text:
Physiologic and molecular consequences of endothelial Bmpr2 mutation
© Majka et al; licensee BioMed Central Ltd. 2011
Received: 28 June 2010
Accepted: 22 June 2011
Published: 1 December 2011
Pulmonary arterial hypertension (PAH) is thought to be driven by dysfunction of pulmonary vascular microendothelial cells (PMVEC). Most hereditary PAH is associated with BMPR2 mutations. However, the physiologic and molecular consequences of expression of BMPR2 mutations in PMVEC are unknown.
In vivo experiments were performed on adult mice with conditional endothelial-specific expression of the truncation mutation Bmpr2delx4+, with age-matched transactivator-only mice as controls. Phenotype was assessed by RVSP, counts of muscularized vessels and proliferating cells, and staining for thromboses, inflammatory cells, and apoptotic cells. The effects of BMPR2 knockdown in PMVEC by siRNA on rates of apoptosis were assessed. Affymetrix expression arrays were performed on PMVEC isolated and cultured from triple transgenic mice carrying the immortomouse gene, a transactivator, and either control, Bmpr2delx4+ or Bmpr2R899X mutation.
Transgenic mice showed increased RVSP and corresponding muscularization of small vessels, with histologic alterations including thrombosis, increased inflammatory cells, increased proliferating cells, and a moderate increase in apoptotic cells. Expression arrays showed alterations in specific pathways consistent with the histologic changes. Bmpr2delx4+ and Bmpr2R899X mutations resulted in very similar alterations in proliferation, apoptosis, metabolism, and adhesion; Bmpr2delx4+ cells showed upregulation of platelet adhesion genes and cytokines not seen in Bmpr2R899X PMVEC. Bmpr2 mutation in PMVEC does not cause a loss of differentiation markers as was seen with Bmpr2 mutation in smooth muscle cells.
Bmpr2 mutation in PMVEC in vivo may drive PAH through multiple, potentially independent, downstream mechanisms, including proliferation, apoptosis, inflammation, and thrombosis.
KeywordsPulmonary Arterial Hypertension mouse model pulmonary vascular disease
Pulmonary arterial hypertension (PAH) is a lethal disorder characterized by pulmonary vasoconstriction and vascular remodeling, leading to progressively worsening right ventricular strain, and eventual right heart failure[1]. Early molecular and physiologic events in the development of the idiopathic form of PAH (IPAH) are not known. However, in most theories of etiology, the pulmonary microvascular endothelial cell (PMVEC) plays a central role, either because of aberrant signaling to the smooth muscle[2], excess proliferation which fills in vessels[3], or excess apoptosis which causes vessels to drop out[4].
The familial form of PAH is usually caused by mutations in the type 2 receptor for the bone morphogenetic protein signaling pathway, BMPR2[5, 6], and there is substantial evidence that suppression of the BMP pathway is involved in all idiopathic PAH[7]. However, detailed effects of BMPR2 mutation in endothelium, either in vivo or in vitro, are not known.
BMPR2 signals through multiple downstream pathways[8]. In canonical signaling, when a dimer of BMPR2 receptors forms a complex with a dimer of type 1 receptors in the presence of ligand, the type 2 receptors phosphorylate and thus activate the type I receptor, allowing the type 1 receptor to phosphorylate SMAD transcription factors 1, 5, or 8. The SMAD transcription factor complexes with SMAD4, and enters the nucleus to transcribe targets. However, BMPR2 also possesses a long cytoplasmic tail, well conserved by evolution but found in no other TGFb superfamily receptor. This tail domain is not required for SMAD signaling[9], and signals through other, currently poorly understood, pathways[10, 11]. Mutations in human patients can fall anywhere in the gene[12]. Determining the common molecular consequences of SMAD dependent and cytoplasmic tail dependent signaling is thus important to determining how BMPR2 mutation predisposes to disease.
We have previously reported physiologic and molecular consequences in smooth muscle of inducible transgenic overexpression of Bmpr2 mutants with defects in SMAD-dependent (and cytoplasmic tail-dependent (Bmpr2R899X) signaling[9, 13, 14]. These transgenic mice rely on the fact that the Bmpr2 receptor works as a dimer; thus, overexpression of a mutant protein inhibits expression of the native protein in two ways - by acting as a decoy receptor, and by binding native receptor into a nonfunctional complex.
The goal of this study is to complete a first description of the physiologic and molecular consequences of the different classes of Bmpr2 mutation in endothelium. Yelle et al have already reported physiologic consequences of our Bmpr2R899X mutant under an endothelial promoter[15]. In this report we thus express our doxyycline-inducible Bmpr2delx4+ mutant in adult mice under the control of a previously published Tie2 promoter[16–18] and perform hemodynamic and histologic examination of consequences. To determine molecular consequences, we cultured PMVEC from control, Bmpr2R899X, and Bmpr2delx4 mice, and performed expression analyses using Affymetrix arrays. We found that Tie2-rtTA x TetO7-Bmpr2delx4 mice developed variably elevated RVSP, associated with increased muscularization particularly of small arteries, increased proliferation, infiltration of a variety of inflammatory cell types, increased thrombosis, and increased apoptosis. Expression analysis found that Bmpr2delx4 and Bmpr2R899X mutations caused very similar changes in proliferation, apoptosis, adhesion, and metabolism, but were quite different in their effect on inflammatory and thrombotic genes.
Generation of Tie2-rtTA x TetO7-Bmpr2delx4+mice
Both transgenes used for this double-transgenic mice have been independently previously published. The Tie2-rtTA mouse was originally generated for study of neovascular retinopathy, and was shown to be endothelial specific in that context[16–18]. The TetO7-Bmpr2delx4+ mice were first described and validated crossed to a smooth muscle-specific SM22-rtTA second transgene[14]. Both mice were on an Fvb/n strain background. Double transgenic mice are doxycycline inducible; they express very little transgene until fed doxycycline, with expression levels in whole lung roughly equivalent to expression in the lungs of our previously published SM22-rtTA x TetO7-Bmpr2delx4+ model[14].
Mouse Phenotyping Protocol
Mice were given Avertin (500 mg/kg IP) to induce a surgical plane of anesthesia. The animals were then shaved to expose the surgical area. Mice were placed on a heated surgical table (Harvard Apparatus 872/1, 872/H, Holliston, MA) and secured with surgical tape. Systemic blood pressure and pulse was measured via a tail cuff and pulse transducer run through a PowerLab NIBP Controller (ADInstruments, Colorado Springs, CO). The surgical site was viewed using a Zeiss OpMi 1 surgical microscope. An incision of approximately 1 inch in length was made extending from the animal's chin down to the right armpit. The thyroid gland was then blunt dissected upwards to expose the underlying tissue and the right jugular vein. The jugular vein was then separated from surrounding tissue using dissecting forceps until the body of the vessel was completely free from adherent tissues. The cranial end of the jugular was tied off completely and a loose tie was then made at the caudal end of the exposed jugular using 4-0 braided silk suture. 4" Micro-dissecting scissors were then used to make a small incision in the medial aspect of the right jugular vein. A Millar 1.4 French pressure/volume micro-tip catheter transducer (SPR-839, Millar Instruments, Houston, TX) connected to a PowerLab/8S (ADInstruments) was then inserted through the incision and gently threaded down into the right ventricle. Proper placement within the ventricle was determined through observation of the pressure volume loop obtained from the catheter. The loose caudal suture was then tightened to secure the catheter in place. Once the catheter is properly placed, data was collected using Chart 5 for Windows (ADInstruments). Once blood pressure and volume data was collected, the caudal suture was re-loosened and the catheter removed for tissue inflation. 100U of heparin was then injected through the jugular incision to prevent clotting. The suture was then re-tightened to prevent bleeding. The animals were then removed from the surgical table to a dissecting area. All animal procedures were approved by the UCHSC animal care and use committee (IACUC).
Mouse tissue collection and morphometry
After checking for insensitivity to toe-pinch, a paramedian sternotomy was performed, lungs were flushed with saline injected through the RV, the right lung was tied off, and the left lung was inflated using low-melt agarose injected through the trachea. The left lung was removed and fixed in 10% formalin and paraffin embedded. The right lung was flash-frozen in liquid nitrogen. Hearts were collected for weighing to determine right ventricular muscularization. Lung sections were stained for smooth muscle actin (SMA), counterstained with hematoxylin, and imaged using a Zeiss Axioscop 2 microscope. Twelve 20x microscopic fields were collected from each animal from which counts of fully or partially (less than 50%) muscularized vessels and vessel diameter were collected.
