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Denon MCX8000
The Denon MCX8000 is a standalone DJ controller that gives you total command of Serato DJ software when using the controller with a laptop, however the Denon MCX8000 also features Denons new ground breaking engine software which means you can use this controller without the need for a computer or laptop!
The MCX8000 is the first true DJ hardware/software controller that offers total command of both Serato® DJ and Denon DJ Engine technology in the same instrument. The DJ can use the MCX8000 with its USB drives by opting to use Denon DJ Engine-without a computer connected! Engine enables the DJ to directly import from Serato DJ crates with all hot cues-as well as create their own-with Denon DJ's Engine software.
With MCX8000 DJs looking for a complete, high-performance set-up with flexible operation and freedom from computer-related restrictions will find an MCX8000 with Engine system fulfills all their requirements: 4-deck Serato control and Engine-provided freedom from a computer, plus the ability to switch to local USB dives when using either Serato or Engine. With the MCX8000 two DJs can seamlessly hand off from one to another combining Engine and Serato DJ. Metal construction and high quality op amps ensure rugged and high-quality performance.
Here we have the Denon MCX8000 in brand new condition and in B-Stock. The B-Stock versions would have been on display at a training evening or trade exhibition only and will come boxed 'as new' with all accessories and a 1-year warranty.
- Includes revolutionary Denon DJ Engine standalone technology with integrated Serato Cue point support
- 2 USB inputs for Engine playback in standalone mode
- Includes 4-deck Serato DJ software
- 2 high-definition displays show Engine and Serato DJ operation
- Professional 4-channel digital mixer with 2 microphone inputs and Dual XLR outputs
- 3 built-in instant pre/post fader effects for Engine playback and line inputs
- Velocity-sensitive performance pads for cues, rolls, slicer and samples
- Stage LinQ network connection to control lighting and video
- Serato DVS Upgrade ready
- Metal construction
Unit Dimensions & Weight:
- 2.39 (W) x 1.42 (D) x 0.23 (H) in feet
- 28.68 (W) x 17.04 (D) x 2.76 (H) in inches
- 728.47 (W) x 432.82 (D) x 70.10 (H) in mm
- 8.34 kg / 18.39 lbs.
Firmware 2.0 Updates:
- Added support for Engine Prime prepared USB drives
- Added Mic 1 attenuation control utility menu option (0dB to -20dB)
- Added Mic 2 attenuation control utility menu option (0dB to -20dB)
- Added ability to change USB Recording Level
- Added ability to change Talk Over Level from (-10dB to -40dB)
- Added Talk Over level utility menu option (-10dB to 0dB)
- Added Talk Over resume type utility menu option (fast, normal)
- Added Booth Receive mic signal utility menu option (on, off)
- Added Beat Grid support for Engine Prime prepared USB drives
- Added Beat Grid slide feature via SLIDE button
- Added Quantize feature via ADJUST button
- Added Quantize options 1/8, 1/4, 1/2, 1-beat and off for setting / triggering hotcues and loops
- Added Beat Sync feature
- Added pitch range lock (soft takeover) to prevent drastic pitch jumps when moving the pitch fader at large pitch percentage ranges
- Improved Slicer quantization, triggering accuracy, and drifting when in sync mode
- Improved Needle Drop accuracy
- Added 12 oclock platter LED reset for cues
- Added BPM sorting when browsing songs through BPM Filter
- Expanded support for sorted folder names from 32 to 64 characters
- Extended display time for letter popup when using first letter search
- Removed preview stutter when using needle drop
- Changed the Default Auto Loop Beat setting name to Default Auto Loop Size
- Changed Auto Cue Level to Track Start Position. Tracks will now load to the Initial Cue position or Track Start
I recently bought a dennon mcx8000 controller and what a pieace of kit this is. Up there with the best. I actually bought this from my cousins recommendation as he has one and he was spot on. Going back to webuynew to see if I can get a deal on a set of speakers, covers and case. Wish me luck.
Excellent controller. So much better than my last controller. Professional layout and excellent sound quality
I purchased the Denon mcx8000 from why buy new because my current controller got damaged. I needed my replacement asap due to upcoming gigs. The controller was posted first thing the very next day, I received updates via email every step of the way. Great service. Thanks again folks.


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bis 1.500 bar HP 1500RL-E-24V
The HP 1500RL–E–24V high-pressure gun is suitable for spray work up to 1500 bar.
With pneumatically driven rotary transmission and an electric remote control, this can be used in a very wide range of applications. The high-pressure gun, fitted for example with a nozzle carrier head, provides a high-performance jetting tool that guarantees extremely efficient area processing or surface cleaning due to the rotation of the nozzle carrier head. The high-pressure gun is notable for its ergonomically formed hand grip and low weight. The gun is fitted with trigger guard and safety catch as well as plastic hand grip and forged steel pressure housing as standard. The HP 1500RL–E–24V high-pressure gun is especially suitable as an accessory for WOMA devices in the Advanced and Expert series.
Technical data
|Operating pressure (bar)
|max. 1500
|Max. flow volume (l/min)
|Inlet temperature (°C)
|max. 50
|Rotational speed (rpm)
|Air consumption (m³/min)
|Air pressure (bar)
|Weight (kg)
|Dimensions (L × W × H) (mm)
|800 x 220 x 100
- General cleaning of parts and surfaces
- Removing paint from ship hulls
- Preparation of surfaces and substrates
- Screen and filter cleaning
- Cleaning of ship hulls and buoys
- Heat exchanger cleaning

subset: commoncrawl


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Chip producer Broadcom Ltd made a spontaneous $103 billion offer for Qualcomm Inc. on Monday, setting the stage for a takeover fight that could reshape the business at the core of the cell phone hardware industry. Qualcomm said it would audit the proposition, yet the San Diego-based organization is expected to dismiss the offer as too low and loaded with risk. Additionally, controllers may dismiss it or take too long to favor it, individuals comfortable with the issue told Reuters.
Broadcom Chief Executive Hock Tan, who turned a small chipmaker into a $100-billion organization situated in Singapore and the United States, revealed to Reuters he would not preclude an intermediary battle to persuade investors to supplant the board and acknowledge the offer.
"We are well advised and know what our options are, and we have not eliminated any of those options," said Tan, who has pulled off a string of deals over the past decade. "We have a very strong desire to work with Qualcomm to reach a mutually beneficial deal."
A joined Broadcom-Qualcomm would turn into the predominant provider of chips utilized as part of the 1.5 billion or so cell phones that are anticipated to sell the world over this year. It would up the ante for Intel Corp, which has been enhancing from its fortification in PCs into cell phone innovation by providing modem chips to Apple Inc.
"We think very long term," Tan said. "What we perceive is that the business environment has steadily improved. As we want to grow our business in the long term, that makes it easier to choose to redomicile in the U.S. to sustain our growth trends."
Yet, Mr. Tan and his group have officially made moves to address some of those issues. Broadcom said the arrangement to move its legitimate central command to the United States was fixed on Republican endeavors to update the assessment code, including the cutting of corporate duty rates. But getting on the side of the Trump organization — which will have a say in takeovers like the Qualcomm offer — was another factor.
"Now it's a game of high-stakes poker for both sides," said GBH Insight analyst Daniel Ives, who believes bullish investors were hoping for $75 to $80 per share. The offer represents a premium of 27.6 percent to Qualcomm's closing price of $54.84 on Thursday.


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About IC Realtime
IC Realtime was established in 2006 and is an American-owned manufacturer of digital surveillance and technology equipment. They are the leading IP video surveillance camera brand in the custom residential market and additionally serve a variety of government establishments like the military, commercial and residential channels. IC Realtime's mission statement is to innovate, deliver and support global video technology; greatly supporting the UL 2802 performance testing standard for camera image quality.
Notable technological breakthroughs include the introduction of the pioneering Cloud Video Recorder™, and as of lately the ground-breaking IC720 360° x 360° situational awareness video surveillance camera.
The company is well-known within the professional security sector for its products' performances. They offer a broad collection of video surveillance cameras and recorders that natively integrate with complex home automation systems.
Residential and commercial IC Realtime camera users are offered a cloud storage service called ICMyCloud, which enables them to stream and record high-definition video footage from their cameras 24/7. This service is also used to connect with IC Realtime's Echo Show cameras. In order to activate the Echo Show, you simply assign a name for the camera within Alexa and ask for it to start recording.
IC Realtime continues to expand the market with innovations for people to capture and interact with their video surveillance in new ways. Ranging from home security and crime/forensics to 360-degree lifestyle videos.


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Created for: The Sims 3
According to current and totally reliable investigations, 4 out of 5 Gunthers prefer Goth Mansion 2.0 to the original.
And why not? It has more of goth, space, comfort and style and less of stupid unnecessary teeny pointless rooms.
It has all the modern comforts, such as tv's and computers, cleverly blended into the anything but modern, stylish, somewhat oldish decor.
Be sure to leave for work in time, as there is a long, winding trail with stairs to climb down.
2 bedrooms( for a total of 4 sims), 3 bathrooms, 2 living rooms, a kitchen with a dining section, a work out area, a home office, several balconies and two fireplaces.
Needs Supernatural, Ambitions and World Adventures
Made by Prickly Hedgehog
Short URL: https://www.thesimsresource.com/downloads/1351281
ItemID: 1351281
Filesize: 46 MB kB
No stuff packs
Credits: EA, TSR & TSR artists
Expansion packs: World adventures,Ambitions,Supernatural
Please report any offensive comments so we can remove them. Use of bad language, unsuitable links or flaming may result in deletion of your account.
This Creation requires what's listed below in order to work properly.
Note that those downloads are not required for the Creation to work!
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Anyone using an Ad-blocker plugin will be forced to wait 180 seconds instead of 10 on the "please wait" page.
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many engines were added in 1.4, currentyl there are:ap20,b16b,b18,boxer4,ca18, cht16,fiat five, ford 6, gsm vr6, honda k, i4 pack, k20, jzgte, ka24det, ls3, 13b, seriesK, sr, sr20de, 2uz, vr38. These engines were added and the stock jack are still there. Some new maps were added, some removed for stability, new decals, new parts, double brake discs were added back in 1.3, new cars and much more.
The progression in garages and roc are all functional.
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NETHERLANDS/JAPAN: Mitsubishi moving production of PSA EU-bound SUVs
Mitsubishi Motors Corporation (MMC) is to transfer production of all EU-bound Outlander SUV derivatives it supplies to PSA Peugeot Citroen from its Mizushima Plant (Kurashiki, Okayama Prefecture) in Japan, where they are currently built, to its European production hub, Netherlands Car (NedCar) in Born, The Netherlands.
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We are turning Information to Inspiration in 2019! Launching our new theme, In News. With Expert Correspondents in each category we'll make sure you stay up to speed on the latest workplace productivity news!
In News will encompass the following:
- Informational content/stories to keep you inundated with the latest tips on workplace productivity and how to make your workplace great.
- Breaking News to bring infectious laughter and delight.
- Monthly "Play Along" trivia questions and opportunities for you to WIN fun prizes! Play along at innews.innovativeos.com
The Making of the Catalog Cover
Since 2011, Innovative Office Solutions has been featuring company employees on the catalog cover and marketing initiatives used throughout the year. Each summer the team heads to the studio for a full day of outfit changes, makeup, and model shots! Take a behind the scenes look at the fun we had creating this masterpiece!


