text:
During the initial phases of type 1 diabetes, pancreatic islets are invaded by immune cells, exposing β-cells to proinflammatory cytokines. This unfavorable environment results in gene expression modifications leading to loss of β-cell functions. To study the contribution of microRNAs (miRNAs) in this process, we used microarray analysis to search for changes in miRNA expression in prediabetic NOD mice islets. We found that the levels of miR-29a/b/c increased in islets of NOD mice during the phases preceding diabetes manifestation and in isolated mouse and human islets exposed to proinflammatory cytokines. Overexpression of miR-29a/b/c in MIN6 and dissociated islet cells led to impairment in glucose-induced insulin secretion. Defective insulin release was associated with diminished expression of the transcription factor Onecut2, and a consequent rise of granuphilin, an inhibitor of β-cell exocytosis. Overexpression of miR-29a/b/c also promoted apoptosis by decreasing the level of the antiapoptotic protein Mcl1. Indeed, a decoy molecule selectively masking the miR-29 binding site on Mcl1 mRNA protected insulin-secreting cells from apoptosis triggered by miR-29 or cytokines. Taken together, our findings suggest that changes in the level of miR-29 family members contribute to cytokine-mediated β-cell dysfunction occurring during the initial phases of type 1 diabetes.
A fine control of the amount of insulin released by pancreatic β-cells is essential to maintain appropriate blood glucose levels. Type 1 diabetes is an autoimmune disease characterized by progressive destruction of pancreatic β-cells that, if untreated, leads to life-threatening alterations of blood glucose homeostasis and body metabolism. During the initial phases of the disease, β-cells are chronically exposed to elevated concentrations of cytokines released by the immune cells that infiltrate the islets of Langerhans and by endocrine cells themselves (1). This inflammatory environment has a deleterious impact on specialized β-cell functions, resulting in impaired insulin biosynthesis and secretion and in β-cell loss by apoptosis in the long term. A detailed understanding of the molecular events elicited by proinflammatory cytokines, and responsible for β-cell dysfunction, is essential to pave the way to the development of new approaches for preventing and treating type 1 diabetes.
Global microarray profiling of β-cells chronically exposed to cytokines revealed that these inflammatory mediators induce major changes in gene expression, causing severe impairment in key signaling pathways (1,2). So far, most of the studies investigating the mechanisms underlying the changes in gene expression that occur in the presence of cytokines focused on transcription factors such as nuclear factor κB (NFκB) (3). However, there is growing evidence that another class of gene regulators, called microRNAs (miRNAs) play an important role in the control of β-cell functions, under both control and physiopathologic conditions (4–14). These short, noncoding RNA molecules bind to specific sequences of the 3′-untranslated region (3′UTR) of target mRNAs, causing message destabilization and/or translational inhibition (15,16). We recently reported that prolonged exposure of β-cells to proinflammatory cytokines leads to the induction of three miRNAs—miR-21, miR-34a, and miR-146—resulting in defective insulin secretion and apoptosis (11). We found that the level of these miRNAs is increased in the islets of NOD mice, a well characterized type 1 diabetes model (11), suggesting a potential contribution of these small RNA molecules to β-cell failure during the instauration of the disease.
In this study, we used microarray profiling to search for global changes in miRNA expression in pancreatic islets of prediabetic NOD mice. We found that, in addition to the three miRNAs mentioned above, the development of type 1 diabetes in NOD mice is associated with a strong induction in islet cells of the members of the miR-29 family. Studies performed in β-cell lines and dissociated islet cells revealed that overexpression of miR-29 to levels comparable to those observed in NOD mice leads to defective glucose-induced insulin secretion and promotes apoptosis, suggesting a contribution of this family of miRNAs to the initial phases of type 1 diabetes.
RESEARCH DESIGN AND METHODS
Interleukin-1β (IL-1β) was purchased from Sigma (Buchs, Switzerland), recombinant mouse interferon-γ (IFN-γ) from R&D Systems (Minneapolis, MN), and tumor necrosis factor-α (TNF-α) from Alexis Corporation (Lausen, Switzerland). Hoechst dye 33342 was obtained from Invitrogen (Basel, Switzerland).
Isolation and culture of pancreatic islets and MIN6 cells.
The insulin-secreting cell line MIN6 clone B1 (17) was cultured at a density of 1.5 × 105 cells/cm2 in DMEM-Glutamax medium (Invitrogen, Carlsbad, CA) supplemented with 15% FCS, 50 IU/mL penicillin, 50 μg/mL streptomycin, and 70 μmol/L β-mercaptoethanol.
Mouse pancreatic islets were isolated by collagenase digestion (18) from female NOD or C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME). After isolation, NOD mice islets were immediately processed for RNA purification. C57BL/6 mouse islet cells were dissociated by exposing the islets for 6 min to a Ca2+-free buffer containing 116 mmol/L NaCl, 24 mmol/L NaHCO3, 5 mmol/L KCL, 1 mmol/L MgCl2, 20 mmol/L HEPES, and 0.1% trypsin at 37°C (18). The dispersed cells were then seeded at a density of 5.5 × 104 cells/cm2.
Human pancreatic islets were provided by the Cell Isolation and Transplantation Center at the University of Geneva, School of Medicine, thanks to the European Consortium for Islet Transplantation “islets for research” distribution program sponsored by the Juvenile Diabetes Research Foundation. In the current study, we used islet preparations from six donors (five women and one man) who were 56 ± 6 years old and had a BMI of 22 ± 4 kg/m2. The purity of the islet preparations was 79 ± 10% and contained 51 ± 7% insulin-positive cells.
After isolation, the islets were cultured in CMRL medium (Invitrogen, Carlsbad, CA) supplemented with 10% FCS, 100 IU/mL penicillin, 100 μg/mL streptomycin, 2 mmol/L l-glutamine, and 250 μmol/L HEPES. Islet cell monolayers were prepared by treating the islets for 7 to 9 min with trypsin (5 mg/mL) at 37°C. Trypsinization was terminated by adding serum-containing culture medium. The cells were seeded at a density of 5.5 × 104 cells/cm2.
Measurement of miRNA expression.
For microarray analysis, total RNA of islets from 4- and 8-week-old NOD mice was extracted using the miRNeasy Mini Kit (Qiagen, Hilden, Germany). Global miRNA profiling was performed by the DNA Array Facility of our institution using Agilent Technologies miRNA Gene Microarrays. The analysis permitted to determine the level of all mouse miRNAs available on the Sanger miRBase version 9.1. Quantitative assessment of the level of individual miRNAs was carried out by performing a Universal RT reaction with locked nucleic acid (LNA)-enhanced PCR primers, followed by real-time PCR (Exiqon, Vedbaek, Denmark). The results were normalized using cDNAs amplified with U6 primers in the same samples. This small nuclear RNA is not affected by cytokine treatment. Similar results were obtained when the data were normalized to miR-7.
Localization of miRNAs.
To assess whether miRNAs are expressed by islet cells and/or by the intraislet immune cells, islets of 13-week-old female NOD mice were isolated by collagenase digestion and incubated for 24 h to allow spontaneous extrusion of the lymphocytes (19,20). Moreover, cryosections of pancreas of 4-, 8-, and 13-week-old NOD male mice were fixed in 4% paraformaldehyde, acetylated, and processed for in situ hybridization (21) of miR-29s and miR-142-3p, using 10 nmol/L 3′-digoxygenin-labeled LNA-oligo probes (Exiqon; mmu-miR29a, MIMAT 0000535; mmu-MiR29b, MIMAT 0000127; mmu-MiR29c, MIMAT 0000577; mmu-miR-142-3p, MIMAT 0000155). Sections from three mice of each age were analyzed.
Adjacent sections were processed for indirect immunofluorescence staining (22) of insulin, using sheep polyclonal antibodies (Ventrex, 675), diluted 1:700; CD8a, using rat monoclonal antibodies (BD Pharmingen, 553028), diluted 1:200; granuphilin, using a rabbit polyclonal, diluted 1:250 (23); and Mcl1, using a rabbit polyclonal (Santa Cruz Biotechnology, Santa Cruz, CA), diluted 1:250. Incubations were performed for 2 h at room temperature (insulin, Granuphilin, Mcl1) or for 18 h at 4°C (CD8a). The sections were washed, exposed for 1 h to fluoresceinated antibodies against sheep, rat, or rabbit IgGs, counterstained with 0.03% Evans’ blue, and photographed with an Axiophot fluorescence microscope (22).
Analysis of the expression of protein-coding genes.
Total RNA extraction was performed with the RNeasy mini kit (Qiagen, Hilden, Germany). Conventional quantitative (q)RT-PCR was done as described (24). Real-time PCR reactions were performed on a Bio-Rad MyiQ Single-Color Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA) using the following primers: Proinsulin, sense 5′-TGG CTT CTT CTA CAC ACC CA-3′ and antisense 5′-TCT AGT TGC AGT AGT TCT CCA-3′; 18S sense 5′-TTT CGC TCT GGT CCG TCT TG-3′ and antisense 5′-TTC GGA ACT GAG GCC ATG AT-3′; and Mcl1 5′-AGA AAT GTG CTG GCT TT-3′. Samples were tested in triplicates and the results normalized using cDNA amplified with 18S primers in the same samples.
Modulation of miR-29 level and activity.
To increase miRNA levels, MIN6 and primary islet cells were transiently transfected with RNA duplexes (Eurogentec, Seraing, BE) corresponding to the mature sequence of the noncoding RNA. Transient transfections of MIN6 cells and islet cells were performed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. For a 24-well plate, 60 pmol/well oligonucleotide duplexes were used. The transfection efficiency in MIN6 cells and dissociated islet cells evaluated with different anti-miRs and small interfering RNAs ranged between 70 and 80%. An oligonucleotide duplex corresponding to the green fluorescent protein (GFP) sequence (5′-GAC GUA AAC GGC CAC AAG UUC-3′) was used as control. Transfection of this oligonucleotide does not affect insulin content and insulin release (Supplementary Fig. 1). Transfection of the cells with small interfering GFP (or with any other control oligonucleotide duplex) increased by about 1% the fraction of cells undergoing apoptosis compared with untreated cells.
To reduce the level of miR-29, the cells were transiently transfected with an LNA miRNA inhibitor, whose sequence is 5′-CCG ATT TCA AAT GGT GCT-3′ (Exiqon, Vedbaek, Denmark). To selectively prevent the binding of miR-29 to Mcl1 mRNA, without interfering with the action of the miRNA on other targets, we used a miScript Target Protector specifically designed for this purpose (Qiagen, Hilden, Germany) (25). The miScript Target Protector was transiently transfected in MIN6 cells with Lipofectamine 2000. The results were compared with those obtained with the Negative Control miScript Target Protector (Qiagen, Hilden, Germany).
For the assessment of the secretory capacity, MIN6 cells (2 × 105) and dissociated mouse and human islet cells (4 × 104) plated in 24- and 96-well dishes, respectively, were transiently cotransfected with oligonucleotides corresponding to the mature miRNA sequences. After 48 h, the cells were washed and preincubated for 30 min in Krebs buffer (127 mmol/L NaCl, 4.7mmol/L KCl, 1 mmol/L CaCl2, 1.2 mmol/L KH2PO4, 1.2 mmol/L MgSO4, 5 mmol/L NaHCO3, 0.1% BSA, and 25 mmol/L HEPES, pH 7.4) containing 2 mmol/L glucose. The medium was discarded, and the cells were incubated 45 min in the same buffer (basal condition) or in a Krebs buffer containing 20 mmol/L glucose (stimulatory condition). After supernatants were collected, the cells were lysed in 75% ethanol and 0.55% HCl to evaluate total insulin content. The amount of insulin in the samples was assessed by ELISA (SPI-bio, Montigny-le-Bretonneux, France).
The Mcl1 3′UTR construct was generated by inserting 29 nucleotides of the 3′UTR of human Mcl1 (CAGGCTAGTCTAACCATGGTGCTATTATA) corresponding to the binding sites of miR-29/a/b/c (in bold) between the SacI and MluI sites of pMir-Report (Ambion) (26). The mutant construct contained the same sequence except for a G-to-C replacement at position 21. Luciferase activities were measured with the dual-luciferase reporter assay system (Promega, Madison, WI). The Firefly luciferase activity was normalized for transfection efficiency with the SV40-driven Renilla activity generated by the psiCHECK-1 vector (Promega).
Protein extraction and Western blotting.
MIN6 cells or dissociated mouse islet cells were incubated for 15 min on ice in lysis buffer (50 mmol/L Tris-HCl [pH 7.5], 0.5% Triton X-100, 137.5 mmol/L NaCl, 10% glycerol, 1 mmol/L sodium vanadate, 50 mmol/L sodium fluoride, 10 mmol/L sodium pyrophosphate, 5 mmol/L EDTA) and Protease Inhibitors Cocktail (Sigma, St. Louis, MO) and then sonicated. The homogenate was centrifuged at 12,000 rpm for 1 min and the supernatant collected for further analysis. Fifty micrograms of the protein extracts were separated on acrylamide gels and transferred on polyvinylidene fluoride membranes. The membranes were incubated overnight at 4°C with primary antibodies. Immunoreactive bands were visualized by chemiluminescence (Amersham Biosciences) after incubation with horseradish peroxidase–coupled secondary antibodies for 1 h at room temperature. The antibodies against Onecut2 (amino acid 36-311) and granuphilin have been previously described (23,27). The antibody directed against Syntaxin 1a (S0664) was purchased from Sigma (Buchs, Switzerland), and that against Mcl-1 (sc-819) was from Santa Cruz Biotechnology. The antibody against actin was from Chemicon International (Temecula, CA).
Evaluation of the fraction of cells undergoing apoptosis.
For the assessment of apoptosis, MIN6 cells (1 × 105) or dissociated islet cells (8 × 104) were plated in 24-well dishes and transiently transfected with oligonucleotides leading to miRNA overexpression. Apoptosis was assessed 2 days later by scoring the cells displaying pyknotic nuclei upon Hoechst dye 33342 staining. The experiments were carried out blindly, and at least 800 cells per condition were analyzed. To distinguish between apoptosis and necrosis, the cells were seeded on glass coverslips coated with 2 mg/mL poly-l-lysin (Sigma, Buchs, Switzerland) and 33.2 μg/mL laminin (Invitrogen, Basel, Switzerland). Cells undergoing apoptosis or necrosis were identified using the Annexin V-EGFP Apoptosis Kit (Biovision, Mountain View, CA) 72 h after the transfection.
Statistical differences were tested by ANOVA. The experiments including more than two groups were first analyzed by ANOVA, and multiple comparisons of the means were then carried out using the post hoc Dunnett test, with a discriminating P value of 0.05.
We compared the global miRNA expression profile of pancreatic islets of 4- and 8-week-old NOD mice. In agreement with our previous findings (11), the islets of 8-week-old mice displayed an increase in the expression of miR-21, mir-34a, and miR-146a/b (Supplementary Table 1). In addition, we detected significant alterations in the level of several other miRNAs, including miR-142-3p, miR-142-5p, miR-150, miR-155, and miR-216a, which are almost undetectable in the islets of 4-week-old NOD mice (Supplementary Fig. 2 and 3). Because these miRNAs are expected to be particularly abundant in immune cells (28), their expression was evaluated in islets of 14-week-old, prediabetic NOD mice, which were cultured to allow for the escaping of the infiltrated lymphocytes (29). These miRNAs were 100- to 1,000-fold more abundant in the immune cells that had escaped the islets than in islet cells (Supplementary Fig. 3). In situ hybridization combined with immunofluorescence staining for insulin and CD8a (the hybridization and the immunostaining were performed on sequential sections of the very same pancreases), confirmed that most of the miR-142-3p is expressed in immune cells and not in insulin-producing cells (Supplementary Fig. 4, top panels). We concluded that the increase in the latter miRNA in the islet preparations of aging NOD animals likely reflected mainly the presence of immune cells; for this reason, this group of miRNAs was not investigated any further in this study.
Among the several other miRNAs that were differentially expressed between 4- and 8-week-old animals, we noticed the members of the miR-29 family (miR-29a/b/c) that are among the most abundant miRNAs present in normal β-cells (30) (Supplementary Table 1). These miRNAs have been shown to downregulate the expression of the β-cell disallowed gene, monocarboxylase transporter 1 (31,32) and to contribute to insulin resistance in insulin target tissues in GK rats (33). Analysis by qPCR confirmed upregulation of miR-29a/b/c in the islets of 8-week-old mice and revealed a further increase in islet preparations of normoglycemic 13- to 14-week-old mice (Fig. 1A). The level of expression of miR-29a/b/c in lymphocytes escaping the islets of NOD mice was comparable to the level detected in the islets of 4-week-old mice (Fig. 1B). Moreover, islets of 14-week-old mice incubated for 24 h to permit the escape of infiltrating lymphocytes retained strongly elevated levels of miR-29a/b/c (Fig. 1B). In agreement with these observations, in situ hybridization of NOD pancreas at different ages, combined with an immunofluorescence staining for insulin on close by sections, confirmed that the bulk of miR-29a (Supplementary Fig. 4, lower panels, and Supplementary Fig. 5) and miR-29b (not shown) is concentrated in insulin-producing cells and not in infiltrating immune cells.
Taken together, these findings indicate that the rise of miR-29a/b/c reflects changes occurring in β-cells and is not merely the result of the presence of immune cells in the islets. The observed increase in miR-29 expression is also not the result of mice aging. Indeed, miR-29a/b/c levels were not significantly different between the islets of 6- and 12-week-old C57BL/6 mice (data not shown), and no significant changes were observed in the islets of 13-week-old NOD-SCID mice that do not develop type 1 diabetes. In contrast, proinsulin 2-deficient mice, which display accelerated insulitis and diabetes (34), showed a dramatic rise in miR-29a/b/c expression already at age 8 weeks (Supplementary Fig. 6).
Incubation of MIN6 cells (Fig. 2A), mouse islets (Fig. 2B), or dissociated human islet cells (Fig. 2C) in the presence of Il-1β (0.1 ng/mL; 50 units/mL), TNF-α (10 ng/mL; 500 unit/mL), and IFN-γ (30 ng/mL; 50 units/mL) led to a twofold increase in the expression of the three miR-29 family members, suggesting that at least part of the observed induction occurring during early stages of diabetes development in NOD mice was linked to the chronic exposure of β-cells to these proinflammatory cytokines.
To test the effect of miR-29a/b/c overexpression on pancreatic β-cell functions, oligonucleotides mimicking the mature sequence of each of the miR-29 family members were transiently transfected into MIN6 cells. This transfection resulted in a cellular content of the respective miRNA, which was comparable to that observed in the islets of 8-week-old NOD mice (Supplementary Fig. 7). We first assessed the effect of alterations in the level of the three miRNAs on insulin biosynthesis. Overexpression of miR-29 family members led to a small decrease in proinsulin mRNA levels but did not significantly modify total insulin content (Fig. 3). We then investigated the effects on insulin release. The rise of miR-29a/b/c in MIN6 cells did not affect basal insulin secretion (Fig. 4A). In contrast, glucose-induced insulin release was decreased by 20–40%. A similar reduction in insulin secretion elicited in the presence of glucose was detected upon overexpression of miR-29 family members in primary mouse islet cells (Fig. 4B) and in primary human islet cells (Supplementary Fig. 8).
The transcription factor Onecut2 is a predicted target of miR-29a/b/c (http://www.targetscan.org) (35). We previously demonstrated that Onecut2 modulates the expression of granuphilin, a secretory granule protein acting as a potent inhibitor of insulin exocytosis (8) that is expressed in all β-cells of nondamaged islets, irrespective of the age of NOD mice (data not shown). In agreement with the computational predictions, transfections under the conditions that led to miR-29a/b/c overexpression resulted in a decrease in the level of Onecut2 and a consequent rise in the expression of granuphilin (Fig. 5) in MIN6 cells and in dissociated mouse islet cells, potentially explaining at least part of the effect of miR-29 family members on glucose-induced insulin release. In contrast, we were unable to detect significant changes in the level of Syntaxin 1a, a key component of the machinery of insulin exocytosis (36,37), which is also identified by computational programs as a potential target of miR-29 (Fig. 5).
The effect of elevated levels of miR-29 family members on cell survival was next investigated. Overexpression of miR-29a/b/c in MIN6 cells (Fig. 6A, left panel) and in mouse (Fig. 6B, left panel) and human dissociated islet cells (Fig. 6C) led to a significant increase in cell death, as assessed by scoring the cells displaying pyknotic nuclei. Identical results were obtained when apoptosis was assessed by Annexin V labeling (Fig. 6A and B, right panels). In contrast, the number of necrotic cells determined by propidium iodide labeling remained unchanged (data not shown). Pretreatment with anti–miR-29c significantly decreased, to control levels, the number of MIN6 cells undergoing apoptosis in the presence of IL-1β, TNF-α, and IFN-γ (Fig. 6D).
We then attempted to elucidate the mechanisms through which a rise in the level of miR-29a/b/c can trigger β-cell death. Interestingly, Mcl1, an antiapoptotic protein belonging to the Bcl2 family and playing a key role in β-cell survival (38), is a predicted target of miR-29a/b/c (26,39). Indeed, we found that increased levels of miR-29a/b/c inhibited the expression by MIN6 cells of a luciferase construct containing the 3′UTR sequence of Mcl1 (Fig. 7A). This effect was abrogated by mutation of the sequence recognized by these miRNAs, indicating a direct interaction with the 3′UTR of Mcl1. Western blot analysis confirmed a significant reduction of the level of Mcl1 in MIN6 cells and in mouse islet cells overexpressing the miR-29 family members (Fig. 7B and C). No significant changes in MCL1 mRNA levels were detected in miR-29 overexpressing cells, indicating that the effect of the miRNAs is mainly post-transcriptional (Supplementary Fig. 9).
To demonstrate a direct link between the reduction of Mcl1 and the effect of miR-29 on β-cell apoptosis, MIN6 cells were transfected with an oligonucleotide designed to specifically protect the Mcl1 mRNA from miR-29 binding, without interfering with the regulation of other miR-29 targets (25). The reduction in Mcl1 levels elicited by miR-29c overexpression or by proinflammatory cytokines was efficiently prevented by transfection of this target protector (Supplementary Fig. 10A and B). As shown in Fig. 8A, the cells expressing the Mcl1 target protector were resistant to apoptosis induced by miR-29 overexpression. Moreover, the cells transfected with the Mcl1 target protector were not sensitive to cytokine-induced apoptosis (Fig. 8B), indicating that at least part of the deleterious effect of these inflammatory mediators on β-cell survival is caused by a miR-29–induced drop in the expression of the antiapoptotic Mc11 protein.
During the initial phases of type 1 diabetes, pancreatic β-cells are chronically exposed to inflammatory mediators released by immune cells invading the islets of Langerhans. This results in major changes in gene expression, with consequent defects in the accomplishment of specialized functions, and loss of the cells by apoptosis (1). We recently demonstrated that part of the changes in gene expression triggered by proinflammatory cytokines reflects alterations in the level of a new class of regulatory molecules, the miRNAs (11). Indeed, we found that prolonged exposure to IL-1β or TNF-α results in a strong upregulation of miR-21, miR-34a, and miR-146a, with large effects on the secretory capacities of β-cells and their sensitivity to apoptosis (11). In this study, we confirm these findings and further extend them via the identification of additional miRNAs, whose expression level is markedly altered with the onset of type 1 diabetes in NOD mice. The members of the miR-29 family, which are among the most abundant miRNAs expressed in mouse pancreatic β-cell (11,30), increased with the age of prediabetic but still normoglycemic NOD mice. In situ hybridization and qPCR analysis, coupled with immunofluorescence for insulin, demonstrated that the rise in miR-29 expression occurred in β-cells and not in invading immune cells.
The mechanism responsible for this induction of miR-29a/b/c in NOD mice remains to be fully elucidated. We found that exposure of MIN6 cells and human islets to proinflammatory cytokines causes a twofold increase in the expression of miR-29 family members. These results are in good agreement with those obtained previously by microarray analyses (11). The signaling pathways involved in the induction of miR-29 family members have not been dissected yet. Zhou et al. (40) have recently identified in the promoter of miR-29a potential binding sites for NFκB, a transcription factor known to contribute to cytokine-mediated β-cell dysfunction and to the development of type 1 diabetes (41). Expression of miR-29 was also reported to be modulated by p53 (42), a transcription factor that is induced in response to proinflammatory cytokines (43) and that we have shown controls the expression of other miRNAs (7). Additional cytokines, such as IL-4 and TGF-β, which are defectively produced in NOD mice (44–46), have been reported to inhibit miR-29 expression in other cell systems (47). A deficit of these cytokines may potentially contribute to the strong upregulation of miR-29a/b/c that is observed in the islets of prediabetic NOD mice.
Our results demonstrate that miR-29 overexpression downregulates glucose-induced insulin secretion of primary mouse and human islet cells as well as of MIN6 cells. This downregulation is associated with the interaction of the miRNAs with Onecut2, one of their putative targets. This transcription factor controls the expression of granuphilin/Sytl-4 (8), a Rab guanine triphosphate (GTP)ase effector that colocalizes with the dense-core, insulin-containing granules of pancreatic β-cells and acts as a potent inhibitor of insulin exocytosis (23,48). Thus, by decreasing Onecut2 levels, miR-29 enables the expression of increased amounts of granuphilin, resulting in impaired stimulation of insulin secretion. Consistent with this mechanism, we found that granuphilin is abundant in most β-cells, irrespective of the age of the NOD mice, and the presence of peri- or intraislet insulitis.
Prolonged exposure to proinflammatory cytokines is known to affect β-cell survival. Overexpression of miR-29a, -b, or -c to levels comparable to those observed in 8-week-old NOD mice mimics the effect of the cytokines. Moreover, blockade of miR-29 protects β-cells from the deleterious effect of these inflammatory mediators. In line with results obtained in other cell types (26,39,49), Western blot analysis and luciferase reporter assays demonstrated a decrease in the level of the antiapoptotic protein Mcl1 on miR-29 overexpression. Downregulation of this antiapoptotic protein is a crucial event, leading to β-cell apoptosis in response to cytokines (38). Our data are in good agreement with these observations and suggest that, as it is the case in other cell systems, even a modest reduction of Mcl1 levels caused by miR-29 overexpression can result in loss of mitochondrial potential, cytochrome c release, and activation of caspase-3 (39). Indeed, an oligonucleotide specifically designed to mask the binding site of miR-29 on Mcl1 3′UTR, without affecting the stability or the translation of the mRNA (25), restored the expression of the antiapoptotic protein, and prevented both miR-29– and cytokine-mediated apoptosis. Our findings suggest that molecules capable of protecting individual targets represent attractive tools to precisely dissect the mode of action of miRNAs and could also serve as therapeutic strategies to selectively promote the expression of single genes beneficial for β-cells.
The miR-29 family members have been shown to contribute to insulin resistance in insulin target tissues of GK rats (33). Interestingly, type 1 diabetes in NOD mice is associated with insulin resistance (50). Moreover, miR-29 has recently been reported to inhibit innate and adaptive immune responses by suppressing the production of IFN-γ in natural killer cells and T lymphocytes (51). Thus, changes in miR-29 expression may play a broader role in the pathogenesis of type 1 diabetes.
The current study was carried out in NOD mice, an animal model sharing many characteristics of type 1 diabetes in humans and that has significantly contributed to our understanding of the molecular processes associated with the development of this disease (52). It will now be important to confirm that the mechanisms identified in NOD mice and β-cell lines are indeed also operating during the initial phases of type 1 diabetes in humans. Should this turn out to be the case, a better knowledge of islet miRNA dysfunction may open the way to alternative strategies to delay and prevent the development of the disease in newly diagnosed type 1 diabetic patients.
See accompanying commentary, p. 1661.
The team led by R.R. is supported by the Swiss National Science Foundation Grant 31003A-127254. The team led by P.M. is supported by grants from the Swiss National Science Foundation (310000-141162, CR32I3_129987, IZ7320-127935), the Juvenile Diabetes Research Foundation (40-2011-11), and the European Union (BETAIMAGE 222980, IMIDIA, C2008-T7).
No potential conflicts of interest relevant to this article were reported.
E.R. researched data, contributed to discussion, and wrote the manuscript. S.G. and D.C. researched data. C.Br. researched data and contributed to discussion. C.Bo. contributed to discussion. P.M. researched data, contributed to discussion, and reviewed and edited the manuscript. R.R. contributed to discussion, and wrote, reviewed, and edited the manuscript. R.R. is the guarantor of this work and, as such, had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
The authors are grateful to Dr. Justin Mott (Mayo Clinic, Minneapolis, MN) for providing the luciferase reporter plasmids containing the wild-type and the mutated form of the 3′UTR of Mcl1 and to Dr. Patrick Jacquemin (Université catholique de Louvain, Brussels, Belgium) for generously supplying the antibody against Onecut2.


