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Secretory factors that drive cancer progression are attractive immunotherapeutic targets. We used a whole‐genome data‐mining approach on multiple cohorts of breast tumours annotated for clinical outcomes to discover such factors. We identified Serine protease inhibitor Kazal‐type 1 (SPINK1) to be associated with poor survival in estrogen receptor‐positive (ER+) cases. Immunohistochemistry showed that SPINK1 was absent in normal breast, present in early and advanced tumours, and its expression correlated with poor survival in ER+ tumours. In ER− cases, the prognostic effect did not reach statistical significance. Forced expression and/or exposure to recombinant SPINK1 induced invasiveness without affecting cell proliferation. However, down‐regulation of SPINK1 resulted in cell death. Further, SPINK1 overexpressing cells were resistant to drug‐induced apoptosis due to reduced caspase‐3 levels and high expression of Bcl2 and phospho‐Bcl2 proteins. Intriguingly, these anti‐apoptotic effects of SPINK1 were abrogated by mutations of its protease inhibition domain. Thus, SPINK1 affects multiple aggressive properties in breast cancer: survival, invasiveness and chemoresistance. Because SPINK1 effects are abrogated by neutralizing antibodies, we suggest that SPINK1 is a viable potential therapeutic target in breast cancer.
The paper explained
In breast cancer treatment, resistance to prevailing drug treatments prompts a need to find novel molecular targets for antibody‐based or small molecule‐based therapeutics. Secretory factors that drive cancer progression are, therefore, attractive immunotherapeutic targets. Towards this end, we mined the expression profiles of 760 primary breast cancers from multiple patient cohorts to select genes encoding extracellular proteins, secreted growth factors and cell surface receptors that are consistently associated with poor patient survival for potential therapeutic targets. SPINK1 emerged as the most promising candidate.
Integrated analysis of microarray profiles of clinically well‐annotated breast tumours identified SPINK1 to be associated with poor survival in estrogen receptor‐positive (ER+) cases. Immunohistochemistry showed that SPINK1 was absent in normal breast, present in early and advanced tumours, and its expression correlated with poor survival in ER+ tumours. Using multiple breast cancer cells as model systems, we show that SPINK1 is essential for cell survival and its forced expression confers chemoresistance via reduction in pro‐apoptotic caspase‐3 protein and induction of the pro‐survival gene, BCL2. Intact protease inhibitor function of SPINK1 is necessary for this function. Further, SPINK1 increased invasion of breast cancer cells in vitro and in mouse tail vein assays.
Our data shows that SPINK1 significantly associates with poor prognosis in ER+ versus ER− patients. Further, high SPINK1 expression in the well‐differentiated ER+ lymph node‐negative (LN−) group is a highly significant predictor of poor outcome. On a molecular level, SPINK1 affects multiple aggressive properties in breast cancer: survival, invasiveness and chemoresistance. Because SPINK1 effects are abrogated by neutralizing antibodies, we suggest that SPINK1 is a viable potential therapeutic target in breast cancer.
In breast cancer treatment, resistance to prevailing drug treatments prompts a need to find novel molecular targets for antibody‐based or small molecule‐based therapeutics. Towards this end, we mined the expression profiles of 760 primary breast cancers from multiple patient cohorts to select genes encoding extracellular proteins, secreted growth factors and cell surface receptors that are consistently associated with poor patient survival for potential therapeutic targets. The most promising candidate was the serine protease inhibitor Kazal‐type 1 (SPINK1).
SPINK1 is a 6 kDa secreted factor and its primary function is trypsin inhibition in the pancreas and small intestines (Paju & Stenman, 2006). Multiple variants of SPINK1 have been previously shown to be a strong causal factor for chronic pancreatitis possibly due to inhibition of its trypsin inactivation function (Bagul et al, 2009; Derikx and Szmola, 2009). In addition, inactivation of SPINK1 in the pancreas results in autophagy of exocrine pancreatic cells, suggesting that SPINK1 may be important in cell survival. Interestingly, mice deficient in SPINK3, the mouse homolog of human SPINK1, die due to excessive autophagy of the pancreatic acinar cells and, similarly, autophagy has recently been demonstrated by silencing the SPINK1 homolog in Hydra. Autophagy is mediated by ATG5/12 and can be regulated by Bcl2 expression (Djavaheri‐Mergny et al, 2010). In liver cells, Hepatitis B and C viruses induced expression of SPINK1, thus protecting liver cells from serine protease dependent cellular apoptosis (SPDCA; Lamontagne et al, 2010). Taken together, SPINK1 appears to play an important role in both cell survival and prevention of apoptosis by several different pathways in normal tissues.
In cancer, SPINK1 was first discovered in the urine of ovarian cancer patients, but has since been found to be strongly associated with poor prognosis in multiple cancers including that of the prostate, ovary, bladder, pancreas and colon (Gaber et al, 2009; Gouyer et al, 2008; Kelloniemi et al, 2003; Tomlins et al, 2008). In some cell lines derived from these tissue types, SPINK1 enhanced proliferation and cell invasion. It has been suggested that SPINK1 induces cellular invasion through pathways such as phosphoinositide 3‐kinase (PI3K), Rho‐like GTPases and protein kinase C (PKC). This process appears to require its trypsin inhibitory property as a single mutation at the trypsin binding site fails to induce cellular invasion of colon cancer cells (Gouyer et al, 2008). These data suggest that SPINK1 confers some oncogenic properties to cancer cells.
Since SPINK1 expression associated with poor survival outcome in breast cancer, we comprehensively altered SPINK1 levels using siRNA‐mediated knockdown, stable overexpression and exogenous application of SPINK1 to characterize its molecular functions. We show that SPINK1 not only affects invasion, motility and cellular survival as reported previously, but, in addition, confers chemoresistance to multiple drugs by decreasing caspase‐3 levels and increasing basal levels of anti‐apoptotic factors, Bcl2 and phosphor‐Bcl2. Moreover, the protease inhibitor activity of SPINK1 is essential for such chemoresistance but unlike in prostate and colon cancer, it is dispensable for cell invasion. Further, we show that both SPINK1 RNA and protein levels are predictive of patient survival specifically in ER+ cases. Our data identifies novel and breast cancer specific mechanisms that may affect cancer progression and suggest that SPINK1 is an important therapeutic target.
SPINK1 transcript levels are positively correlated with breast cancer metastasis
To discover novel targetable oncogenes in breast cancer, we developed a whole‐genome data‐mining approach that leverages large independent microarray studies for the systematic discovery of genes correlated with distant metastasis‐free survival (DMFS) of patients. In an initial screen involving 760 tumours derived from four independent breast cancer cohorts (Loi et al, 2007; Miller et al, 2005; van de Vijver et al, 2002; Wang et al, 2005), we performed Cox proportional hazards regression on each of 44,928 gene probe sets (Affymetrix U133A and U113B Genechips) to identify candidate oncogenes by their statistically significant and reproducible associations with metastatic recurrence. The genes were then cross‐referenced with the Gene Ontology database (www.geneontology.org) to identify all metastasis‐associated genes encoding known and predicted transmembrane receptors and secreted growth factors (Table S1 of Supporting information). Of the small number of candidate genes we identified, SPINK1 was positively correlated with metastatic recurrence in all four cohorts. We next extended our meta‐analysis to all publicly available breast cancer expression array datasets (annotated for survival outcomes; Chin et al, 2006; Loi et al, 2007; Miller et al, 2005; Minn et al, 2005; Schmidt et al, 2008; van de Vijver et al, 2002; Wang et al, 2005; Zhang et al, 2009) as of 2009, and found that the expression of SPINK1 was reproducibly correlated with metastatic recurrence in 8 of 10 breast cancer studies (overall hazard ratio = 1.19; confidence interval [1.10, 1.28]; p < 1E−05; Fig 1). Therefore, SPINK1 and its clinical and biological effects were analysed further.
SPINK1 associates with poor prognosis mainly in ER+ LN− breast cancer
To study the association of SPINK1 with prognosis in specific subtypes of breast cancer classified by Perou et al (Perou et al, 2000), we extended the analyses to a 'mega cohort' of 2034 publicly available breast cancer expression profiles from 14 independent cohorts (Table S2 of Supporting information; Chin & Gray, 2008; Desmedt et al, 2007; Ivshina et al, 2006; Loi et al, 2007, 2008; Miller et al, 2005; Minn et al, 2005, 2007; Pawitan et al, 2005; Schmidt et al, 2008; Wang et al, 2005; Yau et al, 2007; Zhang et al, 2009). We found that SPINK1 expression was linearly associated with poor DMFS in ER+ patients (p < 0.001), but did not significantly correlate with the survival of the ER‐negative (ER−) group (Fig 2). However, stratification of ER− patients into deciles based on the intensity of SPINK1 expression showed that very high SPINK1 levels marginally displayed a minor trend towards poor prognosis in ER− patients (Fig S1 of Supporting information). This suggests that SPINK1 exerts its greatest differential impact on ER+ cancers, with only marginal effect on ER− breast cancers. Interestingly, the impact of SPINK1 on prognosis was highest in the less aggressive clinical subsets of breast cancer (ER+/LN−, Table 1), Further, we studied if SPINK1 was a predictor of outcome or distinguished any intrinsic subtypes within the ER+ tumours. We analysed SPINK1 expression–survival associations in LumA, LumB, Normal‐like, HER2‐enriched, No Subtype and Basal‐like groups, within ER+ breast cancer only (Table 2). By Cox analysis, in the Normal‐like ER+ tumours, SPINK1 expression was significantly correlated with DMFS (p = 0.02), while positive trends for association were observed in both LumA and 'No Subtype' ER+ cases (p = 0.10 and 0.13, respectively). In the LumB ER+ subgroup, the Cox p‐value was 0.62 which essentially argued no survival association. Together, these observations suggest that SPINK1 along with ER status was an important predictor of outcome irrespective of further sub‐classification into intrinsic subtypes. SPINK1 did not show differential expression between ER+ and ER− samples, nor was its expression biased in any intrinsic molecular subtypes of breast cancer (Fig S2 of Supporting information). This is consistent with the notion that the SPINK1 association with survival is not a function of differential expression in the different tumour subtypes.
SPINK1 expression differs in normal and breast tumour tissue
To investigate the localization of the SPINK1 protein in both benign and malignant breast tissues, we immunostained 98 clinically annotated cores on a tissue microarray (TMA) using an anti‐SPINK1 antibody. Normal pancreas served as a positive control and specificity of the staining was evaluated by pre‐neutralizing the antibody with recombinant SPINK1 protein (Fig S3 of Supporting information). The expression of SPINK1 was negligible in normal breast tissue, but most tumours were immunoreactive (95%; Fig 3, panels A–C). This suggested that SPINK1 was probably expressed early in tumour development and continued to be present in advanced disease. Of the 98 cores stained, 81 breast cancer samples were annotated for patient survival data. Analysis (see Materials and Methods Section) showed that high SPINK1 protein levels correlated with overall lower disease‐free survival. Upon sub‐division of patients into ER+ and ER− cohorts, this correlation was most significant in the subset of ER+ breast cancer cases (Fig 4), supporting our initial findings that the SPINK1 transcript displayed positive association with poor prognosis predominantly with ER+ cases. Moreover, SPINK1 expression did not associate with grade, age, ethnicity, stage and cERBB2 (Table S3 of Supporting information). Interestingly, SPINK1 subcellular localization seemed to vary across tumour tissues, with some expressing SPINK1 in the cytoplasm and others in the nuclei. To confirm that this nuclear localization is not an artefact of the immunohistochemical staining of paraffin processed tissue, we performed immunofluorescence on MDA‐MB‐231 cells treated with soluble recombinant SPINK1 bearing a 6Xhis tag. Using an antibody against the tag as well as an antibody against SPINK1, we confirmed that exogenously applied SPINK1 localized in the nucleus within 24 h of application (Fig 3D).
Loss of SPINK1 results in reduced cell survival
