text:
Transforming growth factor-β signalling controls human breast cancer metastasis in a zebrafish xenograft model
DOI: 10.1186/bcr3573
© Drabsch et al.; licensee BioMed Central Ltd. 2013
Received: 12 July 2013
Accepted: 21 October 2013
Published: 7 November 2013
The transforming growth factor beta (TGF-β) signalling pathway is known to control human breast cancer invasion and metastasis. We demonstrate that the zebrafish xenograft assay is a robust and dependable animal model for examining the role of pharmacological modulators and genetic perturbation of TGF-β signalling in human breast tumour cells.
We injected cancer cells into the embryonic circulation (duct of cuvier) and examined their invasion and metastasis into the avascular collagenous tail. Various aspects of the TGF-β signalling pathway were blocked by chemical inhibition, small interfering RNA (siRNA), or small hairpin RNA (shRNA). Analysis was conducted using fluorescent microscopy.
Breast cancer cells with different levels of malignancy, according to in vitro and in vivo mouse studies, demonstrated invasive and metastatic properties within the embryonic zebrafish model that nicely correlated with their differential tumourigenicity in mouse models. Interestingly, MCF10A M2 and M4 cells invaded into the caudal hematopoietic tissue and were visible as a cluster of cells, whereas MDA MB 231 cells invaded into the tail fin and were visible as individual cells. Pharmacological inhibition with TGF-β receptor kinase inhibitors or tumour specific Smad4 knockdown disturbed invasion and metastasis in the zebrafish xenograft model and closely mimicked the results we obtained with these cells in a mouse metastasis model. Inhibition of matrix metallo proteinases, which are induced by TGF-β in breast cancer cells, blocked invasion and metastasis of breast cancer cells.
The zebrafish-embryonic breast cancer xenograft model is applicable for the mechanistic understanding, screening and development of anti-TGF-β drugs for the treatment of metastatic breast cancer in a timely and cost-effective manner.
Transforming growth factor-β (TGF-β) system signals via serine/theronine kinase receptors and intracellular Smad transcriptional mediators to regulate a large number of biological processes [1]. Alterations of the TGF-β signalling pathway are implicated in many human diseases, including cancer (reviewed in [2]). Prior to tumour initiation and during the early stages of cancer, TGF-β often acts as a tumour suppressor; however at later stages it functions as a tumour promoter. As tumours develop they switch their response to TGF-β and utilise this factor as a potent promoter of cell motility, invasion, metastasis, and tumour stem cell maintenance (reviewed in [3, 4]). Multiple signal transduction pathways, involving a range of signalling molecules, determine the effects of TGF-β influence on multiple aspects of tumour growth and progression. Further research on how this cytokine is capable of being a tumour suppressor turned into a tumour promoter is important for the development and informed use of potentially powerful TGF-β targeted therapies [5].
Over the past decade, zebrafish (Danio rerio) has become an important animal model for cancer, immune and stem cell research [6–8]. Zebrafish are ideal for identifying clinically relevant genes and compounds that regulate tumour progression. There is conservation between zebrafish and mammals of many molecular and cellular components that operate during tumourigenesis. In addition, the physiological responses in zebrafish embryo to a wide range of pharmacologically active compounds are comparable to those in mammalian systems [9]. High resolution in vivo analysis of tumour progression and the interactions between tumour cells and the host microenvironment [10, 11] can be readily performed due to the transparency of zebrafish, in combination with the availability of various tissue-specific fluorescent reporter transgenic lines [12, 13]. Several tumour transplantation assays with human and mammalian cells to study different aspects of tumour malignancies in embryo and adult zebrafish, such as tumour cell migration, proliferation, angiogenesis and tumour cell extravasation [6, 12, 14–16] have been developed. Many of these assays are simplistic and are limited to one selected step of tumour development, and thus, do not represent the full complexity of tumourigenesis in one model. A rapid and reproducible zebrafish embryonic xenograft model for simultaneous formation of a localized tumour and experimental micrometastasis, by intravascular injection of tumour cells into the blood circulation of zebrafish embryos, has been recently described by the group of Snaar-Jagalska [17]. They have shown that with non-invasive high-resolution imaging, the critical steps of tumour progression, including tumour vascularisation and tissue invasion, can be characterized.
We applied this xenograft model and focused our studies on the effect of misregulation of TGF-β signalling components in breast cancer invasion and metastasis. We have used breast cancer cell lines of which, in previous studies, we and others have shown that the invasive and metastatic behaviour in spheroid invasion and mouse xenograft models is dependent on TGF-β [18]. We demonstrated that the invasive and metastatic behaviour, corresponding with the cell grade of malignancy can be recapitulated within the zebrafish. Moreover, the effects obtained after inhibiting with TGF-β receptor and Smad function in fish mimicked the effects observed in mice. Importantly, an effector role for matrix metalloproteinases (MMPs) in invasion and metastasis was demonstrated in this model. The differences in invasive properties upon dysregulation of TGF-β signalling components and its effectors are seen with clarity unprecedented in other animal models, making it applicable in a pipeline for new drugs discovery.
Material and methods
Reagents and cell culture
Human cell lines (293 T, 3 T3, and MDA-MB-231) were maintained cultured at 37°C in DMEM-high glucose containing L-glutamine, 10% FCS and 1:100 Penicillin/Streptomycin (Pen/Strep) (Gibco, Invitrogen, Blijswijk, Netherlands). The MCF10A-derived breast epithelial cell lines M1, M2, and M4 (MCF10A or M1, MCF10AT1k.cl2 or M2, and MCF10CA1a.cl1 or M4) were maintained as previously described [19]. Zebrafish cell lines, ZF4 and PAC2 were maintained according to the American Type Culture Collection (Manassas, VA, USA) recommendations. Briefly, zebrafish cells were cultured at 28°C in DMEM containing L-glutamine, 10% FCS and 1:100 Pen/Strep (Gibco, Invitrogen, Blijswijk, Netherlands). The 293 T, 3 T3, MDA-MB-231, ZF4 and PAC2 cells were originally obtained from American Type Culture Collection. MCF10CA1a.cl1 (M4) cells were kindly provided by Dr Fred Miller (Barbara Ann Karmanos Cancer Institute, Detroit, MI, USA).
Luciferase experiments
Cells were plated on day 0 in 24-well plates and transfected with constant total concentration of DNA in a given experiment on day 1. Cells were transfected with a TGFβ/Smad3-responsive promoter, firefly luciferase transcriptional reporter construct, CAGA12-luc [20], in conjunction with a β-galactosidase control for transfection efficiency. Cells were also transfected with zTGF-β1, reverse zTGF-β1, or an empty vector control. The same day, cells were either untreated or treated with TGF-β 1 ng/mL for 24 h. Cell lysates were harvested on day 2 and luciferase activity was measured. Experiments were performed in triplicate and repeated twice. The CAGA12 promoter-driven luciferase activity was normalized to the β-galactosidase activity, averaged, and plotted. Luciferase results were analysed by one-way analysis of variance (ANOVA). Representative experiments are shown.
Fluorescent cell labelling
Non-fluorescent cells were labelled with the fluorescent cell tracker CM-DiI (Invitrogen, Blijswijk, Netherlands) according to the manufacturer's instructions. Briefly, cells were grown to confluence in a T25-cm2 dish, and trypsinized. Subsequently, cells were washed with PBS, transferred to 1.5 ml Eppendorf tubes and centrifuged 5 min, at 1200 rpm. Cells were re-suspended in PBS containing CM-Dil (4 ng/ul final concentration). Cells stained with CM-Dil were incubated for 4 minutes at 37°C and then 15 minutes at 4°C. After this period cells were centrifuged for 5 minutes at 1,200 rpm, the supernatant discarded and cells re-suspended in media, centrifuged again and washed two times with PBS. Cells were suspended in DMEM or PBS for injection into the embryos.
Lentiviral transduction
Lentivirus was produced by co-transfecting an mCherry PLKO plasmid and helper plasmids pCMV-VSVG, pMDLg-RRE (gag/pol), and pRSV-REV into HEK293T cells. Cell supernatants were harvested 48 h after transfection and used to infect cells or stored at -80°C. For stable cell lines, cells were infected at 20% confluence for 24 h with lentiviral supernatants diluted 1:1 with normal culture medium in the presence of 5 ng/mL polybrene (Sigma, Zwijndrecht, Netherlands). At 24 h after infection, cells were placed under puromycin (1 μg/ml) selection for one week, or collected for injection at 1 day after infection.
Zebrafish maintenance
The Institutional Committee for Animal Welfare of the Leiden University Medical Center (LUMC) approved this study. Zebrafish and embryos were raised, staged and maintained according to standard procedures. The transgenic line Tg(fli1:GFP) was used in this study [11, 12].
Embryo preparation and tumour cell implantation
Dechorionized 2 days-post fertilisation (dpf) zebrafish embryos were anaesthetized with 0.003% tricaine (Sigma) and positioned on a 10-cm Petridish coated with 3% agarose. Single cell suspensions of fluorescent mammalian cells were re-suspended in PBS, kept at room temperature before implantation and implanted within 3 h. The cell suspension was loaded into borosilicate glass capillary needles (1 mm O.D. × 0.78 mm I.D.; Harvard Apparatus) and the injections were performed using a Pneumatic Picopump and a manipulator (WPI, Stevenage, UK). Approximately 400 cells (manually counted) were injected at approximately 60 μm above the ventral end of the duct of Cuvier (DoC), where the DoC opens into the heart. After implantation with mammalian cells, zebrafish embryos (including non-implanted controls) were maintained at 33°C, to compromise between the optimal temperature requirements for fish and mammalian cells [21]. For each cell line or condition, data are representative of at least three independent experiments with at least fifty embryos per group. Experiments were discarded when the survival rate of the control group was less than 80%.
In vivotoxicity test of chemical compounds
Chemical compounds were added to the eggwater at 2 dpf for toxicity tests, or 24 h post implantation (hpi) for treatment, and refreshed every second day. Chemical compounds were: LY-294002 (Cell Signaling, Leiden, Netherlands), SB-431542 (Sigma, Zwijndrecht, Netherlands) and Gm6001 (Calbiochem, Amsterdam, Netherlands). For toxicity tests, embryo survival or malformation was scored daily. For treatment, after 5 days embryos were fixed overnight in 4% buffered paraformaldehyde (PFA) at 4°C. Embryos were placed in a glass-bottom 96-well plate (Greiner Bio One GmbH, Frickenhausen, Germany), and imaged as described.
Microscopy and analysis
Fixed embryos were imaged in PBS-Tween. Fluorescent image acquisition was performed using a Leica MZ16FA stereo microscope, or a Leica SP5 STED confocal microscope. Confocal stacks were processed for maximum intensity projections with Leica software or Adobe Photoshop CS4 software. Images were adjusted for brightness and contrast using Adobe Photoshop CS4. Overlays were created using Adobe Photoshop CS4.
Whole-mount immunohistochemistry of zebrafish was carried out as described [22]. Primary anti-phospho-Smad2 (p-Smad2) antibodies (Millipore, Amsterda, Netherlands) (1:200 dilution) and secondary antibodies (Alexa 647 anti-mouse; Invitrogen, Blijswijk, Netherlands) (1:200 dilution) were used for detection. Immunohistochemistry of zebrafish cell lines required cells to be plated in 12-well plates on coverslips. The methods for immunohistochemical staining of p-Smad2 are described in the manufacturer's instructions. In brief, the slides were incubated with p-Smad2 antibody (1:2,000) overnight at 4°C, washed with PBS, and incubated with the secondary antibody (Alexa 488).
Invasion and micrometastasis formation of human breast cancer cell lines in zebrafish
The phenomenon of mammalian cell motility is important in the progression of cancer. Interestingly, M2 and M4 cells displayed a different invasive and metastatic profile than that seen with MDA-MB-231 cells. MDA-MB-231 cell invasion is visible as singular cells into the collagen matrix of the caudal tail, whereas M2 and M4 cell invasion is visible as a collective group within the caudal haematopoietic tissue (CHT) (Figure 1E). To explore the specificity of the events we observe with the invasive breast cancer cells, we analysed the behaviour of non-tumourigenic 3 T3 mouse embryonic fibroblasts and weakly tumourigenic 293 T human embryonic kidney cells within the zebrafish embryonic model. Upon transplantation into the DoC of 2-day-old zebrafish embryos, as above, neither the 3 T3 (n = 187), nor the 293 T (n = 162) cell lines showed invasion at 6 dpi (Figure 1F). MDA-MB-231 (n = 174) cells, transplanted as a positive control, showed invasion after 12 h, and 54% of fish displayed invasion at 6 dpi. The negative control, 293 T (n = 162), did not display invasion or micrometastasis. These data imply that the process of cell invasion and metastasis within a zebrafish model occurs in a specific manner and is not merely an artefact, nor is it a passive process. Importantly, the behaviour of relatively benign M1, to pre-malignant M2 and metastatic M4 and MDA-MB-231 cells as determined in mouse and spheroid invasion assays [18, 19, 24] correlates with the relative state of aggressiveness of these four cell lines in the zebrafish embryo xenograft assay.
Zebrafish and human TGF-β act cross species
