text:
Good Morning and welcome to another fabulous challenge here at ABC!!! Many thanks to everyone who played along with the "V is for Violet" challenge. We had some wonderful creations this time. Picking and choosing our favourites was particularly hard as always..., but we did manage to narrow it down so without further ado in no particular order here are our Top 5 :
#9. Sai Jirawadee
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#9. Sai Jirawadee
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#38. Melanie-Jane
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#66. Coops
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text:
Sony Alpha DSLR-A900 Review
|8 Previous
9 Displays
|10 Next
Record mode display
Although it does have an LCD info panel on the top, the Alpha 900 still uses the LCD monitor as a record mode display for camera settings, exposure etc. In record mode you can press the Display button to toggle between the full recording mode display, 'enlarged' recording mode display (which has less information) or no display.
|Detailed recording mode display
|Enlarged recording mode display
Using the Quick Navi menu
Once again you can now change most settings directly from the info screen by pressing the 'Fn' button, which turns the screen into a 'Quick Navi' menu - one of the Alpha 700's standout features that thankfully hasn't been changed in the Alpha 900. Using the multi-commander you simply move the highlight from one setting to another. Pressing the central button brings up a mini menu for that item, allowing you to change settings. It works well and my only complaint is that you can't change the button used to activate the Quick Navi menu - odd when so much customization is on offer - and the Fn button is too much of a stretch for your thumb (so changing settings is impossible if you're not holding the camera with both hands (and it feels unnecessarily fiddly). There is, however, a way to overcome this (see next section).
|Pressing the Fn button turns on the Quick Navi screen. The left, right, up and down arrows on the multi-controller are used to move from one setting to another.
|Press the middle button on the controller and a menu appears allowing you to change the setting.
|Selecting a Creative Style
|Changing Exposure Comp. setting.
Using the direct access buttons
By default you see a dedicated menu - as shown above - when you press one of the external buttons (ISO, DRIVE, WB and +/-) on the top of the camera. An option in the custom menu allows you to change settings directly on the Quick Navi screen by pressing one of these buttons and turning the front control dial. For settings - such as white balance - with additional options, the rear dial cycles through the parameters. The table below shows the (default) function of each dial/button combo:
|Front Dial
|Rear Dial
|Change ISO (1 EV steps)
|Change ISO (0.3 EV steps)
|Cycle through WB presets
|• Presets: Change WB adjustments (+3 to -3)
• Kelvin: Change Kelvin value and color filter
• Custom WB: choose from 3 saved settings, create new setting
|Cycle through drive, bracketing, self timer, remote control modes
|• Continuous: Hi or Lo
• Self timer: duration
• Bracketing: EV step and number of frames
|AE compensation
|AE compensation (same as front dial)
One big advantage of using the Quick Navi screen in this way is that once you've pressed one of the direct access buttons you can use the multi-commander to move to any of the other on-screen settings (thus overcoming the rather awkward positioning of the Fn button).
| ISO (not all settings shown)
• turning front or rear dial
WB fine tune (not all settings shown)
Intelligent Preview
The Alpha 900 doesn't offer live view (we've yet to get a definitive answer from Sony on exactly why) - in its place is 'Intelligent Preview' - a sort of pre-capture test shot. Pressing the DOF preview button (by default, though it can also be assigned to the Focus Hold button) takes a picture using the current settings and puts the raw data into the buffer. The Intelligent Preview interface (shown below) allows you to experiment with different exposures, DRO settings and white balance until you're happy with the result. At this point you have to actually take the picture by pressing the shutter (the Intelligent Preview shot cannot be saved) - any changes made to the settings are used for the actual photo.
In the studio the system works pretty well, though it must be said that it's no substitute for a good live view mode (we use live view in the studio a lot - though rarely anywhere else), chiefly because it can't be used to check critical focus (you can't magnify the preview image). And obviously it's only really useful when the camera - and subject - are relatively static - I suspect you may start 'missing the moment' if you try to use it for sports photography.
You get three stops either side of the current exposure to play with, though if the preview is massively under or over exposed you'll hit the limits of raw headroom before then (in other words, if you're in P mode and the AE compensation is set to +3.0 when you take the preview shot, you can't see what -3.0 will look like).
|This is what you see after pressing the DOF button; a small preview image, histograms and your current settings.
|Changing the exposure (not all settings shown here) - you can also do this in Manual exposure mode, though obviously you won't see depth of field changes.
|Changing the white balance (again, not all options are shown in this animation).
|And changing DRO settings.
|Press the Display button to toggle between the original settings and your new settings.
- 1 Introduction
- 2 Specifications
- 3 What's new
- 4 Body & Design
- 5 Body & Design
- 6 Operation & Controls
- 7 Operation & Controls
- 8 Operation & Controls
- 9 Displays
- 10 Displays
- 11 Menus
- 12 Menus
- 13 Timings & Sizes
- 14 Features
- 15 Features
- 16 Features
- 17 Features
- 18 Features
- 19 Software & Raw
- 20 Photographic tests (Noise)
- 21 Photographic tests (Noise)
- 22 Photographic tests (Noise)
- 23 Photograpic tests (DR)
- 24 Photograpic tests (DR)
- 25 Photograpic tests (Fall off)
- 26 Photographic tests
- 27 Compared to...
- 28 Compared to (JPEG)
- 29 Compared to (JPEG)
- 30 Compared to (JPEG)
- 31 Compared to (RAW)
- 32 Compared to (RAW)
- 33 Compared to (RAW)
- 34 Compared to (High ISO)
- 35 Compared to (Resolution)
- 36 Conclusion
- 37 Samples
Oct 21, 2011
Oct 21, 2011
Oct 22, 2008
Dec 2, 2010
I own it
I want it
I had it
Discuss in the forums
The new model definitely is worth the money. It is a big upgrade compared to the A700!
- Canon EOS M58.8%
- Panasonic G85/G803.3%
- Panasonic FZ2500/FZ20001.9%
- Panasonic LX10/LX151.2%
- Panasonic GH5 development3.6%
- Sony a99 II15.9%
- Nikon KeyMission 170 and 801.0%
- Fujifilm GFX 50S development28.3%
- Olympus E-M1 II development18.7%
- Olympus E-PL80.1%
- Olympus 25mm F1.2 Pro1.5%
- Olympus 12-100mm F4 IS Pro1.9%
- Olympus 30mm F3.5 Macro0.1%
- Sigma 85mm F1.4 Art3.6%
- Sigma 12-24mm F4 Art2.6%
- Sigma 500mm F4 DG OS HSM Sport2.4%
- GoPro Hero50.8%
- GoPro Karma drone2.2%

subset: commoncrawl


text:
Any time a person is actually interested in investing in a car, they will frequently contemplate buying one of the used cars that are offered to be able to make sure they'll be getting a great deal and to be able to make sure they are not squandering their money on a car who's value will decline significantly when they drive it off the lot. They are nonetheless making a large purchase, therefore no matter if they're enthusiastic about fast cars, classic cars or they simply need something to be able to take to the workplace, they are going to want to not rush prior to investing in a car in order to ensure they obtain just what they want for a reasonable cost.
The first thing an individual should consider will be the price range. If they'll be paying for the automobile at the start, they will desire to be aware of exactly how much they may have to commit. This may include thinking of both the total they might prefer to spend and also the complete quantity they're ready to invest in case they can't uncover the right car or truck for the smaller amount. In the event they're planning on financing the car, they'll desire to think of both the complete quantity and the month to month sum to guarantee they remain inside their spending budget every month.
Once a person has a price range under consideration, they must start to think about the sort of car or truck they desire plus the age. Some may want to keep with somewhat new cars in order to get one they know will likely be reliable. In the event they'd desire something a little older, on the other hand, they will need to take their time to be able to make sure the car or truck is actually in excellent form previous to obtaining it. They may need to have an individual with them that knows a lot regarding automobiles in case they are not a mechanic themselves to be able to assist them to assess just about any cars involved.
Making the effort to thoroughly plan precisely what they really are prepared to spend and also in order to look at the vehicles they're interested in might help somebody locate the ideal one to be able to satisfy their demands. Furthermore, it can help to ensure they are purchasing a car that is going to last without over spending on the original price of the vehicle. Invest time to consider the above if you're searching for the next vehicle to actually make certain you're going to find the perfect one for you.


