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Synergistic inhibition of human cancer cell growth by cytotoxic drugs and mixed backbone antisense oligonucleotide targeting protein kinase A
Communicated by Paul C. Zamecnik, Worcester Foundation for Biomedical Research, Shrewsbury, MA (received for review July 1, 1997)
Protein kinase A type I plays a key role in neoplastic transformation, conveying mitogenic signals of different growth factors and oncogenes. Inhibition of protein kinase A type I by antisense oligonucleotides targeting its RIα regulatory subunit results in cancer cell growth inhibition in vitro and in vivo. A novel mixed backbone oligonucleotide HYB 190 and its mismatched control HYB 239 were tested on soft agar growth of several human cancer cell types. HYB 190 demonstrated a dose-dependent inhibition of colony formation in all cell lines whereas the HYB 239 at the same doses caused a modest or no growth inhibition. A noninhibitory dose of each mixed backbone oligonucleotide was used in OVCAR-3 ovarian and GEO colon cancer cells to study whether any cooperative effect may occur between the antisense and a series of cytotoxic drugs acting by different mechanisms. Treatment with HYB 190 resulted in an additive growth inhibitory effect with several cytotoxic drugs when measured by soft agar colony formation. A synergistic growth inhibition, which correlated with increased apoptosis, was observed when HYB 190 was added to cancer cells treated with taxanes, platinum-based compounds, and topoisomerase II selective drugs. This synergistic effect was also observed in breast cancer cells and was obtained with other related drugs such as docetaxel and carboplatin. Combination of HYB 190 and paclitaxel resulted in an accumulation of cells in late S-G2 phases of cell cycle and marked induction of apoptosis. A cooperative effect of HYB 190 and paclitaxel was also obtained in vivo in nude mice bearing human GEO colon cancer xenografts. These results are the first report of a cooperative growth inhibitory effect obtained in a variety of human cancer cell lines by antisense mixed backbone oligonucleotide targeting protein kinase A type I-mediated mitogenic signals and specific cytotoxic drugs.
Protein kinase A (PKA), a signal-transducing protein playing a key role in the control of cell growth and differentiation, is present in mammalian cells in two distinct isoforms, type I (PKAI) and type II (PKAII) (1, 2). PKAI is directly involved in cell proliferation and neoplastic transformation (1, 3), is required for the G1 > S transition of the cell cycle (3), mediates the mitogenic signals of different growth factors including epidermal growth factor and transforming growth factor type α (4–6), and is overexpressed in the majority of human cancers, correlating with worse clinicopathological features and prognosis in ovarian and breast cancer patients (7, 8). Conversely, PKAII is preferentially expressed in normal tissues and seems to be involved in cell growth arrest and differentiation (1, 9, 10). It has been shown that the selective down-regulation of PKAI by the site-selective cAMP analog 8-Cl-cAMP leads to inhibition of cancer cell growth in a wide variety of cancer cell types in vitro and in vivo (1, 4, 5, 11, 12) and is accompanied by inhibition of expression of different oncogenes and growth factors (1, 4, 5, 11). Several studies have also demonstrated that different antisense oligodeoxynucleotides targeting the RIα subunit of PKAI expression cause cell growth arrest and differentiation in a wide variety of cancer cell lines (13, 14). A recent study has shown that an RIα antisense phosphorothioate oligodeoxynucleotide (PS-oligo) is able to inhibit the growth of human colon cancer xenografts in nude mice (15).
A large number of in vitro and in vivo studies have demonstrated that PS-oligos complementary to the mRNA of proteins involved in the process of neoplastic transformation and progression are effective in inhibiting cancer cell growth (15–21). However, toxicity studies conducted with PS-oligos in animal models and humans have shown dose-dependent side effects, which may be due to the polyanionic structure of PS-oligos and to mitogenic immune response (22–24). Presently, PS-oligos are being tested for their therapeutic potential in human clinical trials. Although PS-oligos have shown promising results as the first generation of oligonucleotides, to further improve their therapeutic potential we have studied mixed-backbone oligonucleotides (MBOs). MBOs have appropriately placed segments of PS-oligo and segments of modified oligodeoxy- or oligoribonucleotides (24). The MBO that we have used in the present study contains five methylphosphonate linkages in the middle of the PS-oligo. These centrally modified oligonucleotides have shown a significant reduction of side-effects in vivo compared with PS-oligos (24).
We have used a MBO targeting the RIα subunit of PKAI, alone or in combination with a series of cytotoxic drugs, to determine its antiproliferative effect in vitro and in vivo on a variety of human cancer cell lines. We have demonstrated that the RIα antisense MBO inhibits the growth of different human cancer cell lines at submicromolar concentrations and has a synergistic growth inhibitory activity with various classes of cytotoxic drugs, including taxanes, platinum-derived agents, and topoisomerase II-selective drugs. Finally, we have observed in absence of toxicity a cooperative antitumor effect of the antisense with paclitaxel in nude mice.
Cell Lines.
LS 174T and GEO human colon cancer, MDA-MB-231 and MDA-MB-468 human breast cancer and OVCAR-3 human ovarian cancer cells were purchased from American Type Culture Collection. Docetaxel was a kind gift of Rhone-Poulenc Rorer (Antony Cedex, France). 5-Fluorouracil, methotrexate, cisplatin, camptothecin, doxorubicin, etoposide, paclitaxel, carboplatin, and vincristine were purchased from Sigma. All drugs were diluted in appropriate solvents and used as ×100 concentrated stock.
The two oligonucleotides used in the study are HYB 190, GCGTGCCTCCTCACTGGC; [targeted against the N-terminal 8–13 codons of the RIα regulatory subunit of PKA (15)] and HYB 239, GCATGCATCCGCACAGGC. HYB 190 and HYB 239 contain phosphorothioate and methylphosphonate internucleotide linkages. These linkages are identified by normal (phosphorothioate) and bold (methylphosphonate) face type for the nucleosides flanking each position. HYB 239 is a control oligonucleotide and contains four mismatched nucleosides as underlined. The two oligonucleotides have been synthesized by the protocol descibed earlier (24). The identity and purity of the oligonucleotides was confirmed by [31P]NMR, capillary gel electrophoresis, hybridization melting temperature, and A269/mass ratio.
Cell Growth Experiments and Antisense Treatment.
LS 174T, MDA-MB-231, and MDA-MB-468 cell lines were maintained in DMEM. OVCAR-3 cells were grown in a 1:1 mixture of DMEM and Ham's-F12 medium. GEO cells were grown in McCoy medium. All media, purchased from Flow Laboratories, were supplemented with 10% heat inactivated fetal bovine serum/20 mM Hepes, pH 7.4/5 mM glutamine/100 units ml penicillin/100 μg/ml streptomycin (Flow Laboratories). Cells were maintained in a humidified atmosphere of 95% air/5% CO2 at 37°C. For cell growth experiments in soft agar 104 cells/well were seeded in 24 multiwell cluster dishes as described (4) and treated with different concentrations of the indicated cytotoxic drug (day 0). The HYB 190 or HYB 239 antisense RIα MBO were added after 12 h (day 1) and on days 2, 3, and 4. Twelve days after the last treatment, cells were stained with nitroblue tetrazolium (Sigma) and colonies larger than 0.05 mm were counted.
Flow Cytometric Analysis of Cell Cycle by Propidium Iodide Staining.
Cells seeded in monolayer in 6-well dish clusters were treated with the indicated cytotoxic drug. After 24 hrs (day 1) either HYB 190 or HYB 239 antisense RIα was added and the treatment was repeated on days 2, 3, and 4. At the indicated time points cells were harvested, fixed in 70% ethanol, stained with a propidium iodide solution (Sigma), and their DNA content was analyzed in duplicate by a FACScan flow-cytometer (Becton Dickinson) coupled with a Hewlett–Packard computer, as described (4). Cell cycle data analysis was performed by the cell-fit program (Becton Dickinson) (4).
Flow cytometric analysis of apoptotic cell death was performed on cell pellet fixed in ethanol 70%, washed in PBS, and mixed with RNase (Sigma) and propidium iodide solution following the method reported (25). DNA content was analyzed by a FACScan flow-cytometer (Becton Dickinson) coupled with a Hewlett–Packard computer, and the percent of apoptotic cells was calculated using the lysys software (Becton Dickinson).
In Vivo Studies with Antisense in Nude Mice.
