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Online Home entertainment is acquiring appeal these days. The Internet has altered the means individuals spend their time. They can play games, conversation, share data, videos and photos with their friends. Online entertainment supplies the ease of playing games, accessing the internet, shopping online and communicating with buddies. There are several methods to obtain captivated online. Below are some instances:
Video gaming: Pc gaming is the most up to date craze. This sort of on-line amusement provides multi-level gaming experience where gamers take on each other for enjoyment. On-line video gaming consists of various genres such as racing, activity, technique, card, arcade, word-power and also parlor game. Video gaming sites supply free video gaming alternatives such as flash gaming, 3D games as well as software downloads. Most prominent gaming web sites likewise offer a variety of gadgets such as computers, game gaming consoles, smart phones, handheld tools and others that make the gaming experience more fascinating.
Amazon Prime Video Clip: Amazon Prime Video is an additional type of on the internet home entertainment. It is similar to viewing TV shows in the residence, yet you can do so from throughout the globe with simply a web connection. On-line vendors offer a selection of movies, shows as well as various other programs of this kind at budget-friendly rates. Amazon Prime Video makes use of flash innovation to deliver streaming services. The business asserts that the delivery of its audio and video streams are faster than any totally free over the air TV solutions. This firm provides complimentary as needed delivery and membership solutions for its movies and shows.
Social media site: With the development of social media sites, the show business has actually progressed right into a platform where brand names can contact their target audience, clients as well as companions. Social media site allows businesses to communicate with their target audience. Its fantastic capacity is yet to be checked out totally. Streaming service providers will include in the richness of web content being provided through these social media websites.
Applications: In this age of smart devices, tablet computer systems as well as various other portable devices, the entertainment industry has located a friend in the form of apps. These apps are made to improve the user experience of individuals. For example, some of the applications in the smartphone market enable customers to lease or buy tickets, access maps, check movie listings, receive information concerning trailers as well as routine approaching screenings, among others. Other prominent apps in the mobile phone market to help in navigation, communication as well as enjoyment. The systems for these apps are boosting each day.
These are several of the major technologies taking place worldwide of web, mobile as well as ecommerce. They have changed the way people as well as services obtain entertaining. As long as people continue to invest in the various kinds of amusement, the ecommerce and internet marketing will certainly thrive. They will certainly likewise remain to broaden their horizons as the target audience rises.
On-line entertainment is the most recent buzzword in e-entertainment. With the arrival of high speed web and broadband links, individuals have ended up being a lot more interactive than ever before. Enjoyment has actually moved from being an experience in a physical kind to an interactive experience with the help of computers and also the net. Today, individuals not just access their favored web sites on the internet, yet can additionally play on the internet video games, see their favored movies, listen to songs tracks and so forth. All this can be done from the convenience of your home!
On the internet home entertainment consists of a variety of activities such as online video games, on the internet shopping, downloading and install applications, talking online, playing video games, watching video clips, listening to songs, social networking, downloading and install ringtones, and numerous such tasks. A few of these activities are also carried out on smartphones! The current buzzword in the round of on-line entertainment is smart devices. A lot of youngsters are going with these phones for a selection of factors. A few of the most fascinating realities regarding smart devices are mentioned listed below:
It is the best method to catch up with your preferred celebs while taking a trip abroad. There are a great deal of channels on which you can catch up with your preferred celebs and also view their different types of video clips. You can also buy tickets for your overseas excursions from the ticketing agencies. As far as the investing in of tickets is worried, you will have a wide variety of choices. Online amusement is obtaining popular with people selecting different sorts of enjoyment such as viewing live online video clips, playing online games, downloading and install ringtones, downloading live wallpapers, getting electronic books, listening to live performances, and so on.
The web tv is one more latest fad in the field of on-line amusement. You can find a number of hours of your favorite TELEVISION reveals streaming survive on to your computer system. Besides watching the TV collection, you can also see several hours of motion pictures and also trailers. If you have actually been searching for the very best option to conserve money on worldwide flights, this is the right one for you. If you wish to conserve few more bucks, you can simply download unrestricted varieties of tracks from several different kinds of artists offered online.
Among one of the most prominent types of on the internet entertainment is YouTube. With almost 2 billion customers, it is thought about to be one of the most popular type of media sharing. Countless individuals check out YouTube every day and there are several hrs worth of video being published on the site. There are numerous popular channels on which you can find videos being streamed onto your mobile devices such as ipads, apples iphone and Android phones, as well as computers. 안전놀이터
Besides being a tool of online enjoyment, Amazon.com Prime Video clip is also proving to be a fantastic choice for advertising and marketing and also connecting to your target audience. It is also considered to be the second most popular resource of video viewing on the net. Actually, according to an estimate, HotStar is accountable for greater than forty percent of Hotmail email sales. To capitalize on this fantastic opportunity, just have a look at the current HotStar clip by seeing Amazon prime video clip.


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Ms. Merle Evelyn Ratner and Honorable Bishop Thomas John Gumbleton, co-presidents of Vietnam Agent Orange Relief and Responsibility Campaign (VAORRC) in the US, were awarded ‘For Vietnamese AO Victims’ insignia in Hanoi on January 23.
The award recognizes the two US citizens active contribution in the struggle for justice for Vietnam’s Agent Orange victims.
While in Ho Chi Minh City, the delegation met with veterans and visited Tu Du Peace Village, where children infected from Agent Orange are being raised; the Cu Chi Tunnels; and the Thien Phuoc Nurturing Center for disadvantaged children.
Recently, at a meeting with former Vietnamese political prisoners, who are also victims of Agent Orange, the delegation heard emotional stories about the brutal torture that the prisoners suffered in the past as well as the current difficulties in their lives.
The delegates stated that they would continue to apply to the US Government to pay compensation to AO victims, introduce a bill on support and compensation to AO victims and encourage Catholic followers and the American people to actively assist them however they can.
Speaking at the occasion, Lieutenant General Nguyen Van Rinh, President of Vietnam Association for Victims of Agent Orange (VAVA),
said that VAORRC has operated effectively in supporting the AO victims’ lawsuit against American chemical companies. Ms Ratner and Bishop Gumbleton have worked diligently to help heal the wounds of wartime and conduct campaigns to support Vietnamese AO victims.
Among their noteworthy activities were arranged trips to the US for six delegations of AO victims and VAVA leaders and trips to Vietnam for US war veterans to raise funds for AO victims.
Ms Ratner expressed the great honor in receiving the insignia and thanked the Vietnamese Government, State and people for their warm welcome to Americans who have returned to Vietnam.
She pledged to continue to make greater efforts in the struggle for justice for Vietnamese AO victims, adding that her colleagues at VAORRC and she will continue to promote the campaign to ask the US Congress’ approval of the bill.

domain: news


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Executive chef Matt Gruber helps Jim Legere browse the selections of fish at the Blue Water Seafood Company and Wine Market in Hendersonville.
- Filed Under
There are more than 600 miles between the mountains of Western North Carolina and the waves of the Gulf Coast, but the consequences of the BP oil spill are already trickling up.
Area seafood retailers and restaurateurs are seeing prices rise after the disaster April 20. An explosion at a BP drilling platform left a busted oil well that's since been spewing massive amounts of oil into rich fishing waters.
"I have never seen anything on this scale," said Doug Parsons, executive chef and general manager of The Lobster Trap on Patton Avenue. "When a hurricane comes through, it affects one alley; it ...


