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Phosphoinositides are emerging as general regulators of the functionally diverse transient receptor potential (TRP) ion channel family. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) has been reported to positively regulate many TRP channels, but in several cases phosphoinositide regulation is controversial. TRP melastatin 3 (TRPM3) is a heat-activated ion channel that is also stimulated by chemical agonists, such as pregnenolone sulfate. Here, we used a wide array of approaches to determine the effects of phosphoinositides on TRPM3. We found that channel activity in excised inside-out patches decreased over time (rundown), an attribute of PI(4,5)P2-dependent ion channels. Channel activity could be restored by application of either synthetic dioctanoyl (diC8) or natural arachidonyl stearyl (AASt) PI(4,5)P2. The PI(4,5)P2 precursor phosphatidylinositol 4-phosphate (PI(4)P) was less effective at restoring channel activity. TRPM3 currents were also restored by MgATP, an effect which was inhibited by two different phosphatidylinositol 4-kinase inhibitors, or by pretreatment with a phosphatidylinositol-specific phospholipase C (PI-PLC) enzyme, indicating that MgATP acted by generating phosphoinositides. In intact cells, reduction of PI(4,5)P2 levels by chemically inducible phosphoinositide phosphatases or a voltage-sensitive 5′-phosphatase inhibited channel activity. Activation of PLC via muscarinic receptors also inhibited TRPM3 channel activity. Overall, our data indicate that TRPM3 is a phosphoinositide-dependent ion channel and that decreasing PI(4,5)P2 abundance limits its activity. As all other members of the TRPM family have also been shown to require PI(4,5)P2 for activity, our data establish PI(4,5)P2 as a general positive cofactor of this ion channel subfamily.
Transient receptor potential (TRP) melastatin 3 (TRPM3) is a member of the TRP ion channel family. It is expressed in several different tissues, including the kidneys, eyes, sensory neurons of the dorsal root ganglia, and pancreatic β cells (Oberwinkler and Philipp, 2014). TRPM3 has been proposed to play roles in a variety of physiological and pathophysiological processes. It is activated by high temperatures and was shown to function as a noxious heat sensor in dorsal root ganglion neurons (Vriens et al., 2011). The neurosteroid pregnenolone sulfate (PregS) activates TRPM3 in pancreatic β cells (Wagner et al., 2008), and the channel has been proposed to play important signaling roles in those cells (Thiel et al., 2013). A missense mutation in TRPM3 was recently shown to underlie inherited cataract and high-tension glaucoma in humans (Bennett et al., 2014). In mice, genetic deletion of TRPM3 caused impaired pupillary light reflexes (Hughes et al., 2012).
TRP channels are activated by a wide range of stimuli and play roles in a variety of physiological and pathophysiological processes (Wu et al., 2010). Given their diversity, general principles in their regulation are difficult to establish. As the majority of TRP channels have been reported to be regulated by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), it is possible that phosphoinositides are general regulators of all TRP channels.
PI(4,5)P2 regulates many different mammalian ion channels. It usually acts as a positive cofactor; for example, its presence is required for the activity of all members of the K+ inwardly rectifying (Kir) and KCNQ K+ channel families (Suh and Hille, 2008; Logothetis et al., 2015). In contrast, PI(4,5)P2 regulation of TRP channels is complex; both positive and negative effects of this lipid have been demonstrated on several members of the TRP vanilloid (TRPV) and TRP classical (TRPC) families (Rohacs, 2014). In contrast, the picture on the TRPM family is simpler. Six of the eight mammalian TRPM channels, TRPM2 (Tóth and Csanády, 2012), TRPM4 (Nilius et al., 2006), TRPM5 (Liu and Liman, 2003), TRPM6 (Xie et al., 2011), TRPM7 (Runnels et al., 2002), and TRPM8 (Rohács et al., 2005), have been shown to be positively regulated by PI(4,5)P2, and no negative effect of the lipid has been reported on any TRPM channel (Rohacs, 2014).
Here we set out to test the effects of PI(4,5)P2 on TRPM3. One of our motivations was to assess whether PI(4,5)P2 is a general positive regulator of the TRPM family. TRPM1 is very difficult to study in expression systems; thus, the only remaining member of the TRPM family on which functional effects of PI(4,5)P2 are reliably testable, but has not been demonstrated yet, is TRPM3. We used an array of approaches that included testing endogenous and exogenous phosphoinositides in excised inside-out patches and various inducible phosphatases in whole-cell patch clamp experiments. All of these techniques point to the same conclusion: PI(4,5)P2 is required for TRPM3 activity. Overall, our work establishes that PI(4,5)P2 is an important cofactor for TRPM3 and, together with data in the literature, suggests that PI(4,5)P2 is a general positive regulator of the TRPM family.
Xenopus laevis oocyte preparation
Xenopus oocytes were prepared as described earlier (Rohacs, 2013). In brief, frogs were anesthetized in 0.25% ethyl 3-aminobenzoate methanesulfonate solution (MS222; Sigma-Aldrich) in H2O, pH 7.4. Bags of ovaries containing multiple oocytes were removed surgically from the anesthetized frogs. Individual oocytes were obtained by overnight digestion at 16°C in 0.1–0.2 mg/ml type 1A collagenase (Sigma-Aldrich), dissolved in a solution containing 82.5 mM NaCl, 2 mM KCl, 1 mM MgCl2, and 5 mM HEPES, pH 7.4 (OR2 solution). The next day the collagenase-containing solution was discarded and the oocytes were washed multiple times with OR2 solution. The oocytes were maintained in OR2 solution supplemented with 1.8 mM CaCl2, 100 IU/ml penicillin, and 100 µg/ml streptomycin at 16°C. 40 ng linearized cRNA transcribed from the human TRPM3 (hTRPM3) cDNA clone (Grimm et al., 2003) in the pGEMSH vector was microinjected into individual oocytes. The injection was performed with a nanoliter-injector system (Warner Instruments). Oocytes were used for electrophysiological measurements 48–72 h after microinjection. The hTRPM3 clone in a mammalian expression vector was provided by C. Harteneck (Eberhard Karls University Tübingen, Tübingen, Germany). The cDNA of hTRPM3 was subcloned into pGEMSH using standard molecular biology techniques.
Excised inside-out patch clamp and two-electrode voltage clamp (TEVC) electrophysiology
Excised inside-out patch clamp measurements were performed as described earlier (Rohacs, 2013). In brief, oocytes were placed in bath solution (97 mM KCl, 5 mM EGTA, and 10 mM HEPES, pH 7.4) in the recording chamber. The vitelline layer was manually removed with a pair of forceps, and then gigaohm seals were formed with borosilicate glass pipettes (World Precision Instruments) containing pipette solution (97 mM NaCl, 2 mM KCl, 1 mM MgCl2, 5 mM HEPES, and 100 µM PregS, pH 7.4). Pipette resistance was 0.8–1 MΩ. Macroscopic currents were recorded with a −100- to 100-mV ramp protocol applied every second (0.25 mV/ms); holding potential was 0 mV. The currents were measured with an Axopatch 200B amplifier and analyzed with the pClamp 9.0 software (Molecular Devices). Once the excised inside-out patch configuration was established, the test compounds, dissolved in bath solution, were applied through a custom-made perfusion system to the cytoplasmic face of the membrane patch. The dioctanoyl (diC8) forms of phosphoinositides, phosphatidylinositol 4-phosphate (PI(4)P), PI(3,4)P2, PI(4,5)P2, PI(3,5)P2, and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3), were purchased from Cayman Chemicals. The naturally occurring arachidonyl stearyl (AASt) forms of PI(4)P and PI(4,5)P2, purified from porcine brain, were purchased from Avanti Polar Lipids, Inc., and they were sonicated on ice for 10 min before experiments with an ultrasonic homogenizer (model 3000 V/T; Biologics Inc.). All phosphoinositides were dissolved in water, aliquoted, and stored at −80°C. Poly-l-lysine (Poly-Lys; molecular mass 4–15 kD), LY294002, PregS, and Gö6976 were purchased from Sigma-Aldrich, dissolved in DMSO, and stored at −20°C. LY294002 was added into the MgATP solution from a 100 mM stock, and DMSO was used in control recordings at the same final concentration (0.3%). The concentration of DMSO for PregS-containing solutions did not exceed 0.2%. Phosphatidylinositol-specific PLC (PI-PLC) was purchased from Sigma-Aldrich as a glycerol-containing stock solution and stored at 4°C; glycerol (0.5%) was added to control solutions in experiments with PI-PLC. Compound A1, provided by T. Balla (National Institutes of Health, Bethesda, MD; Bojjireddy et al., 2014), was dissolved in DMSO. For the MgATP experiments, the pH of the bath solution had to be readjusted to pH 7.4; for details see Zakharian et al. (2011).
Data analysis for inside-out patch clamp.
In Fig. 1, data have been normalized to the peak current immediately after excision. In the remaining figures, the responses have been normalized to the current induced by the 25 µM diC8 PI(4,5)P2 applied after the current had run down. For the dose–response measurements in Figs. 1 and 2, repetitive pulses of 25 µM diC8 PI(4,5)P2 were applied to compensate for any decrease in responsiveness of the patch. Amplitudes evoked by different concentrations of either diC8 PI(4,5)P2 or PI(3,4,5)P3 were normalized to the mean of the amplitudes evoked by the two nearest 25 µM diC8 PI(4,5)P2 applications. Data were then fitted with the Hill equation, using the Origin 9.0 software. For plotting the data in the figures, data points were renormalized to the maximal current obtained from the Hill fits.
TEVC measurements were performed as described earlier (Lukacs et al., 2007); in brief, oocytes were placed in extracellular solution (97 mM NaCl, 2 mM KCl, 1 mM MgCl2, and 5 mM HEPES, pH 7.4), and currents were recorded with thin-wall inner-filament–containing glass pipettes (World Precision Instruments) filled with 3 M KCl in 1% agarose. Currents were measured with the same ramp protocol as mentioned above for excised inside-out patch measurements. For experiments with wortmannin, oocytes were pretested for PregS-induced TRPM3 currents and then incubated for 2 h in wortmannin-containing OR2 solution. PregS-induced TRPM3 currents were measured again, and the currents before and after wortmannin incubation were compared in the same oocytes. The currents were recorded with a GeneClamp 500B amplifier and analyzed with the pClamp 9.0 software (Molecular Devices).
Whole-cell electrophysiology in HEK cells
Whole-cell patch clamp measurements were performed in Human Embryonic Kidney 293 (HEK293) cells (ATCC) transiently transfected using the Effectene reagent (QIAGEN), with the mouse TRPM3α2 (mTRPM3α2) cloned in the bicistronic pCAGGS/IRES-GFP vector (Oberwinkler et al., 2005; Vriens et al., 2011), provided by S. Philipp and V. Flockerzi (Saarland University, Homburg, Germany). The cells were maintained in MEM (Life Technologies) supplemented with 10% (vol/vol) fetal bovine serum, 100 IU/ml penicillin, and 100 µg/ml streptomycin. GFP-positive cells were used for measurements. For the experiments with the voltage-sensitive phosphatase from Ciona intestinalis (ci-VSP), the cells were cotransfected with ci-VSP in the pIRES2-GFP vector (Hossain et al., 2008), provided by Y. Okamura (Osaka University, Suita, Osaka, Japan) and Kir2.1 in pCDNA3 vector to keep the membrane potential in the negative range to avoid any basal activation of the phosphatase. The cells were used for measurements 48–72 h after transfection. Measurements were performed in an extracellular solution containing 137 mM NaCl, 5 mM KCl, 1 mM MgCl2, 10 mM HEPES, and 10 mM glucose, pH 7.4. For measurements in Fig. 7, the same solution was supplemented with 0.5 mM EGTA, and for Fig. 8, the solution was supplemented with 2 mM CaCl2. The recording pipette was filled with intracellular solution containing 140 mM potassium gluconate, 5 mM EGTA, 1 mM MgCl2, 10 mM HEPES, and 2 mM Na-ATP, pH 7.3, adjusted with KOH. For experiments in Fig. 8, this solution was supplemented with 0.2 mM GTP. Once a gigaohm seal was achieved, the whole-cell configuration was established, and the currents were recorded at a constant holding potential of −100 mV. To activate ci-VSP, a 5-s pulse to 100 mV was applied. For measurements with the various rapamycin-inducible phosphatases, cells were cotransfected with mTRPM3α2 and the components of either the rapamycin-inducible 5′-phosphatase (Varnai et al., 2006), provided by T. Balla (National Institutes of Health, Bethesda, MD), or pseudojanin (Hammond et al., 2012), provided by G. Hammond (University of Pittsburgh, Pittsburgh, PA). Experiments were performed with a ramp protocol from −100 to 100 mV applied once every second, and the currents at −100 and 100 mV were plotted. The currents were measured with an Axopatch 200B amplifier, filtered at 2 kHz, digitized through Digidata 1322A, and analyzed with pCLAMP 9.0 software (Molecular Devices).
Statistical analysis was performed using Student's t test or analysis of variance: *, P < 0.05; **, P < 0.01; ***, P < 0.005. The error bars in all of the figures represent SEM.
Online supplemental material
Fig. S1 shows that PregS activates hTRPM3 expressed in Xenopus oocytes in a concentration-dependent manner. Fig. S2 shows that intracellular Mg2+ inhibits hTRPM3 after patch excision. Fig. S3 shows that intracellular Mg2+ inhibits the current increase after washout of MgATP in excised patches and Poly-Lys inhibits MgATP-induced currents in excised patches. Fig. S4 shows that the PKC inhibitor Gö6976 does not inhibit the effect of MgATP in excised patches. Fig. S5 shows that the rapamycin-inducible 5′-phosphatase inhibits hTRPM3 expressed in Xenopus oocytes.
