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We used the RIP1-Tag2 (RT2) mouse model of islet cell carcinogenesis to profile the transcriptome of pancreatic neuroendocrine tumors (PNET) that were either non-invasive or highly invasive, seeking to identify pro- and anti-invasive molecules. Expression of multiple components of desmosomes, structures that help maintain cellular adhesion, was significantly reduced in invasive carcinomas. Genetic deletion of one of these desmosomal components, desmoplakin, resulted in increased local tumor invasion without affecting tumor growth parameters in RT2 PNETs. Expression of cadherin 1, a component of the adherens junction adhesion complex, was maintained in these tumors despite the genetic deletion of desmoplakin. Our results demonstrate that loss of desmoplakin expression and resultant disruption of desmosomal adhesion can promote increased local tumor invasion independent of adherens junction status.
Author Summary
The ability of a tumor to invade into the surrounding normal tissue is one hallmark of a malignant cancer. We sought to identify factors that either restrict or promote tumor invasion in a genetically engineered mouse model of pancreatic neuroendocrine cancer by characterizing the transcriptional profiles of the non-invasive and invasive pancreatic neuroendocrine tumors (PNET) that develop in this model. This analysis demonstrated that multiple genes encoding components of desmosomes, cellular structures dedicated to the maintenance of cell-cell adhesion, were expressed at much lower levels in invasive PNETs, suggesting that loss of desmosomal adhesion contributes to the development of an invasive phenotype in these tumors. Genetic deletion of one of these desmosomal components in PNET-bearing mice resulted in increased local tumor invasion. These results are important since the development of an invasive phenotype is associated with a poor prognosis for many human cancers and is often a precursor to the development of distant metastases. Our findings demonstrate one mechanism by which tumors can acquire such an invasive phenotype and may prove useful in evaluating the malignancy of human cancers.
Citation: Chun MGH, Hanahan D (2010) Genetic Deletion of the Desmosomal Component Desmoplakin Promotes Tumor Microinvasion in a Mouse Model of Pancreatic Neuroendocrine Carcinogenesis. PLoS Genet 6(9): e1001120. doi:10.1371/journal.pgen.1001120
Editor: Bruce E. Clurman, Fred Hutchinson Cancer Research Center, United States of America
Received: April 13, 2010; Accepted: August 12, 2010; Published: September 16, 2010
Copyright: © 2010 Chun, Hanahan. 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: DH is an American Cancer Society Research Professor. This work was supported by a grant from the National Cancer Institute (to DH; 5R01CA45234-24) and by a graduate research fellowship from the National Science Foundation (to MGHC). 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.
The ability of a tumor to invade into the surrounding normal tissue marks a critical step in the transition from benign to malignant tumor growth. The acquisition of this hallmark of cancer is associated with poor prognosis for many human cancers and is often considered a precursor to the development of metastases [1]. As such, considerable effort has been directed towards identifying invasion promoting and suppressing molecules and the mechanisms by which they modulate a tumor's invasive phenotype [2].
Amongst the discernible barriers to the acquisition of an invasive growth phenotype is cell-cell adhesion, and cellular alterations that result in disrupted, reduced, or otherwise functionally altered cellular adhesion are strongly associated with the progression to a malignant tumor phenotype [3]–[5]. The importance of sustaining cellular adhesion for homeostasis, particularly in epithelial tissues, is evident in the number of distinct structures whose primary function is to maintain cell-cell interconnections, which include the adherens junctions (AJs), desmosomes, and tight junctions [6], [7]. These complexes share many structural similarities, including the presence of transmembrane proteins – typified by the cadherins – that mediate adhesive connections with neighboring cells as well as intracellular molecules – exemplified by the catenin and the plakin families – that connect these transmembrane components to the cytoskeleton [6], [7]. In particular, changes in the expression and/or function of AJ components have been associated with malignant cancers, and numerous studies have focused on the role of AJs in restricting invasive growth [3], [8], [9].
In this study, we utilized the RIP1-Tag2 (RT2) mouse model of cancer to identify the mechanisms by which tumors acquire invasive growth capabilities. RT2 mice develop multiple pancreatic neuroendocrine tumors (PNET) by 12–14 weeks of age due to the expression of the SV40 T antigen oncoprotein (Tag) in the pancreatic β cells [10]. This model has proven useful in characterizing many aspects of tumorigenesis due to its relatively synchronous and predictable progression through distinctive lesional stages that culminate in invasive carcinomas [11]–[13]. We used this model to identify pro- and anti-invasive molecules in an unbiased fashion by comparing the non-invasive islet tumors to highly invasive carcinomas using microarray profiling of the mRNA transcriptome. We identified several components of desmosomes whose expression was significantly decreased in invasive tumors, implicating attenuation of desmosomal function in malignant progression. To assess this hypothesis, we engineered into the oncogene-expressing cancer cells in RT2 mice a genetic deletion of desmoplakin (Dsp; MGI: 109611), an intracellular protein critical for desmosomal stability [14]. Loss of Dsp led to an increased incidence of invasive carcinomas providing strong evidence that desmosomal adhesion acts as a distinct barrier to invasive tumor growth.
Expression of desmosomal components is lost in invasive RT2 tumor lesions
We chose to use the RT2 mouse model of cancer to characterize mechanisms governing the switch from benign to invasive tumor growth since a broad spectrum of invasive tumor lesions develop in end-stage RT2 animals. These include the non-invasive islet tumor (IT), the focally invasive carcinoma type-1 (IC1), and the broadly invasive carcinoma type-2 (IC2) [15].
To evaluate potential mechanisms regulating invasive tumor growth in this model, we isolated tissue from IT and IC2 lesions in end-stage RT2 animals by laser capture microdissection and then profiled the mRNA transcriptome. The IC2 class showed widespread transcriptional changes as compared to the IT class (Dataset S1). We chose to focus our attention on differentially expressed genes encoding components of two cell-cell adhesion structures, namely adherens junctions and desmosomes (Table 1), since elements of each were prominently downregulated. The expression of cadherin 1 (Cdh1, also known as E-cadherin; MGI: 88354), a molecule previously demonstrated to restrict invasive growth in this and other models [8], [16], was decreased in IC2 lesions as expected. Interestingly, Cdh1 was the only member of AJs that was significantly altered in IC2 lesions (Table 1). In contrast, multiple genes encoding components of desmosomes were significantly reduced in IC2 lesions (Table 1). Moreover, the expression of several desmosomal genes in addition to Cdh1 was progressively reduced in the distinctive stages of PNET tumorigenesis in RT2 mice as well as in human PNETs as compared to normal human pancreatic islets, when total lesional stages, in particular ungraded tumors, were analyzed (Figure S1) [13]. Although the expression of these genes was reduced in ungraded whole tumors in comparison to normal islets, their levels were further reduced in the microdissected invasive IC2 lesions (Table 1). Based on these results, we sought to determine what role desmosomal adhesion might play in regulating invasive tumor growth in this mouse model of cancer.
To confirm the microarray results, we performed immunohistochemistry for multiple desmosomal components. Staining for Dsp and for one of the desmosomal cadherins, desmoglein 2 (Dsg2; MGI: 1196466), as well as for Cdh1 demonstrated that these molecules are expressed in the pancreatic islets as well as in the pancreatic ducts and the exocrine pancreas of wild-type animals (Figure 1 and Figure S2). In tumors of end-stage RT2 animals, the expression of all three molecules was maintained in IT lesions and was largely extinguished in IC2 lesions (Figure 1 and Figure S2). In contrast to Cdh1, expression of catenin beta 1 (Ctnnb1; MGI: 88276), another component of AJs, was maintained in both IT and IC2 lesions, comparable to wild-type islets (Figure S3). This result is consistent both with the microarray result demonstrating that Cdh1 was the only AJ component to show any change in expression and with a previous study suggesting that Ctnnb1 does not contribute to RT2 tumorigenesis [17]. Collectively, these data confirm the microarray results and suggest the hypothesis that loss of desmosomal adhesion might contribute to the development of an invasive phenotype.
Expression of desmoplakin (Dsp) and cadherin 1 (Cdh1) is lost in the IC2 but not the IT grade of PNET in RT2 mice. (A–C) H&E staining of a normal islet from a wild-type B6 mouse and of an IT and an IC2 tumor from an end-stage RT2 mouse. Dashed lines demarcate tumor margins. (D–F) Immunofluorescence staining with DAPI to visualize cellularity. (G–I) Immunofluorescence staining for Cdh1. (J–L) Immunofluorescence staining for Dsp. (M–O) Merge of Cdh1 and Dsp immunofluorescence staining (G–L). (P–R) Higher magnification of the boxed regions in M–O. Scale bars represent 200 µm (A–O) and 100 µm (P–R).
β cell specific deletion of Dsp in RT2 animals
To address the hypothesis raised by the microarray and immunohistochemistry results, we asked whether functionally disrupting desmosomal activity in vivo would promote invasive tumor growth in RT2 mice. To accomplish this, we chose to genetically delete Dsp since there is a single Dsp gene as compared to other components of desmosomes for which there are multiple non-allelic genes [6]. Furthermore, ablation of Dsp in vivo has previously been shown to impair desmosome function [14]. Since the Dsp whole body knockout is embryonic lethal [14], we employed the Cre/loxP system to ablate the Dsp gene specifically in the pancreatic β cells, the same cells that express the Tag oncogene in RT2 mice. In combination with a DspFlox allele [18], we used a mouse line in which a tamoxifen-regulatable Cre recombinase is controlled by the pancreatic duodenal homeobox gene 1 promoter (Pdx1-CreER) [19]. Pdx1 is expressed in all pancreatic lineages during development and is variably expressed in the adult pancreas, in particular being widely expressed in β cells [20], [21].
