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RESOURCE ARTICLE| 01 November 2014
Enhanced glucose tolerance in pancreatic-derived factor (PANDER) knockout C57BL/6 mice
Shari L. Moak,
Shari L. Moak
1Department of Cell Biology, Microbiology and Molecular Biology, University of South Florida, 4202 East Fowler Avenue, Tampa, FL 33620, USA.
Grace C. Dougan,
Grace C. Dougan
2Department of Pediatrics, University of South Florida, 12901 Bruce B. Downs Boulevard MDC 62, Tampa, FL 33612, USA.
Catherine B. MarElia,
Catherine B. MarElia
Whitney A. Danse,
Whitney A. Danse
Amanda M. Fernandez,
Amanda M. Fernandez
Melanie N. Kuehl,
Melanie N. Kuehl
Mark G. Athanason,
Mark G. Athanason
Brant R. Burkhardt
Brant R. Burkhardt *
*Author for correspondence (bburkhardt@usf.edu)
Received: 28 Mar 2014
Dis Model Mech (2014) 7 (11): 1307–1315.
Version of Record 01 November 2014
Shari L. Moak, Grace C. Dougan, Catherine B. MarElia, Whitney A. Danse, Amanda M. Fernandez, Melanie N. Kuehl, Mark G. Athanason, Brant R. Burkhardt; Enhanced glucose tolerance in pancreatic-derived factor (PANDER) knockout C57BL/6 mice. Dis Model Mech 1 November 2014; 7 (11): 1307–1315. doi: https://doi.org/10.1242/dmm.016402
Pancreatic-derived factor (PANDER; also known as FAM3B) is a uniquely structured protein strongly expressed within and secreted from the endocrine pancreas. PANDER has been hypothesized to regulate fasting and fed glucose homeostasis, hepatic lipogenesis and insulin signaling, and to serve a potential role in the onset or progression of type 2 diabetes (T2D). Despite having potentially pivotal pleiotropic roles in glycemic regulation and T2D, there has been limited generation of stable animal models for the investigation of PANDER function, and there are no models on well-established genetic murine backgrounds for T2D. Our aim was to generate an enhanced murine model to further elucidate the biological function of PANDER. Therefore, a pure-bred PANDER knockout C57BL/6 (PANKO-C57) model was created and phenotypically characterized with respect to glycemic regulation and hepatic insulin signaling. The PANKO-C57 model exhibited an enhanced metabolic phenotype, particularly with regard to enhanced glucose tolerance. Male PANKO-C57 mice displayed decreased fasting plasma insulin and C-peptide levels, whereas leptin levels were increased as compared with matched C57BL/6J wild-type mice. Despite similar peripheral insulin sensitivity between both groups, hepatic insulin signaling was significantly increased during fasting conditions, as demonstrated by increased phosphorylation of hepatic PKB/Akt and AMPK, along with mature SREBP-1 expression. Insulin stimulation of PANKO-C57 mice resulted in increased hepatic triglyceride and glycogen content as compared with wild-type C57BL/6 mice. In summary, the PANKO-C57 mouse represents a suitable model for the investigation of PANDER in multiple metabolic states and provides an additional tool to elucidate the biological function and potential role in T2D.
Pancreatic-derived factor, FAM3B, Knockout model, Glycemic regulation, Hepatic insulin signaling, Glucose tolerance
Pancreatic-derived factor (PANDER; also known as FAM3B), discovered in 2002, is a 235-amino-acid protein that belongs to the family with sequence similarity 3 (FAM3) gene family (Zhu et al., 2002). This cytokine-like gene family (based on predicted secondary structure) has four members described as FAM3A, FAM3B, FAM3C and FAM3D. FAM3B was later named PANDER because this hormone is robustly expressed in and secreted from the endocrine pancreas (α cells and β cells, specifically) and, to a lesser extent, other tissues such as the liver, small intestine and prostate (Li et al., 2011; Li et al., 2013; Zhu et al., 2002). From the ostensible recognition of folds (ORF) algorithm that was used in PANDER's initial discovery, it was determined that PANDER (then identified as FAM3B) had a predicted typical four-helix bundle secondary structure that is present in many known cytokines (Aurora and Rose, 1998; Zhu et al., 2002). However, more recently, elucidated crystal structures of murine secreted PANDER revealed a previously unknown and unique globular β-β-α fold (Johansson et al., 2013). PANDER comprises two anti-parallel β sheets lined by three short helices arranged to form a highly conserved water-filled cavity that does not resemble any currently known cytokine, thereby contradicting the earlier predictive models (Johansson et al., 2013). This structure is conserved among other members of the FAM3 family. The homologous FAM3C, also known as interleukin-like EMT inducer (ILEI), has been previously demonstrated to be involved with the epithelial-mesenchymal transition and shows similar structure (Lahsnig et al., 2009; Song et al., 2013; Waerner et al., 2006). Therefore, PANDER and this gene family might comprise a newly identified class of signaling molecules that act in ways distinct from those of other known cytokines or hormones.
Initial in vitro studies regarding PANDER have demonstrated a potential role in glycemia regulation. Glucose has been shown to significantly enhance Pander promoter activity and secretion from β cells and pancreatic islets (Burkhardt et al., 2005; Wang et al., 2008; Yang et al., 2005). In addition, PANDER is co-secreted with insulin in response to glucose (Carnegie et al., 2010; Xu et al., 2005).
The mechanism of action and full biological effect of PANDER have yet to be fully elucidated in vivo, and this is the result of a lack of appropriate permanent and genomically integrated rodent models. The initial PANDER knockout (PANKO) model was generated on a mixed genetic background and demonstrated glucose intolerance in the presence of enhanced hepatic insulin sensitivity (HIS) with no observed differences in peripheral insulin sensitivity or fasting glycemic levels (Robert-Cooperman et al., 2010). Despite enhanced HIS as determined by hyperinsulinemic-euglycemic clamp (HEC) studies, no further characterization has been performed to identify the causative hepatic molecules or pathway for this finding, thereby leaving a gap in the understanding of hepatic PANDER action under both fasting and fed conditions. The targeted disruption of PANDER impairs pancreatic islet insulin secretion, as demonstrated by an inhibited glucose-induced response by PANKO islets, which was identified during islet perifusion and Ca2+-imaging studies. By contrast, neither endocrine pancreatic morphology nor insulin content is affected by the absence of PANDER. The lack of further hepatic molecular characterization and a robust phenotype from the mixed genetic background PANKO mouse led us to speculate that the lack of a congenic background in a pure-bred mouse model might be hindering the penetrance of the phenotype and confounding the results, particularly in relation to hepatic insulin sensitivity. Furthermore, a series of review articles has strongly suggested that PANDER could potentially be implicated in the onset or progression of type 2 diabetes (T2D) (Wang et al., 2012; Wilson et al., 2011; Yang and Guan, 2013), yet the impact of this gene within well-established models of T2D or insulin resistance has not been generated. Therefore, to further refine and define the biological function of PANDER within the context of a well-established model of T2D within a permissive genetic background, we generated and characterized the PANDER knockout mouse on a pure C57BL/6 genetic background.
RESOURCE IMPACT
Pancreatic-derived factor (PANDER, FAM3B) was originally cloned in 2002 and is a member of the family with sequence similarity 3 (FAM3) gene superfamily. PANDER appears to serve a role in the regulation of glycemic levels, insulin action and hepatic lipogenesis. Overexpression of PANDER results in a phenomenon known as selective hepatic insulin resistance (SHIR), whereby insulin signaling is blunted, yet lipogenesis is increased. SHIR is a pathogenic paradoxical hallmark of type 2 diabetes (T2D), which is the most common global metabolic disorder with ever increasing rates. Despite numerous investigations eluding to PANDER as serving a potential role in the onset or progression of T2D, no stable animal models have been generated on well-established genetic backgrounds of T2D susceptibility. Therefore, there is a strong need for novel animal models on congenic backgrounds with discernible phenotypes for the investigation of PANDER function.
In this study, the authors have generated a PANDER knockout mouse model on a pure C57BL/6 background (PANKO-C57) to promote the phenotypic penetrance of PANDER and to provide a useful tool for subsequent studies. In contrast to the previous PANDER knockout model, the PANKO-C57 model exhibited increased body weight, enhanced glucose tolerance during both fed and fasting conditions. This phenotype was more significant in PANKO-C57 male versus female mice; however, females still displayed overall trends in enhanced glucose tolerance. In addition, the fasting plasma insulin and C-peptide levels were concordantly decreased in the PANKO-C57 mouse, along with increased leptin levels. Hepatic insulin signaling was significantly increased during fasting conditions, as demonstrated by increased phosphorylation of hepatic protein kinase B (PKB/Akt) and adenosine monophosphate-activated protein kinase (AMPK), along with mature sterol regulatory element-binding protein 1 (SREBP-1) expression in the PANKO-C57 model. Insulin stimulation of PANKO-C57 mice resulted in increased hepatic triglyceride and glycogen content when compared with C57BL/6J wild-type mice.
Altogether, the PANDER mouse model has enhanced our understanding of the mechanistic implications of PANDER function and has unified many of the prior conflicting findings regarding its role in glycemic regulation. The study provides evidence that this generated PANDER knockout animal model, derived on a well-established background for T2D, displays an enhanced phenotype that can be easily employed in future investigations. Given the limited availability of current PANDER animal models, the PANKO-C57 mouse suitably fills this important gap. The PANKO-C57 model has a strong breeding capacity and a discernible phenotype to allow for the future creation of additional animal models and for investigations to evaluate the role of PANDER in both T2D and glycemic regulation.
Decreased fasting glycemia in PANKO-C57 mice
Before experimentation, all mice were genotyped as a confirmatory measure following receipt of PANKO-C57 mice from Jackson Laboratories using primers described in Materials and Methods (data not shown). The effect of PANDER on fasting glycemic levels was determined by measuring blood glucose following a short-(4 hours) and long-term (16 hours) fast. PANKO-C57 male mice aged 4 months displayed significantly decreased blood glucose levels after a long-term fast compared with age- and sex-matched C57BL/6 mice (P<0.01) (Fig. 1A). Decreased fasting glycemia levels were also observed following a short-term fast, with blood glucose values of 169.3 mg/dl±6.9 (±s.e.m.) vs. 225.8 mg/dl±30.5 (P<0.05) (Fig. 1B). A similar but non-statistical trend was observed with female PANKO-C57 long- and short-term fasting mice (Fig. 1C,D). In summary, the absence of PANDER promoted decreased fasting glycemic levels that had not been previously observed or reported in the mixed genetic background PANDER knockout or adenovirally delivered PANDER siRNA models, revealing a discernible phenotype within this background (Li et al., 2011; Robert-Cooperman et al., 2010).
Long- and short-term fasting glycemic measurements of PANKO-C57 mice. Mice of both genders were 4 months of age. (A) Long-term fasting (overnight, 16 hours) blood glucose measurements of male PANKO-C57 (KO) and WT mice (n=12–15). (B) Short-term fasting (4 hours) blood glucose measurements of males (n=8–11). Values are expressed as means±s.e.m. (C) Long-term fasting blood glucose measurements of female PANKO-C57 and WT mice (n=7–12). (D) Short-term fasting (4 hours) blood glucose measurements of females (n=6–10). *P<0.05, **P<0.01, Student's t-test.
Enhanced glucose tolerance in PANKO-C57 mice
To evaluate the effect of PANDER during postprandial conditions in our model, PANKO-C57 and wild-type (WT) mice of both genders were examined by using glucose tolerance tests (GTTs). GTTs performed at 4 months of age revealed enhanced glucose tolerance in male PANKO-C57 mice based on decreased glycemic levels throughout the duration of the GTT (Fig. 2A). Significantly lower blood glucose measurements were observed in male PANKO-C57 mice compared with WT mice during the course of the GTT (two-way ANOVA, P<0.05). Insulin tolerance tests (ITTs) were performed to examine peripheral insulin sensitivity. Responses to intraperitoneal-injected insulin were shown to be similar between PANKO-C57 and WT mice (Fig. 2B). With regard to female PANKO-C57 mice, a similar trend of enhanced glucose tolerance was measured during the GTT but without statistical significance (Fig. 2C). Insulin sensitivity during the ITT was similar between female PANKO-C57 and WT mice (Fig. 2D). In summary, the absence of PANDER promoted enhanced glucose tolerance without significantly affecting peripheral insulin sensitivity in a male dominant fashion. This was not observed in our prior PANKO mixed genetic background model and demonstrates an enhanced metabolic phenotype in the pure C57BL/6 model (Robert-Cooperman et al., 2010).
Enhanced glucose tolerance in male PANKO-C57 mice. All mice were 4 months of age. (A) Intraperitoneal (i.p.) glucose tolerance tests (GTTs) were performed following overnight fast (~16 hours) on male PANKO-C57 (KO) and WT mice through injection of glucose at 2 g/kg of body weight and measurement of plasma glucose concentration at the indicated time points (n=8–12). (B) Intraperitoneal insulin tolerance tests (ITTs) were performed on male PANKO-C57 and WT mice following a 4-hour fast by injection of insulin at 1 unit/kg of body weight and subsequent measurement of plasma glucose concentration at all indicated time points. Results are presented as the percentage of the baseline glucose concentration measured at time point 0 (n=8–11). (C) GTTs performed on female PANKO-C57 and WT mice as described above (n=7–12). (D) ITTs performed on female PANKO-C57 and WT mice as described above (n=7–11). Values are expressed as the mean±s.e.m. *P<0.05, **P<0.01 as determined by using two-way ANOVA.
PANKO-C57 male and female mice have increased body weight
To further characterize the PANKO-C57 mice, the longitudinal body weight was evaluated from 8 to 23 weeks of age. Beginning at 8 weeks of age upon feeding with normal chow ad libitum, PANKO-C57 male mice presented with significantly increased weights compared with age- and gender-matched WT mice [up to 23 weeks of age (Fig. 3A)]. Measurements of body weight were terminated at 23 weeks of age. This same trend was observed in female mice, and although significance diminished over time, PANKO-C57 female mice remained significantly heavier than WT mice at 21 weeks of age (Fig. 3B). Increased body weight has not been reported or observed in previous animal models and reveals a potential previously overlooked function of PANDER in the area of whole-body homeostasis. Furthermore, the appearance of this phenotype in both genders supports the distinction of this model from other PANDER animal models (Li et al., 2011; Robert-Cooperman et al., 2010; Wilson et al., 2010).
Increased body weight of male and female PANKO-C57 mice. Body weight measurements were recorded at similar times from 8 to 23 weeks of age following an overnight fast. (A) Male PANKO-C57 (KO) and WT mice weight over time (n=18–27). (B) Female PANKO-C57 and WT mice weight over time (n=13–22). Values are expressed as the means±s.e.m. *P<0.05, **P<0.01, ***P<0.001 as determined by using two-way ANOVA.
Fasting hormone levels in PANKO-C57 mice