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Agriculture experts have instructed farmer to grade potatoes and separates the egg-sized potatoes up to 35 to 55 mm in bulk to use as seed. Farmers should cleaned potatoes before filling, and they should use jute bags for filling.
Injured potatoes should always be placed separated from other potatoes. Better and easy way is that the potatoes should be placed in baskets after harvesting and then shift these potatoes at ground to shattered all the soil from them so that grading should be easy.


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Phone: 817-547-2094
Degrees and Certifications:
BA, SLP Assistant
Mrs. Tara Valdez
My name is Tara Valdez and I am a Speech Language Pathology Assistant! I have been a SLPA for 7 years and this is my fourth year with Birdville ISD. I love working with my students and helping them meet all of their goals!
If there is ever anything I can do to help you, or your student please let me know!
2019/2020 Schedule
Richland Elementary


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Are you tired of logging in and out of multiple systems to get your work done? Wouldn't it be nice to search for images you may want or need for a project while you're working on the project?
We understand the greatness of collaboration when it works well and the pain when it doesn't. That's why we've integrated our leading cloud-based digital asset management solution, the Widen Collective, with Workfront's cloud-based marketing work management solution – to make project and asset collaboration more seamless. By integrating Widen with Workfront, enterprise marketing teams and agencies can quickly search for and use their digital assets within the workflow of their projects for a significant boost in productivity and organization.
Key benefits for Widen and Workfront users include:
- A single Hub – The integration provides one place for your teams to collaborate on assets in the context of a project or task.
- Version Control – Marketing teams and agencies can access the latest version of each asset, thereby eliminating the human error and rework that can reduce client satisfaction.
- Faster Review – A streamlined review for your project and asset approval process.
- Accelerated Search – No need to waste valuable work time looking through your servers and desktop folders for digital assets and project files.
- More time doing what you do best – With Single Sign On, you save time uploading, downloading and logging in, so you can focus on getting things done.
To see how the integration works, request a demo to schedule time with one of our DAM Advisors.
View the press release – Widen and Workfront Announce Strategic Partnership and Software Integration.


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Additions and/or corrections to the database are encouraged! Simple Add/Edit Procedure.
Historical Features are physical or cultural features that are no longer visible on the landscape. Examples: a dried up lake, a destroyed building, a hill leveled by mining. The term makes no reference to the age, use, or any other aspect of the feature. A ghost town, for example, is not a historical feature if it is still visible.
|Displaying 1 to 4 of 4 records
|USGS Topo Map
|Bunkers Lake
|Pines Pond
|Upper Glen Lake
|Sea Cliff
|Watts Pond
|Page 1


