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Widespread Dysregulation of Peptide Hormone Release in Mice Lacking Adaptor Protein AP-3
The regulated secretion of peptide hormones, neural peptides and many growth factors depends on their sorting into large dense core vesicles (LDCVs) capable of regulated exocytosis. LDCVs form at the trans-Golgi network, but the mechanisms that sort proteins to this regulated secretory pathway and the cytosolic machinery that produces LDCVs remain poorly understood. Recently, we used an RNAi screen to identify a role for heterotetrameric adaptor protein AP-3 in regulated secretion and in particular, LDCV formation. Indeed, mocha mice lacking AP-3 have a severe neurological and behavioral phenotype, but this has been attributed to a role for AP-3 in the endolysosomal rather than biosynthetic pathway. We therefore used mocha mice to determine whether loss of AP-3 also dysregulates peptide release in vivo. We find that adrenal chromaffin cells from mocha animals show increased constitutive exocytosis of both soluble cargo and LDCV membrane proteins, reducing the response to stimulation. We also observe increased basal release of both insulin and glucagon from pancreatic islet cells of mocha mice, suggesting a global disturbance in the release of peptide hormones. AP-3 exists as both ubiquitous and neuronal isoforms, but the analysis of mice lacking each of these isoforms individually and together shows that loss of both is required to reproduce the effect of the mocha mutation on the regulated pathway. In addition, we show that loss of the related adaptor protein AP-1 has a similar effect on regulated secretion but exacerbates the effect of AP-3 RNAi, suggesting distinct roles for the two adaptors in the regulated secretory pathway.
Published in the journal:
. PLoS Genet 9(9): e32767. doi:10.1371/journal.pgen.1003812
Category: Research Article
doi: 10.1371/journal.pgen.1003812
The regulated secretion of peptide hormones, neural peptides and many growth factors depends on their sorting into large dense core vesicles (LDCVs) capable of regulated exocytosis. LDCVs form at the trans-Golgi network, but the mechanisms that sort proteins to this regulated secretory pathway and the cytosolic machinery that produces LDCVs remain poorly understood. Recently, we used an RNAi screen to identify a role for heterotetrameric adaptor protein AP-3 in regulated secretion and in particular, LDCV formation. Indeed, mocha mice lacking AP-3 have a severe neurological and behavioral phenotype, but this has been attributed to a role for AP-3 in the endolysosomal rather than biosynthetic pathway. We therefore used mocha mice to determine whether loss of AP-3 also dysregulates peptide release in vivo. We find that adrenal chromaffin cells from mocha animals show increased constitutive exocytosis of both soluble cargo and LDCV membrane proteins, reducing the response to stimulation. We also observe increased basal release of both insulin and glucagon from pancreatic islet cells of mocha mice, suggesting a global disturbance in the release of peptide hormones. AP-3 exists as both ubiquitous and neuronal isoforms, but the analysis of mice lacking each of these isoforms individually and together shows that loss of both is required to reproduce the effect of the mocha mutation on the regulated pathway. In addition, we show that loss of the related adaptor protein AP-1 has a similar effect on regulated secretion but exacerbates the effect of AP-3 RNAi, suggesting distinct roles for the two adaptors in the regulated secretory pathway.
In contrast to most proteins which undergo immediate and unregulated secretion after biosynthesis, proteins destined for regulated release require sorting into LDCVs, but the mechanisms responsible for sorting to LDCVs and indeed LDCV formation remain poorly understood. LDCVs bud from the trans-Golgi network (TGN) [1], [2], [3], and previous work has suggested that lumenal interactions such as the aggregation of granulogenic proteins drive their formation [4], [5]. Indeed, sorting to LDCVs has been suggested to occur by default, with proteins destined for other organelles removed during the well-established process of LDCV maturation [6], [7]. However, direct analysis of budding from the TGN has demonstrated the sorting of regulated from constitutive cargo at this early step, before maturation [3]. In addition, LDCV membrane proteins such as carboxypeptidase E and sortilin have been proposed to serve as the receptors for soluble cargo [8], [9]. In contrast to these lumenal and membrane interactions, the cytosolic machinery involved in sorting to LDCVs and LDCV formation has remained poorly understood.
Several membrane proteins contain cytosolic sequences that direct them to LDCVs. For example, the neuronal vesicular monoamine transporter VMAT2, which fills neurosecretory vesicles with monoamine transmitter, depends on a conserved, C-terminal, cytoplasmic dileucine-like motif for sorting to LDCVs [10], [11], [12], and the LDCV membrane protein IA-2β (phogrin) relies on a remarkably similar sequence [13]. Since the requirement for a cytoplasmic motif suggested an interaction with cytosolic sorting machinery, we recently used VMAT as a reporter to screen by RNAi in Drosophila S2 cells for proteins involved in biogenesis of the regulated secretory pathway, identifying multiple subunits of the heterotetrameric adaptor protein AP-3 [14]. Loss of AP-3 results in mis-sorting of VMAT in both S2 and mammalian neuroendocrine PC12 cells, dysregulated secretion, a reduction in the number and alteration in the morphology of LDCVs [14]. Indeed, AP-3 RNAi disrupts sorting at the TGN and impairs the concentration of membrane proteins such as synaptotagmin that are required for regulated release [14]. However, most work in mammalian cells has focused on the role of AP-3 within the endolysosomal pathway, in trafficking from early endosome to lysosome.
Consistent with a role in the endolysosomal pathway, mocha mice (Mus musculus) lacking AP-3 show defects in lysosome-related organelles (LROs) such as melanosomes, platelet granules and synaptic vesicles [15], [16]. In addition to the abnormal coat color and a bleeding diathesis, however, the animals exhibit perinatal lethality, hyperactivity, head tilt, seizures and reduced fertility [17], [18], [19], and it has remained unclear whether a defect in the endolysosomal pathway can fully account for the severe phenotype. Importantly, previous work in S. cerevisiae has indicated a primary role for AP-3 in the biosynthetic pathway [20], [21], [22]. We have thus now used mocha mice to investigate the physiological role of mammalian AP-3 in regulated protein secretion.
The mocha Mutation Dysregulates Release by Adrenal Chromaffin Cells
To determine whether the loss of AP-3 in vivo affects regulated secretion, we cultured adrenal chromaffin cells from control and AP-3-deficient mocha mice, measuring the release of endogenous secretogranin II (SgII) in response to the nicotinic agonist DMPP [23]. Western blotting of the medium indicated that DMPP stimulates SgII secretion from control cells, but SgII was undetectable in the medium of mocha cells (Figure S1). However, the substantial reduction in cellular SgII content of mocha adrenal glands [14] and of cultured mocha adrenal chromaffin cells (Figure S1) made it difficult to determine whether the cells simply do not contain and release enough SgII to detect, or actually exhibit a defect in regulated release.
