text:
All Xarelto lawsuits, including lawsuits to be filed by Maryland residents, will be consolidated before U.S. District Judge Eldon E. Fallon in the Eastern District of Louisiana.
This happened quickly. Xarelto came on to the market three years ago. Now there is a class action type MDL that has been created because there are too many lawsuits. This does not speak well to this drug and it does not speak well to Johnson & Johnson and Bayer, who introduced Xarelto with so much pomp, circumstance and excitement that it might be a drug that could really change the quality of people's lives.
What the MDL Means?
I always call an MDL a "sort of" class action. All federally filed cases — which is the vast majority of these claim and virtually all of the Maryland claims — are consolidated under one federal judge in a single jurisdiction, Louisiana in this case. When a lawsuit is filed, the lawyers have an opportunity to conduct discovery where both sides get a chance to learn about the case. In the Xarelto lawsuits, there are some facts common to all of these claims. The biggest issue is going to be what Bayer and J&J knew or should have known about the bleeding risks associated with Xarelto.


text:
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Click on a product category below to pull up a list of NRHA Vendor Partners with additional product and training information available.
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and it's free to all NRHA members.
April 16, 2012
Home Hardware Stores Limited sponsored a luncheon for the Rocky Mountain Business Seminar (RMBS), Canada's longest-running student-organized conference. Home Hardware Stores Limited, a key sponsor of the HARDLINES Studies in Retail Excellence (SIRE) Program, sponsored a luncheon event for approximately 130 student delegates from across Canada. During the luncheon, Dr. Paul McElhone, executive director of the School of Retailing, gave an overview of the SIRE program. Home Hardware also provided five $500 prizes to cover the delegate fees of five student attendees, which were drawn randomly.
"The future of retail and the home improvement industry is with young people," said Paul Straus, President and CEO of Home Hardware Stores Limited. "Hardware and home improvement is one of the most dynamic retail sectors in this country today. At Home Hardware, we strongly believe in encouraging innovative minds at the university level to consider this field as a viable career option."
The Studies in Retail Excellence Program was developed by HARDLINES in partnership with the University of Alberta to foster awareness and career opportunities in hardlines retailing at the university level. The money from their sponsorship is directed to scholarships and networking opportunities for a student at the Annual Hardlines Conference, being held Oct. 26-27, in Toronto.
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subset: commoncrawl


text:
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You can try these similar gifts instead.
Sugar and Wheat Free Peanut Butter Protein Bars
Satisfy someone's peanut butter craving while doing their body good. Each of the 10 bars in this box is packed with protein and over a dozen vitamins and minerals, making healthy eating easy to swallow.


text: Recently I've noticed problems on the final (12th) frames of rolls run through my Rolleiflex 3.5E. What could be causing this? No issues with the first 11 frames of each roll, and also it didn't use to do this -- it's only on the last few rolls I've shot. Am I screwing up when I unload the film? I'm having the films developed and scanned at Precision Camera in Austin and I find it hard to believe that they'd be messing up. Far more likely that it's me and/or the camera. Thank you for your consideration. Joel Brouwer


text:
Irish stable lad who won €1.2m with bet returns to Cheltenham as trainer
IRELAND's millionaire stable lad is racing back to Cheltenham after an "unbelievable year" – but this time he will be on holiday and, of course, having a few bets.
Conor Murphy (29, right), from Ballineen, Co Cork, was the toast of the festival last year after winning £1m (€1.2m) on a five-horse accumulator.
It has been a whirlwind year for the hard-working former lad as he used his winnings to realise his ambition and set up as a trainer in the US.
"Cheltenham was an absolute dream," he said. "It was great to get a helping hand."
However, he admitted it was hard to leave his job as assistant head lad to English trainer Nicky Henderson, whose five horses on which he had bet £50 landed.
"The horses there were the best I'd ever worked with and probably will ever work with. They were a dream," he said.
After banking his winnings, Conor took time to weigh his options before buying a house in Louisville, Kentucky, and renting a barn at the Skylight Training facility.
"It was great to be able to buy a home without a mortgage and get going without having to watch every penny," he said. "I'm working as hard now as I did in England. You have to keep the show going."
After setting up in business in September, Conor also bought a few horses of his own to sell to clients, including "a really nice horse" called Di-mension, and Bronterre, who he has already sold a half share in.
"I haven't blown all my money yet," he joked, admitting there had been some frustrations as he strove to land a winner. He now has 25 horses at his stables, including six he is training for the track. The remainder are for his new pre-training business.
"I'd love to be at home or in England training jumpers because jump racing is a passion, but the opportunities are better over here," Conor added.
He proved he still has some of his Cheltenham luck when he met Sheikh Fahad Al Thani who sent him six horses.
Another of the major players, Coolmore, has also sent him some horses to break.
Despite being busy in the States, Conor could not resist placing a few ante-post bets ahead of flying over to Cheltenham this week.
"I'll be in Cheltenham for the week," he said. "I'll have a few bets on. Last year was a one off, but you have to try again just in case. You never know."
Irish Independent


text:
I am having some problems trying to store data into a config file in Codeigniter
The ProblemThe session data grows too big and seems to exceed the 4kb which is the cookie max size. As a result for example flashdata stops working.
Why It HappensIt seems that config variables are added 4 times into the session inside of following objects:- CI_Hooks Object (should be turned off)- CI_Utf8 Object- MY_Router Object (used for replacing '_' with '-' in URL)- CI_URI Object
Interestingly the hooks object is present even if the functionality is turned off in the config file: $config['enable_hooks'] = FALSE;
[B]Questions[/B]01 - how do you store config data without it being multiplied in the session?02 - also, why does the hooks object seem to be enabled when it has been disabled in the config?
Other Details- CI_VERSION 2.1.3- Session library is autoloaded- Sessions are stored into db- Session cookie is encrypted- Config files are loaded in the pages where they are used, mainly inside constructors- The config data is not huge and would fit very well to 4kb if it wasn't multiplied
Session data ( print_r($this->session) ) :[hooks] => CI_Hooks Object ( [enabled] => [hooks] => Array ( ) [in_progress] => ) [config] => CI_Config Object ( [config] => Array...
Thanks in advance for your help, it would be much appreciated...


text:
Compression Tactical Bra - Cheata Tactical
Patented technology is designed to provide the stability of 2-3 sports bras,...
"Cajun Justice," a new police reality show featuring deputies from the Terrebonne Parish Sheriff's Department, premieres tonight on the A&E network.
The show follows Sheriff Vernon Bourgeois and the department's detectives and deputies in a jurisdiction covering 2,000 square miles of treacherous swampland located 45 miles south of New Orleans, according to the network. The show's Website lists six episodes.
The show airs at 10 p.m. on Thursdays. Get more details at the "Cajun Justice" website.
Billy @ 6/8/2012 3:56 PM
I usta listen to dem ol boys down there in Terrebonne Parish on the old Sheriff's Association low-band radio back in the day when I was a young deputy with Jefferson Parish. Might be an intersting show...


text:
Results 1 to 2 of 2
03-26-02, 13:02 #1Registered User
- Join Date
- Mar 2002
- Posts
- 4
Unanswered: failed remote connection to Pervasive 2000i server via JDBC
I installed on a remote machine (windows 2000) Pervasive.SQL 2000i SP3 that I downloaded from pervasive site.
I need a J2EE application that reads data from the remote machine and presents on the local machine.
If the J2EE application is also on the remote machine. It works fine.
But when the J2EE application is on a local machine (also windows2000). I can not retrieve the data.
I installed first workstation engine and later P.SQL2000i server on the local machine Neither helped.
I keep getting the following error when I try the url
java.sql.SQLException: Error retrieving error information
Error retrieving error information
I could not find any information on pervasive site.
03-28-02, 06:06 #2Registered User
- Join Date
- Mar 2002
- Posts
- 4


text:
My Nonprofit Reviews
59 profile views
1 reviews
Review for Correctional Association of New York, New York, NY, USA
I interned at CA over a decade ago when I was a college student. I learned so much about local and state politics, about coalition and collaboration, and, of course, about the criminal justice system. The work of CA matters. It is one of the rare organizations that engages in policy and advocacy, but also has hands and feet on the ground.
More Feedback
Would you volunteer for this group again?
For the time you spent, how much of an impact did you feel your work or activity had?
A lot
Did the organization use your time wisely?
Very Well
Would you recommend this group to a friend?
When was your last experience with this nonprofit?
Role: Volunteer


text: Hello all--- CruxPPC64-2.6 provides a useful 64bit chroot environment with 32bit Slackintosh-12.1. I used Crux to update my shipped g5ppc64 kernel, and then I migrated my Slackintosh install to a JFS file system. Maybe someone will find my experience useful. I know that I rebooted more than necessary, but at least nothing broke. The end result: $ uname -a Linux crackintosh 2.6.36-slackintosh #2 Mon Nov 22 22:15:33 EST 2010 ppc64 PPC970FX, altivec supported PowerMac8,1 GNU/Linux Partitioning: My computer still has the typical map, bootstrap, and HFS partitions for OS X. The rest of the hard disk has survived a few previous Linux installations. I used the CruxPPC64 installation disk to allocate the following Linux partitions: 2GB swap, 75MB /boot (ext2), 3GB for Crux root (JFS), 5GB for a temporary Slack root (ext3), 8GB for a permanent Slack root (JFS), and one more partition for /home (JFS, with all my data from a previous Linux install). CruxPPC: Installing CruxPPC is more involved than installing Slack, so follow the handbook (http://cruxppc.org/Handbook2-6#InstallingFromCD-ROM). For now, do not tell the installer about any partitions other than swap, /boot and Crux root. The critical part is compiling the new kernel. The CruxPPC64 environment is pure 64-bit, so the kernel will run on a G5 under Slackintosh. Any modules compiled under Crux will be unavailable to Slackintosh, however, so no hardware support can depend on any modules. My machine required Sungem, Phy, several filesystems, full G5 support, and plenty of crypto to be compiled directly into the kernel; others will also need SMP support. Set up yaboot as normal and reboot to test. If lsmod shows that Crux still depends on modules, it's probably worth compiling another kernel. Slackintosh, Part 1: Install Slackintosh-12.1 to the temporary ext3-formatted root as normal, but only tell the installer about the temporary Slack root partition, and skip the yaboot installation. Reboot into Crux and edit yaboot.conf to boot into the temporary Slack root using the Crux kernel; run ybin -v. Make sure that the temporary Slack installation works as expected with the Crux kernel. Slackintosh, Part 2: Follow the Zipslack FAQ (http://slackware.com/faq/do_faq.php?faq=zipslack) to migrate the root partition from the temporary Slack root to the permanent one: # mkdir /mnt/newroot # mount /dev/sdXX /mnt/newroot # umask 000 # cp -a /bin /boot /dev /etc /lib /root /sbin /tmp /usr /var /mnt/newroot/ # mkdir /mnt/newroot/proc /mnt/newroot/home Edit /mnt/newroot/etc/fstab to reflect the new root partition and correct /boot partition, and add the dangling /home partition. Either reboot into Crux, edit yaboot.conf, and run ybin -v, or copy yaboot.conf from the Crux partition and run ybin -v directly from Slackware. If everything has worked so far, then there won't be much need to boot into Crux anymore. Final notes: Boot into permanent Slackware and compute. I followed the Slackintosh-current ChangeLog to update my toolchain, and then I downloaded new kernel source and upgraded the kernel once again from inside Slack by using the Crux installation as a chroot. Most 13.1 and later SlackBuild scripts now work with no editing. I prefer my current setup to Debian or Crux, but it is not perfect. I get minor warnings whenever I su or quit screen. I would be interested if anyone else is running something similar. Cheers, -derek --