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Handmade Brown Tweed Seed Bead Necklace
Necklace length – 26”; width – 0.25”
Pendant: 2 1/8” diameter
An exciting handmade bead crocheted necklace, of seed beads and glass pearls make a great holder of a hand carved stone, serpentine, snake design, for your fun. The gold plated end caps and clasp make a beautiful finish for when you wear this lovely necklace.
The beads were strung on cotton crochet thread and then crocheted into the necklace. It is soft to the touch and drapes easily.
10 other shoppers are interested in this item
All reasonable offers considered. For U.S. buyers, buy 3 jewelry items or 3 pairs shoe clips and receive free shipping!

domain: design


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SPRINGFIELD, Mass. … The 2006 Rbk Hockey AHL All-Star Classic, set for this coming Tuesday and Wednesday, will bring some of the best young players in hockey to the MTS Centre in Winnipeg to continue a tradition that actually began 64 years ago.
While 2006 marks the 12th installment of the league’s modern All-Star event, the first AHL All-Star Game was held on Feb. 3, 1942, under the support of league president Maurice Podoloff. The game, played at the famed Arena in Cleveland, was a fundraiser for U.S. and Canadian efforts in World War II and featured some of the most notable names in league history, including Les Cunningham and Eddie Shore, two men whose names now grace AHL awards honoring the league’s MVP and outstanding defenseman, respectively.
The AHL All-Star Game was brought back in 1954, and for the next six years the defending Calder Cup champions would be pitted against a team of league all-stars. Many of the AHL’s great players of the era participated; Dunc Fisher and Fred Glover played in all six of these games. Other AHL All-Stars in the 1950’s included future Hockey Hall of Famers Frank Mathers, Emile Francis, Brian Kilrea, Harry Lumley, Pierre Pilote, Gump Worsley and Johnny Bower, who made 47 saves in a 4-0 shutout win in the October 1956 game.
Following the All-Star Game in December 1959, the event was not held again until current league president and CEO David Andrews revived it 35 years later. In Providence on Jan. 17, 1995, a team of All-Stars from the AHL’s six Canadian teams faced off against All-Stars from the league’s 11 U.S.-based clubs.
The annual Skills Competition was added in 1996, and the format was changed in 1997 to the current set-up pitting Canadian-born players against those born outside Canada. In 2003, John Slaney became the all-time scoring leader with his eighth All-Star point.
A sold-out arena and a live international TV audience is set to once again witness the AHL’s best face off on one of hockey’s brightest stages, the 2006 Rbk Hockey AHL All-Star Classic in Winnipeg. The AHL All-Star TV network will reach nearly 100 million households; click here for a schedule of air times.
AHL HALL OF FAMERS SET FOR INDUCTION … One of the highlights of this week’s festivities in Winnipeg will be Wednesday’s induction of the first seven members of the American Hockey League Hall of Fame, taking place during the annual All-Star Classic Luncheon.
The inaugural Class of 2006 consists of Johnny Bower, Jack Butterfield, Jody Gage, Fred Glover (who will be represented at the ceremony by his brother and former teammate Howie), Willie Marshall, Frank Mathers (represented by his wife, Patricia) and Eddie Shore (represented by his son, Ted).
CH-CH-CHANGES … Some late roster changes have been announced for this week’s All-Star Classic. PlanetUSA defensemen Matt Jones of San Antonio (injury) and Danny Richmond of Norfolk (NHL recall) and Canadian forwards Dustin Penner of Portland and Mike Glumac of Peoria (NHL recalls) will miss the event. Added to the PlanetUSA side is Peoria defenseman Mike Mottau, and to the Canadian team is San Antonio forward Layne Ulmer.
Jimmy Roy of the host Manitoba Moose will replace Penner in the Canadian starting lineup.
SATURDAY CROWDS PACK AHL ARENAS … Nearly 100,000 fans took in a full slate of games around the American Hockey League on Saturday night, with every one of the 13 home games drawing crowds above their season averages.
Houston led the pack with a Saturday crowd of 11,672 at the Toyota Center to watch the Aeros post a 5-4 victory over Chicago. In Cleveland, 8,186 fans were at the Quicken Loans Arena, more than triple the Barons’ season average, for the home team’s game against Milwaukee.
Team Saturday Avg. before Sat. Difference Cleveland 8,186 3,175 +151.7% Houston 11,672 5,170 +121.1% Springfield 5,890 3,223 +79.3% Peoria 8,217 4,522 +78.2% Iowa 8,842 4,938 +76.0% Portland 7,159 4,365 +61.5% Bridgeport 5,223 3,236 +58.9% Albany 5,161 3,959 +29.1% Hamilton 6,111 4,784 +26.6% Hartford 5,927 4,965 +18.5% Syracuse 5,917 5,378 +9.5% Hershey 8,061 7,340 +9.4% Wilkes-Barre 8,347 8,147 +2.3% AHL Average 7,286 5,145 +40.8%
ETC. … Bridgeport forward Robert Nilsson scored a highlight-reel, lacrosse-style goal in Wilkes-Barre on Wednesday night; the clip is available on soundtigers.com… Syracuse center Mark Hartigan heads to Winnipeg for the All-Star Classic on a 16-game scoring streak, with 16 goals and 17 assists in that span… In the team’s first eight years of play, no Hartford defenseman had ever recorded four points in a game; All-Star Thomas Pock has done it three times in his last 15 games… Omaha defenseman Steve Bancroft skated in the 1,000th game of his North American pro career on Saturday night; more than half have come in the AHL… Milwaukee’s Scottie Upshall has picked up 10 goals and seven assists for 17 points in his last seven AHL games… Providence had a 10-game home winning streak snapped by Philadelphia on Sunday… Norfolk’s crowd of 7,064 on Friday night was the largest in the team’s AHL history.

domain: sports


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Plays and Playwrights 2003: The Book Party
Tickets and Information
WHAT IS IT ABOUT?
The New York Theatre Experience, Inc. (NYTE) and Vital Theatre Company present Plays and Playwrights 2003: The Book Party, a free event celebrating the publication of NYTE's newest anthology of plays. The centerpiece of the evening will be performances, by members of the original casts, of scenes from seven of the plays featured in the collection. A reception, where the playwrights will be available to sign copies of the book and meet audience members, will follow the readings. Books, priced at $15, will be available at the event for purchase. Plays and Playwrights 2003 is a collection of eleven plays by new writers that premiered off-off-Broadway during the past year. Featured in this anthology are A Queer Carol by Joe Godfrey, Pumpkins for Smallpox by Catherine Gillet, Looking for the Pony by Andrea Lepcio, Black Thang by Ato Essandoh, The Ninth Circle by Edward Musto, The Doctor of Rome by Nat Colley, Galaxy Video by Marc Morales, The Last Carburetor by Leon Chase, Out to Lunch by Joseph Langham, Ascending Bodily by Maggie Cino, and Last Call by Kelly McAllister. The event is open to the general public and is free.
432 W 42nd St
New York, NY 10036
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domain: literature


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BOOK ONE IN THE BROKEN EMPIRE TRILOGY
“Prince of Thorns deserves attention as the work of an iconoclast who seems determined to turn that familiar thing, Medievalesque Fantasy Trilogy, entirely on its head.”—Locus
When he was nine, he watched as his mother and brother were killed before him. By the time he was thirteen, he was the leader of a band of bloodthirsty thugs. By fifteen, he intends to be king...
It’s time for Prince Honorous Jorg Ancrath to return to the castle he turned his back on, to take what’s rightfully his. Since the day he hung pinned on the thorns of a briar patch and watched Count Renar’s men slaughter his mother and young brother, Jorg has been driven to vent his rage. Life and death are no more than a game to him—and he has nothing left to lose.
But treachery awaits him in his father’s castle. Treachery and dark magic. No matter how fierce his will, can one young man conquer enemies with power beyond his imagining?
This morbidly gripping, gritty fantasy tale is not for the squeamish. Prince Jorg was 10 years old when Count Renar's soldiers hung him on a hook-briar while his mother and brother were murdered. Vicious and desperate for revenge, he joined a bandit gang. Four years later, he's their leader and determined to end a multigenerational war by resurrecting the old empire and becoming emperor. Jorg is the epitome of the antihero, killing not just in battle but whenever he feels the need to set an example. Only toward the end of the book does a startling revelation focus his violence on clearly deserving targets. But Jorg is a product of his world, no worse than any other power there, and Lawrence increasingly makes readers believe that Jorg may indeed be able to use small savageries to end the ongoing greater ones.
Usually I don’t like these fantasy war and gore books, but the anti-hero is addicting and downright scary. Would have been a 5 star but for the raping and burning people alive parts, thankfully short lived.
This is a very good read, albeit very dark, but with under tones that emphasize humanity, especially towards the end. Very good storyline, complex plot, interesting style, entertaining dialogue, and strong action. If you liked The Name of the Wind, The Warded Man, or the Night Angel trilogy, you will enjoy this book.
Literally opens with the lead 13 year old boy raping two girls and then setting them on fire.