To assess regulated exocytosis by chromaffin granules, we used total internal reflection fluorescence (TIRF) microscopy to image neuropeptide and LDCV membrane protein reporters fused to the superecliptic pHluorin [24], [25]. The pHluorin is a modified form of green fluorescent protein (GFP) with increased sensitivity to protons that is quenched at the low internal pH of LDCVs and therefore increases in fluorescence with exposure to the higher external pH on exocytosis. Since neuropeptide Y (NPY)-pHluorin has been shown to undergo regulated exocytosis [26], we used lentiviral transduction to express this fusion protein and monitored individual exocytotic events at the plasma membrane of living chromaffin cells. In the absence of stimulation, control cells showed very few spontaneous fusion events over 90 s of imaging, but AP-3-deficient mocha cells exhibited substantially more (Figure 1A–1B). Both control and mocha cells showed a clear increase in exocytosis in response to stimulation by DMPP, but the extent of stimulation relative to baseline reveals an ∼70% reduction in mocha cells compared to controls (Figure 1B). To extend these findings to an LDCV membrane protein, we transduced chromaffin cells with a virus encoding VMAT2-pHluorin, with the lumenal location of pHluorin enabling detection of release events [27], [28]. Similar to NPY-pHluorin, VMAT2-pHluorin also showed a clear increase in basal, unstimulated exocytosis in mocha relative to control cells, and this again resulted in an ∼75% reduction in stimulated release (Figure 1C). The loss of AP-3 thus dysregulates the release of LDCVs as monitored using either soluble or membrane cargo.
The role of AP-3 in defining LDCV membrane protein composition has suggested that loss of the adaptor results in mixing of constitutive and regulated secretory pathways [14]. The resulting constitutive secretion (measured biochemically in the case of endogenous SgII [14]) could thus reflect either the spontaneous release of constitutive secretory vesicles which contain mis-sorted LDCV cargo but no dense core, or the dysregulated release of LDCVs. To distinguish between these possibilities, we again took advantage of TIRF microscopy. Release from constitutive vesicles without a dense core should yield events with reduced amplitude relative to controls, whereas dysregulated LDCV fusion should yield events with a size similar to controls. Analyzing individual exocytotic events, we observed that the basal as well as stimulated events observed in mocha cells have an amplitude similar to those observed in controls (Figure 1D). The heightened basal secretion observed in mocha cells thus apparently results from dysregulated exocytosis of LDCVs rather than the fusion of constitutive secretory vesicles without a dense core.
Dysregulated Peptide Hormone Release by Pancreatic Islet Cells from mocha Mice
The increased basal exocytosis of NPY and VMAT2 from mocha chromaffin cells is consistent with earlier experiments using RNAi in PC12 cells [14], but does the loss of AP-3 also dysregulate release from other neuroendocrine tissues? Pancreatic β cells store insulin in LDCVs morphologically and biochemically similar to chromaffin granules [29], [30]. To assess the regulated release of insulin in vivo, we measured baseline serum insulin levels while fasting and stimulated levels 15–20 minutes after intraperitoneal injection of glucose (Figure 2A). Before glucose administration, we observed a slight reduction in the serum insulin levels of mocha mice relative to controls, but this did not reach significance. After glucose administration, the control animals showed a clear increase in serum insulin but the mocha mice did not (Figure 2A), suggesting a failure of regulated release.
To assess the consequences of dysregulated insulin release for carbohydrate metabolism, we also measured blood glucose. Surprisingly, we observed no clear difference in fasting blood glucose levels between control and mocha mice (Figure S2A). After glucose administration, mocha animals show an increase in glycemia but less than controls (Figure S2A), a surprising effect in light of the lower serum insulin levels which would have been expected to impair glucose tolerance (Figure 2A). The effects of the mocha mutation on blood glucose levels thus do not correlate with the effects on insulin. However, blood glucose levels reflect the combined action of multiple circulating hormones, many of which may be affected by the loss of AP-3. Indeed, the dysregulated release of other peptide hormones may indirectly affect the release of insulin.
To examine insulin release independent of systemic effects, we isolated pancreatic islets and acutely incubated them in medium containing either low or high concentrations of glucose. Figure 2B shows that in contrast to the clear stimulation of insulin release by high concentrations of glucose in control islet cells, mocha cells exhibit increased basal release with little if any stimulation by high glucose. As opposed to the reduced content of SgII in mocha chromaffin cells, cellular insulin levels show no difference between mocha and control islets (Figure 2C), indicating that the change in basal secretion is not secondary to altered expression of the hormone. In the case of insulin, the mocha mutation thus dramatically and selectively impairs regulated secretion.
Since the effects of the mocha mutation on release of other peptide hormones may complicate the observations in vivo, we also examined glucagon, a peptide released from pancreatic α cells that opposes the action of insulin: glucagon raises blood glucose levels in response to starvation. Although we observed no effect of the mocha mutation on fasting serum glucagon (Figure S2B), baseline glucagon secretion was increased in acutely isolated islets (Figure 2D). In addition, the glucagon content of mocha islet cells did not differ from controls (Figure 2E), and the morphology of the islets in situ appears unchanged (Figure S2C). Thus, mocha mice show dysregulated release of two peptide hormones with opposing actions, suggesting a global effect on peptide hormone release that makes it difficult to predict the net consequences for glucose homeostasis in vivo.
Loss of Both Ubiquitous and Neural AP-3 Isoforms Is Required to Recapitulate the LDCV Phenotype of mocha Mice
The heterotetrameric AP-3 complex is known to exist in two isoforms, one expressed by all tissues, and another expressed more specifically by neurons and endocrine tissue including the adrenal gland and pancreatic islets [16], [31], [32]. In metazoan cells, the ubiquitous isoform contributes to trafficking from early endosomes to the lysosome through a pathway that does not involve multivesicular bodies [15], [33]. In contrast, the neural isoform has been implicated in the formation of synaptic vesicles from an endosomal intermediate [34], [35], suggesting that this isoform may also contribute to the formation of LDCVs. To test this possibility, we used pearl mice lacking the ubiquitous isoform of the β3 subunit (β3A) and β3B knockouts lacking the neural isoform of β3. Since the δ subunit of AP-3 exists as only a single isoform, and the loss of one subunit usually destabilizes the complex [17], [36], we first stained cells in culture for δ to assess the effect of the mutations on the complex as a whole. We were surprised to observe no effect of losing either β3 isoform on the levels of immunoreactive δ in chromaffin cells (Figure 3A), particularly considering the abundance of the ubiquitous β3A subunit in most tissues. However, we did observe reduced expression of δ by β3A-deficient, non-chromaffin cells in the culture (Figure 3B), presumably because they do not express the neural isoform and therefore cannot exhibit redundancy. Consistent with the relative abundance of adrenal cortical cells [37] and of the ubiquitous AP-3 isoform, western analysis of adrenal homogenates showed low levels of AP-3 δ in β3A-deficient pearl animals (Figure 3C) but normal levels in β3B knockouts (Figure 3D).
