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As I read the list of new cardinals named yesterday by Pope Ratzinger (a.k.a. Benedict XVI), it struck me that this was the same-old, same-old — another roster of doctrinal conservatives to reinforce the hard-right line on sex and "morality" in a College of Cardinals already stacked with arch-conservatives by his predecessor, Wojtyla. This is especially true of the man who now becomes the most important American in the Vatican, San Francisco Archbishop William Levada, who takes over The Rat's old job as head of the Congregation for the Doctrine of the Faith (formerly known as the Office of the Inquisition, the Vatican's ideological enforcer.)
But, while I try to keep an eye on Vatican politics, I don't pretend to be an expert — so I asked a progressive friend of mine who is the veteran Vatican correspondent for one of the most prestigious European television networks, whose judgment I trust in such matters, for his opinion. Here's what he told me in an e-mail:
"There are no liberals or reformers among this bunch. The three over eighties can be disregarded. Of the remainder by far the most interesting is the Chinese from Hong Kong. This is a delicate political and diplomatic appointment, a prepositioning of the church before the expected gesture from Beijing sometime before the Games offering to reopen official relations with the Holy See. On matters sexual/moral there is to be no letup. As far as the guy from SF is concerned he is an old buddy of Benedict having worked for twelve years in his departments and is seen as completely trustworthy to carry on the Ratzinger line across the road in the Holy Office."
In fact, the San Francisco Board of Supervisors took the unusual step on Tuesday of unanimously passing a resolution unanimously condemning the appointment of Levada, so reactionary is he, saying, "Cardinal Levada is a decidedly unqualified representative of his former home city, and of the people of San Francisco and the values they hold dear." The resolution was motivated by Levada's militant opposition to the right of same-sex couples to adopt, and the Board of Supervisors' resolution went on to say that, "It is an insult to all San Franciscans when a foreign country, like the Vatican, meddles with and attempts to negatively influence this great city's existing and established customs and traditions, such as the right of same-sex couples to adopt and care for children in need."
The resolution blasted the teaching of the Catholic Church that homosexual adoption does "violence" to children as "hateful and discriminatory rhetoric (that) is both insulting and callous, and shows a level of insensitivity and ignorance which has seldom been encountered by this Board of Supervisors.'
When Ratzinger came to the U.S. prior to the 2004 elections to rev up the Catholic Bishops to help make gay marriage a prime issue in that year's presidential campaign and deny the sacraments of the mass to those candidates for public office who favored it, or who believed in a woman's right to an abortion, the Rat's first stop was to see his old pal Levada in San Francisco. Count on Levada to keep this closet-case Pope's vicious anti-gay agenda at the forefront of the Vatican's doctrinal exigencies.
For a bracing antidote to ultramontane Papacy, see Michel Onfray's brilliant essay in the latest issue of New Politics on the 17th century atheist French priest Jean Meslier, who presented the first coherent atheist philosophy — you can read it by clicking here. You can also read my accompanying introduction to Onfray's scintillating synthesis of Meslier's thought.
Doug Ireland, a longtime radical journalist and media critic, runs the blog DIRELAND, where this article appeared March 25, 2006.


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Bay Street at Palms Point in St Pete Beach
- bedrooms 2
- baths 2
- sleeps 5
- pool no
- hot tub no
- pets allowed yes
- type House
- community 1st Palm Point
- rates (USD) $ 2,400 - $ 2,800 / month
vacation rental description
Beautiful 2/2 home with tile and high quality laminate throughout. Garage. Each bedroom has a queen size bed. All linens and kitchenware provided. Washer/dryer. Internet, 3 TVs,dvd player, and landline phone. Tropical landscaped fully fenced private yard with grill. Walk to beach, shopping, and restaurants. Beach is 2 blocks and Bay is across the street. Central heat and AC. Check-in 4pm/ Check-out 11:00a.m. Living room plus family room.
vacation rental location
property availability
- dates with deals
- unavailable dates
Last modified on 08-19-2014 at 10:59 AM.
email owner view full calendar
Kate Schneider
house rental amenities
Garage, grill, private back yard, sun room, beautifully landscaped and decorated. WIFI. Washer/Dryer. 3 minutes to $8 million dollar city pool complex. Our visitors have loved this house. Beach chairs and beach toys too! Bycicle and games.
activities and attractions in St Pete Beach Gulf Coast Central
>Beach, shopping, restaurants and museums.1 1/2 hours to Disney.Pool 3 minutes away at $8 million city waterfront pool complex. Tennis and Putt-Putt minutes away.
vacation rental rates
**Please call or email for dates/rates and availability!**
$200 refundable pet deposit provided no damage.
$2400-2800 per month plus cleaning fee ($75)
$250 deposit holds reservation. Balance upon check-in
Credit card/check or money order
Kate Schneider
accepted payment methods
Prior to paying a reservation deposit, always verify payment instructions by calling the phone number displayed on this listing page. By contacting the owner directly and paying by an approved method - credit card, paypal, check or bank transfer - your reservation payment will be protected up to $1,000.Learn more in our Security Center
Top things to do in St Pete Beach
- Fabulous Beach
- Shopping
- Restaurants
- Busch Gardens/Tampa
- Daytrips to Orlando attractions
Top things to do on a budget
- Island trips
- Parasailing
- Shelling
- City pool & sports complex
- Beach
Top things to do with a family
- Dolphin watching
- Sunset Sailing
- Daytrip to Orlando attractions
- Beach
- Golf
Kate Schneider , owner

subset: commoncrawl


text:
Hopefully they'll have at least ditched Mick McCarthy.
Haha great. Arry joining the BBC team apparently but still take them over the joy that is Southgate and Towsend
Hope the BBC have Clarence Seedorf as a pundit again.
In any event, I intend to avoid ITV as much as possible. Their entire commentary/punditry lineup is thoroughly objectionable.
What's the situation in England with the coverage? Sky and BBC at the same time, or they take different games?
ITV alternate with BBC. Sky get nothing because they like to keep big international tournaments free-to-air.
ITV. Eeeuuuww. Chiles and Red Clive with Townsend and Southgate prevaricating as if their lives depended on it. When Keane sits in it's like Michelangelo has been forced to make ends meet by doing a bit of artexing in between Sistine Chapels.
Cricket Web's 2013/14 Premier League Tipping Champion
- As featured in The Independent.
"The committee discussed the issue of illegal bowling actions, and believed that there are a number of bowlers currently employing suspect actions in international cricket, and that the ICC's reporting and testing procedures are not adequately scrutinising these bowlers."
- Even the ICC's own official press release thinks things must change
Brumby at his finest.
I actually don't mind Southgate but he might have just stood out at the World Cup because the rest of ITV's panel was so ****.
Can't stand Southgate. A very Southern English brand of moron: one who's far too polite to ever say anything of substance. All you get is a load of "looking back on that he'll think he maybe could have done a bit better there."
He's not horribly, infuriatingly wrong all the time like Townsend though. So in that sense he's one of ITV's better pundits, but that's a bit like being the nicest paedophile.
Last edited by Uppercut; 11-05-2012 at 08:27 AM.
I think Southgate is often quite critical and cutting with what he says to be honest. The reason I dislike him so much though is that he's a massive hypocrite. Happy to say how crap the decisions of other managers are, whilst conveniently forgetting that his tenure with Middlesbrough was absolutely disastrous.
Townsend also incredibly irksome, and just a moron. At least Robbie Earle was a nice lad, don't remember a lot about his punditry before his...er...indiscretion, but at least he wasn't extremely annoying. Can't stand Chiles, Clive, Drury, Beglin or Pleat though.
I like Beglin but that's mostly because I associate his voice with watching our UEFA Cup run on ITV4.
ITV's production values are pissweak compared to the beeb as well
Personally I wish sky were allowed to show some games; not exclusively but it'd be nice to have the choice.
The good news is ESPN have none. Their choice of pundit is...well.
ESPN is just cack all-round imo. Ray Stubbs, maybe the most boring man in the world.
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text:
Memorial Children's Hospital received a big check today.
They were presented with a donation of $56,500 to go towards purchasing a neonatal incubator transport unit for the helicopter program.
The funds were raised in the 2013 Four Winds Invitational hosted by the Pokagon Band of Potawatomi Indians and they say there's no where else they would want the money to go.
"Number one, you're helping young people, you're helping families. Number two, what a lot of people need to continue to recognize is this is home for the Pokagon Band of Potawatomi Indians. We have a number of citizens who live here. So it's not like we're helping out somebody else, we're actually helping ourselves by helping the community," says Pokagon Band chairman Matthew Wesaw.
The Pokagon Band also announced they will once again be the title sponsor next year's Four Winds Invitational.
WSJV-TV FOX 2858096 County Road 7, SouthElkhart, IN 46517
Switchboard: 574.679.9758News Phone: 574.679.4545Station Fax: 574.294.1267Email: email@example.com