Previously, we and others have shown that TGF-β has a critical role in driving invasion of M2 and M4 cells [18, 25] and metastasis of MDA-MB-231 cells to bone [26–28]. Considering that the zebrafish outperforms existing mouse models with respect to high-resolution imaging of the dynamic process of cell invasion during cancer progression, and is easily amenable to genetic and pharmacological screening, we therefore set out to investigate whether the xenograft breast cancer assay in zebrafish embryos can be used to determine the role of TGF-β signalling components in breast cancer invasion and metastasis.
Our assay system is heterologous. However, as zTGF-β1 is highly homologous to higher vertebrates (Additional file 1: Figure S1A) [29], the ligands may act cross species; host-derived zTGF-β may signal on human tumour cells, and tumour-cell-derived TGF-β may bind and activate TGF-β receptors on host zebrafish cells (Additional file 1: Figure S1B-D). The zTGF-β1 construct was capable of inducing TGF-β Smad3/Smad4 mediated luciferase output significantly in two human cell lines (P = 0.05) (Additional file 1: Figure S1 B and C). This indicates that the zTGF-β1 interacts with the human TGF-β receptor signalling pathway. Moreover, human TGF-β is capable of inducing phosphorylated Smad2 (pSmad2), and subsequent nuclear localisation of pSmad2, in zebrafish cell lines, ZF4 and PAC2 (Additional file 1: Figure S1D). Together, these data indicate that upon injection of human tumour cells into zebrafish embryos, the two heterologous cell types may mutually communicate with each other via TGF-β receptor signalling. Furthermore, as breast cancer tumour cells, such as MCF10A and MDA-MB-231, express TGF-β themselves [18, 30], we cannot exclude that human tumour cells may also mediate responses in the zebrafish embryos in an autocrine manner.
Small molecular TGF-β type I receptor kinase inhibitors mitigate invasion of human breast cancer cells in the zebrafish model
Human breast cancer cell lines, MDA-MB-231 and the MCF-10 series (M1, M2, and M4) were treated with or without the inhibitors for 24 h prior to transplantation into the zebrafish embryo (SB-431542 = 5 μM, LY-294002 = 5 μM or a vehicle control). Xenografted zebrafish were treated with the doses of the small molecular inhibitors found to be tolerable (SB-431542 = 5 μM, LY-294002 = 2.5 μM, or a vehicle control for 6 dpi). The zebrafish embryo water was changed every second day. Targeting the TGF-β pathway with small molecular inhibitors proved effective and reduced both invasion of the cancer cells, and also the development of micrometastasis (Figure 2B-C and Additional file 2: Figure S2). Furthermore, MDA-MB-231 cells that were not pretreated with the small molecular TRKI, but only treated after transplantation, also showed a reduction of invasion and micrometastasis (Additional file 3: Figure S3).
Phospho-Smad2 expression can be switched off with small molecular inhibitors targeting the TGF-β signalling pathway
Smad4 knockdown in breast cancer cells inhibits their invasion and metastasis in the zebrafish model
MMP inhibition
MMPs are known to have tumour-promoting and tumour-inhibitory effects but unfortunately, several clinical trials of broad-spectrum MMP inhibitors have failed to show promising effects [40]. The specific MMP2/9 Inhibitor II, has been shown to mitigate TGF-β induced invasion of breast cancer cells [18]. Human breast cancer cell lines, MDA-MB-231 and the MCF-10 series (M1, M2, and M4) were treated with or without the 100-nM MMP2/9 Inhibitor II for 24 h prior to transplantation into the zebrafish embryo. The xenografted zebrafish were treated with 50 nM of MMP2/9 Inhibitor II, a dose of the small molecular inhibitor found to cause minimal malformation and death, or a vehicle control for 6 dpi. The zebrafish embryo water was changed every second day. Inhibiting MMP2 and MMP9 halted the invasion of metastatic breast cancer cells in the embryonic zebrafish (Additional file 4: Figure S4).
Invasion and micrometastasis were only observed from breast cancer cells with metastatic potential. Interestingly, we found that M2 and M4 cells formed clusters of invasive cells mainly in the CHT region, where they are capable of proliferation (data not shown). In contrast, MDA-MB-231 cells were visible as single cells that migrated much more aggressively than M2 and M4 cells into the tail fin. It will be an interesting area of future research to determine what determines the phenotypic differences of cell migration. A recent publication has shown that ectopic expression of Slug or Snail is capable of promoting invasion of single, rounded amoeboid cells in vitro [42]. Furthermore, when M2 cells with Snail or Slug overexpression were implanted into the embryonic zebrafish xenograft model, single-cell invasion was also seen. This would suggest that epithelial to mesenchymal transition (EMT) is a potential requirement for single-cell migration into the tail fin. The degree of EMT within the cancer cells may also effect the communication of these cells with their environment.
Importantly, invasion could be seen at the single-cell level in the zebrafish embryo. The differences between invasive phenotypes (cells that cluster tightly together as opposed to cells that do not) are not able to be detected in larger animal models. To date, the most effective system to analyse the differences between the invasive phenotypes uses in vitro methods [43]. It is only due to the small, transparent nature of the zebrafish that such invasion could be seen at single-cell level in an animal model. The ability to view early-stage cell invasion in vivo provides an opportunity to examine the cell-cell junctions of tumours, and their environment.
We found that injected MDA-MB-231 cells showed active TGF-β/Smad signalling in vivo as measured by pSmad2 staining. Furthermore, we found that zTGF-β may act on human tumour cells, and tumour-cell-derived TGF-β may act on the host zebrafish. The confinement of pSmad2 staining to the tumour cells, and ability to inhibit metastasis by blocking TGF-β signalling in a tumour cell autonomous manner suggests that TGF-β produced by tumour cells acts on tumour cells to mediate cell invasion and metastasis. We were surprised not to see elevated pSmad2 staining in the host tissue surrounding the tumour cells if they secrete high levels of TGF-β. If zTGF-β would have contributed to mediating the invasion and metastatic response we would have also expected elevated pSmad2 staining in the host tissue surrounding the tumour. Further studies in zebrafish may highlight the effect that the stromal environment has on the early stages of metastatic development.
The zebrafish model described here allows simultaneous monitoring of tumour cell extravasation, invasion and micrometastasis in vivo within one week of implantation of human cells [17]. In other zebrafish models, cells have been transplanted into the hindbrain [21], liver, yolk [44–51], or the peritoneal cavity [11, 47, 52] in order to determine the invasive or metastatic potential of cancers. Although these techniques have many advantages, they also have limitations. Many of these studies show cell invasion and metastasis, which may be explained, not by the invasive properties of the cell, but by the accidental injection of cells into the blood circulation of the embryo. In some cases, invasion was shown as a singular cell that was located within the dorsal aorta. For the zebrafish embryo to be considered a dependable tool for use in investigating the process of invasion and metastasis of cancer cells, we believe that injecting cells into the DoC provides the most reliable assay. In our model, micrometastasis formation only occurs following the MDA-MB-231 cell invasion into the tail fin from the posterior end of the CHT. Previous work has shown that this invasion site is determined by the physiologic migration of neutrophils, thus supporting the so-called seed-and-soil hypothesis of tumour metastasis [17]. Furthermore, by limiting the area that we look at for invasive cells, the amount of human error is reduced. Transplanting cells into the DoC, which subsequently form a metastasis, has been shown to be controlled by the tumourigenic property of disseminated cells (as shown in Figure 2), and the microenvironment [17]. However, it is important that the conditions are optimised for each cell type, including cell number, in order that the results are reliable and reproducible.
This unique animal system provides a visual window into the metastastic process in a live vertebrate animal, with unprecedented clarity. The experiments described here establish the basis for the future development of a screening methodology for drugs that inhibit invasion and metastasis of breast cancers. Furthermore, the data also shows that transient transfection of siRNAs may be used to examine the effect of invasion and metastasis (Figures 5 and 6). We have previously used the embryonic zebrafish xenograft model to study novel regulators of the TGF-β signalling pathway, TNF receptor-associated factor 4 (TRAF4) [53] and ubiquitin-specific protease 4 (USP4) [54], as well as the tumour suppressor FAF1 [55], which interacts with the FAS ligand. The zebrafish offers a promising future for functional studies of breast cancers.
Studies of the efficacy of pharmacology and toxicology in murine xenograft models normally use tumour growth, body weight loss and mortality as parameters of toxicity. These studies are cumbersome and time consuming, and drug activity against xenografts does not always correlate with its clinical activity [56]. As seen in Figure 2, the parameters of drug dosage can be quickly and easily visualized in the zebrafish. Furthermore, small molecular compounds can be added directly to the environment of the zebrafish, which can be less stressful to the both the animal, and technician, compared to the injection techniques used in rodent models.
One potential therapy using MMP inhibition was analysed in this study using the zebrafish model. MMPs have a critical role in inflammation and tumourigenesis and appear to be ideal as a drug target. Many inhibitors have been developed, and several have gone as far as clinical trials in cancer patients. Unfortunately, though these inhibitors have showed promising effects in preclinical studies, the same agents did not have such a positive outcome in cancer treatment [2, 57]. This highlights the issue of complexity that the MMPs play in cancer progression. In this study, we used GM6001, a broad-spectrum MMP inhibitor. GM6001 has been previously tested in the development of zebrafish [53, 54]. These studies have shown the importance of MMPs during embryonic development [54] and fin regeneration [53]. We were able to show that MMP inhibition was capable of reducing the amount of invasion and metastasis of human mammary carcinoma cells in the zebrafish. It must be noted that MMP inhibition was initiated at the early stage of cancer metastasis. It had been previously hypothesised that, as MMPs contribute to multiple stages of tumour progression, MMP inhibition is most beneficial at the early stages and the therapeutic benefit decreases as the disease progresses [58]. Accordingly, many studies have shown that disruption of the TGF-β signalling early in metastasis can substantially reduce metastasis burden and that the effect becomes less effective when lesions become well-established [59–61]. It has been argued that well-established bone lesions may become less dependent on bone destruction and TGF-β signalling and, as a consequence, become less sensitive to TGF-β inhibitors [59].
The dysregulation of TGF-β is well known in human breast cancer. Thus, the TGF-β pathway is an attractive therapeutic target. With its own advantages and disadvantages, the zebrafish embryo xenograft model represents a novel tool for investigating the tumour invasion/metastasis process. Furthermore, the zebrafish embryo xenograft model is exploitable for drug discovery and gene targeting.
Caudal haematopoietic tissue
Dulbecco's modified Eagle's medium
- DoC:
Duct of cuvier
- dpf:
Days post fertilisation
Epithelial mesenchymal transition
FAS-associated factor 1
Fas (TNF receptor superfamily, member 6)
Fetal calf serum
Green fluorescent protein
- hpf:
Hours post fertilisation
- hpi:
Hours post implantation
Matrix metalloprotease
Phosphate-buffered saline
- pSmad2:
Phosphorylated Smad2
- Tg:
- TGF-β:
Transforming growth factor β
TNF receptor-associated factor 4 (TRAF4)
TGF-β receptor kinase inhibitor
Ubiquitin-specific protease 4
- zTGF-β:
Zebrafish transforming growth factor β.
We are grateful to Fred Miller (Barbara Ann Karmanos Cancer Institute, Detroit, USA) for the gift of MCF10a cell lines. We would like to thank Ken Iwata (OSI Pharmaceuticals, New York, USA) for TGF-β3 and Chung Peng, Department of Biology at York University, Toronto, Canada for the zTGF-β1 plasmid. We would also like to thank Hans van Dam, Yihao Li, and Chao Cui for many useful discussions. This study was supported by Leducq Foundation, Cancerfonden, Centre for Biomedical Genetics and Cancer Genomics Centre Netherlands and in part by the Key Construction Program of the National "985" Project and Zhejiang University Special Fund for Fundamental Research, as well as the Fundamental Research Funds for the Central Universities.
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- Zhang L, Zhou F, Li Y, Drabsch Y, Zhang J, van Dam H, ten Dijke P: Fas-associated factor 1 is a scaffold protein that promotes β-transducin repeat-containing protein (β-TrCP)-mediated β-catenin ubiquitination and degradation. J Biol Chem. 2012, 287: 30701-30710. 10.1074/jbc.M112.353524.View ArticlePubMedPubMed CentralGoogle Scholar
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This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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Florida Beach Indoor Resort Wedding Venues
Elegant Sarasota Indoor Wedding Venues
The Resort at Longboat Key Club offers some of the best wedding and reception venues in Florida, making it a top choice for Sarasota weddings. Let your dreams become reality in any of our indoor wedding venues. Let The Resort at Longboat Key Club create your perfect day down to the last detail.
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You aren't just limited to indoor Florida resort wedding venues at The Resort at Longboat Key Club. We've also got a stunning variety of outdoor options. Being planning your Sarasota wedding at The Resort at Longboat Key Club today or contact us directly at 800.237.8821, ext. 1605.
Read all of our wedding reviews on our The Resort at Longboat Key Club Storefront at