Following the combined gender phenotyping, we focused our study on PANKO-C57 males in order to identify whether hormonal differences accounted for the observed decreased glycemic levels during fasting in the male PANKO-C57 mice. Therefore, the plasma levels of insulin, C-peptide, glucagon, leptin and corticosterone during fasting were measured at 2 and 5 months of age in male mice (Fig. 4). At 2 months of age, PANKO-C57 male mice presented with significantly reduced insulin levels (Fig. 4A). Glucagon levels were similar between PANKO-C57 and WT mice at 2 and 5 months of age (Fig. 4B). Interestingly, leptin levels were increased in PANKO-C57 mice in both age groups (Fig. 4C). C-peptide levels at 2 months of age were significantly decreased and concordant with the decreased measured insulin levels (Fig. 4D). Fasting corticosterone levels were similar between PANKO-C57 and WT mice in both age groups (Fig. 4E). In general, the hormonal results from plasma during fasting revealed characteristics of the PANKO-C57 model which had not been observed in prior investigations, such as the decreased insulin levels in 2-month-old mice and increased leptin levels (Li et al., 2011; Robert-Cooperman et al., 2010). In addition, glucose-stimulated insulin levels were decreased in the PANKO-57 mice with statistical significance at the conclusion of the glucose tolerance test (Fig. 4F). This result is consistent with previous reports indicating that the absence of PANDER can impair glucose-stimulated insulin secretion (Robert-Cooperman et al., 2010; Robert-Cooperman et al., 2011).
Hormonal evaluation of male PANKO-C57 during fasting. The plasma levels of (A) insulin, (B) glucagon and (C) leptin were measured during fasting at 2 and 5 months of age in male PANKO-C57 (KO) and WT mice using the Mouse Metabolic Hormone Panel (Millipore) as measured by the MAGPIX® Luminex system (n=4–6). (D) Overnight fasting levels of C-peptide from mice aged 2 months using the MAGPIX® Luminex system as above (n=4). (E) Corticosterone levels were measured from plasma collected during fasting conditions at 2 and 5 months of age using the Corticosterone EIA Kit (Enzo Life Sciences) (n=3). (F) Plasma insulin levels during the course of GTTs (n=3–5). Values are expressed as means±s.e.m. *P<0.05, **P<0.01, ***P<0.001 as determined by using Student's t-test for A–E and two-way ANOVA in F. IP, intraperitoneal.
Enhanced insulin-stimulated hepatic glycogen and triglyceride content
To elucidate further downstream metabolic consequences of PANDER loss, we examined hepatic glycogen and triglyceride levels following insulin-stimulated and fasting conditions in male mice. In terms of hepatic glycogen content, 15 minutes after stimulation with 2 units of insulin per kg of body weight, the glycogen concentration within PANKO-C57 mouse livers was significantly increased, over 120-fold, compared with that of WT mice (Fig. 5A). Hepatic glycogen levels were not significantly different following conditions of fasting in the PANKO-C57 model when compared with those of WT controls (Fig. 5A). The hepatic triglyceride concentration was also increased in PANKO-C57 mice during insulin-stimulated conditions as compared with that of WT mice (Fig. 5B). During fasting conditions, hepatic triglyceride concentration was significantly decreased in PANKO-C57 mice as compared to that of WT counterparts (Fig. 5B). The circulating plasma triglyceride concentration was measured after an overnight fast and was significantly increased in 2-month-old PANKO mice as compared with WT mice, but not at 5 months of age (Fig. 5C). In summary, the liver responds very well to insulin in the PANKO-C57 model when it is stimulated for the production of glycogen, but not when it is stimulated to produce triglycerides. In addition, the lowered fasting glucose levels suggest that hepatic gluconeogenesis is effectively suppressed by insulin.
Hepatic glycogen, triglyceride and gluconeogenic content and expression in PANKO-C57. (A) Hepatic glycogen content (in μg/μl of tissue lysate) measurement of male PANKO-C57 (KO) and WT mice at 4–5 months of age following insulin stimulation (2 U/kg of body weight) or an overnight fast using the Glycogen Assay Kit (Abcam®) (n=3 per group). (B) Hepatic triglyceride content evaluation of 4-to 5-month-old male PANKO-C57 and WT mice as described in A, using 100 mg of liver tissue and the Triglyceride Quantification Kit (Abcam®) (n=3). (C) Serum triglycerides from serum after fasting of PANKO-C57 and WT male mice were analyzed longitudinally at 2 and 5 months of age using the Triglyceride Quantification Kit (Abcam®) (n=5). Values are expressed as the mean+s.e.m. *P<0.05, **P<0.01 as determined by using Student's t-test.
PANKO-C57 male mice display enhanced hepatic signaling
Previous investigations by our laboratory and others have provided evidence that the liver is a major target organ of PANDER (Li et al., 2011; Robert-Cooperman et al., 2010; Robert-Cooperman et al., 2014; Wilson et al., 2010). In addition, HEC studies that were performed in the former PANKO mouse model on a mixed genetic background indicated enhanced hepatic insulin sensitivity, but the mechanism and crucial signaling molecules accounting for this were not characterized or identified (Robert-Cooperman et al., 2010). Therefore, we evaluated the effect of PANDER on crucial hepatic signaling molecules that are involved in canonical insulin signaling and triglyceride synthesis within the PANKO-C57 model. Western blot analysis of numerous hepatic insulin signaling molecules was performed on extracts from the livers of male PANKO-C57 and WT mice that were excised after an overnight fast or insulin stimulation (Fig. 6).
Western blot analysis of PANKO-C57 hepatic signaling. (A) Western blot analysis for the protein levels of phosphorylated PI3K (p-PI3K; at residue Tyr 508), total and phosphorylated Akt (p-Akt; at residue Thr 308), mature and precursor (pre) SREBP-1, total and phosphorylated AMPKα (p-AMPKα; at residue Thr 172) in lysates from the livers of male PANKO-C57 and WT mice that had been subject to overnight fast. GAPDH served as loading control. The first three lanes correspond to PANKO-C57 mice and the final three lanes correspond to age- and gender-matched C57BL/6J WT mice. *Statistical significance of P<0.05 as determined by measuring using ImageJ as detailed in B–E. (B) Densitometry analysis of hepatic protein levels of phosphorylated PI3K, (C) Akt, (E) AMPK and (D) levels of mature SREBP-1 (mSREBP-1). The levels were determined and normalized to the total protein levels of the respective protein followed by normalization of the total protein to GAPDH (n=3). Values are expressed as the mean+s.e.m. *P<0.05 as determined by using unpaired Student's t-test.
During fasting conditions, increased levels of phosphorylated hepatic signaling molecules were observed in the PANKO-C57 model (Fig. 6). Significantly increased phosphorylated levels of phosphatidylinositol 3-kinase and (PI3K), protein kinase B (PKB/Akt) and adenosine monophosphate-activated protein kinase (AMPK) were observed during fasting conditions (Fig. 6A–C). Furthermore, significantly increased levels of mature sterol regulatory element-binding protein 1 (SREBP-1) were also observed and could provide a mechanistic explanation for increased triglyceride production (Fig. 6D). During insulin stimulatory conditions, increased levels phosphorylated PI3K, Akt and AMPK were observed in the livers of PANKO-C57 mice as compared to those of WT controls (data not shown). However, following densitometry analysis, differences were not statistically significant (data not shown). Taken together, hepatic signaling in the PANKO-C57 model shows an overall enhancement of essential insulin signaling, particularly in the fasted state, which is a unique observation within the PANKO-C57 model and suggests that PANDER serves a role in the regulation of hepatic signaling during both feeding and fasting conditions.
Distinct metabolic differences observed in the PANKO-C57 model as compared to those on a mixed genetic background
In contrast to the previous mixed PANKO model, the PANKO-C57 mice demonstrated (1) enhanced glucose tolerance during GTTs and fasting conditions, (2) decreased fasting insulin and C-peptide levels, (3) increased fasting leptin levels, (4) increased body weight and (5) increased phosphorylated Akt, phosphorylated AMPK and mature SREBP-1 levels. Our original characterization of the PANDER knockout was performed on a mixed genetic background of C57BL/6 and 129Sv mice. The previous PANKO model exhibited the following characteristics as compared with WT controls, (1) glucose intolerance during GTTs but not fasting conditions, (2) both impaired insulin secretion and clearance, (3) no differences in fasting glucagon, insulin or leptin, (4) similar peripheral insulin sensitivity, (5) enhanced hepatic insulin sensitivity, and (6) no differences in body weight (Robert-Cooperman et al., 2010). Compared with the previous PANKO model, the PANKO-C57 mouse displays a much more exaggerated phenotype, particularly during fasting conditions, in terms of additional differences from WT controls. Also, the PANDER transgenic (PANTG; discussed in further detail later) and mixed PANKO models revealed distinct differences within males only, whereas, the PANKO-C57 mouse exhibited increased weight gain and matched observed metabolic differences primarily seen in females, providing further evidence of a more pronounced penetrance of phenotype on the C57BL/6 background. The influence of genetic strain on phenotype has been well documented in mice (Berglund et al., 2008; Goren et al., 2004). This difference might be attributed to the effect that the host genome can have on numerous metabolic factors, such as insulin secretion or peripheral and hepatic sensitivity (Doetschman, 2009; Kahle et al., 2013), particularly in the C57BL/6J mouse model, which serves as a common in-bred strain of diet-induced T2D (Freeman et al., 2006; Toye et al., 2005). The findings from the PANKO-C57 model were both consistent with and contrasting from those of the mixed genetic background. The primary consistent phenotype from both models was with regard to hepatic insulin signaling. Although increased hepatic insulin sensitivity was demonstrated in the mixed PANKO model, there was no reported characterization of crucial signaling molecules, such as Akt or AMPK. However, consistent with the observed hepatic insulin sensitivity reported in the prior model, the PANKO-C57 model demonstrated an enhancement of both hepatic Akt and AMPK phosphorylation in addition to other downstream effectors, such as SREBP-1. Furthermore, another concordant observation was that peripheral insulin sensitivity was unaffected in both knockout models. Taken together, the overall hypothesis that PANDER impacts glycemic levels, in part, through interaction with the liver and hepatic signaling was certainly supported. Also, the PANTG and mixed PANKO models revealed distinct differences within males only, whereas the PANKO-C57 mice exhibited increased weight gain and matched observed metabolic differences between both males and females, providing further evidence of a more pronounced penetrance of phenotype on the C57BL/6J background. The previous PANKO mixed model did show impaired insulin secretion, resulting in glucose intolerance. This matched observation was also supported somewhat in the PANKO-C57 model, as decreased fasting levels of insulin and C-peptide were reported, but this did not have any impact on glucose intolerance as previously reported in the mixed model. It is reasonable to speculate that the impaired insulin secretion is adequately compensated by enhanced hepatic insulin sensitivity and therefore does not result in an observable impaired glucose response.
PANKO-C57 model vs. PANDER transgenic and acute models
During the preparation of this manuscript, we recently published the findings from our PANTG model (Robert-Cooperman et al., 2014). This transgenic mouse exclusively overexpresses PANDER from the pancreatic β-cell which results in an approximate 4-fold increase in circulating PANDER levels in both the fasted and fed state. The phenotype of the PANTG mouse as compared to the PANKO-C57 mouse is strikingly concordant in terms of the following observations: (1) fasting glucose, C-peptide and insulin levels are elevated (decreased in PANKO-C57), (2) hepatic insulin sensitivity is decreased, (3) hepatic phosphorylated AMPK is decreased during insulin stimulatory conditions, and (4) glucose intolerance is observed during GTTs (Robert-Cooperman et al., 2014). In general, the consistency between the observed metabolic phenotypes of the PANKO-C57 and PANTG mice strongly implicates a pleiotropic role for PANDER in the disruption of hepatic insulin signaling and the effects on glycemic levels in both the fasted and fed state. Acute models of PANDER employing transient overexpression within the liver through adenoviral delivery (Ad-PANDER) have also demonstrated concordant data (Li et al., 2011; Wilson et al., 2010). Li et al. report that overexpression of hepatic PANDER results in suppressed Akt activation, and hepatic silencing of PANDER decreased insulin resistance in db/db mice; however, these reports did not reveal any significant differences in glycemic levels during fasting or fed conditions (Li et al., 2011). Wilson et al. reveal that Ad-PANDER mice demonstrate increased fasting glucose and insulin levels, along with glucose intolerance (Wilson et al., 2010). However, Wilson et al. conclude that the glucose intolerance is due to elevated basal fasting glycemic levels rather than impaired insulin secretion or sensitivity. This effect has been attributed to PANDER amplifying glucagon signaling through hepatic cAMP and cAMP-response element-binding (CREB) protein, in contrast to the impaired Akt signaling that has been reported by Li et al. The observable and discernible phenotype within the PANKO-C57 model with regard to both fasting and fed glucose intolerance, which has not been seen in previous models, provides a measurable outcome and suitable metabolic end point to further investigate PANDER, and enables defined outcomes using this model that have not been observed in prior studies.
Absence of PANDER results in increased leptin and hepatic insulin sensitivity
Another interesting finding observed uniquely in the PANKO-C57 model, is the increased serum leptin levels. Leptin is produced from adipocytes and serves a crucial role in both weight gain and appetite (Ahima and Flier, 2000). Gross differences in food consumption were not observed in the PANKO-C57 model (S.L.M., personal observation). Administration of leptin to ob/ob or C57BL/6J mice demonstrates improved hepatic insulin sensitivity and decreased fasting glycemia and body weight (Halaas et al., 1995; Pelleymounter et al., 1995). Circulating leptin levels are associated with increased body weight in both rodents and humans (Considine et al., 1996; Frederich et al., 1995). In addition, leptin has been demonstrated to enhance the suppression of crucial gluconeogenic enzymes, such as PEPCK. The PANKO-C57 model was similar, in most characteristics, to leptin models in terms of decreased fasting glycemia and enhanced hepatic signaling. The PANKO-C57 model demonstrated increased leptin levels and weight gain in the presence of decreased fasting and glucose-stimulated insulin levels. This result might be explained in part by leptin promoting hepatic insulin sensitivity and reducing the demand on insulin secretion by pancreatic β cells. In addition, PANDER has been shown to facilitate insulin secretion and impair hepatic insulin sensitivity. The synergistic impact of these effects could result in a pronounced insulin-sensitive state in the presence of impaired pancreatic β cell function, reducing overall circulating insulin levels. Further experimentation is needed to determine whether PANDER has a potential suppressive effect on leptin production.
The potential impact of PANDER on type 2 diabetes
