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Freshly popped popcorn covered in a light Marmite coating.
Popped to perfection and flavoured with only the freshest herbs and spices. Guzzle & Wolf popcorn will have you dropping all pretences of civilised nibbling and sharing.
- Delicious gourmet popcorn
- Freshly popped popcorn, lightly covered in a Marmite coating.
- Perfect for snacks, parties and entertaining
- Dimensions: 13cm x 22.5cm
- 45g serving
Ingredients :, Popcorn kernels, Marmite Yeast extract ,Water, Butter, Salt, Coconut Oil.
ALLERGEN: COW'S MILK, barley, wheat, oats and rye
MANUFACTURED IN A FACTORY THAT USES TREE NUTS.
Due to product settlement, there might be a slight variation in weight.

domain: food


text:
The business model of low-cost, low-manpower gyms like Anytime Fitness seem to be at odds with classic member retention strategies. However, as this article illustrates, the use of the right technology can effectively mitigate this problem.
This article explores the ways that pricing strategy can affect customer retention rate in the health and fitness industry, and suggests three ways to improve your membership retention rate by altering the way you price.
The real key to success in the fitness industry is member retention, not attraction. This article answers your big questions surrounding membership retention in the industry, including “What is retention?”, “Why retention?”

domain: health


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Plastic by Brandon Moningka
I recently took some photos of actress Marci Miller. The idea was simple. Get some plastic and have Marci interact with it. She has been a favorite muse of mine for a couple of years, mainly because she is open to the concepts I’ve proposed and easy to work with.
Without getting too philosophical about it, plastic is a major symbol in our society. It can be seen as one of the most mundane things. At the same time we rely so much on it. One moment we can’t live without it, the next we disregard it or criticize it’s affect on the environment. Whatever the case I find it an intriguing subject to shoot and was happy with how the interaction with Marci went and how it was captured here.
Model: Marci Miller (@_marcimiller_)
Brandon Moningka is a film photographer who shoots for both work and fun. He has been published for his celebrity / up & coming actor film photos.

domain: artistic


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Guinea pig meat (cuy) has become very popular in Cuzco’s cuisine as well as in national cooking. It is undergoing a boom. In our city it claims a place of preference in the dishes prepared for celebrations.
Thanks to its pleasing flavor, our people have invented a variety of dishes based on it. For example, you can find cuy chactado, pipian de cuy, baked cuy, cuy on a stick, and others.
Mostly, this animal is raised in people’s homes. It lives its life in small corrals, or in the kitchen by the traditional stove. The stoves are designed to have various holes in their lower sections so that these animals can stay warm and not get sick with the strong cold that often strikes our city.
To make the meat tasty there is a process. First, the animal needs good feed. Often, people give their cuys fresh grass, bran, and other types of food that are the secret of every household. They do this to guarantee that their guinea pigs will have great and unique tasting flesh to add to their cooking skills.
Whenever you want to enjoy this meat in which ever dish that strikes your fancy, there are just a few places outside the home where you can do so any day of the week. However, in Urbanización Bella Vista (the Bella Vista neighborhood) lovers of the cuy can find restaurants that specialize in them called cuyerías. Just outside the city, Tipón is another good place. It is twenty minutes distant and very calm and delightful. You can go their with your family to enjoy a good cuy in their company and in the open air of a garden.
The custom in Cuzco is that when people have birthdays or when visitors come, the howewives prepare something special. That is why they almost raise animals intehir yards, such as chickens, cuys, ducks and more. On that day they grab the animals they want that are ready for sacrificing and cooking. They prepare the animal and surprise you with a delicious plate, such as a cuy chactado or a pipián de cuy.
Cuys are much loved, in part, because they reproduce rapidly and in quantity. From every pregnant female cuy come seven or so small ones. They also grow to maturity quickly.
Finally, Cuy meat is found on our most typical and representative dishes, such as chiri uchu. This dish has all the meat and many other ingredients, but it cannot be lacking guinea pig. When you eat it you will realize quickly that it is one of the best dishes of our city and region of Cuzco.


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Birla Precision Technologies, Ltd. v. ERI America, Inc.
MOTION by Plaintiff Birla Precision Technologies, Ltd. for judgment (Conditional Judgment Against Blackhawk Industrial Distribution, Inc.) (Attachments: # 1 Exhibit, # 2 Exhibit, # 3 Exhibit)(Schaefers, Scott)
Case: 1:17-cv-03125 Document #: 46 Filed: 10/10/17 Page 1 of 1 PageID #:323
IN THE UNITED STATES DISTRICT COURT
FOR THE NORTHERN DISTRICT OF ILLINOIS
BIRLA PRECISION TECHNOLOGIES, INC., Plaintiff,
ERI AMERICA, INC., Defendant.
) Case No. 1:17-cv-3125
) Judge Elaine E. Bucklo
) Magistrate Judge M. David Weisman
AFFIDAVIT OF SERVICE
I, Scott A. Schaefers, state:
I am the counsel of record for plaintiff Birla Precision Technologies, Inc. (“Birla”).
On October 10, 2017 at 9:38 a.m., I personally served copies of Birla’s attached
Citation Notice and Citation to Discover Assets to Blackhawk Industrial Distribution, Inc.
(“Blackhawk”) on Blackhawk’s Illinois registered agent, National Registered Agents, Inc., 208 S.
LaSalle St., Ste. 814, Chicago, IL 60604.
Pursuant to 28 U.S.C. § 1746(2) and subject to penalty of perjury, I declare that the foregoing
statements are true and correct to the best of my information and belief.
Scott A Schaefers
Date: October 10, 2017
Case: 1:17-cv-03125 Document #: 46-1 Filed: 10/10/17 Page 1 of 4 PageID #:324
Case: 1:17-cv-03125 Document #: 46-1 Filed: 10/10/17 Page 2 of 4 PageID #:325
Case: 1:17-cv-03125 Document #: 46-1 Filed: 10/10/17 Page 3 of 4 PageID #:326
Case: 1:17-cv-03125 Document #: 46-1 Filed: 10/10/17 Page 4 of 4 PageID #:327
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.
Why Is My Information Online?

domain: law


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by styletraveller, on 2009-09-24 17:20:30
If they say 1970's tourism builds ruined the Spanish costas and islands, in the case of the Hotel Palm Beach they were wrong. This hotel turned into being quite an icon actually. Now with it's revamped 1970-ish interiours it's even all the better. And what grows better and better is the beautiful palm grove in which the hotel is situated. Very unique! Rooms are ok, maybe not completely up to today's standard but hey I here low-tech is into fashion again.... Do like the privileged feeling you get staying here, any moment you expect someone famous to pass by. Mostly German tourists...but hey...imagine. Speaking of German tourists, as this hotel is owned by a German hotel chain the breakfast is fabulous! Served in that -yes- palm grove.

domain: travel


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Today I went on an ArtWalk around the Tophane and Karakoy districts. All said, we visited eight galleries. It was inspiring to see the different works of art, although I have to admit that some of the more conceptual pieces went right over my head. The woman who started ArtWalk, Saliha Yavuz, was interesting. She essentially started this art tour after she had been working in a gallery and tourists kept coming in to ask her about the different galleries in Istanbul. And let me tell you, there are many! Anyway, she decided that this was something she could create and she did it!
At one of the galleries we met a photographer, Osman Demir, who discussed his documentary on the gentrification of neighborhoods. He discussed his trip to Romania and it was fascinating. There are some very passionate people in this world.
Also, I got to practice a bit of my Turkish. One gentleman even complimented my accent! Plus, it was a beautiful day and I got to explore some neighborhoods that I didn’t know about. This city really does have these wonderful little surprises around every corner.
At one of the last galleries, I ended up buying a small print- it is the skull made out of roses in the photos below. I just really liked this piece (it reminded me of Day of the Dead type art) and as I was talking to the art dude, I was telling him that I was feeling pretty good because it was so nice to walk around after my dang ankle injury and that pay day was yesterday. That’s when he said, “You can walk and you can pay!” All in all, what a great day.
What a great “art walk!” I love the photos and am intrigued with the variety of the art. Istanbul definitely does have an interesting art “vibe”.
Glad you are back to “almost ” normal. Getting praised for your Turkish accent is very impressive. Lovely pictures.


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Mountain Biking Highlight
The descent to Geislingen is not for the inexperienced. The first few hairpin bends I would already describe with S3 - then it will be flowiger;)
August 28, 2018
I can only agree with Markus ;-)
January 26, 2020
I didn't manage the hairpin bends down there in part😫
March 31, 2020
Trail to the left of the Ödenturm into the valley is technically demanding in a wonderful landscape
April 20, 2020
The Ödenturm was once used as a powder tower. Today it is one of the landmarks of Geislingen. It is open on some Sundays and you can go up. This is indicated by a flag.
May 31, 2020
Awesome view and the tower is really nice to look at. At the lookout point you have a beautiful view of Geislingen and the Helfenstein ruins
October 23, 2020
In the know? Log-in to add a tip for other adventurers!
Our Tour recommendations are based on thousands of activities completed by other people on komoot.
Trail Wittingen – Geiselstein
Geiselsteinhaus (Rastplatz und Weg)
Flowtrail mit Aussicht
Blick auf Geislingen von Burgruine Helfenstein
Aussicht über Überkingen
Naturteich im Rohrachtal