Although we observed significant correlation of SPINK1 to DMFS mainly in ER+ patients, we investigated the mechanistic effects of SPINK1 in both ER+ and ER− cells. We attenuated SPINK1 transcript levels by siRNA in the breast cancer cell lines MCF‐7 and MDA‐MB‐231 and conducted assays to detect phenotypic changes in cell proliferation, motility, invasiveness and anchorage‐independent growth. Each of the 3 SPINK1‐targeted siRNA constructs reduced SPINK1 levels in MCF‐7 and MDA‐MB‐231 cells by >50%, with C2 siRNA being the most efficient (>95%; Fig 5A). Consistent with a threshold effect, C2 siRNA led to the most significant reduction of proliferation for both MCF‐7 and MDA‐MB‐231 (Fig 5B), whereas, C1 and C3 siRNAs displayed marginal phenotypes. We posited that the observed decrease in proliferation could reflect either cell cycle arrest or apoptosis. Therefore, we studied the activation of apoptotic markers PARP and caspase‐3 using a cell‐based immunofluorescence assay. Attenuation of SPINK1 induced activation of both PARP and caspase‐3 in MDA‐MB‐231 (p < 0.05, Fig 5C, left panel) and MCF‐7 cells (Fig 5C, right panel). Secondly, since loss of SPINK1 leads to excessive autophagy in animal model systems, we studied if siRNA mediated knockdown of ATG5/12 was able to rescue cell death caused by loss of SPINK1. As shown in Fig S4 of Supporting information, interfering with autophagy could not rescue cell death. Taken together, the apparent decrease in cell proliferation upon loss of SPINK1 was due to the induction of cellular apoptosis. Therefore, SPINK1 appears to be essential for survival of breast cancer cells lines regardless of estrogen receptor status.
Rescue of siRNA‐mediated phenotype(s) by exogenous SPINK1
To determine the specificity of our SPINK1 siRNA constructs by a phenotypic rescue, we generated two sources of recombinant SPINK1 by over‐expressing SPINK1 in MCF‐7 and SF9 insect cells (Fig S5, panels A–C of Supporting information). Conditioned media (CM) was collected from both sources (referred to as SPINK1 CM and SF9spink1 CM), as well as from cells transfected with empty vector as controls (vector‐CM and SF9vecCM, respectively). Treatment of C2siRNA cells with SPINK1‐CM resulted in 5‐bromodeoxyuridine (BrDU) incorporation showing that these cells survived and could subsequently divide (Fig 6). The control Vec‐CM was unable to rescue this cell death. This confirms the specificity of the siRNA used as well as the functionality of the recombinant SPINK1. Interestingly, neither MCF‐7 cells over‐expressing SPINK1 (MCF‐7 SPINK1OE) nor various breast cancer cell lines treated with SPINK1‐CM showed increased proliferation (Fig S6 of Supporting information). This further supports the notion that SPINK1 is probably an important survival factor rather than a potent growth inducer in breast cancer.
SPINK1 augments drug resistance
Given the evidence that SPINK1 is a survival factor in breast cancer cell lines, we asked whether SPINK1 could increase cellular resistance to anti‐cancer drugs in either an autocrine or a paracrine fashion. First, we treated MCF‐7 SPINK1 overexpressing cells (SPINK1OE) with increasing concentrations of five commonly used chemotherapeutic drugs, 5‐fluorouracil (5‐FU), suberoylanilide hydroxamic acid (SAHA), tamoxifen (TAM), adriamycin (ADR) and etoposide (VP16). SPINK1OE cells were significantly more resistant to chemically induced apoptosis as compared to control cells, as shown by decreased PARP (left panel) and caspase‐3 (right panel) activation levels (Fig 7). Next, we pretreated MCF‐7 parental cells with exogenous SPINK1‐CM before inducing apoptosis with 5‐FU. Strikingly, 5‐FU‐mediated cell death was abrogated by pretreatment with SPINK1‐CM but not by control media (vector‐CM), however to a degree lesser than that observed in MCF‐7 SPINK1OE cells (Fig S7 of Supporting information). Therefore, increased levels of SPINK1, both by autocrine and paracrine mechanisms, were capable of conferring increased drug resistance in breast cancer cells.
SPINK1 induces resistance to apoptosis through regulation of main apoptotic players
Since SPINK1 protected cells from drug‐induced apoptosis, we studied the expression of a panel of pro‐ and anti‐apoptotic genes including caspase‐3, caspase‐8, PARP, BID, Bcl2 and phospho‐Bcl2 in MCF‐7 cells treated with SPINK1‐CM for 2 h. As shown, apoptosis‐inhibitor Bcl2 levels were upregulated without changing caspase‐3, BID, PARP and caspase‐8 levels (Fig 8A and S8 of Supporting information). Interestingly, in different clones of MCF‐7 SPINK1 OE cells, endogenous Bcl2 and phosphor‐Bcl2 increased and caspase‐3 levels decreased with increasing expression of SPINK1 (Fig 8B). The decrease in caspase‐3 was, however, not evident in cells treated with SPINK1‐CM (Fig 8A), perhaps indicating that prolonged high expression of SPINK1 may lead to higher chemoresistance. Since previous studies showed that the protease inhibitor function of SPINK1 is necessary for some oncogenic properties of SPINK1 (Lamontagne et al, 2010), we generated a SPINK1 K18Y mutant lacking this function (Gouyer et al, 2008; Fig S5D of Supporting information). As shown in Fig 9, pretreatment of cells with SPINK1‐K18Y‐CM (hatched bars) was unable to protect cells from 5 FU‐ (panel A) or SAHA‐ (panel B) induced apoptosis. Together, these data show that high SPINK1 induces chemoresistance and its protease inhibitor function is critical to mediate this effect.
SPINK1 leads to increased invasion of breast cancer cells
As part of our cellular screen for SPINK1 induced phenotypes, we had found that knockdown of SPINK1 attenuated invasion of breast cancer cell lines and that this effect could be rescued using SPINK‐CM but not by Vec‐CM (Figs S9 and 10A of Supporting information). We extended this analysis using recombinant SPINK1 and found that in two invasive breast cancer cell lines, MDA‐MB‐231 and BT549, SPINK1‐CM significantly increased invasion by 2–3 fold (Fig 10B). Non‐invasive MCF10A and MCF‐7 cells do not invade in 24 h. After 48 h, these cells show a relative increase in invasion, however, the total number of cells was extremely low (Fig S10 of Supporting information). Importantly, we found that induction of cellular invasion in MDA MB 231 and BT‐549 cells could be specifically immuno‐neutralized by anti‐SPINK1 antibodies (Fig 10C, Fig S11 of Supporting information). We then tested whether the invasive property of SPINK1 was due to its activity as a trypsin/serine protease inhibitor. We exposed K18YCM to MDA‐MB‐231 cells, and found that K18YCM retained the ability to induce cellular invasion like wild‐type (WT) SPINK1‐CM, suggesting that active protease inhibitor function is not necessary (Fig 10D). Similar effects were observed in BT549 cells (data not shown). Thus, the anti‐apoptotic and the pro‐invasive activity of SPINK1 are separable by its protease inhibitor function.
SPINK1 induces metastasis in vivo
To test if the in vitro invasiveness of MDA‐MB‐231 cells was similarly affected in vivo, we injected MDA‐MB‐231 cells over‐expressing SPINK1 (MB231 SPINK1OE) into the tail‐vein of Balb/c nu/nu mice. Figure 11 shows that SPINK1 expression led to increase in both the number and size of metastatic lesions in the mouse lung. Mice injected with MB231‐vector cells had only 4.1% ± 2.7 lesions larger than 200 µm, while MB231 SPINK1OE cells gave rise to significantly larger lesions with 24.8% ± 8.5 larger than 200 µm (p < 0.05). The MB231 SPINK1OE cells also formed almost threefold more lesions than that of the control cells (p < 0.05). In concordance with our in vitro data, MCF‐7 SPINKOE cells did not show lung metastases in vivo (data not shown). These data suggest that in breast tumour cells with prior capacity to invade, high SPINK1 expression may lead to increased metastasis as compared to the same cells expressing relatively lower amounts. However, due to unavailability of a suitable invasive model system, SPINK1 effects in ER+ cells remain unexplored.
In this study, using genome‐wide profiling of breast tumours from multiple cohorts as well as validation by cell‐based phenotypic assays, we identified for the first time that SPINK1 is a biological driver of aggressiveness in breast cancer. All breast cancer cohorts analysed display a positive association of SPINK1 expression with poor outcome. Further analysis of SPINK1 in 2034 breast tumours from 14 independent cohorts revealed that SPINK1 is significantly associated with poor prognosis only in ER+ tumours but not in ER−. In intrinsic subtype‐based analysis, SPINK1 was prognostic in Normal‐like and LumA groups. Though LumB is clinically the most relevant subtype for poor prognosis, we found no SPINK1‐survival association in this group. Careful analysis of our cohorts showed that ER status was not synonymous with LumA/LumB classification, the ER+ group contained variable numbers of all five intrinsic subtypes as well as a 'no subtype' group (i.e. <10% correlation with any subtype). Secondly, ER− tumours and those whose clinical ER status was unknown, occasionally mapped to LumA and LumB. We suggest that these factors potentially confound the impact of SPINK1 in these subtypes as it relates to clinical ER positivity. However, SPINK1‐survival association was strongest in less aggressive ER+ LN− tumours.
This prognostic association is recapitulated at the protein level: in a panel of clinically annotated breast tumours, high SPINK1 expression was consistently and significantly associated with poor prognosis of ER+ rather than ER− tumours. More detailed analysis of meta‐analytical data, however, showed a small effect by SPINK1 on prognosis of ER− patients: ER− individuals in the highest SPINK1 expressing deciles have a slightly poorer survival than the lowest expressing deciles. However, the numbers of ER− patients in the meta‐analysis were one third those of ER+ patients, thus limiting the power to discern SPINK1 effects in the ER− group. That SPINK1 may be operative in both ER+ and ER− conditions is confirmed by the enforced overexpression of SPINK1 or the addition of exogenous recombinant SPINK1 protein, which induced the same cellular effects in ER+ and ER− breast cancer cell lines. Such dramatic and non‐physiologic overexpression of SPINK1 in cell culture systems serves to exaggerate phenotypic outcomes. We suggest that any association of SPINK1 with DMFS in ER− tumours could be masked by more dominant oncogenic pathways operative in ER− tumours.
With the exception of gastric malignancies, high levels of SPINK1 expression have been associated with poor prognosis in multiple cancer types such as prostate cancer, liver cancer, ovarian cancer and bladder cancer (Gaber et al, 2009; Gkialas et al, 2008; Gouyer et al, 2008; Wiksten et al, 2005; Kelloniemi et al, 2003; Shariat et al, 2005; Tomlins et al, 2008). SPINK1 expression is high in ETS‐rearrangement negative prostate cancer and loss of SPINK1 attenuates invasiveness and tumour growth (Ateeq et al, in press; Tomlins et al, 2008). Recombinant SPINK1 has been described to have opposing effects that are cell type specific, augmenting cell proliferation in gastric and pancreatic (Marchbank et al, 2009; Ozaki et al, 2009) but not in prostate cancer cell lines (Tomlins et al, 2008). Functionally, SPINK1 serves as a motility factor in various colon, prostate and gastric cancer cell lines and this effect can be attenuated in colon cancer cell lines by PI3K, PKC and Rho GTPase inhibitors (Gouyer et al, 2008; Marchbank et al, 2009; Tomlins et al, 2008).
To determine cellular functions of SPINK1 in breast cancer, we analysed the phenotypic effects of SPINK1 attenuation, recombinant SPINK1 treatment and SPINK1 over expression. Our results strongly suggest that SPINK1 does not induce proliferation in breast cancer cells, but rather is required for survival of cancer cells. Further, SPINK1 led to increased invasion of cancer cells without affecting cell proliferation both in vitro and in in vivo tail vein metastasis assays. However, unlike prostate and colon cancer, we found that the protease inhibitor function of SPINK1 was not important for invasion in breast cancer; however, inhibitors of PI3K, PKC and Rho GTPase pathways attenuated this response (data not shown). This suggests that SPINK1 may initiate a slightly distinct molecular pathway in breast cancer cells. Such decoupling of growth from the conjoint phenotype of invasion and survival has been well documented in another cancer‐associated ligand–receptor system such as the GAS6 ligand with its cognate receptor, the tyrosine kinase AXL (McCloskey et al, 1997).
Since SPINK1 was important for cell survival, and SPINK1 expression was associated with poor patient prognosis, we studied if SPINK1 could induce chemoresistance in cancer cells. We show that recombinant SPINK1 not only enhances cell survival, but also induces resistance to apoptosis induced by common cytotoxic agents: SAHA, 5‐FU, TAM, ADR and VP16. Mechanistically, we demonstrate that the presence of SPINK1 greatly reduces caspase‐3 expression and consequent activation upon drug treatment. In addition, SPINK1 also leads to high basal expression of anti‐apoptotic Bcl2 and phospho‐Bcl2. This is compelling as Bcl2 is a strong prognostic factor in node‐negative, ER+ breast cancers that respond poorly to chemotherapy (Paik et al, 2006). Thus a molecular relationship between SPINK1, Bcl2 and chemoresistance may explain the association of SPINK1 with poor outcome in ER+ LN− patients. Whether SPINK1 expression can be utilized to predict response/benefit from chemotherapy in ER+ node negative patients needs further study.
The K18Y mutant fails to protect cells or induce anti‐apoptotic signalling in cells. Taken together, these data suggest that SPINK1 with intact protease inhibitor activity alters 'set point' of the apoptotic machinery towards one favouring survival. Conceivably, attenuation of SPINK1 expression in breast cancer may promote the efficacy of anticancer therapies (Ghobrial et al, 2005; Kasibhatla & Tseng, 2003; Sun et al, 2004). Further experiments are required to identify the downstream effector that links SPINK1 to invasion and survival and explains this differential requirement for protease modification.
