Thursday, March 15, 2012

Rising star headlines Boston gospel fest

For Israel Houghton & New Breed, 5,000 seats may seem like playing in the lobby.

Back in Houston, his home church Lakewood Church Central -- pastored by Joel Osteen -- is set to move this July into what was home to the Houston Rockets basketball team, the renovated Compaq Center coliseum that seats 16 thousand people. According to Forbes Magazine, some 28,000 attend the Rev. Osteen's services every weekend. The church's congregation is one of America's most diverse.

Even so the Stellar male vocalist of the year is excited about performing this Saturday at the Bank of America Pavilion on Boston Harbor. He said that it's his first opportunity to perform at a ticketed …

Free throws lead URI to 86-77 win over Toledo

Kahiem Seawright had 16 points and went 4-for-4 on free throws as Rhode Island beat Toledo 86-77 Tuesday night at Savage Arena.

The Rockets and the Rams had 32 field goals each, including eight three pointers per team. Toledo lost the game on free throws, hitting just 5-of-18 to Rhode Island's 14-of-22. It was the 10th straight game during which Toledo made 10 or fewer free throws.

Rhode Island (11-4) also scored 24 points off turnovers to Toledo's eight. …

Kennedy assails Bork in visit here

Sen. Edward F. Kennedy brought his campaign against SupremeCourt nominee Robert H. Bork to Chicago on Sunday, saying PresidentReagan should not be allowed to "tilt the balance" of the court forthe next generation.

"We must not permit the president to rule our future through thenomination of Robert Bork,' " Kennedy (D-Mass.) said, adding that theReagan Administration has heightened its public relations campaignfor Bork "to change Mr. Bork's spots and portray him as a model ofmoderation."

But the Massachusetts Democrat said, "The public positions ofMr. Bork speak louder than the public relations of the White House .. . and Ronald Reagan is wrong to try to put him …

Wednesday, March 14, 2012

Jury Convicts 4 White-Supremacists

SANTA ANA, Calif. - A jury convicted four leaders of a white-supremacist prison gang Friday on charges they used murder and intimidation to protect their drug-dealing operations behind bars.

The trial is part of what is believed to be one of the largest federal capital cases, with more than a dozen people potentially facing the death penalty.

Barry "The Baron" Mills, Tyler "The Hulk" Bingham, Edgar "The Snail" Hevle and Christopher Overton Gibson were the first defendants to stand trial in the federal racketeering case aimed at dismantling the feared Aryan Brotherhood.

They all were convicted under Racketeer Influenced and Corrupt Organizations law, and offenses …

War Pass Fine After Last-Place Finish

War Pass is still on the Kentucky Derby trail despite his last-place finish in the Tampa Bay Derby.

Trainer Nick Zito told The Associated Press on Sunday that his colt suffered a few cuts on his leg after being jostled at the start of Saturday's race at Tampa Bay Downs.

War Pass had won …

4-legged Iraq war hero headed for life of leisure

He's a hero on four legs, a returning Iraq war vet who patrolled the mean streets of Baghdad with only his keen senses standing between American soldiers and death at the enemy's hand.

War dog Dexter, known to the government as MWD Dexter C067, is moving at the end of the month to far north suburban Spring Grove to start a life of supreme leisure, well-earned after months prowling around the Abu Ghraib prison.

"I wouldn't be here if it weren't for these dogs," said Danny Scheurer, 30, an Iraq war vet, now living in Round Lake. He worked with similar dogs in his basic infantry unit. "These dogs saved my life."

Scheurer, who founded the military dog-saving …

A working memory test battery for MATLAB

We present a battery of four working memory tasks that are implemented using MATLAB and the free Psychophysics Toolbox. The package includes preprocessing scripts in R and SPSS to facilitate data analysis. The four tasks consist of a sentence-span task, an operation-span task, a spatial short-term memory test, and a memory updating task. These tasks were chosen in order to provide a heterogeneous set of measures of working memory capacity, thus reducing method variance and tapping into two content domains of working memory (verbal, including numerical, vs. spatial) and two of its functional aspects (storage in the context of processing and relational integration). The task battery was …

David Cook wins 'American Idol' by 12 million votes

The grown-up rocker triumphed over the smooth-voiced kid as David Cook claimed the "American Idol" title Wednesday, and it wasn't as much of a surprise as it seemed.

While the judges all but crowned 17-year-old David Archuleta the night before, the voters decided otherwise _ and in a huge and unexpected way. Host Ryan Seacrest said before the results that that the margin was 12 million votes, and it turns out they broke in the favor of the 25-year-old from Blue Springs, Mo.

Cook was overcome with emotion, bending toward the stage after his name was announced. When he stood up, his eyes were filled with tears, the second time in as many nights that the …

Indians feast on Adkins Reliever's steady diet of bad pitches in 10th makes Sox look sick

INDIANS 11, WHITE SOX 7

Confidence is the ingredient White Sox manager Ozzie Guillen keepstrying to add to pitcher Jon Garland's recipe for success.

Yet Garland still seems to find ways to stir up the mix.

When he was handed a plentiful batter of four runs in the firstinning Tuesday against the Cleveland Indians, Garland promptly handedback three runs in the top of the second. He gave up the lead by thefourth inning before his teammates tied it in the bottom of theinning.

Worse, the Sox took a cue from Garland, losing the lead and thenthe game 11-7 in 10 innings, their first extra-inning loss of theyear and Cleveland's first win in four extra-inning …

Ray Hargrove to blow in 'A Trumpet Summit'

Ray Hargrove to blow in `A Trumpet Summit'

Roy Hargrove is no novice, but he's a serious young and mature musician who is joining more seasoned jazz artists in Ameritech Jazz at Symphony Center's "A Trumpet Summit" such as Clark Terry, Roy Hargrove, Lew Soloff, Marcus Belgrave, and Hugh Ragin Friday, January 14 at 220 S. Michigan Ave.

Hargrove is truly one of the most exceptional artists in jazz. His technical accomplishment from both the innate perspective and learned development provide the foundation for his creative imagination and skilled innovation.

It was at Joe Segal's Jazz Showcase that one has witnessed his inventive method of performance and also during the …

EU: limited risk to EU children from China scandal

EU food safety experts have found only a limited risk to children in Europe from food imports from China, despite its health scandal over tainted milk.

Even in the worst case scenario adults in Europe would not exceed tolerable intakes of the industrial chemical melamine, said a European Union report released Thursday. Melamine in dairy products has been blamed for sickening 53,000 infants in China and killing four.

Only children who had a "high consumption" of toffee, chocolate or cookies containing high levels of contaminated milk powder would potentially run a risk in Europe, said the report from the European Food Safety Agency based in Parma, …

Beijing strives for plain English

BEIJING--This city has launched a campaign to wipe out"Chinglish," a version of English that results in weird and wonderful--but largely incomprehensible--phrases that amuse tourists but alarmthe authorities.

The "language mandarins" of Beijing have decided Chinglish is ablight and must be obliterated before the city hosts the OlympicGames in 2008.

The targets of the campaign range from the nonsensical to thecharming. A road sign on the Avenue of Eternal Peace, for instance,advised: "To Take Notice of Safe; The Slippery are Very Crafty," awarning that the pavement was slippery.

A sign in a …

Republicans were ground down by the Democratic juggernaut.

Outlook for Bolton nomination grim

The White House and Republican Senate leaders have a little betterthan two weeks to save John Bolton as ambassador to the UnitedNations after last Tuesday's fiasco in the Senate Foreign RelationsCommittee. All that can be promised is that their efforts on Bolton'sbehalf will be tougher and better organized than they have been sofar. That should not be difficult because they could hardly be worse.

Republicans, weak and disorganized, were ground down by theDemocratic juggernaut. Sen. George Voinovich of Ohio was so impressedby Democratic demagoguery that he impulsively dropped his support ofBolton, ending the narrow 10-8 committee tally for sending thenomination to the Senate floor. But since Voinovich is notoriouslyquirky and prone to break his Republican leash, the question ariseswhy the White House was not more attuned to making sure he was safelyon board.

Presidential aides have met with Voinovich since he jumpedoverboard, beginning the difficult task of reeling him in -- as wellas Senators Lincoln Chafee of Rhode Island and Chuck Hagel ofNebraska, Republicans who followed Voinovich away from Bolton. Evenif the committee majority somehow is restored, Chairman Richard Lugarwill have to defeat efforts by Democrats to bring in Bolton for anauto-da-fe.

The grim outlook for Bolton constitutes a major victory for theadversarial style practiced by Senate Democrats, with Sen.Christopher Dodd of Connecticut taking the lead. Bolton's undeniableconservative ideology has antagonized the State Department's liberalcadre and its senatorial defenders. His hard line on Fidel Castro hasalienated Dodd, whose long-term goal has been normalization of U.S.-Cuba relations. Yet, Dodd on Tuesday made the astounding statementthat his opposition to Bolton "has nothing to do with substantivedisagreements," only his personal characteristics.

Dodd, in demanding a postponement of a vote on Bolton, claimed itwas "rare indeed for me to express objection to a nominee." In truth,Dodd has been a serial objector to GOP nominees over the years. Hehas voted against Martin Feldstein (Council of Economic Advisers),James Watt (interior secretary), James Edwards (energy secretary),Raymond Donovan (labor secretary), William Clark (deputy secretary ofstate and interior secretary), Rex Lee (solicitor general), C.Everett Koop (surgeon general), Kenneth Adelman (arms controldirector), Edwin Meese (attorney general), Robert Gates (CIAdirector), Ted Olson (solicitor general), Porter Goss (CIA director),Alberto Gonzales (attorney general), and Supreme Court nomineesWilliam Rehnquist, Robert Bork and Clarence Thomas. He also opposedBolton for his current undersecretary of state position and kept thenomination of anti-Castroite Otto Reich as assistant secretary ofstate from reaching the Senate floor.

The only new element in Dodd's case against Bolton was the claimby Melody Townsel (self-described as a "vocal" outspoken Democrat)that she was mistreated by Bolton in a 1994 dispute in Moscow whenBolton worked in the private sector. Her claims were buttressed byWashington consultant Kirby Jones, and here again the Cubanconnection emerges. Jones is described by Newsweek as having "bettercontacts in Cuba than any other American" and by the New York Timesas "the man to see about business in Cuba."

Voinovich admitted he had not attended previous committee hearingson Bolton and what he knew was based only on what he had heardTuesday from Democrats. Chafee, indicating that he, too, wasswitching on Bolton, gushed about how thrilled he was to hear asenator change his mind after listening to another senator. Thosecomments could invite future demagoguery from Democrats.