What is a mirrorless camera? Mirrorless cameras are a popular and increasingly advanced type of digital camera that offer many benefits for photographers of all levels. But how exactly do these cameras work, and what sets them apart from traditional DSLR (digital single-lens reflex) cameras?
One of the main differences between mirrorless cameras and DSLRs is the way in which the image is captured and displayed. In a DSLR, light enters the camera through the lens and is reflected off a mirror and through a pentaprism or pentamirror, which directs the image to the viewfinder. The photographer looks through the viewfinder to frame and focus the shot, and then presses the shutter button to take the picture. The mirror flips out of the way, and the image is captured by the image sensor.
In a mirrorless camera, there is no reflex mirror or pentaprism. Instead, the image is captured directly by the image sensor and displayed on the LCD screen or electronic viewfinder (EVF). The photographer uses the screen or viewfinder to frame and focus the shot, and then presses the shutter button to take the picture.
One of the main benefits of this design is that it allows for a smaller, more compact camera body. Because there is no reflex mirror or pentaprism, the camera can be made smaller and lighter, making it more portable and easier to carry around.
Download stunning presets for your photography here
Another advantage of mirrorless cameras is their faster autofocus and continuous shooting speeds. Because the image is captured directly by the image sensor, the camera can focus on the subject more quickly and accurately, and can capture multiple images in rapid succession. This can be especially useful for photographers who need to capture fast-moving subjects or action scenes.
Most of the big camera brands now feature mirrorless cameras in their line-ups, but each manufacturer has their own approach to the system. Sony, Canon and Nikon have fantastic ranges of mirrorless cameras, from APSC (smaller sensors) all the way to full frame (35mm sensors). This enables them to sell to a variety of consumers, both professional and amateur photographers.
Other brands, such as Fujifilm have taken a different approach as their main line up of cameras are all APSC cameras (small sensors). This is their X series and features cameras such as the Fujifilm X-T5 and the X-Pro 3.
Fujifilm have missed out on full frame altogether an instead opted for the larger sensors which they sell in their GFX cameras. These include cameras such as the GFX 50R and GFX 100s. They are both built on mirrorless, medium format sensors. See more here
Mirrorless cameras are certainly here to stay and are often much cheaper to purchase than DSLRs, especially if you purchase a used Fujifilm camera from a few years ago.
Find out the best cameras for under £500 here
Whilst many camera manufacturers have kept their DSLR cameras in production, they are quickly becoming less attractive to consumers, and it’s now only a matter of time before DSLRs are a thing of the past and go out of production. Cameras such as the Canon 5D Mk 3, which was one of the most successful cameras for professional photographers now sell in the used market for much less than £500, when just a few years ago they were selling new for several thousand pounds.
Overall, mirrorless cameras offer a number of benefits for photographers, including a compact design, fast autofocus and continuous shooting speeds, and improved image quality in certain situations. While they may not be right for every photographer, they are an increasingly popular choice for those looking to upgrade their gear or try a new type of camera.


