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Most gastrointestinal stromal tumors (GISTs) possess a gain-of-function mutation in c-KIT. Imatinib mesylate, a small-molecule inhibitor against several receptor tyrosine kinases, including KIT, platelet-derived growth factor receptor-α, and BCR-ABL, has therapeutic benefit for GISTs both via KIT and via unknown mechanisms. Clinical evidence suggests that a potential therapeutic benefit of imatinib might result from decreased glucose uptake as measured by positron emission tomography using 18-fluoro-2-deoxy-d-glucose. We sought to determine the mechanism of and correlation to altered metabolism and cell survival in response to imatinib. Glucose uptake, cell viability, and apoptosis in GIST cells were measured following imatinib treatment. Lentivirus constructs were used to stably express constitutively active AKT1 or AKT2 in GIST cells to study the role of AKT signaling in metabolism and cell survival. Immunoblots and immunofluorescent staining were used to determine the levels of plasma membrane-bound glucose transporter Glut4. We show that oncogenic activation of KIT maximizes glucose uptake in an AKT-dependent manner. Imatinib treatment markedly reduces glucose uptake via decreased levels of plasma membrane-bound Glut4 and induces apoptosis or growth arrest by inhibiting KIT activity. Importantly, expression of constitutively active AKT1 or AKT2 does not rescue cells from the imatinib-mediated apoptosis although glucose uptake was not blocked, suggesting that the potential therapeutic effect of imatinib is independent of AKT activity and glucose deprivation. Overall, these findings contribute to a clearer understanding of the molecular mechanisms involved in the therapeutic benefit of imatinib in GIST and suggest that a drug-mediated decrease in tumor metabolism observed clinically may not entirely reflect therapeutic efficacy of treatment. (Cancer Res 2006; 66(10): 5477-86)
Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the digestive tract. GIST cells are believed to emanate from a progenitor of the interstitial cells of Cajal (1, 2), a population of spindle cells that are pacemaker cells of the gut, or from an interstitial mesenchymal precursor stem cell (3). Most GISTs express gain-of-function mutations of KIT, the receptor tyrosine kinase (RTK) encoded by the c-KIT proto-oncogene (4, 5). It has also been noted that ∼10% GIST are wild-type for c-KIT, with 30% of these harboring activating mutations in the platelet-derived growth factor receptor (PDGFR) α (6).
KIT, a 145-kDa transmembrane glycoprotein, is a member of the RTK type III family that includes PDGFR, macrophage colony-stimulating factor receptor, and FMS-like tyrosine kinase 3 (7–10). These receptor kinases share common structural features: an extracellular domain made up of five immunoglobulin-like repeats, a strongly conserved juxtamembrane domain, and a tyrosine kinase domain that is split into two domains by an insertion sequence of variable length (11).
KIT oncogenic activation results in autophosphorylation of tyrosine residues, which serve as docking sites for various signaling intermediates (12, 13). KIT signaling regulates mechanisms that are crucial for differentiation, phosphorylation, apoptosis, neoplastic transformation, and malignant progression (14–17). However, KIT signals have variable biological effects depending on the cellular context. Therefore, the signaling pathway data in various normal and cancer cell models do not predict which KIT signaling pathways are critical for oncogenic behavior in GIST. It has been shown that, depending on the KIT mutation, various KIT oncoproteins differ in their structure and activation of specific downstream signaling pathways (8, 18).
Clinical trials have found that GISTs are sensitive to imatinib mesylate (Gleevec, STI571, Novartis, Basel, Switzerland), which acts as a selective inhibitor for the KIT, PDGFR, and BCR-ABL tyrosine kinases (19–22). In vitro, imatinib mesylate rapidly inhibits KIT phosphorylation and tumor cell proliferation in GIST cell lines (23, 24). Clinically, imatinib mesylate has been successfully tested in clinical trials of GIST patients with metastatic and/or unresectable tumors (25–27).
Cancer cells use glucose abnormally when compared with normal, nontransformed cells (28). The accelerated rate of glucose uptake and metabolism in tumor cells is multifactorial and may occur as a consequence of oncogenic transformation (29, 30). Glucose uptake by tumors can be clinically detected by positron emission tomography (PET) scan using 18-fluoro-2-deoxy-d-glucose (FDG). In GIST, FDG inhibition is a sensitive method to evaluate early response to imatinib treatment (27, 31). In chronic myelogenous leukemia (CML), imatinib mesylate inhibits the oncogenic activity of the tyrosine kinase BCR-ABL (32) and decreases glucose uptake from the medium by BCR-ABL-positive cells (33). Given the above clinical and experimental observations (i.e., that imatinib mesylate decreases glucose uptake by GIST in vivo as observed by PET scanning and in CML cell lines in vitro and that imatinib mesylate is an inhibitor of specific tyrosine kinases), we decided to evaluate the changes in glucose uptake in response to imatinib in GIST. The purpose of our study was to determine the association of altered glucose uptake directly with cell survival as an indicator of therapeutic benefit of imatinib mesylate and to determine the molecular mechanisms that regulate the alteration of glucose uptake and GIST cell survival.
We report that constitutively active KIT is associated with a high rate of glucose uptake in GIST cells. Imatinib mesylate inhibits KIT activity and leads to down-regulation of AKT, resulting in decreased glucose uptake by the tumor cells. We further show that endocytosis of the Glut4 transporter from the cell surface leads to decreased glucose uptake due to inhibition of AKT activity with imatinib mesylate. However, imatinib mesylate–induced cell growth arrest and eventual apoptosis are independent of AKT activity and may therefore be independent of glucose uptake. Most importantly, we show that activation of the AKT pathway, although acknowledged as a survival pathway that is notably down-regulated by imatinib mesylate, is not the mechanism of GIST cell survival. Additionally, this work suggests that decrease in glucose uptake by GIST cells after imatinib mesylate may not be the only mechanism that results in cell death. In summary, we have shown that tumor growth inhibition and induction of apoptosis by imatinib mesylate is not singularly dependent on changes in glucose uptake or on the inhibition of the AKT signaling pathway.
Materials and Methods
Chemicals. Imatinib mesylate (Gleevec, STI571, obtained from the Fox Chase Cancer Center pharmacy, Philadelphia, PA) was dissolved in sterile PBS and stored at −20°C. 2-3H-deoxy-glucose was purchased from Amersham Biosciences (Piscataway, NJ). LY294002 and U0126 were purchased from Calbiochem (La Jolla, CA). All antibodies (except β-actin, anti-Glut1, Glut4, and Na/K ATPase-α1) used in this study were purchased from Cell Signaling Technologies (Beverly, MA) and used according to the manufacturer's instruction. Anti-β-actin antibody was purchased from Sigma (St. Louis, MO). Anti-Glut4 antiserum used in immunostaining was a generous gift from Dr. Morris Birnbaum (University of Pennsylvania, Philadelphia, PA). Anti-Glut4 and anti-Glut1 antibodies used for immunoblots were purchased from Abcam (Cambridge, MA). Anti-Na/K ATPase-α1 antibody was purchased from Novus Biologicals (Littleton, CO). Horseradish peroxidase (HRP)–conjugated secondary antibodies were purchased from Amersham Biosciences. Fluorophore-conjugated secondary antibodies and Vybrant cell-labeling solutions for membrane labeling were purchased from Molecular Probes (Eugene, OR).
Cell cultures. GIST 882 cells, a generous gift from Dr. Jonathon Fletcher (Brigham and Women's Hospital, Boston, MA), possess a homozygous mutation in c-KIT exon 13 and were grown as described previously (24). GIST T1 cells (34) possess a homozygous mutation in c-KIT exon 11 and were maintained in DMEM supplemented with 10% fetal bovine serum and glutamine. For imatinib treatment, imatinib was added directly to the cell medium at a final concentration indicated in each result.
Preparation of whole-cell extract from cells and immunoblot assays. The whole-cell extract was prepared as described previously (24). Whole-cell extract (50 μg) was electrophoresed and blotted onto Immobilon-P membrane (Millipore, Billerica, MA). Membranes were incubated with primary antibodies (1:1,000 dilution for all primary antibodies used in this study) and diluted in TBS/0.5% Tween 20 with 5% bovine serum albumin (BSA) at 4°C overnight followed by HRP-conjugated secondary antibody at room temperature for 1 hour. Protein bands were detected by Western Lighting Plus chemiluminescence reagent (Roche, Indianapolis, IN).
Viability and apoptosis assays. GIST cells were seeded at 4 × 105 per six-well plates and treated with drugs for indicated time points. For cell growth and viability assays, cells were harvested by trypsinization at each time point, washed in PBS, and stained using Guava ViaCount Reagent (Guava Technologies, Inc., Hayward, CA). The IC50 was determined by treating cells with varying doses of drugs for 72 hours, and the value of IC50 was determined at the concentration when there was a 50% reduction in cell number. For apoptosis assays, cells were harvested at 72 hours after treatment and stained using the Guava Nexin kit according to manufacturer's recommendations. Cell populations were analyzed by the Guava Personal Cell Analysis system, and data were processed using CytoSoft software (Guava Technologies).
Glucose uptake assay. GIST cells were seeded at 5 × 105 per six-well plate. After treatment, cells were rinsed twice with prewarmed glucose uptake buffer [118 mmol/L NaCl, 5 mmol/L KCl, 1.3 mmol/L CaCl2, 1.2 mmol/L MgSO4, 1.2 mmol/L KH2PO4 (pH 7.4)]. After rinsing, 500 μL prewarmed glucose uptake buffer was added to each well, and the cells were incubated at 37°C for 10 minutes. 10× uptake buffer (50 μL; 1 mL glucose uptake buffer contains 5 μCi 2-3H-deoxy-glucose and 50 μmol/L glucose) was then added to each well followed by incubation of cells at 37°C for an additional 10 minutes. The uptake was terminated by washing the cells five times with PBS followed by lysis with 500 μL of 0.1% SDS at room temperature for at least 30 minutes. Cell lysates were transferred into scintillation vials, and each well was rinsed with additional 500 μL distilled water. Then, the total volume was collected and subjected to protein quantitation and scintillation counting. Cytochalasin B (10 μmol/L; Sigma) was used as background control for glucose uptake and subtracted from all counts. To determine the percentage (%) of glucose uptake, uptake per minute per mg protein (cpm/min/mg) was calculated based on cpm and total protein concentration. The absolute uptake was then normalized with nontreated samples to determine the percentage of uptake. At least three independent uptake assays were done for each experiment, and experimental variations and significance were determined by SD.
Lentiviral construction. Myristoylated human AKT1 coding region was amplified by reverse transcription-PCR using forward (5′-ATACTCTAGAGCCACCATGGGGAGCAGCAAGAGCAAGCCCAAGGACCCCAGCCAGCGCCGGAGCGACGTGGCTATTGTGAAG-3′), which contains both the Kozac and myristoylation sequences (35), and reverse (5′-ATACGGATCCTCAGGCATAATCGGGTACATCGTAAGGGTAGGCCGTGCTGCTGGCCGAGT-3′) primers, in which a stop codon was incorporated. Total RNA template was isolated from GIST T1 cells using Trizol reagent following the manufacturer's instruction (Invitrogen, Carlsbad, CA). cDNA was synthesized using avian myeloblastosis virus reverse transcriptase (Promega, Madison, WI). The PCR was carried out following the manufacturer's instruction using Expand High Fidelity PCR System (Roche). The PCR product was then cloned in pCR2.1-TOPO vector (Invitrogen), and the DNA sequence was verified and subcloned into pENTR3C vector (ViraPower Lentiviral Expression system, Invitrogen). The myristylated human AKT2 construct in DsRed2 vector was kindly provided by Dr. Jin Cheng (Moffitt Cancer Center, University of South Florida, Tampa, FL) and subcloned into pENTR3C vector. Amphotropic T293FT cells were used to generate viral stock following the manufacturer's instruction (ViraPower Lentiviral Expression system). Lentivirus expressing myristylated AKT1 or AKT2 was used to infect GIST T1 cells generating GIST T1/AKT1 and GIST T1/AKT2 cell lines, respectively. Lentiviral vector expressing LacZ gene as control was provided by the manufacturer.
