Contrary to frequent beliefs, personal trainers are normally not simply confined into the premises of gyms and health and health clubs. Nowadays, such professionals are additionally getting into different venues and establishments to offer their professional services. Many prospective shoppers additionally find consolation to know that such trainers may now be present in leisure, business, recreational, and home settings.
Private trainers are professionals who?re very a lot qualified to assist and information folks into having good health and extra active lifestyle. Other than having earned 4-yr school levels in kinesiology, train science, or different related programs, most personal trainers right this moment are also certified by major bodily coaching organizations in a selected state or area. Thus, they absolutely are maintaining high requirements and expertise to offer purchasers only the most effective and most dependable services.
As talked about, aside from working in gyms, health centers, and health golf equipment, modern personal trainers might take other job choices at the following venues:
Spas and resorts. Spas and resorts are undoubtedly among the hottest recreational facilities to date. Because of the extraordinary competitors, such establishments are often offering extra worth added services. Included in those added offerings are private training services. On the whole, most of the presently sought-in any case-inclusive resorts are hiring trainers to function health instructors that may teach specific exercise classes, that are supplied as extra persuaders to make guests carry on coming. Many personal trainers favor working in spas and resorts for their very own comfort, convenience, and enjoyment.
Cruise ships. Even luxury cruises are actually providing bodily coaching as value-added or bonus choices to guests. Personal trainers who prefer to work in cruise ships ought to conform to go on with the vessel regardless of destinations. Many luxurious vacationers on cruises want to get private coaching throughout idle days, once they have nothing to do however to relax and luxuriate in life. Most cruise ships immediately offer private training providers for competitive reasons.
Company Fitness. After world firms have started specializing in corporate social accountability, there is another rising trend these days. Many businesses are actually aiming to make their staff maintain good health and fitness regardless of lengthy hours of sitting in front of computers or being confined in workplace premises. The corporate sector is undoubtedly turning into one of many fastest-rising areas the place health and well being emerge. Wellness packages supplied by firms embody private training classes, whereby personal trainers could be assigned to every keen employee. Occasionally, trainers additionally meet the complete employees to teach them how one can do indoor and simple physical workout routines to induce active body movements.
In-dwelling training. Private trainers may additionally opt to simply accept in-residence training sessions. By way of this, they would come over to a shopper?s home and facilitate the training program there. Normally, this sort of service is in a premium in comparison with the conventional type. However, shoppers choose it more for safety and convenience reasons. In-residence training works for personal trainers whose shoppers are within their vicinity. In any other case, this could not be a superb option.
Work-for-yourself. Personal trainers might always perform as his personal impartial contractor if he doesn?t want to be affiliated by any health and fitness club, which could impose on him hefty commissions. In many cases, independent private trainers opt to ascertain their own fitness studios eventually.
If you need more data about personal training business, drop by Neuman Toperstein?s website in a jiffy.
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Yahoo's Flickr is about to punish the popular photo-sharing iPhone app Instagram for not releasing an Android version yet. Flickr launched its Android app Wednesday -- complete with Instagram-like filters. The new Android app brings the Flickr photo stream to Google's mobile OS. The company has included a new activity stream for viewing photos -- one that's essentially a newsfeed of activity among your Flickr friends. You can browse photo albums or check out the albums of friends.
[More from Mashable: Yahoo?s Strategy After Bartz? ?Deeply Personal Digital Experiences?]
"A photo is more than just a few pixels," Flickr Product Manager Markus Spiering said at a press event at Flickr's headquarters in San Francisco. "It's also the context."
Viewing an image on Flickr's Android app is a different experience compared to its iPhone app. The Android app adds features such as likes, comments, photo data and a map showing where the photo was taken. Users can swipe left or right to browse a photo album. There is also a fullscreen experience for browsing photos that includes a slideshow feature.
[More from Mashable: AOL, Yahoo & Microsoft Team Up to Sell Ads [REPORT]]
The core feature of the app, of course, is photo-taking. Flickr has developed a new viewfinder screen (see above). But there's one feature that will catch everybody's attention: photo filters.
The photo filters feature is almost identical to that of Instagram. The app comes with 10 filters that Spiering says are designed to "beautify" photos. They're automatically uploaded to Flickr but can also be shared via Twitter, Facebook and email.
The filters are Android-only for now, but we doubt it will be long until they make their way to Flickr's iPhone app.
Flickr has 68 million registered users that have uploaded more than six billion photos. But newcomer Instagram has momentum and a very loyal user base. This should make for an interesting contest.
Email advertising is currently creating a buzz in the online world and is considered to be one of the top online business promotion techniques. This is where you send an advertisement in the form of a message to your clients and potential clients. These advertisements could simply contain information on various products or services of could be of a promotional nature such as those offering discounts or special offers to clients who make certain purchases. This should not be confused with spamming since you can only send these messages to those clients who opted in to your mailing list.
Email advertising is beneficial in many ways especially to those businesses that are still trying to curve out a niche for themselves in the online world. For one, they are an effective way of building customer loyalty. When you send out these emails, you can personalize them in one way or another such as by adding a greeting with the customer?s name. This helps to build the clients trust in you and also since email offers an instant feedback mechanism they can be able to get to you immediately on any clarifications that they may require regarding your advertisement. This therefore increases the chances of them actually visiting your site and taking some action there.
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Posted on 26. Sep, 2011 by BradleyBeechley8322 in Society
There?s a lot to be discovered about health and drugs, typically it is straightforward to get overwhelmed. Most people I do know do not likely take the time to consider the methods they?re caring for their physique and the effects of their decisions on their brief and long run health. I?ve been learning quite a bit about various varieties of alternative medication and I?ve turn out to be convinced that more folks must be knowledgeable that there are alternatives in your health and fitness needs.
