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|By Sam Ganga
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|August 12, 2014 02:00 PM EDT
Science fiction films abound that warn of machines taking control and wreaking havoc on the human race. "2001: A Space Odyssey," "War Games" and "I, Robot" are just a few of the titles that propose what might happen if we hand too much power over to intelligent, interconnected machines.
Decades after the first cautionary tale, the world's machines are more intelligent and more interconnected than even science fiction authors could have predicted. Machine to Machine (M2M) communication and the mobile revolution have led to the phenomenon of Big Data, an influx of structured and unstructured data at volumes and velocities never before heard of. The insightful analysis of all that data is proving to be a blessing to humanity, not the threat that many feared. M2M and Big Data Analytics can help reduce costs and create competitive advantage for a wide variety of businesses.
What Is M2M?
M2M refers to systems and technologies that make it possible for networked devices to exchange information and perform actions on their own, without (or with minimal) human intervention. Gathering sensor data from devices, analyzing it and using it to exercise more intelligent control can drive better outcomes. Everyday examples include:
Smart meters, coupled with predictive analytics, enable utility companies to predict demand patterns, automatically adjust to meet peak demand and avoid over-production when demand is low.
Remote medical sensors can monitor patients, remind them if they've forgotten their medications and alert doctors when intervention might be needed.
Smart buildings have sensors that can analyze environmental data to save energy and improve safety.
Traffic data from networked sensors can be analyzed to predict shifts in traffic patterns. Using this information to control traffic signals can actually prevent traffic jams, not just ease them.
Automated systems like GM's OnStar can alert emergency services when accidents occur, even when the humans involved aren't able to help themselves.
How M2M Came to Be
M2M didn't arrive on the scene overnight; as with anything else, it followed an evolutionary process. Back in the 1980s, Supervisory Control And Data Acquisition (SCADA) systems were introduced to enhance controls for electricity generation, transmission and distribution, and to improve monitoring and control for traffic and transportation systems. In the '90s, Wireless Sensor Networks were introduced to improve monitoring and control in many manufacturing and industrial systems. Wireless made it easier to monitor and control a broader range of devices but only supported limited, short-range connections.
When data modules were introduced in the mid-1990s and early 2000s that could communicate via cellular networks, a major leap forward occurred. These systems were used first to connect point of sale (POS) terminals, vehicle sensors and other remote monitoring and tracking systems, and then were further extended to automatic meter reading, security, elevator control, fleet management, vending and telemedicine.
M2M communication and applications have really exploded in diversity and number since the introduction of the Internet as a backbone for communication. Three major factors have combined to accelerate the recent growth in M2M:
- More data from more devices can be combined and analyzed more quickly due to advances in tools and technologies for big data analysis and predictive analytics. This enables machine-driven actions based on anticipated conditions - not just faster reaction times.
- The "everywhereness" of broadband networks, wireless and Internet has given rise to the Internet of Things (IoT) and has made it easier and cheaper than ever to connect devices. Assign an IP address to a device with Internet access and you can communicate with it anywhere in the world.
- Cheaper and smaller sensors, memory and processing power mean that more devices can be networked, and the devices themselves can be smarter.
M2M Now and in the Future
Gartner Inc. estimates that there are currently just under 30 billion connected devices and projects $309 billion in additional revenue for product and service suppliers by 2020 due to IoT. They also predict $1.9 trillion in total economic impact from improved productivity and cost savings, among other factors.
As an example of IoT's impact, Gartner turns its attention to data centers. The analyst firm predicts that IoT product and service suppliers will generate revenue exceeding $300 billion, mostly in services, by 2020.
How M2M Is Being Applied
With virtually every industry impacted, M2M's technology solutions applications are startling in their breadth and diversity. Machina Research points to benefits as varied as reduced energy costs, improved safety and security, and increased efficiency and faster response times for emergency services and national defense. Here are some examples:
In terms of how far along companies in key verticals are in implementing M2M initiatives, another recent study by Techpro Research offers some insight. Energy, IT and automotive top the list in current implementations, or plans to implement in the next 12 months, followed by Healthcare, Facility Management, Manufacturing and Retail.
M2M Success in the Marketplace
If businesses do thoughtful planning around how to use M2M to achieve their goals, opportunities to boost revenues, cut costs and more effectively serve customers are tremendous. A few recent examples include:
Retail - Nestlé Nespresso SA has equipped its coffee machines used in restaurants, hotels, offices and luxury retail boutiques to transmit operational and performance data from each machine to a cloud platform for tracking and analysis. The system tracks descaling and other maintenance procedures and alerts technical staff if servicing is required. The applications can also be used to remotely adjust water temperature and pressure. The system helps ensure that machines are maintained in excellent condition, that they produce the highest-quality coffee, cup after cup, and that customers are well supplied with their coffee of choice.
Transportation - The automotive industry and the U.S. Federal Government are embracing M2M. The US Department of Transportation recently conducted research that suggests that Vehicle to Vehicle (V2V) technology could prevent the majority of crashes involving two or more vehicles. Sensors can monitor speed and location of nearby vehicles, analyze risks and either warn drivers (near term) or take action on their own (longer term) to avoid accidents. The research could lead to a mandate to use V2V in the future.
Healthcare - Partnering with the University Teaching Hospitals of Grenoble and Toulouse, France Telecom R&D launched a project called "Gluconet" for managing diabetic patients remotely. A special instrument is used to periodically read patient glycemia data. This information gets transmitted automatically to the management center via mobile devices. The doctors can access the information over the Internet. Based on the analysis, doctors send medical advice to patients via SMS or voice messaging. The key advantage here is that both patients and doctors are alerted of any complications well before they become life-threatening.
Consumer - Lexmark, a provider of printing and imaging products, software, solutions and services, deployed M2M for more effective customer servicing. Lexmark uses M2M to collect data from millions of printers. The company analyzes the data to streamline its products to serve customers better, increase revenues and reduce operational costs.
Facilities Management - Commercial real estate services firm Jones Lang LaSalle (JLL) deployed an M2M system called IntelliCommand to collect data from building systems for security and protection against heating, cooling or fire incidents. Information collected by remote sensors is transmitted to a cloud-hosted system for in-depth analysis. When sensors collect data that strays outside of established parameters, alarms are relayed to a control center to alert managers. JLL's pilot installation with four sites enabled clients to cut costs by 15-20 percent. The real estate giant is now extending its deployment to 76 buildings.
How to Begin the Process
M2M possibilities for some organizations are self-evident. An equipment manufacturer might see an opportunity to leverage machine data to provide better service and build loyalty. Another might see an opportunity to add value that can be monetized. Some companies might find themselves threatened by competitors who have already started using M2M to gain advantage. But it's not so cut and dried for some businesses. The "M2M Opportunity Matrix" shown here offers some structure that can be used to think about M2M and identify opportunities that can improve business performance.
Listed across the top of the Matrix are possible business objectives. This isn't an exhaustive list, but you could do a lot of good for your business by finding ways to reduce cost, increase revenue or add value.
Options related to data sources are listed down the left side. Your organization might already have a large database of information that's coming in from POS systems or manufacturing control systems or some other source - Data In-Hand. But maybe you haven't figured out what to do with the information yet. There might be additional data that you could be collecting from existing "sensors" - New Data from Existing Sources. Or there might be new data that you could access with new sensors, or by sourcing from outside your company - New Data from New Sources. Probably, the data you already have in hand is going to be the easiest to tap into to achieve business objectives. But some opportunities might be so valuable that it's worth deploying new sensors to gather new data.
There's a potential M2M opportunity at the juncture of each business objective and data source. So, do some brainstorming. Start the process by thinking of how to leverage different data sources to achieve various business objectives. It can go in a lot of directions from there.
Alternatively, an experienced data consultant can help you look objectively at your situation and help you to identify low-hanging fruit or the really game-changing opportunities that could deliver more transformative results. There are a lot of right answers. The best thing is to get started.
Making the Most of M2M
It turns out that, so far at least, all those cautionary tales about intelligent machines have proven untrue. In fact, interconnected machines and the data they generate are improving the ways we live and do business. Smarter systems that don't need to rely on slower human input and that can more quickly adapt as needed are the upshot of M2M. Even now we are seeing incredible innovations like remote glucose monitoring, more efficient printing and safer buildings. And that's only the beginning. At the risk of imitating sci fi writers who were a bit off-base, we hesitate to predict what other life-enhancing technologies powered by M2M are on the horizon.
You, meanwhile, should not hesitate to take part in the M2M revolution. If you wait for someone else to figure out how to best leverage M2M, you are likely to lose market share or lose the opportunity altogether. It may seem overwhelming to know where to start; if that's the case, work with a data consultant who can help create a plan. Don't let the intelligent machines outsmart you.
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They might be debating how to make the cost controls in the Affordable Care Act more effective, for example, or the merits of moving to a more efficient single-payer system, as every other advanced country has done.
But they're not debating this, because the federal deficit is not what this war is about.
It's about the size of government. Tea-Party Republicans (and other congressional Republicans worried about a Tea-Party challenge in their next primary) want the government to be much smaller.