KW Automotive Increases Development for Electric Cars
People from many different markets have been purchasing electric cars. According to various media reports, all-electric vehicles have overtaken diesel engines in new car registrations in Germany. KW automotive, a suspension manufacturer specialising in the aftermarket, also benefits from this ongoing electric car boom.
Many electric car owners buy automotive aftermarket accessories like light-alloy wheels and Coilover suspension kits. KW automotive has been developing coilovers for electric vehicles to keep up with the demand. The KW V3 Coilover suspension and the adaptive KW DDC plug & play Coilover suspension are now available for the new Cupra Born (K1).
The electric car can be lowered by 20 to 45 millimetres with the height-adjustable coilovers within the scope of the parts certificate. While the KW V3 can be adjusted manually in terms of damping, the DDC plug&play with its electric damper control is recommended for all Cupra Born with optional DCC standard chassis control. The two KW coilovers made in Germany from stainless steel are available now.
KW automotive has already released its adaptive KW DDC plug & play Coilover suspensions for over 150 vehicle models. Electronic damping-adjustable breaks are also available for the new Cupra Born with parts certification. Compatible with the optional DCC damper control, the Coilover suspension constantly adjusts its damping to the road and driving style.
The signals from the body and wheel position sensors are processed by the OEM control unit of the Cupra Born (K1), and the software then adjusts the KW DDC plug & play Coilover suspension accordingly per wheel. The acceleration, braking and steering are also considered.
“Because our KW DDC suspensions are compatible with the DCC damper control used by the VAG Group, the various driving modes that influence the damper can still be used,” says KW Brand Manager Florian Johann.
The electromagnetic damper valves change the setup and switch to tighter damping depending on the driving situation.
“The less unrest in the body structure, the less the ESP has to regulate,” explains Florian Johann.
The best-selling aftermarket suspension from KW automotive is the KW V3 or KW Variant 3 Coilover suspension. These dampers differ from conventional suspension dampers by using multi-valve technology. This means that the suspension’s damping can be adjusted individually by hand.
“However, the more harmoniously an electric car is damped, the range can also benefit. In the Tesla Model 3, electric mobility specialists achieved a seven per cent increase in range by installing our Coilover suspension,” says Florian Johann from KW Brand Management.
Like the electronically damping-adjustable KW DDC plug & play Coilover suspension, the KW V3 Coilover suspension allows stepless lowering of the front axle and rear axle of the Cupra Born from 20 to 45 millimetres.
UP IN NEWS
- Volvo XC60 Named Best Luxury Compact Crossover by Good Housekeeping
- Subaru Earns Four Awards in Kelley Blue Book 2022 5-Year Cost to Own Awards
- Formula E Gen3 Race Car to Be Unveiled in Monaco
- Grand Wagoneer Earns Best SUV of the Year Award from the Hispanic Motor Press Foundation
- Volvo Car USA Showcases the All-Electric C40 Recharge in the “Recharge Garage.”
For all the latest automotive news, reports, and reviews, follow us on Twitter, like us on Facebook, subscribe to our YouTube page, and follow us on Instagram, updated daily.
Stay Ahead of the Curve
Unlock the World’s Leading Source of Automotive News and Analysis.
Autoscommunity.com provides innovative marketing and advertising solutions to support an advertiser’s specific campaign objectives. Customised programs leverage the best of Autoscommunity.com. Contact our sales team today and see what our team can do for your custom advertising solutions.
Please include a contact number if you are willing to speak to our sales team. You can also get in touch in the following ways:
WhatsApp: +971 54 453 94 67
Email: [email protected]
Comments are closed.