To determine whether the loss of β3A or β3B influences the formation of LDCVs, we then examined the effects on SgII. We were surprised to observe that in contrast to mocha cells which showed the reduction previously reported [14], both β3 mutants had normal levels of SgII by immunofluorescence (Figure 3A). By western analysis of adrenal extracts, both β3A-deficient pearl and β3B knockouts also contained normal levels of immunoreactive SgII (Figure 3C–3D). However, loss of both isoforms in the adrenal gland of double mutant mice produced a reduction in SgII comparable to that observed in mocha mice (Figure 3E). With regard to the cellular content of SgII, the two isoforms thus exhibit redundancy.
Reduced Granin Content Reflects Reduced Gene Expression but Not Increased Degradation
The reduced expression of SgII in mice lacking AP-3 might reflect increased basal secretion or an entirely distinct process. LDCV contents have indeed been shown to undergo transcriptional regulation through a variety of mechanisms [30], [38]. We therefore measured adrenal SgII and chromogranin A (CgA) transcripts from control and mocha adrenals by quantitative reverse transcription (qRT)-PCR. Both SgII and CgA mRNA were substantially reduced (by ∼50%) in mocha mice, although not to the same extent as the protein [14] (Figure 4A). PC12 cells showed a similar reduction in SgII mRNA after AP-3 RNAi (Figure 4B).
Since AP-3 influences trafficking within the endolysosomal pathway, loss of the adaptor may also influence SgII levels through increased degradation in the lysosome. To test this possibility, we inhibited lysosomal proteases after AP-3 knockdown in PC12 cells, but did not observe any increase in the levels of SgII (Figure 4C). On the other hand, the level of lysosomal hydrolase precursor procathepsin D dramatically increased in response to the inhibition of lysosomal degradation, indicating the effectiveness of the inhibitors (Figure 4C). The reduction in cellular SgII observed with loss of AP-3 thus reflects reduced expression as well as increased constitutive release, but not increased degradation.
Non-redundant Roles for AP-3 and AP-1 in the Regulated Secretory Pathway
AP-3 resembles AP-1 in terms of sequence, the ability to recognize similar trafficking motifs and subcellular location at endosomes and the Golgi apparatus [39], [40], [41]. In addition, AP-1 associates with immature LDCVs and promotes their maturation through the clathrin-dependent removal of proteins destined for other organelles [6], [42]. In mouse pituitary AtT-20 cells, maturation indeed contributes to regulated release by removing the inhibitory protein synaptotagmin 4 [43]. In PC12 and pancreatic islet cells, however, immature LDCVs can undergo regulated release [44], [45]. To determine whether silencing of AP-1 impairs regulated release from PC12 cells, we initially targeted AP-1 β-adaptin since this is the only mammalian AP-1 subunit without multiple isoforms and hence with reduced potential for redundancy. Despite highly efficient knockdown of the β1 subunit by RNAi, μ1A levels were not reduced (data not shown), raising the possibility that the β2 subunit (of AP-2) stabilized the complex by replacing β1 [46], [47]. We therefore targeted the γ subunit of AP-1, in particular the γ1 isoform which appears to show little redundancy with γ2 [48]. siRNA transfection reduced endogenous γ1 protein by ∼80% (Figure 5A). It also reduced the stimulated secretion of SgII and the intracellular accumulation of SgII, very similar to AP-3 δ RNAi (Figure 5B,C). However, normalizing to the reduced cellular stores of SgII revealed more of an increase in basal SgII secretion than a reduction in stimulated release with AP-1 knockdown, in contrast to AP-3 RNAi (Figure 5D). AP-1 RNAi thus still reduces the extent of stimulation (stimulated/basal release) from 19-fold for control to 10-fold for AP-1 RNAi (p<0.005). AP-1 knockdown also potentiates the effect of AP-3 RNAi on stimulated secretion even after normalization to the reduced intracellular stores of SgII (Figure 5D). AP-1 thus contributes to regulated secretion, and its role appears independent at least in part from that of AP-3.
The results show that mocha mice have a major defect in the regulated secretion of peptide hormones. mocha animals exhibit hyperactivity, poor fertility, seizures and premature lethality [18], [19], but this has generally been attributed to a role for AP-3 in the endolysosomal pathway and the formation of LROs [15], [34]. We now find that mocha animals exhibit dysregulated exocytosis of adrenal chromaffin granules and both insulin- and glucagon-containing granules from pancreatic islet cells. All of the mocha cells examined show increased constitutive release relative to their cellular stores. In addition, they all show a reduced effect of stimulation, with a virtually complete loss of regulation in pancreatic β cells. Consistent with these findings in mocha mice, AP-3 RNAi increases constitutive and reduces stimulated LDCV release in PC12 cells [14]. Considering the parallel effects of AP-3 deficiency on LDCV behavior in chromaffin and pancreatic islet as well as PC12 cells, we infer that the dysregulated release observed in mocha mice reflects a common disturbance in the formation of LDCVs. The dysregulation of release by multiple neuroendocrine populations further suggests that a global defect in regulated protein secretion contributes to the phenotype of mocha mice, although the specific peptides contributing to individual features of the phenotype such as hyperactivity and seizures remain unknown.
In previous work, we found that the loss of AP-3 reduces the amount of SgII stored in PC12 cells and the adrenal gland [14]. This reflects the increased baseline exocytosis of LDCVs, but we now find that multiple granin mRNAs down-regulate as well, indicating unanticipated transcriptional effects of AP-3 deficiency. Indeed, the transcriptional down-regulation of certain LDCV cargo may account for the apparently different effects of AP-3 deficiency on different reporters and in different cells. The down-regulation of SgII mRNA in PC12 cells presumably makes it difficult to detect an increase in the absolute amount of SgII released constitutively, but transfection into PC12 cells of pHluorin-based reporters, which are not subject to this down-regulation, reveal the increased basal secretion [14]. We observe a similar increase in basal exocytosis of VMAT2- and NPY-pHluorin expressed in AP-3-deficient chromaffin cells using a lentivirus. In the case of pancreatic islets from mocha mice, cellular levels of insulin do not fall, presumably enabling us to detect the increase in basal insulin release.
Similar to AP-3 deficiency, loss of the major LDCV protein IA-2 reduces expression of multiple LDCV cargo [49], [50], suggesting that AP-3 may sort IA-2 to LDCVs. In the absence of AP-3, decreased LDCV IA-2 may thus result in reduced granin gene expression. Although AP-3 has a role in the endolysosomal pathway, we also find that the reduced granin content does not reflect increased degradation. The role for AP-3 in regulated secretion thus appears distinct from its well-established role in trafficking to the lysosome.
