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Activating mutations in the Kras gene are commonly found in some but not all epithelial cancers. In order to understand the susceptibility of different epithelial tissues to Kras-induced tumorigenesis, we introduced one of the most common Kras mutations, KrasG12D, broadly in epithelial tissues. We used a mouse model in which the G12D mutation is placed in the endogenous Kras locus controlled by inducible, Cre-mediated recombination in tissues expressing cytokeratin 19 including the oral cavity, GI tract, lungs, and ducts of the liver, kidney, and the pancreas. Introduction of the KrasG12D mutation in adult mouse tissues led to neoplastic changes in some but not all of these tissues. Notably, many hyperplasias, metaplasias and adenomas were observed in the oral cavity, stomach, colon and lungs, suggesting that exposure to products of the outside environment promotes KrasG12D-initiated tumorigenesis. However, environmental exposure did not consistently correlate with tumor formation, such as in the small intestine, suggesting that there are also intrinsic differences in susceptibility to Kras activation. The pancreas developed small numbers of mucinous metaplasias with characteristics of early stage pancreatic intraepithelial neoplasms (PanINs), supporting the hypothesis that pancreatic ducts have the potential to give rise pancreatic cancer.
Citation: Ray KC, Bell KM, Yan J, Gu G, Chung CH, Washington MK, et al. (2011) Epithelial Tissues Have Varying Degrees of Susceptibility to KrasG12D-Initiated Tumorigenesis in a Mouse Model. PLoS ONE 6(2): e16786. https://doi.org/10.1371/journal.pone.0016786
Editor: Robert Oshima, Sanford-Burnham Medical Research Institute, United States of America
Received: August 17, 2010; Accepted: January 11, 2011; Published: February 2, 2011
Copyright: © 2011 Ray et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by NIH grants R21CA123061 (ALM), P50CA095103 (ALM and MKW), R01DK065949 (GG) and P30DK058404 (MKW). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Cancer is thought to arise from a series of genetic and epigenetic changes that confer oncogenic properties on a cell and its descendents (reviewed in [1]). A number of oncogenes can contribute to cancer initiation or progression but none have been found to be sufficient for tumor formation. Rather, other genes must be mutated, silenced or overexpressed, within the cell or its environment, for a tumor to form. Kras is a proto-oncogene that normally relays signals from a variety of transmembrane receptors to intracellular effectors that regulate processes such as proliferation, survival, and migration. While its activity is tightly regulated, somatic mutations occur that render its activity constitutive and thereby oncogenic (reviewed in [2]). Activating mutations in Kras are found in histologically normal tissues as well as in tumors [3] indicating that while these mutations naturally accumulate over the lifetime of an individual, they only rarely lead to tumor formation.
Underscoring the ability of Kras mutations to contribute to tumor formation, they are common in many epithelial tumors including cancers of the pancreas, colon, and lung but are rare in cancers of the oral cavity and stomach [4]. The underlying mechanism for this tissue-specificity remains unexplored. It is possible that different cell types have different susceptibilities to the neoplastic effects of mutant Kras or that different tissues are more or less subject to Kras mutation. Kras appears to play different roles in different cancers, suggesting there are tissue-specific susceptibilities to Kras mutation. For example, in the pancreas, Kras mutation is an early step in tumor initiation [5]. However, in the colon, tumor initiation is thought to arise largely through aberrant activation of the Wnt signaling pathway with Kras mutation playing a later role in tumor progression [6].
One of the strongest correlations between cancer and Kras mutation occurs in pancreatic ductal adenocarcinoma (PDAC) which has Kras mutations in approx. 95% of cases [7]. Mouse models have shown that introducing mutant Kras throughout the pancreas beginning in embryogenesis results in many pancreatic intraepithelial neoplasms (PanINs) [8] that are thought to be the precursors of PDAC [9]. When combined with loss of tumor suppressor genes, these PanIN lesions progress to invasive and metastatic PDAC [10], [11], [12]. The genetic tools used to activate Kras during pancreatic development target mutant Kras to all cells of the pancreas, including both acinar and duct cells of the exocrine pancreas, making ontogeny of tumors impossible to determine. Because PDAC in humans has morphologic and molecular markers of duct-like cells, it was long held that neoplastic transformation of normal pancreatic ducts gives rise to PDAC [13]. However, we and others have recently shown that pancreatic acinar cells are capable of changing their cellular identity, or transdifferentiating, into duct-like cells in response to growth factors or damage [14], [15], [16], complicating the issue of the origin of PDAC. Understanding how PDAC arises is important for identifying appropriate biomarkers of cancer development as well as identifying targets that might be relevant in the treatment of human disease.
We have recently developed a genetic tool that allows us to investigate the susceptibility of different epithelial tissues to KrasG12D-initiated tumorigenesis in adult mice. Because epithelial tumors of some tissues are frequently associated with Kras mutation and those of other tissues are rarely associated, we tested the hypothesis that some tissues are refractory to the effects of Kras mutation, as opposed to being susceptible to its effects but rarely undergoing its mutation, by introducing the mutation into multiple tissues. We found that KrasG12D could induce neoplastic changes in multiple, although not all, assayed tissues. Susceptible tissues included those which in humans rarely have Kras mutations as well as those that frequently do. There was some correlation of susceptibility with exposure to the outside environment, suggesting that environmental factors may play a major role in promotion of KrasG12D-initiated tumors. However, this correlation was not uniform indicating that there are also intrinsic differences in how cells respond to Kras activation. Additionally, we demonstrate that pancreatic ducts are capable of giving rise to mucinous metaplasias with characteristic of early PanIN lesions.
CK19CreERT produces recombination in multiple epithelial tissues
KrasG12D mutation was targeted to multiple epithelial tissues via the CK19CreERT [17] and LSL-KrasG12D [18] genetically engineered insertional alleles. The LSL-KrasG12D allele cannot be expressed until Cre recombinase removes a transcriptional stop cassette, positioning the mutated coding sequence in line with the endogenous Kras promoter. CK19CreERT encodes the Cre recombinase fused to a region of the estrogen receptor that has been mutated to bind tamoxifen. This ERT domain prevents Cre from entering the nucleus and inducing recombination until it binds tamoxifen. For these studies, tamoxifen was injected into adult mice, abrogating embryonic effects. The CreERT coding region was inserted in exon 1 of the Cytokeratin 19 gene, a structural protein found in many epithelial tissues. We have shown previously that CK19CreERT can activate a loxP-flanked reporter in cells throughout the gastrointestinal tract and in ducts of the pancreas, liver, and kidney [17]. Thus, CK19CreERT can activate KrasG12D in cells of all these tissues.
In the course of these studies, we also found that CK19CreERT was active in lung and oral cavity. In the lung, CK19 protein is widely expressed in all epithelium [19], [20]. However, we found that CK19CreERT recombined the R26REYFP reporter largely in bronchi and bronchioles (28+/−10% of cells labeled) and rarely in alveoli (0.22+/−0.08% labeled) (n = 3 mice; Figure 1A, B and data not shown). CK19 protein is a minor component of oral epithelium [21]. Accordingly, when CK19CreERT was cross to the R26REYFP reporter [22], we found a low frequency of labeling in squamous epithelium of both the lingual and buccal surfaces (Figure 1C, D). Because only basal cells are retained for extended lengths of time, we determined the percent of basal cells in lingual and buccal surfaces that were labeled by CK19CreERT and found that 0.82+/−0.42% (n = 3) were positive for the EYFP reporter. Other tissues including the stomach, small intestine, colon, pancreas and liver were labeled as previously described [17].
Bronchus (A), bronchiole (B), lingual epithelium (C), and buccal epithelium (D) from CK19CreERT; R26REYFP mice immunolabeled for EYFP (brown). Arrows in C and D, labeled cells in basal layer of epithelia. Size bars, 50 µm.
Fifteen CK19CreERT; LSL-KrasG12D mice and littermate controls were given tamoxifen at 6–8 weeks of age and then followed for up to 6 months. Mice were monitored weekly for signs of distress including weight loss. Over the six month time period, 9 of 15 CK19CreERT; LSL-KrasG12D mice were euthanized due to loss of at least 20% body weight (Figure 2). No weight loss or other distress was observed in any mouse that had only one of the CK19CreERT or the LSL-KrasG12D alleles.
Mice were monitored weekly for weight and other indications of overall health. Number maintaining at least 80% of highest body weight is plotted as a function of time for CK19CreERT; LSL-KrasG12D (solid line) and for littermates also injected with tamoxifen (dashed line). An additional 3 control mice were also injected with tamoxifen but were only followed for 3–5 months as controls for earlier timepoints and did not lose weight during that time (data not shown).
KrasG12D mutation induced oral papillomas
Loss of weight was correlated with the presence of large masses in the oral cavity. Histological analyses revealed the presence of squamous papillomas of the posterior tongue and/or buccal surface of all mice that lost >20% body weight (Figure 3A, B). Papillomas of the posterior tongue were found in 100% percent of CK19CreERT; LSL-KrasG12D mice while these lesions less commonly involved the buccal surface, occurring in seven of thirteen bigenic mice aged 4–6 months post-tamoxifen treatment. Interestingly, lingual papillomas were limited to the back of the tongue even though CK19CreERT labeled cells randomly across the anterior-posterior axis of the tongue. The proliferative zone in all papillomas was maintained in the basal cell layer (Figure 3C) and no dysplasia was observed. No oral cavity phenotype resulted from tamoxifen injection or from heterozygosity of the CK19 or Kras alleles, as all mice with single genetically engineered alleles injected with tamoxifen had normal phenotype in all tissues examined.
A.H&E staining of papillomas on back of tongue. B. H&E staining of buccal papillomas. C. Immunolabeling for phosphohistone H3 (brown), an M phase marker, revealed that the proliferative zone in papillomas remained in the basal layer. Size bars, 50 µm.
KrasG12D mutation induced lung adenoma formation
At no time during the study was any difficulty in breathing observed in mice even though we found CK19CreERT was active in lung bronchi and bronchioles. However, upon dissection, lungs of CK19CreERT; LSL-KrasG12D mice were enlarged with an average wet weight of 0.676+/−0.053g compared to 0.175+/−0.014 g for control lungs at 6 months post-tamoxifen (p<0.001; n = 5 CK19CreERT; LSL-KrasG12D, 9 single transgene controls). All CK19CreERT; LSL-KrasG12D mice at 6 months post-tamoxifen had visible tumors up to 3 mm in diameter. Upon histological analysis, all CK19CreERT; LSL-KrasG12D mice had multiple benign lung tumors consisting primarily of papillary adenomas that became densely packed in the larger lesions (Figure 4A, B) [23] while all single gene controls were histologically normal.
A, B. PAS and hematoxylin staining of lung adenomas typical of CK19CreERT; LSL-KrasG12D mice. Size bars, 50 µm.
KrasG12D mutation induced gastric metaplasia
All CK19CreERT; LSL-KrasG12D mice exhibited metaplasia in the fundus of the stomach by 4–6 months after tamoxifen administration while single gene controls exposed to tamoxifen were morphologically and histologically normal. The number of affected glands in CK19CreERT; LSL-KrasG12D mice ranged from 10–50% along the lesser curvature of the stomach with a decreasing frequency farther from the lesser curvature. Affected glands were characterized by foveolar hyperplasia as seen by the extended zone of PAS staining for mucin (Figure 5A), reduced presence of parietal cells (data not shown) and a proliferative zone deeper in the affected glands than in unaffected glands (Figure 5B). TFF2, a marker of mucous neck cells, also showed staining deeper in the glands, indicating that the mucous neck region was located near the base rather than the normal location approx. two-thirds the distance from the base (Figure 5C). Staining with alcian blue yielded a light diffuse staining rather than the dark, punctate pattern seen in intestinal metaplasia (data not shown).
A. PAS (pink) showed extensive mucin production deep into affected glands while surrounding normal glands only had staining at the tops of glands. B. Immunolabeling for phosphohistone H3 as an M phase marker. The proliferative zone in affected glands was shifted toward the base of the glands. C. Immunolabeling for TFF2 (brown) showed positive cells near base of affected fundic glands while normal glands had expression in the normal mucous neck region. Arrows, staining in affected glands; arrowheads, staining in normal glands. Size bars, 50 µm.
KrasG12D mutation led to pancreatic ductal lesions
Within the pancreas, CK19CreERT recombines loxP-flanked alleles in ductal cells with less than 1% recombination in islet and acinar cells [17]. Therefore, this allele allowed us to test the hypothesis that ducts could be the origin of pancreatic ductal adenocarcinoma (PDAC) [13]. At dissection, the pancreas appeared grossly normal in CK19CreERT; LSL-KrasG12D mice. To detect any morphological changes, we examined sections every 200 µm through each pancreas. All five bigenic mice examined at 6 months of age exhibited at least one mucinous ductal metaplasia with features of early mouse pancreatic intraepithelial neoplasm, or mPanIN1A, thought to be the earliest stage of neoplasm leading to PDAC (Figure 6A) while control littermates were morphologically and histologically normal. Of the 6 bigenic mice examined at 4.5 months post-tamoxifen treatment, two had these mucinous ductal lesions (data not shown). Six of all lesions were found in the tail, three in the body, and two in the head of the pancreas. Examination of serial sections showed that all lesions were continuous with normal pancreatic ducts (Figure 6B) suggesting that they did indeed arise via activation of Kras in pancreatic ducts. At the interface between morphologically normal and abnormal cells, staining with alcian blue demonstrated that morphologically normal cells near the transition site expressed low levels of mucin whereas alcian blue staining was not observed in any normal ducts not associated with ductal lesions. Thus, a progression could be observed from cuboidal, non-mucinous to cuboidal, mucinous to columnar, mucinous cells within one duct. We also determined whether pancreatic lesions in CK19CreERT; LSL-KrasG12D mice expressed claudin-18, which is highly expressed in PanINs and PDAC but not in normal ducts or reactive ducts that are commonly seen in chronic pancreatitis [24]. As shown in Figure 6C, all mucinous lesions found expressed claudin-18. No other cells in the pancreas including normal ducts had detectable claudin-18 (data not shown). The ducts that were contiguous with mucinous lesions were all the larger interlobular or intralobular ducts that express the CK19CreERT allele. Because CK19CreERT does not produce Cre recombinase activity in the smaller intercalated ducts, we could not determine whether all ducts of the pancreas could give rise to PanIN-like lesions but can clearly conclude that larger ducts are capable of giving rise to mucinous neoplasms characteristic of early PanIN lesions.
H&E staining of a typical lesion in mouse pancreas (A) and its connection to normal ductal epithelium in a nearby serial section (B) with alcian blue staining to denote mucin-producing cells. Arrowhead, junction between nonmucinous and mucinous cells; arrows, junction between cuboidal and columnar cells. (C) Mucinous lesions were all strongly positive for claudin-18 (brown) with highest concentrations along lateral cell borders. Size bars, 50 µm.
KrasG12D mutation induced metaplasia in the colon
In many patches of the ascending colon, villus-like structures were observed surrounded by areas of normal crypt structure in CK19CreERT; LSL-KrasG12D mice (Figure 7A). Similar but less distinct villus-like projections were also seen in smaller numbers (1–3 per section) in more distal parts of the colon (data not shown). These villus-like projections did not have apparent Paneth cells and so may not have been a homeotic transformation to small intestine. The tops of projections were approximately the same height as surrounding crypts and they were not associated with increased proliferation (Figure 7B). These structures are morphologically similar to those recently reported as murine serrated hyperplasia capable of giving rise to serrated carcinoma in a mouse model [25]. Single gene control mice had no intestinal phenotype.