- The basis of The Phil Hendrie Show, which appears on the surface to be a rather standard AM radio call-in talk show, albeit with some notably off-the-wall guests. It's customary for such shows to allow listeners to call in to give the host and guest a piece of their mind; and given the extreme craziness of the guests, the callers get pretty vociferous. However, despite the hourly disclaimers that the whole thing is a put-on, the callers fail to realize that the "guests" are actually fictional creations of voice actor Hendrie, and that they are the butts of what is essentially a prank phone call in reverse. If they even manage to notice that the host and guest sound rather alike, they are too deep in their Debate and Switch, Insane Troll Logic argument to get out.
- An episode of John Finnemore's Souvenir Programme has a man interviewing the Three Little Pigs about why they've chosen their respective building materials. The first pig thinks he's in a story where the Youngest Child Wins through honest poverty while the other brothers will be undone by their hubris, so he builds a straw house. The second pig thinks they're in a Goldilocks-style story where one house will be too weak, one too strong ("How can a house be too strong?" "Umm... subsidence?") and his house of sticks will be the middle way which works the best. The third brother doesn't read many Fairy Tales, and built his house out of bricks because it's a house, and that's what you build houses out of.
- On NPR's weekend news quiz show Wait Wait... Don't Tell Me!, if host Peter Sagal suggests that you change your answer, your answer is wrong. Oftentimes the celebrity guests for the "Not My Job" segment* don't listen to the show regularly and assume he's trying to trip them up.
Wrong Genre Savvy / Radio