Five- to 6-week-old female BALB/c athymic (nu+/nu+) mice were purchased from Charles River Breeding Laboratories. The research protocol was approved and mice were maintained in accordance to institutional guidelines of the University of Naples Animal Care and Use Committee. Mice were acclimated to the University of Naples Medical School Animal Facility for 1 week prior to being injected with cancer cells. Mice were injected s.c. with 107 GEO cells that had been resuspended in 200 μl of Matrigel (Collaborative Biomedical Products, Bedford, MA). After 10 days, when well established tumors of approximately 0.4 cm3 in diameter were detected, seven mice/group were treated i.p. either with paclitaxel (20 mg/kg) (26) (once, on day 1), or with the MBOs HYB 190 (10 mg/kg) or HYB 239 (10 mg/kg) (for 5 days, from day 2 to 6), or with paclitaxel in combination with either HYB 190 or HYB 239 in a sequential schedule; that is, the mice received the cytotoxic drug first (day 1), then, the oligonucleotides were administered for 5 consecutive days (day 2–6). Tumor size was measured twice weekly up to 67 days from tumor cell injection. Tumor size was measured using the formula π/6 × larger diameter × (smaller diameter)2, as reported (27).
Statistical Analysis.
The Student's t test (28) was used to evaluate the statistical significance of the results. All P values represent two-sided tests of statistical significance. All analyses were performed with the bmdp new system statistical package version 1.0 for Microsoft Windows (BMDP Statistical Software, Los Angeles).
Effect of the RIα Antisense MBO in Cancer Cells.
HYB 190, an 18-mer MBO antisense to the N-terminal 8–13 codons of the RIα subunit of PKAI, and the control HYB 239, containing four mismatched nucleotide bases, were tested to study their effect on the growth of OVCAR-3, GEO, LS 174T, MDA-MB-231, and MDA-MB-468 human cancer cells in soft agar. Although HYB 190 showed a dose-dependent inhibition of colony formation at doses ranging between 0.01 and 1 μM in all cell lines (Fig. 1A), the control MBO HYB 239 at the same doses showed a modest or no growth inhibitory effect (Fig. 1B). Western blot analysis after treatment with the antisense RIα MBO in OVCAR-3 ovarian and GEO colon cancer cells confirmed a dose-dependent inhibition of RIα expression in HYB 190- but not in HYB 239-treated cells (data not shown).
Effects of RIα Antisense MBO in Combination with Cytotoxic Drugs.
HYB 190 at a dose of 0.1 μM showed an inhibition of OVCAR-3 ovarian cancer cell growth of approximately 15% (Fig. 1A), whereas HYB 239 at the same concentration caused less than 10% inhibition (Fig. 1B). Therefore, we selected this low dose to study whether any cooperative antiproliferative effect may occur between HYB 190 or HYB 239 and a series of cytotoxic drugs acting by different mechanisms of action in OVCAR-3 cells. When HYB 190 or HYB 239 were added to OVCAR-3 cells treated with different doses of fluorouracil (0.05–0.1 μg/ml), methotrexate (0.05–0.1 μM), vincristine (5–10 ng/ml), or camptothecin (0.05–0.1 ng/ml), which, when used as single agent, showed a growth inhibition between 20% and 60%, an additive growth inhibitory effect was observed on soft agar colony formation (data not shown). A marked synergistic effect was observed when HYB 190 was added to OVCAR-3 treated with paclitaxel, cisplatin, doxorubicin, or etoposide (Fig. 2 A and C). Conversely, HYB 239 at doses ranging between 0.1 and 1 μM had no such effect when combined with the same cytotoxic agents (Fig. 2 B and D). The cooperative antiproliferative effect was not restricted to ovarian cancer cells. In fact, when 0.01 μM HYB 190 , which caused 22% growth inhibition in GEO colon cancer, or at 0.1 μM, which inhibits 5% of the growth of MDA-MB-231 breast cancer cells, were added to paclitaxel, cisplatin, or doxorubicin, a synergistic growth inhibitory effect was observed. For example, in GEO cells, treatment with 0.01 μM HYB 190 in combination with 1 nM paclitaxel, 0.05 μg/ml cisplatin, or 0.05 μg/ml doxorubicin, which used alone showed 7%, 2%, and 25% growth inhibition, respectively, caused a 60%, 62%, and 78% inhibition of colony formation, respectively.
To study whether this cooperative effect occurs also with other drugs of the same classes, we tested the effect of HYB190 when added to OVCAR-3 cells treated with either the taxane docetaxel or the platinum derivative carboplatin. With each drug, HYB 190 caused a synergistic inhibition of growth. In fact, in cells treated with 0.5 or 1 nM docetaxel, which alone cause 20% and 45% growth inhibition, respectively, addition of 0.1 μM HYB 190 caused a growth inhibition of 60% and 85%, respectively. An even more dramatic effect was obtained with carboplatin, where the mild growth inhibitory effect produced by this drug at 0.5 μg/ml (6%) or 1 μg/ml (11%) increased to 70% and 90%, respectively, after the addition of 0.1 μM HYB 190 to OVCAR-3 cells.
Cell Cycle Analysis and Evaluation of Apoptosis.
We have analyzed the effect of the treatment with the MBOs and/or the cytotoxic drugs on the cell cycle distribution of the cancer cells. After 4 days of treatment with HYB 190, an increase of the cells in G0/G1 was observed at all doses between 0.05 and 0.5 μM, whereas 1 nM paclitaxel or 0.05 μM cisplatin alone moderately increased the percent of cells in G2-M phases. Addition of HYB 190 to the cytotoxic drugs caused accumulation of cells between middle S and G2-M phases, preventing their progression into M phase. For example, the percent of cells in S and early G2-M phases was 41% in untreated cells, 32% in cells treated with 0.1 μM HYB 190, 76% in those treated with 1 nM paclitaxel, and 91% in cells treated with both agents. A similar cell cycle distribution was observed at 8 days and 12 days after treatment, except for the fact that the cells treated with the HYB 190 alone began to accumulate in middle S/early G2-M phases. In the same cell population, we conducted a time-dependent analysis of apoptosis by flow-cytometry (Fig. 3). Although apoptosis was present only in 6–9% of untreated cells, a marked percent of apoptotic cells (46%) was present after 4 days of treatment with 0.1 μM HYB 190 alone and remained over 20% in the cell population examined at 8 and 12 days after treatment. Paclitaxel (1 nM) showed a high percent (about 40% or more) of apoptotic cell death throughout the time course, whereas treatment with both agents in combination always showed, at all time points examined, a percent of apoptosis higher than that obtained with each agent alone.
Effect of RIα Antisense in Nude Mice.
To evaluate whether the cooperative effect observed in vitro could be obtained also in vivo, we used nude mice bearing GEO cell xenografts (27). When large GEO tumors of approximately 0.4 cm3 were detected, seven mice/group were treated i.p. with paclitaxel (20 mg/kg, the maximum tolerated dose in mice) (26), HYB 190 (10 mg/kg/dose), and HYB 239 (10 mg/kg/dose) used as single agent, or with paclitaxel in combination with either HYB 190 or HYB 239 in a sequential schedule (Fig. 4). As compared with untreated animals, a significant delay of tumor growth was observed in mice either treated with paclitaxel (two-sided P = 0.05) or with antisense HYB 190 (two-sided P = 0.05), whereas the control oligomer HYB 239 exhibited only a mild growth inhibitory effect. A marked tumor growth inhibition was obtained in mice treated with paclitaxel plus HYB 190, which was statistically significant as compared with control untreated mice (two-sided P = 0.01), or mice treated with either paclitaxel (two-sided P = 0.04) or HYB 190 (two-sided P = 0.01) alone. In contrast, in mice treated with paclitaxel plus the control oligomer HYB 239 the tumor growth inhibition was similar to that determined by the cytotoxic drug alone. Moreover, tumors grew to a size not compatible with normal life in all untreated mice or in those treated with HYB 239 within 40 days after tumor cell injection, and in all mice treated with either paclitaxel, HYB 190 alone, or paclitaxel plus HYB 239 within 60 days. In contrast, all mice treated with the sequential combination of paclitaxel and antisense RIα HYB 190 were still alive and tumor growth was still very slow up to 67 days after GEO cell injection. Finally, no weight loss or other signs of acute or delayed toxicity were observed in this group of mice.