Lu et al (Lu et al, 2008) suggested that SPINK1 inhibits only serine protease‐dependent apoptosis (SPDCA) owing to its protease inhibitor function but has no role to play in caspase‐dependent apoptosis (CDCA) in human hepatoma cells. In breast cancer, we demonstrate that SPINK1 is capable of interfering with caspase‐3 induced apoptosis suggesting additional mechanisms of chemoresistance.
Our work also found some intriguing new observations about SPINK1 biology. Despite SPINK1 being a secreted extracellular protein, we unexpectedly observed nuclear localization of SPINK1, not only in a time‐dependent manner in cell lines in vitro, but also in the nuclei of primary tumours by immunohistochemistry. Soluble secreted growth factors such as the epidermal growth factor and fibroblast growth factor, are transported into the nucleus and are known to mediate transcription (Planque, 2006). However, the implications of such nuclear localization, in breast cancer progression remain unclear, and will need further study.
These data suggest that though SPINK1 may not be a 'driver' oncogene in breast cancer, its high expression critically influences key processes in cancer progression including cell survival, drug response and cell invasion ultimately leading to poor clinical outcome. Its association with ER+ tumours makes SPINK1 a plausible combinatorial therapeutic target in early breast cancer. That most of these aggressive phenotypes—chemoresistance, invasion and anti‐apoptosis—can be reversed with attenuation of SPINK1 levels suggest that SPINK1 may be a reasonable therapeutic target for neutralizing antibodies.
Breast cancer cohorts
SPINK1 expression characteristics were analysed in breast cancer using an integrated 'super cohort' of 2116 breast tumours profiled using Affymetrix GeneChip technology. The super cohort is comprised of 15 distinct breast cancer cohorts for which corresponding microarray data and clinical annotation were publicly accessible from microarray databases including the Gene Expression Omnibus (National Center for Biotechnology Information, USA), ArrayExpress (European Bioinformatics Institute, UK) and caArray (National Cancer Institute, NIH, USA). General cohort details and literature references are presented in Table S1 of Supporting information. Original discovery and metaanalysis were perfomed on a subset of this 'super cohort'.
Microarray data processing and case selection
In all cases, raw data (CEL files) were pre‐processed and normalized using the R software package (R development Core Team, 2010) and library files provided via the Bioconductor project (Gentleman et al, 2004). In order to preserve a consistent normalization strategy across all cohorts, raw data were MAS5.0 normalized on a per‐cohort basis using the justMAS function in the simpleaffy library from Bioconductor (no background correction, target intensity of 600). The specific array platforms employed here were the HG‐U133A, HG‐U133plus2 and HG‐U113A2 gene chips. To ensure equal information content from each chip type, only probe sets common to all chip types were utilized in subsequent analysis. This resulted in the use of 22,268 probe sets that were common to all microarrays in all cohorts. Cross‐cohort batch effects were corrected using the COMBAT empirical Bayes method (Johnson et al, 2007). Of the initial 2116 tumour profiles, 2034 represent primary invasive breast cancers with no exposure to neoadjuvant therapy prior to array analysis. Of these, 1954 cases are annotated with DMFS time and event. Of note, other clinical annotation such as treatment type, estrogen receptor status, nodal status, tumour size, histologic grade and patient age are available for the majority of cases.
Subtype assignment
Intrinsic subtypes were assigned to samples using the Single Sample Predictor (SSP) algorithm described by Hu et al (2006) and utilized by Fan et al (2006). Affymetrix probe sets were matched to the genes comprising the SSP centroids using UniGene annotation. Prior to batch correction, the expression data for each gene were mean centered, and Spearman correlation was used to find the centroid most closely associated with each tumour sample. In cases where a correlation greater than 0.1 was not achieved with at least one centroid, a subtype was not assigned to that sample (n = 92 cases).
Immunohistochemistry and immunofluorescence
TMA blocks containing cores from 98 breast cancer patients were constructed as described previously (Das et al, 2008; Salto‐Tellez et al, 2007) under institutional ethics committee approval for the tissue array program (NUS‐IRB 05‐017). Staining was done with anti‐SPINK1 monoclonal antibody (MoBiTec: PSKAN2‐0100) at a dilution of 1:1000 for immunohistochemistry and 1:50 for immunofluorescence. Other antibodies used for immunofluorescence include anti‐His (Delta Biolabs), Alexa 488 goat anti‐mouse and Alexa 647 goat anti‐rabbit (Invitrogen) at a dilution of 1:500. The TMA slides were digitized using the Ariol SL‐50 platform (Applied Imaging Inc., San‐Jose, CA). Each slide was scanned at 20× objective with three filters: red, green and blue (RGB). The Ariol software then converted these RGB‐channel scans into colour reconstructions for image analysis. For SPINK1 expression, the Ariol analytical algorithm was trained using the positive controls. The positivity of the SPINK1 expression was initially confirmed by two senior pathologists (BP and MST). The training of the Ariol algorithm was then performed by a pathological assistant (OCW) to ensure that stromal matrix and adipose cells are excluded from the image analysis. After the initial program training, the rest of the analysis was performed without human supervision. Absolute values of expression were subsequently provided by the software for all the TMA cores. Previously, we have reported a high level of concordance (κ > 0.9) between the Ariol scoring and human observer scoring methods for immunohistochemistry in a technical journal (Ong et al, 2010). The positivity of SPINK expression was determined by rank‐transforming all the positive scores of SPINK1 expression into two groups based on a 50th percentile cutoff. Associations between SPINK1 expression and clinicopathological features were assessed using the student's t‐test. All statistical analyses were performed using the SPSS package.
Cell lines, stable overexpression and conditioned media
Human breast cell lines, BT‐549, MCF10A, MCF‐7 and MDA‐MB‐231 were obtained from American Type Culture Collection (ATCC) and maintained at 37°C with 5% CO2 with growth medium recommended by ATCC. Full‐length SPINK1 was commercially obtained (Invitrogen: IOH10627). A 6XHis Tag was added using the primers (‐F‐ 5′‐ATGAAGGTAACAGGCATCTTTCTTCTCAGTGCCTT‐3′) and R2 5′‐TCAGTGATGATGATGATGGTGGCAAGGCCCAGAT‐3′). Mutant SPINK1 K18Y was generated by site‐directed mutagenesis kit according to the manufacturer's protocol (Calbiochem: QIA58). All clones were confirmed by sequencing on both strands. PCR products were shuttled into pCR® 2.1‐TOPO® vector (Invitrogen: K4510‐20) and further subcloned in mammalian expression vector pcDNA™3.1(+) (Invitrogen: V790‐20) via restriction sites NheI and EcoRI. SPINK1 and empty control plasmids were independently transfected into MCF‐7/MDA‐MB‐231 cells using Lipofectamine 2000. Stable colonies are selected by G418 selection (Sigma). The clones are referred to MCF‐7 SPINK1OE and MB231 SPINK1OE, respectively, in this study. Insect SF9 cells were obtained from BD Biosciences and maintained in Grace's insect media (Invitrogen) supplemented with fungizone (Invitrogen) and 10% FBS. SPINK1 with a 3′ 6XHis tag and K18Y mutant SPINK1 were cloned into pPK30x vector, and infected into SF9 cells with baculovirus (BD Biosciences) according to the manufacturer's protocol (Fig S4 of Supporting information).
Immunoblot analysis
For Western blot analysis, whole cell lysates were prepared using RIPA buffer. The proteins were separated by SDS–PAGE and transferred to 0.2 µm PVDF membrane (Biorad). SPINK1 antibody (MoBiTec: PSKAN2‐0100) was used to probe the membrane at 500 times dilution, and antibody–protein complex was detected by HRP‐conjugated antibodies and ECL (Amersham Biosciences).
siRNA mediated knockdown
RNAi transfection on breast cancer cell lines was carried out using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. 3 SPINK1 specific siRNAs were used, C2siRNA (5′‐CCAAGAUAUAUGACCCUGUTT‐3′) gave the optimal knockdown of SPINK1, (C1‐5′‐CCAAGGUUUUGAAAUCCCATT‐3′; and C3‐5′‐GAGGCCAAAUGUUACAAUGTT‐3′). Knockdown efficiency of the siRNAs were checked using Polymerase Chain Reaction (after reverse transcription) (RT‐PCR) with (SPINK1‐F 5′‐CATCTGCCATATGACCCTTC‐3′; SPINK1‐R 5′‐GATGCCTGTTACCTTCATGG‐3′; SPINK1‐H 5′‐FAM‐GACGTGGTAAGTGCGGTGCAG‐BHQ1‐3′), GAPDH was internal housekeeping control. ATG 5 and ATG12 specific siRNAs were purchased from Applied Biosystems (#s18158‐s18160; s17464‐s17466).
Cell proliferation and apoptosis assays
Cells were transfected with siRNAs at 100 nM for 48 h. Cells were plated at a density of 5000 cells/well in 96‐well plates, and cell proliferation was measured at various time points in quadruplicate using WST‐1 (Roche: 11644807001) or BrDU (CalBiochem: QIA58) according to the manufacturer's protocol. p‐Values were obtained by doing a two‐tailed t‐test comparing proliferation rates between loss of gene and scrambled control. For the apoptosis PARP and caspase‐3 assay, 5000 cells were plated into each well in a 96‐well plate. 5‐FU was then added for a period of 24 h, with eight replicates per condition. Cells were fixed and blocked with goat serum. Antibodies anti‐cleaved PARP (BD: 552596), anti‐active caspase3 (BD:559565), goat anti‐mouse IgG Alexa 488 (Invitrogen: A21121) and goat anti‐rabbit IgG Alexa 647 (Invitrogen: 21244) were used for staining. Rescue experiments were carried out using SPINK1CM (see below).
Conditioned media
A total of 8–10 ml of serum‐free DMEM was added to each 15 cm plate of MCF‐7 SPINK1 OE cells (Supporting information Fig 1, clone 9). Twenty‐four hours later, SPINK1CM was collected, centrifuged for 5 min at 800 rpm at 4°C. The CM was warmed to 37°C before use. For SF9 cells, the cells were infected with virus (SPINK1 or SPINK1‐K18Y) at the optimal titer. Three days later, CM was collected and centrifuged for 3 min at 1000 rpm at 4°C to remove cells. Vec‐CM was collected in a similar manner from empty vector transfected cells served as a control. Appropriate volumes of these CM were used in relevant assays. To neutralize SPINK1, CM was pre‐incubated for 2 h at room temperature with 50× molar excess of the anti‐SPINK1 antibody OR IgG. The latter served as another control.
In vitro invasion and migration
Transwell migration and invasion assays were performed using Falcon FluoroBlok 24‐Multiwell inserts (BD Biosciences) with 8 µm pores. For invasion assays, the inserts were coated with 20 µg Matrigel (BD Biosciences) in 80 µl serum‐free growth medium. For both assays, 30,000 cells in 200 µl serum‐free growth medium were loaded into each transwell insert with 750 µl complete growth medium or CM or CM with neutralizing antibody in the lower chamber. After 24 or 48 h, cells that had migrated or invaded through the pores of the inserts were fixed with 3.7% formaldehyde, stained with 2.5 µg/ml Hoechst 33342 (Invitrogen) for 15 min, washed with PBS and counted using the Target Activation Bioapplication on an ArrayScan VTI (Cellomics). Field size was 1 mm2. For both invasion and migration assays, experiments were performed with fourfold replication and 10 fields were scanned per experiment.
Tail vein assay
5 × 105 MB231 SPINK1OE cells or empty vector control cells were resuspended in 300 µl of PBS and injected into the tail vein of Balb/c nude mice 12‐week‐old. Four mice were injected per cell line. Mice were euthanized 12 weeks later and lung sections were stained with haematoxylin and eosin. IACUC protocol #070256 was approved by the Biological Resource Centre, Agency of Science, Technology and Research.
Author contributions
This study was designed and written by KVD, ETL and WWS; WWS performed all experiments with assistance from XBC in certain areas; Breast cohort analysis was done by LDM and MAB; CD provided biostatistical support for immunohistochemistry; BP, CWO and MST provided the NUH tissue arrays, performed and scored the immunostaining.
Supplementary Information
Detailed facts of importance to specialist readers are published as "Supporting Information". Such documents are peer‐reviewed, but not copy‐edited or typeset. They are made available as submitted by the authors.
suppdata [emmm_201100150_sm_suppdata.pdf]
This work was funded by the Agency for Science, Technology and Research (A*STAR) of Singapore. WW Soon is a recipient of the A*STAR Graduate Scholarship. We express our gratitude to Keith Rogers, Susan Rogers and Hassan Hall from the Histology Department for immunohistochemical staining, the Confocal and High Content Screening facility of Basement Shared Facilities (BSF), Agency of Science, Technology and Research (A*STAR) for their services.
Supporting information is available at EMBO Molecular Medicine online.
The authors declare that they have no conflict of interest.
FundingAgency for Science, Technology and Research (A*STAR) of Singapore
- Copyright © 2011 EMBO Molecular Medicine