Immunostaining and microscopy. Cells were fixed with 4% paraformaldehyde for 20 minutes at room temperature followed by extensive washing with PBS to remove excess paraformaldehyde. Cells were then permeabilized with PBS containing 0.2% Triton X-100 for 5 minutes at room temperature followed by three PBS washes with gentle shaking. Permeabilized cells were blocked in PBS plus 1% BSA for 1 hour at room temperature. The cells were then incubated with primary antibodies (1:200 dilution for anti-phosphorylated AKT Ser473 antibody and 1:100 dilution for anti-Glut4 antibody) for 1 hour at 37°C. Cells were washed thrice with PBS and then incubated with Alexa Fluor–labeled secondary antibodies (Alexa Fluor 448 for green fluorescence and Alexa Fluor 647 for far-red fluorescence; Molecular Probes) for 1 hour at room temperature. To counterstain nuclei, cells were washed with PBS containing 1 μg/mL propidium iodide for 15 minutes at room temperature. Images were acquired using Nikon (Tokyo, Japan) E800 upright microscope with a Bio-Rad (Hercules, CA) Radiance 2000 confocal scanhead facilitated with LaserSharp2000 software for acquisition. The images were acquired sequentially to avoid emission bleed-through.
Subcellular fractionation and sucrose gradient centrifugation. GIST cells were maintained in growth medium with and without imatinib treatment for 40 hours. After incubation, cells were washed twice with PBS, scraped, and pelleted by centrifugation at 1,500 rpm for 5 minutes. The cell pellets were resuspended in HES buffer [20 mmol/L HEPES (pH 7.4), 1 mmol/L EDTA, 250 mmol/L sucrose] supplemented with protease inhibitor cocktail (Roche) and 250 nmol/L phenylmethylsulfonyl fluoride (Sigma). The cells were homogenized by passing through a 31-gauge needle six times. Nuclei, mitochondria, and plasma membrane were obtained by centrifugation at 14,000 × g for 15 minutes (Sorvall Super T21). The resulting pellet was resuspended in 5 mL HES buffer and layered on 5 mL of 1.12 mol/L sucrose cushion [20 mmol/L HEPES (pH 7.4), 1 mmol/L EDTA, 1.12 mol/L sucrose] and centrifuged at 100,000 × g in a SW41 rotor (Beckman, Fullerton, CA) for 1 hour at 4°C. The white, fluffy, middle layer is the plasma membrane containing fraction. The plasma membrane fraction was then resuspended with 10 mL HES and pelleted by centrifugation at 40,000 × g in a SW41 rotor for 20 minutes at 4°C. The protein concentration of plasma membrane fraction was determined according to Bradford methods. Protein (10 μg) from the plasma membrane fraction was subjected to immunoblotting assays as described previously.
Sensitivity of GIST cells to imatinib as a function of c-KIT mutational status. Recent clinical studies have shown that response to imatinib mesylate in GIST patients is dependent on the mutational status of KIT (36). Using in vitro model systems, we evaluated two GIST cell lines, GIST 882 and GIST T1, for their sensitivity to imatinib. GIST 882 cells possess a missense mutation in exon 13 (K642E), whereas GIST T1 cells contain a 57-nucleotide in-frame deletion in exon 11 (V560_Y579del). Our results showed that imatinib mesylate blocks autophosphorylation of KIT in both GIST 882 and GIST T1 cells but at different drug concentrations. Imatinib mesylate at 0.01 μmol/L concentration significantly inhibits KIT autophosphorylation in GIST 882 cells, whereas GIST T1 cells required treatment with 0.1 μmol/L imatinib mesylate to completely block KIT autophosphorylation (Fig. 1A). Intriguingly, imatinib mesylate concentrations required for the inhibition of KIT phosphorylation did not correlate well with cell viability and growth of GIST cells (Fig. 1B; data not shown). We determined that GIST T1 cells were ∼30-fold more sensitive to imatinib mesylate than GIST 882 cells with regard to cell growth. The IC50 (50% reduction in cell number) was ∼25 nmol/L imatinib mesylate for GIST T1 cells versus ∼750 nmol/L imatinib mesylate for GIST 882 cells as determined by evaluating cell number at varying doses of imatinib mesylate (0-10 μmol/L; data not shown). Next, we showed that exposure to imatinib mesylate effectively induced apoptosis in GIST T1 cells but not in GIST 882 cells as determined by fluorescence-activated cell sorting analysis using Annexin V and 7-aminoactinomycin staining (Fig. 1C). GIST 882 cells treated with imatinib mesylate show no significant increase in either early or late apoptosis during the 72-hour treatment period. In comparison, we observed a 3- to 5-fold increase in apoptotic cells in GIST T1 (Fig. 1C). These results suggest that V560_Y579del mutant KIT is particularly sensitive to imatinib mesylate–induced cell death by clinically relevant concentrations of imatinib mesylate compared with cells with the missense mutation in exon 13 (K642E), supporting findings in clinical studies (37, 38), and that the phosphorylation status of KIT alone was not reflective of biological response to imatinib mesylate.
Imatinib treatment causes dephosphorylation of KIT and AKT and decreases glucose uptake in GIST cells. We next examined the phosphorylation status of purported downstream mediators of KIT signaling [e.g., AKT and mitogen-activated protein kinase (MAPK1/MAPK2)] after imatinib mesylate treatment. As shown in Fig. 2, imatinib mesylate strongly inhibited phosphorylation of KIT in both cell lines beginning as early as 1 hour and persisted for at least 24 hours (Fig. 2A and B). Treatment with LY 294002 (Fig. 2A, and B, lane LY) and U0126 (Fig. 2A, and B, lane U), the specific inhibitors of phosphatidylinositol 3-kinase (PI3K) and MAPK/extracellular signal-regulated kinase kinase (MEK1/MEK2), respectively, did not cause dephosphorylation of KIT, confirming that KIT dephosphorylation is imatinib mesylate specific. In addition, imatinib mesylate caused a decrease in phosphorylated AKT at Ser473 and Thr308 residues. Treatment with LY294002 did not alter the phosphorylation status of KIT, although it caused dephosphorylation of AKT (Fig. 2A, and B, lane LY). This result indicates that AKT is a shared downstream effector of both KIT and PI3K activity in GIST. In GIST T1 cells, imatinib mesylate leads to a transient dephosphorylation of MAPK1/MAPK2 (Fig. 2A). Interestingly, the phosphorylation of MAPK1/MAPK2 recovered over time during imatinib mesylate treatment. This phenomenon could be secondary to activation of another signaling pathway leading to phosphorylation of MAPK1/MAPK2 when KIT-AKT signaling is inhibited. However, in GIST 882 cells, imatinib mesylate inhibits phosphorylation of MAPK1/MAPK2 throughout the observed time course. These data agree with the clinical observations that KIT mutation status of GIST determines response to imatinib mesylate therapy. For all conditions tested, the total levels of KIT, AKT, and MAPK1/MAPK2 remained unchanged throughout treatments.
Published data indicate that FDG-PET is an early and sensitive method to evaluate response to imatinib mesylate treatment (19, 27, 39). Using FDG, we determined that imatinib mesylate directly reduces glucose transport into GIST cells in culture. GIST T1 displayed an imatinib mesylate–dependent decrease in glucose uptake as a function of time, whereas GIST 882 cells showed an initial increase in glucose uptake for up to 5 hours after addition of imatinib mesylate followed by a sharp decrease of glucose uptake (Fig. 2C). This result clearly shows that imatinib mesylate treatment directly leads to decreased glucose transport into GIST cells in vitro similar to what is seen by clinical FDG-PET. It is important to note that activating KIT maximizes AKT phosphorylation and glucose uptake in GIST cell. However, the addition of insulin does not have a synergistic effect on KIT-mediated glucose uptake, although the insulin receptor is expressed in both GIST cell lines (data not shown).
LY294002 treatment causes dephosphorylation of AKT and decreases glucose uptake in GIST cells. To further determine the mechanism by which imatinib mesylate treatment decreases glucose uptake, LY294002, a specific inhibitor of PI3K, was used to examine the role of AKT in glucose uptake. AKT is the major target for PI3K, and inhibition of PI3K directly leads to inactivation of AKTs. Previously, we have shown that LY294002 effectively caused dephosphorylation of AKT at both Ser473 and Thr308 residues in GIST 882 cells (24). In this study, we examined the inhibitory effect of LY294002 on GIST T1 cells in comparison with GIST 882 cells. LY294002 treatment caused strong dephosphorylation of AKT at both Ser473 and Thr308 residues as early as 1 hour after treatment and lasted for at least 24 hours (Fig. 3A and B). LY294002 treatment did not cause dephosphorylation of KIT, whereas imatinib mesylate caused AKT dephosphorylation, indicating that KIT is an upstream mediator of PI3K and AKT in GIST. As expected, 1-hour treatment caused dephosphorylation of KIT and AKT (Fig. 3A, and B, lane IM), and 1-hour U0126 (Fig. 3A, and B, lane U) treatment did not. Finally, LY294002 treatment clearly resulted in decreased glucose uptake in both cell lines as a function of time (Fig. 3C). This result suggests that glucose uptake in GIST cells is mediated by the PI3K and presumably the AKT pathway.
Imatinib-induced apoptosis is independent of AKT signaling. To examine the function of AKT activation in GIST, we infected GIST T1 cells with lentiviral vectors expressing either myristylated AKT1 or myristoylated AKT2 (hereafter referred to as GIST T1/AKT1 and GIST T1/AKT2, respectively), the membrane-bound AKT isoforms that are constitutively active, and the LacZ gene as control (hereafter referred to as GIST T1/Lac). The rationale for introducing myristylated AKT isoforms into GIST T1 cells is to determine whether expression of constitutively activated AKT will reverse imatinib mesylate–mediated apoptosis in GIST T1 cells as one might predict based on previous studies. As shown in Fig. 4A, imatinib mesylate treatment caused dephosphorylation of KIT as early as 1 hour in all three cell lines, consistent with the result from parental GIST T1 cells (Fig. 2A). However, AKT1 and AKT2 remained phosphorylated in GIST T1/AKT1 and GIST T1/AKT2 cells but not in GIST T1/Lac cells. Glycogen synthase kinase-3β (GSK3β), one of the downstream targets for activated AKT, remained phosphorylated in GIST T1/AKT1 and GIST T1/AKT2 cells throughout the treatment. We also evaluated another AKT target, BAD. BAD is a proapoptotic member of the BCL-2 family. AKT has been shown to promote cell survival via its ability to phosphorylate BAD at Ser136 (40, 41). As observed for GSK3β, BAD remained phosphorylated in T1/ATK1 and GIST T1/AKT2 cells treated with imatinib mesylate and LY294002. These results provide additional evidence that the AKT pathway is constitutively active in the AKT1- or AKT2-transduced cells. As expected, U0126 (Fig. 4A , lane U) had no effect on KIT, AKT, GKS3β, or BAD phosphorylation.
We then assayed the role of AKT signaling in the growth response to imatinib mesylate. We expected that GIST T1/AKT1 and GIST T1/AKT2 cells would be resistant to imatinib mesylate–induced apoptosis, as AKT is involved in the survival pathway. Surprisingly, imatinib mesylate treatment resulted in cell death even in the presence of constitutively activated AKTs (Fig. 4B,, bottom). Similarly, parental GIST T1 and GIST T1/Lac cells were also sensitive to imatinib mesylate–induced cell death (Fig. 4B,, top). The cell death is via an apoptotic pathway as determined by Annexin V staining (data not shown), and phosphorylation of BAD by constitutively active AKT did not prevent cells from undergoing imatinib-induced apoptosis (Fig. 4A and B). These results show for the first time that although AKT is involved in the survival pathway in many tumors, constitutive expression of myristoylated AKT1 or ATK2 fails to rescue imatinib mesylate–induced growth suppression and apoptosis in GIST cells.