Emerging novel strategies of cancer treatment are based on the selective down-regulation of specific targets involved in the process of neoplastic transformation and progression. PKAI seems to be a relevant target for such therapeutic intervention, and antisense oligonucleotides against its RIα subunit have shown promising results in inhibiting cancer cell growth in vitro and in vivo (13–15). In the present study, we have used a second generation modified oligonucleotide with mixed backbone structure (18, 24) designed to target PKAI. Such MBOs have shown better pharmacokinetic and toxicology profile in vivo (24). The 18-mer RIα antisense MBO HYB 190 showed a dose-dependent inhibitory effect on different human cancer cell types, including ovary, colon, and breast, at submicromolar concentrations ranging between 0.01 and 1 μM. The effect appeared to be sequence-specific, as HYB 239, a similar MBO with only four mismatched nucleotide bases, at the same concentrations was ineffective in inhibiting cancer cell growth. The HYB 190 is complementary to the same sequence of RIα targeted by an antisense oligonucleotide previously shown effective in inhibiting PKAI expression and function (15). Indeed, HYB 190, but not HYB 239, inhibited the expression of RIα protein in OVCAR-3 and GEO colon cells. To evaluate whether any cooperative effect may exist between cytotoxic drugs and a novel PKAI selective down-regulator such as the HYB 190 MBO, we tested the effect of HYB 190 or its control sequence HYB 239 in combination with cytotoxic drugs of different classes in several cancer cell lines. We have demonstrated that HYB 190 is additive when added to cells treated with the antimetabolites fluorouracil or methotrexate, the vinca alcaloid vincristine, or the topoisomerase I-selective drug camptothecin. A marked synergistic effect was observed when HYB 190 was used in combination with the taxanes paclitaxel and docetaxel, the platinum derivatives cisplatin and carboplatin, and the topoisomerase II-selective agents doxorubicin and etoposide. In contrast, no cooperative effect was obtained when the same drugs were added to the same or higher doses of control MBO HYB 239. It is likely that the inhibition of the PKAI-related mitogenic pathway plays a key role in the observed cooperative effect with the cytotoxic drugs. In fact, we have recently shown that 8-Cl-cAMP, another selective inhibitor of PKAI expression, has a synergistic growth inhibitory effect with taxanes and platinum-derived compounds in a variety of cancer cell types (29, 30).
In the attempt to understand the kinetics of these events, we analyzed the effect on cell cycle and apoptosis of the antisense compounds with or without the cytotoxic drugs. Whereas paclitaxel causes accumulation of cells in G2-M phases, the antisense HYB 190 tends to first accumulate cells in G0/G1 and, later, in S/G2-M phase. These results are consistent with our previous report that PKAI is important in the G1-S transition (3) and with our most recent observation that PKAI, together with the surge at the G1-S transition, has a second peak of expression at the late S/early G2 phase (31). Along with these cell cycle perturbations, apoptosis seems to be present very early after treatment with either RIα antisense or the cytotoxic drug alone. When the antisense MBO is administered in combination with the cytotoxic drug in the sequential treatment, a marked increase in the S-G2 phase is observed throughout the course of the experiment. Interestingly, a consistent amount of apoptotic cells, over 50%, is present for several days after treatment, suggesting that cells undergo several rounds of apoptosis. These data seem in agreement with a recent study suggesting that uncoupling between increase of S phase and inhibition of cell division is a major event occurring after treatment with certain cytotoxic drugs inducing apoptosis (32).
We next evaluated whether the cooperative effect obtained in vitro could also be reproduced in vivo in nude mice bearing GEO human colon cancer xenografts. We have shown that, unlike the control oligo HYB 239, which only mildly inhibited GEO tumor growth, HYB 190 significantly delayed tumor growth as compared with control mice. Moreover, whereas HYB 239 combined with paclitaxel had an effect similar to that of paclitaxel alone, combination of paclitaxel with HYB 190 determined a significantly increased tumor growth inhibition as compared with untreated mice or to mice treated with each single agent. In addition, all control mice or mice treated with HYB 239 died within 40 days whereas mice treated with HYB 190 alone, paclitaxel alone, or paclitaxel in combination with HYB 239 died within 60 days. In contrast, at the same time point, all animals treated with paclitaxel and HYB 190 (i) showed over 50% tumor growth inhibition as compared with mice treated with either agent alone, (ii) were alive at 67 days, and (iii) the tumor growth was still very slow although the combination treatment was completed 15 days after tumor cell injection. Remarkably, HYB 190 significantly increased the activity of paclitaxel but not the toxicity, considering that the taxane was used at the maximum tolerated dose in nude mice (26).
Our study reports that a relevant cooperative effect toward tumor growth inhibition can be achieved in vitro and in vivo by combining specific cytotoxic drugs with second generation oligodeoxynucleotides targeting PKAI. Moreover, we have provided evidence that the inhibitory effect correlates with a marked induction of apoptosis and does not result in increased toxicity. Experiments are ongoing to study whether the synergistic inhibitory effect correlate not only with suppression of RIα, but also with that of other relevant growth regulators such as growth factors and angiogenic factors. Taken together, these data suggest that selective down-regulation of PKAI, a key mitogenic signal transducer, is an important event able to markedly increase the antitumor efficacy of certain conventional cytotoxic drugs. As HYB 190 is a prototype of novel second generation oligodeoxynucleotides (the MBOs), which have shown an improved in vivo pharmacologic profile without toxicity (24), our study provides a rationale for translating this combination treatment strategy into a clinical setting.
This study was supported by the Associazione Italiana per la Ricerca sul Cancro and from the Progetto Finalizzato-Applicazioni Cliniche della Ricerca Oncologica (Consiglio Nazionale delle Ricerche).
↵† To whom reprint requests should be addressed.
- PS-oligo,
- phosphorothioate oligodeoxynucleotide;
- mixed-backbone oligonucleotides;
- protein kinase A.
- Received July 1, 1997.
- Accepted September 15, 1997.
- Copyright © 1997, The National Academy of Sciences of the USA
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text:
|该页面介绍的软件不是 Re-Logic 开发的。该页所列软件未经Terraria Wiki、Curse、Re-Logic,或它们各自的员工所测试或认可。使用风险自负。下载任何第三方软件即视为您承认您不能让任何人对任何对您的设备、游戏、或在线账户造成的不良影响负责。
Map viewers are 3rd-party programs that analyze saved maps and display their contents in one easily-viewable graphic.
Map viewers are useful for finding Floating Islands and other resources, as well as providing a total World view not otherwise attainable. Some programs also provide advanced tools for checking map variables, checking Chest contents, and even altering maps in various ways.
Template:Infobox softwareA powerful map editor and viewer for Windows.
TerraMap lets you pan and zoom an interactive map and dynamically highlight items, items in chests, dungeons, NPCs, etc. It also supports headless, unattended world map image generation via command-line, complete with highlight capability.
Pan and zoom maps with full game textures and lighting applied (textures require a local Terraria installation). Inspect chests and signs with right-click. Quickly jump to spawn, NPC houses, and Chests containing specific items. A variety of Save options to showcase your creations.
If textures do not display and the "Use Textures" option of the "View" menu is grayed out, click Help -> Settings, uncheck "Autodetect Texture Folder", click "Browse...", and locate the
\Content\Images folder of your Terraria installation. Close and restart Terrafirma for this setting to take effect.
A map viewer that generates .PNGs which can be marked for map features, item locations, and Chests containing specific items.
A cross-platform web application version of TerraMap that lets you pan and zoom an interactive map and dynamically highlight items, dungeons, NPCs, etc.
An extensible, open source, tool collection that merges many conveniences of other tools into a GUI. Available in multiple languages, Omnitool creates world backups and maps worlds on launch. It comes with several plugins to provide more uses, and more plugins may be found on the TerrariaOnline forums.
Original mapping program. Must be unzipped into the folder where Terraria keeps its world saves, usually Documents/My Games/Terraria/Worlds. Then click and drag the map file onto the executable.
Generates the map tiles and the data needed to display a Terraria world using the Google Maps API.
Similar to the other World Viewers, this creates a png map of a Terraria world. Written in Python, this viewer is compatible with Windows, Linux, and Mac operating systems. It also supports marking of points of interest, like altars, chests and crystal hearts.