We intercrossed RT2+; DspFlox/WT with Pdx1-CreER+; DspFlox/WT mice to generate the appropriate genotypes, and all expected genotypes and genders were observed in approximate Mendellian ratios (Table S1 and Table S2). To induce Cre activity, all Pdx1-CreER positive mice were given tamoxifen for five consecutive days beginning at 10 weeks of age when incipient tumors are first observed in RT2 mice [22]. In the absence of the RT2 transgene, genetic ablation of Dsp resulted in uniform loss of Dsp expression in the pancreatic islets, as determined by immunohistochemistry (Figure S4). Deletion of Dsp did not cause any change in Cdh1 expression or in the gross morphological appearance of the non-oncogene-expressing islets (Figure S4). Loss of Dsp was accompanied by significantly reduced Dsg2 expression in the pancreatic islets whereas the expression of insulin (Ins), the hormone produced by β cells, did not appear to be affected (Figure S5). These results are consistent with compromised desmosomal adhesion, although we cannot strictly rule out the possibility that some residual desmosomal function persists in the absence of Dsp. Ablation of Dsp in normal pancreatic islets did not affect multiple physiological parameters, such as body mass and fasting glucose levels, and its expression in this tissue compartment is apparently dispensable in adult mice (Figure S6), setting the stage to assess the impact of its loss on PNETs arising from such islets. Lastly, the tamoxifen induction regimen by itself had no obvious effect on any aspect of RT2 tumorigenesis examined, including tumor invasion, when tamoxifen was applied to RT2 mice that lacked the Pdx1-CreER and DspFlox alleles (Figure S7).
Loss of Dsp does not affect tumor growth parameters in RT2 mice
Induced loss of Dsp at 10 weeks of age did not affect any of the tumor growth parameters in RT2 mice that were sacrificed 4 weeks later. No significant changes were observed in the number of tumors that developed nor in the collective tumor burden when comparing RT2+; Pdx1-CreER+; DspFlox/Flox mice and littermate controls (Figure 2A–B). Furthermore, the rates of tumor proliferation and tumor apoptosis, as judged by the levels of the proliferation marker Ki67 and the TUNEL assay respectively, were indistinguishable between groups (Figure 2C–J). Thus, we conclude that the loss of Dsp does not affect tumor growth in this model.
Conditional genetic deletion of Dsp in angiogenic islet dysplasias and incipient solid tumors does not affect tumor formation or tumor growth parameters in RT2 mice. (A–B) Tumor burden and tumor number in RT2+; Pdx1-CreER+; DspWT/WT, RT2+; Pdx1-CreER+; DspFlox/WT, and RT2+; Pdx1-CreER+; DspFlox/Flox mice. Cre activity was induced at 10 weeks, and mice were sacrificed at 14 weeks. Data shown are individual values plus mean. Groups are not statistically different for these metrics. (C–E) Ki67 staining on tumors from RT2+; Pdx1-CreER+; DspWT/WT, RT2+; Pdx1-CreER+; DspFlox/WT, and RT2+; Pdx1-CreER+; DspFlox/Flox mice. (F–H) TUNEL staining on tumors from RT2+; Pdx1-CreER+; DspWT/WT, RT2+; Pdx1-CreER+; DspFlox/WT, and RT2+; Pdx1-CreER+; DspFlox/Flox mice. (I) Quantification of C–E. Data shown are mean plus standard error. Groups are not statistically different. (J) Quantification of F–H. Data shown are mean plus standard error. Groups are not statistically different. Scale bars represent 100 µm (C–H).
Loss of Dsp leads to increased local tumor invasion in RT2 mice
While conditional genetic ablation of Dsp in the angiogenic islet dysplasias and incipient solid tumors of RT2 mice had no discernible effects on tumor formation and subsequent tumor growth parameters, it did lead to an increase in tumor invasion. RT2 mice develop a spectrum of tumor lesions, including non-invasive (IT), focally invasive (IC1), and broadly invasive (IC2) lesions (Figure 3A–F) [15]. Loss of Dsp resulted in a greater frequency of invasive tumors and a concomitant reduction in the percentage of non-invasive IT tumors in mice analyzed four weeks after genetic ablation of Dsp in incipient solid tumors (Figure 3G–H). Whereas ∼40% of total tumors could be classified as invasive carcinomas in control mice, greater than 60% of all tumors fell into this category in RT2+; Pdx1-CreER+; DspFlox/Flox mice (Figure 3G). Interestingly, this shift appears to result from selective progression to the focally invasive IC1 but not to the widely invasive IC2 tumors. Indeed, while there is no significant change in the development of IC2 lesions (approximately 10% of all tumors fall into this class regardless of Dsp status), more than 50% of tumors can be classified as IC1 lesions in RT2+; Pdx1-CreER+; DspFlox/Flox mice versus ∼30% in control mice (Figure 3H).
Conditional genetic deletion of Dsp in angiogenic islet dysplasias and incipient solid tumors increases the rate of progression to focally invasive IC1 tumors in RT2 mice. (A–C) H&E staining of a non-invasive IT tumor lesion, a focally invasive IC1 tumor lesion, and a broadly invasive IC2 tumor lesion from RT2+; Pdx1-CreER+; DspWT/WT, RT2+; Pdx1-CreER+; DspFlox/WT, and RT2+; Pdx1-CreER+; DspFlox/Flox mice. (D–F) Higher magnification of the boxed regions in A–C. T indicates tumor region and Ex indicates exocrine pancreas. Dashed lines demarcate tumor margins. Arrowheads indicate regions of tumor invasion. (G) Quantification of tumor invasiveness represented as the percentage of IT lesions or total IC lesions (IC1+IC2) in RT2+; Pdx1-CreER+; DspWT/WT, RT2+; Pdx1-CreER+; DspFlox/WT, and RT2+; Pdx1-CreER+; DspFlox/Flox mice at 14 weeks of age. A minimum of 36 tumors per group was graded. * p<0.01 by Fisher's exact test. (H) Same as G except IC lesions are separated into the IC1 and IC2 subclasses. * p<0.01 by the Chi-square test. (I) Tumors from RT2+; Pdx1-CreER+; DspWT/WT, RT2+; Pdx1-CreER+; DspFlox/WT, and RT2+; Pdx1-CreER+; DspFlox/Flox mice were genotyped for the presence of the Cre recombinase (∼530 bp), β2 microglobulin (∼290 bp), and the floxed (∼360 bp) or wild-type Dsp allele (∼230 bp). These same tumors were assessed for the recombination status of Dsp: wild-type allele (∼960 bp), non-recombined floxed allele (∼1200 bp), recombined floxed allele (∼650 bp). Scale bars represent 400 µm (A–C) and 200 µm (D–F).
We confirmed that Dsp was in fact lost in these tumors by examining the recombination status of the Dsp allele by PCR. Tumors that were genotypically DspFlox/Flox showed near universal recombination of the Dsp allele, confirming that Dsp was lost in these tumors (Figure 3I). Tumors isolated from control DspWT/WT or DspFlox/WT mice showed no recombination or were heterozygous for the recombined and wild-type Dsp alleles respectively. Thus, we conclude that the conditional genetic ablation of Dsp in incipient tumors of RT2 mice leads to increased local tumor invasion.
Cdh1 expression is maintained in IC1 tumor lesions regardless of Dsp status
We were intrigued that loss of Dsp led to an increase in the IC1 class but not in the IC2 class of invasive tumors. Since Cdh1 also acts as a dominant invasion suppressor in this model, we examined its status in the tumors from RT2+; Pdx1-CreER+; DspFlox/Flox mice and littermate controls by immunohistochemistry. We found that Cdh1 expression was maintained in the IT and IC1 tumors that developed regardless of Dsp status (Figure 4I–L). Tumor margins and regions of invasion were identified by staining for the Tag oncoprotein (Figure 4E–H). Indeed, Cdh1 appeared to be expressed at comparable levels in IT and IC1 tumor lesions regardless of Dsp status (Figure 4M–T). Expression in IT and IC1 lesions of a second component of AJs, junction plakoglobin (Jup, also known as gamma catenin; MGI: 96650), was also unaffected by Dsp status (Figure S8), consistent with AJ function being maintained in these lesions despite the absence of Dsp and impaired/ablated desmosomal function. Lastly, cadherin 2 (Cdh2, also known as N-cadherin; MGI: 88355), a marker of epithelial-mesenchymal transition (EMT), was expressed at readily detectable and comparable levels in IT and IC1 tumors regardless of Dsp status, as well as in the IC2 tumors that did not express Cdh1 (Figure S9), consistent with the results of a previous study investigating determinants of progression to invasive carcinoma [8]; notably, there is no indication that activation of the invasive growth capability in this pathway involves an EMT, as reflected in differential expression of Cdh2 or other markers of EMT. Given that the expression of both Dsp and Cdh1 was lost in IC2 lesions, the most invasive class of RT2 tumors, both in unmodified RT2 mice and in tamoxifen-treated RT2+; Pdx1-CreER+; DspFlox/Flox mice (Figure 1 and data not shown), we infer that loss of Dsp by itself is sufficient to promote the development of focally invasive tumors while the additional loss of Cdh1 is required to develop a more aggressive invasive tumor phenotype.
Cdh1 expression is maintained in the IC1 grade of tumors in both RT2+; Pdx1-CreER+; DspWT/WT and RT2+; Pdx1-CreER+; DspFlox/Flox mice. (A–D) Immunofluorescence staining with DAPI to reveal cellularity in IT and IC1 tumors in RT2+; Pdx1-CreER+; DspWT/WT and RT2+; Pdx1-CreER+; DspFlox/Flox mice. (E–H) Immunofluorescence staining for the oncoprotein T antigen (Tag). (I–L) Immunofluorescence staining for Cdh1. (M–P) Merge of Tag and Cdh1 immunofluorescence staining (E–L). (Q–T) Higher magnification of the boxed regions in M–P. Arrowheads indicate regions of tumor invasion. Scale bars represent 200 µm (A–P) and 100 µm (Q–T).