Principally, alternative drugs deals with all kinds of non-conventional medicines and strategies of remedy for all of the medical conditions people can discover themselves in. Folks have begun to be extra involved currently with the issues that they?re putting into their our bodies, so different drugs has turn out to be considerably of a buzzword that is being thrown around amongst health-acutely aware individuals right now.
I grew to become fascinated with different drugs after I needed to research and write in regards to the latest tendencies in it for an article in my magazine. I had quite truthfully by no means given a lot thought to the doctor?s orders for certain drugs and strategies of remedy until I began researching for this piece. I quickly realized how cautious we should be about anything that we put into our bodies. In any case, we only get one physique to dwell in and handle, so it?s solely our responsibility to be knowledgable about ourselves and to make the very best decisions for our personal health.
I feel this possession issue is the factor I admire most about people who are involved in alternative medicine. They?re principally proactive people who are searching for new methods to treat their signs and who?re convinced that the more pure something is, the higher it?s for them and for their health.
There?s far too much to say about different drugs right here, so my easy goal is just to tell you that you?re accountable for your health. You are responsible for all the foods you eat, the drinks you drink, and the medicines and coverings that you simply choose. So choose wisely. Take time to think about your entire options, and see if various medicine may present a greater answer for you and your health needs.
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A few days ago, I got to sit down with director Brian Taylor for an exclusive interview on Ghost Rider: Spirit of Vengeance.? With a February 17, 2012 release date fast approaching, Taylor is currently in post-production finishing up the movie.? ?However, while the movie is still almost five months from getting released, they already have a few deleted scenes and Taylor says the sequel will be 90 minutes (the original was 114).
During the interview we talked about where they are in the editing process, the experience of post converting to 3D and his thoughts on 3D, what to expect from Idris Elba and Nicolas Cage in the film, the deleted scenes, the run time, what will be on the DVD/Blu-ray (they captured thousands of hours of footage: interviews, features, behind-the-scenes stuff), what it was like to film in Eastern Europe (he tells some crazy stories), will there be other Marvel characters in the sequel, the full trailer, and a lot more.? Trust me, if you?re a fan of Neveldine/Taylor and have been looking forward to Ghost Rider 2, you?re going to like this interview.? Hit the jump to watch.
Finally, if you missed the teaser trailer for Ghost Rider: Spirit of Vengeance, click here. ?You can also check out over 30 images from the film here.? And one last thing, a huge thank you to Brian Taylor for giving me so much of his time.
Since I know some of you don?t have 15 minutes to watch the interview, here?s 10 things to know about the Ghost Rider sequel:
Neveldine and Taylor?s run-and-gun shooting style does not lend itself to the traditional rules of what looks good in 3D.? They ignored those ?rules? and shot exactly the way they wanted to shoot.
Ghost Rider 2 will be post-converted in to 3D, but don?t fear a rush job.? Taylor was focused on making the 3D ?state of the art, cutting edge, the best it could possibly be.?
The runtime for the sequel is about 90 minutes.? (The original was 114 minutes.)
No prior Neveldine/Taylor film has ended up with a true deleted scene.? Ghost Rider 2 will end up with 2-4 on the DVD.
Alongside the deleted scenes, the DVD/Blu-ray will be packed with interviews, features, and behind-the-scenes footage.? Taylor claims they captured ?thousands and thousands and thousands of hours of footage.?
Cage will be unleashed in this movie.? They wanted ?every face of Nic.?? At the end of the shoot, Cage told the directors, ?Guys, I don?t think I have any other ways to be crazy.? I think I?ve done all of it.?
At the same time, Taylor thinks Elba will blow people?s minds with his charisma.? ?That guy could kill babies on screen.? He?s so likeable.?
Neveldine and Taylor came on to the project wanting to reinvent Ghost Rider on screen.? They redesigned the character, made it ?a lot darker, a lot weirder.?? Sony embraced their take.
The teaser trailer barely showed off anything because most of the CG and ?a lot of the most spectacular things? weren?t done.? They saved some images for the full trailer.
Filming in eastern Europe was dangerous.? To illustrate the level of guerilla filmmaking, Taylor describes a scene where both the actor (Elba) and the camera operator (Neveldine) go over a cliff.? It seemed like the rig could fall over with a slight wind.
Here?s the interview and time index so you can watch the parts that interest you.
Brian Taylor Time Index
Taylor decides to show the readers of Collider an exclusive 3D clip from the movie.
:50 ? They?re putting the final touches on the movie in the editing room.
1:30 ? Post-converting to 3D has been ?awesome.?? Taylor acknowledges badly converted movies, and promises better for Ghost Rider 2.
3:20 ? Neveldine/Taylor found that a lot of the advice they got on what looks good in 3D and what doesn?t was bad advice.
4:30 ? ?There are different notches of Nic-ness.?? They wanted all of Cage?s acting styles in the movie.
5:30 ? Taylor believes Idris Elba will blow people?s minds in this movie.
6:20 ? There are no other Marvel characters in the movie.
6:45 ? The DVD is going to be ?insane.?
7:00 ? The studio has been really supportive of the Neveldine/Taylor vision.
7:40 ? They saved some of the best stuff for the full trailer, though he doesn?t know when that will hit.
8:15 ? Ghost Rider does not eat jellybeans in the sequel.
8:30 ? Talks about the guerilla filmmaking aspect of shooting in eastern Europe.
10:10 ? The runtime is ?90 minutes-ish.?
11:00 ? They are always open to working with Jason Statham, even outside of a Crank capacity, but nothing is in the works at this time.
12:10 ? There?s so much stuff for the DVD/Blu-ray.? They captured thousands of hours of footage: interviews, features, behind-the-scenes stuff.
13:30 ? They ended up with 2-4 deleted scenes.
15:10 ? Believes the version of the movie that came out is the version of the movie.
Business Continuity, Disaster Recovery & ROI ? How Do They Fit?
