text:
John Bannon was much more than just a premier, he was a tenacious runner, clocking up 28 Adelaide marathons among 1979 and 2006, say members of the South Australian Road Runners Club.
Peter Slagter ran the inaugural Gawler to Adelaide marathon with Dr Bannon in 1979 and the two became portion of the so-known as Magnificent Seven, a group of guys competing to run the most Adelaide marathons consecutively.
"Back in 2005, him and I and another chap had been the final three left standing who had carried out all the marathons and the year after it was only John and the other guy," Mr Slagter mentioned.
"John attempted to do that one particular soon after a key surgery that he had, some four weeks soon after the significant surgery and Angela, his wife, and I had been standing on the side lines considering 'John, what the heck are you doing?'
"That just shows you the tenacity of the guy. However he only lasted five ks but he wanted to be the last man standing to have completed all the Adelaide marathons."
The South Australian Road Runners Club has paid tribute to Dr Bannon on social media, saying he holds the equal ideal typical operating time more than 25 Adelaide marathons of three hours and seven minutes.
His very best time was in 1983, when he ran a marathon in two hours and 44 minutes.
"Most of us runners try and strive to do a 3-hour marathon and he has accomplished most of these 28 under three hours so as an athlete he was quite excellent. I do not know how he identified the time to do it," Mr Slagter stated.
He stated that when Dr Bannon hit the road, he was just like any other runner.
"The beauty about this running game is it is a hell of a leveller," he said.
"You could be operating effectively in this case, politicians or the dustman next door or anyone, and John was one of these guys who did not want to be up there.
"He was just a regular guy and you could talk to him on any level."
His old operating mate recalled one instance of this being his choice of operating attire.
"His dress sense when it came to running was constantly a funny issue due to the fact him and his black shorts, it was always a topic of conversation at the coffee shop afterwards simply because John never ever wore the up-marketplace Nike style shorts and this kind of issue," mentioned Mr Slagter.
Dr Bannon was a life member of the South Australian Road Runners Club, and Mr Slagter mentioned he would be sorely missed in the operating community.
"We missed him in the final seven years, due to the fact of his illness he wasn't running. I'm sure I can speak on behalf of the running fraternity that we'll miss him even more now."
Subjects: government-and-politics, states-and-territories, death, neighborhood-and-society, sa, adelaide-5000


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After two lost flights in around four months, and facing a slew of refund requests, it's not clear whether the more than 70 year old Malaysia Airlines can restore its damaged reputation and regain travelers' trust.
On Saturday MAS published a statement offering full refunds to customers who wanted to cancel their tickets in the wake of the MH17 disaster. Customers have until Thursday to cancel or change their tickets without financial penalty.
A 25-year old Singaporean woman, who preferred to remain anonymous, told CNBC she had opted for a refund on her MAS flight from Singapore to Paris in three weeks' time.
Read MoreLive blog: Black boxes handed over to Malaysia
"My personal view is that it wasn't their fault, and it could happen to any airline. But it was mainly my parents who wanted me to change it," she said.
The source added that she wouldn't rule out flying MAS again in the future, however.
"If they are still around...I think I would wait at least eight months to a year first though. Even if I saw a really good deal, I would still wait for a while, mainly for my parents' peace of mind," she added.
Read MoreRussian media on downed airliner: The CIA did it
A spokesperson for MAS was unable to confirm how many passengers had asked for refunds.
Meanwhile other regular flyers told CNBC they would be reluctant to use the airline again.
"One missing flight is unlucky but two is just plain irresponsible... Think I've only flown with them once but I wouldn't be in a rush to book with them anytime soon," said Michelle Perry, an editor based in London, United Kingdom.
Read MoreAfter second tragedy, what now for Malaysia Airlines?
"The second incident is less concerning than the first with regards to safety. Although an utterly horrific and a senseless tragedy you can't blame Malaysia Airlines for being shot down by separatists. However, until MH370 is recovered and the situation explained I would avoid the airline and - to be honest - flying any 777-200ER aircraft," said Sam Wildbore, a British born expatriate who lives in Singapore.
Flight MH17 from Amsterdam to Kuala Lumpur was shot out of the sky by a surface-to-air missile on Thursday, in the airline's second major air disaster this year.
In March, flight MH370 from Kuala Lumpur to Beijing disappeared, in a yet-to-be-solved mystery which captivated the world. There were no survivors from either flight.
Read MoreWhy was Malaysia Airlines MH17 flying over Ukraine? Time, money
MAS began price cuts on its websites on Friday, slashing fares on its Singapore-Frankfurt route by around 500 Singapore dollars ($400) overnight, in a bid to lure passengers.
