster care, psychotropic drug prescriptions.
Confronting Doctors
Part 3: Sharyn Alfonsi confronts physicians about risky prescriptions.
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John Sachs
John Sachs was born in London, England. He is known for his work on Dancing on Ice (2006), Popstar to Operastar (2010) and The Brian Conley Show (1992). See full bio »
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Kashmiri Chops
Total Time
Prep 0 mins
Cook 30 mins
Ingredients Nutrition
- 1⁄2 kg mutton chops
- 1 1⁄2 cups milk
- 1 -2 egg
- 1⁄2 teaspoon ginger powder
- 1⁄2 teaspoon ground aniseed
- 1⁄2 teaspoon cardamom powder
- 1⁄2 teaspoon black pepper
- 3⁄4 teaspoon salt
- oil (for frying)
- in a pressure cooker place the chops.
- add in the milk, ginger powder, aniseed powder, cardamom powder and pepper powder.
- pressure cook this for 10 minutes or till chops become tender.
- remove the cooker lid and on low flame dry the milk.
- add salt, cook for few mins then keep aside.
- beat the eggs in a bowl with a pinch of salt in it.
- heat oil in a pan.
- dip the chops in the egg and deep fry till golden brown.
Most Helpful
The chops turned out very good...my family just loved it.


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Japan, China, South Korea: Asian Bonds and Currency Preview
The following events and economic reports may influence trading in Asia's bonds and currencies today. Bond yields and exchange rates are from the previous trading session unless stated otherwise.
Japan: Chief Cabinet Secretary Yukio Edano and Finance Minister Yoshihiko Noda will hold media briefings after a Cabinet meeting in the morning. Edano will have another press briefing at 4 p.m. in Tokyo.
The trade ministry may report that Japan's tertiary industry rose 2.7 percent in April from March, when it dropped 6 percent, according to the median estimate of economists in a Bloomberg News survey. The report is due at 8:50 a.m.
The Bank of Japan will release at 8:50 a.m. its report on the corporate goods price index for May. The index gained 2.5 percent from a year earlier, a separate survey showed.
The yield on the 1.2 percent government bond maturing in June 2021 was at 1.13 percent, according to Japan Bond Trading Co. The yen traded at 80.39 per dollar as of 7:03 a.m. in Tokyo.
South Korea: The central bank will keep its benchmark rate unchanged at 3 percent, according to nine of 17 economists in a Bloomberg News survey, while eight forecast an increase of a quarter of a percentage point.
The won was at 1,082.98.
China: The trade surplus jumped to $19.3 billion in May from $11.4 billion in April, according to the median estimate of economists in a Bloomberg News survey before data due today. The customs bureau report will also show that export growth slowed to 20.4 percent last month from a year earlier, compared with 29.9 percent in April, according to the poll.
China Development Bank will sell 20 billion yuan ($3.1 billion) of 10-year bonds.
The yield on the 3.83 percent bond due March 2021 was 3.83 percent, according to Chinabond, the nation's biggest debt-clearing house. The yuan was at 6.4759.
India: A government report today will show industrial output grew 5.6 percent in April from a year earlier, after gaining 7.3 percent the previous month, according to the median estimate of economists in a Bloomberg News survey.
The government plans to raise 120 billion rupees ($2.7 billion) selling bonds maturing in 2018, 2021 and 2027.
The yield on the 7.80 percent bond due April 2021 was 8.25 percent. The rupee was at 44.76.
Taiwan: The government will sell NT$100 billion ($3.5 billion) of 364-day debt.
The yield on the 1.375 percent bond due March 2021 was 1.47 percent, according to Gretai Securities Market. The Taiwan dollar was at NT$28.780.
Thailand: The central bank will sell 50 billion baht ($1.6 billion) of 14-day bills and 15 billion baht of floating-rate notes due 2014.
The yield on the 3.125 percent debt due December 2015 was 3.47 percent. The baht was at 30.45 per dollar.