"My goal," says conservative guru Grover Norquist, "is to cut government in half in 25 years, to get it down to the size where we can drown it in the bathtub."
What's behind this zeal to shrink government? It's not that the U.S. government has suddenly become larger. In fact, non-military government spending relative to the size of the U.S. economy remains the smallest of any other rich nation.
Apart from the military, Medicare and Social Security account for almost everything else the federal government does -- and these programs continue to be hugely popular, as Republicans learn every time they threaten them.
The animus toward government has more to do with the growing frustrations of many Americans that they're not getting ahead no matter how hard they work.
Government is an easy scapegoat, utilized by much of corporate America to convince average Americans to cut taxes, spending, and regulations -- and divert attention from record-high corporate profits and concentration of income and wealth at the top.
The median wage continues to drop, adjusted for inflation, even though the economy is growing. And the share of the economy going to wages rather than to profits is the smallest on record.
Increasingly it's looked like the game is rigged, especially when people see government bailing out Wall Street (the Tea Party movement grew out of the bailout, as did the Occupiers), and handing out corporate welfare to big agriculture, big pharma, oil companies, and the insurance industry, to name but a few of the recipients.
The outrage grows when average working people are told -- falsely -- that a growing portion of Americans don't pay taxes and live off government handouts.
The battle over the fiscal cliff is over, but the trench warfare will continue.
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Definición de entitle en inglés:
verbo[with object] (usually be entitled)
1Give (someone) a legal right or a just claim to receive or do something: employees are normally entitled to severance pay [with object and infinitive]: the landlord is entitled to require references
Más ejemplos en oraciones
- If she is entitled to receive it for professional services rendered, then she must be paid.
- In particular, would she be entitled to claim any compensation if I asked her to move out either now or in the future?
- He is entitled to claim that money back from the Commons as long as the office is not used for party politics.
qualify, make eligible, authorize, allow, permit;
enable, empower
2Give (something, especially a text or work of art) a particular title: an article entitled "The Harried Society."
Más ejemplos en oraciones
- De Botton presents us Xavier de Maistre, a Frenchman who, in 1790, undertook a journey around his bedroom, later entitling an account of what he had seen 'Journey Around My Bedroom'.
2.1 [with object and complement] archaic Give (someone) a specified title expressing their rank, office, or character: they entitled him Sultan
title, name, call, label, head, designate, dub
formal denominate
Late Middle English (formerly also as intitle): via Old French from late Latin intitulare, from in- 'in' + Latin titulus 'title'.
Palabras que riman con entitlemistitle, recital, requital, title, vital
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A highly recommended Documentary Wedding Photographer based in Belfast , Northern Ireland – affordable & relaxed
– Snappitt Wedding Photography Belfast
Affordable – Relaxed – Quirky – Professional – Fun
" To be a good wedding photographer , you need to be good with people "
"Thanks for stopping by. I am Laurence a documentary wedding photographer based in Belfast , Northern Ireland . I will document weddings throughout Ireland and the UK . My style is fun and relaxed and a little quirky , I tend to make the day lighthearted for you , your friends and family when appropriate and I keep things simple as possible and with one main objective…to tell your story as it unfolds and to leave you with amazing memories from your day. Take your time to look through some of the images and wedding stories that I have told and I would love to hear from you about your wedding plans and how we can work together to capture your story."
Everyone has a story – What is your story ? ♥
Lucie and James
Lots of stories → Wedding Posts Lots more photos → Photo Gallery
Lauren & Gavin at Slieve Donard Hotel Continued →
Aisling and Kevin Continued →
Laura & Mark at the Dunadry Hotel
Emma and Joe at St Josephs , Glenavy Continued →
Aisling and Kevin June 2016 Continued →
Jo and Jonny , Punk meets Pirate Wedding Continued ↔
The grounds of the Dunadry Hotel , Templepatrick
Valerie and Chris at the Cityhall July 2016 Continued →
Emma and Joe , Glenavy
Bryan and Neil at the Old Inn Crawfordsburn June 2016
Ellen & Conor Continued →
Pamela and John have Halloween themed wedding at Parkanaur Continued →
Kerri the MUA does the reveal for Valerie in Jurys Hotel Belfast
Aisling has time for a quick lippy moment at the Ballymac
Laurence is a full time wedding photographer who has experience in many fields of photography . His passion , though , is for wedding photography ! He says it is such a great opportunity and pleasure to capture the fun and emotion of a couples special day .. with so many emotions on view , so much fun , and all those little unpredictable moments … " it's just a fantastic day !" he says. The final joy is when the couple get to see all their wonderful images .
Laurence draws on his wide photography experience to use whilst documenting weddings… He maintains , that it takes a certain type of person to cope with the difficulties of documenting a wedding … not everyone is up to the challenge , you have to have a particular type of personality as well ! A range of photography disciplines is a huge help , but having a good sense of humour and a quirky personality is a must ! Combining the two is a winning combination, producing , natural and relaxed pictures . Laurence sincerely believes that " To be a good wedding photographer , you really do need need to be good with people " !
Recent review : "Laurence was absolutely amazing! We had a fantastic day and he made all our guests feel so comfortable. He also made my very camera shy husband love getting his photo Taken! I've been trying for 4 years! He just fitted right into the day and everyone loved him. " Catherine and Andy
Catherine and Andy at Larchfield Estate – Continued →
Michelle and Charles at Ballygalley Castle Hotel Continued →
Bryan and Neil – get to know me session at Strangford Continued →
Helen and Paul at the Orange Tree House Continued →
Parkanaur Halloween wedding continued…..
Judith and John at Saul Continued →
Black & White Weddings continued
A Big Day at Belfast Castle continued…
ROCK N ROLL Bride magazine feature Punk Princess meets Pirate – Real Alternative wedding photography. Here .
Jo & Jonny Friday 13th November 2015
Jo and Jonny – Full story here
Top Tip ψ – Why Client Reviews Are Important
Source : Bride Box Pro
" One of the most popular channels from which potential clients derive information is online reviews. It is extremely helpful for potential clients to see what past and current clients are saying about you so that they can have an idea of what to expect from you in terms of professionalism, services, experience and quality of work. By reading past client experiences, they can determine whether or not hiring you as their wedding photographer is the right choice. It is true that they can gather some of this information from your website, but you can't deny that there are often discrepancies between what is marketed to you at face value versus what you actually receive. Reviews from actual customers with experience are the best way for a potential client to get a feel for what can be expected if he was to hire you. "
Laurence has built his business on this belief , and has built up a large collection of client reviews from previous couples going back several years.. Reviews , are really worth a visit.
Here are a few to get you started …
Shelley and Nathan Belfast Castle August 2015 ( Google )
" Without doubt Snappitt Wedding Photography, run by the lovely Laurence King, was the perfect choice for our wedding on Friday 21st August 2015 at Belfast Castle. Laurence provided an excellent service from start to finish, we couldn't fault it. He was professional throughout, providing us with wonderful photographs which we will cherish, but just as importantly he was fun and made us feel relaxed. Laurence made the difficult task of finding a wedding photographer so much easier for us. He responded to our initial enquiries very quickly and we soon chatted on the phone- at this stage I knew he was the one for us! We met in person a few months before the wedding to discuss our expectations and of course to meet, it certainly helped to put a name to the face and to see how our personalities might work together on the day. Laurence was also very patient with my last minute panic emails regarding photo lists and various other worries. On the day, Laurence was there waiting for us as we arrived- happily taking photos and chatting with guests. He maintained a perfect balance between professionalism and guest interaction. We particularly appreciated how helped to put us and the guests at ease, it made the photo session feel like fun rather than a chore. We were pleased that Laurence joined us for a meal after the ceremony. Laurence promised us a photo preview and to our delight we were able to view some of our wedding photos later that evening, yes- Laurence had actually put up some of our photos within 3 hours of leaving our ceremony! The guests really enjoyed looking through these whilst we had our evening meal. We couldn't have asked for anything more, a perfect end to a perfect day. If you want a friendly, professional and fun service with excellent communication and a speedy turn around then don't go anywhere else. I don't see why anyone would fork out ££££ for wedding photography when you can get the best for less! You won't regret booking with Snappitt Wedding Photography."
Catherine and Andy 25th March 2016 ( Google )
"Laurence was absolutely amazing! We had a fantastic day and he made all our guests feel so comfortable. He also made my very camera shy husband love getting his photo Taken! I've been trying for 4 years! He just fitted right into the day and everyone loved him.
Thank you so much. The photos are incredible!! "
Ellen & Conor Continued →
Ellen and Connor August 1st 2015 Google Review
" Anybody that knows me, knows how obsessed I am with photographs (maybe a bit too much!) Anyways keeping that in mind it was a big task to find a photographer I liked as well as their style of photographs. I searched the Google religiously checking out portfolios and galleries, that's when I found Laurence. I loved the style of photographs he took, I had family members prewarned that I didn't want any overly staged photographs, I wanted them natural. That's exactly what I got! Laurence was a pleasure to deal with from the first moment we met up with him. He came the morning of the wedding to capture the wedding preparations, and he was very bubbly interacting with the bridal party and family members. He got along great with everyone throughout the day, I'm constantly bumping into wedding guests that are pinpointing how great he was at his job! I'm over the moon with the photographs from our wedding and I'm so glad I spent so much time researching, but I'm constantly recommending Laurence for family to use in the future. Thank you Laurence for capturing every moment of our special day."