We next tested if glucose uptake was dependent on AKT signaling in GISTs. We showed that glucose uptake in GIST cells is dependent on both AKT1 and AKT2 isoforms (Fig. 4C). The PI3K-AKT pathway is the major mediator for glucose transport via the insulin receptor pathway in insulin-responsive cells. Here, we show that, in the absence of insulin stimulation, GIST cells expressing constitutively active AKT1 or AKT2 are able to maintain high levels of glucose uptake even when KIT activation is inhibited by imatinib mesylate. This result indicates that glucose uptake in GIST is AKT dependent, in contrast to imatinib mesylate–induced effects on cell growth and apoptosis, which can be AKT independent.
PI3K mediates the therapeutic effects of imatinib independent of AKT. KIT phosphorylation at the Tyr719 leads to the activation of PI3K (13). Mutation of this residue completely disrupts PI3K binding to KIT and reduces PI3K-dependent AKT activation by 90% (12). To establish whether PI3K activity has an essential role in KIT signaling and whether it is essential for imatinib mesylate–mediated effects on GIST cell growth and apoptosis, we again examined the inhibitory effect of LY294002. In GIST T1/AKT1 cells, it was very clear that LY294002 had no inhibitory effect on myristoylated AKT1 at both Ser473 and Thr308 residues (Fig. 5A,, middle) but inhibited the endogenous levels of phosphorylated AKT at these two residues (Fig. 5A,, arrowheads). The doublet bands of phosphorylated AKT represent the myristoylated AKT1 and endogenous AKT (Fig. 5A,, arrowheads). Likewise, LY294002 did not inhibit myristoylated AKT2 phosphorylation in GIST T1/AKT2 cells (Fig. 5A,, right). In addition, GSK3β and BAD also remain phosphorylated throughout treatment due to constitutively active AKT1 or AKT2 (Fig. 5A and B). As expected, LY294002 strongly inhibits phosphorylation of endogenous AKT in GIST T1/Lac cells (Fig. 5A , left). These results confirm that myristoylated AKT isoforms remain phosphorylated in transduced cells even with LY294002 treatment.
Similar to the imatinib mesylate–induced apoptosis (Fig. 4B), we noted that GIST T1/AKT1 and GIST T1/AKT2 cells also display LY294002-induced growth arrest. We determined that the IC50 for LY294002 of GIST T1 cells is 5 μmol/L (data not shown). Additionally, parental T1 and GIST T1/Lac, when treated with LY294002, showed similar degrees of arrest due to PI3K inhibition as well as decreased levels of phosphorylation of AKT at Ser473 by immunoblots (Fig. 5C,, top, insets). It is then surprising that LY294002 also caused cell arrest in GIST T1/AKT1 and GIST T1/AKT2 cells despite there being no inhibition of phosphorylation of AKT (Fig. 5C , bottom, insets). However, these results are consistent with the studies of imatinib mesylate, suggesting that PI3K inhibition, independent of AKT inhibition, may help mediate the effects of KIT inhibition by imatinib mesylate.
Next, we examined whether PI3K signaling was involved in glucose uptake in GIST cells. Unlike imatinib mesylate, LY294002 preferentially inhibits glucose uptake in myristoylated AKT1 cells better than in myristoylated AKT2 cells. The rate of glucose uptake remained high throughout treatment in GIST T1/AKT2 cells. However, glucose uptake in GIST T1/Lac and GIST T1/AKT1 cells was inhibited by LY294002 (Fig. 5D). This result suggests that the AKT2 isoform may have a dominant role in glucose uptake in GIST.
Mechanistic evaluation of imatinib mesylate on the glucose transporter Glut4. Finally, we examined the mechanism that regulates glucose transport in GIST cells. We focused our studies on the expression and localization of two prominent glucose transporters, Glut1 and Glut4. Parental GIST 882 and GIST T1, GIST T1/Lac, GIST T1/AKT1, and GIST T1/AKT2 cells were treated with imatinib mesylate for 48 hours followed by immunoblot assays of the plasma membrane fraction (Fig. 6A and B). Densitometry scanning showed that, in GIST 882, GIST T1, and GIST T1/Lac cells, the amount of Glut4 transporter on the plasma membrane fraction was reduced in imatinib mesylate–treated cells compared with nontreated cells (see relative plasma membrane level, Fig. 6A). The reduction of the plasma membrane-bound Glut4 by imatinib mesylate leads to decreased glucose uptake (Figs. 2C and 4C). Treatment with imatinib mesylate caused Glut4 to translocate, via endocytosis, from the plasma membrane to the cytosol as evidenced by lower levels of plasma membrane-bound Glut4. By contrast, GIST T1/AKT1 and GIST T1/AKT2 cells have comparable amounts of plasma membrane-bound Glut4 that was not reduced by imatinib mesylate treatment. Na/K ATPase-α1 antibody was used as a plasma membrane marker and a loading control. The relative level of plasma membrane-bound Glut4 for each sample was derived by normalizing Glut4 intensity with Na/K ATPase-α1 intensity. For purity control, we also blotted the membrane with β-actin, the marker for cytosolic compartment (data not shown). We have shown that Glut1 is also expressed in GIST cells, with the majority sequestered in cytosolic membrane vesicles (data not shown).
We further confirmed the imatinib mesylate induced Glut4 translocation by immunofluorescent staining. GIST 882 and GIST T1 cells without imatinib mesylate displayed staining of the plasma membrane as well as intracellular microsomes of membrane-bound Glut4 (Fig. 6C,, 1-3 and 7-9), whereas imatinib mesylate–treated cells had prominent cytosolic staining of intracellular microsomes (Fig. 6C,, 4-6 and 10-12). The prominent staining of intracellular microsomes in imatinib mesylate–treated cells could be due to endocytosis of Glut4 from plasma membrane or sequestering of intracellular microsomes from exocytosis. In combination with our data above, our results suggest that imatinib mesylate increases Glut4 endocytosis, resulting in decreased plasma membrane-bound Glut4 molecules and leading to reduction of glucose transport into cells. Likewise, GIST T1/Lac cells also display imatinib mesylate–mediated decrease of plasma membrane-bound Glut4. However, expression of constitutively active AKT1 or AKT2 did not result in imatinib mesylate–induced endocytosis of Glut4 (see Supplementary Data). These results agree with the glucose uptake assays shown in Figs. 4 and 5.
The findings presented here show a novel insight into the molecular exploration of the therapeutic effects of imatinib mesylate in the treatment of GIST cells specifically with regard to drug-induced tumor cell killing. Imatinib mesylate leads to the reduction of glucose uptake in GIST cells in an AKT-dependent manner and inhibition of cell growth and induction of apoptosis in an AKT-independent manner. Importantly, we have shown that reduction of glucose uptake, which is quantified by FDG-PET and is prognostic of tumor response to imatinib mesylate, may not absolutely correlate with decreased cell growth and tumor shrinkage via apoptosis. Initially, we established that GIST cell lines harboring mutations in c-KIT display differential sensitivity to imatinib mesylate and that inhibition of KIT phosphorylation is not the sole predictor of the biological response. These results are consistent with clinical trial results of imatinib mesylate, which found that GIST tumors with KIT mutations in exon 11 (e.g., GIST T1) have the highest response rate and disease-free survival compared with mutations outside this region (e.g., GIST 882; ref. 36). In our current study, we have shown that both GIST cell lines undergo imatinib mesylate–mediated inhibition of KIT autophosphorylation and growth arrest but that drug treatment of GIST T1 cells elicits a more efficient apoptotic response.
It was suggested in a recent report that imatinib mesylate caused a decrease in glucose uptake and that this enhanced the cytotoxic effect of imatinib mesylate only in an imatinib mesylate–sensitive tumor. An imatinib mesylate–resistant tumor showed no changes in FDG uptake (42). That report agrees with conventional observations, that is, a decrease in glucose uptake by tumor is a direct reflection of the therapeutic effect of drug treatment. However, that study used KIT-transfected myeloid progenitor cells as a GIST model, which may not accurately mimic de novo response of GIST. In our study, we examined whether imatinib mesylate has influence on glucose uptake by GIST cells and the association of this glucose uptake with analytic measurement of GIST cell survival. We have shown that imatinib mesylate directly decreases glucose uptake, in an AKT-dependent manner, in both GIST cell lines despite the fact that one cell line is sensitive and the other is resistant to imatinib mesylate–mediated apoptosis. Furthermore, constitutively active AKT1 or AKT2 isoforms did not rescue GIST T1 cells from imatinib mesylate–induced apoptosis, suggesting that AKT activation may not be the sole mechanism for GIST cell survival after addition of imatinib mesylate. However, a separate AKT-mediated mechanism is responsible for glucose uptake in GIST cells as shown by constitutively active AKT1 or AKT2 isoforms capable of reversing the inhibitory effect of imatinib mesylate on glucose uptake. Collectively, our results show that, when treated with imatinib mesylate, the cell growth and glucose uptake in GIST are two mutually exclusive events, with AKT-independent cell growth and survival and AKT-dependent glucose uptake. Most importantly, we have shown that the therapeutic effect of imatinib mesylate in GIST may be independent of AKT despite glucose uptake being dependent on AKT. These results suggest that loss of FDG uptake as detected on PET scan may be a surrogate for but not pathognomic of the actual tumor cell killing.
In addition, we show in vitro that gain-of-function mutations in KIT lead to constitutive activation and result in increased glucose transport in GIST cells in an insulin-independent manner (data not shown). The high rate of glucose uptake in GIST is therefore a biological phenomena regulated by the constitutively active KIT/PI3K/AKT pathway, which maximizes AKT activity. These results indicate that the high rate of glucose uptake in GIST tumor cells may correlate closely with AKT-dependent tumor survival mechanisms.
Finally, we have shown a mechanism that mediates glucose transport in GIST. We show that the glucose transporter Glut4 and, to a lesser extent, Glut1 are prominent on the plasma membrane of GIST 882 and GIST T1 cells (Fig. 6; data not shown). It has been shown previously that Glut4 transporter mediates the entry of glucose into certain tissues (43, 44), and Glut4 expression has been found in breast, gastric, and other malignancies (45–51). In our study, we show that the expression of Glut4 in GIST cells is located in both plasma membrane and cytoplasmic compartments. However, on imatinib mesylate treatment, there is a significant decrease of membrane-bound Glut4 molecules in GIST 882 and GIST T1 cells, indicating that the endocytosis pathway of Glut4 is activated after imatinib mesylate treatment of GIST cells. Overall, these collective findings contribute to a clearer understanding of the molecular mechanisms involved in the therapeutic benefit of imatinib mesylate in GIST.
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).
Grant support: NIH grant CA106588-01, a supplement to grant 3 U10 CA21661-27, and Ovarian Cancer Specialized Program of Research Excellence grant P50 CA83638, by an appropriation from the Commonwealth of Pennsylvania (A.K. Godwin); National Research Service Award Postdoctoral Fellowship (C. Tarn); and the Pennsylvania Department of Health.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Dr. Morris Birnbaum for generously providing anti-Glut4 antibody, the Image Core Facility (Fox Chase Cancer Center) for image processing and analyzing, the Biosample Repository and Tumor Bank Core Facilities (Fox Chase Cancer Center) for specimens, Tania Stutman, and the GIST Cancer Research Fund.