Exogenous phosphoinositides reactivate TRPM3 currents in excised patches after current rundown
The neurosteroid PregS is a well-established activator of TRPM3. Both the human and the mouse TRPM3 have several splice variants, summarized in Oberwinkler et al. (2005). Most experiments documenting the activating effect of PregS on TRPM3 have been performed on the α2 isoform of the mouse TRPM3 (mTRPM3α2; Wagner et al., 2008; Vriens et al., 2011; Frühwald et al., 2012). Here we used the isoform of the human TRPM3 (hTRPM3) described in Grimm et al. (2003) for electrophysiological recordings in Xenopus oocytes. First we tested whether PregS is a reliable activator of hTRPM3 in TEVC experiments. We found that PregS induced outwardly rectifying currents in TRPM3-expressing oocytes in a concentration-dependent manner, with an EC50 of 19.3 µM at 100 mV and 28.5 µM at −100 mV (Fig. S1).
Our central hypothesis is that PI(4,5)P2 is required for the activity of TRPM3. PI(4,5)P2 is found in the inner leaflet of the plasma membrane; thus, we tested the effects of phosphoinositides applied directly to the cytoplasmic side of excised inside-out patches. In the cell-attached configuration, with 100 µM PregS in the patch pipette, hTRPM3 currents were relatively small, and they increased substantially immediately after excision (Fig. 1, A–C and E), reflecting the loss of a cytoplasmic inhibitory factor. Intracellular Mg2+ ions inhibit TRPM3 (Oberwinkler et al., 2005). Intracellular free Mg2+ concentrations in Xenopus oocytes were reported to be ∼0.3 mM (Gabriel and Günzel, 2007), which is within the range of that in most mammalian cell types (0.25–1 mM; Grubbs, 2002). To test whether current amplitudes increase after excision into the Mg2+ free bath solution because of the loss of Mg2+, we also excised patches into a bath solution containing 0.3 and 1 mM Mg2+. As shown in Fig. S2, the increase after excision was somewhat smaller in 0.3 mM Mg2+ than in 0 Mg2+, and no increase was detected when the patch was excised into 1 mM Mg2+. These data suggest that relief from Mg2+ inhibition contributes to the current increase but is unlikely to be solely responsible for it.
After the current had reached the peak, it decreased (rundown) to <10% of the initial current within 2–5 min in most patches at 100 mV. In some patches rundown was slower, but even there current amplitudes decreased to 10–20% of the original current level. Current rundown at −100 mV was generally more complete, usually reaching zero. At negative voltages, however, reliable differentiation from leak was not always possible because of the small current amplitudes; thus, most statistical analysis was performed for excised patch measurements at 100 mV. Nevertheless, we show the inward currents in the figures because those are the physiologically relevant currents for nonselective cation channels. Rundown in excised inside-out patches generally is caused by the loss of a positive cofactor when the patch is removed from the cellular environment. In many cases, the lost cofactor is PI(4,5)P2, which is dephosphorylated by endogenous phosphoinositide phosphatases in the patch membrane (Hilgemann, 1997).
To test whether PI(4,5)P2 is the lost cofactor for TRPM3, we perfused excised inside-out patches with various phosphoinositides after the current had run down. As shown in Fig. 1 (A and D), application of the natural long acyl chain AASt PI(4,5)P2 (10 µM) restored the TRPM3 currents to levels higher than those immediately after excision. The effect developed slowly; the time to reach 50% of the effect was 96 ± 18 s. This slow effect is consistent with the slow incorporation of long acyl chain PI(4,5)P2 micelles in the membrane (Rohács et al., 2002). Stimulation of PI(4,5)P2-sensitive ion channels by AASt PI(4,5)P2 is generally not reversible on the 2–5-min time scale (Rohács et al., 2002; Zakharian et al., 2011), but can be inhibited by Poly-Lys, a known chelator of phosphoinositides and other negatively charged lipids (Lopes et al., 2002). We found that 30 µg/ml Poly-Lys decreased the TRPM3 currents close to baseline level after AASt PI(4,5)P2 (Fig. 1, A and D). PI(4)P, the precursor of PI(4,5)P2, is found in the plasma membrane at similar quantities to PI(4,5)P2 and may also activate ion channels such as TRPV1 (Lukacs et al., 2007; Klein et al., 2008; Ufret-Vincenty et al., 2011). AASt PI(4)P increased the TRPM3 current to a smaller extent compared with AASt PI(4,5)P2 (Fig. 1, B and D). The effect developed slowly, with a similar kinetics to AASt PI(4,5)P2; the time to reach 50% of the effect for AASt PI(4)P was 114 ± 12 s.
We also tested the effects of the synthetic diC8 PI(4,5)P2 and PI(4)P. These short acyl chain water-soluble phosphoinositide analogues are routinely used to study ion channel regulation because their effective concentrations can be easily controlled and their effects are readily reversible (Rohács et al., 2002; Nilius et al., 2006; Stein et al., 2006). As shown in Fig. 1 (A–D), application of diC8 PI(4,5)P2 stimulated TRPM3 currents after rundown in a quickly reversible fashion, whereas 25 µM diC8 PI(4)P only had a marginal effect (Fig. 1, C and D). DiC8 PI(4,5)P2 reliably stimulated TRPM3 currents in the same patches, both before and after the application of PI(4)P, showing that the lack of effect of the latter was not caused by the lack of responsiveness of the patch. We also determined the concentration dependence of the effect of diC8 PI(4,5)P2 by applying the lipid consecutively at different concentrations to the same patch. PI(4,5)P2 stimulated TRPM3 activity with an EC50 of 18 µM (Fig. 1, E and F). Note that the effect of diC8 PI(4,5)P2 was smaller than that of AASt PI(4,5)P2 (Fig. 1 D), even at the highest concentration tested (100 µM; Fig. 1 F).
We also tested whether other phosphoinositides can stimulate TRPM3. Fig. 2 (A–D) shows that 25 µM diC8 PI(3,4)P2, PI(3,4,5)P3, and PI(3,5)P2 also activated TRPM3 after current rundown. At this concentration PI(3,4)P2 was slightly less active than PI(4,5)P2, PI(3,5)P2 was somewhat more active, and PI(3,4,5)P3 had the highest activity. We tested the concentration dependence of the effect of PI(3,4,5)P3 in patches, where we also applied 25 µM diC8 PI(4,5)P2 as a normalizing stimulus to make the results quantitatively comparable with those obtained with diC8 PI(4,5)P2 (Fig. 2, E and F). Based on the Hill fits, PI(3,4,5)P3 had an approximately twofold higher maximal activity and a left-shifted concentration dependence (EC50 = 9.4 µM) when compared with PI(4,5)P2 (Fig. 2, E and F).
In addition to the marked rundown of current activity after excision, we also observed a moderate reduction in responsiveness to diC8 PI(4,5)P2 in many, but not all, patches. We quantitated this phenomenon in 13 patches where diC8 PI(4,5)P2 was applied at least four times. In those patches, on average, the second pulse of diC8 PI(4,5)P2 gave 82 ± 6% of the response evoked by the first application. The third and fourth applications gave 67 ± 8% and 66 ± 9% of the first response, respectively. This reduced responsiveness is a relatively small effect compared with the almost complete rundown even by the first application of diC8 PI(4,5)P2, and we did not further examine its mechanism. Similar phenomena have been observed with many well-established PI(4,5)P2-dependent ion channels, for example Kir2.1 (Rohács et al., 2003), TRPM8 (Rohács et al., 2005), and TRPV6 (Cao et al., 2013a).
The effects of endogenous PI(4,5)P2 in excised inside-out patches
Our data so far show that exogenously applied PI(4,5)P2 and other phosphoinositides stimulate TRPM3 after current rundown. However, exogenous lipophilic molecules can potentially cause membrane perturbations (Hilgemann, 2012; Ingólfsson et al., 2014); thus, we also tested whether endogenous PI(4,5)P2 would have a similar effect. PI(4,5)P2 can be replenished by perfusing excised membrane patches with MgATP, which is required for the activity of phosphoinositide kinases in the patch membrane (Hilgemann and Ball, 1996; Sui et al., 1998; Zakharian et al., 2011). As shown in Fig. 3 A, application of 2 mM MgATP increased TRPM3 currents slowly. Upon washout of MgATP, current amplitudes increased transiently, followed by a slow decrease. The peak of the transiently increased current was measured as MgATP-activated TRPM3 current for statistical analysis. Free Mg2+ in the MgATP solution was calculated to be 0.3 mM using the MaxChelator program. When the MgATP solution was switched to bath solution containing 0.3 mM Mg2+, the current increase was not observed, signifying that it was caused by relief from inhibition by Mg2+ (Fig. S3 A). The slow decrease in current after the peak is likely caused by dephosphorylation of PI(4,5)P2 and PI(4)P by lipid phosphatases in the patch, similar to that observed during rundown after patch excision.
PI(4,5)P2 is generated from phosphatidylinositol by the sequential action of phosphatidylinositol 4-kinases (PI4Ks) and PI(4)P 5-kinases (PIP5Ks; Balla, 2013). PI(4,5)P2 can be further phosphorylated to PI(3,4,5)P3 upon activation of phosphoinositide 3-kinases (PI3Ks). To demonstrate that MgATP stimulated TRPM3 currents through increased production of PI(4,5)P2, we tested the effects of MgATP in the presence of LY294002. This compound is known to inhibit PI3Ks at low micromolar concentrations and type III PI4Ks (PI4KA and PI4KB) at high micromolar concentrations (Balla, 2001). When 300 µM LY294002 was applied along with MgATP, the ability of MgATP to reactivate TRPM3 currents was lost (Fig. 3 B). The effect of MgATP was not inhibited by 10 µM LY294002 (Fig. 3 C), indicating that 300 µM LY294002 inhibited the TRPM3 currents via inhibiting PI4K. When Poly-Lys was applied after MgATP, it quickly inhibited TRPM3 currents (Fig. S3, C and D), again suggesting that MgATP acted via generating PI(4,5)P2.
Recently, novel, more potent, and more specific PI4K inhibitors became available (Bojjireddy et al., 2014). We tested one of these novel compounds (A1), which inhibits PI3K enzymes at higher concentrations than PI4K and shows preference for the PI4KA enzymes over PI4KB. As shown in Fig. 3 (D–F), compound A1 completely inhibited the effect of MgATP at 100 nM and exerted an ∼70% inhibition at 10 nM. The IC50 of A1 was reported to be ∼50 nM for PI3K, but 0.15 or 3.1 nM for PI4KA and 19 and 63 nM for PI4KB in two different assays (Bojjireddy et al., 2014). Given the ∼70% inhibition of the effect of MgATP at 10 nM, it is likely that MgATP exerted its effect predominantly via PI4KA. However, a firm conclusion is difficult to draw because the pharmacological profile of Xenopus PI4K enzymes is not known.
In addition to pharmacological inhibitors, we also used a bacterial PI-PLC, which selectively hydrolyses phosphatidylinositol, thus removing the precursor for PI(4)P and PI(4,5)P2 (Hilgemann and Ball, 1996; Sui et al., 1998). Pretreatment with PI-PLC almost completely prevented the effect of MgATP, showing again that the latter acted via generating phosphoinositides (Fig. 4). MgATP is also a cofactor for protein kinases; thus, we also tested the effect of a PKC inhibitor. Fig. S4 shows that 1 µM Gö6976 did not inhibit the effect of MgATP. Collectively, our data suggest that endogenous PI(4,5)P2 is important for the activity of TRPM3.
Inhibition of PI4K inhibits TRPM3 currents in intact cells
Next we tested whether reducing the concentration of PI(4,5)P2 in intact cells limits TRPM3 activity. In these experiments, we used another inhibitor of type III PI4 kinases, wortmannin. Wortmannin inhibits PI3K at low nanomolar concentrations and type III PI4 kinases at micromolar concentrations. We recorded PregS-induced currents from Xenopus oocytes expressing hTRPM3 in TEVC experiments (Fig. 5 A). After measuring TRPM3 currents, the oocytes were incubated in either 35 nM or 35 µM wortmannin for 2 h, and then the PregS-induced currents were measured again in the same oocytes. As shown in Fig. 5 (B and C), current amplitudes decreased significantly in oocytes incubated in 35 µM wortmannin, but not in oocytes incubated in 35 nM wortmannin, showing that the effect is caused by inhibition of type III PI4Ks, indicating that PI(4,5)P2 is vital for the optimal functioning of TRPM3.
TRPM3 currents are inhibited by rapidly inducible phosphoinositide phosphatases
All of our experiments so far were performed in oocytes heterologously expressing hTRPM3. However, most of the studies on TRPM3 used the mTRPM3α2 clone. To test whether PI(4,5)P2 is also necessary for the function of mTRPM3α2, we expressed these channels in HEK293 cells and studied the effects of various inducible phosphoinositide phosphatases on channel activity. The experiments were performed in the absence of extracellular Ca2+ to avoid desensitization of PregS-induced TRPM3 currents (see last paragraph of Results). First, we depleted membrane PI(4,5)P2 using ci-VSP. At negative voltages (−100 mV), ci-VSP is inactive, probably because its phosphatase domain is located away from the cell membrane. When the cell is depolarized, the phosphatase is activated and dephosphorylates PI(4,5)P2 to PI(4)P (Okamura et al., 2009). To test the effect of ci-VSP activation on TRPM3, we recorded PregS-induced TRPM3 currents from HEK cells in the whole-cell configuration at a holding potential of −100 mV, where ci-VSP is inactive. In the presence of PregS, the membrane was depolarized with a short pulse (5 s) of 100 mV to activate the phosphatase, and then potential was returned to −100 mV. As shown in Fig. 6 A, upon activation of ci-VSP by depolarization, PregS-induced TRPM3 currents were inhibited. When the holding potential was returned to −100 mV, TRPM3 currents recovered to the same levels as before the depolarizing pulse within ∼20 s, similar to data obtained with PI(4,5)P2-dependent KCNQ channels (Falkenburger et al., 2010). In cells transfected with the C363S mutant of ci-VSP lacking phosphatase activity (Okamura et al., 2009), TRPM3 currents were not significantly different before and after the depolarizing pulse (Fig. 6, B and C).