Do Androids Dream of Electric Vespas?
Well, apparently they do because their dream has come true! Introducing the first-ever Vespa Elettrica -- the future comes to the scooter world courtesy of the world's premiere scooter maker. The Elettrica features up to 100 km (62 miles) of range -- it's the perfect scooter for running around the neighborhood visiting your
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Previous studies have highlighted a role for the Notch signalling pathway during pronephrogenesis in the amphibian Xenopus laevis, and in nephron development in the mammalian metanephros, yet a mechanism for this function remains elusive. Here, we further the understanding of how Notch signalling patterns the early X. laevis pronephros anlagen, a function that might be conserved in mammalian nephron segmentation. Our results indicate that early phase pronephric Notch signalling patterns the medio-lateral axis of the dorso-anterior pronephros anlagen, permitting the glomus and tubules to develop in isolation. We show that this novel function acts through the Notch effector gene hrt1 by upregulating expression of wnt4. Wnt-4 then patterns the proximal pronephric anlagen to establish the specific compartments that span the medio-lateral axis. We also identified pronephric expression of lunatic fringe and radical fringe that is temporally and spatially appropriate for a role in regulating Notch signalling in the dorso-anterior region of the pronephros anlagen. On the basis of these results, along with data from previous publications, we propose a mechanism by which the Notch signalling pathway regulates a Wnt-4 function that patterns the proximal pronephric anlagen.
Recent research in the field of nephrology has determined the Notch signalling pathway as an important regulator of renal development(Dressler, 2008; Kopan et al., 2007; Liu et al., 2007; Taelman et al., 2006). Aberrant Notch signalling during development or in adulthood is a major cause of renal disease, specifically affecting glomerulus function(Barisoni, 2008; McCright et al., 2001; Mertens et al., 2008; Niranjan et al., 2008). Despite these studies, the mechanism by which the Notch signalling pathway regulates nephrogenesis remains unknown.
The Notch signalling pathway is a paracrine signalling pathway composed of the large transmembrane proteins Notch, Delta and Serrate (or Jagged in mammals and zebrafish). In the core pathway, Notch receptors bind Delta and Serrate ligands on opposing cells, which causes cleavage of the Notch receptor(Brou et al., 2000; Kopan and Goate, 2000),liberating its intracellular domain (Notch-ICD)(Schroeter et al., 1998; Taniguchi et al., 2002). Notch-ICD then translocates to the nucleus where it binds to the CSL transcription factor, switching it from a transcriptional repressor to a transcriptional activator (Jarriault et al., 1995). Consequently, altered gene expression directs the cell towards a specified cell fate. In addition to Notch-ICD canonical signalling,Notch-ICD independent and ligand-dependent signalling have also been reported to be important in the overall signal transduction of this pathway(Ascano et al., 2003; Ikeuchi and Sisodia, 2003; Kolev et al., 2005; LaVoie and Selkoe, 2003).
The pronephros, the embryonic kidney, develops from nephrogenic mesenchyme within the intermediate mesoderm lateral to the anterior somites(Brändli, 1999; Dressler, 2006; Jones, 2005). The pronephros is a paired organ, consisting of four distinct compartments; the glomus,coelomic cavity, nephrostomes and tubules (which are further characterized as proximal, intermediate, distal and connecting tubules), together these components form a non-integrated nephron(Nieuwkoop and Faber, 1994; Reggiani et al., 2007; Saxén, 1987; Vize et al., 1995). The pronephros is a simple organ to study, showing both morphological and physiological similarities to more complex kidney forms, the meso- and metanephros, which make it an ideal model to study with reference to kidney development (Vize et al.,1995).
McLaughlin and co-workers (McLaughlin et al., 2000) were the first to highlight the role of the Notch signalling pathway in Xenopus laevis pronephrogenesis. This study showed that expression of components of the Notch signalling pathway in the pronephros appears in two phases. The early phase is characterized by notch1, serrate1 and delta1 expression in the dorso-anterior region of the pronephric anlagen between stages 21 and 32. This is then followed by late phase expression of notch1 and serrate1more ventrally, in the developing proximal tubules. Knowledge of how the Notch signalling pathway regulates pronephrogenesis was further advanced by Taelman and colleagues (Taelman et al.,2006), who provided experimental evidence that early-phase pronephric Notch signalling promotes glomus formation, whereas late-phase pronephric Notch signalling promotes proximal tubule development.
In this study, we show that early-phase pronephric Notch signalling is required for medio-lateral patterning, a function that we suggest indirectly mediates proximal-distal patterning and permits the glomus and proximal tubules to develop in isolation. We provide evidence highlighting a novel role for Wnt-4 in establishing the glomus and nephrostomes, a function that is probably regulated by the Notch effector gene hrt1 (also known as hey1). Furthermore, we report pronephric expression of X. laevis homologues of the Drosophila fringe gene, lunatic fringe and radical fringe, and propose a role for their gene products in proximal pronephrogenesis.
MATERIALS AND METHODS
Whole mount in situ hybridisation
Whole-mount in situ hybridisation was carried out as described elsewhere(Harland, 1991). The embryos were fixed in MEMFA (0.5 M MOPS, pH 7.4, 100 mM EGTA, 1 mM MgSO4,4% formaldehyde) and linearized plasmid from Na+/K+-ATPase (SmaI/T7), slc5a2(EcoRI/T7), odf3 (EcoRI/T7), nephrin (SmaI/T7),Wnt-4 (XhoI/T7), Notch-1 (ClaI/SP6), Delta-1(NdeI/T7), Serrate-1 (HindIII/T7), lunatic fringe(NcoI/T7), radical fringe (BamHI/T7), ClC-K(NotI/T7), GATA-3 (SmaI/T7), Xbrachyury (SacII/T3), MHC(NcoI/SP6), HRT-1 (Not-1/T7) and Lim-1 (XhoI/T7)was used to generate digoxigenin-11-UTP-labelled (Boehringer Mannheim)antisense RNA probes from the polymerases indicated. Probes were visualised using anti-DIG-alkaline phosphatase secondary and NBT/BCIP for the colour reaction according to the manufacturer's recommendations (Boehringer Mannheim).
mRNA synthesis, morpholino antisense oligonucleotides and micro-injection
Capped RNAs were synthesised in vitro from Notch-ICD, DeltaSTU,Su(H)DBM (Chitnis et al.,1995), HRT-1-hGR(Taelman et al., 2006), Wnt-4,Lfng and Rfng using the SP6 Message Machine Kit (Ambion). Typically 600 pg notch-ICD mRNA, 1 ng deltaSTU mRNA, 1 ng su(H)DBM mRNA, 1 ng hrt1 mRNA, 1 ng wnt4mRNA, 500 pg lfng mRNA, 2 ng rfng mRNA and 400 pg beta-galactosidase mRNA was injected. The lfng, rfng, wnt4and hrt1 antisense morpholino oligonucleotides used were: lfng MO1 5′-gcttcttcccccagttcttcagcat-3′; lfng MO2 5′-gcgctaccttctctcctcctctata-3′; rfngMO 5′-tgaggccaacgtaagtgactcttcat-3′; wnt4 MO 5′-gtactctggggtcatcctgctgctg-3′; and hrt1 MO 5′-tagtcgtgtccccgcttcatggctg-3′; (Gene Tools LLC). The control morpholino is a random sequence of the same length. Typically 20 ng of rfng MO, hrt1 MO and control MO, 10 ng of wnt4 MO and 5 ng lfng MO1 or lfng MO2 was injected. Embryos were dejellied and injected with mRNA alone or in combination with MO (as specified in the text) into a V2 blastomere of an eight-cell-stage embryo to target the future pronephros (Dale and Slack,1987; Moody and Kline,1990), under 5% Ficoll in BarthX.
Whole-mount immunohistochemistry was performed using standard methods on MEMFA-fixed embryos. The primary antibodies used were monoclonal antibody 3G8,which detects the proximal tubules, and 4A6, which detects the intermediate and distal tubules (Vize et al.,1995). The secondary antibody was alkaline phosphatase-conjugated goat anti-mouse (Sigma). BCIP/NBT (Boehringer) or Fast Red TR/Napthol AS/MX(Sigma) was used for the colour reaction, according to the manufacturer's recommendations.
In vitro translations of lfng and rfng mRNA
Either mRNA alone, or mRNA with a MO, was translated in vitro in the Rabbit Reticulocyte Lysate System (Promega) with 35S-Met according to the manufacturer's protocol. Reactions (25 μl) were denatured at 95°C in 2×SDS loading buffer (Harlow and Lane, 1988) and run on a 10% (w/v) SDS-PAGE resolving gel using a vertical mini-gel apparatus for 2 hours at 100 V. The gel was exposed to Kodak X-ray film overnight at room temperature, before being developed.
Early phase pronephric Notch signalling promotes formation of the nephrostomes
In order to mis-activate the Notch signalling pathway we injected notch-ICD mRNA, a truncated form of the Notch receptor that is constitutively active, or a dominant-negative form of the Delta ligand,DeltaSTU (Chitnis et al.,1995). Both constructs were targeted to the lateral mesoderm including the future pronephros by injection into a V2 blastomere of embryos at the eight-cell stage, with beta-galactosidase mRNA to act as a lineage tracer. Embryos were cultured to various stages of development and effects on different markers of distinct regions of the pronephros were observed. Pronephric phenotypes were determined by comparing the injected and uninjected sides. All data presented here that produced pronephric phenotypes were analysed statistically, using the Chi-squared test, and were significantly different to embryos injected with control mRNA within 95%confidence limits. Experiments were repeated several times, yielding similar data.
Overexpression of notch-ICD mRNA in embryos harvested at stage 41 and whole-mount antibody stained with 3G8, which detects nephrostomes and proximal pronephric tubules, and 4A6, which detects intermediate and distal pronephric tubules (Vize et al.,1995), showed an increase in size of the proximal region of the pronephros at the expense of the distal pronephric region. A total of 76% of embryos had ectopic 3G8 staining, whereas 4A6 staining was reduced in 97% of embryos (n=34; Fig. 1A). Injection of deltaSTU mRNA inhibited proximal pronephros formation but had no effect on distal pronephros development. 33% of embryos had reduced 3G8 staining, with only 5% having reduced 4A6 staining (n=36; Fig. 1B), suggesting that the Notch signalling pathway has no role in distal pronephros cell fate determination.
To investigate this phenotype in more detail, we injected notch-ICD mRNA and deltaSTU mRNA and cultured embryos to stage 34. In situ hybridisations for two markers of pronephric tubules; Na+/K+-ATPase, an ion transporter expressed in the proximal, intermediate and distal tubules, and slc5a2, a solute carrier expressed in the proximal tubules(Raciti et al., 2008; Reggiani et al., 2007), was then performed. Injection of notch-ICD mRNA reduced Na+K+ATPase (also known as atp1a1)expression in 96% of embryos (n=24; Fig. 1C), and similarly reduced slc5a2 expression in 95% of embryos analysed (n=38; Fig. 1E), thus tubulogenesis was completely inhibited. Injection of deltaSTU mRNA reduced slc5a2 expression in 76% of embryos (n=34; Fig. 1F), but had a localised effect on Na+K+ATPase expression. In 23% of embryos Na+K+ATPase expression was completely reduced, however the most prominent phenotype, observed in 47% of embryos, was a reduction in only the proximal tubule region of the Na+K+ATPase expression pattern (n=43; Fig. 1D).
Since injection of notch-ICD mRNA inhibited proximal tubule formation (slc5a2 reduction), but caused ectopic 3G8 staining, we concluded that this 3G8 staining must be completely nephrostomal, because 3G8 only stains nephrostome and proximal tubule domains. To confirm this, embryos were injected with notch-ICD mRNA and deltaSTUmRNA as previously described, cultured to stage 34 and in situ hybridised for expression of outer dense fibre 3 (odf3), a marker of multi-ciliated cells in the epidermis and nephrostomes. Overexpression of deltaSTU inhibited nephrostomal odf3 expression in 89% of embryos (n=28; Fig. 1H). However, as expected, we observed ectopic nephrostomal odf3 expression in 74% of embryos injected with notch-ICDmRNA (n=27; Fig. 1G). As the percentage of embryos showing an increase in odf3 expression was equivalent to those with ectopic 3G8 staining, we conclude that notch-ICD overexpression resulted in excess nephrostomal tissue.
McLaughlin and co-workers (McLaughlin et al., 2000) and Taelman and colleagues(Taelman et al., 2006) showed that early misactivation of the Notch signalling pathway induced ectopic glomus formation, a result we have confirmed; 81% of embryos injected with notch-ICD mRNA had increased staining of the glomus marker nephrin, as observed by in situ hybridisation (n=27)(Fig. 1I). deltaSTU mRNA inhibited nephrin expression in 88%of embryos (n=34; Fig. 1J). In conclusion, early phase Notch signalling inhibits tubulogenesis and promotes glomus and nephrostome formation.
DeltaSTU is presumed to act as an anti-morph to inhibit Notch signalling, but its exact mechanism of action is unknown. To confirm that the phenotypes observed after deltaSTU overexpression resulted from inhibited Notch-ICD signalling we targeted a dominant-negative Suppressor of Hairless construct, Su(H)DBM, to the pronephros. Overexpressing su(H)DBM produced exactly the same pronephric phenotypes as deltaSTU overexpression (see Fig. S1 in the supplementary material), confirming that the pronephric phenotypes were a consequence of reduced Notch-ICD signalling. To further control for the specificity of these results, we have shown identical phenotypes using hormone inducible Notch constructs (see Fig. S2 in the supplementary material), which are consistent with previous publications(McLaughlin et al., 2000; Taelman et al., 2006).
Overexpression of wnt4 affects pronephrogenesis in an identical manner to overexpression of notch-ICD
During development, the Notch and Wnt signalling pathways frequently, if not always, act in an integrated manner to promote specific cell fates(Hayward et al., 2008). The Wnt gene wnt4, is expressed in the proximal tubules and nephrostomes during stages of development when the Notch signalling pathway is inducing nephrostome and glomus cell fate decisions(Saulnier et al., 2002). We hypothesised that these two pathways could be integrated and thus aid cell fate decisions in the dorso-anterior region of the pronephric anlagen. In order to investigate this possibility, wnt4 mRNA and beta-galactosidase mRNA were co-injected as previously described, and embryos were cultured to various stages of development to determine the effect of wnt4 overexpression on different markers of distinct regions of the pronephros.
wnt4 mRNA induced ectopic 3G8 staining in 69% of embryos, with 4A6 staining reduced in 77% of embryos (n=26; Fig. 2A). Expression of the tubule markers Na+K+ATPase and slc5a2was reduced in 83% (n=23; Fig. 2B) and 92% (n=36; Fig. 2C) of embryos,respectively. wnt4 mRNA induced ectopic nephrostomal odf3expression in 76% of embryos (n=34; Fig. 2D) and increased the domain of nephrin expression in 75% of embryos (n=32; Fig. 2E). Thus, wnt4overexpression induced ectopic nephrostome and glomus formation at the expense of tubules, an identical phenotype to notch-ICD overexpression.
Notch-ICD signalling induces expression of wnt4 in the pronephros
To observe whether Notch signalling affected wnt4 expression in the pronephros, embryos were injected with notch-ICD mRNA or deltaSTU mRNA into the V2 blastomere, cultured to stage 28, and analysed for wnt4 expression by in situ hybridisation. Of embryos injected with notch-ICD mRNA, 64% had ectopic wnt4expression (n=69; Fig. 3A). Injection of deltaSTU mRNA inhibited wnt4 expression on the injected side in 70% of embryos(n=71; Fig. 3B). In conclusion, activation of the Notch-ICD dependent pathway induced wnt4 expression. Conversely, suppression of Notch signalling inhibited wnt4 expression.
To establish whether pronephric wnt4 overexpression could upregulate components of the Notch signalling pathway, wnt4 mRNA was injected into the V2 blastomere and embryos were cultured to stage 28 where whole mount in situ hybridisation for expression of notch1, delta1and serrate1 was performed (Fig. 3C,D). wnt4 overexpression had no significant effect on notch1 (data not shown), delta1 or serrate1expression levels. delta1 expression was reduced in only 6% of embryos (n=32; Fig. 3C). Expression of serrate1 was reduced in 14% of embryos(n=36; Fig. 3D). However, we did observe a change in the anterior-posterior register of gene expression of both delta1 (84%) and serrate1 (61%). Frequently, elements of the expression pattern were more distal (see Fig. 3B, marked with bars), and different from delta1 or serrate1 expression on the uninjected side. In summary, wnt4 overexpression alters the expression pattern of delta1 and serrate1, but does not seemingly inhibit or upregulate the expression levels of these genes.
Wnt-4 depletion prevents ectopic proximal pronephrogenesis caused by injection of notch-ICD mRNA
Given that notch-ICD overexpression induced wnt4expression, but wnt4 overexpression had little effect on delta1 and serrate1 expression levels, we hypothesised that Wnt-4 could be acting downstream of the Notch signalling pathway. To investigate this possibility we simultaneously overexpressed notch-ICD and depleted endogenous wnt4 translation using a morpholino oligonucleotide (MO) (Saulnier et al., 2002). Embryos injected with notch-ICD mRNA and wnt4 MO were cultured to stage 32, where they were analysed for nephrin expression.
Single injections of notch-ICD mRNA induced ectopic expression of nephrin in 94% of embryos (n=47, data not shown). 74% of embryos injected with the wnt4 MO had reduced nephrinexpression (n=27; Fig. 4A). Co-injection of notch-ICD mRNA and the wnt4MO reduced expression of nephrin in 50% of embryos, with no embryos showing ectopic nephrin expression (n=28; Fig. 4B). We also repeated this experiment to determine the effect on 3G8 and 4A6 staining, and observed a 71%reduction in both 3G8 and 4A6 staining when notch-ICD mRNA and the wnt4 MO are co-injected (n=24, data not shown). Again, none of these embryos had ectopic 3G8 staining. Consequently, ectopic proximal pronephrogenesis normally caused by overexpression of notch-ICD mRNA was completely prevented by the wnt4 MO. In conclusion, this result suggests that Wnt-4 acts downstream of the Notch signalling pathway in the proximal pronephros to influence medio-lateral patterning.
We next attempted the reciprocal experiment; overexpressing wnt4with concomitant inhibition of Notch-ICD signalling. Co-injection of wnt4 mRNA with either su(H)DBM mRNA (57%increased, n=21; Fig. 4C) or deltaSTU mRNA (67% increased, n=36; see Fig. S3A in the supplementary material) caused ectopic nephrin expression, again suggesting Wnt-4 functions downstream of Notch signalling. We also observed the effects these co-injections had on proximal tubulogenesis. Co-injections of wnt4 mRNA and su(H)DBM mRNA caused ectopic slc5a2 expression in 43% of embryos (21% of embryos had reduced slc5a2 expression; n=28; Fig. 4D). This result is surprising because single injections of either wnt4 mRNA or su(H)DBM mRNA reduced slc5a2 expression(Fig. 2A; see Fig. S2C in the supplementary material). Similarly, co-injection of wnt4 mRNA and deltaSTU caused ectopic proximal tubulogenesis in 15% of embryos, with only 8% of embryos having reduced slc5a2 expression(n=26; see Fig. S3B in the supplementary material).
hrt1 acts upstream of wnt4 in the pronephros
The Hairy-related transcription factor gene hrt1, encodes a downstream mediator of Notch signalling that has been shown to be responsive to Notch signalling in numerous tissues(Pichon et al., 2002; Rones et al., 2002). HRT-1 has been shown to regulate glomus formation and patterning of the pronephros anlagen (Taelman et al.,2006). We aimed to determine whether hrt1 overexpression and MO knockdown affected wnt4 expression in the pronephros in order to position the activity of HRT-1 upstream or downstream of Wnt-4 during proximal pronephrogenesis.
We targeted mRNA encoding a hormone-inducible hrt1-hGR construct to the future pronephros as described previously. Embryos were left to develop to stage 18, where half of the injected embryos were switched into a dexamethasone containing culture medium to activate the message. Embryos were fixed at stage 28, stained for lineage label and analysed for wnt4expression. Embryos injected with hrt1-hGR mRNA alone displayed no phenotype (3% reduced, n=38; Fig. 5A). Of embryos incubated in dexamethasone, 29% displayed ectopic wnt4 expression (Fig. 5B), 10% had reduced wnt4 expression, and the remainder had no phenotype (n=41). We also knocked down hrt1translation using a MO previously shown to specifically deplete endogenous HRT-1 (Taelman et al., 2006). Injection of this hrt1 MO to the pronephros caused a reduction in wnt4 expression in 69% of embryos scored (n=26; Fig. 5C).
To place HRT-1 in the genetic hierarchy, we examined the effect of notch-ICD and wnt4 overexpression on hrt1expression. Mis-activation of the Notch signalling pathway induces hrt1 expression (Taelman et al.,2006). We reproduced this phenotype; 80% of embryos injected with notch-ICD mRNA had ectopic hrt1 expression (n=70; Fig. 5D). However, following wnt4 overexpression, no embryos showed regions of ectopic hrt1 expression. A total of 92% of embryos showed a slight phenotype,of which 73% had disrupted hrt1 expression, with the remaining embryos having reduced hrt1 expression (n=28; Fig. 5E). Thus, overexpression of wnt4 did not significantly affect hrt1 expression. We suggest the disrupted pronephric expression profile of hrt1 after wnt4 overexpression is a product of the overall effect Wnt-4 has on pronephrogenesis (as observed in Fig. 2A). Thus HRT-1 acts upstream of Wnt-4 and is required for wnt4 expression.