A business continuity/disaster recovery group on LinkedIn posted an interesting question about whether business continuity/disaster recovery was purely an insurance issue – or whether there is real return on investment. So how do finances fit?
We have been involved in BC and DR technologies that provide what is called ?fault tolerant? computing for several years. These not only address the technical aspects of continuous processing, no matter what the circumstances, but also have a compelling ROI angle also. Hopefully the next paragraph or two will explain why…
Fault tolerant computing means literally that – should components of a multi server infrastructure have a fault, or indeed should a full host server have a complete outage – there is no impact at all on the computing environments – ie zero downtime. Even multiple faults can occur on separate servers in a protected pool at the same time, and again there is no impact on the processing environment. (example – two servers working together in our simplest of architectures – we can tolerate losing the complete disk array on server one, at the same time as losing the network connection on server two – and there is no impact at all on the computing environments that rely on this two server setup, not a single ping is dropped, no data is lost, users do not know any faults have occurred). This whole locally protected environment can then be replicated real time off site to complete the end-to-end local (BC) and off site (DR) protection. Most importantly from the pure technical BC and DR perspective, local technical outages such as disk failure or loss of network connection are treated as such and are protected – and only full site outages (ie a true site disaster) invoke the DR failover to a second site (hopefully never ! – so I accept the DR aspect of what I am describing is more of an insurance with less ROI argument). This technology also involves server virtualisation techniques – hence environments that traditionally live in a physical world – ie one operating system / computing environment per physical server, can be concatenated and operate on a much smaller hardware (and hence carbon and energy spend) footprint.
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So what you may say – sounds like VMware or similar right? why get involved in the discussion and where is the point ref ROI etc? Well no it isn?t – it is much more cost effective and easy to use than VMware, and does not rely on expensive shared storage. This is on standard hardware with no shared storage.
So the question of getting senior management to buy into a BC / DR strategy – when there is no apparent ROI but perception is purely that BC / DR is an insurance….. Certainly, in the technologies we implement for clients – this is not the case. Critical environments such as security access, prison CCTV systems, banking and trading hall systems, shop floor manufacturing systems, on line ticketing / booking systems, casino systems, etc stay up and running with absolute zero downtime – (ie the primary objective). However, server farms previously of (eg) 50 physical servers can be consolidated down to just 6 or 8 host machines. It doesn?t take a genius to work out there are huge savings in terms of energy spend, air conditioning requirements, cost of lifecycle replacements, plus reduced and simpler maintenance and support overheads to look after such a reduced and simpler infrastructure. This is NOT cloud computing by the way – this is within the client?s own server room or data centre environment.
So at least as far as our customers are concerned – ROI comes into the discussion very early on, and tends to get senior management very interested indeed. In fact – it?s pretty much a 50 / 50 split with the entry point of project sponsorship between the IT manager/director responsible and the FD or MD. There are attractive benefits for all.
Tracy Barlow has worked in the field of marketing for more than 17 years and is Marketing Director of 24/7 Uptime. She is a Chartered Marketer and ran her own successful marketing consultancy for 11 years.
People all over the world today are vying for free music downloads. Sometimes it is for entertainment and fun. At other times it is for learning. For yet others it is for the purpose of storing for future uses. However in either case there could be legal hassles and barriers depending on the nature of music you download and related laws of the land. While there is absence of any uniform international legal patterns, laws vary widely according to geographical divergences and you have to be careful not to violate them including the much talked about copyright provisions.
Fact remains that while everybody likes free music nobody likes to face litigations. People who are seeking to find free music downloads online could get simply the desired music from some friendly website but that could also bring up the letter from a lawyer that might not be as friendly. However there are also experts online who can advice you suitably on downloads that could be made without facing legal hurdles. These experts have long professional backgrounds and they can really show you the way. Line between the legal and illegal is always murky. Some of the experts will advice you for paying the licensing fees to the artists concerned. However in the rapidly changing legal scenario that might not be sufficient.
Problem lies in the fact that law seems to be lagging behind the advancement of technologies all along. Despite all these advances there are certain gray areas where the so called free music downloads are neither clearly legal nor clearly illegal. Sometimes people can take advantages of it with little fear of lawsuits. However it will be necessary to be abreast of the legal developments all the time since what was legal a few days back might already have turned otherwise with the legal developments coming up. It will never be the nicest of the experience to remain on the wrong side of the law.
Shifting law of the land has caused problems for many downloading people in the past. Basically the legal concept revolves round the principles of fair use. Ethically nothing could be fair without the permission of the copyright holder. Yet people can legally transfer CDs to their iPods after the judgment delivered in Sony Corporation of America versus Universal City Studios. The ruling allows copying of even copyrighted materials for sheer entertainment and noncommercial uses. So when one downloads with the aim of using it in a family party, he or she cannot be sued for violation of copyright with such free music downloads.
Some tricky legal questions will always crop up in the process. For instance if you have paid and bought the right to hear, will it be material whether you have transferred the music from your vinyl record or free music downloads online sites to your iPod? Downloading from online sources could be a violation while fact remains that you have also paid for the record. Once again this is gray area and has never ever been covered by judicial pronouncements.
Whether it is vinyl records or free music downloads from online sources, you will have a comprehensive overview of the legal aspects when you visit bearshare.com. The website has a comprehensive database of legal experts and agencies and online advice and consultation on free music downloads are available on the site.
Periodically, BizEngine will run the story of a small business. Don?t miss this feature!
In his nearly 11 years in business as the president of a coffee equipment and supply distributor, Hupp has applied that mantra in his daily work at DC Coffee Products in Divernon, Illinois. Before striking out on his own, he worked for coffee giant Bunn, and was allowed to work at home as a distributor after the birth of his second child. When he went into business on his own as a sole proprietor, he continued to sell Bunn, but added companies as diverse as Cecilware, Newco and Wilbur Curtis to his stock. Today, he sells equipment, coffee, flavored syrups and other supplies directly to coffee lovers across the United States.