A. H&E staining shows villus-like architecture in an area of the ascending colon. Normal colonic crypt structure resumes on upper right side of each panel (arrowhead). B. Immunolabeling for Ki67 (brown) indicates that the proliferative zones associated with villus-like structures are similar in cell number to normal colonic crypts. Size bars, 50 µm.
Some tissues appeared unaffected by KrasG12D activation
We have reported previously that the CK19CreERT allele is active in hepatic ducts, renal papillary ducts and throughout the small intestine. However, upon examination of random sections through each of these tissues in each CK19CreERT; LSL-KrasG12D mouse at 4–6 months post-tamoxifen, we failed to find any morphological abnormalities in liver, kidney or small intestine. Because CK19CreERT has a high rate of recombination in the small intestine but no morphological phenotype, we also immunolabeled this tissue for markers of proliferation, phospho-histone H3 and Ki67. All intestinal crypts had a normal number of proliferating cells (data not shown). Thus, KrasG12D mutation alone is not sufficient to alter homeostasis in this tissue.
One reason for tissue-specific differences in KrasG12D susceptibility could be a difference in the extent of recombination of the LSL-KrasG12D allele. While we had shown that CreERT produced from the CK19 allele could recombine the R26REYFP allele in all these tissues, different alleles may have different rates of recombination due to loxP flanking sequences or higher level chromatin structure. Therefore, we performed PCR analysis of DNA extracted from each of the tissues that had little or no phenotype in CK19CreERT; LSL-KrasG12D mice, as well as from stomach which had a strong phenotype, and from tail DNA as a negative control. As expected, the KrasG12D allele was recombined in stomach but was also recombined to some extent in small intestine, colon, liver, pancreas, and kidney while no recombination was detected in tail DNA (Figure 8A). The amount of recombination was barely detectable in liver and kidney perhaps due to the small number of duct cells in each of those tissues that had shown recombination of the R26REYFP allele [17]. Because the intestine had a seemingly high amount of recombination of the KrasG12D allele but no apparent phenotype, we compared the amount of recombined allele present in the small intestine to that in oral papillomas, where the majority of epithelium should have recombined the KrasG12D allele (although there was some submucosa present in which CK19CreERT is not active). As expected, the amount of recombination (upper bands in Figure 8B) was much higher in isolated papillomas than in total oral tissue. The amount of recombination in intestine was higher than in total oral tissue but less than that seen in the papillomas. This is consistent with the observed activity of CK19CreERT being high in intestine but not recombining in every cell. In conclusion, CK19CreERT was able to recombine the KrasG12D allele in multiple epithelial tissues even when no resulting phenotype was observed.
A. DNA was extracted from the indicated tissues of three different mice and subjected to 35 cycles of PCR that would detect both the wildtype Kras allele (271 bp) and the recombined LSL-KrasG12D allele (310 bp) but not the unrecombined LSL-KrasG12D allele. Tissues examined: 1) tail, 2) small intestine, 3) liver, 4) pancreas, 5) kidney, 6) stomach, and 7) colon. Strong recombined bands were detected for small intestine, pancreas and colon and weaker bands for stomach, kidney and liver, while recombination was never detected in tail DNA. B. The amount of recombination of an unaffected tissue, the small intestine, was compared to the amount in an oral papilloma in which all or nearly all of the epithelium should have a recombined Kras allele. For three different mice, DNA was prepared from total oral tissue (lanes 1–3), isolated papilloma tissue (lanes 4–6) and total small intestine tissue (lanes 7–9) and subjected to 30 cycles of PCR. Little recombination can be detected in total oral tissue while the recombination is abundant in the isolated papilloma (upper band in each lane). The amount of recombination in the small intestine was intermediate between these two populations.
Environmental impact on KrasG12D-initiated tumorigenesis
While the CK19CreERT mouse can activate KrasG12D in many epithelial tissues of the mouse, this activation only induced widespread neoplasia in three tissues, the oral mucosa, stomach, and lung. Of the tissues targeted by CK19CreERT, these tissues are among the most exposed to the outside environment. This suggests that environment-induced damage or mutation may cooperate with Kras mutation to promote tumor formation. However, anterior tongue and the small intestine did not develop detectable neoplastic changes in response to KrasG12D mutation. Therefore, environmental exposure cannot be the only factor in susceptibility. Rather, cells must also have intrinsic differences in their response to the KrasG12D mutation.
Some tissue susceptibility may arise from differences in Kras mutation rate
In two cases, KrasG12D initiated neoplastic changes in tissues that rarely have Kras mutations in corresponding human cancers. In both the oral cavity and stomach, frequent and profound hyperplasia resulted from KrasG12D expression, yet cancers of these tissues in humans rarely carry Kras mutations. No information exists on the actual rate of Kras mutation in normal human tissues and we cannot rule out species-specific differences. However, our results combined with other studies discussed below, suggest that some epithelial tumors or hyperplasias do not carry activating mutations in Kras because of selection for other mechanisms that upregulate Kras activity. This may reflect a lower rate of occurrence of Kras mutation in these tissues or a higher rate of other mutations/events that affect Kras activity.
One of these tissues is the oral mucosa. Caulin et al. [26] also showed that KrasG12D can induce squamous papillomas in oral mucosa, using an inducible Cre transgene regulated by the CK14 promoter to activate the LSL-KrasG12D allele. Although activating mutations in Kras are found in only 4–7% of squamous cell carcinoma of the head and neck (HNSCC) in humans [27], [28], Kras protein is often elevated in this cancer suggesting that its activity plays a critical role in tumor initiation or progression. Supporting this role, a low level of expression of microRNA let-7d and a polymorphism in a let-7 microRNA binding site in the Kras 3′UTR are associated with poor survival from HNSCC [29], [30] and let-7 has been shown to regulate Kras expression [31].
In stomach cancer, there have been reports that Kras may also be regulated by microRNAs and/or by gene amplification [32], [33]. We showed that KrasG12D can induce frequent metaplasia in gastric fundus. These metaplastic glands displayed characteristics of foveolar hyperplasia with an extended zone of PAS-positive surface mucous cells, oxyntric atrophy, and basal expression of TFF2. Foveolar hyperplasia is associated with conditions such as Menetrier's disease rather than gastric cancer [34]. It will be interesting in future studies to determine whether these Kras-initiated hyperplasias in stomach mucosa can progress to invasive or metastatic disease.
Cell of origin in lung and pancreatic cancers
In lungs, the CK19CreERT allele produced recombination mostly in bronchi and bronchioles leading to the papillary adenomas observed in the CK19CreERT; LSL-KrasG12D mice. These tumors were quite different than those observed when the LSL-KrasG12D allele was activated by nasal installation of adenovirus encoding Cre recombinase [18], [35]. The difference likely reflects different cells of origin for tumor formation. Since we determined that CK19CreERT is active primarily in bronchi and bronchioles, it is likely that inhaled viral delivery targets mainly other cell types such as alveolar cells.
The cell of origin of pancreatic ductal adenocarcinoma has been widely debated. While these lesions in humans consist entirely of ductal cells, recent studies in mice have pointed to an amazing plasticity in the adult pancreas, showing that acinar cells readily give rise to ductal epithelium, notably to mucinous, hyperplastic epithelium in the case of transgenic overexpression of the growth factor TGFα [14]. Furthermore, studies have shown that using an acinar-specific CreERT allele to activate KrasG12D results in a low frequency of mPanIN1A lesions by 2 months post-tamoxifen treatment and progression to later stage PanINs as mice age [36], [37]. Similarly, expression of KrasG12V in acinar cells also led to mPanIN and PDAC but only in conjunction with chemically induced pancreatitis [38]. Thus, at least in the mouse, acinar cells are capable of giving rise to PanIN-like lesions and PDAC.
Until now, the tools to direct KrasG12D mutation to pancreatic ducts have been lacking and testing the ductal compartment as the cell of origin for PanIN or PDAC was not possible. We now show, using the CK19CreERT allele to activate the KrasG12D allele, that pancreatic ducts can give rise to lesions with the morphological and molecular characteristics of early PanINs. While these cells have the characteristics of early PanIN lesions, we cannot conclude at this point that they are true "PanINs," because they did not give rise to PDAC in the course of our analysis. Future studies will determine the ability of these lesions to progress to invasive or metastatic disease when combined with loss of tumor suppressor genes or with induction of pancreatitis.
Because CK19CreERT is expressed in a small percentage of acinar cells [17], it is possible that those few cells gave rise to the lesions reported here. This is unlikely for two reasons. One, our model had recombination in <1% of acinar cells while the acinar-specific Elastase-CreERT: LSL-KrasG12D model had 50% of acinar cells recombined and yet had a similar number of PanIN lesions arise as we did in our study [36]. Since CK19CreERT labels <1% of acinar cells [17], we would expect to see a much lower frequency (∼1/50th the number) of lesions than those seen from elastase-driven activation of KrasG12D if acinar cells were the cell of origin in our study. Two, the PanIN-like lesions resulting from CK19CreERT activation were continuous with normal ductal epithelium. Comparing our results and those described above, we conclude that both duct and acinar cells are capable of giving rise to mucinous lesions with PanIN-like characteristics and therefore potentially to PDAC.
Some tissues in the adult are refractory to KrasG12D-initiated tumorigenesis
We did not observe hyperplasia in the intestinal tract although CK19CreERT is widely expressed in this mucosa [17] and a substantial amount of recombination of the KrasG12D allele was seen (Figure 8). Jacks and colleagues [18], [39] reported that activation of the LSL-KrasG12D allele via the FABP-Cre transgene resulted in extensive hyperplasia along the crypt-villus axis throughout the intestine. Rather, we found only induction of altered crypt architecture in the colon and no phenotype in the small intestine. One difference between that study and ours is the timing of KrasG12D activation. FABP-Cre is active embryonically [40] whereas we did not activate CK19CreERT until 6–8 weeks of age. In the pancreas, embryonic activation of the KrasG12D allele resulted in widespread early neoplasms with occasional progression to more advanced lesions [8]. However, activation in adult acinar cells [36], [37] or adult ducts (reported here) gave only rare lesions unless further injury was induced [38]. A similar phenomenon may occur in the intestine, where KrasG12D in the embryonic epithelium induces changes that result in a higher susceptibility to tumorigenesis. Perhaps adult tissues are intrinsically more refractory to the effects of activated Kras or else the embryonic environment (e.g., the abundant mesenchyme that often regulates epithelial development) promotes oncogenic events that do not necessarily become apparent until after birth.
The studies presented here clearly show that different tissues have different susceptibilities to KrasG12D-initiated tumorigenesis. This differential susceptibility is most evident in the small intestine which had a high rate of recombination of the LSL-KrasG12D allele with no apparent resulting phenotype. This could be due to multiple mechanisms. It could be that Kras regulates different signaling pathways in different cells and that some pathways are more oncogenic than others. Supporting differences in Kras function, high level of KrasG12D may induce tumorigenesis in some cells but will induce senescence in other cells [41], [42]. Alternatively, susceptibility may depend upon the level of Kras expression. While Kras expression has been found in all tissues examined [43], [44], [45], its relative expression level among different tissues varies [45]. Those experiments were limited in that they did not look at expression in each cell type, but clearly indicated that Kras expression, while ubiquitous, has some tissue-specific regulation. For example, liver has a lower level of Kras expression than many other tissues while gut has among the highest level of Kras expression. Low expression of Kras may explain the inability of KrasG12D to induce liver tumors but does not explain the inability of KrasG12D to induce intestinal tumors. The activity of oncogenes is frequently abrogated by the expression of tumor suppressor genes. Tissues may vary in which tumor suppressors are expressed, how many are expressed, and how readily they are silenced. It is likely that future studies will uncover multiple mechanisms that vary from tissue to tissue, allowing some cells to maintain homeostasis in the face of Kras activation while others become transformed.
Materials and Methods
All experiments were performed with approval from the Vanderbilt Institutional Animal Care and Use Committee, Animal Welfare Assurance number A3227-01, protocol numbers M/06/078 and M/09/394. CK19CreERT [17], LSL-KrasG12D [18] and R26REYFP [22] mice were previously published and were bred for at least 8 generations into the C57BL6/J background. At 6–8 weeks of age, mice were administered tamoxifen or corn oil vehicle 3 times over the course of 5 days at a dosage of 1 mg in 50 µl each time. Mice were weighed weekly and euthanized if weight decreased by more than 20%. Fifteen bigenic CK19CreERT; LSL-KrasG12D mice were analyzed as well as 15 littermate controls that were wildtype or had either the CK19CreERT or the LSL-KrasG12D allele but not both.
Tissues were fixed in 4% paraformaldehyde for 4–5 h at 4°C, except tissues for EYFP detection which were fixed overnight, washed in 70% ethanol and embedded in paraffin after dehydration through a series of increasing ethanol concentrations and Histoclear (National Diagnostics, Atlanta, GA). Paraffin sections were cut at a depth of 5 µm. Antibodies used were rabbit anti-GFP (Novus Biological, Littleton, CO), mouse anti-TFF2 (a kind gift of Nick Wright), rabbit anti-phosphohistone H3 (Millipore, Telecuma, CA), rabbit anti-claudin-18 (Zymed/Invitrogen, Carlsbad, CA) and mouse anti-Ki67 (Novocastra, Newcastle upon Tyne, UK). Mucin was detected by staining with periodic acid-Schiff reagent (Sigma, St. Louis, MO) as per manufacturer's instructions or with Alcian blue, pH 2.5 (Sigma).
Detection of LSL-KrasG12D recombination
Tissue was scraped from slides and digested with proteinase K in DirectPCR lysis buffer (Viagen Biotech Inc, Los Angeles, CA). Lysates were heat-inactivated, extracted with phenol and chloroform, precipitated with isopropanol, resuspended and 100 ng used in polymerase chain reaction (PCR) as described [26] with primer sequences KRasLox-F2: 5′CAGTGCAGTTTTGACACCAGCT3′, and KRasLox-R1: 5′GCATAGTACGCTATACCCTGTGGA3′ and 30–35 cycles of amplification as indicated.
The authors thank James Goldenring and Robert Coffey for helpful discussions, Nicholas Wright for the TFF2 antibody, and Heidi Moreno for technical assistance.
Author Contributions
Conceived and designed the experiments: ALM. Performed the experiments: KCR KMB JY ALM. Analyzed the data: KCR KMB GG CHC MKW ALM. Contributed reagents/materials/analysis tools: GG ALM. Wrote the paper: ALM GG CHC MKW.
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- 8. Hingorani SR, Petricoin EF, Maitra A, Rajapakse V, King C, et al. (2003) Preinvasive and invasive ductal pancreatic cancer and its early detection in the mouse. Cancer Cell 4: 437–450.SR HingoraniEF PetricoinA. MaitraV. RajapakseC. King2003Preinvasive and invasive ductal pancreatic cancer and its early detection in the mouse.Cancer Cell4437450
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subset: commoncrawl