text:
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2. Referrals
When you buy a package from our website we guarantee that you will get the direct referrals you've payed for.You will receive the direct referrals in maximum 30 days since you've ordered.All the referrals will be real people from different countries, we don't make accounts for you, we don't pay people to sign up and we don't use robots, we just advertise your link around the world.If you are not satisfied by our services you have 40 days to cancel your order.
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subset: commoncrawl


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|CD Review: Moving Forward by Bernie Williams
|Thursday, 28 May 2009 09:23
By Ken Volpe – GJD Contributor
Sometimes, we are blessed to experience an individual that possesses greatness in two completely different arenas. Bernie Williams definitely fits into that rare mold. His latest release, Moving Forward, showcases Mr. Williams' talents as a composer and a performer. In addition, he allows some of the industry's top session musicians to lend their own creativity and to share in the spotlight, which I believe to be a sign of humility and artistic integrity.
The CD starts with the title cut, "Moving Forward." It is a feel good tune with great melody lines and masterful guitar layering. Bernie's steel-string guitar sounds sit wonderfully in the mix. His tone is crisp and warm with a bell like timbre. The track also features an interesting bridge and some amazing percussion.
The third cut titled, "Songo" features more great acoustic lines and some Holdsworthian shredding courtesy of Scott Henderson. I must admit I would have liked to have Scott's guitar be up a bit higher in the mix.
A little further down the line Bernie lets it rip on an instrumental Praise tune called "He Reigns." I always enjoy Praise tunes that are well done like this one because the music showcases the majesty of the Holy One. Also, I really enjoyed Michael Landau's solo at the end of the song.
Next I sampled, "Chillin in the West," which is another well-crafted gem. Bernie's sense of melody and blending of rhythm is top notch. Towards the end of the CD, Bernie does a beautiful rendition of "Take Me Out To The Ball Game." Any true Yankee fan will appreciate the intro by legendary announcer Bob Sheppard. Bernie kind of reminds me of the great Tommy Emmanuel on this cut.
Moving Forward, contains 14 total songs with a variety of styles including some vocal tunes by Bruce Springsteen and John Secada. There is also a huge Latin influence throughout that fits perfectly with Bernie's liquid guitar tones. Some of the tunes have a Rippingtons-like flavor; but at the same time are very unique in their own way. Bernie's compositions are both complex and simple and contain very natural transitions. Bernie weaves in his signature riffs throughout on a variety of Electric and Acoustic guitars. Additionally, the recording quality and production are off the chain.
As a third generation Yankee fan and a lifelong musician, this project has a special place in my heart. The thing that really struck a chord with me is how similar Bernie's music and his baseball skills are to each other. Both exhibit dedication, passion, grace, and class. Moving Forward is so good, that even a Boston Red Sox fan will love it.