An alternative approach to ci-VSP is the rapamycin-inducible translocation of a 5′-phosphatase to the plasma membrane, based on the heterodimerization of FKBP12 and the FRB fragment of the mammalian target of rapamycin (Suh et al., 2006; Varnai et al., 2006). Fig. 6 (D–F) shows measurements where the components of the inducible 5′-phosphatase (Varnai et al., 2006) were coexpressed with the mTRPM3α2 in HEK293 cells. Application of rapamycin induced a slowly developing ∼80% inhibition in cells expressing the active phosphatase, whereas there was essentially no inhibition in control cells expressing all components of the translocation system, without the phosphatase (Fig. 6, D–F). Rapamycin-induced inhibition was not reversible on the time scale of the experiments, consistent with earlier studies (Suh et al., 2006; Varnai et al., 2006). Rapamycin also induced a small (∼20%), but statistically significant inhibition of hTRPM3 in Xenopus oocytes expressing the 5′-phosphatase, but not in control oocytes (Fig. S5). This small inhibition is consistent with the lower efficiency of the rapamycin-inducible system in oocytes (Lukacs et al., 2007).
To evaluate the possible contribution of PI(4)P to channel activity, we also tested the effects of rapamycin in HEK cells expressing pseudojanin, an inducible combined 5′- and 4′-phosphatase that reduces both PI(4,5)P2 and PI(4)P levels (Hammond et al., 2012). Application of rapamycin inhibited TRPM3 currents in these cells (Fig. 7) to a similar extent to that observed in cells expressing the 5′-phosphatase in experiments in Fig. 6 D; see Fig. 7 E for comparison of the time course of inhibition by the different phosphatases. Even though pseudojanin has the same phosphatase domain (INPP5E) as the rapamycin-inducible phosphatase used in Fig. 6, its 5′-phosphatase activity is somewhat lower (Hammond, G. and T. Balla, personal communication), probably because it is a larger construct, also containing the 4′-phosphatase sac1, which may lead to poorer expression. Therefore, we also used a variant of pseudojanin, in which the 4′-phosphatase domain is inactivated with a point mutation (PJ 5′-Ptase). This construct is the same size as pseudojanin; thus, its 5′-phosphatase activity is expected to be the same. Rapamycin application in the cells expressing the PJ 5′-Ptase inhibited TRPM3 currents significantly less than in cells expressing pseudojanin (Fig. 7, B, D, and E). In control experiments, we used a pseudojanin clone in which both the 4′- and 5′-phosphatases were inactivated (Fig. 7 C). Current levels in these cells also showed a small decrease after application of rapamycin, but the decrease induced by rapamycin was significantly larger both in cells expressing the intact pseudojanin or the PJ 5′-Ptase (Fig. 7 D).
PLC activation inhibits TRPM3 currents
To test whether activation of PLCβ enzymes inhibits TRPM3 current, we coexpressed mTRPM3 channels and human muscarinic M1 receptors (hM1) in HEK cells. We performed these experiments in the presence of extracellular Ca2+, which promotes desensitization in many TRP channels (Gordon-Shaag et al., 2008) and also renders PLC activation more efficient upon GPCR activation (Horowitz et al., 2005). PregS-induced currents displayed clear partial desensitization, i.e., decrease in current amplitude in the continuous presence of PregS (Fig. 8). Application of carbachol induced a fast and essentially complete inhibition of PregS-induced currents (Fig. 8).
TRPM3 is a phosphoinositide-dependent ion channel
Regulation of TRP channels by PI(4,5)P2 is complex and in some cases controversial. Although the majority of them are positively regulated by PI(4,5)P2, negative effects of this lipid have also been described on several members of the TRPC and TRPV families, in some cases concurrent with positive effects (reviewed in Rohacs [2014]). The heat- and capsaicin-sensitive TRPV1 is a prime example of these ongoing debates (Cao et al., 2013b; Lukacs et al., 2013; Senning et al., 2014; reviewed in Rohacs [2015]).
These controversies and complexities are not unique to the TRP field, and some of the discrepancies may arise from the differences in the techniques used by different groups (Hilgemann, 2012). Voltage-gated K+ (Kv) channels, for example, were suggested to be regulated by PI(4,5)P2 based on application of phosphoinositide micelles to excised patches (Oliver et al., 2004; Decher et al., 2008), but experiments with inducible phosphatases in the whole-cell configuration suggested no dependence on PI(4,5)P2 for most Kv channels, with the exception of the Kv7 (KCNQ) family (Kruse et al., 2012).
To avoid inherent problems with relying on one experimental setting, here we used a wide array of approaches both in excised inside-out patches and in the whole-cell configuration. We found that TRPM3 currents show marked rundown in excised patches and channel activity could be restored both by application of natural long acyl chain PI(4,5)P2 and synthetic diC8 PI(4,5)P2. Application of MgATP after rundown also restored channel activity. This effect was eliminated both by inhibiting PI4K enzymes and by removal of phosphatidylinositol using the bacterial PI-PLC enzyme, demonstrating that MgATP acted via increasing endogenous phosphoinositide levels. In whole-cell patch clamp experiments, channel activity was inhibited by four different inducible phosphoinositide phosphatases, as well as activation of PLC. Overall, we concluded that TRPM3 channels, similarly to other members of the TRPM family, require phosphoinositides for activity.
Quantitative considerations and phosphoinositide specificity
Even though our results clearly point to PI(4,5)P2 being an important cofactor for TRPM3 activity, some quantitative discrepancies between the results obtained with different approaches require discussion. TRPM3 channels displayed an almost complete rundown in excised patches, indicating a strong dependence on phosphoinositides. Rundown in most cases was relatively fast, indicating low or moderate apparent affinity for PI(4,5)P2. Consistent with this, the EC50 of ∼18 µM for diC8 PI(4,5)P2, indicates an intermediate affinity of TRPM3 to PI(4,5)P2 between high-affinity channels (EC50 < 5 µM), such as Kir2.1 (Du et al., 2004) or TRPV1 (Klein et al., 2008) and low-affinity channels such as KCNQ-s (EC50 > 40 µM; Zhang et al., 2003; Li et al., 2005).
It was calculated that application of ∼40 µM diC8 PI(4,5)P2 to excised patches results in a mole fraction of 0.01 (1%) of diC8 PI(4,5)P2 in the inner leaflet on the plasma membrane, which is considered the physiological concentration there (Collins and Gordon 2013). Based on our dose–response measurements, this physiological level of diC8 PI(4,5)P2 (40 µM) induced an ∼67% saturation of TRPM3 currents (Fig. 1 F). This is comparable with our AASt PI(4,5)P2 measurement, where the maximal current induced by this lipid was ∼1.6 times that of the current after excision (Fig. 1 D), which we assume to reflect the effect of endogenous physiological levels of PI(4,5)P2. Thus, based on the AASt PI(4,5)P2 measurements, endogenous PI(4,5)P2 induced ∼62% of the maximum effect. Overall, the data with diC8 and AASt PI(4,5)P2 indicate that endogenous PI(4,5)P2 exerts 62–67% saturation of TRPM3 activity, which is overall consistent with a moderate apparent affinity for PI(4,5)P2.
Given the preference of the channel for PI(4,5)P2 over PI(4)P, its full dependence on PI(4,5)P2, and its moderate affinity for the lipid, it is expected that the inducible 5′-phosphatases inhibit the channels robustly and quickly. Inhibition by ci-VSP was incomplete, however, and the effect of the rapamycin-inducible 5′-phosphatase developed slowly. Slow or incomplete inhibition by PI(4,5)P2 depletion is often associated with high apparent affinity for PI(4,5)P2, which seems to be at odds with the excised patch data, which indicated low/moderate affinity.
The inducible 5′-phosphatases used in this study in whole-cell patch clamp experiments convert PI(4,5)P2 into PI(4)P. When the effects of diC8 phosphoinositides were tested in excised patches, PI(4,5)P2 clearly activated the channel, but PI(4)P had a negligible effect. The natural AASt PI(4)P was also less effective than AASt PI(4,5)P2, but it induced a quite substantial activation, especially at positive voltages, where it induced ∼60% of the effect evoked by AASt PI(4,5)P2. The concentration of PI(4)P is generally thought to be comparable in the plasma membrane with that of PI(4,5)P2. Thus, it is possible that the contribution of PI(4)P to channel activity accounts for incomplete inhibition by the inducible 5′-phosphatases, especially that the concentration of this lipid is expected to acutely increase after activation of a 5′-phosphatase. To test this possibility, we used the combined 4′- and 5′-phosphatase pseudojanin that depletes both PI(4,5)P2 and PI(4)P (Hammond et al., 2012). Application of rapamycin in pseudojanin-expressing cells did not induce a faster or more complete inhibition than the 5′-phosphatase used in Fig. 6 (Varnai et al., 2006). As mentioned in Results, however, pseudojanin may have a lower 5′-phosphatase activity than the rapamycin-inducible phosphatase we used first. Therefore, we compared the effect of pseudojanin with its variant, in which the 4′-phosphatase is inactivated (PJ 5′-Ptase). In cells transfected with PJ 5′-Ptase, rapamycin induced a smaller inhibition than in cells with the intact pseudojanin. This suggests that PI(4)P contributes to TRPM3 activity, which may explain the slow and partial inhibition by the 5′-phosphatases in whole-cell patch clamp experiments.
Overall, both our excised patch and whole-cell data demonstrate that PI(4,5)P2 is a positive cofactor for PregS-induced TRPM3 activity and that PI(4)P may also contribute to supporting channel activity in a cellular context.
Potential physiological relevance
PI(4,5)P2 is the substrate for PLC enzymes. We found that stimulation of PLCβ via hM1 receptors robustly inhibited TRPM3 activity. The inhibition was faster and more complete than that induced by any of the inducible phosphoinositide phosphatases, raising the possibility that in addition to PI(4,5)P2 depletion, other Ca2+-sensitive factors, such as calmodulin (Holakovska et al., 2012), may also contribute to this inhibition. It also remains to be seen whether endogenous PLC-coupled receptors also inhibit the channel.
In the presence of extracellular Ca2+, PregS-induced TRPM3 currents displayed marked desensitization, i.e., reduced currents despite the continuous presence of PregS. Desensitization of several TRP channels, such as TRPM8 (Rohács et al., 2005), TRPV1 (Lukacs et al., 2007), and TRPV2 (Mercado et al., 2010), has been shown to proceed via Ca2+-induced activation of PLC and the resulting depletion of PI(4,5)P2. Because TRPM3 is Ca2+ permeable and its activity depends on PI(4,5)P2, this mechanism is feasible for its desensitization. We indeed found that application of PregS to cells expressing TRPM3 induced activation of PLC, even though to a lesser extent than carbachol (unpublished data).
PI(3,4,5)P3, the product of PI3K, had a larger effect on TRPM3 than PI(4,5)P2. The concentration of PI(3,4,5)P3 in the plasma membrane is thought to be <10% of that of PI(4,5)P2 even in stimulated cells (Balla, 2013). PI(3,4,5)P3 stimulates many phosphoinositide-sensitive channels, usually to a comparable extent with PI(4,5)P2 (Rohács et al., 2003). Given its much lower concentration, the more abundant PI(4,5)P2 is likely to override its effect. For TRPM3, however, PI(3,4,5)P3 had both higher apparent affinity and substantially higher maximal stimulating effect than PI(4,5)P2. This raises the possibility that PI(3,4,5)P3 generated by PI3K is capable of further stimulating TRPM3, but testing this hypothesis will require further experiments.
Concluding remarks
Using several different techniques both in excised patches and in whole-cell patch clamp experiments, we demonstrate that PI(4,5)P2 is an important cofactor of TRPM3. Given that all other members of the TRPM family, with the exception of TRPM1, which is hard to study both in native and recombinant systems, have also been shown to be positively regulated by PI(4,5)P2, our work establishes TRPM channels as a bona fide PI(4,5)P2-dependent ion channel family.
The human TRPM3 clone was provided by Christian Harteneck and the mouse TRPM3α2 by Drs. Stephan Philipp and Veit Flockerzi. The ci-VSP clone was provided by Dr. Yasushi Okamura, the rapamycin-inducible 5-phosphatase system and compound A1 by Dr. Tamas Balla, and pseudojanin by Dr. Gerald Hammond.
This work was supported by National Institutes of Health grants R01NS055159 and R01GM093290 to T. Rohacs.
The authors declare no competing financial interests.
Sharona E. Gordon served as editor.
- AASt
arachidonyl stearyl
- ci-VSP
voltage-sensitive phosphatase from Ciona intestinalis
- diC8
- Kir
K+ inwardly rectifying
- Kv
voltage-gated K+
phosphatidylinositol 3,4,5-trisphosphate
phosphoinositide 3-kinase
phosphatidylinositol 4,5-bisphosphate
phosphatidylinositol 4-kinase
phosphatidylinositol 4-phosphate
phosphatidylinositol-specific PLC
- Poly-Lys
- PregS
pregnenolone sulfate
two-electrode voltage clamp
transient receptor potential
TRP melastatin
TRP vanilloid