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Wednesday, May 9
It didn’t take long to fix the starboard spud. Timmy disconnected the old cable and we threaded the stainless steel cable into place. The end had to be trimmed to attach it to the winch, and I wound it tightly and took up the slack. Timmy tied a rope from the top of the spud to the flat boat to pull, and I stayed on board to push the spud up and operate the winch to eventually rotate it back to its upright position. The pins were reinserted and locked and we were ready to pump again. Hopefully, that would be one less cable to worry about.
We could dredge as soon as the discharge was moved to a new position. For this, we needed the airboat. A quick trip back to the headquarters to drop Timmy off, and I headed back in the flat and tied it to the bank. Timmy came by to pick me up in the airboat. We headed off to the Cole’s Bayou airboat trail and took the route around by LSU’s old study site into Deep Lake to the study site.
On the way, we detoured through Coles Bayou to look at the recently completed terrace project. Marsh birds were thick in the existing marsh, and Timmy counted 19 Least Bitterns that we flushed on our trip around the canal.
At the study site, we were greeted by a flock of least, semi-palmated and stilt sandpipers that were reluctant to leave the rich pickings of the fresh mud. They kept swirling about as the airboat was cranked up or moved about.
It took several tries to move the pontoon and the discharge hose. The pontoon was at the end of a large curve of hose, and moving it meant pulling against a lot of heavy, thick mud. The pull ropes were tied to the airboat, and the first tug wouldn’t budge it. The second tug broke the rope. (AARGH!)
The only way to get to it without getting lost and covered in mud was by airboat. Timmy drove around to the south side of the cell and carefully entered the cell. I stepped out onto the slippery 4’x4’ pontoon deck and tied a new, hefty rope through the handles and around the hose. Then straight across the mud of the cell we went, the pontoon began to move – then we hit the marsh island, where our momentum was slowed just a bit and we got stuck. We eventually got enough slack in the rope to turn the airboat around, and Timmy gunned it and pulled the outfall almost to the NW corner of the cell. We’ve been trying to avoid bringing the airboat into a cell full of mud, not knowing how it would affect the fill. Surprisingly, the airboat passage had not left a ditch as we had feared, but skated across the top of the mud merely smoothing it out. Hmmm, this might be a new technique – mud sculpting by airboat!!!
All of this was completed early and we started dredging at 10 am. We stopped after 10 minutes to add a section of hose to the canal side to give the dredge swing some slack. After an hour of dredging, Karen took the pirogue to the pond to assess our progress. The water level was -1″ ML in the pond and +2” ML in cell 4.
There were several places where the effluent was escaping into Cell 2: at each end of the plywood containment 4b, under the reed containment 4a through a blow-out, and through the marsh itself. Even with this loss of material, the cell was contained well enough to hold 3 inches higher than the pond, and the outfall position was creating a delta of heavier material in the NW corner as we intended, filling in the lowest part of the cell and make a semi-dam to keep more material in the cell.
We stopped dredging at 12:00 to again move the outfall to the east, making a large curve in the hose. With the cell so close to being full, we were trying to move the discharge every hour to spread the heavier material and make a series of mounds. It seemed to be working. At almost 2, we headed back to the house for lunch.
Timmy had some things to do, so Karen went back to dredge alone and started dredging again at 3:00. Twenty minutes later, one of the neighbors, Mike Sagrera, stopped by to see what I was doing, so I showed him how the dredge worked while I pumped. After he left and I finished a swing across the canal and back, I shut down the dredge at 4:10 and headed to the pond again. The one hour of dredging into the shallow cell had made another delta roughly +2-3” ML. I managed to move the outfall over between the last 2 deltas by hand. Well, by part of me anyway! I wrapped the rope around the weighty-est part of my body, set my feet solidly, then leaned back to pull, ease up to pull the slack around, and leaned back again. Inch by inch, I pulled the pontoon toward me. Ya’ learn to use what ya’ got to get the job done! After that, I had just enough energy to take some pictures, pack up and get back to the house at 5:30.
My day was done, but Timmy’s was not. The carpenters working on the new camp next door called from the boat landing on their way home, to report that gar fishermen were putting in. We’ve had trouble in the past with gar poaching, so Timmy went on alert and called the local Wildlife and Fisheries enforcement agents as well. At 4 am. Timmy was out on patrol, and sure enough found evidence of their trespassing, but was too late and missed them.

domain: geography


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HOUSTON - Airport officials say a Northwest Airlines passenger jet landed safely after blowing a tire on takeoff from Houston.
NWA Flight 3492 was headed to Minneapolis-St. Paul on Monday morning. Airport spokeswoman Marlene McClinton says the plane circled the airport so the control tower could check damage from the ground. Some rubber from the blown tire was scattered along the runway.
The jet returned safely to Houston's Bush Intercontinental Airport, and the airline was rebooking passengers.


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Screw Yer Roommate goes hybrid in response to COVID restrictions
“Screw Yer Roommate,” the mass blind date event hosted each year by the Rice Program Council was held again this year, despite COVID-19-specific challenges. This year the event had a hybrid format, with both an in-person and virtual option to allow remote students to participate.
RPC Socials committee co-chairs Amy Barnett, Baker College sophomore, and Yasmin Givens, Baker junior, were responsible for planning Screw this year. Givens said they had to be flexible in preparation for the event since they were unsure how many students would actually participate.
“Given the volume of interest that we received for the event, we adapted our original plan to include waves at multiple locations in order to accommodate the number of students interested and to minimize the number of students at a location at any given time,” Givens said.
After being paired off by their roommates in the usual Screw fashion, in-person pairs signed up for the event online and were then assigned to one of nine locations on campus, in either the 5 or 5:30 p.m. wave. Time and location designations were emailed to students on the day of the event.
Luke Stancil, a freshman at Baker, said he enjoyed his first Screw and that he felt the event was safely organized.
“We were told to go walk around with our popsicles and dates for 30 minutes [and] then leave the location. I think everyone followed that, and everyone wore masks when not eating,” Stancil said. “It was actually kind of cool because we had this structured time to get to know our date in a very relaxed way.”
Eric Torres, a senior at Wiess College, has attended Screw all four years now and said this year ended up being similar to past experiences after all.
“I dressed up, met my date and walked with a group to pick up food at Hopdoddy’s, then brought it back to eat at Rice. We all had really long conversations and it was a nice time to catch up with friends,” Torres said. “The only main difference is that we ate at Rice instead of at a restaurant.”
According to Givens, turnout for the in-person event was relatively on par with attendance for Screw in previous years, with 450 students registered.
Virtual Screw, or “speed-friending” according to the Facebook event, was a series of blind dates over Zoom and was attended by 25 students, according to Givens.
“Despite a relatively small turnout, the virtual version of Screw went really well,” Givens said. “Everyone who participated had a chance to meet and talk with others that they wouldn’t typically have a chance to meet with, across both grades and colleges.”
Rishi Shridharan, a sophomore at Will Rice College, said he was especially pleased with how it was organized.
“[Givens] set up breakout rooms of two to three people and suggested casual talking prompts, like ‘What’s the craziest thing you’ve done?’ and ‘What’s your biggest pet peeve?’” Shridharan said. “The prompts gave our conversations a loose structure, but we often went off in enjoyable tangents. Every few minutes, we’d return to the main room and join a new breakout room.”
Shridharan said the virtual event was overall a success in his opinion.
“Remote Screw gave me the unique opportunity to meet new people that I probably wouldn’t have ever interacted with in person. All in all a great experience,” Shridharan said.
More from The Rice Thresher
Sophomore distance runner Grace Forbes followed up her conference title with a first-place finish at the NCAA South-Central Cross Country Regional on Friday in Waco, TX, helping the Rice women’s team to a second place finish. The result earned them a spot in this Saturday’s NCAA championships for just the seventh time in program history.
Students demanded that Rice University remove William Marsh Rice’s statue from the Central Academic Quadrangle to a new, non-prominent location by the end of this academic year, during the Nov. 15 Student Association Senate meeting. The SA will vote on the resolution Nov. 29, according to SA President Kendall Vining.
Rice will be providing one final vaccine clinic on Dec. 3, partnering with the Houston Health Department to have 400 vaccine slots available for the Rice community. Three previous clinics were held on Nov. 4, Nov. 5 and Nov. 12.