Republicans always expect the worse from Chafee. But Voinovichtook the party by surprise. That surprise validates the opinion ofsenior Republican senators who consider this administration'scongressional outreach the worst they have seen.

The only serious Republican defense of Bolton on Tuesday was byfirst-term Sen. Norm Coleman of Minnesota. This passivity not onlyleads Democrats to believe they will prevent Bolton from going to theUnited Nations but also shows them the way to replicate this triumph.

Tuesday, March 13, 2012

Yuppies give Downer North's Moran boost

Hobnobbing with yuppies at a beach volleyball court isn't whatmost teenagers do during their formative years. Usually, they aremore interested in playing organized sports or hanging out withfriends.

But for Downers Grove North volleyball star Omar Moran, the FourLakes apartment complex in Lisle has provided more than a roof overhis head. It's given him a head start in sports, especiallyvolleyball, and fond memories of some caring adults who offeredunexpected friendship.

"I started playing volleyball the summer before my freshmanyear," said Moran, who moved from Peru to the United States at age 9. "I watched the people at Four Lakes playing volleyball and all of asudden they let me play. Then they helped me out. None of themplayed in college. They just played for fun. I still see some ofthem over the summer."

"Four Lakes is a young adult mecca," Downers North coach RayButkus said. "A lot of the men and women play volleyball there andthey take it pretty seriously. The adults took Omar under theirwing and he has a following of people who come to watch him play.

"He always has been the kind of kid who seems like a naturalwhen it comes to volleyball. But I think it's because he got goodtraining at a young age."

Moran has been a starter since his freshman year. So talentnever has been an issue. Butkus says the primary growth in his 6-1outside hitter is his ability to help others raise their level ofplay.

"When you're used to being the best, sometimes it's hard to keepyour head. But Omar has really matured," Butkus said. "In thepast, he never shared his skills with others. But now he hasdeveloped into a team player, not only by being a powerful player butby helping the others."

Moran's skills have improved, too, thanks to Butkus and a yearof training under coach Rick Butler at the Sports PerformanceVolleyball Club in West Chicago.

"My footwork was backwards but they (at Sports Performance)fixed it and I'm learning how to be quicker on my spike approach,"said Moran, who was an all-state selection last season.

Moran hopes to play volleyball in college, then coach the sport.

"I've been fortunate to be at Downers North," he said. "It'sone of the few schools that had a volleyball program when I was afreshman. And coach Butkus is always positive and always helps meto get back on my feet whenever I'm having a problem. He reallysupports me."

Iran digging graves for US troops if they attack

TEHRAN, Iran (AP) — Iran has dug mass graves in which to bury U.S. troops in case of any American attack on the country, a former commander of the elite Revolutionary Guard said.

The digging of the graves appears to be a show of bravado after the chairman of the U.S. Joint Chiefs of Staff, Adm. Mike Mullen, said last week that the U.S. military has a contingency plan to attack Iran, although he thinks a military strike is probably a bad idea.

The U.S. and some of its allies accuse Iran of using its civilian nuclear program as a cover to build nuclear weapons. Iran has denied the charges, saying its nuclear program is geared merely toward generating electricity, not bomb.

Gen. Hossein Kan'ani Moghadam, who was the Guard's deputy commander during the 1980s, said graves have been dug in Iran's southwestern Khuzestan province, where Iran buried Iraqi soldiers killed during the ruinous 1980-88 war between the Islamic republic and Iraqi leader Saddam Hussein's regime.

"The mass graves that used to be for burying Saddam's soldiers have now been prepared again for U.S. soldiers, and this is the reason for digging this big number of graves," Moghadam told The Associated Press Television News late Monday. He did not say how many were prepared.

Footage obtained by APTN showed a large number of empty, freshly dug graves in a desert region of Khuzestan. The digging of the graves was first reported earlier this week by Iran's semiofficial news agency Fars.

Moghadam repeated warnings that Iran will retaliate against U.S. bases in the Gulf if there is an attack on Iran. The U.S. Navy's 5th Fleet headquarters is based just across the Gulf from Iran in Bahrain.

If U.S. forces attack, "Iran will have no choice but to strike the American bases in the region," he said. "The heavy costs of such a war will not be just on the Islamic Republic of Iran. America and other countries should accept that this would be the start of an extensive war in the region."

The war of words has intensified between Iran and the United States after the U.N. Security Council imposed a fourth round of tougher sanctions in June in response to Iran's refusal to halt uranium enrichment, a technology that can be used to produce nuclear fuel or material for an atomic bomb.

The U.S. and Israel have said military force could be used if diplomacy fails to stop what they suspect is an Iranian nuclear weapons program.

Jets Bomb Tamil Rebels' Naval Center

Sri Lankan air force jets bombed the Tamil Tiger rebels' naval headquarters Saturday while the group's sea wing leaders were holding a meeting there, the military said.

Pilots confirmed hitting the base near Puthukudiyiruppu village in rebel-held Mullaitivu district but did not provide any details about any damage, military spokesman Brig. Udaya Nanayakkara said.

The base was bombed because officials had received information that the sea wing leaders were meeting there, Nanayakkara said.

Earlier, a defense official said the rebels' powerful naval chief, known as Soosai, was at the base during the bombing. The official spoke on condition of anonymity, citing government rules.

Nanayakkara said, however, that Soosai's presence was possible but had not been confirmed.

The Tigers did not immediately comment on the government's claims.

The air raid was part of the government's campaign to kill the guerrillas' top brass and crush their decades-old separatist war.

In November, the air force killed the Tigers' political wing head, S.P. Tamilselvan, believed by many to have been the rebels' No. 2 leader.

The military later claimed that top rebel leader Velupillai Prabhakaran had been injured in another air raid. The guerrillas, however, denied he was hurt.

Fighting has raged around the rebels' de facto state in the north since the government announced earlier this month that it was pulling out of a Norwegian-brokered cease-fire, which has long been ignored by both sides.

More than 500 people have been killed since the cease-fire officially ended, according to the military.

A series of battles across Sri Lanka's north killed 17 guerrillas and one soldier Friday, the military said.

Clashes along the front lines in Vavuniya district, just south of the rebels' de facto state, left 13 Tamil Tigers dead, a defense ministry official said.

Separate clashes in nearby Mannar district killed four insurgents and one soldier, he said on condition of anonymity because he was not authorized to speak the media.

It was not possible to independently verify the military's claims because the fighting took place deep in the northern jungles, where access is restricted. Both sides often release inflated casualty figures for their opponents while lowering their own.

The Tigers have been fighting since 1983 for an independent state in the north and east for the country's ethnic Tamil minority after decades of being marginalized by Sinhalese-dominated governments. The fighting has killed more than 70,000 people.

Curiosity shop: Once home to sweets and sodas, Mount Hope's Bon Bon Hardware Store is rich in history

DAILY MAIL STAFF

MOUNT HOPE - Floyd Bonifacio has served more sodas, ice creamcones and Cokes than he can count during his days behind the fountainat his Bon Bon Hardware Store here.

Both the combination of soda fountain and hardware store and theunusual name garner attention, though the name really doesn't haveanything to do with candy.

"People don't think about a hardware store having a soda fountainin it, but that's how we started," Bonifacio said.

"I've probably served thousands of ice cream cones," he said."When the kids were in school across the street, they'd come here andget a soda and a hotdog for 25 cents or a chocolate malt or a Cokefor a nickel.

"And ice cream? Well, we served vanilla, chocolate, maple nut,strawberry and sometimes chocolate ripple," Floyd remembered. "AfterWorld War II we couldn't get regular ice cream so we ran soft-servefor a while."

The store was started by his father, Sam, in 1920.

"He was a shoemaker in Glen Jean and at the time Mount Hope was aboomtown," Bonifacio said. "He decided to move here and open a shoeshop. The store also had a soda fountain in it so he partitioned offthe shoe shop to one side with a separate entrance and opened up thesoda shop in the other."

He believes his father played off the family name in coming upwith a name for the store.

In the early days, there were plenty of hard-working miners inMount Hope and, therefore, lots of customers for the store.

"We stayed busy," said Floyd, who was born above the store in1924. "I was 6 years old when I got my first job shining shoes. I got10 cents a shine and did pretty well.

"My dad fixed a lot of shoes in those days because the town was soprosperous," he said. "We'd have miners coming in each day for a sodaor to have their shoes fixed or shined."

When World War II broke out, Floyd was a junior in high school andhe went into the service.

"They were taking just about anyone who wanted to go. I went inthe Navy and was shipped to the Pacific."

His first job after the war was running the soda fountain.

"My dad died in 1941 and my mother, Maria, didn't want to continuethat part of the business," he said. "After I came back in 1946, weclosed up that part and remodeled the store, expanding theconfectionary.

"We sold hot dogs, ice cream, magazines, things like that," herecalled.

"We had marble-topped tables and eight wooden booths and a 1946jukebox that everyone in town loved. I wish I still had it."

The 20-foot-long soda fountain structure is made of marble and hasnickel working parts. "We have found some parts of the fountain thatare stamped 1900 so we guessed that's when it was made," he said.

The marble counter top has a beautiful patina from years of beingshined and polished.

In the 1940s, Cokes were 5 cents and hot dogs were 10 cents.Business was booming from the kids, the families and the coal minerswho frequented the place.

"We got Coke syrup in 50-gallon wooden barrels and we'd set it upwith a wooden faucet. We'd draw up a gallon at a time and fill up thepumps in the fountain," he said.

"We'd pour in a shot (about 3 inches up in a 6-ounce glass) andthen fill the rest of the glass with soda. We could add cherry,vanilla, chocolate or whatever the customer wanted to the glass tomake that soda different."

As the business grew, Floyd decided to branch out and buy themovie theater across the street. The Princess Theater was owned by afilm distribution company that leased it to Bonifacio and his wife,Phyllis.

"After a while, we decided to buy," he said. "I ran the projectionroom while Phyllis worked the front, taking up the money."

The 500-seat theater was nearly always full in the 1950s and 60sand sometimes he or Phyllis would have to run across the street tothe store to get more chairs.

The Bonifacios were having so much fun they decided to buy anothertheater up the street.

"You have to remember that the town was booming at the time," herecalled. "The other place was the Mount Hope Theater. A town thatsupported two movie theaters was a big deal.

"We were showing six pictures a week across the street and six upthe street at the other one. We'd book 40 pictures a month in boththeaters. You have to remember that that's all people had to dothen."

As the town dwindled in size, the mines played out and the minersdied off, the Bonifacios sold the Princess Theater. About 12 yearsago, they sold the Mount Hope Theater.

In addition to his business ventures, Floyd has served 22 years incity government, six years as recorder, four years as city councilmanthen 12 years as mayor.