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The son of Green Bay Packers coordinator Joe Philbin is missing after a weekend boating trip in Wisconsin. Michael T. Philbin, 21, went missing on Sunday morning after heading out with friends to an area near the University of Wisconsin-Oshkosh campus.
Divers recovered a body from the Fox River on Monday, but police have not publicly identified it, and will not do so until Tuesday. The Winnebago County Sheriff's Department told reporters that a man at a local business heard a man on the ice in the river yelling for help on Sunday morning.
Philbin has been the Packers' offensive coordinator since 2007, and with the team since 2003. Since becoming the team's coordinator, Philbin has overseen a late-career renaissance by Brett Favre and the development of Aaron Rodgers, who guided Green Bay to its first Super Bowl title since 1996 and is a prohibitive favorite to win the 2011 NFL MVP award.
For more on this story and the Packers, visit Acme Packing Company.


text:
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Museum and Wandschneider Park Information
Welcome to the Downers Grove Park District Museum
CLICK HERE to check out the virtual tour of the Museum!
Preserving History's Future! The Downers Grove Park District Museum provides leisure and educational experiences for the community through collecting, preserving, exhibiting and interpreting materials that explain and document the history of Downers Grove and its relationship to local, state, regional and national history.
Time lapse video of the 1846 Blodgett House moving to the Museum property (provided by the Downers Grove Heritage Preservation Corporation)
Make it a Historic Birthday!
Click here to view History Themed Birthday Parties!
To view the calendar for
Museum Events, click here.
The Museum is located near downtown Downers Grove on the grounds of Wandschneider Park in the historic Blodgett House. The gracious Victorian structure was built in 1892 as the 11-room home of the Charles Blodgett family, descendants of one of the Village's first settlers.
Step across the threshold of the Blodgett House for a glimpse of the past. Enter a bygone era when families rested on fainting couches instead of recliners, stored food in iceboxes instead of refrigerators and entertained themselves with parlor games instead of cyber games.
A treasure trove of fascinating facts and fun, the Museum is a great place for a family outing, a resource for local research and a memorable way to spend a part of the day.
Experience your heritage at The Downers Grove Park District Museum. Take a glimpse back into a past which nostalgia has gently etched in our memories. Exercise your mind and expand your knowledge of the community around you. Through varied exhibitions and programs, you'll discover your museum and the history of your village.
Through an agreement between the Downers Grove Park District and the Downers Grove Heritage Preservation Corporation, the 1846 Blodgett House was relocated onto the Museum campus in 2008. The 1846 Blodgett House was built by Israel and Avis Blodgett. Not only does the house have architectural historical significance as the second or third oldest house in the Village, but it also has great cultural significance as an active stopover point on the Underground Railroad. The Downers Grove Heritage Preservation Corporation has been founded with a mission to save and restore this historic house and create a museum focusing on the underground railroad. Click here to learn more.
The Museum
Admission: FREE - donations appreciated
Office and Research Hours
Open Hours
Group Tours
Downers Grove Seed Library
Downers Grove Park District Museum is now home to Chicagoland's first seed lending library. To join the library, residents must attend an orientation event, held on April 12 or April 26, 2014 from 2-3 pm at the Downers Grove Museum. Vicki Nowicki, an award winning organic gardener and co-owner of the Land Office, has stocked the library with a collection of thousands of seeds sourced from the Seed Savers Exchange and The Pepperfield Project, including several rare and endangered plants. After the orientation, gardeners will be able to pick out up to five varieties of heirloom seeds, including beans, peas, tomatoes, and lettuce, to 'borrow' and plant. Members will also make a commitment to bring back some of the new seeds in the fall, to restock the library for the next season.
Wanschneider Park
831 Maple Avenue
This 4 acre park contains the Downers Grove Park District Museum, including the 1892 Blodgett House (main museum and offices), the 1846 Blodgett House, and the Annex Building, which houses special collections and featured exhibitions. Working in conjunction with the Museum, Wandschneider Park provides leisure and educational experiences for the community through collecting, preserving, and exploring parts of Downers Grove. The museum uses the park to help with exhibiting and interpreting materials that explain and document the history of Downers Grove and its relationship to local, state, regional and national history, including site features such as the "Wildflower Walk" showcasing native prairie plants and the Heritage Garden showcasing heirloom vegetable gardening!
Dawn to Dusk
|* Gazebo
|* Drinking Fountain
|* Guided Tours
|* Natural Area
|* Picnic Tables
|* Wildflower Walk Gravel Pathway
|* Heritage Vegetable Garden
Pauline Wandschneider was a long time resident of Downers Grove and was the founder and leader of the Downers Grove Historical Society. She dedicated her life to collecting, preserving, displaying and interpreting the history of Downers Grove. In addition to this, she also aided in finding a permanent home for all of these materials. In 1977, in honor of her contribution, the Park District named the site around the Park District Museum, Wandschneider Park. Pauline died in 1993, but the park carries on her name and contribution to the Downers Grove community.
Upcoming Improvements/Construction
No improvements are currently planned for the upcoming construction season
Special Event Rentals
Wandschneider Park is a beautiful setting for your next special occasion. Complete with gazebo, gardens, walkways and open space, the Museum Campus offers a private sactuary of nature and history in one. For more information on events and rentals, contact Julie Bunke at 630-963-1309.
"It was a great day and we are truly thankful for all that you did to make our daughter's wedding reception a success. We still are getting compliments on how wonderful the location the reception was." - Mother of the Bride, Wedding Reception held August 2012
Public Research
The Downers Grove Park District Museum has historical materials in the research library and archives. Materials include birth and death certificates, cemetery surveys, census records, telephone directories, school yearbooks, published genealogies, newspaper clippings, and various other documents, such as letters and photographs.
The Museum is available for public research during our office hours, which are Monday through Friday from 8:30 a.m. to 4:30 p.m. Reservations are not necessary, but the staff suggests you call in advance to determine if there are any materials relevant to your research topic. This will save you travel and time.
For individuals who are unable to travel to The Museum, a staff researcher is available for limited help. To obtain assistance from our staff researcher, you must write a detailed letter with your questions, including as much background information that is available. Letters can either be faxed to our 24-hour line at (630) 963-0496, or mailed to: Staff Researcher, The Downers Grove Park District Museum, 831 Maple Avenue, Downers Grove, Illinois 60515. Inquiries can also be e-mailed to firstname.lastname@example.com
Donating Artifacts
What does The Museum collect?
It is the mission of the Downers Grove Park District Museum to collect, preserve, exhibit, and interpret materials that explain and document the history of Downers Grove and its relationship to local, state, regional and national history. All objects accepted into the collections must have a direct connection to Downers Grove, or be able to help tell the story of the community.
How can you donate to The Museum?
If you are clearing out your attic or closets and find things of interest, contact the Museum. The staff will be able to tell you whether or not the object(s) would be considered for it's collection.
What happens to donated objects?
Donated objects accepted by The Museum are given one of three designations: Permanent Collection, Education Collection, or Property. Items are placed in the appropriate category based on a variety of factors, including provenance, intended use, availability, and condition.
Are object donations tax deductible?
YES! Under the terms of the Federal Omnibus Budget Reconciliation Act of 1993, charitable gifts of appreciated property are fully deductible. Contributions of $250 and over will require a receipt for all donations, including property and cash.
Does The Museum staff appraise objects for donation?
NO. The value of an object donation must be appraised by a qualified person. The staff is prohibited from providing appraisals of the monetary value of materials offered as gifts, brought in for identification, or submitted for any other purpose. Therefore, the donor is responsible for obtaining the services of an appraiser.
Did you know?
The Museum is a green facility in many ways:
- Compact Fluorescent Light bulbs (CFLs) or LED lights for lamps and can lighting
Lighting Upgrades - OH bulbs at carriage house
Turn off lights, computers, monitors at end of day or when employees leave desk
Turn off copiers, printers
Windows & doors adequately sealed
No dripping faucets, fountains in the buildings (waste reduction)
Native plant landscaping
Rain barrel/rain garden – rain barrel at carriage house
Bike rack for employees and visitors
Entrance mats of appropriate length at entrances
Natural, unbleached, recycled content paper towels, tissue & napkins
Use washable tablecloths, cleaning rags, etc. to reduce paper use
Furniture/furnishings reuse - exhibits
Effective recycling program in place (plastic, paper, cardboard, aluminum, steel, glass)
Additional waste reduction activities
Composting program in place - Coffee grounds
Encourage employees/clients to use both sides of paper
Print defaults as two-sides
Use second side as scratch paper
Print only when necessary