The analysis of isoform-specific knockouts indicates redundancy between the ubiquitous and neural isoforms of AP-3 with regard to LDCV formation. Using SgII as a reporter for a defect in the regulated secretory pathway, we find that only the loss of both ubiquitous β3A and neural β3B causes a reduction in adrenal SgII levels. However, the loss of these isoform-specific subunits has differential effects on other trafficking phenomena. In neurons, loss of β3B mimics the effect of the full mocha mutation, with reduced presynaptic expression of proteins such as the zinc transporter ZnT3 and the chloride carrier ClC-3 [35]. Loss of β3A, on the other hand, increases presynaptic expression of these proteins [35]. The redundancy of neural and ubiquitous AP-3 forms in LDCV formation thus contrasts with the opposing roles of the two isoforms in delivery of specific proteins to the nerve terminal.
How does AP-3 promote regulated secretion? In PC12 cells, the loss of AP-3 reduces the number of LDCVs and changes their morphology [14]. They appear less dense by gradient fractionation and larger by electron microscopy (EM). Consistent with this, previous work in mocha mice has shown enlarged chromaffin granules by amperometry and EM [31]. In addition, we found that AP-3 deficiency affects the membrane proteins required for regulated exocytosis: the calcium sensor synaptotagmin 1 shifts from LDCVs to lighter membranes [14]. An assay for budding from the TGN further shows that AP-3 deficiency impairs LDCV formation [14]. Despite the importance of AP-3 for LDCV formation, however, its role may be indirect, and previous work has indeed localized AP-3 primarily to endosomes [33], [41].
Several observations have suggested a role for AP-3 at the Golgi complex. In yeast, AP-3 contributes to a direct pathway from the Golgi to the vacuole [20], [22], [51]. Although this has been considered specific to yeast, work in mammalian cells has more recently supported a role for AP-3 in delivery of membrane proteins from the biosynthetic pathway to the lysosome [52], [53]. Biochemical studies have further demonstrated the specific binding of AP-3 to membranes derived from the Golgi or to artificial membranes containing the Golgi lipid phosphatidylinositol-4-phosphate (PI4P) [54], [55]. Ultrastructural studies with immunogold have also demonstrated a small pool of AP-3 at the Golgi complex [33], [41]. However, it remains possible that the role of AP-3 in LDCV formation is indirect, helping to recycle critical LDCV membrane proteins to the Golgi, or adding these proteins to LDCVs during their maturation [56].
Despite the complete loss of AP-3 and increased basal secretion, adrenal chromaffin cells from mocha mice still show residual stimulated release, raising the possibility that another system also contributes to LDCV formation. Interestingly, previous work has implicated the related adaptor AP-1 in the formation of secretory granules by other cell types, such as glue granules of the Drosophila exocrine salivary gland, and the Weibel-Palade bodies of mammalian endothelial cells [57], [58]. AP-1 also has a clear role in LDCV maturation [6], [42], but immature LDCVs can undergo release from PC12 cells [45]. We were therefore surprised to find that loss of AP-1 impairs regulated release by PC12 cells. It remains possible that maturation promotes regulated secretion even if it is not absolutely required. Alternatively, AP-1 may promote regulated release independent of LDCV maturation.
We also find that the loss of AP-1 exacerbates the dysregulation of release by AP-3 RNAi, suggesting that the two adaptors act through distinct mechanisms. If AP-1 promotes regulated secretion through its role in LDCV maturation, it may indeed act to remove proteins that interfere with regulated release, a process that occurs in AtT-20 cells [43]. We speculate that a proofreading role for AP-1 may become even more important in the absence of AP-3 to concentrate the membrane proteins required for regulated secretion.
Materials and Methods
Ethics Statement
All procedures involving animals were approved by the UCSF Institutional Animal Care and Use Committee.
The rabbit SgII antibody was obtained from Meridian Life Science, the mouse actin monoclonal antibody from Millipore, the mouse δ SA4 monoclonal antibody from the Developmental Studies Hybridoma Bank, the goat cathepsin D antibody from Santa Cruz, the mouse HA.11 monoclonal antibody from Covance, the mouse adaptin γ monoclonal antibody from BD Transduction Laboratories, the mouse insulin monoclonal antibody from Sigma, the guinea pig glucagon antibody from Linco, the mouse glucagon monoclonal antibody from Sigma and the rabbit somatostatin antibody from Thermo Scientific.
Silencer Select rat Ap3d1 (sense, 5′-CAUGGAUCAUGACCAAGAA-3′) and corresponding non-targeting control siRNAs were from Ambion. ON-TARGETplus rat Ap1g1 (sense, 5′-CAUAAAUAUUCUUGGUCGA-3′, 5′-GUGUGGAGAUGCACGCUUA-3′, 5′-UGUAACAGUGAUAACGAUA-3′, 5′-GGACUGGAAUUCACGGCAA-3′) and corresponding non-targeting pooled control siRNAs were from Dharmacon.
Molecular Biology
The sequences of NPY-pHluorin (a generous gift of R. Holz, U. Michigan) and VMAT2- pHluorin were amplified by PCR to add 5′ BamHI and 3′ EcoRI sites, then subcloned into the FUGW lentiviral expression vector, replacing the EGFP coding sequence.
Cell Culture and Lentivirus Production
PC12 cells were maintained in DMEH-21 medium supplemented with 10% horse serum (HS) and 5% cosmic calf serum (CCS; HyClone) in 5% CO2 at 37°C. siRNA transfection was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. HEK293T cells were maintained in DMEH-21 medium with 10% fetal bovine serum (FBS) in 5% CO2 at 37°C. Lentivirus was produced by transfecting HEK293T cells with FUGW, psPAX2 and pVSVG and Fugene HD (Roche) according to the manufacturer's instructions [59].
Chromaffin Cell Isolation and Culture
Mouse adrenal chromaffin cells were isolated and cultured as previously described [60]. Briefly, adrenal glands were dissected and placed in cold Ca++-, Mg++-free (CMF) Hank's balanced salt solution (HBSS). The surrounding fat and cortex were removed and the medullae transferred to tubes containing 300 U/ml Collagenase I (Worthington) in CMF-HBSS. Medullae were dissociated by shaking for 40 min at 37°C. Collagenase solution was then replaced by CMF-HBSS containing 200 µg/ml DNAse I (Sigma) and 1% heat-inactivated FBS (Gibco), the tissue triturated first with a P200 pipette tip, then with a 23 gauge needle. The cells were pelleted at 300 g for 8 min at room temperature and resuspended in pre-warmed culture medium. Cells were maintained in DMEH-21 medium supplemented with 10% FBS and antibiotics. For lentiviral transduction, freshly isolated chromaffin cells were plated in viral supernatant, and fresh medium was added the following morning.