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Patterning of cell fates along the sea urchin animal-vegetal embryonic axis requires the opposing functions of nuclear β-catenin/TCF-Lef, which activates the endomesoderm gene regulatory network, and SoxB1, which antagonizes β-catenin and limits its range of function. A crucial aspect of this interaction is the temporally controlled downregulation of SoxB1, first in micromeres and then in macromere progeny. We show that SoxB1 is regulated at the level of protein turnover in these lineages. This mechanism is dependent on nuclear β-catenin function. It can be activated by Pmar1, but not by Krl, both of which function downstream of β-catenin/TCF-Lef. At least partially distinct, lineage-specific mechanisms operate, as turnover in the macromeres depends on entry of SoxB1 into nuclei, and on redundant destruction signals, neither of which is required in micromeres. Neither of these turnover mechanisms operates in mesomere progeny, which give rise to ectoderm. However, in mesomeres, SoxB1 appears to be subject to negative autoregulation that helps to maintain tight regulation of SoxB1 mRNA levels in presumptive ectoderm. Between the seventh and tenth cleavage stages, β-catenin not only promotes degradation of SoxB1, but also suppresses accumulation of its message in macromere-derived blastomeres. Collectively, these different mechanisms work to regulate precisely the levels of SoxB1 in the progeny of different tiers of blastomeres arrayed along the animal-vegetal axis.
The correct specification and patterning of cell fates along the animal-vegetal axis of sea urchin embryos depend on the balance between the vegetalizing activity of nuclear β-catenin and the opposing activities of a set of transcriptional regulators, termed the animalizing transcription factors or ATFs (reviewed by Angerer and Angerer, 2000). Activation of the endomesoderm gene regulatory cascade begins when β-catenin enters the nuclei of micromeres, shortly after they form at the vegetal pole by the asymmetric fourth cleavage (Davidson et al., 2002a; Logan et al., 1999). Nuclearβ -catenin, functioning as a transcriptional co-activator with TCF-Lef, is required both for the differentiation of micromeres and for their acquisition of signaling properties (Logan et al., 1999). During several subsequent cleavages, β-catenin also enters nuclei of the overlying macromere progeny, where it is required for these blastomeres to respond to micromere signals, to activate downstream genes in the endomesoderm network and to relay the inductive cascade (Davidson et al., 2002b). Among the signals that mediate this series of inductions are Delta, Wnt8 and Nodal. Delta/Notch signaling is required for specifying various secondary mesenchyme cell types (Sherwood and McClay, 1999; Sweet et al., 2002; Sweet et al., 1999); Wnt8 is required for some aspects of both primary mesenchyme cell (PMC) and endoderm differentiation (Wikramanayake et al., 2004); and Nodal is required for oral ectoderm specification (Duboc et al., 2004). In addition to these defined signaling pathways, an early signal (ES), whose molecular identity is not known, is transmitted from micromeres, primarily between the 16- and 60-cell stages, and is required for normal endomesoderm development (Ransick and Davidson, 1995). The property of the endomesoderm gene regulatory network (GRN) that is most significant for the present study is the fact that nuclearβ -catenin transcriptional function currently stands alone at the top of the hierarchy, because zygotic expression of all members of this GRN depends on its function (Davidson et al., 2002b)
Opposing this β-catenin-dependent vegetal inductive cascade are the ATFs, whose prototypical and most extensively studied member is SoxB1 (Kenny et al., 1999; Kenny et al., 2003). Maternal SoxB1 protein is uniformly distributed among all nuclei through the eight-cell stage, but then rapidly begins to disappear from micromeres soon after they form at the fourth cleavage division. Beginning around the seventh cleavage, SoxB1 also progressively clears from nuclei of macromere progeny. This clearance is necessary because forced continued accumulation of SoxB1 protein in vegetal blastomeres by injection of SoxB1 mRNA at the one-cell stage blocks activation of genes in the endomesoderm GRN (L.M.A., R.C.A. and E. Davidson, unpublished) and prevents all vegetal development (Kenny et al., 2003). This phenotype is indistinguishable from that produced by loss of β-catenin nuclear function achieved through cadherin overexpression (Howard et al., 2001; Logan et al., 1999; Wikramanayake et al., 1998). Conversely, morpholino antisense knockdown of SoxB1 protein leads to a large increase in β-catenin-dependent transcriptional activity, as measured by a β-catenin/TCF-Lef-dependent reporter transgene (Kenny et al., 2003). Thus, an important function of SoxB1 in the early embryo is to limit the range ofβ -catenin activity along the AV axis.
An essential aspect of endomesoderm differentiation, in turn, is the activation of mechanisms for the temporally and spatially controlled downregulation of SoxB1 in vegetal blastomeres. Elimination of SoxB1 from these vegetal lineages is completely dependent on nuclear β-catenin function (Wikramanayake et al., 1998; Logan et al., 1999; Howard et al., 2001). In micromeres, the rapid disappearance of SoxB1 is most likely to be regulated cell-autonomously, whereas in the overlying macromere lineages, clearance at around seventh cleavage depends, at least in part, on a signal from micromeres, because it is inhibited when they are removed (Oliveri et al., 2003). Generation of this signal can be stimulated by Pmar1, a micromere-specific transcription factor, because cells that lack nuclear β-catenin, but are supplied with Pmar1 mRNA, can induce clearance of SoxB1 protein in neighboring cells (Oliveri et al., 2003). We have reported that SoxB1 protein levels also are sensitive to the presence or absence of the transcriptional repressor Krl. Like Pmar1, Krl is a β-catenin target gene but, unlike Pmar1, Krl is expressed in both micromere and macromere progeny (Howard et al., 2001; Minokawa et al., 2004).
Here, we show, using loss-of-function assays, that two different nuclear,β -catenin-dependent, mechanisms operate to downregulate SoxB1 by reducing the level of its mRNA and by promoting turnover of the SoxB1 protein. Neither of these mechanisms depends on Krl, as shown by morpholino knockdown of its translation, suggesting that the previously demonstrated effects of Krl on SoxB1 levels (Howard et al., 2001) are quite indirect. The finding that SoxB1 is selectively degraded in vegetal blastomeres was unexpected and demonstrates a new kind of regulatory mechanism that mediates the antagonism between SoxB1 andβ -catenin/Tcf-Lef activity in the endomesoderm GRN. This output may work through Pmar1, as misexpression of this factor can promote SoxB1 turnover in all cells of the embryo. Lastly, we provide evidence for at least two different lineage-specific degradation mechanisms, as the turnover of SoxB1 in macromere progeny, but not in micromere progeny, depends both on its nuclear localization and on sequences in its C-terminal domain. The existence of these two degradation pathways may reflect cell-autonomous regulation of SoxB1 in the micromere lineage and non-autonomous regulation in macromere descendants.
Materials and methods
Embryo cultures
Adult sea urchins (S. purpuratus) were obtained from Charles M. Hollahan (Santa Barbara, CA). Microinjected fertilized eggs were cultured in artificial seawater (ASW) in injection Petri plates at 15°C.
Construct preparation
SoxB1-GFP was constructed using SOE (splicing by overlap extension) PCR (Horton et al., 1989) to link the SoxB1 protein-coding region (amino acid residues 1 to 344) in frame with GFP. The SoxB1 fragment was generated by PCR with a forward primer containing an XhoI site and N-terminal SoxB1 codons, and a reverse primer containing C-terminal SoxB1 and N-terminal GFP codons. The GFP fragment was generated from a pSP64T.clone construct containing GFP sequence originally derived from Green Lantern (Promega), using an N-terminal GFP forward primer and a reverse primer starting just downstream of the polyadenylation sequence. After linking the SoxB1 and GFP fragments by three rounds of PCR, the fusion was amplified with the outside primers and the amplimer was ligated into pET-15b between the XbaI and EspI (blunted) sites. This construct served as the parent plasmid for producing the series of SoxB1-GFP variants described below, all of which lack 5′ and 3′ UTR sequences. All constructs encoding SoxB1-GFP variants were verified by sequencing across junctions and by translation of synthetic mRNAs in vitro; translation of full-length proteins in vivo was verified by green fluorescence.
SoxB1DBD (DNA-binding domain)-GFP was produced by synthesizing SoxB1DBD by PCR as described previously (Kenny et al., 2003). This fragment was then ligated in frame at the EcoR1 site of GFP/pSP64T.clone. The template for production of SoxB1GFP RNA was made by PCR using a 5′ primer equipped with an Sp6 promoter followed by N-terminal SoxB1 sequence (-12 to +9 with respect to AUG; GCT CAG ACT GAC CAA AAT GTC TGT T), and a 3′ primer downstream of the polydT tract (CCG GAA TTC TGT CTT CTT CAA CAG GGT CTT; the EcoR1 site is underlined). For the templates containing a partial SoxB1 sequence C-terminal from the DNA-binding domain (NDBD SoxB1GFP and 3′NLS SoxB1GFP), the forward primers encoded an Sp6 promoter, followed by a translation initiation site (Kozak, 1989), and five SoxB1 codons, starting at either amino acid residue 146 or residue 127, respectively. These constructs lacked any SoxB1 5′ or 3′ UTR sequence. The construct encoding SoxB1 lacking the DNA-binding domain was made by deleting codons for 44 amino acid residues from the DBD of the SoxB1GFP/pET-15b plasmid template (residues 83-126) using SOE PCR (Horton et al., 1989). The resulting fragment, retaining the two nuclear localization signals, was amplified using the primers described above for SoxB1GFP. SOE PCR was used to create 3′ SoxB1-GFP deletions in the C-terminal half of the protein: deletions 1, 2 and 1+3 lack amino acid residues 138 to 252, 254 to 344 and 138 to 252 + 326 to 344, respectively. The templates for producing Krl (Howard et al., 2001), cadherin (Lee and Gumbiner, 1995) and SoxB1 MO-immune (Kenny et al., 2003) mRNAs have been described previously. SoxB1MO-immune mRNA contains no 5′ SoxB1 UTR sequence and approximately 1 kb of 3′UTR sequence. The plasmid containing full-length Pmar1 cDNA sequence in pBK-CMV was a gift from P. Oliveri and E. Davidson (California Institute of Technology, USA).
Microinjection and confocal image analysis
Fertilized eggs were microinjected with synthetic mRNAs or SoxB1MO as described previously (Kenny et al., 2003). Capped, polyadenylated mRNAs were transcribed using the mMessage mMachine kit (Ambion) and purified according to the manufacturer's instructions with DNase digestion and LiCl precipitation. RNA concentrations were chosen that generated sufficient GFP signals, but that were well below the levels that inhibit β-catenin function (Kenny et al., 2003), so that the embryos developed normally. For all SoxB1 variants, approximately 250,000 mRNA molecules were injected. Injection solutions containing RNA, 10 mM Tris-HCl (pH 8.0) and 30% glycerol were filtered by centrifugation through 0.22 μm filters. Shortly after hatching (20-23 hours post-fertilization), at least 30 embryos were examined by fluorescence microscopy for SoxB1-GFP distributions. They were then collected in a thin capillary pipette and deciliated by passage sequentially through two 100-μl aliquots of cold, hypertonic seawater (Stephens, 1997) in a depression slide. The embryos were then equilibrated in ASW, deposited into a drop of ASW underneath a coverslip and immediately photographed using a Leica TS confocal microscope. Fluorescent GFP signals were overlaid on DIC images using Adobe Photoshop.
Immunostaining and whole-mount in situ hybridization
Embryos were fixed with 4% paraformaldehyde, 1×PBS (phosphate-buffered saline), 0.2% Tween 20, for 15 minutes at room temperature, and stained with a rabbit polyclonal SoxB1 antibody and a CyIII-conjugated mouse anti-6e10 antibody, as described previously (Kenny et al., 2003). Embryos that were analyzed for both SoxB1 mRNA and protein were first hybridized with digoxigenin-labeled SoxB1 antisense RNA. The template consisted of a 415-bp BalI/NdeI fragment of the SoxB1 coding sequence inserted into the EcoRV site of pBluescript, which was digested with XhoI. RNA was synthesized with T3 RNA polymerase in vitro. Hybridization and hybrid detection were carried out according to Minokawa et al. (Minokawa et al., 2004) with the exception that the hybridization time was shortened to one day. After staining with anti-digoxigenin linked to alkaline phosphatase, embryos were immunostained by incubation for 2 hours with anti-SoxB1antibody (1:1000) and 1 hour with goat anti-rabbit fluorescein isothiocyanate (FITC)-conjugated antibody (1:500; Zymed).
Vegetal cells regulate SoxB1 protein at a post-transcriptional level
Post-transcriptionally regulated clearance of SoxB1 from vegetal blastomeres was detected when all the SoxB1 protein in the embryo was translated from uniformly distributed microinjected mRNA. To create this situation, we eliminated translation of both maternal and zygotic endogenous transcripts by injecting a SoxB1 morpholine-substituted oligonucleotide (SoxB1MO) and co-injecting SoxB1 synthetic mRNA that lacked sequence complementary to the MO (MO-immune). Immunostaining of embryos injected with SoxB1MO alone showed that, by 12 hours of development, maternal SoxB1 protein was undetectable (data not shown). As previously reported, a reproducible loss-of-function phenotype was produced, in which the archenteron failed to form and the ectoderm was radialized (Kenny et al., 2003). Although SoxB1 transcripts normally are abundant during the 3 days of development to the prism stage (Kenny et al., 1999), the SoxB1MO very effectively inhibits translation throughout this period (Fig. 1A). Surprisingly, when embryos were co-injected at the one-cell stage with MO and MO-immune SoxB1 mRNA, SoxB1 protein accumulated in the wild-type pattern, in which it was mostly confined to all of the presumptive ectoderm (Fig. 1B). This observation indicates that the protein must be relatively stable in these cells, as the injected messages turn over by the gastrula stage (2 days; data not shown). By contrast, and as in normal embryos, SoxB1 protein was absent from most macromere derivatives, including nearly all of the archenteron and secondary mesenchyme (Fig. 1B; coelomic rudiments, pharyngeal muscle fibers derived from secondary mesenchyme, and non-primary mesenchyme cells in the blastocoel are indicated by white arrowheads). Interestingly, a low level of SoxB1 protein reappears in a portion of the foregut, which is a normal site of expression after the gastrula stage. The simplest interpretation of this observation is that some translation of endogenous SoxB1 message escapes suppression at later stages by diminishing concentrations of the MO, and that the post-transcriptional mechanism is no longer active in these cells at the late gastrula stage. As we have documented previously (Kenny et al., 2003), rescue of the SoxB1 loss-of-function phenotype by uniformly distributed SoxB1 mRNA is surprisingly effective in these doubly injected embryos. As we now show, this is attributable to the fact that embryos have a robust post-transcriptional mechanism that effectively downregulates accumulation of the SoxB1 protein in vegetal lineages, thereby permitting activation of the endomesoderm GRN.
Differential post-transcriptional regulation of SoxB1 in animal and vegetal tissues of these late embryos might simply be a late aspect of their differentiation that does not operate earlier when cell fates are being specified. In normal embryos, SoxB1 is cleared from vegetal blastomeres by the mesenchyme blastula stage. To determine whether the post-transcriptional mechanism is active in earlier embryos, we examined similar doubly injected embryos at 17 hours post-fertilization. We compared the distribution of SoxB1 mRNA, which comes from both endogenous and exogenous sources, analyzed by whole-mount in situ hybridization, to that of the SoxB1 protein translated from the MO-immune transcripts, determined by immunostaining. As shown in Fig. 1C, SoxB1 protein is undetectable in the nuclei of vegetal cells, which, however, maintain the same concentration of mRNA as in the rest of the embryo. This indicates that the post-transcriptional mechanism is very robust during the period when SoxB1 protein normally disappears from macromere-derived blastomeres (seventh-eighth cleavage). Furthermore, it demonstrates that the post-transcriptional mechanism operating at this stage does not involve differential turnover of the injected mRNA.
A SoxB1GFP fusion protein is selectively degraded in vegetal cells
The mechanism that prevents accumulation of SoxB1 protein in vegetal cells could be translational or post-translational. To begin to discriminate between these alternatives, we injected mRNA encoding GFP-tagged SoxB1 lacking the 5′ and 3′ UTR sequences, in which translational control sequences nearly always reside. In all experiments in which SoxB1 mRNAs were injected, the dose was approximately 10-fold lower than the minimum dose that interferes detectably with endomesoderm development, yet high enough to detect GFP fluorescence. Live embryos accumulated this SoxB1-GFP fusion protein in a very reproducible pattern: it was detectable in all nuclei of the embryo beginning at about the eight-cell stage and was partitioned to nuclei of mesomeres, macromeres and micromeres of the 16-cell embryo, in a manner similar to endogenous SoxB1 protein (Fig. 2A). It was cleared from the descendants of micromeres and from most of the macromere progeny by the early mesenchyme blastula stage. A confocal image of a representative embryo is shown in Fig. 2B. This result strongly suggests that the post-transcriptional mechanism that clears SoxB1 from vegetal cells is selective turnover, not translational control.
Preferential turnover of SoxB1 protein in endomesoderm requires nuclear β-catenin function
Embryos in which nuclear entry of β-catenin and, consequently, development of endoderm and mesenchyme are blocked by injection of C-cadherin mRNA accumulate abnormally high levels of SoxB1 protein in all their nuclei and arrest as epithelial spheres of cells that resemble poorly differentiated ectoderm (Howard et al., 2001). To determine whether the molecular signatures of this animalized phenotype include failure of SoxB1 to be degraded in vegetal cells, we co-injected mRNAs encoding SoxB1-GFP and C-cadherin, and analyzed the distribution of the GFP reporter in embryos at the temporal equivalent of the mesenchyme blastula stage. Again the phenotype was consistent in the several dozen embryos analyzed, one of which is shown in Fig. 2C. The fusion protein accumulated uniformly in the nuclei of all cells, indicating that spatially regulated turnover of SoxB1 is an output of the β-catenin-driven endomesoderm GRN. The converse experiment, in which nuclear β-catenin activity is upregulated by microinjection of mRNA encoding a stabilized form, results in vegetalized embryos (Wikramanayake et al., 1998). Increasing doses of stabilized β-catenin progressively restrict endogenous SoxB1 protein to a region around the animal pole (Howard et al., 2001). To determine whether turnover of SoxB1 protein contributes to this spatial regulation, we co-injected SoxB1-GFP mRNA (again at a very low dose that does not produce a detectable phenotype) and message encoding stableβ -catenin at a level sufficient to vegetalize the embryos. Clearance of SoxB1 in these embryos was compared with that in embryos from the same batch injected with SoxB1-GFP mRNA alone (Fig. 2, control), when the latter had reached the mesenchyme blastula stage. As shown in Fig. 2D, upregulation of nuclear β-catenin activity expands the vegetal region in which SoxB1-GFP is preferentially degraded. We conclude that nuclear β-catenin is both necessary for SoxB1 protein turnover in normal embryos and can cause ectopic turnover when expressed in more animal blastomeres, although cells closer to the animal pole are more resistant to this effect. It is important to note that because perturbations of nuclear β-catenin concentrations cause dramatic changes in cell fate, the regulation of SoxB1 turnover byβ -catenin may be indirect.
SoxB1-GFP degradation in vegetal cells does not depend on Krl, but can be promoted by Pmar1
We next examined the potential roles of two candidate genes, Krl and Pmar1, in mediating SoxB1 turnover. Krl mRNA is expressed in the cells in which SoxB1 is selectively degraded in response to nuclear β-catenin, and accumulates in a vegetal-to-animal wave like that of nuclear β-catenin. MO-mediated loss of Krl function causes endogenous SoxB1 protein to accumulate to higher levels throughout most of the embryo, including some cells within the endomesodermal territory that normally would downregulate this protein. Embryos arrest at a stage that resembles a mesenchyme blastula (Howard et al., 2001). Conversely, mis/overexpression (MOE) of Krl by mRNA injection eliminates SoxB1 protein from almost all cells of the embryo, with the exception of a few near the animal pole (Howard et al., 2001). We therefore tested the effect of altering Krl levels on SoxB1-GFP distributions by injecting Krl mRNA or by blocking its translation with a KrlMO. As shown in Fig. 2E,F, neither perturbation detectably affected the clearance of SoxB1-GFP.
Pmar1 is a second candidate for mediating β-catenin-dependent SoxB1 turnover. Embryos mis/overexpressing Pmar1 are extremely vegetalized, with most cells of the embryo being transformed to a PMC-like phenotype by the mesenchyme blastula stage (Oliveri et al., 2002). Micromeres lacking β-catenin nuclear function but supplied with Pmar1 do not accumulate SoxB1 and can induce its downregulation in nearby cells (Oliveri et al., 2003), although it is not known whether this regulation operates at the level of SoxB1 mRNA or protein. To test whether Pmar1 can affect the stability of SoxB1 protein, mRNAs encoding Pmar1 and SoxB1-GFP were co-injected. As shown in Fig. 2G, SoxB1-GFP is not stable in these embryos, indicating that ectopic expression of Pmar1 can promote SoxB1 turnover. The effect of eliminating Pmar1 was not tested because of Pmar gene redundancy (P. Oliveri and E. Davidson, personal communication).
SoxB1 turnover in macromere derivatives requires functionally redundant sequences within its C-terminal region
SoxB1 contains a highly conserved HMG-box DNA-binding domain (DBD) that is flanked by a short N-terminal (58 amino acid residues) and a much longer C-terminal (207 residues) sequence. At the borders of the DBD are motifs matching the two separate nuclear localization signals that are found in this class of transcription factor (Fig. 3A). To begin mapping the sequences that are required for SoxB1 degradation, we injected mRNAs encoding mutated SoxB1 proteins, each tagged at its C terminus with GFP. At least 30 embryos expressing each construct were analyzed and the results were very reproducible. SoxB1-GFP chimeras lacking most of the sequence C-terminal to the DBD were stable in all cells except the PMCs (Fig. 3B). This suggests that the essential destruction sequences in SoxB1 that are recognized in macromere-derived endomesoderm reside C-terminal from the DBD, and that a different, or additional, degradation mechanism functions in micromere derivatives. Interestingly, each of several different peptides that were partially deleted in the C-terminal region of SoxB1 was degraded preferentially in both macromere and micromere progeny (Fig. 3C-E). Although a number of putative phosphorylation motifs are distributed throughout the C-terminal region, the three regions that can mediate turnover do not share a common candidate motif. These results suggest the existence of functionally redundant sequences mediating SoxB1-GFP decay. Although these data do not rule out the formal possibility that turnover is in some way compromised when the GFP and SoxB1 DBD domains are closely spaced, such an effect would be restricted to turnover in endomesoderm derived from macromeres and not from micromeres.
Turnover of SoxB1GFP in macromere progeny requires its entry into nuclei, but not binding to DNA
SoxB1 accumulates primarily in nuclei except during mitosis, when it is released to the cytoplasm (Fig. 4). To test whether SoxB1 turnover requires its nuclear localization, we injected mRNA encoding a GFP fusion lacking the SoxB1 N-terminal region, which bears the DBD, flanking nuclear localization signal (NLS) motifs and the short N-terminal peptide. As shown in Fig. 5A, this peptide remains cytoplasmic and it is degraded only in the PMCs. By contrast, a peptide lacking only the DBD but retaining the NLS motifs is cleared from both macromere and micromere progeny (Fig. 5B). It is not the case that the NLSs themselves contain motifs sufficient to target SoxB1 for degradation, because, as shown above (Fig. 3B), a fusion protein that contains the NLSs but lacks the C-terminal half of SoxB1 is stable in macromere-derived endomesoderm. In addition, introduction of two point mutations that reduce binding of SoxB1 to DNA by two orders of magnitude (Kenny et al., 2003) does not detectably alter the timing or extent of SoxB1 degradation in vegetal cells (data not shown). A construct retaining only the 3′NLS gave intermediate results: both nuclear localization and turnover in macromere derivatives were less efficient (Fig. 5C). These results suggest that, within the macromere progeny, SoxB1 is either degraded in the nucleus, possibly through ubiquitin-dependent nuclear proteasomes, or must be modified there for recognition by the degradation machinery in the cytoplasm. In either case, vegetal clearance of SoxB1 does not require its DNA-binding function.
In summary, SoxB1 turnover in macromere progeny requires nuclear entry and at least one of several functionally redundant sequences in the C-terminal region of the protein, whereas turnover of SoxB1 in the PMCs, derived from micromeres, requires neither.
SoxB1 may provide a negative feedback on its own mRNA accumulation
Previous studies showed that the distribution of SoxB1 mRNA modulates from its uniform maternal pattern to a non-vegetal pattern during blastula stages, around the seventh to eighth cleavage (Kenny et al., 1999). If SoxB1 were a positive regulator of its own transcription, then its loss from vegetal cells could account for reduced SoxB1 transcription in these cells. To test this possibility, embryos were injected with SoxB1MO and SoxB1 mRNA accumulation was analyzed semi-quantitatively by whole-mount in situ hybridization. As shown in Fig. 6, loss of SoxB1 did not lead to a decrease in signal. Instead, the signal was strongly increased in non-vegetal cells. This result corroborates real-time PCR measurements, which indicate that SoxB1 mRNA levels in whole embryos increase about tenfold in SoxB1MO embryos (C. Livi, L.M.A. and E. Davidson, unpublished). Thus, these results show that SoxB1 does not positively regulate accumulation of its mRNA and raise the possibility that it functions either directly or indirectly in negative-feedback regulation.
Downregulation of SoxB1 mRNA in vegetal cells requires nuclear β-catenin, but not Krl
To test whether the reduction in SoxB1 mRNA in vegetal cells of blastulae requires nuclear β-catenin function, we compared the distribution of this message in embryos injected with C-cadherin mRNA to that in controls injected with glycerol only (Fig. 7A). All cells accumulated approximately equivalent concentrations of SoxB1 mRNA in the experimental embryos, demonstrating that nuclear β-catenin function is required to downregulate SoxB1 mRNA in vegetal blastomeres. This effect probably reflects regulation at the level of transcription rather than differential mRNA stability, because, as shown in Fig. 1C, injected SoxB1 mRNA persists at similar concentrations in animal and vegetal cells of 17-hour blastulae. (However, a caveat to this argument is that the injected mRNA did not contain its complete 3′UTR.)
Repression of SoxB1 transcription in vegetal cells of normal embryos could be mediated by β-catenin-dependent production of a repressor, by blocking production of an essential activator, or by both of these mechanisms. A good candidate for mediating repression is Krl, because it is expressed in precisely the right cells at the right time, its production isβ -catenin-dependent and it has repressor activity (Howard et al., 2001). If Krl had this function, then its MO-mediated knockdown should increase the abundance of SoxB1 transcripts, at least in the vegetal cells that express Krl. However, neither knockdown of Krl activity nor its MOE by mRNA injection altered the downregulation of SoxB1 mRNA in vegetal blastomeres (Fig. 7B). Surprisingly, although signals were somewhat variable, knockdown of Krl usually caused a decrease in SoxB1 mRNA abundance in animal blastomeres, whereas Krl MOE resulted in a dramatic increase in expression in the same domain. Thus, Krl does not repress SoxB1 transcription; instead it can elevate SoxB1 mRNA levels. It is not clear why the regulatory effect of Krl on SoxB1 protein is negative, while on SoxB1 mRNA it is positive, given that Krl does not affect SoxB1 vegetal turnover. One possibility suggested by the phenotype of Krl MO embryos (Howard et al., 2001) is that loss of Krl leads to developmental arrest and a general delay in turnover of maternal SoxB1 protein throughout the embryo. Elevated levels of SoxB1 would then lead to reduced mRNA accumulation through negative autoregulation. Misexpression of Krl could have nonspecific effects on multiple genes leading to loss of SoxB1 and consequent increase in SoxB1 mRNA concentration.
In previous work, our laboratory has demonstrated that tight temporal and spatial regulation of SoxB1 levels is essential for correct specification and/or differentiation of the major tissue types arrayed along the animal-vegetal axis of the sea urchin embryo (Kenny et al., 2003). Misexpression of SoxB1 blocks all vegetal differentiation, while loss of SoxB1 function strongly suppresses endoderm differentiation and gastrulation. In addition, both perturbations disrupt ectoderm patterning along the orthogonal oral-aboral axis. Consistent with this stringent regulatory requirement, we provide evidence that at least five different mechanisms regulate SoxB1 levels in micromere, macromere and mesomere lineages. Collectively, these form a regulatory system whose major effects are: (1) to rapidly reduce SoxB1 levels initially in micromeres; (2) to produce a subsequent, more gradual reduction in macromere progeny; and (3) to maintain an essentially constant concentration in nuclei of the remainder of the embryo throughout most of development. Our current understanding of the known mechanisms operating within this regulatory system is summarized in Fig. 8. Although these mechanisms are illustrated in sequence according to the stages at which they are most clearly observed, more than one mechanism may operate at the same stage.
Downregulation of SoxB1 protein concentration is first observable in micromeres, shortly after these blastomeres form at the fourth cleavage (about 5.5 hours). Kenny et al. (Kenny et al., 1999) reported lower SoxB1 concentrations in nuclei of micromeres (see Fig. 8A) and noted that this difference most likely reflected, in part, the ∼4-fold lower cytoplasmic volume inherited by micromeres versus macromeres through asymmetric cleavage. In Fig. 4, we document the fact that most SoxB1 protein is released to the cytoplasm during mitosis, and re-enters nuclei during interphase. Thus, SoxB1 is distributed unequally in proportion to cytoplasmic volume. Similarly, we have not detected any inhomogeneity in the concentration of SoxB1 mRNA in 16-cell embryos, when most transcripts are of maternal origin, and therefore micromeres initially also have a decreased capacity for zygotic translation of new SoxB1 protein.
Our results show that SoxB1 expression also is spatially regulated at the level of protein turnover (Fig. 8B,C). When SoxB1 protein was synthesized from uniformly distributed microinjected mRNA transcripts and detected by immunostaining, it was found to accumulate in animal, but not vegetal blastomeres at the hatching blastula stage (about 17 hours) (Fig. 1C), thus demonstrating post-transcriptional control. When mRNA encoding a SoxB1-GFP protein chimera was injected, fluorescence initially was detectable in all cells of eight- to 16-cell embryos (Fig. 2A), but the protein turned over in both micromere and macromere progeny at the early mesenchyme blastula stage. Analysis of the behavior of partial SoxB1 peptides similarly tagged with GFP provided evidence for a separate protein turnover mechanism that functions only in micromeres: turnover in macromere derivatives requires signals in the C-terminal region and the NLS sequences, whereas neither of these is required for selective degradation in micromere derivatives (Fig. 3B, Fig. 4). Either of these mechanisms could account for the observation by Kenny et al. (Kenny et al., 1999) that the amount of SoxB1 DNA-binding activity per microgram of total protein is about 5-fold lower in whole-cell extracts of micromeres, when compared with extracts of macromeres plus mesomeres. Together, these observations suggest that the micromere-specific mechanism may be activated as early as the 16-cell stage. However, we have not been able to demonstrate this directly by the available assays because turnover of GFP-tagged peptides was not observable until the early mesenchyme blastula stage (Figs 3, 5). Potential explanations for this delay include the possibility that the level of SoxB1-GFP that is required for detection overloads the turnover mechanisms, or that the conformation of the SoxB1-GFP fusion proteins somehow slows the rate of turnover. As misexpression of SoxB1 blocks all vegetal development (Kenny et al., 2003), an early micromere-specific mechanism for removal of SoxB1 could be a crucial feature of activating the endomesoderm GRN.
In normal embryos, SoxB1 mRNA begins to be downregulated in macromere progeny between seventh and eighth cleavages (10-15 hours) (Fig. 8D). This presumably reflects regulation at the level of transcription, as there is no evidence at the present time for a lower stability of SoxB1 mRNA in vegetal blastomeres. At the same time, SoxB1 protein begins to disappear from the presumptive secondary mesenchyme and endoderm, starting within the more vegetal blastomeres (Kenny et al., 1999). The exact timing of transcriptional repression versus selective protein turnover, and the relative contributions of these mechanisms to the establishment of polarized SoxB1 distributions are not yet clear. However, it is important to note that the protein turnover mechanism is active and robust in the early blastula. We showed that SoxB1 protein is cleared from embryos that have uniformly distributed microinjected SoxB1 MO-immune mRNA that is ∼3-fold higher than the level of endogenous mRNA in the egg and throughout cleavage (Fig. 1C). The functional significance of SoxB1 protein turnover is suggested by the fact that normal vegetal differentiation proceeds on schedule in these embryos, despite the persistence of elevated levels of uniformly distributed SoxB1 mRNA. Thus, the embryo appears to have an excess capacity to downregulate SoxB1 in vegetal blastomeres at the level of protein turnover. These observations also can explain why even higher levels of microinjected SoxB1 message, i.e. about 10-fold above normal endogenous levels, are required to evoke the animalized misexpression phenotype (Kenny et al., 2003). Together, the available data strongly suggest that the post-translational mechanism serves to clear SoxB1 protein from endomesoderm more rapidly than could be achieved by downregulating mRNA abundance. However, definitive evaluation of the relative importance of the transcriptional and post-translational mechanisms in regulating SoxB1 distributions and normal patterning of fates along the AV axis will require specific inhibition of SoxB1 turnover, which is not yet possible.
Our previous work has shown that reduction of SoxB1 mRNA levels in vegetal blastomeres is dependent on the function of nuclear β-catenin. In the present work, we have demonstrated that differential turnover of SoxB1-GFP is blocked in embryos in which β-catenin function is blocked by overexpressing C-cadherin. As SoxB1 turnover follows entry of β-catenin into nuclei of macromere progeny, this process could be cell-autonomous. However, available evidence suggests that the link between nuclearβ -catenin function and SoxB1 turnover in macromere derivatives is indirect and, at least partially, non cell-autonomous. Oliveri et al. (Oliveri et al., 2003) have reported that efficient SoxB1 clearance from endomesoderm requires a signal from micromeres, as it is diminished when micromeres are removed after the fourth cleavage. This signal was reported to be mediated by Pmar1 function, as micromeres carrying C-cadherin mRNA could downregulate endogenous SoxB1, both in themselves and in nearby blastomeres, if supplied with exogenous Pmar1 mRNA. Although these experiments demonstrate a possible function for Pmar1 upstream of a signal that promotes loss of SoxB1 in macromere progeny, tests of loss of Pmar1 function will be required to confirm that it is necessary in the normal embryo; to date such tests have been confounded by the multiplicity of Pmar1-related genes. Interestingly, injection of Pmar1 mRNA also conferred on mesomeres the same capacity, although mesomere derivatives never express Pmar1 in normal embryos. This observation suggests a cell-autonomous role for Pmar1 in the clearance of SoxB1 from micromeres. Our experiments do not reveal whether the early micromere-specific protein turnover mechanism depends on nuclearβ -catenin and Pmar1, because over accumulation of SoxB1 mRNA in cadherin-expressing embryos could overwhelm this mechanism by the early mesenchyme blastula stage, when we carried out our analyses.
Finally, SoxB1 appears to exert a potent negative feedback on its own transcription because SoxB1 mRNA levels are elevated about 10-fold in SoxB1MO-knockdown embryos (Fig. 6, Fig. 8E) (C. Livi, E. H. Davidson and L.M.A., unpublished). As SoxB1 is a DNA-bending `architectural factor' without detectable ability to activate transcription independently (Kenny et al., 2001), it might interact with other regulatory factors to directly repress its own expression, or to activate production of an intermediary repressor. The fact that SoxB1 mRNA increases dramatically in abundance in presumptive ectoderm of SoxB1MO-knockdown mesenchyme blastulae, but is undetectable in all endomesoderm (Fig. 6B, Fig. 8E), suggests that this feedback loop is restricted to ectoderm, whereas other,β -catenin-dependent, mechanisms downregulate SoxB1 transcription in vegetal blastomeres. We think it likely that this feedback loop serves to limit SoxB1 accumulation in presumptive ectoderm after cleavage, as the rate of cell division decreases. Transcription of several ectoderm-specific genes has been found to decrease significantly during this same period (Gagnon et al., 1992; Lee et al., 1992).
Modulation of transcription factor concentrations via regulated turnover is a relatively unusual developmental mechanism. An example closely related to the selective degradation in early blastomeres described here is provided by the early C. elegans embryo, in which germline regulatory protein degradation is activated in somatic blastomeres during the first few asymmetric cleavages (Pellettieri et al., 2003). An example analogous to the antagonism between SoxB1 and nuclear β-catenin has been described in Xenopus embryos: in that system selective proteolysis of the homeodomain repressor Xom is activated on the future dorsal side of the embryo, thus preventing it from inhibiting activation of dorsal-specific genes, including goosecoid (Zhu and Kirschner, 2002). Targeted protein turnover has also been shown to be a response to various signals, as is probably the case for SoxB1 degradation in macromere progeny. For example, the dorsal/Nfκb cytoplasmic tether cactus/Iκb is degraded in response to signaling through Toll/cytokine receptors (reviewed by Belvin and Anderson, 1996) and the stability of β-catenin itself is regulated through Wnt signaling (for a review, see Nelson and Nusse, 2004). Our understanding of β-catenin-dependent pathways has expanded dramatically in recent years with most emphasis placed on regulatory target genes, encoding either transcription factors or signaling molecules. To our knowledge, this is the first description of a β-catenin-dependent pathway that promotes the turnover of a key developmental regulator of transcription, the SoxB1 protein of the sea urchin embryo.
Note added in proof
Using GFP-tagged β-catenin, Weitzel et al. (Weitzel et al., 2004) recently demonstrated a pattern of protein stability along the animal-vegetal axis of sea urchin embryos that is reciprocal to that of SoxB1.
We thank David Oleksyn for technical assistance, Carolina Livi and Eric Davidson for helpful discussions, and Paola Oliveri for donating pmar-1 cDNA. This work was supported by an NIH grant (NIGMS25553) to R.C.A. The information and views contained herein are not the product of any government and are not necessarily those of the US Government, DHHS or NIH.
- © 2005.