Site map : Home – Reviews – Pricing and Packages – Recent Posts – Highlight Gallery – Contact Page – Faq's
Choosing a wedding photographer – Story Book Albums
For a recommended Northern Ireland wedding photographer please feel free to contact us here
Member of the PPANI www.ppani.co.uk
Relaxed – Affordable -Professional
wedding photographer northern ireland , alternative , affordable , fun , quirky ,relaxed , informal,non-traditional,easy to work with , honest &professional


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The G20 helped steer the world through the worst of the economic storm; now it must show it can set in motion a new governance for the post-crisis world. That's a task our organisation stands ready to help with, says the OECD's G20 Sherpa, Gabriela Ramos.
Sweden has developed an extensive and sound policy framework to limit greenhouse gas emissions.
The aim of the OECD Green Growth Strategy is to provide a clear framework for how countries can achieve economic growth and development while at the same time preventing costly environmental degradation, climate change and inefficient use of natural resources.
OECD Green Growth Strategy Workshop: we asked Nathalie Girouard, the Green Growth Strategy Co-ordinator, for her views on some of the topics being discussed.
To ensure that green growth policy recommendations are relevant to countries' needs, the OECD organised a consultation to review the first draft of the Synthesis Report on 10-11 February 2011.
At this roundtable, M. Gurría concluded that the topic of fairness and intergenerational solidarity is an essential part of our responsibility today and will be essential for the creation of a stronger, cleaner, fairer world economy tomorrow.
This meeting explored the role of policy in addressing issues related to climate change, agriculture and land use, both in the context of GHG mitigation and of farmer adaptation to climate change.
The Common Agricultural Policy (CAP) is an important policy for the European Union and accounts for about 40% of the EU budget. Ever since its inception in 1958, the CAP has been regularly reviewed and adjusted to improve its performance and adapt to changing circumstances. At a time when the post-2013 future of the CAP is being discussed and major challenges such as food security and climate change lay ahead, it is important to review the impact of past reforms and to draw lessons for the design of future policies.
While the studies in these proceedings often take account of national and international market effects of agricultural policies, they tend to focus on the impact of policies on farms and at the regional and local levels. Today, the European Union is composed of very diverse regions that are affected very differently by any given farm policy, depending on the structural characteristics of the farms' and regions' economies.
This report collects papers presented at the OECD Workshop on Disaggregated Impacts of CAP Reforms, held in Paris in March 2010, which focused on recent reforms. In particular, it examined the implementation of the single payment scheme since 2005 and the transfer of funds between different measures. Special attention was also paid to reforms of the sugar and dairy sectors with respect to the quota system and the restructuring of both these industries. The papers also look at the impact of the new direct payment system on land use, production and income.
Climate change will affect fisheries, fish stocks and coastal communities. Fisheries policymakers must strengthen global governance, use a rights-based management system, protect ecosystems, end environmentally harmful subsidies and focus on demand for sustainably caught seafood.
The limits for extraction of natural resources have largely been reached and climate change is expected to continue lowering natural water endowments markedly in future especially in dry areas of the country


text:
Today's Runes for Stanley Kubrick
spread reveals the dynamic forces at work in a situation. It is the spread of choice for understanding a hidden conflict.
Gold Runes
are most commonly used for questions about business, career, and property.
The bottom rune represents the foundation that forms
the basis of the issue
can be easily recognized as the antlers of the elk that it represents. The elk can represent victory, but is much more appropriately associated with the thrill of the hunt itself. This rune therefore can portend vigor and success in active endeavors. Also, this rune seems symbolic of a hand with outstretched fingers - a protective hand. This hand may suggest that you will be shielded from things negative - the problems still exist, you are spared the brunt of their force.
The left rune represents
one of the forces acting on the issue
at hand.
is the rune representing water. Laguz is a strongly feminine rune, and like the ocean contains much power of an uncontrollable nature. Here the rune is seen in the reversed, symbolic of the ebbing tide. This may suggest power being drawn away, or may perhaps the start of a journey. Since this is a rune of sexual power, the reversal can also suggest sexual problems or dissatisfaction.
The right rune represents
another of the forces acting on the issue
at hand.
refers to a god or the spoken word of God. It is the voice of reason, law, prophecy and truth. It is the truth heard in the wind. This rune represents wisdom obtained or knowledge delivered. It is the rune of absolute good advice and true council - the message we all must strive to hear.
The top rune represents
the conclusion
to which your strivings can carry you.
means gift, and like any gifts, the rune may be understood on many levels. Gifts are generally positive things, for both the giver and the recipient. In many cultures however, gifts and favors carry with them an obligation to respond in kind. It is for this reason that gifts, and hence the rune Gebo, are frequently symbolic of friendships, marriages, alliances, mergers, and other bonds between people or organizations. Gebo is a strong rune and the unions represented are strong as well. Moreover, Gebo is not reversible, as true friendships are not easily undone.
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text:
LeBron James to Heckler: "I'd Kick Your Ass Right Now"
Don't be talking about LeBron James' mama unless you want to find yourself on the end of a physical threat from the 6'8" superstar. According to an NBC Sports journalist's Twitter, James apparently got into a bit of verbal smack down with a fan heckling him at Friday's game against the Washington Wizards.
NBC Sports writer and self-proclaimed "Mila Kunis' butt double" Sarah Kogod saw a fan at the Wizards game exchange words with LeBron during the game, and then went to get his side of the story. She then Tweeted what the fan told her.
Wanted to give Heat PR time to comment on the LeBron fan altercation. I'll update you if they end up doing so, but here's the story...
...I saw a Wizards fan and LeBron in a heated conversation on the sideline during the game Friday night. I spoke to the fan and got his side...
...LeBron was waiting alone in the backcourt when fan Bob Moore made a comment to LeBron about Delonte West and LeBron's mom...
...Accrdng to Moore, LeBron said "The only reason you talk shit is because you know I can't come off the court right now and beat your ass"...
...Also according to Moore, LeBron continued "But if I had a free pass, I'd kick your ass right now."...
...Moore replied "I'm right here baby. Let's go" before security stepped in.
Of course, no word yet from Heat PR, but you can't blame a guy for getting upset when someone talks smack about his mama.
[via Deadspin]
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I am looking to replace the compressor on my 98 blazer ls however it is very expensive and am looking at doing it myself. It seems like a relatively simple job. But, I need to know the correct steps to take after purchasing the compressor.
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A Pirate Ship Gold Diggers Rush to Battle for Ancient Treasure - FREE Game !
By Hotel Romeo Media LLC
Open iTunes to buy and download apps.
You and your crew of pirate scavangers need to get your hooks on as mich gold as possible. Watch out because the ship you're raiding is haunted with the bones of pirates lost. You'll also have to watch out for sharks, and octopi down there.
The more gold you colect, the more help you'lll get from other pirate characters you can unlock.
Here's how the game works:
- Gameplay is simple and becomes more challenging as you avoid skeletons, sharks and octopi
- Tap the screen to swim upward, let go to drop downward
-Tap the right side of the screen to throw your sword
- Collect gold coins to unlock more pirate partners
- Collect the treasure chests and treasure maps for a burst of speed
- The longer you play, the faster the game moves. Each time you play A Pirate Ship Gold Diggers Rush to Battle for Ancient Treasure it changes so you never play the same game twice
- Gamecenter Leaderboards let you see how you score compared to other players by how long you're able to survive and collect gold
- Challenge your friends to beat your score!
-DISCLAIMER FOR KIDS MODE IN APP PURCHASE FEATURE- Kids mode is an In App Purchase feature within the game that allows the user to have unlimited health. Also this feature disables the ability for the character to collect in game coins and the ability to shoot down obstacles within the game. The purpose of this feature is to allow the user and/or kids for that matter to be able to play the game for an extended period of time without being hit by any in game obstacle. The user will also have the option to turn KIDS MODE OFF as well.
Customer Reviews
Gorgeous this thing
This game is quite great. Forget the babysitter, I gave them this
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- Free
- Category: Games
- Released: Sep 15, 2013
- Version: 1.0
- Size: 38.4 MB
- Language: English
- Seller: Hotel Romeo Media LLC
- © Hotel Romeo Media LLC
Compatibility: Requires iOS 5.0 or later. Compatible with iPhone, iPad, and iPod touch.