text:
President Trump has said he will not release his tax returns, despite repeated promises during his election campaign to do so and a petition by American citizens that collected well over a hundred thousand signatures almost immediately after being published.
Trump swore in members of his cabinet that did not need Senate confirmation, including his son-in-law Jared Kushner as senior adviser.
Protests took place against Trump's presidency in all 50 states. At some, hundreds of thousands of people gathered.
Trump will meet with Mexico's president (cancelled by Mexican president after Trump said wall construction would begin, and commenters asked if he had any choice given the rhetoric and stance of Trump) and Canada's prime minister soon to renegotiation NAFTA, which Trump has said he will cancel. Mexico is considered to strongly fear the end of NAFTA because the country and its people have benefited a great deal from it. Mexico has some power in the negotiations due to the large amount of trade going on between the two countries, but they have much less leverage than the U.S. because they are the weaker power and they have more to lose if NAFTA is cancelled.
Trump then signed pipeline projects back into action: the 1100-mile long Keystone XL from Alberta, Canada through Montana, South Dakota and Nebraska which Obama halted in 2015, and the North Dakota Access Pipeline through North Dakota, South Dakota, Iowa and Illinois. The Standing Rock Sioux tribe has protested the Dakota pipeline, saying it endangers culture and water, and it has been a meeting point for several native groups and environmental groups. As he signed the Keystone XL, the President told reporters. "Lot of jobs. 20,000 jobs. Good construction jobs." The chief of the Standing Rock tribe responded to the moves by saying they'd focus on waging legal battles.
Trump also asked the commerce department to come up with a plan so that pipelines would all be built with American-built products. American steel, essentially.
He then met with auto executives to brief them that his office was going to look at cutting red tape for them, and would give them permits quickly or tell them they wouldn't give them permits quickly. "It's out of control," said Trump to the execs.
Trump also began the border wall this week. While Mexico won't pay for the wall up front, Trump said, America will be reimbursed from "whatever transaction we make" in the future. He said construction would begin immediately.
Trump also stripped funding from "sanctuary cities" that house illegal immigrants without turning them in, and increased border patrol agents. Some mayors responded favorably and some said they would refuse.
Trump then tweeted that if Chicago didn't cut down it's gun violence and murder rates, he would "send in the Feds."
U.S. stock markets continue to surge.
House Republicans have created a timeline over a 2-day meeting. By August, they want something concrete for two of their biggest projects: How to repeal Obamacare and how to replace the tax code. Speaker Paul Ryan also said that they wanted legislation passed on these things by the end of the year – a goal considered 'optimistic' by analysts.
Theresa May visited Trump. They were outspoken about the points they disagree on (UK opposes Russia friendliness and use of torture with prisoners). She also said that Trump had said he was 100% in favor of NATO.
Trump made moves toward reducing refugees and toughening acceptance of immigrants.
Nikki Haley visited the UN for the first time, and she told them the U.S. would be standing with its friends but would be "taking names" of those who "didn't have our backs."
There was another large protest in Washington, this time for anti-abortion. There was also a pro-abortion protest. Trump's pick for the empty Supreme Court seat is being looked at as a step towards reversing Roe V. Wade. Dissenting analysts consider that decision more of a political action than a sound legislative one.
Analysts have seen the first week of the Trump presidency as both following through on campaign promises and also unnerving in how much change is happening, and the unsettling nature of the change.
Israel is going ahead with building over 500 homes in three settlements. The permits had been on hold during the Obama administration, which opposed any new settlements by Israel in the Palestinian territory. Days later then announced another building plan: 2,500 new homes. Chicago's mayor said he'd welcome help by federal law enforcement.
Islamic violent attacks continued in several countries, including a notable bombing in Ethiopia.
UK's prime minister, Theresa May said at a conference that the UK, which recently voted to leave the European Union, and the U.S. had "the opportunity — indeed, the responsibility — to renew 'the special relationship' for this new age. We have the opportunity to lead together again." May has spoken well of Trump.
The Gambian president (Abdalla Barrow) returned to the country today after it was secured by West African military forces. The president stayed out while Gambia was occupied by its longtime ruler (Yahya Jammeh).