text:
Know Your Star: Bipasha Basu
Bipasha Basu goes glam for 'Humshakals'
Bipasha Basu has redefined the image of the Bollywood actress, by completely dispelling the myth that fair means beautiful!
She is now being hailed as one of Bollywood's sexiest women to date.
Here is a pictorial ode to the bold and beautiful Bengali stunner, who will be seen next in 'Humshakals'.
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Weight: 0.81
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International customers, please view our shipping page for information on additional shipping fees. CLICK HERE
LIQUITEX-Gesso. This surface protector seals; stiffens and provides surface 'tooth' for painting surfaces. It is permanent; non- yellowing; flexible; and water resistant when dry. For use with oil; acrylic; pastels; and other paint media. Apply it to raw canvas; harboard; paper; wood; and other non-oily porous surfaces. Sand hard; shiny surfaces before applying. Can be tinted with acrylic colors but is not to be mixed with oil paints. This package contains 8oz/237mL of Gesso. Conforms to ASTM D4236. Imported.


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Thread: help me some problem
Mar 4, 2013, 21:20 #1
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help me some problem
I have problem with my code by AS3. Who have code about MP3 Player by as3? Please shared me. My english is not very good. Thanks!
Mar 5, 2013, 05:38 #2
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check this code for the AS3 for simple Mp3 player
// First we define a variable of type URLRequest and attach our mp3 file.
var soundRequest:URLRequest = new URLRequest("JeffWofford_Trouble.mp3");
// Then we make a new sound instance and naming it sound_
var sound_:Sound = new Sound();
// Also a sound control to control and adjust later on.
var soundControl:SoundChannel = new SoundChannel();
// last variable is a soundtransform instance, this is to control the volume up and down function. (and we set them to 1,1 thats the right and left speakers).
var transform1:SoundTransform = new SoundTransform(1, 1);
// now we attach the sound file to the sound_ instance we made before.
// here is four eventlisteners, the control all our buttons, and "listen" and calls functions if the buttons are clicked.
playButton_.addEventListener(MouseEvent.CLICK, playSound);
stopButton_.addEventListener(MouseEvent.CLICK, stopSound);
soundUp_btn.addEventListener(MouseEvent.CLICK, soundUp);
soundDown_btn.addEventListener(MouseEvent.CLICK, soundDown);
// This is the function to control the play button, only thing happening is that we attach the soundcontrol to the sound and calls its play method.
function playSound(event:MouseEvent):void
soundControl = sound_.play();
//the stop function also uses the coundControl to stop the mp3 file from playing.
function stopSound(event:MouseEvent):void
//Now this is a bit more tricky (not much but more then just stop and play).
//At the top of our code we declared a soundtranform named transform1
//this is a value, and by saying ++ we add one to the original volume number then attach it to the soundcontrol.soundTransform
function soundUp(event:MouseEvent):void
transform1.volume += .1;
soundControl.soundTransform = transform1;
//this is the same as above, we just use -- to subtract from the volume number.
function soundDown(event:MouseEvent):void
transform1.volume -= .1;
soundControl.soundTransform = transform1;
Mar 5, 2013, 19:51 #3
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ok, thank you. can you show me how to get total times of MP3 player? I try calculated by this code:
var mySound:Sound= new Sound();
var sl:Number;
//calculate the total number of minutes
function LoadComplete(e:Event):void
var a:Number;
var b:Numer;
a= Math.round((sl/1000)%60);
b= Math.round((sl/1000)/60);
//time when play Mp3 file
var R:Number;
var Q:Number;
Q= ((myChannel.position/1000)/60);
my problem is :Although playing file Mp3 complete but (a) not equal(R) and (b) not equal(Q).
Can anyone help me. Thanks !


text: Sponsored by the Six District Educational Compact, the Festival takes place at Kent State University in May every year. The 2014 Festival will be held on on May 8, 2014, with featured author Gabrielle Zevin. Students will get to meet the guest author and participate in sessions involving book cover art, drama, poetry, and various writing contests, all pertaining to the Festival Books.


text:
Griffin's offers blend of whimsy and comfort foodBy Gordon Dritschilo
Staff Writer | May 27,2013Albert J. Marro / Staff Photo
Lucas Wilson cooks at Griffin's Publick House on Center Street in Rutland.Griffin's Publick House, its proprietors will tell you, is not a bar — not even a high-end one.
Co-owner Don Hubert Jr. said he was tired of owning a bar, which was why he shut down the Downtown Tavern and, after extensive renovations, opened the space as Rutland's first gastropub.
Ever seen one of those episodes of "Top Chef" where they take junk food or fast food and make similarly structured dishes with much better ingredients? A gastropub does that with traditional bar food.
Griffin's french fries are cooked in duck fat, unless they are served alongside a burger, in which case they are seasoned with truffle salt. Their poutine is made with a short rib ragout. Pizzas — referred to as "artisanal flatbreads" on the menu — come with toppings ranging from broccoli and wild mushrooms to Korean barbecue beef.
"This was a niche that wasn't being filled," he said. "It's been a long time since this place has had good, locally produced food in a casual atmosphere."
"Local" has, of course, become a leading trend in area restaurants. Numerous local menus boast about what area farm some of their ingredients come from, and Griffin's is right around the corner from a restaurant whose entire concept was local food. The causal atmosphere, Hubert said, is what will set Griffin's apart.
"I want it to be the sort of place you can come to if you have a softball team," he said. "At the same time, I want it to be a place where doctors and lawyers will come. ... I want it to be a place professionals feel like they can come, have a drink or some food, and stay."
Anyone passing by on Center Street will surely feel the kitchen window beckoning, as the action at the grill and wood oven is clearly visible. Inside, exposed brick combines with blacks and browns to give an old-world feeling. The liquor and draft beer selection is extensive.
Hubert also said Griffin's boasts Rutland County's only nitrous-driven draft wine system, which keeps wine away from its two mortal enemies, light and air, until it is served.
"You never get a bad glass of wine," he said. "You never get a corked bottle."
Chef de cuisine Brian Garvison, who came to Griffin's from Burlington, resisted efforts to categorize his food.
"It's an eclectic mix," he said. "We've got comfort food, fine dining, some whimsical things. It's done in a very non-pretentious way. People are going to try stuff that's new."
That whimsy has resulted in "hog wings" — pork shank prepared like Buffalo (or barbecue, or Jamaican jerk, or Thai curry) chicken wings — and an "apple pot pie" made with cheddar, bacon and gelato.
Plans include live music and a menu of "pre-Prohibition" drinks.
Also in the works is an iPad menu system co-owner Darwin Harder says will increase the information available at customers' fingertips while enabling the restaurant to change the menu instantly without waiting for new copies from the printer. A smartphone app downloadable from the restaurant's website will let customers at home peruse the menu and make reservations.MORE IN National / World BusinessWASHINGTON Average long-term U.S. Full StoryNEW YORK To win the hearts of frequent business travelers, United Airlines is going through... Full StoryHOFFMAN ESTATES, Ill. Sears Holdings Inc. Full Story
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- RICHARD'S POOR ALMANACK: On this day in 1915, the New York World publishes scoop: Thom. Edison diverts chemical from war production to help German pharmaceutical company make aspirin; on this day in 1935, Will Rogers, Wiley Post die in Alaska plane crash.
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- TODAY'S WEATHER MINUTE: Climatologists might not know as much about El Nino as they thought they knew. New studies show 10,000 years ago, El Nino was active, and polar ice sheets were rapidly melting — just like today.