One source, who preferred to remain anonymous, told CNBC she was considering a MAS flight from Singapore to Paris, which had been cut to 900 Singapore dollars ($719) from more than 2,000 Singapore dollars ($1,599) following the crash last week.
"Malaysia Airlines didn't do anything wrong. Before this year, its safety record was much better than most airlines," the source told CNBC.
Read MoreCondemnation of Russia bad and likely to get worse
"You won't get a cheaper fare to Europe right now. I admit I'm a bit of a risk taker, but I think the main risk here is that MAS might go out of business before I would have had a chance to use the ticket," she added.
Other sources also told CNBC they wouldn't necessarily avoid Malaysia Airlines as a result of the recent incidents.
"I think they've just been unlucky....surely lightening can't strike three times?" said 33-year old Rachel Wolstenholme, who lives in London, U.K..

subset: commoncrawl


text:
Hakalau Surf Forecast and Surf Reports
(Big Island, USA)
Hakalau Spot ID
Photo credit: Devera
Type: river
Reliability: fairly consistent
Best: Swell ENE | Wind WSW
Sea Temperature Today is: 24.7 °C
|Thursday 17
|Wave (m)
|Period (s)
|Wind (km/h)
|Wind State
View detailed 48hrs and full 7 days forecasts
Hakalau in Big Island is a river break that has fairly consistent surf. Winter is the optimum time of year for surfing here. Works best in offshore winds from the west southwest. Most of the surf here comes from groundswells and the best swell direction is from the east northeast. It's sometimes crowded here. Take care of - Rips / undertow - Rocks - Man-made danger (buoys etc.) - Localism - Pollution - Sharks.
Tide Times:
At Hakalau (0 km).
|IS AT (local time)
|high tide
|low tide
Hakalau Tide Times and Tide Chart
Live Weather:
At HAKALAU Hilo wx, 3 km from Hakalau.
|Air temperature
Overall Hakalau Surf Consistency and Wind Distribution in January
The graph shows the combination of swells directed at Hakalau through an average Januar... (show all text)
The rose diagram illustrates how often and how strongly the wind blows from different d... (show all text)
The two graphs above are also combined in the single Hakalau surf consistency graph.
Also see Hakalau surf quality by season and Hakalau surf quality by month.
Surf photos from
the gallery
|Roads & Rivers:
|Select a surf spot from the menu
Use this relief map to navigate to HAW - Big Island surf spots and tide stations that are in the area of Hakalau
Visitor Reviews of Hakalau
"Hakalau Neach closed still due to high levles of Lead metal contamination. Not safe to get in water or touch soil in park or in water."
Ikaika Conner from UNITED STATES - 08 Jan 2019
Other Nearby Surf Breaks closest to Hakalau:(click location name for more details)
- Closest break1 mi
- Second closest break9 mi
- Third closest break10 mi
- Fourth closest break11 mi
- Fifth closest break11 mi
Wave buoys nearest to Hakalau:
- Closest wave buoy13 mi
- Second closest wave buoy114 mi
- Third closest wave buoy135 mi
- Fourth closest wave buoy196 mi
- Fifth closest wave buoy202 mi
Tide stations nearest to Hakalau:
- Closest tide station12 mi
- Second closest tide station24 mi
- Third closest tide station47 mi
- Fourth closest tide station54 mi
- Fifth closest tide station57 mi
Ski Resorts closest to Hakalau:
- Closest ski area2436 mi
- Second closest ski area2436 mi
- Third closest ski area2444 mi
- Fourth closest ski area2453 mi
- Fifth closest ski area2455 mi
- The closest passenger airport to Hakalau is Hilo International Airport (ITO) in Usa Hawaii Isl., 22 km (14 miles) away (directly).
- The second nearest airport to Hakalau is Bradshaw Aaf Airport (BSF), also in Usa Hawaii Isl., 47 km (29 miles) away.
- Third is Waimea Kohala (Kamuela) Airport (MUE) in Usa Hawaii Isl., 58 km (36 miles) away.
- Upolu (Opolu) Airport (UPP) in USA is 87 km (54 miles) away.
- Kona International At Keahole Airport (KOA) in Usa Hawaii Isl. is the fifth nearest airport to Hakalau and is 98 km (61 miles) away.