The focus of the store slowly changed to hardware as the fountainbusiness slacked off. The Bonifacios used to come in at 4 a.m. tostart their day, but now wait until 6:30 and close at 4 p.m. And eventhe hardware business is slow these days.

"People come in now and want four nails instead of dozens,"Bonifacio said. "Business is not what it used to be."

While Bonifacio, 82, has had a few health problems recently, heconsiders himself blessed. He and Phyllis have been married 50 yearsand they have three sons.

"As long as my health holds out, I'll keep going," he said. "Mymother lived to be 101. Maybe I'll make it that long."

Contact writer Mary Childress at maryc@dailymail.com or 348-4879.

S. Side firm makes lots of bread by making McDonald's croutons

David N. Moore and George L. Johnson started from scratch andnow are making a lot of dough.

Johnson, 41, and Moore, 33, were on the fast track atMilwaukee-based Miller Brewing Co. in 1986, when opportunity camecalling in the form of David P. McDuffie, home office manager forbusiness development at Oak Brook-based McDonald's Corp. McDuffie waslooking for a minority-owned company or aggressive individualswilling to start a business. The company then would be given theopportunity of suppling McDonald's restaurants with croutons for thefast-food chain's salads. McDuffie said he got Johnson's and Moore'snames and those of several other likely candidates through social andbusiness connections.

"There wasn't a guarantee, but there was an opportunity to dobusiness with McDonald's," he said.

It was the chance Johnson and Moore had been looking for.Since the two had met several years earlier at a Miller employeegathering, they had talked repeatedly about starting a business.They seized the opportunity and have built a successful company intwo important aspects. Their business, Quality Croutons, 825-29 W.37th Pl., not only is booming financially, it also has a reputationas place where employees love their work and encourage others to jointhem.

At the time of McDuffie's call, Johnson was working for Millerin San Francisco as sales manager for Miller's Northern Pacificregion, and Moore was market development manager in Los Angeles.Both had been with Miller nine years.

The weekend following their conversation with McDuffie, Johnsonand Moore flew to McDonald's headquarters. It was the first of threetrips the two would make to Oak Brook in order to talk to thecompany's executives.

They also talked with McDonald's suppliers, and they readarticles about the fast-food chain's operations. When they made thedecision to leave Miller's corporate womb, they also went back toschool.

The two, who didn't have any experience in the baking industry,studied at East Balt Commissionary Inc., a South Side bakery thatsupplies hamburger buns to McDonald's.

McDuffie said Johnson and Moore learned all phases of the bakerybusiness and took home a report card with filled with A's. "They werevery astute. They picked up the baking business even quicker than wehad expected," he said.

Their ability to quickly learn also helped them get the supportof East Balt Commissionary in getting bank financing, McDuffie said.Moore said they received $2 million in financing from AmericanNational Bank & Trust Co.

The investment in Johnson and Moore by McDonald's, East Balt andAmerican National Bank has already paid off and is expected to showmore returns in the future.

A year after Quality Croutons opened its doors in May, 1987, ithad revenue of more than $3 million. This year, the two expect salesto exceed $6 million, and in 10 years they hope to do $35 million inbusiness.

Before Quality Croutons' success began showing up on the balancesheet, it began showing up on the company's shop floor and in itsoffices. It's a place where people will do almost anything to gethired.

For example, when Diane Simmons interviewed for the job ofoffice manager at Quality Croutons, she was so impressed with thecompany's management and employees, Simmons used an unorthodox andunproven method to get hired.

"I told George, I was going to talk him into hiring me bycommunicating with him telepathically," she joked.

Simmons got the job and even took a 12 percent pay cut from herold job in order to work at Quality Croutons.

"I never liked working until I came here," said Simmons. "Theyrespect my opinion and I have input."

A number of Quality Croutons' 39 other employees also saidworking for the company is the best job they've had because of theiraccess to management, the firm's family atmosphere, its innovativebenefits program and the high quality of its products.

"This company is fantastic. We put out a quality product and aslong as we're doing that, the business will keep growing," said JimAlexander, Quality Croutons' production supervisor.

Ironically, Moore, who serves as the company's vice president,said hiring employees was their hardest job.

"It's very difficult to find a good employee. When we opened,we wanted people with baking experience, but we couldn't findanyone," he said.

Moore, a Milwaukee native, said they later dropped that idea andstarted looking for employees who were trainable, intelligent,energetic and willing to work with others as a team.

The two also put together a benefits program that would make thecompany attractive to job hunters.

Besides profit-sharing, major medical and dental coverage and a401(k) plan, Quality Croutons gives each of its employees six paidsick days a year. But, "At the end of the year, the employeesreceive a bonus of 70 percent of their base pay for each sick daythey don't use," Moore said.

The company also operates four days a week, 10 hours a day fromSeptember to February so employees are guaranteed one day off duringthe week to take care of personal matters.

"We made a commitment to hire single mothers, but we realizedthey needed a day off to go to the doctor, dentist or their child'sschool," said Johnson, who serves as the company's president. "If awoman has to run those errands, then taking care of those needsbecomes more important than coming to work. So this type of workschedule has helped to cut down on absenteeism."

When Quality Croutons gets into its busy season and beginsoperating sometimes six days a week, employees can use their threepersonal days to take off from work. Moore said crouton sales, andthus production, increase during the summer because people eat moresalads.

Johnson said he and Moore came up with their approach toemployees in order to create a family atmosphere at Quality Croutons.

It seems to be working.

"I have been in the baking business since 1962," said RexHigdon, Quality Croutons' general manager, "and this is where I wantto be."

Bernadette Hill, a machine operator, said she likes workingthere because Johnson and Moore are available to discuss bothpersonal and professional problems.

Quality Croutons happy/productive atmosphere has helped thecompany increase the number of McDonald's restaurants it supplies.It also has helped the company pick up new customers.

When the company opened its doors, it supplied 50 percent of thecroutons to 6,000 McDonald's restaurants east of the MississippiRiver, and in Texas and Oklahoma. The company has now grown tosupply McDonald's with 85 percent of its croutons.

Quality Croutons also sells its product to supermarket chains,including Lucky Stores, Jewel, Treasure Island and Kroger. The firmsupplies Pizza Hut, United Airlines and Houston's Restaurants.

Bob Lynn, vice president of research and development forHouston's, an Atlanta-based company, says Johnson and Moore representa growing trend in business "where people are willing to work hardand pay more for a product that's high quality."

Johnson said he has made that his goal: "I want to be recognizedby the industry and consumers as a quality manufacturer." Moore saidQuality Croutons are better than its competitors because they aremade from fresh bread, not stale or rejected bread.

As a result of its growing business, Quality Croutons is lookingfor additional space because it's busting out at the seams of 37,000square-foot factory/headquarters.

Solar eclipse will darken Europe

Solar eclipses are both routine and predictable. The shadow ofthe moon streaks across the Earth about once every 18 months andastronomers can forecast its precise path centuries ahead.

But next Wednesday's total eclipse has Europe talking.

The main reason is that the eclipse path happens to cover a moredensely populated swath of the globe than any of its predecessors.There may also be a touch of millennial feeling about the last greatcelestial event of the century.

The track of totality - in which the face of the moon blots outthe whole of the sun - is just 75 miles wide. It starts out atsunrise off the coast of Nova Scotia in Canada and dashes eastwardacross the north Atlantic at 1,550 m.p.h., making landfall inCornwall, England.

The path then crosses France, Belgium, Luxembourg, Germany,Austria, Hungary, Romania, Turkey, Iran, Pakistan and India, beforedying at dusk in the Bay of Bengal. Cities covered include Plymouth,Rouen, Stuttgart, Munich, Bucharest and Karachi.

A partial eclipse, in which the moon bites away more than half thesolar surface, will be visible over all of Europe and the westernhalf of Asia.

Many people will find the partial solar eclipse boring. All theexcitement and romance comes with totality, which tens of millionswill experience.

Assuming the sky is clear, the real action begins when just a thinsliver of sun is left. The temperature will have dropped by a coupleof degrees and the wind will abate. Confused birds and animalsprepare to roost. There may be weird light effects on the ground,with the leaves of trees and bushes acting like pinhole cameras andprojecting tiny images of the crescent sun; refraction patternscalled shadow bands may race across the Earth.

In the final moments before totality, the last direct rays ofsunlight will shine through one or more lunar valleys, creating adiamond ring effect known as Bailey's beads. Then "the celestialflower opens its white petals," as eclipse specialist Francisco Diegoof University College London puts it.

The "petals" are the corona, the rarefied solar atmosphere that wecannot normally see because it is outshone by the sun. The sun iscoming into the most active phase of its 11-year cycle, so there is agood chance of seeing loops and streamers of glowing gas heading outinto space.

Although modern astronomers have sophisticated observatories forstudying the sun from Earth and space, including instruments forblotting out the solar disc and creating an artificial eclipse, somestudies of the corona are best made during an eclipse.

Americans need not feel left out entirely. Although a partialeclipse will be visible only in the extreme northeastern part of thecountry, cable television viewers will get to see the total eclipseon the Discovery Channel. It will carry live coverage from 5 to 8a.m. Wednesday Chicago time.

The last total solar eclipse in the mainland United States tookplace in 1979. The next is due Aug. 21, 2017.

Contributing: Associated Press

NZ central bank again holds key interest rate

WELLINGTON, New Zealand (AP) — New Zealand's central bank held its key interest rate at 3 percent Thursday as a weak, slow recovery continues, and the bank signaled it's in no hurry to hike rates.

This second consecutive rate hold follows two successive quarter-point increases that lifted the rate to 3 percent from its record low of 2.5 percent, where it was held for 13 months to counter the worldwide recession.

The Reserve Bank said some domestic data had turned "out weaker than projected," as "continued household caution has seen consumer spending and housing market activity remain muted, and many firms have become less optimistic about their future prospects."

Disappointing gross domestic growth figures for the June quarter showed the economy grew a mere 0.7 percent in the year to June 30, and the consensus of observers suggests the economic recovery remains patchy and fragile.

Reserve Bank Governor Alan Bollard said that continued high export prices, along with reconstruction and repairs after a magnitude-7 earthquake in the southern city of Christchurch that damaged 50,000 homes, would support activity over the coming year.

While expecting inflation to move higher because of this month's rise to 15 percent of the good and services tax, up from 12.5 percent, Bollard said the subdued state of domestic demand suggested the resulting inflation spike would have limited impact. Currently, annual inflation is at 1.5 percent.

He warned that downside risks continue to cloud the global growth outlook with high public and private debt inhibiting recovery in many developed economies. But he noted that strong growth continues in China, Australia and emerging Asia, offsetting the wider risk.

Observers noted the dovish outlook of the review, as the bank delayed further lifting its key rate into 2011.