Total Internal Reflection Fluorescence (TIRF) Microscopy
For TIRF microscopy, control or mocha chromaffin cells were plated onto glass chambers coated with poly-L-lysine, immediately transduced with lentivirus encoding either NPY- or VMAT2-pHluorin and imaged live 4–7 days later. Images were typically collected for 40–50 ms at 10 Hz and room temperature using an inverted TIRF microscope (TE2000E; Nikon) with 100× Plan Apo 1.49 NA oil objective, a 1.5× tube lens and an electron-multiplying charge-coupled device camera (QuantEM; Photometrics). Basal exocytosis was measured in Tyrode's solution containing (in mM, 119 NaCl, 25 HEPES-NaOH, pH 7.4, 30 glucose, 2.5 KCl, 2 CaCl2, 2 MgCl2) over 90 s, and release stimulated for 60 s in Tyrode's containing 5 µM 1,1-Dimethyl-4-phenylpiperazinium (DMPP; Sigma). Individual exocytotic events were quantified manually using NIS-Elements software (Nikon). The amplitude of individual exocytotic events was measured by placing 2×2 pixel ROIs manually over the center of events, and the mean ROI intensity prior to an event subtracted from the maximum event intensity.
Glucose Tolerance Tests and Serum Insulin Measurements
Glucose tolerance was assessed and serum insulin levels measured using 5–15 week-old mocha mice and age-matched controls. Mice were fasted overnight (∼16 h), weighed the following morning, and blood samples collected for baseline glucose and insulin levels. Mice were then injected intraperitoneally with glucose at 2 mg/g body weight and blood samples collected from the tail vein at the time points indicated. Blood glucose levels were measured using the FreeStyle glucometer (Abbott). To measure serum insulin, the blood was allowed to clot, sedimented at 2000 g and 4°C for 20 min, and insulin levels determined using the Ultra Sensitive Mouse Insulin ELISA kit (Crystal Chem).
Pancreatic Islet Isolation and Insulin Secretion
Islets were isolated as previously described from 9–16 week-old mocha mice and age-matched controls [61]. Briefly, islets were purified on a Ficoll gradient and allowed to recover for 1 h at 37°C. Five islets were aliquoted into tubes containing HEPES-buffered RPMI medium supplemented with either 2.8 mM glucose (basal) or 28 mM glucose (regulated) and incubated for 1 h at 37°C. The islets were then sedimented and the supernatants collected to measure insulin secretion. The pellets were resuspended and sonicated in 2 mM acetic acid, 0.25% RIA-grade BSA to extract intracellular insulin. Finally, the nuclei were lysed by additional sonication in 67 mM ammonium hydroxide, 0.2% Triton X-100. Secreted and cellular insulin were quantified by ELISA (Mercodia) according to the manufacturer's instructions, and islet DNA quantified to confirm that the amount of islets per tube was similar between conditions. Glucagon was measured by ELISA (R&D Systems).
Chromaffin cells were fixed by adding an equal volume of 4% formaldehyde in CMF-PBS to the culture medium and incubating for 20 min at room temperature. Cells were blocked and permeabilized in CMF-PBS containing 2% BSA, 1% fish skin gelatin and 0.02% saponin. Primary antibodies were diluted in blocking solution at 1∶500 (SgII) and 1∶100 (δ SA4). The following secondary antibodies conjugated to Alexa Fluor dyes (Invitrogen) were used at 1∶1000 in blocking solution: goat anti-rabbit IgG 488 and goat anti-mouse IgG 594. Images were acquired using a Zeiss LSM 510 confocal microscope and 100× oil objective.
Adrenal Gland Granin Content
pearl and mocha mice were obtained from the Jackson Laboratory, and mocha animals were backcrossed to C57BL/6 to remove grizzled and Pde6brd1 alleles. Ap3b2 KO mice were obtained from V. Faundez (Emory) and S. Voglmaier (UCSF). Double mutant pe/pe; Ap3b2−/− mice were generated by crossing pe/pe; Ap3b2+/− males to pe/+; Ap3b2+/− females. Adrenal glands from 3–6 week-old mice were homogenized in 150 mM NaCl, 50 mM Tris-HCl, pH 8.0, 1% NP-40, 0.5% sodium deoxycholate, and Complete protease inhibitors (Roche) with 1 mM EGTA and 1 mM PMSF. After sedimentation at 14,000 g to remove nuclei and cell debris, 20–40 µg protein was separated by electrophoresis through polyacrylamide, transferred to nitrocellulose, and the membranes immunoblotted for AP-3δ and SgII, with actin as loading control and the appropriate secondary antibodies conjugated to IRDye800 (Rockland Immunochemicals). The immunoreactivity was quantified by imaging with an Odyssey system (LI-COR Biosciences) and ImageJ (National Institutes of Health), and the signals normalized to actin. For western analysis of the SgII secreted from chromaffin cells, Tyrode's solution was collected, sedimented at 300 g for 3 min at 4°C, and the supernatant mixed with SDS-PAGE sample buffer before electrophoresis through polyacrylamide. Chromaffin cells were directly lysed by the addition of sample buffer. In this case, SgII and actin were detected using ECL plus (GE Healthcare).
Total RNA was isolated from PC12 cells and mouse adrenal glands using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. To improve the yield of adrenal RNA, ultrapure glycogen (Invitrogen) was added to the TRIzol as carrier. In addition, adrenal RNA was treated with RNAse-free DNAse I (NEB) to remove contaminating genomic DNA. cDNA was synthesized using oligo(dT) or gene-specific primers and a Transcriptor First Strand cDNA Synthesis kit (Roche). qPCR was performed with SYBR Green (Applied Biosystems) on a Stratagene Mx4000 machine. The following primers were used: rat SgII fwd: 5′-ACAATATAAGACAGAGGAAAATTTT-3′, rev: 5′-TGGATAAGAAGCAGAACTG-3′; rat β-actin fwd: 5′-CCGTGAAAAGATGACCCAGATC-3′, rev: 5′-CAGGGACAACACAGCCTG-3′; mouse CgA fwd: 5′-CCAACCGCAGAGCAGAG-3′, rev: 5′-AGCTGGTGGGCCACCTT-3′; mouse SgII fwd: 5′-AAGTGCTGGAGTACCTCAACC-3′, rev: 5′-TTACATGTTTTCCATGGCCCG-3′; mouse GAPDH fwd: 5′-ATGGTGAAGGTCGGTGTGAAC-3′, rev: 5′-TCCACTTTGCCACTGCAAATG-3′.
Lysosomal Inhibition
Two days after the second siRNA transfection, PC12 cells were incubated for ∼24 h in complete medium supplemented with vehicle or a cocktail of lysosomal protease inhibitors (Sigma) including (in µM) 10 antipain, 10 leupeptin and 5 pepstatin A. Cells were washed on ice with cold PBS and lysed by the addition of 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100, and Complete protease inhibitors (Roche) plus 10 mM EDTA and 1 mM PMSF. Samples were analyzed by quantitative fluorescent immunoblotting.
Secretion Assays
Cells were transfected with siRNA (100 nM) on days 1 and 3 after plating, washed 2 days later and incubated in Tyrode's solution containing 2.5 mM K+ (basal) or 50 mM K+ (stimulated) for 30 min at 37°C. The supernatants were collected, cell lysates prepared as described above, and the samples analyzed by quantitative fluorescent immunoblotting.