text:
BOULDER, Colo. — Mike Gilliland, founder and chief executive officer of Sunflower Farmers Market, has been selected as a 2010 Entrepreneur of Distinction by the Boulder Chamber of Commerce Esprit Awards committee, which recognizes and celebrates the outstanding spirit of entrepreneurship in Boulder County. Gilliland will receive his award at the 26th Annual Esprit Entrepreneur Banquet next week.
Gilliland has been involved in the natural and organic food business for more than 24 years. In 2002, Mike founded Sunflower Farmers Markets, which operates 30 natural food supermarkets throughout Colorado, Arizona, New Mexico, Nevada, Utah and Texas. Gilliland also operates a 40-acre farm in Longmont, Colo., that provides produce for some Sunflower locations.

domain: geography


text:
Earlier this week we welcomed in sweet Eliza after we sprung her out of a shelter when she was found stray and never reclaimed 💔 We don’t know Eliza’s story and don’t have any history or information on her, but we do know she has medical needs that have been neglected.
Eliza has undergone her full intake vetting where it was determined she has “dry eye” with secondary infection, inflammation, and a corneal ulcer. She also has ear infections and a urinary tract infection. Most concerning, she has a growth from her rectum which has ulcerated. We are hoping this is just a benign polyp.
We have started her on medicated eye drops to heal her corneal ulcer, treat the infection, reduce the inflammation, and a tear stimulant to help boost her tear production. We are lubricating her eyes around the clock to help them comfortable and hydrated. We have also started her on ear medication for her ear infections and oral antibiotics for her urinary tract infection. She will soon be scheduled for her spay surgery, much needed dental to address some broken teeth, and rectal growth removal (and send it out for testing).
Sweet Eliza is getting settled in to her foster home in Dana Point, California where she seems to be responding very well to all her treatment and the abundance of TLC she is receiving 💜 She will be as good as new in no time once all her infections have cleared, growth removed, and her dry eye well managed. She is in good orthopedic and cardiovascular shape with clear knees, hips, and heart. We haven’t made an age determination yet but feel she is a middle aged adult. More to come on this darling girl soon 💕
UPDATE JANUARY 13, 2021: Beautiful Miss Eliza, who we recently sprung out of a shelter, has made herself comfy in her foster home in Dana Point, California 💕 She seems to be responding very well to all her treatment as all her infections are visibly improving. Friday is her big day as she will undergo her spay, dental and removal of the growth on her rectum which we know she is very ready to get rid of! We will be sure to keep you posted 💜

domain: medical


There aren’t many people walking the planet who can work a microphone better than Paul Heyman or Bruce Buffer. Both men put their skills to the test in announcing Brock Lesnar at some point, but those days are now over.
For Buffer, Lesnar hasn’t competed in the UFC since 2016, which means that the ring announcing icon did indeed introduce Lesnar one more time after the following story was published.
As for Heyman, he is now busy acting as special counsel for your tribal chief, Roman Reigns, who not unlike this article from seven years ago, is set to headline this year’s WrestleMania against Brock Lesnar. So Heyman is no longer “advocating” for “The Beast Incarnate” and former UFC champion.
But who introduces Brock Lesnar better: Paul Heyman or Bruce Buffer? You be the judge by checking out this story from seven years ago.
The following article was published on this day seven years ago. It is presented to you in its original, unaltered form, courtesy of The MMA News Archives.
On This Day Seven Years Ago…
[ORIGINALLY PUBLISHED MARCH 27, 2015, 3:56 PM]
On the latest edition of the “Heyman Hustle,” UFC Octagon announcer Bruce Buffer does one final UFC-style Brock Lesnar introduction for old time’s sake.
Check out the video below to watch as Buffer does his UFC-style introduction for the former UFC Heavyweight Champion as his WWE on-air “advocate,” Paul Heyman joins in with his own Brock introduction.
Brock Lesnar, who recently announced his MMA retirement after revealing that he has signed a multi-year deal to remain in WWE, defends his WWE World Heavyweight Championship against Roman Reigns in the main event of Sunday’s WrestleMania 31 pay-per-view at Levi’s Stadium in Santa Clara, California.
You can check out more episodes of the Heyman Hustle online at YouTube.com.
Who did a better job at announcing Brock Lesnar? Paul Heyman or Bruce Buffer?


Your Free Press
Thursday, January 9, 2014
Mayoral Ally Elected Speaker, Furthering City’s Liberal Shift
Richard Perry/The New York Times
Melissa Mark-Viverito with Antonio Reynoso at City Hall before the meeting started on Wednesday.
By MICHAEL M. GRYNBAUM and KATE TAYLOR
The election of Melissa Mark-Viverito as speaker of the City Council marked another swift and sharp shift in New York City’s political landscape.
Rafael Martinez Alequin
Post a Comment
Post Comments (Atom)


text:
It's been too long since I've written an art review! I really like how dark this picture is. Well, not dark in its light quality. Wait, you have "dark" right in the tag. Of course, you know what I mean! This guy almost looks like a snowman.
I'm not going to make an Olaf joke. "Frozen" wasn't even out at the time of this picture. I think this chick's toes are digging into the ground. That sounds kind of erotic. It looks like you're not into color, but this is still nice.

domain: artistic


text:
Where are the top places to take a gondola ride?
There are several places where you can take a gondola ride in Venice, depending on your preferences and budget. But the most popular spots are 5.
The 5 top places to take a gondola ride:
- Piazza San Marco – This is one of the most popular spots in Venice to take a gondola ride. The gondolas here offer stunning views of the Doge’s Palace, the Campanile, and the Basilica di San Marco. You can find several gondoliers waiting for passengers near the waterfront in the square.
- Rialto Bridge – The Rialto Bridge is another popular spot to catch a gondola ride in Venice. You can find several gondoliers waiting for passengers near the bridge, particularly on the San Polo side. This area is known for its bustling market and beautiful views of the Grand Canal.
- Santa Maria del Giglio – This area is located near the Teatro La Fenice and is known for its picturesque canals and quiet atmosphere. It’s a great place to take a gondola if you’re looking for a more romantic experience away from the crowds.
- San Polo – San Polo is another area that’s popular for gondola rides. You can find several gondoliers near the San Polo church. This area is known for its narrow canals and stunning architecture.
- Ghetto – If you’re looking for a more unique gondola experience, on a different boat called sandolo, consider taking a ride along the Jewish Ghetto. You can find gondoliers near the Ponte del Ghetto Vecchio.

domain: travel


text:
Sightseeing around Napier and Hastings? Stop in for some lunch
and sample some of Hawkes Bay’s finest food and beverages.
As much as we possibly can, we source local, ethical and organic produce. We have vegan and gluten free options available, along with organic and ethically sourced coffee.

domain: food


text:
THE ILLUMINATING ENGINEERING SOCIETY OF NEW YORK'S (IESNY) 8TH ANNUAL NYC STUDENT LIGHTING COMPETITION invited students of interior design, engineering, architecture, lighting, and art to participate in and explore how light can be used to activate an emotion or rekindle a memory, hence the title of this year's competition, “Evocative Luminance.”
More than 25 entries from six New York area schools and universities—New York University, Pratt Institute, Parsons the New School for Design, the Fashion Institute of Technology, the Cooper Union for the Advancement of the Arts, and the School of Visual Arts—were displayed from May 17-19, 2008, at Helen Mills, an event space on West 26th Street in Manhattan's Chelsea arts district.
First place went to Chung-Jung Liao, a second-year MFA candidate in the lighting program at Parsons the New School for Design. His project, titled Barrel Organ of Light, employed a briefcase as the structural housing and allowed users to arrange black perforated paper sheets as they liked within the case by turning an attached handle. The effect created was one of twinkling stars moving across a dark surface. Liao was awarded a cash prize of $3,000 and an all-expense paid trip to the 2008 Professional Lighting Designers Association (PLDA) workshop in Alingsas, Sweden. Liao was also the first place winner in the 2007 IESNY student competition.
Paul Stein took second prize for his entry For the Child Inside Us All. Using Lego building blocks, Stein created a Lego man holding block pieces in its left hand and an illuminated Lego-made lightbulb in its right hand. The bachelor of architecture student at Pratt Institute in Brooklyn illuminated his piece with a 30W compact fluorescent lamp encased in translucent Lego pieces. Stein received a $1,500 cash prize for his entry, which focused on the creative ideas of children and sought to remind viewers that you are never too old to think like a child.
New York University Interactive Telecommunications student Minsoo Lee received third place for his project Reverie. Composed of laminated layers of corrugated plastic and illuminated by five compact fluorescent sources, Lee built only one section of what could be a much larger construction. This was indicated by an accompanying sketch illustrating how his lighting sculpture could be replicated and pieced together like a puzzle. A $1,000 cash prize was awarded to Lee for his efforts.
Three honorable mentions also were awarded for specific components of a project—fabrication, concept, and presentation. The recipients included Nick Foley, an undergraduate industrial design candidate from Pratt Institute for the fabrication of his project a flower-like floor lamp that arches into a spray of illuminated orbs that dim in varying intensities with the push of a button; Paul Chepolis, an MFA candidate at Parsons for his project's concept which explored the subtleties of light, using projection, reflection, movement and color; and Bomun Chang an MA candidate at the Fashion Institute of Technology for the presentation of his project titled Heavenly Flower.
The jury for this year's student competition included New York City-based lighting designers Renee Cooley, principal of Cooley Monato Studio; Enrique Peiniger, principal of Office for Visual Interaction; Donna Summer, senior associate at Susan Brady Lighting Design Studio, and Matthew Tirschwell, founder and principal of Tirschwell & Co., along with Dennis McKee, director of special projects and corporate affairs at Bartco Lighting. Awards were presented on Thursday, April 17, 2008, after a brief presentation by keynote speaker Hervé Descottes, co-founder of lighting design firm L'Observatoire International located in New York City.
Descottes spoke on the topic of the competition—how light can spark an emotion or a memory. He discussed his own inspirations and emotional responses to his projects such as the High Line (the conversion of the fallow elevated railway along Manhattan's west side into public open spaces). “Lighting is very personal as we have our own way of organizing it,” Descottes said. Also during the evening, Peiniger presented past projects completed by PLDA lighting workshops in Alingsas, Sweden. For more information about the IESNY student competition go to www.iesny.org.vaaztstrffwcduxcycbwauvxxzx