"While it is appropriate to keep the OCR (official cash rate) on hold today, it remains likely that further removal of monetary policy support will be required at some stage," Bollard noted.

The New Zealand dollar response was muted, rising 15 points to US$0.7449 half an hour after the release, from US$0.7434 ahead of the announcement.

Using molecular beacons to quantify low levels of type I endonuclease activity

The ability to quantify residual type I endonuclease has been hampered by the lack of methods that possess the required performance, sensitivity, sample throughput, and economy. A novel use of molecular beacons may be the answer to this problem.

Detecting and quantifying type I endonuclease activity is important in several areas of biopharmaceutical product development. Measuring the activity of a product such as the recombinant human DNase Pulmozyme, used to treat cystic fibrosis, is one such application (1). Another important application is quantifying trace residual endonuclease levels in biopharmaceuticals that use a type I endonuclease to remove residual host-cell DNA during processing. One representative of type I endonucleases, benzon nuclease, is a broad-specificity extracellular protein isolated from Serratia marcescens that degrades both single- and double-stranded DNA and RNA with no apparent sequence specificity (2). Residual testing for endonucleases added to processes for removal of excess DNA is now critical in light of several recent publications suggesting that permeabilized mammalian cells treated with benzon nuclease showed signs of chromosomal aberrations (3-5). This finding underscores the need for residual endonuclease assays with high sensitivity.

A number of assays are described in the literature for quantifying type I endonuclease activity. The standard hyperchromicity assay described by Kunitz for quantitating DNase I activity is a widely accepted assay but requires extensive manipulations, including an acid precipitation step before reading (6-7). The measurement step for a hyperchromicity assay is an A26o determination, which requires that samples and standards be read individually in a quartz cuvette. That requires extensive hands-on time during analysis and cannot easily be adapted to a high sample throughput format. Additionally, the method is relatively insensitive, and in our tests its lower limit of quantitation (LLOQ) was 0.7 U/mL of benzon nuclease.

Several alternatives to hyperchromicity assays have become available in the past few years. A higher throughput colorimetricbased assay described by Sinicropi exploits a color shift in methyl-green labeled DNA on enzymatic degradation (8). This assay has superior sensitivity to the hyperchromicity assay, but the preparation of the substrate is time consuming, and the prepared substrate has a brief storage life. It does have a higher sample throughput, but in our tests it lacked the desired sensitivity to be used as a trace residual assay, and it had problems overcoming sample matrix interference. A commercially available ELISA kit uses an antibody specific for benzon nuclease in a capture-type format with a stated lower limit of quantitation of 0.5 ng/mL, equivalent to about 0.5 U/mL of benzon nuclease. Unfortunately, the kit is prohibitively expensive and offers no significant increase in sensitivity over the hyperchromicity method.

This article describes a homogeneous format (with no wash or separation steps), microplate-based quench fluorescence assay that uses molecular beacon technology to quantify type I endonuclease activity in a number of sample matrices. We then compare the sensitivity of this method with those of existing assays.

Molecular beacons are described in the literature as sensitive tools for detecting specific DNA target sequences (9). A molecular beacon is an oligonucleotide with a specific target probe sequence flanked on both its 3' and 5' sides by a complementary stem sequence terminating with a fluorophore at the 5'-end and a quench molecule at the 3'-end. When not hybridized to its target sequence, the molecule generates minimal fluorescence because its stem region maintains the fluorophore and quench in close proximity. However, when hybridized to its complementary target sequence, the molecular beacon generates a fluorescent signal proportional to the amount of target sequence present because it separates the quench from the fluorophore.

We hypothesized that a nonspecific nuclease would unquench the fluorescence of the beacon, providing the foundation for the beacon endonuclease (BEN) assay. The original substrate we investigated was a 34-mer with a 7-nucleotide stem region. Figure 1 provides a conceptual view of the BEN assay. Two additional experimental substrates that varied the oligonucleotide length (4 and 8 nucleotides) or eliminated the stem region (no stem or a two-nucleotide stem) were also evaluated in the BEN assay system. The advantage of those smaller oligonucleotides was that their smaller size allowed them to be synthesized less expensively because no extensive purification was required. This assay can quantify low levels of type I endonuclease activity in a number of sample matrices using commercially available reagents. To our knowledge, it is the most sensitive quantitative endonuclease activity assay reported to date.

Materials and Methods

Molecular beacons of three different lengths and sequences (Figure 2) were obtained commercially from the Midland Certified Reagent Company (Midland, TX). Benzonase was obtained from Nycomed Pharma (Uppsala, Sweden), and the Pulmozyme was purchased by prescription from Genentech (South San Francisco, CA). OmniCleave was obtained from Epicentre Technologies (Madison, WI). Stock TrisHCI (1M, pH 7.5) solution was purchased from BioFluids, Inc. (Rockville, MD), and Dulbecco's phosphate buffered saline solution was purchased from Quality Biological (Gaithersburg, MD). HPLCgrade reagent water was used for all solution preparations and was obtained from VWR Scientific (West Chester, PA). Black 96-well OptiPlates came from Packard Instrument Company (Meriden, CT), and 96-tube dilution boxes were obtained from United Laboratory Plastics (St. Louis). The Cytofluor 4000 reader was purchased from PE Biosystems (Foster City, CA). The humanized recombinant monoclonal antibody Synagis came from MedImmune (Gaithersburg, MD). Unless specified, all other reagents were procured from the Sigma Chemical Company (St. Louis). BEN protocol. Endonuclease stock was diluted in assay buffer (20 mM Tris, 2.5 mM MgCl^sub 2^, 0.1 % w/v bovine serum albumin (BSA), pH 7.5) in a sterile 96-tube dilution box in 150 (mu)L volumes to construct a standard curve. Samples, spiked samples, and controls were likewise diluted in assay buffer in the dilution box. The stock molecular beacon (beacon 1) was prepared in phosphate buffered saline (PBS) to a concentration of 14.5 (mu)M and was further diluted to 1:100 in assay buffer to prepare the working solution. Then, each tube in the dilution box received 150 (mu)L of that working solution using a 12-channel pipette. The box was sealed with Parafilm (Structure Probe, West Chester, PA), vortexed, and placed in an incubator in the dark at 37 degC, 100% humidity for the specified incubation period (6 or 24 hours). Before analysis, the box was taken from the incubator, vortexed, unsealed, and 200 (mu)L volumes were transferred to a black OptiPlate.

The plate was read in a Cytofluor 4000 plate reader with a 99-second shake cycle, then read with an excitation wavelength of 485 nm and an emission wavelength of

530 nm. The resulting data were exported to Excel (Microsoft, Redmond, WA) and analyzed with SOFTmax (Molecular Devices, Sunnyvale, CA) to generate a standard curve from which sample concentrations were obtained. The acceptance criteria for the BEN assay were a correlation coefficient >= 0.990 of the 4-parameter-fit standard curve, spike recovery for each sample between 75-125% of theoretical, and a positive control value (0.04 U/mL endonuclease spiked into assay buffer) within 75-125% of the expected value. The assay LLOQ was calculated by first taking the mean fluorescent signal from six wells containing assay diluent, representing the background. Then

10 standard deviations were added to that mean signal to produce the LLOQ signal. That signal was regressed on the endonuclease standard curve to give a concentration that was set as the LLOQ of the assay.

EDTA effects. Endonuclease standard curves were prepared in assay buffer containing 0, 1, or 5 mM ethylenediaminetetraacetic acid (EDTA), the 34-mer beacon reagent (beacon 1) was added, and the completed assay was incubated for 24 hours as described above. The 24-hour incubation was chosen to simulate the most rigorous conditions to ascertain that there was no spontaneous breakdown of the beacon substrate.

DNA fragments. DNA fragments were generated by using benzon nuclease to digest calf thymus DNA for 48 hours at 37 degC in assay buffer. Fragments were harvested by passing the sample through a 1,000 MWCO NanoSep spin filter (Pall Filtron, Northborough, MA). The material was electrophoresed on a 10 X 20 cm 2% agarose gel for two hours and stained with ethidium bromide to verify complete digestion by comparing the digest to a DNA ladder ranging from 8 to 200 base pairs. The fragments were quantified using an A^sub 260^ measurement, and the purity was ascertained by calculating the A^sub 260^/A^sub 280^ ratio. Benzon nuclease was diluted to 0.0625 U/mL in 150 (mu)L of assay buffer, and 10 (mu)L spike volumes of the DNA fragments were added at final concentrations in the assay of 66.7 through 0.52 (mu)g/mL. After the addition of the 34-mer beacon substrate (beacon 1), the dilution box was incubated overnight and read as previously described.

Substrate stability. Beacon 1 (34-mer) was prepared at 14.5 (mu)M in PBS, and 0.3 mL aliquots were prepared in Nunc screw-cap CryoTubes (Nalge Nunc International, Rochester, NY). The tubes were wrapped in aluminum foil and incubated at -20 degC,

4 degC (standard storage condition), or 37 degC for the duration of the study. Tubes were sampled weekly and used as substrate in the standard (24-hour substrate incubation) BEN assay for a 0.01 U/mL benzon nuclease spike in assay buffer (n=3). The tube stored at -20 degC was thawed, sampled, and returned to -20 degC at each sampling. The fluorescent signal was averaged for the triplicate samples and plotted for each temperature/time condition.

Synagis spie recovery. Five separate lots of the formulated, lyophilized, humanized monoclonal antibody Synagis were reconstituted in water to a concentration of 100 mg/mL of total protein (25 mM histidine, 1.6 mM glycine, 3% wlv mannitol pH 6.0), diluted to 1:16 in assay buffer, and spiked with 0.04 U/mL of benzon nuclease. The spiked sample was two-fold serially diluted in assay buffer to a final dilution of 1:256. The BEN assay was then performed as described above, and the sample was incubated for 24 hours before analysis on the plate reader. The percent spike recovery was based on the amount of activity calculated from the spiked sample, minus the activity in the unspiked sample, divided by the theoretical spike value. Substrate incubation. The standard BEN assay (24-hour substrate incubation) was run using each of the three beacon substrates at a 4.45 X 10^sup -7^ M concentration. Each assay LLOQ was calculated as described previously.