?The coffee industry is set up just like the auto industry,? Hupp said. ?You can?t buy your equipment from the industry if you?re not a dealer.?
That means that the equipment needed for zombie-like workers across America to fill their cups with a liquid jolt of caffeine comes in to Hupp, and he deals directly with the customers. At every turn, Hupp said, he attempts to provide customer service his competitors can?t. He is David with a coffee cup, standing against Goliaths.
That leads into his quick answer when asked what the biggest challenge was for his small business.
?The biggest challenge is all the competition that?s out there. There?s a lot of distributors throughout the nation,? he said. ?(Hupp achieves separation) by trying to answer the phone every time it rings and providing great customer service. That?s the only thing that separates us, and now manufacturers have been sending end users my way because of distributors not taking care of their customers.
So that?s the difference maker for DC Coffee Products?
?Yeah, you have focus on customer service. That?s the only thing that will separate you from the next guy,? Hupp responded.
What?s changed in his business since it began more than a decade ago? Many customers are choosing to repair instead of replace busted coffee systems as the economy pinches them. DC Coffee Products has also been replacing ?a lot? of coffee services in offices, replacing the traditional practice of buying metric tons of coffee and using a company?s equipment as a perk.
?Folks are starting to get away from that type of business, and they?re coming right to us for those,? Hupp said.
Business and the economy being what they are, Hupp turned to financing company Direct Capital to help him sort out cashflow issues from ?slow? hospital and government pay orders. As someone who prizes customer service, Hupp said he was pleased with the service he received for his financing, which has gotten him through his cashflow issues at the moment.
Our interview ended after that when I heard another line ringing in the background. I let the polite, well-spoken distributor go knowing he was off to do what he does best: Answer a phone call and win a customer.
Enjoyed this story? Visit Hupp at http://www.dccoffeeproducts.com/.
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DENVER ? The bad blood ended with Jon Jones planting a peck on the cheek of Quinton "Rampage" Jackson after the fight.
That was after Jones punished him so bad that Jackson needed stitches to close a wound above his right eye. It's simply the way respect is earned in the octagon.
Jones defended his light heavyweight title by putting Jackson in a choke hold and forcing the veteran to tap out Saturday night at UFC 135.
The 24-year Jones relied on his quickness and long reach to get inside on Jackson and inflict damage. In the fourth round, Jones put a bleeding Jackson in a rear naked choke hold and Jackson signaled he was done. It was the first time Jackson has tapped out in his UFC career.
The fighters entered the bout in the midst of a squabble. It started when Jackson accused Jones of sending a spy into his camp to steal his secrets, a charge that Jones vehemently denied.
From there, the acrimony only escalated, with Jones and Jackson exchanging barbs up until it was time to brawl.
Afterward, the feud was declared over, replaced by talk of respect. Jones even gave Jackson a quick hug and a peck.
"I told Quinton that I admire him so much, that I respect him so much," Jones said. "Told him, 'You're awesome. It's such an honor to fight you.'"
The feeling was mutual.
"I thought he was all hype," Jackson said. "But he's the real deal."
In the other big fight of the night, welterweight Josh Koscheck knocked out UFC hall of famer Matt Hughes.
For UFC, this was a return to the city where the organization staged its first championship fight 18 years ago. A capacity crowd filled the Pepsi Center and was largely split in their loyalties to Jones and Jackson, who's known for his role as B.A. Baracus in the big-screen version of "The A-Team" almost as much as his MMA career.
The charismatic Jackson put in the work to get ready for this fight, giving up burgers and relocating to the Mile High City to train in the altitude. That's how motivated he was to win back a title he once held.
And while Jackson maintained Jones was the future of the sport, Jackson said he was the present.
It didn't take Jones long to end that notion. Jones opened up a cut above Jackson's right eye with an elbow in the third round and it was pretty much all over from there.
With his win, Jones may just be bringing stability back to the division. The belt has switched hands a number of times since Jackson last won the crown in 2007.
Down the road, Jones will face Rashad Evans, who was taking in the fight from the seats.
Evans knows Jones quite well ? and there's no love lost between them.
He was friends and training partners with Jones before a falling out led to some bitter feelings.
The low-key Jones beat Mauricio "Shogun" Rua last March to become the youngest title holder in UFC history. But he was facing probably the hardest puncher around in Jackson.
However, Jackson never had much of a chance to get inside.
"He insulted me, saying I had no punching power," Jones said after the fight. "We got together and improved my striking."
Koscheck wasn't even Hughes' intended opponent, becoming a late fill-in only when Diego Sanchez broke his hand.
This was Koscheck's first fight since last December when he had the orbital bone in his eye shattered in a title fight against Georges St. Pierre.
Koscheck showed little signs of rust as he jumped on Hughes early. With a second remaining in the opening round, the referee stepped in and halted the fight.
"I just want to say thanks to Matt Hughes for taking this fight late in his career. He's a legend," Koscheck said. "I'm really proud to fight him."
Koscheck was booed when he entered the ring partly because Hughes is so beloved. The 37-year-old Hughes is pondering calling it a career. But he's not quite ready.
"I'm not retiring," Hughes said in the ring. "I'm going to tell the UFC to put me on the shelf."
Meaning, the former welterweight champion is going to take some time to think about what comes next.
UFC president Dana White said he doesn't think Hughes will fight again.
In other bouts:
_ Travis Browne, who stands 6-foot-7, won an unanimous decision over Rob Broughton in a heavyweight clash.
_ Heavyweight Mark Hunt bloodied the face of favorite Ben Rothwell early in the second round en route to a convincing win.
_ Lightweight Nate Diaz made quick work of Takanori Gomi, getting the former Pride world champion to tap out late in the first round. Diaz was returning to the division after experimenting at 170 pounds.