BOBLOV KJ09 Body Camera, 1296P Body Wearable Camera Built-in SD Card 64GB IP66 Waterproof Support External Lens Simultaneous Recording, Include Two Batteries Night Vision Body Camera Police Model. With 120°wide angle lens, up to 1296p video resolution, It allows you to capture more high quality, clear photos and videos. Long press the video key for 2-3 seconds can quickly turn on and recording. 2, Waterproof IP66 and 2m Shock Proof: The waterproof level is up to IP66 which means the body camera is strong enough to resist rough weather.
But can't for storm or leave it in water for a long time. 2m High falling drop resistance, which can protect camera dropping in the process of using.
3, 2800mAh Capacity Battery: KJ09 body camera adopts low power consumption technology. It is equipped with 2 pcs high-quality batteris and one charging base, One in camera, own a great endurance which can meet the needs of most customers. 2800mAh rechargeable battery gives about 10 hours working time (1080p, screen off) and just takes about 3 hours charging time.4, Infrared Night Vision: KJ09 body camera has manual and auto night vision function, it has built-in 1 white LED light, 1 red and 1 blue patrol light, four infrared lamps can capture the face clearly in low light conditions. Very suitable for police patrols, meeting records, driving records. This product does not include external lens. VideoResolution:1296P30,1080P30,720P30,480P30. Protection level:IP66 Audio Format:WAV.
1KJ09 CAMERA 1Long shoulder clip 1Short back clip 1Charging data cable 1Instruction manual. Or it depends(except US). Hope you can wait some more days.
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The seller is "seasonshopping2016" and is located in this country: US. This item can be shipped to United States.


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Jan 01, 2021
From 10 January 2021, Vietnamese students will be eligible to apply for the British Ambassador’s Scholarship valued at VND 800 million per individual for their studies at British University Vietnam (BUV). The BUV Scholarship fund and financial aid with a total value of VND 53 billion for the school year 2021, as well as application portals, will officially launch on the same day, providing many other prestigious scholarship opportunities for students in Vietnam.
To support talented students during their application process, at the official launch of the BUV Scholarship fund on 10 January 2021, Mr. Christopher Jeffery, Chief Academic Officer of BUV will provide information about the different types, application requirements as well as selection criteria for the scholarships. Additionally, previous winners will also share about their own experience applying, preparing documents, and present some strategic tips to impress the Interview Panel and win the scholarship.
The launch of the Scholarship fund also kicks off the first series of events at BUV in 2021, including BUV Experience Days on the 10th and 17th of January. On top of exploring scholarship opportunities, students will also take part in various activities: Choosing from 19 options students can experience a demo class of their favourite or desired subject as well as receive personal consultation on career orientation; take an English proficiency test and build a personal roadmap to conquer international universities; meet and mingle with BUV students who will talk about: subject selection, school selection, adapting to international university life, improving their English and IELTS score; exploring international-standard education to actively design their own futures and become the best version of themselves.
The BUV Scholarship fund and financial aid was established in 2018 with the aim of giving Vietnamese students the opportunity to pursue British undergraduate and postgraduate programmes in an international-standard education environment right here in Vietnam. In 2021, the BUV Scholarship fund will continue to be dedicated to providing students in Vietnam prestigious full scholarships including four British Ambassador’s Scholarships for exceptional well-rounded students and one Lion’s Heart Scholarship for less-fortunate students who have the will to overcome difficulties for outstanding achievements. In addition, the fund also covers many other valuable scholarships including: President’s Scholarship (75% tuition fee), Dean’s Scholarship (50% tuition fee), Talent Scholarship (20-50% tuition fee) for students with outstanding achievements in sports, culture and arts. The MOU Scholarship for excellent students from BUV’s partnered high-schools, alongside other financial aids for deserving students.
According to Mr. Christopher Jeffery – Chief Academic Officer of BUV: “We are looking for smart, talented, passionate students who have the ability to develop and contribute to the community. Be yourself, be confident, be honest and be determined to stand out. You are ‘investing’ in a project worth thousands of dollars, so you should make sure to spend enough time and effort on it.”
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Friday, May 16, 2014
(Worcester, MA) -- Binge drinking is more dangerous than previously thought. Massachusetts researchers found a single binge-drinking episode can lead to bacteria leaking from the gut. They said that leads to increased levels of endotoxins in the blood. Endotoxins are in the cell walls of certain bacteria, and they're released when the cells are destroyed. Binge drinking is defined as five or more drinks in a single sitting for men. For women, it's four or more.