Immunoflourescence and Carstair's Stain
Immunofluorescence was done as previously described using the following antibodies and dilutions. Monoclonal anti-alpha smooth muscle actin antibody was purchased from Sigma (St. Louis, MO) and used at a dilution of 1:1000. Rabbit polyclonal anti-smooth muscle actin was purchased from Abcam (Cambridge, MA) and used at a dilution of 1:250. Rabbit polyclonal anti CD45 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and used at 1:100. Rabbit polyclonal CD3 epsilon was purchased from Abcam (Cambridge, MA) and used at a dilution of 1;200. Mac-3 antibody was purchased from BD Pharmingen (San Jose, CA) and used at a dilution of 1:200. Tissues processed for immunofluorescence were rinsed and placed in either Alexa Fluor 488 or 594 (Invitrogen, Carlsbad, CA) secondary antibody at a 1:1000 dilution for 1 hour at room temperature in the dark. Slides were then rinsed with PBS and coverslipped with Vectashield with DAPI (Vector Laboratories, Burlingame, CA). All staining was evaluated and digital images acquired using a Zeiss Axioskop 2 equipped with an AxioCam (Carl Zeiss Microscopy, Jena Germany). Carstairs' stain[19] for fibrin and platelets was done by IHC tech (LLC12635 Montview Blvd. Ste. 215 Aurora CO, 80045) Briefly, deparaffinized slides were incubated 5 minutes in ferric aluminum, rinsed, stained with Mayer's hematoxylin 5 minutes, rinsed, stained in picric acid- orange G solution for 30 minutes, rinsed, differentiated with 1% phosphotungstic acid, rinsed and stained with aniline blue solution for 1 hr and rinsed before mounting. This is a modification of picro-Mallory staining, resulting in orange to red fibrin, gray-blue platelets, bright blue collagen, red muscle, and red, green, or yellow red blood cells.
Cell culture and siRNA
Murine pulmonary microvascular endothelial cells (MVECs) were isolated by collagenase digest of whole lung tissue and selected by cytometry using PECAM-1. The isolated cells were grown to ~60% confluency on 10 cm cell culture dishes in Endothelial Growth Media 2 (EGM2) containing 5% serum. Following VE-Cadherin and diLDL staining to confirm EC phenotype, transfections were done at passage 3 using 100 nM scrambled siRNA control (Qiagen, Valencia, CA) or siRNA to BMPR2 (Ambion). Lipofectamine and Plus reagents (Invitrogen, Carlsbad, CA) were mixed with Optimem media (Invitrogen) and siRNA as per manufacturer's instructions. EGM2 media was replaced with Optimem before lipid-RNA complexes were added to the cells and incubated for 2 hours at 37°C. A volume of EGM2 media containing 10% serum equal to that already on the plate was then added to the existing transfection media and the cells were incubated for and additional 24-48 hrs. Cells were subsequently harvested using the PARIS kit from Ambion (Austin, TX). To extract the BMPR2 from the membrane, SDS was added to a final concentration of 6%. Equal amounts of protein determined using BCA protein assay (Pierce, Rockford, IL) was loaded onto a 10% polyacrylamide gel with 10% glycerol and transferred to Hybond-P membrane (Amersham, Piscataway, NJ). BMPR2 is detected with a mouse monoclonal antibody diluted 1:150 (BD Transduction Laboratories, San Jose, CA) and visualized by chemiluminescence (ECL, Amersham, Piscataway, NJ).
Apoptosis - Tissue
The fluorescein FragEL DNA fragmentation detection kit (Calbiochem, San Diego, CA) was used to look at fragmented DNA signifying apoptosis. The manufacturer's protocol was followed. Briefly, paraffin embedded lung tissue from Tie2- BMPR2delx4+ and control mice were cut, put onto slides and deparaffinized as described above. Fragmented DNA was labeled using terminal deoxynucleotidyl transferase which binds to exposed 3'-hydroxyl ends left after fragmentation of DNA in response to apoptotic signals and catalyzes the addition of fluorescein-labeled and unlabeled deoxynucleotides allowing for visualization.
Apoptosis - Cells
Plates (60 mm) were seeded with 3 × 105 mouse pulmonary microvascular endothelial cells in Minimum Essential-alpha (MEM-a) media (Invitrogen, Carlsbad, CA) supplemented with 20% FBS and incubated overnight. BMPR2 siRNA or scrambled siRNA (100 nM, Ambion) was transfected using Lipofectamine and Plus reagent (Invitrogen, Carlsbad, CA), cells were recovered with complete media at 2 h. At 72 h cells were trypsinized, washed with ice-cold PBS and resuspended in 1 ml of PBS. Apoptotic cells were labeled with YO-PRO-1, Vybrant Apoptosis Assay (Invitrogen, Carlsbad, CA) which stains apoptotic cells green fluorescent while necrotic cells stain red fluorescent with propidium iodide separating early apoptosis and necrosis using flow cytometry. To prevent apoptosis through the BMPR2 pathway, cells were seeded as described above. After overnight incubation, cells were washed with PBS and media replaced with MEM-a without FBS. At 24 h, 150 ng/ml BMP-2 (R&D systems, Minneapolis MN) was added and at 72 h cells were trypsinized, washed with ice-cold PBS and resuspended in 1 ml of PBS. Apoptotic cells were labeled with YO-PRO-1 (Vybrant Apoptosis Assay, Molecular Probes) and analyzed by flow cytometry.
Generation of murine PMVEC
Immortomouse X Rosa26-rtTA2 X TetO7-Bmpr2R899X or Immortomouse X Rosa26-rtTA2 X TetO7-Bmpr2delx4+ triple transgenic mice (Immorto-Bmpr2R899X) were bred with transgenes verified by PCR genotyping of tail DNA. The immortomouse contains a transgenic insertion of the SV40 large T antigen, tsA58, under control of an interferon-inducible promoter[20]. When cells are grown at 33°C and interferon is added, the transgene is activated and the cells are immortalized and proliferate freely; at 37°C, this transgene is inert. The immortomouse therefore produces cells which proliferate as though they were immortalized at 33°C, but revert to a more normal phenotype when cultured at 37°C. Immorto-Bmpr2R899X PMVEC were collected from adult mice as previously described[21], and verified by staining for endothelial markers VWF, PECAM, and VE-Cadherin.
Affymetrix arrays
Mouse Gene 1.0 microarrays (Affymetrix, Foster City, CA) were performed on pooled RNA from three plates each. Array results were submitted to the NCBI gene expression and hybridization array data repository (GEO, http://www.ncbi.nlm.nih.gov/geo/), accession number pending. Arrays were analyzed using a combination of Affymetrix Expression Console, comparisons within Microsoft Excel, and Webgestalt for gene ontology groupings[22].
Statistical analysis was performed with Statview 5.0 (SAS Institute, Cary NC). Specific tests used are listed in figure legends, with p < .05 considered significant.
Tie2-Bmpr2delx4+mice have increased RVSP, muscularization of vessels, and cellular proliferation
Tissues were collected from additional groups of Tie2-Bmpr2delx4+ with transgene activated 2.5 and 8 weeks to allow more complete analysis of histologic changes. There was an increase in muscularization of very small (<25 mm) vessels by 2.5 weeks, and an increase in both small and mid-sized vessels (25-50 mm) by 8 weeks; larger vessels never had a statistically significant increase in muscularization (Figure 1B-F).
Tie2-Bmpr2delx4+mice have increased pulmonary inflammatory cells and thromboses
Thrombosis and perivascular inflammation could be found together (Additional File 2, Figure S2B), but this was uncommon.
Loss of endothelial BMP signaling results in apoptosis in vivo and in culture
Molecular consequences of Bmpr2delx4+ and Bmpr2R899Xmutations in PMVEC
PMVEC were cultured from double and triple transgenic mice, expressing the Immortomouse transgene[20], the Rosa26-rtTA2 transgene[27], and either no additional transgenes (control PMVEC), the TetO7-Bmpr2R899X, or the TetO7-Bmpr2delx4+ transgene. The immortomouse transgene was added because it is otherwise impractical to collect enough PMVEC from mice to perform experiments. Affymetrix Mouse Gene 1.0 microarrays were used to determine molecular consequences of Bmpr2delx4+ and Bmpr2R899X mutations in PMVEC. Two arrays were run for each cell type, with RNA for each array consisting of a pool of three independently grown plates (total of six arrays).