text:
Psyche Circuit Cuff
Psyche Circuit Cuff
Psyche's Bichos collection is inspired by the work of Venezuelan artist, Gego. Gego's goal was not to create original expression but more to give material form to the most universal of laws – math, geometry, and physics. Creating angles with short circuits, the angles become shapes and forms in space.
With Lena Dunham and FKA Twigs among their growing fan base, emerging line Psyche Jewelry is one to watch. Designer Nina Berenato draws inspiration from the common shapes, objects and discoveries she sees on her morning walks, bringing it back to the Brooklyn studio where pieces are created from ethically-sourced and recycled materials. Imagination is at the heart of each collection, so slip something on and start dreaming.
Do you have questions about this product? We're here to help!


text:
Every time she opens her pretty little mouth these days, Megan Fox seems to say something outrageous.
The Transformers beauty, who in the past has called herself "a tranny," now tells the British edition of GQ that she's also a little on the whorish side.
"When you think about it, we actors are kind of prostitutes," Fox offers. "We get paid to feign attraction and love. Other people are paying to watch us kissing someone, touching someone, doing things people in a normal monogamous relationship would never do with anyone who's not their partner. It's really kind of gross."
More gross than dating Brian Austin Green? Shudder to think!
But despite comparing herself to a common streetwalker, Fox goes on to note that her wild reputation is greatly exaggerated.
"I have this sort of promiscuous image," Fox protests. "People assume I'm really overtly sexually aggressive and that I'm this wild child. And I'm not like that at all."
Gee, Megan, whatever would have given people that idea?
Readers, have your say in the comments section: Would you compare Megan Fox to a prostitute?