Top In-App Purchases
- unlock kids mode feature$3.99
- remove ads$3.99
- coin pack of 100000 coins$2.99


text:
Multiple secretoglobin 1A1 genes are differentially expressed in horses
© Côté et al.; licensee BioMed Central Ltd. 2012
Received: 20 April 2012
Accepted: 18 December 2012
Published: 19 December 2012
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© Côté et al.; licensee BioMed Central Ltd. 2012
Received: 20 April 2012
Accepted: 18 December 2012
Published: 19 December 2012
Secretoglobin 1A1 (SCGB 1A1), also called Clara cell secretory protein, is the most abundantly secreted protein of the airway. The SCGB1A1 gene has been characterized in mammals as a single copy in the genome. However, analysis of the equine genome suggested that horses might have multiple SCGB1A1 gene copies. Non-ciliated lung epithelial cells produce SCGB 1A1 during inhalation of noxious substances to counter airway inflammation. Airway fluid and lung tissue of horses with recurrent airway obstruction (RAO), a chronic inflammatory lung disease affecting mature horses similar to environmentally induced asthma of humans, have reduced total SCGB 1A1 concentration. Herein, we investigated whether horses have distinct expressed SCGB1A1 genes; whether the transcripts are differentially expressed in tissues and in inflammatory lung disease; and whether there is cell specific protein expression in tissues.
We identified three SCGB1A1 gene copies on equine chromosome 12, contained within a 512-kilobase region. Bioinformatic analysis showed that SCGB1A1 genes differ from each other by 8 to 10 nucleotides, and that they code for different proteins. Transcripts were detected for SCGB1A1 and SCGB1A1A, but not for SCGB1A1P. The SCGB1A1P gene had most inter-individual variability and contained a non-sense mutation in many animals, suggesting that SCGB1A1P has evolved into a pseudogene. Analysis of SCGB1A1 and SCGB1A1A sequences by endpoint-limiting dilution PCR identified a consistent difference affecting 3 bp within exon 2, which served as a gene-specific "signature". Assessment of gene- and organ-specific expression by semiquantitative RT-PCR of 33 tissues showed strong expression of SCGB1A1 and SCGB1A1A in lung, uterus, Fallopian tube and mammary gland, which correlated with detection of SCGB 1A1 protein by immunohistochemistry. Significantly altered expression of the ratio of SCGB1A1A to SCGB1A1 was detected in RAO-affected animals compared to controls, suggesting different roles for SCGB 1A1 and SCGB 1A1A in this inflammatory condition.
This is the first report of three SCGB1A1 genes in a mammal. The two expressed genes code for proteins predicted to differ in function. Alterations in the gene expression ratio in RAO suggest cell and tissue specific regulation and functions. These findings may be important for understanding of lung and reproductive conditions.
The mammalian airway epithelium is composed of heterogeneous cell populations grouped into three main types according to morphology and function: basal, ciliated, and secretory cells[1]. Further, eight morphologically defined subtypes are recognized, including non-ciliated, cuboidal and secretory Clara cells[2]. Clara cells synthesize the most abundantly secreted protein in the airway surface fluid, secretoglobin family 1A, member 1 (SCGB 1A1)[3]. The list of names attributed to SCGB 1A1 is extensive and includes Clara Cell Secretory Protein (CCSP), uteroglobin, blastokinin, Clara cell 10 kDa protein (CC10), CC16, polychlorinated biphenyl-binding protein (PCB-BP), and urine protein-1 (UP1). SCGB 1A1 has been ascribed many functions including binding of lipophilic substances, inhibition of leukocyte recruitment, inhibition of phospholipase A2, and other anti-inflammatory roles (reviewed in[4, 5]).
SCGB 1A1 is consistently expressed at high levels in the lung of most mammalian species. More specifically, studies in the horse showed that SCGB 1A1 is detected in non-ciliated lung epithelial cells, but not in goblet or ciliated epithelial cells[6]. SCGB 1A1 comprises 2 to 12% of bronchoalveolar lavage (BAL) fluid proteins[7, 8], and is considered to be an important component of proteins protecting the pulmonary epithelium against deleterious inhaled environmental substances[9]. It was previously reported that horses with recurrent airway obstruction (RAO), an asthma-like chronic inflammatory condition affecting mature individuals, have ultrastructural changes in Clara cells[6], decreased lung SCGB1A1 gene expression[6, 10] and reduced BAL fluid SCGB 1A1 concentration[6]. Expression of SCGB1A1 has also variously been described in extra-pulmonary tissues from diverse species. In horses, SCGB1A1 transcripts were present in uterine and prostatic tissues and absent in liver, kidney, heart, spleen, thyroid, pituitary and adrenal gland tissues[11].
A single SCGB1A1 gene has been described in the genome of multiple mammals, including rabbit, rat, mouse, monkey, and human[12–15]. The general structure of the SCGB1A1 gene includes two introns and three exons coding for a small secreted protein of ~70 amino acids. This organizational structure is remarkably conserved between species; however, the length of the SCGB1A1 genomic locus fluctuates[12–17]. In horses, the first reported sequence was described as a unique cDNA and was ascribed to a single gene[11]. However, the recent availability of the complete Equus caballus genome sequence provided evidence of three highly similar SCGB1A1 gene sequences on chromosome 12, suggesting the horse has diverged from the "single copy" SCGB1A1 consensus. Two distinct SCGB 1A1 protein products were also identified in uterine fluids during early pregnancy[18], further implying that more than one SCGB1A1 gene may be transcribed and translated.
Considering that horses appear to have multiple similar, but not identical, SCGB1A1 gene copies, and that total SCGB 1A1 levels are decreased in the lung of horses with RAO, we hypothesized that SCGB1A1 variants may be differentially expressed and have different functions. Herein, we report on three distinct copies of the SCGB1A1 gene in horses. We developed assays to distinguish each gene, determined tissue- and copy-specific gene expression, and evaluated cell-specific presence of the SCGB 1A1 protein. We further determined that horses with RAO have an abnormal expression ratio of different SCGB1A1 genes.
A partial sequence including a large part of the adjoining 5' and 3' non-coding DNA was extracted from the EquCab2.0 sequence for each predicted copy (~10 kb/sequence) and analyzed by multiple sequence alignment. Bioinformatic analysis confirmed that each gene had comparable exon/intron organization, and covered about 2,650 base pairs (bp) of genomic DNA (Figure 1B). A high degree of pairwise identity (92.7%) was observed in large segments overlapping the SCGB1A1 coding regions and 8,941 identical sites (87.8%) were found among the three genes, suggesting that SCGB1A1 genes developed from an intrachromosomal triplication event. The pairwise identity increased to 97.8% and the number of identical sites rose to 96.7% upon alignment of the predicted complementary DNA (cDNA) sequences. However, SCGB1A1 genes differed from each other by 8 to 10 nucleotides, and were expected to produce different proteins. Therefore, the distinct genes were termed SCGB1A1P, SCGB1A1, and SCGB1A1A; with SCGB1A1P located most proximal to the centromere. These novel sequences were deposited in GenBank with the following accession numbers: JQ951929, JQ951930 and JQ951931.
A long-range (LR)-PCR strategy was developed to amplify individual full-length SCGB1A1P, SCGB1A1, and SCGB1A1A genomic sequences, using three distinct gene-specific primer sets (Figure 1C). The size of the different LR-PCR products ranged from 5.4 to 6.0 kb. Samples from a total of 24 animals were used for amplification, purification, and identification of the SCGB1A1P sequence, compared to SCGB1A1 and SCGB1A1A sequences, which were accurately documented by the examination of 12 sequences due to a reduced amount of polymorphisms between individuals analyzed. The identity of all PCR fragments was further evaluated by restriction enzyme digestion assay (Figure 1D), which confirmed presence of three different SCGB1A1 genes in each animal assessed.
A total of 212 PCR products were amplified from 665 reactions (32% efficiency) using lung cDNA limiting dilutions as template (42-124 amplicons/animal). An additional 86 products were generated from 175 reactions (49% efficiency) using uterine cDNA (28-29 amplicons/animal). Each individual PCR product was purified, sequenced (forward), and identified by the SCGB1A1 signature sequences (Figure 3B). In lung samples, 30% of products were identified as SCGB1A1 cDNA (A--A--A) and 70% as SCGB1A1A (A--G--G). Likewise, in uterus samples, 56% were identified as SCGB1A1 and 44% as SCGB1A1A.
From the 298 amplicons identified, 20 SCGB1A1 and SCGB1A1A cDNAs were randomly selected for further characterization. Each sample was re-submitted for sequencing in both forward and reverse directions, and results were aligned to determine the cDNA sequence delimited by the start and stop codons. Subsequently, SCGB1A1 and SCGBA1A cDNA consensus sequences were obtained by alignment of the 20 gene-specific sequences (accession numbers JQ906259, JQ906260, JQ906261, Additional file1: Figure S1). As expected, strict nucleotide identity was observed between an individual cDNA and the corresponding genomic sequence. However, several disagreements were observed upon comparison with the predicted EquCab2.0 cDNA sequences. Among samples in this study, the partial SCGB1A1 cDNA consensus sequences displayed a non-conservative substitution at position 19 (A to G) and two variable codons (Variant A, TTA or variant B, CTC) at positions 232 to 234. The latter variants both code for the production of a leucine residue. Similarly, our SCGB1A1A consensus sequence showed 5 non-conservative substitutions at position 65 (A to G), 78 (C to A), 81 (T to G), 175 (A to G), and 217 (A to G) compared to the EquCab2.0 SCGB1A1A predicted cDNA sequence. Single-nucleotide insertions or deletions were not detected.