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This analytical report has surveyed two areas of trade-related policy to assess their potential for reducing the impacts of the implementation of response measures via economic diversification: global value chains and innovation. It also explored the idea of green industrial policy. Ultemately, the report found a number of ways in which trade policy and trade-related policies could further the goal of economic diversification, helping to reduce response measure vulnerability, and in the process helping to implement the goals of the Paris Agreements.
Download file: English
Topics: Industry, Trade, Green Jobs/Economy, COP24 List of UN publications
Type of material: Analytical-Technical Document
Publication date: 2018

domain: politics


23 Apr 2012 The Motherlode EP is here!
The Motherlode EP was released yesterday! You can get it on iTunes and Amazon, or buy a physical copy through Rough Trade.
It hits America on the 29th of May.
Hope all of you enjoy it!
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A couple of weeks ago, I came across the article on Journalism.co.uk: “Why all would-be journalists need to blog”.
Have a read of that, and then read on. It was really insightful for me. I’ve always felt that having a blog was beneficial. Not only to keep up my writing and experience in this, but also to learn more about the industry, demands of web-writing and social media. I contacted Sue Greenwood who wrote the article, to ask for some advice that I could share on this blog, and also learn more about the web-based journalism that she teaches. On my NCTJ I don’t feel like I learnt much about writing online, we rarely covered blogs apart from ‘It’s helpful to have a blog’ (which the majority of us did), and our experience in online journalism was a series of three two-hour afternoon workshops. They very much covered the basics of Facebook, Twitter, WordPress, SEO and to be honest, I didn’t feel that we learnt much that we didn’t know already.
Sue is part-time as a senior lecturer in the School of Journalism, Humanities and Social Sciences, and specialises in web-based and entrepreneurial journalism, teaching students how to use the web to communicate, how to value what they produce, and how to make money from their ideas. I asked her a few questions about her experience in journalism and what she teaches to share on the blog.
What initially interested you in journalism?
When I was a kid, my dad would buy The Mirror on his way to work every day and we’d get the Hull Daily Mail delivered every night. He’d give me both to read after he’d finished and always made me feel that he was passing something important on when he handed me each newspaper.
Later at uni, I did film and photography but it was the documentary projects – talking to people and shaping their story, that interested me most. After uni, I volunteered at Viking Radio, in Hull, I was there on the day the station opened and it was massively exciting even through I spent the day filing! That experience led me to do my post-grad.
How did you find your post-graduate diploma in radio and TV journalism?
Fabulous! It was the first year of that particular Diploma so it was a smallish student group and the lecturers had joined straight out of the industry. They brought with them a bit of a maverick approach and, even though we did shorthand, law, public affairs – all the things we still expect journalism students to do – we spent as much time arguing over the rights and wrongs of journalism as we did learning the skills. It was an incredibly intense and full year and being encouraged to question journalism as well as do it has shaped the way I’ve approached the rest of my career.
What roles have you had in journalism?
Definitely not a traditional route! It took me about two years to get a proper job after uni and my first was as a funding officer for film and photography projects at Yorkshire Arts – so nothing to do with journalism. I moved into voluntary sector management running projects for CSV media, which did TV/radio back-up stuff such as running campaign helplines and media training. While I was there I also created a ‘pop-up’ cable station. I recruited around a hundred volunteers, raised cash, borrowed equipment, and broadcast a 48-hour community cable station, backed by Yorkshire TV.
Then I needed to move to back to Hull with my family and used that to try get into journalism directly. I persuaded the then editor of the Hull Daily Mail (HDM), John Meehan, to interview me but wasn’t convincing enough! A few weeks later I applied for and got a page designer’s job putting together advertising supplements and a few months after that, Meehan noticed I was at the HDM and put me in charge of their web team. We won awards and I started moving up – eventually becoming regional managing editor for websites across half-a-dozen of Northcliffe’s newspapers.
But for lots of reasons I really wanted to get closer to news so I took a big pay drop and joined the HDM’s newsdesk as Assistant News Editor. I went from there to Deputy News Editor at the Sentinel, in Stoke-on-Trent, then News Editor at the Telegraph and Argus, in Bradford. After that I moved into teaching journalism half-time while working on other content-led projects or businesses, and I’m also halfway through my PhD.
How would you define web-based journalism and why is it important?
Journalism that happens first on the web and journalism published for the web.
The web is more important than any other media we use right now. We spend more of our time online than we do with other media and it plays a bigger part in the way we gather and share information. The internet as a medium for delivering news and information is global and social in a way newspapers, TV and radio have never been. At the same time, it’s a very different medium for news and information. What makes it unique is that it’s interactive – we don’t decide how long a site visitor stays with a story; and that it has hyperlinks – a visitor can drill down and through a story, even to places we didn’t plan for them to go. The ability to package a story and for a visitor to spend as long as they want with that story and wandering around it, it is what makes the web so important to journalism.
What are some common questions you frequently get from your students?
How do I get a job? Are there any jobs? How do I make my blog better? How do I get people to read my blog? What mark did I get?…
What would you describe a poor blog to be like? How can we avoid these mistakes?
A poor blog is one that’s written as a sounding board for the writer without any understanding of the audience. If you want to write just for yourself, keep a journal, but a blog is public and you need to give people a reason to read it – information, entertainment, ideas – whatever. You’re writing it for someone who doesn’t know you and you have a responsibility to be clear to them what you’re offering and to deliver what you promise. There are other things you can get wrong – poor navigation, poor design, etc. you can create something that is written well but is just too hard to read. But writing for an audience rather than for yourself, is the most important thing to get right.
Can you explain the context behind setting up wreckoftheweek and what it taught students?
I like to try something myself before I teach it and in this case I was developing ideas around how to build an audience and how to monetize a basic blog. I think the idea itself came because I used to love looking at the “wreck of the week” property in the weekend newspapers and then I realized the domain was available. The starting point for optimizing a site in terms of search engines is the name – so I bought the domain and set up the site with a couple of sample posts on it and started playing around with other SEO-related ideas.
At first it was just a way to test what I was teaching in relation to SEO but I then started to really enjoy writing it and, as the audience grew and I started to get emails and feedback, it became even more important. Now it’s another thing I do and that I have fun doing – even when, like today, I’ve spent six hours on one post because I had to start digging around to find out more about a house I wanted to feature! I think that’s back to my point about blogging for an audience. If ‘Wreck’ was just an exercise to help me teach, it wouldn’t be so successful and I’d probably have stopped writing it by now. But now I’m at a stage where I get emails and photographs from people who’ve bought houses I’ve featured and are renovating them and I feel responsible to them.
What is entrepreneurial journalism and what message do you like to get across to your students?
Audience again – thinking about who you’re producing your journalism for. It’s not about getting a story past your news editor or through conference, it’s about understanding who the market is for that story, that information; and the best way for you deliver it to them. The audience is your responsibility too, not just your boss’s. It’s also about a more creative approach – whether you’re working for someone or not. It’s about taking responsibility yourself for getting that content out, or for solving that problem, or for finding work. Being entrepreneurial is about doing it yourself – and that includes finding the right people to help you and work with you.
Why should every journalism student set up a blog?
I’m starting to repeat myself! Audience again. Understanding that you’re writing in order that people will read what you have to say. Too often as a journalist you’re in a workplace where the goal is to impress your news editor/editor with your story. The journalist isn’t expected to think about how many people read their story or shared it. I’m definitely not saying popularity should be the criteria for assessing the worth of a story (its not about Buzzfeed) but I am saying that journalism students have to appreciate the line from them to the audience for the story they produce. Blogging helps that to happen. Plus, blogging keeps you writing, keeps you producing and gives you something (hopefully impressive!) to show a potential employer.
How do you approach the subject of generating money from a blog?
I tell my students they don’t have to monetize their blog but they do have to make a decision one way or the other, based on whether it’s right for their content and their audience at that point. I show them what other people make from blogging (the students usually like that bit!) and I tell them that it’s not about making money but about recognizing that everything they produce has a value and the first person who has to value what they produce is themselves. Just because they’re students, they shouldn’t assume everything they do is unpaid. Even if all they make from their blog is a tenner, that’s a tenner more than they had before and it means they’re earning from what they produce.
Follow Sue on Twitter: @SueGreenwood