text:
Lamb Meatballs With Cucumber Mint Yogurt and Couscous
Recipe by MarraMamba
Chef Ivy Starks creation for "chef on a shoestring budget". prep does not include the 4 hour time to drain yogurt
Top Review by mliss29
Well, I think I dirtied up every pan in the house, but it was worth it! This was really good! I believe my husband said, "wow!", "this is awesome", "this is a really delicious dish", etc. There are really a lot of flavors, all contributing to the overall wow. Mostly I obeyed the directions, but I used ground turkey, 2 tsp dried parsley, no cilantro, and no chili pepper flakes. And I only drained the yogurt & cukes about 1.25 hours (because I forgot to prepare ahead), but they were fine. My husband said the only thing to change "the next time you make this" would be to double the sauce. Thank you so much for this tasty dish! Made for Newest Zaar Tag, 08A.
See Discussion:1 Review
Ingredients Nutrition
- 1⁄2 cup breadcrumbs
- 1⁄4 cup milk
- 1 lb ground lamb
- 1 cup blanched almond, toasted and ground
- 1⁄3 cup chopped onion
- 1⁄3 cup raisins, coarsely chopped
- 1 egg, lightly beaten
- 3 garlic cloves, minced
- 2 tablespoons chopped fresh parsley
- 1 tablespoon chopped fresh cilantro
- 1 tablespoon chopped of fresh mint
- 1⁄4 teaspoon chili pepper flakes
- salt & freshly ground black pepper
- 2 tablespoons olive oil, plus more
- olive oil, for drizzling
- 1 teaspoon chopped parsley
Moroccan Yogurt Sauce (use 2 cups)
- 1 cup yogurt
- 1 cup cucumber, peeled, seeded and coarsely grated
- 1⁄4 cup mint, minced
- 3 tablespoons lemon zest, minced
- kosher salt & freshly ground black pepper
- couscous
Directions See How It's Made
- In a small bowl, soak bread crumbs in milk. In a medium bowl, combine the lamb, almonds, soaked bread crumbs, onion, raisins, egg, garlic, parsley, cilantro, and chili pepper flakes. Season lightly with salt and pepper.
- Drizzle olive oil onto a sheet pan you will place the meatballs on and rub oil into the palm of both hands.
- Form meat mixture into meatballs 1/2-inch in diameter. The recipe makes about 30 golf ball-sized meatballs.
- Heat oven to 400, place meatballs on a baking sheet and cook about 12 minutes until heated through.
- Yogurt sauce:.
- Place the yogurt in a sieve lined with cheesecloth. Allow to drain at least two hours to overnight.
- Toss the cucumber in 2 teaspoons salt and place in a sieve to drain for at least two hours.
- Combine in a medium bowl, the yogurt, cucumber, mint, and the preserved lemon. Season with salt and pepper.
- Serve warm over couscous and drizzled with yogurt sauce and, parsley and sesame seeds.