To date, much of the work on desmosomes in human disease has focused on their role in maintaining heart and skin integrity, where desmosomal defects are associated with cardiomyopathy and skin blistering conditions respectively [23]. More recently, a potential role for desmosomes in cancer progression has been suggested based on a variety of experimental clues [24]. For example, in vitro cell culture assays demonstrated that inhibiting desmosomal adhesion via blocking peptides caused morphological disorganization [25] while introduction of desmosomal components into a nonadhesive cell line resulted in increased cell aggregation and reduced cellular invasion in vitro [26]. These studies suggested that loss of desmosomal function might contribute to tumor invasion and malignancy, consistent with their role in maintaining cellular adhesion. (Our attempts to perform similar in vitro experiments using cell lines derived from RT2 tumors [βTCs] were hindered by the fact that βTC cell lines express desmosomal components at low levels, presumably due to adaptations to culture, and generally perform poorly in migration/invasion assays – data not shown). In further support of the proposed role of desmosomes as a barrier to malignant progression, several pathology studies characterizing human cancers have shown that decreased or altered expression of desmosomal components, including Dsp, correlates with increased tumor invasion, advanced tumor grade, and poor patient prognosis, particularly in oral cancers where expression of desmosomal components are highly expressed in the normal oral mucosa [4], [5], [27]. Additionally, our bioinformatic analysis of human cancer databases confirmed that the expression of desmosomal genes is often decreased in a variety of human epithelial cancers as compared to normal tissues and is occasionally further decreased in more advanced grades of tumors (Table S3). The present study substantively extends this current state of knowledge by demonstrating that desmosomal adhesion can indeed act as a distinct barrier to the development of an invasive tumor phenotype in the in vivo setting of a genetically engineered mouse model of cancer.
We identified several components of desmosomes – Dsp, Dsg2, desmocollin 2 (Dsc2; MGI: 103221), and plakophilin 2 (Pkp2; MGI: 1914701) – whose expression was significantly downregulated in the highly invasive tumor lesions that develop in the RT2 mouse model of PNET. These changes were reflected at the protein level as determined by immunostaining of non-invasive IT lesions and broadly invasive IC2 lesions. The simultaneous decrease in expression for multiple desmosomal genes suggests that there may be coordinated transcriptional regulation of desmosomal components. Prime candidates for such regulation include the transcription factors that regulate EMT, such as the Snail and Twist families of transcription factors [28]. Notably, however, we did not detect significant differential expression of such transcription factors in our microarray analysis comparing non-invasive IT and highly invasive IC2 PNETs (Dataset S1), and the expression of one prominent marker of EMT, Cdh2, was not obviously different between IT and IC2 lesions, consistent with the results of a previous study investigating determinants of the invasive phenotype using this same model of PNET [8]. Thus, the current evidence suggests that the acquisition of an invasive phenotype in this tumor type does not involve a classical EMT. Our results clearly demonstrate that the conditional genetic deletion of a single core desmosomal component, Dsp, promotes increased local tumor invasion in RT2 mice, producing a phenocopy of such inferred transcriptional regulation in the normal circumstances of tumor progression.
While desmosomes play an integral role in maintaining epithelial integrity, they are by no means the only structure involved in cellular adhesion. In addition to desmosomes, several related structures, including AJs, contribute to maintaining cell-cell adhesion [7]. However, while desmosomes and AJs play related biological roles in terms of maintaining cellular adhesion and have similar structural compositions, it is worth noting that there are clear differences in the consequences of impaired desmosome adhesion versus impaired AJ adhesion on tumor phenotypes. An elegant functional genetic study demonstrated that Cdh1, a core member of AJs, acts as an invasion suppressor in vivo; targeting a transgene encoding a dominant-negative Cdh1 molecule to the oncogene-expressing pancreatic β cells markedly accelerated tumor progression and led to significantly increased frequencies of invasive carcinomas and to the development of lymph node metastasis in this same mouse model of PNET [8]. In comparison, deletion of Dsp led to an increase in the frequency of the focally invasive IC1 grade of islet carcinomas but not the more widely aggressive IC2 carcinomas, and distant metastases were not observed (data not shown). One possible explanation for the differences in these phenotypic outcomes is the different roles that Dsp and Cdh1 play within their respective adhesion complex. While Cdh1 is a transmembrane protein that directly links cells together by forming homotypic interactions with other Cdh1 molecules on neighboring cells [29], Dsp is an intracellular molecule that contributes to the overall stability of the desmosomal plaque and links this structure to the intermediate filaments [14]. Therefore, deletion of Dsp may attenuate but not totally abolish desmosomal function; if so, then the specific deletion of one of the desmosomal cadherins, Dsc2 or Dsg2, might have a more pronounced effect on invasiveness. An additional explanation for the increase in the focally invasive IC1 fraction but not the broadly invasive IC2 fraction of invasive tumors following ablation of Dsp involves the observed maintenance of Cdh1 and AJs. Expression of Cdh1 as well as a second component of AJs, Jup, was retained in both the non-invasive IT tumors and in the now more prevalent focally invasive IC1 tumors following genetic deletion of Dsp. It would seem likely, in light of the aforementioned functional study in this same mouse model of cancer [8], that the preservation of Cdh1 expression and of AJ function serves to maintain an additional, stronger brake on tumor invasion. Thus, while loss of Dsp and impairment of desmosomal adhesion leads to the focal invasion observed in IC1 lesions, the development of the broadly invasive phenotype found in IC2 lesions evidently requires the concomitant loss of Cdh1. Indeed, the IC2 tumor lesions that normally develop in RT2 mice show a coordinated reduction in the expression of Cdh1 and multiple desmosomal components (Table 1, Figure 1, and Figure S2). The apparently independent regulation of desmosomal and AJ adhesion is notable since AJ stability has been proposed to affect desmosomal stability and vice versa in other contexts [18], [30], [31], whereas Cdh1 and Jup are evidently not affected by the deletion of Dsp during PNET tumorigenesis in RT2 mice.
Interestingly, the genetic deletion of Dsp had no consequential effects on the other parameters of RT2 tumorigenesis beyond invasion. Although it has been suggested that Dsp and other desmosomal components can affect cellular proliferation and apoptosis [32], [33], we did not observe any changes in tumor growth parameters following the genetic deletion of Dsp (Figure 2). Our results are consistent with one of the earliest studies to examine the role of Dsp in vivo, wherein a skin-specific deletion of catenin alpha 1 (Ctnna1; MGI: 88274), the AJ homologue of Dsp, led to increased skin proliferation and hyperplasia whereas ablation of Dsp did not [34]. Thus, with regards to the RT2 model of PNET and possibly other forms of cancer, it appears that desmosomes primarily serve to maintain cell-cell adhesion and hence suppress the acquisition of an invasive growth capability such that the observed downregulation of desmosomal genes results in the impairment of desmosomal function and a concomitant weakening in cellular adhesion without affecting other parameters of tumorigenesis.
Finally, it is important to set these results into the broader context of knowledge about malignant progression to an invasive growth state in this stereotypical pathway of multistep tumorigenesis. While disrupted cell-cell adhesion caused by the reduced expression of Cdh1 [8] and/or desmosomal genes (this report) clearly promotes invasive tumor growth, other factors are involved as well. Thus for example, increased expression of the type-1 insulin-like growth factor receptor (Igf1r; MGI: 96433) can drive these PNETs to acquire a highly invasive phenotype [15]. Additionally, the recruitment of immune cells to the margins of these PNETs has been shown to promote invasiveness, in part by supplying cathepsin proteases and heparanase (Hpse; MGI: 1343124) [35]–[37]. As such, multiple factors can impact the progression to invasiveness by varying degrees (Figure 5), and future research may well identify additional components. Irrespective, our results demonstrate that loss of desmosomal adhesion, as exemplified by the genetic deletion of Dsp, can enable a tumor to acquire an invasive phenotype. The functional study presented herein establishes desmosomal adhesion as a distinct and ostensibly independent suppressor of invasive tumor growth. This knowledge will likely contribute to a better understanding of the mechanisms governing tumor progression to an invasive growth state and may prove useful in evaluating invasive states of human cancers.
Multiple factors impact the progression to an invasive phenotype as illustrated by the RIP1-Tag2 (RT2) mouse model of pancreatic neuroendocrine tumorigenesis (PNET). This study demonstrates that the genetic deletion of desmoplakin (Dsp) and concomitant loss/attenuation of desmosomal adhesion can promote local tumor invasion, specifically to a focally invasive state typified by the IC1 tumor class. Other factors have also been demonstrated to affect tumor invasion in this model. Activation of heparanase or of cathepsin proteases [35]–[37] supplied by infiltrating immune cells or suppression of cadherin 1 (Cdh1, also known as E-cadherin) [8] can each contribute to invasion. Upregulation of the type-1 insulin-like growth factor receptor (Igf1r) preferentially promotes progression to the IC2 stage, in part via a branched pathway from earlier neoplastic stages such as angiogenic islet dysplasias (AI), bypassing the canonical AI→IT→IC1→IC2 progression [15]. Future research may well identify additional factors that impact tumor invasion.
Materials and Methods
Ethics statement
All mice used in this study were housed and maintained in accordance with the University of California, San Francisco (UCSF) institutional guidelines governing the care of laboratory mice.
Genetically engineered mice
The generation and characterization of the RIP1-Tag2 (RT2) [10], DspFlox [18], and Pdx1-CreER [19] mouse lines have been previously reported. All mice were backcrossed a minimum of six generations into the C57Bl/6 (B6) background (Charles River, Wilmington, MA) and then intercrossed to generate the specified genotypes. To induce CreER activity, mice were injected intraperitoneally with 100 µl of 10 mg/ml tamoxifen (Sigma, St. Louis, MO) suspended in peanut oil for five consecutive days beginning at 10 weeks of age. To relieve the effects of hypoglycemia induced by the insulin-secreting tumors, all RT2 mice received 50% sugar food (Harlan Teklad, Madison, WI) beginning at 10 weeks of age.