domain: medical


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Open Access This article is
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Int. J. Mol. Sci. 2019, 20(4), 838; https://doi.org/10.3390/ijms20040838
Low Endotoxin Recovery—Masking of Naturally Occurring Endotoxin
Microcoat Biotechnologie GmbH, Am Neuland 3, 82347 Bernried am Stanberger See, Germany
LPS (Laboratory Program Support) Consulting Office, Tokyo 160-0023, Japan
Department of Host Defense and Biochemical Research, Juntendo University Graduate School of Medicine, Tokyo 113-8431, Japan
Institute of Physical and Theoretical Chemistry, University of Regensburg, 93040 Regensburg, Germany
Author to whom correspondence should be addressed.
Received: 10 December 2018 / Accepted: 12 February 2019 / Published: 15 February 2019
Endotoxins are cell wall components of Gram-negative bacteria. A release of endotoxins into the human blood stream results in an inflammation reaction that can lead to life-threatening conditions like sepsis. Therefore, control for endotoxin contamination of intravenously administered drugs is crucial. Drugs are usually tested for putative endotoxin contamination with Limulus-based tests. However, validity of the compendial test procedures is questioned in the case of low endotoxin recovery (LER). To assure validity, regulatory authorities request hold-time studies of endotoxin in addition to pharmacopoeial requirements. Within these studies, endotoxin is added (spiked) to an undiluted product. The spiked product is held for a certain period of time and subsequently diluted for endotoxin determination. Due to the known heterogeneity of endotoxin the question has been raised as to which source represents the most adequate endotoxin spike. In the present study, endotoxin hold-time studies were analyzed by using different sources of endotoxin. Highly purified endotoxin, crude endotoxin extracts (Naturally Occurring Endotoxin) from different bacterial species and varied growth conditions as well as endogenous endotoxin contaminations were investigated. The results clearly demonstrate that endotoxin masking—an effect of LER—is dependent on the endotoxin source used. Various parameters such as bacterial strain and growth conditions lead to different masking susceptibilities. Due to these effects it is impossible to predict the susceptibility of bacterial endotoxin contamination to LER. In order to determine whether a sample is prone to LER, an endotoxin spike that is susceptible to LER is required.
Keywords:low endotoxin recovery; endotoxin; lipopolysaccharide; limulus amoebocyte lysate; naturally occurring endotoxin; bacterial endotoxin testing; masking
Endotoxins play an important role in the pathogenesis and manifestation of Gram-negative infections and in particular of septic shock. Lipopolysaccharides (LPS) are the dominating constituents of the outer leaflet of the outer membrane of Gram-negative bacteria (). Therefore, the terms endotoxin and LPS are used interchangeably ([1,2,3,4]). Because the structures of LPS can vary significantly in its O-antigen, core region and lipid A, not all endotoxins possess the same toxicity . In order to detect endotoxins, diverse detection methods like the rabbit pyrogen test [6,7], the monocyte activation test ([8,9,10], and the Limulus-based tests methods [11,12,13,14] are used. These methods have been used for decades in the pharmaceutical industry to test for potential contaminations of drug products with bacterial endotoxins. Due to their simplicity and high sensitivity [15,16], the Limulus-based tests are the most commonly used methods [15,16]. However, the effectiveness of the current test procedures is under debate regarding Low Endotoxin Recovery (LER). In 2013, Chen and Vinther presented LER, a masking effect of endotoxin in a matrix containing a chelator and a surfactant . They showed that endotoxin spiked into an undiluted sample could not be recovered by dilution or by magnesium replacement, two methods commonly used to overcome test inhibition. After public recognition of the LER phenomenon, some publicized [4,18,19,20,21,22], and many not publicly accessible endotoxin recovery studies of biopharmaceutical drug products and their formulations were performed by endotoxin test providers, contract labs, and pharmaceutical companies. LER is defined as the inability to recover more than 50% activity over time when endotoxin is added to an undiluted product. LER cannot be overcome by dilution. LER and the associated masking effects have been widely confirmed, especially when tested drug products contained surfactants and chelators. Hold-time experiments are usually performed using standardized endotoxins such as the reference standard endotoxin (RSE) or the control standard endotoxin (CSE) as spike. However, since bacterial endotoxin tests are commercially used, the source and preparations of appropriate standard endotoxin are under debate . The primary standard in bacterial endotoxin testing (BET) is RSE, which is endotoxin from E. coli O113:H10:K negative. RSE is purified in several steps including hot phenol-extraction . For better handling, lactose and polyethylene glycol are added. This standard is accepted worldwide and sets the baseline for secondary standards. Because of limited RSE-availability, vendors of Limulus-based test systems distribute secondary standards which are calibrated against RSE. The preparations of such secondary standards are inspired by RSE, but the source can differ from E. coli O113. Exact production processes and formulations are not published. Since LER hold-time studies are widely performed, the source of the endotoxin is controversially discussed. It is especially debated, whether LER is dependent on the source of endotoxin [18,19,22,24,25]. Obviously, depending on source, preparation, and degree of purification, the LPS itself and the accompanying matrix components can vary respectively [26,27]. For instance, endotoxins from different bacteria can differ in their molecular structures [28,29]. There are differences in the lipid A (e.g., acylation), core region (e.g., substitution of sugar units) and O-antigen (e.g., distribution of sugar units). Moreover, depending on the preparation, endotoxin suspensions may vary in their compositions. For example, crude endotoxin extracts of bacteria, also known as Naturally Occurring Endotoxin (NOE), may contain substantial amounts of lipids and proteins, whereas phenol-extracted endotoxin preparations may only contain limited amounts of hydrophobic matrix components. Some experiments have proposed that the detectability of selected endotoxins in complex samples might be more robust compared to the detectability of commercially available standard endotoxins [18,19,30]. Therefore, endotoxins from different bacteria, grown under manipulated conditions, crude and highly purified endotoxins, as well as endogenous endotoxins were analyzed with regard to their detectability in a polysorbate/citrate matrix in the present study.
2.1. Preparation and Characterization of Naturally Occurring Endotoxin (NOE)
In order to study the masking susceptibility of endotoxins from different bacteria, crude suspensions of bacterial endotoxin (NOE) were prepared from E. coli, E. cloacae, S. marcescens, P. aeruginosa, B. cepacia, S. maltophilia and R. pickettii. All bacteria were grown under equal, controlled, and defined conditions (See material and methods “prep a” for details). After 18 h of growth, the absorbance at OD (optical density) = 600 nm of all bacterial suspensions was determined as growth control (Table 1). While the bacterial culture of E. coli and E. cloacae showed the highest absorbance values at 600 nm (>1.7), lower absorbance values were obtained for S. maltophilia and B. cepacia (<0.7) (Table 1). This result indicating varying growth characteristics of different bacteria under fixed growth conditions.
Endotoxins are usually incorporated into the bacterial cell wall. During active proliferation, however, substantial amounts of endotoxins are released into the growth media via exocytosis. Thereby outer membrane vesicles (OMV), LPS monomers or fragments of the bacterial outer membrane resulting from disrupted bacterial cells can be released. All of these particles, besides the actual bacteria, contribute to the endotoxicity (detectable endotoxin activity with bacterial endotoxin test) of the sample. In order to prepare sterile endotoxin spike material, sterile-filtrated supernatants of bacterial suspensions were used and endotoxin activity determined. As a consequence, bacterial cells and bigger breakdown products are removed and their activity does not contribute under given conditions. The measured endotoxin activity of the filtrate of different bacteria suspensions spanned a broad range from approximately 400 to 400,000 EU/mL, although equal growth conditions were used for all stains. Only a weak correlation between the measured OD 600 and endotoxin activity values was observed.
The endotoxicity of different species varies, indicating that endotoxicity seems to be species-dependent.
Endotoxins were applied to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and detected by silver staining (Figure 1). The typical ladder pattern of LPS could be observed in most lanes. The varying intensities of the bands reflect different concentrations, which were approximately in agreement with the detected activities (Figure 1 and Table 1). Upon closer examination, variations in the arrangement of bands between the different endotoxin samples could be observed.
These reflect different molecular structures and again confirm the heterogeneity of endotoxins. Taken together, crude preparations of endotoxin from different species grown at the same growth conditions show a variable endotoxicity and are heterogeneous in their structure.
2.2. Analysis of the Masking Ability of Different NOE
Recent publications have questioned the LER phenomenon. They showed that a “laboratory-derived endotoxin from E. coli” was not masked after spiking it into three different batches of a monoclonal antibody formulated in a polysorbate 80/citrate buffer . Similar results were obtained using a Natural Occurring Endotoxin from Enterobacter cloacae [25,31]. To challenge these observations, NOEs derived from eight different bacterial strains (E. coli O55:B5, E. coli O113, E. cloacae, S. marcescens, P. aeruginosa, B. cepacia, S. maltophilia and R. pickettii), grown under similar conditions, as described in the chapter above, were used as an endotoxin spike for masking experiments in a polysorbate 20/citrate matrix (Table 2 and Figure 2). Crude extracts of endotoxin from E. coli, E. cloacae and S. maltophilia resulted in low recovery already at day 0. In contrast, endotoxin from S. marcescens, P. aeruginosa, and R. pickettii showed a gradual loss of recovery over time. The endotoxin from B. cepacia could be detected over time and showed no trend in reduced activity.
These results show, in clear contrast to the aforementioned data, that NOEs from different bacteria, but grown and prepared under equivalent conditions, exhibit different masking susceptibilities and can be masked in a polysorbate 20/citrate matrix.
2.3. Analysis of the Masking Ability of Different NOEs Depending on Their Growth Conditions
Every preparation of LPS is a heterologous mixture of various LPS molecules [32,33]. While all LPS molecules contain the lipid A moiety and polysaccharides, the degree of acylation, substituents (e.g., aminoethanol) as well as the sugars (e.g., O-antigen) varies. Additionally, bacteria might adapt to the environmental growth conditions, which results in modifications of the LPS molecule. One potential change due to environmental conditions is the modification of an LPS-phosphate group with an amino-arabinose group . In order to exclude that the observed masking effects of NOEs in a polysorbate 20/citrate matrix were caused by growth conditions, we performed a similar experiment with endotoxin derived from two different growth conditions. Three bacterial strains, E. coli O113, P. aeruginosa and B. cepacia were grown under conditions including either rich-nutrition media (100% LB) at elevated temperatures (37 °C) or low-nutrition (1% LB) at room temperatures (prep b). NOEs were prepared as described before. A buffer containing 10 mM sodium citrate and 0.05% (w/v) polysorbate 20 was spiked with 100 EU/mL from both preparations and held for up to 7 days, respectively (Figure 3a). The endotoxin from E. coli O113 was not recovered independent of growth conditions after one day of masking. In contrast, the recoveries of crude extract from B. capecia showed no significant decline (Recovery >50%), independent of growth condition (Figure 3b). Recovery of endotoxin from P. aeruginosa depend on growth conditions (Figure 3c). For both P. aeruginosa preparations the same recovery trends are observed, but the recovery dropped below 50% already within one day (bacteria grown at 37 °C using 100% LB media). For the second preparation, the recovery dropped below 50% only after 2 days. Thus, modified growth conditions resulted in diverging masking kinetics.
Taken together, this result demonstrates that masking can be dependent on the growth conditions and the nutrition content in the media.
2.4. Comparison of the Masking Behavior of Purified Lipopolysaccharides (LPS) and NOE