text:
TDE x KitKat Chocolatory Card Holder & Lunar New Year Pouch
In stock
It's our pleasure to share our limited release collaboration with The Daily Edited. This gift set features both our card wallet & Lunar New Year silk pouch.
TDE x KitKat Chocolatory Card Wallet
Made with real leather in iconic KitKat red. Double cardholder with space for four cards & notes in the middle. Make it yours with up to four characters embossed in gold.
Make It Yours
Now: Personalise. We'll personalise it for you - just follow the steps. Allow up to 3-4 business days for your purchase to arrive.
Later: Add To Cart. We'll include a card for you to visit any The Daily Edited boutique or concession nationally and personalise your purchase.
- Made from 100% leather.
- Holds 4 cards & bank notes
- Dimensions: H 8cm x W 10cm
Made from 100% leather
Personalisation is done individually on each item, slight imperfections are normal
Use a damp cloth to clean and keep it away from chemicals, perfumes and oils
|TDE x KitKat Chocolatory Card Holder & Lunar New Year Pouch
Contains Milk, Soy and Wheat. May contain Peanuts and Tree nuts.
|Store in a cool, dry place.
|Know Your Portions
|8 serves per pack
|Nutrition & Ingredients
Sugar, Full Cream Milk Powder, Wheat Flour, Cocoa Butter, Vegetable Fat [Emulsifier (Soy Lecithin)], Cocoa Mass, Cocoa, Glucose Syrup, Emulsifiers (Soy Lecithin, 476), Yeast, Raising Agent (Sodium Bicarbonate), Salt, Flavours.
Product contains 70% Milk chocolate and 30% Wafer fingers.
Milk chocolate contains minimum 22% Cocoa solids, 25% Milk solids.
Nut Free: No
Gluten Free: No
Vegetarian: Yes
|Country of Manufacture