This is the Surf-Forecast page for Hakalau, HAW - Big Island. Use the tab navigation above to view Hakalau sea temperature, Hakalau photographs, Hakalau detailed swell predictions, wind and weather forecasts, Hakalau webcams, current wind from HAW - Big Island live weather stations and Hakalau tide predictions. The United States swell maps link will open up the big picture showing United States and the surrounding seas. These swell maps can be animated to show the different swell components, wave energy, wave period, wave heights, together with wind and weather forecasts, current weather and sea state observations from United States wave-buoys, passing ships and coastal weather stations. Each of the forecast pages for this surf break feature a global and regional wavefinder tool to locate the best surf conditions in the area around Hakalau.


text:
Getting fresh daily news content onto a website is the bane of all webmasters. It's a lot of work. You can now put Scuttlebutt Europe onto your website with very little effort intially, and zero ongoing effort.
If you have any questions on implementation, feel free to write us:
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text:
Kumkum Bhagya 2nd May 2017 Written Episode Update
Kumkum Bhagya 2nd May 2017 Written Episode Update on teleshowupdates.com
The Episode starts with Nikhil telling Pragya that Tanu asked him to keep eye on her and says you are caught in colored clothes and says you are cheap than us, but you didn't get any advantage. You thought nobody will catch you and thought that you will fool Aaliya and Tanu, but not me. He asks her to thank you for saving him. He asks her to go else he will kick her out. Pragya says I will kick you out and says she will marry only Abhi. She says she is married to Abhi and doing this as he lost his memory. She says Aaliya, Tanu and you can't stop my wedding. Aaliya is coming there and asks some girls to ask Pandit ji to do Abhi's tilak. Dadi says he is thinking again about marriage. Dasi says what will happen to Pragya then. Dadi says he will refuse Tanu, but marry pragya. Dasi says Abhi and Pragya will unite. Abhi says they are getting late. Dasi says they will come. Purab asks him to come. Pragya asks Nikhil not to come inbetween them and says she will not let Tanu marry him. She says Tanu was pregnant with your baby and was giving name to baby of Abhi. She says Tanu is using this.
Nikhil says Tanu is marrying Abhi with my wish and says I am letting her marry him. Pragya tries to emotional blackmail him and says you must have feelings for him. She asks him to do one good thing and says your sins will be washed. Nikhil sees Abhi far and takes Pragya to room. Abhi is sad. Pragya's hand kerchief falls there. He takes her inside. Aaliya comes and stops Abhi from going to room. She asks him to come. Nikhil keeps hand on Pragya's mouth. Aaliya asks what he was talking to Dadi. Abhi says nothing and says it was about marriage. Aaliya says you would have asked Pandit ji and asks him to come. Pragya tries to push him.
Abhi thinks he feels as if Pragya is here. Pragya tries to kick on the door to make noise. Nikhil closes the door. Aaliya asks what happened? Abhi says something had fallen. He sees Pragya's handkerchief there and picks it up. He gets emotional. Pragya asks God to send someone and not to separate her from Abhi. Aaliya asks what happened? Abhi keeps handkerchief in his sherwani and asks Aaliya to come. Dasi tells that Abhi went down. Dadi says marriage will happen now. Purab says Aaliya took Abhi holding his hand. Purab says Aaliya will get them married today. Dadi says this is God's justice. They celebrate without thinking about Pragya.
Nikhil tells Pragya that everything is finished now and tells that all her talks are nonsense, and says if God wanted then Abhi would have been here.. Pragya pushes him and tries to hit him with showpiece. He falls. Pragya is about to go. He pulls the floor mat and makes her fall down. He holds her and pushes her. She picks a vase and is about to hit him. He holds her hand and hits vase on her head making her unconscious. Pragya faints and falls down.
Mrs. Mehta waits for Abhi and Tanu. Mitali says what is going on and tells Tai ji that Abhi will not return. Abhi comes back with Aaliya and sits on the mandap. Aaliya says she will bring the bride now and asks Tanu's mum to make sure that Abhi sits here. She agrees.
Nikhil looks at unconscious Pragya and thinks to check outside. He throws her dupatta and peeps outside the room, sees Dadi, Dasi and Purab. Purab sees someone standing behind the door. Dadi tells that everyone is in the hall. They take Purab. Nikhil thinks to send Pragya to some place from where she couldn't return atleast today. He calls Damru and asks him to reach upstairs. Damru says okay. He hears someone coming.
Aaliya thinks where is Tanu? Nikhil calls her and asks her to come. He takes her inside the room. Nikhil says I want to tell the plan. Aaliya says I am going to go and tells that she will kick him out after marriage. He asks him to listen. He holds her and takes her inside. Aaliya slaps him and asks how dare you touch me. Nikhil says I have no interest in you. Aaliya says how marriage will happen without Tanu. I am not getting Tanu. Nikhil tells him that bride is here. Aaliya sees Pragya and is shocked.