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- Home
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Sand Dollar offers some of the most spacious and comfortable studio, one, two and three bedroom condominiums on Bonaire. Private porches offer striking views of the Caribbean Sea. Read more…
PADI 5 Star Dive Friends Bonaire, Sand Dollar's on-property dive partner, brings you the best diving and services that Bonaire has to offer for shore and boat diving as well as dive courses.
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Our Bonaire condominium resort complex includes a pool & pool bar, restaurant, sea side lounging areas across the expansive waterfront, two tennis courts, deli, ice cream parlor, grocery store and bakery. Read more….
Our reservations specialist have the expertise to help you plan your trip, book your condo, diving and truck rental and practically pack your suitcase for your ideal Bonaire vacation package. Contact us today.


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About Desertrain
Weather and nature mostly.
By: Desertrain , 6:09 PM GMT on July 13, 2014
Monsoon rainfall 1.98" to date.
Nearby areas, (within a few miles), have had much more.
Usually we receive nearly half of the monsoon rain total here in the first 2 weeks, sometimes have had 6-8" by now, then areas north and west receive theirs.
Last year Tucson had a mediocre monsoon while Douglas Arizona to the southeast had its wettest on record. In my quarter+ century of witnessing the AZ version of the Southwest monsoon, no two years have been alike. Often during the 2 months of the heart of the monsoon persistent patters will seem to hold throughout with occasional exceptions. A valley will experience repeated heavy rainmakers, or will a particular mountain range. You may receive repeated quarter inch rainfalls and 8 miles away might see repeated 2-3" or more deluge.
Being a gardener in an arid landscape often has me wanting more. Ive been working on terracing the land to retain more H2O to keep the cottonwoods and other native trees alive along with fruit trees.
The views of the author are his/her own and do not necessarily represent the position of The Weather Company or its parent, IBM.
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Receive the latest in Footwear, Fashion, Music and Creativity in our newsletters.
Us Versus Them shows off its upfront Californian sensibility in this latest lookbook for 2013 spring/summer. Shot in the laid back customary surroundings of Southern California — corner stores, beach fronts and car parks – this season's garments again showcases the brand's blend of urban and beatnik design cues. While graphic-heavy designs across a number of T-shirts and vests are prominent, a selection of plaid and pinstripe shirts offer more dapper options. Rounded up by a number of trucker caps and snapbacks, the capsule delivery is another street-ready dispatch fitting for the summer months ahead. Head over to the Us Versus Them online store for the full collection.