text:
This is a proposal. It is not yet in effect.
General Terms
- Austria and Yugoslavia are restricted from invading or declaring war on Poland, France, Czechoslovakia, or Germany for ten years.
- All current military conflict between the two sides must end.
Territorial Changes
- A very small piece of land in Northeastern Austria goes to Czechoslovakia. This includes the town of Poysdorf.
- A piece of land at the Western edge is ceded to Germany. This includes the cities of Bregenz, Dornbirn and Feldkirch.
- Yugoslavia must withdraw from Albania.
- Austria must pay 5% reparations to Czechoslovakia, and 1% reparations to Germany, France and Poland (Total of 8% reparations).
- Yugoslavia must pay 2.5% reparations to Czechoslovakia, and 0.5% reparations to Germany, France and Poland.
- This treaty also creates a formal alliance between Germany, France and Poland.
- Austria, Poland, Yugoslavia, Czechoslovakia, France, and Germany must all be in the League of Nations for the next ten years.
- Poland- Vatonica (talk) 02:45, February 25, 2015 (UTC)
- France-
- Germany-
- Czechoslovakia-
- Yugoslavia-
- Austria-
Dutch Proposal
General Terms
- All hostilities will end.
- Yugoslavia will sign a ten-year NAP with Albania and Czechoslovakia.
Territorial Changes
- Austria will be annexed by Germany entirely.
Other Terms
- Yugoslavia will cede 2.5% of its monthly income to Albania and Czechoslovakia.
- Half of the Austrian treasury will be ceded to France and Czechoslovakia and the other half will be kept by Germany.
- France-
- Germany- Oskar von Hindenburg
- Czechoslovakia- Leader X "This is not your grave but you are welcome in it." 20:58, February 28, 2015 (UTC)
- Yugoslavia- Milan Strojadinovis
- Albania- Leader Y, probably King Zog "This is not your grave but you are welcome in it." 20:58, February 28, 2015 (UTC)
- Poland- Andzej Poplawski
Second Polish Proposal
I compromised a lot here, and based it on the Dutch proposal.
General Terms
- All hostilities in Austria, Czechoslovakia and Albania will end.
- Yugoslavia will agree to recognize Albania as independent, and withdraw.
- Yugoslavia signs a ten-year NAP with Czechoslovakia and Germany.
Other Terms
- Austria is placed under complete German control.
- Yugoslavia gives 2.5% of its monthly income to Czechoslovakia and 5% to Albania.
- Austria gives 50% of its treasury to Germany, 25% to Czechoslovakia, 15% to France and 10% to Poland.
- Poland and Germany agree to an unconditional ten-year alliance.
- Yugoslavia is removed from the League of Nations.
- Germany-
- Poland- Andrzej Poplawski
- Yugoslavia-
- France-
- Albania-
- Czechoslovakia-
Austrian Proposal
General Terms
- All hostilities in Austria, Czechoslovakia and Albania will end.
- Yugoslavia will agree to recognize Albania as independent and withdraw.
- Yugoslavia signs a ten-year NAP with Czechoslovakia and Germany.
Other Terms
- Austria will be completely occupied by Germany. (NOTE: THIS DOES NOT MEAN ANNEXATION!)
- Austria will tie its remaining military assets and its treasury to Germany.
- The Austrian People's Assembly will all be imprisoned.
- Hans Markert will become wanted in all countries participating. (He fled to the Soviet Union at the height of the war.)
Yugoslavia will give its treasury to the allied forces.(I will not give up all of my money).
Yugoslavia will be removed from the League of Nations.(You guys don't control the League of Nations)
- Germany-
- Poland-
- Yugoslavia-
- France-
- Albania-
- Czechoslovakia-
- Austria- People's Assembly of Austria
- Soviet Union-
Only Germany can take land because they were the only ones launching an offensive war. Second, Poland and the Dutch weren't involved in the official version. Why are they making treaties? In dust you began....... and in dust you shall end........
Germany is the only one taking land in my version. Also, I just asked to be allowed to as the Dutch felt they had a good treaty already. Hail Sean! (Get a free potato here)
20:14, February 27, 2015 (UTC)
First of all, Poland declared war on Austria, so we have the right to. Second, Czechoslovakia has the right to take land as part of a treaty, as they won the war and were the main subject of the war. Finally, Czechoslovakia didn't launch an offensive and thus has no right to take land. Hail Sean! (Get a free potato here)
21:07, February 27, 2015 (UTC)
I'm sorry, but I'm not signing a ten year alliance. I'm willing to aid you if the Soviet Union declares war on you and outright invades you but I'm not going to get dragged into a war that frankly can be avoided. Also on your map you still show Danzig as a free city but you agreed to my annexation of Danzig so that city now belongs to Germany.
On another matter, to Austria, no not an occupation, The only way you're staying autonomous is if you agree to enter an economic union with the Reich, the other terms you lay down, and for purposes become an autonomous part of the Reich. With Blood and Iron (talk) 11:26, February 28, 2015 (UTC)
Also stop acting as if Poland has to sign anything. They can make a treaty, but they weren't involved in the algo, so they wouldn't need to sign. Hail Sean! (Get a free potato here)
12:32, February 28, 2015 (UTC)
There were so many attempts at algo, IDK how many count. I had withdrawn most of my forces from this war (to put them on border with Soviets and put down Communist uprising) by the time the last algorithm was put out, but I was involved earlier in the war. I joined the war to prevent the spread of Communism, just as I am preparing to do in case of a Soviet invasion. Also, it is impossible for this treaty to end all hostilities in Yugoslavia, as Italy is still at war with them. In terms of relations with Germany, I completely understand. However, I need an agreement so that I have a port city, so that I can be involved with global trade. This could be Danzig, or full access to the Wisla river even within your borders. Also, I am upset that you have not proven your concern about the Soviet Empire and their Communist imperialism. I hope this changes in the future, and Poland will live strong without a German ally. Vatonica 14:05, February 28, 2015 (UTC)
Poland tentatively agrees to the Dutch Proposal. But if you erase my name because we are "not needed", then that would infuriate us. WE DECLARED WAR. Germany and France were our allies, and we were completely against Communism. Vatonica (talk) 14:42, February 28, 2015 (UTC)
Sign if you want, I guess. Always good to have another nation agreeing. Also can this stuff hurry up? We're gonna need a mod to sign for Czech and Albania and LL needs to hurry too. Hail Sean! (Get a free potato here)
16:19, February 28, 2015 (UTC)
Just a heads up, Poland was involved in every algo, and did declare war. We were also the ones who called talks for a peace treaty, not the Netherlands. Vatonica(talk) 16:45, February 28, 2015 (UTC)
Its not the time for war against the soviets yet im gearing up for war but i still need time poland. also you would have free access to the port of Danzig but the city is german and will remain as such. With Blood and Iron (talk) 00:38, March 2, 2015 (UTC)
Poland wasn't involved in the offical algo. In dust you began....... and in dust you shall end........
- http://althistory.wikia.com/wiki/Talk:Axis_vs_Allies_Resurrection_(Map_Game)#Eon.27s_Offical_Version_of_The_Austrian-Czech_War_.28Touching_this_without_permisson_gets_you_banned_for_a_week.29
- http://althistory.wikia.com/wiki/Talk:Axis_vs_Allies_Resurrection_(Map_Game)#Austrian_Reconquest_of_Czechoslovakia
- http://althistory.wikia.com/wiki/Talk:Axis_vs_Allies_Resurrection_(Map_Game)#German_Invasion_of_Austria
Poland is mentioned in all three. That's more than France. Vatonica (talk) 03:00, March 2, 2015 (UTC)


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"Waiting Time - Cataract"
About: Furness General Hospital Furness General Hospital Barrow-In-Furness LA14 4LF
Posted by Anonymous
My father is 84 years old and finally after more than a 6 week waiting time got an appointment to see the Doctor. He was told yesterday that his first eye will be done the second week in June, second one minimum 6 weeks later. I think this waiting time is absolutely disgraceful. He is confined to the house as he cannot drive the car. He is slowly deteriorating with his Parkinsons and now has to wait in total of over 8 months before he can drive again. This is a joke. He has worked every day of his working life and now has to hang around waiting for 8 months for an operation in what could be his final years.
Response from Furness General Hospital 2 years ago
We are really sorry that your father's experience at our hospital wasn't what you would expect. We would need some more information from you in order to investigate fully and provide you with a response, and we'd therefore ask you to contact our Patient Experience team on 01539 795497 to allow us to carry this out. Thank you
- {{helpful}} of {{total()}} people think this response is helpful
- Was this response helpful? Yes | No
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One of the more overlooked features of the new iPhone 3.0 is support for a new open standard for live video streaming over HTTP, which promises to open up standards-based video broadcasting to a wide audience while giving mobile users an optimized picture as they roam between WiFi and mobile networks. Apple is not supporting Mozilla's efforts to use the obsolete Ogg Theora on the web, which lacks silicon support for hardware acceleration on mobiles and appliances. For examples of HTTP Live Streaming, see iphone.akamai.com. Viewing live streams requires iPhone 3.0 or Snow Leopard QuickTime X.
More info: AppleInsider


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|Message view
|« Date » · « Thread »
|« Date » · « Thread »
|"William A. Rowe, Jr." <wr...@rowe-clan.net>
|Fw: SHA1 and Base64
|Wed, 29 Nov 2000 01:03:49 GMT
From: <firstname.lastname@example.com> To: "Greg Stein" <email@example.com> Cc: <firstname.lastname@example.com> > > > We can stratify and create as many layers in Apache as we want to put up > > with. But when we're talking about a *portability* library, then it should > > focus on just that. > > All of the code we are talking about is portable code, it may not have > portability issues itself, but programs that use this code do have > portability issues, and have an MD5 hash or SHA1 and Base64 encoding do > allow those programs to be more portable. As a poor, downtrodden Win32 guy... I entirely agree. There is lots of stuff implemented as unix/v or freebsd libraries that just aren't portable without cygwin. We aren't trying to be cygwin, but trying to offer some cross platform fn's with native optmizations. There is nothing wrong with translations, hashing, tables or encodings implemented in APR, there just has to be someone who might need the features. I'm questioning if/how much of/ iconv we should be distributing. But any which way, we should be providing these simple data types to make c coding easier and more portable across platforms. We rejected a mission statment that said apr was nothing but portability... the fns we are discussing are all very reasonable tools to include in APR (including base64/sha1). Put them into a 'lib' or 'util' section, or whatever, but arguing is pointless.
|View raw message


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HOUSTON, June 26, 2013 (GLOBE NEWSWIRE) -- Cardtronics, Inc. (Nasdaq:CATM), the world's largest retail ATM owner, today announced that Silicon Valley-based Technology Credit Union (Tech CU) is actively helping its members save money, having recently debuted FeeAlert from Cardtronics. With FeeAlert, members using out-of-network, fee-charging ATMs receive a personalized email alert from Tech CU, pointing them to the nearest in-network, and therefore fee-free ATMs. "Tech CU is always looking for ways we can use technology to save our members time and money—and FeeAlert from Cardtronics is a great example," said Joe Anzalone, chief banking officer for Tech CU. "An easy-to-use, unobtrusive use of a smart technology, FeeAlert helps to further improve the Tech CU banking experience." "The idea behind FeeAlert from Cardtronics is taking every opportunity to help cardholders find nearby fee-free ATMs," said Rick Updyke, president, North America Business Group, Cardtronics. "Before a transaction, ATM locator tools, whether built into a website or mobile banking app, are tremendously effective in helping members find the nearest fee-free ATM. But sometimes they forget to look and end up paying a fee even though they could've used, for example, a surcharge-free Allpoint ATM right around the corner. FeeAlert can't read minds, but it can send helpful reminders after a transaction, giving cardholders an opportunity to save the fee the next time around." How it Works: FeeAlert @ Tech CU
- Member uses out-of-network/non-affiliated ATM
- Daily ATM transaction info sent from Tech CU to FeeAlert platform
- FeeAlert generates a list (using geo-location technology) of nearby in-network, fee-free ATMs
- Tech CU member receives an email (next-day delivery), detailing available free ATMs for future reference.
- FeeAlert from Cardtronics – http://bit.ly/FeeAlert