Kumkum Bhagya 3rd May 2017 Written Episode Update Precap : Aaliya asks Nikhil to tell his goons to take Pragya somewhere. Purab asks Dadi where is Pragya? Aaliya comes to washroom and sees Tanu unconscious.
Kumkum Bhagya Details
Kumkum Bhagya is a Hindi-language Indian soap opera which premiered on April 15, 2014 and is broadcast on weeknights on Zee TV. The show is losely based on the novel Sense and Sensibility by Jane Austen.
Seasons : 1 | Channel : Zee TV| Picture Format : 576i , 1080i | Run Time : 24 Min


text:
Vernee Thor's Screen Compared With the iPhone 6S (Video)
by Mar 29, 2016 1:22 am0
Vernee is marketing its mid-range Thor smartphone pretty aggressively. The company will release the phone sometime in April, which is just a few weeks away. Today, it released a new video comparing the $129.99 Vernee Thor with the iPhone 6S, claiming that its phone delivers a stellar display performance.
In this regard, Vernee released an on-screen comparison of the devices, as well as an off-screen comparison to show the performance of its upcoming handset.
You can check out the video below for the on-screen comparison between the Thor and the iPhone 6S. In the video, the phone does pretty well, coming with higher color saturation and warmer tones. The video tests different colors, dark and light shades, viewing angles and a number of other parameters.
Coming to the off-screen test, Vernee Thor is pitted against the iPhone 6S and the Moto X flagships. The company seems to have performed a thorough off screen analysis of the three phones, showing that the Vernee Thor does a good job in the comparison.
Here's the official comparison released by the company:
Off-screen Comparison:
Scenario 1:
Crosswise comparison in highlight environment:
With screen reflection totally avoided, we let the 3 devices display the unobservable off-screen integration.
We can see from the close shot that iPhone 6s has the best off-screen integration, Vernee Thor comes second, and the Moto X the last.
Scenario 2:
360°light environment, symmetrical contrast with equal light projecting on each device
From top to bottom: Moto X, Vernee Thor, iPhone 6S
In this scenario, Vernee Thor and iPhone 6s have almost the same off-screen integration, and Moto X lags much behind.
Scenario 3:
With light projection on the top of the three 2.5D screens, iPhone 6s and Vernee Thor both win also in terms of viewing effects.
From top to bottom: Vernee Thor, iPhone 6S, Moto X
Scenario 4:
Single-side highlights projecting equally on the three devices, and the result is obvious.
From top to bottom: iPhone 6S , Vernee Thor, Moto X
From top to bottom:Moto X, Vernee Thor, iPhone 6S
From top to bottom:Moto X, iPhone 6S, Vernee Thor
So, what do you think about the Vernee Thor? It costs $129.99 and comes with the following specs:
- MT6753 8-core LTE chipset
- 5.0 inch on-cell HD arc display
- 5.0mp front camera
- 13.0mp rear camera
- 2800mAh with 5V2A quick charge
Buy Links:


text:
Write some software
Budget $750-1500 CAD
I need you to develop some software for me. I would like this software to be developed for Windows using C or C++. We need a animated screensaver done in Unity 3d that would be a canyon with a camera fly through.
There will be a clock displaying day month and time. It will be designed as a installer for PC and android phone. It will have a reminder function and will play local radio or playlist to be set in options.
13 freelancers are bidding on average $1281 for this job
Dear Employer! I want to work for your project. I'm very interested in your project. I'd like you to call me on chat. Please give me your project detail. If you give me the task, I'm very glad with you. I'll give you More
I have read your proposal carefully and I have the confident that I will complete project on time. As you can check my reviews and portfolio on my profile page, I am passionate developer that have the strongest skills More
Hello, My name is Dragoș M. and I represent IGID Studio, a game and software development studio based in Bucharest, Romania. I have a team of professional developers and designers ready to deliver high-end 3D and More
[login to view URL] [login to view URL] Hello Sir/Madam I am very happy to meet your project on my skill. I am a senior unity developer with 5+ years history and have the full expe More
Ready to start the work to develope the 3d screensaver unity 3d ,we can discuss more over chat,thanks regards Arjun S.
[login to view URL] (puzzle game) [login to view URL] (Subway Runner) Dear! How are you? I have carefully r More
Dear hiring manager, I am a professional C/C++ expert who you are looking for. I have 10 years experience in C/C++ Programming and mastered in there. I am also good at Unity 3D. I read your job description carefully an More


text:
Route from Homs to As Sawram
The following map shows the route from Homs to As Sawram.
For more detailed information about any of the places please click on the place name.