Overexpression of notch-ICD inhibits formation of the lateral pronephric mesoderm
Overexpression of notch-ICD caused highly significant pronephric phenotypes, increasing the size of the glomus and nephrostomes at the expense of the proximal tubules. To establish the exact spatial relationships between these two differentiated tissues and other internal structures, the injected embryos were analysed in section.
Embryos injected with notch-ICD mRNA and stained for nephrin expression by in situ hybridisation at stage 32, marking the medial pronephric region, were sectioned(Fig. 6A). In these embryos,the medial pronephric mesoderm extended into the normal region of lateral pronephric mesoderm after notch-ICD mRNA injection. The only other gene whose expression is increased after overexpression of notch-ICDis odf3, a marker specific for the nephrostomes in the pronephros. Despite their position on the lateral side of the coelom, nephrostomes form from medial pronephric mesoderm (Howland,1916). Thus it is perhaps unsurprising ectopic glomus formation is accompanied by ectopic nephrostome formation. In conclusion, we suggest that lateral pronephric mesoderm does not form in embryos overexpressing notch-ICD, the entire anlagen switches to medial pronephric fates.
Embryos, injected with notch-ICD mRNA and co-injected with gfp mRNA lineage label, were sorted for left- or right-injected sides at stage 26. At stage 41, embryos were fixed, wax-embedded, sectioned and analysed histologically with haematoxylin and eosin. Sections through the proximal pronephric region showed an abnormal mass of cells without tubular structure on the injected side (Fig. 6B). Normal pronephric development of the proximal pronephric region could be clearly seen on the uninjected side of this embryo. The glomus, indicated by dispersed podocytes, the coelomic cavity, and the proximal tubules all formed in their correct positions. In sections through the distal pronephric region ectopic glomus and nephrostomal tubule formation was clearly visible on the injected side(Fig. 6C), whereas a single distal tubule was visible on the uninjected side.
lunatic fringe and radical fringe are expressed in the dorso-anterior region of the proximal pronephros at tail bud stages of development
Fringe proteins are glycosyl transferases that extend carbohydrate chains on the EGF repeats of the extracellular domain of the Notch receptor. In Drosophila, such post-translational modifications promote Notch interactions with its Delta ligand(Bruckner et al., 2000; Moloney et al., 2000). Previous studies have highlighted roles for downstream Notch effector genes during proximal pronephrogenesis (Rones et al., 2002; Taelman et al.,2006), yet an understanding for how Notch signalling is regulated,and how this regulation affects pronephros development, has not been investigated. If X. laevis homologues of the Drosophila fringe gene, lunatic fringe (lfng) and radical fringe (rfng), were temporally and spatially expressed appropriately in the pronephros, this finding would further understanding of the mechanism by which Notch signalling is regulated in the proximal pronephros.
We performed whole-mount in situ hybridisations on uninjected embryos using 3′UTR-specific DIG-labelled antisense RNA probes to detect the temporal and spatial expression of lfng and rfng in X. laevis (Fig. 7). A previous study investigated the expression pattern of lfng in X. laevis (Wu et al., 1996);however, pronephric expression was not detected. Using an improved in situ hybridisation protocol, we observed pronephric expression of both lfng and rfng. lfng is expressed in the dorso-anterior region of the proximal pronephros between stages 22 and 32. Pronephric rfng expression was detected in the same region between stages 26 and 32. The temporal and spatial expression of lfng and rfngtherefore suggests they might have a role in regulation of the Notch signalling pathway because notch1, serrate1, serrate2, delta1 and wnt4 are all expressed in this pronephric region at a similar time during development (McLaughlin et al.,2000; Rones et al.,2002; Saulnier et al.,2002; Taelman et al.,2006).
Overexpression of Radical fringe causes ectopic formation of the entire proximal pronephros
In order to observe whether mis-expressing lfng and rfngcaused pronephric phenotypes, morpholino oligonucleotides (MOs) were designed to specifically knockdown translation of endogenous lfng and rfng transcripts (for MO sequences see Fig. S4A-C in the supplementary material), and overexpression experiments were performed. In vitro translation of lfng and rfng mRNA, in a rabbit reticulocyte lysate system using 35S-methionine, could be knocked down by each specific MO (see Fig. S4D-F in the supplementary material).
We observed gross developmental defects upon mis-expression of lfng (see Figs S5-S7 in the supplementary material). Consequently, we were unable to perform functional analysis of Lfng in the pronephros. Furthermore, targeted injection of the rfng MO to knockdown endogenous rfng expression had no statistically significant effect on development of any region of the pronephros. Na+K+ATPase, slc5a2 and nephrinexpression were normal in these embryos (data not shown). Antibody staining of the mature pronephros with 3G8 and 4A6 caused a statistically insignificant 6%decrease in 3G8 staining and 2% decrease in 4A6 staining (n=47; see Fig. S5F in the supplementary material). Normal levels of rfng are therefore not required for normal development of the pronephros during embryogenesis.
By contrast, overexpression of rfng had a dramatic effect on pronephros development: rfng mRNA overexpression caused ectopic expression of the early pronephric marker lim1 in 94% of embryos at stage 26 (n=17; Fig. 8A). Expression of hrt1 was also increased by rfng overexpression in 77% of embryos (n=35; Fig. 8B). Unlike injection of notch-ICD mRNA, rfng overexpression caused ectopic 4A6 staining (63%, n=41), in addition to ectopic 3G8 staining (93%, n=41; Fig. 8C). Frequently, the proximal pronephric domain appeared to be completely duplicated in more distal regions (white arrow in Fig. 8C). Expression of tubule markers Na+K+ATPase (n=23; Fig. 8D) and slc5a2(n=22; Fig. 8E) was increased in 67% and 59% of embryos, respectively. Ectopic formation of the glomus was observed in 81% of embryos examined by in situ hybridisation for nephrin expression (n=21; Fig. 8F). Ectopic nephrostome formation was also observed, with the domain of odf3 expression increased in 62% of embryos (n=21, data not shown). In situ hybridisation detecting expression of gata3, a marker of the distal tubules, was also observed. gata3 expression was reduced in 84% of embryos (n=112; Fig. 8G). In conclusion, Rfng promotes formation of the proximal pronephros and suppresses distal pronephrogenesis.
In vertebrates, Fringe proteins promote Notch-ligand interactions(Haines and Irvine, 2003; Hicks et al., 2000; Tsukumo et al., 2006). We have shown that Wnt-4 acts downstream of Notch signalling (see above), thus we wished to confirm that Rfng also acted upstream of Wnt-4. Co-injection of rfng mRNA with the wnt4 MO reduced nephrinexpression in 65% of embryos (n=23; Fig. 8H) and also inhibited slc5a2 expression in 82% of embryos (n=22; Fig. 8I). This result indicates Rfng acts upstream of Wnt-4, most likely by mediating Notch-ligand interactions in the proximal pronephros.
Overexpression of rfng induces expression of components of the Notch signalling pathway and wnt4
Fringe proteins are enzymes whose activity is dependent on available substrate (Haines and Irvine,2003). To understand the mechanism by which Rfng induces ectopic pronephrogenesis, we injected rfng mRNA as previously described and performed in situ hybridisation for expression of wnt4, delta1 and serrate1 at stage 32 (Fig. 9). Injection of rfng mRNA caused ectopic wnt4expression in 73% of embryos (n=62; Fig. 8A). Expression of delta1 and serrate1 was ectopic in 84% and 80% of embryos,respectively, when rfng was overexpressed (n=51, Fig. 9B; n=65, Fig. 9C). We suggest that rfng overexpression caused ectopic pronephrogenesis by promoting Notch signalling, as has been described in other systems(Hicks et al., 2000; Tsukumo et al., 2006). Promotion of pronephric Notch signalling activates wnt4 expression,the protein product can be secreted in more distal positions than those found normally, thus producing ectopic proximal pronephros cell fates in distal regions.
We have established three novel findings that extend our knowledge of the mechanism by which the Notch signalling pathway regulates proximal pronephrogenesis in X. laevis. We show that early-phase Notch signalling patterns the medio-lateral axis of the dorso-anterior region of the pronephros anlagen. This activity is mediated by regulation of a downstream Wnt-4 function. We also identify lfng and rfng expression in this pronephric region, which most likely act as regulators of Notch signalling in the proximal pronephros. These findings further the mechanistic understanding for how the Notch signalling pathway regulates proximal pronephrogenesis.
Misactivation of the Notch signalling pathway induces ectopic nephrostome and glomus development, but inhibits tubulogenesis
Early mis-activation of the Notch signalling pathway in the pronephros, by injection of notch-ICD mRNA, caused increased nephrostomal and glomus formation but inhibited tubulogenesis completely. This novel identification of a nephrostomal phenotype has not been observed previously because pronephric markers used to indicate the effects of mis-expressing components of the Notch signalling pathway failed to distinguish between nephrostomes and proximal tubules. McLaughlin and co-workers(McLaughlin et al., 2000)reasoned that the increase in 3G8 staining and lim1 and pax2expression they observed after mis-activation of the Notch signalling pathway was due to an increase in proximal pronephric development (glomus,nephrostomes and proximal tubules). However, complete inhibition of tubulogenesis, indicated by loss of Na+K+ATPaseexpression, suggested that the ectopic 3G8 staining observed after notch-ICD overexpression was completely nephrostomal. This theory was confirmed by notch-ICD mRNA injection inducing ectopic odf3expression. We therefore extend the results currently in the literature to show mis-activation of early phase pronephric Notch signalling induces ectopic glomus and nephrostome development, at the expense of tubules. Indeed,mis-activation of early phase pronephric Notch signalling caused the entire anlagen to acquire a medial pronephric fate.
Overexpression of wnt4 reproduces the phenotypes observed after notch-ICD overexpression
Overexpression of wnt4 highlighted a novel role for the Wnt signalling pathway in pronephric anlagen patterning. Consistent with this, wnt4 is expressed in the dorso-anterior pronephros during early tail-bud stages of development (Saulnier et al., 2002). We overexpressed wnt4 and showed that it caused identical phenotypes to notch-ICD overexpression: ectopic nephrostome and glomus formation, accompanied with inhibited tubulogenesis. Intriguingly, Saulnier and colleagues showed that overexpression of wnt4 disrupted nephrostome formation and proximal tubulogenesis without affecting more distal tubule development(Saulnier et al., 2002). These findings are similar to our own, although they failed to observe ectopic nephrostome formation or inhibition of distal tubule development. We suggest that this difference is due to the different amounts of exogenous message injected in these two experimental series. We injected 1 ng of wnt4mRNA compared with 0.25 ng wnt4 mRNA injected by the other researchers (Saulnier et al.,2002); hence, the phenotypes they observed were less pronounced. They also knocked down translation of endogenous wnt4 transcripts using a MO and showed this inhibited proximal tubule development, but left distal tubulogenesis largely unaffected(Saulnier et al., 2002). We have confirmed this result and extended these observations to show a reduction in glomus size. Thus, wnt4 MO knockdown produces an identical phenotype to deltaSTU and su(H)DBMoverexpression. The Wnt signalling pathway is therefore required for proximal pronephrogenesis and the correlation between the phenotypes observed following overexpression of both wnt4 and notch-ICD, and inhibition of these pathways using deltaSTU mRNA and wnt4 MO injection, suggest these two pathways might be integrated.
The Notch-ICD dependent pathway is required for wnt4expression and Wnt-4 acts downstream of pronephric Notch signalling
We aimed to see whether the Notch and Wnt signalling pathways were integrated by observing the effect mis-activation of either pathway had on expression of components of the other pathway. We found that injection of notch-ICD mRNA caused ectopic wnt4 expression on the injected side. Suppression of the Notch signalling pathway by injection of deltaSTU mRNA inhibited wnt4 expression on the injected side, a phenotype that correlates well with the inhibitory effects caused by overexpressing deltaSTU on proximal pronephrogenesis.
Overexpression of wnt4 did not have an obvious effect on overall levels of notch1, delta1 or serrate1 expression, indicating that Wnt-4 does not either induce or suppress expression of components of the Notch signalling pathway. This result is informative because it suggests there is no positive-feedback loop between the Notch and Wnt signalling pathways in the proximal pronephros and the Notch-ICD-dependent pathway is required upstream for wnt4 expression. We have provided evidence that Wnt-4 acts downstream of pronephric Notch signalling since co-injection of a wnt4 MO with notch-ICD mRNA prevented the ectopic glomus and nephrostome phenotype observed when notch-ICD overexpression is performed alone. Additionally, overexpression of wnt4 and concomitant suppression of Notch signalling still caused ectopic glomus formation. This result suggests that Wnt-4 acts downstream of Notch signalling, and also illustrates that the major role of Notch signalling in the proximal pronephros is to regulate Wnt-4 function, therefore suggesting that Wnt-4 is the integral protein that patterns the proximal pronephric anlagen.
We also investigated the role of HRT-1 in regulation of wnt4expression. HRT-1 has previously been shown to regulate glomus formation and patterning of the proximal pronephros(Taelman et al., 2006). We show that HRT-1 is capable of inducing wnt4 expression, and MO depletion of HRT-1 prevented wnt4 expression, although we do not know whether these effects are either direct or indirect. By contrast, we observed a very slight disruption of hrt1 expression following wnt4overexpression, which we believe is due to the overall effect of wnt4overexpression on pronephrogenesis. Thus, we conclude that Notch-ligand interactions promote hrt1 expression, which then promotes wnt4 expression, and Wnt-4 functions to pattern the proximal pronephros.
A role for Fringe proteins during pronephrogenesis
lfng and rfng are expressed in the dorso-anterior pronephros, suggesting that Fringe proteins are active in regulation of the Notch signalling pathway in the pronephros. Interestingly, lfng, rfngand manic fringe are expressed in the proximal compartment of the mammalian nephron, although their functional role is unknown(Leimeister et al., 2003). Misexpression of lfng caused gross developmental defects, thus we were unable to confirm a role for Lfng in pronephrogenesis. However, rfng mRNA was able to rescue the effects of lfng depletion(see Fig. S8 in the supplementary material), supporting previous studies that suggest it has functional homology with Lfng(Rampal et al., 2005). Overexpression of rfng increased only the size of the proximal pronephros region at the expense of the distal pronephros, a result that correlates with the predicted proximal pronephric phenotypes we expected to observe given the expression profile of this gene in the pronephros. rfng overexpression also caused ectopic expression of delta1,serrate1 and wnt4, suggesting that Fringe proteins mediate Notch signalling in this pronephric region, which then regulates wnt4expression and ultimately proximal pronephrogenesis.
Does early phase pronephric Notch signalling establish a developmental boundary between the medial and lateral pronephric mesoderms?
We initially expected an asymmetrical Notch-ligand interaction mechanism for Notch-mediated boundary formation - as has been described in the Drosophila imaginal wing disc(Johnston et al., 1997) - to be conserved in the pronephros anlagen. This hypothesis was strengthened by our novel observations that early phase pronephric Notch signalling regulates the medio-lateral patterning of the pronephros anlagen and that both lfng and rfng are expressed in this dorso-anterior pronephric compartment. However, discrete spatial expression of Delta and Serrate genes across the medial-lateral axis of the proximal pronephros anlagen is not observed. Previous studies have outlined expression profiles for wnt4 (Saulnier et al.,2002), delta1, serrate1, notch1(McLaughlin et al., 2000), hrt1 and serrate2(Taelman et al., 2006). All these genes are expressed in the dorso-anterior region of the pronephros anlagen during the stages of lateral and medial pronephric mesoderm separation. Surprisingly, all these genes (apart from hrt1, which is initially expressed in both the lateral and medial pronephric mesoderms,before localising to the lateral pronephric mesoderm around stage 25) are expressed on the lateral side of the dorso-anterior pronephric mesoderm. None of these genes is expressed in the medial pronephric mesoderm where the future glomus develops. This lack of medial expression of genes involved in Notch signalling suggests that differential Notch-ligand interactions do not mediate separation of the lateral and medial pronephric mesoderms in X. laevis. Therefore, medio-lateral patterning of the dorso-anterior pronephros must occur by a novel Notch-mediated mechanism.
Taelman and co-workers (Taelman et al.,2006) showed that early phase pronephric Notch signalling promoted glomus development and later proximal tubulogenesis. Overexpression of rfng seemingly overcame these temporal effects and promoted formation of the entire proximal pronephros. We suggest one explanation for this phenotype could be the new-found complexity of the Notch signalling pathway(Bray, 2006; Fiuza and Arias, 2007; Kadesch, 2004). Misactivation of Notch signalling by overexpression of notch-ICD will only affect the Notch-ICD-dependent pathway; theoretically all cells in the anlagen will undergo gene expression activated by the CSL transcription complex. However,Notch-ICD-independent pathways are known to exist and are being realised as an important part of the overall signal transmitted by Notch-ligand interactions(Brennan and Gardner, 2002; Bush et al., 2001; Karsan, 2008). Unlike injection of notch-ICD mRNA, rfng overexpression would probably have a more global effect on Notch signalling because vertebrate Fringe proteins promote Notch-ligand interactions rather than one specific aspect of Notch signal transduction (Hicks et al., 2000; Tsukumo et al.,2006). Thus, we suggest that Notch-ICD-independent pathways have a role in development of the proximal pronephros. In addition, upon inhibition of Notch-ICD dependent signalling by overexpression of su(H)DBM, we have shown that Wnt-4 was able to promote proximal tubulogenesis. This surprising result is important because it indicates that Wnt-4 is capable of promoting formation of all proximal pronephric domains. Since we have shown that Wnt-4 is the integral gene regulated by Notch signalling that controls proximal pronephrogenesis, it is likely that the temporal effects of Notch signalling identified by Taelman and colleagues (Taelman et al.,2006) mediate Wnt-4 function such that Wnt-4 promotes glomus formation early, and proximal tubulogenesis later.
Collating the data we have presented here with published results, we propose the following model for Notch-mediated pronephric patterning(Fig. 9D). Notch signalling begins in a pool of cells on the lateral side of the dorso-anterior pronephros anlagen around stage 22, with Notch-ligand interactions promoted by fringe proteins. The Notch-ICD signal induces expression of wnt4, which is then secreted by these cells, perhaps acting as a morphogen, generating glomus cell fates in the medial pronephros. This signal is maintained throughout tail-bud stages of development until the lateral and medial pronephric mesoderms physically separate, around stage 28, after which medio-lateral patterning is not required and proximal pronephric Notch signalling then promotes proximal tubulogenesis.
In summary, the mechanism for Notch-mediated boundary formation observed in the Drosophila imaginal wing disc does not separate the medial and lateral pronephric mesoderms in X. laevis. Instead, we propose a pool of cells, under the control of the Notch signalling pathway regulates medio-lateral patterning in a temporal fashion. We suggest that future experimentation should focus on the function of Fringe proteins, whether Wnt-4 acts temporally, and the effect Notch-ICD-independent pathways could have on renal patterning. Given the apparent conservation of mechanisms associated with renal development, such investigations are likely to uncover novel aspects of nephrogenesis that could explain the causes of nephropathy associated with aberrant Notch signalling in humans.
We thank Peter Vize for helpful comments and the nephrin probe, Anna Philpott for Notch-ICD, DeltaSTU and MHC constructs, Kelly McLaughlin for the Notch-1 probe, Eric Bellefroid for the HRT-1 in situ probe and inducible constructs, Surinder Bhamra for performing histological sections and Paul Jarrett for maintenance of frogs. We thank unknown reviewers for improving the manuscript. This work was supported by BBSRC grants G1988, G12713.