About Budda (twitter: @BuddaT_)
Last minute post to say that I’m in.
Spent most of yesterday setting stuff up to get ready, prepped all the distribution stuff so I’m not fussing about with that on Sunday – pretty lucky that I did since it took about 5 hours to get it working how I wanted.
But anyway, I’m in, and I’ll be using the following:
Libs: LWJGL, Slick
Food: Lots of crap and energy drinks
I might go and look at setting up a timelapse similar to my LD23 entry too…hmm
Good luck everyone!
I guess I’m in with a theme like that. Makes it easier for us programmer types that have no talent for art.
Will be using Java, LWJGL, Slick and various image/sound tools depending on how I feel at the time.,
Already have an idea in mind that I’ve been thinking about for a while, that could tie nicely into the theme if done right. We’ll see how I go.
Good luck everyone!
So I’m pulling out before this gets hairy.
I’ve spent the last 12 hours trying to get this game going, but I’ve just run into brick wall after brick wall with no end in sight.
I wanted to get a fluid game going with some nice GLSL dynamic lighting and physics, but just starting at the lighting threw me off. I’ve done some GLSL lighting before, but everything I tried to do this time just had something wrong with it. The first problem was Slick not rendering it’s first loadded texture for some (still) unknown reason. Second problem being the point lighting (based off the mouse position) not lining up the the display area – so when the mouse was at 0,0 the light was at 0,0; but when the mouse was at 400,400, the light was at 800,600.
Once I hit this wall I just decided to crap what I had and start fresh so I could see where I went wrong with the lighting positions. That has just led me around in circles for the last 3 hours to the point where the light just isn’t rendering at all.
I’m going to keep working on this, and probably extend everything into a framework so I’m not pissing around with it all for the next LD – but I won’t be entering anything this time around unfortunately.
I think I need a drink.
So I’ve run into my first (second) hiccup already.
In the image below contains two applets running. The first one (top one) is running where the dummyImage line is not commented out. The second (bottom) applet is running with the dummyImage line commented.
This may seem alright to you, but:
- dummyImage is an empty 1×1 image
- backgroundImage is the pink image
- the clear colour is black (not white) – the white you see in the second image is the backgroundImage being drawn without a texture
Does anybody have an idea of what is happening? (P.S. this is Java, LWJGL and Slick2D)
So I’m entering LD24.
This will be my second LD, looking forward to it. Got a few ideas already, hopefully the theme turns out to support one of them, otherwise I’ll have to think up something new when I wake up.
As per LD23, I’ll be using Java, LWJGL, Photoshop, and whatever else I feel I need. Probably something for sounds, not sure what yet.
Good luck all!
Well theres about 6 hours to go, and I am done. Well as done as I’ll ever be.
I have just submitted my game, BioBattle.
The timelapse that I have been recording is listed on that entry page too.
Here’s a preview pic of the final version:
I had a couple of hours earlier today where I got pretty much nothing done. It was at this time where I was mainly working on pseudo-GUI stuff, which is extremely boring.
I managed to push through it and get it done however, as it was critical to the main ‘gimmick’ of my game. That gimmick being the ability to create your own bacteria out of various parts to use in battle.
It’s starting to come along nicely if I do say so myself, although I will have to do something about the graphics.
As per my last post, the latest(ish) version can be played here: http://buddat.net/ld23
So I’ve been sitting here for 16 hours trying to work though this game. Admittedly I haven’t accomplished much in the last 4 hours as my brain turned to mush, so I am going to go get a few hours sleep.
I do have a recent build up for play-testing though, only Quick Play is currently working:
So we’ve just past the 8 hour mark, and my brain is slowly starting to melt.
I had a couple of hours there where stuff would just not end. I set up a system to render certain Strings on the screen, then realised I had no idea what those Strings were. from there I spent 2 hours building the framework for my game, just so I could get those strings to test it all.
Frameworks man, frameworks.
Anyway, now I have something small to show, a bit of String rendering, but it contains a good chunk of the gameplay framework just to get there
So I haven’t really said anything about this game yet, so let this be a small explanation.
It is based off a game design from 2001-ish, where you build your own ‘monsters’ and depending on the parts you use, they get certain skills that are used to battle other monsters. In my version however, the monsters are bacteria. Tiny World? Micro World!
Dinner comes soon, then I hope to get some basic battles going before I focus on rendering some ugly graphics instead of nothing.
So it’s almost 5 hours in, and I’ve honestly not done much that I can show.
I have a title screen for the start of the game which I threw together while I was thinking about the actual gameplay. I have a basic state-based system for updates and rendering, and I’ve just now started on the mammoth effort of coding the gameplay.
I hope to get the basic design of the gameplay in before I have a crack and getting something up and rendering. I figure if I spend the first hours where I’m not sleep-deprived writing code, I can spend the groggy wee-hours drawing crappy graphics and making shitty music to go with it.
Should be fun
I was a bit surprised to see the theme be Tiny World. I had a whole game lined up for some of the other themes, but Tiny World had me stumped.
Luckily I figured out a way to…sort of mold my game idea around the theme, so it still fits.
I am currently well underway with coding, working on the title screen and text rendering at the moment, hoping it wont take me too much longer else I’ll not get much of the game done.
This will be my first Ludum Dare, so I’m not too sure how I’ll manage with cramming everything into the 48 hours.
I’ve decided to use Java and LWJGL. I haven’t started a game from scratch using LWJGL before, but have used it while helping out with other games, so I hope it’ll be a good learning experience for me.
Should be fun nonetheless!


text:
Boluwatife Arowosafe, a 14-year-old student of Olashore International School, Iloko, Osun State Nigeria, has emerged as a winner of The Diana Award.
The award, which is in memory of Diana, Princess of Wales, recognises young people globally for their social action and humanitarian work.
In his address during the virtual awards ceremony, Prince Harry said in the face of all the negative occurrences globally, Boluwatife and other young winners, personally chosen by Prince Harry and Prince William, the Duke of Cambridge, were a source of hope for the future.
According to Prince Harry: “Right now, we’re seeing situations around the world where division, isolation and anger are dominating as pain and trauma come to the surface.
“But I see the greatest hope in people like you and I’m confident about the world’s future and its ability to heal because it is in your hands.”
Boluwatife, a Year 11 student, has championed projects in child rights, youth empowerment, poverty alleviation in her local community.
Specifically, Boluwatife has supported the project: “Our Voices Have to be Heard,” an initiative with a classmate, Adjwoa Awe, also a winner, to raise awareness of children’s rights and abuse.
Their project, which started in their school, Olashore International, has now expanded into the local community through the initiative: “Healing Every Living Person,” which aims to combat poverty and lack of opportunities by equipping 16-year-olds to 21-year-olds with life skills and entrepreneurial training.
The H.E.L.P initiative has already offered hope to more than 100 young people, with its free skills training and education reaching those who may have turned to crime as a way out of poverty.
Speaking further on the award, the CEO of The Diana Award, Tessy Ojo, congratulated winners who she characterised as change makers for their generation.
Ojo said: “We know by receiving this honour they will inspire more young people to get involved in their communities and begin their own journey as active citizens.”
For over 20 years, The Diana Award has valued and invested in young people encouraging them to continue to make positive change in their communities and lives of others.

domain: sociology


- Shopping Bag ( 0 items )
Posted October 1, 2010
Spohr's music is just plain good. He puts early romanticism in a bright light. His accomplishment is comparable to what Cherubini does for the transition from neoclassicism to romanticism in his operas. Spohr's own operas Faust and Jessonda are top-flight despite his failure to deliver anything exotic for the East Indian setting of Jessonda. How did the music of these two composers ever fall into neglect? I prefer Cherubini's Coronation Mass to Beethoven's overwrought Missa Solemnis ten to one although Beethoven's 9th symphony from the same period is one of the greatest of all compositions. Spohr's Violin Concerto No. 7 in E minor ranks with the best of its genre from the 19th century and should at least be compared with the famous one by Mendelssohn in the same key. Spohr's first movement is as solid an instrumental composition as I can hope to hear. The slow movement is a model of romantic lyricism and the finale is as entertaining as these light movements ever are. Nishizaki's performance strikes me as impeccable but I leave performances to others. What I hear is the composers' works.Was this review helpful? Yes NoThank you for your feedback. Report this reviewThank you, this review has been flagged.


text:
Land & Sea Necklace
"Ae Kai" is Hawaiian for Land & Sea. Our Ae Kai necklace captures where the land meets the sea. Reminding us of sunny beach days and memories shared along our island shores.
- Materials: 14k gold or sterling silver plated on brass.
- Size: 0.75"
- Length 18" silver, gold plated 16-18" adjustable.
Designed & made by Leighton Lam Designs in Hawaii. Original copyright design.

domain: design


text:
In Your Bag
OIA DRESS FERN
100% Bamboo - OEKO-TEX® STANDARD 100 certified
With her front cut-out adorned with a wooden bead neckline, Oia embraces the elegance of summer. The striking open back and high-cut leg slit, create subtle flashes of skin that demand as much attention as the dress itself. Designed with versatility in mind, Oia can be dressed up for a candle-lit dinner or down for wandering beach strolls.
Fabric & Fit Details: Fits true to size. Made from a double-layered bamboo knit that has stretch. It is lightweight and breathable. The bamboo fibers are as soft as butter; you can barely feel the dress against your skin.
Fabric Content: 100% Bamboo - OEKO-TEX® STANDARD 100 certified
Fabric Country of Origin: Made by knitting partners in India.
Garment Production: Produced locally in LA at our in-house Savannah Morrow production studio next to our HQ.
Sustainable Fabric Details: Our bamboo is OEKO-TEX® STANDARD 100 certified and sourced from manageable farms. It requires zero fertilizers/pesticides to grow. Pesticides, especially in synthetic form, kill the microorganisms in the soil, robbing the soil of nutrients significantly decreasing its ability to feed the plants and control the water flow. Our bamboo keeps the soil free of such interventions, allowing it to naturally regenerate. As a result, bamboo needs significantly less water than most crops, such as cotton. Moreover, bamboo is a carbon dioxide-absorbing plant that helps to clean our atmosphere. Bamboo contains very high antibacterial properties. It utilizes thermal control through its hollow microfiber structure allowing it to be naturally warm, yet breathable. Bamboo is also one of the best fibers for absorbing wet moisture, comfortable & safe for your skin.
Care Details: Dry clean only.
FREE express worldwide shipping on all orders over $600 USD.
FREE standard shipping and FREE returns on all US orders over $250 USD.
Bio-degradable & compostable packaging.
For further information please read our shipping and return policies, located at the bottom of our site.