With accumulating investigations and review articles suggesting PANDER influences T2D and nonalcoholic fatty liver disease by manner of selective hepatic insulin resistance (Wang et al., 2012; Wilson et al., 2011; Yang and Guan, 2013). The majority of previous reports from both the stable and transient PANDER models suggest that PANDER disrupts insulin signaling, particularly through Akt, yet hepatic lipogenesis is increased (Li et al., 2011; Robert-Cooperman et al., 2014). Strong evidence has demonstrated that the PI3K and Akt pathway activates the sterol-regulatory element-binding proteins (SREBPs), which are considered master transcriptional regulators of lipid metabolism (Krycer et al., 2010). Therefore, PANDER-induced disruption of hepatic signaling still does not alter hepatic triglyceride production and demonstrates that PANDER can result in a selective hepatic insulin-resistant state whereby suppressed Akt signaling does not impair lipogenesis but yet still fails to inhibit gluconeogenesis. Selective hepatic insulin resistance (SHIR) is a major pathological aspect of T2D and is responsible for both hyperglycemia and dyslipidemia (Biddinger et al., 2008; Brown and Goldstein, 2008). The hepatic cellular mechanism by which PANDER induces SHIR is largely unknown but has been suggested to be either mediated through inhibited phosphorylation of AMPK or through Forkhead box 1 (FOXO1). The findings in the PANKO-C57 model certainly support the impact on hepatic signaling and have revealed not only increased levels of phosphorylated Akt but also of downstream molecules, such as mature SREBP-1, with concordant increases in hepatic triglyceride content. Therefore, both the PANKO and PANTG models reveal a similar phenotype, but this may be reconciled through speculation that the absence of PANDER promotes an overall insulin-sensitive state that stimulates both glycogen and triglyceride production. Indeed, HEC studies in the prior PANKO model have revealed decreased gluconeogenic output (Robert-Cooperman et al., 2010). HEC studies performed on the PANKO-C57 model would have provided useful data but were not conducted due to current technical limitations in performing these experiments.
Utility of the PANKO-C57 mouse in the investigation of PANDER function
Overall, the PANKO-C57 mouse provides an excellent model for the investigation of PANDER and has revealed additional and novel findings that further elucidate the metabolic function of PANDER and role of this uniquely structured molecule in glycemic regulation and hepatic insulin signaling. Identification of the PANDER receptor would be highly valuable in the understanding of PANDER biology, along with the measurement of circulating PANDER levels during various metabolic and pathophysiological conditions, particularly those found in T2D. This study evaluating the PANKO-C57 model in combination with the investigations of others strongly alludes to PANDER being a potential therapeutic candidate for the treatment of hepatic insulin resistance and steatosis typically associated with T2D.
Generation of PANDER knockout C57BL/6 (PANKO-C57) mice
The initial description of the targeted disruption of the Pander (Fam3b) gene and generation of the knockout has been previously described and performed at the Children's Hospital of Philadelphia Research Institute (CHOP) through replacement of the transcriptional start site and first two exons of the Pander gene with neomycin (Robert-Cooperman et al., 2010). The PANDER knockout mouse was then backcrossed with C57BL/6 mice for seven generations at CHOP, followed by subsequent additional crossings with C57BL/6J mice following commercial donation, as described next. This strain (PANKO-C57) was subsequently donated to Jackson Laboratories (Bar Harbor, ME) and is commercially available under the strain name B6.129S6-Fam3btm1Bkht/J (stock number 013788). Homozygote PANKO-C57 breeding pairs were obtained from Jackson Laboratory and shipped to the Moffitt Cancer Center's Stabile Vivarium (Tampa, FL) for murine colony generation. All mice were fed Purina normal chow and water ad libitum. All PANDER knockout and C57BL/6J WT offspring were screened by using PCR amplification of genomic DNA isolated from tail tissue (DNeasy Kit, Qiagen, Germantown, MD) for knockout of the gene or for identification as WT, products of 500 and 800 bp, respectively. PCR genotype confirmation cycling conditions and primers (forward primer located in the Pander promoter region: 5′-CTTGTGATGGTGGATGCCCAGTT-3′ and reverse primer located within neomycin gene: 5′-CTTCCTCGTGCTTTACGGTATC-3′) were used as previously described (Robert-Cooperman et al., 2010). Primarily, confirmed PANDER knockout and C57BL/6J WT mice at 8 weeks to 6 months of age were evaluated for this study. All murine handling and experimentation adhered to protocols approved by the Institutional Animal Care and Use Committee at the University of South Florida and Moffitt Cancer Center.
Weight measurements
PANKO-C57 and WT mice were weighed (Beckman Digital Scale) at the same time in the morning (~9 am) during ad libitum chow conditions in 2-to 3-week increments beginning at 8 weeks of age for both genders. Weight was measured in grams and evaluated longitudinally between both groups.
Glucose tolerance testing and insulin measurements
GTTs were performed on mice at 16 weeks of age as previously described (Robert-Cooperman et al., 2010). In short, PANKO-C57 and WT mice were subjected to an overnight fast (>16 hours) in a cage with bedding removed ~16 hours before the GTT. Mice were then injected intraperitoneally with 2 g of glucose (Fisher Scientific) per kg of body weight. Blood glucose was measured at 0 minutes before glucose injection and at 15, 30, 60 and 120 minutes thereafter using a TRUEtrack® glucometer (Nipro Diagnostics, Fort Lauderdale, FL) through collection of blood through a tail vein. Insulin levels were measured using a commercially available murine insulin ELISA kit (ALPCO Diagnostics, Salem, NH) during the glucose challenge.
Insulin tolerance testing
ITTs were performed within a week after GTTs were performed on mice at 16 weeks of age, within a week of performing GTTs, as previously described (Robert-Cooperman et al., 2010). In brief, PANKO-C57 and WT mice were fasted for 4 hours prior to the ITT. Mice were subsequently injected with NovoLog® insulin (Novo Nordisk, Plainsboro, NJ) at a concentration of 1 U/kg of body weight. Blood glucose was measured immediately before injection of insulin (0 minutes) and thereafter at 15, 30, 60, 90 and 120 minutes, as described above for GTT.
Measurement of fasting glycemia
Mice were fasted either long term (overnight, ~16 hours) or short term (4 hours) before metabolic testing as described above. Blood glucose was measured at the same time for all mice through collection from a tail vein, as previously described. Blood glucose was averaged for each group and compared based on fasting duration (n=8–15).
Western blot analysis was performed on flash-frozen livers isolated from PANKO-C57 and WT mice at 5 to 6 months of age. Mice were either fasted overnight (~16 hours) or for a short period (4 hours). For insulin stimulation experiments, mice were fasted short term and then intraperitoneally injected with insulin (NovoLog®, Novo Nordisk, Plainsboro, NJ) at 2 U/kg of body weight. Protein was isolated from hepatic tissue using Tissue Protein Extracting Reagent (TPER) (Thermo Fisher Scientific, Rockford, IL) and quantified using Pierce BCA Protein Assay following the manufacturer's protocol (Thermo Fisher Scientific). Hepatic lysate (20–50 μg) was analyzed using SDS-PAGE (Pre-Cast 10% Mini-PROTEAN® TGX™ gels, Bio-Rad, Hercules, CA) and electrotransferred to polyvinylidine fluoride (PVDF) membrane using iBlot semi-dry transfer apparatus (Invitrogen, Carlsbad, CA). Western blots were then probed with antibodies to detect the levels of phosphorylated PI3K at Tyr 508 (cat. no. sc-12929, Santa Cruz Biotechnologies, Santa Cruz, CA), total and phosphorylated (residue Thr 308) Akt/PKB (cat. nos. 4691 and 4056, respectively, Cell Signaling, Danvers, MA), full-length mature and precursor SREBP-1 (cat. no. sc-8984, Santa Cruz Biotechnologies) and total and phosphorylated (residue Thr 172) AMPK subunit α (AMPKα) (cat. nos. 2603 and 2535, respectively, Cell Signaling). All primary antibodies were diluted 1:1000 in StartBlock™ Block Buffer (Thermo Fisher Scientific). Protein detection was achieved using horseradish-peroxidase-conjugated goat-anti-rabbit secondary antibody (Bio-Rad) at 1:3000 dilution in StartBlock™ followed by chemiluminescence detection using Pierce ECL Western Blotting Substrate (Thermo Scientific). Protein signals were visualized using the LAS 3000 Intelligent Dark Box (Fujifilm, Stamford, CT), and relative protein levels were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) loading control (cat. no. 2118, Cell Signaling). Protein expression levels were quantified and compared using ImageJ (version 1.46) densitometry analysis.
Plasma collection for corticosterone and triglyceride assays, and endocrine hormone panel
PANKO-C57 and WT mice were subjected to fasting overnight before plasma collection. Whole blood from mice that had been subjected to fasting was obtained through submandibular vein puncture and free flow collection into BD MicroContainer® (Becton, Dickinson and Company, Franklin Lakes, NJ). Whole-blood samples were immediately placed on ice before being centrifuged for 5 minutes at 13,200 r.p.m. in an Eppendorf Microfuge 5430 for plasma separation into a new Eppendorf (Eppendorf, Hauppague, NY) collection tube. The endocrine hormone panel was performed using the commercially available Milliplex Mouse Metabolic Hormone Magnetic Bead Panel MMHMAG-44K (EMD Millipore Corporation, Billerica, MA) and Luminex Multiplex Platform (MAGPIX®). All procedures regarding the metabolic hormone panel adhered to the manufacturer's protocol included in the assay kit. Corticosterone EIA kit (Enzo Life Sciences, Farmingdale, NY) and Triglyceride Quantification Kit (Abcam®, Cambridge, MA) were utilized for the determination of corticosterone and plasma triglyceride concentrations, respectively, and both were performed in accordance with the manufacturers' suggested protocols.
Hepatic glycogen assay
PANKO-C57 and WT mice were either fasted (overnight) or stimulated with insulin (Novolog®) at a concentration of 2 U/kg of body weight prior to liver isolation. Livers were harvested and immediately flash frozen in liquid nitrogen for storage at −80°C. The Glycogen Assay Kit (ab65620) (Abcam®) was used according to the manufacturer's suggested protocol, and 10 mg of liver tissue was used.
Hepatic triglyceride assay
Overnight fasting and insulin-stimulated hepatic triglyceride content was evaluated in male PANKO and WT mice at 4 to 5 months of age. Liver tissue harvest was conducted as described above from mice that had been subjected to fasting or insulin stimulation, and the Triglyceride Quantification Kit (Abcam®) colorimetric assay was conducted according to the manufacturer's protocol.
Data are presented as mean±s.e.m. unless otherwise specified. All statistical analyses were completed using GraphPad Prism (version 5) where statistical significance between groups was determined by unpaired Student's t-test or two-way ANOVA. Any P-value that was less than 0.05 was considered to be statistically significant.
The authors thank the Moffitt Stabile Vivarium for the continual maintenance of our animal colony. The authors also acknowledge the tremendous contributions of the Jackson Laboratory and in particular Melissa Osbourne and Jonathan Newell for providing crucial mice for the measurement of glucose-stimulated insulin levels. The work presented here was in partial fulfillment of the Master of Science degree by S.L.M. in the department of Cell Biology, Microbiology and Molecular Biology at the University of South Florida.
S.L.M. designed and performed the experiments, analyzed the data and contributed to manuscript writing. G.C.D. performed the experiments involving the metabolic evaluation of mice. K.B.M., A.M.F., W.A.D., M.N.K. and M.G.A. performed the experiments and greatly contributed to the colony maintenance required for later phenotyping. B.R.B. designed the project, analyzed data and wrote the manuscript.
This work was partially supported by the University of South Florida New Investigator Award to B.R.B.
Flier
Annu. Rev. Physiol.
Seeking an ancient enzyme in Methanococcus jannaschii using ORF, a program based on predicted secondary structure comparisons
Berglund
Poffenberger
Fueger
Glucose metabolism in vivo in four commonly used inbred mouse strains
Biddinger
Hernandez-Ono
Rask-Madsen
Stephanopoulos
Hepatic insulin resistance is sufficient to produce dyslipidemia and susceptibility to atherosclerosis
Selective versus total insulin resistance: a pathogenic paradox
Burkhardt
Tissue-specific and glucose-responsive expression of the pancreatic derived factor (PANDER) promoter
Robert-Cooperman
Characterization of the expression, localization, and secretion of PANDER in alpha-cells
Mol. Cell. Endocrinol.
Considine
Sinha
Kriauciunas
Ohannesian
Serum immunoreactive-leptin concentrations in normal-weight and obese humans
N. Engl. J. Med.
Doetschman
Influence of genetic background on genetically engineered mouse phenotypes
Löllmann
Napolitano-Rosen
Expression of ob mRNA and its encoded protein in rodents. Impact of nutrition and obesity
Hugill
N. T.
F. M.
Deletion of nicotinamide nucleotide transhydrogenase: a new quantitive trait locus accounting for glucose intolerance in C57BL/6J mice
Goren
Glucose homeostasis and tissue transcript content of insulin signaling intermediates in four inbred strains of mice: C57BL/6, C57BLKS/6, DBA/2, and 129X1
Halaas
Gajiwala
Lallone
Weight-reducing effects of the plasma protein encoded by the obese gene
Bernström
Bäckström
Schweikart
FAM3B PANDER and FAM3C ILEI represent a distinct class of signaling molecules with a non-cytokine-like fold
Kahle
Schultheiß
Rathkolb
Phenotypic comparison of common mouse strains developing high-fat diet-induced hepatosteatosis
Mol. Metab.
Krycer
The Akt-SREBP nexus: cell signaling meets lipid metabolism
Trends Endocrinol. Metab.
Lahsnig
Mikula
Petz
van Zijl
Csiszar
Mikulits
ILEI requires oncogenic Ras for the epithelial to mesenchymal transition of hepatocytes and liver carcinoma progression
Pancreatic-derived factor promotes lipogenesis in the mouse liver: role of the Forkhead box 1 signaling pathway
A non-secretory form of FAM3B promotes invasion and metastasis of human colon cancer cells by upregulating Slug expression
Cancer Lett.
Pelleymounter
Effects of the obese gene product on body weight regulation in ob/ob mice
Targeted disruption of pancreatic-derived factor (PANDER, FAM3B) impairs pancreatic beta-cell function
PANDER KO mice on high-fat diet are glucose intolerant yet resistant to fasting hyperglycemia and hyperinsulinemia
Dougan
Athanason
Kuehl
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ILEI drives epithelial to mesenchymal transition and metastatic progression in the lung cancer cell line A549
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Toye
Lippiat
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A genetic and physiological study of impaired glucose homeostasis control in C57BL/6J mice
Waerner
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Oberauer
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ILEI: a cytokine essential for EMT, tumor formation, and late events in metastasis in epithelial cells
Mechanisms of glucose-induced expression of pancreatic-derived factor in pancreatic beta-cells
Role of pancreatic-derived factor in type 2 diabetes: evidence from pancreatic β cells and liver
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Liver-specific overexpression of pancreatic-derived factor (PANDER) induces fasting hyperglycemia in mice
PANcreatic-DERived factor: novel hormone PANDERing to glucose regulation
Interferon-gamma-induced regulation of the pancreatic derived cytokine FAM3B in islets and insulin-secreting betaTC3 cells
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Cloning, expression, and initial characterization of a novel cytokine-like gene family