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Click here to view 2017 Exhibiting and Sponsorship Opportunities for CLEAN GULF in Houston, TX!
The need to be prepared for a spill isn't an option…it's a necessity. CLEAN GULF is the annual event that brings together the entire spill prevention, preparedness and response community, including State and Federal Regulators, to establish and build the relationships you need in place before an incident on water or land occurs. It's your opportunity to come together with all the people you work closely with on a response and connect with those who will play a role in future incidents.
The 2016 conference agenda covered best practices and lessons learned in spill prevention, preparedness and response for the exploration & production, transportation, and storage of oil & hazardous materials. The agenda consisted of six conference tracks covering planning and preparedness, response, inland preparedness and response, subsea, emerging trends and information/response technologies and research. Download a PDF of the 2016 conference program here.
"Clean Gulf remains the best venue for professional networking, actively engaging the experts, and learning about best practices, lessons learned, and emerging technologies." – Joe Leonard, Jr., CDR, USCG (ret), Senior Consultant, CTEH"
"Clean Gulf provides me with the latest information on emerging response technologies,critical incident lessons learned and preparedness trends." – Zachary Pickett, U.S. Coast Guard


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Whole Wheat Zucchini or Carrot Bread
Total Time
Prep 10 mins
Cook 1 hr
You won't believe it's an all whole wheat quick bread! Lower in sugar (honey) that most recipes, this zucchini bread is fabulous. Low fat seekers can sub 1 cup of applesauce for oil. I have not made the carrot bread variety. From Whole Foods for the Whole Family.
Ingredients Nutrition
- 1 1⁄2 cups zucchini or 1 1⁄2 cups carrots, grated
- 1⁄2 cup oil or 1⁄2 cup butter, melted
- 1⁄2 cup honey or 1⁄2 cup brown sugar
- 2 eggs, beaten
- 1 teaspoon vanilla
- 1 1⁄2 cups whole wheat flour
- 1⁄2 teaspoon baking soda
- 1⁄2 teaspoon baking powder
- 1⁄2 teaspoon salt
- 1 1⁄2 teaspoons cinnamon
- 1⁄2 cup nuts, chopped
- 1⁄2 cup raisins (optional)
- 1⁄2 teaspoon nutmeg
- Beat together oil and honey, then add eggs, vanilla and zucchini.
- Sift dry ingredients and add to zucchini mixture.
- Add nuts last.
- Bake in a greased 9" loaf pan at 350F for 45 to 60 minutes.
Most Helpful
I did this one with both 2 cups of zucchini and 2 mashed overripe bananas, and used pumpkin pie spice instead of individual cinnamon and nutmeg (I know, I know). It turned out really moist and delicious, and not overly sweet. Probably my favorite version ever. I used honey and coconut oil.
Delicious recipe-- better than I expected actually :-) good texture (in spite of all whole wheat flour) and great flavor
Great recipe! Want to try more healthy alternatives such as coconut oil, applesauce, and brainless flours like coconut or almond flour.. Made a double recipe and used up all of my juicer pulp. Kale, chard, carrot, apple, strawberry, Asian pear, kiwi, beets, and broccoli! Along with walnuts, I added about 1/4 cup of flaxseeds to round off the cup of nuts/seeds. Every one loves it! It's going fast and is great with my morning juice!