subset: commoncrawl


text:
Judge fines Johnson & Johnson $572 million in landmark case
An Oklahoma judge has fined Johnson & Johnson $572 million for fueling the opioid crisis in a landmark ruling that could open the doors for more lawsuits.
The AP's Sean Murphy had the news:
An Oklahoma judge on Monday found Johnson & Johnson and its subsidiaries helped fuel the state's opioid crisis and ordered the consumer products giant to pay $572 million, more than twice the amount another drug manufacturer agreed to pay in a settlement.
Cleveland County District Judge Thad Balkman's ruling followed the first state opioid case to make it to trial and could help shape negotiations over roughly 1,500 similar lawsuits filed by state, local and tribal governments consolidated before a federal judge in Ohio.
"The opioid crisis has ravaged the state of Oklahoma," Balkman said before announcing the judgment. "It must be abated immediately."
An attorney for the companies said they plan to appeal the ruling to the Oklahoma Supreme Court.
Before Oklahoma's trial began May 28, the state reached settlements with two other defendant groups — a $270 million deal with OxyContin-maker Purdue Pharma and an $85 million settlement with Israeli-owned Teva Pharmaceutical Industries Ltd.
CBS News quoted the judge:
In making his ruling, Judge Balkman said Johnson & Johnson bears responsibility for helping to fuel the state's opioid epidemic by aggressively marketing painkillers. Judge Balkman took about a month to make this judgement after a seven-week trial.
"We showed how the company repeatedly ignored warnings by the federal governments and its own scientific advisers about the dangers of its drugs and the risks of marketing its products the way it did," said Oklahoma Attorney General Mike Hunter during a press conference on Monday. "We have proven that Johnson & Johnson have built its billion dollar brand out of greed and on the backs of pain and suffering of innocent people."
A brief statement by Johnson & Johnson said, "Johnson & Johnson plans to appeal the opioid judgment in Oklahoma."
Known for infant shampoo and other family-friendly products, Johnson & Johnson has a lesser-known pharmaceutical division, Janssen Pharmaceuticals. Johnson & Johnson said its opioid products account for less than a percentage of the Oklahoma market, but the state of Oklahoma disputed that.
Reuters' Heide Brandes and Nate Raymond reported the fine was much smaller than expected:
An Oklahoma judge on Monday ordered Johnson & Johnson (JNJ.N) to pay $572.1 million to the state for its part in fueling an opioid epidemic by deceptively marketing addictive painkillers, a sum that was substantially less than investors had expected, driving up J&J's shares.
The state's attorney general had filed the lawsuit, seeking $17 billion to address the impact of the drug crisis on Oklahoma. It had been considered a bellwether for other litigation nationwide over the opioid epidemic.
"The expectation was this was going to be a $1.5 billion to $2 billion fine," said Jared Holz, healthcare strategist for Jefferies & Co. "$572 million is a much lower number than had been feared."
J&J said it would appeal the decision.


text:
DJI has partnership with National Geographic offers free rent of DJI Mavic Pro. Through September 5th to October 31st, you can rent DJI Mavic Pro for free for about two weeks. DJI said that they have hundreds of brand new Mavic Pros for free rent.
You can rent Mavic Pro at your local DJI Store. All you need just follow the steps below to rent the Mavic Pro for free:
- Fill out the form to sign up for a DJI Mavic Pro rental FREE.
Selection will be conducted in a random manner.
- Applicants will receive a confirmation email within one week of registering. Please show staff your confirmation email and pick up code at your local DJI store.
- The Mavic Pro is yours for two weeks! Tag your best Instagram photos with #MyMavicContest and #NatGeoTravel to enter the contest and National Geographic will announce the lucky winners. Find out more HERE.


text:
2 Line Inspirational Shayari In English
|2 Line Inspirational Shayari In English
Card houses are not built in the air,
Crying does not make you impaired,
Cheer up to win the world -e-friend,
No victors are made by one victory.
Man is alone in conflict,
The world is with him in success,
The one on whom this world laughed
He has created history.
When the world fingers you
When people get in your way
So do not give up in front of these difficulties
Prove yourself, winner, you flip over
Why do you get upset with people like children?
People go out saying anything to anyone
When things change, then people's words change.
|two line Shayari
Like happiness, the gum is also a custom
When darkness falls everywhere, the time comes to burn
Never show your dreams a world of reality
Try yourself in this world
Make your identity the most different in the world.
Do good things in the river
Otherwise, you will be trapped in the storm due to the burden of righteousness
Because the boat moves faster, reducing the load
|2 line Shayari
When steps get tired, encouragement
When everyone turns away, God supports.
Move to a new way of thinking
Touch your dream heights with courage
Which was confined till today in thoughts
Make those dreams come true.
Remember to break up freshly,
Do not get the throne without effort,
Find your destination in the dark,
Because firefly is never enchanted by light…
Will continue on the path,
I will become an expert on walking !!
Either floor will be found,
I will become a good traveler !!
|mirza ghalib shayari in hindi 2 lines
Man is alone in conflict,
The world is with him in success,
The one on whom this world laughed
He has created history…
Ordinary-looking people are the best people in the world: this is why God creates so many people ..!
Change over time
Or learn to change time
Don't force compulsions
Learn how to walk
The storm does not always work,
Difficulties do not always happen.
You will get your destination,
You try at least
|do line shayari
It is not difficult in some world,
You try at least
The dream will change into reality,
You try at least
If you want to get the destination then
Be your own leader,
They often go astray
Those who have support
Keep the courage, that will also come
Thirsty will also come to the sea,
Do not sit tired, traveler of the floor,
The floor will also be found…
And will also enjoy meeting.
Just remember when you start breaking
There are no throne-o-taj achieved without hard work,
Find your friends on the dark floor,
Because firefly is never a lover of light.
|2 line love Shayari in Hindi
If you do not get the floor, it is a matter of luck
It is a wrong thing to not even try.
Write this script on every milestone,
The destination is not met with failed intentions.
Let's adopt the character of the moon, we friends
Keep the stain with you and distribute the light.
|Inspirational Shayari In English
Hope and safety are two words to say
But this is the story of the world.
Tomorrow this dream will turn into reality,
Today, the dream is seen as a dream.
The one who is smiling must have brought up the pain
There will be a blister in the leg of what is going on,
A human cannot shine without struggle,
Whatever is lit will be lit in that lamp.
Witnesses of my hand's streak,
I have carved myself like a stone.
|very sad 2 line Shayari
Keep encouraging me … my crutches,
All the floors continued on the head of Sir Uni.
When you have come to play the character on the ground,
Let us do something to give an example.
|2 Line Inspirational Shayari In English
Read more Click Here >> Two Line Inspirational Shayari In Hindi
|2 Line Inspirational Shayari In English
Last Line: You all liked in our 2 Line Inspirational Shayari In English (Two Line Inspirational Shayari In English), you will say about our Shayari by commenting and if you visit the puzzle bar on our website, then you will definitely have Bell Icon Show at the bottom or either above you I will tell you to allow it, do not forget to press it, you will have the benefit of this. Whenever Shayari posts Quotes Dps, you will get the notification of that new post.