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Infection with the hepatitis B virus (HBV) leads to either acute, self-limiting, or fulminant hepatic disease, or to chronic hepatitis B (CHB) infection with a wide spectrum of viral activity, clinical manifestations, and disease progression.1–3 The factors and molecular events that influence the course and the outcome of chronic infection remain poorly understood. Persistent HBV infection of the hepatocyte is characterized by the presence of a covalently closed circular (ccc) DNA episome, whereas the degree of viral replicative activity is primarily determined by the transcriptional state of cccDNA molecules and the production of pregenomic (pg) RNA.4–6
On entry of the HBV virion into the hepatocyte, the viral genome is translocated to the nucleus and converted into a supercoiled cccDNA viral minichromosome.7–9 The cccDNA episome is the template for the HBV mRNA transcripts; and the 3.5-kb pg RNA, template for reverse transcription and synthesis of the viral genome. Transcription of pgRNA from cccDNA is a key event in HBV replication, and the process is under the control of the HBV core promoter (CP),10 which directs the synthesis of two species of overlapping 3.5-kb viral transcripts that only differ at their 5′ initiation sites.11, 12 Translation of the longer precore (preC) mRNA leads to the synthesis of the precore protein, precursor of the HBV e antigen (HBeAg).13 The shorter pg RNA serves as mRNA for the core and the polymerase genes and, after encapsidation, as the template for reverse transcription to generate viral DNA. Intracellular cycling of DNA-containing nucleocapsids to the nucleus is necessary for cccDNA pool maintenance.9, 14
During the course of chronic HBV infection and during prolonged antiviral therapy, development and selection of mutations in the HBV genome may become clinically relevant and influence the outcome of infection.15–17 CP mutations clustering in the basic CP (BCP) region are commonly found among chronically infected patients.18, 19 CP mutations may affect HBV gene expression, replication, and pathogenicity by altering the levels and balance between pgRNA and precore mRNA production20–23 and affecting the transcriptional activity of HBV cccDNA molecules. HBV cccDNA is believed to be responsible for viral persistence and reactivation.24–26 However, the factors that determine the transcriptional state of cccDNA in hepatocytes are not well understood, and widespread HBV mutant strains have different transcriptional and replicative activities.20, 23, 27, 28 Therefore, measuring in vivo pgRNA levels in conjunction with cccDNA levels could provide a more precise method for evaluating HBV persistence and activity in infected patients than measuring cccDNA alone.
Despite the generation and publication of methodology on HBV cccDNA detection,29–34 few in vivo data are currently available from human liver samples,32–35 and most of the existing knowledge is derived from animal models.36–39 Even less in vivo information is available on cccDNA replicative activity and pgRNA production,22, 37 because most HBV expression studies have been performed in vitro or on heterologous systems.
We have designed and used quantitative assays for the measurement of both concentration and, for the first time, the replicative activity of intrahepatic cccDNA in liver biopsy samples of patients with CHB. Our data show that the combined measurement of cccDNA and pgRNA levels provides valuable information by monitoring the presence, as well as the activity of intrahepatic HBV cccDNA in chronically infected patients.


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Fund Your Franchise with Guidant
Learn how to invest your IRA or 401k into a franchise. ($50k min)
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Company Profile
Franchising Since
1984 (32 Years)
Corporate Address
1305 Hope Mills Rd., #A Fayetteville, NC 28304
Cheryl Young
Parent Company
Stork News of America, Inc.
About Stork News of America Inc.
Stork News of America Inc. was founded in 1983 and began franchising in 1984. It has more than 135 locations in the United States and Canada. Stork News has an arrangement with Babies "R" Us that places Stork News displays in all Babies "R" Us units that are located in an area where a Stork News franchisee is present, thus offering franchisees automatic advertising. Stork News of America is based in Fayetteville, North Carolina.
Financial Requirements
Initial Investment
$11,530 - $75,700
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$8,000 - $30,000
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The Franchise 500 is not intended to endorse, advertise, or recommend any particular franchise. It is solely a research tool you can use to compare franchise operations. Entrepreneur stresses that you should always conduct your own independent investigation before investing money in a franchise.
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2004 Subaru Legacy Wagon L AWD 4dr Wagon (2.5L 4cyl 5M)
Very well build car. Exceptional on snowy/slushy roads. Great road drivability. Bought just several months ago but love it so far. Not too keen with the outdated design though.
Superb road handling in winter conditions thanks to AWD as well as low center of gravity. I love it!
Very outdated exterior and not too shocking interior. Interior paneling (i.e. side walls in trunk area) needs to be improved; panels very cheap and way too delicate; some misalignment of interior parts. If changing design please do not over do it. No need to remind of XXI century!
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Outstanding six bedroom luxury villa with pool in Hvar
This outstanding six bedroom villa with pool is located on the waterfront and set in one 1000 square metres of mature garden and is within easy reach of Hvar town centre. Lavishly decorated, no expense has been spared when it comes to interior décor where contemporary designed furniture sits amidst attractive colour palates. Fascinating works of art, some containing adult themes, are on display from some of the best contemporary Croatian artists.
The villa is divided into two apartments and is approached via a coastal road. Guests enter via the main gate up a flight of stairs, passing through the extensive garden along the way to the ground floor entrance which leads directly into the lounge of the lower apartment. In this apartment the colour theme is monochrome mixed with bold colours. In the main lounge area there is a dining area, sofas and a large sliding door leading out onto a sea facing terrace.
An archway from the lounge leads to a kitchen with breakfast bar, two double bedrooms one of which has an en-suite bath room and doors to a terrace, a twin bedroom, a separate shower room and separate bathroom with a bath. Stairs lead to the brightly coloured upper apartment. This consists of a main lounge area with doors leading out onto a sea view terrace, two double bedrooms and a twin room all with doors leading out onto one long balcony with garden views.
There are two separate bathrooms, one with a shower and the other with a bath. An indoor kitchen and a very comprehensive outdoor summer kitchen, terrace and dining area, ideal for those who like to enjoy outdoor entertaining, make up the rest of this apartment.
Undoubtedly the villas finest attributes are its outdoor facilities and services. The extensive mature garden contains an outdoor heated pool in winter and a sundeck. Swimming is possible on the beach in front from the rocks or a short walk away (approx 400m) is the Pokojni Dol beach - ideal for children. Guests staying at the villa are able to make use of a 20 minute boat service (charged at Euros 7 per person) to take them to the island of St Clement, part of the Pakleni islands which sit opposite the town of Hvar.
On this island guests can swim on the lovely beaches and dine in one of two excellent restaurants serving Mediterranean food. The return boat service back to the villa is also available whatever time of day or night you choose to come back. Hvar town centre is a 20 minute walk away from the villa and is considered one of Croatia's most attractive cities. With its abundant cafes, restaurants and nightlife it is an excellent place to experience Mediterranean atmosphere Croatian style at its best.For more information about Hvar Island watch video.WATCH THE VIDEOWATCH 360° PANORAMA
Suitable for:
£5,355.0002 October 2016 - 09 October 20167 nights
- Family / Friends
- Groups / Special Occasions
- Nightlife / Entertainment
- Island Holidays
- Distance to beach20 m
- Nearest airportSpit
- Distance to airport2-3 hours including ferry crossing.
- Nearest townHvar town centre
- Distance to town20 minutes walk
- -
- -
- Details on nearest shopsGrocery store
- Distance to nearest shops500 m
- Distance to nearest restaurant500m
- Read more
- Sleeps in bedrooms: 12
- Sleeps total (includes sleeping capacity on sofa/extra beds): 14
- Minimum stay: 7 nights for June-August, 3 nights for all other dates.
- Arrival days: Saturday for June-August, any day for all other dates.
- Check-in: 16:00
- Check-out: 10:00
- Last renovated: 2007
- Area: 1,000m300m2
- Garden: 1000m2
- Frequency of linen/towel change: Weekly
- Car park: Off street parking available for 2 cars.
- House pets allowed (on request)
- Sea view
- Whole house air-conditioned
- Sun deck
- Heating
- Parking
- Children
- Air-conditioning (some of the rooms): 4 units
- Cot/crib (available on request)
- Pool size: 7.5m x 3.5m
- Pool private to the property
- Heated pool
- Wifi
- Each apartment is equipped with a kitchenette. Oven available in the summer kitchen.
- Stove
- Dishwasher
- Dryer
- Refrigerator
- Microwave
- Stereo
- Toaster
- Washing machine
- Satellite TV
- DVD player
- Laundry room, with w/m available in the villa. Separate laundry service available on request and payable locally
- Furniture in terrace/balcony/garden: Table, chairs and sun recliners.
- Kitchen
- Balcony with sea view
- Kitchen table
- Terrace with sea view
- Balcony
- Terrace
- Lounge/living room
Bedroom details
- Number of double bedrooms: 4
- Total number of bedrooms: 6
- Number of twin bedrooms: 2
Bathroom details
- All bathrooms have toilets
- Number of bathrooms with shower: 3
- Number of bathrooms with bathtub: 2
- Total number of bathrooms: 5
- Number of toilet only rooms: 1
- Additional information on bathrooms: All bathrooms have massage showers .
Staff on site /available
- Caretaker
- Maid is available once a week free of charge , for basic cleaning of the house only.Extra maid services available on request , charged at 10 euros per hour ,payable locally
- Chef service available on request.
Information on Location
- Distance to beach: 20 m
- Nearest airport: Spit
- Distance to airport: 2-3 hours including ferry crossing.
- Nearest town: Hvar town centre
- Distance to town: 20 minutes walk
- Details on nearest shops: Grocery store
- Distance to nearest shops: 500 m
- Restaurant within walking distance
- Distance to nearest restaurant: 500m
- On the seaside
- Car recommended
- Information on transfers: Available on request.
- In city
Price includes
- Electricity
- Gas
- Water
- Provision of linen/towels
- Heating
- Internet
Separate charges
- Final cleaning: €150
- Maid service: €10 per hour
- A Chef is available on request at a cost of 30 euros per hour + food cost, payable locally.
Security deposit
- The following must be produced on arrival: €800
Pricing & availability:Price bandsProperty per night
01 May - 06 June£ 765.00 06 June - 04 July£ 963.00 04 July - 29 Aug.£ 1,241.00 29 Aug. - 19 Sept.£ 963.00 19 Sept. - 10 Oct.£ 765.00 10 Oct. - 01 Jan.£ 590.00Prices are in UK Pounds (£)
*Rental prices do not include Residence Tax: £ 0.92 (per person per night)
Pricing and booking information
Pricing Information
Pricing is calculated per property per night in GBP Sterling. For rentals in Croatia an additional tourist tax applies of 92 pence per adult per night. Children ages 12-17 pay 50% of this amount. There is no tourist tax charge for children under 12.
Booking Information
A 30% deposit is required at time of booking. Full balance is due 10 weeks prior to arrival.
If booking within 10 weeks of arrival, the full cost of the villa must be paid at the time of booking.
Holding an Option on a villa
Please Contact Us should you wish to place an option on a property for 24 hours whilst you book your flights and/or make other arrangements.
Payment Information
For online bookings, payment can be made by credit or debit card. Credit card payments (Visa or Mastercard) incur a credit card fee of 2% of the total amount of your booking. There is no surcharge for Debit card payments. All major currencies are accepted when paying online by credit card.
Payment by bank transfer (In sterling or Euros), UK online banking or cheque in Euros or sterling can be accepted. Please Contact Us if you wish to make a payment in this way.
Our full terms and conditions can be read here:
Select your travel datesFree Occupied Your selection£ 5,355.0002 October 2016 - 09 October 2016for 7 nightsON REQUEST
Customer reviews:
Impressed with: Beauty friendliness of people
Not impressed with: Ferry arrangements and Split airport
Recommend: Boat trip via owner
Favourite villa moment: Terrace
Stayed August 2016
The catamaran tickets were always booked up, it would have been useful to have a representative from the villa buy our tickets in advance. Quentin was very helpful in sorting out a private boat, but it was expensive in comparison to other private boats.
Impressed with: Beautiful sea and place
Not impressed with: The food, it was very salty.
Recommend: Carpe Diem Bar/Club.
Favourite villa moment: BBQ outside and drinks
Stayed June/July 2012