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With mayor Subrata Mukherjee piloting the move, legislation is on the anvil to make tenants and slum-dwellers pay for the civic amenities they enjoy.
It is necessary for the survival and rejuvenation of the city and it will act as a deterrent to the drift of have-nots from the districts and from Bihar, Orissa and Bangladesh, the mayor explained.
Municipal affairs minister Asok Bhattacharya said he was yet to receive such a proposal from the mayor. However, if the civic authorities suggest such a tax and submit a proposal, we will explore its legal validity, he said.
Former mayoral council member Sudhanshu Sil, now CPM councillor and legislator, backed the mayor. He said citizens enjoying civic amenities should share a part of the cost required to provide them.
In defence of the legislation, senior councillor and mayoral council member Pradip Ghosh pointed out: Occupiers tax is not a new concept. Earlier, the civic authorities would collect tax from the tenants, too. Ghosh holds the civic portfolio of slum development.
The recent imposition of water tax can be an eye-opener, the mayor said. Earlier, the state government, the civic authorities and the political parties had always unanimously opposed the imposition of a tax on filtered water. But the deplorable financial condition of the civic body had made the levy inevitable, he added.
Hence, users of civic amenities should have to pay for them, now that the Calcutta Municipal Corporation (CMC) coffers are empty. It was magnanimity on the part of the tax-payers that they had allowed the CMC to provide services to those with little stake in the city, said mayor Mukherjee.
He pointed out that Calcutta was perhaps the only city in the world where the cost of providing civic amenities to 10 million people was borne by 300,000 house-owners only.
Since the tax-payers are the minority, the civic authorities, too, are less bothered about their welfare. As an example, the mayor cited slum development. The civic body has a full-fledged department and a mayor-in-council member to look after the welfare of the slums, though they contribute nothing to the CMC coffers.
Mukherjee said this unfair practice should have been stopped a long time ago. It should have struck some predecessor of mine that even the slum-dwellers should be made to pay for something that they use. They cannot expect others to foot the bill. The time has come to get matters rectified, he asserted.


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Python Web Graph Generator
Python Web Graph Generator is a threaded Web graph (Power law random graph) generator. It can generate a synthetic Web graph of about one million nodes in a few minutes on a desktop machine. It supports both directed and undirected graphs. It implements a threaded variant of the RMAT algorithm. A little tweak can produce graphs representing social networks or community networks. It can also output connected components in a graph. (Source: http://freecode.com/)
References in zbMATH (referenced in 1 article )
Showing result 1 of 1.
- Huo, Liang'an; Lin, Tingting; Huang, Peiqing: Dynamical behavior of a rumor transmission model with psychological effect in emergency event (2013)


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All Android applications categories
This is an interesting screen lock.It simulate the tape cutting to unlocking the screen!
Use the knife to cut the tape and unlock the screen!
How do you get the inspiration of this kind of screen lock?
Usually,we will cut the tape and open the box to take the goods out!
So why not using the take to lock our phone screen.
They really have something in common!
We provide a knife to this screen lock to cut the tape.
How do you unlock the screen?
The tape is placed on the phone screen vertically.There is a gap in the middle of the phone screen.But it is coverd by the tape.
There is a knife at the top of the phone screen.
Drag the knife down to cut the tape.
OK.The screen is unlocked.
Lock Feature List:
---This application is provided for free.
---It could be used by Android phone and tablets.
---The way to unlock screen is new and interesting.
---Time and date are displayed on the unlocking interface.
---It is vivid and easy to unlock the phone screen!
In addition,this screen lock could also supported vibration and sound effect while unlocking.
You could use the full screen while locking.But if you want to check some recent notifications,you could cancel the full screen and enable to expand the status bar.
If you like this screen lock,please share it with your friends and family.
I'd be happy to receive your feedback email!!!
==========================Important Notification=====================
If there are any problems, please contact me by email, and we can troubleshoot the problem together. I am unable to follow up or reply to comments left on the market.
Many carriers do not provide unlocking codes, and some who do force the consumer to pay a fee. Despite this, unlocking a SIM without carrier consent may be legal in some jurisdictions.Beyond that, it is often legal for carriers to force SIM locks for certain amounts of time, varying by region.
from 3 reviews
Download Tape Cut Unlock Screen
Free - V41001 - 749K
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This app is no longer available.Explore Android
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text:
|Property type
|Minimum Stay
|3 nights
Save $600. Easy walking distance to the VITRAN- no need to rent a vehicle.
This Caribbean cabana, also affectionately known as the Christmas Cottage, offers the intrepid traveler an unforgettable St. John experience. Nestled comfortably on the valley floor of Coral Bay village in a quiet residential neighborhood, the cabana presents rustic simplicity, with irresistible Caribbean charm. The Cabana has been a 'honeymoon' cottage to the owners for the last five years while they constructed the main house on the property. Cozy yet airy, the cabana makes available all the outdoor living experience of camping with the convenience of electricity, refrigeration and laundry facilities. Situated on level ground makes for easy access and parking. Local grocery store, mail service, restaurants, includes local landmarks, Skinny Legs, Island Blues and Donkey Diner. Enjoy plenty of nightly entertainment a simple 5 minute walk. Yoga classes convene several times a week at the Agricultural pavilion, a short walk from the house. Or if Pilates is your preference, classes are available at the same convenient location. Catch the VITRAN to world-class beaches for the day, or hike the many trails on the eastern end of St. John. Head to the bustling town of Cruz Bay via the VITRAN for a dollar. Bars and restaurants, quaint boutiques and island ferry opportunities are all conveniently located to the cabana.
Keywords: Economical, Private, Eco-Cabin
Island Roots Charters- Explore inlets and beaches of St. John. Snorkel in the most popular as well as out-of-the-way spots. Spend a day in the British Virgin Islands exploring the Caribbean archipelago. Community ultimate frisbee and soccer fields also within walking distance.
|Rate Period
|Weekend Night
|Monthly *
My Standard Rate
3 night minimum stay
For renters staying only 3-4 nights, the rate is $100 per night year-round plus a $50. housekeeping surcharge.All listed rates are based on double occupancy; $100. per additional guest will be charged per week.
For stays 3 & 4 nights there is a $50. housekeeping surcharge.
To Reserve: a 50% deposit is required to secure dates. The remaining balance is due (1) before arrival. All deposits are fully refundable up to a month of arrival date and at that point non-refundable- with few exceptions
* Approximate monthly rate. Actual rate will depend on the days of the month you stay.


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American Government
American History
World at War
World History: Ancient & Medieval Era
World History: The Modern Era
Ancient and Medieval History Online:
Focuses on Egypt, Mesopotamia, Greece, Rome, Africa, Europe, the Americas, Asia, and Islamic Empire from prehistory through the 1500s.
American History Online:
Spans more than 500 years of political, military, social, and cultural history to cover the American experience.
Modern World History Online:
Covers the people, places, and events in the broad expanse of history - from the mid-15th century to the present.
Selecting History will reveal many reference books on a variety of topics. One can search within these printed books
E-Library Curriculum Edition
Site Map
Printable View
© 2008 - 2017 Stamford High School
| City of Stamford


text:
Question: The Amundsens contracted with Wright doing business as J Wright
The Amundsens contracted with Wright, doing business as J. Wright Homes, to build a house for them. The Residential Construction Contract contained an arbitration clause in which the parties agreed to submit any and all disputes that might arise between them to binding arbitration pursuant to the "procedures established and maintained by the Central Oklahoma HomeBuilder's (sic) Association." As it later turned out, however, the Central Oklahoma Home Builders Association does not have, and has never had, procedures established for arbitration. In other words, the specific arbitration procedures agreed to by the Amundsens and Wright did not exist. A dispute arose between the parties and the Amundsens filed suit against Wright. Wright filed a motion to compel arbitration, based on the arbitration clause in the contract. Will the arbitration clause be enforced?
View Solution:
View Solution:
Answer to relevant QuestionsThe Bassos contracted with Dierberg to purchase her property for $1,310,000. One term of the contract stated, "the sale under this contract shall be closed . . . at the office of Community Title Company . . . on May 16, 1988 ...Dove had been employed by Rose Acre Farm, operated by Rust, its president and principal owner, in the summers and other times from 1972 to 1979. Rust had instituted and maintained extensive bonus programs. In June 1979, Rust ...Richard Burnett agreed to purchase a mobile home with a shed from Betty Jean Putrell, Executrix of the Estate of Lena Holland. On Saturday, March 3, Burnett paid Putrell $6,500 and was given the certificate of title to the ...On February 28, 2005, David Dobrovolny purchased a 2005 F-350 pickup truck which had been manufactured by Ford Motor Co. The truck caught fire in Dobrovolny's driveway on April 16, 2006. No one was physically injured, and no ...James Shelton is an experienced musician who operates the University Music Center in Seattle, Washington. On Saturday, Barbara Farkas and her 22-year-old daughter, Penny, went to Shelton's store to look at violins. Penny had ...
Post your question