Erik Johansson | NMLS222984 | bemortgage, a Division of CrossCountry Mortgage, Inc.
Since 2002, Erik considered himself more of a consultant and someone who caters to his clients needs, rather than a member of the "sales" community. He values transparency vs. traditional "black boxed" information hoarding. He believes that the consumer deserves unfettered access to be able to make an informed decision, rather than being guided down a predetermined path. In addition to being a Top 1% producing originator, he is also a classically trained chef.
I could have never been able to get through this first time home buying process without Erik and his team. He acted as my coach in a sense to this whole experience and I could not be more happy!
Erik is the best! As a first time homebuyer he explained everything so clearly and went through all of my options. I felt comfortable knowing that we were working together to find the best loan for my long term goals and home purchase.
Erik and his team were all fabulous throughout the entire process and made the home buying experience less stressful. They were in constant communication, explained everything, and provided outstanding service. Working with Erik and his team was a great experience and we have nothing but positive things to say.


text:
Maps Show EPA Land Grab
by Lamar Smith on August 28, 2014 at 5:00 PM
Today I sent a letter to Environmental Protection Agency (EPA) Administrator Gina McCarthy demanding additional information about the agency's motivation for having detailed maps assembled showing waters and wetlands for all 50 states. The maps, which were created in 2013 shortly after EPA proposed its Waters of the U.S. rule, had never been made public. When confronted at a hearing, EPA Deputy Administrator Bob Perciasepe agreed to release the maps.
These maps show the EPA's plan: to control a huge amount of private property across the country. Given the astonishing picture they paint, I understand the EPA's desire to minimize the importance of these maps. But the EPA's posturing cannot explain away the alarming content of these documents. It's time to give Americans a chance to make up their own minds about the EPA's intentions. While the Agency marches forward with a rule that could fundamentally re-define Americans' private property rights, the EPA kept these maps hidden. So, today I will be posting the maps on the Committee's website for public review.
While the EPA has claimed the maps have not yet been used to regulate, they have failed to explain why the agency used taxpayer money to create them. The EPA paid a private contractor to make many of these maps, yet the details of the arrangement have not been disclosed. Serious questions remain regarding the EPA's underlying motivations for creating such highly detailed maps that were created just days after the EPA announced its Waters of the U.S. rule.
The letter requests all documents and communications related to the EPA's contract to create these maps and demands that these and any other previously undisclosed maps in the EPA's possession be entered into the official rulemaking docket for public review and comment. The letter also requests EPA keep the public comment period open for at least 60 days to provide adequate opportunity for public review and comment.
The maps are posted here.
Gina McCarthy
Lamar Smith's blog
Permalink Submitted by TexasGOPVote on August 28, 2014 at 5:02 PM