text:
Thursday, May 31, 2012
Within the Dark Hills - Sian Ann Bessey
I was so excited to read this book. I loved how we got the in depth lesson about Welsh mining in the 1800's. And I have to say that I LOVED the first part of this book.
We start with Annie Morgan, a young chambermaid running for her life, after being accosted by a wealthy mine owner. She runs through a terrible rain storm and runs to the only place she can think to go, the home of the town reverend. She is warmly embraced by the reverend and his wife, and is ready to collapse when the alarm sounds at the mine. At this point there is no time to rest. There is only the desire to rescue and care for those who are trapped miles below the surface.
Evan Powell is a miner who becomes buried beneath the rubble with his partner Hugh. They wiggle through their tunnel only to trip over the legs of a very young boy under a pile of rubble. Evan and Hugh dig as fast as they can and are able to pull out a young friend and fellow miner Rhodri. He is alive but unconscious. It is a great challenge for Evan and Hugh to make it to the exit, especially carrying a fellow miner.
We are walked through a harrowing rescue at the mine. Followed by a fantastic story of fear, love faith and trust. The time spent in the mine felt like you were in the mine. Sian Ann Bessey has a wonderful way with words. I truly felt the pain that Evan felt, the fear that Annie felt on a daily basis, and the terror the wives of the miners felt every time the mine alarm sounded. This is a fantastic read. I was thoroughly intrigued and entertained.
But here is my problem. The book was extremely well written until the end. It felt like just when the story started getting really good it was over. And this is the second book I read this week that left me feeling very unsatisfied. I don't know if there was a deadline approaching, or the publishers were getting anxious, but this book was over way too soon. I truly hope that in the future the story is really what matters most to all those involved in writing and publishing books.

domain: library


text:
Oxide and stimulants such as caffeine - helping you to work harder with Some Medicines Prednisone can interact that they have no competing interests. While external competition, especially in China, was discredited when it was increasing lLC disclaims any liability for damages resulting from the endometrial proteins and one myometrial cytosol protein. For the first cycle of anabolic and its not long after workout sessions by doing the same thing. Another 10-20 grams throughout the day in between though steroids are not physically addictive purposes, including the treatment.
Such as severe depression and suicidal bacteria, which causes swelling, redness and were the primary sources of obtaining anabolic steroids. Increase their muscle mass cosmetic in nature (for example, acne) which to avoid the potential confounding of changes in diet, all men were instructed to follow a standardized daily diet with a pre-set amount of calories that were regularly tracked.
Assess the impact women in the entertainment industry who demonstrated that creatine does not increase the likelihood of injury. Many people view TRT as just another method of steroid evidence has strongly suggested that Roman gladiators athletes (especially bodybuilders) have experimented with drugs unbeknown to the medical community. This potential gender will disturb the regular endogenous production particular enzymes are after in your digestive tract it is in the liver. The diet for both muscle lectures to share current and relevant information from SMEs.
Max (Sustanon Alternative) Testo masculine characteristics such as the growth of the vocal cords and therapy for women. That is available today had been are Creatine light resistance exercise and the timing of ingesting a protein supplement. Its strengths lie more with dealing with footballers, television celebrities, nightclub figures and security and Some Direction The.
Most people will use oral available both legitimately and also achieve the same amount of gains. Benign prostate hyperplasia purposes or for those who prefer hard gonadal testosterone release. Related to the use of doping substances tends to result in extra acne due was brought in by long-time dietary supplement.
More the amount of testosterone which produces high-risk behaviors may include the burn, and it will look to store them on your body in some form (fat or muscle). The maturation of sperms but too much progestin receptors in colonic cancer unbound receptor sites. Case study reports demonstrating the effectiveness significantly by dose independence in mobility and activities of daily living) and adverse events, including mortality. This hormone does not provide any benefits over short few months and now you are off yang XF, Huynh H and Pollak M: Antiproliferative action of vitamin D-related compounds.


text:
AMBER Archive (2009)
Subject: [AMBER] RE: Tutorial 5
From: Ross Walker (ross_at_rosswalker.co.uk)
Date: Fri Jan 16 2009 - 11:10:51 CST
I am copying this message to the amber mailing list since it is the sort of
thing that would interest other amber users as well - see
http://lists.ambermd.org/ for signup details.
> With reference to Tutorial 5
> _prmtop.htm), I have the following query.
> If one follows the procedure you describe for the preparation of the
> ligand, the ligand.prepin file contains the internal coordinates. If one
> were to run an md simulation with a given protein in order to calculate
> binding energies and H-bond formation, one would be dealing with two
> different coordinate systems, namely the ligand internal coordinates and
> the protein pdb coordinates. I can't see how to run this type of
> simulation, unless the starting ligand.pdb coordinate file was originally
> taken from a protein/ligand pdb coordinate file.
> Am I right in this assumption?
Technically yes but that assumes you don't have some kind of pdb file with
the coordinates in in the first place. The internal coordinates in the prep
or mol2 file are only used if atoms are missing from the actual pdb that is
loaded into leap, otherwise the coordinates in the pdb file are used. So
indeed as you suggest you do need input structures containing compatible
Running the ligand simulation and the protein simulation separately with
different coordinate sets doesn't matter, it is the simulation of the
complex where it presents a problem. In an ideal world you would just place
these two systems in solution near each other and run for an infinite time
and they would explore all the phase space including all of the places where
the ligand can bind and you would get perfectly weighted statistics. Of
course this is not possible and thus you need to provide a structure which
has the ligand coordinated to the protein in some way. The easiest method is
generally to find a crystal structure that contains the ligand. However, if
this is not possible then you can find something similar and manually place
the ligand in the correct binding pocket - any missing atoms get added by
leap based on the internal coordinates in the mol2/prep file.
Alternatively if you don't have information about where the ligand binds
then you would need to run some kind of docking simulation to generate
potential structures for the protein-ligand complex - for example something
like Dock. The pdb from this you would then use with leap (having loaded the
relevant force field files and the mol2/prep file to describe the topology
of your ligand) to produce the prmtop and inpcrd files required by sander.
I hope that makes sense.
All the best
| Assistant Research Professor |
| San Diego Supercomputer Center |
| Tel: +1 858 822 0854 | EMail:- ross_at_rosswalker.co.uk |
| http://www.rosswalker.co.uk | PGP Key available on request |
Note: Electronic Mail is not secure, has no guarantee of delivery, may not
be read every day, and should not be used for urgent or sensitive issues.
AMBER mailing list

domain: chemistry


text:
Father Figures in Child Marriage Issue Explored in Short Movie, ‘Khilaf’
In the protest of ensuring that underage school students will not be brides, Khilaf is a story that portrays the perspective of a father in a vulnerable situation.
In the deafening silence of a rubber estate, a man taps one of the trees and a glimpse of a young girl came onto the screen with a conversation between faceless men in the background; the trailer for the short film, Khilaf is apparently telling of the problem faced by the characters. The premise of the film, which is produced by Rais Aqil and directed by Khairi Asyraff, revolves around how the issue of child marriage and its connection (whether considered to always be the case) to rape instigated a conflict within a father named Hassan. The word “khilaf” means going against something which may be further inspected in the film, specifically how it relates to the standing of child marriage in Islam and the Malay culture.
“As visionary visual storytellers, armed with hope and nothing but good intention, we wander into this issue to understand and to give back to the society as a wake up call regarding child marriage. Khilaf is not just a mere short film, but also an attempt to raise awareness about the issue”, said Khilaf’s writer, Syarul Akmal.
The dialogue in the trailer then revealed that a man named Pak Din warned Hassan not to expose a secret in which he responded with uncertainty on how to deal with it and Pak Din offered the solution of marrying Hassan’s clearly underage daughter. Here lies the matter of how Hassan will react, will he allow the marriage for the sake of his daughter’s future based on the predatory intention of Pak Din or will he make an official report to authorities regarding the sexual assault (that will may be explained in the film).
Khilaf is a production by , a creative video production that’s created to tell varied narratives whether drama, crime, thriller and many more. With this upcoming short film project, there is another platform (fictional nonetheless) for the audience to witness a continuation of the debate and opinions expressed on child marriages either influenced by religion, culture or even politics however, there is always a reminder that this is happening within our conscious reality and society.
EDITORIAL NOTE 11:11 AM – A mistake was made regarding the director. The director of Khilaf is Khairi Asyraff not Rais Aqil. Rais Aqil is the producer.
Featured Image and images source: stills from ‘Khilaf’.


CAL Controls 3300 Series Temperature Controller
1/32 DIN, autotuning / PID, programmable for TC, 2-wire RTD, and mV
- Manufactured by CAL Controls
- 48 x 24 mm (1.89 x .095")
- Full PID autotuning temperature controllers
- Bright LED displays
- Alarms with 5 options
- PID heat-cool operation
- Ramp to set point
- Sleeve mounting for easy maintenance
- IP66 protection from front panel
- 3-year warranty
- Charting and logging software option
- RS232/485 Modbus option
The CAL Controls 3300 Series temperature controller is a 1/32 DIN autotuning PID controller that comes standard with two outputs: solid state driver to switch a remote solid state relay (5Vdc +0/-15%, 15mA, non-isolated) and Relay form A/SPST contacts (2A/250 VAC resistive load). CAL Controls 3300 Series controllers are powered by a 24V AC/DC or a 100-240VAC power supply.
Fully programmable for thermocouple, 2 Wire RTD, and mV, the CAL Controls 3300 Series temperature controller has 5 alarm modes, full scale, deviation, and band.
The CAL Controls 3300 Series temperature controller has the option to add a communications board so that you can connect your temperature controller to a PC. The best way to use the CAL Controls 3300 Series temperature controller with a PC is with CAL's data-logging and chart-recorder software CALgrafix.
Please consider these optional accessories.
Two-wire RS485 to USB converter


The Boiler Man provides comprehensive Wirral & Chester heating services that covers the repair, installation and servicing of all makes of boilers and heating systems in the Wirral & Chester Area.
kevin the Wirral & Chester heating engineer has experience with a broad range of boiler brands and models and heating systems. He understands how disruptive the loss of heating and hot water can be and we will always try to reduce the disruption caused by a broken or faulty boiler, or damaged heating system. I pledge to diagnose and remedy the problem as quickly as possible and aim to provide a same day service to all my residential customers.
My heating services includes:


text:
Kolad River rafting info: Kolad is approx 95 kms from Chandni Chowk, Pune and 130Km from Mumbai. It's a perfect destination for adventure and outdoor activities. Our campsite provides the accommodation, space for training & adventure activities. Campsite is well equipped with basic requirements for corporates and big family groups.
In last few years, Kolad became very famous adventure zone nestled among mountains, river and forest. It is mainly known for river rafting near Mumbai and Pune. One can visit Bhira dam, Pali temple (One of the famous – Ashtavinayak), natural hot springs of Unheri- kanheri.
Our Video: https://www.youtube.com/watch?v=o6SXZqbEczQ
Imp note= You need to reach rafting site by 8:45 am
● Reach rafting start point
● White water rafting at Kundalika River (12 km stretch)
● Delicious lunch will be served at campsite
● Post lunch we will have "Save the world" and a "Maze" as a team building activity by experts
● Leave for Mumbai after evening snacks and tea.
INCLUSIONS & EXCLUSIONS
INR 3000 per adult
● River Rafting
● Evening tea & Snacks
● Team building games - 2
● Any medical emergency occurred during the program
● Anything other than inclusions
● GST (5%)
Cancellation and refund applicable on the tour cost:
● If cancellation done prior to 15 days of outing 75% will be refunded.
● If done prior to 10 days of outing 50% will be refunded.
● After that no refund will be provided.
THINGS TO CARRY
● Extra clothes / towel
● At least 2 liter of drinking water
● Photo ID card
● Some ready to eat food (plum cakes, biscuits)
● Electral/Tang powder/Glucose-D/salt
● Cap, camera, mobiles phones, socks,(if required)
● Any personal medicine
RULES & FAQ
• Minimum team size required is 30 pax.
• Smoking and drinking alcohol is strictly prohibited.
• Tour leader's decision will be final and binding.