Tissue preparation, tumor analysis, and histology
Pancreata were isolated from 14-week-old mice and embedded in OCT (Sakura Finetek, Torrance, CA) on dry ice. Tumor number and tumor volume were quantified as previously described [12]. For histological analysis, frozen tissues were sectioned at 10 µm thickness, and every tenth section was stained with hematoxylin and eosin (Surgipath Medical Industries, Richmond, IL) using standard methods. Tumors were classified as a non-invasive islet tumor (IT), a focally invasive carcinoma type-1 (IC1), or a broadly invasive carcinoma type-2 (IC2) using a previously defined grading scheme [15].
Laser capture microdissection and RNA purification and amplification
Fresh-frozen pancreatic sections (10 µm) from 14-week-old RT2 B6 mice were fixed in cold 70% ethanol for 16 hours prior to laser capture microdissection (LCM). Sections were stained using a modified hematoxylin and eosin stain that preserves RNA integrity while allowing for the microscopic visualization of pancreatic structures [38]. LCM was performed using an Arcturus PixCell II laser capture microscope system (Molecular Devices, Sunnyvale, CA). Total RNA was isolated using the Arcturus PicoPure RNA Isolation kit (Molecular Devices, Sunnyvale, CA) and DNase I treated (Qiagen, Valencia, CA). Equal amounts of RNA (8 ng/lesion) from three independent IT or IC2 tumor lesions were pooled, and then cDNA was generated, amplified, and biotinylated using the Ovation Biotin System (NuGen, San Carlos, CA). Three independent pools per tumor class were generated for subsequent microarray analysis.
Microarray analysis
Labeled cDNA was hybridized to Affymetrix Mouse Genome 430 2.0 arrays (Affymetrix, Santa Clara, CA) according to the manufacturer's specifications. Data were analyzed by the UCSF Helen Diller Family Comprehensive Cancer Center Biostatistics and Computational Biology Core. The data were normalized using a robust multi-chip averaging method utilizing the freely available R language. Linear models were fit for each pair of groups to be compared with log2 expression as the response and the tumor phenotype indicator as the independent variable using the limma package in Bioconductor. Moderated t-statistics were used, and p-values were adjusted by controlling the false discovery rate. A change in gene expression was identified as significant if the false discovery rate was less than 0.05, meaning that fewer than 5% of false findings would be expected among the genes declared to be differentially expressed.
Immunohistochemical staining and analysis
Frozen tissues were sectioned at 10 µm thickness. For immunofluorescence staining, sections were fixed in cold acetone. For colorometric staining, sections were fixed in 10% Zn-buffered formalin (Medical Chemical Corporation, Torrance, CA), subjected to antigen retrieval using the Antigen Unmasking Solution (Vector Laboratories, Burlingame, CA), and blocked for endogenous peroxidase activity. Antibodies used in this study were as follows: rat anti-cadherin 1 (Invitrogen, Carlsbad, CA); mouse anti-desmoplakin I/II, mouse anti-desmoglein 1/2 (Fitzgerald, Concord, MA); mouse anti-catenin beta 1, mouse anti-cadherin 2, mouse anti-junction plakoglobin (BD Biosciences, San Jose, CA); guinea pig anti-insulin (Millipore, Billerica, MA); rabbit anti-T-antigen (Hanahan laboratory preparation); rabbit anti-Ki67 (Novus Biologicals, Littleton, CO); rhodamine red-X-conjugated donkey anti-mouse IgG, rhodamine red-X-conjugated donkey anti-rabbit IgG, FITC-conjugated donkey anti-rat IgG, FITC-conjugated donkey anti-guinea pig IgG, biotin-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA). For mouse antibodies, non-specific binding was blocked using the Mouse on Mouse Blocking Reagent (Vector Laboratories, Burlingame, CA). Fluorescently labeled tissues were mounted with Vectashield mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA) to visualize cell nuclei. The TdT-mediated dUTP-digoxigenin nick-end labeling (TUNEL) assay was used to assess tumor apoptosis as previously described [15]. For colorometric staining, signal was amplified using the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA), visualized using Nova Red substrate (Vector Laboratories, Burlingame, CA), and counterstained with hematoxylin. For Ki67 and TUNEL quantification, two to three random fields were obtained using a 40× objective lens from at least two tumors per mouse and at least five mice per group. The proliferation or apoptosis index was calculated as the percentage of total cells per field that were Ki67- or TUNEL-positive respectively using the MetaMorph software package (Molecular Devices, Sunnyvale, CA). For all other immunohistochemical analysis, two to three tumors per mouse from a minimum of five mice per indicated group were analyzed per staining condition. All images were captured using an Axio Imager bright field microscope or an Axio Scope fluorescence microscope and the AxioVision LE software package (Carl Zeiss, Thornwood, NY).
Statistical analysis
Fisher's exact test and the chi-square test were used to compare tumor invasion metrics. The Mann-Whitney test was used to compare tumor burden, tumor number, tumor proliferation rates, tumor apoptosis rates, and body mass metrics. The Mann-Whitney and the Wilcoxon matched pairs test was used to compare fasting glucose metrics. For all statistical tests, a p-value of p≤0.05 was considered significant. All statistics were performed using the Prism software package (GraphPad Software, La Jolla, CA).
Fasting glucose measurements
Animals were fasted overnight for 14–16 hours prior to the first tamoxifen injection and one week following the final tamoxifen injection. Fasting glucose levels were measured using a FreeStyle Freedom glucose meter (Abbott Laboratories, Abbott Park, IL).
Tumor genotype analysis by polymerase chain reaction (PCR)
Tumor tissue was isolated directly from OCT embedded tissues, and genomic DNA was isolated using the QIAmp DNA Micro kit (Qiagen, Valencia, CA). PCR was performed using standard methods. Primers used were as follows: Cre (forward: 5′-CATGTTCAGGGATCGCCAGG-3′ and reverse: 5′-TGCGGTGCTAACCAGCGTTTT-3′); β2 microglobulin (forward: 5′-CACCGGAGAATGGGAAGCCGAA-3′ and reverse: 5′-TCCACACAGATGGAGCGTCCAG-3′); Dsp-WT/Flox (forward: 5′-GGTTGGGCCTCTCGAATCATGAGTGTCTAGCG-3′ and reverse: 5′-TGTCTGTTGCCATGTGATGCC-3′); Dsp-Recombined/Non-Recombined (forward: 5′-ACAGGCCAGATGAGATCACC-3′ and reverse: 5′-TGTCTGTTGCCATGTGATGCC-3′).
Real-time quantitative PCR
Normal islets were isolated from six-week-old wild-type B6 mice, and hyperplastic islets were isolated from six-week-old RT2 B6 mice as previously described [39]. Angiogenic islets were isolated from nine-week-old RT2 B6 mice by selection based on their red, hemorrhagic appearance following collagenase digestion of pancreata [39]. Islet tumors were excised from the surrounding exocrine pancreas from 14-week-old RT2 B6 mice. Total RNA was purified using the RNeasy Mini kit (Qiagen, Valencia, CA) and DNase I treated (Qiagen, Valencia, CA). cDNA was synthesized using the iScript cDNA Synthesis kit (Bio-Rad Laboratories, Hercules, CA). Real-time quantitative PCR was performed using a 7900HT system (Applied Biosystems, Foster City, CA) (see Table S4 for a complete list of primers used in this study) according to the manufacturer's specifications.
Supporting Information
Dataset S1.
Microarray data for comparison of IT and IC2 classes of RIP1-Tag2 tumors.
Figure S1.
Expression of adherens junction and desmosomal components is decreased during PNET tumorigenesis in RT2 mice and in human pancreatic neuroendocrine tumors. (A) Real-time quantitative PCR values for AJ components (cadherin 1 [Cdh1], catenin alpha 1 [Ctnna1], catenin beta 1 [Ctnnb1], catenin delta 1 [Ctnnd1], junction plakoglobin [Jup]) during the stages of RT2 tumorigenesis - normal, hyperplastic, angiogenic, and tumor stage. Notably, in this analysis, whole ungraded RT2 PNETs were analyzed without knowledge of their invasiveness in contrast to the analysis presented in Table 1, which involved microdissected tissue from either widely invasive IC2 tumors or from non-invasive IT tumors. (B) Same as A except for desmosomal components (desmocollin 2 [Dsc2], desmoglein 2 [Dsg2], desmoplakin [Dsp], plakophilin 2 [Pkp2]). (C) Same as A except for the insulin-like growth factor 2 (Igf2), a gene whose expression is known to increase at the mRNA level during the later stages of RT2 tumorigenesis. (D) Real-time quantitative PCR values for Cdh1, Dsg2, and Dsp in pools of normal human pancreatic islets and individual human pancreatic neuroendocrine tumors (PNET). PNETs include ungraded primary and metastatic insulinomas, glucagonomas, and non-functional neuroendocrine tumors. Values are shown as the percent expression of the housekeeping genes ribosomal protein L19 (L19) (A–C) or glucuronidase beta (Gusb) (D).
Figure S2.
Desmoglein 2 expression in RT2 PNETs. Expression of desmoglein 2 (Dsg2) is strongly reduced in the IC2 but not the IT grade of PNET in RT2 mice. (A–C) H&E staining of a normal islet from a wild-type B6 mouse and an IT and IC2 lesion from an end-stage RT2 mouse. Dashed lines demarcate tumor margins. (D–F) Immunofluorescence staining with DAPI to reveal cellularity. (G–I) Immunofluorescence staining for Cdh1. (J–L) Immunofluorescence staining for Dsg2. (M–O) Merge of Cdh1 and Dsg2 immunofluorescence staining (G–L). (P–R) Higher magnification of the boxed regions in M–O. Scale bars represent 200 µm (A–O) and 100 µm (P–R).
Figure S3.