_ Lightweight Tony Ferguson may have had the heaviest punch of the night, landing an uppercut that broke the jaw of Aaron Riley.
Some of the celebrities at the event Saturday included actor Steven Seagal and Denver Nuggets forward Chris "Birdman" Andersen, who may be paying a visit to the Pepsi Center for the final time for a while as the NBA and the players try to work out the lockout.
1School of Molecular Bioscience, University of Sydney
2Department of Surgery, Royal Prince Alfred Hospital
3Department of Anatomical Pathology, Department of Anatomical Pathology
4Department of Medicine, Concord Repatriation General Hospital
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Sample Filtration279
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Sample Preparation for Cell Capture392
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Antibody Microarray Cell Capture498
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Fluorescence Multiplexing with Conjugated Antibodies641
The current prognosis and classification of CRC relies on staging systems that integrate histopathologic and clinical findings. However, in the majority of CRC cases, cell dysfunction is the result of numerous mutations that modify protein expression and post-translational modification1.
A number of cell surface antigens, including cluster of differentiation (CD) antigens, have been identified as potential prognostic or metastatic biomarkers in CRC. These antigens make ideal biomarkers as their expression often changes with tumour progression or interactions with other cell types, such as tumour-infiltrating lymphocytes (TILs) and tumour-associated macrophages (TAMs).
The use of immunohistochemistry (IHC) for cancer sub-classification and prognostication is well established for some tumour types2,3. However, no single ?marker? has shown prognostic significance greater than clinico-pathological staging or gained wide acceptance for use in routine pathology reporting of all CRC cases.
A more recent approach to prognostic stratification of disease phenotypes relies on surface protein profiles using multiple 'markers'. While expression profiling of tumours using proteomic techniques such as iTRAQ is a powerful tool for the discovery of biomarkers4, it is not optimal for routine use in diagnostic laboratories and cannot distinguish different cell types in a mixed population. In addition, large amounts of tumour tissue are required for the profiling of purified plasma membrane glycoproteins by these methods.
In this video we described a simple method for surface proteome profiling of viable cells from disaggregated CRC samples using a DotScan CRC antibody microarray. The 122-antibody microarray consists of a standard 82-antibody region recognizing a range of lineage-specific leukocyte markers, adhesion molecules, receptors and markers of inflammation and immune response5, together with a satellite region for detection of 40 potentially prognostic markers for CRC. Cells are captured only on antibodies for which they express the corresponding antigen. The cell density per dot, determined by optical scanning, reflects the proportion of cells expressing that antigen, the level of expression of the antigen and affinity of the antibody6.
For CRC tissue or normal intestinal mucosa, optical scans reflect the immunophenotype of mixed populations of cells. Fluorescence multiplexing can then be used to profile selected sub-populations of cells of interest captured on the array. For example, Alexa 647-anti-epithelial cell adhesion molecule (EpCAM; CD326), is a pan-epithelial differentiation antigen that was used to detect CRC cells and also epithelial cells of normal intestinal mucosa, while Phycoerythrin-anti-CD3, was used to detect infiltrating T-cells7. The DotScan CRC microarray should be the prototype for a diagnostic alternative to the anatomically-based CRC staging system.
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Figure 1. Work flow for preparation of a suspension of live cells from a surgical sample of CRC.
1. Clinical sample disaggregation
All samples were collected from the Royal Prince Alfred Hospital (Camperdown, NSW, Australia) and Concord Repatriation Hospital (Concord West, NSW, Australia) with informed consent under Protocol No. X08-164.
Collect fresh colorectal cancer (CRC) or adenoma specimens, and normal intestinal mucosa at least 10 cm from the tumour. Store samples in Hank?s balanced salt solution pH 7.3 (HBSS) at 4?C for up to 12 h after resection.
Follow safety regulations for human pathogens, process all clinical samples in a biological safety cabinet class II. Dissect the samples into 2 mm cubes in a Petri dish using two scalpel blades.
Incubate tumour and normal tissue in separate Eppendorf tubes with occasional gentle mixing for 60 min at 37?C with an equal volume of RPMI 1640 medium containing 2% (v/v) collagenase type 4 (Worthington, Lakewood, NJ, USA) and 0.1% (w/v) deoxyribonuclease I from bovine pancrease (DNAse I; Sigma-Aldrich).
Force semi-digested tissue through a fine wire mesh strainer using a plunger from a 10 mL syringe; wash cells through with HBSS.
Pass resulting cell suspension through 200 ?m and 50 ?m Filcon filters (BD Biosciences) to remove cell aggregates. Most of the DNA, mucus and cell aggregates are removed in this series of filtrations.
Centrifuge cell suspensions at 400 x g at 20? for 5 min.
Resuspend cell pellets in heat-inactivated FCS containing 10% dimethyl sulphoxide (DMSO), freeze slowly in cryovials and store at -80?. The freezing process tends to reduce mucus in the sample and lyses red blood cells.
2. Sample preparation for cell capture
Thaw out samples quickly in a 37? water bath and resuspend cells in 10 mL of HBSS to wash out the DMSO.
Centrifuge cell suspensions at 410 x g at 20? for 5 min.
Decant the supernatant and resuspend the cell pellet in 500 ?L of HBSS.
Treat the sample with 0.1% (w/v) DNAse I for 20 min at room temperature.
Mix 10 ?L of each cell suspension with an equal volume of trypan blue and load 10 ?L of the mixture into a hemocytometer. Using a light microscope at 100-fold magnification, count viable cells, which appear clear due to trypan blue exclusion, while dead cells take up the dye. A minimum 4 x 106 viable cells is required for cell capture on the microarray.
Following DNAse treatment, resuspend the cell suspension in 10 mL HBSS and centrifuge at 410 x g at 20? for 5 min.
Decant the supernatants and resuspend cell pellets in RPMI 1640 to a final volume of 200 ?L.