subset: commoncrawl


text:
Can someone help me with GitHub in the configurator ?
Is there a guide that I can follow ?
I followed this guide: https://www.home-assistant.io/docs/ecosystem/backup/backup_github/
But it only works in the SSH Server Terminals and this with the username and password.
When I want to do this in the configurator I get an error.
I do the following steps in the configurator:
Git Init
Git Commit
Git Push --> Error
Cmd('git') failed due to: exit code(128)
cmdline: git push --porcelain origin
stderr: 'fatal: could not read Username for 'https://github.com': No such device or address'
If I want to do this in the Visual Studio Code on my laptop I get also an error.
Can someone help me with this ?
Kind regards,
Bart Engelen
How to get GitHub working?


text:
Lana Efremova
Lana Efremova
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So many options on the house you could build on this lot. 2.3 Acre size lot and is on the corner at the front of the neighborhood. NO HOA, but yes there are just a few covenants. Maybe even could build your home and have a mini-farm or even that family that needs a shop or work from home area. Homes in this subdivision are 1500 to over 4000 SF so bring your ideas and your builder to create the home of your dreams. Completely wooded lot so you can have as much privacy as you need. Just a few minutes to downtown Monroe, real close to get to Hwy 78 or Hwy 83 back towards Lake Oconee. Well located property outside the hustle and noise, but easy enough to get to Athens or Atlanta.Read All Less
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**Here is a purchase page of 5 beverage pouches (including 5) at a time** **Here is a purchase page of 5 beverage pouches (including 5) at a time** **Here is a purchase page of 5 beverage pouches (including 5) at a time** **Please fill in the subscript note the required color** **If you do not fill out the random shipments :)** **ʕ • ͡ᴥ • ʔ Trouble Select the desired quantity and write down your favorite color at the note** **ʕ • ͡ᴥ • ʔ Each color can be optional Oh, not all the same color** **ʕ • ͡ᴥ • ʔBuy more than five cups, a beverage bag for 210 yuan, not five drinks bag 210 yuan!** **ʕ • ͡ᴥ • ʔ purchase of five or less, do not bid on this, if mistaken mistakes store, need to make up the difference https://www.pinkoi.com/product/RJbhvTDG?category=5** / Secretly shouting my hand tailor room / Each piece of fabric through my hand, into your heart and baby! Finally launched in late 2017 ☺ three points leaked to help the polar bear ☺ environmental portfolio! This combination has a total of: beverage bag / coaster / tableware storage bag three works "You're a bit, I'm a bit, he's a little bit, and everyone works together for the best of the planet!" Will everyone's efforts, "three points do not leak" into embroidery little embroidered in the works Remind us that there is only one planet on earth. No one is an outsider on the matter of environmental protection https://c1.staticflickr.com/5/4586/24803413928_27426f3afd_z.jpg https://c1.staticflickr.com/5/4534/38675872611_b301b4229f_z.jpg ʕ • ͡ᴥ • ʔ About drinks bag In order to make the most practical cup, repeated version of the version several times, improved some details, and finally make the most perfect look / Can hold most major beverage stores hand cup, convenience store coffee cup, breakfast tea black bean milk can also be put! // wide and narrow version of the type, you can make the lower edge of the cup sleeve is also close to the cup /// Inside thick pound canvas, so that ice is not easy to wet cup sets ʕ • ͡ᴥ • ʔ on coasters Each time you buy a drink on the table, have to take a piece of toilet paper padded drinks will not wet the table, but feel good waste, this time is the chance of a coaster appearance! ! ! Cute and not wet the table coasters! ! ! ʕ • ͡ᴥ • ʔ About tableware storage bags This is a long time to launch the work, this time with the cup coaster became a family, launched a series of tableware bags! Length can be loaded into chopsticks, spoons, forks, green straws are no problem Yeah ~ ~ ~ introduced each piece, then we will look at all the colors! A little red https://c1.staticflickr.com/5/4536/38675877011_cc1dd34157_z.jpg Sakura season https://c1.staticflickr.com/5/4543/37958591824_236c5cfe61_z.jpg Fuji apple https://c1.staticflickr.com/5/4524/37788260695_f1a4bc9a54_z.jpg lightning https://c1.staticflickr.com/5/4552/26900727219_f8163e2770_z.jpg Milk tea https://c1.staticflickr.com/5/4543/38620634726_f26039a55e_z.jpg Matcha Melaleuca https://c1.staticflickr.com/5/4546/38675878781_17d736131a_z.jpg Tree in the mountains https://c1.staticflickr.com/5/4586/38675875081_1703e150d8_z.jpg Alley in Kyoto https://c1.staticflickr.com/5/4524/37788252415_356da5521d_z.jpg Blue sky and white clouds https://c1.staticflickr.com/5/4564/37788261735_b341287bd1_z.jpg time Square https://c1.staticflickr.com/5/4553/37788256735_ebd351b3e4_z.jpg latte https://c1.staticflickr.com/5/4578/37788266015_8975299808_z.jpg Here is the first point: drink cup cover purchase site ~ remember! Three o'clock can not be missed! ! ! If you want to take three at a time, here ☞ https://www.pinkoi.com/product/mpeghWQy?category=5 If you only want to take the second place ☞ https://www.pinkoi.com/product/Z2r2UtKH?category=5 If you only want to take the third pouch holder ☞ https://www.pinkoi.com/product/Wm5sT6ZR?category=5 About each piece, if you like fabric production, we welcome the private information we customize :) Origin / manufacturing methods Taiwan Tainan design tailor
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4584 Lomax Ct. 1b, Murrells Inlet
Charming 2 bedroom 2.5 bath townhome in desirable Wynbrooke subdivision! The updated kitchen features stainless steel appliances. Each of the 2nd floor bedroom include a full private bathroom. Enjoy community amenities including a large pool, beautiful ponds, and relaxing fountains. Book your showing today!
26A Myrtle Beach Area--south of 544 & west of 17 b
Cable Available, Electricity Available, Phone Available, Sewer Available, Water Available
© Copyright 2020 of the Coastal Carolinas Association of REALTORS® MLS.
IDX information is provided by the Coastal Carolinas Association of REALTORS® for consumer's personal, non-commercial use and may not be used for any purpose other than to identify prospective properties consumers may be interested in purchasing, data is deemed reliable but is not guaranteed accurate by the MLS.


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GfG has developed a powerful and positive relationship with The College of Westchester! They provide donated space for our bi-monthly workshops & activity sessions, as well as provide our girls with a glimpse into various
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Adamtroup got their new logo design by running a design contest:
Help Inspire Video with a new logo
Check out Adamtroup's Logo design contest…
The Brief
Wedding Service
About us
I specialise in producing high end, cinematic wedding films although I also do a wide range range of video production including corporate work. Although I selected wedding service as my business industry I'm not looking for a wedding specific logo.
Logo types to explore
The Brief
Design style
The Brief
Other notes
I have been happy with my current logo but there are a couple of things that need to change, the word video needs as much prominence as inspire, I also want to drop the word production. The film strip on the logo isn't obvious what it is because of the very small holes along the edges of it when printed small and also on material. I would like a complete re imagining of it though. I think I would like to keep red as the colour but I am open to other colours, would have to see it! I do offer a premium service and I want this conveyed, even if its just with an elegant design, I don't necessarily want it to shout super expensive though! I am open to gradients etc but I would also like it to be useable in a solid colour too. My general clients tend to be between 25 and 35 in age, educated and successful. Wedding wise I tend to have more enquiries from females. In general work, conferences, promotional video etc. they can come from both male and female.
360 £ Silver package
Every design category has flexible pricing for all budgets. Logo design starts at €269.
File deliverables
Full copyright with production-ready files for digital and/or print.
It all began with a design brief.
A quick, interactive guide helped them understand their design style and captured exactly what they needed in their logo design.
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design entries
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Work with talented, professional Logo design designers to turn your ideas into reality.
Select your favorite custom Logo design (Or two! Or three!). And the design is all yours.
Adamtroup collaborated with designers to refine their ideas
Rate designs
When design entries come in, you can rate them so designers know what you're looking for in your logo design.
Give feedback
99designs has great collaboration tools so you can pinpoint and capture your ideas
And then… they selected a winner!
Client review
Alexandra is brilliant. I didn't have a clear vision for my new brand logo, but based on my brief, Alexandra created the perfect concept and fell in love with it.
She was super helpful, creatively collaborative, and a great communicator throughout the entire process.
I hope she'll be creating designs for my company for many years to come.
Along the way, they met lots of talented designers…
We think contests are a super fun way to get design.
Recently completed contests:
Wedding Show 24/7
We are creating an online wedding exhibition, where suppliers can create stands and exhibit their services, like venues,
Events hire logo design
We hire products to people getting married. Cars, lights, photo booths, ect
Modern, minimalist logo for wedding videos
I film wedding videos. my target group are couples who are getting married soon.
Wedding Venue Logo needed with a Horse/Ranch theme!
Sweet Blessings Ranch will be a wedding venue that will have a horse/ranch theme.
Modern, luxurous wedding photography logo needed
Luxury wedding photography. Target audience = 24-35 years old, located in USA, higher end clothing, educated, enjoys ar
AS Monogram For Wedding Design
Looking to have a wedding monogram created for a couple getting married on 2.26.20 Need artists to look at examples we
Fun logo for a wedding industry business
This is a wedding communication platform for brides, grooms, and wedding planners to use
Design a modern eye catching logo
We decorate venues for weddings etc. The company will be based in East Africa. We aim for exclusive and classy clients.
BrittLee Chapel
This is going to be a wedding venue chapel on top of a hill overlooking mountains
Elegant upscale logo design for tent and event rental company. (weddings and all type of events)
Magnif-A-Tent is a tent and event rental company based out of Long Island, NY.
Fine Art wedding biz needs beautifully designed on-boarding doc
Fine art wedding paintings, work with warm sensitive types of brides, typically more high end but also some people who a
Wedding venue looking for a logo with chances to do much more!!
We are a rustic wedding venue, in the Adelaide Hills. Exclusive but accessible. Beautiful old heritage buildings made ou


text: Bushman's Revenge is normally a Norwegian power trio who all seamlessly meld jazz improvisation with rock dynamics and prog rock and roll. The music group was founded in 2003 by guitarist Also Helte Hermansen, bassist Rune Nergaard, and drummer Gard Nilssen. After building a near rabid live pursuing in night clubs and garnering some celebration invites, the band's debut, Cowboy Music, was documented for saxophonist Jon Klette's Jazzaway Information in 2007. After a protracted EUROPEAN tour and performances at jazz celebrations, Bushman's Revenge agreed upon to Rune Grammofon. Their debut for the label, You Shed Me at Hello in '09 2009, garnered near general acclaim from critics. This year 2010, the music group released Jitterbug, which highlighted keyboardist Stale Storløkken within a visitor capacity and uncovered the music group turning even more toward the rock and roll aspect of their persona; they also included a cover of Metallica's "Harm." A small amount of Big Bonanza rather than Brain the Botox implemented in 2012. The trio toured not merely in European countries but also in Japan. The live record Electric Komle premiered in 2013, accompanied by Thou Shalt Boogie in early 2014, with concert performances in European countries, Asia, and america. Upon time for Europe, they performed the celebration circuit and done new materials in the studio room. The activity led to two simultaneous produces in 2016: a studio room providing entitled Jazz, Fritt Etter Hukommelsen that opened up having a reading of McCoy Tyner's "Contemplation," as well as the LP-only live arranged Bushman's Fire.