text: Craig Partridge wrote: > Hi folks: > > I'm currently working on a challenge related to how one modularizes > problems of looking up addresses, assigning (or learning of) addresses on > interfaces, at the like. > > To make this problem concrete, consider the requirement that a multihomed > host accept any datagram with any one of the host's IP addresses as the > destination. That presumes the weak host model; there are cases where that's not what you want (e.g., to group sets of tunnels to associate then with different virtual hosts/routers inside a real host/router). > If address control is done in the MAC/device driver layer, > then a datagram could trigger one lookup per interface. If address control > is done by the IP layer, then there are challenges of mapping IP addresses > to MAC layers/interfaces (required, as I recall, on outbound where the > source IP address usually must but that of the outbound interface). When is this 'address control' happening? There's already one lookup per packet on arrival to see if it matches one of the interfaces. On departure, that typically happens only for packets whose source addresses are unassigned. I thought RFC1122 talked about this in detail. > Somehow, in the back of my head, there's the idea that someone looked at > this problem in the early 1990s. There's text in the Router Requirements > RFC and Host Requirements sketching out the problems, but not espousing a > particular software modularity. Did anyone write something on this problem? I thought it implied that most of this happened at the network layer, not the link layer. Joe > > Thanks! > > Craig > > E-mail: craig at aland.bbn.com or craig at bbn.com -------------- next part -------------- A non-text attachment was scrubbed... Name: signature.asc Type: application/pgp-signature Size: 250 bytes Desc: OpenPGP digital signature Url : http://mailman.postel.org/pipermail/end2end-interest/attachments/20061023/4895c206/signature.bin


text: What does Shrimplick mean?January 29, 2020 AZ DictionaryShrimplick in Community Dictionary Male with a shrimp sized penis just who quickly completes in a single swing.(one pump chump) by Lynwood CowanReport definitionLink to this pageHow would you define Shrimplick?All the definitions on AZdictionary were written by people just like you. Now's your chance to add your own! Your Full NameYour definitionPost Definition


text:
Everything I Need To Know About Motherhood I Learned From My Mom
I used to wonder why my mom loved high-waisted jeans. I used to wonder why she took so much time getting ready to go on a date with my dad. I used to…Continue Reading
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I used to wonder why my mom loved high-waisted jeans. I used to wonder why she took so much time getting ready to go on a date with my dad. I used to…Continue Reading
Life with little people is amazing. It's also unquestionably hard. Minutes add up to countless hours of mental overload and emotional exhaustion.…Continue Reading
When you have a boy, you'll find underwear hanging off the dining room chandelier, and a crocodile suspended off the light switch by its…Continue Reading
Hey kid, My love for you can't be earned. I felt it long before you were born. I recognized it when I saw two pink lines. When I first beheld your…Continue Reading
It starts with an umbilical cord. Blood and nutrients pumping into an unborn babe, connecting the baby to its mother. The first thread of life. And…Continue Reading
Moms love trying to fold laundry while little bodies flail back and forth in the clean pile, rolling like pigs in a sty as their tiny arms whisk out…Continue Reading
Right now, I'm raising artists. Both of my children never sit down faster than when it is time to play with Play-doh, their chubby fingers adeptly…Continue Reading
Far too often, I let my feelings dictate my thoughts. When I pick up the same toys for the third time that day, knowing it won't be the last time .…Continue Reading
I'm going to be honest and admit that at times, I have been a terrible pet owner. Other times, I have managed to balance it all with grace; still…Continue Reading
During the week, I stay home with the kids while my husband works. I change their diapers in the morning, get them dressed (sometimes), feed them…Continue Reading
Want more stories of love, family, and faith from the heart of every home, delivered straight to you?


text:
Salient. Victoria University of Wellington Student's Newspaper. Volume 31 Number 2. March 12, 1968
Films — I love Lilith
I love Lilith
In the first issue of Salient I mentioned worthwhile films being shown by the Roxy/Princess, and the fact that some of these have been recommended to the management by the V.U.W. Film Society.
I am pleased to report our efforts have unearthed one movie of outstanding merit-Robert Rossen's "Lilith", a delicate, mysterious, extraordinarily beautiful film which by any standards must be considered one of the undisclosed masterpieces of the cinema.
"Lilith" comes as a revelation in the light of Rossen's previous work, excellent though some of his other films have been.
In the period 1937-1964 he wrote the scenarios for 23 films (including "They Won't Forget", "The Roaring Twenties", "A Walk in the Sun", and "The Treasure of Sierra Mad re"), and directed ll of these.
"The Hustler" (1962), Rossen's best film after "Lilith", was screened by the Film Society last year.
It is undeniably a very skilful piece of work, both in the pyrotechnics of the billiard scenes and the subtle observation of the Piper Laurie-Paul Newman relationship.
Nothing in Rossen's output, however, has prepared us for "Lilith", a film that is in so many ways superior to the rest in conception and technical execution.
This story of the love of a sanitorium orderly for a gifted patient is a blend of fragile, baroque lyricism and those Gothic qualities that imbue every foot of "The Night of the Hunter".
It is a fairy tale vision of the beauty and destructiveness of madness, presented with sympathy and insight.
The bewitching dream of this nightmare world is depicted in a stream of alluring, hypnotic visuals-"the enchantment of horror lurks beneath the images that beguile and bewilder the rational, unsuspecting viewer.
Eugene Shuftan's monochrome photography, ranging from high contrasts to muted whites and greys, fits perfectly the intermingling of reality and symbol.
Rossen's way in "Lilith" is stealthily quiet (lingering close-ups—a slow, constant internal rhythm), but with an underlying tension that occasionally errupts on to the screen as outbursts of frenzied camera movement and cutting-like in the jousting sequence at the 'carnival' (an excursion into the 'real' world that makes reality look like a madhouse) and the use of the hand-held camera near the end, an effective indication of Vincent's imminent disintegration.
Kenyon Hopkins' music is quite weird, seeming to be unrelated to what is happening in the film, yet at the same time creating its own aura of suspense and ambiguity.
Warren Beatty does well as Vincent, although he has not yet (even in "Bonnie and Clyde") been able to discard the self-consciousness that is characteristic of much of his acting.
But as C. A. Lejeune once said of Stewart Granger, "he doesn't need to bother about being a bit out of practice in acting, he looks so scrumptious".
Kim Hunter, Jessica Walter, and Gene Hackman lend outstanding support.
Jean Seberg, whose debut in "St. Joan" (1957) was one of the classic flops in films, and who later starred in Godard's "Breathless", gives under Rossen's direction an outstanding performance, even better than her too brief appearance in "A Fine Madness".
She relates in "Cahiers du Cinema" how Rossen and his team were sadly disappointed at the reception given the film by critics and public alike.
Only an enthusiastic reception by the "Cahiers" group, and a later, grudging acknowledgment by "Sight and Sound", have rescued "Lilith" from total obscurity.
Rossen was already seriously ill when he made this, his last film, and one can sense how much of himself and his life went into its making.
"Lilith" is quite unlike any of his other films In its own haunting way it is quite unlike any other film ever made.
—Rex Benson.


text:
Abel's Tackle Box- Islamorada, Florida Keys
Listed in Florida Keys Alcohol, Grocery, Food, Drug, Liquor, Wine, Beer stores, Florida Keys Fishing Bait, Tackle & Supplies, Florida Keys Fishing Charter Boats & Commercial Fishermen, Florida Keys Marina
84341 Old Overseas Hwy. Islamorada, FL 33036
Description: Abel's Tackle Box is a bait shop in Islamorada, Florida Keys. Read more...


text:
St. Lucia's department of Health and Wellness has received confirmation of the association of H1N1 Influenza, in 1 of the recent respiratory related deaths among males.
This confirmation was received from the Laboratory at the Caribbean Public Health Agency (CARPHA) in Trinidad.
H1N1 influenza has also been confirmed in other persons who only developed mild flu like illness and who recovered completely with treatment.
H1N1 influenza (also referred to as Swine Flu) is caused by a virus. This virus was the cause of the influenza pandemic in 2009.
Since then, it has become one of many respiratory viruses which circulate each year during the flu season. Therefore, its confirmation is not unusual.


text:
Come to the Northwoods for your next vacation getaway and stay at the beautiful Chanticleer Inn on the Chain of 28 Lakes! With shared amenities like docks, a game room, a playground, a tennis court, and pickleball and basketball, you'll have all the entertainment you'll need at your fingertips. This dog-friendly suite has a refrigerator, a coffee maker, and a flatscreen TV, as well as free WiFi. Come experience true Northwoods living!
What's nearby:
The Chanticleer Inn has an on-site dining room, bar, and even boat rentals, so you can travel by water to downtown Eagle River (three miles away). National events like the Upper Mississippi Steamboaters Meet and Pond Hockey National Championships are hosted here, perfectly blending accommodations and entertainment. The Chanticleer Inn is located close to the World Championship Derby Complex as well as the Snowmobile Capital of the World trail system.
Things to know:
Free WiFi
Dogs welcome
This property is managed by Vacasa Wisconsin LLC.
You must be 21 years or older to rent this property.