text:
Thu 2nd May at 7:00pm - 3:00am
English Sporting Defeat
@ 12 Bar Club, London
Also appearing: Eleanor Penfold
This event has been and gone!
Catch the next one:
Check out the info below for future tour dates, and track them to receive alerts when they tour near you
Never miss another 'English Sporting Defeat' show
12 Bar Club Presents: English Sporting Defeat + Eleanor Penfold
Info provided by the Event Organiser
English Sporting Defeat
This four piece band draw influences from punk, post-punk and the garage-rock revival period of Robert Scott (guitar/vocals) and Harry Pearce's (bass/vocals) shared teenage years and from Jessica Watson's (strings/keys/vocals) background in folk, film and classical music.
The trio recruited drummer Kieran Brown on the basis of his incredible reputation on local music circuits.
English Sporting Defeat tour dates
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Venue Facts
Venue: 12 Bar Club
Website: 12barclub.com
Email: ...
→ Drayton Park 7 min walk
→ Holloway Rd 3 min walk


text:
Mardi Gras is coming up Tuesday, and for many people that's a good reason to down a Hurricane or maybe an Abita beer or six. But for anyone looking for a more refined taste of New Orleans, you can't beat the Vieux Carré.
Fortunately, you can get an excellent version at the Table, Jim Stump's new restaurant in San Jose's Willow Glen neighborhood.
Vieux Carré, pronounced "voo karay," is French for "old square" and is one of the more classy nicknames for the French Quarter. The cocktail was concocted in 1938 by Walter Bergeron at New Orleans' famed Hotel Monteleone.
"It's one of my favorite cocktails," said Stephen Shelton, bar manager at the Table. "I've always wanted to put it on a menu."
Shelton got his chance after a customer requested one, and pretty soon it became a regular offering. His version is pretty traditional, with Maison Surrenne cognac, Sazerac brand rye whiskey, Benedictine, Carpano Antica sweet vermouth and a combination of Peychaud's and Angostura bitters. It's served in a thick-bottomed Old Fashioned glass, with a single, large ice cube (which takes longer to melt and, therefore, dilutes the drink less).
It's amazingly smooth for a whiskey drink, no doubt because of the herbal aromatics of the Benedictine liqueur and the influence of the cognac. It's a drink I would recommend to people who want to like the Manhattan but find it too harsh.
The Vieux Carré ($11) holds its own on the Table's cocktail menu alongside such classics as the Negroni ($8) and the Sidecar ($11). There are also drinks Shelton has developed himself, including the Temptation ($9) — a well-balanced mixture of Diplomatico Reserva Exclusiva dark rum and citrus flavors — and a Manhattan that perks things up with the addition of a five-spice syrup that's house-made. If you want to stick to the flavors of the Big Easy, there's also a Sazerac ($11) on the menu.
The restaurant fills up for dinner after 6 p.m. on a pretty regular basis, mostly Willow Glen residents out for dinner. There never seems to be a huge crowd at the small, concrete-topped bar, which is good because it seats only about 10 people. Cocktail samplers would be wise to stop by for happy hour during the week from 4 to 6 p.m. (there's also a late-night happy hour from 10 p.m. to midnight Thursdays through Saturdays). The happy hour menu has food and drink specials, including a few that aren't listed on the regular menu.
Following Stump's lead, most of the bar selections are off the beaten path. The beers on tap are mostly from California, including the terrific Scrimshaw Pilsner from Fort Bragg's North Coast Brewing and the Cat's Meow Continental Ale, made at Stump's other venture, Los Gatos Brewing Co. The wine list is sizable, too, but I decided to save that for a future visit.
While I would have been thrilled for barbecued oysters or a cup of gumbo to go with my Vieux Carré, the starters at the Table are great accompaniments. The chipotle-spiked meatballs ($10) were a tangy snack and paired well with the caramelized polenta fries ($5 on the happy hour menu), which are served as four fish-stick sized pieces.
Of course, you can always just order drinks with your dinner and enjoy the Vieux Carré on Mardi Gras or any other night of the week.
Details: The Table, 1110 Willow St., San Jose, www.thetablesj.com; 408-638-7911.
Open until 10 p.m. Monday through Wednesday, until midnight Thursday through Saturday, and until 9 p.m. Sunday.


text:
Theater Department Presents Oleanna June 27-29June 24, 1997
The Department of Theater Arts at Baylor University will present the provocative play Oleanna June 27-29 in the Mabee Theater at the Hooper-Schaefer Fine Arts Building. Curtain times are 7:30 p.m. June 27-28 and 2:30 p.m. June 29.
Written in 1992 by David Mamet, Oleanna focuses on the relationship between Carol, a young woman who has returned to college, and John, a professor. The play deals with many of the aspects of political correctness from gender relations to sexual harassment.
"The young woman goes to a professor's office because she is failing the class," said Patricia Cook, professor of theater arts and director of the Baylor production. "Something happens to her in that office, but if you want to find out what, you must come see the play.
"This is a very powerful and volatile play. It makes people in the audience angry and points out that in the world of political correctness, nobody wins. This play proves that human relations can't be legalized."
Oleanna features Stanley Denman, lecturer in theater arts, in the role of John and Christine Henry, a graduate student in the theater department, in the role of Carol.
Tickets for the play are $6 and can be purchased from 9 a.m. to 4 p.m. at the Hooper-Schaefer box office. For more information, contact Cook at 755-1861.
More News ...


text:
- Photo
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- View Full Specifications
Available now
- Year:
- 1991
- Length:
- 40'
- Engine/Fuel Type:
- Twin / diesel
- Located In:
- Hull Material:
- Fiberglass
- 70074-2436576
- Current Price:
This Monte Carlo 40 Express is the epitome of an Express Cruiser and/or an ideal tender for a large yacht. The Monte Carlo 40 is powered by fresh 550HP Detroit Diesel 6V92TAs with only 50 hours since major overhaul(SMOH), cruises at 30+ knots and has a top speed of 38 knots.
Her cabin layout includes a large center line queen stateroom forward, double lavatories amidships (one with shower & sink - one with head & bidet), comfortable salon with convertible dinette starboard and a compact galley to port.
The air-conditioned cockpit accommodates eight and has two drop-down bolstered helm seats, a rear seat for three and a port side seat for three. There is also a built in refrigerator and a separate sound system with amplifier for cockpit and sun pad entertaining.
The helm is loaded with updated electronics, Her electrical systems are designed for both US and European standards and she is the ultimate fast express cruiser ready to be delivered to the port of your choice.
Her current owner is truly motivated to sell and will consider a trade ... so bring on an offer.
For more information or to schedule a viewing please contact JIM CLARK at (727)692-1712 -email@example.com
You can also contact Bob Fillingham at toll free (888)830-1421 or (727) 460-5687 or Shar Fillingham at (727)403-0520 or visit our website, www.fillinghamyachts.com.
Certified on-the-water, at-the-helm powerboat training available. Go to our website for details.
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Additional Specs, Equipment and Information:
Builder: Offshore Marine
Designer: Syntheis
LOA: 40 ft 0 in
Beam: 12 ft 0 in
Maximum Draft: 2 ft 10 in
Dry Weight: 23150 lbs
Total Power: 1100 HP
Engine 1:
Engine Brand: Detroit Diesel
Engine Model: 6V92TA
Engine Type: Inboard
Engine/Fuel Type: Diesel
Engine Hours: 50
Drive Type: Direct Drive
Engine Power: 550 HP
Engine 2:
Engine Brand: Detroit Diesel
Engine Model: 6V92TA
Engine Type: Inboard
Engine/Fuel Type: Diesel
Engine Hours: 50
Drive Type: Direct Drive
Engine Power: 550 HP
Cruising Speed: 39 mph
Maximum Speed: 43 mph
Fresh Water Tanks: (75 Gallons)
Fuel Tanks: (450 Gallons)
Holding Tanks: (25 Gallons)
Number of double berths: 1
Number of cabins: 1
Number of heads: 1
Outside Equipment/Extras
Electric windlass
Displacement: 23150 dry
Total Power: 1100
Fuel: 450
Fresh Water: 75
Holding: 25
The Monte Carlo 40 Express with an air conditioned cockpit that seats eight, and a interior that sleeps four. The master stateroom is forward with a center line queen and the plush salon has a convertible dinette starboard.
- Cruisair Air Conditioning
- Hot Water Heater
- Princess (2) burner stove
- Kenmore Microwave
- Vitri Refrigerator / Freezer
- Pressurized Water System
- SS Sink
- Furuno 1610CF GPS Chartplotter
- Furuno 851 Radar
- Northstar 951X GPS
- Rudder Angle Indicator
- Autohelm Depth Sounder
- Cetrek Auto-Pilot
- Shipmate VHS
- Whitestar Compass
- Alpine AM/FM/Cassette/CD (Salon)
- Sony TV
- Optimax Amplifier
- Bose & Pioneer Speakers (cockpit)
- ONAN 6.5KW Generator
- 6 Batteries w/ parallel switch
- 12V DC - 24V DC - 220V AC Systems
- Battery Charger
- Twin 550HP Detroit Diesel 6V92TA (50Hrs.since major overhaul)
- ARNESON Rocker Plate Trim Tabs
Hull & deck are predominantly white with blue and red feature stripes. The hull is a deep-vee configuration with a triple stepped bottom.
- Life Raft in stern compartment
- Lofrans Windlass w/ remote
- Danforth Anchor & rode
- Powered rear boarding ladder (Mediterranean style)
- Swim Platform
- Stainless Steel Ports Throughout
- Fully enclosed Air Conditioned Helm & Cockpit
- Fresh Water Wash down
- Built-in fender compartment inside gunwales
- Fenders - Lines
- Powered rear swim ladder with hand rails
- Bimini & Sun Pads for aft deck
- COAST GUARD APPROVED Jackets & Life Rings
The Company offers the details of this used boat or used sail boat for sale in good faith but cannot guarantee or warrant the accuracy of this information nor warrant the condition of the boat or yacht. A buyer should instruct his agents, or his surveyors, to investigate such details as the buyer desires validated. This boat is offered subject to prior sale, price change, or withdrawal without notice.
Visit Website


text: My name is Joseph Youngs but everyone calls me José. I'm a die hard Red Sox fan and I was born and raised in Warwick, Rhode Island. I am well versed in social media and marketing technology, tools and trends. Multi-faceted and creative professional with audio, video, Microsoft word and web content management experience. Excellent problem solving skills. Organized, efficient and absolutely willing to go the extra mile for on time delivery.