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I recorded some videos of OCTGN league play a few weeks ago, and finally got one uploaded. These assume you have a basic understanding of how to play Doomtown.
Let me know if these are at all helpful!
This particular game is my 108 Blessed vs shekky_ducky's Desolation Row. Went a little over an hour, but it would have easily been less than 55 minutes had I not been talking strategy/thoughts. Ended up being a very good game!
Great video so far - and a great example of why you might not want to go into a gunfight round 1 with your extra even though it only costs you a GR!
Aaand the danger of staying in a shootout with no cards in your hand!
And for the bit more strategic discussion - why didnt Sloane stay for one more round of the shootout after the round he lost, when he knew he was going to draw his joker in the next hand? (41:00)
On why you may not want to go into round 1, even if its only a 1 GR chump: I was probably wrong, here - that's 2 casualties I didn't have for later fights with important dudes, and even if Jaels was free, its still a lost cheatin' res goods (although shekky basically never cheats, so its not as good against him).
Lesson learned: throwing away any dude, even if that dude was free, is not smart. Exceptions include: if you have a chance to inflict some real damage, or if you can get rid of some shootout or cheatin res cards from their hand for a follow up fight.
I'll save the 3rd question for @shekky_ducky
That's an easy one: I didn't realize that. I was focused more on my empty bag o' tricks than my future draw hand. As Pr0digy points out, without cards I am nigh powerless.
Locally or online, it's 'know your opponent'.
One of my locals plays base Lawdogs plus updated tech (Judge Harry, Faster on the Draw, etc.). I know he'll tend to avoid shootouts until he has a Sun in Yer Eyes or a Pistol Whip to dictate the terms.
Where have I heard that phrase... 'Play the man, not the game...'
Which works fine untill you sit in a foreign country with opponents you dont know
That's when you start reading their lowball hands and discard pile a lot more than you usually would to try and figure out their plan
Second one uploaded: https://www.youtube.com/watch?v=MUk5XUZTOCc
#3 is up: https://youtu.be/zwlkgMwgB0Y
Relatively short one, #4 - Slide vs Aggro https://youtu.be/fmzB2ClfdMg
Follow up to #4, the next one plays the same deck (PTPT Slide) but against a less crazy aggressive deck: https://youtu.be/zGbNaVFi7T4
aka: Playing Other Peoples Decks Poorly!
Great game showing how you can win shootouts, but still narrowly lose the game. This is why I love Doomtown!