Statistical Analysis
Statistical analysis was performed using the Student's two-tailed t-test unless otherwise indicated.
Supporting Information
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Hayes owned Leopard Head for just about a month and a half when his life came to a gruesome and untimely end. We really try to be good lizard owners, but happy, healthy lizards just don't seem to be in the cards for the Geldmachers. Remember Skid's tail incident? Please don't turn us in to whatever animal protection agency out there might find this alarming. We really do try. The boys love the lizards kind of like Tommy Boy loves his sale/roll/pretty new pet. Anyway, Dax, Hayes and a friend were in their room last week when one of them came screaming-I mean really, really screaming down the hall, "we killed Leopard Head! He's DEAD!" I ran to the room to find Dax and the friend sobbing. And yes. A very severely injured Leopard Head. Apparently someone-not sure who- was holding the lizard and he jumped out of the hand to the ground. I also don't know if Dax jumped out of the way, or tripped, or what exactly but he stepped on the lizard. And squished it. With his bare foot. And I don't mean invisible-to-the-naked-eye internal injuries here, either. This was gruesome blood and actual guts stuff. Dax and the friend were seriously traumatized. Sobbing, upset, traumatized. Hayes never cried or really acted upset. And to his credit he never blamed Dax or got mad. I think he could tell Dax felt bad enough already. He just asked me to help dig a hole in the flower bed, which I did, although I too was slightly traumatized at the sight of the guts. Hayes colored a few pictures for Leopard Head and made him a paper headstone. I called Tyler and asked why does this kind of stuff never happen when you're home??? Dax cried some more. He felt very sorry and responsible. Hayes just kept telling Dax that it was ok and accidents happen. The next day Dax came home from school with this:
It's about the size of half of a poster board, so it's not small. I asked Dax when he made it and he told me he just asked Mrs. Moody if he could do this while the rest of the class worked on something else. Okaaayyy...... The guy with the tear on his face just about breaks my heart. You can't tell in the picture, but the drawing of the water bowl says "I will buy you a new lizard." Not sure if that's a good idea. But I do like the use of just a little glitter and there was also a candy glued to it. Awww.
So there you have it. Rest In Peace, Mr. Leopard Head.

subset: commoncrawl


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The Story
Founder of Roc Ya Spirit, Brian K. Chesson was born in Little Rock, Arkansas. Raised by my mohter Pamela Chesson and step father Fred Chesson who adopted me at the age of five.
I lived in Buffalo,NY the majority of my childhood until I turned eighteen. I grew up to witness my dad live a very fast life in the streets, he did whatever it took to make money, but he caught a lucky break and began a executive career in the music & entertainment industry. After I attended highschool and college in 1994 my dad introduced me to the music and entertainment business, which was a blessing and a curse at the sametime.
By 1997 after three years at the age of 23 I was managing a multi million dollar concert and touring promotions company, but I was never aware of all the temptations and addictions that came along with the entertainment industry such as making fast money, promiscuous behaving, stealing, drugs, un healthy relationships. Between the years 1994 -1997 I became completely addicted to these behaviors and lost myself to this secular entertainment world and its evil ways, I even considered taking my life because I felt there was no way to deal with the pain, depression and pressure this industry inserted in my life.
When I completely lost my soul and direction,1998 thats when GOD revealed his face to me, I remember it like it was yesterday he said SON DO YOU WANT TO COME WITH ME? I cried for hours after I said yes. I left the secular entertainment industry to follow christ and life has been great ever since, god has supplied me with everything I need and more to survive in this falling world.
Its 2010! many years later, I realized I never thanked and praised him enough from saving me from satan and myself, So now Im choosing to Share with the world through my story & gifts on how great God is. God will break satan's chains off of you so you can be free in this falling world and live a awesome meaningful life. ALL YOU HAVE TO DO IS SAY YES I WILL COME WITH YOU MY HEAVENLY FATHER. Only then you will experience the true love your seeking and deserve in this falling world.
Brian Chesson
Roc Ya Spirit Founder


text: Saw Thanos Part 2 last night, and it was the perfect end to a decade of awesomeness I would have never imagined could happen 11 years ago. :D\n\nRemember when x-men became a movie? And we all thought it was a actually pretty good? After we grew up and were resigned we'd never get more then cartoons or crappy movies like spawn?


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Tag Archives: namaste
A Beautiful Mess
To advance in life is to let go. It is to be conscious. It is to be spiritually awake. It is to love yourself. It is to be open to life's challenges. It is to smell the roses. It is to see the sun. It is to have knowledge. Knowledge is truth. Truth is awakening.
When your inner spirits awaken, your soul awakens. It is a beautiful mess. It is seeing your garbage. It is seeing your own hate. It is a acknowledging your mess. It is throwing out the garbage. It is cleaning your inner house. It is crawling out of the dark shadows and walking into the light.
It is a beautiful mess.
It is releasing your inner toxins by crying it out. It is sweating out your negative particles. It is enduring the inner pain to feel renewal. It is walking out your demons.
It is renewal.
It is life.
It is your life.
It is your beautiful mess ❤
Selfless Love
Truly, truly, truly people, you should embrace yourselves for a bounty of a positive life if you can love selflessly. If you ever "feel" possessive, jealous or wanting someone all to yourself, that is your EGO talking! That is NOT love! Love is selfless when you are really wholeheartedly in love with your mate. It will take you down a road of beauty and freedom! It will allow you to share yourself with no judgments, no complaints and no jealousy! It is truly a hard concept to live by, but if you can do it, it will take you to a higher level of consciousness and your universal vibrations will go up. It is the same as living a positive life, it is a challenge at first, but you must always be conscious as our ego wants to constantly take over!
If you can honestly attempt to experience selfless love with a mate, you will see a whole other world! I challenge you! Let's get rid of possessiveness, it only leads to "more" wants. It is time to decrease all of our "needs" and live selflessly to bring happiness to others. In turn, your karma will be good and love will come back to you with no ties!
Today, live to bring happiness to your mate, family and friends!
©cami*star 2015


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Comment on What is the best wine for a romantic evening? by J.
The best thing to do is to ask her and ask what she likes for wine. The worst thing is to buy a nice bottle and end up having both of you guys hating it.
The most romantic wine is Champagne. It is a great wine for celebration, fun to drink and goes well with food as well as well with hot tub/fire place and all those activities. Honestly, if you want to try Champagne, I would spend the money to get something nicer – at least something in $40-50's range (include Taittinger or Krug) but may be something better (Dom Perignon $140, La grande Dame $150). You can get them at better price at Sam's or Costco's, but you should consider spending money at least for one time.
As for Zinfandel, here's the scoop. Zinfandel is a type of grape grown primarily in California. Two types of wines are produced from Zinfandel – White Zinfandel and Red Zinfandel (typically known only as Zinfandel) You need to know which type of Zinfandel she likes.