Catenin beta 1 expression in RT2 PNETs. Expression of catenin beta 1 (Ctnnb1) is maintained in both the IT and IC2 grades of PNET in RT2 mice. (A–C) Immunofluorescence staining with DAPI to reveal cellularity of a normal islet from a wild-type B6 mouse and an IT and IC2 tumor from an end-stage RT2 mouse. (D–F) Immunofluorescence staining for Cdh1. (G–I) Immunofluorescence staining for Ctnnb1. (J–L) Merge of Cdh1 and Ctnnb1 immunofluorescence staining (D–I). (M–O) Higher magnification of the boxed regions in J–L. Scale bars represent 200 µm (A–L) and 100 µm (M–O).
Figure S4.
Genetic deletion of desmoplakin does not affect cadherin 1 expression in the pancreatic islets. Expression of Cdh1 is maintained following conditional genetic deletion of Dsp in the pancreatic islets of control mice lacking the RT2 oncogenic transgene. (A–C) H&E staining of pancreatic islets in Pdx1-CreER+; DspWT/WT, Pdx1-CreER+; DspFlox/WT, and Pdx1-CreER+; DspFlox/Flox mice at 14 weeks. Cre activity was induced at 10 weeks. (D–F) Immunofluorescence staining with DAPI to reveal cellularity. (G–I) Immunofluorescence staining for Cdh1. (J–L) Immunofluorescence staining for Dsp. (M–O) Merge of Cdh1 and Dsp immunofluorescence staining (G–L). Scale bars represent 100 µm (A–O).
Figure S5.
Genetic deletion of desmoplakin leads to decreased desmoglein 2 expression but not insulin expression in the pancreatic islets. Expression of Dsg2 but not insulin (Ins) is strongly reduced in the adult pancreatic islets following conditional genetic deletion of Dsp in mice lacking the RT2 oncogenic transgene. (A–C) Immunofluorescence staining with DAPI to reveal cellularity in pancreatic islets in Pdx1-CreER+; DspWT/WT, Pdx1-CreER+; DspFlox/WT, and Pdx1-CreER+; DspFlox/Flox mice at 14 weeks. Cre activity was induced at 10 weeks. (D–F) Immunofluorescence staining for Ins. (G–I) Immunofluorescence staining for Dsg2. (J–L) Merge of Ins and Dsg2 immunofluorescence staining (D–I). Scale bars represent 100 µm (A–L).
Figure S6.
Genetic deletion of desmoplakin in the pancreatic islets does not affect multiple physiological parameters. Conditional genetic deletion of Dsp has no effect on the physiological parameters of body mass and islet function in regulating glucose levels. (A) Body mass of Pdx1-CreER+; DspWT/WT, Pdx1-CreER+; DspFlox/WT, Pdx1-CreER+; DspFlox/Flox, RT2+; Pdx1-CreER+; DspWT/WT, RT2+; Pdx1-CreER+; DspFlox/WT, and RT2+; Pdx1-CreER+; DspFlox/Flox mice at 14 weeks. Cre activity was induced at 10 weeks. Groups are not statistically different. (B) Fasting glucose levels in Pdx1-CreER+; DspWT/WT, Pdx1-CreER+; DspFlox/WT, and Pdx1-CreER+; DspFlox/Flox mice. Cre activity was induced at 10 weeks. Mice were fasted for 14–16 hours. Glucose levels were measured immediately prior to the first tamoxifen dose and one week following the last tamoxifen dose. Pre- and post-tamoxifen glucose levels within and between groups are not statistically different.
Figure S7.
Tamoxifen does not affect the parameters of RT2 tumorigenesis. Tamoxifen does not affect PNET tumorigenesis in unmodified RT2 transgenic mice. Cohorts of male RT2 mice that were DspWT/WT and that lacked the Pdx1-CreER allele were treated with five consecutive daily doses of tamoxifen or vehicle at 10 weeks of age and sacrificed 4 weeks later. (A–C) Tumor burden, tumor number, and body mass at time of sacrifice for RT2 mice treated with tamoxifen or vehicle. Data shown are mean plus standard error. Groups are not statistically different for these metrics. (D) Quantification of tumor invasiveness represented as the percentage of IT lesions or total IC lesions (IC1+IC2) in RT2 mice treated with tamoxifen or vehicle. A minimum of 76 tumors per group was graded. Groups are not statistically different. (E) Same as D except IC lesions are separated into the IC1 and IC2 subclasses. Groups are not statistically different.
Figure S8.
Genetic deletion of desmoplakin does not affect junction plakoglobin expression in RT2 PNETs. Junction plakoglobin (Jup, also known as gamma catenin) expression is maintained in the IC1 grade of tumors in both RT2+; Pdx1-CreER+; DspWT/WT and RT2+; Pdx1-CreER+; DspFlox/Flox mice. (A–D) Immunofluorescence staining for DAPI to reveal cellularity, Cdh1, Jup, and merge of Cdh1 and Jup staining in an IT PNET from a RT2+; Pdx1-CreER+; DspWT/WT mouse. (E–H) Same as A–D except for an IC1 PNET from a RT2+; Pdx1-CreER+; DspWT/WT mouse. (I–L) Same as A–D except for an IT PNET from a RT2+; Pdx1-CreER+; DspFlox/Flox mouse. (M–P) Same as A–D except for an IC1 PNET from a RT2+; Pdx1-CreER+; DspFlox/Flox mouse. (Q–T) Same as A–D except for an IC2 PNET from an unmanipulated RT2+ mouse. Arrowheads indicate regions of tumor invasion. Scale bars represent 200 µm.
Figure S9.
Genetic deletion of desmoplakin does not affect cadherin 2 expression in RT2 PNETs. Cadherin 2 (Cdh2, also known as N-cadherin) expression is maintained in the IC1 grade of tumors in both RT2+; Pdx1-CreER+; DspWT/WT and RT2+; Pdx1-CreER+; DspFlox/Flox mice. (A–D) Immunofluorescence staining for DAPI to reveal cellularity, Cdh1, Cdh2, and merge of Cdh1 and Cdh2 staining in an IT PNET from a RT2+; Pdx1-CreER+; DspWT/WT mouse. (E–H) Same as A–D except for an IC1 PNET from a RT2+; Pdx1-CreER+; DspWT/WT mouse. (I–L) Same as A-D except for an IT PNET from a RT2+; Pdx1-CreER+; DspFlox/Flox mouse. (M–P) Same as A–D except for an IC1 PNET from a RT2+; Pdx1-CreER+; DspFlox/Flox mouse. (Q–T) Same as A-D except for an IC2 PNET from an unmanipulated RT2+ mouse. Arrowheads indicate regions of tumor invasion. Scale bars represent 200 µm.
Table S1.
Genotyping of Pups Resulting from Intercross between RIP1-Tag2+; DspFlox/WT and Pdx1-CreER+; DspFlox/WT Mice.
Table S2.
Gender Distribution of Pups Resulting from Intercross between RIP1-Tag2+; DspFlox/WT and Pdx1-CreER+; DspFlox/WT Mice.
Table S3.
Bioinformatic Assessment of Desmosomal Gene Expression in Human Cancers.
Table S4.
List of primers used for quantitative PCR.
We thank: Elaine Fuchs (Howard Hughes Medical Institute, Rockefeller University, New York, NY) and Douglas Melton (Howard Hughes Medical Institute, Harvard University, Boston, MA) for the DspFlox mice and the Pdx1-CreER mice respectively; Eric Nakakura (UCSF, San Francisco, CA) for the human PNET RNA samples; The J. David Gladstone Institute Genomics Core (Chris Barker, director) for assistance with the microarrays; the UCSF Helen Diller Family Comprehensive Cancer Center Genomics Core; the UCSF Diabetes Center Microscopy and Islet Isolation Cores; Susan Cacacho, Ehud Drori, Marina Vayner, and Annie Wang for superior technical support; Johanna Joyce for critically reviewing the manuscript; and Matthias Hebrok, Martin McMahon, and members of the Hanahan laboratory for advice and encouragement at all stages of this project.
Author Contributions
Conceived and designed the experiments: MGHC DH. Performed the experiments: MGHC. Analyzed the data: MGHC DH. Wrote the paper: MGHC DH.
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Cinema Guild announced the acquisition of all U.S. distribution rights to Lisandro Alonso's new film "Jauja," which was produced by and stars Viggo Mortensen.
READ MORE: Cannes: International Federation of Film Critics Announce Their Winning Selections
"Jauja" premiered in Un Certain Regard at the 2014 Cannes Film Festival, where it won the FIPRESCI Prize. It also screens next month at both the Toronto and New York Film Festivals.
"Lisandro Alonso has created a work of immense beauty and power that will be studied and talked about and adored for years to come," said Cinema Guild's Ryan Krivoshey. "It's a film that needs to be seen by as wide an audience as possible and that's exactly what we intend to do."
From the film's synopsis, courtesy of Cinema Guild: "A remote military outpost in Patagonia, 1882, during the so-called 'Conquest of the Desert,' a genocidal campaign against the aboriginal population of the region. Acts of savagery abound on all sides. Captain Gunnar Dinesen has come from Denmark with his fifteen year-old daughter to take an engineering job with the Argentine army. Being the only female in the area, Ingeborg creates quite a stir among the men. She falls in love with a young soldier, and one night they run away together. When he wakes up the Captain realizes what has happened and decides to venture into enemy territory to find the young couple. 'Jauja' is the story of a man's desperate search for his daughter, a solitary quest that takes us to a place beyond time, where the past vanishes and the future has no meaning."
No release date for "Jauja" has yet been set.

subset: commoncrawl


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[Note: Over a year and a half after this article, kids are still having wild fun on our trampoline with zero serious injuries. See this article for more.]
We just installed a trampoline at our house, and it's been a great success. Every day, at least one group of neighborhood kids visits to play with my kids on it. One day, we had four different groups come to play on it, and it's the middle of the winter! I think the trampoline has finally enabled us to reach a goal I've been aiming at for the last year: to make my yard into a neighborhood hangout for kids.