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Idiopathic Parkinson's Disease (IPD) is the second most common neurodegenerative disease, affecting an estimated 500,000 Americans;there are 35,000 new cases of IPD annually in the United States. The disease follows a progressive course, culminating in death or severe disability after about 20 years. Although there are medications that help to alleviate the symptoms of IPD by increasing central levels of the neurotransmitter dopamine, there remains no treatment to slow the relentless progression of the underlying degenerative process. A number of potential mechanisms of cell death have been illuminated by studies of pathology, epidemiology and experimental toxin-induced parkinsonism in animals. These include oxidative stress, protein aggregation, mitochondrial dysfunction, indirect excitotoxicity and apoptosis. A number of putative neuroprotective agents including propargylamines, complex I cofactors, substances affecting the mitochondrial energy potential, amantadine and others are emerging from the laboratory or early clinical trials. The Movement Disorders Group, of the Department of Neurological Sciences, Rush Medical College proposes to serve as one of 42 clinical centers for the Parkinson's Disease Neuroprotection Clinical Trial. The Rush Clinical Center will (1) participate in the design and implementation of pilot studies and the large simple clinical trials of 2 or more potential neuroprotective agents (2) recruit up to 12 subjects with early untreated Parkinson's Disease for pilot studies and 2 subjects per month for 36 months in the large simple trial and (3) collaborate with the Coordination and Statistical Centers in timely and accurate data collection and transmission.
|Wills, Anne-Marie A; Elm, Jordan J; Ye, Rong et al. (2016) Cognitive function in 1736 participants in NINDS Exploratory Trials in PD Long-term Study-1. Parkinsonism Relat Disord 33:127-133
|Wills, Anne-Marie A; Pérez, Adriana; Wang, Jue et al. (2016) Association Between Change in Body Mass Index, Unified Parkinson's Disease Rating Scale Scores, and Survival Among Persons With Parkinson Disease: Secondary Analysis of Longitudinal Data From NINDS Exploratory Trials in Parkinson Disease Long-term Study 1 JAMA Neurol 73:321-8
|Derkinderen, Pascal; Shannon, Kathleen M; Brundin, Patrik (2014) Gut feelings about smoking and coffee in Parkinson's disease. Mov Disord 29:976-9
|Elm, Jordan J; NINDS NET-PD Investigators (2012) Design innovations and baseline findings in a long-term Parkinson's trial: the National Institute of Neurological Disorders and Stroke Exploratory Trials in Parkinson's Disease Long-Term Study-1. Mov Disord 27:1513-21