3. Antibody microarray cell capture
Moisten the DotScan antibody microarray by dipping the nitrocellulose section into phosphate buffered saline (PBS) for approximately 20 s. Carefully wipe the glass edges of the microarray with folded Kimwipes, avoiding touching the nitrocellulose section.
Add water to the microarray incubation tray to provide a humid chamber. Place the microarray into the chamber and pipette the cell suspension in RPMI 1640 onto the moist nitrocellulose section. Pipette drops on each corner of the nitrocellulose to ensure an even spread of cells.
Incubate the microarrays at 37?C for 1 h. The incubation allows cells to settle and come in contact with antibodies on the microarray. Cells expressing surface antigens corresponding to the antibodies they land on will be captured.
After incubation, dip the microarrays gently and vertically into three troughs containing at least 15 mL PBS to wash off unbound cells (20 s per wash).
Prepare 3.7% (w/v) formaldehyde in PBS to fix the cells and antibody by cross-linking. Gently pipette approximately 1 mL to cover the nitrocellulose section of the microarray. Incubate for 20 min at room temperature.
Next dip microarrays into 3 changes of PBS (15 mL; 30 s each) to wash out excess formaldehyde.
Wipe the edges and back of the glass slide with Kimwipes and scan the microarray using the DotScan scanner while, the nitrocellulose section is moist. The optical scan provides the antigen expression pattern of a mixed cell population e.g. CRC cells, leukocytes and other stromal cells of the tumour.
4. Fluorescence multiplexing
Remove the microarray from the scanner and apply 200 ?L of blocking buffer (2% w/v BSA, 2% heat-inactivated human AB serum, PBS, pH 7.3). Incubate it in the microarray tray at room temperature for 20 min.
Prepare the multiplexing solution in an Eppendorf tube covered with aluminum foil: 20 ?L Phycoerythrin-anti-CD3 (Beckman Coulter, Gladesville, NSW, Australia, #IM12824; 1/7.5 final dilution), 10 ?L Alexa Fluor 647-anti-EpCAM (Biolegend, San Diego, CA, USA; 1/15 dilution), 2 ?L of heat-inactivated human AB serum (Sigma-Aldrich, Castle Hill, NSW, Australia) and 118 ?L of blocking buffer.
Drain off excess blocking buffer from the microarray and pipette the multiplexing solution onto the nitrocellulose section, spreading uniformly. Incubate for 30 min in the dark at room temperature.
Dip the microarray vertically into three troughs of 15 ml fresh PBS; (30 s each).
Let the microarrays dry in the dark and store at 4?C in a slide box. The microarray can be stored in the dark for up to 3 months without loss of fluorescence.
Scan the dry microarray using a Typhoon FLA 9000 scanner (GE Healthcare, Rydalmere, NSW, Australia) with resolution set to 50 (532 nm laser, 580 BP30 emission filter for PE. 633 nm laser and 670 BP30 emission filter for Alexa 647). The microarrays are scanned with the nitrocellulose side facing downwards on the glass scanner tray.
Save the fluorescent images as TIFF files and using Photoshop set image size to 17 x 25 cm and resolution to 72 pixels/cm. Import the image into the DotScan analysis software to analyse the intensity of dots.
The DotReader captures a digital image of the dot binding pattern and quantifies the density of cell binding on each antibody dot on an 8 bit greyness scale (1-256 U). Occasional non-specific isotype control binding was subtracted from binding values for antibodies with corresponding immunoglobulin isotypes. Dot fluorescence intensities for each microarray were normalized against the brightest dot set at 100% intensity. Signal/spot strength was recorded in an .xml file (raw data) or represented as a bar chart in an .pdf file (final report)
Microarray heatsmaps and hierarchical clustering were conducted using MultiExperiment Viewer (MeV) version 4.4 from the TM4 Microarray Software Suite (http://www.tm4.org/mev.html). Hierarchical clustering was performed on background-adjusted data using MeV with complete linkage analysis. Euclidean distance was used for similarity measure. The 2-tailed Student?s t-test with equal variance was used to determine the statistical significance of the results.
5. Representative Results:
Results from the DotScan microarray should show consistent cell binding patterns between duplicate arrays. Strong alignment dot binding (CD44/CD29) enables a grid to be placed over the array area. Figure 2 shows an example of optimal cell capture and multiplexing. Figure 3 shows some common problems encountered during cell capture and the possible solutions.
The microarray cell binding results can be quantified by measuring dot intensities expressed on a greyness scale ranging from 1 to 256. Figure 4 shows numerical data from 58 surgical CRC samples, stained with EpCAM-Alexa 647 antibody, as a heatmap with hierarchical clustering. Even though the number of samples is limited, CRCs of the same stage tend to cluster in the same group.
Figure 2. Cell binding pattern of clinical colorectal cancer tumour (Australian Clinic-Pathological Staging, ACP stage B1). (a) DotScan antibody key showing locations of antibodies for the left half of the duplicate microarray (outlined). The top section contains the original 82 antibodies of the DotScan leukemia microarray. An additional 40 antibodies, corresponding to specific surface antigens found to be up-regulated in the literature, were added as a CRC ?satellite? microarray. The bottom section consists of isotype control antibodies (b) Optical image of CRC cells binding to the microarray. (c) CD3 fluorescence image showing T-cells. (d) EpCAM fluorescence image showing CRC cells. Figure 3. Examples of poor DotScan results and possible solutions. (a) Low cell binding; solution: make sure at least 4x106 viable cells are on the array (b) Isotype control binding and non-specific cell binding; solution: add heat-inactivated human AB serum to sample before incubation on microarray to minimise isotype control binding. Occasionally, a small amount of non-specific binding of cells to the nitrocellulose occurs with CRC samples and does not significantly affect the results. (c) Nitrocellulose drying out during incubation; solution: ensure sample covers the whole nitrocellulose section and microarray is incubated on a flat surface. (d) High background artifacts; solution: ensure the microarray is thoroughly washed following incubation. Figure 4. DotScan analysis software generated bar charts representing cell binding densities on a greyness scale ranging from 1 to 256. Numbers on the axis refer to CD antigens. Other abbreviations are TCR, T-cell receptor; ?, ?, immunoglobulin light chains; sIg, surface immunoglobulin; DCC, deleted in colorectal cancer protein; EGFR, epidermal growth factor receptor; FAP, fibroblast activation protein; HLA-A,B,C HLA-DR, human leukocyte antigens DR and A,B,C respectively; MICA, MHC class I chain-related protein A; MMP-14, matrix metallopeptidase 14; PIGR, polymeric immunoglobulin receptor; TSP-1, thrombospondin-1; Mabthera, humanised anti-CD20. Click here to view larger image.