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This Trident rubber head strap is low priced and sure to provide safe and lasting hands-free convenience. Order online or call today at 1-800-975-0986.
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Streamlight flashlights, batteries, spot lamps, headlamps, and accessories are trusted tools in the lighting industry.
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Synthesis and Excited-state Photodynamics of a Chlorin–Bacteriochlorin Dyad—Through-space Versus Through-bond Energy Transfer in Tetrapyrrole Arrays
Version of Record online: 15 JAN 2008
DOI: 10.1111/j.1751-1097.2007.00258.x
© 2008 The Authors. Journal Compilation. The American Society of Photobiology
Photochemistry and Photobiology
Volume 84, Issue 3, pages 786–801, May/June 2008
Additional Information
How to Cite
Muthiah, C., Kee, H. L., Diers, J. R., Fan, D., Ptaszek, M., Bocian, D. F., Holten, D. and Lindsey, J. S. (2008), Synthesis and Excited-state Photodynamics of a Chlorin–Bacteriochlorin Dyad—Through-space Versus Through-bond Energy Transfer in Tetrapyrrole Arrays. Photochemistry and Photobiology, 84: 786–801. doi: 10.1111/j.1751-1097.2007.00258.x
Publication History
- Issue online: 15 JAN 2008
- Version of Record online: 15 JAN 2008
- Received 10 October 2007, accepted 1 November 2007
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In Bruges: 8 reasons you should check out Europe's fairytale city
If you were to picture the city where an aspiring Disney director might choose to film Cinderella or Beauty and the Beast, you could do a lot worse than Bruges.
It's official, guys: here are the 6 hottest destinations in 2016
Bring out the bucket list: here are six destinations that are gonna be hot, hot, hot in 2016.
Bosnia & Herzegovina: 7 reasons to look beyond the headlines
People tend to associate Bosnia and Herzegovina with war and destruction, but a lot has changed since the 1990s.
Need for Swedes: 7 reasons why Sweden is Europe's new capital of cool
Today is the day you realise IKEA is the LEAST cool thing to come out of Sweden.
Behind the masquerade: The untold stories behind the world's great festivals
When we think of festivals, we often picture trampled city streets shot through with bright streamers. We hear the rumble of drums and the hooting crowd.
Beyond Berlin: 5 German stops to include on your ultimate Eurotrip
Like many before me, I had a limited view of how Germany worked. It turns out there is a world beyond Berlin.
7 reasons Sardinia is Europe's hottest new sailing destination
Johnny Depp gets it. When his character Jack Sparrow stood at the helm of the Black Pearl and said 'Bring me that horizon', he was talking about the sailor's endless quest for new waters and unchartered shores (or at least shores unchartered by most major cruise liners).
Photo proof that Switzerland is the most beautiful country in Europe
Warning: may include dangerous levels of natural beauty. Consume at own risk.
A walking tour of Paris' best foodie haunts (with handy step-by-step map)
When I landed in Paris for the first time, I spent a happy few hours Googling the best baguette, the most celebrated croissant, a critically acclaimed salted caramel.
Introducing Europe's newest independent countries?
The number of official countries in the world currently sits at 206, with the latest to join the party being Kosovo in 2008 and South Sudan in 2011. That doesn't include the strange, tiny countries (Population: 1) that some people set up in their backyards, complete with Monopoly money currency and a flag knitted from their mother's old quilt.


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N.J. SUPERIOR COURT, APPELLATE DIVISIONNew Jersey Law Journal
S.P. v. Newark Police Department
NEGLIGENCE — Domestic Violence
The immunity provisions of the Tort Claims Act apply because the police officers did not have a ministerial duty to arrest pursuant to the Prevention of Domestic Violence Act.
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Focus: Functional Discussions
In this issue, we've asked several makers to describe not only their process for making one of their functional pieces, but the value they hope their work carries that is beyond its basic function—and we hope you value their efforts as much as we do.—Sherman Hall, Editor
All-access and Web-edition Subscribers can view/download this issue here.
Buy this back issue—$4.99 (PDF only).
On the cover: Peter Pincus' Blue Line Shift, to 11 in. (28 cm) in height, colored porcelain, fired in an electric kiln to cone 6.


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Take your itinerary with you using our My Alaska Trip feature and the TravelAlaska app.
Alaska's only year round 100+ year old hot springs resort. 80 lodge rooms, full service restaurant and lounge, activities galore, Aurora Ice Museum 11AM, 1PM, 3PM, 5PM & 7PM. Northern Lights viewing, Aurora Viewing tours, dog kennel, horses, massage, guided ATV tours, guided snowmachine tours, 1980 acres of beautiful wilderness, wildlife, RV park and camping, Yurt rental, year round Alaska Resident Rates. Daily Geothermal Energy Tours 2PM & 4PM. Check out our Geothermal projects. Visit the Activity Center for specialty coffees and snacks in the Aurora Cafe. Book your activities at the Activity Center Desk.
River, Sea and Air: 5% Off Normal Package Price Listed Below for May, June, August, September


text:
Full name William Williams
Born April 12, 1861
Died April 14, 1951, Hampton Wick, Middlesex (aged 90 years 2 days)
Major teams Middlesex
Batting style Right-hand bat
Bowling style Legbreak
Fielding position Wicketkeeper
|First-class span
|1885 - 1902
Billy Williams, who died at his home at Hampton Wick on April 14, aged 90, was a fine allround sportsman. Born on April 12, 1860, he was a member of MCC from 1900. In the seasons of 1885 and 1886 he appeared as wicketkeeper for Middlesex, and after an absence of 14 years returned to the county side as a bowler of legbreaks, playing occasionally till 1905. During the winter of 1896-97 he toured the West Indies with Mr. Arthur Priestley's team and, with 67 wickets at an average cost of 9.62, finished second in the bowling averages to A. E. Stoddart. He often assisted MCC and was credited with taking 100 or more wickets a season in all matches for 55 years. In his last summer as a player, he turned out at the age of 74 for MCC against the House of Lords and, after dismissing Lord Dalkeith, Lord Tennyson and Major L. George for 16 runs, was presented by the Marylebone Club with the ball. A good story is told of when, at the age of 65, he visited the West Indies during the tour of the Hon. F. S. G. Calthorpe's M.C.C. team in 1925-26. In Georgetown he challenged a West Indian friend against whom he played 30 years before to a single-wicket match for £25 a side. Winning the toss, Williams severely punished his opponent's bowling, completed a century and declared. Then, with a googly, he bowled his exhausted victim first ball.
Apart from his cricket, Williams, as he was known to everyone for many years, was celebrated as the man who, in the early part of the century when a member of the Middlesex County Rugby Union Committee, ended a long search for a suitable site for a National Rugby Union ground by discovering a cabbage field of 10 acres which has since developed into the famous Twickenham enclosure. For a long time the ground, later extended by 18 acres, was known to Rugby football followers as "Billy Williams's cabbage patch." Formerly a player for the Harlequins RFC, he was a Rugby referee for 21 years. An honorary member of the Wimbledon Park Golf Club, he played a daily round until his last illness, and was a regular attendant at Lord's and at Rugby matches in the London area, particularly Richmond.
Wisden Cricketers' Almanack
India's unbeaten streak and Santner's double
Stats highlights from the first Test between India and New Zealand in Kanpur
Ravindra Jadeja, maker of differences
A 41-run stand for the last wicket, and three tail-end wickets in one over. That is the very definition of "crucial" in a low-scoring contest
The debate about Pujara's strike-rate
Cheteshwar Pujara's strike rate has been a worry over the last 30 months, but much of it is to do with his inability to score against pace when playing overseas
New DRS rule for lbws is great news for bowlers
The new DRS protocol for reviewing not-out lbw decisions came into effect on September 22. Life is going to get harder for batsmen, and here's why
An all-time India Test XI chosen by our readers
A modern look to India's all-time XI selected on the occasion of the country's 500th Test
Vote for your all-time India Test XI (199)
In September 2010, as part of ESPNcricinfo's All-time XI series, a jury had selected an All-time India Test XI. Six years later, would you make some changes?
The debate about Pujara's strike-rate (81)
Cheteshwar Pujara's strike rate has been a worry over the last 30 months, but much of it is to do with his inability to score against pace when playing overseas
An all-time India Test XI chosen by our readers (55)
A modern look to India's all-time XI selected on the occasion of the country's 500th Test
Ravindra Jadeja, maker of differences (48)
A 41-run stand for the last wicket, and three tail-end wickets in one over. That is the very definition of "crucial" in a low-scoring contest
How do New Zealand play the straighter one? (37)
New Zealand have done many things right while facing R Ashwin and Ravindra Jadeja, but they are yet to find an answer to the two spinners' biggest weapon: the one that looks like it will turn, but does not turn