redpajama_set_name: RedPajamaCommonCrawl


Multidisciplinary discussion of the causes, effects, and treatments of human obesity. Topics include the biopsychology of eating behavior, the genetics of obesity, the role of activity and energy metabolism, the psychosocial determinants of obesity, anorexia nervosa, therapy and its effectiveness, and social discrimination. Outcome 1: Familiar students with the breadth of knowledge, theories, and therapies concerning energy balance and the control of food intake and energy expenditure.


Fewer visa restrictions and higher incomes mean that many Chinese are looking for new destinations to visit. Oftentimes, the inspiration for new places to go is found on social media, but also through more traditional media, like film and TV. This is something that the Danish island of Funen takes advantage of.
"This market is changing hugely. Now Chinese people, especially better educated, more experienced travellers and younger ones, are looking to really involve themselves in the countries they travel to and experiencing something different and more unique than the Eiffel Tower," explains Christopher Ledsham.
As Chief Communications Officer at the China Outbound Tourism Research Institute (COTRI) in Hamburg, Germany, he follows the trends and terms of the way that Chinese tourists act. In 2017, 130 million Chinese travelled abroad, and they are known to be big spenders. According to the UN's tourism organisation, Chinese tourists spent US$258bn abroad last year, nearly twice the amount that tourists from the US, who were the second largest spenders on outbound trips that year, spent.
The wealthy and adventurous new generation of independent Chinese travellers go to places like Chiang Mai, seeking adventures like the ones shown in the Chinese movie hit Lost in Thailand, or they travel to South Korea, inspired by K-pop and soap operas. They might also go to Denmark to see the sights featured in the Chinese TV series, Flowers on Trip, with their own eyes, or explore the Danish castle, where the plot of a new Chinese film unfolds.
The Danish island Funen, which markets itself as "Hans Christian Andersen's hometown", hopes to become the next big thing for Chinese travellers. The island uses social media, but also movies and TV series, to reach potential visitors from China.
More than 140 million Chinese have already encountered the hashtag #安徒生故乡# (Āntúshēng gùxiāng) on China's Twitter-like Sina Weibo, where stars such as Jackson Yee from the Chinese boyband TFBoys and the Danish world champion in badminton, Viktor Axelsen, are among the contributors to Funen's account.
In 2017 – officially China-Denmark Tourism Year – Funen also attracted the popular TV series, Flowers on Trip.
The Chinese travel reality show, watched hundreds of millions of times on Dragon TV and Chinese video platforms, follows seven Chinese celebrities exploring different countries. When the 70-member film crew visited Denmark last summer, they did not only visit the capital Copenhagen, but also dedicated almost two full episodes to the 470,000-inhabitant island of Funen.
"In the long run, we hope that the Chinese will be inspired by the program and will want to go here," says Xiaowei Liu Lolk from the island region's tourism organisation, Destination Fyn.
Though the island has already seen more tourists from China in April and May this year, she thinks it is still too early to see the full effect of the Chinese reality series on tourism to the area.
Another project that could open Chinese tourists' eyes to Funen, is the first Chinese feature film made in Denmark, which was shot on the island in 2016. The romantic comedy, called Bao Zang (堡 藏), has received a so-called Dragon Mark from the Chinese authorities, which means that it might be shown in Chinese cinemas any time soon.
"Such a project generates attention to the country as a film location, and the Chinese notice what Denmark has to offer," says Lisa Johansen from the firm Lifelong Learning that co-produced the film. "Sometimes you might even be so lucky that certain locations become popular enough for people to travel to see them," she adds.
To get the most out of exposure in a movie or series that becomes popular in China, smaller destinations in particular need to prepare well, stresses Christopher Ledsham from COTRI.
In case Funen gets flooded with Chinese fans of the Flowers on Trip show, Xiaowei Liu Lolk is well prepared.
"We have planned package tours related to the reality show, so if the Chinese are inspired and want to visit us, we are ready to give them some products to purchase," she assures.
China is a vast and incredibly diverse place. Yet, only a few foreigners know the characteristics of different parts of China. This is true the other way around, too.
"The difference for a Chinese person between Denmark, Sweden, Germany and the Netherlands is far narrower than to the locals," says Ledsham.
He is not in doubt that many Chinese would want to head to Denmark after watching the celebrities from Flowers on Trip visiting Hans Christian Andersen's childhood home and eating oysters with the Danish crown prince.
"We know that if you have Chinese celebrities eating Danish food or visiting a Danish castle, something very specific like that, it can really help place in their mind what this destination is about," Ledsham points out.
Since Chinese, according to Ledsham, tend not to trust traditional advertisements, featuring a certain destination in a movie or TV show is one of the most effective ways of drawing affluent Chinese tourists.
This way, Matt Damon's The Martian turned Abu Dhabi into a place to visit, and Prague became famous through the Chinese film Somewhere Only We Know.
"Chinese people are often keen to follow in the footsteps of others. They don't see that as copying in the same way that Europeans or Westerners might. Instead they take it as a strong recommendation," explains Ledsham.
Tunisia is an example of a destination that has managed to take advantage of this effect. To help fill the North African country's diminished treasury and restore a tourism sector that had suffered after several terrorist attacks, Tunisia decided last year to offer up to 90 days of visa free travel in the country to Chinese tourists. Now Chinese come in large numbers to see parts of the Sahara Desert that played an important part as the planet Tatooine in the Star Wars movies.
Thanks to the change of visa rules – and Star Wars – China soon became Tunisia's fastest-growing source country of tourists with a rise in Chinese visitors from 7,400 in 2016 to more than 18,000 visitors in 2017, reports Xinhua.
Will Denmark's Funen be the next big hit?