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It's too hot to do much, but I did some deadheading just now. Deadheading
performs one extra benefit. It gives you a chance to look more closely at
things. In two cases today it was a good thing I did. The dime-a-dozen
Bloody Cranesbill had engulfed the Double Tangerine Poppy. This is a
fantastic little poppy that blooms it's head off most of the season, The
brilliant flowers are supported way above the leaves on wiry stems. But the
leaves were completely covered up by the geranium, so I ripped out a chunk
to give the poppy some breathing room. Just a few feet away was my Anemone
'Bodnant Burgundy', a shortish, fall blooming variety. It didn't move at
all the first few years, but now it's starting to spread abit, which I'm
thrilled about. However, on closer inspection it was coming up through
Thymus 'Doretta Klaber' which had just begun to bloom. This is the
teeniest, tinyest thyme I've ever seen and I don't want to lose it. So, out
came the anemone. Fortunately the stems hadn't mixed so no damage to the
thyme and I got plenty of anemone root so I could pot them up.
neIN, Z5
To sign-off this list, send email to email@example.com with the
message text UNSUBSCRIBE GARDENCHAT


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Join Us
Unity Campaign '16 Youth Electoral Organizing Initiative
Black Youth Vote! (BYV!) is the youth and young adult civic leadership development and organizing arm of the National Coalition. The BYV! #Vote4Justice Organizing Initiative's goal is to continue it's legacy of engaging black youth in civic engagement, issue organizing, voter empowerment and voter protection. When BYV! began in 1996, voter turnout for young people was extremely low averaging 17-34 percent. However, over the past decade black youth have led voter turnout in presidential election years, voting at 45 percent in 2012. BYV! Is one of the leading youth led movements focused on training a new generation of black civic leaders, elected officials, advocates, activists and organizers. BYV!'s priority is to connect to protest to public policy through the power of the vote.
Tactics used for the #Vote4Justice will include: ❏ HBCU Campus/Community Voter Engagement Challenge ❏ Voter Registration efforts, particularly on college campuses ❏ Ensuring that Registered Voters and Potential Voters have Proper Identification ❏ Nonpartisan Voter Education and Training ❏ Identification Polling Locations ❏ Get Out the Vote Efforts (GOTV) on targeted campuses and communities ❏ Distribution of Voter Protection Resources (Websites, poll workers, poll monitors, social media interaction) ❏ Sparking circular community engagement (students to families)
Target States ❏ Alabama ❏ Florida ❏ Georgia ❏ North Carolina ❏ Maryland ❏ Michigan ❏ Ohio ❏ Pennsylvania ❏ Virginia ❏ Washington DC
TAKE ACTION with #Vote4Justice
CONNECTING with #Vote4Justice via SOCIAL MEDIA
We will energize, create excitement, motivate and galvanize Black youth to get out the vote in 2016 in record numbers through various tactics to include social media by:
1. Promoting the work of our #vote4justice partners and BYV! State Coordinators 2. Connecting black youth in our targeted states to tools and resources preparing for Election Day 2016. 3. Building the narrative online that black youth vote!
HBCU/Community Voter Engagement Challenge
The goal of the HBCU/Community Voter Engagement Challenge is to REGISTER, EDUCATE, and MOTIVATE students/youth to vote at HBCU's other colleges/universities and surrounding communities; and help to PROTECT voters on campus and surrounding communities by volunteering to be poll workers or poll monitors.
✓ Criteria of the HBCU/Community Challenge will be a percentage-based contingent upon the population of students who are registered at the respective institution and registration will be tallied through "Pledge Cards" (which consists of a signature that states you will go out and vote on or before Election Day 11/8/16 and volunteer to be a poll worker or poll monitor).
✓ All schools will be recognized by the National Coalition on Black Civic Participation (NCBCP) for participating in the HBCU/Community Challenge on their campus.
✓ The HBCU/Community Challenge winner that proportionally has the most students, registered to vote and sign up to volunteer to be poll workers or poll monitors in their local communities in 2016 will receive the Black Youth Vote! Campus of the Year Award and recognized by the NCBCP and our media partners (Radio One, Essence, NNPA)
✓ The HBCU/Community Challenge Activities include: ○ Campus Voter Registration, GOTV, Voter Protection ○ Absentee Ballot Campaign ○ Poll Worker Recruitment ○ Poll Monitor Recruitment ○ Daily Engagement via hashtags #Vote4Justice, #HBCUunity16 ○ Social Media Promotion ○ Promote HBCU/Community Challenge on Campus and in Community
The HBCU/COMMUNITY Challenge LAUNCH DATE is AUGUST 6th.
#BLACKMENVOTE -- "From PROTEST to POLICY and to the POLLS"
The Black Youth Vote! BMI will mobilize moving from them PROTEST to POLICY and NOW to the POLLS 2016 through use of voter engagement and the collection of our BYV! BMI Pledge Card. Collected cards received will provide the BYV! Coordinators and partners to mobilize other black men based upon pledge commitments to include: voter registration, voter education, voter protection and GOTV.
Click this link to sign the BYV! Black Men Vote "Pledge Card" https://goo.gl/OybhwR
For additional information about #Vote4Justice Campaign please contact the Black Youth Vote! Team 202.659.4929
Deven D. Anderson Unity '16 Senior Advisor & BYV! Lead Organizer firstname.lastname@example.com
Brianna Patterson Black Youth Vote! Civic Engagement National Coordinator
Daniel Bradley BYV! Black Male Initiative National Coordinator
Sherman Justice BYV! Black Male Initiative Fellow
Savannah Campbell BYV! Collegiate Interns
Darrell Coles BYV! Collegiate Interns


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Publisher's Description+
What's new in this version:
*Address entry for credit card payments.*Fixed consolidated closeout error.*Fixed drawing of cut appointments when pasting into a different day or week.*Fixed note entry on iPads.*Fixed some crashes sending email.*Fixed stretched week view for 15 minute intervals.*Fixed transaction gift certificate not showing discounts.*Localized dollar signs for international currencies (does not affect entered amounts).*Localized time to display 24 hour time for those regions.*Localized weekday headers on cal... See all new features »