Modified hypechromicity. The benzon nuclease standard curve was prepared in sample diluent (PBS containing 0.1%o w/v of BSA) in a total volume of 0.5 mL per concentration point. Samples were prepared either undiluted or diluted at 1:2 in sample diluent, and 40 (mu)L of standard or sample was then transferred to a 1.5 mL microfuge tube. All tubes received additions of 250 (mu)g of sonicated herring sperm DNA, Tris/magnesium buffer ([Mg^sup 2+^] = 2.5 mM), 500 (mu),g of bovine serum albumin (BSA), and benzon nuclease spike and were incubated in a 37 degC water bath for six hours. The reaction was quenched by adding 0.8 mL of cold 5% v/v perchloric acid to each tube. After vortexing, the tubes were microcentrifuged at 9,000g for five minutes, and 0.4 mL of the supernatant was transferred to a glass tube containing 1.0 mL of PBS. The A^sub 260^ was determined for each sample on a UTV spectrophotometer zeroed against PBS. Benzon nuclease activity levels were calculated based on a 4-parameter regression to the best fit curve, and percent spike recoveries were calculated based on the theoretical spiking level. Assay acceptability criteria include a correlation coefficient of >= 0.995 for the standard curve and spike-recovery levels of 80-120%.

Methyl green. The methyl-green protocol described by Sinicropi involves using salmon sperm DNA dissolved in buffer (25 mM HEPES, 1 mM EDTA, pH 7.5) and stirring for four days at room temperature (RT) at a concentration of 2 mg/mL (8). An aliquot of 11.5 mL of 0.4% crystal violetfree methyl green was added to 192 mL of the dissolved DNA, with the addition of 46 mL of buffer C (25 mM HEPES, 4 mM CaCl^sub 2^, 4 mM MgCl^sub 2^, 0.1% BSA, 0.01% thimerosal, 0.05% Tween-20, pH 7.5). A benzon nuclease standard curve and samples were prepared in buffer C, and 100-(mu)L aliquots were transferred to a 96-well clear microplate. Each well then received an addition of 100 (mu)L of the DNA substrate, was mixed with a multichannel pipette, and then was sealed and incubated for 24 hours at 37 degC. The absorbance was measured at 620 nm, and the nuclease activity was determined from the 4-parameter regressed standard curve. The assay LLOQ was determined by averaging the signal from six blank wells (assay diluent and beacon) and subtracting 10 standard deviations from that mean signal value. The next higher standard curve point with a coefficient of variation (CV) of =<15% was designated as the assay LLOQ and was determined for each assay mate

Assay Comparison. Benzon nuclease standard curves were constructed and run int he BEN and methyl-green assays using the protocols above. Each standard curve point was run in triplicate in the methyl-green assay and in quadruplicate in the BEN assay. Six replicates of the blank (no benzon nuclease) were also run in each assay in order to calculate the LLOQ.

Results

Assay Comparison. Benzon nuclease standard curves were constructed and run in the BEN methyl-green assays using the protocols above. Each standard curve point was run in triplicate in the methyl-green assay and in quadruplicate in the BEN assay. Six replicates of the blank (no benzon nuclease) were also run in each assay in order to calculate the LLOQ.

Results

Figure 3 shows typical benzon nuclease standard curves comparing the 6- and 24-hour versions of BEN to the modified hyperchromicity assay 3. The LLOQ for both incubation time variations of the BEN assay typically ranges from 0.0005 to 0.002 U/mL, whereas the LLOQ for the modified hyperchromicity method was determined to be 0.7 U/mL based on formal validation of the method. Figure 4 compares the standard curves of benzon nuclease analyzed in the BEN and methyl-green assays with resultant LLOQ values calculated as described previously. The LLOQ for benzon nuclease in the methylgreen assay was 0.3 U/mL, whereas the LLOQ for the BEN assay was 0.001 U/mL. Beacon signaL To better characterize the BEN assay and its limitations, a series of experiments assessed performance parameters. To verify that beacon signal generation in the assay was due to the action of benzon nuclease and not the spontaneous degradation of beacon by other routes, the effect of EDTA on standard curve signal generation was examined. Because the benzon nuclease enzyme has a requirement for Mg^sup 2+^ for enzymatic activity, the addition of the divalent canon chelator EDTA would be expected to inhibit the enzyme, reducing the signal generated as a result of enzymatic degradation of the beacon substrate (2). Figure 5 shows that increasing levels of EDTA in the assay buffer containing Mg^sup 2+^ result in a decrease in the signal generated, suggesting that the signal response is generated solely by the benzon nucleasemediated degradation of the beacon substrate. Only the 24-hour BEN assay was used in the EDTA study because it represented the most stringent test conditions.

Enzyme activity. To investigate whether the benzon nuclease digestion products inhibit enzyme activity, a spiking study was performed with a fixed concentration of benzon nuclease run in the BEN assay eithe: unspiked or spiked with increasing levels of DNA fragments generated as described previously. Table 1 shows that DNA fragment levels as high as 66.7 (mu)g/mL had no inhibitory effect on the enzyme, indicating that no fragment-induced inhibition of the enzyme took place when compared with the no-fragment-added control. The lack of product inhibition most probably accounts for part of the sensitivity of the assay and the ability to achieve maximal results and sensitivity within six hours of incubation, Shorter incubation times were tried (1-3 hours) but were unacceptable because of the increased variability observed for the standard curve (data not shown). Typically, an assay incubated overnight (16-24 hours) facilitates efficient work flow in the laboratory.

Stability. To assess substrate stability at various temperatures over time, as well as the effects of multiple substrate freeze-thaws on assay performance, a stability study was executed for the 34-mer beacon substrate (beacon 1) as described above. As shown in Figure 6, the mean fluorescent signal generated by the overnight incubation of 0.1 U/mL of benzon nuclease with the beacon was remarkably stable over the four week duration of the experiment under all conditions tested. Additionally, the beacon sample stored at -20 degC underwent a freeze-thaw cycle before each weekly test but showed no loss of signal-generating potential even at four weeks. These data suggest that the beacon substrate has exceptional stability over a wide range of storage conditions and that the beacon should have an extremely long shelf life when stored frozen. Additionally, rehydrated beacons stored in the dark at 2-8 degC for over one year have been successfully used in the BEN assay (data not shown). For those reasons, the 4 degC storage condition was chosen to be optimal for this study, and the variability in signal observed for that condition was considered normal. The test conditions of -20 and 37 degC showed no greater variability over the course of the study than that of the beacon stored at 2-8degC.

Sensitivity. Figure 7 shows the results obtained when beacons with variable nucleotide and stem region lengths were used as substrate in the 24-hour BEN assay to quantify benzon nuclease activity. Figure 2 compares the three beacons examined with the bolded nucleotides representing the stem region of the molecule. As shown in that figure, the 34-mer beacon (beacon 1 ) comprises a 7 base-pair stem region and a 27-nucleotide loop region, whereas the 8-mer beacon (beacon 2) has a 4-nucleotide loop and a 2-nucleotide stem region. Beacon 3 has a 4-nucleotide loop region with no stem region. Because it has been reported by the manufacturer that the end product of DNA digestion with benzon nuclease consists primarily of 2- to 4-mers, it would be expected that the sensitivity of the assay would decrease with decreasing nucleotide lengths. As Figure 7 shows, the substrate yielding the most sensitive assay was the 34-mer beacon (LLOQ = 0.001 U/mL), followed by the 8-mer (LLOQ = 1 U/mL), and then the 4-mer (LLOQ = 30 U/mL) beacons.

Impurities. Because the BEN assay is homogenous, with no wash or separation steps required, determining the effect of extraneous sample matrix proteins on assay performance is important. To determine the ability of the assay to quantify endonuclease in potentially interfering protein solutions, five lots of a 100 mg/mL concentration of humanized monoclonal antibody (Synagis) were spiked with 0.04 U/mL of benzon nuclease and tested at various twofold serial dilutions in the BEN assay under standard conditions. Table 2 shows the calculated spike-recovery rates for five different lots of the Synagis material. It shows that acceptable spike-recovery (75-125%) levels were obtained for samples diluted at 1:16 or greater, corresponding to a maximal Synagis concentration of 6.25 mg/mL. That particular dilution ratio was selected as a starting point based on previous work (data not shown) that indicated that materials at that concentration (100 mg/mL) required a minimum dilution of 1:16 to obtain acceptable spike-recovery results and therefore to maximize detection sensitivity.

Enzyme sore. Figure 8 shows representative standard curves generated in the BEN assay using three commercially available sources of type I endonucleases (Benzonase, Pulmozyme, and OmniCleave). All three endonucleases show a similar LLOQ, which demonstrates that the BEN assay can quantify type I endonuclease activity regardless of the enzyme source. The calculated LLOQ values were

0.00195 U/mL for OmniCleave, 0.000244 U/mL for Pulmozyme, and 0.000975 U/mL for benzon nuclease.

Discussion

A number of methods are reported in the literature to quantify type I endonuclease activity. Our version of the hyperchromicity assay had a determined LLOQ of 0.7 U/mL of benzon nuclease based on formal assay validation studies, which corresponds to about 0.7 ng of enzyme. That assay is undesirable for a number of reasons, but primarily because of its lack of sensitivity, cumbersome manual processing, use of hazardous reagents, and single cuvette reading steps. A commercially available ELISA-based assay kit to quantify residual levels of benzon nuclease, recently introduced by the manufacturer of that product, offers a significant improvement in sample throughput without the use of hazardous chemicals. The claimed sensitivity of that assay is 0.5 ng/mL (0.5 U/mL), which offers only a modest increase in sensitivity when compared with the hyperchromicity method. Moreover, a severe limitation of the ELISA assay is that because a benzon nuclease-specific antibody is used, only benzon nuclease can be quantified. Thus, from a production standpoint, if that source of endonuclease became unavailable, a new assay would need to be put in place to measure the new endonuclease level. Of more importance, the ELISA format lacks enzyme activity data, which is crucial to address the safety concerns surrounding low levels of endonuclease present in final products.

A recent article described a 96-well platebased homogenous assay to quantify enzyme activity in the type I endonuclease Pulmozyme (8). This method has the advantage of being homogenous (requiring no wash or separation steps), unlike the previous hyperchromicity and ELISA methods. In our tests, we obtained greater sensitivity with this method than with the hyperchromicity method, but the sample matrix interference made the assay unusable as a trace residual assay. Also, the preparation of the DNA-dye substrate takes three to four days, and the final substrate has a storage life at 4 degC of less than 30 days. Several drawbacks to the methyl-green assay method center on the physical properties of the dye-DNA complex. The authors reported that the dye-DNA complex was less stable in certain buffers, such as Tris, and that phosphate buffer was a potent inhibitor of the methyl-green assay.

In our experiments to develop an assay with improved sensitivity for endonuclease activity, our initial studies used a 34-mer beacon with the loop region using the sequence of the mouse beta-actin gene. We chose that sequence because it was well characterized by the company that synthesized the beacon reagents and because of the sequence-independence of type I endonucleases. Our initial experiments centered solely on benzon nuclease, and the BEN assay was characterized primarily using that nuclease. A large increase in sensitivity (nearly 1,400-fold) was gained by using the molecular beacon as a substrate when compared with the modified hyperchromicity method (Figure 3). Also, only modest sample dilutions needed to be performed to eliminate sample matrix interference with the assay, based on the spike-recovery studies (Table 2).