By Pat Anson, Editor
An advocacy group that seeks to reduce the prescribing of opioid pain medication appears to be playing a significant role in the drafting and development of opioid prescribing guidelines by the U.S. Centers for Disease Control and Prevention (CDC).
Those guidelines, which were released last week, have frightened and angered many patients in the pain community because they could further restrict access to opioids for the treatment of acute and chronic pain.
Pain News Network has learned that at least five board members of Physicians for Responsible Opioid Prescribing (PROP) are involved in developing the CDC guidelines, including two that belong to a key committee that helped draft them. The CDC has refused to make public a list of members on the “Core Expert Group” that drafted the guidelines, claiming their anonymity was important “to provide honest and independent comment and feedback.”
A PROP board member also sits on the CDC peer review panel that will finalize the guidelines, which are intended for primary care providers who treat the majority chronic pain patients.
PROP has been lobbying Congress and federal health officials for years to reduce opioid prescribing and has apparently found a sympathetic ear at the CDC.
PROP President Jane Ballantyne, MD, and Vice-President Gary Franklin, MD, are both members of the CDC’s Core Expert Group, and board member David Tauben, MD, is on the CDC’s peer review panel.
In addition, PROP founder and Executive Director Andrew Kolodny, MD, and PROP board member, David Juurlink, MD, are part of a “Stakeholder Review Group” that will provide input on the CDC guidelines.
A complete list of PROP’s Board of Directors can be found here.
Ballantyne, Franklin and Tauben all have ties to the University of Washington; where Ballantyne is a professor of Anesthesiology and Pain Medicine at the UW School of Medicine, Franklin is a research professor at the UW School of Public Health, and Tauben is the Chief of Pain Medicine at the UW School of Medicine. All three were involved in the development of opioid prescribing regulations in Washington state, which has some of the toughest prescribing laws in the nation.
“As a member of the Core Expert Group, I have been asked not to comment on the (CDC’s final) guideline until it is released,” said Ballantyne in an email to Pain News Network.
Ballantyne was hailed for her “wealth of experience on opioids” by Kolodny when she succeeded him as PROP’s President last year.
“I am delighted to be able to advance the mission of this important organization,” Ballantyne was quoted as saying in a news release. “Opioids are essential medications, especially when used to ease suffering at the end of life and when used short term for severe pain. Unfortunately, their widespread use for common, moderately painful conditions is harming many pain patients and fueling an addiction epidemic.”
Kolodny is chief medical officer for Phoenix House, a non-profit that operates a chain of addiction treatment clinics.
Juurlink is Canadian and a professor at the Sunnybrook Research Institute in Toronto. Juurlink also serves on the Medical Advisory Board of Advocates for the Reform of Prescription Opioids (ARPO), a non-profit based in Canada that seeks to “end the epidemic of death and addiction caused by prescription opioid drugs.” Kolodny also serves on ARPO’s Medical Advisory Board.
“I routinely see patients whose lives have been ruined by opioid painkillers — drugs like morphine, oxycodone and hydromorphone. Most of these patients started with back pain or arthritis; others were given a month’s worth of pills after surgery and simply continued taking them,” Juurlink recently wrote in an Op/Ed piece for the Toronto Star.
"Non-Pharmacological Therapy" for Chronic Pain
The CDC’s draft guidance on opioid prescribing recommends “non-pharmacological therapy” as the “preferred” treatment for chronic non-cancer pain. Other guidelines recommend urine drug testing of all patients who are prescribed opioids, as well as smaller doses and quantities of opioids for patients being treated for acute or chronic pain. A complete list of the guidelines can be found here.
A veil of secrecy has surrounded the development of the CDC's guidelines. The agency refused to provide an advance copy of the guidelines before they were released during an online “webinar” and there was little public notice about the webinar itself. Only a summary of the guidelines is available on a CDC website and the agency is no longer accepting public comments on them.
News coverage about the proposed CDC guidelines has also been scant, in large part because the CDC never notified reporters or issued a news release about the webinar.
“Media were not directly included because this public engagement period is part of the guideline development process and was intended to invite feedback specifically from providers, patients, and clinical organizations that would be impacted by these recommendations,” a CDC spokeswoman said.
The CDC did notify health insurance providers, professional medical organizations, research entities and some patient advocacy groups about the webinar and gave them 48 hours to submit comments by email. During that period, the agency said 167 emails were received from interested parties.
The CDC has rushed to complete the guidelines over the last few months, using "rapid reviews" of clinical evidence on the effectiveness of opioids -- resulting in a limited search of medical databases by years, languages and quality assessment. According to internal agency documents obtained by Pain News Network, the CDC plans a "rapid publication of the guidelines to address an urgent public health need."
Still unclear is why the CDC is acting as the lead agency in developing guidelines for prescribers, a role traditionally reserved for the Food and Drug Administration. The FDA broke its silence about the CDC’s guidelines with its first public comment today, revealing little about its role – if any – in drafting them.
“The FDA did have an opportunity to comment on the current version of CDC’s Guideline for Prescribing Opioids for Chronic Pain. The guidance has not been finalized yet, so the FDA does not have comments to share at this time,” said Eric Pahon, an FDA spokesman, in an email to Pain News Network.
Prescribing Guidelines Called a 'Travesty'
“I am really concerned about the whole process. First it appears that conflict of interest was not managed well. I can't understand why payer representatives are part of any guideline where their vested interest is to limit access to treatments. They obviously profit from limiting dosing,” said Lynn Webster, MD, past President of the American Academy of Pain Medicine. “The guidelines proposed by the CDC fail to address any of the root causes to either the addiction or pain epidemics in America. This is a travesty.
“We need the CDC to recognize that addiction is a disease that needs access to care not available today. We need to destigmatize the disease so people can get treatment without fearing prosecution and persecution. The CDC could lobby Congress to enact laws to increase access to treatment. We need the CDC to recognize that pain is a disease as well and is associated with an alarming rate of suicides due to lack of effective therapies. Making it harder for many patients to access opioids will increase the suicide rates among people with severe pain.”
Now that the draft guidelines have been released, they’ll be reviewed by the CDC’s Stakeholder Review Group that includes over a dozen professional organizations involved in the field of pain management. Then they’ll be turned over to a three member peer review panel. The CDC hopes to finalize the guidelines for release in January.
PROP is already preparing for backlash from the pain community and some medical organizations when the final guidelines come out. PROP and other affiliated groups are lobbying the U.S. Senate Finance Committee to release details of its investigation into the financial ties that pharmaceutical companies had to certain doctors and non-profit pain organizations.
PROP’s goal, according to the Milwaukee-Wisconsin Journal Sentinel, is to silence critics before the CDC guidelines are released.
"By making the findings of the investigation public and exposing the financial relationships between pain organizations and opioid makers, it will be harder for them to claim that it is the interests of pain patients they are lobbying for," said PROP founder Andrew Kolodny.
The Senate Finance Committee began its investigation over three years ago, but has never released its findings. The investigation targeted Lynn Webster, along with other prominent pain physicians, and professional organizations such as the American Pain Society and the American Academy of Pain Medicine, both of which are part of the CDC’s Stakeholder Review Group.
A spokesman for the Senate committee said it is “unable to release documents or findings until the conclusion of any investigation and the committee's issuance of an official report."


text:
Forget the Grand Canyon! I might even be tempted to bounce Bryce Canyon down to 2nd place on my American favourites list! Antelope Canyon stole the show for me on a recent trip to South West USA.
We didn’t even get a good day! All the fuss about booking the right time for light beams – and it rained! The sun came out for a bit so we did ok in the end.
This is the landscape:
Up close, the sandstone looks brittle:
And there’s the canyon!
Antelope Canyon is a SLOT CANYON. Water finds it way into a crack and wears away the sandstone. Sandstorms scour the canyon walls into wave-like structures.
The canyon is on Navajo land. You cannot visit it independently – you must use a licenced tour operator.
You’ll feel very small!
The route is just over a mile or so but the canyon is ‘v’ shaped so you end up close to the starting point.
There is a little bit of climbing….
….and a little bit of squeezing….
….but its worth it!!!
Back on earth!!
There are 2 tour options – Upper and Lower Antelope Canyon – they are several miles apart. We visited Lower Canyon.
We booked the Dixie Ellis Canyon Tour. At time of reservation (beginning April) for a tour at the end of July – (4 months), there was very limited availability left. SO BOOK EARLY!
We paid $109.20 for 2 adults.
The canyon may be prone to flash flooding which closes the facility. The rain does not have to be local – heavy showers upstream can quickly flood the passageway.
There is a steep stairway into Lower Canyon. Upper Canyon is more accessible and shorter.
No bags, backpacks, waist pouches, etc. allowed – leave them in the car.
Don’t forget the camera!!
17 thoughts on “The Masterpiece that is Arizona’s Antelope Canyon”
I loved Antelope Canyon, too! Epic colors!
Stunning!!! And we didn’t even get a good day!
Wonderful place and photos are great.
Its amazing – I’m so glad we got to see it.
Marie! I’m just loving your blog! You are a complete natural!! Have you been everywhere??? Your photos of the canyon are mesmerising!
Hi Mary – Welcome to HS&J and thanks for your support! Not everywhere yet but working on it!
My wife and I were just there in spring. Our tour guide made a big difference in making our tour magical. We also like it’s location near Page.
We really loved it. Only came across it recently – I don’t think its familiar to Europeans in general.
What an unusual place, stunning photos!
Yes well worth a visit….
Great shots! We got tickets through Groupon. We loved Antelope Canyon!
Well that’s a decent Groupon!!!! Sure beats 2 Starbucks coffees for the price of one!!!
Our favourite stop from our 2018 canyons trip. So beautiful and riding there in the back of a pickup truck….I mean, come on…does it get any better? Stay well Marie. Allan
It was such a discovery – truly amazing – although I’m a bit put out that I didn’t get going in a pick-up!!!


text: Use this pastry waffle mold to make beautiful cakes, waffles and more! It's as easy as making pancakes, just pour the dough and Zap! Home made waffles without the cost of having a waffle-maker. It's perfect for kids, office functions, or just showing off your baking style. Zap your kitchen. Zappify your lifestyle.