Election laws that make sense demand that voters provide identification upon voting. It is required by law for voters to provide identification in federal elections in the United States. Every election ought to require identification. If not, anyone could show up with a postcard notification and cast a ballot under any name under the sun.
To maintain secure elections, voting must be accompanied by identification. Liberals, though, seek to do away with the requirement. They label as racist everyone who thinks identification should be required. That implies that people of color are too dumb to obtain an ID in some way.
Of course, that is a false assumption. Democrats want ID restrictions eliminated so they can likely engage in various unethical cheating methods. One will be to let everyone who entered the country illegally to cast a ballot in federal elections without restriction.
Election officials will have to work diligently to stop this scheme. But Democrats are also big supporters of universal mail-in voting. Likewise, they want to eliminate any identification requirements on the ballot.
That would allow basically any person to take a ballot and mail it in. Certainly, a sane person can see how wide open this is for fraud. Do Democrats care? But of course they don’t. Democrats can use dead people’s votes, illegal migrant votes, and any other old votes they can.
The liberal fight against election integrity legislation isn’t to fight voter suppression. No, sir. They’re trying to grab every single opportunity available to them to cheat. They cannot win on their policies, so they have to scam their way into office.
Democrats focus heavily on key swing states. They used this crooked strategy to “supposedly win” critical battleground states in the 2020 presidential election. Their scam worked. Joe Biden, the guy who campaigned from his basement, “supposedly” got the most votes in history.
He didn’t. Biden cheated. But there are other important races in the 2022 midterms. Republicans are battling to save the country. The radical leftist agenda must be stopped before it totally destroys the nation.
Republicans can do it if they take back control of Congress. By all accounts, Republicans are poised to seize control of the House of Representatives and the Senate. That could effectively freeze the radical leftist agenda in its tracks.
Numerous states are in play for a “Republican red wave” to materialize. The numbers indicate huge midterm wins in store for conservative candidates. But Democrats, similar to the 2020 presidential election heist, need to cheat in specific states to hold on to power.
With a call to blow up the longstanding U.S. Senate filibuster, control of this chamber may be extremely important. Democrats are looking at four or five races where they could cheat out a seat. Pennsylvania is one of those states.
The Republican nominee Dr. Mehmet Oz is clearly the best choice. John Fetterman is a radical liberal with a terrible track record. Fetterman will be a senate rubberstamp for liberal leftist policies. But Fetterman had a stroke in May. He’s clearly not fit for office.
Democrats have panicked. They’re looking at an obvious loss of yet another senate seat. So, what do they do? Pennsylvania Democrats are using every trick in the book to cheat. Mail-in ballots are their go-to strategy where state ID requirements are in place.
Pennsylvania Democrats mailed out over 250,000 unverified ballots. These ballots went to individuals whose identities couldn’t be authenticated by social security or driver’s license numbers. Without verification, these ballots never should have even been mailed.
But they were. Pennsylvania Republican Representatives sent a letter to the acting Secretary of the Commonwealth. All 15 representatives endorsed the letter to Leigh Chapman. It demands that these illegal, unverified ballots be set aside. This is the Pennsylvania law.
Before the ballot is tabulated, the individual assigned to the ballot must verify that the ballot is rightfully his or hers to cast. Identification is required. That’s potentially over a quarter of a million illegal votes. You can bet most of them “check the box for John Fetterman”.
There’s also a reasonable likelihood that a large portion of them will not be verified. That’s because they can’t be. They’re not verifiable voters. Election officials need to confiscate every one of the ballots. They must be secured ahead of the election pending verification.
If they’re not verified, they must not be counted. But Pennsylvanians can bet that Democrat operatives will work feverishly to sneak them in. This is a red flag. Liberals, especially John Fetterman, are going to lose. Democrats are going to try to cheat. You can bet “the House” on it.


We audited the Commonwealth of Massachusetts’ (State) Small Cities Community Development Block Grant (CDBG) program because the State was the largest recipient of CDBG funds in New England. HUD awarded the State more than $88 million in CDBG funding for program years 2015, 2016, and 2017. In addition, we had not audited any of the State’s community planning and development programs in the last 10 years.
PK Management, LLC, Richmond Heights, OH, Did Not Always Maintain Documentation Required to Support Housing Assistance Payments
We audited PK Management, LLC, based on (1) media coverage of problems associated with Essex Village, an apartment complex in Virginia that it managed, and (2) issues identified in our prior audit of PK Management in Birmingham, AL. Our audit objective was to determine whether PK Management assisted eligible tenants and maintained documentation to support the housing assistance payments it received for reside
August 02, 2019
Hamilton County, OH, and People Working Cooperatively, Inc., Did Not Always Comply With HUD’s Requirements in the Use of Community Development Block Grant Funds for a Housing Repair Services Program
We audited Hamilton County’s subrecipient agreement with People Working Cooperatively, Inc. (corporation), for a Community Development Block Grant-funded housing repair services program based on a request from the U.S.
September 27, 2018
The Cuyahoga Metropolitan Housing Authority, Cleveland, OH, Generally Administered Its Public Housing Program in Accordance With HUD’s and Its Own Requirements
We audited the Cuyahoga Metropolitan Housing Authority’s public housing program based on an anonymous complaint to our hotline. Our objective was specific to the allegations in the complaint and was to determine whether the Authority (1) engaged in nepotism when hiring staff, (2) used program funds for inappropriate or unreasonable travel expenses, (3) failed to comply with the U.S.
August 28, 2018
The Youngstown Metropolitan Housing Authority, Youngstown, OH, Did Not Always Comply With HUD’s and Its Own Requirements Regarding the Administration of Its Housing Choice Voucher Program
We audited the Youngstown Metropolitan Housing Authority’s Housing Choice Voucher program based on the activities included in our 2016 annual audit plan and our analysis of risk factors related to the public housing agencies in Region 5’s jurisdiction. Our audit objective was to determine whether the Authority appropriately managed its Family Self-Sufficiency program and Housing Choice Voucher program files in accordance with the U.
July 07, 2017
The City of Springfield, MA, Needs To Improve Its Compliance With Federal Regulations for Its Community Development Block Grant Disaster Recovery Assistance Grant
We audited the Community Development Block Grant Disaster Recovery (CDBG-DR) assistance grant provided to the City of Springfield, MA, by the U.S.
October 17, 2016
The Jefferson Metropolitan Housing Authority, Steubenville, OH, Did Not Always Ensure That Its Section 8 Housing Choice Voucher Program Files Complied With HUD’s and Its Own Requirements
We audited the Jefferson Metropolitan Housing Authority’s Section 8 Housing Choice Voucher program based on a request from the U.S.
September 09, 2015
The Department of Housing and Community Development, Montpelier, VT, Did Not Always Operate Its Disaster Recovery Programs Effectively and Efficiently
We audited the State of Vermont, Department of Housing and Community Development’s disaster recovery programs. We initiated this audit because of the significant funding awarded and the Boston Office of Community Planning and Development’s concerns about the State’s capacity to administer this funding. Our audit objective was to determine whether the State administered its disaster recovery programs effectively and efficiently in a
September 29, 2014
The Adams Metropolitan Housing Authority, Manchester, OH, Generally Used Public Housing Program Funds in Accordance With HUD’s and Its Own Requirements
We audited the Adams Metropolitan Housing Authority’s public housing program as part of the activities in our fiscal year 2014 annual audit plan. We selected the Authority based on a request from the U.S. Department of Housing and Urban Development’s (HUD) management. Our objective was to determine whether the Authority used public housing program funds in accordance with HUD’s and its own requirements.
July 31, 2014
The State of Mississippi Did Not Ensure That Its Subrecipient and Appraisers Complied With Requirements, and It Did Not Fully Implement Adequate Procedures For Its Disaster Infrastructure Program
We audited the State of Mississippi’s Community Development Block Grant Hurricane Disaster Recovery Program. The Gulf Coast Regional Infrastructure Program was selected for audit based upon a congressional request, and it was also included in the U.S.
December 29, 2013