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Handmade With Organic Love
SALVATION: The state of being saved or protected from harm, risk, etc.
You will celebrate the revival of your skin with our Salvation Salve. Created out of need for a natural products that treats skin with the utmost care.
Made with Comfrey, Calendula & St. John's Wort infused organic sweet almond oil. Your skin will be calm & moisturized. Great for people with very dry or irritated skin.
Our goal is to provide nourishing all natural hair and skin care products.


text:
Changes at Old Dominion Brewery
I'm pleased to say that I might be wrong (so far). The annual beer festival is gone (the usual site right by the brewery is now a construction zone), but they haven't gooned up the beers yet. The did change the menu a bit, and added a pretty good sunday brunch. I can still get Hop Pocket Ale, and if it's out I'll be happy to settle for New River Pale Ale.


text:
Steve Klinsky and Maureen Sherry/
NET WORTH: Unknown
SOURCE OF WEALTH: New Mountain Capital
FUNDING AREAS: Education & Youth
OVERVIEW: Steve Klinsky founded Modern States Education Alliance, which is dedicated to free, universally accessible college education for motivated students. The couple doesn't appear to have a formal charitable vehicle.
BACKGROUND: Steve Klinsky received a B.A. from the University of Michigan in 1976, and an MBA and J.D. from Harvard in 1979 and 1981, respectively. Klinsky was a co-founder of the leveraged buyout group of Goldman Sachs, and worked at Forstmann Little, rising to general partner. In 1999, he established New Mountain Capital.
EDUCATION & YOUTH: Klinsky has been involved in the education space for years. In 1999, he founded New York's first charter school, the Sisulu-Walker Charter School of Harlem. He had previously opened an after-school program in the city in honor of his late brother, called the Gary Klinsky Children Centers. In addition to Gary's influence, Klinsky's wife Maureen, who also worked on Wall Street, has tutored at inner-city schools. Maureen is an active board member of charities focused on families in poverty, equality in education and environmental sustainability. Klinsky also chairs the board of Great Oaks Charter Schools.
More recently, Klinsky has focused on his Modern States Education Alliance, "a non-profit dedicated to making a high quality college education free of cost and accessible to any person who seeks one." The outfit's first program, Freshman Year for Free, offers online college courses free of charge to prepare students for AP and CLEP exams that can count toward college credits. The nonprofit is covering exam fees for the first 10,000 test takers. If students pass enough exams, they could shave a year off college for a fraction of the cost.
LOOKING FORWARD: For now, expect Klinsky to remain focused on Modern States but down the line expect other interests to emerge, as well. Klinsky has mentioned an interest in health. Maureen has transitioned from Wall Street to writing, and is outspoken on issues of sexism in finance. These issues to may come into play.


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Toggle navigation
50K Cycle
30K Cycle
why enter?
Hurley Hoey
10K Charity Run / Jog / Walk
Saturday 9 Mar 2019
Doora-Barefield GAA Complex
Run/Walk: 11:00 A.M.
50K/30K Cycle:10:00 A.M.
Hurley - Hoey 2013 - What were the funds raised used for?
20 January 2014
In advance of the formal launch of the 2nd Hurley-Hoey Run/Walk this coming weekend and in light of very legitimate public concerns about the use of charitable donations, we in the Hurley-Hoey Committee want to be completely transparent in terms of outlining what the monies that were raised at last year's run/walk were used for. In summary €28,000 was raised for two local causes at last year's event which was an incredible figure for an event in it's first year. The money was divided between two very worthy local causes with €14,000 going to St. Anne's Special Needs School in Ennis and €14,000 going to The Sunshine Ward for Sick Children in the Regional Hospital in Limerick. The €14,000 that was allocated to the Sunshine Ward was used to a) purchase 10 special purpose beds for parents of sick children staying in the ward overnight and b) purchase an Activity/Play Unit for children coming into the Sunshine Ward for Day Procedures. The €14,000 that was allocated to St. Anne's School was used for a) structural work for a Sensory Room for the children in the school and b) to purchase equipment for the sensory room. A picture of some of the equipment purchased is included with this story. The Hurley-Hoey Committee had 100% visibility on all purchases from the monies raised including all related invoices. We are absolutely satisfied as a committee that the monies donated by the public were used exclusively for the items listed above. The Hurley-Hoey Committee members operate on a completely voluntary basis and no member of the committee received any monies from their involvement with this charity.


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3635 N. Beltline Rd Suite 110Sunnyvale, TX 75182ph: 972-216-1500fax: 972-216-1300
I am pleased to announce the opening of my new office in Sunnyvale!! It is conviently located just north of Town East Boulevard on North Beltline Road. It is a brand new modern facility. I have been a family physician serving Dallas, Mesquite, and Sunnyvale patients in solo private practice since 1984. I offer 18 years of inpatient hospital experience combined with 29 years of outpatient office experience. I want you to know providing quality healthcare in a friendly personal compassionate manner is my goal. I believe everyone should have a doctor that can provide continuity of care . I am an independent sole proprietor beholden only to my patients.
Copyright 2013 Sunnyvale Family Medicine. All rights reserved.
Web Hosting by Yahoo!


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The African honeybee, introduced in Brazil to increase honey production and hardiness in local hives, instead produced a super aggressive hybrid known as the Africanized Honeybee (AHB) or Killer Bee. Since the 1950's the AHB has migrated from Brazil, through South America, Central America, and Mexico. Today they are common in every southern state from California to Florida and they continue to move north, recently reported in Colorado, Arkansas, and Virginia.
AHB's are a major problem. Since the first U. S. Killer Bee fatality in Harlingen, Texas in May 1991, governmental authorities and the medical emergency community are increasingly dedicated to AHB control measures. All efforts to stop the spread of AHB have been futile. In other words, the AHB is here to stay.
The developer of the BeeAlert Stinging Insect Control System is Will Baird. Like most residents of Texas, Will kept an eye on the Africanized Honeybee (AHB) or Killer Bee problem since news reports first warned of the coming invasion in the 1970's. In the summer of 2004, Killer Bees moved from the pages of newspapers to reality for him. A friend, mowing the yard at his weekend home outside of Houston, came too close to a set of bee boxes which he kept as a hobby. Unknown to him one of the hives had become Africanized. In seconds the AHBs, stirred up by the vibration of the tractor, launched an attack killing Will's friend. A month later, another friend, an 80-year-old rancher in South Texas, was nearly stung to death while working in a field he had plowed every year of his adult life.
Baird began research to create a method to stop the threat of these horrific incidents. After interviewing experienced EMS first responders and bee experts, gaining a full understanding of their needs, he began to develop the BeeAlert Stinging Insect Control System.
The purpose of the system is two-fold. First, fend off an attack of stinging swarms and allow victims time to exit the hot zone.
The BeeAlert Bee Control System uses a patented spray system and non-toxic solution that knocks the insects down without harming the victim, the operator, or the environment.
The BeeAlert Stinging Insect Control System has several applications for use in an attack. These devices are Tractor/Dozer Roof Mounted Tank, Tractor/Dozer Roll Bar Mounted Tank, and Aerosol Spray Can in three convenient sizes; TWO PACK 4oz, 10oz and 14oz.
NOTE: Currently, BOTH Tractor/Dozer units are NOT available for purchase. We are in the process of improving the devices to better serve our customers. Please contact us if you would like to be informed when these units are available.


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I've been fantasising about going on an extended cycle tour for a long time. In 2008 I rode around New Zealand on and off for a few months but I haven't done any extensive touring since then.
I recently found out that a friend of mine, Tom Donhou has been building custom bicycle frames for a while now. I didn't have a clue that he was in that line of work. With one look at all the glorious bike porn on his website I knew I had to get him to build me a frame for an expedition bike which I had been planning on building. You should take a peek at his site: Donhou Bicycles. Tom is based in Norwich and recently won the "Best in Show" category at the Bespoked Bristol UK Handbuilt Bicycle Show 2011. I challenge you not to drool over his designs!
I put the deposit down as soon as I had the chance. Setting up recipients with Lloyds TSB when you're living in South Korea is a pain in the arse. It takes 7 days to register a new phone for internet banking..
Today I got the draft design emailed to me and I'm getting feverishly excited about it! Now I just have to figure how I am going to get it to South Korea. I can either ship it directly to Korea, or try to get my sister to bring it with her to Malaysia where I'll be spending Christmas with my family. Go on Pip! I'll pay for the extra costs!
Tom and I first met in Marseille in 2002. I was skateboarding at the infamous skatepark on the beach whilst he was riding his BMX. He was riding towards me, so I spoke to him in French as I guessed he was a local, to which he replied, "I thought you were English". Correct assumption Tom! We got chatting and little by little figured out that not only did we both live in Sheffield at the time, but we both studied in the same building and we had a bunch of mutual friends, most notably Lucien, "The Panda". When I was living in London I shared an apartment with another mutual friend of ours George Marshall, so I got the regular pleasure of having Tom round for drinks.
I've spent hours ogling at the bicycles on his site and suggest that you do aswell! I'll keep you posted on the development of my expedition bike. I've got a lot of parts to think about and would welcome any advice.
Below are some photos that I've lifted straight from the Donhou Bicycles website.