Surprisingly, SCGB1A1P cDNA was not detected by EPLD-PCR. Since complete genomic/cDNA nucleotide identity was observed for SCGB1A1 and SCGB1A1A, SCGB1A1P similarly was assessed using the predicted cDNA sequences extracted from EquCab2.0 and our consensus genomic sequences (accession numbers JQ951929, JQ951930, JQ951931, Additional file1: Figure S1). Analysis revealed 98% pairwise identity between the predicted cDNA and the genomic DNA (272/276 nucleotides). Three non-conservative substitutions were identified at positions 14 (T to G), 19 (G to A), and 220 (A to T). Most strikingly, the A to T substitution detected at position 220 in the variant sequences B and C represented 54% of the horses analyzed (13/24), independent of their genetic background. This polymorphism is expected to replace an AAG codon encoding lysine, to a TAG codon, encoding a stop codon. Therefore, these results demonstrate the presence of a SCGB1A1P gene variant that may encode a truncated protein in a large proportion of animals.
Semi-quantitative analysis of SCGB1A1 RT-PCR amplicons
Relative amplicon density
Expression category
3.50 ± 0.94
3.65 ± 0.72
2.25 ± 0.59
2.02 ± 0.61
Fallopian tube
2.67 ± 1.60
1.49 ± 1.59
Mammary gland
0.67 ± 0.67
nd - 0.33
nd - 0.65
nd - 0.07
nd - 1.67
0.42 ± 0.55
nd - 0.32
0.25 ± 0.38
0.01 ± 0.01
nd - 0.07
0.16 ± 0.20
nd - 0.49
0.04 ± 0.01
nd - 0.19
0.81 ± 0.73
nd - 0.26
nd - 0.43
0.09 ± 0.08
0.08 ± 0.02
Nose epithelium
nd - 0.17
0.04 ± 0.03
0.06 ± 0.04
nd - 0.03
nd - 0.07
nd - 0.04
nd - 0.02
nd - 0.01
nd - 0.02
nd - 0.01
nd - 0.05
Small intestine
0.02 ± 0.01
Large intestine
0.03 ± 0.02
nd - 0.01
nd - 0.02
Salivary gland
nd - 0.01
Adrenal gland
nd - 0.01
Seminal vesicle
nd - 0.42
0.02 ± 0.01
Eyelid gland
Cardiac muscle
Lymph node
Bone marrow
Results in the first group corresponded to cDNA samples that generated abundant PCR amplicons with high OD (>0.50) for both SCGB1A1 genes. Samples from lung, uterus (non-pregnant), Fallopian tube, and mammary gland (non-lactating) consistently and reproducibly met this criterion.
The second group included cDNAs that produced a faint PCR product for either SCGB1A1 or SCGB1A1A, and had relatively low (0.01 to 0.50) OD values. Brain, pituitary gland, eye, nose epithelium, tongue, parotid salivary gland, trachea, aorta, liver, spleen, small and large intestine (cecum), adrenal gland, kidney, skin, bladder, urethra, prostate, epididymis, seminal vesicle, testis and ovary were included in this group.
A third group included cDNAs that did not produce gel-detectable PCR amplicons for any sample tested, and therefore, were unsuitable for quantitative assessment. Eyelid gland, thyroid, bone marrow, cardiac muscle, pancreas, stomach and lymph node were considered negative for SCGB1A1 expression.
Analysis of the extra-pulmonary tissues revealed that epithelial cells in the uterus, ovary, Fallopian tube and mammary gland also expressed SCGB 1A1. Within the uterus, large coiled glands composed of columnar epithelial cells stained most strongly positive (Figure 7B), while in the ovary only epithelium from the wall of residual cysts showed some degree of staining, and luteal cells were negative. The ciliated cells of the Fallopian tube stained positive, with a strong apical signal extending to the cilia (Figure 7C). Immunoreactive SCGB 1A1 was not detected in pancreas, liver, kidney, adrenal gland, large intestine, striated and smooth muscle, duodenum, adipose tissue, nerve, nose epithelium, skin, stomach, aorta, cartilage, bone marrow, brain and pituitary gland.
In this study we report the identification and characterization of three equine SCGB1A1 genes. A large segment of genomic sequence was amplified for each SCGB1A1 gene and the partial coding sequences leading to the mature secreted proteins were sequenced. We found that certain nucleotides from each of the three genes differed from the other two genes, and that this pattern was conserved amongst individuals. The distinct SCGB1A1 genes were predicted to produce slightly different proteins, and were therefore referred to as SCGB1A1P, SCGB1A1 and SCGB1A1A based on the recommended systematic gene nomenclature system[20]. A non-contiguous region composed of three distinct nucleotide variants was chosen as a signature sequence to generate assays specific for each individual SCGB1A1 gene.
Analysis of the predicted SCGB1A1 sequences extracted from EquCab2.0 (NCBI) revealed more than 90% pairwise identity between SCGB1A1P, SCGB1A1, and SCGB1A1A genes, including large segments of 5'- and 3'-flanking regions. This high level of sequence identity between both the coding and non-coding regions of SCGB1A1 genes created a challenge for isolation and assessment of individual genes. Thus, more than 10 kb of non-coding sequence surrounding each SCGB1A1 gene was interrogated to identify anchor regions for gene-specific primers. Primers selected included at least three nucleotides unique to each SCGB1A1 gene. Sequence analysis of products confirmed specific amplification of desired targets with lack of cross-amplification.
Comparative analysis of the coding region of SCGB1A1 genes revealed some disparity with the predicted EquCab2.0 sequences. Our sequences differed at three positions in SCGB1A1P, one position in SCGB1A1, and five positions in SCGB1A1A, corresponding to 1.1, 0.4, and 1.8% of difference, respectively. These differences may result from copy number variants and chromosomal rearrangements hindering automated sequence assembly of the equine genome[21]. This is consistent with recent studies reporting equine chromosome 12 as a "hotspot" for genomic rearrangements, such as enrichment in copy number variants and single nucleotide polymorphisms (SNP)[22, 23]. A SNP was detected in SCGB1A1P and two SNPs in SCGB1A1, but neither affected the predicted translated products. Altogether, we identified several sequence differences to the EquCab2.0 genome, which highlighted the importance of developing gene-specific assays.
The three SCGB1A1 genes had similar gene structure and highly conserved intron/exon organization, suggesting that each contained all the elements required for expression. Thus, the transcriptional state of each gene was evaluated by EPLD-PCR. SCGB1A1 and SCGB1A1A, but not SCGB1A1P, were specifically detected in EPLD of lung and uterus. These results are consistent with detection of two differently migrating SCGB 1A1 proteins in uterine washes from mares in early pregnancy[18]. Furthermore, the sequence of each gene between start and stop codon was identical to our genomic sequence. The equine genome draft was derived from DNA of a single Thoroughbred mare, while our EPLD-PCR data were generated from animals of various genetic backgrounds and may thus be more representative of this challenging genome region.
SCGB1A1P transcripts were not detected in lung or uterine tissue. Reasons for lack of SCGB1A1P gene transcription were unclear, especially since the gene structure is virtually identical to that of the other SCGB1A1 genes. However, the SCGB1A1P sequence was more variable between individuals, had a shorter promoter region (which may imply lack of regulatory elements) and contained a putative stop mutation in a significant percentage of individuals. These characteristics suggest that SCGB1A1P may be a pseudogene.
The non-synonymous nucleotide variations observed between SCGB1A1 and SCGB1A1A result in 12 amino acid (AA) substitutions among the 70 residues of the mature secreted proteins. Seven of the variable AAs are concentrated between position 26 and 36 (protein including the signal peptide). This region borders the SCGB 1A1 central cavity that binds hydrophobic ligands (reviewed in[24]), and AA with hydrophobic properties comprise the cavity[25]. Since some conserved AA, such as phenylalanine 27 (F27), also have ligand-binding properties, substitution of F27 to L27 in SCGB 1A1A suggests 1) a change in ligand-binding specificity, and 2) that SCGB 1A1 and 1A1A may have independently evolved to bind distinct substrates. Other substitutions in this region largely maintain hydrophobic properties (A28 to V28, I31 to V31, G33 to A33, F35 to Y35), implying minor change in ligand affinity. There is high sequence identity in other regions of each protein with preservation of critical structural residues such as C24 and C90 needed for homodimer interaction, and K63, D67 and A58 required for protein stability. However, also of interest, the predicted isoelectric point (pI) of SCGB 1A1 and 1A1A proteins are 5.1 and 6.3, respectively, which corresponds to the SCGB1A1 variants described in uterine washes of pregnant horses[18].