Using a requirement of a minimum of a 1.4x change, a minimum expression intensity of the higher of the pair of at least 128 (arbitrary Affymetrix units), and average expression values separated by more than the sum of their standard deviations (which equates to roughly a p < .05 for difference), we found 840 unique genes with gene ontology information differentially expressed in Bmpr2delx4+ compared to controls (Additional File 3, Table S1), and 1692 differentially expressed in Bmpr2R899X mutants compared to controls (Additional File 4, Table S2).
In contrast, expression of Bmpr2delx4+ and Bmpr2R899X mutations in PMVEC had very different effects on inflammatory and thrombotic genes. Bmpr2delx4+ mutation resulted in induction of multiple cytokines not upregulated in Bmpr2R899X (Figure 7E). There were many of these, but examples upregulated only in Bmpr2delx4+ include the angiogenic chemokine Cxcl12[38], the interleukin 27 receptor Il27ra, and the inflammatory cell adhesion molecule CD24a. Some inflammatory genes were changed only in Bmpr2R899X, including downregulation of MAP kinase p38δ (Mapk13) and upregulation of the TH1 inducer interleukin 18 (Il18). We also tested expression of interleukin 6, which we had previously found to be regulated by Bmpr2delx4+ mutation in smooth muscle; we found it upregulated in Bmpr2delx4+ mutant PMVEC, but not in Bmpr2R899X (Additional File 5, Figure S3).
Effects of the two mutations were also very different in thrombosis related genes (Figure 7F), with increases in prothrombotic genes in Bmpr2delx4+ but not Bmpr2R899X; examples include 4x increases in platelet/endothelial cell adhesion molecule (PECAM1) and platelet chemokine ligand Ccl5, a 28x increase in platelet adhesion molecule selectin P (Selp), and an 18x increase in Entpd1, a nucleotidase involved in response to thrombosis[39]. In contrast, the anticoagulant thrombomodulin (Thbd) is downregulated, but only in Bmpr2delx4+.
Endothelial markers are not consistently changed by Bmpr2 mutation.
Post Hoc (p < .05)
Delx4 > Control
Delx4 > Control > R899X
Endothelial-Specific Marker 1
Delx4 = R899X > Control
Flk1 (VEGF receptor 2)
Delx4 = Control > R899X
Flt1 (VEGF receptor 1)
Control > R899X > Delx4
Delx4 > Control = R899X
Multimerin 2
Delx > Control = R899X
Selectin, endothelial cell
Delx4 = Control > R899X
Delx4 > R899X > Control
Delx4 > Control = R899X
R899X > Control = Delx4
Von Willebrand factor
This study analyzed the physiologic and molecular effects of Bmpr2 mutation in endothelium. We found that most Tie2-Bmpr2delx4+ mice developed elevated RVSP, although with large variability in degree. This was associated with increased muscularization of small arteries (Figure 1), an increase in proliferating cells (Figure 2), inflammatory cells(Figure 3), thrombosis (Figure 4), and apoptosis (Figure 5). Molecular changes substantially matched the physiologic changes, with an increase in gene ontology groups related to proliferation (Figure 7B), apoptosis (Figure 7A), and thrombosis (Figure 7F). The increase in inflammatory cells seen is probably related both to the increase in cytokines (Figure 7E, Additional File 5) and the decrease in cell-cell adhesion genes (Figure 7C). BMPR2 has been previously shown to regulate endothelial barrier function in the context of leukocyte transmigration[40]. We also saw large scale alteration in metabolic genes, including response to oxidative stress (Figure 6); this matches our previous findings in smooth muscle and whole animals[27]. Unlike expression of Bmpr2delx4+ mutation in smooth muscle, expression in PMVEC did not cause loss of differentiation markers. Overall, this is the first report of the molecular effects of Bmpr2 mutation in PMVEC, with results consistent with both our current physiology and our previous findings.
Physiologic results from this mouse were similar to results from an endothelial-specific BMPR2 knockout mouse described by Hong et al[41], despite having very different methods of generation. The only difference was higher penetrance in our model (~80% as opposed to ~30%). Histologic details such as thromboses and perivascular inflammatory cells and levels of increased proliferating cells were all almost identical between the models, which strongly increases confidence that both models accurately represent the consequences of complete suppression of signaling through BMPR2 in the endothelium.
Both of these models are quite different in histologic detail than expression of a different Bmpr2 mutation, Bmpr2R899X, in endothelium[15]. Endothelial expression of Bmpr2R899X, which retains intact Smad signaling[9], results in vascular pruning and increased RVSP, but without the obvious increase in thromboses. This also matches our molecular data; increases in thrombotic pathways were not seen in PMVEC with Bmpr2R899X mutation (Figure 7F). Our molecular results imply that neither increased thrombosis nor increased inflammation are required for the development of Bmpr2-mediated PAH, although obviously both could exacerbate disease.
Broadly, we believe that the sum of the data to date suggests that the normal role of BMPR2 in the adult pulmonary vasculature is as a master switch mediating injury response. After injury, suppression of BMPR2 signaling through its cytoplasmic tail results in a decrease in cell-cell adhesion to improve migration and leukocyte recruitment, a metabolic shift towards glycolysis to support production of proteins and lipids needed for proliferation, and an increase in propensity for both proliferation and apoptosis to support tissue remodeling and angiogenesis or angeogenic repair. Suppression of BMPR2 signaling through SMAD results in an increase in thrombosis and an increase in expression of cytokines, and in smooth muscle but not endothelium, dedifferentiation. In those with intact BMPR2, this naturally resolves as the injury is repaired. In the case of BMPR2 mutation, these injury responses can never be successfully resolved, leading to a pathologic continuation of the pro-thrombotic and pro-proliferative pathways which would have been adaptive in acute injury.
Decades ago, long before molecular origins of PAH were known, there was controversy over whether the initiating events in the disease involved inflammation, proliferation, thrombosis, or vasoconstriction through defective endothelial-smooth muscle communication[42]. The discovery of BMPR2 as the familial PAH gene had promised a resolution to this controversy. However, the current study, as well as its predecessors in the literature, indicate that all of these are consequences of BMPR2 mutation alone, and our expression arrays suggest that they may all be relatively direct consequences of BMPR2 mutation, not an in vivo cascade. Our study does not address the issue of whether any are dispensable for the development of PAH. However, since each of these individually is capable of driving PAH in animal models[43], it is possible that inflammation, proliferation, thrombosis, and vasoconstriction in the context of pulmonary vascular injury can not be easily separated; it is not that one is the cause and the other are bystanders, but rather they are inextricably linked.
We present here a relatively comprehensive first look at the physiologic and molecular consequences of Bmpr2 mutation. These data support an important role for Bmpr2 signal in maintaining endothelial homeostasis, and support our broad hypothesis that a normal role for Bmpr2 is in injury response. Future studies will be needed to determine the specific roles of and alterations in pathways identified, including, p53-mediated proliferation, MSX1-mediated apoptosis, a shift to glycolysis, and a loss of cell-cell adhesion.
List of abbreviations
- Bax:
BCL2-associated X protein
- Bnip3:
BCL2/adenovirus E1B interacting protein 3
- Cadm2:
cell adhesion molecule 2
- Ccl5:
chemokine (C-C motif) ligand 5
- Ccnb2:
Cyclin B2
- CD24a:
CD24a antigen
- Cldn10a:
Claudin 10a
- Cxcl12:
chemokine (C-X-C motif) ligand 12
- Dap:
Death Associated Protein 1
- Dgka:
diacylglycerol kinase a
- Dgkh:
diacylglycerol kinase h
- Eno2:
enolase 2, gamma neuronal
- Entpd1:
ectonucleoside triphosphate diphosphohydrolase 1
- Gfpt2:
glutamine-fructose-6-phosphate transaminase 2
- Il6:
Interleukin 6
- Il18:
Interleukin 18
- Il27ra:
interleukin 27 receptor, alpha
idiopathic pulmonary arterial hypertension
- Itga1:
integrin, alpha 1 [
- Lhx2:
LIM homeobox protein 2
- Mapk13:
mitogen activated protein kinase 13
- Msx1:
homeobox, msh-like 1
pulmonary arterial hypertension
proliferating cell nuclear antigen
- Pecam1:
platelet/endothelial cell adhesion molecule 1
- Pgk1:
phosphoglycerate kinase 1
pulmonary microvascular endothelial cell
- Ptges:
prostaglandin e synthase
- Ptprv:
protein tyrosine phosphatase, receptor type, V
- Selp:
selectin P
- SerpinE1:
serpin peptidase inhibitor, clade E, member 1
- Sesn2:
Sestrin 2
- TGFbeta:
transforming growth factor, beta 1
- Thbd:
- Vav3:
vav 3 oncogene
- Vcam1:
vascular cell adhesion molecule 1.