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Inder Residency, udaipur
Accomodation :
144 rooms.
Amenities :
Swimming pool, Gymnasium, Steam and Sauna Beauty Salon & Spa, Business Centre, Concierge, Travel desk, In-house Laundry, Doctor on call Money Exchange, In-house Bakery & Cake Shop, In- room dining (Round the clock) Shopping Arcade with Boutique Gift / Book Shop Deluxe Rooms (88), Super Deluxe Rooms (35), Hawa Mahal (14), Maharaja Suite (1), Presidential Suite (1)
Dining : Laziz –
The Fine-Dining Restaurant,
Tashkeen –
The Coffee Shop,
Harbour –
The Bar, Pool side Bar-be-que Restaurant.
Location :
Railway Station – 3 Kilometers, Bus Stand – 3 Kilometers, Lake Pichola – 3 Kilometers.
Hotel Inder Residency
It is a good luxurious and perfectly located hotel of the City of Lakes: The Inder Residency – Udaipur. Surrounded by lovely gardens, misty hills and breathtaking lakes and ponds of Udaipur, this grand five-star hotel is sprawled across 3.75 lakh sq. ft. area and is ideally located, with the airport and the railway station within easy reach.
Address :
Goverdhan Vilas, Ekling Nagar, Shikharbadi Road, Udaipur 313001 India.
Tel :
+91 - 294 - 2489888 +91 - 9414155139
Fax :
+91 - 294 - 2488733
Website Address :
Holiday packages covering udaipur
Five-star Hotels
First-class Hotels
Budget Hotels
Oberoi Udaivilas
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Paras Mahal
Trident Oberoi
Oriental Palace
Swaroop Vilas
Garden Hotel
Taj Lake Palace
Jagat Niwas palace
Leela Palace Kempinski
Jaiwana Haveli
Karni Fort Bambara
Lotus Resort
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Lake Pichola
Lalit Laxmi Vilas Palace
Karohi Haveli
Rajputana Resort
India International
Rockwood Palace Resort & Spa
Royal Retreat
Cambay Resort
Ravla Khempur
Inder Residency


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Brett Eldredge 'The Elf' Stage Bombs Couple Singing His Song
Last weekend Brett Eldredge put on his best "Buddy the Elf" costume and started popping up at honky tonks around Nashville, taking the stage where and when he could. No, it doesn't make perfect sense, but the videos are sort of awesome.
The first video shows a couple singing Eldredge's hit song "Beat of the Music" karaoke style. Eldredge stands next to them, helping out before assuring them he can take the second verse solo. It appears no one involved — including the videographer — knew that there was a famous country singer seated nearby in Elf regalia. Of course when he started to sing, his cover was blown.
A second video was captured by Nashville musician Chad Bearden at the Stage on Lower Broadway in Nashville. Eldredge sings "Silent Night," but also played a few originals before signing off, and Facetiming the singer's daughter, Bearden writes.
"Don't forget to tip the band," Eldredge says at the end of this 45-second clip.
Bearden tells Taste of Country that the "Mean To Me" singer regularly joins him on stage during his Saturday night gigs at the Stage, and he knew he would be there on that night. His band played a few of Eldredge's hits (sonically they compliment each other), and sure enough the 29-year-old couldn't resist joining them for "Don't Ya," "Mean To Me" and "Silent Night." After the show, Eldredge and his team called it a night.
As for why Eldredge decided to dress up as an elf and hit the town? That's not clear. In fact the only explanation may be because he's Brett Eldredge and he likes to do things like this:
Last week, Eldredge also wrapped up 2015 by performing a private set of Frank Sinatra songs for Music City insiders. He wrapped up his Suits and Boots Tour with Thomas Rhett earlier this year. "Drunk On Your Love" is the current single from his Illinois album.
Is Brett Eldredge Country's Sexiest Male? Find Out:


text:
Shipping Canada Post
Q: What is your shipping policy?
A: See our shipping policy listed below. It contains complete details regarding the different delivery services that are available.
Q: Can I have my online order delivered to an address other than my own?
A: Yes. You can have your online order delivered to any address you choose, as long as it is within Canada. This option is perfect for sending gifts! Simply enter the address into the "Delivery Address" field at checkout.
Shipping policy
All products sold by Langevin Forest can be delivered to your residence. This service is only available within Canada. For orders outside of Canada, please contact our customer service via email at firstname.lastname@example.com"> email@example.com or by telephone at 1-800-889-2060 .
3 easy steps!
- Place your order online at www.langevinforest.com or by telephone at 514-322-9330 (local) or 1-800-889-2060 (long distance).
- Make a secure payment for your purchases.
- Receive your order within a few days!
- Langevin Forest offers you the most economical and efficient delivery solutions.
Delivery via Canada Post
Coverage area: Canada
>Delivery delay: 1 to 3 business days
>Applicable products: This service is used for smaller parcels
- Boxes with dimensions up to 12" x 12" x 48" or 36" x 36" x 12"
- Panels with dimensions up to 24" x 48"
- Materials for sculpting, turning, artisan work, etc…
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For any products that cannot be delivered by Canada Post, a note to that effect is included on the "details" page for that product.
Canada Post Restrictions
Parcel size and weight restrictions:
- Maximum length: 2 metres (78 3/4")
- Maximum circumference: 3 metres (118")
- Length + (Height x 2) + (Width x 2 ) < 3 metres
- Maximum weight: 30 kg (66 lbs)
Hazardous products:
We are not permitted to ship hazardous materials.
Liquids such as strippers, solvents, thinners, paints, lacquers, and varnishes can only be delivered by Canada Post in formats of 500 ml or less.
Winter delivery period
We do not ship items that are sensitive to freezing during winter months.
Delivery charge for shipments by Canada Post
Shipping fees vary depending on the amount of the order.
|Orders amount
|Shipping cost
|Orders up to 20 $
|8 $
|20,01 $ to 50 $
|50,01 $ to 100 $
|12 $
|100,01 $ to 150 $
|14 $
|Orders over 150 $
|16 $
Additional charges : From 12$ to 16$ of additional charges applies for heavy or oversize items.
Remote areas and products not covered by Canada Post services
We make every effort to deliver everywhere within Canada. However, home delivery of oversized parcels to some remote areas is not always possible and/or may incur additional delivery charges – particularly in remote communities accessible only by air.
Orders weighing more than 66 lbs (30 kg) or having a length in excess of 78" (200 cm) cannot be delivered by Canada Post but can be shipped using a common carrier.
In such cases one of our customer service representatives will contact you to discuss the best way ship your order.


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text:
INSERM and UNIMAN (University of Manchester) are the two major players in the development of the Kidney and Urinary Pathways Ontology (KUPO): INSERM provides the biological expertise and UNIMAN the ontology engineering know-how. Julie Klein (INSERM) visited UNIMAN on May 19-21, 2009 to attend the Introduction to OWL course and discuss KUPO development issues with Robert Stevens and Simon Jupp.
The meeting participants agreed that instead of being created from scratch, the KUPO would be seeded by merging relevant portions of the following source ontologies: Gene Ontology, Mouse Anatomy Ontology, Cell Type Ontology, Human Disease Ontology and the Protein Ontology. They identified simple modeling patterns that will be used to develop an initial prototype of the KUPO; this will be presented to EuroKUP members at the next working group meeting early November.
In the meantime, an initial working version will be made available for KUP specialists to browse online using the Web Protégé application; this tool will also allow EuroKUP members to give their feedback on the ontology. For more in-depth discussions, a forum and/or a wiki will be set up on the EuroKUP/e-LICO collaboration portal, as well as mechanisms through which experts can provide definitions about domain entities and their relations, or suggest terms for inclusion in the ontology.


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In Canada the popularity of the ruling Liberal Party, led by Justin Trudeau, has fallen over the past year, following several gaffes, most recently by the finance minister, Bill Morneau, in relation to a botched tax reform. New leaders for the Conservatives and the New Democratic Party, elected during 2017, will compete more strongly for attention ahead of the next election in late 2019.
The biggest risk to Mr Trudeau's re-election prospects may prove to be the renegotiation of the North American Free-Trade Agreement (NAFTA). Canada wants to see the inclusion of chapters on e‑commerce, the environment and gender and indigenous rights. These will be approved. However, there is strong resistance to Canada's wish for more open government procurement, the preservation of its supply management rules for the dairy and poultry industries, and the retention of dispute resolution mechanisms. The US, for its part, wants tighter rules-of-origin requirements, greater access to Canadian and Mexican government contracts and an option for all members to leave the agreement at five-yearly intervals. Canada and Mexico are opposed to these changes.
The economic consequences of a US withdrawal from NAFTA would be far greater for Canada and Mexico than for the US. However, if it became likely that if Mr Trump was minded to remove the US from the agreement, an enormous lobbying effort in all three countries would kick in, and the US Congress would consider the as-yet-unresolved question of whether the president has the legal authority to remove the country from a trade agreement that has been ratified by the legislature. Given the widespread opposition among business and politics to withdrawal, we expect a revised agreement to be signed, but after the current deadline of March 2018. The changes will be less extensive than the US desires, and its effects on the US-Canadian trading relationship will be marginal.


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1 1/2" Suede Strip
$6.29 - $6.99 30% off Leather & Leather Kits
- Brand: Realeather
Use this Suede Strip to trim picture frames, make belts, and for a variety of other leather craft projects.
- Length: 42"
- Width: 1 1/2"
Item not available in Hobby Lobby Stores.
Check your local store for availability.
Send me an email when it's back in stock.
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I was able to do a bit of painting on Sunday before going to sleep, mainly because I was so excited about working on Eiryss. I think that she is coming out as my best model yet!
I was able to lay down the blacks and fine tune some of the details I initially missed the first go around.
Even at this stage I was feeling good about the job and scheme. Nary a bit of splash over at this point. Everything is going great. I'm thinking maybe I should keep the white cloak because this Black/White, Silver/Gold scheme looks pretty good…
But no! The plans were for a little Red Riding Hood of Ios!
Now at this point I nervously start to lay down the reds. In the pictures it looks a bit orange-ish, but that is P3 Khador Red base after just two thin coats.
Finally before calling it a night, I gave the metallics a wash, the silver getting Badab Black and the gold getting Devlan Mud. When I come back to her, I'm going to give the red a few coats more and attempt to smooth it out best I can. I have some highlighting and cleaning up to do (don't I always) and also another idea for finishing the cloak. Her base is planned out and started but I'll need my green stuff to finish it completely, so looks like I'll be waiting on that (arrrgh).
This morning I was reading on Hand Cannon Online's Getting Started in Warmachine series and they give an example order for painting. Going forward I'm going to keep this in mind. Any tips are welcome on my way to learning to paint these miniatures professional looking!
This week marks the start of 15 point games in the Slow Grow league and this is the list I'll be rocking in case you are curious/forgot. Right now at the end of the first phase, I'm 2 for 3 games and am sitting at 10 league points. The top person, however, has 31! All off games. If I intend on placing at the end of this thing, I'm going to need to start playing a ton more games…