text:
This Steinway Model O Grand has a Traditional case and is finished in High Gloss Ebony. This piano had a new soundboard installed 15 years ago.
Watch videos of some Restored Model O pianos: Steinway O videos
Testimonials of Steinway Purchases: Watch Here
|Serial #:149802
|Length: 5'10"
|Veneer:High Gloss Ebony
Once your piano is delivered, we give you 30 days to play it and make sure everything is exactly as you wish. If not, simply let us know, and we will pick up the piano and give you a full refund. Additionally, when you buy a restored piano from Lindeblad you can, at any time in the future, exchange that piano for another of our pianos and apply the full price you paid to the exchange.
Cleveland, OH From our standpoint, it was everything we would want as a customer. Watch Video
East Orange, NJ | 1912 Steinway D I went to the factory. I saw what they do—the work they do. My Steinway D was a complete rebuild, and it's just incredible. I've got to find some more superlatives: "incredible, great, amazing!" Watch Video


text:
– Hi everyone, Kristina here. Welcome to Watercolor
Week, or welcome back to Watercolor Week. Yesterday I started things off with a fun color swatching and testing
out some new watercolors, so if you want to check that out, look for the link. I think it's in that corner (laughing). It'll come down and you can
click over to that video. So for today's video,
I thought I would try giving myself a challenge, and I mentioned I would give myself a
challenge in last week's video, so here I am, this is the challenge. I am going to be painting or watercoloring with only one color. We're gonna see what
happens, kind of exploring the different contrasts
you can get by using a more saturated color versus something that's more tinted or pale. I'm gonna experiment
with that a little bit, so let's get going! I've decided to use Mama
Elephant's Animal Parade stamp set for this card. I thought the different images
would be really fun to paint. I was specifically looking for a stamp set that had a lot of open images to paint because I want to challenge myself to really explore the
different shades of one color. So I'm using some Arches
cold press watercolor paper and I'm stamping the
images and diversifying onyx black ink. This particular ink is
very, very stark black, so that's what I love when
I'm doing black stamping and it's also waterproof,
so this is gonna be great for painting over the
top, it's not gonna smear or smudge or bleed once I start adding my watercolor paint on top. And because this watercolor
paper has a little bit of texture to it, I wanted
to stamp it twice in a row to make sure that those stamped lines really penetrate the paper. Sometimes the texture
will prevent the images from stamping completely,
so its always best to stamp 'em a couple times. And I did the same
thing with this greeting from the stamp set. You have a few different greeting options in the stamp set. I chose the one that says happy birthday, and I did end up stamping it twice because one time was enough, but I did stamp it twice eventually. So I'm going to be using
the watercolor paints that I showed you in yesterday's video and I've chosen the color C33. I thought that would be a nice, deep blue and then I would have
some good opportunity for the background and also
getting some deep colors kind of in the shaded areas
of each of these images. So as I color these, I'm gonna start. Well, this first one I'm
starting with the dark and then I'm gonna work
kind of lighter to that, but then I realized that a better method was actually to start with a medium shade and let that dry and then come in with the darker shades to add shading. I'm not sure why, this
is the first time I went straight to the darkest color. I'm not sure why I did,
but I did figure out with the fox and also the
other two little characters that it was easier to
paint them if I started with kind of a lighter tone and then added the darker shades on top. That penguin kills me, it's so cute. Anyway, so I'm adding
these lighter shades in and thinking about where
the shadow will be. I decided to have the light
coming from the right side of the scene, so the
backside of these animals will be in shadow. So I'm painting with that light in mind and I'm making sure that
I add the darkest shades on the backside of the animals. It's interesting, as I was painting this, I thought it would be a lot more difficult than it actually was. I think I found it almost
a little bit easier because I didn't have to
worry about changing colors and deciding which colors
to use and which colors would be best in shadow
and things like that. I found the restriction of only one color, it was a challenge because I had to think about how I was going to
use it and yet it was also a little bit easier, if that makes sense. I think it would be interesting
to take this challenge one step further and possibly paint your entire scene in black and white just using a black paint or even just a dark brown and
then once everything's dry go back each of the sections
with very pale tints of different colors and just
add a little bit of color on top and I think that would
look really, really cool. You might have a little bit of a look like those old black and white photos that they then tented with markers or with paints. I think it would kind of look like that. That'd be a fun technique. So I might have to do that. So I think that would
be really interesting. So I finished up all of the characters and then I went onto this banner, and to just to keep it simple,
I did some darker shades at the bottom of the
banner and near the pole and then faded it out from there
and I did just a little bit of shading on that pole
so that it was finished. So I'm gonna work on the background and I'm taping it down
to my cardboard here so that I can do a lot of painting, but I realized before
I paint the background, I need to determine where
each of these images will be in the scene so that I
know how to paint the ground. So I used three of the dyes according, with the stamp set, but
I didn't use the dye for the bear with the
banner because the dye wasn't matching up completely. I think when I mounted the
stamp to my Misti tool, it moved the banner a little
bit, so the dye itself doesn't match up, but I kinda
figure that that was user error on my part, so I
just used some scissors and trimmed out that
image the best I could, just cutting out around the edges and fussy cutting everything. So I positioned all of those
images on the background piece where I thought
roughly, where they would be and I determined where I
wanted the ground to be. Put a little tick mark
with a pencil on the tape on the side, and then
used a ruler to draw in that horizon line. I wet the entire piece
of watercolor paper. By the way, this is that same Arches cold press watercolor paper. I wet that down so that
I have a nice wet surface to work on, and then I
started dropping in some of this color, and I'm
bringing it in mostly at full strength because
I really want the color to wick away from those edges and have a lot of contrast. I wasn't sure how I felt
about this sky background at first, but I really
love how it turned out. So it was one of those moments for me when I was like oh yeah,
trust the watercolor, trust the watercolor,
it will come through. As long as you're thinking
to keep some white space free so that it looks like clouds,
I think skies are so fun to paint and they're almost foolproof. I can't say they're completely foolproof because I've painted some really bad skies in the past, so I can't
say that they're completely foolproof, but just remember
to keep some white space in your sky and it will
probably turn out beautiful. So I just painted a
dark blue at the bottom. And to me right now, this
look like an ocean scene. I didn't even think that it
would with all of the blue, but it totally looks like an ocean scene, so I'm gonna bring in
those images once more and put 'em in the
places where I want them, and then I'm gonna paint in
some slightly darker areas for shadows underneath each of these and still looks like ocean
to me, but that's okay. The whole entire card is
blue, but it'll make a little more sense once things are put together. So I placed all those images
back on the card front or on this background and I
placed it in my Misti tool and then I stamped some additional images from the stamp set. These were images that
were a little bit too small to cut out on their own, and
so I thought it would be best to have them on the actual background. So I've got three little music notes and also some juggling
balls for above the penguin. So I stamped those images
in the same black ink and then I brought in some white gouache. This is the one area of the card where I veered away from just
that one watercolor paint and that's because I just
wasn't thinking ahead, and if I would have been thinking ahead, I would have figured out
the spacing of these animals before painting the background,
stamped those images and then used a bit of liquid
masking fluid over these so that I could preserve
the white color underneath, but I wasn't thinking that far ahead, and so I did end up
having to use a little bit of an opaque white paint just to get some of the brightness
back in those images. So I prepped my card base out of some Neenah Classic Crest
solar white card stock. This is the 110 pound version. I just squared that up
five-and-a-half to create a a side-holding card and
I put some foam tape on the back of my
painted background piece. And the last thing to
do is put some foam tape on the back of all of
these really cute images and place them onto the card. So I'm making sure I have
them in the right spot, right above those shadow
spots that I added earlier and I'm also being very
conscious of where that penguin is beneath the balls. I want to make sure that
they're on the right position and that pretty much
finishes the card for today. I thought this challenge was a really interesting take on it. It was still a challenge,
but I had a lot of fun and I found it really freeing to only use one color of paint. Thanks so much for watching today's video. I hope you guys enjoyed this challenge and if you decide to take the challenge, tag me on Facebook or
on Instagram or whatever because I really want to see your take on this particular challenge. I think it'd be really, really fun. Onscreen right now are two
other watercolor videos for you to check out. Don't forget to give
this video a thumbs up if you enjoyed it and
share it with a friend if you think they would enjoy it, too. Thanks so much for watching and I'll catch you guys in the next video.


text:
The Taipei Fubon Braves bounced back from a tight loss in their last game as they repeated over the Macau Black Bears, 106-93, Friday night at the Taipei Heping Gymnasium. It was a more comfortable win for the newest team in the ASEAN Basketball League as they found a way to throttle the usually explosive three-point shooting of the Black Bears.
The turning point of the match happened in the third quarter where Mikh McKinney was called for his fifth personal foul on a Tsai Wen-Cheng and-one opportunity. After committing four fouls in the first half which included a technical foul, Macau head coach Charles Hantoumakos risked playing McKinney to open the third. It worked out at first as last season's leading scorer combined with Kenny Manigault to keep Macau in the game amidst the fiery shooting of OJ Mayo but McKinney was disqualified from the contest with still 3:21 remaining in the period.
To make matters worse, Hantoumakos was also called for a technical foul, which was quickly followed by a mini-run for the Braves to establish a 15-point lead, 77-62, after two Charles Garcia free throws.
The Braves built their biggest lead of the match at 18 points, 91-73, after a Tsai basket. Jenning Leung tried to power one last rally for Macau as he heated up from downtown to inch his team close. Two free throws from the guard got them to within five, 97-92, with 1:51 left in the game.
However, a dunk from Sim Bhullar and a triple from OJ Mayo slammed the door on the Black Bear's uprising to seal Taipei Fubon's second win in three games.
Garcia was the Best Player of the Game with 25 points, seven rebounds, five assists, three steals, and two blocks as he shot 9 of 12 from the field. Mayo led them in scoring with 26 points with Bhullar adding 21 markers with only a single miss in 10 shot attempts. Tsao led the Braves' locals with 13 points along with five rebounds and six assists.
Taipei Fubon banked on their might inside as they dominated inside scoring 58 to 36 while also showing better chemistry with a 27 to 16 advantage in assists as Macau's offense took a turn for the worse late in the game without their best creator in McKinney.
Manigault led Macau in scoring with 31 points, which is the third time he got to at least 30 in his first four ABL games. Leung added 16 points to their cause while Brandon Edwards chipped in 15 points and seven rebounds, which is 13 short of the caroms he picked up in their first meeting with the Braves.
The Braves are in for another quick turnaround as they face Hong Kong Eastern on the very next day while the Black Bears will try to score a bounce-back win against the neighboring Formosa Dreamers as they continue their road tour of Sunday.