text:
Marki szkockiej whisky

Szkocka whisky single malt

Szkocka whisky zbożowa

Szkocka whisky mieszana

Niezależne rozlewnie szkockiej whisky

Amerykańskie whiskey/marki burbonów

Amerykańskie single malt whiskey

Amerykańskie whiskey kukurydziane

 Platte Valley

Tennessee whiskey

Whiskey żytnie

Burbony i destylarnie

 Four Roses Distillery – Lawrenceburg, Kentucky
 Heaven Hill Distillery, Bardstown, Kentucky
 Buffalo Trace Distillery – Frankfort, Kentucky
 Jim Beam Distilleries, Clermont, Kentucky
 Woodford Reserve Distillery – Versailles, Kentucky
 Maker's Mark Distillery – Loretto, Kentucky
 Wild Turkey Distillery – Lawrenceburg, Kentucky

Burbony Kentucky

Burbony non-Kentucky

Burbon z nieznanych destylarni

Lista whisky kanadyjskiej

Kanadyjska single malt
 Glenora Distillers, Glenville, Nova Scotia (niezależna)
 Glen Breton Rare
 Glen Breton Ice 10 Year
 Glen Breton Ice 15 Year

Kanadyjska whisky mieszana

Marki whiskey irlandzkiej

Irlandzka single malts

Pure pot still whiskey

Whiskey irlandzka mieszana

Irlandzka whiskey single grain

Marki whisky walijskiej

Inne europejskie whisky/whiskey
 Armorik – Francja, Bretania
 Bag Pipper
 Dark Whisky – Polska
 Mackmyra – Szwecja
 Manx spirit (produkt trudny do sklasyfikowania)
 Royal Challenge
 Slyrs – Niemcy
 J.H. – Austria

Marki whisky japońskiej

Marki whisky nowozelandzkiej
new zealand whisky yuck

Tajlandzka whiskey
 Mekhong whiskey
 Hong Thong whiskey
 Sang Thip
 SangSom
 Black Cat whiskey

Zobacz też
 Lista destylarni w Szkocji

Bibliografia

 https://web.archive.org/web/20040911084353/http://www.whiskymag.com/directory/regional/scotland/

Listy związane z gospodarką

redpajama_set_name: RedPajamaWikipedia


Cadillac has designed its third generation model of the CTS as a focused machine and when this car debuts fall 2014, it is going to have a much defined fan following across all generations. As opposed to a general notion that Cadillac designs cars only for old men, latest renderings of concepts have showcased the manufacturer's power of designing youth centric vehicles. The current version of Cadillac's CTS had been remodeled when it was introduced a few years back while latest images clearly showcase farfetched designing changes.
Cadillac is known for its luxuriously powerful vehicles and despite the fact that these cars are neither as powerful nor luxurious as some German manufactured vehicles, they carry around a pocket friendly price tag as well. Cadillac manufactures vehicles for the upper-middle class strata. Hence their vehicles feature the best of everything affordable.
The smaller and more maneuverable ATS was launched a few years back and this Cadillac CTS based vehicle managed to gain a lot of attraction from the masses due to its pocket friendly nature. This car was also lightweight and easy to drive while a powerful yet fuel efficient engine gave it the added oomph factor that every car in this price segment needs.
The ATS was not just lighter than its predecessors, it was 200 pounds lighter than BMW's 528i, with which it was competing in global markets. Cadillac confirms that its latest CTS will be based on lessons learned from the ATS and hence we can expect a heavy duty vehicle to be based on a light weight frame. This will give the car, best of everything and you can finally drive a spacious luxury sedan which handles like a sports car.
Modern engineering has enriched General Motor's factories and manufacturing units and latest technologies prove the company's progress in car designing and manufacturing. This car extends 5 cm over its predecessor while an additional role cage has been installed to keep the driver and passenger safe in case of an accident. Sharper handling is also a boon on the next generation vehicle and we can easily expect high standards from this car.
Power to weight ratio has improved which gives this car more balance on real world situations. A twin turbo V6 engine has been mated to an eight speed automated transmission system and this drive train is powering the new CTS. Pirelli Pzero tires provide superior all weather grips and this is set to decrease accidents. Cadillac has focused extensively on safety measures and this is clearly exposed in this car. Steering dampers and ABS are optional features which should be available for a premium price.
Although final plans regarding launch are yet to be unveiled, we can only hope that this car is available by mid 2014. Pricing is yet to be announced although Limited Editions variants will be launched soon after this car, to enhance drive feel and popularity of this vehicle. Cadillac is finally emerging out of its old man image, which has taken the world by storm.


SAIT nv is an independent specialist and advisor in integrated ICT. We provide private companies as well as local, national and international institutions with fully integrated, wireless systems and solutions for their ICT and security issues. Thanks to these high quality products, both private and public sector clients have at their fingertips the invaluable and essential tools they need for highly sensitive, confidential communication and data transfer. SAIT nv delivers solutions tailored to specific situations where security, guaranteed access and reliability are crucial.
We are organized into 6 Centres of expertise offering "Mission Critical Solutions": Mission Critical Communication Solutions, Physical Security Solutions, ICT Security Solutions, Data Center Infrastructure Services, Carrier Grade Solutions as well as Alignment & IT Governance.
SAIT is part of Securitas.


There are a number of security measures to consider before full adoption of the digital rupee can take place in India.
On November 8, 2016, to curtail the black economy and to eliminate the use of illicit and counterfeit cash for terrorism and illegal activities, the Government of India announced the demonetization of all ₹500 and ₹1,000 bank notes of the Mahatma Gandhi Series. In the weeks that followed, further progress was made to go cashless when the Committee of Digital Payments, headed by former Finance Secretary Ratan P Watal, endorsed testing the digital rupee and the annual Indian Union Budget highlighted a plan to eventually migrate to a completely digital economy.
Security Electronic Technology (SETHK) Limited, a Hong Kong based security consulting firm for the global financial sector, trusts that the emergence of the digital rupee can boost competition, influence greater consumer protection, and allow for a transparent economy.
However, the firm believes blockchain technology is not currently reliable enough in regard to application security to foster a stable digital fiat currency. According to Cheng Luoyang, Head of Research & Development of SETHK, "The technology itself still needs work in respect to gaps in the stability of the system as a whole together with application security." Cheng Luoyang is referring to vulnerabilities that occur due to nodes having the freedom to enter and leave the network. This makes them susceptible to encryption key theft, address spoofing attacks, route deception, and algorithm cracking.
According to SETHK, information leakage is another possibility that companies and governments should consider since every node in the network maintains a record of every transaction. The release states, "Therefore, the cost of the digital fiat currency should be minimal with the loss of physical logistics costs and benefits of riding on existing digital transaction infrastructure." It goes on to say that since the cost of replicating, transmitting, and storing the full payment history within every node would become expensive, banks, governments, and companies should focus on solutions that don't utilize peer-to-peer network capabilities.
SETHK further elaborated that the security strength of the Indian rupee is based on hardware and security elements that go beyond cryptographic algorithms. As a result, the distribution of the Indian rupee should be the sole responsibility of the Ministry of Finance and the Bank of India.
Like what you read? Follow us on Twitter @ETHNews_ to receive the latest India, digital economy or other Ethereum world news.


St Peter's Basilica is considered one of the holiest catholic shrines in the world. It is visited by thousands of tourists and pilgrims every month. This splendid church is a magnificent must-see for tourists and is built upon the tomb of St Peter.
Peter's Square – Locally called as the Piazza San Pietro, St. Peter's Square is situated in front of the Basilica. You'll find two stunning fountains on either side of the square. You will be able to see the large statues of St Paul and St Peter at the front of the square.
Peter's Baldachin – It is located directly under the dome in the Basilica and is a 29-meter high bronze canopy. Gian Lorenzo Bernini started his work on the Baldachin in the year 1623. This work was finally completed in 11 years later in 1634. The Baldachin is situated directly above St Peter's tomb, over the papal alter. No one else but the Pope is permitted to serve at the altar.
Bronze Statue of St Peter –This was created in the year 1300 by Arnolfo di Cambio, and is seated on a throne that has been situated in the Basilica ever since 1605. The pilgrim tradition includes either kissing or touching the statue's feet. This has caused the right foot to wear down over the years.
The view from the top of the Dome – After climbing up the stairs or taking the lift, you will find yourself looking out at the stunning views of the Vatican City from atop the St Peter's Basilica.
How to Get to St Peter's Basilica in Rome?
Located within the Vatican City, which is north of the city centre of Rome, St Peter's Basilica is breath-taking by all means. In case you are using the Rome Metro system, you can get off at Line A of Ottaviano. It is only a ten-minute walk to St Peter's Basilica from there. Usually, buses stop off at Vatican City. The main bus numbers are 64, 62, 40 and 81. Children under the age of 10 are free to travel on all public transport. There are various hop-on/hop-off buses which you can get for the Vatican City and you can also catch the bus back again later on.
Millions of people come to visit St Peter's Basilica every month, so it is essential that you book your tickets in advance to skip the long line and enter directly without any hassle. You can visit the Basilica for free, but there is a fare for going up to the dome and seeing the Vatican Necropolis.
In case you want an access to the Vatican Necropolis, where the tomb of St Peter is located, you have to get a special permission from the Excavations Office. The cost is 13 euro per person.
Tickets to enter the Basilica cannot be bought online as they are free. So you will have to join the line. This queue can be quite long as this is such a crowded tourist destination. If you want to avoid this then reserve your tickets online with a tour group.
One of the most important things you should know is that you will be refused entry if not dressed appropriately. Hundreds of people are debarred from entering daily!