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The Sigma 150-500mm f/5-6.3 APO DG OS HSM is one of a growing number of "affordable" telephoto zoom lenses is the $800-$1500 price range. While not as fast or as sharp as a premium telephoto prime, these lenses often provide good value for money and they have a number of advantages over a prime lens. They are smaller and lighter, they zoom and they're significantly less expensive. With optical stabilization they can be handheld and with higher ISO setting becoming available on more cameras, the fact that they may be 1 or 2 stops slower than a prime lens is less important than it used to be.Lenses of this type are useful for wildlife photographers and sports shooters, enabling them to obtain images without getting too close to their subjects. The "APO DG OS HSM" designation indicates that it is a Sigma DG lens (full frame coverage), with OS (Optical Stabilization), an HSM (HyperSonic Motor), and is an "APO" (Apochromatic) design to minimize chromatic aberration.
The finish of the lens is semi matte black and, at around 4.2lbs, it's light enough to handhold fairly easily. The stabilization (OS, 3-4 stops) means that it's a reasonable proposition to handhold this lens, even at 500mm and f6.3.
The lens comes with a hood, hard carrying case, and two straps(one for the lens and one for the case). It's covered by a 1 year Sigma warranty, but for US purchasers who buy the lens from a Sigma-authorized US dealer, the warranty is extended to 3 years
|Focal Length
|150-500mm (240-800mm equivalent on Canon APS-C)
|Maximum: f/5-6.3 Minimum: f/22
|Format Compatibility
|Full Frame, APS-C
|Angle of View
|16.4° to 5° (150mm to 500mm, full frame)
|Minimum Focus Distance
|7.22' (2.20 m)
|Maximum Magnification
|Filter Thread
|86 mm
|Diaphragm Blades
|21/15 (including 3 Special Low Dispersion (SLD) elements)
|Dimensions (DxL)
|3.7" x 9.9" (9.40 x 25.15 cm)
|4.19 lb (1.90 kg)
|Camera Mount Type
|Canon, Nikon, Pentax, Sony, Sigma
|Lens Hood
|Supplied with lens
|Approx. Price (02/15)
|$870 -
The marked focal length span of the Sigma 150-500mm f/5-6.3 APO DG OS HSM is, of course, 150mm to 500mm. Remember that focal lengths are always specified for the lens focused at infinity, and it's not unusual for the actual focal length to be up to +/-5% different from the nominal values. A change (decrease) in effective focal length is also pretty common when a telephoto zoom is close focused. Bearing this in mind...
When set to maximum zoom (nominally 500mm) I measured the actual focal length of the Sigma 150-500mm f/5-6.3 APO DG OS HSM set to be around 480mm focused at infinity and around 450mm focused at 10 m (about 30 ft). At the closest possible focus (~2.2 m), magnification is around 0.2x, which implies a focal length of roughly 300mm. None of these numbers are out of the ordinary for a lens of this type.
I did observe some zoom creep. The lens extended from 150mm to 500mm when held pointing downward and retracted from 500mm to 150mm when pointing up. There is a zoom lock at the 150mm position to ensure the lens doesn't accidently extend when being carried.
The maximum aperture of the Sigma 150-500mm f/5-6.3 APO DG OS HSM varies from f/5 between 150mm and 173mm to f5.6 between 174 and 313mm to f6.3 from 314mm to 500mm.
The time required to focus from the closest focus distance (2.2m) to infinity was around 0.95 seconds and that did not change with focal length. The HSM focusing motor noise is very quiet. It's inaudible in normal use. Focusing was generally positive (no hunting) and accurate, with the great majority of the images I shot being well focused.
At 500mm I found the optical stabilization (OS) system enables me to get pretty sharp handheld shots most of the time down to a shutter speed of around 1/125s, occasionally as slow as 1/60s. That's about 2 stops of stabilization most of the time, 3 stops if you are lucky and/or shoot a burst of shots and pick the sharpest. Two stabilization modes are available, one for static subjects and one for moving subjects where the camera is panning to follow them. Potential buyers of the lens for Pentax and Sony cameras should be aware that models in those mounts are not optically stabilized because both system use sensor shift stabilization which is built into the camera body. The Nikon, Sigma and Canon mount lenses all have optical stabilization.
I tested the Sigma 150-500mm f/5-6.3 APO DG OS HSM by shooting both resolution test targets and normal pictorial subjects, using both APS-C crop sensor and full frame DSLRs. I won't attempt to reproduce all the images here, but I'll give my overall conclusions and a few samples. Critical sharpness tests using resolution targets were done using Live View and manual focus in order to separate the intrinsic sharpness of the lens from the accuracy of the AF system. Normal pictorial shoots were taken using AF, which in fact proved to be accurate and consistent.
At 150mm the Sigma 150-500mm f/5-6.3 APO DG OS HSM shows high sharpness from center to edge at f5 and little is gained by stopping down. Chromatic aberration is very well controlled.
At 200mm image quality is very similar to that at 150mm with very good sharpness and negligible chromatic aberration.
At 300mm there's a very slight drop in sharpness wide open. Stopping down does improve sharpness a little, but on normal pictorial targets you'd be hard pressed to see the difference. A very small amount of chromatic aberration can be detected at the edges of the frame.
At 400mm center sharpness remains high the edges are slightly less sharp with the lens wide open. There's a small improvement stopping down to f8, but not so much that I'd feel that I had to do so to get a sharp shot. Chromatic aberration is low, but visible at the edges of the frame.
At 500mm wide open (f6.3) the image is slightly softer and lower in contrast than at 500mm. I'd guess what I'm seeing is probably a small amount of uncorrected spherical aberration, but whatever the cause, stooping down by 2/3 stop to f8 bring up the sharpness and contrast. The edges of the frame aren't as sharp as the center and show a small amount of chromatic aberration. At 500mm I'd probably try to shoot at f8 with this lens if I wanted maximum image quality, however I wouldn't do so if I needed the extra shutter speed that f6.3 would allow.
My bottom line conclusion is that the Sigma 150-500mm f/5-6.3 APO DG OS HSM is a pretty good lens, especially considering its price (02/15 around $870). Sharpness at 150mm is very good from center to edge, even with the lens wide open and on both APS-C and full frame sensors. Sharpness at 500mm is a notch lower wide open at f6.3, but stopping down (by 2/3 stop) to f8 brings the sharpness back up close to that observed at shorter focal lengths.
Here's an example of the sharpness that the Sigma 150-500mm f/5-6.3 APO DG OS HSM is capable of at 400mm and f8. First the whole image (EOS 7D):
And now a 100% crop of the cormoramt's head. This shot was taken handheld at a shutter speed of 1/250s.
Now here are two similar shots, this time taken at 500mm wide open at f6.3. First the whole frame (EOS 7D)
And now a 100% crop of the head and eye of the cormorant:
While sharpness certainly isn't bad, I think you can see it's not quite as good as it was at 400mm and f8.
Chromatic aberration is visible between 250mm and 500mm but it's at a fairly low level. Here's an example at 370mm, shot using and EOS 7D DSLR. First the whole frame:
And now the area at the right edge of the frame indicated by the red box in the image abobe. This crop is at 100%.
as you can see there is a small amount of red/green fringing on high contrast edges. This can be minimized in post processing if it becomes an issue, but that's only likely if you're making large prints for close viewing. The CA seen at the edges of a full frame shot at 500mm isn't very different from this.
Vignetting is observed with the Sigma 150-500mm f/5-6.3 APO DG OS HSM, especially when the lens is used with a full frame DSLR, wide open or at 500mm. The images below show the extent of corner darkening when shooting with a full frame camera
At 150mm vignetting is negligible if the lens is stopped down to f11, but at 500mm the corners are darker even with the lens stopped down to f11.
While this could be an issue, usually it's not. Vignetting is fairly easy to digitally correct in post processing. Also, unless the corners of the frame contain areas which are uniform in brightness (e.g. sky), vignetting even wide open may not be noticeable. If shooting with an APS-C crop sensor camera, vignetting, even wide open at 500mm is very unlikely to be a problem. The image below is the full images captured using an EOS 7D with the Sigma 150-500mm f/5-6.3 APO DG OS HSM wide open (f6.3) at 500mm.
The worst case scenario is shown below. A full frame shot at 500mm and f6.3 with a uniform sky. The top corners are clearly darker.
However with a little digital processing the image is much more uniform as shown below:
The Sigma 150-500mm f/5-6.3 APO DG OS HSM isn't a macro lens. Maximum magnification is 0.19x (about 1/5 life size) but nevertheless it's fine for closups of flowers and detail shots of larger objects. On a full frame camera at maximum magnification the area in the image will measure about 5" x 7.5". On an APS-C crop sensor camera like the EOS 7D, the frame will cover about 3.1" x 4.7".
Distortion is low at all focal lengths, as would be expected from a telephoto zoom of this type. It would be very unlikely to be a problem, in fact you'd probably have a hard time seeing it, even in architectural shots.
The is a very nice lens, especially considering the price which is currently around $870 (02/14 and includes a $200 instant rebate). The optical sharpness is excellent at shorter focal lengths and still very good through 400mm. The harpness does drop a little when the lens is used at 500mm wide open, though stopping down to f/8 makes it almost as sharp at 500mm as it is at 400mm. The AF speed is average for a lens in this price range. The dedicated telephoto prime lenses from the major manufacturers are faster, but that's to be expected based on their much higher cost.
The exhibits low distortion and chromatic aberration is well controlled, especially at shorter focal length settings. Even zoomed out all the way to 500mm, chromatic aberration is not really problematic and if it really bothers you, digital correction can remove most of it. On the downside there is noticeable vignetting when used with a full frame camera, but that can be easily corrected digitally and even the best and most expensive telephoto primes show some vignetting. The bottom line is while it may not be as fast, sharp, or quick to focus as a 500mm prime lens from Canon or Nikon, it's still a good performer and a good deal at around 1/10th of the cost...plus it's a zoom too!
None of the major camera manufacturers (Canon, Nikon, etc.) make a telephoto zoom that's this long or that's close to the price (~$870 as of 02/15) so the alternatives are lenses made by Sigma themselves and by Tamron, unless you're willing to settle for a reduced focal length range with something like the ($2200) and pay about 2.5x the price.
Sigma has three lenses that are similar to the . There's the new Sigma 150-600mm f/5-6.3 DG OS HSM, which adds 100mm to the focal length, two FLD elements (a glass similar to Fluorite), and an accelerometer to further stabilize the lens when panning. There are two versions of this lens, a "C" version (Contemporary) and an "S" version (Sports). The Sports version, , should have better performance, with extra elements (24 vs. 20), and is weather sealed. The cost of the "S" version is more than $1000 higher than that of the 150-500mm, and at the time of writing (November 2014), the lens has not yet arrived in stores. There's no price or availability information yet on the "C" version, , though presumably it will be less expensive than the "S" model. There's also the , which adds 100mm to the short end of the range along with an extra low dispersion element and a price that's around $650 higher.
From Tamron there's the . It's only around $200 more than the Sigma 150-500mm and it adds 100mm to the long end of the range. Though it was announced quite a while ago (I reviewed last year - Tamron 150-600 Review), it's still in limited supply as of 02/14, though the wait times for an order to be fulfilled are getting shorter. It's a very nice lens though, so worth the wait! For me it would be worth paying the extra $200 over the Sigma 150-600 as the slightly longer reach would be useful for wildlife work.