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Kiwi music legends Tim Finn, Bic Runga and Dave Dobbyn will again take the stage together as the nationwide MORE FM Winery Tour announces its Summer 2010 line-up.
It will have been almost a decade since the legendary trio first shared the stage performing their own and each others' hits, together on stage throughout the entire performance. The MORE FM Winery Tour is delighted that they will reprise that hugely successful show this Summer.
Tim, Bic and Dave have already started rehearsals and promise a two-hour, back to back selection of all of their famous hits, songs that have secured them not only scores of awards but have cemented them as New Zealand music icons.
Also joining Winery Tour 2010 line-up is Boh Runga, performing songs from the Stellar* songbook as well as her solo material. Boh will be joined on stage by Supergroove front man and solo star Che Fu, bringing his own hits such as "Fade Away" and "Misty Frequencies" to the set. This will be the first time these two powerful vocalists will have performed together, and it is sure to be a hit-laden highlight of the tour.
The growing popularity of the MORE FM Winery Tour sees it visit 16 venues for 18 shows in February and March 2010, adding shows in Whangarei, Palmerston North and Taupo regions for its fourth season.
Tickets (and bus/ticket packages) go on sale on Monday 02 November via the Winery Tour website www.winerytour.co.nz and Ticketmaster. For Arrowtown and Blenheim shows, tickets can be purchased via Winery Tour website www.winerytour.co.nz, Ticketmaster and Ticket Direct. For Palmerston North show, tickets can be purchased via Winery Tour website www.winerytour.co.nz and Ticket Direct.


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Women's "Country Chic" Style
Tweed is iconically British and always look at home in the field as they do in the city - a tweed field coat like Joules' feminine, fitted jacket teamed with a silk scarf (in the style of Audrey Hepburn) will result in one of the smartest feminine styles and can be worn by all ages.
For a more contemporary "on trend" chic then a belted Trench mac (we love Aigle's Trenchy in linen) and a soft, oversized scarf like the cotton Aigle Pralig in blush pink or if you can't resist a bit of cashmere our British made cashmere shawls are stunning.
Country Chic is a fusion of conventional, fashion and practicality.
Joules Silk Clarence Scarf - Navy Horse & Hound
Aigle Ladies Duffleclan Duffle Coat - Dark Navy
From £150.00
Joules Women's Kingston Shirt - Cornflower


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A kid's accidental death, and a trauma surfaces in its wake
You may have heard about it. Late last Thursday, a 15-year-old kid named Brody Hurst, who lived in Newport on Sydney's northern beaches with his family, became the focus of a search.
Brody had gone missing without explanation. His mum posted details on social media: where he'd been last seen, what he was wearing and carrying. This was "totally out of character", were her words.
Brody's bike was found locked to a rail at the base of the stairs leading onto a path that winds around the top of the headland between Newport and Bilgola Beach. The next morning, his body was found at the base of the headland cliff, on the Bilgola side.
A real tragedy, especially for Brody's family and friends.
But another kind of tragedy lurked behind the flood of responses. The police had said "no suspicious circumstances". Nothing more was reported. Yet almost everyone — on social media, and the many people I bumped into on the street and down at the beach over the long weekend — seemed to think it was suicide.
Think about that for a second. A community in one of the wealthiest and most superficially attractive places in the world has become so inured to the idea of young people killing themselves, it's their first reaction to an unexplained death. They assumed it. They expected it.
Youth suicide is a huge burden on Sydney's northern beaches. This century so far, the Pittwater area has had one of the highest rates of suicide between the ages of 15 and 24 of anywhere in greater Sydney, higher than the eastern suburbs, Cronulla/Sutherland, the inner west, Parramatta or Penrith.
Until very recently, when state government funds have begun to flow into various programs, it's been ill-served by government resources of any kind.
Two years ago, Avalon's Paris Jeffcoat, who'd lost two friends from her year group at Barrenjoey High School to suicide, decided to find out what help was available. On the ground, she found … nothing. Lifeline, and a Headspace in Brookvale, and that was it. In response, Paris and a friend, Leanne Westlake, founded an organisation called One Eighty. Its aim is prevention — or as One Eighty's mission statement goes, a future free of youth suicide.
She's not surprised at the response to Brody's death. "With young people around here, that's the context and the conversation, there's no escaping it, whatever the facts are," she told CW.
Neither Paris or Leanne had any background in mental health work. They had to do a lot of their own research, delving into established organisations like Lifeline, the Black Dog Institute, Origin, and the Australian Bureau of Statistics latest Census data, along with NSW Department of Health policy and research through Paris's job with the department at the time.
Community fundraising along with private sector and government grants has allowed One Eighty to set up a range of programs in schools and community centres. Some, like Tomorrow Man and Tomorrow Woman, are run by trained professionals and focus on helping young people express themselves more freely. Others, like Open Up, are peer-to-peer support groups, run meeting-style and designed for adults.
They also co-ordinate with Lifeline's Accidental Counsellor training programs, and help run the Avalon Youth Hub, a crisis support centre targeted at young people — though Paris says parents often come in looking for information. And kids of primary school age who she says are starving for knowledge about the teen world they're about to face.
"The community's response has been incredible, which shows how much it was needed," she said.
But this is an area full of clubs and busy social lives. There's a lot of apparent support on show. "I think it's in that word, 'apparent,''" Paris told us. "There's still a lot of disengagement, a lot of pressure to achieve, and also to fit in. This is one of the least diverse areas in Australia. If you don't surf…"
That's it, isn't it. That's where we come in. Surf cultures can be crushingly conformist. Everybody's perfect. Everybody is beautiful, gleaming, travelling the world, #vanlyfe, Instagram, Yoga, wooo. Maybe you know it's all bullshit, maybe you don't.
What if you don't fit in?
Thing is, that wasn't this story. Brody surfed a lot. He had a bunch of good friends. He wasn't a current member of the local boardriders' club, though the younger members knew him.
His death appears to have been completely unintentional – just a terrible accident.
His friends were left stranded in their grief. On Monday night, the last night of this long summer break, one of them tagged Newport Surf Club from head to toe. It was ugly yet at the same time, wrenchingly poignant. "RIP Brody", it read, "miss you mate", "where are you BH".
Where are we, more like.
If you, or a young person you know, is struggling with symptoms of mental illness please contact your local headspace centre here or chat to them online, here, or call Kid's Helpline on 1800 551 800. If you are over the age of 25 and suffering from symptoms of mental illness please contact your local GP for a Mental Health Assessment Plan or call Lifeline Australia on 13 11 14.
If you or someone you know is in need of urgent mental health treatment, call 000 immediately. Help and support is available.


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If a horn on your car has worked perfectly fine, then suddenly won't make sound, what's the first thing to check?
The easiest fix should always be what you start with, and in this case, it's a fuse. If there was a problem with the horn or wiring, you likely would have experienced intermittent problems before.