This work was supported by National Institute of Health HL82694, HL071596, HL079315, HL014985, and HL091105.
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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


text:
Magento store for clothing store
This project received 101 bids from talented freelancers with an average bid price of £629 GBP.Get free quotes for a project like this
Project Budget£250 - £750 GBP
Total Bids101
Project Description
I am a project manager for an upcoming magento ecommerce website.
The basic requirement for this project is to setup magento site, configure with provided template and components.
Setup mailchimp integration for newsletter blast.
Integrate paypal as payment processor
Last but not least, prepare an user manual.
You need to have solid understanding of magento setup and be able to assist us in running a successful magento store.
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Can a vegetarian person get a good meal at this restaurant?
Does this restaurant have a TV?
Is this a seafood restaurant?
Is this restaurant appropriate for Kids?
Does this restaurant offer takeout or food to go?
Is this restaurant good for bar scene?
Can a gluten free person get a good meal at this restaurant?
Is this restaurant good for business meetings?
Does this restaurant serve Bar / Pub food?
Is this restaurant good for dinner?
Thanks for helping!
Made a mistake and reserved a table on the Naples restaurant instead of fort Myers, Sarah was so demeaning and making fun I made a mistake, I will never. return to that place
We had seen the reviews online and decided to try this place while on vacation. We weren't disappointed. The spinach souffle was the best I've ever had anywhere. There were 4 in our party and everyone had very good food. The service was a little slow but not too bad. This is not fine dining but it is quality overall.
The food, wine and atmosphere at the Stonewood do not disappoint whenever I go there. There are good menu selections to choose from and the wait staff is always friendly. Prices are on the slightly higher end, but I always feel that the value for the money is worth the price paid. I enjoy dining in the bar area or...
Had dinner here and it was a good choice. Not too crowded after 8 PM, good menu choice available and specials. Prices are mid-range to upper mid-range.
Service was adequate and we'll come back.
From entry to exit, the experience was wonderful. Our server Bob was attentive and knowledgeable and presented me with a complimentary dessert with burning candle. Burger and salad were delicious. Atmosphere is "business casual," and carpeting keeps the noise level pleasant. Highly recommend!
Nice location for traveling from Cape Coral to the SWFL airport. Great food, nice bar area and happy hour deals.
2 of us showed up for dinner at 5:30pm on a Monday. It was recommended by the party I was with. Since we did not have a reservation(the place was nearly empty), we were told we could eat in the bar area. We did get a nice booth and enjoyed our meals. Would definitely return but would make a reservation....
I've had nearly every appetizer on the menu and they are all great, but the Chips with Blue cheese and Balsamic vinegar reduction are amazing!!! The smoked salmon is also off the charts luscious. I haven't had an actual dinner at Stonewood because we end up sharing the happy hour apps and never make it that far. I love the...
It's been a few years since we visited the restaurant. The decor is upscale,the service very friendly but the most important thing is the food was very good and the wine reasonably priced. The cost of the meals were also lower than comparable restaurants. All in all good value.
Went here with friends and met in the bar area which was very nice. Service a little slow on the drinks and server was friendly. All of our meals were very good and they even understood my no carb lifestyle and accommodated me.
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Would like to see full menu.......
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text: The Register – As soon as the idea of a WLAN phone was floated, the critics came out of the woodwork, claiming that telephony over IP didn't offer high enough voice quality and that Wi-Fi coverage wasn't complete enough. Soon those claims will be tested in the market, as Mitsubishi has released the first commercially available 802.11b-based phone. The Japanese giant has beat expected pioneer Cisco to the punch.


text:
An Incredible WordPress Website
- Status Closed
- Budget $30 - $250 CAD
- Total Bids 44
Project Description
Hey Everyone!
I am looking for a fun to work with a Wordpress designer that can design a gorgeous website on a budget.
I will also give you a testimonial and case study in it as well.
I am thinking that the best template to work off of is Thesis, but please let me know what your thoughts are on this!
I am looking for a design as rich and beautiful as [url removed, login to view]
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22 March 2016
Holy Week 2016: Codwch
Join us during Holy Week as we reflect on what it is to arise from the sorrow and darkness of Good Friday and into the light of Easter Day.
by Daphne Godwin, leader of Ffald-y-Brenin along with her husband Roy
Codwch means arise in Welsh. Click here to see a full size version of the reflection in it's original form alongside the picture.
The cube, the box
It's strong
It's deep
It's dark
The walls are thick,
The stone is hard
The stone is black
Inside the box
The lid is off
Who lifted it?
The light is in
The stone is not black
But clean and good
There's hope, I feel
My spirit rise
I see I am good
And caring
To help me out
Dare I
Leave my cosy box
It hides a lot
The light
The light will highlight everything
Dare I
Dare I
Take that hand….?
You might also be interested in
- 27 Mar 2016 Holy Week 2016: Snap
- 26 Mar 2016 Holy Week 2016: Arise out of waiting
- 25 Mar 2016 Holy Week 2016: Good Friday
- 24 Mar 2016 Holy Week 2016: The good thief
- 23 Mar 2016 Holy Week 2016: Arising out of disappointment


text:
Unfair criticism fired up Leicester - VardyFootball
Edinburgh 17-18 Cardiff Blues
Lloyd Williams scores a late try as Cardiff Blues recover from 11 points down to snatch victory over Edinburgh.
Lloyd Williams scores a late try as Cardiff Blues recover from 11 points down to snatch victory over Edinburgh.
Edinburgh recruit Darcy Graham, "one of the most exciting prospects in Scottish rugby", on a two-year professional contract.
Richard Cockerill says he has a "little bit of sorting out to do" and will prioritise local talent when he takes over as Edinburgh head coach.
Pro12 club Edinburgh appoint former Leicester Tigers director of rugby Richard Cockerill as head coach from next season.
Cardiff Blues recover from 11 points down to snatch victory over Edinburgh courtesy of Lloyd Wiliams' try.
With Alan Solomons gone, it is up to caretaker Duncan Hodge to revive a stale Edinburgh side, writes Tom English.
Tom English assesses the outlook for Edinburgh following the departure of Alan Solomons and Duncan Hodge's appointment as acting coach.
Samoan centre Sasa Tofilau is set for a Pro12 debut after switching from the third tier of Scottish club rugby to Edinburgh.
BBC Scotland's Tom English reviews an entertaining 2015-16 Pro12 season, which saw Connacht crowned champions.
Scotland's record cap holder Chris Paterson looks ahead to a season-defining few weeks for Glasgow and Edinburgh.
Six Nations misery, glory for Glasgow and World Cup heartache: Tom English reviews Scotland's year in rugby.
Back from the long-term injury that dashed his captaincy chance, Scotland's Grant Gilchrist is pushing for a World Cup place.
Edinburgh hooker Stuart McInally is training to be a pilot as he bids to win a place in Scotland's World Cup squad.
How to get into rugby union - into the thick of the 15-player game or try rugby sevens, which made its Olympic debut in 2016.
Get latest scores and headlines sent straight to your phone, sign-up to our newsletter and learn where to find us on online.
Enjoy the best debate, analysis and interviews with BBC 5 live and World Service plus our rugby union commentary listings.
This week's sport on the BBC includes England's ODI series with West Indies, European Indoor athletics plus FA Cup & Premier League football.


text:
Hundreds of birds die in western Ky.
There are 81703 comments on the The Cincinnati Enquirer story from Jan 5, 2011, titled Hundreds of birds die in western Ky.. In it, The Cincinnati Enquirer reports that:
MURRAY, Ky. - State wildlife officials say "several hundred" dead birds were found near the Murray State University campus last week.
Join the discussion below, or Read more at The Cincinnati Enquirer.
#75594 Nov 23, 2012
You ppl need a job, a hobby, a something. How in the world do u come up with all this random babbling day in and day out, day after day after day after day??
#75595 Nov 23, 2012
I am Thankful that I have electricity out here on the rock farm at all. I recall the "Good Old Days" when we did not and trust me, they just were not all that good. LOL
#75596 Nov 23, 2012
Most of us have had our turns at a job already, and we have a hobby. It is to randomly babble day in and day out. What is yours?