For this study we analyzed NOEs from different species. However, in the pharmaceutical industry purified LPS preparations are used as reference standard endotoxin and as control standard endotoxin. These standards are purified by phenol-chloroform extraction and further steps (e.g., size exclusion chromatography). They are the endotoxin of choice when masking is analyzed in hold-time studies. As most of the studies that are used to analyze masking and its mechanism are performed using purified LPS preparations [20,21,35], masking for purified LPS preparations is well established. Purification of spike material is the basis for standardization because of its required identity and known purity . To further evaluate the impact of endotoxin purification, endotoxins from one bacterial species (E. coli O55:B5) were prepared by different methods and subsequently analyzed. Phenol-extracted endotoxin and crude supernatants of bacterial suspension were spiked into a polysorbate 20/citrate matrix and incubated for up to six days at room temperature (Figure 4a). Both preparations showed no recovery after one day of incubation, which confirms the pronounced masking susceptibility of endotoxin from E. coli.
Due to the fast kinetics of masking, the experiment was also performed at decreased incubation temperature 2 to 8 °C (Figure 4b). The reduced incubation temperature was chosen, as endotoxin masking can be decelerated, allowing a better resolution of slight differences in masking susceptibilities (Figure 4b). After 2 days of incubation, 50% and 14% after 14 days of the initial endotoxin content could be detected within the NOE at 2 °C to 8 °C. In comparison, the recovery of phenol-extracted endotoxin was already low after one day of incubation (27%) and no significant endotoxin content was detectable after three days of incubation. At lower incubation temperature the detectability of NOE decreased slower, compared to phenol-extracted endotoxin. However, both preparations of endotoxin were affected by masking.
In consequence, endotoxin-masking kinetics can be affected by the extraction method of endotoxin, but the kinetics rather depends on the source of bacteria than on the purification state of the endotoxin.
2.5. Masking Behavior of an Endogenous Endotoxin Contamination
For all of the examples above, the source of endotoxin was known. Endotoxins were consciously added to samples containing surfactants and chelators. In order to examine a real endotoxin contamination, the detectability of an endogenous contaminated monoclonal antibody was analyzed. The lyophilized antibody was solubilized in four different chelator-containing buffer systems (A: 25 mM sodium citrate pH 6.5, B: 10 mM sodium citrate pH 7.5, C: 160 mM trehalose and 10 mM sodium phosphate pH 6.2 and D: 10 mM sodium phosphate pH 7.5). An average endotoxin activity of 135 EU/mL and 114 EU/mL was determined before and after sterile filtration, respectively (Figure 5a). The different buffer systems as well as the filtration had no major effects on the detectability of the endogenous endotoxin contamination of the antibody. Only small differences between the activity before and after the filtration step were observed. As expected, no masking was observed in the buffer systems containing only the chelating agent. As is known from previous studies on the mechanism of LER [20,21], masking is driven by a simultaneous presence of a surfactant (e.g., Polysorbate 20 or Polysorbate 80) and a chelator (e.g., sodium citrate). Therefore, 0.07% (w/v) of the surfactant Polysorbate 80 was added to the antibody in buffer A, 0.05% (w/v) Polysorbate 80 to the antibody in buffer B, 0.04% (w/v) Polysorbate 20 to the antibody in buffer C and 0.05% (w/v) Polysorbate 20 to the antibody in buffer D, respectively. The endotoxin concentration was directly determined after adding the detergents (day 0). Comparable activities as determined directly after filtration were observed (Figure 5a). After an incubation of 3 days at room temperature, the endotoxin content was determined again. A significant decrease (90% to 98%) of the endotoxin activity could be observed in all four samples (Figure 5b).
Endotoxin masking was observed in all four antibody preparations, independent of the composition of the masking buffer. According to previous publications, all of these buffers have masking capabilities [20,21]. Thus, the results are consistent with the above mentioned data and literature, no further replicates or statistical analysis of the data were performed. The present data show a series of experiments, in which NOEs can be masked under LER-conditions, but with various masking kinetics. Individual masking kinetics are consistent when reproduced (Figure 3) or compared to other experiments (Figure 2, Figure 3 and Figure 4). Finally, the data clearly demonstrates that an endogenous endotoxin contamination, which reflects a real naturally occurring endotoxin, is masked in a commonly occurring formulation buffer of biologicals.
Endotoxins are complex molecules originating from the outer membrane of Gram-negative bacteria containing a hydrophobic lipid moiety and a polysaccharide moiety. The polysaccharide part of the endotoxin is highly diverse and varies in number of sugars within a unit, the nature of the linkages of the sugars, as well as the number of repetitive units. O-antigen sugars appear to be most variable, core structures appear to be less variable. In contrast, lipid moieties of LPS are considered highly conserved within a genus [1,37]. Gram-negative bacteria exhibit only minimal growth requirements and can thus be found in clean water, processed water, or buffers , required for the production of biopharmaceuticals. As endotoxins are strong stimulators of the human immune system mediated by the Toll-like receptor 4 complex, monitoring of endotoxins is a crucial step in the production of biopharmaceuticals. While the Limulus-based tests are the method of choice to test for a potential endotoxin contamination, these have been challenged under the light of the low endotoxin recovery phenomenon. To evaluate whether a sample is affected by LER, regulatory authorities request hold-time studies to evaluate detection of an endotoxin spike in an undiluted drug product over time, but the kind of endotoxin used as spike was under debate. Recent studies suggested the use of NOE as standards for hold-time studies instead of purified endotoxin. Moreover, it is hypothesized that masking of endotoxin is mainly induced by the use of purified endotoxin [39,40]. The authors claim that the use of NOEs is closer to contaminations observed during biopharmaceutical drug production. Purified endotoxins are not observed in nature and exhibit different properties due to the extraction process and missing pieces like proteins or lipids. Hence, to investigate whether the phenomenon of LER is limited to standardized endotoxins from E. coli, detectability of NOEs from different bacterial species was studied in a typical biopharmaceutical drug product matrix containing polysorbate and sodium citrate. Such a matrix has been shown to induce LER. In this work, it could be shown that some NOEs were masked as well. However, masking of NOEs was shown to depend on species and growth conditions. While no masking was observed over a 7-day period for NOEs from B. cepacia and R. pickettii, masking was observed for crude extracts of E. coli, E. cloacae, S. marcescens, P. aeruginosa and S. maltophilia. These results show endotoxins which are rapidly affected by LER (e.g., E. coli O113) and endotoxins which are less affected by LER. Endotoxin from B. cepacia was not affected by masking under given conditions. Regarding B. cepacia, it is described in the literature that the LPS possess an unusual structure. The bacteria lower the anionic charge of the cell surface by the substitution of 4-amino-4-deoxyarabinose residues bound to phosphates of the lipid A backbone [41,42,43]. This might explain the observed limited masking susceptibility. These results would prove NOEs unsuitable for endotoxin hold-time studies. The absence of masking in a drug product could be due to the analyzed species and its respective growth conditions. However, masking would be observed in this exact drug product, if a different species had been used. It has to be pointed out that not all endotoxins are similarly affected when growth conditions are modified. A change of masking susceptibility cannot automatically be observed when comparing recovery kinetics of endotoxins from the same source, but derived from different growth conditions.
These results can be explained with an adaptation of the bacteria to an unfavorable environment to ensure viability. It is known that bacteria are able to modify their primary LPS structure under certain growth conditions. Thereby they can reinforce the external membrane to assure optimal protection against the environment . Recent publications have claimed that the endotoxin extraction method is the reason for LER, as the endotoxin is extracted from its native environment and can adopt non-natural aggregation states. The present findings, using highly purified endotoxin and NOE from the same species (E. coli O55:B5), show that masking is not caused by the purification process itself, as both preparations were similarly masked. At decreased temperature, the masking process of the NOE was slower than observed for the purified endotoxin. These results indicate a change in the masking kinetics, probably due to the additional elements like proteins and lipids within the crude extract. However, masking characteristics remained unchanged, independent of the purification process. Taken together, masking characteristics of a given endotoxin preparation are mainly influenced by growth conditions, not by the purification process. With the exception of Schwarz et al. , all published studies either used a purified endotoxin standard or a lab-derived NOE to analyze masking and to simulate a possible contamination. Within this study, the masking behavior of an endogenous endotoxin contamination affecting a commercial antibody was analyzed, as this represents a realistic case. The contamination is observed in the final product and the bacterial species and source of the contamination is unknown. This represents a scenario that pharmaceutical quality control departments face during product release. The masking behavior of this contamination was analyzed using four commonly used buffers for drug product formulation. Within all four buffers, rapid masking was observed in the tested product. Less than 10% of the initial endotoxin activity was measured after three days. These results clearly show that endotoxin masking is not linked to the purification process, but rather to the molecular structure. This emphasized that NOEs are no solution to overcome LER, as masking was also observed for an endogenous endotoxin contamination. The use of NOEs in hold-time studies could instead be misleading, as masking would not be observed with the analyzed product, but might indeed be observed with an endogenous contamination.
The origin of LPS is inevitably connected to the respective bacteria. However, an intrinsic heterogeneity is certain. It is impossible to predict source and cause of potential bacterial endotoxin contaminations of a sample. As a consequence, the masking susceptibility of a potential endotoxin contamination is unknown, too. In order to ensure reliable detection of endotoxins, the masking capability of a sample has to be evaluated. To analyze the masking capability of a sample, endotoxin hold-time studies have to be performed with endotoxin spikes, that are susceptible to masking. The results above have shown that standardized endotoxins from E. coli exhibit a pronounced susceptibility to endotoxin masking and represent an appropriate source for endotoxin hold-time studies. With regard to the heterogeneity of endotoxins and the diversity of sample compositions, a panel of different endotoxins might be the safest way to determine the masking capability of a sample and to ensure detectability of a potential contamination. A reference standard and an additional control standard from different species could be used as such a panel. Lastly, as soon as a sample is identified with the capability of endotoxin masking, a suitable detection method needs to be developed in order to properly detect endotoxins and to avoid the underestimation of a contamination.
4. Material and Methods
4.1. Chemicals and Materials
Polysorbate 20, polysorbate 80, sodium chloride, sodium azide, trisodium citrate, phosphoric acid, sodium dihydrogenphosphate, potasium dihydrogenphosphate, disodium hydrogen phosphate-heptahydrate, 2-amino-2-(hydroxymethyl)-1,3-propanediol (tris), 2-mercaptoethanol, isopropanol, d(+)-glucose, sodium chloride, calcium dichloride and magnesium dichloride were obtained from Sigma-Aldrich Chemie GmbH, Steinheim, Germany. Ammonium hydroxide, formaldehyde and d(+)-trehalose-dihydrate were obtained from AppliChem GmbH, Darmstadt, Germany. Acetic acid, glycerol, periodic acid, sodium hydroxide, silver nitrate, sodium dodecylsulfate (SDS) and yeast extract (powdered) were obtained from Carl Roth GmbH & Co. KG, Karlsruhe, Germany. Bromophenol blue sodium salt was obtained from Merck Chemicals GmbH, Darmstadt, Germany. 20× Tris-tricine/SDS electrophoresis buffer were obtained from Serva Electrophoresis GmbH, Heidelberg, Germany. The mouse monoclonal antibody (MAK33) was obtained from Roche Diagnostics Deutschland GmbH, Mannheim, Germany. Tryptone Bacto TM was obtained from Becton Dickinson GmbH, Heidelberg, Germany. Depyrogenated water, depyrogenated borosilicate glass tubes and recombinant Factor C tests, EndoZyme® and EndoLISA® were obtained from Hyglos GmbH, Bernried, Germany. Prior to the experiments, all relevant materials were tested for endotoxins and proven to contain less than 0.05 EU/mL.