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Cheque Cashing Blackpool
23 results for Cheque Cashing in Blackpool
Starstruck Promotions Ltd30, Greenfinch CourtFY3 8FG Blackpool 01253 390 393
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Pawnbroker services including Cheque Cashing PayDay Loans Jewellers Blackpool Rochdale Birmingham
Happytots Childminding63 Valentia RoadFY2 0RN Blackpool 01253 500 532
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text:
DONETSK, Ukraine (AP) – France coach Laurent Blanc remained tightlipped over his future after the team's 2-0 loss to Spain on Saturday in the European Championship quarterfinals.
Blanc's contract with the French Football Federation expires at the end of the month. He wanted a new contract before Euro 2012, but FFF President Noel Le Graet preferred to wait until after the tournament before making a decision.
''We're out of the competition and we're upset, so now is not the time to talk about staff and players,'' Blanc said. ''We will analyze this Euro in the coming days. There will be some satisfaction and some disappointment, and you will find out what happens after.''
Some English media recently linked Blanc to the vacant position at Premier League team Tottenham.


text:
A trade group says Facebook needs 'measurement transparency'
The call for an audit of Facebook's metrics comes a week after the social network acknowledged inflating its video metrics.
A trade group says Facebook needs 'measurement transparency'
The call for an audit of Facebook's metrics comes a week after the social network acknowledged inflating its video metrics.
Google bolsters its ad targeting and analytics tools
The tools build on the vast amount of information Google knows about consumers.
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The best web sites provide lots of data in easily digested ways, a panel of retailers told the Shop.org Summit in Anaheim, CA, this week. Among the best practices: simplicity, alternative views, product comparison, easy ways to contact the company.
Posted 10/07/2004Paul DemeryPost a comment
As e-retailers upgrade their e-commerce platforms from outdated or homegrown versions, there are five key best practices they should follow when purchasing and implementing new technology applications, according to a new Forrester Research report.
Posted 08/16/2007Paul DemeryPost a comment
A decade of commercial use has given rise to best practices in marketing e-mail, but not every marketer uses them. Only one of 18 identified best practices was used by all marketers studied.
Posted 07/13/2006Paul DemeryPost a comment
In his keynote address at the Internet Retailer 2005 Conference & Exhibition, Tom Beckwith, vice president of Amazon Services, will cover best practices in e-retailing and reveal details about one of the retailers that operates on the Amazon platform.
Posted 05/26/2005Kurt PetersPost a comment
Experts share their tips at IRWD 2012 conference.
Posted 02/15/2012Kevin WoodwardPost a comment
Related Content:Nathan Decker
Citing the need to build consumer trust, the Direct Marketing Association has issued standards for affiliate marketing and online advertising.
Posted 06/26/2006Kurt PetersPost a comment
When an unusually large number of packages came back to The Vitamin Creek, receiving staff let other personnel know. That helped the e-retailer avoid losses from a scheme involving orders that criminals asked to have reshipped to illegitimate addresses.
Posted 04/08/2010Paul DemeryPost a comment
Taking action based on web analytics data can dramatically improve business for Internet retailers, but launching web analytics requires a skill set that many e-retailers do not have, Forrester Research says in new report.
Posted 07/27/2006Paul DemeryPost a comment
Retailers can limit their exposure to e-payment security risks by following techniques like PC fingerprinting and IP geolocation, Tom Donlea, executive director of the Merchant Risk Council, will explain during a presentation at IRCE 2008 next week.
Posted 06/04/2008Paul DemeryPost a comment
Sometimes plain text works better than HTML and sometimes a retailer is better off not sending an e-mail at all, says a new report on e-mail marketing from Jupiter Media Metrix. And success is measured not just on response rates, Jupiter says.
Posted 05/02/2002Kurt PetersPost a comment


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Boston, MAUSA
Hometown: Boston, MA
October 15, 2013
Safari or Chrome, depending on the day. Webkit 4 lyfe... or until something better comes along.
OSX, with iOS a close second.
Front-end UX • IA • IxD Designer/Developer
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Britney Spears might have said no to the "X Factor" but it looks like she's saying yes to Las Vegas.
The "Hit Me Baby One More Time" singer is looking at a residency at one of the Caesar properties. According to TMZ, "The deal is a go. There are several stipulations that could throw a monkey wrench in the deal, but it's basically done."
The singer recently split from fiancee Jason Trawick, which would give her a clean break to relocate to Vegas and perfect her show. The singer said of the split, "Jason and I have decided to call off our engagement. I'll always adore him and we will remain great friends."
Trawick chimed in, "As this chapter ends for us a new one begins. I love and cherish her and her boys and we will be close forever."


text:
Free personalized radio that
plays the music you love
Think Straight Again
Features of This Trackbasic rock song structures
ska influences
a subtle use of vocal harmony
call and answer vocal harmony (Antiphony)
mixed acoustic and electric instrumentation
major key tonality
vocal duets
These are just a few of the hundreds of attributes cataloged for this track by the Music Genome Project.show more
Similar Tracks
2 Cds (that I know of)
1 All nighters
2 New Invasion
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Weight Loss Without Meal Replacement
Make Green Go Green is honored and excited to have Elizabeth Stanley host a special webinar on Weight Loss Without Meal Replacement. Elizabeth is founder of Healthy Girl Holistic Lifestyle, an accredited Holistic Health Coach, certified Personal Trainer, co-host of Wellness Talk Radio, and a Melaleuca Marketing Executive. She will be sharing with us her […]Read More


text:
Free personalized radio that
plays the music you love
Artifacts Of Sound
Features of This Tracka subtle use of vocal harmony
mild rhythmic syncopation
demanding instrumental part writing
mixed acoustic and electric instrumentation
a vocal-centric aesthetic
electric guitar riffs
These are just a few of the hundreds of attributes cataloged for this track by the Music Genome Project.show more
Similar Tracks
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BBB Accredited Business since
BlueWagon Landscape & Design, Inc.
Phone: (925) 330-3587 Fax: (925) 283-3890 1166 Monticello Rd, Lafayette, CA 94549 http://www.bwland.com View Additional Web Addresses
BBB Business Reviews may not be reproduced for sales or promotional purposes.
This company offers residential and commercial landscape design, construction and maintenance.
Request a Quote
BBB Accreditation
A BBB Accredited Business since
BBB has determined that BlueWagon Landscape & Design, Inc. meets BBB accreditation standards, which include a commitment to make a good faith effort to resolve any consumer complaints. BBB Accredited Businesses pay a fee for accreditation review/monitoring and for support of BBB services to the public.
BBB accreditation does not mean that the business' products or services have been evaluated or endorsed by BBB, or that BBB has made a determination as to the business' product quality or competency in performing services.
Reason for Rating
BBB rating is based on 13 factors. Get the details about the factors considered.
Factors that affect the rating for BlueWagon Landscape & Design, Inc. include:
- Length of time business has been operating
- No complaints filed with BBB
Customer Complaints Summary
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This business is in an industry that may require professional licensing, bonding or registration. BBB encourages you to check with the appropriate agency to be certain any requirements are currently being met.
These agencies may include:
Contractors State License Board
9821 Business Park Dr, Sacramento CA 95827
Phone Number: (800) 321-2752
The number is 889808.
Type of Entity
Business ManagementMr. Kevin Bates, CEO
Contact Information
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Landscape Contractors Landscape Contractors Landscape Maintenance Patio & Deck Builders