White Zinfandel is a rose wine or pink wine, started at Sutter Home in 70's. It is a sweet simple fruity wine that is not very overpowering. It is relatively cheap and available from various wineries such as Kendall Jackson, Sutter Home, etc. for $10-15.
Red Zinfandel is a dark red wine, medium in body, and fill with fruit flavor – lots of berry and plum, with a touch of spiciness. Over past few years, California tend to make Zinfandel with ihgher alcohol content, up to 14-15% now (normally for wine 12.5-13.8%). My favorite brands of Zinfandel include Rosenfel (various types, including cheap Cuvee $12, other single vineyard Zinfandel costing $20-30, very highly rated and wonderful to drink) and Ridge (various vineyard also, personal favorite is Lytton Spring $30).
Good luck. Thanks for the good work at Iraq.
J Also Commented
What is the best wine for a romantic evening?
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Pick up a few bottles of Italian prosecco…. chill well, serve in flutes and, my GI friend, Bacco will tell you (both) what to do after a few glasses. The rest of the bottles of prosecco will help keep your throats from drying out throughout the evening.
What is the best wine for a romantic evening?
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Night Train! lol no on a serious note Kendal Jackson Cabernet is wonderful.
What is the best wine for a romantic evening?
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Recent Comments by J
What are some wine substitutes to use when cooking?
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One of the reasons why alcohol is used in cooking is that certain flavor compounds are alcohol soluble, but not water soluble. Alcohol substitutes aren't going to liberate those alcohol soluble flavors. Your dish will not be identical to the original recipe, but it will still be lovely.
When you decide what to use to substitute for the wine, think about what the wine is in the dish for.
If it is to provide acidity in a marinade, fruit juice may be a good substitute. If you substitute juice, it's going to add a great deal of sweetness to the dish. You might do part juice and part red wine or rice vinegar if you don't want additional sweetness.
If the wine is to add needed (and flavorful) moisture in a dish, I'd use stock. Any kind of stock – vegetable, chicken, beef, veal, you name it. But don't use bullion from cubes – it will over salt your dish. Don't have any stock? Use water - it won't add any flavor to the dish, but it won't hurt the flavors that are already there.
If wine is called for to deglaze a pan, again, I'd use some stock that would compliment the dish.
Happy eating to you!
What are some wine substitutes to use when cooking?
Content courtesy Caffeinated Content
Depending on the dish, any kind of stock, chicken, beef, vegetable, could be substituted. The wine usually adds some flavor, but in some dishes it actually tenderizes the meats.
But like Emerel always says, "This aint rocket science" So just substitute anything for the wine…even water ,depending on the recipe
Good luck, oh..and BabBaBaBAMMMMMM!!!!
Should I keep bottled wine in a wine cooler or in a dry place to store it?
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If you are talking storage, keep it in the basement, out of the light, and dry. Make sure it is laying on its side, and try not to move them very much.
In the cooler go the whites you will drink over the next two weeks, or the reds you will drink in the next 5-7 days.
What are some wine substitutes to use when cooking?
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First of all, alcohol doesn't steam out of anything however it will burn off if caught on fire.
I recommend a non alcoholic wine. I don't drink either and use alchohol free wine.
Should I keep bottled wine in a wine cooler or in a dry place to store it?
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The location should have good humidity and a temperature around 55 degrees.
Store with the wine touching the cork so the cork will not dry out.
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Visited the homepage, and got a series of redirects through a bunch of sites without me doing anything, and I discovered a very suspicious script in the html for the home page:
<script type="text/javascript" src="http :// kokosina.in/1"></script>
It's near the bottom. It is obfuscated, so likely malicious. Checking my system out now to see if I received an unwelcome drive-by install.


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Tower helps tourists view Roman remains
A metal tower designed by Austrian studio Marte.Marte is helping tourists to locate excavated Roman ruins.
Stefan Marte created the structure between the remains of two Roman villas, close to the town of Brederis in Austria.
Few traces of the original buildings remain, so the new installation provides the only landmark above ground level.
"The tower-like sculpture is designed to make the excavation site visible for miles around," said Marte. "It acts like a magnifying glass, offering an insight into history."
Made from Corten steel, the ten-metre tower has a glazed lower section that exposes a hollow centre, allowing visitors to look down to the underground remains.
A platform extends from one side of the structure to form a standing area, while displays of replicas of Roman objects are on the wall. Both were also constructed from pre-weathered steel sheets riveted together, giving the structure a rusty look.


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What's This? A Seed's Story
by Caroline Mockford (author) (UK edition)
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Share the excitement of a small girl who discovers how a tiny seed grows into a magnificent sunflower! Watch how the sunflower makes new seeds for the coming year – and follow simple guidelines to grow your very own! A captivating picture book that glows with colour, and includes helpful tips for parents and teachers on how to cultivate seeds.
"A simple story that works on many levels as a lovely picture book, and an educational book about nature and how things grow." Junior
"Beautifully illustrated… the colours leap off the page and Caroline Mockford's drawings really do capture the wonder and magic of the natural world." Schoolsnet.com
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Discussion Platform SPACES of Negotiation
What about the role and format of art projects in public and unconventional spaces? From art in protest space to reclaiming the city and urban curating. Inputs by, amongst others, Yuriy Kruchak and Yulia Kostereva, Elke Krasny, Romana Schmalisch, Stefan Rusu.
More information on the Programme
SPACES of Negotiation was organised by CSM.
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Contact us - HQI
Business development outside Québec
Do you have questions about
- our investment approach?
- our technological innovations?
- our exports?
Please fill out a short form and we will be happy to get back to you as soon as possible.
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Camfil APC
Camfil Air Pollution Control manufactures industrial dust, fume and mist collectors to improve air quality and workplace safety. Camfil was looking to build brand awareness and generate more qualified leads, so they reached out to Schubert b2b for an integrated digital marketing program.
Working closely with the Camfil team, we bring together many marketing channels – content marketing, advertising, SEO, public relations, social media, video production – to find B2B buyers, drive them to a network of landing pages and convert them into sales-qualified leads.
Camfil's program includes:
- Messaging and positioning strategy
- Digital marketing
- Content creation
- Advertising
- PR and social media
- Website development
- Video production
Camfil's new, responsive website is designed to increase conversions and improve usability.
Camfil's Filterman character is a fun and engaging way to promote aftermarket sales.
A part of Camfil's corporate culture for many years, Schubert b2b helped create an updated version of Filterman for 2018.
Digital marketing content was created to support a critical product launch in the North American market.
Product videos show the unique benefits and capabilities of Camfil's dust collectors and filters.
Lead nurturing workflows build connections with prospects and generate more qualified leads.
Educational video content positions Camfil as a thought leader in the industry.
Print advertising raises awareness in niche markets and expands Camfil's marketing presence and reach.