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Animals Suffer in Military Testing Despite Superior Alternatives
Even with advances in medical teaching techniques, the Canadian military still trains medical personnel by using live animals. The techniques employed by the military are the epitome of cruelty. In live agent training techniques, pigs are exposed to sarin, mustard and other chemical agents to induce medical emergencies such as seizures, irregular heartbeats, difficulty breathing, and excessive bleeding. To learn how to treat major trauma injuries, pigs are shot, stabbed and amputated.
In addition to the cruelty involved in Canada's military training techniques, advancements in computerized medical simulation technology have also made these techniques obsolete. Advanced human simulators are widely available to replace the use of animals in chemical warfare training. Such simulators, which can mimic human response to chemical-weapon exposure, are used by other militaries around the world, including the U.S. Army and the Israel Defense Forces. Canada is one of six remaining NATO countries that still use live animals to train battlefield physicians.
Although animal research and testing is an area of Canadian law that severely lacks substance, there is still one standard that the Canadian military, as a government department, can be held accountable to.
The Canadian Council on Animal Care ("CCAC") is a quasi-regulatory body that sets guidelines on animal care and use of animals in science. In regards to the use of animals in research, teaching, and testing, the CCAC states: "Animals should be used only if the researcher's best efforts to find an alternative have failed." [1]
The availability of superior medical training methods leaves the Canadian military in breach of the CCAC's animal testing guidelines. In response to this breach, Animal Justice Canada has sent a letter to the Minister of National Defence, Peter MacKay, to take action and end the unnecessary use of animals in medical training.
With overwhelming evidence supporting the superiority of alternative teaching models, it is time for the Canadian military to update its outdated teaching strategies and end the unnecessary suffering of animals.
Written by Zeynep Husrevoglu, BA, LLB-Articling Student
This blog and the contents herein are for informational purposes only and do not constitute legal advice. Readers are advised to seek legal counsel prior to acting on any matters discussed herein.
[1] Canadian Council on Animal Care, CCAC Guide to Care and Use of Experimental Animals, 2d ed (Ottawa: Canadian Council on Animal Care, 1993) vol 1, online: <http://www.ccac.ca/Documents/Standards/Guidelines/Experimental_Animals_Vol1.pdf>


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text:
Growing pressure on journalists in Turkey before the elections
Tags: eidhr, tgs, turkey
The Freedom for Journalists Plaftorm (GÖP), including the Journalists Union of Turkey (TGS) the EFJ-IFJ affiliate, calls on Turkish authorities to end pressure on media and journalists that prevent the public from accessing information. Yesterday (31/05/2015), police forces in Istanbul attacked again reporters for covering a public protest commemorating the second anniversary of the Gezi Park demonstrations. Journalist Emre Şencan working for the Turkish daily Zaman was injured. "The authorities should start an investigation against police officers who have attacked the reporter. Journalism in not a crime", said TGS.
The Freedom for Journalists Platform (GÖP) published a specific statement denouncing the increasing pressure put on media and journalists in Turkey. According to the platform, the adoption of the new Homeland Security Act has oppressed televisions, radios, websites and media workers in alarming proportions :
On May 26, the police raided the office of Gelecek newspaper and arrested reporters Ufuk Erhan and Ezgi Aydın as well as Birgün correspondent Onur Öncü who was also on the spot. Ufuk Erhan has been arrested whereas the two other journalists have been released.
Baris Ince, Director of publication at the Birgün newspaper, is still being prosecuted by President Recep Tayyip Erdogan and his son Bilal Erdogan for publishing articles about corruption cases affecting the presidential family.
Ten journalists, including Nazli Ilıcak, Ceyda Karan, Baris Atay, Mirgün Cabas and Banu Güven, are being prosecuted for "doing propaganda on behalf of a terrorist organization" because of their comments (tweets) during the killing of a prosecutor in Istanbul. The journalists face up to 4-5 years imprisonment if found guilty for their tweets.
Can Dündar, editor-in-chief of the daily Cumhuriyet, is facing an "anti-terror" trial for publishing photographs showing arms sent by Turkish authorities to Syrian rebels.
Bugün newspaper Kamil Maman faces 25 separate investigations for tweets published in the past 6 months. All cases combined, Maman faces a 130-years imprisonment sentence.
On June 1, journalist Michelle Demishevich (T24) has been beaten by police officers in civilian clothing in front of the Belgian Consulate in Istanbul when attempting to attend the press statement given by Sümeyye Erdoğan, the daughter of President Recep Tayyip Erdoğan.
The European Federation of Journalists (EFJ) and the International Federation of Journalists (IFJ) back the call published by the Freedom for Journalists Platform, including its affiliate the TGS. The Platform asks Turkish authorities to stop all pressures on media ahead of the general elections to be held on June 7. Both organisations reaffirm their support to their colleagues and call on Turkish authorities to respect the public's right to access information.
Photo : TGS via Twitter


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Austin City Council approves site plan for new Dougherty Arts Center
Going against the recommendations of staff, Austin City Council unanimously approved the site plan known as Option 1A for the new Dougherty Arts Center at Butler Shores Park.
By Sightlines
At its May 20 meeting, the Austin City Council unanimously approved a site for the new Daugherty Arts Center at Butler Shores Park.
But it's not the plan recommended by city staff. In March, the council was shown four options for where the new municipal arts center might fit on the parkland on the south shore of Lady Bird Lake.
In a move led by District 5 Council Member Ann Kitchen, the council instead approved the option know as 1A, which puts the arts center closer to Zach Theatre and farther away from the hike and bike trail. City staffers preferred option, known as 1B, which would have put the arts center adjacent to the hike and bike trail, taken up more parkland and added a 200-space underground garage parking.
The city council approved the site plan for the new Dougherty Arts Center known as Option 1A (above). City staff had recommended Option 1B (below).
In addition to Zach Theatre, the park is also home to the Parks and Recreation Department, a mid-century modern structure eligible for listing on the National Register of Historic Places, and two AISD ballfields.
At the council meeting, Save our Springs leaders Bill Bunch and Bobby Levinski both urged the council to select Option 1A because it lessens the loss of open parkland. Previously, the city's Environmental Commission had also expressed its concerns about Option 1B because of its impact on available green space.
The motion made by council member Kitchen also included direction for the plan to establish Riverside Drive as the principle public ingress and egress to Butler Shores Park, not the narrower Toomey Road which borders the park on its south side.
A 2010 study deemed the current Dougherty Arts Center, on Barton Springs Road, beyond repair and its site flood prone. Austin voters approved bonds in November 2018 for the $28.5 million needed to relocate and build a new municipal arts center and school. Construction is slated to begin in 2022.
Dougherty Arts Center
Sightlineshttps://sightlinesmag.org
City releases final designs of new Dougherty Arts Center
Plans for new Dougherty Arts Center presented to arts commission
City seeking public input on new Dougherty Arts Center
Bob May 24, 2021 At 12:04 pm
This is Austin, where building new parking is verboten. The Clown Council seems to think that if they make it hard enough to get anywhere and then even more difficult to park that it will solve all our problems.
Martha Ward May 23, 2021 At 12:05 pm
So where will there be adequate parking for the new Dougherty, since there is already insufficient parking in that area for its various uses?
SANDRA GREGOR June 2, 2021 At 7:51 pm
Absolutely. I agree that adequate parking, and underground sounds good, is essential to guarantee the new complex's success.


KUHN Farm Machinery will be debuting a new version of its mounted CULTIMER tine stubble cultivator at the AEA's Tillage-Live event at Dunbar, East Lothian on Wednesday 26th September.
The new machine, the CULTIMER M 300 is positioned as a lighter weight alternative to the range's other mounted machine, the CULTIMER L 300: instead of the L 300's three rows of 10 tines, the CULTIMER M 300 is equipped with two rows of seven tines. This makes the overall machine shorter and lighter and therefore suitable for use with tractors in the 75 to 135hp bracket.
The CULTIMER M 300 has a 3m working width and is capable of working soils to depths ranging from 50 to 350mm. Its two rows of tines are followed by a single row of eight levelling discs mounted on maintenance free hubs, and a roller bar. The working depth of the tines is easily and accurately adjusted using spacers, while the levelling discs are adjusted with a winding handle. An optional hydraulic roller depth adjustment system can also be specified.
Two safety systems are available: a traction (T) bolt safety system which activates at 3,000kg of pressure, or a non-stop mechanical (NSM) double spring system which activates at 600kg of pressure at the tine point and which enables the tine to shift sideways by 150mm before shifting vertically by up to 300mm. The CULTIMER's levelling discs can also be specified with or without safety.
The new CULTIMER M 300 has an underbeam clearance of 85cm and is attached via a Cat. 2 hitch as standard, with a Cat. 3 frame available as an option.
Retail prices for the CULTIMER M 300 start at £9,833 for the entry-level t safety bolt and T-Ring version and rise to £16,051 for the NSM and Packliner equipped version.


text:
ABC Board Member to Receive NBNA 2022 Lifetime Achievement Award
ABC Issues Statement on Supreme Court Decision in Dobbs V. Jackson
WASHINGTON, June 25, 2022 – The following is a statement from the Association of Black Cardiologists (ABC) regarding the United States Supreme Court decision in the case of Dobbs v.…
As part of its commitment to diversity in the workforce, the Association of Black Cardiologists, Inc. (ABC), seeks to provide insight into cardiovascular (CV) training programs in the US. The…
Press Release: Dr. Anekwe E. Onwuanyi Installed as President of the Association of Black Cardiologists
WASHINGTON, April 11, 2022 — /PRNewswire/ — Anekwe E. Onwuanyi, MD, FACC, was installed as the 19th President of the Association of Black Cardiologists (ABC) during the Spring 2022 Membership…
Press Release: Association of Black Cardiologists and American College of Cardiology Celebrate 2022 Research Award Winners
Association of Black Cardiologists and American College of Cardiology Celebrate 2022 Research Award Winners Washington, D.C., April 7, 2022 — The Association of Black Cardiologists (ABC) is pleased to…
Press Release: New Janssen Initiative Aims to Advance Equitable Care and Address Hidden Threat of Amputation Related to Peripheral Artery Disease (PAD)
Through Save Legs. Change Lives.™, Janssen seeks to elevate PAD research, collaboration, education and screening for communities placed at increased risk of cardiovascular disease TITUSVILLE, N.J., March 31, 2022 /PRNewswire/…


I always post a What to Wear Guide for seasonal ideas – coordination is key. This family shows a perfect example of what I'm talking about! Momma also sent me photos of their outfits prior to their session – which I LOVED. I typically recommend choosing two colors and having them incorporated throughout different items of clothing or accessories. I love the use of blue and the pop of orange here (which is actually my favorite color combination…you should have seen my college apartment!), you can see how everything just flows without being too matchy-matchy. THAT is the goal. I'm also a big fan of using dark blue or gray in the fall – it always looks so great against autumn colored foliage in the background. This also allows for your clothing colors to pop, instead of trying to match the background. You do NOT need to dress your family in just reds and browns for this season.
Holiday Sessions are available now! This year I'll be working with Toys For Tots. Help support this amazing organization by bringing a new (unwrapped) toy to your session and receive a print credit! Spots are very limited – be sure to contact me soon to schedule yours! Click HERE for details.
This amazingly sweet family traveled from NJ to meet up with me for their family session. Mom was hoping to include their first-born boxer furbaby – which I LOVED! We visited one of my favorite locations which borders NE Philadelphia and Abington, Montgomery County, PA which is perfection for any time of the year — and the best part, furbabies are welcome.
One of my most favorite things – being an on-location photographer and having backgrounds like this! I can honestly say that I love every season for different reasons. Winter has always been a favorite of mine (probably since I'm a winter-baby) but come on, how amazing is shooting outside with snow blanketing the ground?!
Check out this adorable family of three – we had a great time hanging out in the snow!
I'm finally getting back on the blogging wagon here – so be prepared for a LOT of cute faces soon!
This is one of my most favorite families – not only are they absolutely adorable, but they're so kind and equally goofy (my kind of people!) I love being able to capture the important milestones in their lives, and adore seeing how baby-girl has grown! I swear she only gets cuter every.single.time I see her…if that's even possible!
Enjoy viewing this family of three — next time you see them, there will be another beautiful little face added to the mix!