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Well, Thanksgiving dinner has been cooked, hallelujah. All that's left to be done is to pick up the rotisserie turkey in the morning (yeah, we outsourced that), and for Julie to make the celery root remoulade. Tonight I listened to old Rolling Stones, and the Avett Brothers, and made (with a little help from Nora and Julie):
- Christopher Kimball's awesome toasted cornbread and pecan dressing
- Brussels sprouts with onions and bacon (see above)
- Gratin dauphinois (scalloped potatoes boiled in milk, salt, pepper, and nutmeg, then doused in heavy cream and slow-cooked in the oven)
- Roasted beet salad with honey-lime vinaigrette
- Cranberry sauce
Our guests are bringing pies for dessert. We're going to drink Muscadet. People like Chardonnay on Thanksgiving, but I don't care for buttery California Chardonnay. The ideal, for me, is white Burgundy, which is made with the Chardonnay grape, without all the over-oaking. But it's not cheap. We ran across a delicious and affordable Muscadet (La Fruiterie) at Whole Foods not long ago, and I think it'll be perfect. The acid will cut all the heaviness in the Thanksgiving food.
We are pondering whether or not to make Dorie Greenspan's French-style carrot salad, which is really delicious, and usually part of our Thanksgiving table, but for some reason we forgot to work it in this year. Maybe, since we're so far ahead, we'll do it in the morning.
What are you cooking? Open thread.
UPDATE: One of our guests brought a homemade pumpkin pie that, as best as I can figure, she had laced with barbiturates. That is the only way I can explain the fact that I stepped away from the gathering around 4:30 to make a phone call from the bedroom, and woke up four and a half hours later, having had the most epically satisfying nap. Thanksgiving for the win!
And by the way, that nap came after I had had two cups of strong coffee in a failed effort to rally.


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After the cancellation of the over $500 million deal with Indian firm GMR for Male airport in 2012, a promise to grant two plots of land by the the previous Nasheed government to Indian firms Tata Housing and 18SG Group for development has been left hanging, says 18SG owner Sanyogita Atrey.
The plots in Nadhee and Odeon in Male island were promised as free sale land by the then government of President Mohammed Nasheed in 2010 to be developed into high-end housing by the Indian firms.
The land was promised as part of a deal inked by Tata Housing and 18SG to construct 350 houses in Male, in a government buy-back initiative. The houses have been completed and handed over to the Maldivian government.
After the coup, in which Nasheed was deposed and went into exile, everything changed. The housing project got delayed. They gave us the land then, and now the new government has taken it back, Atrey told IANS.
Ratan Tata has filed a case and demanded that the present government of President Abdulla Yameen settle the issue.
"We approached the Ministry of External Affairs (MEA) over the issue. The land was free sale, and in exchange for all the money we have put in for the housing project. But it has been terminated," she said.
The MEA has helped us a lot. Brotin Banerjee, MD of Tata Housing, met External Affairs Minister Sushma Swaraj. She was very supportive," Atrey said.
"They said what belongs to us should be given to us."
"Ratan Tata has told the Maldivian government to give us compensation, like they did in the GMR case."
"The government project is done, and they have taken delivery of the houses, but our personal land promised to us for free sale has been taken away."
"We wanted to meet the President of Maldives to arrive at a settlement. It has been so many years we have been involved in the project. Let's see what turn the case takes," she added.
Full details are available from the link below:
Source URL: Google News


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The Life of Myrtle Williams Hensley
Myrtle Williams Hensley Funeral services for Myrtle Williams Hensley, 85 year old Eakly, Okla. resident, will be held on Friday, Jan. 13, 2012 at 11 a.m. in 'The Chapel' of Lockstone Funeral Home with Rev. Louis Brown officiating. Burial will be held on Monday, Jan. 16, 2012 at 2 p.m. in the Rose Hill Memorial Park Cemetery in Massillon, Ohio.


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Vishay Asia Honored With TTI Supplier Excellence Award for Eighth Consecutive Year2018/05/03 Up Teks Co., Ltd.
Platinum Level Recognition from TTI Asia Pacific
MALVERN, Pa. — May 3, 2018Vishay Intertechnology, Inc.(NYSE: VSH) today announced that Vishay Intertechnology Asia Pte Ltd has received the 2017 TTI Asia Supplier Excellence Award. TTI is an authorized specialty distributor of passive, connector, electromechanical, and discrete semiconductor components and the distributor of choice for industrial, military, aerospace, and consumer electronics manufacturers worldwide. Vishay is one of the world's largest manufacturers of discrete semiconductors and passive electronic components.
This is the eighth year in a row that Vishay has won the TTI Asia Supplier Excellence Award, earning the company Platinum Level recognition. The awards program centers on quality measurements that include on-time delivery, receiving quality, customer quality, administrative quality, sales support, and business systems. Winners are determined by performance points for these criteria.
Anthony Chan, president, TTI Asia Pacific, presented the Supplier Excellence Award to Victor Lim, senior director of distribution sales, Vishay Asia, in a ceremony at electronica China 2018.
Vishay Intertechnology, Inc., a Fortune 1000 Company listed on the NYSE (VSH), is one of the world's largest manufacturers of discrete semiconductors (diodes, MOSFETs, and infrared optoelectronics) and passive electronic components (resistors, inductors, and capacitors). These components are used in virtually all types of electronic devices and equipment, in the industrial, computing, automotive, consumer, telecommunications, military, aerospace, power supplies, and medical markets. Vishay's product innovations, successful acquisition strategy, and "one-stop shop" service have made it a global industry leader. Vishay can be found on the Internet at http://www.vishay.com.
News content originated from:http://www.vishay.com/company/press/releases/2018/2017TTIAsiaAward/


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Last week, my son, Ollie, took part in a music festival. We were so proud of him. He'd only taken up the bass clarinet this year, and here he was, playing in an orchestra with over 200 other intermediate school children at the town hall.
When the concert was over, Richard and I and our respective parents were standing at the top of the stairs waiting for Ollie to come and meet us. He dragged his huge instrument case up the stairs, while we congratulated him on his amazing performance, telling him how impressed we were.
Ollie stopped. He smiled. Then he announced, "I met a girl!"
When we got home, I told his sister about it, who responded "You know I've got a boyfriend too, right?"
And just like that, we've fallen into the next phase of our children's lives.
I hadn't anticipated that this phase would happen so quickly. Both have had "the talk" at school, and are learning about romantic relationships as part of their physical education syllabus. Amelia is well and truly through puberty and out the other side, but Ollie hasn't even sprouted his first hair at this stage!
He's always enjoyed the company of girls over the company of boys, even from a very young age. At about 5 or 6, he told us that he knew who he was going to marry (the very pretty blonde daughter of a good friend). When we go away skiing, he's always surrounded by a flock of girls. He's kind, he picks them up when they fall into the snow, and he likes that he doesn't feel like he needs to compete with them. He's always more relaxed with girls.
I've talked to Amelia a great deal about the challenges of adult relationships. About being treated with respect. About walking away from boys who are verbally, physically or mentally abusive. About setting your own timeline for progressing to a sexual relationship. About the importance of taking control of your own fertility. About how we will always be available when she wants to talk, or if she finds herself in trouble, night or day.
As a former teenage girl myself, I understand the minefield that is female teenage romance and sexuality. But I'm a bit in the dark with teenage boys.
I'm not sure how to construct a narrative that isn't inherently negative. How to teach him to treat women respectfully, without him feeling that I suspect he may do the opposite (which I don't, by the way). How to stay away from a girl who's had too much to drink. How to use his brain first and his penis second.
But equally importantly, how to make sure he's treated with respect. To make sure he walks away from girls who are verbally, physically or mentally abusive. To make sure he sets his own timeline for progressing to a sexual relationship. To make sure he knows the importance of taking control of your own fertility. To make sure he knows we will always be available when he wants to talk, or if he finds himself in trouble, night or day.
I guess the narrative is the same for both children, regardless of gender. They are both navigating the same challenges, just from opposite sides of the coin. But many of the challenges they face will be exactly the same. Their hearts will be broken, they will make mistakes, they will treat someone badly and be treated badly in return (although hopefully not too badly). And we will always be available when they want to talk, or if they find themselves in trouble, night or day.
In the same way that I would hate for either of them to have their behaviour judged on the basis of their gender, so I need to make sure we treat their emerging sexuality without gender bias.
I just thought I'd have a bit more time.
I created this dish as yet another way to get vegetables into my children's diets (yes, even as they head into their teenage years, they are still a challenge diet-wise). It's a bit lighter than a traditional bolognese sauce, so is well suited to our emerging spring days. Find the recipe here


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I modelled this little chap a few months ago but due to lack of free time recently have been unable to do much more until today. He is rigged, textured and raring to go! Hopefully get some nice little animations out of him soon.
Modelled and rendered using Autodesk Maya 2011.