The spectrum of tissues expressing SCGB1A1 has not been extensively studied in horses. Expression nonspecific for individual genes was previously identified in lung, uterus and prostate, and absent in liver, kidney, heart, spleen, as well as thyroid, pituitary and adrenal gland tissues by Northern blot analysis[11]. We selected a gene-specific RT-PCR approach to characterize the distribution of SCGB1A1 and SCGB1A1A transcripts in a total of 33 tissues. This assay amplified the coding region of the entire mature protein (second and third exons) to reduce potential genomic DNA targeting, and specificity was verified by random purification and sequencing of amplicons. As expected, SCGB1A1 and SCGB1A1A transcripts were strongly detected in lung and uterus, but also in Fallopian tube and mammary gland tissue. Fewer transcripts of either gene were present in brain, pituitary gland, eye, nose epithelium, tongue, salivary gland, trachea, aorta, liver, spleen, small and large intestines, adrenal gland, kidney, skin, bladder, urethra, prostate, epididymis, seminal vesicle, testis and ovary, and no transcripts were detected in eyelid gland, thyroid, bone marrow, cardiac muscle, pancreas, stomach and lymph node. Detection of transcripts in a greater range of tissues by PCR than by Northern blotting may reflect higher sensitivity of the former, and greater specificity due to exact primer match. Some tissues had more of one relative to the other gene product, suggesting tissue-specific expression patterns. While the regulatory mechanisms that could selectively drive the expression of individual SCGB1A1 genes remain to be elucidated, this finding is consistent with unique functions of different SCGB 1A1 proteins.
Immunohistochemical analyses were carried out to evaluate the correlation of SCGB1A1 gene and protein expression, and to determine cell-specific expression within tissues. The antibody employed recognized an epitope shared by all SCGB 1A1 proteins. Strong staining was detected in tissues expressing the highest number of transcripts such as lung, uterus, Fallopian tube and mammary gland. However, within these tissues, SCGB 1A1 was present in only specific epithelial cell populations, and absent in all other cell types (Figure 7). This distribution likely contributed to variation of SCGB1A1 transcript intensity, since the proportion of epithelial cells and subtypes in tissues selected was variable[26].
SCGB 1A1 may be selectively taken up and targeted for degradation upon binding to a transmembrane protein called cubilin[27]. Interaction of SCGB 1A1 with lipocalin-1 interacting membrane receptor (LIMR) has also been reported, but the effect of this interaction is unclear[28]. Of note, LIMR is expressed in tissues positive for SCGB1A1 transcripts, including lung, mammary gland, trachea, prostate, testis, pituitary gland, adrenal gland, cerebellum, kidney, and colon[29]. SCGB 1A1 protein was not detected in tissues with low transcript expression such as bladder, trachea, liver, nose epithelium, pituitary and intestine. Possible reasons are lesser sensitivity of IHC compared to PCR, and transient gene expression insufficient to produce detectable protein, as has been reported in studies of rabbit tissues[30].
Horse and human have a similar pattern of SCGB1A1 transcript distribution in lung[31], uterus[13, 32], Fallopian tube[33], prostate[34, 35], trachea, thyroid, mammary gland, brain, pituitary, thymus, aorta, heart, stomach, spleen, adrenal gland, kidney, liver, small intestine, ovary and testis[36]. In humans, the SCGB1A1 gene was ultimately considered to be ubiquitously expressed in most cells of epithelial origin and to inactivate inflammatory mediators on surfaces exposed either directly or indirectly to the external environment[37, 38]. Conversely, it was also reported that decreased SCGB1A1 expression could contribute a tumor microenvironment permissive of inflammation and hence tumor progression[38, 39].
Since total SCGB1A1 expression was reported as decreased in RAO, we sought to evaluate if both SCGB1A1 and SCGB1A1A genes were similarly affected. Our analysis demonstrated that the ratio of SCGB1A1A/SCGB1A1 was significantly different in RAO affected-animals compared to controls. This finding may arise as a result of chronic inflammation with preferential transcription of SCGB1A1A, or may signal inherent differences in transcriptional regulation of SCGB1A1 genes. Segregation analysis previously revealed a complex genetic background influencing expression of the RAO phenotype[40]. SCGB1A1 was among candidate genes[10], suggesting that further evaluation of specific gene expression may be warranted. The latter observation also raises the question whether SCGB1A1 genes are controlled by different regulatory mechanisms and whether they have different physiological functions. Such hypotheses are difficult to address due to the absence of multiple SCGB1A1 copies in other mammals except other equidae, such as donkeys and Przewalski horses (unpublished data). SCGB1A1 isoforms in equid species remain to be characterized, but may yield insight into SCGB1A1 gene origin and ancestral gene triplication.
Three equine SCGB1A1 genes were isolated and characterized. SCGB1A1P appears to have evolved into a pseudogene, which no longer generates a detectable transcript and includes a non-sense mutation in the majority of animals. The distribution of SCGB1A1 and SCGB1A1A gene transcripts and proteins indicates highly specific expression in specialized epithelial cells of lung and reproductive organs. Gene specific assessment of transcripts showed approximately 2.5 fold higher expression of SCGB1A1A than SCGB1A1 in lung and uterus of control animals, and an increased ratio in lung tissue of animals with RAO, an asthma-like condition. Future studies will assess the function of different SCGB 1A1 proteins and attempt to elucidate their anti-inflammatory properties.
Animal procedures were approved by the University of Guelph Animal Care Committee (Protocol R10-031) and conducted in compliance with guidelines of the Canadian Council on Animal Care. All horses belonged to the institutional research herd; horses with and without RAO were of similar age ranging from 12 to 20 years. Horses with RAO had a history of recurrent cough, difficulty with exhaling air, and neutrophilic inflammation in BAL fluid samples. Control horses had no history of lung disease, had normal physical exam findings, and no abnormalities on airway bronchoscopy or pulmonary function testing, as described before[6].
Horses were restrained in stocks and percutaneous lung biopsies were obtained under sedation with romifidine (10 mg/mL, IV), as previously described[6]. Samples were immersed in RNAlater™ solution (Qiagen, Mississauga, ON) and stored at -80°C until RNA preparation.
SCGB1A1 sequences were obtained from the National Center for Biotechnology Information (NCBI) database. Alignment of the Equus caballus chromosome 12 genomic contig (EquCab2.0) [GenBank: NW_001867370.1][19] and the equine secretoglobin precursor mRNA [GenBank: AY885564.1] was performed through a nucleotide megablast analysis for highly similar sequences using the following parameters: expected threshold, 10; word size, 16; match-mismatch scores, 1, -2; gap cost - linear; filter - low complexity. Further data processing, including sequence extractions, multiple sequence alignments, and statistical analysis, were accomplished with Geneious Pro software (version 5.5.3, Auckland, NZ) with occasional minor manual adjustments[41].
Blood samples or buccal swabs were available from 24 adult horses including 6 breeds (Standardbred, Thoroughbred, Icelandic, Canadian sport, Lipizzaner, and Quarter horses) and animals of mixed breeding. Genomic DNA was extracted according to the manufacturer's protocol (DNA Mini kit, Qiagen). LR-PCR primers were as follows: SCGB1A1P primer forward UG1-F (5′-ACA GAG CCA GCC CAA GCA ATG-3′) and reverse UG1-R (5′-GAT TAC CTT GGC GGT TGC CTA GAG-3′), SCGB1A1 primer forward UG2-F (5′-CAC CTA ACA GCC TCA TCT C-3′) and reverse UG2-R (5′-GTG AGA GCT CTC ATC TGG TA-3′), and SCGB1A1A primer forward UG3-F (5′-ACA CAG ATC TGA TGC CCA AG-3′) and reverse UG3-R (5′-AGT GCA GCT CTC TCA GGC AT-3′), amplifying 5559, 6029, and 5442 bp of genomic DNA, respectively. Primers were obtained from Sigma-Aldrich (Burlington, ON). LR-PCR amplifications were carried out using a Platinum Taq polymerase PCR kit (Invitrogen, Mississauga, ON). Each reaction was performed in a final volume of 50 μL, including 5 μL of 10X PCR buffer, 0.2 mM dNTPs, 2 mM MgSO4, 0.3 μM of each primer, 2 U of Platinum Taq, and 5 μL of template DNA (100 ng). Conditions for amplification were 1 min at 94°C followed by 35 cycles of 94°C for 30 s; gene-specific annealing T°C for 30s; and 68°C for 6:30 min, followed by final elongation for 10 min at 68°C. Gene-specific annealing temperatures for SCGB1A1-1, -2, and -3 were 62°C, 55°C and 60°C respectively. Twenty μL of each PCR product was separated by electrophoresis in a 1% agarose gel stained with SYBR Safe (Invitrogen). The amplified DNA bands were cut out and the DNA was extracted, purified (QIAquick, Qiagen) and quantified using a NanoDrop 2000 photometer (Thermo Fisher Scientific, Mississauga, ON). To validate the gene-specificity of the PCR products, each purified DNA fragment was digested with HindIII (Invitrogen), separated by electrophoresis and monitored for the appropriate digestion pattern.