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Community Preschool of Whitefish Bay Sponsoring Community Book Drive for Next Door Foundation
Community Preschool of Whitefish Bay is sponsoring a community book drive for the Next Door Foundation's Books for Kids program. New and gently used children's books for babies through young adults will be accepted gratefully. However, the greatest need is infant/toddler books and early read-to-me-type books by authors such as Eric Carle, Bill Martin Jr., and Dr. Seuss.
Donations can be made directly at Community Preschool of Whitefish Bay from Mondays through Thursdays, starting Monday, January 28th – Thursday, February 7th between 9:00 am – 3:00 pm. A collection bin will be located outside the classroom.
The Next Door Foundation's Books for Kids program was established in 1990 with the hope that all children in Milwaukee's central city, regardless of family income or education levels, could have books to call their own. To learn more about the Books for Kids program, visit www.nextdoormil.org/NextDoor/Our-Programs/Books-for-Kids.htm.
What: Community Book Drive to support Next Door Foundation
Where: Community Preschool of Whitefish Bay
School is located in Garden Level of Christ Church
5655 N Lake Drive, Whitefish Bay
Best parking on Beaumont Avenue or in Sendik's parking lot
When: Mondays – Thursdays, 9am – 3pm
Monday, January 28th – Thursday, February 7th
Community Preschool of Whitefish Bay is a non-profit parent cooperative preschool, which provides families with the opportunity to be an integral part of their child's first school experience.
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- Concordia University Wisconsin's Early Childhood Literacy Festival features Grafton author
- Concordia University Wisconsin Computer Science Tech Camp unleashes children's creativity
- Cigar Networkers of Milwaukee Present Smoke on the Water 2!
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- You Are Here:
- Home
- Your Government
- Economic / Community Development
- Housing & Citizen Services
Housing & Citizen Services
Date of Publication: September 30, 2016
City of Pueblo
One City Hall Place
Pueblo, CO. 81003
These notices shall satisfy two separate but related procedural requirements for activities to be undertaken by the City of Pueblo.
On or after October 17, 2016 the City of Pueblo will submit a request to the U.S. Department of Housing and Urban Development for the release of Community Development Block Grant and HOME Investment Partnership Act funds under Title I of the Housing and Community Development Act of 1974 P.L. 93-383, and Title II of the National Affordable Housing Act of 1990, as amended, to undertake the following project:
Community Development Block Grant Program Funds
CD1609 ADA Ramps-Eligible Census Tracts $400,000
Construction of sidewalk ramps at approximately 39 street intersections to meet Americans with Disability Act requirements.
No. Street Intersection Corners
1 Lamar Ave & Midway Ave NW, NE, SE
2 Lamar Ave & King Ave NE, SE, SW
3 Berkley Ave & Corwin Ave NE, NW, SE, SW
4 Colorado Ave & Orman Ave N, S, E, W
5 Colorado Ave & Grant Ave N, S, E, W
6 Colorado Ave & Pitkin Ave N, S, E, W
7 Colorado Ave & Routt Ave N, E, W
8 Colorado Ave & Evans Ave N, S
9 Colorado Ave & Abriendo Ave S
10 Prairie Ave & Lakeview Ave NE, NW, SE, SW
11 Prairie Ave & Columbine St NE, SE
12 Columbine St & Lakeview Ave NW, NE
13 Prairie Ave & Wheatridge Dr. NW, SW
14 Prairie Ave & Tucci Ln NE, NW, SE, SW
15 Prairie Ave & Oneal Ave NE, NW, SE, SW
16 Prairie Ave & Holmes St NE, SE
17 Prairie Ave & Aster St NW, SW, NE
18 Jackson St & Pitkin Ave N, E
19 Jackson St & Routt Ave N, S, E, W
20 Jackson St & Evans Ave S, E, W
21 Jackson St & Abriendo Ave N, S, E, W
22 Jackson St & Abriendo Ave 2 Ea @ both medians
23 Abriendo Ave Quincy St. N, S, E, W
24 Abriendo Ave Quincy St. 2 Ea @ both medians
25 Broadway Ave & Routt Ave S, E, W
26 Broadway Ave & Pitkin Ave S, E, W
27 Broadway Ave & Grant Ave N, S, E, W
28 Broadway Ave & Orman Ave N, S, E, W
29 Broadway Ave & Adams Ave N, S, E, W
30 Mesa Ave & Van Buren Ave NE, NW, SE, SW
31 Mesa Ave & Acero Ave NE, NW, SE, SW
32 Mesa Ave & Gaylord Ave NE, NW, SE, SW
33 Santa Fe Ave & 14th St NW
34 Greenwood Ave & 10th St NE, NW, SE, SW
35 Greenwood Ave & 11th St NE, NW, SE, SW
36 Greenwood Ave & 12th St NE, NW, SE, SW
37 Greenwood Ave & 13th St NE, NW, SE, SW
38 Greenwood Ave & 14th St NE, NW, SW
39 Greenwood Ave & 15th St SE, SW
CD1610 Portland Park Shelters $68,209
1700 Block of East 19 Street – Eastside Neighborhood.
Installation of one park shelter and the accompanying ADA accessible route, relocation of irrigation system, electrical service and lighting, installation of new picnic tables.
CD1612 St Anne's Park Ball Field Rehabilitation $115,993
2700 E. 12 Street -Eastside Neighborhood
The project will undertake the rehabilitation of the ballfield to include the removal of substandard structures to provide improved playing conditions to area residents and youth baseball teams. Improvements to be undertaken are construction of the backstop, removal and replacement of broken chain-link fencing, construction of a new announcer's booth and new infield mix.
CD1613 Installation of Streetlights $6,000
2500 & 2600 Blk. E. 9 St & 2600 Blk. E. 10 St
Installation of three cobra head streetlights on the above listed streets. At present there are no streetlights on these blocks. Improvements will provide for safer pedestrian and vehicular transit.
On or after October 17, 2016 the City of Pueblo will submit a request to the U.S. Department of Housing and Urban Development for the release of Community Development Block Grant and HOME Investment Partnership Act funds under Title I of the Housing and Community Development Act of 1974 P.L. 93-383, and Title II of the National Affordable Housing Act of 1990, as amended, to undertake the following project:
This is a multiyear project 2016 to 2019 that will undertake property acquisition, disposition, clearance, and rehabilitation of single-family homes. Properties are yet to be identified. The funding sources are identified below.
CD1611 Abatement of Unsafe Structures $100,000
The project will undertake the clearance of approximately 2 unsafe abandoned and condemned structures in low and moderate-income neighborhoods on a spot slum/blight basis.
CD1614 Emergency Repairs and Essential Services for Seniors $45,000
Repairs to 9 Income Eligible Senior Households - Citywide
CD1615 Minor Repairs Program $75,000
Repairs to 15 Income Eligible Households- Citywide
CD1616 Owner-Occupied Rehabilitation Program $50,000
Rehabilitation of 2 Homes of Income Eligible Households –Citywide
HO1630 Pueblo County Owner Occupied Rehabilitation Program $53,558.99 Rehabilitation of 2 Homes of Income Eligible Households – Outside City Limits
The City of Pueblo has determined that the project will have no significant impact on the human environment. Therefore, an Environmental Impact Statement under the National Environmental Policy Act of 1969 (NEPA) is not required. Additional project information is contained in the Environmental Review Record (ERR) on file at the Department of Housing and Citizen Services, 2631 E. 4 St., Pueblo, Colorado, 81001 and may be examined or copied weekdays 8 A.M to 4:30 P.M.
Any individual, group, or agency may submit written comments on the ERR to the Department of Housing and Citizen Services, 2631 E. 4th St., Pueblo, CO., 81001. All comments received by October 17, 2016 will be considered by the City of Pueblo prior to authorizing submission of a request for release of funds. Comments should specify which Notice they are addressing.
The City of Pueblo certifies to the U.S. Department of Housing and Urban Development that Stephen G. Nawrocki in his capacity as President of the City Council consents to accept the jurisdiction of the Federal Courts if an action is brought to enforce responsibilities in relation to the environmental review process and that these responsibilities have been satisfied. The U.S. Department of Housing and Urban Development approval of the certification satisfies its responsibilities under NEPA and related laws and authorities and allows the name of grant recipient to use HUD program funds.
The U.S. Department of Housing and Urban Development will accept objections to its release of funds and the City of Pueblo's certification for a period of fifteen days following the anticipated submission date or its actual receipt of the request (whichever is later) only if they are on one of the following bases: (a) the certification was not executed by the Certifying Officer of the City of Pueblo; (b) the City of Pueblo has omitted a step or failed to make a decision or finding required by HUD regulations at 24 CFR part 58; (c) the grant recipient or other participants in the development process have committed funds, incurred costs or undertaken activities not authorized by 24 CFR Part 58 before approval of a release of funds by the U.S. Department of Housing and Urban Development; or (d) another Federal agency acting pursuant to 40 CFR Part 1504 has submitted a written finding that the project is unsatisfactory from the standpoint of environmental quality. Objections must be prepared and submitted in accordance with the required procedures (24 CFR Part 58, Sec. 58.76) and shall be addressed to the U.S. Department of Housing and Urban Development, Region 8 Office, at 1670 Broadway Street, Denver, Colorado, 80202-4801, 303-672-5440. Potential objectors should contact the U.S. Department of Housing and Urban Development to verify the actual last day of the objection period.
Mr. Stephen G. Nawrocki
President of the City Council
One City Hall Place
Pueblo, Colorado, 81003
Minor Repairs Program
City of Pueblo, Department of Housing and Citizen Services has funds available for Minor Repairs applications. To qualify for the program, applicants must of low to moderate-income homeowners. Have owned the property for the past two years, property taxes must be current and the property must be free from all liens. The property must be the applicant's primary residence. Eligible Minor Repairs include Mechanical: Repair or replacement of non-functioning heat system, Electrical System: repair or replace unsafe wiring, outlets, breakers, Plumbing System: repair or replacement of water heater, Safety and Security: Exterior doors, exterior door locks and dead bolts, window locks and broken windows, Weatherization: Insulation, door and window weather-stripping, doors and window replacement, roofing, Handicap accessibility: Grab bars, rails, ADA compliant, Exterior Residence Appearance: Exterior painting.
Applications are available from July 5, 2016 thru July 19, 2016 at the Department of Housing and Citizen Services, 2631 East 4th Street, Pueblo, CO 81001 or online, Minor Repairs Application. You may call, 719-553-2850, to request an application be mailed to you.
Addressing the Community's Needs
- Economic development
- Education
- Human services delivery systems
- Infrastructure
- Neighborhoods
- Planning
NSP Single Family Homes For Sale
For more information please contact our Loan Coordinator:
Kevin Ortiz
The City of Pueblo Housing and Citizen Services Department is being recognized for a project it developed that helps an often ignored segment of our population.
Housing Colorado NOW! has presented an "Eagle Award" to the city's Housing and Citizen Services Department for the homeless veterans housing project it completed in January of 2013, on East 4th Street.