• Homs
• As Sawram
Map optionsCurrent route: Homs -> As Sawram
Show reverse route: As Sawram -> Homs
DistanceRoute by foot
Please note: Some route options are not available.


text:
You probably already know that Julia gave birth to her puppies earlier in July. What you might not know is these puppies now have names. Meet Prague, Bohemia, Moravia, Jana, and Garnet—the Czech puppies!
This litter theme and names were chosen by Julia's breeder caretakers, Susan and Bill. They recently returned from a trip to the Czech Republic, where they were visiting their friends Robert, Jana, Katrina, and Christina.
The pups were actually born while they were overseas. The puppies were whelped in the Bohenick family's home, and they lived there until Bill and Susan returned. Thank you to the Bohenick family for taking care of Julia and the puppies during their first days!
SSD Garnet was named after the garnets that are mined in the Czech Republic. She's wearing a purple collar. If you look closely, you'll see the garnet ring on her toes.
SSD Bohemia is named after one of the large general regions in the Czech Republic. In this picture, he's posing with a postcard of a town and castle that Susan and Bill visited during their visit. Bohemia, a male pup, is wearing a black collar.
SSD Moravia is named after another of the large general regions in the Czech Republic. The family that breeder caretakers, Susan and Bill, visited lives in Moravia. The puppy Moravia is a male and is wearing a red collar.
SSD Jana is named after Susan and Bill's friend Jana (pronounced Yah-na). The pup is wearing a pink collar.
SSD Prague is, of course, named after the capital city, which has a rich history. In this photo, he is posing next to a book about that history. He's wearing a yellow collar.
In case you're wondering, the basket that all five puppies are lying in was made in the Moravian region out of grass. The pink baby blanket comes from a friend.


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Muscular Dystrophy NSW
Muscular Dystrophy is a devastating neuromuscular disorder, which results in the progressive deterioration of muscle strength and function. It affects 24,000 people in Australia and is more prevalent than Cystic Fibrosis, Huntington's Disease and Tay Sachs Disease combined. The most common form is Duchenne Muscular Dystrophy, which primarily affects boys. Onset of this condition is between 3 and 5 years of age and the disorder progresses rapidly with most boys completely dependent on an electric wheelchair by their early teens. Life expectancy for this disorder is usually only until their early thirties.
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text:
Patience Is a Virtue in Technology Development
From Cotton Grower Magazine – June 2014
There's no denying it. Technological advances in seed traits and chemistries have done wonders for production agriculture over the last two decades.
It was only 20 years ago, in 1994, that the first genetically modified crop was commercialized. Since that time the concept has been rapidly adopted, becoming the preferred method of production agriculture in the United States. The end result has been significantly improved yield across the board.
But that doesn't mean the process of bringing forth a new piece of trait technology is any easier today than it was 20 years ago. In fact, if a new trait were conceived and invented today, representatives at leading trait development companies say it would be at least another decade before farmers could see the product sold commercially. Often, half of those years are spent testing genetic traits in-house, before they are ever submitted for review to agencies like USDA and EPA.
"Initial work is done in our Discovery Groups – phase one and phase two. That takes approximately five years," says Marianne Malven, Monsanto's molecular characterization team lead in the regulatory group. "And then regulatory analysis and submissions are averaging approximately six years. So yes, it takes quite a while. And I think 10 years is probably a conservative time frame."
Through the years, Malven has been heavily involved in product testing and regulation management at Monsanto – most recently with XtendFlex technology. In cotton, the product will be featured in varieties which are tolerant to dicamba, glyphosate and glufosinate. She says her company leaves no stone unturned when testing trait products in-house.
"We're looking at the gene and the protein," Malven says. "We're looking for efficacy, we're looking for safety. We're making sure that there is a history of being exposed to this gene or protein previously. It's making sure that the event that goes on for further testing is going to be safe."
As of press time, XtendFlex was under review of a USDA environmental impact statement, although company representatives say they expect to receive approval to commercialize the product in time for the 2015 production season.
USDA won't be the only regulatory organization Monsanto must satisfy with the company's new technology. Key U.S. trade partners are granted authority to regulate products that may ultimately be consumed within their own borders. This is how American consumers occasionally hear of agriculturally-specific products being held up by regulators in, for instance, Japan.
"For cotton, the critical import countries are Mexico, Canada, Japan and others," Malven says. "We still make submissions to all countries that have functioning regulatory processes in place. It's just that we must have approval from those critical import countries before we can launch."