In this video, we demonstrate how the DotScan antibody microarray can be used in a simple, semi-quantitative way to study surface antigen profiles for cell populations from CRC tissue.
Obtaining a viable single cell suspension from tissue is critical to the success of the experiment, because energy-dependent processes (eg., antigen capping and/or pseudopodia formation) appear to be required for firm binding of whole cells to antibody dots during incubation, while dead cells are subsequently washed off. Type 1 collagenase was initially employed for tissue disaggregation8, but was replaced by collagenase 4 which causes less damage to cell membranes, as it contains fewer protease contaminants. This change did not affect cell yield, viability or binding patterns. Mucus from some tumours and control samples reduced cell yield and interfered with cell capture. This stickiness of the mucus was minimized by storage of disaggregated cell suspension in 10% DMSO/FCS at -80?C, presumably due to changes in mucus properties after freezing and thawing9. Subsequently, all samples were stored frozen in 10% DMSO/FCS after disaggregation and were rapidly thawed to produce viable cell suspensions, with consistent binding patterns. Samples with <50% viability or showing poor binding on alignment/housekeeping dots (CD44/CD29 ) were omitted from the analysis.
A few antibody clones showed reduced affinity when bound to the nitrocellulose possibly due to a change in conformation, e.g., the prominent CRC marker CD15s had very little or no cell binding. Antibodies that exhibited consistently negative results should be replaced with different hybridoma clones. Another possible cause of poor activity of some antibodies was interference to binding by bovine serum albumin (BSA). BSA-free antibodies should be used on the microarray where possible.
Although large patient cohorts are required for statistical analysis of microarray data, hierarchical clustering of our results (58 clinical samples) has been encouraging. Normalization of data using approaches described by Yang10 should provide improved statistical significance.
While the DotScan antibody microarray enables determination of new patterns of expression of known CD antigens, it is most effective when used in combination with proteomic discovery techniques, such as 2-dimensional gel electrophoresis and LC-iTRAQ-MS, to identify novel differentially abundant proteins. Such novel proteins are potential markers; corresponding antibodies could be added to the array, and validated with clinical CRC samples.
The use of fluorescently-labeled antibodies to profile sub-sets of captured cells provides a powerful DotScan platform for analysis of mixed populations of cells.
No conflicts of interest declared.
We thank staff at the Anatomical Pathology Laboratories of the Royal Prince Alfred and Concord Repatriation Hospitals for collecting fresh samples of CRC and normal intestinal mucosa. The work was funded by a Cancer Institute New South Wales Translational Program Grant.
532 nm laser, 580 BP30 emission filter for PE. 633 nm laser and 670 BP30 emission filter for Alexa647
MultiExperiment Viewer v4.4
TM4 Microarray Software Suite
Open ? source software (Ref 11)
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Xiao, G.G., Recker, R.R., & Deng, H.W. Recent advances in proteomics and cancer biomarker discovery. Clin. Med. Oncol. (2008).
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Zhou, J., Belov, L., Solomon, M. J., Chan, C., Clarke, S. J., Christopherson, R. I. Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing. J. Vis. Exp. (55), e3322, DOI: 10.3791/3322 (2011).
Holiday traditions ?r? ?m?ng th? m??t followed customs th?t exist ?n families around th? world. Th?t ?? wh? ?t ?? vital t? carefully maintain th?m ?n such a way th?t th?? ??n easily b? passed down fr?m one generation t? th? next. Holiday scrapbooking ?? a creative way t? d? th??, ?nd m?k?? th? overall process f?nt??t?? fun.
Whether relaxed simple, ?r complexly involved, traditions ?r? a way t? leave behind th? sentiment ?f a particular occasion, ?nd wh?t better way t? maintain th? memories th?n t? chronicle th?m ?n ?n holiday scrapbooking album th?t w?ll b? talked ?b??t f?r years t? come. Y?? m?? ?h???? t? commemorate each ritual b? showcasing ?n array ?f pictures arranged ?n ?n artful design, ?r take ?t a step additional b? incorporating ?th?r forms ?f ornamentation th?t w?ll personalize ???r autograph album, b?t ??th?r way, b? sure t? add ???r ?wn special touches t? m?k? ?t truly yours. Below ?r? ??m? tips th?t w?ll h?l? ??? g?t ?t?rt?d.
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Wh?n choosing photographs, select pictures th?t ?r? ?? closely related t? a particular tradition ?? possible. F?r example, ?f ??? ?r? chronicling th? annual family tree trimming, b? ??rt??n ??? include photos ?f everyone involved. Y?? m?ght ?l?? ?h?w th? innumerable stages ?f th? tree ?? ?t ?? trimmed, ?nd ?nd th? segment b? presenting images ?f th? f?n??h?d product. If ??? ?r? missing ??rt??n pictures, try substituting th?m w?th information documenting wh?t ?? missing ?nd explaining wh?t took ?l??? ?? ?? t? ?t?rt a mental image.