#75597 Nov 23, 2012
I have not gotten that rare letter either, but I understand that it will be less than 2 %.
#75598 Nov 24, 2012
You WILL get a letter in December from the SSA. Your percentage will be 1.7%. Dad said about 37.50 for them.
Funny, the clerk at The Road Ranger in Tollesboro told me tonight they had just had an increase of 10% across the whole store. But, to make up for the big jump in cigarette prices, they are selling 2% Milk, 2 for $6. Hurry, supplies can't last long.:-)
#75599 Nov 24, 2012
Seen the say thing in my hometown birds die,,the air pollution kill them in flight above my Town
#75600 Nov 24, 2012
I guess I won't be hurrying. At the price of gas, it would not be very cost effective for me to come to Tollesboro even if the milk was free. LOL
You are right, I confirmed the 1.7% with someone else that should know.
#75601 Nov 24, 2012
Good Morning folks. This will be a great day to get warmed twice by my wood. Once when I split and stack it outside my back door and the second time when I put it in the stove. It warmed me the third time yesterday when I cut it and brought it down off the hill and loaded it into the truck.
@Hempburn.. eat your heart out. I will be using the hydraulic wood splitter hehehe
#75602 Nov 24, 2012
G'day mates, looks like a nice day brewing. I need to hit a store or two but dread the crowds, best go early. I've learned a couple of things in my time about shopping: Never get between a fat chick and a sale table. Never stop at the new invention table for the product that slices, dices and crawls on it's bellie like a reptile. Never ever touch the empty cart in isle 6, it's not abandoned it's a trap.
Have a good one,,,,, trolls, not so much.
Since: Apr 09
#75603 Nov 24, 2012
Lol. Couldn't have said it better myself. We have already worked our arses off over the years, now this is all we are able to do.
Since: Apr 09
#75604 Nov 24, 2012
Thanks, any raise is better than none. Lol.
#75607 Nov 24, 2012
Good Sat, to you all. I , know all about the wood thing got to cut a load today , but lucky I have kids to stack it . Take care shopping , Ill be working the next few weekends , to pay for all I spent Thursday night. U, be good , you may be tuff, but I think your a teddy bear in hiding, really with a great big heart.. See yall , Happy Weekend...
#75608 Nov 24, 2012
Sad news this morning, RIP Larry Hagman, who IMO starred in one of the greatest roles as a villain ever as J.R. Ewing on Dallas.
"Is who I am"
Since: Aug 08
#75609 Nov 24, 2012
I heard that. It's sad. No more J.R..
Since: Mar 11
Location hidden
#75610 Nov 24, 2012
Watch out for that hydraulic wood splitter. My neighbor was using one a few years back and it malfunctioned. It nearly caused an accident. I don't know much about them. Hubby used to split our wood the old fashioned way. Tough work, but good exercise. We don't have a wood stove down here, but we are thinking of finishing off one of our outbuildings and putting a wood stove in there, just in case we lose power like we did in the ice storm a couple of years back.
Since: Mar 11
Location hidden
#75611 Nov 24, 2012
Una, that's fluoride, not chloride, LOL. Conspiracy theorists believe that fluoride in the drinking water gradually makes people less aggressive and causes brain damage over time. But it's not a conspiracy if it is true. BTW, Una, you are quite clever in many of your remarks that are not filled with you know what. I think that the smut belies an intelligence that you are trying to hide. Why don't you let the real UNA shine through the fog emanating from your bowels.
Since: Mar 11
Location hidden
#75612 Nov 24, 2012
After spending 35 years working overtime, I am happy to engage in random babbling for a change. Lighten up.
#75614 Nov 24, 2012
Flurries in Eastern , Ky this morning. Got the shopping finished in an hour flat. Didn't get assaulted by any buggys( shopping carts). Increase in electric bill this month by $47 bucks. Those of you with wood burning stoves need to chop your logs,but I'd rather do that than pay some electric bills. I had family with $300-400 bills last year:( Stay warm and good day to all the decent posters...
#75617 Nov 24, 2012
Going out could be dangerous on this wild and wooly day when anything can happen. You'll need to expect the unexpected and take special care in public places. Your friends and associates may need your assistance but they may also be a drain on your resources and your sanity. Romance can be exhilarating but may be tinged with just a touch of obsession.
#75620 Nov 24, 2012
I hope everyone stays warmm, decent or indecent LOL. Burning firewood is my 'job' and I can save $300-$400 or more in utility bills which I can't afford living on that monthly Social Security payment. I believe it was Ben Franklin's "Poor Richard's Almanac" that said a penny saved is a penny earned.
I can cut, split and stack that natural resource and with about 8 hours work get about a month's supply. I already had enough yesterday to last through January and with an afternoon of work yesterday and a little more than that today, I think I will be able to make it until spring and anything I do after today will just be fore recreation and exercise. Plus with a power outtage, I can cook on my stove if need be. Coffee break and babbling time now. LOL
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text:
Medical Waste Shredder With Ce - Chinaprice: contact company for price
Brand Name: ENERPAT
Model Number: GWS
Shredding Champer: 250x250 mm
Hopper Opern: 400x400 mm
Blade Width: 20 mm
Blade Diameter: 380 mm
Throughput: 100~500 kgs per hour
Machine Size: 800x500x1500 mm
Machine Weight: 0.4 tons
Enerpat have USA, UK, CHINA, AUSTRALIA service company, supply the 2 days fast
After sales service, we have the complete spare parts supply chian by DHL, which supply spare parts to customers hand fast in 3 days from uk and china
( 1) Enerpat is the UK company. We have the good credit
( 2) Enerpat is the CE certificated company
( 3) Enerpat have the ISO And CE production management system
( 4) Enerpat have ENERPAT UK, ENERPAT AUSTRALIA, ENERPAT USA, ENERPAT UKRAINE ENERPAT GERMANY, we supply the biggest sales guarantee
Q1: What is the Warranty for our machines
A1: For all enerpat china recycling machines have 12 month warranty, If any quality problems on our side occured in this period , we will take on the shipping cost and replacement.
Q2: What is the relationship between ENERPAT CHINA And Enerpat Group Uk
A2: Enerpat china is a wholly owned subsidiary of ENERPAT GROUP UK LTD, we play as the Asia production base
from 1986 years on.
Q3: : How can i trust ENERPAT CHINA?
A. ENERPAT is the UK baded company located in London
B. ENERPAT is the SGS certificated company
C. ENERPAT have the USA, UK, Australia, Germany company, and more than 450 agents
D. WELCOME VISIT ENERPAT ANY COMPANY in the fixed time.
Q4: Where is ENERPAT CHINA located in china
A4: Enerpat china located in NO 1 Xin Tai Road, Chang Qing Town, Nantong City, Jiangsu province, which is 2.5 hours from shanghai city. you can choose Taxi and Bus to our city.
Q5: What machines ENERPAT CHINA main produce?
A5: We main produce scrap metal baler, scrap shredders, recycling plant, Horizontal Balers and Vertical balers
Q6: How to choose the right scrap metal baler?
A6: When you plan to buy one enerpat brand metal baler, we suggest you consider 3 issues as below:
( 1) How many tons scrap metal you get for processing per day
( 2) The size of the stove, because you will choose the right bale size for stove
( 3) Choose china made machinery parts or Europen made machienry parts
Q7: Is there cheap shipping cost to import to our country?
A7: For small order, express will be best. And for bulk order, sea ship way is best but take much time.
For urgent orders, we suggest via air to airport plus our ship partner send to your door.
Q8: Can we get support if we have our own market position?
A8: Please inform us your detailed mind on your market demand, we will discuss and propose helpful suggestion for you, to find the best solution for you.
Company Contact:
- Posted By: NANTONG ENERPAT MACHINE CO.,LTD
- Phone: 051355087511
- Address: Changqin industry zone,rugao, Nantong, China
- Email:
- Website: http://www.enerpatgroup.com
Published date: April 12, 2016 -
- Business Description: ENERPAT is a British multinational engineering and recycling company headquartered in London and Manchester. It is UK' S largest engineering company and maker of scrap recycling equipment and its recycling plant, which generates about 12 percent of the company' S total sales, is its second-most profitable unit behind the industrial automation division
Enerpat' Principal activities are in the fields of industry, energy, environment and recycling. It is organized into three main divisions: Industry, Energy, recycling, Enerpat and its subsidiaries employ around 50, 000 people across nearly 20 countries and reported global revenue of approximately EURO 28.3 billion in 2012. The company has been the subject of a number of controversies in its history.