To ease the regulatory process, companies like Monsanto have on-site staff to handle regulatory requests within the borders of the critical import countries. Oftentimes, these foreign regulatory processes are going on simultaneously with USDA and EPA regulatory reviews.
Recently, researchers like Malven have begun advocating for a uniform set of regulatory requirements from each of the foreign agencies from which they seek approval. The process of satisfying multiple, differing sets of requirement regulations can take a significant toll on time and resources, they say. Genetic researchers know that standardizing international regulations could take some time, however.
"We are speaking with different country regulators to about standardization of regulations across countries," Malven says. "I don't know if it will happen, but it would benefit farmers."


text:
Ham on the Sunday menu?
If a ready-cooked ham is on your Easter menu, here are three resources.
Don't know how to carve a ham? Check out the videos produced by Jones Dairy Farm's president Philip Jones at jonesdairyfarm.com/aboutus/videos.aspx.
For money saving coupons on HoneyBaked ham, log on to honeybaked.com and click on the Promotions tab.
Penn Dutch, which has stores in Hollywood (3950 N. 28th Terrace, 954-921-7144) and Margate (3201 N. State Road 7, 954-974-3900) smokes all of its own hams in its Hollywood facility. Visit Penn-dutch.com
John Tanasychuk


text:
21 February 2017
Despite of this, Israel is always apprehensive whenever Iran goes for military drills or missile test. Every country follow the same practice as Iran is following. According to the details, the Islamic Revolution Guards Council has recently conducted military maneuvers by the title of ' The Holy Prophet 11'. Different models of state of the art Smart Guided Missiles have been tested.
The drills exhibited the strength of the defence capability that the Islamic Republic is equipped with; Different units of the ground forces have displayed the military might in its first level. In other levels, the intelligence capabilities will also be displayed as the drill continues.


text:
[Skip to content]
The re-covering of roofs with tiles is seen under the regulations as the replacement or renovation of a thermal element that requires the submission of an application under the Building Regulations.
In addition if tiles that are either heavier or lighter than the originals are used, there may be structural implications requiring the installation of additional roof members.
Contact details
Telephone 01202 633250
Text Relay 18001 01202 633250
Address Building ConsultancyRoom 206Civic CentrePooleBH15 2RU


text:
Pulmonary tuberculosis (TB), caused by the intracellular bacteria Mycobacterium tuberculosis, is a worldwide disease that continues to kill more than 1.5 million people every year worldwide. The accumulation of lymphocytes mediates the formation of the tubercle granuloma in the lung and is crucial for host protection against M.tuberculosis infection. However, paradoxically the tubercle granuloma is also the basis for the immunopathology associated with the disease and very little is known about the regulatory mechanisms that constrain the inflammation associated with the granulomas. Lipocalin 2 (Lcn2) is a member of the lipocalin family of proteins and binds to bacterial siderophores thereby sequestering iron required for bacterial growth. Thus far, it is not known whether Lcn2 plays a role in the inflammatory response to mycobacterial pulmonary infections. In the present study, using models of acute and chronic mycobacterial pulmonary infections, we reveal a novel role for Lcn2 in constraining T cell lymphocytic accumulation and inflammation by inhibiting inflammatory chemokines, such as CXCL9. In contrast, Lcn2 promotes neutrophil recruitment during mycobacterial pulmonary infection, by inducing G-CSF and KC in alveolar macrophages. Importantly, despite a common role for Lcn2 in regulating chemokines during mycobacterial pulmonary infections, Lcn2 deficient mice are more susceptible to acute M.bovis BCG, but not low dose M.tuberculosis pulmonary infection.
Citation: Guglani L, Gopal R, Rangel-Moreno J, Junecko BF, Lin Y, Berger T, et al. (2012) Lipocalin 2 Regulates Inflammation during Pulmonary Mycobacterial Infections. PLoS ONE 7(11): e50052. https://doi.org/10.1371/journal.pone.0050052
Editor: Bernhard Ryffel, French National Centre for Scientific Research, France
Received: June 18, 2012; Accepted: October 15, 2012; Published: November 20, 2012
Copyright: © 2012 Guglani et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by funds from Children's Hospital of Pittsburgh to SAK, and a Research Advisory Committee Grant from Children's Hospital of Pittsburgh of the University of Pittsburgh Medical Center Health System to YL. It was also supported by grants from National Institute of Health, USA, A1083541 and HL105427-01 to SAK. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Pulmonary tuberculosis (TB), caused by the intracellular bacteria Mycobacterium tuberculosis, is a widespread disease that continues to kills more than 1.5 million people every year worldwide. Furthermore, approximately one-third of the world's population is infected with M.tuberculosis and about 5–10% of these individuals will develop clinical disease over their lifetime. Induction of inflammatory molecules in response to infection mediates cellular accumulation, formation of the tubercle granuloma and host protection against M.tuberculosis infection. However, the generation of the tubercle granuloma is also thought to be the basis for the immunopathology associated with the disease. Although it is known that the granuloma is dependent on the recruitment and localization of lymphocytes [1] and that both inflammatory chemokines [2], [3], [4], [5] and homeostatic chemokines [1], are involved in cellular accumulation, very little is known about the regulatory mechanisms that restrain chemokines and regulate inflammation associated with TB granuloma formation.