text:
Police say a Chinese employee of Foxconn Technology Group has jumped to his death from a company-rented apartment building in southwest China's Sichuan province.
Foxconn, the world's largest contract maker of electronics including iPhones, has been besieged with reports of poor working and living conditions for its workers at factories and dorms. At least a dozen Foxconn employees have jumped to their deaths or tried to do so since 2010.
A public security bureau in Chengdu city announced the death Wednesday night on a popular Chinese microblogging site. The post identifies the employee only by the surname Xie and says the deadly jump occurred Wednesday afternoon.
Police say the cause is under investigation.
China's official Xinhua News Agency reported Thursday that the man was 23 years old.


text: Home » » IMG_1108 IMG_1108 January 07.2017. 0 Comment lulzsalad Please follow and like us: Tweet About Author lulzsalad A mixed bag of observations, commentary, information and various sundries from the wildly diverse set of topics, experience and interests which, taken collectively, account for my mind. There will also be recipes. Leave a Reply Cancel reply


text:
Hedgehog signaling through its receptor, Patched, activates transcription of genes, including Patched, that regulate the fate of various progenitors. Although Hedgehog signaling is required for endodermal commitment and hepatogenesis, the possibility that it regulates liver turnover in adults had not been considered because mature liver epithelial cells lack Hedgehog signaling. Herein, we show that this pathway is essential throughout life for maintaining hepatic progenitors. Patched-expressing cells have been identified among endodermally lineage-restricted, murine embryonic stem cells as well as in livers of fetal and adult Ptc-lacZ mice. An adult-derived, murine hepatic progenitor cell line expresses Patched, and Hedgehog-responsive cells exist in stem cell compartments of fetal and adult human livers. In both species, manipulation of Hedgehog activity influences hepatic progenitor cell survival. Therefore, Hedgehog signaling is conserved in hepatic progenitors from fetal development through adulthood and may be a new therapeutic target in patients with liver damage.
- Indian hedgehog
- liver
- Patched
- progenitor cell
- Sonic hedgehog
adult tissues routinely turnover, and this tissue renewal involves cells that span a wide spectrum of differentiation. Hyperplastic growth by diploid cells and hypertrophic growth by polyploid cells play important roles in this process (34, 46, 49). Ultimately, tissue renewal is fueled by cells of the stem cell compartment. Stem cell compartments have long been known to exist in adult skin and the gastrointestinal tract (10, 11) and have been assumed to exist in fetal, but not adult, livers (70). In adults, hepatic progenitors are thought to contribute to liver regeneration and turnover only when there is a significant loss of mature liver cells or when replication in mature hepatocytes is blocked (17, 69). Under these circumstances, the presence of a hepatic stem cell compartment becomes evident. This compartment is characterized by the appearance of hepatic progenitors, which are small cells (<10–12 μm in diameter) that coexpress hepatocytic and biliary antigens and accumulate in the periportal zone of the liver (20). The existence of a hepatic stem cell compartment in adult liver tissue has been supported further by the characterization of the antigenic and biochemical profiles of some of the hepatic progenitor populations in murine and human liver models (1, 49, 60, 65). Moreover, the Canals of Hering have been identified as the anatomic structures in which these progenitors localize in adult livers (62). In recent years, it has become apparent that subpopulations of precursors from extrahepatic sites can also acquire a hepatic fate (44, 61). However, a subsequent study (21) has questioned the contribution of such cells to liver regeneration in all but unusually severe liver injury. Thus consensus is emerging that in most settings, resident hepatic progenitors are a greater source of replacement hepatocytes than extrahepatic precursors (66). As in the skin and gut, chronic injury or oncogenic insults in the liver increase the local accumulation of progenitors (51, 69). The latter observations suggest that these cells play a greater role in tissue renewal after hepatic damage than in the normal, healthy liver.
To optimize progenitor cell participation in liver regeneration, it is necessary to understand the mechanisms that regulate their fate. One strategy to identify such mechanisms is to utilize knowledge gained from studying the hepatic specification of endodermal progenitors during embryogenesis. During gastrulation, Indian hedgehog (IHH) ligand is expressed in cells undergoing differentiation to become visceral endoderm (5, 19). Thereafter, endodermal cells that are destined to form the liver bud begin to express the DNA binding protein, GATA-4 (32). Some of these multipotent progenitors also express Patched (PTC), a receptor that engages ligands of the Hedgehog (Hh) family (57). Interaction between IHH or Sonic hedgehog (SHH) ligands and PTC activates Hh signaling and promotes the proliferation and/or differentiation of hepatic progenitor populations that ultimately form the liver bud (15). As the liver matures, Hh signaling abates in mature hepatocytes and cholangiocytes. Ultimately, most of the cells in adult livers do not express Ptc, a transcriptional target of the Hh pathway (6). These differentiation-dependent variations in the expression of Hh signaling components during liver development suggested to us that this pathway might identify and regulate a subset of residual, relatively primitive, intrahepatic progenitors in adult livers.
The purpose of the present study was to determine if murine and human liver precursor populations harbor such a reservoir of Ptc-expressing cells spanning from the time of endodermal differentiation to adulthood. In addition, we wanted to characterize the Ptc-expressing populations and determine if regulation of Hh signaling altered the viability of progenitors from fetal and adult livers.
Herein, we demonstrate that the Hh pathway regulates liver progenitors and that this process is conserved within the liver from embryogenesis through adulthood. Blockade of Hh activity in such liver progenitors decreases their survival, whereas stimulation of Hh activity inhibits endogenous apoptotic activity in these cells. Because Hh signaling is required for the optimal viability of liver progenitors throughout life, this pathway is likely to modulate hepatic epithelial renewal in health and disease. Therefore, our work identifies Hh pathway components as novel therapeutic targets in chronic liver disease.
Animal care.
Adult male C57BL/6 mice were obtained from Jackson Laboratory (Bar Harbor, ME) and housed in a barrier facility. Adult male Ptc-lacZ reporter mice were obtained from Dr. P. A. Beachy (Johns Hopkins University; Baltimore, MD), and mouse embryos acquired from pregnant dams at 11.5 days postconception were obtained from Dr. E. Meyers (Duke University Medical Center; Durham, NC) (18). All animal experiments fulfilled National Institutes of Health, Johns Hopkins University, Duke University, and University of North Carolina (Chapel Hill, NC) requirements for humane animal care.
Embryonic stem cell culture and endodermal lineage restriction.
Murine embryonic stem (ES) cells were cultured and endodermally lineage restricted as previously described (16). Media were changed daily, and cells were allowed to grow for 7 days before RNA isolation.
Culture of murine cell lines.
A murine hepatic progenitor cell line (OV) was kindly provided by Dr. B. E. Petersen (University of Florida; Gainesville, FL) and cultured as previously described (44, 45). A well-differentiated murine hepatocytic cell line (AML-12, Hep) was obtained from the American Type Culture Collection (ATCC; Manassas, VA) and cultured according to ATCC instructions.
Two-step real-time RT-PCR.
Total RNA was extracted from cells with the RNeasy kit followed by RNase-free DNase I treatment (Qiagen; Valencia, CA). Primers were designed using Genbank sequences or as previously described (Table 1) (6, 25, 26, 45, 54). Amplicon products were separated by electrophoresis on a 2.0% agarose gel buffered with 0.5× Tris-borate-EDTA (TBE). Products were visualized using an AlphaImager 3400 Gel Analysis System (Alpha Innotech; San Leandro, CA) with ultraviolet transillumination and were recorded using a 12-bit charge-coupled device camera that was controlled by AlphaEaseFC 4.0.1 software (Alpha Innotech). For each experiment, total RNA was reverse transcribed to cDNA templates and amplified using Ready-To-Go You-Prime First-Strand Beads (Amersham Biosciences; Piscataway, NJ) with pd(N)6 first-strand cDNA primers (Amersham Biosciences). For quantitative RT-PCR, 1.5% of the first-strand reaction was amplified using iQ-SYBR Green Supermix (Bio-Rad; Hercules, CA), an iCycler iQ Real-Time Detection System (Bio-Rad), and specific oligonucleotide primers for target sequences as well as the β-glucuronidase housekeeping gene. The PCR parameters were as follows: denaturing at 95°C for 3 min followed by 40 cycles of denaturing at 95°C for 15 s and annealing-extension at the optimal primer temperatures for 45–60 s. Threshold cycles (Ct) were automatically calculated by the iCycler iQ Real-Time Detection System. Target gene levels in the treated cells are presented as a ratio to levels detected in the corresponding control cells according to the ΔΔCt method as previously reported (35). These fold changes were determined using point and interval estimates.
Patched-lacZ staining and reporter assay.
We studied mice in which one allele of Ptc is replaced in-frame with the β-galactosidase gene by homologous recombination to evaluate Hh signaling in the liver. Because Ptc is a transcriptional target of Hh signaling, expression of β-galactosidase (lacZ) indicates activation of the Hh pathway (18, 58). Staining and quantification of gene expression were performed as previously described using the β-Galactosidase Detection Kit (Promega; Madison, WI) (67). Images were visualized using an Axiovert 200 microscope (Carl Zeiss) at ×10–100 with halogen illumination and were recorded using an AxioCam HRC camera (Carl Zeiss) that was controlled by Axiovision 4.4 software (Carl Zeiss).
Immunofluorescent staining of mouse livers.
Sections of formalin-fixed, paraffin-embedded mouse livers (7 μm thick) were used for immunofluorescent staining. Slides were deparaffinized in xylene and serially rehydrated in sequential ethanol (100–70%). Epitope retrieval was performed by microwave-incubating slides in 10 mM sodium citrate (pH 6.0)-0.05% Tween 20 for 10 min. Slides were then rinsed twice in PBS-0.05% Tween 20 for 5 min. Sections were then blocked with 2% goat and donkey serum for 10 min followed by incubation with primary antibodies for 45 min at room temperature. The primary antibodies were mouse monoclonal anti-β-galactosidase IgG2a(k) (1:400, Promega) (23, 47, 71) and rabbit polyclonal anti-human callus cytokeratin antibody (1:200, DakoCytomation; Carpinteria, CA) (48). The secondary antibodies were Alexa 568-conjugated goat anti-mouse IgG (1:75, Molecular Probes; Carlsbad, CA) and FITC-conjugated donkey anti-rabbit IgG (1:50, Jackson ImmunoResearch Laboratories; West Grove, PA). Slides were coverslipped after the completion of staining. Negative controls were performed by omitting the primary antibody from the protocol and revealed minimal background staining (data not shown). Images were visualized using an Axiovert 200 microscope (Carl Zeiss) at ×63–100 magnification with epifluorescent illumination.
Isolation of murine liver cells.
The nonparenchymal cell (NPC) fraction was isolated by in situ portal vein perfusion with pronase-collagenase (Sigma Aldrich; St. Louis, MO) to destroy mature hepatocytes and release NPCs from the liver matrix (66). NPCs were collected by density gradient centrifugation through OptiPrep (Accurate Chemical), which separates NPCs from nonviable cells and other liver cell populations. Cell fractions were pooled from two to six mice.
Sourcing of human liver cells.
Human liver tissues were obtained following approval by the Institutional Review Board for Human Studies at the University of North Carolina. Prenatal livers were obtained from fetuses at 16–20 wk gestational age (Advanced Biological Resources; San Francisco, CA). Donated postnatal livers, not suitable for orthotopic liver transplantation, were obtained from federally designated organ procurement organizations.
Processing of human livers.
Human fetal livers were processed as previously described (36) while cells from postnatal human livers were isolated by enzymatic digestion as previously described (9). The freshly isolated cell suspensions were subjected to immunoselection for cells expressing epithelial cell adhesion molecule (EpCAM), which has been previously noted to be one of the surface markers identifying hepatic progenitor cells in human livers (14, 49) (U.S. Patent Appl. 20050148072 and 20020182188. Human Liver Progenitors. L. Reid et al. 2002.). Immunoselection was performed using monoclonal anti-EpCAM antibody coupled to magnetic microbeads according to the manufacturer's instructions (Miltenyi Biotec; Auburn, CA).
Cytospins of freshly isolated, immunofluorescently stained human hepatic progenitors.