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In the top of the fourth inning, Cardinals reliever Fernando Salas induced a leadoff pop-up from Nelson Cruz. Matt Holliday dropped the ball, and Cruz wound up scoring.
In the top of the fifth inning, Cardinals reliever Fernando Salas induced a leadoff pop-up from Josh Hamilton. David Freese dropped the ball, and Hamilton wound up scoring.
It didn't glance off the edge. It's as if Freese loaded his glove with tiny springs.
Anyway, Hamilton reached first base safely and scored when Michael Young followed the error with a double to the left-center gap. Hamilton didn't run like a guy playing through an injury, racing all the way around three bases.
The double made it 4-3 Texas. It could've and should've been worse. Adrian Beltre and Cruz made outs, but then Salas was ordered to intentionally walk Mike Napoli. That brought up pinch-hitter David Murphy - in for Craig Gentry - and Murphy drew a walk to load the bases.
Bases loaded, two outs, one-run game, World Series. Derek Holland had been throwing in the bullpen. Ron Washington allowed Colby Lewis to bat.
I literally don't know if I've ever seen a manager make a worse decision than that.


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Although long-term devotees to the original Alien series may be disappointed by this prequel, Prometheus as a stand-alone project is pretty good. As someone who has not seen any other films from the Alien franchise, I can vouch for the fact that you do not need any prior knowledge of the concept to enjoy Prometheus, although it does mean some of the links to the other movies are likely to go unnoticed. In some ways it may be better to go into the cinema without any standards against which to measure the film, as I definitely was not sure what to expect.
One of the best things about the film is its excellent cast. Big names such as Michael Fassbender, Charlize Theron, Guy Pearce and Noomi Rapace feature in the film. Believable performances help to suck you into the drama and make you feel as though you are involved, as does the powerful atmosphere created by Scott. The movie offers more than just action and thrills, there are also minor romantic plots woven into the main storyline, which is itself woven into the storyline of the saga. Well shot and directed, the whole film comes together well because of the contribution from all areas of production, including the screenplay, actors and the direction.
I found Prometheus to be an enjoyable film to watch, if a bit scary in some places, although what I deem to be frightening may be seen as child's play by some. With an age rating of 15 compared to the usual 18 in the Alien series, the movie involves less gore than the others, although that aspect is still present, and is in some ways less scary, although there are still moments that will make you jump. It seems that in Prometheus, there is more focus on the concept and the story, rather than on the actual action with the aliens themselves. There are a few moments in the film that seem to drag at times, and as with any film, it is down to personal taste. Individual's reception of the film probably also depends on expectations people have and what they want the film to be. In my opinion, it is well worth a watch.


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About the AuthorYesterday Studios
AimeeStarr Denver, Colorado
Yesterday Studios specializes in life event photography. From weddings to senior and family portraits, we will capture the soul and essence of the story you're telling. Yesterday Studios consists of two principle photojournalists. Our wedding photography is always shot with both of us, as we firmly believe that a single photographer simply cannot capture the entire event as it should be documented.
Publish Date August 12, 2007
Dimensions Standard Landscape 130 pgs Standard Paper
Category Wedding
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- Rika Awakihara '14
- Hometown: Hiroshima, Japan
- Major: Sociology
As a global salesman, I would like to increase the exchange of goods and skills as well as cultural exchange so others could enjoy a better life.
- Ashley Christensen '16
- Hometown: Champlin, MN
- Major: Criminal Justice and Sociology
Sociology Internships and Hands-On Learning
Students have the opportunity to get involved in the more than 80 organizations at Mount Union including the Sociology Club and Pi Gamma Mu, a national social science honorary.
The Department of Sociology and Criminal Justice frequently brings sociology graduates of Mount Union back to campus to discuss opportunities in the field with current students.
Out of the Classroom Activities
Mount Union believes that there are some things students can't learn in the classroom, which is why experiential learning is important to the overall college experience. The Department of Sociology and its faculty encourage students to intern in local social service agencies and organizations and conduct their own independent research.
Students can intern at hospitals, police and probation departments; county prosecutor's and public defender's offices; agencies that treat alcoholism, drug abuse and mental illness; business corporations; non-profit organizations; retirement centers and nursing homes; and state and private agencies that work with juvenile delinquency, spousal abuse and children.
Helpful links from the American Sociological Association
- A Valuable Preparation
- Prospects for the BA/BS Sociology Major
- The Employer's Perspective
- Prospects for the MA/PhD Sociology Graduate
- Sociological Roles Relating to Business, Industry, and Work
- Careers in Sociology
- Undergraduate Student Resources
- 21st Century Careers with an Undergraduate Degree in Sociology
What Skills Do Sociology Majors Learn and What Is the Pathway to Using Them on the Job?


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NOGALES (Tucson News Now) - Another drug tunnel has turned up in this Arizona border town.
Immigration and Customs Enforcement says they discovered it Wednesday afternoon at the end of Nelson avenue in downtown Nogales, Arizona about 200 yards east of the Morley Pedestrian Gate.
7831 N. Business Park Drive
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Results from tuberculosis testing at a local school were supposed to be released Monday, Oct. 14th.
Students at A. R. Johnson were tested last week after one of their peers tested positive.
Results were expected to be read today.
We have put in calls and emails to get those but have yet to hear back.
This is the third school within the last year to undergo this scare.
1336 Augusta West Parkway
Telephone: 706.722.6664
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Five suspects were arrested Friday afternoon and a sixth is being sought after Central Precinct Tactical Investigation Unit and Crime Suppression Unit officers raided a home in part of Savannah.
It happened around 2 p.m. at a shot house near the intersection of Barnard and West 31st Streets. Agents confiscated cash, drugs and drug paraphernalia, illegal alcohol and cigarettes and identifying an illegal rooming house. The raid was initiated after complaints from neighbors..
"We started 2014 off the same way we finished 2013," said Capt. DeVonn Adams, Central Precinct commander. "Our mission is to improve the quality of life in the Central Precinct. We are here to help people whenever and wherever we can, but we will continue to enforce the laws.
"This shot house not only was illegal, but it put innocent people at risk and robbed them of their sense of security. I am proud of the way our officers conducted this operation."
Arrested without incident were Bobby Ray Robinson, 32, of the 500 block of East 49th street, - sale of controlled substance / crack-cocaine (two counts); Angel Gail Richardson, 53, 1500 block of Barnard Street – distributing, selling, or otherwise dealing in alcohol without a license (four counts); Eddie Snyder, 73, 1500 block of Barnard Street – distributing, selling, or otherwise dealing in alcohol w/without a license (three counts); Arsinio Larnon Robinson, 24, 100 block of West 32nd Street, – sale of marijuana (two counts); and Rodney Jones, 57, of the 100 block of West 42nd Street – possession of marijuana and possession of drug-related object.
Central detectives still are searching for Michael Anthony Kelsey, 54, 800 block of East 41st street. charged with sale of marijuana. Anyone with information on his location is asked to call Crimestoppers at (912) 234-2020 or text CRIMES (274637) using the keyword CSTOP2020. Tipsters to Crimestoppers remain anonymous and may qualify for a cash reward.
1430 East Victory DriveSavannah, GA 31404
Telephone: 912.651.0300
Fax: 912.651.0320
Email: email@example.com


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Elevate provides prospective students (and families) a chance to get to know Bryan College through experiences like campus tours, chapel, dining in the cafeteria, sitting in on classes, meeting with professors or coaches, and the most valuable thing we offer, a chance to meet and interact with our current students.
Please remember that we are located in the EASTERN TIME ZONE.
Can't stay overnight? Join us for the day on Friday to tour the campus, visit class, and eat lunch in the dining hall. You can also learn valuable information about the admissions/financial aid process.
Overnight Information
The overnight in the residence halls are restricted to prospective students aged 16 and older. Parents and other family members may stay in a local lodging, many of which offer Bryan College discounts.
Students staying in the residence halls will want to bring:
- Bedding (sleeping bag and pillow)
- Pillow
- Personal toiletries
- Towels
NOTE: Our visit events are free of charge.
If for any reason you need to cancel please notify as soon as possible Campus Visit Coordinator Amanda Peckman via e-mail or call her at 423.775.7211.