Lipocalin 2 (Lcn2), also known as neutrophil gelatinase-associated lipocalin, siderocalin, 24p3 or uterocalin is a member of the lipocalin family of proteins and possesses a barrel shaped tertiary structure with a hydrophobic pocket that can bind lipophilic molecules [6]. The best-characterized role of Lcn2 is its ability to bind to siderophores that are made by pathogenic bacteria, such as Escherichia coli, and its resultant ability to restrict bacterial growth [7], [8], [9]. Accordingly, Lcn2 gene deficient mice (Lcn2KO) exhibit higher mortality rates than control mice when challenged with pathogenic E.coli [7], [8], [9]. Furthermore, Lcn2 is an acute phase protein, as it is induced under inflammatory conditions [7], [10] and is implicated in control of cell growth and differentiation, tissue involution and apoptosis [7], [11], [12]. Accordingly, high Lcn2 levels have been associated with malignancy in various cancers [13], [14], [15].
Recently it has been documented that Lcn2 can also bind to soluble siderophores of mycobacteria, namely carboxymycobactins [16], and that Lcn2 can inhibit the in vitro growth of M.tuberculosis [17], [18] as well as the intracellular replication of M.tuberculosis in cultured macrophage cell lines [19]. Furthermore, Lcn2 can also inhibit the growth of attenuated mycobacterium strain, M.bovis Bacille Calmette Guerin (M.bovis BCG), in cultured murine alveolar epithelial cells [18]. In addition, in response to acute intratracheal high dose infection with virulent M.tuberculosis H37Rv, mice deficient in Lcn2 are more susceptible than wild type (WT) infected mice [18]. However, it is not known whether Lcn2 plays a role following a more physiological exposure to M.tuberculosis, such as pulmonary infection with a low dose of aerosolized M.tuberculosis. Moreover, since the expression of Lcn2 is limited to mucosal tissues, such as the trachea, lung, stomach, salivary gland and colon [20], it is likely that Lcn2 plays an inflammatory role at mucosal surfaces. In this study, we reveal a new role for Lcn2 in regulating lung inflammation and T cell accumulation during mycobacterial pulmonary infections, by restraining CXCL9 induction. Accordingly, absence of Lcn2 results in enhanced induction of lung CXCL9 mRNA and increased inflammation following both acute M.bovis BCG pulmonary infection and low dose chronic pulmonary M.tuberculosis infection. Importantly, despite a common role for Lcn2 in regulating CXCL9 expression during mycobacterial pulmonary infections, Lcn2KO mice are more susceptible to acute M.bovis BCG pulmonary infection, but not low dose M.tuberculosis pulmonary infection.
Materials and Methods
C57BL/6 (WT) mice were purchased from Taconic Farms, Inc. (Hudson, NY) and Lcn2KO mice [21] were obtained from Dr. Tak W Mak, University Health Network, Toronto, Canada and were bred and maintained at Taconic Farms. The mice used for all the experiments were age- and sex-matched and were infected between the ages of 6 to 8 weeks in accordance with the University of Pittsburgh IACUC guidelines under protocol 1106203.
Experimental M.tuberculosis and M.bovis BCG Infection
M.tuberculosis H37Rv strain or M.bovis BCG was grown in Proskauer Beck medium containing 0.05% Tween 80 to mid-log phase and frozen in 1 ml aliquots at –70°C. For aerosol infections with M.tuberculosis, the animals were infected with ∼100 bacteria using a Glas-Col (Terre Haute, IN) airborne infection system as described before [22]. In some experiments, mice were intratracheally infected with 5×106 M.bovis BCG in 50 µl of PBS as reported before [23]. All experiments included 4–5 mice and were repeated atleast twice independently.
Determination of Bacterial Load
Mice were euthanized by CO2 asphyxiation, and the organs were excised and individually homogenized in 0.9% physiological saline, and serial dilutions of the organ homogenates were plated on nutrient 7H11 agar. Bacterial colony formation was counted after 3 wk of incubation at 37°C and Log 10 CFU per organ calculated.