text:
Office-Condominium Market Growing
By PENNY SINGER
Published: July 3, 1988
OFFICE condominium means never having a lease run out and never having to make a deal with a landlord. It also means independence and tax benefits for professionals and businessmen such as Jeffrey H. Nagelberg, a 43-year-old food broker who is president of Panorama Produce Sales in Mamaroneck.
Eighteen months ago, Mr. Nagelberg moved his business from rented offices in Fort Lee, N.J., into a 1,200-square-foot office condominium he bought in the Mamaroneck Harbor Professional Office Center at 933 Mamaroneck Avenue.
''It's been one of the smartest moves we've made,'' Mr. Nagelberg said. ''Now we can control our costs and, more important, control our future. We don't have to worry about losing a lease or having to relocate. In addition, the good rental buildings I looked at had minimum space requirements, which meant we would have had to take more space than we needed for our staff of five full-time employees and three part-timers.'' All Suites Sold
The nine office-condominium suites in the 14,500 square foot Mamaroneck Harbor professional center, averaging about $200 a square foot, sold out during construction, according to Richard A. Pink, a partner in the Midland Equities and Development Corporation, the Mamaroneck-based developer of the project.
''We put a sign on the building when it was half completed and we sold out,'' he said. ''Our new project - a 17,000-square-foot condominium we call the Larchmont Professional Center now under construction on the Post Road - has only the steel up, but over 90 percent of the space has been committed before we began any formal sales or marketing.''
Such response, Mr. Pink said, proves the strong demand for office condominiums. ''We figure that there are at least 11,000 potential office-condo buyers in Westchester - such as doctors, dentists, lawyers, accountants, architects, engineers and small-business men who need about 1,000 square feet of office space,'' Mr. Pink said. ''Yet only about 400,000 square feet of office-condo space exists in Westchester. And that's about 4 percent more than was available five years ago, when there was practically none.''
The market, he added, is getting bigger all the time, with the National Association of Home Builders projecting that nearly one billion square feet of office condominiums will be built nationally by the year 2000.
Mr. Pink, who is 44 years old, and his partner, Victor R. D'Ortona, 38, are alumni of Morgan Guaranty Trust Company in Manhattan. Mr. Pink spent 16 years at Morgan as a senior officer in corporate finance departments concentrating on real-state project financing. 'Look at the Market'
''I took a conservative, analytical approach to lending,'' Mr. Pink said. ''Those were difficult days for commercial projects. The market was soft and overbuilt. When I left the bank to go into business for myself, which was something I always wanted to do, I applied the same basic rule. 'Look at the market, let it show the way.' When we started Midland there was a glut of rental office space around. Rather than build another leased property, we decided to take a rifle approach and build for a special market: condominiums for the local professionals.'' Before forming Midland in 1983, Mr. D'Ortona served as a corporate officer with Morgan Guaranty and the Chase Manhattan Bank. He started out to become an architect. ''But in 1972, when I graduated from Pratt Institute, the market for architects was terrible,'' he recalled. ''I joined Morgan Guaranty in the real-estate department.''
After 11 years of experience in real-estate management and appraisal, and the acquisition of hundreds of millions of dollars of commercial and industrial properties for the bank's customers, Mr. D'Ortona wanted to try his hand at running his own company. ''We went into business with a good concept,'' he said. ''Rather than compete with the big office-park developers, we found a special niche and became one of the few companies specializing in building office condos from the ground up.'' A Custom-Built Office
Building an office condominium is different from converting an existing office building to condominium ownership, Mr. Pink emphasized. ''It's as different as building a custom home is from building one in a tract development,'' he said. ''Office condominiums are built with the individual owner in mind. The suites are self-contained; unlike offices in rental buildings, they all have individual lavatories and we put in rough plumbing for private dining rooms or kitchens or second-lavatory use. They all have individual heating and air-conditioning systems and separate metering for each owner. A common conference room is provided and parking spaces come with each suite.''
The condominium offices have the greatest appeal for local professionals who have well-defined businesses and stable space requirements, Mr. D'Ortona said. ''The kind of professionals who want to be right in the community, not in some office park off the highway are perfect condominium owners,'' he said. ''All our condo owners live within a five-mile radius of the office.''