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Loan-Anh Johnson didn’t play much last year. In fact, she played so little that coach DoBee Plaisance would not have been totally surprised if she decided to transfer during the offseason.
In 24 appearances, Johnson averaged just 6.2 minutes per game. Of the 13 Nicholls players who recorded minutes in 2018-19, she finished with the third fewest. Only six times did she record 10 or more minutes.
But considering who she was behind in the depth chart, Cassidy Barrios with her 38.5 minutes per game and Airi Hamilton with 32.9 mpg, Johnson was fortunate to see even that much time on the court.
Ultimately, though, Johnson did choose to stay in Thibodaux. Not only that but she dedicated herself to getting better and more athletic during the offseason. And with Barrios graduating at the end of last year, by the time Nicholls reported back for preseason, Johnson was expected to be a primary contributor, if not full-time starter.
Now, a year after she could hardly step on the court, Johnson rarely steps off. At 25.1 minutes per game, including 17 starts, few Colonels saw a bigger year-to-year jump in their individual production.
“I just had to come in really confident,” Johnson said. “I knew there was a lot of room in terms of points that had to be made up and as far as leadership, so it was just stepping into what we needed as a team.”
Johnson’s rapid improvement could not come at a better time for Nicholls, either. By the time the Colonels got into the meat of their conference schedule, they were down three starters due to injury and resigned to playing, at most, eight players in any given game.
That meant Johnson was forced into not just a key contributor role but into a full-fledged leadership position on top of finding her footing in the starting lineup. Fortunately, Plaisance said she came into the season in game shape and was able to focus on her new role.
She responded well with 10.3 points and 3.8 rebounds per game, both third on the team. She’s also fourth with a 71.65% clip from the field among players with more than 20 shot attempts on the season.
For her work, Johnson was named Southland Player of the Week on Jan. 13 after she record 20-plus points in back-to-back games against Lamar and Southeastern Louisiana.
“She’s probably one of the most explosive and strongest players on the team,” Plaisance said. “We really worked with her on how to be a non-one-dimensional player and to use that explosiveness to better enhance her overall game.
“Last year she was basically going to get the ball and go to the rim. Now she’s doing a better job of pulling up, assessing what’s going on, her jumper is there and it’s going to get better. And then defensively she does a real good job for us there. And then explosiveness, she’s probably one of our best finishers around the rim.”
Of course the pressure of filling Barrios’ shoes were a challenge of its own. Barrios left Nicholls as the unquestioned greatest player in program history and one of the best to ever grace the Southland.
Johnson knew and respected that. She didn’t want to be the next Cassidy Barrios, only to make her own way in the program.
Knowing she had the trust of her coach and fellow teammates, Johnson said, went a long way in giving her the confidence to succeed.
“It was hard at first because we knew it was a big piece of our team that had left,” she said. “We all had to make an orchestrated effort to bring back what we needed as a team.”
Unfortunately Johnson’s play hasn’t been able to keep Nicholls from struggling through the season due to the aforementioned injuries that continue to severely limit the options of what they can put out on the court.
She’s been crucial to the success they have experienced to date, and the Colonels are far from removed from postseason contention with nine games remaining to the regular season, but Nicholls is in a hole at 3-8 in SLC play and the deadline to begin a run is quickly approaching.
Nicholls is back in action on Wednesday at home against Stephen F. Austin. The Ladyjacks currently lead the Southland with a 9-2 record in conference and 16-4 record overall.
Johnson said the Colonels haven’t started feeling the pressure just yet, “Because I feel like a lot of us are sitting in the same place. Definitely down the line it’s going to get tighter and more worrisome. We just have to keep our cool and play our game.”