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Candy Club - Birthday Bears
Three layers of multicolored fruity flavor and a head-to-toe dusting of sour sugar make these sour gummies make the bear-y best birthday gift! Celebrate sweetly with jumbo sour bears in a tasty mix of orange, strawberry, lemon, and apple flavors.
Made in United States of America
NET WEIGHT: Small: 6 oz

domain: food


text:
The next sections will outline each one of the 5S steps with some ideas relating to software and benefits. Extra Benefit: You will be helping the economic system by reusing your older pc as a result of you can buy new pc components, software, accessories, displays, speakers, mice, computer books, and different computer related devices to make your old computer usable once more. By buying laptop components, and pc associated merchandise, you might be creating jobs because the cash goes again to the employees, firms, and stores which produce and sell the merchandise you purchase.
WOT lastly made it public and Google and Mozilla went ballistic pulling the WOT extension (add-on) from their websites. I’m pretty livid but they do not listen to you or me. We cherish them and depend on them for extra things day by day. We really feel profitable with expertise now. For me one of … Read More

domain: economics


UH-Maui to get $210,000 award
The U.S. Economic Development Administration will award $210,000 to the University of Hawaii for the Maui Food Innovation Center, which is used by agribusiness and food entrepreneurs to develop and manufacture products, U.S. Sen. Brian Schatz has announced.
The funding will create a packaging and marketing support center at UH-Maui College.“This funding will help MFIC meet its goals to create jobs, lower the amount of imports to Maui, and help businesses on the island export their products,” said Schatz, a member of the Senate Appropriations Committee. “We’re excited to see what Maui businesses can do with the extra federal support.”
The Food Innovation Center is a first in-state incubator focused on helping entrepreneurs in the food and agribusiness sector, according to a news release.
The center also assists the community by providing technological expertise, innovative training, consultation, a manufacturing facility and a resource hub for new and established food entrepreneurs working to bring their products to market locally, regionally and nationally.


The Montlake Bridge will be closed on Sunday, March 30 from 5 a.m. to 9 a.m. for filming activity. All traffic including pedestrians and bicyclists will be redirected to the University Bridge. The westbound SR520 off-ramp to Montlake Boulevard East will also be closed.
The bridge will also be closed for brief periods intermittently during the day, but these will require no detours.
During the morning closure, traffic coming from the south on 24th Avenue East will be detoured to the University Bridge by way of Boyer Avenue East. Traffic from the north will be detoured to the University Bridge using Northeast 45th Street and Roosevelt Way Northeast.
Streets south of Northeast 45th Street and north of Boyer will remain accessible to local traffic as far as the Montlake Bridge.
The commercial filming is part of the City of Seattle’s Commercialize Seattle initiative to attract high wage commercial film work to the region.


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Vizag, on Thursday night, reported its first positive of coronavirus. As per sources, the patient is currently undergoing treatment at the Chest hospital in the city.
Reportedly, the 65-year-old man, who belongs to Allipuram, had arrived in Vizag from Saudi Arabia on 12 March. The samples of the patient, which were sent for tests a few days ago, have turned positive to register the first case of coronavirus in Vizag. Reports further claim that test results of a few more suspect cases in the city are currently awaited.
The latest case has taken the total number of coronavirus confirmed cases in Andhra Pradesh to three. On Thursday morning, it was reported that a youngster who had returned from London, tested positive for coronavirus in Ongole, Prakasam district. The State had registered its first case on 12 March when a person had tested positive for the infection in Nellore.
Earlier in the day, Andhra Pradesh Tourism Minister Muttamsetti Srinivasa Rao said that there was no need for the public to panic as the government was taking the necessary measures. He further stated that people must not neglect the precautions and act responsibly.
As a precautionary measure to contain the spread of the virus, the Andhra Pradesh State Government, on Wednesday, called for shut down of educational institutions. The authorities have also restricted darshan to devotees at the holy shrine of Tirupati amid the current global health crisis.

domain: medical


text:
A woman who rammed her boyfriend’s car and also produced a scissors in the course of a domestic dispute has been fined €500. Clonakilty District Court had heard in May that Katie Kehoe, aged 21, of Coach Rd, Dunmanway, Co Cork, admitted to gardaí that in the course of the incident she rammed the other car while she had two children in her own vehicle, including a baby.
At the previous court sitting, Judge James McNulty had heard that a domestic dispute had spiralled “out of control” leading to Kehoe repeatedly ramming her boyfriend’s car, with him jumping onto the bonnet of the moving vehicle to avoid serious injury.
Kehoe, a mother of one, had pleaded guilty to three charges, including the production of a scissors in the course of a dispute and dangerous driving. Garda Liam Galvin had told Judge James McNulty that at 11pm on November 11, 2018, he and a fellow officer were on duty when they received a call in relation to an alleged domestic dispute on Kilbarry Rd in Dunmanway.
On arrival at the scene, gardaí noted damage to the left-hand side of one car and a flat tyre on the same vehicle, with another car parked a few feet away. The judge was told in May that the injured party told gardaí that Kehoe had rammed the side of his vehicle and was waving around a pair of scissors at him and his mother. During the incident, Kehoe had to be restrained.
In addition to admissions at the scene and later in Garda interview, CCTV showed the injured party at one point standing between the two cars and, according to a Garda witness, “having to jump on the defendant’s bonnet while [it was] colliding with the side of his vehicle, narrowly missing being seriously injured”.
Kehoe’s solicitor, Eamonn Fleming, said at the previous hearing that the couple have since got back together and at sentencing yesterday reiterated this was the case. The man had earlier wished to withdraw his complaint relating to criminal damage.
With Kehoe having previously pleaded guilty, Judge McNulty fined her €300 for dangerous driving, €100 for producing a scissors in the course of a dispute, and another €100 for a public order offence.
Mr Fleming said his client needed a driving licence and would be appealing the dangerous driving charge, having sought to have it reduced to careless driving only for gardaí to reject that proposition. Kehoe faces a two-year disqualification from driving and lodged an appeal on her own bond of €100, no cash.

domain: law


The aerial photo shows the Beijing Daxing International Airport that is scheduled to start operation soon.
The Beijing Daxing International Airport has opened its doors to global passengers on Sept. 25, 2019.
46 kilometers away from Tian’anmen, traditionally considered the center of Beijing, the new mega airport is seemingly remote.
However, it takes as rapidly as19 minutes to arrive at the airport from downtown Beijing, thanks to a newly constructed subway line that has completed test runs and is able to operate as fast as 160 kilometers per hour.
Apart from the subway line, a “tridimensional” transportation system featuring rail transit, inter-city railways and expressways will be serving the airport.
The system enables passengers to arrive at the airport from Beijing and Xiong’an New Area within half an hour, and those from the key cities of the Beijing-Tianjin-Heibei region, such as Shijiazhuang, Tangshan and Baoding can also reach the airport in less than an hour.
Besides, multiple bus routes will be put into use to facilitate the transportation of the airport, covering major cities around it, including Tianjin, Tangshan and Baoding.
Now, the Beijing Daxing International Airport Expressway, a two-way eight-lane motorway that spans approximately 27 kilometers has been put into operation. It starts from the South Fifth Ring Road of Beijing to the northern perimeter of the Beijing Daxing International Airport. With 86 percent of the expressway built on bridges, the new road will shorten the transportation time from the South Fifth Ring Road to the airport to around 20 minutes.