Enerpat offers a wide range of recycling engineering- and scrap processing-related products and services. Its products can be broadly divided into the following categories:
Car Recycling Plant, Tyre Recycling Plant, E-Waste Recycling Plant, Municipal waste recycling plant. Waste Shredders, Waste Balers, And Waste Machines. And transportation and logistics-related products
Nantong Enerpat Machine Co., Ltd is the wholly owned subsidiary of ENERPAT
Which also Enerpat' ' S Asia production base.
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Introducing the OtterBox for iPhone 3G Impact Series! The Impact Series merges durability, impact absorbing features and style in a single, slim-fit Silicone layer. All buttons, ports and functions are accessible right through the case, and the inner corners have been engineered to dissipate impact AWAY from most Apple iPhone. Also, the OtterBox for iPhone 3G Impact Series has an ergonomic design that your hands will love to be wrapped around!
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text:
Translation 3 pages + SEO
This project was successfully completed by Handyman84 for $309 USD in 30 days.Get free quotes for a project like this
Project Budget$300 - $1800 USD
Completed In30 days
Total Bids41
Project Description
Jetico is currently looking at someone with SEO and Google AdWords
experience to translate (German, French, Italian, Russian and Finnish) and optimize 3 webpages.
We require:
- Translation (German, French, Italian, Russian and Finnish)
- Website analysis to find out the best suitable keywords for each language/related market
- On-page optimization
- Off-page optimization
- Monitoring and adjustment (monthly fee to agree)
- Monthly report (guaranteed keywords ranking in first page to agree)
If successful, we will post a second project which will include translation and optimization of the following languages:
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Job Overview
A career with the Forest Service will challenge you to manage and care for more than 193 million acres of our nation's most magnificent lands, conduct research through a network of forest and range experiment stations and the Forest Products Laboratory, and provide assistance to State and private forestry agencies.
It's an awesome responsibility - but the rewards are as limitless as the views.
This position is temporary and has a Not-to-Exceed date. However, an extension of the appointment may be possible without further competition. The appointment may also end early due to lack of work or funds.
This position is located in a Forest Service unit. The position is a general technical assistant performing routine tasks under a higher graded technician or professional.
These positions are located in Region 6 on the Rogue River-Siskiyou National Forest in Central Point, Oregon.
For additional information about the duties of the position, please contact: John B. Justin at firstname.lastname@example.com or 541-858-6101.
TEMPORARY APPOINTMENT: Some positions will have varying work schedules. Some positions may have irregular and protracted hours of work. Tours of duty include full-time or less than full-time and will involve working weekends or irregular work hours. Considerable travel may be required. Some positions involve work that requires travel away from the official duty location to remote worksites within commuting distance (49 miles).
Positions will be filled as a temporary appointment up to 6 months (1039).
The USDA Forest Service has legislative authority to recruit and fill Permanent (Career/Career- Conditional), Temporary, and Term Appointments under the USDA Demonstration Project. Under this authority, any U.S. citizen may apply.
Assists technical or professional personnel in performing various routine field and/or laboratory tests employing standard biological techniques. Independently does routine repetitive tasks on experiments or tests.
Receives training and performs a variety of simple and routine tasks.
Receives training and assists in experiments that are more complex or field work.
Travel Required
- Not Required


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Khalsa Wills
reducing inheritance tax
Khalsa Wills may discuss with clients ways to reduce inheritance tax.
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We offer a dedicated and professional service to you to assist YOU in making your will, deciding if you need
Khalsa Wills speaks both English AND Punjabi, delivering personal service to its clients.
Khalsa Wills writes professional wills based on its client's needs.
We offer expert legal advice to our clients with a personal and professional service, 7 days a week.
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text: I just noticed this comment, because of NAFTA very rarely is duty charged on items coming across the border from the US, with all the things I've bought on Head-Fi I think I may have paid duty once or twice but that would be on items coming from other than the US like China. Alberta may not have to pay any provincial sales tax but they should still have to pay all other charges like GST and brokerage. Prince Edward Island is like this, at least until the new year when HST goes into effect, for whatever reason PST is not applied to packages from outside of Canada so until HST goes into effect here next year we only pay 5% GST and whatever brokerage and, in rare cases, duty that are applied. It's going to hurt having to pay an extra 9%.


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"I Found A Way" to Get Involved
If you want to find a way to help animals in distress, whatever the cause, you can do so right here by taking the following actions. Also, don't forget to sign up for IFAW's action alerts to get all the news on the animals and issues you care about most.
Donate Now
Our work depends on your generous support. Please give what you can to help the animals.
Our Animal Action Education programme helps children learn and care about animals
Congress is considering a joint resolution (H.J. Res. 69 / S.J. Res. 18 ) to eliminate important protections for bears, wolves and other species on national wildlife refuges in Alaska.
In 2016, the U.S. Fish and Wildlife Service (FWS) issued a rule (with IFAW's individual and coalition support) to protect native wildlife—bears, wolves, and other iconic species—on national wildlife refuges (NWR) in Alaska from inhumane and nonselective hunting and trapping practices.
The rule prohibits certain unsporting and irresponsible practices authorized by the Alaska Board of Game for taking native wildlife on the state's national wildlife refuges. These practices include: killing black bears, including young cubs and mothers, at den sites; using bait to attract and kill brown bears; relying on cruel body-gripping traps and snares to kill black and brown bears; aerial tracking and gunning of black and brown bears; taking wolves and coyotes during the denning season when they are likely to be caring for young; and killing native predators even where such take is in consistent with refuge purposes. Each of these practices threatens target and non-target animals with inhumane capture and killing, disrupts ecosystems, and runs counter to traditional fair chase principles.
Now, H.J. Res. 69 / S.J. Res. 18 threatens to reopen the door to these cruel and ecologically damaging practices.
Rescinding the Alaska NWR rule and turning over wildlife management decisions to the State of Alaska would make hunting a priority above all others on Alaska's refuges, despite the fact that the vast majority of visitors to national wildlife refuges are not hunters. It would interfere with the rights of all Americans to enjoy land and wildlife in national wildlife refuges that are supposed to be managed for our shared benefit.
The U.S. House passed H.J. Res. 69, despite fierce opposition from the public and many members of Congress. The measure will now go to the Senate, where we will continue the fight to defeat it. Please contact your senators today and urge them to oppose H.J. Res. 69 / S.J. Res. 18.
Rising seas. Melting ice caps. Drought. Raging wildfires. Animals and humans are already losing habitat and their homes to the early effects of global climate change, and scientists say that the next decade is our last, best chance to avoid reaching the point of no return.
But there is hope, and there is a clear path toward a clean energy future that benefits everyone. We need to show President Trump that Americans of all stripes care about this crucial issue. Stand with us and add your voice to the call for climate action.
We've prepared a template e-mail for you to send. We encourage you to personalize this copy to make it more effective. Please ensure that you are polite at all times.
We will then submit all letters together to President Trump so make your voice heard today!
Australia's new Environment Minister Josh Frydenberg signed the death warrant on the Ballina 200 on the very day he was sworn into office.
Please email Minister Frydenberg to let him know that, despite the road being built, Ballina's koalas still have friends who will be watching out for them as the bulldozers rumble in.
We've prepared a template e-mail for you to send. We encourage you to personalize this copy to make it more effective. Please ensure that you are polite at all times.
© Friends of the Koala
Earlier this month, The U.S. Fish and Wildlife Service released strong new rules that (with proper implementation and enforcement) all but shut down interstate ivory sales, imports and exports of ivory.
This is a huge win for elephants. The new regulations close loopholes that previously allowed ivory from recently poached elephants to be sold. They also reduce the amount of elephant trophies than can be brought into the U.S. We are the largest importer of elephant trophies in the world!
The new regulations could not have come at a better time. Elephants are facing an unprecedented poaching crisis driven by demand for their ivory tusks. By shutting down the U.S. market for ivory, FWS is helping reduce global demand and therefore pressure on elephants halfway across the world in Africa.
The rules come on the heels of ivory bans in several states including New York, New Jersey, California, Washington State, and most recently Hawaii. Efforts are underway in Oregon, Massachusetts, and other states. The federal and state rules also set an example for international action, and it is clear that momentum is building to end the elephant poaching crisis.
While FWS's new rules are very strong, they are not perfect. U.S. citizens will still be allowed to import a limited number of sport hunted elephant trophies – up to 2 elephants, per hunter, per year. IFAW believes that even one trophy is too many, and elephants deserve to live, not be a grotesque ornament on a wall.