After enzymatic digestion of fetal livers, 106 freshly isolated cells were centrifuged at 600 g for 5 min at 4°C. Cells were then resuspended in PBS and 4% paraformaldehyde (1:1). After fixation for 10 min at room temperature, cells were centrifuged as previously described. The supernatant was aspirated, and cells were then blocked in PBS with 2% Triton X-100 (Sigma Aldrich), 10% goat or donkey serum (depending on the secondary antibody), and 2% teleostean gelatin (Sigma Aldrich). Cells were then incubated for 1 h at room temperature and then centrifuged as described above. The primary antibodies were employed for 100 min at room temperature as follows: mouse anti-human EpCAM (1:250, Lab Vision; Fremont, CA), goat anti-SHH (1:50, Santa Cruz Biotechnology; Santa Cruz, CA), rabbit anti-IHH (1:100, Santa Cruz Biotechnology), and rabbit anti-PTC (1:100, Santa Cruz Biotechnology). Cells were then washed twice in PBS-2% Triton X-100. The appropriate secondary antibodies were employed for 60 min at room temperature with 4′6-diamidino-2-phenylindole (DAPI) as follows: Alexa 488-conjugated goat anti-rabbit IgG (1:1,000, Molecular Probes), Alexa 568-conjugated goat anti-mouse IgG (1:1,000, Molecular Probes), Texas red-conjugated donkey anti-mouse IgG1 (1:500, Molecular Probes), and Alexa 488-conjugated donkey anti-goat IgG (1:1,000, Molecular Probes). Cells were then washed twice in PBS-2% Triton X-100, centrifuged, resuspended in blocking buffer, and cytospun at 800 rpm for 3 min using precoated cytoslides (Shandon Anatomical Pathology; Pittsburgh, PA). Cells were then mounted in aqueous glycerol gelatine mounting media (Sigma Aldrich) and coverslipped. Images were visualized using a Leica SP2 AOBS laser scanning confocal microscope (Leica Microsystems; Bannockburn, IL) at ×189 that was controlled by Leica SP2 TCS software (Leica Microsystems). Negative controls were performed by omitting the primary antibody from the protocol and revealed no staining (data not shown).
Human liver immunohistochemistry.
Serial sections of formalin-fixed, paraffin-embedded human livers (10 μm thick) were used for immunohistochemical staining. Sections were incubated twice in PBS with 0.3% Triton X-100 (Sigma Aldrich) for 10 min and then in 0.3% hydrogen peroxide solution to block endogenous peroxidase activity. Sections were then blocked with serum and treated with an avidin-biotin blocking kit (Vector Laboratories; Burlingame, CA) to inhibit nonspecific binding in the tissue. Sections were incubated with primary antibody overnight at 4°C. Protease digestion using the Auto/Zyme Reagent Set (Biomeda; Foster City, CA) was employed for cytokeratin-19 (CK-19) staining before blockade with serum. The primary antibodies employed were as follows: mouse anti-human CK-19 (1:100, Novocastra Laboratories; Newcastle, UK), mouse anti-human EpCAM (1:200, Miltenyi Biotec), rabbit anti-IHH (1:100, Santa Cruz Biotechnology), and goat anti-SHH (1:50, Antibodies Incorporated; Davis, CA). Sections were then incubated with biotinylated secondary antibody and ABC reagents of the Vectastain Elite Universal ABC kit according to the manufacturer's instructions (Vector Laboratories). The secondary antibody was then detected using the avidin-biotin-peroxidase method with 3,3′-diaminobenzidine as the substrate (Vector Laboratories). For each antibody, negative controls were performed by omitting the primary antibody and/or using isotype control antibodies. These controls revealed no or minimal background staining (data not shown).
Colonies of human hepatic progenitors.
After immunoselection, EpCAM+ cells from human fetal livers were plated at densities of 103–104 cells/100-mm2 plate in serum-free media tailored for hepatic progenitors and referred to as "Kubota's medium" (29, 36). These clonogenically derived colonies expand for weeks at the rate of approximately 1 division/day and coexpress EpCAM, CK-19, telomerase, and other markers of human hepatic progenitors (L. M. Reid and E. Schmelzer, unpublished observations) (49).
Immunofluorescent staining of human hepatic progenitor colonies.
After 2–3 wk of culture, the medium was removed from the colonies of human hepatic progenitors. Adherent cells were rinsed three times with Hank's balanced salt solution (HBSS). Cells were then fixed with fresh acetone-methanol (1:1) for 2 h at room temperature. Fixed cells were then rinsed three times with HBSS and were then incubated with 20% goat serum in HBSS for 4–6 h at 4°C. Cells were then rinsed three times with HBSS before incubation with primary antibody for 6–12 h at 4°C. The primary antibodies employed were as follows: goat anti-SHH (1:100, Antibodies Incorporated), rabbit anti-IHH (1:100, Santa Cruz Biotechnology), and goat anti-PTC (1:100, Santa Cruz Biotechnology). After incubation with primary antibody, cells were rinsed three times with HBSS, and secondary antibody was added for 75 min at room temperature. The appropriate secondary antibodies were employed as follows: Alexa 488-conjugated donkey anti-goat IgG or phycoerythrin-conjugated goat anti-rabbit IgG (1:800, Molecular Probes). Cells were then rinsed three times with HBSS and preserved with 2% formaldehyde in HBSS. Images were visualized using an Olympus IX70 microscope (Olympus Imaging America; Melville, NY) at ×4–10 with epifluorescent illumination and were recorded using a Nikon BM-2 camera (Nikon Instruments; Melville, NY) that was controlled by Nikon View 6.0.1 software (Nikon Instruments). Negative controls were performed by omitting the primary antibodies from the protocol and revealed no or minimal background staining (data not shown).
Pharmacological regulation of Hh signaling.
Colonies of human hepatic progenitor cells and the murine hepatic progenitor cell line were treated with regulators of Hh signaling. Human hepatic progenitor cell colonies were treated with cyclopamine (10 μM, Calbiochem; San Diego, CA) (7, 8, 22, 28). The murine hepatic progenitor cell line was treated with recombinant Shh-N ligand (10–20 nM, Stem Cell Technologies) (6).
Cell death assays.
Three- to four-week-old colonies of human hepatic progenitors from three different fetal livers were cultured as previously described. Imaging of the colonies was performed before treatment with cyclopamine (Calbiochem) and every 24 h after treatment. After 48 h, control and treated colonies were stained using Vybrant Apoptosis Assay Kit No. 8 (Molecular Probes) according to the manufacturer's instructions. Briefly, colonies were washed twice with binding buffer and incubated for 15 min at 37°C with R-phycoerythrin annexin V (1:50) and SYTOX green (0.01 μM) diluted in binding buffer. Colonies were then washed twice, and fluorescent microscopy was performed. Numbers of dead cells stained with SYTOX green (i.e., necrotic cells) or R-phycoerythrin annexin V (i.e., apoptotic cells) were assessed by morphometric analysis with the MetaMorph Imaging System (Molecular Devices; Downington, PA). Immunofluorescently stained cells were quantitated in four representative fields of figures under ×10 magnification. For the murine hepatic progenitor cell line, apoptotic activity was measured with an Apo-ONE Homogeneous Caspase-3/7 Apoptosis Assay (Promega) according to manufacturer's instructions (56, 59). Briefly, the murine cell line was cultured for 24 h and then treated with Shh-N-supplemented medium or control medium for 48 h. In these replicate experiments (n = 4), fluorescence measurements were then performed using a FLUOstar OPTIMA microplate reader (BMG Labtech; Durham, NC).
Statistical analysis.
Comparisons between groups were made using Intercooled Stata 8.0 (Stata; College Station, TX). For replicate experiments, data are reported as means ± SD. Comparisons between groups were performed used the Student's t-test for real-time RT-PCR analyses; changes in target mRNA levels are represented by using point and interval estimates. Significance was accepted at the 5% level.
Hh ligands and receptors are differentially expressed in ES cells and mature hepatocytes.
ES cells are pluripotent and can be propagated indefinitely in culture (30). ES cells require Hh signaling for differentiation into ectodermal (37, 38) and endodermal (37, 55) tissues. Recently, it has been reported that mouse ES cells treated for 7 days with acidic fibroblast growth factor can be differentiated into cells that are lineage restricted to the endoderm (16). When such cells were injected into a mouse model of hemophilia B that lacked clotting factor IX, these putative endodermal precursor cells engrafted within the livers and acquired hepatocyte functions, including hepatocyte-specific production of clotting factor IX, and the latter corrected hemophilia in recipient mice. In the present study, we evaluated murine ES cells and ES-derived putative endodermal precursors for expression of Hh ligands and receptors. Mouse ES cells expressed Hh ligands (Shh and Ihh) and the Hh receptor Ptc both initially (ES day 0) and when cultured under conditions that promote endodermal lineage restriction (ES day 7; Fig. 1A). Interestingly, as the ES cells differentiated toward an endodermal lineage, Shh expression fell, Ihh expression increased (Fig. 1B), and Ptc expression declined slightly (Fig. 1C). This is consistent with earlier reports (5, 19) showing that Ihh is expressed in the visceral endoderm of ES cell embryoid bodies and that Ihh is involved in visceral endodermal differentiation. In contrast to undifferentiated ES cells and endodermal progenitors, the well-differentiated hepatocytic cell line AML-12 did not express Ptc but continued to produce Hh ligands (Fig. 1). Therefore, well-differentiated hepatocytic cells did not exhibit evidence of active Hh signaling, whereas undifferentiated and endodermally lineage-restricted ES cells transcribed mRNA for the Hh receptor and gene target Ptc (57).
Hh-responsive cells exist in pre- and postnatal murine livers.
Ptc-lacZ mice, which carry a transgene encoding β-galactosidase driven by Hh-responsive elements in the Ptc promoter, have been used to identify cellular Hh activity (18, 43). To more definitively localize Hh-responsive hepatic progenitors in fetal livers, we examined whole mounts of Ptc-lacZ mouse embryos at 11.5 days postconception, which roughly corresponds to 16 wk of gestational age in human prenatal development. At this time point, the developing neural tube, notochord, and muscularis propria of the stomach had β-galactosidase-expressing cells, as expected (data not shown). Small cells interspersed between unstained fetal liver cells also demonstrated reporter activity (Fig. 2A). Thus, in late-gestational mouse embryos, Ptc is expressed by neural tissue, cells in the muscularis propria of the gut, and cells lining the hepatic sinusoids, but not by more mature epithelial cells of the stomach and liver.
We then examined four healthy Ptc-lacZ mice (10–14 wk old) to determine if, like fetal livers, adult livers harbored cells that expressed Ptc. As expected, the gallbladder wall from these mice had numerous β-galactosidase-positive cells (data not shown). Consistent with an earlier report (6), as well as our RT-PCR analysis of the hepatocytic cell line (Fig. 1A), Hh pathway activity was not detected in mature hepatocytes or cholangiocytes at ×10 magnification (Fig. 2B) or ×100 magnification (Fig. 2B, inset). However, at ×100 magnification, we found rare β-galactosidase-positive cells in the periportal zone of adult livers (Fig. 2C). These cells were much smaller in size than adjacent mature hepatocytes. The location and size of the Ptc-expressing cells suggested to us that, like hepatic progenitors, these small cells may reside among the hepatic NPC fraction. To verify this suspicion, we perfused the livers of two additional adult Ptc-lacZ mice and isolated the hepatocyte and NPC populations. Protein extracted from the NPC fraction exhibited β-galactosidase activity more than 45,000-fold higher than the hepatocyte fraction (P < 0.001; Fig. 2D), suggesting that the Hh-responsive cells in the periportal zone (Fig. 2C) coisolate with liver progenitors that reside within the NPC fraction of adult livers.
To determine if these Ptc-expressing cells colocalized with cells expressing a hepatic progenitor marker in the adult liver, we performed immunofluorescent staining using anti-β-galactosidase antibody to mark Ptc-positive cells and polyclonal anti-cytokeratin antibody (48) to mark committed, bipotential hepatic progenitors. β-Galactosidase-positive cells were heterogeneous in nature (i.e., some, but not all, were pancytokeratin positive by immunohistochemistry). As previously noted, β-galactosidase-positive cells were mostly located in the periportal zones of the liver. Some were "periductal null" cells (31) (i.e., ductular-like cells separate from the biliary tree) and others were within bile ductules (40, 41, 62) (Fig. 2E). Rare clusters of small hepatocyte-like cells that coexpressed β-galactosidase and cytokeratins were also observed within the hepatic parenchyma (Fig. 2F).
Fetal human hepatic progenitors secrete Hh ligands.
Although Ihh is known to regulate ES cell differentiation into the endoderm (19) and Shh is known to play an essential role in the hepatic specification of embryonic endodermal progenitors (15), the specific identity, phenotype, and tissue localization of Hh-secreting cells in the fetal liver are unknown. To clarify these issues, we used immunohistochemistry to examine fetal human livers. Our results demonstrated that IHH (Fig. 3A) but not SHH (data not shown) was expressed in the stem cell compartments of fetal livers (e.g., ductal plate and adjacent hepatoblasts) (33, 62). Furthermore, these IHH-secreting cells colocalized with multiple markers of hepatic epithelial progenitors, namely, CK-19 (Fig. 3B) (12, 13), EpCAM (Fig. 3C) (14), and α-fetoprotein (AFP; data not shown), suggesting that progenitors identified in the stem cell compartments of human livers express IHH but not SHH. These findings in human fetuses complement and extend our findings from fetal and adult mouse livers and together demonstrate that Hh regulation of hepatic progenitors is conserved across vertebrate species.