In this article, we will take a look at some of the best chosen Fpv Drones Under 50. We would recommend using HR Drone For Kids With 1080p HD FPV Camera,Mini Quadcopter For Beginners With Altitude Hold,One Key Start/Land,Draw Path,2 Modular Batteries,Remote Control Toys Gifts for Boys Girls as it is a high-quality product.
The best Fpv Drones Under 50
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Holy Stone HS340 Mini FPV Drones with Camera for Kids 8-12 RC Quadcopter for Adults Beginners with One Key Take Off/ Landing, Gravity Sensor, Headless Mode, Waypoint Fly, Throw to Go, Indoor & Outdoor
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- 【Reduce damage】：effectively protects the arm and reduces the probability of damage to the arm under extreme conditions.
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- 【In The Box】:DJI FPV Drone Arm Bracers (pair) ×1+Screwdriver ×1, equipped with screwdriver for easy installation or removal.
- 【Satisfied Customer Service】: Providing a 100% satisfaction experience is our main priority to our customers. Feel free to message us through “contact sellers” or if any questions about the product. and We have served more than 200,000 customers, 99.99% satisfied with us.
NEHEME NH525 Foldable Drones with 1080P HD Camera for Adults, RC Quadcopter WiFi FPV Live Video, Altitude Hold, Headless Mode, One Key Take Off for Kids or Beginners with 2 Batteries
$47.99 in stock
2 used from $47.51
- ✈HD CAMERA & FPV FUNCTION: NEHEME Mini Selfie Drone equipped with 45° adjustable 1080P HD camera, which can broaden your views and record every unforgettable moment in your life. Meanwhile, with advanced FPV real-time transmission tech, you can enjoy real-time images through App for beautiful scenery.
- ✈SAFETY & ULTRA-LONG FLYING TIME: NEHEME NH525 drone is equipped with 4 Propeller Guards to protect you from spinning propellers and ensure the drone's propellers from the collision; 2 intelligent batteries for up to 22 minutes ultra-long flight time. Besides, it also can reach up to 100m control distance giving you more extensive flight experience.
- ✈USER-FRIENDLY & DESIGNED FOR BEGINNER: Just one key is all it takes to take off and to land the drone, keep it simple to control for even beginners. Other functions, like Headless Mode, Speed Adjustment functions, Altitude Hold Mode, Hand Gesture Photo/Video. Best choice with enough fun for beginners.
- ✈MULTIPLE FUN FLIGHT: After improving several times, NEHEME NH525 is a versatile drone, can perform stunts like 360°flip, high-speed rotation and etc. And you can draw a route on the App screen and the hover drone will fly following the path accordingly. Last but not least, there are 3 ratio options available to meet all kinds of needs, you can have fun with this mini foldable drone.
- ✈FOLDABLE DESIGN & GREAT GIFT: Foldable blades with a gift box design make this quadcopter drone small, you can take it anywhere you like. More user-friendly design, it comes with a detailed manual that provides accurate instructions about the function and usage of this HD drone. ADD TO CART NOW and buy it as a great gift for your friend, son, grandson, etc.
DJI FPV Drone Arm Bracers
- Easy to assemble and disassemble, the DJI FPV Drone Arm Bracers effectively enhance drone arm strength and reduce the risk of drone arm damage.
- In the box: DJI FPV Drone Arm Bracers (pair) ×1 Screwdriver ×1
- Compatibility: DJI FPV Drone
Drone with Camera for Adults, Foldable RC Quadcopter Kids Toys, 1080P HD FPV Video Drone for Beginners, 2 Batteries,Carrying Case,One Key Start,Altitude Hold,Headless Mode,Waypoints Function,3D Flips
- 【Multifunction Drone 】 Drone comes with one key start/landing, Altitude Hold, Headless Mode, Speed Adjustment functions, adapts to all experience levels even for kids and beginners. Through the APP Gestures Control Trajectory Flight and Gravity Sensor Control can be activated and allows you to capture selfie easily by Gesture Control.
- 【 Drone With 1080P HD Camera 】 The E58 drone is equipped with a high definition camera with adjustable angle. With the 120° wide-angle setting, Capture a HD footage and directly saved, share the amazing view to social media with just one click.
- 【 Real-time WIFI FPV Transmission 】 With a 120° Wide-angle 1080P HD Camera Wi-Fi real-time transmission FPV system can connect to your phone with the drone and the view will be shown directly on your phone, thus enjoy the world above the horizon, accurately capture photos and record videos for extraordinary moments.
- 【 Portable and Safe 】 The four propellers of the drone can be folded for you to put in our storage bag,So it is convenient for you to take it out to fly! With 4 Propeller Guards that fully protects the propellers and improves flight safety.
- 【 Long Flight Time and Quality service 】 2 batteries can support a 20 Minutes flight which makes it easy to replace battery and safe to charge battery.If you have any problems, please message us at anytime, we can reply quickly.
Copter Drone - A Guide to Quadcopters, Drone Helicopters, FPV and Remote Control Drones
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- Be the first to know all about Drones - download Copter Drone - A Guide to Quadcopters, Drone Helicopters, FPV and Remote Control Drones App, download now!
FPV Drone Symphony 2.0
NEHEME NH530 Drones with Camera for Adults Kids, FPV Drone with 1080P HD Camera, RC Quadcopter for Beginners with Gravity Sensor, Headless Mode, One Key Return/Take Off/Landing, Drone with 2 Batteries
$54.99 in stock
1 used from $54.44
- Drone with 1080P HD Camera: Simply click to capture High-quality aerial photos/videos and directly save on your phone album; easy gesture selfie and video recording, definitely a fabulous remote control drone flying indoor/outdoor. Note: If you want to use the drone outdoors, please adjust the speed to the highest to reduce wind interference.
- Multifunction Mini Drone: The NH530 rc drone comes with voice control, 3-speed adjustment, circle fly, 3D Flips and high-speed rotating functions, fulfill your flying time with multiple joy! 2 modular batteries provide up to 18-20 mins long flight time, easy to replace and safe to charge. The size of NH530 mini drone is 6.9 x 6.9 x 1.6 inch (with guards), which makes it very easy to carry.
- Best Drone for Kids and Beginners: User-friendly features like One Key Start/Landing, Headless Mode, and Altitude Hold, support an easy and stable flight. 4 Propeller Guards free your worries of a collision or injury. Ideal toy drone choice for boys and girls. Great drone for kids to practice.
- APP Intelligent Control: Control the drone by your smartphone to explore extra surprise. Gravity Sensor Control, Trajectory Flight, FPV Live Video and VR Mode will show you an extraordinary new world!
- Great Gift Choice: The NH530 fpv drone has entry-level speed and operations for kids and beginners, various novel features for skilled pilots. Perfect gift for lover, friends, family for Christmas/Birthday/Holiday. Comes with a warranty, never worry about any possible issues with the drone.


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521.OX.2611.LR Hublot. The Hublot Classic Fusion Chronograph 521.OX.2611.LR is a luxurious and stylish timepiece. The 45.00 mm 18K Rose gold case with a fixed bezel gives the watch an elegant and refined look. The opalin dial with gold tone hands and index hour markers adds to the overall sophistication of the watch. The scratch resistant anti-glare sapphire crystal on the front side protects the dial from scratches and enhances the clarity of the display. The date window is positioned at the 6 o'clock position, which adds to the functionality of the watch. This watch features two chronograph sub-dials located at 3 and 9 o'clock positions, which allows for accurate timing of events. The black crocodile leather with rubber strap and double-locking foldover clasp provides a comfortable and secure fit on the wrist. The automatic movement with a power reserve of 42 hours ensures reliable and accurate timekeeping. This watch is also water resistant up to 50m/150ft, which makes it suitable for water activities such as swimming and snorkeling. Overall, the Hublot Classic Fusion Chronograph 521.OX.2611.LR is a high-end timepiece that combines style, functionality, and durability. It is a perfect choice for those who appreciate luxury and fine craftsmanship.
521.OX.2611.LR Hublot 18K Rose Gold Watch.

domain: design


Financial feasibility of green-water shrimp farming associated with mangrove compared to extensive shrimp culture in the Mahakam Delta, Indonesia
MetadataShow full item record
This paper presents a post-hoc assessment of the introduction of intensive shrimp farming strategies, with and without green-water (GW) technology, in the Mahakam Delta where extensive systems (ES) dominate. The study also assesses the potential of integrated mangrove GW shrimp production (MGW). The method section describes the systems considered, the cost-benefit analysis applied and the assumptions for different scenarios. The data for the GW and non-GW systems were based on a survey in the Philippines. Assessing cultured shrimp yields from the total farm area showed that production from non-GW was 10% higher than from GW farms. Compared to these two systems, the MGW system produces about 20% of the total shrimp, but provides complementary livelihood options and ecosystem services. Per unit area covered, MGW system produces 20 times more shrimp than ES, while income for farmers doubles and opportunities for livelihoods enhancement associated with the mangrove area increase. Low operating costs make the ES interesting for resource poor farmers, but risks to producers and societal cost are underrated. Transferring from ES to MGW system will increase the contribution to the national economy whilst maintaining ecosystem services, that would otherwise be lost, were intensive culture systems to predominate.
Paper presented at the 9th Asian Fisheries and Aquaculture Forum April 21-25, 2011, Shanghai, China
CitationBosma, R. H., Tendencia, E. A., & Bunting, S. W. (2012). Financial feasibility of green-water shrimp farming associated with mangrove compared to extensive shrimp culture in the Mahakam Delta, Indonesia.
PublisherAsian Fisheries Society
Preparation of this paper was supported by the MANGROVE project which received research funding from the European Community’s Sixth Framework Programme [Contract: INCO-CT-2005-003697], and from the RESCOPAR project funded by the INREF programme of Wageningen University. This publication reflects the authors’ views and the European Community is not liable for any use that may be made of the information contained herein.
Showing items related by title, author, creator and subject.
ArticleJH Primavera, JP Altamirano, MJHL Lebata, AA delos Reyes Jr. & CL Pitogo -
Bulletin of Marine Science, 2007 - University of Miami, Rosenstiel School of Marine and Atmospheric ScienceThe capacity of a natural mangrove system in Ibajay, Aklan province, central Philippines to process shrimp pond culture effluents was assessed through analysis of mangrove community structure and 24-hr monitoring of water quality parameters (NH3-N, NO3-N, PO4-P, sulfide, and total suspended solids). Results from the latter showed decreased nutrient levels within 6 hrs after daytime draining of effluents into the mangrove stand, but only nitrate reduction was statistically significant. Based on nitrate loss, volume of water drained, mangrove area, and shrimp farming data (e.g., N loss from ponds, feed composition, feeding rate), calculations show that 1.8–5.4 ha of mangroves are required to remove nitrate wastes from 1 ha of shrimp pond. N uptake by the mangrove macroflora was supported by data showing longer nipa palm leaflets and faster mangrove seedling growth in the experimental mangrove receiving effluents compared to a control mangrove, but not from mangrove biomass measurements. These results have significant implications for the Philippine brackishwater pond culture industry to conserve or rehabilitate mangroves as potential pond biofilters, to implement legally mandated 20- and 50-m greenbelts, and to reverse the national 0.5 ha mangrove: 1.0 ha pond ratio.
magazineArticleThis article briefly summarizes the techniques developed, verified and/or refined during the implementation of the Project on the Promotion of Mangrove-Friendly Shrimp Aquaculture in Southeast Asia, which was implemented by the SEAFDEC Aquaculture Department from 2000 to 2005. Conducted under the ASEAN-SEAFDEC FCG collaborative mechanism, the project which received generous funding from the Government of Japan through its JTF Program, aimed to develop sustainable culture technology packages on shrimp farming that are friendly to mangroves and the environment.
Conference paperAT Triño & JH Primavera - In LMB Garcia (Ed.), Responsible Aquaculture Development in Southeast Asia. Proceedings of the Seminar-Workshop on Aquaculture Development … Southeast Asia organized by the SEAFDEC Aquaculture Department, 12-14 October 1999, Iloilo City, Philippines, 2001 - SEAFDEC Aquaculture DepartmentThe SEAFDEC Aquaculture Department studies on mangrove-friendly aquaculture (MFA) can be categorized under two models: a) mangrove filters where mangrove forests are used to absorb effluents from high-density culture ponds, and b) aquasilviculture or the low-density culture of crabs, shrimp and fish integrated with mangroves. In a study using the first model, shrimp pond effluents were retained in an enclosed mangrove area prior to release to receiving waters. Nutrients and other water quality parameters, and bacterial levels were monitored in the untreated effluents and post-mangrove water. In the second MFA model, mangrove pens and ponds installed in old growth and newly regenerating mangrove sites in Aklan, central Philippines were stocked with mud crab Scylla olivacea/S. tranquebarica and shrimp Penaeus monodon. Investment costs, survival and production, and cost-return analysis for the pens and ponds are reported in the paper. Aside from the aquasilviculture trials in collaboration with local government units, other activities in the Aklan mangrove sites are the survey and mapping of the 75-ha area in Ibajay, construction of a treehouse, and the educational use as field site by Coastal Resource Management trainees of SEAFDEC Aquaculture Department and field biology students of the University of the Philippines in the Visayas.