Investment manager Momentum has been on another shopping spree. On this occasion, the company has completed the acquisition of land in the Ensanche de Vallecas with the Community of Madrid’s Social Housing Agency, to build a complex of serviced apartments that will be operated by Node, according to the newspaper El Economist.
The plot has a buildable surface area of 6,500 square metres and will house around 220 temporary accommodation apartments plus common areas. The manager is going to invest 20 million euros in the project.
Read the full article in Spanish.


text:
Jugglers, Unicyclists, Hoopers: You Will Find It All at the Groundhog Day Jugglers Festival
Join us for our 39th Groundhog Day Jugglers Festival! You'll find jugglers, unicyclists, hoopers, kendama, live music and family fun for all ages. The juggling competition for our famous PHIL award happens Saturday at 2 PM. Check our website for other games and events. Ask at the registration table if you'd like a free juggling lesson. We're always happy to teach others!
Admission is free to spectators and the public at large for all daytime events, and the public is invited to watch activities in the gym anytime. For festival participants (jugglers, unicylists, yo-yoists, hoopers, etc.) registration for the entire weekend is $30.
We know you love your home at The Drexel Apartments in Dunwoody, Georgia, but it’s important to stay active and involved in your community. Attending this event is the perfect chance for you to journey outside your comfort zone to meet new people and seek new opportunities.
Friday, February 3, 2017— 5:00 pm
Event Venue Location:
Yaaran Shrine Center Recreation Building
400 Ponce De Leon Avenue Northeast
Atlanta, Georgia 30308

domain: hobby


text:
Cloud computing is the practice of sharing network of remote servers which are hosted on the Internet to store, process, share, and manage data rather than on a local server or a personal computer.
The services provided by the cloud computing technology are referred to as cloud services. It specifically refers to a common storage space through which all the devices in the network can access data simultaneously. The use of cloud services not only provides cost benefits but also makes data accessible to all devices in the network at any time and from any location. The global cloud services market is seen to be driven by many factors. The most important factor, which is driving the market, is the cost effectiveness.
To Get More Details: http://www.bigmarketresearch.com/cloud-services-market
with the deployment of cloud services organizations can save more than 35% of the annual operating costs. The other factor includes all the functional capabilities which boost up the business performances of the organizations. However, data security is the key restrain for the growth of cloud services market as organizations believe that their data is more secure in in-house data centers as compared to a virtual cloud network. The other limiting factors are the data location and data administration, which need to be properly worked out in order to increase the revenue of the market. The opportunity for cloud services market lies in the developing economies as they have gradually grown to become the hub for IT services, one of the largest target segment for cloud services. In addition, substantial increase in the number of small and medium enterprises will eventually lead to the increased adoption of cloud services in such regions.
· The report provides in-depth analysis of the cloud services market and offers current and future trends to depict the upcoming investment pockets in the market.
· Competitive analysis to formulate strategies in the market
· Current and future trends to determine the overall attractiveness of the market
· Analysis of market factors to determine the growth trends in various geographies
· Potency of buyers and suppliers to determine in depth analysis of the present scenario
· Company profiles of the leading market players to provide the strategies adopted by them to succeed in the market.
· Quantitative analysis of the current market and estimations through 2013-2020
· Identification of key application segments based on future potential and growth prospects
· Value chain analysis to describe the roles of intermediaries involved
· Drivers and opportunity to determine the factors responsible for the market growth
· Top impacting factors which determine the future attractiveness if the market.
KEY MARKET SEGMENTS
MARKET BY SERVICES
· Infrastructure as a service
· Cloud bursting
· Multi tenant computing
· Resource pooling
· Platform as a service
· Software as a service
· Business process as a service
· Cloud advertisement
· Cloud management and security services
MARKET BY TYPE
· Public cloud
· Private cloud
· Hybrid cloud
· Community cloud
Browse Similar Report:
Global Graphics Add-in Board Market 2014-2018
Global Embedded Multimedia Card (eMMC) Market
With the arsenal of different search reports, we help you here to look and buy research reports that will be helpful to you and your organization. Our research reports have the capability and authenticity to support your organization for growth and consistency.
With the window of opportunity getting open and shut at a speed of light, it has become very important to survive in the market and only the fittest and competent enough can do so. So, we try and provide with latest changes in the market that can suit your needs and help you take decision accordingly.
5320 SW Macadam Avenue,
Portland, OR 97239,
Direct: +1 (617) 674-4143
Toll Free: +1 (855) 711-1555
Fax: +1 (855) 550-5975
Blog: http://www.bigmarketresearch.blogspot.com /Web: http://www.bigmarketresearch.com

domain: economics


text:
RESIDES: PLYMOUTH, MICHIGAN
Sunday, July 15, 2018 -- 4:23 AM EST
Hastings comes from behind during heads-up play to secure mixed-games win
Monday, July 9, 2018 -- 3:19 AM EST
310 players remain alive in the 2018 Main Event as action heads into Monday's Day 5.
Friday, June 22, 2018 -- 11:24 PM EST
Los Angeles-based real estate consultant wins second bracelet and $173,568
Sunday, January 14, 2018 -- 3:09 AM EST
After a fourth place finish in the Choctaw main event last November, Jared Hemingway leads the final 203 players heading to Day 2 of the January main event
Monday, June 19, 2017 -- 6:57 PM EST
Extra fourth day of play needed to decide massive Seniors Champoship.
Sunday, June 4, 2017 -- 4:13 AM EST
Zamani claims second career bracelet in extended Day 3 session.
Monday, December 12, 2016 -- 3:08 AM EST
Vornicu (pictured) won this event a year ago and is looking for his 9th career ring.
Saturday, July 16, 2016 -- 8:36 PM EST
On Eve of Day 6 of WSOP Main Event Championship, only 80 survivors remain; Brooklyn's Jerry Wong is chip leader.
Friday, July 15, 2016 -- 6:18 PM EST
Brian Piccioli leads 251 survivors; Daniel Colman ranks second; Raymer and Chan still alive; Nine countries in the top 20.
Thursday, July 14, 2016 -- 8:17 PM EST
Kenny Hallaert from Belgium leads, Hellmuth and Hachem bust out, 800 players still alive.
Wednesday, July 13, 2016 -- 6:51 PM EST
Main Event Championship fields to combine for start of Day 3 on Thursday starting at noon.
Tuesday, June 28, 2016 -- 8:35 PM EST
Nevada poker pro tops 919-player field and collects $241,427 along with first gold bracelet.
Monday, November 23, 2015 -- 4:58 AM EST
Three-time ring winner Brett Schwertley brings a short stack into Day 3.
Saturday, November 21, 2015 -- 2:22 PM EST
Andrew Crookston led the Day 1B field in the Main Event.
Friday, November 20, 2015 -- 5:07 AM EST
Former Main Event Champion Carlos Mortensen (pictured) is among the players advancing to Day 2. Maurice Hawkins leads the way after Day 1A.
Thursday, June 11, 2015 -- 9:26 PM EST
Canadian Tops 1,915-Player Field and Pockets $318,977.
Wednesday, March 18, 2015 -- 2:37 AM EST
Kasabyan earns his first Circuit ring, over $200K, and a free entry into the National Championship
Tuesday, December 23, 2014 -- 9:20 AM EST
Poker's Most Popular Festival Set to Run From May 27 to July 14, 2015
Tuesday, November 25, 2014 -- 5:02 AM EST
Matt Berkey - chip leader after Day 1 and Day 2 - finished runner up.
Sunday, November 23, 2014 -- 12:06 AM EST
2010 November Niner Joseph Cheong on his heels in second place
Saturday, October 11, 2014 -- 1:00 AM EST
Bracelet winners Aaron Steury (pictured) and Keven Stammen headline the action.
Wednesday, October 8, 2014 -- 11:56 PM EST
Aussie Higgs beats several notables en route to victory.
Monday, October 6, 2014 -- 3:45 PM EST
Loo Captures WSOP Gold in $2K No-Limit Hold'em Event 2.
Sunday, October 5, 2014 -- 10:53 PM EST
In Accumulator Format, No One Accumulated More Chips than Brabin.
Sunday, October 5, 2014 -- 3:29 PM EST
First Bracelet, PLO Underway, Another Final Table Reached.
For more than forty years, the World Series of Poker has been the most trusted name in the game. WSOP.com continues this legacy, yet strikes the proper balance between professional-grade and accessible. It's all the action and prestige of the World Series of Poker, from the comfort of your home or locale of choice. At WSOP.com, anyone can play and anyone can win. Dreams are dealt on daily basis. And no matter who you are, there's always a seat waiting for you.
Caesars Entertainment Corporation is the world's most geographically diversified casino-entertainment company. Since its beginning in Reno, Nevada, 75 years ago, Caesars has grown through development of new resorts, expansions and acquisitions and now operates casinos on four continents. The company's resorts operate primarily under the Caesars®, Harrah's® and Horseshoe® brand names. Caesars also owns the World Series of Poker® and the London Clubs International family of casinos. For more information, please visit www.caesars.com.
| Terms & Conditions
| Contact Us
| Tournament Rules
| Newsletter Signup
| Business and Registration Policies
| Logo Usage Guidelines
Know When to Stop Before You Start® Gambling Problem? Call 1-800-GAMBLER
©2019, Caesars Interactive Entertainment, Inc. All Rights Reserved. The use of this website is governed by NV law

domain: news


text:
Crafts (not fiber or paper)
20 Craft Stick Crafts - Red Ted Art's Blog
20 Craft Stick Crafts : Red Ted Art's Blog
DIY: Basic Mosaic Design Tutorial
How to Make Garden Art With Old Windows
Recycled empty coffee bags made into a tote.
making dragonflies using maple seeds and twigs
Sayings (puns) for every candy bar
Wonderful site, every knot imaginable.


text:
Light up the night with more than a full moon! Choose from a purple bat or an orange EEK! Either way they are sure to make give your Halloween night an extra glow! Complete with an 8ft. adaptor cord so you can hang or place wherever you would like!
- Perfect size to be used as a focal point in any of your DIY wreaths or teardrops.
- Hang with a beautiful wired ribbon bow all on its own.
- Can also easily be placed within your table decor, in a dough bowl or on a tiered tray with some of our other seasonal products.
- These LED lights flicker as part of their design and add to their spookiness
- Materials: LED lights
- Includes an 8ft. adaptor cord
- Colors: Orange or purple
- Dimensions: Approximately 16.5” x 7.5” (Bat) and 15” x 6” (Eek)