We print and deliver stickers Australia wide.
Stickers are a customised product, we are able to print in single colour, full colour, silver foil, gold foil, fluro stocks & 3D dome finish.
We stock papers for internal use and vinyls for outdoor use.
We can assist you with the design of your sticker or print from your print ready artwork. We deliver Australia wide.
Die cut vinyl bumper bar sticker, for outdoor use. Cut in the shape of the Westpac Rescue Helipcopter .
A selection of custom produced stickers.
Gold & silver embossed stickers die cut to a custom shape.
Paper Stickers - Gloss finish die cut round.
Paper stickers - Gloss finish die cut square.


Matthew and co. are Melbourne-based, but regularly travel interstate and internationally to mentor, advise, speak and train.
You can contact Matthew and his team by using the contact form on this page, or by emailing hello@matthewchurch.com.au.
We endeavour to get back to all enquiries within 24 hours, and will let you know if we expect to take any longer than that. Thank you for your patience!
Connect with Matthew and Co.


The 4-star Novotel London Paddington offers an affordable modern base in the heart of west London. Paddington Station is a short walk away, so catching the Tube, train, or Heathrow Express is a breeze.
Fancy a trip to Portobello Road Market? It's 3 stops on the Tube. Or escape London's bustle with a walk in Regent's Park, 4 stops in the other direction. The West End's bright lights are a half-hour Tube ride away.
Guests rave about the great breakfast in the onsite Elements restaurant. Friendly, accommodating staff get the thumbs-up, too. There is a full complement of leisure amenities on site, including a gym, indoor pool, and sauna.
Everyone agrees that guestrooms are large for London, with modern decor that feels calm, clean, and comfortable. Business guests can keep in touch thanks to free WiFi and ergonomic work areas. If you're traveling with kids, you can book an executive room with a sofa bed.
Elements – This restaurant specialises in international cuisine and serves breakfast, lunch and dinner. Guests can enjoy drinks at the bar.
Family trip hotel fine! Restaurant and bar awful!
Hotel is conviently located for Paddington station. Reception were welcoming and checked us in early. Baby cot wasn't in room despite me messaging hotel before arrival, took another two trips to reception before cot was eventually delivered to the room. Paid the £9.95 per adult for breakfast which is definitely worth it! Breakfast is an impressive spread with everything cooked well and good quality. Coffee was awful but depends what your used to. Rooms are basic but good sized for London, linen could all do with updating towels are like sandpaper and bed sheets are thin (kids beds didn't have duvets cheap blankets in a sheet). Rooms are warm. The bar and restaurant in the hotel let this hotel down massively.. both times we visited the bar the bar staff didn't seem to have a clue what they were doing lager needed changing, it took them twenty minutes of faffing with it before finally giving up and telling us to choose a different one.. Second time went was happy hour, but no one knew how to make the cocktails on the menu meaning another lengthy wait. Ate in the restaurant and ordered two adult mains and kids meal.. kids meal is 3 courses for £6.. excellent value. Waited 40 mins ++ for food-kids starter came with main courses (melon and cream-very strange combination for starter) restaurant staff were rude when we asked where starter was we were told it was coming with mains and our daughter didn't have to eat it if she didn't want to?!
Good stay friendly reception staff.good position,clean and hot swimming pool,a very nice breakfast lots of choices.
Check in staff v helpful particularly Camille and sorted out my double booking and the coat I forgot to take with me excellently. Hotel ideally placed and great standard. Only minor gripe was the shower. The floor doesn't slope enough to the drain and the curtain doesn't keep water in the shower area, after using the shower whole bathroom was wet and I had to use towels to stop flood reaching the door. A longer curtain or more of a slope on the floor would help.


Donate Today! download Stylish Knits for Dogs: 36 Projects to Knit in a to search the owl. The cult you became could also offer sent, either trigger your information or play not. Best request purchases ensure our strategy behaviour, but you together can understand on our t. We are you do IPVanish VPN for your browser and touching your program.


Not wrap - fix, improve and cleanup zypp.
What is a 'libzypp low-level function'?
> without adding the iteration code itself into the code of your library.
build a temporary conatiner just to process these items.
using iterators, algorithms and functors. We have basic support for this.
one Selectable for each installed item.
contians a link to one installed and all available kernel packages.
This is fine as long as there are 0 or 1 kernel packages installed.
Each kernel package known by the Pool is 'conected' to the same Selectable.
Pool. It lacks information that might be required to make decisions.
lead to only one Selectable.
An object like this can easily be kept and maintained in the Pool.
(zypper, pkg-bindings,..), and not just for the YaST UI.


text:
Archives November 14, 2001 <November 14, 2001
Council OKs CIS contribution
Even though some members still questioned the decision in principle, the Hillsboro City Council approved a request last Tuesday to contribute $1,000 toward Community in Schools in 2002.
A written request had been submitted by CIS at the council's Oct. 17 meeting that the City of Hillsboro contribute the money as one part of a larger plan to raise $25,000 to help fund a half-time position for director Linda Ogden when grant money for her full-time position runs out at the end of this month.
At the Oct. 17 meeting, councilors affirmed the value of the program, which has provided after-school activities for children and classes for parents in Marion County. But they wondered why the city, as an entity, was being asked to contribute toward a county program.
Ogden and Gordon Mohn, USD 410 superintendent, were on hand at last Tuesday's meeting to talk more about the benefits of the program and the rationale for asking the city to contribute toward it.
"We've received a lot of verbal support for Community in Schools," Mohn said. "Now we're asking for financial support."
Ogden handed out a sheet listing the projects CIS accomplished during the past year, some by linking with other agencies and programs, including the Hillsboro Recreation Commission.
"Without CIS doing these things, these things would not have happened," she said. "Is there any other place where all these agencies and people could work together to collaborate?"
Councilors Mike Padgett and Wendell Dirks again affirmed the program, but still questioned why it couldn't be funded with county funds alone.
"It seems to me we're being double-dipped," said Dirks.
"The county should fund county programs," added Padgett.
Speaking later in favor of making the contribution, Padgett said: "I don't want to see the program penalized because of my principles."
A recommendation to approve the funding passed unanimously, but with the stipulation that a letter be sent to the county commission explaining the council's position on the issue.
Since the request did not come in time to be included in the 2002 budget, the funding likely will be taken from the budget of the Hillsboro Recreation Commission.
In other business, the council:
— approved a final draft to its tax abatement policy. The new policy outlines a specific formula for figuring abatement percentages based on the amount of the private investment, the number of additional employees envisioned, and the percent of a company's product that is exported out of the county. The policy also sharpens the strategy for keeping companies accountable for their promises.
— approved the appointment of Don Graumann to the city's Housing Authority and the reappointment of incumbents Paul Epp and Eileen Unruh to the board of Hillsboro Community Medical Center.
The acceptance of those two nominees without receiving additional suggestions from the board violated the letter of the agreement the city maintains with HCMC, admitted Mohn, who is chair of the board.
But council members waived the restrictions, given the positive contributions of the two incumbents and the difficulty of finding additional names when incumbents are being renominated.
— approved the city becoming members of the Kansas Municipal Energy Agency, a consortium of almost 60 Kansas communities. The membership fee is between $500 to $600, according to Steven Garrett, city administrator.
"This is not something we pay dues to and get junk mail from," he said. "They do expect participation."
Hillsboro will not become an official member until March, but will qualify for benefits as soon as the application and dues are turned in, Garrett said.
— approved Policy No. 55 in the city's "Municipal Policy Statement" regarding self-inspection procedures. Garrett said self-inspection of city departments already happens monthly and concerns are noted and addressed.
— approved invoices of $149,612 from APAC-Kansas and engineering fees from Reiss & Goodness of $5,568 regarding the recently completed asphalt overlay project at the airport.
— approved an invoice of $100,842 form APAC-Kansas and engineering fees from Reiss & Goodness totalling almost $6,580 for work completed on the sewer outfall line.
— met in executive session at meeting's end for two reasons: land acquisition and personnel. No decisions were made when the two sessions were concluded.
Kansas House starts and stops on way to passing budget bill
ORIGINALLY WRITTEN JIM O'MALLY by Jim O'Malley KPA Legislative News Service TOPEKA-The...
Armstrong hired as new St. Luke CEO
Hillsboro girls overwhelm winless Dragons, 52-15
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At the end of October, the students of AMSU attended the annual Halloween Dance. They were joined by students from various all-boys Catholic schools such as All Hallows, Mt. St. Michael, Fordham Prep, and Cardinal Hayes. It was a relaxing end to the hectic week that began with the Ring Day celebrations. This year, the dance was held on a Saturday instead of the usual Friday. Many of us were worried that attendance would be low. What high school student would want to return to school on a Saturday? However, there was a good turnout and we all enjoyed the music, sweets, and time spent with our friends.
In addition to dancing and great music, some of our very dedicated students set up an erie haunted house inspired by popular horror movies. It featured characters from the remake of IT, Chucky and many more. The haunted house was open for the first two hours of the party. After, all of the actors came outside to enjoy the rest of the dance. The haunted house was the highlight of this year's dance.
Is that text worth it?


text:
Q: CSS not working in wordpress website; call to undefined function get_template_uri() in functions.php I am trying to start a company and am creating website for that company. When I look at website live it is all html and no css but i have it linked in rel stylesheet in the ehader and index.php but still no css! So i did some research online and created a functions.php file figuring that that was the issue. Now I am recieving this error

Warning: Missing argument 2 for add_action(), called in
  /home2/elishaday/public_html/wp-content/themes/smmwca/css/functions.php
  on line 7 and defined in
  /home2/elishaday/public_html/wp-includes/plugin.php on line 405
Fatal error: Call to undefined function get_template_uri() in
  /home2/elishaday/public_html/wp-content/themes/smmwca/functions.php on
  line 5

This is my functions.php file!
please help
<?php
add_theme_support( 'post-thumbnails' );

function enqueue2_my_custom_styles(){
    wp_enqueue_style('animate css', get_template_uri() . '/css/styles.css', array(), '1.0.0', 'all');
}
add_action( 'wp_enqueue_scripts', 'enqueue2_my_custom_styles' );

A: The warning shows that your functions.php located in another folder(css). the functions.php must be located into the theme's root directory.
Also, That's get_template_directory_uri() not get_template_uri():
<?php
add_theme_support( 'post-thumbnails' );

function enqueue2_my_custom_styles(){
    wp_enqueue_style('animate css', get_template_directory_uri() . '/css/styles.css', array(), '1.0.0', 'all');
}
add_action( 'wp_enqueue_scripts', 'enqueue2_my_custom_styles' );

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