Denon is adding a new member to its Heos line of wireless speakers. The new HomeCinema is a soundbar, with an external subwoofer, that can improve TV sound and connect to the wireless Heos sound system.
Better sound for your TV
As TVs have become thinner soundbars have become more popular. Consumers want great sound to match great pictures. Denon promises to address that concern with its latest soundbar that doubles as a music speaker.
The soundbar has built-in stereo speakers but can also imitate surround, says Denon. It supports Dolby Digital Plus and DTS and can be connected to a TV via HDMI. If your TV supports HDMI ARC you can control the volume with the TV remote, too. The soundbar also offers optical and coaxial audio connectors, and the subwoofer is a separate box that connects wirelessly to the soundbar.
HomeCinema can connect wirelessly to Denon’s Heos multi-room system. This means that you can listen to music on the soundbar and other speakers from a phone or tablet, and have your music playing in several rooms at once. It is not yet possible to get 5.1 surround from the TV by adding extra Heos speakers, which Sonos is offering with its Playbar.
Heos HomeCinema will be available starting in July for 799 USD / Euro.
HEOS HomeCinema Key Features
Amplifies TV sound and works as a HEOS speaker
Wireless subwoofer included
Dolby Digital, Dolby Digital Plus, and DTS
HDMI input/output, HDMI ARC
Optical and coaxial inputs and AUX in
USB input for mass storage
Music and Movie DSP modes
Dialog enhancer and Night mode
TV sound can be distributed throughout the HEOS ecosystem
Shelf or wall mount
Built-in keyholes for wall mounting, including wall mount template
HDMI and optical cable for TV connection included
Installation assistant via app, including IR TV remote learning
IR blaster in case TV sensor is hidden by the soundbar


It proposes to demolish the existing building, on the corner of St Cross Road and South Parks Road, to make way for a new (provisionally named) Life and Mind Building. The building will accommodate the Department of Experimental Psychology and a new Department of Biology, combining the existing Departments of Plant Sciences and Zoology.
As part of this redevelopment, various enabling works are proposed in relation to adjacent and adjoining buildings, as well as infrastructure works and the demolition of the current building. At the consultation event the University will set out its proposals for and invite feedback on these and on the emerging design concepts for the new building.
Read more (University of Oxford website)


text:
Interferon-Induced Transmembrane Protein 1 Regulates Endothelial Lumen Formation During Angiogenesis
Objective—It is well established that angiogenesis is a complex and coordinated multistep process. However, there remains a lack of information about the genes that regulate individual stages of vessel formation. Here, we aimed to define the role of human interferon-induced transmembrane protein 1 (IFITM1) during blood vessel formation.
Approach and Results—We identified IFITM1 in a microarray screen for genes differentially regulated by endothelial cells (ECs) during an in vitro angiogenesis assay and found that IFITM1 expression was strongly induced as ECs sprouted and formed lumens. We showed by immunohistochemistry that human IFITM1 was expressed by stable blood vessels in multiple organs. siRNA-mediated knockdown of IFITM1 expression spared EC sprouting but completely disrupted lumen formation, in both in vitro and in vivo xeno-transplant model. ECs lacking IFITM1 underwent early stages of lumenogenesis (ie, intracellular vacuole formation) but failed to mature or expand lumens. Coimmunoprecipitation studies confirmed occludin as an IFITM1 binding partner in ECs, and immunocytochemistry showed a lack of occludin at endothelial tight junctions in the absence of IFITM1. Finally, time-lapse video microscopy revealed that IFITM1 is required for the formation of stable cell–cell contacts during endothelial lumen formation.
Conclusions—IFITM1 is essential for the formation of functional blood vessels and stabilizes EC–EC interactions during endothelial lumen formation by regulating tight junction assembly.
- Received September 13, 2013.
- Accepted February 17, 2014.
- © 2014 American Heart Association, Inc.


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Easy to digest, lower calorie cat food for older cats. Natures Menu senior cat food is veterinary approved and complete and balanced for the needs of our senior feline friends.
A grain free, seriously meaty raw ready meal for cats. Made with succulent Chicken and Duck. 95% real meat.Individual Pack Size 400g.❄ This is a frozen product
Made with 95% ethically sourced Chicken and full flavoured Rabbit. A complete and balanced raw ready meal for cats.Individual Pack Size 400g.❄ This is a frozen product
A delicious combination of responsibly sourced Chicken and Salmon. Complete and balanced raw ready meal. Grain Free.Individual Pack Size 400g.❄ This is a frozen product
A naturally nutritious complete and balanced meal for older cats. Low in fat and packed with real cuts of Chicken, Salmon and Cod, plus all the essential vitamins and minerals your cat needs to stay fit and healthy.Individual Pack Size 100g.
Threxton Road Industrial Estate,Watton, Norfolk, IP25 6NG,United Kingdom
+44 (0)1953 883770
Copyright: Natures Menu 2016All Rights Reserved
Reg. No: 1548073VAT. No: GB595 4890 79
© 2017 Natures Menu All Rights Reserved.
Maintained by Affinity Agency.


Motorola Inc. is advising users of several models of its Oncore Global Positioning Satellite (GPS) receiver units that a software glitch will cause the devices to display the incorrect date for a one-second period towards the end of November.
The problem will occur when, according to Universal Time (UTC), Nov. 27 becomes Nov. 28. While the time output from the receivers will remain correct, the date reported by the units will be incorrect for the first second of the new day. Instead of advancing from Nov. 27 to Nov. 28, the time out put will jump to Nov. 29 before correcting to Nov. 28 from one second past midnight. Positioning functions will not be affected.
Motorola has issued a technical application note to its users and is advising them to check any systems that rely on the GPS date signal, although the company says it thinks the glitch will cause few problems.”We are in the process of analyzing that now,” said Tim McCarthy, director of Motorola’s GPS Product Group. “For the vast majority, 99 per cent of users, it won’t cause any problem at all but we’re digging into the details.”
The reason for the problem is an usually long gap between leap seconds, or extra seconds inserted into UTC to ensure it does not stray too far from the Earth’s rotational time. The software in some models of Motorola GPS units keeps count of the number of elapsed weeks since the last leap second and stores this in an 8-bit long field, which allows for a count of up to 256 weeks. This number will cycle over on Nov. 28 when the 257th week since the last leap second begins.
“It will be the first time 256 weeks have elapsed without a leap second needing to be inserted in Universal Time,” said McCarthy. “No one imagined this would happen and no one thinks it will happen again.”
The problem affects the VP-, UT-, GT- and M12 Oncore units. Motorola wouldn’t disclose the number of affected units shipped. However, in September 2002 the company announced total shipments of all of the company’s GPS units had reached five million units.
The modules are typically used in two broad classes of application, McCarthy said.
The first is in systems that use the GPS system for navigation and location finding purposes and the second is in systems that use the GPS clock as an accurate source of time. The former includes applications such as car navigation systems and Qualcomm Inc.’s OmniTracs system, while the latter includes such apparatus as cellular base stations where customers include Telefonaktiebolaget LM Ericsson and Lucent Technologies Inc.
“I am absolutely sure there will be no problem,” said


text:
Mental Game Tip: Relaxed and Poised
One of the keys to playing well is the ability to relax and play without stress and tension. Once you are relaxed, the key to playing at your best is your ability to become poised to enter the zone. This is quite an undertaking for the average golfer, but poise under pressure is what all great golfers have in common.
Unfortunately, many golfers find it hard to relax because they are too stressed out trying to play well and shoot a low score. Some golfers who can relax complain that it is hard to play well because they are too relaxed. That’s because they don’t take the next step and get poised before executing their shot.
Learn and develop this four-step routine as mental keys to improve your golf:
1) feedback response, 2) relax, 3) preparation and 4) instinctive execution.
Once you make your shot, whether it was good or bad, learn from the feedback. Since it’s over and is now just history, take the time to clear your mind and relax before you prepare for your next shot.
Now that you are relaxed, it’s time to prepare for the shot you’re facing. First, you have to take in all the inputs necessary for your mind to calculate the requirements to execute the shot at hand.
Then, it is time to become poised so you can prepare your mind and body to execute while in the zone, which is when you play at your best.
So how do you become poised? The answer to this question is the key to excellence in golf and life. Once you are relaxed and have taken in the inputs, you focus intently on your target expecting to hit it without any concern about the outcome. While doing this, slow down and take several deep breaths. It’s similar to putting yourself into a trance like state of mind. By making this practice the most important part of your pre-shot routine, you will start to get a feel for what is it like to be poised and ready to execute in the zone.
You will be in the present without any thoughts about the past or future. You will not have a care in the world except your total focus on your target. Your mind will have its full intention on hitting the target without being attached to achieving the outcome. Your brain’s chemistry will be at its optimum and
you will be poised to execute your shot instinctively. Once you are poised, trust your triangle and hit the ball.
It takes time and effort to train yourself to become poised. Once you master this art, your game will never be the same because you will fully realize that controlling your state of mind is the key to increasing your ability and probability of making the shot.
So if you want to lower your score, forget about scoring. Focus on becoming relaxed and poised. This is what great players do.

domain: psychology


text:
As a self professed LOVER of actual “techno”, it gets me IRKED that people still refer to general electronic dance music [EDM] as techno, when in actuality there are MAJOR differences between the sub-genres. So here I am, to enlighten you….
While performing her 1994 song “Human Nature” on her
MDNA tour in Istanbul, this past week, her Madgesty reminds us:
She’s still not sorry. Skip to 2:50 for it!

domain: artistic


text:
The Not So Secret Life of the Faculty
Professors teach classes— everyone knows this—but how do they spend their time outside of that MWF 8:00am seminar? Get a glimpse into the roles of Southern Methodist University professors as mentors, writers, researchers, administrators, and more through this exhibition of faculty letters, manuscripts, photographs, memorabilia, publications, and artifacts from the SMU Archives, DeGolyer Library.
This exhibit is hosted in honor of SMU’s Year of the Faculty.
Exhibit: The Not So Secret Life of the Faculty
Location: DeGolyer Library
Dates: August 20 to October 1, 2014; Monday – Friday, 8:30 a.m. to 5 p.m.
Reception: Thursday, September 18th at 6:00 p.m.
The reception will include a book signing and lecture by Professor Emeritus Darwin Payne, author of No Small Dreams: J. Erik Jonsson—Texas Visionary. For additional information or to RSVP: 214-768-3637 or firstname.lastname@example.org.
The reception is co-sponsored by DeGolyer Library and the Friends of the SMU Libraries.

domain: library