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I highly recommend viewing Penny's website documenting her show LOSS: An Exhibit of Quilts from August 20-26, 2016. Her quilts eloquently investigate the process of grieving for her son, Jeremy Gold Amor, who died in July 2004.
kK: I noticed on the memorial page for Jeremy that you included a recording of his voice. Many people discuss a fear of forgetting the sound of someone's voice. Other people talk about fear of forgetting that way someone smelled. Were there other visceral things that you wanted to preserve or were able to preserve?
Penny Gold: I know many people wonder what to do with clothing, those physical items in such close contact with the body of the beloved. After my mother died (16 months before my son), I kept the items of clothing and jewelry that I thought I would use myself (we were close to the same size), and I wore something of hers each day for a year. I still have several things that I wear from time to time; what I couldn't wear, I gave away to Goodwill. I thought I would do the same with Jeremy's clothing, but he was bigger than me, and there was little I could wear. Soon after the funeral, when his close friends were at the house, I let them each choose something from his clothing. What was hanging in his closet at the time of his death, I left there, and it remains there still. The rest I packed away in a large suitcase in the attic. I can't bring myself to give any of it away, and really, there is no reason to.
At some point after Jeremy's death, I purchased a large wooden jewelry box, and I have put there a miscellaneous collection of various small objects from Jeremy's life, kind of like a reliquary. His wallet, some Magic cards, special photos, his baby teeth, and other things. The box sits on a dresser in the corner of my bedroom; I like having it there.


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Join us for our year-long celebration of the USA World Premiere Movie in conjunction with Made-for-TV Mayhem! Check out out previous entries on THE FORGOTTEN here, MURDER BY NIGHT here, THE TICKET here, SNOW KILL here, DEADLY GAME here, ACCIDENTAL MEETING here, HITLER'S DAUGHTER here, ARE YOU LONESOME TONIGHT? here, AS GOOD AS DEAD here and MURDER ON SHADOW MOUNTAIN here, TRUCKS here, OUR MOTHER'S MURDER here , THE HOLLYWOOD DETECTIVE here, ULTIMATE DECEPTION here, NIGHTMARE ON THE 13TH FLOOR here and WRITER'S BLOCK here, THE CHIPPENDALES MURDER here, RUBDOWN here, THE SUBSTITUTE here and THE CHINA LAKE MURDERS here, DIAMOND FLEECE here, RUNNING AGAINST TIME here, MY STEPSON, MY LOVER here and THE HAUNTING OF SEACLIFF INN here!, MURDER 101 here, TAINTED BLOOD here, BURIED ALIVE here, PSYCHIC here, SCANDALOUS ME: THE JACQUELINE SUSANN STORY here, INTO THE BADLANDS here, LIES OF THE TWINS here and CABIN BY THE LAKE here, VOYAGE here, RETURN TO CABIN BY THE LAKE here, WHITE LIE here and SINS OF THE MIND here, DUPLICATES here and MATERNAL INSTINCTS here, DEAD HUSBANDS here and THE HAUNTING OF SARAH HARDY here, CHILD OF DARKNESS, CHILD OF LIGHT here and HIGH DESERT KILL here, I'M DANGEROUS TONIGHT here and THE COVER GIRL MURDERS here!
RATTLED, a killer critter flick that first aired on February 14, 1996, certainly starts out with promise. The credits state that the film is written by the Wheat Brothers, of AFTER MIDNIGHT and PITCH BLACK fame! It's directed by Tony Randel, of HELLBOUND: HELLRAISER II and TICKS! There are explosions! Rattlesnakes – real ones, mind you, not the CGI phonies that populate the land of SyFy Original Movies – actually burst out of the ground! Clint Howard's presence is promised! Clearly, RATTLED will be B-movie gold!
Eden Valley is set to be a nifty new housing community with architect Paul (William Katt!) putting all the pieces together. Unfortunately, things don't exactly go as planned when the explosions used to clear the area end up releasing hordes and hordes of rattlesnakes. These displaced serpents soon make their way into the surround area (their first stop: construction crew foreman Clint Howard) and wreak snakely havoc onto the nearby locals.
Among the closest of these locals is ophidiophobiac Paul, who lives with his newly-formed family – his wife Krista (Shanna Reed) and his step-kids Michelle (Monica Lacy) and Adam (Michael Galeota). Adam is having some issues with the transition, and he doesn't feel like they're in their "home." Michelle, meanwhile, is having boy questions, and Krista helps her daughter out to determine whether some boy likes her or likes her. Krista has her own problems, as she discovers that she's pregnant. Paul tries to become more involved, but Adam just wants to skip school and feels abandoned by his birth father, who OH CHRIST I DON'T CARE WHEN ARE THEY GOING TO GET TO THE FIRESNAKES FACTORY?!?
RATTLED is a case in point for one of the bigger issues that many made-for-television movies have. When you create a storyline involving a nuclear family unit, you're immediately constricting yourself as to the type of story you can tell and the drama you can bring out of it – there are only a certain number of combinations of family-related stressful relationship issues that can be used as half-assed character development in a horror film, and they've all been done, so you'd better make damn sure you can do something, anything, different with the conflict. You're also setting up a situation where it's basically unheard of for any actual harm to befall the lead family unit (save for the occasional eccentric aunt or grandmother – see AMITYVILLE IV: THE EVIL ESCAPES), so you'd better make sure you've got enough weirdness and/or auxiliary characters to torment and fill up the running time.
RATTLED forgets to do these things. It goes through the motions of family drama in a bland, unexceptional way. There are occasional close calls where the kids (and Adam's irritating best friend) are in conceptual danger from wandering snakes, but we know that a bland, inoffensive TV-movie isn't going to kill of a child, so there's nothing really that feels like "tension." There are a couple of additional characters (the corporate exec and his wife, played by Ed Lauter and Bibi Besch, a gardener, an electrician and a scientist played by Ian Abercrombie), but their interactions with the snakes are few and way too many commercial breaks between.
It's not to say Randel doesn't give it the old college try when it comes to actually creating tension in the snake attack sequences – it's just that the stakes are so low and the characters so dully conceived that you have a tough time even being engaged on a campy level. The best moment, in fact, comes without a human in sight, as an innocent Ii'l frog finds himself on the wrong end of a rattler's grasp.
RATTLED is such a disappointing exercise in blandness that you could probably absent-mindedly come across it while flipping channels and just figure it's a mid-'90s family drama for a half hour before realizing that it's supposed to be about killer snakes. By the time the snakes actually descend upon the family's house and cause (inevitably unremarkable) problems for our leads, you may have already fallen asleep.
There's nothing wrong with a PG-13 level creature feature – I'll be the first to defend TREMORS or ARACHNOPHOBIA – but RATTLED feels like a PG-13 creature feature stripped of wit or fun, punctuating commercial breaks not with snake attacks but with family drama. Heck, even the VHS box art seems slapdash probably the only instance in which a killer snake film's marketing doesn't really feature a snake at all. In which case, it's a perfectly appropriate campaign, as anyone looking for a film in which William Katt and Shanna Reed look vaguely perplexed will be richly rewarded.
- [THE BIG QUESTION] WHAT'S YOUR FAVORITE FEMALE ENSEMBLE IN MOVIES? - July 22, 2016
- [IN THEATERS NOW] THE BOY (2016) - January 24, 2016
- Cult Movie Mania Releases Lucio Fulci Limited Edition VHS Sets - January 5, 2016
Tags: Clint Howard, Ed Lauter, Ian Abercrombie, Rattlesnakes, Snakes, USA World Premiere Movie, William Katt


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JH Life Serves Jackson Heights, East Elmhurst, Corona, Elmhurst, and Woodside, Queens, NYNew user? Please read The Rules.
Is it safe to assume that each building will have enough storage units for each resident or first come first serve?
Sponsor attorney said our apartment should be ready my 10/17. How long does it usually take from that point to closing date? We're cutting pretty close to our due date
Hi, everyone.As a fairly new resident of Washington Plaza with a two-and-a-half-year-old, I was wondering if children make the rounds within the complex for trick-or-treating. It'll be our first Halloween here, and we wanted to know if residents are open to having kids come around. If not, where do the children of the neighborhood go? Any insight would be much appreciated. Thanks!
Hello Washington Plaza owners,Hope all is well. I have a question. We received a Depart of Finance An Taxation Star credit application but it's asking me for the lot and building number. Does anyone know what that is for WP?Thanks!


text: Spiced Biscotti Recipe 2/28/2016 A great treat to go with your cup of coffee! What's great about this recipe is that there aren't unhealthy calories from high fat dairy or saturated fats like butter or cream. However, keep in mind it still contains some sugar so enjoy this treat in moderation in a well-balanced diet! Ingredients1 cup vegetable oil3/4th cup sugar½ cup orange juiceOrange zest 1 tsp baking soda1 tsp ground cinnamon½ tsp nutmeg1 tsp ground anise seeds½ cup chopped almonds3 cups of all purpose flourProcedure 1-Mix the oil and sugar together, then add orange juice, orange zest, baking soda, and spices. Continue mixing.2- Gradually add the flour and nuts and mix until the dough holds together and is not too stick (it can be a little wet). 3. Divide dough into 4 equal parts on a lightly floured surface and roll them out in 4 log shapes. 4. Place onto a pan (covered with a cookie sheet to avoid sticking) and flatten into a rectangular loaf ( about 7 wide and 2 cm thick). Then using a knife make slices into the loaves of about 2 cm in size. Do not slice all the way through. 5. Bake for 25 minutes or until golden brown. Remove and cool slightly. Slice the biscotti all the way through and turn them to the side. Place them into the oven again and bake for 5 min on each side. Serving Size: approximately 50 biscottiCalories per serving: 87 Author: Massar Dabbous Interested in a NutriWorks Diet Plan? BOOK NOW


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A Havurah (חבורה Hebrew: "fellowship," plural: havurot) is a small group of Temple members who meet regularly, with or without children, to share Jewish experiences. They learn, pray, play and create Jewish memories. Most havurot depend on the leadership and participation of all the group members, rather than looking to clergy or other Jewish professionals to create the programming.
Havurot do not replace synagogue life. Instead, they extend that sense of community by creating "a family within the Temple family," smaller friendship groups within the Temple community. They promote friendship and community among members. Typical gatherings might include discussion-based learning, group singing, participatory prayer, food and fellowship.
If you are interested in joining a havurah, call the Temple at (904)733-7078. As soon as there are a sufficient group of interests people/families, a new havurah will be formed.