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Book Writing
- Status Closed
- Budget $15 - $25 USD / hour
- Total Bids 16
Project Description
A non-fiction English book of 150 pages is to be rewritten - the book pertains to biography, religion, history and culture. The current form is meant for academic and research purposes and the language is therefore quite complex. The new version to be produced is intended for lay readers and hence need more simple English.
We will need from you:
1. Sample writings, if possible of non-fiction
2. Detail of earlier similar work undertaken
3. Approximate time required for the project
4. Rate
I have yet no idea of the prices/rates and so am keeping it open-ended.Get free quotes for a project like this
Skills Required
Looking to make some money?
- Set your budget and the timeframe
- Outline your proposal
- Get paid for your work
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text: KRUSH OFFICIAL Krush Music New York City KRUSH is a music production & songwriting house out of New York City. We produce and write for artists, labels, DJs, Film, TV & licensing companies. For all enquiries, please send an email to: firstname.lastname@example.com KRUSH OFFICIAL's tracks B.o.B - Ray Bands (Krush Trap Remix) by KRUSH OFFICIAL published on 2012/08/28 01:39:14 +0000


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If you want to pay your bill, please visit Dillards.com/payonline.
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Comments about Tween Diva Big Girls 7-16 Lace Popover Dress:
Attached necklace fell apart. Returned it.
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post #34126 of 122416
8/30/12 at 8:05pm
Not my size, but still awesome.
Mostly odd jackets and trousers, sometimes with jeans on the weekends. Not too slim cut. Don't the Indy's look a bit casual for a jacket and tie?
I think I'd probably need a 10E or 10.5E in the Aberdeen, and both are sold out.


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Southern District of Intellectual Property Cases
Cases filed
Cases 1 - 10 of 383
ACCU Steel, Inc v. Legacy Building Solutions, Inc.
as 1:2016cv00050
Plaintiff: ACCU Steel, Inc.
Defendant: Legacy Building Solutions, Inc.
Counter_claimant: Legacy Building Solutions, Inc.
Counter_defendant: ACCU Steel, Inc.
Cause Of Action: Patent Infringement
JM Enterprises, Inc. v. The Ames Companies, Inc.
as 4:2016cv00626
Plaintiff: JM Enterprises, Inc.
Defendant: The Ames Companies, Inc.
Cause Of Action: Patent Infringement
RCD Enterprises, Inc v. Stubby's Real Pit Bar-B-Q
as 4:2016cv00595
Plaintiff: RCD Enterprises, Inc
Defendant: Stubby's Real Pit Bar-B-Q, Scott Stubblefield, John Does 1-25
Cause Of Action: Trademark Infringement
Confluence v. Confluence Consulting, Inc.
as 4:2016cv00539
Plaintiff: Confluence
Defendant: Confluence Consulting, Inc.
Cause Of Action: Trademark Infringement
Confluence v. Confluence Design Group, LLC
as 4:2016cv00537
Defendant: Confluence Design Group, LLC
Plaintiff: Confluence
Cause Of Action: Trademark Infringement
Golden et al v. Clear Advantage Marketing et al
as 4:2016cv00529
Plaintiff: G2 Database Marketing, Inc. , Glenn Golden
Defendant: Clear Advantage Marketing , Mark McKamey , Tim Bollmann and others
Cause Of Action: Copyright Infringement
Type: Intellectual Property › Copyrights
Freed et al v. Bagel Labs, Inc.
as 4:2016cv00499
Plaintiff: Jeffory D. Freed , Herbert O. Dixon, Jr. , Burton D. Johnson and others
Defendant: Bagel Labs, Inc.
Cause Of Action: Patent Infringement
The Iowa Clinic, P.C. v. Iowa Pain Clinic, LLC et al
as 4:2016cv00484
Plaintiff: The Iowa Clinic, P.C.
Defendant: Iowa Pain Clinic, LLC, Central States Medicine, PLLC
Cause Of Action: Trademark Infringement
Gary W. Clem, Inc. v. Oxbo International Corporation
as 4:2016cv00398
Plaintiff: Gary W. Clem, Inc.
Defendant: Oxbo International Corporation
Cause Of Action: Patent Infringement
Pella Corporation v. Clopay Building Products Company, Inc.
as 4:2016cv00294
Plaintiff: Pella Corporation
Defendant: Clopay Building Products Company, Inc.
Cause Of Action: Trademark Infringement (Lanham Act)
Disclaimer: Justia Dockets & Filings provides public litigation records from the federal appellate and district courts. These filings and docket sheets should not be considered findings of fact or liability, nor do they necessarily reflect the view of Justia.


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Permanent Account Number(PAN) is a unique number, issued to all the entities identifiable under Indian Income Tax Act 1961 .It is equivalent to a National Identification Number and also serves as an Important identity proof.
PAN Card is issued by Indian Income Tax Department under the supervision of Central Board of Direct Taxes.
PAN Service is also offered under CSC Scheme through apna.csc.gov.in portal. SCAs /VLEs should register to apna.csc.gov.in portal to access the PAN Service.
Process Flow:
i. Fill Pan Application Form.
ii. Upload the Pan Form, Photo, Signature and the Supporting document as per scanning guidelines.
iii. Make Payment.
iv. Generate Acknowledgement Receipt.
v. Create Batch on regular intervals having at least one Application.
vi. Send courier, having all the required Hard Copies and the Batch Detail Sheet (of all the Batches created), at the end of every month to the address given below.
For any queries and information, please contact on Toll Free no:
1800 3000 3468


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Whenever I set the table in our dining room, I feel as though a band should start playing or a choir should start singing. Why? Because sadly, we hardly ever use it. I can count on one hand the number of times we eat in there per year. That said, there is nothing I love more than putting together a welcoming yet simple tablescape. That's what I'm sharing today along with some tips and sources. These Easter table setting ideas use seasonal items but the basic concepts can be used year round. A source list can be found at the end of the post.
The easiest place for me to start is with the fabric and flowers. Once I have those in place the rest seems to be easier. I started with my yellow buffalo check runner and added blue ball jars with yellow tulips. For spring, I don't think you can get a fresher look than combining blue and yellow. Actually any pastels would be lovely.
It literally took me 10 minutes to trim the tulips and set them in the jars. It was easy and absolutely anyone can do this. You don't need vintage jars…any sort of jar will work. If you use clear jars, tying a ribbon around the neck can add the perfect pop of color. Next I turned to the place settings. I love to layer and I think it's essential for a pretty table. You don't need five or six layers…just a few can totally make the same statement.
I didn't use a placemat because I love the way the white charger pops off the wood table. The chargers have felt circles on the bottom so they don't scratch my table. White plates, patterned salad plates, vintage napkins and cute place card baskets finish everything off.
I had the little baskets from a few years ago and filled them with blue eggs. I made the name cards on my computer, printed them on card stock and cut them out. If you choose a pretty, script font your guests will think you hand wrote them yourself!
I didn't have a real centerpiece but to add some height I used a cake plate for a bunny nest. I layered a clump of greenery, eggs, carrots and bunnies.
So…when you are getting ready to set your next table just remember these simple things: fabric and flowers first, layer the table settings and then add the finishing, seasonal touches. Use what you have and add a handmade touch if you can.
(some are affiliate links)
Wood chargers
White plates (not exactly the same but very close)
Cake plate
Carrot bunches
Blue speckled eggs
I could not find any ceramic baskets but these links would work well:
Mini buckets
Mini picnic baskets
Place card font: Contribute
Napkins – vintage
Table Runner fabric – Country Curtains
Salad plates – Cynthia Rowley from TJMaxx
Flatware – no longer available
Glasses – vintage
Since it's Sunday, I'll leave you with these wise words.
Click to download Sunday printable.
Graphics by angiemakes.com.
Have a lovely day and a beautiful week!