The 34-mer beacon substrate (beacon 1) demonstrated exceptional stability in the tests we performed. The EDTA studies showed that the signal generated in the assay was due to specific degradation of the substrate by benzon nuclease, and not by spontaneous breakdown of the beacon over the course of the assay. The beacon showed exceptional stability at a number of temperatures, even after four freeze-thaw cycles (Figure 6). Because of that stability, large amounts of beacon can be obtained and stored frozen for an extended period of time, yielding consistent results in the assay. Because the BEN assay uses covalently coupled fluorophore and quench on the nucleotide structure, the substrate is relatively resistant to buffer and pH changes compared with the noncovalent dye-DNA complex of the methyl-green assay. Thus, the BEN assay allows a greater subset of sample types to be successfully tested in the method. Another advantage of the BEN assay over existing assays is its high sensitivity. In comparison with the methylgreen assay read at 24 hours, the BEN assay reaches a 300-fold greater sensitivity after only six hours of substrate incubation. The lack of feedback inhibition on benzon nuclease activity (Table 1) may be partially responsible for the rapid kinetics observed and the ability to perform extended incubations resulting in that level of sensitivity.

The majority of existing assay methods have both low and high sensitivity formats so that an assay can be tailored to the type of samples being analyzed, Increased sensitivity is usually achieved by increasing the incubation time of the sample with the substrate. Our studies concluded that varying the size of the beacon substrate could modulate assay sensitivity independent of the incubation time. Studies performed with beacons of different sizes and stem region composition were compared with the 34-mer standard beacon. Substrates of similar size to the theoretical size of the final digest fragment size (2- to 4-mers) would be expected to be poor substrates for benzon nuclease and would result in decreased assay sensitivity. The substitution of an equimolar amount of a 4-mer with no stem region (beacon 3) for the standard 34-mer showed a 30,000-fold decrease in assay LLOQ in the 24-hour BEN assay when compared with the 34-mer substrate (Figure 7). Interestingly, the use of an equimolar amount of an 8-mer with a 2-nucleotide stem region (beacon 2) gave the expected intermediate assay sensitivity, but the maximal fluorescent intensity was significantly reduced when compared with the 34-mer and 4-mer substrates (Figure 7). We hypothesized that perhaps the nuclease could not efficiently digest the 2-nucleotide stem region or that dimerization of the beacons was occurring that resulted in a mixed population of substrate molecules. Experiments using the 34-mer beacon to determine the role of the stem and loop regions in signal generation by endonuclease used BAL31 and mung bean nucleases. They failed because of the nonspecific nature of those nucleases for single rather than double-stranded DNA (data not shown). We did not examine beacon substrates larger than 34 nucleotides long because of the cost associated with their synthesis and because we did not need further sensitivity.

One of the advantages of an activitybased assay is that theoretically any type I nuclease activity could be quantitated with minimal changes to the assay. We demonstrated this by running standard curves in the standard BEN assay at the same activity levels with the commercially available endonucleases Benzonase, Pulmozyme, and OmniCleave (Figure 8). The curves could be nearly superimposed, indicating that the BEN assay could be used to quantify activity levels for any of those commercial preparations. Optimal assay performance was observed with the benzon nuclease standard curve because the dynamic range of the curve's linear portion was broader than either of the other nucleases. Presumably, that was because the BEN assay buffer was designed for the optimal performance of benzon nuclease based on its pH and ionic requirements. We hypothesize that the Pulmozyme and OmniCleave curves could be optimized in the BEN assay by changing the formulation of the assay buffer. Despite that shortcoming, the LLOQ values were within the typical range observed for benzon nuclease (Figure 8).

Perhaps, molecular beacon technology could be exploited as a rapid method for determining enzyme activity of sequencespecific restriction endonucleases (type III]. The stem region, which is the only doublestranded portion of the beacon, could be synthesized with the recognition sequence for a particular restriction enzyme. Incubation with that enzyme under the proper buffer conditions may result in cleavage and subsequent signal generation. That would allow development of a rapid homogenous assay to measure restriction enzyme activity in commercial restriction enzyme products.

Acknowledgments

We would like to thank the Process Biochemistry Department at MedImmune, Inc., as well as James Young and Gail Folena-Wasserman for their support of this project.

[Reference]

References

[Reference]

(1) S. Shak et al., "Recombinant Human DNase I Reduces the Viscosity of Cystic Fibrosis," Sputum. Proc. Natl. Acad. Sci. 87, 9188-9192 (1990).

(2) K. Biedermann et al., "Purification and Characterization of a Serratia marcescens Nuclease Produced by Escherichia coli," Carlsberg Res. Commun. 54, 17-27 (1989).

(3) G. Obe, C. Schunck, and C. Johannes, "Induction of Sister-chromatid Exchanges by Alu I, DNase I, Benzon Nuclease and Bleomycin in Chinese Hamster Ovary (CHO) Cells," Mutat Res. 307, 315-321 (1994).

[Reference]

(4) C. Johannes and G. Obe, "Induction of Chromosomal Aberrations with Benzon Nuclease in Chinese Hamster Ovary (CHO) Cells," Mutat. Res. 325, 113-116 (1994).

(5) C. Johannes and G. Obe, "Ultrasound Permeabilizes CHO Cells for the Endonuclease Alu I and Benzon Nuclease," Mutat. Res. 374, 245 251 (1997).

[Reference]

(6) M. Kunitz, J. Gen. Physiol. 33, 349-362 (1950).

(7) M. Kunitz, J. Gen. Physiol. 33, 363-377 (1950).

(8) D. Sinicropi et al., "Colorimetric Determination of DNase I Activity with a DNA-Methyl Green Substrate," Anal. Biochem. 222, 351-358 (1994).

(9) 5. Tyagi and F.R. Kramer, "Molecular Beacons: Probes That Fluoresce Upon Hybridization," Nat. Biotechnol. 14, 303-308 (1996). BP

[Author Affiliation]

Corresponding author Robert J. Strouse is Group Leader, fadi 1 Hakki and Sheau-Chiann Wang are Associate Scientists, Anthony W. Defusco is a Research Associate 11 in immunoassay development, Jennifer L. Garrett is a QC Analyst in quality control, and Mock A. Schsnermon is Director of Analytical Biochemistry at MedImmune, Inc. 35 West Watkins Mill Road, Gaithersburg, MD 20878, (301 ) 527-4488, fax (301 ) 527-4200, strouser@medimmune.com, www.medimmune.com.

Monday, March 12, 2012

Moldovan Released After 15 Years

TIGHINA, Moldova - A political prisoner who was jailed for 15 years on charges of terrorism in Moldova's separatist state of Trans-Dniester was released Saturday after serving his term.

Andrei Ivantoc, 46, was a member of the Popular Moldovan Front, a political movement that called for the reunification of Moldova with neighboring Romania. Separatist authorities arrested him in 1992, and he and the three others were sentenced on charges of committing terrorist acts against citizens of Trans-Dniester a year later. The group's members were seen as martyrs by some in Moldova and Romania for their opposition to the separatists.

Trans-Dniester authorities drove Ivantoc to the border with the rest of Moldova, where he was freed and expelled. After he was released, he tried to return to the separatist republic but was stopped by authorities there.

He was later forcibly bundled into a car, in which his wife and a Moldovan official were also traveling. The car set off for the Moldovan capital. No statements were made and there was no immediate explanation for his action.

About 50 people came to greet Ivantoc, carrying roses and wild flowers to this border town some 37 miles southeast of Chisinau, the Moldovan capital.

Russian-backed Trans-Dniester, bordered by the Ukraine, fought a war with Moldova in 1992 and does not recognize its authority. The enclave, also known as Transnistria, is not internationally recognized.

The European Court for Human Rights in 2005 ordered the separatists, Moldova and Russia to free Ivantoc and one of the other three sentenced along with him. It also ordered Moldova and Russia to pay the four a total of more than $1 million in compensation for the deprivation of their freedom, torture and inhumane treatment while in custody.

Car bomb kills 20 in Pakistani city of Peshawar

A car bomb devastated a busy street in northwest Pakistan on Friday, killing at least 20 people, injuring scores more and unnerving a region already dangerously on edge after the attacks on India's commercial capital last week.

Escalating violence is destabilizing Pakistan's northwest just as the country faces accusations from archrival India that the gunmen behind the carnage in Mumbai were trained in Pakistan and steered by militants based there.

Neither the motive nor the culprits behind the Friday evening blast in Peshawar were clear. But provincial government chief Haider Khan Hoti said "external forces" could be to blame _ a comment understood in Pakistan to mean India.

The bomb went off near Peshawar's famed Storytellers Bazaar early Friday evening, wrecking a Shiite Muslim mosque and a hotel and setting a string of vehicles and shops ablaze.

Mohammed Bilal, a 28-year-old goldsmith being treated at a city hospital for a gash on his face, said he saw a white van explode in the street as he was walking home.

"Something struck me in face, and I fell down. There was fire and smoke and the cries of the injured people," Bilal told an Associated Press reporter.

Television footage showed survivors frantically carrying bloodied victims through the rubble to private cars and ambulances as fire crews sought to douse the flames.

Khizer Hayat, a senior doctor at the city's main hospital, said 20 bodies as well as over 60 wounded people were brought there.

Police chief Malik Naveed Khan said the bomb seemed to contain chemicals designed to spread fire.

While police were guarding government and religious buildings, "it is not possible to prevent this kind of terrorism unless you have extremely credible information," he said.

Pakistani forces have stepped up operations against Taliban and al-Qaida strongholds in the northwest from where militants have been mounting attacks in both Afghanistan and Pakistan.

Insurgents have responded with a campaign of gun and bomb attacks that have raised concern that they could cut a key supply line for NATO and U.S. troops through the Khyber Pass into Afghanistan.

There has also been a rash of kidnappings and attacks on foreigners in the northwest, including the Nov. 12 shooting death of a U.S. aid worker in Peshawar.

Earlier Friday, a suicide car bomber killed six people at a checkpoint in the Orakzai tribal region, just south of Peshawar, after police and local tribesmen waved for him to stop.

In the nearby town of Bannu, police said militants armed with guns and rockets killed two officers manning another checkpoint.

Militants recently vowed to step up attacks on Pakistani forces in retaliation for cross-border U.S. missile strikes into the region, which is considered a likely hiding place for al-Qaida chief Osama bin Laden.