Please sign our petition to thank FWS for shutting down the U.S. ivory trade, but also make it clear that the U.S. should not allow any more of these gruesome trophies to enter our country. All you have to do is fill out your information on the right, and we will add your name to the list of elephant champions who have made their voice heard. Feel free to personalize the email, but please be courteous at all times.
Trophy hunting affects people, animals, and habitat worldwide. IFAW's Killing For Trophies: An Analysis of Global Trophy Hunting Trade report found that as many as 1.7 million hunting trophies may have been traded between nations between 2004 and 2014, with at least 200,000 of those being threatened species. The United States accounted for a staggering 71% of the import demand, or about 15 times more than the next highest nations on the list. We are importing thousands of trophies annually from animals threatened with extinction, a practice that is far out of step with modern day animal welfare ethics.
Therefore, we ask you to write your U.S. representative and senators and urge them to support the Conserving Ecosystems by Ceasing the Importation of Large (CECIL) Animal Trophies Act (S. 1918 / H.R. 3526).
If enacted, this bill will amend the Endangered Species Act (ESA) to help protect animals that have not yet received a final ESA listing from trophy hunts by restricting their import into the U.S.
This bill would help to protect imperiled wildlife around the world. Now, it is more important than ever to let your voice be heard. Ask your U.S. representative and senators to co-sponsor H.R. 3526 and S. 1918, (respectively). A simple note will help protect imperiled species from being hunted and brought to the U.S. as "trophies."
Feel free to personalize your own email, or use the template below.
If you've already taken our pangolin action, please take a moment to sign our petition to list Elephants as "Endangered".
Last July, IFAW co-authored and submitted a technical petition to list a little known, but amazingly unique, animal as "Endangered" under the US Endangered Species Act (ESA): the pangolin. Pangolins are medium-sized, scale-covered, insect-eating mammals that live in several areas of Africa and Asia. But while pangolins are fascinating and play an important role in their ecosystems as insect regulators, they are one of the most imperiled species on the planet.
In March, pangolins received some good news from the US Fish and Wildlife Service (FWS).Our petition received an official, positive "90-day finding." Now, the US government is conducting an investigation, based on our petition, to find out if pangolins will receive "Endangered" status.
And there is no time to waste. Pangolins are the most illegally trafficked mammal in the world. Over 1 million pangolins are estimated to have been poached in the last decade in order to satisfy demand for their meat and scales. The rate at which they are being killed is completely unsustainable and cruel, and may lead to extinction if nothing is done. The U.S. can take a big step in stopping the trade of pangolins through an "Endangered" designation. According to government data, thousands of pangolin products are imported or smuggled into the U.S. every year.
Pangolins need the best protection they can get as soon as possible. You can help by signing our petition showing support for listing all seven unlisted pangolin species under the ESA (there is only one species that is currently protected). All you need to do is fill out the form here, and will submit this letter with your signature. Make your voice heard today!
If you've already taken our elephant action, please take a moment to sign our petition to list Pangolins as "Endangered."
The US Fish and Wildlife Service (FWS) is considering our petition to list African elephants as "Endangered" under the US Endangered Species Act (ESA). In March, the petition passed the first hurdle by receiving a positive 90-day finding from FWS. But now, the US government is conducting a comprehensive investigation to decide if elephants will receive full "Endangered" status. Currently, African elephants are only listed as "Threatened," which sets a lower bar for protective measures.
That elephants need our help is clear. African elephant populations have declined dramatically in the last decade due to extremely high levels of poaching. It is estimated that an elephant is killed every 15 minutes, on average, for its ivory tusks – that is 96 animals a day! At this rate, some regional populations of elephants may be wiped out in the next decade.
Trophy hunting – overwhelmingly conducted by wealthy Americans – is another inhumane threat to these magnificent animals, because hunters target the healthiest animals and destroy their tight family bonds. An "Endangered" listing would be a huge step towards stopping trophy hunting and the trade of elephants in the US once and for all.
The Fish & Wildlife Service needs to improve protections for elephants as soon as possible. You can help by signing our petition showing support for listing elephants as "Endangered." All we need is for you to fill out the form here, and we will submit this letter with your signature. Make your voice heard!
Reflecting broad public support for big cat protection, both the U.S. House and the U.S. Senate have introduced bills to bring an end to the private ownership of dangerous wild cats. We now have an opportunity to let members of Congress know that their constituents support big cat protections and to ask that they cosponsor the Big Cat Public Safety Act, H.R. 3546/S. 2541.
By advancing H.R. 3546/S. 2541, Congress can help end the mistreatment of captive wildlife and greatly reduce the unnecessary risks to the public from improperly managed big cats.
Tell members of Congress that you care about big cats and support H.R. 3546/S. 2541.
A quick note to your members of Congress could help protect big cats nationwide. We have prepared a template email for you to send. If you prefer, you can personalize this copy to make it more effective. Please ensure that you are polite at all times.


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The man who shot the polar bear that killed a schoolboy and savaged four others in Norway has been hailed as a hero.
Adventurer Michael 'Spike' Reid, 29, was one of two leaders of the expedition which came under attack.
Horatio Chapple, 17, an aspiring medical student, was killed and four others were injured by the bear yesterday morning.
The group was on a British Schools Exploring Society (BSES) expedition to the Arctic.
Mr Reid, from Plymouth in Devon, suffered serious head and neck injuries when he was mauled by the animal.
He is reported to be in a serious, but stable, condition after being airlifted to a hospital in the Norwegian town of Tromso.
His family said they were told by British Embassy officials that it is believed he shot the animal.
Peter Reid, Michael's father, said: "We got a phone call from the BSES in the morning. We were devastated and very worried.
"We have been told everyone was saying it was Michael who shot the bear and he was a hero. It was very moving.
"A lot of the day, we've been thinking about the family of the boy who died."
The attack on the campsite, near the Von Post glacier, about 25 miles (40km) from Longyearbyen, took place early yesterday.
Mr Reid said the incident had come as a complete shock.
He said: "In all honesty, we were more worried last year when Michael went to Afghanistan to climb.
"This was such a rare and unlikely event, it was inconceivable."
Mr Reid added his son was one of the team leaders responsible for science experiments and had also been involved in training.
He said: "It's what he loves to do. He was so excited to be going when we spoke to him four days ago.
"He was having a terrific time and was really in his element.
"We were shaken by the news, but we have a son alive and under very good medical care in Norway.
"There's a family in Wiltshire who have lost their son. For us, their grief must be unimaginable."
BSES Expeditions said the others injured were fellow team leader Andrew Ruck, 27, believed to be from Aberdeen, and youngsters Patrick Flinders, 16, from Jersey, and Scott Smith, 17, of Cornwall.
The British Embassy in Norway said its priority was to reunite the patients with their families in a hospital in Britain.
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Register to recognize inspiring companies at Top Workplaces Dec. 4 reception and Dec. 5 section
SANTA ANA, Calif. – Dec. 3, 2013 - The Orange County Register will announce the 100 small, midsize and large companies ranked highest by local employees at the newspaper's annual Top Workplaces awards night on Wednesday, Dec. 4 at the City National Grove of Anaheim. The 100 Top Workplaces also will be featured in a special, 52-page glossy magazine in the newspaper on Dec. 5.
"The 100 companies honored at the Top Workplaces reception and within the section provide inspiration to people who aspire to create and work for thriving businesses in Orange County," said Aaron Kushner, the Register's publisher and co-owner. "It is an honor each year to award local businesses with the exclusive distinction as Top Workplaces, and celebrate the accomplishments of employers who make a meaningful impact in our community."
Twenty large companies, 30 midsize and 50 small workplaces (those with 35 to 100 employees based in Orange County) made the 2013 list of Top Workplaces. Rankings were calculated by the independent survey firm WorkplaceDynamics.
More than 60,000 employees from more than 1,000 companies across Orange County participated in the 2013 Top Workplaces survey, and responded anonymously to 19 statements about their workplace experience and leadership at their companies. Employees ranked their bosses in areas such as ongoing training, culture and atmosphere, pay and benefits, inspiration from managers, and meaningfulness of their work.
For the first time, the survey also asked employees to rank their companies as to how "socially responsible" they are. The top-rated winners include companies that donate time and resources to the homeless, hospitals, schools, senior centers and animal shelters.
Creatively dubbed the "Oscars of H.R.," the annual Top Workplaces reception is attended by more than 400 people from top-nominated businesses and other employers interested in networking and modeling the successful approaches implemented by the Register's Top Workplaces. The Dec. 4 event is open to the public, and hosted in partnership with Professionals In Human Resources Association.