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Publication: theday.com
Norwich – The St. Vincent de Paul Place soup kitchen is moving after all to the St. Joseph School on Cliff Street for six months during repairs of the current location in the former train station behind Main Street.
The move was announced in early June, but then was canceled when the facility couldn't obtain a building permit to occupy the former cafeteria at St. Joseph School.
Now, with structural repair work expanding at the train station, and the state building official approving a temporary waiver of handicapped accessibility requirements at the school, the move is back on.
The soup kitchen will move Wednesday to the new location for about six months, Diocese of Norwich spokesman Michael Strammiello said today.
Soup kitchen officials also will decide soon whether to stay permanently at the train station, move permanently to the school or even to a third unidentified location.
For now, the move to the school is temporary, and the school building would need handicapped accessibility before the facility would be allowed to remain there beyond six months, city Director of Inspections James Troeger said this morning.
The Big Day 2014
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Front OfficeBack To Front Office
Title: DIRECTOR OF TICKET SALES
Email: firstname.lastname@example.com
Originally from Queensbury, New York, he moved to Oklahoma while serving in the United States Air Force. A combined 10 years Active Duty and Air Force Contractor in the Power Production/Aircraft Arresting System career field. Since leaving the Air Force, he has had General Management experience in the Hospitality and Restaurant industry. He is a certified massage therapist and currently holds a 4th Degree Black Belt with the World Tae Kwon Do Federation. In his spare time he is passionately involved with the development of Youth Hockey as a Coach for the Oklahoma City Youth Hockey Association. GO BARONS!!!


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SAPD chief: Woman dies after 'violent crime'
Passersby found woman in front of duplex in 5700 block of Canterbury
SAN ANTONIO - A San Antonio woman in her 40s was found bleeding in her front yard Friday morning on the city's West Side, police said.
According to San Antonio police Chief William McManus, passersby found the woman in the front yard of a duplex in the 5700 block of Canterbury.
McManus said she was the "victim of a violent crime" and that there was a "violent scene inside the house."
He said the EMS units responded and took the woman to an area hospital, but she had died by the time of their arrival.
McManus said police are looking for a suspect but would not confirm the relationship between the woman and the suspect.
He said investigators have taken some witnesses downtown for questioning and is cautiously optimistic that investigators will "close this case sooner, rather than later."
Copyright 2013 by KSAT.com All rights reserved. This material may not be published, broadcast, rewritten or redistributed.


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Hi guys
Just wondering if the 7 year rule applies to a change of use. Basically we have been operating a very small office from a house for approximately 8 years. No clients visiting, no passing trade just one person working in an office. We are now wondering if we need to apply for planning permission, for example if a neighbour made a complaint about this (they haven't to date) would we be covered?
Thanks in advance:confused:


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Linda B said about: Low Woods Furnishings
Very good selection of fabrics and furnishing. Helpful and pleasant staff and lovely coffee and cake
Added August 2014
Jenny T said about: Low Woods Furnishings
I love low woods furnishings because they have a really extensive range if fabrics for many different users so much choice and a beautiful cafe and excellent friendly service
Added August 2014
Sarah K said about: Robert J Dutton Ltd
Fantastic service. Professional and efficient and good value for money....
Added August 2014