A genomic DNA band of 526 bp coding for the full-length mature secreted protein (exon 2 to 3) was amplified by nested PCR using LR-PCR purified DNA products as template and the forward UGn-F (5′-GCT TCT GCA GRA ATC TGC CAG AG-3′) and reverse UGn-R (5′-CTA AGC ACA CAG TGG GCT CTY TRC-3′) primers. PCR amplifications were carried out in duplicate using the Taq DNA polymerase Native PCR kit (Invitrogen) in a final volume of 25 μL of PCR buffer (2 μL of 10X PCR buffer, 1.0 mM dNTP, 1.5 mM MgSO4, 0.6 μM forward and reverse primers, 1 unit Platinum Taq, and 1 μL of template DNA). Cycling conditions were 1 min at 94°C followed by 30 cycles of 94°C for 30 s; 62°C for 30 s; and 72°C for 90 s with a final elongation of 7 min at 72°C. Twenty μL of each PCR product was subjected to electrophoresis; bands of appropriate size were excised from the gel, purified and submitted for automated sequencing (Laboratory Services Division, Guelph, ON). Amplicons were analyzed in duplicate using reverse and forward primer sequencing strategies. The consensus sequence for each copy was determined using multiple sequence alignments.
Total RNA was isolated from fresh or frozen lung and uterus tissues (RNeasy, Qiagen) according to the manufacturer′s recommendations. RNA integrity was verified through capillary electrophoresis in a 2100 Bioanalyzer (Agilent Technologies) prior to analysis. Complementary DNA (cDNA) was synthesized following the Superscript III Reverse Transcriptase kit′s protocol (Invitrogen), with an additional 15 min DNAse I treatment (Qiagen) at room temperature. A cDNA band of 256 bp delimited by the start (ATG) and termination (TAG) codon within each of the SCGB1A1 genes was targeted with the following primers: forward UGm-F (5′-GTC CAC CAT GAA ACT CGC CA-3′) and UGm-R (5′-CTA AGC ACA CAG TGG GCT C-3′). End-point limiting dilution (EPLD)-PCR assays were performed in a volume of 25 μL of PCR reaction mix (2 μL of 10X PCR buffer, 1.0 mM dNTP, 1.5 mM MgCl2, 0.6 μM forward and reverse primer, 1 unit of Platinum Taq and 1 μL of template cDNA). A broad range of serially diluted cDNA concentrations was tested (0.25 to 0.000156 ng/μL) to determine the optimal limiting-dilution for each sample (0.0005 to 0.0006 ng/μL). PCR products were separated by electrophoresis, purified, sequenced and identified via their SCGB1A1 gene-specific signature sequences.
SCGB1A1 and SCGB1A1A relative transcript levels were evaluated by semi-quantitative reverse transcriptase-PCR (RT-PCR) in 33 different tissues from normal adult horses including cortical brain, pituitary, eye, nose epithelium, tongue, thyroid, trachea, lung, aorta, cardiac muscle, liver, spleen, small and large intestine, stomach, kidney, pancreas, skin, bladder, urethra, prostate, epididymis, seminal vesicle, testis, uterus, ovary, Fallopian tube, bone marrow, lymph node, as well as mammary, salivary, adrenal and eyelid glands. Total RNA isolation and cDNA synthesis were performed as described above. A specific cDNA band of 200 bp was amplified for each SCGB1A1 gene using the following primers: SCGB1A1 forward UGrt-2 F (5′-GCT TTG CAG ACA TCA TTC AAG GCC-3′) and reverse UGrt-2R (5′-CTA AGC ACA CAG TGG GCT CTT TG-3′) as well as SCGB1A1A forward UGrt-3 F (5′-GAT TKG TAG GCA TCG TTC AAG CCC-3′) and reverse UGrt-3R (5′-CTA AGC ACA CAG TGG GCT CTC TA-3′). A 254 bp equine glyceraldehyde dehydrogenase (GAPDH) gene product served as an internal control using forward GAP-F (5′-GTT TGT GAT GGG CGT GAA CC-3′) and reverse GAP-R (5′-TTG GCA GCA CCA GTA GAA GC-3′) primers. PCR amplifications were carried out using the HotStar Taq Plus PCR kit (Qiagen) in a 20 μL Master mix (10 μL of 2X PCR buffer, 0.4 μM of copy-specific forward and reverse primers, and 1 μL of template cDNA). Conditions for amplification were 5 min at 95°C followed by 30 cycles of 95°C for 30 s; 61°C for 30 s; 72°C for 1 min followed by a final elongation of 7 min at 72°C. PCR products were analyzed by electrophoresis in a 1% agarose gel and stained with SYBR Safe. PCR-grade water was distributed as template in negative control reactions. No amplifications were detected in samples with water instead of template DNA.
SCGB1A1, SCGB1A1A and GAPDH primers were as described above. The equine 18S ribosomal RNA gene was retained as an additional internal control using forward 18S-F (5′- ATG CGG CGG GGT TAT TCC-3′) and reverse 18S-R (5′-GCT ATC AAT CTG TCA ATC CTG TCC-3') primers. Quantitative PCR amplifications were performed in a Master mix containing 10 μL of SYBR Green 2X PCR buffer (Qiagen), 8 μL of PCR-grade water, 0.4 μM of each forward and reverse primer, and 1 μL of cDNA template (1 ng/μL). Conditions for amplification were 7 min at 95°C followed by 45 cycles of 95°C for 15 s; 61°C for 15 s; 72°C for 20 s using a LightCycler® 480 instrument (Roche, Montreal, QC). SCGB1A1 primer specificity and identity of the PCR products was confirmed with a melting curve (95°C for 5 s; 45 to 95°C; 40°C for 10 s) and sequence analysis, respectively. For each gene, a series of purified cDNA PCR product dilutions (100, 10, 1, 0.1, 0.01, 0.001 ng/μL) was amplified and the average crossing point of each dilution was used to derive a standard curve. SCGB1A1, SCGB1A1A, GAPDH, and 18S cDNA were amplified in triplicate for each sample along with standard curve calibrators. Data were analyzed using LightCycler® 480 SW 1.5 software (Roche). Statistical analysis was carried out using Prism5 (GraphPad Software, San Diego, CA).
Expression of SCGB 1A1 protein was investigated through immunohistochemical staining of the 33 adult horse tissues described above. Fresh tissues were fixed in 10% neutral buffered formalin overnight, embedded in paraffin, and sectioned to 5-μm thickness. Sections were deparaffinized in xylene, rehydrated in graded alcohols and incubated consecutively for 10 min in Dako endogenous dual enzyme blocker (DakoCytomation), 30 min with Dako protein block (serum-free), 30 min with SCGB 1A1 primary antibody (1:400 dilution), and 30 min with horseradish peroxidase-labeled secondary IgG (1:2000, Dako EnVision HRP). Bound antibodies were detected with Nova Red (Vector Laboratories, Burlington, ON) chromogen, and slides were counterstained with hematoxylin. SCGB 1A1 primary antibody was raised in a rabbit immunized with a 21mer peptide[6]. Pre-immune rabbit serum was used for negative control slides. Images were acquired on a Leica DMRA2 microscope (Leica Microsystems, Concord, ON) using Open Lab software (PerkinElmer, Waltham, MA).
Values were expressed as means ± standard deviations. Unpaired two-sample Student's t-test was used for statistical analysis; p ≤ 0.05 was considered significant.
End-point limiting dilution PCR
Long-range PCR
Recurrent airway obstruction
Semi-quantitative PCR.
The authors thank Heindrich Snyman, Brian Stevens and veterinarians at the Toronto Zoo for assistance with tissue procurement, and Nina Jones for critical review of the manuscript. This work was supported by operating funds from Equine Guelph and the Natural Sciences and Engineering Research Council of Canada (NSERC); animal resources were supported by the University of Guelph and the Ontario Ministry of Agriculture and Rural Affairs. O.C. is the recipient of a graduate award from the University of Guelph.
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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I have product list and every product has create date in DateTime type. I want to take some products that created after my entering time in string type.
I enter EnteredDate in string type, like this format : 05/16/2012
1. var dates = from d in Products
2. where d.CreateDate >= DateTime.ParseExact( EnteredDate, "mm/dd/yy", null )
3. select d;
In second line I got error as String was not recognized as a valid DateTime for "mm/dd/yy". I also tried DateTime.Parse(), Convert.ToDateTime() and got same error. How can I filter this product list by create date?