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9 Ways On How To Get The Most From This Abyssinian Kittens For Sale Price | Abyssinian Kittens For Sale Price
Abyssinian Kittens For Sale Price
– Burmese cats are sometimes called copper cats, and that we think is a pretty cool nickname. They are generally famous for their playful and affectionate nature, and as we all know many cat breeds are just not very comfortable around people, that's not true with the Burmese.
Incredible Abyssinian Kittens For Sale Price
Burma (formerly Myanmar) is a large nation in south eastern Asia which borders on Thailand and China, and also has close ties to India via the sea. The country has a long and turbulent history, and has been colonized by the British never long ago. It is via British colonization and ties to the western world that Burmese cats found their way to houses around the world. In fact that the colonization of much of the planet in the 19th century did much to disperse the majority of the cat breeds, without ties to the western world we wouldn't like these cats now.
Interestingly enough, all of those Burmese cats in the USA are supposed to have only one common ancestor, that kitty's name is Wong Mau, and he was brought in the U.S. sometime in the 1930's by a guy named Joseph Thompson. Abyssinian Kittens For Sale Price
If you have one of these cats, then maybe you will want to build a small makeshift altar to your little friend's great ancestor, the illustrious Wong Mau. It is known that ancient Aztec people worshipped these cats and worshipped their ancestors, who knows this could make your cat feel right at home.
There are three distinct breeds of Burmese-the Contemporary, the Traditional, and the European; they're all a little bit different. The European variety is much slimmer, for instance, along with the Traditional look most like the first cat attracted over from Burma (the famous and successful Wong Mau). All of these cats are rather muscular and have a brief coat, making great sense knowing that Burma is a tropical country where a long haired cat would not be advantageous.
Buying Burmese kittens in most areas of north America ought to be rather simple as it is a fairly common breed, the single thing that we ask of you is that you simply check to see if the breeder is licensed and reputable because sometimes in the pet industry there is some shadiness happening. Abyssinian Kittens For Sale Price
In closing, these Burmese are very human like, and thus extremely popular cats all around the world.


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Tutorials/Combination locks
Combination locks are a type of redstone circuit. They generally have a number of components which must be set in the right combination in order to activate something such as a door. Combination locks can be very useful in creating adventure maps. Note that if you are playing in survival multiplayer, other players will still be able to break into the mechanism and cause it to activate without knowing the password.
Order-insensitive combination locks[edit]
Lever lock[edit]
A lever lock is one of the easiest redstone locks to create. Each lever doubles the number of combinations the lock has. The possibilities can be found using 2 ^ n, where n is the number of levers used.
When constructing, you will probably first want to place the levers and put them into the desired positions. Next, you should place the redstone torches and repeaters. Torches go behind levers that are down and repeaters go behind levers that are up. When you are done, all of the torches and repeaters should be turned off. If not, double check any components that are turned on.
- Step 1
First, place an 11 block row of any block, and make it two blocks high. Then, add a row of 7 branching off both sides.
- Step 2
Add levers/switches on the bottom row of the 11 blocks, followed by signs on the top row. Each sign should have a number in order (1, 2, 3 etc.). Alternatively, you can make the signs have letters or words on them instead.
- Step 3
Place a repeater in front of each block of the row that has the levers.
- Step 4
According to the password you want, you need to place a block in front of the switches that make up your password and put a redstone torch on top of each. For example, if your password for 1-2-6 (like in the example), each block and redstone torch is placed in front of switches 1, 2 and 6.
- Step 5
Next, place a block in front of each block that you just placed. Fill all the spaces in between the blocks with repeaters.
- Step 6
Place a row of redstone dust in front of the repeaters and blocks.
- Step 7
Place a strand of the redstone, starting from the middle, down to the 2nd last row. At the last row, place one block in front of the redstone.
- Step 8
Add one redstone torch on top of the block just added. Then, add another block on top of the torch. Once you've done this, add a door on top of that block.
- Step 9
Your passlock door is finished. To activate, flick all of the levers of which you had the password made up of, but no others. For example, if your password is 1-2-6, turn the levers below the signs that say 1, 2, and 6, on, but all the others should be off. The door should open.
- Final Touches
Cover all the redstone with blocks, so that players can't see it.
- Notes
The design shown in these directions can have up to 29 inputs. Larger amounts are more secure; the number of possibilities is 2^c, where c is the number of levers used. You can't have more than 29 inputs with this design as the redstone signal won't travel far enough.
|Demonstration video (view on YouTube)
Item frame lock[edit]
Item frame combination locks are a step up in complexity from lever locks. Items in item frames can be rotated into 8 different positions. This rotation can be detected by a comparator which will output a signal strength from 1 to 8 depending on the position. Since each item frame has 8 possible states, the number of possible combinations can be found with 8 ^ n, where n is the number of item frames.
If you want to make it extra tricky, it is also possible to detect an empty item frame by when a comparator doesn't output anything. Simply omit the redstone torch and leave a repeater connected to the comparator.
|Tutorial video (view on YouTube)


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In 1985 The Cosby Show was number one in the evaluations, the Live Aid concert happened, Back to the upcoming was the highest-grossing movie, I was wearing a very short, asymmetrical New Wave hairdo and Michael Graves became the first American designer to create an object for Alessi. It was a smart teakettle designed for mass production, and thus far it's outsold every other Alessi item. It is a slick design with a whimsical bird whistle at the end of the spout. You'd be amazed at the selection of kitchen fashions it fits right into; have a look below while I do the math on how many poor hair trends its own style has outlasted (really big perms came next) …
Alessi 9093 Kettle – $184
The teakettle's layout gives a postmodern wink; it's a slick mirrored-steel kettle of quite careful geometry and proportions, but then it's this practical blue plastic handle letting you know that it's OK to touch this part, along with a red cartoonish bird that screeches at you if the water is prepared. I wonder if those colour choices were Graves' way of injecting a small bit of USA to the iconic Italian company's product lineup.
Old World Kitchens & Custom Cabinets
This kitchen seems as if it could have been inspired by Graves' kettle, with its stainless steel elements and gorgeous use of blue.
A+B KASHA Designs
The kettle is right at home being one of the only cosmetic objects in a kitchen that is minimalist.
Mal Corboy Design
Holly Marder
The kettle consists of 18/10 polished stainless steel and measures 8 1/2 inches in diameter and 9 inches high. The tapered design is efficient for boiling water fast.
Mauricio Nava Design, LLC
Ronan Rose Roberts Architects
Additionally, it works well in modern kitchens filled with warm wood accents.
Kindred Construction Ltd..
A shiny steel backsplash reflects the kettle here. Another new classic we've explored recently will be the chairs in this kitchen. They're Hat Trick Chairs with a starchitect and product-designer colleague of Graves, Frank Gehry.
Rusk Renovations
The 9093 also adds a pop of charm charm to transitional kitchens.
Archer & Buchanan Architecture, Ltd..
Reico Kitchen & Bath
The kettle holds its own at a country-style kitchen perhaps as it's a tiny critter atop its spout.
Ike Kligerman Barkley
Birdseye Design
The holding-its-own statement is true whether the kettle is in a room with full size country charm or a single with subtle country touches, such as this one. Chances are, one of these would work in your kitchen also; at 28 years old, it's the most popular teakettle found in kitchen photographs.


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Balcones 60in Friendship Table with Leaves
Leaves are placed at the ends of the table between the middle and very ends. When you remove the leaf, the end of the table remains intact to keep the tapered edge.
Table extends to 84-inches with both leaves in place
Will seat eight guests with leaves and six without leaves
Height is adjustable from 30H to 36H
Finish: Top: A timeworn saddle brown
Base: An anthracite black with light dusty-wax hang up
Item Material: Hardwood and Poplar Solids with White Oak and Walnut Veneers with Resin
Dimensions: L: 60.5in W: 32in H: 36in


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This blog is mine, my thoughts, my moods my musings. Comments are welcome and un-moderated however vile comments, verbal abuse of others, will be removed.
Monday, 21 December 2009
Labour Lies and Spin
Well it transpires that our leader and Mr 21 out of 23 Bob Ainsworth have within a week of the historic visit to the troops and the general rehashing of the defence budget been proven to be lying once again.
I a nutshell...........the announcement was made on defence spending.
I blogged here and here on the subject.
I felt the announcement was weak...............VERY WEAK.
Much hype was made of the fact 22 Chinooks where to be purchased.
But...........Never in modern history, has the Government raided the budget of the Ministry of Defence to fund a continuing military operation".
And had Gordon Brown as chancellor not shown complete contempt for our armed forces this decision would not have been necessary if he had not, against all advice, cut £1.4bn from the helicopter programme in 2004.
In excess of 100 servicemen have been lost this year alone primarily due to road side IED's. Another died over the weekend. Fatalities to IED's are preventable on choppers. Total losses stand at 240.
So basically delivery 2013 meaning on average another 300 people may die before the first Chinook even turns a blade. Estimate about 600 deaths.
But that is not the truth..............please see here.
And I quote" As part of a significant re-balancing of Britain's immediate-term defense spending, Gordon Brown's Labour Party government plans to buy 10 new CH-47 Chinook helicopters for delivery in 2012-2013, with the intent to buy another 12 Chinooks later. The RAF's Chinook fleet would increase in size from 48 – 70 airframes, including 8 "Mk3 Chinooks" will finally enter service after a costly and controversial program, plus purchased replacements for 2 Chinooks destroyed during operations."
STOP.............F*****g do what, only 10 with an option on another 12. That is where the 22 figure came from.................an option!!!!!!!!!!!!!!!.
Still I suppose a shitty written letter and a stamp is cheaper than a chopper isn't it Gordon.
But you must be happy as long as you have the troops there.............you can kiss ass to the Americans and help then get the oil line out to the Caspian Sea.
Merry F******g Christams !!
Posted by The Last Of The Few at 08:43
Labels: afghanistan, ainsworth, brown, USA
2 comments:
- subrosa said...
Aye Mark, I was told about this on Saturday. No surprise of course but typical of the lies from the MoD.
- 21 December 2009 at 12:16
- The Last Of The Few said...
I think we all knew a twist was on its way.
But to be sooooo pathetic and blatant as that.
Looking at the MSM not 1 of them or the other parties have made a move on the story.
Pathetic all of em!
- 21 December 2009 at 12:25
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Trail Maintenance was carried out on BKB on Holmbury hill. This trail has had very little done to it in the last few years, and has suffered further from the tree thinning work carried out on this part of the hill earlier in the year.
The "dip" on the very first section was rebuilt as it had become a bit of a wheel sucker, and then the first section was repaired. This section is quite flat requiring drainage holes to be dug alongside the trail.
The entrance to BKB (by the road) was then cleaned up, but this will require more work to it as it is quite flat and suffers from standing water. It is currently a bit of a mess again.
Finally, the bottom of the "roll in" on the second section was cleared out. This is essentially a drainage ditch, so a large amount of debris and mud had built up.
The next build day will be in January 2012.
Thanks to all those who have helped so far.
Hurtwood Mtb.