U?? bits ?nd pieces associated w?th th? entire family. If ??? children brought home hand m?d? ornaments ?n th? past th?t ?r? n? longer hung ?n th? tree, consider adding th?m t? ???r n?w holiday autograph album. Th?? w?ll add a nice touch ?nd invoke many fond memories.
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NEW YORK ? Two American hikers held for years in an Iranian prison came home Sunday, declaring that they were detained because of their nationality, not because they might have crossed the border from Iraq.
Joshua Fattal and Shane Bauer arrived in New York on Sunday morning, ending their diplomatic and personal ordeal with a sharp rebuke of the country that sentenced each to eight years in jail for espionage and illegally walking into Iran. They say they may never know if they actually stepped across the border while hiking and getting lost.
"From the very start, the only reason we have been held hostage is because we are American," Fattal said at a news conference at a Manhattan hotel. "Iran has always tied our case to its political disputes with the U.S."
The two 29-year-olds were freed last week under a $1 million bail deal and arrived Wednesday in Oman, greeted by relatives and fellow hiker Sarah Shourd, who was released last year.
The men's families said Sunday they don't know who paid the bail.
The men's saga began in July 2009 with what they called a wrong turn into the wrong country. The three say they were hiking together in Iraq's relatively peaceful Kurdish region along the Iran-Iraq border when Iranian guards detained them. They always maintained their innocence, saying they might have accidentally wandered into Iran.
The two men were convicted of spying last month. Shourd, to whom Bauer proposed marriage while they were imprisoned, was charged but freed last year before any trial.
A beaming Shourd faced reporters and cameras that packed a conference room at the Parker Meridien hotel.
"There's a huge burden lifted off of all of our chests ? so much joy," she said. "Shane and Josh and I are beginning our lives again, and there are so many new joys that await us; I've never felt as free as I feel today."
But her face darkened when she was asked whether the men had been mistreated in captivity. She said Bauer was beaten and Fattal forced down a flight of stairs.
The men took turns reading statements, surrounded by relatives and Shourd. They didn't take questions from reporters.
Fattal said he wanted to make clear that while he and Bauer "applaud Iranian authorities for finally making the right decision," they "do not deserve undue credit for ending what they had no right and no justification to start in the first place."
The two countries severed diplomatic ties three decades ago during the hostage crisis. Since then, both have tried to limit the other's influence in the Middle East, and the United States and other Western nations see Iran as the greatest nuclear threat in the region.
The hikers' detention, Bauer said, was "never about crossing the unmarked border between Iran and Iraq. We were held because of our nationality."
He said they don't know whether they had even crossed into Iran: "We will probably never know."
The irony of it all, Bauer said, "is that Sarah, Josh and I oppose U.S. policies towards Iran which perpetuate this hostility."
The two also detailed the difficult conditions in the Tehran prison where they were held in near-isolation.
"Many times, too many times, we heard the screams of other prisoners being beaten and there was nothing we could do to help them," Fattal said.
Added Bauer: "How can we forgive the Iranian government when it continues to imprison so many other innocent people and prisoners of conscience?"
They said their phone calls with family members amounted to a total of 15 minutes in two years, and they had to go on repeated hunger strikes to receive letters. Eventually, they were told ? falsely ? that their families had stopped writing them letters.
"Solitary confinement was the worst experience of all of our lives," Fattal said. "We lived in a world of lies and false hope."
They kept in shape physically and mentally by lifting water bottles, discussing books and asking each other questions, family members said. And they ripped slivers of cloth from prison blindfolds to secure their footwear so they could run for exercise.
The two managed to hold on to reality by reading letters from family members that included news of what was happening in the world, Bauer's mother, Cindy Hickey, told The Associated Press.
Fattal said their release last week came as a total surprise.
On Wednesday, he said, they had just finished their brief daily open-air exercise and expected, as on other days, to be blindfolded and led back to their 8- by-13-foot cell.
Instead, the prison guards took them downstairs, fingerprinted them and gave them civilian clothes. They weren't told where they were going.
The guards then led them to another part of the prison, where they met a diplomatic envoy from Oman.
His first words to them: "Let's go home."
Hours later, the gates of Tehran's Evin prison opened and the Americans were driven to the airport, then flown to Oman.
The days following their sudden release, Fattal said, made for "the most incredible experience of our lives."
Shourd was with the families to greet the two on the tarmac at a royal airfield near the airport in Oman's capital, Muscat. At about 20 minutes before midnight Wednesday, Fattal and Bauer, wearing jeans and casual shirts, bounded down the steps from the blue-and-white plane. The men appeared very thin and pale, but in good health.
The first hint of change in the case came last week when Iranian President Mahmoud Ahmadinejad said Fattal and Bauer could be released within days. But wrangling from within the country's leadership delayed efforts. Finally, Iranian defense attorney Masoud Shafiei secured the necessary judicial approval Wednesday for the bail ? $500,000 for each man.
Iran's Foreign Ministry called their release a gesture of Islamic mercy.
Until their release, the last direct contact family members had with Bauer and Fattal was in May 2010, when their mothers were permitted a short visit in Tehran, which Iranian officials used for high-profile propaganda.
Since her release, Shourd has lived in Oakland, Calif. Bauer, a freelance journalist, grew up in Onamia, Minn., and Fattal, an environmental activist, is from Elkins Park, Pa., a Philadelphia suburb.
Bauer and Shourd were living in Damascus, Syria, when Fattal came to visit and the three went hiking.
On Sunday, the men's families told reporters that they hadn't made plans for what they would do next ? except for carving out some private time together. They would not say where they were sleeping on Sunday night.
Finally, if you missed the teaser trailer for Ghost Rider: Spirit of Vengeance, click here. ?You can also check out over 30 images from the film here.? And one last thing, a huge thank you to Brian Taylor for giving me so much of his time.
Here?s the interview and time index so you can watch the parts that interest you.