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Banana Chip Pancakes Recipe
- 2 cups biscuit/baking mix
- 1 egg
- 1 cup milk
- 1 cup mashed ripe bananas
- 3/4 cup swirled milk chocolate and peanut butter chips
- Maple syrup and additional swirled milk chocolate and peanut butter chips, optional
- Place biscuit mix in a large bowl. Combine the egg, milk and bananas; stir into biscuit mix just until moistened. Stir in chips.
- Pour batter by 1/4 cupfuls onto a greased hot griddle; turn when bubbles form on top. Cook until the second side is golden brown. Serve with syrup and additional chips if desired. Yield: 12 pancakes.
Reviews for Banana Chip Pancakes
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I use semi sweet choc chips and always have to do a double batch, they are my favorite simply because they are sweet enough to just hold in hand and eat!! My 5 kids love them!
These were okay. I made a 3/4 batch because that's how much biscuit mix I had on hand, but still used 1 egg (who want's to split an egg up into 3/4s?). I only had 1/2 cup banana too, but didn't alter the milk ratio. I used mini semi-sweet chocolate chips and thought anything more than 1/4 cup was exorbitant. So, I guess I really just made a new recipe... Either way, we didn't like the taste or texture of banana in pancakes, so we won't be making these again.
Yummy! I used Reese peanut butter chips...it turned out GREAT! You can taste the peanut butter flavor. Recommend it!
Absolutely delicious. The combination of banana and peanut butter is amazing.
This was a big hit with my whole family.
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- Alzforum Recommends
Coen K, Flannagan RS, Baron S, Carraro-Lacroix LR, Wang D, Vermeire W, Michiels C, Munck S, Baert V, Sugita S, Wuytack F, Hiesinger PR, Grinstein S, Annaert W. Lysosomal calcium homeostasis defects, not proton pump defects, cause endo-lysosomal dysfunction in PSEN-deficient cells. J Cell Biol. 2012 Jul 9;198(1):23-35. PubMed.
Please login to recommend the paper.
When Lee et al. published their study in 2010, it made a big impact in the field because it described a novel role for PS1 in lysosomal acidification and—even more importantly—suggested that mutations in PS1 attributed to familial AD (FAD) can cause similar defects in patients' fibroblasts. This finding would have nicely tied together PS mutations in FAD, Aβ formation, and pathologically observed lysosomal and autophagosomal deficits (Pickford et al., 2008; Nixon et al., 2005; Boland et al., 2008).
In 2011, Neely et al. published a study that explored the role of PS on autophagosomal-lysosomal degradation. It found that PS1 and PS2 are "important for overall degradation through autophagy in multiple cell types and indicate that presenilins are key regulators of autophagolysosome formation or lysosome function." However, the authors did not find signs of altered lysosomal acidification and conclude "presenilins might function at the lysosome in another manner besides lysosome acidification." Given the high-profile nature of the study by Lee et al. and the potential implications for the treatment of FAD patients, many independent groups have tried to repeat and expand on the Lee findings.
Now, two new studies (Zhang et al., 2012; Coen et al., 2012) support the findings of Neely et al. in that a lysosomal acidification defect is absent and thus appears not to be the primary cause of PS1/2-mediated autophagosomal-lysosomal deficits. The Zhang study systematically reexamines the findings from the Lee paper, and the authors cannot reproduce many of the acidification-related findings, both in vitro and in vivo. While they discuss the possibility that some clonal drift might have occurred in the cells studied by Lee et al., causing the differences in results between the two studies, the Coen paper uses many of the same cell lines as the Lee study and also fails to observe acidification deficits. Currently available data suggest that acidification defects are unlikely to be involved in PS-mediated autophagosomal-lysosomal deficits.
Nevertheless, all these studies collectively strongly support an important role for PS1/2 in maintaining proper flux and degradation capacity in the autophagosomal-lysosomal system. Further studies will be needed to elucidate this crucial cellular degradation pathway and its role in AD/FAD—whether PS acts by "selective impairment of autolysosome acidification and cathepsin activation" (Lee), by "mediating autophagosome-lysosome interaction or lysosome function" (Neely), by "calcium storage/release" (Coen), or by "regulating lysosomal biogenesis" (Zhang). This latest rush of partially contradictory findings will undoubtedly stimulate more exciting research, and future studies will show if PS contributes to some or all of the proposed functions.View all comments by Philipp Jaeger
Controversy makes for better news than consensus but we need to keep in mind that there is considerable agreement about the links between PS1 and autophagy defects in AD. Four different lab groups agree that autophagy is disrupted by PS-deletion and/or PS1FAD mutations (1-4). Each of these groups also implicates defects in lysosomal function directly or indirectly as a basis for autophagy dysfunction. The one exception is the report from a fifth group led by Sam Sisodia, which concluded that PS has no role in autophagy at all (5). A careful look at that report will show that no direct assessment of autophagic protein turnover was actually made in the Zhang et al. study. The points of agreement among the four different labs are of paramount importance for AD pathogenesis and therapy since autophagy, particularly the proteolytic clearance steps, are clearly disrupted in all forms of AD and AD models (6-8). Selectively enhancing lysosomal function in AD mouse models by any of several mechanisms has been shown to have marked therapeutic effects on amyloid and lysosome pathology, synaptic function, and behavior (9-12). Conversely, AD-related pathologies can be produced experimentally by inducing selective lysosomal proteolytic dysfunction (8,13). Autophagy impairment provides a basis for the amplified amyloid and lysosomal system neuropathology and the earlier clinical onset seen in PS-FAD versus sporadic AD (14,15).
Divergence arises in views on the mechanism(s) underlying PS-related lysosomal dysfunction. Coen et al., in one of the two studies that addresses mechanism (1,4), challenge the mechanism we reported, which is based on impaired lysosomal acidification due to PS1-dependent vATPase hypofunction. Instead they show abnormal calcium release from lysosomes, although they do not explain the mechanism of this defect or how the defect is mechanistically linked to PS or PS loss. In extensive ongoing studies, we observe the same calcium abnormality but we can show that the effect is secondary to impaired lysosomal acidification and can be corrected by normalizing lysosomal pH. This is not surprising given the very close relationship already known to exist between pH and calcium regulation in lysosomes (16,17). What is more surprising to us, based on the literature, is why Coen et al. did not see a lysosomal pH alteration in the face of such significant lysosomal calcium release.
Why, then, is it that we can consistently measure abnormal pH elevation in lysosomes of PS1-deficient blastocysts, PS1-FAD fibroblasts, PS-deficient neurons, and, in our newer analyses, in all of the cell lines reported in the recent studies, while three groups claim to see no differences in lysosomal pH values? A closer look at the data in two of the papers (3,5), reveals that these groups did not actually measure lysosomal pH.
The Zhang, Sisodia et al. study used an unconjugated dye that enters all vesicular compartments of the cell, not lysosomes specifically. The value for "vesicular pH" of 6.6, which is the basis for the claim in the report's title that lysosome acidification in PS models is normal, has no relevance to the pH of lysosomes, which is well established to be below 5.0. Of significant concern is that the majority of the pH values measured in the wild-type cells were above pH 7.0 and even above the range of the standard curve. Inexplicably, these values were excluded from the calculation of "vesicular pH," which is astonishingly high even without including these arbitrarily omitted values. Clearly, this analysis bears little relationship to pH of even the acidic vacuolar system of the cell, much less the lysosome itself.
In the earlier Neely et al. report (3), pH was only assessed indirectly by LysoTracker intensity. Although LysoTracker is a pH-sensitive dye, it is mainly an organelle marker and not a substitute for dyes designed to measure pH rigorously. LysoTracker signal intensity does not consistently reflect acidity of lysosomes (18,19). Importantly, unless LysoTracker signal is calculated per lysosome, rather than per cell as in this report, the size and/or number of lysosomes confound the signal intensity as a readout of acidity.
Coen et al. used several methods to assess pH, including LysoTracker—at 250 times the manufacturer's recommended concentration. They also used dextran-conjugated fluorescein, which exhibits pH dependent changes in fluorescence intensity within the pH range of 5-9 and is most appropriately used to assay pH values closer to neutrality due to its extremely low sensitivity at acidic pH, according to the manufacturer's technical bulletins. This group also used LysoSensor Yellow/Blue DND-160, which was not targeted specifically to lysosomes, and obtained ratiometric values but did not calculate pH values, thus limiting any evaluation of whether relevant lysosomal pH values were obtainable with this method.
In contrast to these approaches, we measured lysosomal pH with a dextran-conjugated ratiometric dye specifically targeted to lysosomes and optimally sensitive to the pH range of lysosomes (1). The elevation of pH in PS1 models was supported by demonstrating predicted changes in a broad range of lysosomal pH-dependent phenomena, including but not limited to (1) decreased in situ activity of cathepsin D and of cathepsin B in lysosomes of living cells (2) decreased activity and specific activity of cathepsins D, B, and L measured in vitro (3) impaired maturation of cathepsin D (4) failed dissociation of MP6R from cathepsins (5) accumulation of autophagy substrates in autolysosomes and pathological accumulation of autolysosomes confirmed by double immunofluorescence labeling and EM (6) delayed clearance of autophagy substrates from autolysosomes after induction of autophagy and (7) recapitulation of the above phenotypic features by bafilomycin 1 treatment (1).
These findings are consistent with the loss of lysosomal vATPase we reported. Our new soon to be submitted work will provide, in our opinion, compelling further evidence implicating vATPase hypofunction in PS cell models and AD mouse models. A similar mechanism has now been shown to explain autophagy dysfunction in Parkinson's Disease due to mutations of another lysosomal proton pump, ATPase13a (20). Space limits do not permit a detailed discussion of concerns about the vATPase data in the new reports but the exclusive use of vATPase over-expression in the Zhang et al. study is not expected to yield definitive insight into the behavior of the protein at endogenous levels and, in the Coen et al. report, the atypical molecular weight of the vATPase band monitored, >20 kD lower than the molecular weight reported by all other labs, is quite puzzling (21, 22). Moreover, Coen et al. report that vATPase V0a1 and V0a2 knock down in PC12 cells had no effect on lysosome acidification, citing the work of Saw et al. (23) as support. In fact, this finding actually directly conflicts with Saw et al., who reported lysosomal acidification defects induced by V0a1 or V0a1/V0a2 knockdown using the identical knockdown construct and PC12 cells (23).View all comments by Ju-hyun Lee