Hepatic progenitors from human livers express components of the Hh pathway and are Hh responsive in vitro.
To further investigate the significance of our findings showing that Hh ligands and receptors are expressed in pluripotent ES cells, endodermally lineage-restricted ES cells, and resident hepatic progenitors within fetal and adult livers, the relative mRNA expression pattern of Hh pathway components was compared between freshly immunoselected human liver cells lacking EpCAM expression (i.e., mature hepatocytes and nonepithelial liver cells) and EpCAM+ cells (i.e., hepatic progenitors) (2, 14) isolated from the livers of several (n = 8) young and old individuals (age range: 2 days–58 yr). In contrast to mature liver cells (EpCAM− fraction), EpCAM+ progenitor fractions were consistently enriched with Ihh (P < 0.05) and Ptc (P < 0.05) transcripts (Fig. 4, A and B), whereas both fractions had similar expression levels of Shh. Although it appeared that the progenitor cells tended to have more Ptc expression, EpCAM− fractions also contained cells that expressed Ptc transcripts. This may be explained by the occassional failure rate of EpCAM immunoselection (L. M. Reid and E. Schmelzer, unpublished observations) as well as by recent evidence showing that hepatic stellate cells, EpCAM− liver mesenchymal cells, also express components of the Hh pathway (54). To determine if, in addition to expressing mRNA for Hh pathway components, EpCAM+ progenitor cells produce SHH, IHH, and PTC proteins, we performed immuofluorescent staining to colocalize these proteins and EpCAM. We found that freshly isolated cytospins of EpCAM+ cells also expressed all three Hh components (Fig. 4, C–N).
To corroborate our findings showing that freshly isolated cytospins of liver cells include cells that coexpress EpCAM and Hh pathway components, clonally derived colonies of EpCAM+ cells were established in culture for 2–3 wk to eliminate contaminating cells, and immunofluorescent staining was performed for Hh ligands and PTC (n = 2 plates of colonies/primary antibody). Staining demonstrated colony expression of SHH (Fig. 5A), IHH (Fig. 5B), and PTC (Fig. 5, C and D). SHH was most strongly expressed in cells along the edge of the colony and colocalized with areas that most strongly expressed PTC. IHH expression was fairly uniform throughout the colonies.
Next, we evaluated the effect of treating EpCAM+ colonies with cyclopamine, a potent and highly specific blocker of Hh signaling (8). Upon initiation of the experiment, representative colonies from the control group (n = 3 plates of colonies; Fig. 5E) and the treatment group (n = 3 plates of colonies; Fig. 5I) appeared morphologically similar. However, after 2 days of treatment with control medium or 10 μM cyclopamine-supplemented medium (7, 28), only cyclopamine-treated colonies (Fig. 5J) developed large bare fields within their centers as compared to control colonies (Fig. 5F). Therefore, cyclopamine significantly inhibited the viability of EpCAM+ cells in vitro. To determine if this effect was due to cellular necrosis or apoptosis, fluorescent staining was performed on the same colonies. In the control colonies, there was minimal cellular necrosis (Fig. 5G) or apoptosis (Fig. 5H). In contrast, the cyclopamine-treated colonies had a slight increase in the number of necrotic cells (Fig. 5K) and substantially higher numbers of cells undergoing apoptosis (Fig. 5L). Cyclopamine-induced cell death was morphometrically quantified and demonstrated a twofold increase in cell necrosis (P < 0.05) and a threefold increase in apoptosis (P < 0.01; Fig. 5M). Together, these findings demonstrated that Hh signaling modulated the apoptosis of human hepatic progenitors and that Hh activity was necessary for optimal viability of these cells in culture.
Murine hepatic progenitors are regulated by Hh signaling in vitro.
To verify the possibility that Hh activity regulated hepatic progenitor cell viability, we examined a murine hepatic progenitor cell line (OV) (44, 45). This murine progenitor line strongly expressed embryonic (muscle) pyruvate kinase (Mpk), Afp, and Ck-19, all markers of hepatic progenitors (64) (Fig. 6A). These committed progenitors also expressed Ihh and Ptc but not Shh (Fig. 6B). The latter finding was consistent with our earlier data demonstrating that IHH is present in human hepatic progenitors. In addition, the expression of Ptc was >35,000 fold higher than in our well-differentiated hepatocytic cell line (P < 0.01; Fig. 6C) but only 4.5% of the level in endodermally lineage-restricted ES cells, suggesting that there is a progressive decrease in Ptc gene expression with differentiation.
In our earlier experiments using colonies of human EpCAM+ cells, we demonstrated that Hh signaling regulated the fate of hepatic progenitors and is required for optimal viability. Recently, another group (63) demonstrated that the binding of SHH ligand to PTC prevented the induction of apoptosis mediated by caspase-3, and to a lesser extent caspase-7, in neuroepithelial cells. Therefore, we investigated if treating our murine hepatic progenitor cell line with recombinant Shh-N ligand would decrease caspase-3/7 activity, an indicator of cellular apoptosis. As expected, the presence of supplemental Hh ligand significantly decreased endogenous caspase activity in a dose-dependent fashion (P < 0.05; Fig. 6D). Therefore, as in neuroepithelial cells, viability was optimized by the addition of SHH to Ptc-expressing murine hepatic progenitors.
Hh signaling, long known to be important in endodermal differentiation (5, 19) and liver organogenesis (15), has been found to persist in the maintenance of hepatic progenitors throughout life. Hepatic progenitors freshly isolated from human fetal, infant, and adult livers express Hh pathway components, as do ES cells lineage restricted toward an endodermal fate and a well-characterized murine hepatic progenitor cell line. Furthermore, both human and mouse liver progenitors depend on Hh signaling for optimal viability in culture. Our studies localized cells that secrete Hh ligands to the same stem cell compartments that harbor Hh-responsive cells in fetal and adult livers. The comparison of pluripotent ES cells, endodermally lineage-restricted ES cells, and hepatic progenitors with mature hepatocytic epithelial cells suggests that Hh activity is extinguished as progenitors differentiate toward mature liver parenchymal cells. Therefore, although mature liver epithelial cells lack Hh pathway function, resident hepatic progenitors throughout life retain Hh activity and express components of this well-known fetal morphogenic pathway.
To our knowledge, this is the first evidence that, like the skin (10), intestine (11), and bone marrow (3), the adult liver possesses a progenitor compartment that is regulated by Hh signaling. Previous work by Zaret and colleagues (15) has established the requirement for Hh signaling during hepatogenesis. The present work demonstrates simultaneous expression of Hh ligands and the Hh receptor in cells within the hepatic progenitor compartment, suggesting that liver progenitors maintain their viability via autocrine and/or paracrine Hh signaling throughout fetal and adult life. Similar localized progenitor compartments that depend on Hh signaling have been described in the brain (42) and the foregut (68). In those tissues, PTC and Hh ligands cooperate to maintain a population of transiently amplifying Hh-responsive progenitors. Because PTC is the only known receptor for Hh ligands, PTC expression marks cells that retain sensitivity to SHH. However, PTC is a double-edged sword (Fig. 7). When it is unoccupied by the ligand, the receptor spontaneously activates caspase-3 and -7, assuring a high constitutive level of apoptosis that limits the size of the progenitor compartment (63). Conversely, when the ligand availability increases, binding of SHH to PTC prevents this default induction of caspase-mediated apoptosis, activates further downstream signals that promote progenitor cell viability, and therefore expands populations of PTC-expressing cells. Thus populations of PTC-expressing progenitors amplify or shrink according to the availability of Hh ligand. This mechanism maintains a small reservoir of transiently amplifying progenitors that aid in life-long maintainence of tissue integrity while minimizing the risk for neoplasia (50).
Our studies of cultured murine hepatic progenitors suggest that they might be regulated by similar mechanisms. Like other Hh-sensitive progenitors, cultures of hepatic progenitors derived from adult mouse livers exhibited endogenous caspase-3/7 activity, and this was reduced significantly by providing SHH. In addition, cyclopamine-mediated blockade of Hh-initiated signals downstream of PTC caused cell death in colonies of human hepatic progenitor cells. Collectively, these results demonstrate that Hh signaling is necessary to maintain the optimal viability of hepatic progenitors from fetal development to adulthood.
Our findings are important because they indicate that the liver is similar to other tissues that rely upon Hh signaling for homeostasis (4). For example, mucosal expression of Hh ligands and their receptor, Ptc, have been demonstrated in healthy adult intestines, and Hh activity increases in several inflammatory disorders of the bowel (39). Androgen deprivation of the prostate ablates prostatic epithelium but enriches the gland with prostatic progenitors. Subsequent blockade of Hh signaling by cyclopamine or Shh neutralization prevents prostatic regeneration (27). Regeneration of airway epithelium after acute lung injury is marked by the induction of Shh and Gli1, a transcriptional gene target of Hh signaling, suggesting that airway regeneration also requires Hh activation (67). The present demonstration of Hh activity in hepatic progenitors complements a recent report showing that hepatic stellate cells produce Hh ligands (54) and extends earlier findings of increased Ptc and Smo transcripts in chronically injured human livers (52). Together, these results demonstrate that cells with the capacity for Hh signaling persist and thrive in several adult tissues, including the liver, and suggest that Hh-regulated regenerative mechanisms are probably conserved among adult tissues that were derived from the endoderm.
This discovery opens novel areas for liver research that are likely to have broad implications, because transiently amplifying progenitors provide the seeds for both healthy regeneration and neoplasia (24, 40). Consistent with the latter concept, overactivation of the Hh pathway has been implicated in the formation of tumors in several other endodermally derived tissues (57), including the pancreas, gallbladder (6), prostate (27), and lung (67). A role for Hh signaling had not been considered in hepatocarcinogenesis in prior studies, because mature liver parenchymal cells lack Ptc. However, the present finding that Hh-responsive cells reside among progenitors in adult livers challenges that assumption. Furthermore, we (53) have recently shown that human hepatocellular carcinomas and liver cancer cell lines express the Hh pathway and are regulated by Hh signaling. Thus resident populations of Hh-sensitive progenitors may play a role in the genesis of liver cancer, as they do in malignancies of other endoderm-derived organs (4).
In conclusion, these findings have exciting therapeutic implications for patients with various types of chronic liver disease. For example, they suggest that targeted ablation of Hh activity in malignant liver cells might prove useful as a treatment for hepatocellular carcinoma. On the other hand, supplementing Hh activity in healthy hepatic progenitors might help to improve recovery from liver damage. Actualization of these putative therapeutic advances will require a better delineation of how the Hh pathway interfaces with other autocrine and paracrine signals that control the fate of resident hepatic progenitor populations during health as well as during states of accelerated tissue remodeling after liver injury.
This work was supported by National Institutes of Health Grants RO1 AA-010154 (to A. M. Diehl), RO1 DK-053792 (to A. M. Diehl), RO1 AA-012059 (to A. M. Diehl), RO1 DK-52851 (to L. M. Reid), RO1 AA-014243 (to L. M. Reid), RO1 IP30-DK-065933 (to L. M. Reid), and T32 DK-007713 (to J. K. Sicklick) as well as United States Department of Energy Grant DE-FG02-02ER-63477 (to L. M. Reid) and sponsored research grants from Vesta Therapeutics Incorporated (to L. M. Reid) and the International Taiwanese Research Institute (to L. M. Reid).
The authors thank Dr. P. A. Beachy for the kind gift of adult Ptc-lacZ mice used in these studies as well as criticisms; Dr. E. Meyers for the kind gift of Ptc-lacZ mouse embryos used in these studies; Dr. B. E. Petersen for the kind gift of a murine hepatic progenitor cell line; Dr. W. Chen for technical assistance; and D. F. Sandler for assistance with manuscript preparation.
↵* L. M. Reid and A. M. Diehl contributed equally to this work as senior authors.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2006 the American Physiological Society