domain: news


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George Clooney is one actor who is using his popularity for a good cause. After becoming famous for his roles on the TV series ER and films like Ocean's Eleven and O Brother, Where Art Thou, Clooney won an Oscar for his performance in Syriana. As one of Hollywood's most handsome bachelors, he has been named People Magazine's Sexiest Man Alive - twice.
In spite of the celebrity frenzy that follows him, Clooney has spoken out on issues that are important to him. He traveled to Darfur to headline the Save Darfur rally and help end the genocide, and he has been equally passionate about environmental issues, driving eco-friendly vehicles. In January 2008, Clooney was named a United Nations Messenger of Peace.
Clooney was born in Kentucky and attended both Northern Kentucky University and the University of Cincinnati, though he never graduated. He has only been married once, to actress Talia Balsam, but the couple divorced in 1993. Actresses Michelle Pfeiffer and Nicole Kidman each bet $10,000 that he would have a child by the time he was 40. Clooney won.

domain: entertainment


The Fayetteville-Manlius School District Board of Education has appointed a new assistant superintendent for business services, and it’s someone many in the district already know.
At its April 4 meeting, the F-M Board of Education approved hiring Brad Corbin, who served as the district’s treasurer for nearly four years before leaving in 2013 to accept the business administrator position for the Tully Central School District. Corbin will begin his F-M assistant superintendent position Sept. 1.
“The Fayetteville-Manlius School District has been a long-time leader in education that has been known for its academic excellence and strong community support,” Corbin said. “I feel very fortunate that I was selected as the next assistant superintendent for business services, and I am very excited to be coming back to F-M. I look forward to being a part of such a great team of administrators, faculty, staff and the Fayetteville-Manlius community.”
In addition to his work with school districts, Corbin has experience as a financial analyst, an auditor with both large and small firms and worked as an accountant in the private sector.
“We look forward to welcoming Brad back to the F-M community,” F-M Superintendent Craig J. Tice said. “He brings with him a wealth of experience in the financial and education industries that will benefit our district, community and, ultimately, our students.”
Corbin, a Camillus resident, will replace William Furlong, who plans to retire as of Sept. 30. Furlong has served F-M for five years and has over 25 years of experience in the education field.
“I have truly enjoyed my time serving the entire Fayetteville-Manlius school community,” Furlong said. “It has been an honor and a privilege to work for the F-M school district, and I would like to thank the Board of Education for the opportunity to do so. I will miss working with the dedicated administrators, faculty and support staff that make F-M such an outstanding educational institution and school district of choice in Central New York.”
The assistant superintendent for business services supervises F-M’s Business Office, which is responsible for financial reporting, accounting, auditing, payroll, purchasing, records management, capital project tracking, contract negotiations and the coordination of employee benefits.
The assistant superintendent for business services also supervises the district’s transportation, buildings and grounds, information technology support and food service departments and develops an annual school budget for the consideration of the district’s board of education and voters.


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CALL OF DUTY delivers the gritty realism and cinematic intensity of World War II's epic battlefield moments like never before through the eyes of citizen soldiers and unsung heroes from an alliance of countries who together helped shape the course of modern history. Play through the chaos of battle as part of a well-trained squad that lays down covering fire and pulls it's wounded to safety. In addition to authentic squad movements and tactics, each soldier's distinct personality and training comes out on the battlefield.
Call of Duty is a Shooter/First-Person Shooter video game for N-Gage developed by Infinity Ward and published by Activision on November 1, 2004. It's rated Teen by the ESRB and no users have rated it yet.
Our newest cheats for Call of Duty were added December 2, 2010. We also have the game available on PC, in addition to N-Gage.
As we beta test our new user reviews feature, we're giving away a free Xbox, PlayStation, or Wii console to one lucky reviewer every month! Each accepted review is another contest entry (reviews must meet our publishing guidlines to be accepted). So write a Call of Duty review today for your chance to win!
If you like Call of Duty for N-Gage, then you might also like playing:

domain: game


View Full Version : Sherry Birkin RE6 China Outfit Gun Holster Assistance
09-21-2012, 10:10 PM
09-24-2012, 02:54 AM
you can maybe notch the holster to fit the scope and laser sight
09-25-2012, 05:39 PM
09-26-2012, 12:11 AM
they have holsters that fit the under rail laser sights i've seen them on amazon and ebay.
09-28-2012, 05:45 PM
Gun holster you might be looking for Police Holster (http://www.ebay.ca/itm/Uncle-Mikes-Reflex-HOLSTER-Paddle-Belt-GLOCK-Mod-17-19-22-23-NEW-/320984934318?pt=BI_Security_Fire_Protection&hash=item4abc3167ae)
10-04-2012, 04:47 PM
10-14-2012, 12:52 PM
You might have to take some liberties with Sherry's holster, if you want to holster the gun. There's no enclosed holster that I've ever seen that will fit a gun with both a laser and a mounted scope.
What you would probably need for her gun is something like this:
That's a high speed competition shooter's holster. It will let the gun fit with the laser and the scope still mounted.
If you want to be accurate with her holster, you will probably have to heavily modify an existing one, or just tuck your gun in your belt when you don't want to carry it.
10-14-2012, 07:23 PM
10-29-2012, 08:51 PM
Raven, Birkin's Triple Shot appears to be an unholy amalgam of a Beretta M93R, Springfield XD and the Sig P220 SL Sport. Honestly, unless you can get someone to cast the slide of a P220, with the hammer, rear, trigger and foregrip of the 3R with the frame of an XD, just stick with your standard cheapo airsoft gun. I usually don't tell people to buy cheapo airsoft, but with Resident Evil 6, most of the firearms are all two or three weapons combined. Take the "Bear Commander" for example. Some argue that it's based on the FN FAL paratrooper variant, but if one looks closely, the weapon is chambered in 5.56, while the FAL is chambered in 7.62, and certain parts of it look closer to what one would imagine a Chinese knockoff of an H&K G36 to look like.
So, yeah... best bet is to get a Glock 17 or Beretta M9 holster, leave the holster empty and grab yourself one of those cheap Remington 870 airsofts with the weaver rails on top for a holo sight.
11-01-2012, 06:41 PM