text:
Recent evidence suggests that protease release by neutrophils in the bone marrow may contribute to hematopoietic progenitor cell (HPC) mobilization. Matrix metalloproteinase-9 (MMP-9), neutrophil elastase (NE), and cathepsin G (CG) accumulate in the bone marrow during granulocyte colony-stimulating factor (G-CSF) treatment, where they are thought to degrade key substrates including vascular cell adhesion molecule-1 (VCAM-1) and CXCL12. To test this hypothesis, HPC mobilization was characterized in transgenic mice deficient in one or more hematopoietic proteases. Surprisingly, HPC mobilization by G-CSF was normal in MMP-9–deficient mice, NE × CG-deficient mice, or mice lacking dipeptidyl peptidase I, an enzyme required for the functional activation of many hematopoietic serine proteases. Moreover, combined inhibition of neutrophil serine proteases and metalloproteinases had no significant effect on HPC mobilization. VCAM-1 expression on bone marrow stromal cells decreased during G-CSF treatment of wild-type mice but not NE × CG-deficient mice, indicating that VCAM-1 cleavage is not required for efficient HPC mobilization. G-CSF induced a significant decrease in CXCL12α protein expression in the bone marrow of Ne × CG-deficient mice, indicating that these proteases are not required to down-regulate CXCL12 expression. Collectively, these data suggest a complex model in which both protease-dependent and -independent pathways may contribute to HPC mobilization.
The use of hematopoietic progenitor cells (HPCs) to reconstitute hematopoiesis following myeloablative therapy has significantly improved the clinical outcome for patients with a variety of diseases. Recently, mobilized peripheral blood HPCs instead of bone marrow–derived HPCs have been used because of reduced engraftment times and relative ease of collection. Although the great majority of HPCs reside within the bone marrow, a small number of HPCs also circulate in the peripheral blood. The number of circulating HPCs can be dramatically increased, or mobilized, by a wide variety of stimuli including hematopoietic growth factors, chemotherapy, and chemokines.1,2 Currently, granulocyte colony-stimulating factor (G-CSF) is the most commonly used agent to mobilize HPCs because of its potency and lack of serious toxicity. However, the mechanisms that mediate G-CSF–induced HPC mobilization are incompletely understood.
We previously showed that expression of the G-CSF receptor (G-CSFR) on HPCs is not required for their mobilization into the blood in response to G-CSF.3 This observation suggests that G-CSF induces HPC mobilization indirectly through the generation of trans-acting signals. Recent studies suggest that hematopoietic proteases released by neutrophils into the bone marrow microenvironment may represent such a signal. A highly proteolytic microenvironment is induced in the bone marrow during HPC mobilization by G-CSF.4 In particular, matrix metalloproteinase-9 (MMP-9 or gelatinase B), neutrophil elastase (NE), and cathepsin G (CG) accumulate in the bone marrow of mice during treatment with G-CSF with kinetics that mirror that for HPC mobilization.4 MMP-9 is highly expressed in neutrophils, monocytes, and macrophages.5 In neutrophils, MMP-9 is stored in gelatinase granules and can be rapidly released following activation.6 Evidence for a direct role for MMP-9 in HPC mobilization is provided by the observation that neutralizing antibodies to MMP-9 partially block HPC mobilization by interleukin-8 (IL-8) in nonhuman primates.7 Moreover, a recent study reported that HPC mobilization by G-CSF was impaired in MMP-9–deficient mice.8 Collectively, these data suggest the hypothesis that MMP-9 is a key mediator of HPC mobilization by G-CSF or IL-8.
Neutrophils express 3 serine proteases: NE, CG, and proteinase 3.9 These proteases are stored in primary granules of neutrophils and can be released following neutrophil activation.9 We previously reported that during HPC mobilization by G-CSF, NE and CG are released into the bone marrow microenvironment and are capable of proteolytic cleavage of vascular cell adhesion molecule-1 (VCAM-1).10 Importantly, during mobilization with G-CSF, VCAM-1 expression in the bone marrow stroma is strongly reduced. VCAM-1 is thought to be the major ligand for α4β1 integrin (very late antigen-4 [VLA-4]), a key integrin expressed on HPCs.11 These observations suggest a model in which proteases released by activated neutrophils cleave VCAM-1 on stromal cells leading to the disruption of its key adhesive interaction with VLA-4 on HPCs.
More recently, evidence supporting an alternative mechanism by which NE and CG may regulate HPC mobilization has been generated.12,13 NE and CG are capable of proteolytic cleavage of both CXCL12 (stromal cell–derived factor-1) and its receptor, CXCR4.12 The interaction of CXCL12 with CXCR4 on HPCs is thought to be a key signal regulating HPC trafficking in the bone marrow.14,15 We and others showed that CXCL12 protein expression in the bone marrow is significantly decreased following HPC mobilization with G-CSF.12,13,16 Moreover, during G-CSF–induced HPC mobilization the amino-terminus of CXCR4 on HPCs is cleaved.12 Collectively, these data suggest the hypothesis that G-CSF may induce HPC mobilization through the proteolytic inactivation of both CXCL12 and CXCR4 by NE and CG. In support of this hypothesis, Petit et al13 showed that treatment of mice with an NE inhibitor partially blocked HPC mobilization by G-CSF.
To test these hypotheses, neutrophil mobilization by G-CSF was characterized in transgenic mouse lines deficient in one or more hematopoietic proteases or in mice following treatment with a broad-spectrum metalloproteinase inhibitor. We show that HPC mobilization by G-CSF is normal in mice lacking MMP-9 or functional neutrophil serine proteases.
Materials and methods
MMP-9–deficient, NE-deficient, CG-deficient, and dipeptidyl peptidase-I (DPP-I)–deficient mice, all inbred onto a 129 genetic background, were produced as previously described.17-21 NE- and CG-deficient mice were intercrossed to produce NE × CG-deficient mice using standard breeding strategies. Southern blot analyses were performed to confirm the presence of homozygous null alleles for NE and CG (data not shown). The lack of functional NE and CG in these mice was confirmed using NE- or CG-specific chromogenic substrate assays, as previously described (data not shown).22 In some experiments, DPP-I–deficient mice backcrossed for 10 generations onto a C57BL/6 background were used. All mice were housed in a specific pathogen–free environment and examined twice weekly by veterinary staff for signs of illness. All studies were approved by the animal studies committee at Washington University, St Louis, MO.
Mobilization regimens
Recombinant human IL-8, a generous gift from Searle (now part of Pfizer, New York, NY), was given by a single intraperitoneal injection at a dose of 30 μg per mouse and peripheral blood was analyzed at the indicated times. Recombinant human G-CSF (Amgen, Thousand Oaks, CA) diluted in phosphate-buffered saline (PBS) with 0.1% low endotoxin bovine serum albumin (BSA; Sigma, St Louis, MO) was administered by daily subcutaneous injection at a dose of 250 μg/kg per day for 5 days. Mice were analyzed 4 hours after the final injection of G-CSF. Cyclophosphamide (CY; Sigma) was reconstituted in sterile water and administered as a single intraperitoneal injection at a dose of 200 μg/kg.
Administration of MMI270
MMI270, a broad-spectrum metalloproteinase inhibitor, was a generous gift from Novartis Pharma KK (Ibaraki, Japan). MMI270 was administered by continuous subcutaneous infusion using an implantable miniosmotic pump (Alza Pharmaceuticals, Palo Alto, CA) at a rate of 30 mg/kg per day for 6 days. Briefly, MMI270 was reconstituted in a 1:1 ratio of dimethylsulfoxide (DMSO)/polyethylene glycol 300 at a concentration of 37.5 mg/mL, loaded into a miniosmotic pump, and surgically inserted into the subcutaneous tissue on the backs of mice. Serum levels of MMI270 were analyzed at the time of animal harvest using a previously described high-perfomance liquid chromatography (HPLC) assay.23
Gelatin zymography
The amount of MMP-9 present in the serum of mice at the indicated times after treatment with IL-8 or G-CSF was measured by gelatin zymography, as described previously.24
Colony-forming cell (CFU-C) assay
Blood, bone marrow, and spleen cells were harvested from mice using standard techniques, and the number of nucleated cells in these tissues was quantified using a Hemavet automated cell counter (CDC Technologies, Oxford, CT). Blood (10 μL-20 μL), nucleated spleen cells (1 × 105), or nucleated bone marrow cells (2.5 × 104) were plated in 2.5 mL methylcellulose media supplemented with a cocktail of recombinant cytokines (MethoCult 3434; Stem Cell Technologies, Vancouver, BC, Canada). Cultures were plated in duplicate and placed in a humidified chamber with 6% CO2 at 37° C. Colonies containing at least 50 cells were scored on day 7 of culture.
In vitro cleavage of recombinant human VCAM-1 by bone marrow extracellular fluid
Bone marrow (BM) extracellular fluid was obtained by flushing both femurs of a mouse with a total of 1 mL ice-cold PBS and then collecting the cell-free supernatant following centrifugation at 500g for 2 minutes. BM extracellular fluid (10 μL) was mixed with an equal volume of PBS containing 0.1 μg recombinant extracellular domain of human VCAM-1 (R&D Systems, Minneapolis, MN) and incubated at 37° C for 30 minutes. Digestions were stopped by adding 20 μL of 125 mM Tris-HCl, pH 6.8, 20% glycerol, 4% sodium dodecyl sulfate, and boiling for 5 minutes. Samples were separated on 10% polyacrylamide gels, transferred onto nitrocellulose, and stained with a goat antihuman VCAM-1 serum (R&D Systems) as previously described.10
VCAM-1 immunohistochemical staining
Bone marrow was flushed from femurs directly onto poly-l-lysine–coated glass slides and processed as previously described.10 Following fixation in ice-cold acetone, slides were air-dried and kept at room temperature until staining. Prior to staining, slides were rehydrated in PBS Tween 20 (PBST) for 2 hours and blocked for 2 hours in PBST containing 5% donkey serum (PBSTDS). Slides were incubated overnight at 4° C with either M/K2-7 hybridoma supernatant mixed with an equal volume of PBSTDS or 2 μg/mL nonimmune rat IgG1 in 50% PBSTDS/50% hybridoma growth medium. Following 4 washes with PBSTDS, slides were incubated for 2 hours at room temperature with 1:250 biotinylated donkey F(ab′)2 fragments antirat IgG non–cross-reacting with mouse proteins (Jackson ImmunoResearch Laboratories, West Grove, PA) in PBSTDS. Following 4 washes in Tris-buffered saline Tween 20 (TBST), slides were incubated for 1 hour in TBST, 0.1% BSA, 1:400 alkaline phosphatase–conjugated streptavidin (Amersham). Following 4 washes in TBST, slides were stained with fast red TR, naphtol AS-MX phosphate, and levamisole, then counterstained with hematoxylin and mounted with Aquamount (Fronine Laboratory Supplies, Riverston, Australia) as previously described.10 Microscopy was performed using a Zeiss Axiokop 2 microscope (Carl Zeiss Microimaging, Thornwood, NY) with a Zeiss Plan-Neoflur × 20 objective (numerical aperture 0.5). Digital images were obtained using a Diagnostic Instruments Spot RT Slider camera (Sterling Heights, MI) and analyzed with Spot Software Windows version 4.0.2 imaging software (http://www.spotsoftware.nl).
CXCL12α ELISA
CXCL12α protein concentration in BM extracellular fluid was measured by enzyme-linked immunosorbent assay (ELISA), as described previously.16 BM extracellular fluid was diluted 1:3 in ELISA diluent (0.1% BSA, TBST). Recombinant human recombinant CXCL12α was used to generate a standard curve (data not shown). All ELISA reagents were purchased from R&D Systems.
Progenitor transmigration assay