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A ceasefire failed in Syria, although it was meant to allow for the observance of the Muslim festival Eid al-Adha. The ceasefire, negotiated between rebel and government forces, broke down on Friday and the fighting has continued into the weekend. A death toll of 150 people was reported on the first day of the ceasefire alone.
The ceasefire was brokered by an international peace envoy, but lasted only a few hours. On Friday, a suicide bomb rocked Damascus near a playground and sparked a downward spiral of fighting that has lasted into Sunday. The AP is reporting continued fighting in eastern Damascus suburbs that are currently held by rebel forces.
Syrian President Bashar al-Assad made a rare appearance on state television on the first day of the ceasefire, in which he was shown attending prayers at a mosque and talking with other worshippers. Soon after, state TV acknowledged the Damascus attack and reported, "the Armed Forces are firing back and confronting the armed terrorist groups."
The uprising in Syria has become a longstanding fight and has essentially spiraled into a full on civil war. Recently, fighting has spilled over into neighboring Turkey and Lebanon, heightening tensions in the region as a whole.
Hizbollah, a Shia armed force that fills an ad-hoc governmental role in Lebanon, has been a strong supporter of Bashar al-Assad's government, but some important figures in its ranks are now questioning their position. There are a large number of Sunnis and Christians in Lebanon and some see the future of Syria as directly related to the future of Hizbollah's influence and strength in Lebanon.
Syria has been a topic of great interest during the presidential election, particularly for its far-reaching implications in the Middle East. The American administration has struggled with balancing support of rebel forces and direct involvement, and has endured the critique that it has not done enough to stop the escalating death toll. Throughout the campaigning cycle, candidates have been asked to define an opinion on how to best approach the Syrian conflict, although both have been avoiding any resolution that would include a full-on US military intervention.
The Americans also have a great interest in the conflict because Iran has been playing an important role in the conflict, a growing concern to the US government. However, Iran appears to have scaled back some of its involvement due to scrutiny back in Tehran.
Russia and China have come under fire by the West for their lack of condemnation of the Syrian government. This silence is seen as direct support for the al-Assad regime. Both Russia and Syria hold places on the UN Security Council and their positions are currently blocking the council from intervening militarily in the region.
A diplomatic resolution to the Syrian conflict is highly, but the next US president will have urgent choices to make as violence spreads to Syria's neighbors and Iran's entanglement becomes more apparent.


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Saturday, May 6, 2017
"And when they heard of the resurrection of the dead, some mocked: and others said, We will hear thee again of this matter. So Paul departed from among them. Howbeit certain men clave unto him, and believed: among the which was Dionysius the Areopagite, and a woman named Damaris, and others with them" (Acts 17:32-34 KJV).
How true even today!
Verse 16 says: "Now while Paul waited for [Silas and Timotheus, verse 15] at Athens, his spirit was stirred in him, when he saw the city wholly given to idolatry." Idols are everywhere in the city. Paul is excited. What an opportunity to preach the Gospel of Jesus Christ to all these lost and dying heathen! He discourses with the synagogue Jews and other religious persons; also, in the marketplaces, where large crowds exchanged ideas and products, Paul speaks with individuals (verse 17).
Philosophers of the Stoicks and Epicureans hear him, and wanting to learn more, they bring him to Mars' Hill, a place where religious ideas were offered and debated (verse 18-21). Paul preaches to these idolaters from verse 22 to 31. Notice his final remarks: "[29] Forasmuch then as we are the offspring of God, we ought not to think that the Godhead is like unto gold, or silver, or stone, graven by art and man's device. [30] And the times of this ignorance God winked at; but now commandeth all men every where to repent: [31] Because he hath appointed a day, in the which he will judge the world in righteousness by that man [Jesus Christ] whom he hath ordained; whereof he hath given assurance unto all men, in that he hath raised him from the dead."
Then comes today's Scripture. Some mock Paul's "resurrection" comments—Greek religion denied it, and so do they. Others want to discuss it more at some later time, but they too are more interested in learning more of the "new thing" (verses 18-21) than believing God's Word. Paul left them, not bothering to waste time. Still, a few men and women trusted Christ as Saviour and thus joined Paul. We see such scenarios even today. Some want to hear; others do not. Some believe; others laugh. We should not be surprised or discouraged! 🙂


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It is not what you think! I am not getting a day job again, at least not at the moment - though I did wake up this morning feeling like my holiday is finally over. Frankly, I was relieved! The past two months I have been a bit slow, a bit tired. Though I have been hard at work on my book I have also been sleeping in. Not as productive as I was at my day job (then again, the past 3 years I have been keeping a day job while going to school, blogging and writing a book on the side. It has been crazy.)
Today I woke up and just knew that this slump is over. I am energised for no reason at all. My eyes opened on their own at 6.50 am. I wasn't tired, sleepy or wanting to stay in bed. I had breakfast and got to work on my book. I think my book is finally finally done. Acknowledgements written, appendixes written (including an introduction to energy management for what I call novice Healers). I did one last check to make sure everything was consistent from an UPPER CAPS/lower caps point of view and I think I am all set to go! This is exciting!
What is next? There are 2 or 3 books that I feel like I need to read asap. One is on the biology and science of cancer, its called the Emperor of all Maladies. Though my book is on complementary therapies like Reiki and Energy Healing and how they can help, having a good understanding of the disease process will help me tap into the body at an intuitive level. Last year I attended a workshop at the School of Intuition and Healing where we learned how to tune into the organs and see how they are feeling (read more here). I also have to play catch up on a distance learning anatomy and physiology class.
You can also find her on: Amazon - Facebook - Youtube
Regina Chouza is a qualified Energy Healer, Angel Medium and author of Self-Healing, Cancer & Love and Chakra Healing & Magick. A graduate of the School of Intuition & Healing in London, she is developing a new technique that combines natal astrology, energy healing and crystals. Subscribe for an introduction to grounding, clearing and shielding your personal energy HERE.


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We're often asked for advice on lifestyle insurance (such as life insurance or income protection) and what's the best way to ensure you're covered if the worst happens. We've always recommended using a specialist broker, as lifestyle insurance can be complex and when you set out as a contractor or freelancer it's crucial you get the right cover in place based on your circumstances. It was difficult finding specialists that understood our customers as well as we do.
We're pleased to say that we've found a broker offering Lifestyle Protection Insurance that is a small business, freelancer and contractor specialist. Their story is similar to ours: experts in their field that came together to offer a niche service based on sound financial advice, and to look after clients for the long term.
Oviso is a small team of experts with big ideas and they've won the 'Best Protection Independent Financial Adviser', Financial Times Adviser Magazine, in 2012 and 2013. They are approachable and knowledgeable about insurance, so we think they are a perfect fit for us as our recommended broker.
We caught up with Fiona Todd, Protection Specialist with Oviso to talk protection and why using a broker is best when arranging the right insurance cover. Click here to read the article.