text:
Sheff Wed – Fulham Match Prediction | 22-09-2019 10:00
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With that in mind, a 1-1 draw is our correct score pick here. We dont expect that to change as the Cottagers chase back to back wins against Wednesday for the first time since the early 80s. Sheffield Wednesday manager Garry Monk arrived at the perfect time, as he has never lost a Championship match against Fulham (P4 W1 D3 L0).
With this, we always try to provide gratis all the tools you need to bet responsibly and we hope winning! Under you found the prediction processed by our proprietary software and reviewed manually by our editors of the football match Sheffield Wednesday-Fulham, match of the championship England Championship. Even stats, standings, news and information on the football league England Championship and on teams Sheffield Wednesday and Fulham.The prediction 1x2 or under/over or goal/nogoal, with odds of bookmakers comes complete with the correct score.
It has to be noted that he scored 23 goals in the English second tier in the 2015/2016 season. Winnall helped Barnsley eliminate Walsall in the 2015/2016 League One semis as he scored a brace in the first leg. He provided 8 assists for his teammates during the season and he finished the campaign as the third highest goalscorer in the league, just behind Nicky Ajose and Will Grigg.
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The Cottagers, on the other hand, played out a 1-1 draw with WBA in their last league fixture, with the team extending their winless run in the English second tier to three games. Anyhow, a close encounter is on the horizon and we believe that there is a big value in betting on draw. Serbian attacker Aleksandar Mitrovic has been scoring goals for fun in all competitions of late and we predict that the former Newcastle United man is going to prove a handful for the opposition defenders.
They have had little problem going forward especially with Aleksandar Mitrovic already in great form but at the back Parkers men are lacking. The Cottagers have conceded in four of their last five Championship matches, while they have failed to keep a clean sheet across their away trips this term. Fulham have failed to win their last four games, failing to keep a clean sheet in each of them.
Wednesday are at home to Fulham, with the Cottagers aiming to return to form and boost their own promotion aspirations. Garry Monk took Sheffield Wednesday to a victory in his first game in charge, but will he repeat the trick in this clash? The former Birmingham boss has made a bright start to life with the Owls, but this weekend brings a substantial test.
Coach Slavisa Jokanovic and his men are eager to return to winning ways and we predict they will go all guns blazing at Sheffield Wednesday when the two teams take on each other at Craven Cottage this weekend. Fulham impressed in the 2016/2017 Championship campaign, but they eventually failed to secure promotion to the Premier League. The Cottagers, though, have yet to record a win in the new Championship campaign, with the team sharing the spoils with the likes of Norwich City, Reading and Leeds United in the first three games of the season. It is well known that Fulham are a real force to be reckoned with at home and we believe that the time has come for the capital club to record their first win of the season. The Owls have been struggling in front of goal at the start of the season and they are widely expected to return home empty-handed.
He went on to score his first goal for Barnsley on 23 August 2014 in a 4-1 win over Gillingham. He was on loan at Burton Albion, Hereford United, Inverness CT and Shrewsbury Town and in the 2013/2014 campaign he signed a contract with Scunthorpe United. Sam Winnall scored 23 goals in 45 appearances for the club and, as a result, secured a lucrative move to Barnsley in 2014.


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A cropping systems research manager at the Foundation for Arable Research, who has worked to support cropping farmers in the areas of economic and environmental sustainability since 2010, has been bestowed the organisation's top honour for 2019.
FAR CEO Alison Stewart says awarding Diana Mathers the 2019 Researcher of the Year award recognises the significant impact she's had in her areas of work.
"Working with growers around the adoption of new technologies is a current focus for Diana, who helped to test and calibrate DIY Quick N testing kits as part of the Ministry for Primary Industries' Sustainable Farming Fund project Nitrogen - Measure it and Manage it.
"The results of this project have confirmed that Quick N testing strips are an efficient and inexpensive tool to help growers understand exactly how much nitrogen is available to their crops at any given time, and therefore, how much they need to apply to reach target yields."
The judges' citation for awarding Diana the accolade says she has been, and continues to be, actively involved in a number of national and regional research projects, which focus on ensuring high quality information is made available to farmers in a format that is easy to understand and apply.
"She's led a number of environmental and biodiversity projects such as the Good Management Practices For Winter Dairy Grazing On Arable Cropping Land, the Building Better Biodiversity on Arable Farms, and the Trees for Bees projects.
"Diana was instrumental in developing a Farm Environment Plan template for cropping farms, which was released in 2015 and has been extremely well received by growers and regional councils. Its success comes from Diana's determination that it should able to be completed easily by the landowner and form a valuable information base for the farm business."
The FEP is now being delivered to farmers through ongoing workshops and will become an increasingly more important tool for growers as the Government increases its environmental compliance requirements, say the judges.
"Diana has also had significant involvement in benchmarking Overseer for crop farms and delivering information on nutrient management on crop farms to arable and vegetable crop farmers through a series of nutrient management workshops across NZ.
"In all these projects she's focused on using data collected on-farm and farmers' own records to inform improved on-farm decision making."
The judges say Diana also provides valuable input to a number of regional council groups throughout NZ where she helps to ensure policy is practically aligned with farm practices, particularly in relation to nutrient management. "She's a strong advocate for the implementation of good farm management practices and works closely with growers to support their adoption and uptake.
"This award is fitting recognition of Diana's empathy with growers and her determination to help them understand and negotiate the increasingly complicated issues associated with on-farm environmental management."


text:
Price: £3.75
Quantity: 1 available
Book Condition: Average
. lvi + 380 pages. A little wear and tear to extremities; a very few minor instances of fairly neat red pen underlining.
Title: The Epistles of St John: The Greek Text with Notes and Essays
Categories: Commentaries, Greek and Hebrew,
Edition: Third Edition
Publisher: Cambridge, MacMillan and Co: 1892
Binding: Cloth
Book Condition: Average
Item: 1.00 Item
Seller ID: 014809
Keywords: Epistles, John, Greek New Testament, Bible, Commentaries, Christianity,


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The Federal Government is finalising arrangements to raise about N25 billion through its third green bond issuance.
This was revealed by the Minister of Environment, Dr. Mohammad Abubakar, during a courtesy visit to the Nigerian Stock Exchange (NSE) in Lagos.
Nigeria had issued its maiden green bond of N10.69 billion in 2017 and followed this with second issuance of N15 billion in 2019.
Abubakar said the government was planning a bigger issuance under the third series to accommodate more projects as it continues to seek ways of improving sustainable growth.
"We want to embark on more projects that are climate sensitive that will help in eliminating climate problems," Abubakar said.
He outlined that the net proceeds of the green bond will be used to finance power, aforestation, deforestation, water, energy and agriculture among other projects saying that government was moving towards smart agricultural production to produce more foods with minimal impact on the environment.
According to him, the third green bond will further demonstrate government's commitment to the reduction of greenhouse gas emissions by 20 per cent unconditional and 45 per cent conditional by 2030, as outlined under the Paris Agreement signed on September 21, 2016.
He pointed out that green bond and climate change are the defining issues of today and the future.
"Green project are what we need today because of the climate issue we as a country is facing, we need to change our environment through solid waste management, turning waste to wealth, wasting to resources, these are all the things we need green bond to finance," Abubakar said.
The Federal Government had in December 2017 launched Nigeria's maiden sovereign green bond as part of efforts to diversify government revenue and deepen the domestic capital market.
Nigeria's first sovereign green bond was oversubscribed by about N100 million as investors staked N10.791 billion on the N10.69 billion maiden bond.


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Solo Mt. Willard adventure day 2/16/20 & being honest with yourself
Hey folks! Happy Tuesday. I'm currently writing this post from my couch, drinking turmeric tea with vanilla hemp milk + cinnamon + local honey, and looking up every so often watching the snow fall outside. Sunday was a "solo" frolic day to Mt. Willard. I quoted solo because I was very much not alone, me […]Read More


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Prince Of Wales, Gorton - pub details
|[image 2]
Address: 410 Abbey Hey Lane, Gorton, Manchester, Lancashire, M18 8RQ [map] [gmap]
Tel: 0871 951 1000 (ref 9790) - calls cost 10p per minute plus network extras
Gorton (0.3 miles), Ryder Brow (0.9 miles), Belle Vue (0.9 miles)
Pub facilities/features:
- Wireless internet access (provided by The Cloud)
Are you the Licensee? Click here. ** SPECIAL Royal Summer Sizzler offer! **
> Current user rating: 6.7/10 (rated by 3 users)
> Hotels nearby: Search
> Log in to rate this pub or upload a picture of it.
> Post a comment > Mail a friend
> Post a comment > Mail a friend
other pubs nearby:
Royal Oak, Gorton (0.2 miles), Angel, Gorton (0.4 miles), Vale Cottage, Gorton (0.4 miles), Band Stand, Gorton (0.4 miles), High Bank Inn, Openshaw (0.4 miles) - see more nearby pubs
user reviews of Prince Of Wales, Gorton
please note - reviews on this site are purely the opinion of site visitors, so don't take them too seriously.
|It's been a few years since I've been there. I enjoyed a cold boddies back then.
harleymike - 25 Oct 2008 21:40
|Great pub - Deb, Bobby and gang doing a great job.
Jim X - 19 Sep 2004 06:39


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Hello loyal followers of Jasper's blog. It's Jasper's mummy here. I am afraid to say that Jasper has had an accident. He was run over this morning by a four-wheeled drive car and trailer outside our house. He was naughty and got out of the property and was chasing a car. He dragged under both the car and trailer. You will be glad to know that after an emergency trip to the vet (on a public holiday, so you can imagine how much the vet bill is) he has been pronounced as being OK. We were all very worried about him. As you can see from the photos he did not look very well. There is a lot of grazing around his groin and he had a few stitches to close up the cuts on his legs. He lost half a front tooth. But for the for grace of god it could have been a lot worse and our family would have been devastated if anything had happened to Jasper because we all love him dearly. He is such a good dog, so think of Jasper and hopefully he will be back to his usual self soon. Love Jasper's mummy
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The Most Important Life Lesson Ever Learned
There are many lessons that we learn from God's Word – The Bible. We can make ourselves dizzy just trying to remember them all, let alone putting them into practice.
At times, it is a good idea to just step back and take a look at what is truly fundamental in our relationship with our Lord and Savior Jesus. Anna B. Warner did exactly that when, in 1860, she wrote these words: "Jesus loves me – this I know, for the Bible tells me so; little ones to Him belong – they are weak, but He is strong." We of course know this as the first verse to that children's hymn – Jesus Loves Me.
These words are simple, direct, and fundamental to our relationship with our Lord and Savior. So, the next time that you have the opportunity to sing this hymn, do so with the gusto and innocence of the Child of God that you are.
Know that you are loved in spite of what you may have done in the past, what you are doing, and what you might do in the future. Allow that love to bring you to the confession of your sins and to guide you this day and every day that follows.
Scripture: Ephesians 3:14-18