text:
I was wondering whether anybody uses compression at the recording stage on guitars & vocals. I read somewhere that the steadier level makes it easier to adjust the gain, which makes perfect sense to me.
I was wondering, however, how much compression should really be applied at that stage, bearing in mind that more compression may be added during mixing. What is common practice? How much do you apply? Thanks.


text: I'm trying to think of names which don't evoke an image of one specific region— names which are equally common in many parts of the world, but outside the US top 100. Variations of spellings are okay, as long as the name doesn't fundamentally change from language to language. For example, Bruno, Felix, and Raphael are all quite popular in Europe and South America, but not so much in the US or UK. However, they wouldn't feel unusual or foreign on an American or British kid. For girls, Laura and Lena fit the bill. I've been combing through international top 100s to flush out the list, but turn to you for more ideas.


text:
Alton Brown Live - Eat Your Science
Cadillac Palace Theatre
151 W. Randolph Chicago
"Alton Brown Live: Eat Your Science", the follow-up to the smash "Edible Inevitable" tour, will hit the road in 2016. Fans can expect more comedy, talk show antics, multimedia presentations and music (yes, he sings), but Brown is adding a slew of fresh ingredients including new puppets, songs, bigger and potentially more dangerous experiments and what every cook needs in his kitchen - fire! Brown has a knack for mixing together a perfect base of science, music and food into two hours of pure entertainment. Critics and fans rave about the interactive fun when Brown invites an audience member on stage to serve as his assistant. This year's show has even more protective ponchos for the first few rows when things get even more messy. According to Alton, "there will be plenty of new therapy inducing opportunities during our audience participation segments. I don't want to give too much away, but we're also going to play a little game with the audience," says Brown.
Price: $22-$97
Show Type: Comedy
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text:
Matchup: Forrest Griffin (9-2-0) vs. Stephen Bonnar (6-1-0)
Date: April 09, 2005
Location: Las Vegas, Nevada
Recap: Everyone should already know about this fight, however: The Ultimate Fighter® Finale bout between Forrest Griffin and Stephan Bonnar was a battle that showed incredible heart and skill that went to a difficult judges' decision. Where in the end both fighters earned a contract in the UFC®.
Outcome: Griffin by Unanimous Decision
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Actor Channing Tatum thrilled a group of female fans in New York City on Saturday by making a cameo at a local bachelorette party.
The hunky star, who recently stripped off in saucy movie 'Magic Mike,' was dining with a pal at Manhattan hotspot Toy when he became swarmed by a crowd of women, celebrating the impending nuptials of their best pal.
And Tatum made sure the bride-to-be had a night to remember by agreeing to take photos with the lucky lady.
A source tells the New York Post, "He was approached by a group of girls in a bachelorette party asking him to pose for photos with the bride-to-be. (He wished) the blushing gal the best at her wedding."


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We're really sorry but we've sold out of this product and are unable to obtain more stock. In most cases this is usually because the manufacturer has discontinued the product. We don't want you to miss out though so here's some of our best selling products which are similar to what you are looking for. Take a look and you could just find the perfect item. Just a small point to note - they might not have the exact features as the product you initially wanted so please be sure to check the features before you buy.
The Plantronics M175 mobile headset offers hands-free convenience for your active lifestyle. Keep both hands on the wheel while driving and enjoy additional productivity in the office and on the go. Its convertible design can be worn as headband or earloop on either ear. You choose between greater stability and convenient small size. The adjustable noise-canceling microphone keeps your voice crystal clear, even in noisy environments. Includes one-touch volume and mute. Comfortable, durable, and lightweight, it also works with headset-ready cordless phones.


text: This 32 in. Wrought Iron Table Lamp in traditional rusty black finish is adorned with scroll ironwork accents. Lamp measures 32H with a 9 1/2 in. base. Accepts a 150-watt three-way bulb. Shade included. Add regal elegance to your room decor with simple ornamentation. ~ Ships from the manufacturer. Allow 4-6 weeks. Cannot express ship or ship to Alaska or Canada.