4.2. Endotoxins and Bacteria
Endotoxin from Escherichia coli O55:B5 (gel-filtrate) was obtained from Sigma-Aldrich Chemie, Steinheim, Germany. Freeze-dried bacteria from E. coli O55:B5 (DSM 4779), Enterobacter cloacae (DSM 30054) and Pseudomonas aeruginosa (DSM 500 71) were obtained from Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany. Freeze-dried bacteria from E.coli O113 (Ecor 30) was obtained from Escherichia coli Reference Collection, East Lansing, USA. Freeze-dried bacteria from Burkholderia cepacia (2008 B02-12.20.164) and Stenotrophomonas maltophilia (DSMZ 50 170) were obtained from Robert Koch-Institute, Wernigerode, Germany. Bacteria from Ralstonia pickettii (isolate) were a kind gift from Hyglos GmbH, Bernried, Germany.
4.3. Preparation of Crude Endotoxin Extracts (Naturally Occurring Endotoxins, NOEs) (Prep a)
For bacterial growth, 5 mL lysogeny broth (LB) media (10 g/L sodium chloride, 5 g/L yeast extract and 10 g/L tryptone) were inoculated with the desired bacterial strain, followed by incubation overnight at 37 °C in a shaking incubator (Platform shaker: Innova 2300, New Brunswick Scientific Co, Enfield, USA; Incubator: Wärmeschrank für Plattformschüttler, Mytron Bio- und Solartechnik GmbH, Heilbad Heiligenstadt, Germany). Afterwards, 200 µL of the preparatory culture were transferred into 500 mL of media (12.8 g/L disodium hydrogenphosphat-heptahydrat, 3 g/L potassium dihydrogenphosphat, 0.5 g/L sodium chlorid, 1 g/L ammonium chloride, 0.01 g/L calcium dichloride and 0.4 wt% glucose) and incubated for 24 h at 37 °C. Growth of bacteria was monitored by light absorption at 600 nm using a spectro photometer (V550 Jasco Germany GmbH, Gross-Umstadt, Germany). Bacterial growth was stopped by temperature reduction to 4 °C. Crude bacterial extracts (Natural occurring endotoxins) were prepared by centrifugation of the culture at 4500 rpm (Heraeus Multifuge 3 S-R) and sterile filtration (0.2 µm, Pall GmbH, Dreieich, Germany) of the supernatant. For conservation 0.05% (v/v) sodium azide were added. Required endotoxin concentrations for endotoxin recovery studies were adjusted by dilution with depyrogenated water.
4.4. Preparation of NOEs from Bacteria Grown under Low-Nutrition Conditions (Prep b)
Five mL LB media (10 g/L sodium chloride, 5 g/L yeast extract and 10 g/L tryptone) were inoculated with the desired bacterial strain, followed by incubation overnight at 37 °C in a shaking incubator (Platform shaker: Innova 2300, New Brunswick Scientific Co, Enfield, USA; Incubator: Wärmeschrank für Plattformschüttler, Mytron Bio- und Solartechnik GmbH, Heilbad Heiligenstadt, Germany). Afterwards, the preparatory culture was transferred into 20 mL of LB media or a minimal media (1% LB media in depyrogenated LRW) and incubated for 18 h at room temperature. Bacterial growth was stopped by temperature reduction to 4 °C and sterile filtration (0.2 µm) of the bacterial suspension. For conservation 0.05% (v/v) sodium azide was added. Required endotoxin concentrations for endotoxin recovery studies were adjusted by dilution with depyrogenated water.
4.5. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Silver Staining
The NOEs of all different species were vortexed for 30 s. 40 µL of each sample were mixed with 10 µL sodium dodecyl sulfate (SDS) loading buffer and boiled at 100 °C for 10 min. 18 µL of these samples were loaded on a 20% gradient Tris-Tricin gel (Anamed Elektrophorese GmbH, Rodau, Germany). The sodiumdodecylsulfatepolyacrylamid gelelectrophoresis was performed in Tris-Tricine/SDS buffer at 130 V for 90 min. The gel was fixed overnight in fixation solution (25% (v/v) isopropanol and 7% acetic acid) and oxidized for 5 min using an oxidation solution (0.06 g/mL periodic acid, 0.33% (v/v) Isopropanol and 0.09% (v/v) acetic acid. The gel was washed 5 times in depyrogenated water and silver stained for 10 min in staining solution (350 µL sodium hydroxide (8 M), 1 mL concentrated ammonium hydroxide (28% (w/v), 2 mL silver nitrate (20% w/v) and 75 mL depyrogenated water). The stained gel was washed 5 times with depyrogenated water and developed for 10 min with the development solution (1 mg/mL citric acid, 0.037% formaldehyde). The development was stopped using acetic acid (10% v/v).
4.6. Sample Masking Preparation for Masking Kinetics
Samples were prepared in 5 mL glass tubes with sample volumes of 1 mL per sample. Unless otherwise described, samples were spiked with a 10-fold or concentrated stock solution of the respective endotoxin. The pH of buffer components was adjusted to 7.5 if not otherwise described. Before adding the spikes to the sample, the stock solution was shaken at 1400 rpm for 10 min. After spiking, the samples were incubated at defined temperatures and for defined periods of time. Immediately after incubation, the samples were mixed at 1400 rpm for 2 min again, and diluted in depyrogenated water in order to avoid test interference. Necessary dilutions of the particular sample compositions were determined prior to the actual experiment.
Samples with different incubation periods were measured on the same microtiter plate, to avoid variation from test to test. Therefore, endotoxin recovery kinetics was performed in a reverse manner. The particular sample was aliquoted and all aliquots were stored under equal conditions over time. The aliquot with the longest endotoxin incubation period was spiked first (e.g., 7 days prior to the measurement). Further aliquots with shorter incubation periods were spiked later in accordance with the respective incubation period. The zero-time point aliquot was spiked immediately before measurement. Resulting kinetics consists of various time points and reflects one experiment, if not otherwise indicated.
The validity of the actual measurement was controlled by spiking the defined endotoxin amounts into the diluted samples (Positive Product Control (PPC)). Endotoxin determination in a diluted sample was considered valid, if 50% to 200% of the spiked endotoxin (PPC) was recovered. To control the accuracy of the endotoxin spiked into the undiluted samples, equal amounts of endotoxin were spiked into depyrogenated water (water control), mixed and identically incubated as the actual sample. The spike into the undiluted sample was considered valid if the water control was in the range of 50% to 200% of the theoretical expected spike concentration.
For calculation of the recovery, the determined endotoxin concentrations in the tested samples were compared to the endotoxin concentrations at time zero in water controls and stated as percent.
4.7. Sample Preparation using an Endogenous Endotoxin Contamination
The lyophilized monoclonal antibody (MAK33) was solubilized at a concentration of 10 mg/mL in buffer A (25 mM sodium citrate, pH 6.5), buffer B (10 mM sodium citrate, pH 7.5), buffer C (160 mM Trehalose, 50 mM sodium phosphate pH 6.2) and buffer D (10 mM sodium phosphate, pH 7.5). The endotoxin concentration of the natural contamination was determined before and after sterile filtration using the EndoZyme® assay. After filtration, 0.07% (v/v) polysorbate 80 were added to the sample in buffer A, 0.05% (v/v) polysorbate 80 were added to the sample in buffer B, 0.04% (v/v) polysorbate 20 were added to the sample in buffer C and 0.05% (v/v) polysorbate 20 were added to the sample in buffer D, respectively. The endotoxin content was determined immediately after the addition of polysorbate (day 0) and after incubation at room temperature (19–25 °C) after 3 days (day 3) using the EndoLISA® assay. For calculation of endotoxin recovery, the determined endotoxin concentrations in the actual sample was compared to the determined endotoxin concentration in the water control stated as percentage.
4.8. Endotoxin Detection
For bacterial endotoxin detection, Limulus-based tests (rFC) were used according to manufacturer’s instructions. The amount of fluorescence substrate (amino-methylcoumarin) released was measured fluorometrically at 440 nm (Excitation: 380 nm) with a FLx800 fluorescence microplate reader (BioTek Instruments GmbH, Bad Friedrichshall, Germany). All samples were measured in duplicate and average values were used for further calculations. Standard curves were fitted using a four-parameter logistic non-linear regression model. The detection limit of the assay was 0.005 EU/mL (EndoZyme®) and 0.05 EU/mL (EndoLISA®). If not otherwise indicated, EndoZyme was used for endotoxin detection.
Microsoft Excel 2010, Version 14.0.7015.1000 was used to calculate endotoxin recovery, plot graphs and to simulate endotoxin recovery kinetics. Gen5 Data Analysis Software Version 2.05 from BioTek Instruments GmbH, Bad Friedrichshall, Germany was used to calculate standard curves for determination of endotoxin concentrations.
J.R. was responsible for measurements, reading and writing. F.A.W. was responsible for data curation and writing. H.T. and I.N. were responsible for details of the study concept, reading and writing. H.M. was responsible for the general study concept and supervision.
This research received no external funding.
The authors wish to express their gratitude to the participating organizations, which enabled a charitable and comprehensive, collaborative study between academia and industry.
Conflicts of Interest
The authors declare no conflict of interest.
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Figure 1. Analysis of crude bacterial extracts. A sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the crude extracts was silver-stained. The lanes reflect endotoxins from E. coli O55:B5 (1), E. coli O113 (2), E. cloacae (3), S. marcescens (4), P. aeruginosa (5), B. cepacia (6), S. maltophilia (7) and R. pickettii (8).
Figure 2. Hold time study using NOEs of different species. Endotoxin recovery over seven days at room temperature are shown for different species (E. coli O55:B5 (a), E. coli O113 (b), E. cloacae (c), S. marcescens (d), P. aeruginosa (e), B. cepacia (f), S. maltophilia (g) and R. pickettii (h)). For spiking, a 10-fold concentrated stock solution was prepared. The concentration of the water controls corresponding to the activities (EU/mL) are shown in (i). For the recovery experiments, the crude extracts were spiked into 0.05% (w/v) polysorbate 20 and 10 mM sodium citrate. The recoveries were determined after 0, 1, 2, 5 and 7 days. All recoveries were calculated based on the corresponding water control at time point 0.
Figure 3. Masking ability of different NOEs depending on the growth condition. Endotoxin recovery was plotted as function of incubation time. 100 EU/mL endotoxin from different bacteria ((a) E. coli O113:H21, (b) B. cepacia and (c) P. aeruginosa) were spiked into samples containing 10 mM sodium citrate and 0.05% (w/v) polysorbate 20 and incubated at room temperature (RT). The used endotoxin extracts were derived from two different bacterial growth conditions. Squares reflect recovery of endotoxin from bacteria grown at 37 °C using 100% LB media. Triangles reflect recovery of endotoxin from bacteria grown at room temperature using 1% (v/v) LB media. Each endotoxin was prepared in triplicate. The corresponding endotoxin measurements of three repetitions were analyzed on the same microtiter plate. For calculation of the data points the mean value of the three individual preparations was used. The error bars represent the standard deviation of the three replicates.
Figure 4. Differences in the masking behavior of purified endotoxins and NOEs. Recovery of endotoxin from E. coli O55:B5 is shown over incubation time. 100 EU/mL of gel-filtrated endotoxin (black bars) and sterile filtrated bacterial suspension (grey bars) were spiked into samples containing 10 mM sodium citrate and 0.05% (w/v) Polysorbate 20. The samples were incubated up to 14 days at RT (a) and 4 °C (b).
Figure 5. Masking of an endogenous endotoxin contamination. Endotoxin activity of an unknown endotoxin contamination under different buffer conditions is shown. 10 mg/mL monoclonal antibody (MAK33) were solubilized in 25 mM sodium citrate, pH 6.5 (buffer A); 10 mM sodium citrate, pH 7.5 (buffer B); 160 mM trehalose, 50 mM sodium phosphate pH 6.2 (buffer C) and 10 mM sodium phosphate, pH 7.5(buffer D). Endotoxin content was determined before and after sterile filtration (0.2 µm) (a). For endotoxin detection EndoZyme® was used. After filtration 0.07% (w/v) and 0.05% (w/v) polysorbate 80 were added to the sample in buffer A and B, respectively. 0.04% (w/v) and 0.05% (w/v) polysorbate 20 were added to the sample in buffer C and buffer D, respectively. Endotoxin activity was determined immediately after addition of polysorbate (day 0) and after incubation of three days (day 3) at RT (b). For endotoxin detection the EndoLISA® was used.
Table 1. Correlation of bacterial growth behavior and endotoxin activity. To monitor the growth of different Gram negative bacterial species (E. coli O55:B5, E. coli O113, E. cloacae, S. marcescen, P. aeruginosa, B. cepacia, S. maltophilia and R. pickettii), the OD 600 was determined before harvesting and correlated with the activity of the prepared crude extract.
|E. coli O55:B5||1.9||146,174|
|E. coli O113||1.7||402,789|
Table 2. Hold time study using naturally occurring endotoxins (NOEs) from different species. The hold time study was performed over 7 days using the prepared NOEs for different species. All recoveries in percentage were calculated based on the water control at t(0).
|Masking Time in Days||Endotoxin Recoveries Based on the Water Control t(0) (%)||Activity of the Water Control (EU/mL)|
|E. coli O55:B5||32||7||8||2||0||40.9|
|E. coli O113||42||0||0||0||0||64.7|
© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