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Some of the protections are explained below.
Researchers follow the general ethical guidelines, some of which are listed below, proposed by various regulatory and oversight organizations.
All federally-supported research is governed by Federal regulations that are overseen by the Office of Human Research Protections (OHRP). Participation in studies is specifically regulated by Title 45 CFR 46.
Research that involve products that are regulated by the FDA follow Title 21, CFR 50
The NIH has several sets of guidelines, including some that incorporate the provisions above, for those who conduct and support clinical research funded by the NIH. Information about some of these guidelines is included below:
Back to "Steps involved in clinical research efforts" landing page.


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I was admittedly in no rush to see this but finally got around to the conclusion of the dinosaur saga on Father’s Day and it was, more or less, exactly what I expected. Jurassic World Dominion clearly wanted to cash in on nostalgia, but it struggles with tone as it doesn’t seem to know what kind of movie it wants to be.
I typically summarize the overall idea myself, but I wanted to highlight something by using the IMDB summary that reads, “Four years after the destruction of Isla Nublar, dinosaurs now live–and hunt–alongside humans all over the world. This fragile balance will reshape the future and determine, once and for all, whether human beings are to remain the apex predators on a planet they now share with history’s most fearsome creatures in a new Era.” Yeah…no. That is not what this movie is about.
Are there some dinosaurs that made it off the island and do now co-exist alongside humanity? Sure…at least to some degree…but that so-called “fragile balance” is barely an element of this narrative at all. It is less than 5% of the story and there’s no struggle for dominance atop the food chain. In fact, there’s yet another human-controlled dinosaur habitat where the creatures live in relatively plush captivity, but captivity nonetheless. That doesn’t sound like a power struggle to me.
I understand Colin Trevorrow, Emily Carmichael, and Derek Connolly not wanting to go the full Planet of the Apes route with the story, but there was room to adapt and apply some aspects of that here. Sprinkle in some Godzilla-esque humanity vs. nature and you’ve got a good template that raises some tactical and moral questions about what to do with a situation that was created due to our hubris as a species. Even half of that is a far more interesting idea than the kidnapping action/adventure rescue mission we end up on, against the social commentary backdrop of evil tech/big pharma.
The idea of a genetically manipulated global food crisis is actually very interesting and timely if you read the news. It drives home the point that Campbell Scott’s frighteningly familiar villain, Lewis Dodgson, is super evil despite his supposedly philanthropic exterior but a manufactured food shortage seems out of place with the world we are presented with.
That disconnect grows larger as a motorcycle chase through the streets of Malta gets us away from the familiar tropical setting, at least briefly. Visually, it looks strikingly similar to scenes in The Bourne Ultimatum, Skyfall, and a couple of the Mission Impossible movies. As if the filmmakers said, “Let’s do that! But with dinosaurs!” It’s a solid action sequence with clear intent that they are trying to appeal to those other crowds, but it’s missing the point. What was missing from this franchise wasn’t action and those other deficiencies are more noticeable in contrast.
By this point, it’s abundantly clear that the studio and the filmmakers don’t have a solid understanding of what made the original Jurassic Park a great film. If they do, they don’t have the ability/willingness to articulate and adapt that. That left this trilogy hopelessly handcuffed to GCI boosted dino-action and nostalgia. With the current studio model, it’s all about return on investment. As long as these movies continue to gross billions of dollars, there is no incentive for them to actually be any good. Audiences continue to show up to watch dinosaurs do dinosaur things and the humans are just sort of there. That’s essentially the exact opposite of the original film, where you care about the humans and the dinosaurs are the catalyst. So, it makes even less sense to bring back the original trio of stars and not know how to use them.
On the bright side, Jeff Goldblum is the best part of the film besides the visual effects. Watching him essentially make fun of the movie while it’s happening is the kind of self-awareness that could have benefitted the script as a whole. I don’t know if it was just his delivery as a performer or if it was written that way, but he’s the only one who knows what kind of movie he’s in. Sam Neill and Laura Dern try their best but what should be a touching and heartfelt reunion was shoehorned in and doesn’t give their history any respect. There’s a lot of tension between their characters in the original and when we are reintroduced to them again here, it’s like starting from scratch. You may as well not even set it up if you’re not going to have it pay off.
Chris Pratt and Bryce Dallas Howard never had much romantic chemistry and that hasn’t evolved over the course of the three films. He has better chemistry with newcomer Kayla Watts and this must have been pretty obvious because his and Howard’s whole character dynamic was shifted to the role of adopted parents for Maisie Lockwood, a clone from the end of Fallen Kingdom, and they work better in tandem like that. Unfortunately for them, at his point in the series, they’re along for the ride with everyone else. There’s no character development and no real depth. They are mostly there as bodies connecting plot points.
That’s already six characters that all need writing but, for some reason, this film decided to introduce several new ones and bring back a couple more. The story was either lacking some connective tissue and the extra characters were a stopgap to help explain certain things or having a big ensemble cast was always the plan and there were too many mouths to feed. Either way, the cast bloat is a real problem. There are some cool new additions like DeWanda Wise who plays Kayla Watts, a pilot who smuggles dinos, but she’s just kind of thrown in with no reasonable backstory to justify her participation in the story. It was as if they drew concept art for the character, thought it looked cool, and just went for it. Dichen Lachman’s dino-gangster, Soyona Santos, suffers from all the same issues and would feel much more at home as a Bond villain.
For lack of a better term, there’s so much meat left on the bone with this film and the franchise as a whole. The sizeable cast and dipping into the nostalgia pool require a lengthy runtime and never address the underlying themes of the series. The DNA of the original adaptation is such fertile ground but, despite attempting to walk us back through many of the once iconic moments, the Dominion and the rest of the Jurassic World trilogy spent most of its screentime fundamentally missing the point.
Recommendation: If you are a fan of the franchise, then you’ll get plenty of dinosaur action and the kind of character callbacks you’d expect from the trailers.
Thanks for reading! I still believe word of mouth is the best way to help, so if you enjoy what I’m doing, please tell somebody. And if you have a comment, I’d love to hear it! Liking, subscribing, and sharing go a long way too. As usual, be well, be safe, and have fun no matter how you get your movies!


Today the Obama administration announced a small but significant change to immigration law that will affect thousands of people and prevent the heartbreaking separation of families that takes place on a daily basis.
Pressure from legal and civil rights organizations about the racial profiling of black motorists in Fayetteville, N.C. led the Fayetteville City Council on Tuesday to informally vote to approve a temporary moratorium on consent searches.
During the four-month moratorium, the police department’s policies on searches, data collection and citizen complaints will be reviewed by an outside party, the Fayetteville Observer reported Wednesday.
Recent revelations, following a racial discrimination lawsuit, reveal that the U.S. Customs and Border Protection (CBP) agency operated a program in which they categorized those they arrested as: “Mexican”, “Other than Mexicans” or “Alien from a Special Interest Country.”
A coalition of professional organizations of police officers of color and a women’s police organization has thrown their support behind the End Racial Profiling Act of 2011. In a letter to Lamar Smith (R-TX), the chairman of the House Judiciary Committee and John Conyers (D-MI), the ranking member of the Committee, which held hearings in the fall on racial profiling, the National Council of Law Enforcement Officers Association (NCLEO) indicated strong support for passage of H.R. 3618, the End Racial Profiling Act.
New York college student Nicholas Peart discussed the impact of the NYPD’s stop-and-frisk policies on his life in an op-ed in Sunday’s New York Times. At 14, Peart’s mother told him: always carry ID, never panic if stopped by an officer and never run from the police because you might get shot.
Peart, now 23, has had to take that advice to heart.
FOR IMMEDIATE RELEASE Contact: Keith Rushing, Communications Manager, krushing@Rightsworkinggroup
(p) 202.591.3305, (c) 202.557.4291
The federal government jails U.S. citizens without probable cause. How does this happen? Watch a video about the case of Antonio Montejano, an American citizen, who was jailed for days and forced to sleep in a jail on an “immigration detainer” because of Immigration and Customs Enforcement’s Secure Communities (SCOMM) Program.