text: AdChoices Terms of Use Privacy Policy Hide TOP 10 Top 10 Better Man 7 Ways To Wear A T-Shirt Share Tweet 0 Shares 7. Underneath A Classic White Shirt Macy's Enviable style isn't about having the latest labels or trying out every trend. In fact, great taste is often found in a simple yet timeless ensemble. For example, it's hard to beat the sporty sophistication of white pants and a white short-sleeve woven cotton button down for summer. So keep things laid-back but refined by picking up a white button-down that skims your body, like this one from Kenneth Cole Reaction, along with white cotton or linen pants. For a more daring look, add some contrast 2012-style by slipping Kenneth Cole Reaction's EDV cobalt T-shirt underneath your white top. Next Page TOP 7 6 5 4 3 2 1 Comments Share your opinion Your name More Like This Top 10 Dating 10 Scientific Reasons Why Having More Sex Is Good For Your Health Read More Top 10 Travel 10 Honeymoon Destinations That Will Win You Brownie Points For Life Read More Top 10 Entertainment 10 Blockbuster Flicks That Sank Worse Than The Titantic Read More Top 10 Dating Here's How You're Going To Become A Master Flirter Read More Top 10 Dating Are You Good Boyfriend Material? Only If You Possess These 10 Traits Read More Top 10 Entertainment The World's 10 Worst Theme Parks, Ranked Read More Around the Web Special Features Special Feature The Ultimate Guide To Hooking Up Online Read More Guy Gear The Best Watches Under $500 Read More


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It's down more than 20% on over 750,000 shares traded in pre-market. Piper Jaffrey has just downgraded to neutral with a $10 price target. I'm not sure you are seeing the bigger picture and are kidding yourself if you think this is just some manipulation to get cheap shares. The briefing doc calls into question whether or not an Adomm will recommend for approval, not something anyone would have anticipated 24 hours ago, especially after the positive AEGR briefing docs. This is going down today and tomorrow, especially if the adcomm goes well for AEGR tomorrow. If the adcomm does go well for ISIS on Thursday, you'll see a big pop, but then get out as the REMS suggested by the FDA is VERY limiting. ISIS is still a good long-term play, so think of anything under $10 as a buying opportunity, but I wouldn't buy before the adcomm unless you have a very high risk tolerance.


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The average salary of a(n) Mechanical Engineering Manager in United States is $128,675.
The average hourly rate of a(n) Mechanical Engineering Manager in United States is $61.86.
The average bonus of a(n) Mechanical Engineering Manager in United States is $9,329.00.
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CHICAGO – The stage play that Harry Lennix is in town to direct – "A Small Oak Tree Runs Red" – is in its last weekend, and is giving the actor/director the best notices of the theater part in his long and successful career. For more information about the play, and ticket availability, click here.
Steve Little
TV Review: HBO's 'Eastbound & Down,' 'Life's Too Short' Offer Unique Takes on ComedySubmitted by BrianTT on February 19, 2012 - 9:54am
CHICAGO – HBO brings back a few of their most larger-than-life personalities with the third (and final) season premiere of "Eastbound & Down" and the stateside premiere of "Life's Too Short."
Blu-Ray, DVD Review: 'Eastbound & Down,' 'Tracy Morgan: Black and Blue'Submitted by BrianTT on August 9, 2011 - 2:52pm
CHICAGO – HBO brought two of their most outgoing personalities to Blu-ray and DVD last week with the releases of "Eastbound & Down: The Complete Second Season" and "Tracy Morgan: Black and Blue." Both are a step down from the best that these men have offered the comedy world but they also feature enough quality laughs for fans of Danny McBride and the controversial star of "30 Rock."
DVD Review: HBO's Hilarious 'Eastbound and Down' Gets Great ReleaseSubmitted by BrianTT on July 2, 2009 - 7:00am
CHICAGO – Danny McBride ("Pineapple Express," "Land of the Lost") gave one of the best TV performances of the last several years as the over-the-top anti-hero of HBO's hysterical "Eastbound & Down," a sitcom about a raging a-hole sports star brought back to Earth and forced to work as a gym teacher in the small town he grew up in, now available on DVD.


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Thursday night at the Crowne Plaza Garden Pavilion 710 employers and their staffs from 60 companies took part in a raucous celebration of the workplaces in the region with the most satisfied and engaged workers. more
By PBN Staff
Rhode Island's seasonally adjusted unemployment rate was 5.4 percent in May, unchanged from the revised April rate, but seven-tenths of a percentage point lower than it was in May 2015, the state Department of Labor and Training said Thursday. more
By Emily Gowdey-Backus
Employer costs for employee compensation in March were higher in New England than any other geographic region in the country, according to data from the federal Bureau of Labor Statistics released this week. more
By PBN Staff
With Father's Day this weekend, Rhode Island has been named the 16th best state for working dads. more
By Lori Stabile
When General Electric Co. in 2015 announced plans to move out of its longtime Connecticut headquarters, many were surprised when Rhode Island was named as a possible new location. more
By Eli Sherman
(Updated 12:12 and 12:37 p.m.) General Electric Co. plans to open a new GE Digital information technology center, which will bring "hundreds" of new technology jobs to Providence, Gov. Gina M. Raimondo said Thursday. more
By PBN Staff
The Providence-Warwick-Fall River metropolitan area ranked 91st among 358 metro areas nationwide for its 6 percent construction job growth over the year in April, the Associated General Contractors of America said Wednesday. more
By PBN Staff
Jobless claims fell for a second week, indicating the surge at the start of May reflected temporary dismissals. more
By Victoria Stilwell and Michelle Jamrisko
Employers gathered at the Roadmap to Reentry Community Workforce Development Summit Wednesday morning to learn about how ex-offenders are prepared for a smooth re-entry into the mainstream workforce and incentives available to employers who take the risk to hire those with a criminal record. more
By Emily Gowdey-Backus
In the last year, more and more companies have started offering their employees cash to pay off student loans as part of benefits packages. more
By Rebecca Greenfield


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Jul 27, 2016 - Independent game developer, Bivis today introduces Blicke 1.0, the company's new challenging puzzle game for iOS devices. Blicke is a unique puzzle game that encourages you to guide colored blocks across the board to take each one of them to their target. There is no time or move count limit. The blocks slide until they hit a wall or another block, challenging the player to figure out how to strategically use their pieces as support to create new paths and beat all 72 levels.
Jul 26, 2016 - Nex Game Studios announces Arcane Quest 3, a strategic RPG for all mobile platforms. Featuring real-time multiplayer quests with cross-platform functionality across all devices, Arcane Quest 3 is an RPG with classic tabletop style, based on the original board games such as HeroQuest and Dungeons and Dragons. The game offers dice rolling, hero leveling and powerful spells, with a unique mix of strategy, action and role-play, incredible 3d graphics with an epic fantasy story line.
Jul 26, 2016 - OrionArts today announces Petagon: Rise of the Dragonlord 1.1, its twelfth mobile game developed exclusively for iOS devices. Enemies and challenges become increasingly difficult across three realms. Players are able to recruit more powerful allies, turn enemies into friends with benefits, and even fight and tame dragons. Players can expect quality artwork, a storyline with humorous characters, heroes with special skills and dragons that can be tamed as companions.
Jul 25, 2016 - Berlin-based indie developer, David Zobrist today introduces Pixel Mage 1.0, his new retro oriented game title for iOS and Android devices. Pixel Mage is a geniusly simple-to-pick-up platform game with the twist. Players switch between flying or walking instead of jumping, creating 2 lines to switch between as engage battles with goblins. The game surprises with beautiful art, atmospheric levels and high quality voices. The quirky humour makes you grin straight through all 12 levels.
Jul 22, 2016 - Funcity Game Studio today announces SquareStack 1.12, an update to the company's iOS casual puzzle game developed for iPhone, iPad and iPod touch devices. Similar to Hayao Miyazaki's "Castle In The Sky" animation and 3D Tetris, in SquareStack players use three dimensional blocks to build a Tower of Babylon. This easy-to-play game features beautiful animated graphics with more than twelve completely different themes. Version 1.12 offers a much improved user interface and minor bug fixes.
Jul 20, 2016 - UK based Full Fat today releases Golf Island 1.3, an update to its popular game title for iOS and Android devices. Welcome to Golf Island! Join Terry the Toucan on a golfing adventure with tropical islands, cherry topped ice cream mountains, dancing trees and more. The new update brings a new caddy, new Wild West levels, new themed balls, an extended reward ladder along with bug fixes and other minor improvements to gameplay. Grab yer shootin' irons, we're off to the Wild West. Yee-Haw!
Jul 19, 2016 - China-based development team, GameInLife today announces Color Rush 1.0, their colorful and quick-paced arcade game for iOS devices. Color Rush challenges a player's reflexes and alertness by offering a colorful, fast-moving challenge. Stay alert, as the colorful ball travels to the next obstacle. Be sure to tap the correct color button to match the ball's color to that of the upcoming barrier, or it's game over. The challenge increases as the game progresses, so stay at the ready.
Jul 14, 2016 - Texas based developer, Magnin & Associates today introduces Mirror Maze Challenge 1.0, their new game title on iOS, Apple TV, Android, and Windows 10 Phone and PC. Mirror Maze Challenge recreates the excitement of a carnival or boardwalk style glass and mirror maze. Find the exit as quickly as possible. Use the on-screen dPad controller to move forward, backward, turn left or right. Instant replay displays a top-down view showing how efficiently you made you were through the maze.
Jul 14, 2016 - Pterosaur Design today introduces Doppler Ball 1.0, a new retro-style, action arcade game for iOS devices. A modern interpretation of classic paddle-style games from the 1970s and 80s, Doppler Ball challenges the player to use a humming, hyperactive ball to knock down blocks and clear levels. The game features immersive, 3D sound and visual effects that simulates what a game of squash or racquetball might look like if the game ball were moving at close to the speed of light.