domain: biology


text:
A Joann's fabric store is closing in a nearby town. Poor town. I'd almost feel bad for them except that the reason they are closing is so that they can relocate their store to my town! Mine! Yay. I took advantage of their close-out sales, and came home with some of this sweet stuff.
There are good things and bad things about close-out sales, it's all at a great price, but it has been nit-picked to death. I wasn't able to find black or white thread for instance.
And this little trinket is the reason for the given title of this post. My daughter loooves wallets. She loves digging through them and pulling out all the cards so she can hide them under the rug. I felt it high time I made her a play-wallet for all of those oh so insignificant and unused old cards you always have stuffed in your wallet taking up space.
As I was picking the colors I thought I wanted it to be bright, and this is a little on the warm side I think. The colors remind me so much of candy starbursts.
Red, pink, yellow and orange. Yum!!!
She loves it.
And the winner of the Easter goodie-bag giveaway is:
Jamie, if you could please contact me at firstname.lastname@example.org that would be wonderful!

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text:
Anyone following the Guantánamo military commissions would do well to read Bob Loeb and Helen Klein's trenchant take on last Friday's D.C. Circuit decision in In re Khadr, in which the Court of Appeals declined to issue a writ of mandamus even while agreeing that there may be a serious question "whether the civilians who serve as judges on the U.S.
Latest in Karen LeCraft Henderson
This morning, a three-judge D.C.
As I explained on Sunday, one way to understand the diffference between the majority and dissenting opinions in last Friday's D.C. Circuit decision in al Bahlul v. United States is as reflecting two different methodological approaches to the question of whether Congress can empower non-Article III military commissions to try "domestic" offenses like inchoate conspiracy.
At their simplest, both Judge Henderson's 85-page dissent from the D.C. Circuit's decision in al Bahlul v.


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