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Contact Us
Have a specific query? Get in touch using the form below.
Aongatete Forest itself is at the top end of Wright Road, Aongatete
The Project's address for service is 2771 State Highway 2, RD2, Katikati 3178
For general enquiries, please phone 07 808 0792
Want to be kept up to date with the latest news?
Provide your email address in the box below to receive our quarterly newsletter


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HB CDEM Group coordinates emergency management in Hawke's Bay - call 0800 422 923 for urgent access to essential household goods and services (7am - 7pm) || visit COVID19.govt.nz or call the free Government Helpline on 0800 779 997 (8am - 1am) || call Healthline on 0800 358 5453 for free COVID19 health advice || Find out more »
A detailed business case for a redefined National Aquarium was presented to central government in December 2019. This will identify the costs and the benefits. If all goes well, Napier could have a new aquarium sometime around 2025.
Project Shapeshifter Business Case
The detailed business case, put together by Terra Moana, to redefine the National Aquarium of New Zealand is now complete and has been submitted to the Ministry of Business, Innovation and Employment (MBIE). This is the culmination of several months of national and international information gathering, including getting feedback from environmentalists, educators, researchers, iwi, youth and the general public as well as engaging US aquarium architects EHDD and fundraising consultancy AskRIGHT.
Click here for the Detailed Business Case, architect's concept designs, Colmar Brunton survey and other reports.
Project Shapeshifter is not just an expansion of the current aquarium. The proposal is to build a new, nationally significant National Aquarium and Oceans Centre. The project's vision is creating a place where land and sea are joined, where the stories of the ocean are told by hapū, conservationists, scientists, volunteers, and most importantly by the species that make the ocean their home.
The facility will seek to inform and raise awareness of environmental issues and empower visitors to actively support the conservation of species and ecosystems through smaller actions or larger commitments.
If the business case is approved by MBIE, a revenue generation strategy will be created that will seek philanthropic donations, sponsorship opportunities and grants from Lotteries, charitable trusts and foundations, and other government sources, to help offset project costs.
Project Shapeshifter is subject to a gateway process, where the Government is currently considering the submitted detailed business case. If approved by Central Government, further gateways including external funding and public consultation, would be required before the project would commence. We anticipate being advised on the outcome of the detailed business case submission in the first half of 2020.
The existing aquarium cannot continue in its current state. The ongoing operating budget required for the Aquarium to meet and maintain minimum standards for animal welfare is not feasible.
When considering modern animal welfare standards the building itself is no longer fit for purpose. For example, there is limited staff accessibility to maintain tanks and exhibits, and limited space for quarantine and vet care facilities. This makes maintaining the animal's welfare challenging, and it is not financially sustainable in the long term.
Furthermore, when considering the purpose of modern aquaria - which is to educate and inspire positive behavioural change - the layout of the existing building provides for an outdated form of aquarium, where animals are exhibited for people's entertainment.
Any closure of the aquarium would be up to five years away. Before any decisions are made, we need to hear back from MBIE and we also need to consult with Napier residents. This will provide clarity on the direction we will take.
Read about the history of Napier's Aquarium from 1957 to 2017.
The proposed expansion of the aquarium is known as Project Shapeshifter: Redefining our National Aquarium. This name is emblematic of Māui – the 'shapeshifter' and great East Polynesian ancestor-explorer of the Pacific Ocean. Our challenge is to be bold and adventurous like Māui – to be a shapeshifter and game-changer.
Central government has asked us to prepare a business case for redefining the National Aquarium of New Zealand, which is located in Napier. Key partners in the project include Air New Zealand, University of Waikato, Hawke's Bay Tourism, Hawke's Bay Regional Council, and local iwi.
Our vision is to create an iconic destination that all New Zealanders will be proud of. A memorable experience that centres Aotearoa New Zealand as the environmental champion for Papatūānuku planet earth and her oceans.
Redefining our National Aquarium gives us a unique opportunity to tell New Zealand's freshwater and marine story through themes of sustainable utilisation and conservation. Located in the heart of Te Matau-a-Māui Hawkes Bay, the National Aquarium is best positioned to tell the story of Aotearoa New Zealand from mountain top to deep trench, promoting the best of environmental science with leading indigenous knowledge systems and Te Ao Māori, the Māori worldview.
The main level of the current aquarium is 5.5 metres above sea level. With the expected rise in sea levels in the coming decades due to climate change, the current aquarium site is at risk of flooding during 100-year flooding events.
To account for this, the design will include strategies to minimize damage and protect the aquarium's collection. Incorporating these design strategies means the aquarium can be built to survive future storm surge events with minimal damage.
The current 100-year flood level along Napier's coast is 5.7 metres above sea level. Research suggests that sea levels will rise 0.6 metres in the next 50 years, bringing a 100-year flood event to 6.3 metres. The main floor of the new aquarium would be 7.3 metres above current sea level – 1 metre above the 100-year flood level adjusted for sea level rise.
To minimise damage, mechanical, electrical and telecommunication rooms and associated equipment would be located on the upper floor or the building's roof to avoid flooding.
The building will be designed to withstand high wind speeds during storms. In general, aquariums are very robust buildings due to the stress load requirements of the wet exhibits.
The total capital cost of the preferred option is $77.5m, which includes $65.6m of construction costs, $7.0m in contingency and $4.9m associated with cost escalation during the construction period. Real fit-out replacement costs equal $1.5m every five years and real exhibition refurbishment costs equal $3.2m every 10 years. revenue in the first full year of operations of $6.6m (approx. 196,000 visitors) against operating costs of $9.6m (inflation of 2.8% p.a.). Further details are in the business case (pg 83).
We are looking for new and innovative ways to externally fund the construction and ongoing operational costs of the aquarium. We are mindful that the cost cannot fall solely on Napier ratepayers. The business case seeks to position the aquarium as an attraction of truly national significance, in much the same way as Te Papa is. As such, it will be funded through a mix of channels – ranging from central government through to private funding from various channels, both here and overseas.
No, as other projects have their own budgets allocated as part of the Long Term Plan. Project Shapeshifter does not impact on any other work Council is undertaking.
Napier residents gave strong support for the National Aquarium expansion during the Long Term Plan 2018-2028 consultation. A more formal consultation process with Napier residents will be undertaken in 2020 if the business case is approved by central government.
The community will be able to feed in ideas and share their thoughts on what they might like to see (including programmes and activities). Napier residents, our current Friends of the Aquarium members, youth, school audiences and a range of community groups will be key to this process.
The first phase of the new project is planned to be just north of the existing site, to be closer to the central city. The original 1970s building will need to be demolished, and the 2002-built extension will be re-purposed as an education centre and administrative offices for the new facility.
The closure of the National Aquarium will need to be considered. A scaled-back refurbishment is not an option because the ongoing operating budget required for the Aquarium to meet and maintain minimum standards for animal welfare is not feasible.
When considering modern animal welfare standards, the building itself is no longer fit for purpose. For example, there is limited staff accessibility to maintain tanks and exhibits and limited space for quarantine and vet care facilities. This makes maintaining the animal's welfare challenging and it is not financially sustainable in the long term.
Furthermore, when considering the purpose of modern aquaria - which is to educate and inspire positive behavioural change - the layout of the existing building provides for an outdated form of aquarium, where animals are exhibited for people's entertainment.
Aquariums are often located on coastlines so they can connect with the sea. The water from the ocean is also vital for the life support systems needed by the animals. The current aquarium site is 5.5m above sea level.
A detailed seismic assessment has been commissioned and the findings will be incorporated in the project. Most buildings in close proximity to the sea would be challenged by a significant tsunami. The aquarium has animal rescue procedures in place for natural hazard events.
An aquarium has been in Napier since 1957 when local fish-keeping enthusiasts gathered together to use space in the lower level of War Memorial Hall to display their fish. The Napier Aquarium was expanded in the early 2000s and opened as the National Aquarium of New Zealand in 2002. Read about the history of Napier's Aquarium from 1957 to 2017.
Hawke's Bay is known by its Māori name of Te Matau-a-Māui The Fish Hook of Māui, which carries significant meaning for the National Aquarium. Being associated with Te Matau-a-Māui supports the National Aquarium being in Napier, drawing on a 1,000 year legacy of navigation, voyage and migratory movement of species.
Admission charges are likely to increase to $39 per adult and $21 per child, in line with admission for Kely Tarlton's, but there will be much more to see and do. There is a significant discount planned for Hawke's Bay residents.
People can also join the Friends of the Aquarium by paying an annual membership. From 1 July 2019 the Friends of the Aquarium annual fee will be $65 per adult, $95 for an adult and one child, $150 for two adults and up to two children, and $25 per extra child, per year. Council reviews fees and charges every year for all facilities.
Anyone is able to join the Friends of the Aquarium, which will still continue if the expansion goes ahead. We welcome our community to feed in their ideas of what they might like from membership in future too.
Friends can visit every day the aquarium is open. They also receive special invites to events and new exhibit openings. From 1 July 2019 the Friends annual fee will be $65 per adult, $95 for an adult and one child, $150 for two adults and up to two children, and $25 per extra child, per year.
We are looking at the possibility of setting up a charitable trust for the aquarium and will investigate this has been investigated in the business case (page 74).
The first phase of the new project will be just north of the existing site, meaning the current facility can remain open until the new one is ready. Consideration has been made for the noise and vibrations from construction in order to ensure there is no impact on the animals in the current aquarium.
The National Aquarium of New Zealand is a member of the Zoo and Aquarium Association of Australasia (ZAA). As a member, the aquarium is required to undergo accreditation where we must clearly demonstrate how we meet their standards of, and our commitment to, positive animal welfare. The aquarium expansion is an opportunity to continue to show visitors ways that we can all care for the environment and its inhabitants. The aquarium will continue to have a range of animals to visit, but there will be greater use of digital technologies for visitors to experience. This means visitors will have an opportunity for close up experiences with species that they may never otherwise see.
We will be focusing on animals that are native to New Zealand and the Pacific, in a range of large scale, immersive exhibits. We will still have our Little Penguins. But the main focus of the new facility will be to encourage connection to wildlife and nature, educate and inspire positive behavioural change, rather than focusing on exhibiting animals set up for people's entertainment. Real life issues such as climate change and plastics in our oceans will be addressed. Other animals, not suitable to be held in captivity, will be present digitally/virtually.
We have received funding from the government's Provincial Growth Fund to prepare a detailed business case for a new redefined National Aquarium of New Zealand. This was submitted in December 2019. If the business case is successful and we manage to find funding from other sources, construction will likely begin in 2022 and the new aquarium may open in late 2025.
Watch quick summaries of the thoughts of people who helped us to define the Project Shapeshifter Business Case.
Project Shapeshifter attempted to create a compelling movement as the business case for change was developed. To better understand how we may redefine our National Aquarium, we ensured there was national input for what will eventuate. Project Shapeshifter undertook an extensive engagement programme across Aotearoa New Zealand to build momentum on our vision. Workshops were held with Māori leaders, environmental experts, the tourism industry and research and education providers. These workshops brought together diverse thinkers who contributed to the development of the business case and helped to redefine what could be on offer. You can read a summary of their ideas here.
Our aquarium has the potential to be a national centre showcasing research, education, environmental stewardship, indigenous knowledge and science. This will provide a unique experience that would not be found anywhere else in the world. A more formal consultation process with Napier residents will be undertaken in 2020 if the business case is approved by central government.
The detailed business case for a redefined National Aquarium has been presented to the Government. We are excited by this project and the opportunities it presents to the entire region.
In 2018, Napier City Council submitted an Indicative Business Case to the Government for a redefined National Aquarium of New Zealand. There was strong support given for this by ratepayers in the Long Term Plan 2018-2028 consultation, with an allocated project budget of $10.2m. Following the submission of the Indicative Business Case to the Government, NCC received funding from the Government's Provincial Growth Fund to complete a Detailed Business Case for a new expanded aquarium. This was presented to the Government in late 2019.
Disclaimers and Copyright
While every endeavour has been taken by the Napier City Council to ensure that the information on this website is accurate and up to date, Napier City Council shall not be liable for any loss suffered through the use, directly or indirectly, of information on this website. Information contained has been assembled in good faith. Some of the information available in this site is from the New Zealand Public domain and supplied by relevant government agencies. Napier City Council cannot accept any liability for its accuracy or content. Portions of the information and material on this site, including data, pages, documents, online graphics and images are protected by copyright, unless specifically notified to the contrary. Externally sourced information or material is copyright to the respective provider.
© Napier City Council - www.napier.govt.nz / +64 6 835 7579 / firstname.lastname@example.com