In short, a trampoline is the best kid-magnet feature you can install in your yard. I should know – besides a trampoline, I've installed a huge playhouse, a swing set, a huge whiteboard, a sandbox, a fountain, and a basketball hoop in my yard.
In this article, I've compiled a few lessons I've learned for purchasing, installing, and managing a trampoline:Where to Buy?
There are a lot of choices of companies to purchase a trampoline from, and unfortunately, there are no obvious brand names. Companies that I found that seem good (I didn't buy from these, but I spent time on their web sites and called them) are: JumpSport and FunSpot. These are conventional trampolines. JumpSport emphasizes its innovative spring technology, for getting safe and high bounces, while FunSpot sells heavier duty trampolines that, one would guess, last longer and support more weight.
Also, I looked into the SpringFree trampoline, which can be purchased online or at your local Costco store. I have read great reviews of this product, which is touted for its very safe design, but I believe that this added safety may come at the expense of strength and durability.
I decided to choose a firm not in this list because I chose an in-ground design – see below.
Rectangular or Octagonal?
From these trampoline companies' web sites, it seems that octagonal trampolines are better sellers than rectangular ones. I don't understand why this is the case. I chose a rectangular design because a rectangle fits in the layout of my back yard better than an octagon. If you think about it, all spaces in your yard are rectangles because your yard and your house are both rectangles. When you try to fit an octagon into a rectangular space, you waste space in each of the rectangular corners.
Above Ground or In-Ground?
The vast majority of families install trampolines with the frame resting on the ground, so that the trampoline surface is about three feet above the ground.
I decided to install our trampoline in-ground, so that the surface is at ground level. It was a tremendously consequential decision, and I would recommend that everyone who can pay the additional cost of installing an in-ground trampoline to do so. Here are its advantages:
- safety: The American Academy of Orthopedic Surgeons published a list of recommendations for trampolines, and one of these is that they be installed in-ground. They are safer than above ground trampolines that are surrounded by nets for two reasons: first, they have no hard poles to fall into, and second, nets get old and tear, especially if they're exposed to harsh weather.
- accessibility for kids: Any kid who can walk can walk on to our trampoline. Getting on an above ground trampoline requires climbing a shaky ladder. This enables my two-year-old to get on our trampoline a lot more.
- spontaneity: Anyone who's in our back yard can step on to our trampoline instantly. Kids can play games that spill over from our back yard lawn to the trampoline seamlessly.
- easy for adults to help kids: Adults don't have to climb up a shaky ladder and squeeze through a narrow net opening to help kids on a trampoline. Thus, it's a lot easier to help young kids who are just getting used to a trampoline.
- good social atmosphere: Kids and adults who aren't jumping on the trampoline can sit around it and talk to those who are on it jumping. With an above-ground trampoline, there is nowhere comfortable for people to sit who aren't on the trampoline jumping.
The downside to an in-ground trampoline is cost. In order to install one, you need to have a hole dug about three feet deep, and you need to install some sort of retaining wall all around the edge of the hole to keep it sturdy. I had a landscape contractor do this for substantially more money than the cost of the trampoline (about $2,000 delivered).
Also, if you decide to go this route, I suggest that you buy a trampoline design that is customized for in-ground installation, rather than buying an above-ground trampoline and lowering it into the hole. I bought the in-ground design from WeBounce and I'm very happy with it.
Enhance the Area Around the Trampoline
In order to be a hangout for kids and their parents, a trampoline needs to be surrounded by comfortable seating. As I mentioned above, the seating will enable social interaction between jumpers and non-jumpers if the trampoline is in-ground. I have beanbag couches lining two sides of our in-ground trampoline. Even if the trampoline is above-ground, seating will provide comfort for those who aren't jumping. For instance, jumpers need to take a rest once in a while, and parents who don't jump with their kids at all may want a comfortable place to sit.
The other important thing to consider about the area around the trampoline is that it is where jumpers fall if they fall beyond the outer perimeter of the trampoline. Since in-ground trampolines typically have no surrounding net, in that case the area around the trampoline must be entirely soft to cushion a fall. My in-ground trampoline is surrounded on two sides by thick pads and beanbag couches, on one side by grass, and on another by soft playground wood mulch.
Start Them Young
My 5-1/2 and 2, and they can't jump very high on the trampoline. That makes it very unlikely that they'll get hurt on it right now. So, they'll get a lot of experience on it before they're old enough and strong enough to do any real damage to themselves.


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JACKSON, Miss. (AP) -- Julysses Nobles scored 20 points - including a 3-pointer at the final buzzer - to lift Jackson State over Alabama State 70-68 Monday night.
The lead changed four times in the last 1:11.
Jackson State (5-9, 1-1 Southwestern Athletic) was up by seven with 1:32 on the clock. But Alabama State got back-to-back 3s from DeMarcus Robinson and Bobby Brown to draw within one with 1:11 left. Jamal Waters hit a pair of free throws to give ASU a 1-point lead with 33 seconds remaining, but Brandon West's layup with 16 seconds left put JSU back up one.
Waters' jump shot with just 3 seconds left seemed to put Alabama State (7-6, 1-1) in a good position to win - until Nobles' heroics.
Derell Taylor had 11 points for Jackson State while West added 10 points and 10 rebounds.
Robinson finished with 27 points and Brandon Graham added 11.


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The District Court of Appeals ruled that Universal Marketing Research was an illegal lottery, and that the promissory note should be nullified. Universal Marketing sold Childress a home central vacuum system for a promissory note and the names of 16 friends who might also be interested in the system. For each referral that purchased a vacuum system and also supplied the names of 16 friends, the Childress received a credit of $50 toward their vacuum system. They would also receive credit for those referrals of referrals and so on that purchased systems. Childress defaulted on the note, and the assignee sued for its value. Childress claimed that the note was void because it was issued to participate in an illegal lottery, activity that the assignee knew about when it took assignment of the note. Both sides disagreed if the Universal MLM program met all the common law terms of a lottery, consideration, chance and prize; or if the program was a legitimate referral sales program. The court held that a determination of the common law elements was unnecessary because the legislature had declared that pyramid clubs were lotteries as a matter of law. The structure of the compensation plan used by Universal was the same as a pyramid club. The court held that it was an illegal lottery as a matter of law and that the promissory note could not be enforced against Childress.
The case and case summary can also be viewed online at: http://www.mlmlegal.com/legal-cases/Lippincott_v_Childress.php
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Reference Info:State, 11th Circuit, Florida | United States
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I used to worry that my daughter was damaging her fragile ear drums with a volume set to "too loud." Thankfully, we now have LoudEnough Volume-Limiting Earphones so when I yell DO YOU HEAR ME? I know that if I don't get an answer it's only because she's ignoring me.
These kid-size earphones use some fancy high-tech function to keep the volume on mp3 players and portable
gaming devices at a safer level. I don't
know how they do it, but given that these people have worked on the sound equipment
for The Jonas Brothers and Mariah Carey, I take it that they know a thing or two about volume
and sound.
I too know something about volume and sound, namely that I have had too much for too many years. I slip these earphones on, figuring that the 6+ age recommendation must include people as old as me. With soft, removable tips in various sizes, turns out there is one that fits me just fine. Can I ignore my kids now? –Christina
Congratulations to LoudEnough earphones winner, Jeanne M!


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Wigtownshire wind farm rejected
- 28 September 2011
- From the section South Scotland
Councillors have rejected plans for a wind farm in Wigtownshire due to concerns over its "significant adverse impact" on the landscape.
Developer RES wants to construct 11 turbines at Glenchamber between New Luce, Kirkcowan and Glenluce.
A planning committee meeting of Dumfries and Galloway Council decided to refuse the scheme.
A company spokesman said it would review the decision before "considering the next step".


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Dillagi: AVOID YAAR
So do read what she has to say - and then banish this snoozefest from taking up any real estate in your brain. Unless you want my DVD of it (free to the first taker!), in which case, I warned you.
You know, it was the ANTITHESIS of the filmi Curse of the Second Half. It suffered from the Curse of the First Three Quarters, but the last quarter was okay...once Hema stopped sucking on lemons (and got offscreen), and Garam Dharam got to show off his comedy chops.
I'm about to say that same thing about Thoda Pyaar Thoda Magic! :)
You guys! *stomps foot*
I really liked Dillagi (cutesy harmless romcom, with emphasis on rom rather than com!) and I *love* Thoda Pyaar..
*crawls back to her hole*
Hi Beth....dont mind...but I dont know in what state of mind you watched this one...its surely not that bad...I like your views but this one...it sounds...you were writing with full rage...I advise you watch it one more time...maybe you would like to update your post afterwards...
Hmmmm, I'll probably avoid this unless veracious does a really convincing post on why I should give it a chance, but I'm definitely trying TPTM.
veracious - Really? I can't quite call this harmless because I felt like it wasted some cute potential. I had thought it would be sort of calm but full of joys and laughs like the Hrishikesh Mukherjee movies I've seen and was really let down. Then again, I also chose this because of the year and who was in it, so it's not like I made an informed decision about the director or having heard interesting discussion of it. Serves me right.
Sanjay - I don't mind! :) To each their own. I don't know, I was really unengaged with this one, so it would take a lot for me to try it again anytime soon.
Ajnabi - Wise choice ;)
I really liked this one. It has a very under-stated humour, like in most of other Basu chatterjee films..
Surprising you didnt like it. But i guess as u said, Different people.. different perceptions...
Jeebus, how many fricking Dillagis are there anyway? I thought for a second that you meant the Sunny Deol directorial debut thingy which was horrendous and fully deserving of the Golden Teat Award (for movies that like to suck, obvs) but I see you meant the Deols senior.
Harsh - That's somehow the vibe I had from it when I bought it (not knowing anything about it), but it just didn't work for me. Yawn!
Amrita - You have spared me ever watching such a thing! Why do people think they can direct?