The Top Workplaces reception begins at 5 p.m. Wednesday at the City National Grove of Anaheim, at 2200 E. Katella Ave., Anaheim. The evening's host is Orange County Register & Freedom Communications Vice President Steve Churm. The keynote speaker is Chapman University Business Dean Reggie Gilyard.
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P0885 - TCM Power Relay Control Circuit/Open
- Transmission Control Module (TCM) Power Relay harness is open or shorted
- Transmission Control Module (TCM) Power Relay circuit poor electrical connection
The transmission control relay is used to supply power to the solenoid pack when the transmission is in normal operating mode. When the relay is off, no power is supplied to the solenoid pack and the transmission is in Limp-in mode. The relay output is fed back to the Powertrain Control Module (PCM). It is referred to as the Transmission Control Relay Output circuit or switched battery.
Immediately after a controller reset (ignition key turned to the run position or after cranking engine), the PCM verifies that the relay contacts are open by checking for no voltage at the switched battery terminals. After this is verified, the voltage at the pressure switches is checked. There should be no voltage on the pressure switches at this time. The PCM will then activate the relay.


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Fuel Tank Cleaning
I have a 91 YJ V6 and it started acting up on me last night. We checked the fuel pressure and it was 5 lbs. I had a shop look at it and they said it was my fuel pump and they would charge me $400 to fix it. I didn't believe them at the time so I changed the fuel filter and that did nothing. So, tonight I'm changing the fuel pump.
One of my friends told me I should clean out the tank along with the replacing the pump. Any advice on proper methods or chemicals to use to clean it out?
Is it a stock engine since they only had an inline 6 or a four cylinder that year?
When you drop your tank and pull out the fuel filter look inside the tank, most likely you'll find out it is just fine. If you do decide to wash it out just use soap(dishsoap) and water, just make sure to get all the soap out and let it dry completely before putting it back together. Remember if you clean the tank and leave chemicals or water behind you are going to cause more problems than you would solve.
It is a stock inline 6. Thanks for the advice, VHunter. Just wanted to make sure I didn't need to do anything specific (with chemicals) to it.
Took out the tank Thursday night to replace the pump and realized I had no gas in the tank all along. my gauge was reading that I still had a quarter tank left but I was getting no fuel pressure to the engine. Go figure, I just had no gas. The worst part was I had the Jeep towed from a gas station. You live and you learn!
Does anyone know if the gauge is reading incorrectly, could it still be that the fuel pump is sending the information to the gauge incorrectly or is there a different sending unit on the Jeep?
The sending unit is a seperate componet from the fuel pump.
Never clean a fuel tank with a chemical cleaner, it will loosen years of rust and sediment and varnish which will get through the fuel filter to the engine and cause serious problems.
Just use a high quality detergent fuel and change the filter often.
So, if my guage is reading I have a quarter tank when I'm actually empty then what could the possible problems be?
I can only narrow it down to either a bad sending unit or the gauge. I just wanted to make sure there couldn't be another part that could go bad.
If the guage still shows 1/4 tank when empty then i would replace the sending unit as they fail after time.
FULL TANK? Faulty Gauge? idk HELP !!!! tnx
Jeep Wrangler 1989 Base. My fuel gauge is ALWAYS FULL even though i know the tank is empty. What are ALL possible reasons why this is? engine 258 6cyl. everything is stock. Just need my baby running once again. THANKS !!!! - boonew88
I would start with the gauge. If it like my boat, you should be able to ground out the lead to gauge, or test the resistance with an ohmmeter. Sorry, but thats as technical as I can get....I have no idea how to actually do either of those things. But good luck!
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It's Time for a Fresh Approach — Rhythm Can Help Rhythm is inextricably tied to language. The human heartbeat shares a time signature with one of the most universal linguistic patterns known to man, the iambic meter ("Shall-I com-PARE thee-TO a-SUM mer's-DAY?"). Speechwriters frequently use rhythm to their advantage, hoping to stir listeners with a specific cadence or tempo. Numerous ...Read More »
Author Archives: Saga Briggs
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30 Academic Resources on Learning – for Educators and Innovators
Many of us read blogs about learning and discuss the learning process with our colleagues, but few of us actually bother keeping up with the latest science. Maybe it's because, if we do manage to identify a source or two, we are immediately turned off by all the dense scientific jargon. Or maybe it's not always clear which resources provide information specifically on learning and not just education in general.Read More »
Now-ism: Why Perfect Scores Don't Matter And What To Focus On Instead
"Education is what people do to you; learning is what you do to yourself." These are the wise words of Joi Ito, Director of the MIT Media Lab. In a recent TED Talk, Ito reveals a revolutionary style of innovation that he and his colleagues discovered during a time when they had to take learning into their own hands.Read More »
12 Must Read Books on Education for 2015
Few things are more satisfying than finally getting your hands on a book you've been meaning to read. In 2015, you're going to want to make room in your schedule for a lot of satisfaction.Read More »
10 Most Powerful Uses of Technology for Learning
Regardless of whether you think every infant needs an iPad, I think we can all agree that technology has changed education for the better. Today's learners now enjoy easier, more efficient access to information; opportunities for extended and mobile learning; the ability to give and receive immediate feedback; and greater motivation to learn and engage. But if we had to pick the top ten, most influential ways technology has transformed education, what would the list look like?Read More »
15 Surprising Discoveries About Learning
What are some of the most encouraging known facts about learning? From taking a walk to learning a new language, there are countless things we can do to improve the way we learn. Below we list fifteen steps toward a better brain:Read More »
16 Ways Your Brain Is Sabotaging Your Effort To Learn
Our own brains regularly deceive us in order to make sense of the world we live in. Our brain will tell us we're smarter and better looking than everyone else, and that any fault brought to our attention should probably be blamed on someone else.The human brain is our best friend, and our worst enemy, and unless we keep one eye peeled, it can hijack our learning completely. In this article I'd like to examine some of the "traps" the brain sets for us during the course of our academic careers, and what we can do to avoid them.Read More »
20 Things Educators Need To Know About Digital Literacy Skills
Widely understood to be essential to success in the workplace and modern life, digital literacy is beginning to emerge as a necessary component of curricula across the globe. As current undergraduates have never known a life without the internet, it's only natural that universities should nurture their familiarity with technology, encouraging its use in teaching and learning.Read More »
How Educators Around The World Are Implementing Mobile Learning
In less than a decade, mobile technology has spread to the furthest corners of the planet. Of the estimated 7 billion people on Earth, 6 billion now have access to a working mobile phone. Africa, which had a mobile penetration rate of just 5% in the 1990s, is now the second largest and fastest growing mobile phone market in the world, with a penetration rate of over 60% and climbing.Read More »


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Associate Professor, Eppley Institute
Courtesy Appointment:
Tel: 402-559-5558 (Office)
Research Interests
Molecular mechanisms and therapeutic targets of cancer metastasis and chemoresistance
Summary of Research
Metastasis is the cause of 90 percent of deaths from cancer, and yet little is known about its underlying mechanisms. Therapies to effectively treat metastatic disease are scarce. Traditional chemotherapy and radiotherapy are limited by their lack of specificity. Molecular targeting of oncogenic signaling molecules has the potential to be a more effective approach for cancer treatment than those presently available. However, it has so far had a very limited clinical impact. In addition, drug resistance is one of the main reasons of colon cancer recurrence and therapeutic options are very limited. Thus, there remains a need for the identification of potential drug targets and development of novel therapies to improve cancer treatment. Understanding the mechanisms of metastasis and chemoresistance will provide important information necessary for the development of new drugs and efficient therapeutic strategies. Metastatic cells acquire abnormal characteristics that facilitate their deadly behavior, including the ability to:
The research in my lab focuses on understanding the molecular mechanisms of cancer metastasis and chemoresistance and identification and validation of novel targets associated with cell survival, invasion, angiogenesis and metastasis. The ongoing research projects include:
- Identify and validate new targets associated with metastasis and drug resistance;
- Investigate the role of microRNAs, a special category of oncogenes or tumor suppressors, in cancer metastasis and drug resistance (using human cancer cells and in vivo mouse models) as well as their regulation by different signaling pathways;
- Investigate the role of cancer stem cells (or tumor initiating cells) in cancer metastasis and drug resistance.
- Investigate the crosstalk between LGR5, TGFβ and Wnt signaling pathways and their role in colon cancer.