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Lakeside Cabin Rentals
The Hayward lakes Region is an enjoyable four-season recreation area. We are 2 hours from Minneapolis, 4.5 hours from Madison, and 5.5 hours from Chicago. Whether it's boating on one of the areas pristine lakes, hiking one of the many well-maintained trails or golfing at the areas top rated public course, you can find what you are looking for during the summer months.
Lodging on Chippewa Flowage
Gander's Nest
This newly constructed 3 bedroom 2 bath chalet is located on Chief Lake of the vast and famous Chippewa Flowage at 17000 acres. This body of water is most widely known for producing the world record Muskie caught by Louis Spray in 1949. Since then the flowage has consistently produced 50 plus inch muskies and is great walleye, crappie and bluegill fishing. Wildlife abounds along the pristine and wild shores and there are countless islands that one …..read more….View Home Details »
Hayward Haven
Looking for a sandy beach on the Chippewa Flowage? Look no further! Hayward Haven is a comfortable 5 bedroom/3 bath cabin with 138' of sandy frontage on the famous Chippewa Flowage! 2 docks, large deck, level frontage, fire pit area and great views of the lake can be all yours for your family's next summer vacation. Located at the end of a quiet, dead end road you can enjoy a peaceful morning or afternoon walk. Besides the awesome …..read more….View Home Details »
Tall Pines Lodge
Welcome to Tall Pines Lodge, a perfect escape located in the heart of the great Northwoods. This property is surrounded by national forest, snowmobile & ATV trails and 17,000 acres of the Chippewa Flowage. Located on the Hay Lake side of the Chippewa Flowage, you have a short walk to access your shared private dock to keep your boat, fish & swim! Experience world-class muskie, walleye & crappie fishing right off the dock!! This dog friendly home …..read more….View Home Details »
The Rowan Tree
This well appointed Northwoods square log home is located on the north central portion of the Chippewa Flowage. You'll feel like your in Canada as you motor or paddle the endless wild shorelines and islands of the Big Chip. Home of the World Record Musky and premier walleye, smallmouth and panfishing. Resting in the Chequamegon National Forest, The Rowan Tree has a huge wrap around deck and level frontage leading to a sweeping view of the …..read more….View Home Details »
The quiet time during the fall offers spectacular foliage tours or fishing for trophy Musky, Walleye, and trout. When winter arrives, get out the snowmobiles, or rent a snowmobile and ride hundreds of miles of groomed trails and lakes into the early spring or bring along the skis and enjoy the hundreds of miles of groomed trails. To have your vacation be perfect stay in one of Property Management of Hayward's rental homes like Lac Courte Oreilles .
Lakeside cabin rentals in the Hayward lakes Region of Northern-Wisconsin.
Lake Home Rentals
Lakeside Cabin Rentals Hayward Wisconsin
Many of our vacation homes have WIFI (look for the icon) and some of our homes are pet friendly (watch for the icon)
Four Season vacation home house rentals like The Rowan Tree are available on several Hayward area lakes. Many of our vacation home are located near the Birkie Ski Trails. If your summer travels bring you to Hayward Wisconsin you will enjoy renting a private vacation home like Balsam Lodge or Island View from Property Management of Hayward.


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Our Terms and conditions
- To enable us to meet your requirements, we require at least 24 hours notice for online bookings.
All other requests please phone the number at the top of the page.
- Web Box Solutions Ltd do not store or collect credit card details nor do we share customer details with any 3rd parties.
Request a Call Back »
Have a query or need help with your booking? Enter your details and we will call you back.
Special Prices
From or to ST.ANDREWS & DUNDEE


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There are more than 1500 kinds of Chinese snack recipes here. Friends who like DIY and delicious food must not miss them. Collect them quickly. When you are free, try it. If you have a passion for Chinese cuisine, you should be thrilled to see this page. XD
"Today, my children said they wanted to eat Hainan powder. I said I would cook it for you at noon.Did she say that what you did tasted good?I said try it.I searched all over the world for gourmet food, only two or three recipes of Hainan powder, and then went to the headlines to search. I saw several good practices, then combined with the Hainan powder I usually eat, I started.This time, it was quite successful. It tasted delicious. The children ate two bowls and kept saying that Mom had done a great job.It's the best Hainan powder she eats!"
|Hainan powder
|Bean sprouts
|a kind of edible bamboo shoot
|soy sauce
|Appropriate amount
|Fermented bean curd
|2 pieces
|corn starch
|Appropriate amount
|Appropriate amount
|Maotai flavor
|time consuming
Raw bacon, sour bamboo shoots.
Raw material: Soybean sprouts are boiled in water and fried peanuts.
It's a sauce. It's easy to make. Heat the sauce in the pot, then add soy sauce, sugar, two pieces of sufu, starch and water.I didn't put salt, because the curd is very salty, and then put salt is too salty.The viscosity of starch can be controlled by oneself. Add it little by little, and you can see that the consistency is about the same.
Hainan flour is fragrant only when fried sesame seeds are added.
Chop scallion and coriander.
Hainan powder I bought forgot to take photos. I used boiled water to pass over Hainan powder first. Then I started the bowl and put Hainan powder first. If there were pickles, it would be better. I forgot to buy it today.Bean sprouts, sour bamboo shoots, fork-roasted meat, scallion and coriander, peanut and sesame, sauce.Mix well and eat it. It tastes very fragrant.It's better than buying it!
This is Hainan flour mix, I did not think this time too successful, the taste is particularly delicious! _______
Add another plate of Hainan flour.
Works from the side of the world where gourmet food is served _______