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HTC Looking to Buy More Patents to Bolster Defenses
Oct 21, 2011, 8:51 AM by Eric M. Zeman @phonescooper
HTC is highly interested in increasing its patent portfolio, chairman Cher Wang told the Wall Street Journal. The company believes it can adequately defend itself against patent-related litigation, but admits that owning more intellectual property would be helpful. "There's so many innovations out there in Silicon Valley and different places, and that's the exuberance of the mobile experience, I would love to buy many more small companies," she said. Though she admitted that "sometimes we get in and the price goes really high," forcing the company out of some patent auctions. Patent-related litigation is rife in the smartphone industry, with nearly all the players involved in one degree or another. As recently as July, HTC made a patent-protection purchase when it bought S3 for $300 million. Wang also indicated that HTC is taking a wait-and-see approach with respect to creating its own operating system to better compete with Samsung, Apple, and Motorola. "We have a very strong team in OS...and I think that it's not difficult for us to have our own OS, but the most important thing is how do you cooperate with the partners," she said to the Journal. HTC uses Google's Android platform, Microsoft's Windows Phone platform, and Qualcomm's BREW MP in its hardware designs.
more info at Wall Street Journal »
No messages


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Berg, 23, struck out 10 batters and walked 11 in 28 1/3 innings, going six innings or more in three of his five starts this season. Opponents batted .264 against Berg.
"His record is not indicative of how he's pitched here," Tennessee pitching coach Dennis Lewallyn said of Berg. "He's throwing the ball well and he has matured a lot as a person."
Lewallyn outlined some of the improvements that Berg has made this season as opposed to a year ago in Berg's first stint above Single A ball.
"One of the things that he was really bad at was holding runners," Lewallyn said. "He's much better at that now and is quicker to the plate with runners on base.
"His focus is better this year," added Lewallyn. "He can get people out in the big leagues if he throws the ball over the plate. He just needs to be consistent."
Berg made 26 starts a season ago at Double-A, going 7-7 with a 4.95 ERA in 140 innings. He walked 69 batters and struck out 69. The Cubs acquired Berg from the New York Yankees in August, 2005, for OF Matt Lawton.
"We've been very happy with him," said Cubs Assistant to the General Manager Gary Hughes. "All he needs is (more) command and to keep making positive steps."
To coincide with the move, Russell was promoted to Tennessee from Class High-A Daytona. In five starts with Daytona, he was 1-1 with a 2.30 ERA in 27 1/3 innings. The southpaw struck out 14 batters and walked seven.
Russell, 22, was the Cubs' 14th-round selection in the 2007 draft.
No other roster moves are known at this time.
InsideTheIvy.com staff writer Maria M. Cornelius contributed to this report.


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Something mindblowing - Dark/Serious/Memorable Anime wanted! :)
Hey Hey,
I'm looking to find some more anime to watch together with my boyfriend. We both haven't watched much in the last years and we are trying to get back into watching some quality shows. It doesn't really have to be a recent show. We're just after something mindblowing & memorable. :) Would be nice to finally find an anime again where we have to keep watching episode after episode cause it's so damn good! Here is a list of stuff we'd like to be included: - interesting setting - memorable characters - serious theme - dark enviroment - violent - psychological drama - an ongoing storyline. (kinda find it hard to get into shows that are episodic) - decent animation quality (would be a nice bonus) Here is some Anime we really enjoyed: - Berserk - Claymore - Monster - Death Note - Gantz Other Stuff we watched and liked / found ok: Black Lagoon, Elfen Lied, Basilisk, Evangelion, Chrono Crusade, Last Exile, Full Metal Alchemist, Saikano, Gantz, Hellsing, Witch Hunter Robin, GunGrave, Texhnolyze, Mononoke, Clannad & Clannad Afterstory, Eden of the East, Shigurui, Trust & Betrayal... Recently Finished: - Guilty Crown (liked, although a bit confusing at points and the ending was a bit too simplistic) Currently Watching: - Phantom - Requiem for the Phantom So ya ^^ suggestions please! Thank you. :) |
Psycho Pass
Madoka Nanoha Berserk Higurashi... Fate/Zero |
Guilty Crown ~ It'll blow your mind.
Baccano! - action with great style of story telling.
Code Geass - just like Death Note, it has an anti MC. Fate/Zero - great action series with memorable cast and dark story. Ga-Rei: Zero - dark setting, psychological, memorable main heroine... Series start from the end, then go into flashback and lead us to the start of the show. Aggressive at first, comedy later and then aggressive again. Slightly reminds to Teresa's arc in Claymore. Ghost Hunt - great series about ghost hunting. Knows how to build the atmosphere. Series are more arc/case based though but they all are connected to each other. My fave arc is Bloodshed Labyrinth, probably the darkest of them all while second arc is the creepiest. Mahou Shoujo Madoka★Magica - despite looking all cute, series are dark and cover some psychological topics. #at least give first 3 episodes a try. Phantom: Requiem for the Phantom - MC is given a choice: get killed or kill and survive. He chooses to become a professional assassin. Series are not scared to cover some dark and important topics. Rainbow: Nisha Rokubou no Shichinin - great series about friends who meet in prison. Series follow their life while in prison and after. Covers variety of topics like bullying, rape, abuse, lost dreams, lost love and etc. Shiki - villagers suddenly start to die and epidemic is suspected, but is it really the case? Steins;Gate - shows how tiny changes in the past can change everything. Gets better in second half. Not my fave but you could also try Another, especially if you like Final Destination movies. |
Psycho Pass Madoka Fate/Zero Steins;Gate Code Geass Fate/Zero Suggesting: Shoujo Kakumei Utena Mawaru Penguindrum |
I'll start with a couple of Asian imperial epics, the classic Junni Kokki or Twelve Kingdoms, and Seirei no Moribito. The first is bloodier than the second, but the sporadic fighting scenes in Moribito are remarkably well-animated. Both tell serious stories, though they have a good dose of the supernatural in each case.
Next i'm going to suggest a show that will make you wonder whether I read your initial posting. I did, but I'm going to suggest you give Hikaru no Go a try anyway. All the battles in this show take place across a Go board, but that doesn't mean they aren't exciting or dramatic. If you find you like Hikaru, I'll suggest the recent Chihayafuru and the somewhat older Shion no Ou. The latter has a parallel mystery concerning the murder of young Shion's parents. The reason I suggested Hikaru is its very effective use of cliff-hangers. It's a hard show to stop watching. Another show that might fit your interests is Ghost Hound. The main character is a thirteen-year-old boy who is suffering from PTSD. He was kidnapped with his sister as a young boy and watched her die next to him. He connects up with a couple of other boys who also have some issues. They all discover they have a knack for "astral projection" which they use in an attempt to discover the identity of the kidnapper. Psychiatry and neuroscience meet mysticism in this show. You might also enjoy the recent Lupin III/Mine Fujiko remake. It's quite a dark tale though you may want to consider whether you want your boyfriend watching a show with a seductive female lead who appears nude or nearly nude in every episode. Mouryou no Hako is a fascinating show in many respects though it's a bit slow going in the middle episodes. It's a bit hard to find out there since it was subbed piecemeal by a variety of groups. Finally I'll suggest Welcome to the NHK!. There isn't any bloodshed or even much in the way of violence in NHK, but it's got characters with psychological issues by the boatload. It starts off hilariously enough, but soon you discover there is a much deeper and darker story behind the laughs. The streams at Funimation and Hulu are both dubbed, but there are fansubs floating around in the ether. |
Maybe you may like these Anime!
~Kekkaishi ~Nura Rise of the Yokai Clan ~Yuu Yuu Hakusho ~Flame of Recca ~Rurouni Kenshin ~Vampire Hunter D |
Wow thanks alot for all the suggestions :) I really appreciate it.
There are lots of interesting shows there. We'll work our way through all of those and keep you updated. |
- interesting setting: might not seem interesting at first since the setting is just a rural village but the anime does a very good job of making the most out of it - memorable characters: with so many moraly ambiguous characters I think you'll find some of them quite interesting - serious theme: quite serious,the anime spawned lots of debates about the characters' action,there is no clear good and evil here. - dark enviroment: i'd say the ost sets the tone - violent: the 2nd half of this show is a bloodbath with some quite graphic deaths - psychological drama: again,lots of morally ambiguous characters. - an ongoing storyline. (kinda find it hard to get into shows that are episodic): the first episode is really slow paced but it really starts to get going afterwards with plenty of cliffhangers - decent animation quality: it's just decent,nothing amazing, the chara designs are a bit weird by they contribute to the athmosphere of the show,here's the OP Be sure to watch the OVA's as well they help give closure to some of the characters' stories Madoka Magica : Quote:
- interesting setting: it might not seem so at first but the staff really tried to make everything about the "witches" the girls have to fight unique and interesting,can't really be described with words - memorable characters: If anything, the magical creature Kyube has become one of the most iconic characters in recent history - serious theme: Is it really a good idea to ask 14 year old girls to fight deadly monsters? - dark enviroment: all promotional material for this was misleading on purpose,even the character designer wasn't told how dark this would be when she started working on it , the ED's the only thing that hints at the true nature of the show - violent: well,not very gory but there is violence - psychological drama : all the girls here are going to go through psychological breakdowns - an ongoing storyline. (kinda find it hard to get into shows that are episodic): it really starts off on episode 3 after the first couple serve as an introduction,after that it's a roller coaster ride - decent animation quality: again nothing amazing but they went out and experimented with plenty of different styles. |
Based on your favorites I think Gen Urobuchi's Fate Zero and the unfinished Psycho Pass will be right up your alley.
Madoka Magica
Mirai Nikki Shiki Those would be my suggestions |
I totally recommend Madoka Magica and Psycho-Pass : both are created by the creative mastermind of horrors named Gen Urobuchi, and they're both good.