The region is also bedeviled by sectarian tensions between extremists from the majority Sunni Muslim community and minority Shiites, and mosques have been repeatedly targeted in what officials say are tit-for-tat attacks.

The United States is seeking to calm tension between Pakistan and India, nuclear-armed neighbors who have fought three wars, in part to ensure that Islamabad remains focused on fighting militants in the northwest.

But Hoti, the provincial leader and a member of the party in the federal coalition government, pointed to "elements" in the lawless tribal belt who "act at the behest of external forces" when asked who carried out the attack in Peshawar.

"In today's tragic incident, the possibility of external involvement is very much there," he said.

Bahamian found guilty in shooting of US policeman

NASSAU, Bahamas (AP) — A jury in the Bahamas has convicted a 19-year-old man of attempted murder in the 2008 shooting of a New Jersey police officer who tried to prevent a robbery while on vacation.

Ebenezer Sherman is expected to be sentenced in November. His lawyer, Godfrey Pinder, says he will appeal the verdict.

The jury on Friday found another suspect, 23-year-old Bradley Saunders, not guilty in the shooting. Jurors convicted both defendants of attempted robbery.

Authorities said Bergenfield policeman John Casper was shot in the chest when he tried to stop the two men from stealing a woman's purse. The shooting occurred as Casper and several relatives walked near the home of former Bahamian Prime Minister Perry Christie.

Barr defends subpoenas for House bank records

WASHINGTON House leaders have no constitutional right to withholdrecords from investigators in the House banking scandal, AttorneyGeneral William Barr contended on Sunday.

"When it comes to these kinds of transactions, (congressmen are)no different than anybody else," Barr maintained in a confrontationbetween Congress and the Bush administration.

Retired federal Judge Malcolm Wilkey, who was appointed March 20by Barr to investigate possible criminal violations at the Housebank, last week subpoenaed all bank records, not just those of the325 current and former House members who wrote penalty-freeoverdrafts at their now-closed bank. About 170 lawmakers had nooverdrafts at the bank.

If the House refuses to comply, Wilkey could seek a court orderto have the subpoenas enforced or ask for a contempt citation. TheHouse also could go to court to seek to have the subpoenas quashed.

House Speaker Thomas S. Foley (D-Wash.) has said that, inresponse to a request for the records, he and House Minority LeaderRobert H. Michel (R-Ill.) wrote Wilkey last week that the Houseparliamentarian determined that House rules did not allow them tocomply with a request for the records. The letter did not explainwhy.

"I added that, whatever the rules might dictate, I could not inconscience comply with the sweeping and unprecedented scope of hisdocumentary request," Foley said.

The next day, Wilkey subpoenaed the records. He said in aletter to Foley, "There is no other source which contains nearly ascomplete a picture of the bank's operations, which is first priorityand fundamental to our inquiry."

Wilkey promised to return data from the accounts of the 170members who did not write overdrafts.

On Friday, Foley sent a letter to House members, complainingthat, rather than continue to negotiate over how to provide access tothe records of members who had overdrafts without infringing onprivacy rights, Wilkey went forward with the subpoenas.

The letters capped a month of high-level negotiations overWilkey's investigation, including a meeting among Foley, Wilkey andBarr.

Foley has indicated he plans to discuss the matter with othercongressional leaders when they return from a break this week.

Contributing: Baltimore Sun

2 dead, 9 missing following Senegalese shipwreck

An army spokesman says a fishing vessel capsized in the sea off the coast of Senegal, killing at least two people.

Col. Ousmane Sarr says nine people are still missing and are feared dead. Only three survivors were found of the 14 aboard the Artemis, the fishing trawler which capsized early Wednesday.

It's unclear why the boat capsized. It overturned in open water 40 miles (70 kilometers) off the coast of the Senegalese village of Popenguine. Sarr said the search continues for the missing with the help of a French military plane.

Field Tests of Aerobic Capacity for Children and Older Adults

ABSTRACT

Physical therapists frequently interact with patients, including children and older adults, who are deconditioned due to decreased physical activity associated with sedentary lifestyle, injury, disease, or disability. Objective quantification of aerobic capacity provides a baseline of patient performance, informs the evaluation and intervention process, and establishes the efficacy of interventions. The purpose of this article is to review field tests of aerobic capacity, including walk tests, step tests, and shuttle tests, for use in the pediatric and geriatric populations. Aerobic capacity of healthy children can be evaluated using walk tests, shuttle run tests, and step tests. To determine aerobic capacity in children with cystic fibrosis or who are severely ill, time-based walk tests, step tests, and shuttle walk tests are appropriate. Field tests of aerobic capacity for healthy older adults include walk tests, step tests, and shuttle walk tests, whereas for older adults with cardiorespiratory, neurologic, or orthopedic impairments time-based walk tests and shuttle walk tests are used. Field tests are safe, easily administered, and require little equipment. Using standardized administration, these field tests provide valid and reliable estimates of aerobic capacity for pediatric and geriatric patients that can be used to inform exercise prescription and quantify the efficacy of therapeutic interventions.

INTRODUCTION

Many of the patients that physical therapists interact with are deconditioned due to decreased physical activity associated with injury, disease, or disability.1,2 Increasingly, however, low activity levels are becoming the norm even within the 'healthy' population. Decline in physical activity has been observed in every segment of the population, including pediatric and geriatric populations. A recent study of healthy children ages 4 to 1 8 years found that 60% to 80% had fitness levels below the 25th percentile.3 Similarly, more than 50% of persons 65 years and older are reported to have a sedentary lifestyle.4

The importance of physical activity in maintaining and improving cardiopulmonary fitness and reducing the risk of type II diabetes mellitus and cardiac events, such as myocardial infarction, stroke, and sudden death, can not be overstated. Indeed, recent studies have demonstrated that individuals with average cardiopulmonary fitness have 50% reduced incidence of mortality and morbidity from cardiac events as compared to those with low fitness levels.1 As health care professionals with education and experience in the evaluation of a patient's functional capacity, physical therapists are ideally situated to identify individuals with low cardiorespiratory fitness and provide positive management interventions.

To optimize a patient's functional recovery and prevent the sequelae of low cardiorespiratory fitness, objective quantification of aerobic capacity is essential. Field tests of aerobic capacity provide baseline information that permits the identification of improvements in cardiorespiratory fitness resulting from therapeutic interventions. Determination of aerobic capacity is ideally performed in an exercise testing laboratory utilizing specialized equipment and maximal exercise protocols on either a treadmill or a cycle ergometer to measure maximal oxygen consumption (VO^sub 2^max), the 'gold standard' measurement of cardiorespiratory fitness. However, the availability of these specialized testing environments is often limited to specific patient populations, such as individuals with cardiac disease or athletes. Additionally, many deconditioned patients may not tolerate the metabolic demands of maximal exercise testing. Alternatively, cardiorespiratory fitness determined by submaximal testing using either treadmill or cycle ergometer protocols has been found to be highly correlated with measurements obtained using maximal protocols. Thus, reliable and valid submaximal treadmill and cycle testing protocols have been developed and used in numerous clinical settings and with various patient populations.5 Unfortunately, many physical therapists do not have access to treadmills or cycles in their clinical settings.

In this regard, exercise tests requiring minimal equipment are available for the determination of cardiopulmonary fitness. These field tests provide reliable and valid correlates of aerobic capacity and thus provide a standardized measurement that can be used in most clinical settings with many patient populations.6,7 Utilization of standardized measurements of aerobic capacity allow for appropriate exercise prescription and provide an objective measure of the efficacy of a physical therapists' intervention. The purpose of this article is to review field tests for the determination of aerobic capacity with the goal of providing physical therapists with easily implemented measurement tools with which to quantify the aerobic capacity of pediatric and geriatric patients. The field tests of aerobic capacity reviewed in this article include walk tests, step tests, and shuttle tests.

WALK TESTS

Introduction

Walk tests are simple, inexpensive, safe, and reliable tests for the measurement of functional exercise capacity and the monitoring of treatment effectiveness. Most commonly used walk tests are the time-based walk tests that measure the distance covered in the specific time period. These include the 12-min walk test (12MWT),8 6-min walk test (6MWT),8-10 and the 2-min walk test (2MWT).8 Alternatively, distance-based walk tests, such as the 400-m walk test,11,12 and the 1-mile walk test,13 measure the time taken to complete the specified distance.

The advantages of walk tests include the ease of administration, minimal equipment requirements, and a high degree of validity and reliability. Patients from 5 years14 and older10 are able to perform the 6MWT. These tests employ a functional activity that patients are familiar with. Patients who use assistive devices are also able to perform these tests. Corridor walking is generally more acceptable to older participants than is treadmill walking15 as they may experience physical difficulty and anxiety when walking on a treadmill.11,15,16 This can result in an underestimation of functional capacity as compared to walking in a hall or corridor.17

Standardization of the testing procedure is essential for obtaining accurate results.9 The use of standardized instructions is recommended as verbal encouragement has been shown to influence the results of the 6MWT.18 A practice may be considered, but is not thought to be essential as the resulting increase in walk distance ranges from 0% to 1 7%.9 If a practice test is performed, it is recommended to wait one hour prior to performing another test.9 Disadvantages of the time-based walk tests include the reliance on patient motivation to achieve a valid test.19 The 400-m walk test is thought to be intrinsically more motivating owing to the patient focusing on finishing the distance and not walking for a set time.11 Indeed, comparison of the 6MWT with the 400-m walk test demonstrated that individuals aged 70 to 79 years achieved a 20% greater walking speed (m/sec) during the 400-m walk test than with the 6MWT.11

The 6MWT is the most commonly used of the walk tests. Following the introduction of the 12MWT,8 the test was subsequently shortened to 6 minutes without a loss of validity or reliability.8 The 6MWT has been used with many patient populations, including pediatric20-22 and geriatric patients,10,15,23-27 and has been the subject of several recent reviews.7,9,28 Recently, the 2MWT has been gaining popularity for use with patients who are unable to sustain exercise for 6 minutes. The 2MWT has been used in severely ill children,14 the frail elderly,26 and individuals with respiratory symptoms,8,29 cardiac surgery,30 neurological disorders,31 and lower limb amputation.32 With respect to the distance-based walk tests, the 400-m walk test appears to obtain superior performance in older individuals as compared with the 2MWT and the 6MWT11,12 and is able to discriminate walking ability and fitness.11 The 1 mile walk test is not used very often in the pediatric or geriatric populations because many of these patients are unable to walk 1 mile.