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Shenipsit State Forest
- Address: 166 Chestnut Hill Rd, Stafford Springs, CT 06076, USA, Ellington, United States
- Ticket Price: Free
Shenipsit State Forest - Review
During your vacation plans to United States, you should also consider visiting Shenipsit State Forest located in the city of Ellington. You can cross check the address and the exact location from the map given on this page. In case of any troubles reaching Shenipsit State Forest, or confusion regarding the visiting hours you can also call on the number provided.
While planning your visit, be sure to check if a ticket needs to be purchased for visiting Shenipsit State Forest and also account for the usual time required to cover the place so that you do not miss out on anything. You can also explore the various tours offered by our partners on this page which would help enhance your experience of Ellington.
Being well connected, Shenipsit State Forest has bus station(s) and railway station(s) nearby and if you prefer to use your own vehicle, information on gas stations and parking lots can also be found on this page.
On your visit to Shenipsit State Forest, you can also pay a visit to the Tourist Information Center and Public safety station for more information on the place. You can alternatively use our Ellington trip planner to explore the nearby areas to plan the day perfectly. Areas near Shenipsit State Forest, Ellington also has easy access to public essentials such as Pharmacies, ATMs and Banks and Internet Cafes, details of which can be seen on this page.
There are also a lot of options for the foodies near Shenipsit State Forest. From places offering quick bite foods such as pizzas and burgers to multi-cuisine restaurant’s, you will not regret visiting this place. There are also some specialty food places offering steaks, barbeques, sea food etc. In case you want to have it light, you can visit any of the dessert and coffee places available which should leave you content with the variety in their offerings. In case you are one of the kind who prefers a drink where ever they go, there is also an option to go to bars, breweries, cafes and hotel lounges. Also check other things to do in Ellington nearby that can be covered in case you still have time left to kill.
There are many accommodation options ranging from budget to luxury available near Shenipsit State Forest, Ellington. To avail the best hotel deals, you can go to our hotels section and look for hotels powered by our partners.
Make sure you also go through a couple of reviews on this page to get a fair idea of what you have planned to visit. You can also refer to some of the trip plans people have made when visiting Shenipsit State Forest and Ellington. This will help you account for some other places which might have skipped while planning your trip. Check out our country trip planner to start planning your own trip or Ellington vacation packages to get more ideas.
You can head to cost of trip to get an estimate of the entire trip budget including your accommodation, food and other miscellaneous things.
Shenipsit State Forest Map
Shenipsit State Forest Trips
Shenipsit State Forest, Ellington Reviews
Great place to come hike or just drive up the mountain for the view/ride. I love coming here, this is one of my favorite places to come and relax when I'm in the area. There are picnic tables at the top of the mountain to sit and eat or just hang out (which it seems like a lot of kids do because of the many drawings/writings on the tables). There are trails but I haven't gone down many of them, I like riding up the dirt road from across the street because it's a great surface for my dirt tires. I wish that the picnic tables were stained with something that would make it impossible for people to ruin but I guess that's just how it is, some people don't appreciate what we have but I'm telling you it's worth checking out!!!
Very nice place to hike and picnic. Great views of the surrounding hills. Wish the tower was open for even better viewing.
Nice area with some great hiking trails and a few nicely graded dirt roads to explore. Unfortunately the tower on Soapstone mountain is closed but there are a few nice picture areas with great views to the east.
Shenipsit State Forrest , Soapstone Mountain and the Valley Falls Trail Way are excellent places for recreation and exercise. When I'm in Connecticut this is where I head to for exercise and relaxation.
I flew over this place on my way home from Florida and Google asked me to leave a review. So here ya go! I have been here before and it's a nice hike with great views.
Find places near Shenipsit State Forest, Ellington
Workers' Fcu ATM 201 Chestnut Hill Rd0.488 km away
Clubhouse Cafe 51 Old Springfield Rd1.939 km away
Sub Surface Informational 961 Main St2.321 km away
Lake View Cafe 51 Old Springfield Rd1.907 km away
Pond House Bed & Breakfast 19 Crystal Lake Rd2.346 km away

domain: geography


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Amazon is now making use of electric three-wheelers for deliveries in India, as part of it’s drive to to produce more eco friendly vehicles to its fleet. The company has deployed almost 100 auto rickshaws, made by Indian electric car manufacturer Mahindra Electric, across seven cities including New Delhi, Bengaluru, Hyderabad, Ahmedabad, Bhopal, Indore and Lucknow.
Mahindra’s electric rickshaw is built to carry cargo. The “Treo Zor” range Amazon is using can bear a 500kg load on the back, offers 125km of range on a single charge of just under 4 hours, and has a top speed of 50 km/h.
That target also includes a global commitment of 100,000 electric vehicles in its delivery fleet, of which 10,000 are earmarked for India by 2025.
India is home to over a billion people, Amazon sees it as a massive growth opportunity. The move also fits into the retailer’s broader climate pledge of using only renewable energy by 2030. The compact Treo Zor is seemingly well-equipped for deliveries on India’s busy roads and could feasibly be expanded in the future.
TECH NEWS>>>>Spotify Expands Its Branches To 85 New Market


Here is the new addon pack for NINJA Massive. Nearly 4.000 new games are added, resulting in a total amount of 21 console systems and a total games compilation around 8.000 games…. Also more skins were added… GUI performance was boosted again….
most recent version = R3 – APRIL 2018
(included in Massive Pack 2)
bugfix for .XBE, so they go up one level when exiting Consoles
recoded, so Amiga WHD for CoinOPS is working correctly in NINJA
Correct Aspect Ratio now working with scanlines for FBA, Mega Drive, PC Engine and SNES
File Manager fixed
Smooth Right position tweaked
Big thanks to @BritneysPAIRS, @jonny555 , @rusjr1908, @smithis, @Pipmick, @Randyslim76 ,@EmperorB10S and @XeNoMoRPH for work on this pack. Amiga games will be released on standalone pack when, we can fix controls of mouse games. –ToniHC.


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I have this packet of neutral coloured pom pom for quite some time not knowing what to do with them. Yes. I bought them simply because I like them. (^_~) Recently when I was making this one-piece for Haru, I suddenly thought why not use the pom pom instead of the ribbon in the original design?
This is the outcome. Not too bad, I think. (^_^)

domain: design


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Cucumbers grown in the wrong conditions can become bitter or tasteless. Here is a simple tried and true tip for having the sweetest cucumbers in town.
Want Sweeter Cucumbers?
If you want your cucumbers to be sweeter, plant them alongside sunflowers. As they grow, the cucumber tendrils will use the stout sunflower stalks for climbing support, and you’ll have the sweetest cucumbers you’ve ever eaten. This is known as companion planting. When you plant and combine certain plants, they benefit each other, which impacts the plants’ health, taste, and yield.
What Makes them Sweeter?
When planted using this method, each plant then selects and rejects certain nutrients that alter its flavors. In other words, the companion plant makes the cucumbers sweeter by absorbing the nutrient that normally makes them bitter.
Cucumbers contain naturally occurring compounds known as cucurbitacins. The higher the level of cucurbitacins, the more bitter the cucumber will taste. Weather conditions that are too hot, or too cold can cause an increase in cucurbitacin levels, as can a lack of proper watering.
Sunflowers release an enzyme called elastase which naturally reduces the amount of cucurbitacins within the cucumber.
Cucumber Growing Tips
To ensure that your homegrown cucumbers aren’t high in bitter cucurbitacins, always plant them with sunflowers. Space them at least 8″ apart to avoid overcrowding (or grow them as we do in weed-free self-watering hydroponic grow buckets!).
Cucumbers need plenty of water, never allow the soil to go completely dry between waterings. Again, you can easily prevent this issue altogether with the self-watering grow bucket method. The buckets always have a constant, maintenance-free water supply.
Temperatures that are consistently in the ’90s or higher will cause natural stress and increase the chances of higher levels of cucurbitacins. Planting with sunflowers provides natural afternoon shade, filtering the harsh sun.
For more Companion Planting tips on getting the most from your garden, check out our Companion Planting Guide.
If you’ve purchased cucumbers and want to avoid the bitterness that they sometimes contain, try this simple Bitter Cucumber Cure.
© Can Stock Photo Inc. / fotostok_pdv

domain: agronomy


Throughout this summer, we’ve seen some excellent numbers coming out of the box office. Mission Impossible: Fallout brought in well above expectations, currently beating out the previous installment thus far. It was announced a few days ago the Black Panther just passed the $700 million in domestic, and Incredibles 2 joining the $1 billion club. This brings us to a new segment simply predicting the top five films weekend box office numbers. Without further ado, let’s get started!
According to Box Office Mojo, last week’s top five consisted of the following:
- Mission Impossible: Fallout – $35,323,815
- Disney’s Christopher Robin – $24,585.139
- The Spy Who Dumped Me – $12,103,043
- Mamma Mia! Here We Go Again – $9,003,030
- The Equalizer 2 – $8,755,907
These numbers were quite impressive considering Fallout is still holding the number 1 slot while it seems that Christopher Robin underperformed. As for the other 3 films, those hit in the ballpark on where I was predicting the previous week. Only 2 of the 3 are a new release from last week but with The Meg around the corner, it’ll be an interesting week.
1. Mission Impossible: Fallout – $25 Million
The reason Fallout will come in at the top is because of two reasons. For one, the percentage drop after it’s opening weekend was around the expected range, therefore I only see it dropping about 29% from last week’s 42.3%. The other reason this film will stay in this range is due to the lack of competition. Sure, you have The Meg coming out, but if it sticks to the same formula and pattern as previous shark films, Fallout may remain in the lead.
2. The Meg – $21 Million
Although I am excited about this movie, I don’t see it making that much. It’s projected to make around $23-24 million, but it’s hard not to think about some previous shark movies such as The Shallows opening at $16.8 million and 47 Meters Down coming in at $11.2 million opening weekend. Personally, I love shark movies, therefore I wouldn’t be surprised if it made above $21 million, however, I don’t think it’ll beat out Fallout if that film makes $25 million.
3. Disney’s Christopher Robin – $14 Million
Not having seen the movie yet, I have heard that the newest Disney reboot is quite charming. That being said, there have been reviews stating that the film has some narrative issues. This could very well be why it didn’t crack above $30 million, however, I do think it’ll have a normal percentage drop considering it’s one of the few family films out right now.
4. Slender Man – $13.5 Million
The only reason I place this film in the fourth spot is that it’s a horror flick. Say what you will about horror films, but these types of movies make money very quickly; whether they have a good weekend or not. Granted that if the film has good word of mouth, it could even take the third spot this weekend. I personally don’t know anything about the film as I try to stay away from most trailers and marketing material until I see the film. This is to give me the best viewing experience possible. It’s also important to note that horror films have been doing good recently but it’s all down to if the movie is receiving good responses. The only way I see this film having trouble is Spike Lee’s BlacKkKlansman.
5. BlacKkKlansman – $13 Million
I believe there will be a close tie between Slender Man and BlacKkKlansman. There are interesting arguments to be made whether one should take the number four slot. BlacKkKlansman is opening to over 1,500 locations but has received the Grand Prize of the Jury award at Cannes Film Festival, which has had people giving it some praise. As with Slender Man, it’s opening to about 2,300 locations which does potentially give it some legs to stay in the top five for a week or two.
Do you agree with this list? What are your predictions for the top five this weekend? Do you think ‘Slender Man’ will have what it takes to stand as a well-received horror flick? Sound off below!
Sources: Box Office Mojo