Shiki, Rurouni Kenshin (you mentioned the OVA, have you not watched the TV series itself?), Yuu Yuu Hakusho (it's not really a 100 % serious/mature work per se but hey you liked Claymore and Death Note), Higurashi, Ga-Rei: Zero, Baccano pretty strongly. Madoka, Fate|Zero, Psycho-pass, Code Geass fit your taste and are very good but I hold the opinion that people rate them too highly. Utena and Penguindrum are good shows but not exactly for everyone and you will miss a lot of stuff if you don't get all the references and/or symbolism. And even if you get everything, it's still a bit nonsensical anyway. Flame of Recca, Nurarihyon, Steins;Gate, Mirai Nikki (crazy, seriously crazy girl warning) etc. are fun shows too. And I will add these recommendations: Now and then, Here and there is a classic. It's serious, dark, mature, tells a good story and has memorable characters. Among the best that anime as a medium has to offer. Haibane Renmei is everything that a serious anime should be. Casshern Sins may not be for everyone but it's dark, serious and memorable as it gets alright. Jinrui wa Suitai Shimashita is a recent show that's all about dark comedy and satire of the human race as a whole. You may want to keep this for after you are more familiar with the Japanese "anime and manga" / "otaku" culture though as some of the best episodes in there are heavy parodies of the said culture. Level E has very memorable characters and is fun enough that you will want to watch it in a go. It's a comedy but it definitely doesn't lack the dark elements. Mushishi is memorable, serious and has interesting characters. You will get your share of the dark and the happy sides of life. Kuchu Buranko (Trapeze) has a quirky psychiatrist as the lead. It is rather unique due to its artstyle and has an actual human nurse with emphasized cleavage as a character (letting you know because you will be watching this with your bf hehe) but definitely worth checking out. Un-Go has interesting characters and a dark, memorable setting. It may be a tad bit uncomfortable to watch for you guys though. And nudity warning for this one as well. I can keep going but these should keep you more than busy for a while :heh: |
Thanks again! :)
So much stuff to keep us going. Thank you, all! Just to keep you guys updated, we watched some episodes of Guilty Crown. We watched some of the openings of the Animes suggested in this thread and we both found it quite interesting based on that. Anyway, so far we haven't been disappointed with the show and we're enjoying it. ^^ |
Well, in that case, you ought to go for Code Geass next. It's right up your alley. I count stuff like Death Note, Code Geass and Guilty Crown to be superficially deep and pretentious myself, and in an increasing order (meaning Guilty Crown is very superficial and pretentious compared to Death Note but that's not saying much because Death Note itself is somewhat like that, especially the anime version) but that's just me. But yeah, if you liked Death Note and also found Guilty Crown interesting, the best you can go for is Code Geass. It's a very fun (read: fabulous!) ride for sure, just not exactly what I would consider serious and memorable, especially in the face of other titles mentioned in the thread. Matter of taste though and based off your history, it fits yours perfectly.
Also, you may see a repetition of themes and very visually similar images and stuff as you go through these shows. It's because anime world is not that large and make references to previous works very very often. It would be awesome if you went through these works in terms of broadcast date so that you would get the references but that's just too tedious for little benefit I guess. Just don't be surprised when you see essentially the same stuff in different shows :heh: |
I suppose what I like about Guilty Crown (i only watched 4 episodes so far) is that it's catchy and very easy to get into while some other shows just take a while to kick in... I don't deny that it seems a bit shallow / pretentious at times, tho. :) Maybe it's just what we need to get back into Anime atm, before we can move on to more serious / hard to stomach stuff. ^^
I actually noticed the repetition of themes you mentioned. There were moments in the show that just made me think, "hey, this is just typically anime. Haven't I seen exactly this already?" ^^" Now that you mention Code Geass (and since it's been mentioned before), I have to admit that I have given it a go some years ago. I think I ended up watching 2 episodes and then I dismissed it, thinking it was rather childish. I can't even say what I didn't like about it but at that time it just didn't grab me much. I suppose I should have given it more of a chance... I also feel a bit strange about Madoka being mentioned SO often. I looked at pictures, checked the opening and it just seems cute and not serious at all. ^^" Actually that style kind of turns me away from even trying the show, but at the same time I'm curious to find out what all this hype is about. :) Judging by the opening and summary, I feel that I would really enjoy Phantom - Requiem for the Phantom, btw. What I've seen appealed to me right away. Since my bf isn't interested in it (he wants to see Psycho Pass after Guilty Crown) I might end up watching that one alone. ;) |
I know exactly what you mean, this kind of shows are not my stuff either but trust our word. This show is very deceiving! As you said it looks all happy and cute. OP makes you facepalm thinking "Not another dumb show about cute girls" and etc. However, all of that are lies that are meant to make you feel secure. As someone once said "It lures you into a false sense of security and punches you in the face with depression." Series don't even show a proper ED until episode 3 where walls of security start to crumble and ED reveals real colours of the show. As I said try to watch first 3 episodes. If my words are not enough then there is no other choice but to show you this ED: Spoiler for ED:
Does it still look cute? :p Not to spoil anything but song lyrics have far more meaning than you might think at first. They pretty much sum up key things from the show. But you mostly will understand meaning behind them after you watch the show. Hope this sounded convincing :D And Phantom series are aweasome! :p |
Kitten320 already pretty much covered what needed to be,however I do want to mention the OP.You're probably blinded by all the pink and the fanservicy transformation sequence but look closer again and pay very close attention to the pink haired girl at all times,if this was a lighthearted fun show,surely the girl would be shown happy,laughing,smiling...but is that really the case? My favorite part is from 00:24 to 00:34 , the animation distracts you with a few silly illustrations when in fact you should be really be paying attention to the bottom left corner. |
The streams at Funimation and Hulu are both dubbed, but there are fansubs floating around in the ether. http://www.wengmj.com/1a4.jpg
All times are GMT -5. The time now is 01:56. |
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text:
usine à gaz
List of random indices. It is a list [ rM, r(M+1), ..., rN ] where rX is a random number from X to N
> nexts g (l,h) = unfoldr nxt (g,l,h)
> where
> nxt (_,l,h) | l >= h = Nothing
> nxt (g,l,h) = let (r,g2) = randomR (l,h) g
> glh2 = (g2, l + 1, h)
> in Just (r, glh2)
This code has been called "clever". Anytime somebody calls your code "clever", you should begin to worry. I mean just look at that thing! You can positively hear the gears turning and the doodads clicking. This code is what the French call a "gas factory", something incomprehensibly and needlessly complex.
is there no simpler way?
Stop micromanaging! Just tell the computer what you want, not what you want it to do
> nexts2 g (l,h) = map (\x -> fst $ randomR (x,h) g) [l..h-1]
No gears or doodads. Geez... I don't what it is with me and making things more complicated than they need to be.


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No, Giants are always tight against the cap because they have a bunch of talented players.
Usually the teams with a bunch of cap space are those devoid of talent.
The only player that we'll pretty much HAVE to give a new contract to next season will be Cruz b/c he's essentially playing on a UDFA contract right now.
Nicks I think still has one more year after this contract (usually 1st rounders sign for 5 years) and JPP I believe still has two years. They're both being payed like 1st rounders so they're not exactly going poor.
All Hail The New York Giants!!
Lets not get crazy here. He's not a once in a decade talent. He is a very good player but not transcendant. He hasn't broken 1200 yards recieving once (Cruz hit 1500 last year alone) in part because of his injuries. And the injuries will count at contract time. The Giants may franchise him or perhaps he gets Desean Jackson money but if he misses most of this year there is no way they break the bank for him.
Depending on how Barden and Randle progress this year,I believe will give the Giants there bargaining tool.If the two start to shine in this system I believe they will pay Cruz and maybe not low ball Nicks but not offer him what he believes he is worth because of his injury problems.This will give them leverage over Nicks,He probly will walk if he is not paid top receiver dollar,and as the past has shown most x Giants do not flourish in other systems.Although ther is some exception to the rule.
We aren't going to let Nicks go don't worry. Phillips may be lost next year. Osi will be gone. T2 was only gauranteed 1 million from us, all of the other money was incentives and bonuses based on performance. So we aren't wasting or losing any money on T2. Nicks and Cruz are our 2 biggest signing priorities for the next 2 years. No shot we let either go, Reese knows this.
Bennett is extremely happy in NY. We signed him for 1 year and so far I like what I see from him. He'll resign for a discount because of the fact NY suits his lifestyle very well. He is a perfect example of a player who's potential was exposed thanks to Eli.
I predict he will get somewhere in the ball park of V-Jax. He loves the organization. The only place I could see him jetsetting to is the Panthers because of his family. He's a father first.
We can't compare his career to Fitz because Fitz has put together spectacular seasons with garbage QB's AND never gets hurt. Injuries year in and year out do make a difference, no matter what anyone says.
Dude did you watch the playoffs last year????? Nicks is a top 5 talent and only person that had a better playoff run was Fitz he is going to asked for Fitz kind of money...and the only reason why he hasn't hit the 1200 yard mark is he was injured like two games a year.... I don't think the Giants will let him walk cause they know how critical he is to Eli he is not SS this time it works both ways qb need wr and wr needs qb...I'm think he gets a deal like 5 years 40 million maybe more.......
Cruz benefited last season from defenses focusing on Nicks. He's still producing this season without him, but just like any top receiver should. If Hakeem Nicks is on the field the defense had to account for him and it just makes Eli's reads that much easier.
Name a better receiver the Giants have had in the modern era than Hakeem Nicks. The short list might include Amani Toomer, Ike Hilliard, and Chris Calloway. Tiki Barber is the third leading receiver in Giants history and he was a running back. Nicks is more talented than any of those players and worth every dollar he asks for in my opinion.
How many receivers have the Giants miffed on? Tim Carter, Sinorice Moss, and then going back to Joe Jurevicius, Ron Dixon, Thomas Lewis, even Ike Hilliard didn't pan out to be anything more than solid. The jury is still out on Ramses Barden, Jerrel Jernigan, and Rueben Randle though none has done anything much as of yet and they could easily be added onto that list.
Toomer, Nicks, and perhaps Manningham are only players drafted by the New York Giants to actually produce to expectations.