In 2010. Lee et al. (Lee et al., 2010) reported that, as a consequence of presenilin deficiency, lysosomes fail to acidify. In their view, presenilin 1 is required for proper glycosylation of the Voa1 subunit of the vATPase, and improper glycosylation in presenilin-deficient cells precludes delivery of the vATPase to lysosomes. This is an attractive model, but the notion that single cells, let alone multicellular organisms (like people), can exist without functional lysosomes needs to be examined very carefully. Indeed, a number of groups—including our own—tested the model, but failed to replicate the findings of Nixon's group (Coen et al., 2012; Zhang et al., 2012; Neely et al., 2011).
In a recent submission to Alzforum, Nixon and colleagues summarily dismiss these more recent findings, stating that "these groups did not actually measure lysosomal pH." This statement is inaccurate and ill informed. In addressing our work, Nixon et al. state that we not only used the wrong concentrations of pH-sensitive probes, but even the wrong probes! This conclusion is seemingly based on the manufacturer's technical bulletins. Nixon and colleagues would have reached a different conclusion had they instead read the original scientific literature in some detail (including our paper). Firstly, their claim that we used LysoTracker at 250 times "the manufacturer's recommended concentration" is simply untrue. As explicitly stated in our paper (Coen et al., 2012), we used 250 nM LysoTracker Red, about three times the concentration recommended by Invitrogen and 2.5 times the concentration used by Lee et al. themselves (Lee et al., 2010). Considering the buffering capacity of lysosomes (upwards of 30 mM/pH), there is no reason to expect nanomolar concentrations of LysoTracker to affect the measured pH. Indeed, we could readily document differences between untreated lysosomes and lysosomes treated with vATPase inhibitors or weak bases, yet never saw a difference between wild-type and presenilin-deficient cells.
Nixon et al. also criticized our use of fluorescein-conjugated dextran to measure lysosomal pH, again based on a manufacturer's technical bulletin. Since the pioneering work of Ohkuma and Poole (Ohkuma and Poole, 1978), fluorescein-conjugated dextran has been the method of choice to measure lysosomal pH, as attested by dozens of publications from multiple laboratories (e.g., Barriere et al., 2007; Trombetta, 2003; Ohkuma and Poole, 1978; Yoshimori et al., 1991; Carraro-Lacroix et al., 2011). The pKa and dynamic range of this probe make differentiation between acidic and neutral lysosomes trivial (the 485 nm/440 nm fluorescence ratio changes by >fivefold). Accordingly, we could easily distinguish untreated lysosomes from those exposed to ammonium (which is not a vATPase inhibitor, but a membrane-permeant weak base) or concanamycin (a bona fide vATPase inhibitor). Still, we did not detect differences between wild-type and presenilin-deficient cells.
In another attempt to dismiss our findings, Lee et al. suggest that we inappropriately cited a coauthor's manuscript and presented data that are in direct conflict with his previously published work (Saw et al., 2011). The record needs to be set straight on this account as well. We indeed utilized PC12 cells with stably knocked-down expression of the Voa1 and Voa2 subunits of the vATPase, and failed to detect any lysosomal acidification defects (in direct contradiction with the model of Lee et al.). This is by no means in conflict with the findings of Saw et al. (Saw et al., 2011), who described altered acidification of dense core vesicles, which Lee et al. would surely agree are secretory organelles, distinct from lysosomes.
In summary, none of the claims made by Lee et al. on Alzforum regarding our pH determinations are correct. Careful reading of the original scientific literature would have avoided such misstatements. We are confident in our data, which are consistent with those of others and do not support the claims made by Nixon's group.View all comments by Sergio Grinstein
We would like to continue this collegial, public discussion of why our respective studies are at odds. We believe such discussion is important as numerous labs independently attempt to replicate and build on published work, so that the field as a whole develops robust advances. We agree with Nixon and coworkers that "controversy makes for better news than consensus." On the other hand, some controversy is truly needed to prevent dogma. After all, the first level where consensus can be reached is the scientific data, not necessarily the interpretation of those data. Collectively, we should be able to agree at least on the scientific data at hand.
We point out here that in their comment on the three independent studies that failed to reproduce their original data (Lee et al., 2010), Nixon and coworkers make errors both in interpreting consensus and in their critique of the science.
We and others have shown that PSEN1 regulates (in a γ-secretase-independent way) organelle and protein turnover (1,2), but stating that this function is linked to autophagy defects in AD is premature. Autophagy is a trendy topic, not least in neurodegenerative diseases, including AD. However, just because autophagic vacuoles accumulate in AD does not mean that one can claim autophagy defects in the disease. To our knowledge, autophagosomes may accumulate in people with AD, but the fact that they do form indicates that there is no defect whatsoever with respect to the molecular mechanism of autophagosome formation. Furthermore, the profiling data of Zhang et al. (3) and ours (unpublished), as well as the study by Neely et al. (5), confirm that in PS-deficient cells the formation of autophagosomes is not corrupted.
Autophagy is, in the first place, a "rescue pathway" for the cell to dispose of aberrant or accumulating organelles. As such, autophagy in PS-deficient cells and AD should, in our opinion, not be regarded as the cause but the consequence of transport dysregulation elsewhere in the cell/neuron. Both our group and Kim Green/Frank LaFerla come to exactly the same conclusion—that the defect is located at the level of lysosomal fusion efficiency, not in the autophagy limb of degradation (4,5). We prefer to focus in our future work on unraveling the causes, not being distracted by the consequences.
With respect to Nixon and colleagues' list of what they consider technical flaws in our data, these "flaws" do not reflect a profound knowledge of the literature or are simply erroneous. Therefore, they contribute to the controversy about the original paper by Lee et al.
Many of the reports discussed here (4-6) highlighted our previous findings that PSEN1 has a role in the degradative machinery of the cell, whether it be in neurons or any other cell type used so far (1,2). The novelty of our report lies in uncovering the underlying mechanism, namely defective storage and release of lysosomal calcium (4), leading to the accumulation of autophagic vacuoles that are unable to be cleared from the cells (1). We therefore provide a mechanism that likely explains the observed endolysosomal defects and impaired lysosomal fusion capacity in PSEN-deficient cells and neurons. How lysosomal calcium is distorted is the next question, and we are currently addressing that. We are pleased to notice that two independent laboratories confirmed this lysosomal calcium deficit (Nixon's and Green's labs; see Kim Green's comment). This consensus makes us confident of the quality of our experimental approaches.
Nixon and coworkers express surprise that the pH is not altered in the face of observed lysosomal calcium deficit. It is correct that pH and calcium in lysosomes are close partners, but either one can be independently affected. This was demonstrated in the case of Niemann-Pick disease type C patient fibroblasts. There, the late endosomal/lysosomal dysfunction originates from sphingosine accumulations, thereby compromising lysosomal calcium storage and release, and, hence, lysosomal fusion without concomitant lysosomal acidification defects (7).
Nixon and coworkers argue that the failure to demonstrate pH defects is based on the lack of quantitative measurements or technical flaws. This is a surprising statement, given that the original work by Lee et al. (2010) predominantly used the qualitative LysoTracker probe as a readout for acidification defects. In only one single case (Panel I, Figure 3) did they use LysoSensor, and then only to compare wild-type (WT) with PS1 knockout blastocysts (without including any rescue cell lines). The most surprising aspect of their original methodology is that they did not use LysoSensor to quantify pH in individual lysosomes (as it should be done), but, on the contrary, in trypsinized cells, measured total fluorescence of cell suspensions using fluorometry. Molecular Probes, the maker of these reporters, recommends that LysoSensor and LysoTracker should not be used to measure pH by flow cytometry or fluorometry.
The approach by Lee et al. strongly contrasts with ours. We utilized strategies generated independently by two different labs (ours and the Grinstein lab). Besides basic LysoTracker experiments, we used LysoSensor to evaluate pH at the organellar level. Secondly, we included a robust, well-established, live-imaging approach that uses pH-sensitive fluorescein derivatives. Our ability to monitor pH continuously also enabled us to study "online" the effects of reversible alkalinizing drugs or proton pump blockers. This allowed us to extract exact values for different parameters related to proton pump function in addition to the basal pH, including buffering power and rates of H+-leakage from lysosomes. None of these quantitative parameters revealed differences between wild-type and PSEN-deficient mouse embryonic fibroblasts and embryonic stem cells, or fibroblasts from familial AD patients. In an additional approach, we used DQ-BSA, a probe of acid proteases, to assess the activity of enzymes that function optimally in media of low pH. These experiments failed to show any indication of differential pH between the wild-type and mutant/PS knockout cells. Finally, the comment that we exceed LysoTracker concentrations 250 times above the manufacturer's recommendation is false. We used 200 nM for 30 minutes, as opposed to Lee et al., who used 100 nM for one hour. For live imaging, our collaborators (Grinstein lab) used 250 nM for eight minutes. All experiments were therefore done well within the range used in the literature.
We would also like to comment on the criticisms regarding V0a1 trafficking. Using three independent approaches, we demonstrate that endogenous V0a1 exits the endoplasmic reticulum (ER budding assay), traffics to the cell surface (surface biotinylation), and localizes to endosomal/lysosomal compartments (cell fractionation) in both WT and PSEN-deficient cells, neurons, or blastocysts (the latter unpublished). In other cases, we used stable viral transduction of V0a1 to minimize overexpression artifacts—here, also, V0a1 localized normally to LAMP1-positive organelles.
We used exogenous expression to demonstrate that there is only one N-glycosylation site on V0a1. These experiments, as well as the in-vivo experiments in Drosophila, demonstrate that glycosylation of V0a1 is not important for its trafficking or function in post-Golgi compartments. This is an important finding, as it directly challenges the hypothesis put forth by Lee et al. According to them, PSEN1 chaperones the glycosylation of V0a1 during its translocation in the rough ER. In PSEN-deficient cells, V0a1 fails to become glycosylated, impeding its traffic to lysosomes and consequently affecting proton pump function and lysosomal acidification.