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Coconut flour is a gluten free flour made from powdered dried coconut. The flour is popular for gluten free and low-carb baking, since it is low in carbohydrates and very high in fiber, and it is becoming more widely available at markets. Many gluten free "flours" – such as almond flour/meal – are easy to work with because they can be substituted for part of ... Read More »
Tag Archives: gluten free
Gluten Free Buttermilk Pancakes
I've noticed that gluten free pancakes are popping up on the menus of lots of the breakfast restaurants that I like. I am a sucker for pancakes of any kind – and I've been impressed with how much the gluten free pancake offerings at many of these places have improved over the years. But as is the case with most pancakes, they're even better when ... Read More »
Girl Scouts Gluten Free Chocolate Chip Shortbread Cookies, reviewed
This year, the Girl Scouts added a gluten free cookie to their cookie lineup. There have been requests for gluten free cookies in past years from girl scouts who can't eat the regular cookies and from consumers, and it's great to see that the Girl Scouts are listening! The cookies are being test marketed this year in about 20 different locations around the country. My ... Read More »
Girl Scouts Add Gluten Free Chocolate Chip Shortbread Cookie to 2014 Lineup
Every year, the Girl Scouts launch their fundraising cookie sale to raise money both for local troops and for the regional Girl Scout councils. Some cookies have been included in the sale for decades, such as the Trefoil shortbread cookies and Thin Mints, but new cookie are also added to the lineup every year, often reflecting the food trends of the rest of the country. ... Read More »
Flourless Chocolate Almond Cookies
A batch of fudgy brownies is something that everyone can enjoy, and these Flourless Chocolate Almond Cookies pack the same kind of rich chocolate flavor as brownies, but in a totally different package. They have a crisp, meringue-like exterior and a moist, chewy interior that really holds a lot of chocolate flavor. They also happen to be completely gluten free. Best of all, they're easy ... Read More »
What is Coconut Flour?
Coconut flour is a gluten free "flour" that is essentially dried coconut in powdered form. It is made from the coconut solids that are left over after the meat been used to produce coconut milk. The solids are ground into a very fine, flour-like powder. The flour is popular for gluten free and low-carb baking, since it is low in carbohydrates and very high in ... Read More »
Rustic Amaranth Chocolate Cake
You never know what you'll see posted on Facebook or Twitter, but once in a while you find something that really inspires you. The other day I found a recipe that I knew I wanted to try immediately. Rick Bayless posted a recipe for Rustic Chocolate Cake that looked easy and sounded delicious, so I got together the ingredients to give it a try. The ... Read More »
Flourless Chocolate Cakelets
Flourless chocolate cakes are one of the most indulgent types of cakes that there is, since they usually deliver a generous dose of chocolate in every single bite and there aren't a lot of other ingredients to get in the way of that chocolate flavor. I tend to bake them when I'm really in the mood for indulgence (or for the birthday of a chocolate-loving ... Read More »


text:
This morning, following Buzzfeed's Friday scoop about the introduction of an algorithmic timeline and hundreds of thousands of tweets from enraged and confused users, the company quietly announced a new feature. The headline, "Never miss important Tweets from people you follow," is pretty clear about the tone they want to set: this is for you, and it's going to make your Twitter life a lot better. But frankly the announcement feels rushed, squeezed as it is between the Buzzfeed scoop and earnings, which will be announced this afternoon.
As I wrote yesterday, there are a lot of implications to such a shift. I'm not going to repeat my points from yesterday's post in detail, but here they are in summary form, updated with what we're able to glean from the brief announcement. Here's what brands need to know and consider, at least at first:
The basics:
Implications for brands:
I'll update this as more information becomes available. What do YOU think about this change?
Lots of changes happening at Twitter, here's what the latest means for brands that use the platform.
A new report from eMarketer predicts that by 2017, Instagram ad sales will cross $2 billion in ad sales.
Was Twitter wrong for taking down "Politwoops?"
The implications of Twitter turning off the tap for one of its biggest data partners.
How content marketers can take advantage of the new partnership that allows tweets to show up in Google search results.
Real-time content synchronization between offline and online media has become the darling of social TV, frequently serving as its very definition for companies looking to marry traditional and digital marketing experiences.
Co-written with: Susan Etlinger, Rebecca Lieb, Andrew Jones, Linda Saindon, Brian Solis, and Ed Terpening The not-so-long awaited Twitter S-1 is out and now the intense scrutiny begins.


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Are you looking for Digital Tv Antennas? Hotfrog showcases more than 600 popular businesses related to Digital Tv Antennas! To find more business related to Digital Tv Antennas, use the left navigation menu.
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9014 9419 FITZROY Northcote Richmond Heidelberg, Digital TV Antenna Phone ADSL & Service Installation and Repairs: TV Antennas for Digital,…
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DIGITAL TV ANTENNA INSTALLATIONS and TUNING SURVEILLANCE CAMERAS and SYSTEMS SERVICING UPPER COOMERA AND SURROUNDS …
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SkyFox Digital Tv Antennas is part of the SkyFox Group and has been in
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Literary Learning: Teaching the English Major (Scholarship of Teaching and Learning)By: Sherry Lee Linkon (author)Hardback
1 - 2 weeks availability
DescriptionLiterary Learning explores the nature of literary knowledge and offers guidance for effective teaching of literature at the college level. What do English majors need to learn? How can we help them develop the skills and knowledge they need? By identifying the habits of mind that literary scholars use in their own research and writing, Sherry Lee Linkon articulates the strategic knowledge that lies at the heart of the discipline, offering important insights and models for beginning and experienced teachers.
Create a review
About AuthorSherry Lee Linkon is Professor of English and American Studies and Co-director of the Center for Working-Class Studies at Youngstown State University. Her books include Teaching Working Class; Steeltown USA: Work and Memory in Youngstown (with John Russo); and New Working-Class Studies (edited with John Russo).
ContentsPreface One: The Literary Mind Two: Making Literary Thinking Visible Three: Course Design for Literary Learning Four: Analyzing Students' Learning Bibliography Index
Product Details
- publication date: 15/09/2011
- Format: Hardback
- Number Of Pages: 182
- weight: 431
Delivery Information
- Saver Delivery: Yes
- 1st Class Delivery: Yes
- Courier Delivery: Yes
- Store Delivery: Yes


text:
12 College Green, Gloucester, GL1 2LX
Suggested donation - £5
7:30am-18:00pm daily
We look forward to welcoming you to our wonderful cathedral! There is plenty to see, with exhibitions and guided tours so you can make the most of your visit.
Normal opening times are listed above. In term time the building is closed between 08:45 and 09:15 for school assembly.
There may be days when access to some areas of the Cathedral is restricted due to services and other major events.
Sunday services are held at 7.40am, 8.00am, 10.15am and 3.00pm.
You are most welcome to join these services, but please be aware that Cathedral access will be restricted.
Recommended Sunday viewing times: 11.45am-2.45pm.
Times and prices are subject to change. Contact venue before setting out.
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Home Inn (Wuhan Dadongmen)
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Best Tours to do in Maldives – Explore the B
... why not bask in the scenery while trying your hand at fishing. All you need to do is bait your hook, drop your line direct from a 'Dhoni' to the reef edge & hope for a nibble as you watch the spectacular colors of sunset & ocean waves lap the surrounding islands. The memories you will be creating are sure to last a lifetime. The best part about fishing in Maldives is that your catch can be prepared to your liking by night ...
Am I Allowed Here?
I'm now on the part of my trip that is not so new as I've only got Wuhan and Hong Kong left and I've been to both before. We have a delayed start to our trip to Wuhan as it takes us nearly 3 hours to travel the 3km from the train station to our hotel. This is because I am not allowed to: 1. Walk the 3km 2. Take the subway 2 stops and walk 800m 3. Use an unregistered taxi So we're 100th in the queue for a registered taxi, like they're any ...
City guide of Wuhan
... of entertainment options all over the city. Nowadays, visitors and locals alike can enjoy a great selection of nighttime entertainment options including movies, dancing, karaoke and plenty of pubs and bars. With so many international, leisure and cultural options, visitors will have plenty of great nighttime entertainment options to choose form in Wuhan. Make sure to leave room in your itinerary to visit Ji Qing Yie Street for some incredible after-hours ...
Yangtze River Cruise - Badong
From Badong dock we took a smaller boat for the 15 km up the Goddess Stream or Shennv Xi, Shennong Stream which flows from goddess Peak at Badong. It is a tributary of the Yangtze and with the dam the water levels have risen between 70-100 metres in depth depending on the season. There are steep cliffs where you can see the high water mark plus some farms which have been cut out of terraces. Up high ...


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You guys really take GREAT pictures! I have always taken ugly ones,
but perhaps it is time to buy a new camera.
This year I'm determined to get some GOOD pics of whatever has the
smarts to grow and bloom in my garden.
I am leaning toward a digital, and a USB port would be nice. I'd also
like to keep the price under 500.00, and it will be used primarily
for photographing iris...but also a few folks here and there too.
I'd like to try to get colors as true as possible, as well as detail
such as veining.
It is VERY confusing when I go to a camera store..so please, send me
some suggestions.
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