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Our major source of financial support comes from:
Environment Canada's Aboriginal Fund for Species at Risk
These funds play an important role in the recovery of species at risk on Aboriginal lands. The goal is to contribute to the conservation and protection of Canada's biodiversity by supporting species recovery planning, habitat protection, and overall conservation and capacity-building-initiatives by Aboriginal people in Canada. For more info visit Environment Canada's website.
And in the past we have received funding from the Habitat Stewardship Program. As part of Canada's national strategy for the protection of species at risk, the federal government established the Habitat Stewardship Program (HSP) for Species at Risk. The HSP became operational in 2000-2001 and allocates up to $10 million per year to projects that conserve and protect species at risk and their habitats. We are thankful for their support
The Alderville Black Oak Savanna is an active member of the Rice Lake Plains Joint Initiative . Members of this initiative are dedicated to the restoration and protection of sustainable tallgrass prairie and oak savanna habitat through co‐operative efforts in conservation science, land stewardship, public outreach, and legal protection of land.


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Cresta Mall robbery investigated
Johannesburg - Police are investigating a robbery that took place at Cresta Mall, Johannesburg, in which one person was shot and injured, the shopping centre's management said on Wednesday.
"The SAPS are investigating the incident and, together with our security advisers, we're undertaking our own investigation," manager Deborah Nathan said in a statement.
"We are also supporting the SAPS in their investigation, including providing CCTV footage of the incident."
An elderly man was shot and wounded when three armed men robbed iStore at the mall.
Police said three men entered the iStore and ordered the employees to lie down.
They took an undisclosed number of cellphones and iPads and fled the scene.
The men then started shooting randomly in the parking lot of the mall as they got away, injuring the elderly man.
Nathan said the mall had extensive security measures in place.
Assessment and an update of security measures were conducted on an ongoing basis.


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Legal Question in Traffic Law in Ohio
Un-signed traffic ticket
I received a speeding ticket over the weekend and the officer never asked me to sign the ticket. I did not refuse to sign it. He just handed it to me and told me that it was waiverable and if I wanted to show up in court to fight it I could. That was it. So, I have an unsigned traffic ticket and I was wondering if I was still liable because of his mistake.
1 Answer from Attorneys
Re: Un-signed traffic ticket
You have been issued a petty misdemeanor traffic citation. It is not legally required that you acknowledge receipt of the citation by signing for it. That is sometimes done and there is provision on the citation for your signature, but your signature is not required.
If you ignore the ticket there will be serious consequences. If you are doubtful, then go before the Judge and ask him to dismiss the citation for lack of your signature. At the same time, be prepared to pay the fine and court costs.
Good luck.
Related Questions & Answers
Proper police procedure? I was driving in a three lane highway. i was in the middle... Asked 11/21/00, 5:58 pm in United States Ohio Traffic Law


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Throw Pillows For Kids
9 Stylish Throw Pillows Made Just For Kids
9 Stylish Throw Pillows Made Just For Kids
You have the modern nursery bedding, the best-looking storage bins, even the awesome wallpaper: now it's time to add the final touch to your hip kids' room with a too-cool (and easy-on-the-wallet) throw pillow. We tracked down nine options for every kind of kid. Whether he loves Star Wars or she can't get enough of wild animals, we have the throw pillow that's sure to delight both you and your little one. Keep clicking to find your favorite.
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Andouille gumbo and tortilla soup, fried mushrooms with truffled dipping sauce, enchiladas con queso and steak frites, crabcakes with chipotle coulis, pierogi, Asian pot stickers with ginger sauce, chicken piccata, cheeseburgers served with a bottle of Chimay Red, miso soup and Hawaiian tuna salad. And sushi (on request) from the sushi bar next door.
Magnolia -- the three-year-old brainchild of Peter and Mara Soutiere, partner Tim Ackerman and chef Chris Pierce -- is a restaurant with a serious identity crisis. It's everyone's favorite Cajun-Creole-Belgian-Mexican-Asian-Polish-fusion-steak-and-seafood restaurant with a sushi bar attached. A culinary gangbang, like Ecstasy Night at the U.N. commissary, like a primer on world cuisine written by Mark Bittman off his ADD meds.
And that's just tonight. Tomorrow or next week or next month, it'll be Indo-Asian-French-Italian fusion with English bangers and tacos, then Bulgarian-Lyonnaise-Argentine with handmade Piedmontese pastas, pho and Kansas City barbecue. Plus edamame. Plus California rolls. With a wine list featuring producing regions as varied as the cuisine, and a custom cocktail lineup that torpedoes all the chocolate martinis and tortured cosmos and sugar-rum weirdness out there with something as simple as a Cherry Coke: cola and cherry-infused vodka, served tall, with a straw.
1381 Forest Park Circle, Lafayette, 303- 665-3080. Open at 5 p.m. nightly.
For the menu alone, I might have mocked this place mercilessly had my meals gone just a little differently -- taken the lineup apart piece by piece, then veered off on some ill-considered food-history jag about Escoffier and H.L. Mencken on a coach tour of the best Asian restaurants in the Pyrenees, or Anthony Bourdain smuggling pierogi out of Warsaw in his underpants. But once I tried a few things, I knew I couldn't. Pierce's housemade andouille sausages were really very good -- stiff, smoky, precisely grill-marked but not overdone at all, served split lengthwise and in a skin with just the right amount of snap. No, they weren't brats (which would be the historically and regionally appropriate sausage to serve with a pierogi), but I don't like brats all that much and I do like andouille, and I love a nice pierogi, so, oddly, this plate worked for me. The skin on the pierogi was pan-seared an ideal golden-brown, and it was stuffed with a lovely, smooth potato-and-onion filling cooled out with a big dollop of crème fraîche off to the side -- because if a big serving of sausage and dumplings aren't enough to blue-light you straight into the nearest cardiac emergency unit, then eating sour cream by the spoonful should certainly put you over the edge.
At Magnolia, I found myself eating steak frites, a plate of New Orleans-style beer-BBQ'd shrimp with a spicy red-pepper broth and really tasty sweet potatoes, and vegetable risotto topped with a lace of pepita oil. And I barely flinched at the Disneyland-food-court, Parade-of-Nations, It's-a-Small-World-After-All weirdness of it. Or the fact that I was eating all of it in the middle of Lafayette, where Taco Bell is a reasonable choice for Mexican and Olive Garden is haute-fucking-cuisine.
The strip mall where Magnolia sits is one of those prefab retail outposts that get dropped into some farmer's front forty just ahead of the condo complexes going in -- larval sprawl, on a street that didn't even exist five years ago, vanguard of the developers' advancing horde. Magnolia's front door (and I am not making this up) looks out over a dog bakery -- where they sell dog treats, I assume, not baked goods made from dogs -- and a franchised tutoring outlet called Mathnasium. Around the corner was a deli called Blue Sky that didn't make it -- the "For Lease" signs up in the windows, the equipment under wraps -- and already there's another deli squatting in one of the strip mall's other units. And next door, Sushi Mara (which shares the same building, the same owners, the same front door and the same hostess stand as Magnolia) is Lafayette's only sushi restaurant, which is on par with being the only Algerian restaurant in Toledo or the only hamburger stand on Mars.
The Soutieres and Ackerman are restaurant veterans. Q's in Boulder, the long-gone Two Bitts Bistro -- they've been around. But Magnolia is their first shot at ownership, the first time they've had a place of their own. Peter Soutiere is a manager and a sommelier, Mara a trained chef (a former sous in Q's kitchen) who now mostly handles the books, Ackerman a manager and waiter. Pierce came with a fine-dining background and even did a few months at Q's himself before he was poached by the partners. They decided to make their stand in Lafayette because they knew the town had no upscale eateries that could compare to their vision of Magnolia.
In order to get out of the radius of chain restaurants crowding the Lafayette landscape -- and because the deal on the space vacated by the Platinum Grill was too good to pass up -- they put Magnolia in this out-of-the-way location. They added Sushi Mara about a year later, putting the sushi bar in an unused section of the originally even more massive dining room. And they kept adding more dishes. Crabcakes and sausage, pierogi and pot stickers. The groundless menu offers all things to all people -- because without Magnolia, there'd be nothing for nobody.
And Magnolia's loyal customers appreciate that. They appreciate that they can have a sushi restaurant. That they can have a restaurant that doesn't feature as its main draw unlimited salad or free balloons or Riblets. That there's a place that offers everything they could possibly want -- if not right now, then next week -- in comfortable surroundings. There's a fun bar and patio seating and rooftop seating and acres of dining room, not even counting the private rooms. The tables are made of brushed stainless or polished wood, depending on where you sit, and the walls are done in a marzipan palette of pastel shades that defy pigeonholing as Mexican or American or contemporary or classic. Pieces of suitably abstract art are hung on some of them. There are steel railings and accents that bring to mind an urban Chipotle, as well as little wood sculptures of ducks and other knickknackery that give Magnolia a down-homey, Squat-n-Gobble kind of feel. It's multiple specificity -- using many different defining styles of decor to make a room as comfortable as possible to as many different people as possible without ever committing to a single, over-arching theme.
And the menu is the same way, of course, which would drive me crazy if not for the fact that everything I tried on it was done so surprisingly well. Pierce is taking chances in a way that I can't help but respect because of the skill he and his crew bring to the table, and even if this kitchen has no grounding in anything even approaching a singular cuisine, what it does have is a thrust -- an aggressive reaching for interesting combinations that provide customers with a tour d'appétit to keep them coming back again and again.
The whole roast beef tender was deceiving because it wasn't tenderloin and it wasn't really a roast. It was ten ounces of tournedos, cut from the chuck (a good cut -- bloody and flavorful and tender, as promised) and fanned as though it were tenderloin over a mound of creamy mushroom risotto that anywhere else might have been a button-mushroom throwaway but here was intelligently constructed of chanterelles and oysters fully giving up their flavor to the rice. On the side, there was grilled asparagus that looked awful and tasted beautiful -- smoky, green and earthy -- and horseradish freshly shaved off the root. At every step, intelligent choices and inarguable technique.
The Flatiron steak was similar in butcher's gimmickry to the tender: a top blade cut, like a ribeye but not a ribeye. Magnolia proudly serves hanger instead of sirloin just because hanger is better -- not as sexy, but twice as tasty. The burgers are made with Coleman Natural beef, and that housemade andouille sausage showed up again in a bowl of sausage and rock-shrimp linguine.
I asked for the seafood presentation du jour, but the server said it was sold out. The last of the crabcakes went as I sat there as well, sign of a house that's turning tables at a rapid clip, serving maybe everyone in Lafayette two or three times a week. So instead, I ordered the salmon, which was squeezed on the menu between the oven-roasted chicken, the enchilada plate and the Caesar salad.
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I don't really like salmon. In the culinary world, salmon is the girl with self-esteem issues, the one who'll go home with anyone and try anything once just to get a little love. Salmon can be pickled, poached, grilled, baked or seared. It can be turned into a mousse, a puff, a paste. It can be cured or smoked and paired with just about anything under the sun. You can do things to salmon that no one would consider doing with any other fish in the world, and the next day, salmon will always be there waiting for you, just hoping to be used again, altogether too eager to please.
But when salmon is handled with even the tiniest amount of respect, it can be great. It takes to flavors like a drunk to cheap bourbon and holds them like a dream, a quality that Pierce wisely exploited with the salmon filet on the dinner menu, infusing it with smoke (cedar, maybe, or applewood) and pairing it with artichokes, swiss chard, smoked bacon and a red verjus -- "green juice," as in the juice of unripe wine grapes, which is used to make a sauce taken from some obscure page of the French canon that most chefs probably haven't ever read. And with the exception of the Swiss chard -- which is hard to like under the best circumstances, being all fibrous and tough, but flat-out impossible to enjoy when a little past its peak, as it was here -- all of these disparate elements labored beneath one perfectly cooked piece of salmon to elevate the dish to excellence. The verjus was like a red-wine-vinegar broth but gentler, though still bitter and astringent enough to cut through the smoke and oiliness of the fish like a razor. The brunoise of smoked bacon lent an earthy base to the broth, the artichoke hearts (cut into sticks, but with less precision than I would have liked) a pleasant nuttiness.
And though I was fully stuffed by this point, I'd luckily saved some room for dessert. Magnolia has its own pastry chef, Gina Scott, and she'd made a warm apple bread pudding topped with burnt-sugar ice cream and a smooth, thick caramel-whiskey sauce that I would've kicked myself for missing had I staggered to the door without a final course. Sure, she also offered the ubiquitous flourless chocolate cake, as well as fresh-baked chocolate-chip-cookie ice-cream sandwiches with handmade whipped cream and chocolate sauce. But burnt-sugar ice cream? That's just brilliant.
As a matter of fact, that's a good way to describe Magnolia in general. It's brilliant because it is all things to all people, all under one roof, and all at the right time and place. And it's all good.