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Talk:Brahmi numerals
|WikiProject Mathematics
|(Rated Start-class, Low-priority)
Sh letter[edit]
This article says:
For instance, chatur 4 early on took a ¥ shape much like the Kharosthi letter ch; panca 5 looks remarkably like Kharosthi p; and so on through shat 6, sapta 7, and nava 9 (Kharosthi sh, s, n).
However, the Kharosthi article doesn't reveal an sh letter. What's wrong here?? Georgia guy (talk) 18:22, 20 August 2014 (UTC)
- Both ś and ṣ are "sh", though I don't know which one this article means. Shreevatsa (talk) 18:29, 20 August 2014 (UTC)
- And this can be improved by replacing the Unicode characters at Kharosthi (which show up as boxes on my computer) with images. Georgia guy (talk) 18:32, 20 August 2014 (UTC)


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Centura Health has more experts in more places throughout the region. Start your health journey by finding the right one for you.
If you have questions or if you would like to make an appointment, please feel free to contact our genetic counselor:
Melissa Gilstrap, MS, CGC
Genetic Services Coordinator
Phone: 303-269-4348
Fax: 303-269-4964
Melissa Gilstrap
The Parker Adventist Hospital Genetic Counseling Clinic is held at:
Cancer Center
9395 Crown Crest Boulevard
Parker, CO 80138
Appointment line: 303-269-4975
Our search tools require a zip code to match the location nearest you.


text:
THIS RESTAURANT IS CLOSED Kyoto Bowl
1111 S. Rural Rd. (Apache Blvd.) Send to Phone
480-894-9888 | Make Restaurant Reservations
OpenLunch & Dinner daily
- Parking lot
- Dress code: Casual
- Heart-healthy dishes
- Kid-friendly
* Click here for rating key
THIS RESTAURANT IS CLOSED Kyoto Bowl Restaurant Review: This local chain brings Americas favorite Japanese flavors to Tempe. Choices include Americanized favorites such as chicken teriyaki and California rolls. Selections are clearly described on the menu, portions are generous and prices are reasonable.
Find out which restaurants and chefs top the list of GAYOT's 2016 Annual Restaurant Awards.


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Keep the indulgence in "free from" baking: a vegan, refined sugar-free or gluten-free cake should still be a comforting and delicious treat
I am seeing a lot of books and articles on "healthy" and "free from" baked goods. Dairy-free, Vegan. Gluten-free, No Refined Sugar etc. And I applaud the effort. Some of them are great. I am a big fan of Henrietta Inman's new book Clean Cakes, for example – Free From Cake as Art. Many innovators have been writing about these issues and developing recipes for a very long time. For me personally, Alice Medrich's Flavor Flours is my go-to book when looking at finding gluten-free alternatives. The glorious Jude Blereau and her wholefood thinking sits on the top of my She-roes list. The Minimalist Baker and Oh She Glows are superb online "free from" magicians. The Flying Apron in Seattle should be added to the list of great American pioneers, as far as I am concerned. I have written about my own relatively new-found passion for "free from" baking and the satisfaction of baking outside of the conventional box. And I love, really LOVE, the moment when a customer with a tricky combination of food allergies finds something wonderful on my market stall that they can eat and enjoy. Elation is the only way I can describe seeing the current popularity of my vegan products – chatting to the non-vegan customers who are trying them and appreciating their deliciousness using only pure plant-based ingredients.
But for the record, my cake DNA is about PLEASURE first and foremost. I really can't help it. I am blessed and cursed with a hedonism that defies logic and any good sense. I understand that a person can get used to no sugar, that making healthier food choices is overall a good direction to go in and that too much cake is bad for you. But I am not so sure I understand the current trend for cakey treats to be "clean" when the result doesn't really taste much like a treat. What drives me forward is the journey to the little patch of ground where natural baking meets delectable.
Yet I have to live in the real world and on my stall at Berwick Street Market I get a lot of requests for sugar-free cakes from customers across the board: GF, Vegan, those that will eat anything but want to cut down on sugar. I have struggled with my indulgent nature on this one and have yet to make a cake without any sweetener at all that I like – I would struggle to call that beast a "cake". And I can't bear the taste of artificial sweeteners in baking. (Don't get me wrong. I am no stranger to a Diet Coke. It is only in baking that this seems to be a problem for me.) So I find that once again, thanks to my persistent clientele, I am discovering something new. Currently happily working away in the land of maple syrup and honey. I developed this cake as an option for everyone. It is full of wholesome ingredients and contains maple syrup and fresh fruit to sweeten. It is very popular. I confess that I eat it for breakfast. It is all about texture; could be made with different fruits and berries and nuts. I did a nut-free version with coconut milk and Summer berries that was delicious.
Blueberry Cake with lemon, almond & thyme – Gluten-free, refined sugar-free, Vegan
4 tbls flaxseeds + 8 tbls water
225 grams soya or sunflower margarine (gluten and dairy-free)
275 grams pure maple syrup
2 tsp gluten-free baking powder
½ tsp bicarbonate of soda
2 tbls psyllium husks (optional)
½ tsp salt
Juice and zest of 2 lemons
380 grams of GF flours
I use: 140g oat flour, 140g millet, 70g sweet rice sorghum, 30g cornflour
250 ml unsweetened almond milk (you can also use coconut milk)
350 grams fresh blueberries, washed and tossed in 1 tbls of gf flour
50 grams oat flour
50 grams GF oats
50 grams soya margarine (or sunflower or coconut oil)
¼ tsp salt
2 tbls maple syrup
75 grams slivered almonds
A few sprigs of fresh thyme, remove leaves and discard stems
Preheat oven to 180c (160c for fan assisted).
Prepare a 23 cm springform tin by greasing with a little soya or sunflower vegan and gluten-free margarine sides and bottom. Line the bottom and sides with baking parchment.
Combine the flaxseeds and water in a small bowl and set aside. Combine the flours, salt, baking powder, bicarb of soda and psyllium husks (if using) in a medium bowl and set aside. In the bowl of a stand mixer, mix together the margarine and maple syrup until well blended. It will look a bit curdled. Add the flax eggs and the flour mixture and mix by hand with a spatula. Add the almond milk and the lemon juice and mix on medium setting until the batter is smooth, about 2 minutes. Scrape down the sides and bottom with the spatula then add the lemon zest and blueberries, gently combine by hand. Pour into the prepared tin and bake for 35 minutes (initially).
Prepare the crumble by combining all of the ingredients by hand until you have a pebbly mixture. Don't be tempted to replace the fresh thyme with dried. If you don't have fresh …the cake will be fine without. Put in the fridge while the cake is baking. Once the cake has been in the oven for 35 minutes, gently and quickly take it out of the oven, sprinkle the crumble on top, return to oven and bake for a further 30-40 minutes – ovens vary.
The cake is ready when a toothpick comes out clean and the crumble has browned nicely.
Allow to cool in the pan for 20 minutes then remove the springform and allow to cool completely on a wire rack. To serve: sprinkle with fresh blueberries and thyme leaves, if you are sticking to no sugar. Delicious with a low-sugar lemon sorbet.


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Amsale / New Learn More
Amsale 'Nicole'
/ Resell It & Make Up To: $1,080 Learn More
This is a gorgeous Amsale dress. It is a label size 6 and is brand new, never worn! No alterations have been made to the dress. It has been stored in a clean, pet-free, smoke-free home. It is a stunning ivory dress with a modified sweetheart neckline. It is a classic designer dress made of silk faced satin. There is a belt attached with a crystal pin, but the dress can also be worn without the belt.
- Condition: new
- Alterations: no
- US Street Size: 04
- Dress Tag Size: 6
- Bust: 32
- Waist: 28
- Hips: 32
- Tallest Wearable Height: 6' 0"
- Fabric: silk
- Silhouette: trumpet
- Neckline: sweetheart
- Features: beading
- Dress Length: full
Once you have placed your order the Seller will be notified to pack and ship the gown. The seller will then ship the item to us and our inspection and authentication team will check that it is authentic and matches to the seller's description. The item will then be shipped to you upon approval.
Dress Q & A
Have a question not answered here? Contact us below.
Try It On - No Risk Return Policy: Have your cake & eat it too. Returns accepted on all items. More details