Administration

Time-based tests (12MWT, 6MWT, 2MWT)

Distance is the primary outcome measure obtained with these tests. Using standardized instructions,8,9 the patient is instructed to walk as far as possible in the specified time. The patient is permitted to slow down, stop, and rest as necessary. The American Thoracic Society (ATS) recommends an indoor course of 30 m with marks every 3 meters, but corridors of longer and shorter distances and the use of indoor tracks have been cited in the literature.9,27

Distance-based tests (400-m walk, 1-mile walk)

Time is the primary outcome measure obtained with these tests. These tests use standardized instructions11-13 to direct the patient in performing the required distance as quickly as possible at a pace that they can maintain. The course for the 400-m walk test is 20 m (with marks every meter) and the patient performs a 2-min 'warm up walk,' by walking at a pace that they can maintain for the 2 minutes. After a 60-second rest period, the patient begins the 400-m walk test by performing 10 laps of the course.

Determination of Aerobic Capacity (VO^sub 2^)

The energy expenditure during the walk test can be determined from the walking speed that is calculated from the distance walked during the specified time period. Since the patients are instructed to perform a maximal effort during the test, the energy expenditure correlates well with measures of maximal aerobic capacity measured on the cycle or treadmill.23,29 The walking speed obtained from any of the walk tests (both time-based and distance-based) is used to calculate aerobic capacity as shown in Table 1.

Normative Values

There are several published reports of normative values for distance walked for the 6MWT for community dwelling elderly individuals and those residing in retirement homes (Table 2).

STEP TESTS

Introduction

Step test are field tests of aerobic capacity that require the participant to step up and down from a platform. Aerobic capacity determined by step tests has been shown to correlate well with aerobic capacity determined by treadmill33-35 and cycle ergometer exercise36-37 protocols. Step tests use the post-exertional heart rate response to predict oxygen consumption. Step tests are submaximal tests, though they often impose higher metabolic costs than self-paced walk tests and thus result in higher heart rates.38 Step tests can be single-stage or multistage; externally paced or self paced; have continuous or discontinuous work loads; and have predetermined, arbitrary end points. Externally paced step tests require the participant to maintain a set stepping rate for a specified time period and thus this test is relatively free of the variability associated with patient motivation as occurs in a self-paced step test or walk test such as the 6MWT.39 Other advantages of the step test include safe, simple, quick administration and portability.

The Harvard Step Test40 was one of the earliest reported step tests. This maximal effort test used a single stage work level that required participants to step up and down a 20-inch bench at a rate of 30 steps per minute for 5 minutes. The recovery heart rate was used to determine aerobic capacity.40 Due to the high exertion level and the demands on the leg muscles resulting from the high step height, the Harvard Step Test has subsequently been modified. Currently employed externally paced step tests use step heights between 6 and 20 inches, stepping rates between 18 and 36 steps per minute, and time periods between 2 and 6 minutes.34,37,39,41 These tests have been validated in healthy individuals between 7 and 18 years36,42 and over the age of 65 years38,43 as well as in children with cystic fibrosis39,41,44 or who are blind36 and older adults with chronic obstructive pulmonary disease.45

Studies have examined the relevance of step height in the accurate prediction of oxygen consumption.35,46 The findings demonstrated that varying the step height to accommodate the individual's stature may35,47 or may not46 result in more accurate prediction of aerobic capacity. To account for this inconsistency, predictive equations often incorporate step height into the calculations.48

In healthy children ages 7 to 17 years, predicted VO^sub 2^max using the Canadian Step Test49 and the Canadian Home Fitness Test50 has been shown to have good correlation with VO^sub 2^max as determined by treadmill testing.36,51 More recently, a 3 minute single stage externally paced step test was developed to assess aerobic capacity in children with cystic fibrosis.41 In one study of children with cystic fibrosis (age 7-18 years; FEV^sub 1^ = 17-67% predicted), this 3 minute step test produced a greater rise in HR and a greater fall in SaO^sub 2^ than the 6MWT, especially in children with more severe lung disease.39 Therefore, the step test was suggested to be rigorous enough to be a valuable assessment tool when assessing a child's suitability for lung transplantation.39

For adults older than 65 years, the single stage43,45 and multistage50,52 step tests have been shown to be valid. Recently, a self-paced step test for individuals over 65 years was introduced and was found to be highly correlated with aerobic capacity measured on a treadmill.38 This test was able to identify individuals of differing functional capacities and was found to be sensitive to the changes in fitness levels.

Administration

Single stage externally paced step tests

The most commonly used step test for children and older adults is a single stage externally paced step test using a 3-minute time period, cadence of 26 steps/min (adults) or 22 steps/min (children), and a step height that is appropriate for the individual's stature. The equipment required for the step test includes a step and a timer. Participants step up and down a single step such that the feet both go up and then both return to the initial level (ie,"up-up-down-down"). The stepping rate is indicated by a metronome set at 4 times the cadence so that each foot movement is associated with a sound. The stepping procedure is demonstrated to the patient prior to the test so that they understand the stepping sequence. Standardized encouragement may be given.44 The test is completed when the patient chooses to stop, the SaO^sub 2^ drops below accepted standardized levels, or the 3-minute stepping period is completed. The HR is measured for 15 seconds within 5 seconds of the completion of the test.42

Single stage self paced step test

This relatively new step test consists of stepping up and down 2 small steps [each 20 cm (8 inches) in height)] 20 times at a self selected pace.38 The step pattern is up-up-up-down-down-down using alternate legs. The arms are freely hanging at the patient's side and no railings are used. The test begins with a familiarization period in which the participant steps up and down the steps 10 times at a 'slow' pace to ensure that they are able to perform the stepping rhythm. Following a rest period of 5 minutes or until the HR has returned to within 5 beats per minute of the resting rate, the participants perform 20 step cycles at a 'normal' pace. Immediately following the 20th cycle, the time (in seconds) to complete 20 step cycles and HR (counted for 15 seconds) are determined.

Normative Values

Single stage externally paced step tests

Due to the nature of this test being a predictor of maximal aerobic capacity, there are no normative values beyond those for VO^sub 2^ for the specific age group or patient population.

Single stage self paced step test

In a recent study of 108 females and 92 males ≥ 65 years (average 72 �4 years),38 time to complete 20 step cycles at a 'normal' speed was 122 � 4 sec for females and 126 � 4 sec for males.

SHUTTLE TESTS

Introduction

The Shuttle Walk Test (SWT) is a standardized, incremental walking test that measures functional capacity by exercising an individual to a symptom limited maximal performance.53-55 The SWT was originally developed by Singh and colleagues53 and was based on the 20-m Shuttle Run Test.56 These incremental exercise tests require patients to walk (10-m test) or run (20-m test) at increasing speeds back and forth a 10-m or 20-m course. The speed of walking is increased every minute and is controlled by audio signals played from a tape.53-55 The external pacing of this test overcomes the motivation limitations that have made other self-paced step tests and walking tests, including time-based tests such as the 2-, 6-, and 12-minute walk tests,8 less attractive as objective measures of functional capacity.53,54,56

The SWT was designed to assess functional capacity in patients with chronic obstructive pulmonary disease (COPD)" and has been validated in the elderly (≥ 70 years)55 and in older patient populations with cardiac pacemakers,57 heart failure,58-61 patients awaiting heart transplant,62 idiopathic pulmonary fibrosis,63 and younger adult populations with cystic fibrosis.64 (See Cardiopulm Phys Ther J. 2005;16(2):21-23). The SWT has also been used to evaluate children under the age of 7 years.65,66 The Shuttle Run Test has been validated for healthy children66-70 and children with cystic fibrosis65 and asthma.71

Administration

Standardized instructions" for the test are provided on an audiocassette tape (available from the developers at sally.singh@glenfield-tr.trent.nhs.uk). The test uses a quiet corridor or treatment area at least 12 meters (10-m test) or 22 meters (20-m test) in length. Patients are required to walk (10-m test) or run (20-m test) back and forth, turning around 2 cones that are placed 9 meters (10-m test) or 19 meters (20-m test) apart, providing a shuttle distance of 10 or 20 meters, respectively, while keeping pace with a prerecorded auditory signal.

The patients aim to set a pace to complete a shuttle as each bleep sounds. Every subsequent minute, the audio signal sounds at increasingly shorter intervals. Each shuttle is indicated by a single bleep on the tape, whereas speed increases every minute are indicated by a triple bleep. The velocity of the 10-m shuttle test is set at 0.5 m/s for the first level and increases by 0.17 m/s every level thereafter. The 20-m shuttle test begins with a velocity of 4 km/hr at level 1 and increases by 0.5 km/hr every subsequent level. There are 12 levels with each level requiring the patient to perform successively more walks (shuttles) within the 10-m or 20-m course.

Distance walked in the SWT is recorded in meters (calculated as the number of shuttles x 10 m/shuttle). No encouragement may be provided to the patient. The test is terminated when the participant chooses to stop or when on 2 consecutive paced signals the patient is more than 1 m away from the closest marker.53 The SWT and Shuttle Run Test can be carried out in any clinical setting without the need for special facilities and can be completed in 15 minutes.

SUMMARY

Physical therapists are encountering more patients who are deconditioned due to the increased prevalence of a sedentary lifestyle. The associated decrease in aerobic capacity is occurring across the lifespan, including the pediatric population. Physical therapists are uniquely suited to identify deconditioning and to prescribe interventions to promote the attainment of increased fitness and health. Field tests of aerobic capacity, including walk tests, step tests, and shuttle tests, provide the physical therapist with valid, reliable outcome measures to quantify the aerobic capacity of their patients in most environments. These tests are safe, require little equipment, and employ functional activities. Aerobic capacity of healthy children can be evaluated using walk tests, shuttle run tests, and step tests. To determine aerobic capacity in children with cystic fibrosis or who are severely ill, time-based walk tests, step tests, and shuttle walk tests are appropriate. Field tests of aerobic capacity for healthy older adults include walk tests, step tests, and shuttle walk tests, whereas for older adults with cardiorespiratory, neurologic, or orthopedic impairments time-based walk tests and shuttle walk tests are used. Using standardized administration, these field tests provide valid and reliable estimates of aerobic capacity for pediatric and geriatric patients that can be used to inform exercise prescription and quantify the efficacy of therapeutic interventions.

ACKNOWLEDGEMENTS

PJO is supported by grants from the American Lung Association and the Interdisciplinary Research and Creative Activities Fund at the University at Buffalo.

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[Author Affiliation]

Patricia J. Ohtake, PT, PhD

Associate Professor, Program in Physical Therapy, Department of Rehabilitation Science, University at Buffalo, Buffalo, NY

[Author Affiliation]

Address correspondence to: Patricia J Ohtake, PT, PhD, Department of Rehabilitation Science, 515 Kimball Tower, University at Buffalo, Buffalo, NY 14214 Ph: 716-829-3141 ext 142, FAX: 716-829-3217 (ohtake@buffalo.edu).