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Remainders: Malarkey
The definition of 'malarkey,' per the Oxford English Dictionary.
Joe Biden and Paul Ryan clear the bar — barely.
Most media outlets believe the debate was a draw...
...but The New York Times gives it to Biden.
Fox News won cable coverage of the veep debate.
Mitt Romney seeks newspaper endorsements.
President Obama will appear on "The Daily Show."
And Candy Crowley shows off her pad.


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Well, I have a conundrum. The more I read about tilling, perhaps I shouldn't be doing so every year, but... I have been adding my city's compost and well rotted horse manure for many years. Every year I till my veggie plots before planting. Every year my soil seems as hard as before. Today I spent 3 hours turning over soil in one plot and breaking up the clods with the shovel. I only turned over 6'X 4'. It is so rough, I don't know how I would plant seeds there. Folks, I'm 68 years old, and an active female, but this is nuts! I'll never get the friable soil I dream of this way. So, do I keep adding stuff and keep tilling? Perhaps I should till only shallowly instead of as deep as I can go with my Mantis. The topic might bring a storm of tillers and non tillers, but I'd like input here. I want soil that crumbles in my hands! You know - Martha Stewart soil!
There are 30 steps from my driveway to the garden, but I'm willing to spend more money to get what I want. The compost and manure is free, but not the labor to get it up to my garden. All your help is appreciated.
Compost & horse manure and still poor soil
Well, I have a conundrum. The more I read about tilling, perhaps I shouldn't be doing so every year, but... I have been adding my city's compost and well rotted horse manure for many years. Every year I till my veggie plots before planting. Every year my soil seems as hard as before. Today I spent 3 hours turning over soil in one plot and breaking up the clods with the shovel. I only turned over 6'X 4'. It is so rough, I don't know how I would plant seeds there. Folks, I'm 68 years old, and an active female, but this is nuts! I'll never get the friable soil I dream of this way. So, do I keep adding stuff and keep tilling? Perhaps I should till only shallowly instead of as deep as I can go with my Mantis. The topic might bring a storm of tillers and non tillers, but I'd like input here. I want soil that crumbles in my hands! You know - Martha Stewart soil!
I'm paralyzed from the waist down, and the native soil here is gooey, thick clay. Since I can't run a rototiller, I cheat.
My first tactic is to build raised beds, because it's easier to pile good raw materials on top of the clay than it is to incorporate it into the clay.
Next, I plant cover crops wherever I want to really bust up the clay & bring minerals up from below. Clovers are very good at driving their roots very deeply into the subsoil. This opens up channels for water & microorganisms to get down there & expand those pathways. I also plant Daikon radishes in the nastiest clay patches, & I never pull them; they rot in place over the winter & leave big holes behind.
At the same time, I scatter all the spent coffee grounds I can get my hands on. It's like ringing the dinner bell for worms. Worms do a fantastic job of aerating soil, and they leave behind castings that plants love.
I didn't get good soil quickly, but I did eventually get it. Now I just maintain the conditions needed to keep forming more: plants on top, plant litter & organic matter at the surface, occasional water, and that's it. Worms & microorganisms do the rest for me.
Here's the scoop on how topsoil gets created: http://managingwholes.com/new-topsoil.htm
This message was edited Apr 18, 2011 2:07 PM
I wish I could make raised beds. The 32 steps are due to a slope that I am on.
The other tactics still work wonders on areas where I have no raised beds.
A good discussion on letting nature do your work for you: http://davesgarden.com/community/forums/t/800670/
The benefits of cover crops: http://davesgarden.com/guides/articles/view/1708/
Choose the right cover crops for your situation: http://calshort-lamp.cit.cornell.edu/bjorkman/covercrops/decision-tool.php
The importance of microbes to building good soil: http://davesgarden.com/guides/articles/view/1936/
A balanced diet for your soil: http://davesgarden.com/guides/articles/view/2351/
32 steps? Up and down?! Ouch!
I thought that enough compost and manure would soften anything, but I noticed that my clay stayed gooey when I only added compost. Then I added coarse sand, and that seemed to make it more friable, even though many people say "sand doesn't help clay".
Now I add medium pine bark mulch, screened if I have the time and energy. And sand if its in the budget.
You might think about mechanical compaction and drainage.
If you walk on top of soil with any clay in it, it probably will pack down hard and tight.
Especially if you walk on them when wet.
Make narrow beds, and ALWAYS walk around them, never ON them.
Since walking around a long bed takes time, make them narrow and fairly short.
3x15 feet? 2.5 x 20 feet?
You can even till "from the side" though it is harder.
If narrow enough, and some of the plants are short, you can step over them. Or set two cinder blocks on their sides in the center as a stepping stones.
It's better to have 50 square feet of GOOD aerated soil than 150 square feet of hypoxic clay pudding.
Even heavy rain seems to pound clay down into a harder state. Keep some coarse mulch on top. Pine bark mulch has become my hero.
Wet clay is hopeless - you want very good drainage. At least you have a slope! Why does that argue against raised beds? Think of them as terraces.
If you have great drainage, the soil won't stay as wet as long. That may encourage to compact itself less over the months.
This is speculative, I haven't built beds on anything this steep, but I've been thinking about it for a steep, shady spot I have.
Chop a WIDE staircase into the hill, where each 'step' is a rasied bed. You might use the removed soil to form the next step down, probably adding manure, compost, sand, crushed rock or bark as you go.
Or, if you have infinite energy or helpers, wheelbarrow it away to somewhere you can screen it and amend it before bringing it back.
(And consider filling the new beds or the top half of the new beds with much better imported soil or LOTS of organics. Like 3-5 parts compost and amendments to one part clay. The lasagna method of building soil lays down corrugated cardboard or many layers of newspaper on the floor, before adding lots of organic material.)
Each step or raised bed would cut into the slope 4-6 feet, for a bed 3-4 feet wide and a walkway 1-2 feet wide. Depending on how far you're willing to walk to get around each bed, maybe run them for 15-20 feet of length (this dimension is at a constant height on the hill; I'm not explaining this very well). Probably start with just one bed near the top of the slope.
You want a 1-2 foot wide walkway BELOW each bed, so you don't have to stoop as much.
If the walkway is BELOW the 'floor' of the bed, it has better drainage, especially if your lower walls have some holes or notches on the bottoms, or you chop a few drainage cuts where the floor of the bed meets the RB wall.
The "floor" of each RB should be mostly level, so the RB has fairly uniform depth, but should slope downhill just a little for drainage. Maybe just an inch or two of drop over 2-3 feet of bed width.
The wall on the downhill side ought to be 8" to 16" high, so you have some depth of soil.
If your base, unimproved clay is hard enough to be a structural material, the uphill wall might be partly or all clay (like the riser of a staircase). Since the upper wall of one bed would merge into the walkway of the bed above it, that should stay packed pretty hard!
I think the slope of your hill and width of bed plus walkway determines whether the uphill wall is all clay, or clay plus a short wall. One nice thing about RBs on a steep slope: you might only need half as many walls!
I like paving stones stood on end and tilted back an inch or two into the bed for stability. I don't need stakes - they lean on the soil. It also motivates you NOT to walk on the soil in the bed! That tends to topple walls and maybe send you rolling downhill!
An 8x16" paver lets you have an 8" depth, or a 12" depth. But the 12x12" pavers are thicker and give more stability. Either costs around $1 where I live.
Depending on slope, you might have 12" or 16" lower walls, but 4-6" upper walls. Thent he upper walls could be one wooden board, and the lower walls paving stones (or 2-3 boards nailed together).
If you need or want to till with a machine, you can take paving stone walls down, lay them on the walkway, and put them back up easily. I till with a gardening fork and a "sharpshooter" spade. It has a long, narrow blade that goes down 14-16". But once the soil gets soft enough that worms don't need jackhammers to penetrate it, and has enough 'structure' to drain well enough to let air in, I seldom till deeply.
If I can find something I like that grows in deep shade, one of these years I'll let you know how this scheme works in practice! Until then, I hope you can turn your clay into soil!
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It has been a long road for the crew at Empire Access breaking into the Television service business but now it's official with their first installation in Batavia.
Empire Access Executive Bob VanDelinder, says you will get more than great speed and reliability from Empire.
"With this service we're able to provide amazing features, digital TV service, DVR service, whole-home DVR, and we have a watch-TV-anywhere feature, which allows you to watch TV from anywhere in the world as long as you have an internet connection. As most people know we're a local company so we provide great customer service. You call a live call center when you call and talk to us."
Dozens more are already signed up awaiting to receive TV services from Empire in Batavia.
For more on the story check the article by our news partner The Batavian.
For more information visit www.Empireaccess.com.