Transwell migration of HPCs in response to CXCL12α was performed, essentially as described previously.12 In brief, mononuclear cells from the bone marrow or peripheral blood were isolated using a Lympholyte-Mammal density gradient, per manufacturer's recommendations (Cedarlane Laboratories, Hornby, ON, Canada). Cells were incubated in RPMI supplemented with 10% fetal calf serum at 37° C for 1 hour to allow time for resensitization of potentially desensitized CXCR4. Cells (2 × 105) were loaded into the upper chamber of 5-μm pore transwell inserts (Corning Costar, Cambridge, MA). Recombinant human recombinant CXCL12α was added to the lower chamber at a concentration of 200 mg/mL and the chamber was incubated for 4 hours at 37° C. The CFU-C content in the original cell preparation and in cells that transmigrated to the lower chamber was determined using the CFU-C assay.
Statistical analysis
Data are presented as the mean plus or minus the standard deviation (SD). Statistical significance was assessed by 2-tailed Student t test.
MMP-9 is not required for HPC mobilization by IL-8 or G-CSF in mice
Recent data suggest that MMP-9 is a critical mediator of HPC mobilization by G-CSF and IL-8.7,8 To test this hypothesis, we first determined whether treatment of mice with IL-8 or G-CSF results in the release of MMP-9 by measuring the amount of MMP-9 in the plasma of animals after treatment with these agents (Figure 1A). Indeed, at the time of peak HPC mobilization (15 minutes after IL-8 administration or after 5 days of G-CSF treatment), a significant increase in the amount of MMP-9 in the serum was detected relative to untreated mice. The identity of the MMP-9 band was confirmed by its specific loss in MMP-9–deficient mice treated with G-CSF. Moreover, the MMP-9 band was abolished by incubation of the gel in the presence of EDTA (ethylenediaminetetraacetic acid; data not shown). We next measured MMP-9 release in G-CSFR–deficient mice after treatment with IL-8. We previously showed that HPC mobilization by IL-8 is markedly impaired in G-CSFR–deficient mice.25 Interestingly, no significant increase in serum MMP-9 was detected in these mice following IL-8 administration (Figure 1B). Finally, MMP-9 release during HPC mobilization by CY was examined in wild-type mice. Treatment of mice with CY results in robust HPC mobilization that peaks on day 8 after administration. Surprisingly, no increase in serum MMP-9 was detected at the time of peak HPC mobilization by CY (1Figure 1C). Collectively, these data show that an increase in the plasma level of MMP-9 is detectable during HPC mobilization by some, but not all, agents.
To more directly assess the role of MMP-9 in HPC mobilization, HPC mobilization by IL-8 or G-CSF was studied in MMP-9–deficient mice. Of note, the MMP-9–deficient and wild-type mice used in this study were inbred on a 129 genetic background. As reported previously,26 treatment of wild-type mice with IL-8 results in a rapid (peak 15-30 minutes after administration) but modest (3.4-fold increase over baseline) increase in circulating CFU-Cs (Figure 2A). Surprisingly, the kinetics and magnitude (5.0-fold over baseline) of the increase in peripheral blood CFU-Cs after IL-8 administration were similar in MMP-9–deficient mice. In addition, a similar increase in circulation neutrophils was observed in wild-type and MMP-9–deficient mice (data not shown).
We next examined G-CSF–induced HPC mobilization in MMP-9–deficient mice (Figure 2B). As reported previously, treatment with G-CSF (250 μg/kg per day for 5 days) resulted in a significant increase in blood (35-fold) and spleen (19-fold) CFU-Cs in wild-type mice.25 A similar increase in the number of blood (31-fold) and spleen (18-fold) CFU-Cs was observed in MMP-9–deficient mice. Likewise, a similar increase in the number of blood and bone marrow neutrophils was observed in wild-type and MMP-9–deficient mice (data not shown). Of note, no significant difference in the number of CFU-Cs in the bone marrow of MMP-9–deficient or wild-type mice was observed. Collectively, these data demonstrate that MMP-9 is not required for IL-8– or G-CSF–induced HPC mobilization in mice.
NE and CG are not required for HPC mobilization by G-CSF in mice
Neutrophil serine proteases, specifically NE and CG, recently have been implicated as key mediators of G-CSF–induced HPC mobilization.4,10 To test this hypothesis, HPC mobilization by G-CSF was examined in NE × CG-deficient mice inbred on a 129 background. Of note, basal hematopoiesis is normal in these mice.22 G-CSF treatment induced a similar increase in blood and spleen CFU-Cs in wild-type and NE × CG-deficient mice. In the blood, 21-fold and 28-fold increases in CFU-Cs were detected in wild-type and NE × CG-deficient mice, respectively. Likewise, a similar increase in spleen CFU-Cs was detected in wild-type (15-fold) and NE × CG-deficient mice (15-fold).
In addition to NE and CG, neutrophils contain one additional serine protease, proteinase 3. To determine whether the protease activity of proteinase 3 contributes to HPC mobilization in the absence of NE and CG, we studied DPP-I–deficient mice. DPP-I is an enzyme required for the processing of many proteases. Loss of DPP-I leads to the functional inactivation of a broad range of hematopoietic serine proteases, including NE, CG, and proteinase 3.22 Of note, DPP-I–deficient mice inbred on a 129 genetic background have normal basal hematopoiesis.19 These mice were treated with G-CSF for 5 days and the number of CFU-Cs in the blood, bone marrow, and spleen was quantified (Figure 3B). Robust HPC mobilization similar to that seen in wild-type mice was observed. There is considerable heterogeneity in the mobilization response to G-CSF among different inbred strains of mice. To determine whether the contribution of neutrophil serine proteases to HPC mobilization is strain dependent, we studied DDP-I–deficient mice inbred onto a C57BL/6 background. As reported previously, HPC mobilization by G-CSF was reduced in wild-type C57BL/6 mice compared with 129 mice (1113 ± 534 CFU-Cs per mL of blood on day 5 of G-CSF treatment). A similar mobilization response was observed in DDP-I–deficient C57BL/6 mice (1763 ± 531 CFU-Cs per mL). Collectively, these data show that the protease activity of neutrophil serine proteases is not required for HPC mobilization by G-CSF in mice.
Combined inhibition of a broad spectrum of metalloproteinases and neutrophil serine protease activity does not result in impaired HPC mobilization by G-CSF
It is possible that MMP-9 and neutrophil serine proteases provide redundant proteolytic activities required for HPC mobilization. Thus, the loss of a single protease activity may not result in impaired HPC mobilization. To address this possibility, we treated DPP-I–deficient mice with MMI270, a broad-spectrum metalloproteinase inhibitor. MMI270 inhibits MMP1, MMP2, MMP3, MMP9, and MMP13 with an inhibitory concentration (IC50) of less than or equal to 50 nm.23 DDP-I–deficient mice were treated with MMI270 (30 mg/kg) or vehicle alone by continuous subcutaneous infusion for 6 days. G-CSF was given on days 2 to 6 and CFU-Cs were quantified after the final dose of G-CSF on day 6. The plasma concentration of MMI270 at the time of animal harvest was 454 ± 203 nM, well above the IC50 for the indicated metalloproteinases. As shown in Figure 4, treatment of DPP-I–deficient mice with MMI270 had no effect on G-CSF–induced HPC mobilization. Thus, the combined inhibition of a broad spectrum of metalloproteinases and neutrophil serine proteases had no significant effect on HPC mobilization by G-CSF.
G-CSF–induced cleavage of VCAM-1 on bone marrow stromal cells is dependent on NE and CG but is not required for HPC mobilization
We previously showed that NE and CG are able to cleave VCAM-1 in vitro.10 Since interaction of VCAM-1 on stromal cells with VLA-4 on HPCs is thought to be a key adhesive interaction regulating HPC trafficking in the bone marrow,11 this observation suggests that VCAM-1 cleavage by NE or CG may be a key step in HPC mobilization. To further examine this possibility, we studied VCAM-1 cleavage in NE × CG-deficient and DPP-I–deficient mice. The release of proteases capable of cleaving VCAM-1 was assessed by incubating bone marrow extracellular fluid with recombinant human VCAM-1 (Figure 5). As previously reported by our group,10 in wild-type mice, G-CSF induced the release of proteases capable of cleaving VCAM-1 (Figure 5, lanes c, d, k, l). In contrast, no VCAM-1 cleavage was observed in bone marrow extracellular fluid harvested from G-CSF–treated, NE × CG-deficient (Figure 5, lanes g, h) or DPP-I–deficient (Figure 5, lanes o, p) mice. These data confirm our previous observation that NE and CG are the major VCAM-1 cleaving proteases released into bone marrow extracellular fluids during G-CSF treatment.
We next examined VCAM-1 expression on bone marrow stromal cells in vivo by immunohistochemical staining of VCAM-1 in the bone marrow (Figure 6). As reported previously by our group, G-CSF treatment induced a significant decrease in VCAM-1 expression in the bone marrow.10 Interestingly, VCAM-1 expression in the bone marrow was consistently increased in NE × CG-deficient mice at baseline. However, after G-CSF very little change in VCAM-1 expression was detected. These data suggest that VCAM-1 cleavage on bone marrow stromal cells is not required for efficient HPC mobilization by G-CSF.
G-CSF induces a significant decrease in CXCL12α protein expression in the bone marrow and a decrease in functional CXCR4 expression on HPCs that is not dependent on NE or CG
Accumulating evidence suggests that disruption of the interaction of CXCL12α with its cognate receptor, CXCR4, on HPCs is a key step in mobilization. We and others previously showed that the level of CXCL12α protein in the bone marrow decreases following G-CSF treatment.12,13,16 Two recent reports suggested that this decrease in CXCL12 expression is mediated by NE- and CG-dependent proteolysis of CXCL12α.12,13 Moreover, we recently showed that CXCR4 on HPCs is cleaved during G-CSF treatment, resulting in a nonfunctional receptor, and providing further evidence implicating these proteases. To test this hypothesis, CXCL12α protein expression and functional CXCR4 expression on HPCs were examined in NE × CG-deficient mice. As reported previously, a significant decrease in CXCL12α protein was detected in bone marrow extracellular fluid of wild-type mice following G-CSF treatment (Figure 7A). Surprisingly, a similar decrease in CXCL12α protein was detected in NE × CG-deficient mice. Thus, down-regulation of CXCL12α protein expression in the bone marrow by G-CSF is not dependent on NE or CG. Expression of functional CXCR4 on HPCs was measured using a CXCL12α chemotaxis assay. As reported previously, the percentage of CXCL 12–responsive CFU-Cs in the blood following G-CSF treatment was reduced compared with BM resident CFU-Cs (Figure 7B). Similar data were observed in NE × CG-deficient mice. These data show that the decrease in functional CXCR4 expression on HPCs mobilized by G-CSF also is not dependent on NE or CG.
Recent studies have implicated hematopoietic proteases as potentially key mediators of HPC mobilization.4,7,8,10,12,13,27 In the present study, we have examined this hypothesis through the characterization of HPC mobilization in selected protease-deficient mice. There is strong evidence that specific hematopoietic proteases accumulate in the bone marrow during HPC mobilization. We recently showed that NE and CG activity in bone marrow extracellular fluid increased 8- to 100-fold following treatment with G-CSF or CY.4 Importantly, the peak in NE and CG activity coincided with maximal HPC mobilization. Pruijt et al7 showed that treatment of rhesus monkeys with IL-8 induces a dramatic and rapid increase in plasma levels of MMP-9 that correspond with HPC mobilization. We previously showed that the level of MMP-9 also is significantly increased in bone marrow extracellular fluid following treatment with G-CSF or CY.4 However, the kinetics of the increase in MMP-9 were delayed, particularly with CY, where the peak of MMP-9 in the bone marrow extracellular fluid was on day 10 following CY injection when the number of circulating HPCs returns to baseline values.4 Consistent with these observations, in the present study we show that the plasma level of MMP-9 increases following treatment with G-CSF or IL-8 in mice. However, no detectable increase in plasma MMP-9 was detected following CY treatment, despite robust HPC mobilization. These data raise the possibility that MMP-9 may not be required for CY-induced mobilization, even though we previously showed that mobilization by this agent is dependent on G-CSF receptor signals.25