The fact that there is only one glycosylation site—as Nixon and coworkers did not comment on this crucial finding, we assume they agree—has important consequences for the interpretation of the Western blot data. One N-glycan chain maximally gives a mobility shift of 1.5 kDa. We were unable to clearly resolve such a subtle mobility shift in the size range of the protein (100-120 kDa) using the low-resolving 4 to 20 percent precast gels used by Lee et al. Secondly, the primary antibodies Lee et al. used to detect endogenous V0a1 give additional background bands that migrate in the same range as V0a1, complicating further any interpretation of glycosylation differences. We noticed this after selectively downregulating V0a1 with high efficiency—a simple control for antibody specificity that seems absent in the original Cell paper of Lee et al. We invested quite some time to optimize both the gel type and running buffer conditions in order to increase resolution required to properly and indisputably evaluate the glycosylation status of V0a1 in the multiple cell lines used in our study. This allowed us to detect a mobility shift of the endogenous V0a1 and show that these immunoreactive bands indeed represented the endogenous V0a1, and again we controlled by both overexpression and siRNA-mediated knockdown of V0a1. We found that PS knockout had no effect on V0a1 glycosylation.
Secondly, we think most biochemists might agree that the electrophoretic mobility of a protein is a relative measure based on the mobility of commercial reference marker proteins. (The original "p116" name of V0a1 is simply derived from the fact that it runs like the 116 kDa marker). We made an effort to compare six different commercial reference markers in the two electrophoresis conditions (NuPAGE 4-12 percent Tris-Bis gel system using a MOPS-SDS running buffer and Novex 4-20 percent Tris-Glycine gel system using a Tris-Glycine-SDS running buffer, as in Lee et al.). The same sized pre-stained markers not only run at different heights within one gel system (thus different between different companies), but the same set of markers (of one given company) behaves quite differently between the two gel systems. Thus, changing gel systems and running buffers not only alters the electrophoretic mobility of V0a1, but also that of reference markers. This indicates that protocol-based mobility shifts are not a unique feature of V0a1, but are more general than assumed. Again, in these cases, both overexpression and siRNA KD controls rule out any confusion (and such controls are lacking in the work by Lee et al.). Lastly, that proteins behave differently in different gel/running buffer conditions shouldn't be surprising, particularly for AD researchers. Most in the field agree, for example, that amyloid peptides run in a reverse mobility when analyzed on urea gels.
With respect to the note that we wrongly cited the work of Saw et al., again we have to correct this. The paper by Saw et al. (8) was not about lysosomal acidification induced by V0a1/V0a2 knockdown. Saw and coworkers used an NPY-epHluorin probe for quantification of acidification, but NPY is a neuropeptide and thus marks secretory vesicles in PC12 cells, not lysosomes. This is similar to NPY-EmGFP, which showed strong colocalization with secretogranin II, a secretory granule marker. Secretory granules belong to the biosynthetic-secretory route, while lysosomes are the endstage of endosomal/degradative routes.
As a final note, our paper was submitted to Cell as a Matters Arising paper more than a year ago: According to Cell's policy, Nixon and colleagues have had our full dataset (including the lysosomal calcium defect) for over a year. In this period they confirmed our original findings on lysosomal calcium. With respect to pH measurements, they focus only on sparse technical details as the basis for a critique that appears to be erroneous. On the other hand, we see no criticisms on our work demonstrating that glycosylation is not required for V0a1 function in vitro and in vivo, or that V0a1 is normally trafficked and located where it should be. Thus, the relevance of the key element in their hypothesis, namely that PSEN1 chaperones the N-glycan transfer in the rough ER, is brought into question. Why would nature bestow such a fundamental role to PSEN1 when V0a1 doesn't even require it to function?View all comments by Katrijn Coen
We appreciate the responses of Annaert and colleagues. We believe disagreement centers on the definition of autophagy. We would respectfully submit that Annaert and Coen misunderstand fundamental concepts of autophagy and the essential role of successful lysosomal proteolysis in this process. This leads them (and Zhang et al. [3]) to believe that autophagy is completed upon formation of the autophagosome (AP) rather than after autophagosomes and their contents are digested in autolysosomes and the lysosomes are reformed (4,5). Their view of autophagy is at odds with the consensus view of the autophagy community reflected in guidelines on autophagy coauthored by 1,270 investigators in the field (6). When correctly defined as being a much broader, lysosomal degradative process, there is considerable consensus among labs that initial steps in autophagy such as autophagosome formation are normal in PS1 deficiency, but that autophagolysosome clearance by lysosomes is compromised. We reported this (1), and Neeley et al. (2) confirmed.
The main unresolved issue concerns defective lysosomal acidification mechanisms as the main basis for these autophagy defects in PS deficiency. While we were not persuaded by the detailed technical discussion of methods Annaert et al. used to challenge the pH mechanism, this issue will likely resolve itself with forthcoming data. To correct an assertion in Grinstein and Flannagan's comment, lysosomal function is not completely blocked in PS-deficient cells. If true, this would indeed be incompatible with maintaining cell life: Lysosomes have mechanisms for partial acidification that do not depend on vATPase activity.
The lysosome is, in fact, the only organelle common to all forms of autophagy. New evidence even points to additional critical roles of lysosomal proteolysis in regulating upstream events in autophagy. For example, the mTOR complex controlling autophagy induction is located on lysosomes (7) and is regulated by amino acids released during lysosomal digestion of autophagic substrates (8). Moreover, many of the genes regulating autophagy induction and autophagosome formation are transcriptionally controlled by the TFEB-activated CLEAR network of genes that regulates lysosomal biogenesis (9).
The basic misunderstanding of autophagy reflected in the Annaert commentary explains subsequent unsupportable statements that autophagy impairment is not established in Alzheimer's disease and AD models. Work in our and others' labs unequivocally documents autophagy-related pathology in AD and AD mouse models (10-16), and links it to AD-related mechanisms that drive lysosome dysfunction (1,10-14,16-26). The additional finding that PS1 hypofunction selectively disrupts lysosomal proteolysis required for autophagy and for recycling of unneeded endocytosed proteins (1) is just one more facet of a rapidly expanding literature underscoring the pathogenic importance of lysosomal proteolytic failure in AD, PD, and some other neurodegenerative disorders (27,28). As also discussed below, scant evidence has been presented by the Annaert group or others for any alternative mechanism that could explain the autophagy phenomenon or specifically the lysosomal calcium defect they describe. We look forward to new, evidence-based ideas on this important area of AD research.
Annaert and Coen challenge the evidence for autophagy defects in AD and PS-deficient models, mainly by citing a consensus finding from reports by Neely et al. (2), Wilson et al. (28), and Zhang et al. (3) that autophagosome formation is normal in PS-deficient cells. We also reported that autophagosome formation is normal in PS-deficient cells (1). Therefore, we all agree on this point. But it is the second part of the autophagy process that is defective in PS-deficient cells. Here again, there seems to be little disagreement among most labs (1,2,28-30) that some aspect of the lysosomal clearance of autophagosomes is impaired by PS deficiency. It is worth reiterating that our studies of PS deficiency revealed diverse evidence of lysosomal proteolytic dysfunction in addition to acidification deficits and vATPase instability.
While the calcium data reported in Coen et al. clearly imply altered lysosome function (30), Annaert dismisses this, and autophagy deficits in general, as consequences of a hypothetical defect in AP-lysosome fusion. Alteration of AP-lysosome fusion is a reasonable idea, and not be mutually exclusive of pathogenic lysosomal deficits, but where are the data to back up the hypothesis? Coen et al. provide no evidence in their paper to demonstrate such a defect, or to clarify a possible mechanism, or to show how this hypothetical defect is mechanistically linked to either calcium dysregulation or to presenilin. In a previous paper, the Annaert group suggested that AP-lysosome fusion was impaired based on one preliminary finding in PS-deficient cells (29), but fusion impairment was not confirmed by either Wilson et al. (28) or Lee et al. (1). Zhang et al. (3) and Neely et al. (2) didn't investigate AP-lysosome fusion. Specifically, Neely et al. concluded that "our results do indicate that presenilin loss leads to dysfunction at the lysosome." Moreover, neither of Annaert's reports provides evidence linking lysosomal calcium release to this putative defect in AP-lysosome fusion.
It may ultimately turn out that AP-lysosome fusion is not normal, particularly when lysosome pH is altered. However, even if solid evidence for a fusion deficit existed, why would one assume that it is primary rather than secondary to lysosomal defects, which they and we have demonstrated? How does this proposed abnormality in fusion come about? In this regard, we have evidence that calcium defects in PS-deficient cells are secondary to lysosome pH elevation, which is a well known basis for altering lysosomal calcium. If there are alternative molecular mechanisms and supporting evidence, we look forward to learning what they are and integrating them into our current thinking.
We have already pointed out some possible reasons why pH measurements by Zhang et al. and Coen et al. yielded different results from ours. Despite the detailed discussion of their procedures, we remain puzzled why Coen et al. presented LysoSensor data as ratios and not pH values, especially given that a similar procedure used by Zhang et al. yielded pH values that were frequently well above 7.0 when they interpolated pH values from LysoSensor ratios using their standard curve. Also, we have previously expressed our concerns about the vATPase data in Zhang et al. and Coen et al. Our published data are fully supported by our latest findings that will be forthcoming. The diminished V01a levels in PS1-deficient cells are consistent with PS1 holoprotein having chaperone function, which is not necessarily restricted to effects on this vATPase subunit. Consistent with such a function, misfolding/degradation of unchaperoned the V01a subunit is critical for its failure to reach lysosomes.
Finally, we hope Annaert and Coen's last statement in their comment was not meant to imply that our own observations on lysosomal calcium dysregulation were made after reading a limited treatment of their calcium finding in an earlier version of their manuscript submitted to Cell. Given our three decades of work on lysosomes, our longstanding awareness of lysosomal acidification deficits in PS-deficient cells, and the well-known connection between pH and calcium regulatory mechanisms in lysosomes, the logic of investigating lysosomal calcium regulation has occurred to us more than once, certainly dating back more than a year.View all comments by Ju-hyun Lee