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Price: $17.50
Quantity: 1 available
Book Condition: Used - Very Good
Very Good Hardcover with Very Good- Dustjacket. Light to moderate soiling and shelfwear to DJ, including one tiny tear. Light soiling and shelfwear to covers. Spine ends bumped. Pages clean and tight in binding. Pictures available upon request. A locally owned, independent book shop since 1984.
Title: Sister Brother
Categories: Literary Biographies,
Publisher: Putnam Adult: Apr-96
ISBN Number: 399141030
Binding: Hardcover
Book Condition: Used - Very Good
Jacket Condition: Very Good
Item: 2.10 Item
Seller ID: 32952


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I'm sure there have been times that everyone had trouble expressing themselves fully. It's there but you can't put it into words. That is simply a small hiccup in processing language. Almost everyone needs to talk to have fluid communication with people. Though language processing disorder can negatively impact it. Language processing disorder is trouble …
Tag: mental health
What Exactly Is Visual Perceptual/Visual Motor Deficit?
Sight is one of the many things that, humans, usually take for granted. We get most of the information from outside with our eyes. Therefore, it is of no question that our eyes are very important. It would be a true horror for us to have some sort of problem with it. People with visual perceptual/visual motor deficits have issues comprehending their visual information. Most often, kids or people with learning disabilities have this condition sometimes people with brain damage have this condition. These kinds of issues can be subtle or worse. When it is subtle, a person can spend a lifetime before knowing they have such a condition which is precisely why people should be informed to check whether they or the people they know have this condition. By being informed of this we can help these people to make their lives easier and more comfortable. The visual perceptual/visual motor deficit has a lot of signs and symptoms to look for which can make it easier to determine the condition. Simply view this website to know what they are.
Dyscalculia symptoms
Dyscalculia or numlexia is a disability that prevents a child from learning or understanding arithmetic. About 3 – 6% of the population is estimated to suffer from the disability or to manifest dyscalculia symptoms. Dyscalculia symptoms may fall into the general category of difficulty dealing with numbers or numerical concepts. Click here to know some …
Common ADHD Symptoms In Teens
ADHD or attention deficit hyperactivity disorder is a kind of mental disorder. Although people of all ages may be hit by this condition, children, especially teens are more likely to get affected by ADHD. The condition affects teenagers and may continue to adulthood. As such, identifying the signs early and treating the problem is always advised to avoid worsening of the condition. Phone or email a physician once you notice some of the symptoms that are detailed below.


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Last Saturday morning, and for no apparent reason, I could neither send nor receive E-mails. No problem for this Boomer-type computer whiz. I did what all good Geeks do. I turned the computer off and rebooted (that means turning it off and starting again for those out there who are not computer whizzes). Still, no E-mails.
Alright, now for the serious stuff. I "Force Quit" the damned thing and told it to "Quit Mail".
"That'll teach it to screw with me!"
But still no E-mail.
So then I picked up my lap top and shook hell out of it.
That didn't work either, but it made me feel better.
And still no E-mail.
My E-mail account is G-mail, which I think belongs to a fellow named Google. I don't know his first name, but he is a nosey bastard. He knows everything I do and everything I like.
The other day, Mr. Google asked if I had bought that Senior Shaft golf driver I have been looking at. I suppose he could see the drool on my computer screen.
"No, I have not, and it's none of your business."
"It's a nice club. Might get you an extra ten yards off the tee?"
How did he know I'm short off the tee?
The next day he wanted to know if I needed any more barbeque sauce.
"Only if it's Carolina sauce." It was and I bought three bottles.
But now this nosey son of a gun has cut off my email. Why?
"Because you're using too much data space. Did you know you have E-mails on your server dating back to 2013?"
I explained that I couldn't erase them because they sometimes brought back fond memories.
"If you don't reduce your capacity by 20,000 mega-somethings, we're going to cut you off."
So I deleted most of my E-mails from 2013.
Still no E-mail,
Finally we arrived at the crux of the matter. If I paid Mr. Google just $2 a month, he'd give me more capacity and let me read my E-mails again.
"I'm going to call Allen, Allen and Allen," I threatened. " Don't you hate those guys?"
"I'll cut them off, too."
I have now erased all my E-mails since last Tuesday, I bought the Senior Shaft driver, more barbeque sauce, paid Google $2 and I can now send and receive E-mails.
But I am getting 10 more yards off the tee!


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Wonder Woman fears nothing..She is a God..You are not..What do you fear my lady? ….. Welcome to TeTe & Espresso, a CastBox original, I'm TeTe. Ladies all my ladies, help me out, loud and proud, come on, all my ladies. Today we will look at 5 fears of women, based on a facebook poll, and research, I just did.
Ps…Being a woman is the furthest for knowing what that life it is truly like. I tried my best with this podcast, please accept this work as an empowerment to women, in my own way.
What group of people have been treated worst than native Americans or Jews? Women..Oh you poor women throughout history..woooweee …But now is a different time. One of equality. Even backed by science and studies, we know now, beyond a shadow of a doubt, that women and men are equal to each other. And the strength, intelligence, and accomplishments of women are surpassing our societal beliefs every day… To know what we fear, is how we act in the face of the fear, and grow. And it's time my lovelies, it's time to continue the growth of you, the lady…In no particular order, here is my list for 5 fears of women:
Gaining Weight
Something "bad" happening to her child
Something bad happening to her family/loved ones
Being alone romantically, in the long term
Not being able to support herself
This was tough to compile because of the diversity of women friends I have. Mom's, single Moms, Australian, Latinas, everything… And yes there were answers of rape, which I thought would be a higher response. Maybe that's because that was my greatest fear for my sister, Mom, and sweet grandma. And I am happy to hear that women's fears are becoming based around thriving, and no longer about surviving. Because I, and the free society believe in you. Let's create the opposite of these fears for you in affirmation form.
say, "I am in the best shape I can be in. I love my body, I love being the healthiest version of myself possible. I respect myself. My family and loved ones are safe, right now, and that makes me happy. My child has the best chance in history to live a long and happy, healthy life. I am grateful for my child's perfect healthy. Say, "I am worthy of love. The kind fo love I want is on it's way, all I must do is stay patient. I believe in myself, I know I am worthy. I believe in the romance I visualize!..In this day and age, I have so many jobs available to me. So many careers, trade schools, special license, and abundance to thrive…I know the music is done, let's go for 20 more seconds because women are worth it. .. Say, I have more success than I have ever experienced. I am enough, and there is enough for me. I am woman. and I believe in me!
Let the empowerment grow, and subscribe to this podcast
PS… 1 on 1 coaching is available, ask to donate 50% of the fee to the orphanages of Tijuana, Mexico.


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- Join Date
- Dec 2013
- Posts
- 5
Travel with a Britax Pavilion or get a new lightweight car seat? Which car seat carrier?
I have a Britax Pavilion and I'm wondering if I should purchase another, more lightweight car seat for my son for air travel; and if so, which one? We average about 12-16 flights per year. I'll be traveling alone with him in a few weeks and, while I can do it, I'd rather not lug the Pavilion through the plane while managing my 2yo. Also, does anyone have experience with the Gogo Baby Travelmate, Travelmate Deluxe or Britax Travel cart? I'm trying to decide which one would be best to use when hauling the seat through the airport and onto the airplane. Thank you for any input, it's really appreciated!
02-22-2015 09:04 PM # ADSSponsored Ads
- Join Date
- April, 2001
- Posts
- Many
Re: Travel with a Britax Pavilion or get a new lightweight car seat? Which car seat carrier?
I am a fan of a plain old luggage cart (Magna Cart is a good one) or the Traveling Toddler if you are using carry-on luggage.
I also like specific seats for planes, either because they are lightweight OR because of the way they install. I REALLY like the Radian for planes, especially if it is to be used FF. It just works SO well with the tray table (though you do need a belt extender to make it comfortable and usable).
Nearly all the best things that came to me in life have been unexpected, unplanned by me. ~Carl Sandburg
Learning to cope with the unexpected...