Recent evidence suggests a direct role for MMP-9 in HPC mobilization. Pruijt et al7 showed that pretreatment of rhesus monkeys with neutralizing antibodies to MMP-9 partially blocked HPC mobilization by IL-8. In a subsequent report, the authors showed that depletion of neutrophils prior to IL-8 administration also blocked HPC mobilization in mice, suggesting that neutrophils may be the primary source of protease release.28 However, consistent with a previous report,28 we show in the current study that IL-8–induced HPC mobilization is normal in MMP-9–deficient mice. Collectively, these data raise the possibility that species-specific differences in the requirement for MMP-9 to mediate IL-8–induced HPC mobilization may exist.
MMP-9 also has been implicated in G-CSF–induced HPC mobilization in mice. Heissig et al8 reported that HPC mobilization by G-CSF was significantly impaired in MMP-9–deficient mice. Moreover, treatment of mice with CGS27 023A (MMI270), a broad-spectrum metalloproteinase inhibitor, nearly completely blocked HPC mobilization by G-CSF. The authors showed that MMP-9 cleaved Kit-ligand from the surface of bone marrow stromal cells, providing a potential mechanism for its effects on HPC trafficking. In contrast, in the present study, we show that HPC mobilization by G-CSF is normal in MMP-9–deficient mice. Since Heissig et al used a lower dose of G-CSF (50 μg/kg per day for 5 days), we repeated our studies using this G-CSF regimen. Again, no difference in HPC mobilization between wild-type and MMP-9–deficient mice was observed (data not shown). Consistent with our data, a recent report also showed that G-CSF–induced HPC mobilization is normal in MMP-9–deficient mice.29 To begin to address the possibility of redundant protease activity, we treated DDP-I–deficient mice with MMI270, the same broad-spectrum metalloproteinase inhibitor used by Heissig et al. In contrast to their results, treatment with MMI270 had no significant effect on HPC mobilization by G-CSF. In the present study, a plasma concentration of MMI270 of 454 nM was achieved (similar data are not provided by Heissig et al). Although direct proof that MMP-9 was inhibited in our mice is not provided, this plasma concentration of MMI270 is well above the IC50 for MMP-9 (8 nM), and it is above the IC50 (200 nM) for the inhibition of angiogenesis in a rat aorta model, a model that is dependent on metalloproteinases. Of note, treatment of wild-type mice with BB-94/Batimastat30,31 (British Biotechnology, Oxford, United Kingdom), another broad-spectrum metalloproteinase inhibitor, also had no significant effect on G-CSF–induced HPC mobilization (data not shown). Although the reason for the discrepancy between these 2 studies is not clear, several potentially important differences have been identified. First, different dosing regimens for MMI270 were used: 30 mg/kg per day for 6 days by continuous subcutaneous infusion in the present study and 60 mg/kg given subcutaneously every 3 days in the study by Heissig et al. Second, different genetic strains of mice were used. In the present study, 129 mice were used, whereas Heissig el al used mice outbred on a CD1 genetic background. This latter possibility is consistent with previous reports showing considerable differences in G-CSF–induced HPC mobilization among different mouse genetic strains.32 There is nearly a 12-fold difference in the magnitude of HPCs mobilized into the blood between poor responders (for example, C57BL/6 mice) and good responders (for example, DBA mice). 129 mice display an intermediate phenotype. Unfortunately, there are no published reports comparing HPC mobilization in outbred CD1 mice with inbred strains of mice.
We previously showed that VCAM-1 expression on bone marrow stromal cells is markedly decreased following HPC mobilization by G-CSF. VCAM-1 is thought to be a key ligand for VLA-4, a major integrin expressed on HPCs.33 The importance of VCAM-1–VLA-4 interactions to HPC trafficking in the bone marrow has been confirmed by studies showing that antibodies directed against VCAM-1 or VLA-4 lead to HPC mobilization.11,34,35 Moreover, a recent report showed that the inducible deletion of α4 integrin in adult mice results in a 7-fold increase of circulating CFU-Cs that was maintained over 20 weeks.36 We previously showed that NE and CG are able to cleave VCAM-1 in vitro.10 Since NE and CG are induced in the bone marrow during G-CSF treatment, this observation suggested that NE- and CG-mediated cleavage of VCAM-1 on bone marrow stromal cells is a key step in HPC mobilization. In the present study, we show that VCAM-1 cleaving activity in bone marrow extracellular fluid and the down-regulation of VCAM-1 expression on bone marrow stromal cells in vivo is markedly reduced in NE × CG-deficient mice, confirming that NE and CG are the major proteases mediating VCAM-1 cleavage on bone marrow stroma in vivo and directly regulate VCAM-1 expression in this tissue. However, G-CSF–induced HPC mobilization is normal in NE × CG-deficient mice, despite continued high expression of VCAM-1 in the bone marrow. These data strongly suggest that, although the disruption of VCAM-1–mediated interactions in vivo is sufficient to elicit mobilization,11,34,35,37 VCAM-1 cleavage is not required for efficient HPC mobilization by G-CSF in mice.
Accumulating evidence suggests that interaction between CXCL12 and CXCR4 may play a key role in regulating HPC trafficking from the bone marrow. CXCL12 is constitutively produced by bone marrow stromal cells and CXCR4 is broadly expressed on hematopoietic cells.38-40 Studies of mice lacking CXCL12 or CXCR4 have established that CXCL12 is necessary for the normal migration of hematopoietic progenitors from the fetal liver to the bone marrow.14,41 Elevation of CXCL12 levels in the blood by administration of CXCL12 or by injection of an adenoviral vector expressing CXCL12 is associated with a significant mobilization of hematopoietic progenitors.42,43 Finally, recent data show that treatment of humans with AMD3100, a specific CXCR4 antagonist, leads to rapid and robust HPC mobilization.44 These data suggest that CXCL12 may be a key retention signal in the bone marrow for HPCs. We and others previously showed that G-CSF treatment results in a significant decrease in CXCL12α protein in the bone marrow of wild-type mice.12,13,15,16 Evidence that the decrease in CXCL12α protein expression in the bone marrow may be secondary to its proteolytic degradation by NE and CG in vitro and in vivo has recently been provided.12,13 Proteases present in bone marrow extracellular fluid from G-CSF–mobilized but not untreated mice are able to cleave, and inactive CXCL12 and specific inhibitors for both NE and CG abolish this activity.12 Moreover, treatment of mice with a peptide inhibitor of NE partially blocked G-CSF–induced HPC mobilization.13 In the present study, we show that a similar decrease in CXCL12α protein expression in the bone marrow is observed in wild-type and NE × CG-deficient mice. Thus, there are efficient NE- and CG-independent mechanisms to down-regulate CXCL12α protein expression during G-CSF–induced HPC mobilization. We recently showed that the amino-terminus of CXCR4 is cleaved on HPCs during G-CSF–induced HPC mobilization, resulting in receptor inactivation and further disrupting CXCL12-CXCR4 interactions. In the present study, we show that functional CXCR4 expression on mobilized blood HPCs is reduced to a similar degree in both wild-type and NE × CG-deficient mice, indicating that the proteolytic cleavage of CXCR4 also does not require NE or CG. Interestingly, in contrast to the situation with humans, no decrease in functional CXCR4 expression was observed in bone marrow–resident HPCs following G-CSF treatment. Nonetheless, these data are consistent with the hypothesis that perturbation of CXCL12-CXCR4 interactions is a key step in HPC mobilization by G-CSF.
What role do hematopoietic proteases play in HPC mobilization? Our data suggest several possibilities. First, it is possible that functional redundancy exists among proteases contributing to HPC mobilization such that the loss of any single protease is well compensated. Our finding that G-CSF–induced HPC mobilization is normal in DPP-I–deficient mice (which are functionally deficient in all neutrophil serine proteases), or in DDP-I–deficient mice treated with a broad-spectrum metalloproteinase inhibitor, argues against this possibility. Second, other proteases not inhibited in our studies may contribute to HPC mobilization. A recent study showed that MMP-9 expressed on the cell surface of neutrophils is resistant to inhibition by tissue inhibitor of metalloproteinases (TIMPs).45 Thus, it is possible that only partial MMP-9 inhibition was achieved in mice treated with MMI270. Recently, Christopherson et al27 reported that CD26 (dipeptidylpeptidase IV) a membranebound extracellular protease expressed on a subset of HPCs, may contribute to G-CSF–induced HPC mobilization. They showed that CXCL12 is cleaved and inactivated by CD26. Moreover, G-CSF–induced HPC mobilization is defective in CD26 deficient mice or in wild-type mice treated with a specific CD26 inhibitor.27,46 Third, non–protease-dependent mechanisms may contribute to HPC mobilization. Consistent with this possibility, we recently showed that expression of CXCL12 mRNA in bone marrow cells is significantly reduced during HPC mobilization by G-CSF (C.L. Semerad, M. Christopher, F. Liu, et al, manuscript submitted). In fact, the magnitude and kinetics of the decrease in CXCL12 mRNA closely mirrored the decrease in CXCL12α protein. Collectively, current evidence supports a complex model in which multiple protease-dependent and -independent mechanisms contribute to HPC mobilization.
We thank Jill Woloszynek and Jean Hendy for their expert technical assistance. We also thank Shinichi Koizumi and Hiroaki Fukaya (Novartis Pharma KK, Tsukuba Research Institute) for their assistance in obtaining MMI270 and measuring its concentration in mouse plasma samples.
- Reprints:
Daniel C. Link, Division of Oncology, Department of Medicine, 660 S Euclid Ave, Campus Box 8007, St Louis, MO 63110; e-mail: dlink{at}im.wustl.edu.
Prepublished online as Blood First Edition Paper, March 9, 2004; DOI 10.1182/blood-2003-05-1589.
Supported by grants from the National Institutes of Health National Heart, Lung, and Blood Institute (R01 HL60772-01A1 [D.C.L.]; RO1 HL47328 [R.S.]; RO1 AI49261-02 [C.P.]) and from the National Health and Medical Research Council of Australia (080193 [J.-P.L. and P.J.S.]).
J.-P.L. and F.L. contributed equally to this study.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
- Submitted May 16, 2003.
- Accepted February 26, 2004.
- Copyright © 2004 by The American Society of Hematology


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BA drops e-commerce £6 million project
British Airways has pulled the plug on a major Web site development with 25 staff expected to lose their jobs
The Website - codenamed Project Quicksilver - had been under development since August, reports claimed. The site was intended to be an information and e-commerce site offering news on subjects including shopping and travel.
BA blamed the decision on delays to the launch of the site, which was being developed by Affinity Internet. Affinity Internet pulled out of the project last week.
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BA is reported to have invested £6m in the site, to be called Peeled, before shelving it. The airline had intended to market the site to members of its Executive Club Loyalty programme, who would have been rewarded for shopping and surfing on the site.
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