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Ivanka Trump's new business book, aptly titled The Trump Card: Playing to Win in Work and Life, debuted on shelves earlier this year. In it Ivanka details what it's been like growing up in The Donald's shadow and how she has already made a name for herself in real estate and fashion at the age of 27.
Interested in the book, I requested The Trump Card from my local library and patiently waited for a copy to be available. I should add this relevant disclaimer: I did not read every single word on every single page. When it comes to books, I'm a skimmer. That being said, the essence of the book was clear and palpable regardless.
Who is the Book Geared Towards?
Ivanka declares early-on in her book that The Trump Card is intended for young females looking for the kind of jump-starting career advice that would come from an older sister. She's accomplished enough to give a few pointers, but young enough to still remember what it's like to cram for tests and be nervous about job interviews.
With her audience in mind, The Trump Card intentionally doesn't come across as a normal business book. The pages read like a beauty magazine where the pretty and smart girl gets interviewed, sharing a bunch of stories while taking a trip down memory lane. I found the storytelling and chattiness to be likeable, but straight-shooters looking for hard career facts will likely want to skim more than I did, or bypass the book altogether.
Book Review: Is "The Trump Card" a Must-Read?
Was it good? Yes. Was it a must-read? Probably not.
There were definitely parts that entertained and enlightened, but I didn't find anything completely revolutionary in what was shared. Mrs. Trump does give some practical job advice, but the fact that the book is a business memoir lends itself to storytelling and explanations of "here's how it worked for me" rather than "here's how it really can work for you." After all, the voice is that of an individual professional and not a team of researchers compiling studied trends among women in the workforce.
However, Ivanka includes a "Bulletins from my Blackberry" entry at the end of each chapter quoting from her many famous and successful friends. One bulletin is from television-guru Mark Burnett; another is from Zappos' Tony Hsieh, and so on. If you're only going to skim The Trump Card, I'd take the time to read the bulletins as these were my favorite parts of the book.
Source: Trump, Ivanka, "The Trump Card: Playing to Win in Work and Life."


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|Location: I Building/ I-201 (campus map)
|Subject: Sermon Discussion
|Taught by: Randy Leinen 818.776.1500 email@example.com, Mika Kaltoft 818.918.1570 firstname.lastname@example.com
If you are a college student or young adult, this class is for you! We are currently studying the Gospel of Luke. Learn how to study God's Word as we Observe, Interpret and Apply together the truths about Jesus' life and ministry.


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Orlando City officials confirmed striker Dom Dwyer will miss the club's Friday match against Toronto FC.
Dwyer, who has six goals so far this season, did not travel with the team to Toronto. He is not listed in the latest MLS injury report, though he trained separately from the rest of the team most of this week. Club officials said Dwyer has a "lower-body injury."
Dwyer's return to Orlando City's lineup sparked the club's six-match win streak.
The Lions (6-3-1, 19 points) will also be without midfielder Yoshi Yotún due to yellow card accumulation.
Orlando City is coming off a 2-1 loss to rival Atlanta United.
Orlando City plays Toronto FC (2-6-1, 7 points) at 8 p.m. on Friday at BMO Field.


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The perfect excuse for eating chocolate for breakfast is with these delicious chocolate chunk muffins from a cake mix. This recipe has been updated since it first appeared on my site in 2016 with new photos and a new printable recipe card.
Chocolate Chunk Muffins from a Cake Mix
Yes, you can have chocolate for breakfast and these Chocolate Chunk Muffins from a Cake Mix are the perfect recipe to enjoy!
Every once in awhile it's nice to make something sweet and delicious for breakfast for your family. Maybe you're hosting a brunch at your house or you're having overnight guests to your home at some point this summer.
These chocolate chunk muffins are the perfect treat to add to your breakfast menu SOON because they don't require much work to bake them. As a matter of fact, they start out with a cake mix so it doesn't get any easier than that to enjoy a little chocolate for breakfast with your family this weekend.
- Spray the cupcake liners with non-stick cooking spray to make sure the muffin doesn't stick. I started doing this years ago when the paper liners were sticking to whatever I was baking.
- You can also use a Silicon Muffin Tray and you won't need paper baking cups.
- I always bake muffins or cupcakes on the center rack in my oven.
- If for some reason, the muffin pan is not completely filled with muffin batter, fill the empty cups in the pan with water.
- Always follow baking time directions, but you'll know when the muffins are done when you insert a toothpick into the center of the muffin and it comes out clean or with just a few crumbs sticking to it. If you have batter sticking to the toothpick, you need to bake them for a few extra minutes.
How to Make Chocolate Chunk Muffins
Make sure you print out the recipe below and save it for later!
- 1 box chocolate cake mix – 15.25 ounce. This is the current size of a boxed cake mix when this recipe was updated in 2019.
- Additional flour – about two tablespoons to toss the chocolate chunks in before adding to the batter to help them not sink to the bottom of the mix when baking.
- Eggs
- Milk
- Vegetable oil
- One teaspoon baking powder – Adding the teaspoon of baking powder to the mix helps boost the freshness of the cake mix. Because, let's be honest, sometimes we have cake mixes sitting in our pantry for a long time. Or maybe it's just in my house?
- Chocolate Chunks or Chocolate Chips – either works fine!
Let's Get Baking!
- Combine the chocolate chunks or chocolate chips with the 2 tbsp of flour and set aside.
- Mix all ingredients together in a bowl except the chocolate chunks.
- After the batter has been combined, then stir in the chocolate chunks.
- Spoon into prepared muffin cups – I love using a cookie scoop!
- Bake in a 375 degree preheated oven for approximately 20 minutes or until a toothpick inserted into the middle of the muffin comes out clean.
How to Freeze Muffins
Sometimes you have leftovers. Additionally, there are times you need to bake ahead for a special weekend where you're expecting company. Here's a few simple tips on how you can freeze the muffins to enjoy at a later time:
- Bake the muffins according to the directions and allow them to cool completely.
- Place the muffins on a baking tray that's been lined with parchment paper.
- Then place the baking tray in the freezer to flash freeze the muffins for about 30 to 60 minutes. Make sure the muffins are frozen and not still soft otherwise they will stick together when you freeze them.
- Place the frozen muffins in a freezer safe container or plastic bag.
- Muffins frozen like this should last at least two months.
More Delicious Muffin Recipes
- Cheesecake Chocolate Chip Muffins – these are my favorite!
- Homemade Blueberry Muffins – easy basic muffin recipe
- Bakery Style Blueberry Streussel Muffins
The perfect excuse for eating chocolate for breakfast is with these delicious chocolate chunk muffins from a cake mix. Depending on the size of your muffin pan, this recipe should make 12 to 16 muffins. As an Amazon Associate and member of other affiliate programs, I earn from qualifying purchases.
Chocolate Chunk Muffins from a Cake Mix
Recommended Products
Nutrition Information:
Yield: 12
Serving Size: 1
Amount Per Serving: Calories: 221Total Fat: 15gSaturated Fat: 6gTrans Fat: 0gUnsaturated Fat: 8gCholesterol: 52mgSodium: 101mgCarbohydrates: 18gFiber: 1gSugar: 14gProtein: 4g
The perfect excuse for eating chocolate for breakfast is with these delicious chocolate chunk muffins from a cake mix.
Depending on the size of your muffin pan, this recipe should make 12 to 16 muffins.
As an Amazon Associate and member of other affiliate programs, I earn from qualifying purchases.


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Latest News...
New Expanded Scope of Practice for Newfoundland and Labrador Pharmacists
In the last few months the pharmacists have been given expanded roles in health care. Your pharmacist can now administer drug therapy by injection and inhalations, with Medication Manangement extend and adapt a prescription, and prescribe for minor ailments.
Posted on January 13th, 2016


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– Well guys, so this drill is shooting but this is a volley. You come in running. Running, okay, so you're
sprinting a little bit and we have Dylan who's
going to cross the ball, make a pass around and
then I'm going to shoot it with the left foot. It's important. I know it might not be
easy for most of the guys, for most of the soccer players,
but we got to be able to to practice this drill. If you want to improve at something, you have to do something
that is difficult. That challenges yourself to become better. Okay guys? Let's go. [soccer players making volley kicks] Okay, guys so I hope you liked this drill. The most important thing
about this drill is timing because you have to be running and be able to know where the
ball is coming on the side, time your run and also
have to position your body according to the ball. Because if you have your
body going backwards, the ball is going to go up. But you have to go, keep your body down, keep your head above the ball and be able to raise your knee. If you raise your knee,
the ball is going to go down. Also sometimes you have to
adapt your foot to the side because if you have your foot like this, it's going to out of the net. So if you're going to guide
it, the ball into the net, you have to move and hit it a little bit with the outside. So that's really important and key. And repetition again is the
most important in this drill because it doesn't come naturally. You just have to do it and
once you become used to it it's gonna become easier, slowly. At the beginning it's going to
be hard until you get. Okay, make sure you practice it and check our website
at bestsoccertricks.com. Thank you so much. [Best Soccer Tricks]


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U exude sensuality the way U walk the way u t…. Search EuropeDiChan's tweets. Here's my wishlist that some of U asked for. Adult entertainer w hundreds of t. Re-tweeted tweets and favorited tweets are shown so that they are easily spotted!
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