Preparation of Single Cell Suspensions
Lung cell suspensions were prepared by perfusing cold heparinized saline injected through the right side of the heart of the dissected animals until the lungs appeared white [24]. The lobes of the lungs were removed and placed in ice-cold DMEM (Mediatech-Cellgro) followed by sectioning using sterile razor blades. To digest the collagen matrix, the lung tissue samples were then incubated in DMEM containing collagenase IX (0.7 mg/ml; Sigma-Aldrich) and DNase (30µg/ml; Sigma-Aldrich) at 37°C for 30 min [22]. Following this treatment, the digested lung tissue was gently passed through a 70-µm pore size nylon tissue strainer (Falcon; BD Biosciences). The resultant single-cell suspension of all organs was treated with Gey's solution to remove any residual RBCs, washed twice, and counted. Cells from this single cell suspension were used for flow cytometry analyses.
Immunohistochemistry and Morphometric Analysis
Lung lobes were instilled with 10% neutral buffered formalin and embedded in paraffin. Lung sections were stained with hematoxylin and eosin and inflammatory features were evaluated by light microscopy (Research Histology Core, University of Pittsburgh). For immunofluorescent staining, formalin-fixed, lung sections were cut, immersed in xylene to remove paraffin and then hydrated in alcohol, 96% alcohol and PBS. Antigens were unmasked with a DakoCytomation Target Retrieval Solution and non-specific binding was blocked with 5% (v/v) normal donkey serum and Fc block (BD Pharmingen, San Diego, CA). Endogenous biotin (Sigma Aldrich) was neutralized by adding first avidin, followed by incubation with biotin. Sections were probed with Biotinylated rat anti Gr-1 (BD Pharmingen) to detect neutrophils, anti B220 to detect B cells (Clone RA3-6B2, BD Pharmingen. San Diego, CA) and anti CD3 to detect T cells (Clone M-20, Santa Cruz Biotechnology. Santa Cruz, CA) in the inflammatory lesions. Primary antibodies were visualized with a secondary antibody, donkey-anti-rat, conjugated to Alexa-Fluor 488 (Invitrogen, Carlsbad, CA). Slow fade gold antifade with DAPI (Molecular probes, Eugene, OR) was used to counterstain tissues and to detect nuclei. Images were obtained with a Zeiss Axioplan 2 microscope and were recorded with a Zeiss AxioCam digital camera. Caudal lobes underwent morphometric analysis in a blinded manner. The area occupied by cellular infiltrates was determined with the automated, morphometric tool of the Zeiss Axioplan microscope (Zeiss), which determines the area defined by the squared micron value for each granuloma measured, as previously described by us [1], [25].
In Situ Hybridization
Mouse Lcn2 cDNA was RT-PCR amplified with primers BF.mLcn2_F1 (5′- ACCTAGTAGCTGTGGAAACC-3′) and BF.mLcn2_R1 (5′-TCAGCCACA CTCACCACCCA-3′) using reverse transcribed total RNA from cultured murine splenocytes. PCR products were ligated to the pGEM-T vector (Promega) and DNA sequenced. The pGEMT-Lcn2 plasmid was linearized by restriction digest. Gene-specific riboprobes were synthesized by in vitro transcription using a Maxiscript SP6/T7 kit (Ambion) and unincorporated nucleotides were removed using RNA Mini Quick Spin Columns (Roche). Paraffin embedded tissue specimens were pretreated as described [26], following deparaffinization in xylenes and rinsing in ethanol. In situ hybridization with 35S-labeled riboprobes against Lcn2 was performed at 50oC overnight with 0.1M dithiothreitol included in the hybridization mix as described [27]. Tissue sections were coated with NTB-2 emulsion (Kodak) and exposed at 10oC for 7 days. The sections were counterstained with Hematoxylin (Vector) and mounted with Permount (Fisher). Microscopic fields were visualized using a Olympus BX41 microscope and captured using a SPOT RT3 digital camera (Diagnostics Instruments, Inc). CXCL9 mRNA was detected as previously described [26].
Flow Cytometry
Single cell suspensions were stained with fluorochrome-labeled antibodies specific for CD3 (17A2), Gr1 (RB6-8C5) and CD11b (M1/70). For intracellular staining, cells stimulated with Phorbol myristate acetate (50ng/ml), ionomycin (750 ng/ml; Sigma Aldrich) and Golgistop (BD Pharmingen), were surface stained, permeabilized with Cytofix/Cytoperm solution (BD Pharmingen) and stained with anti-IFNγ (XMG1.2).
Real Time PCR (RT-PCR)
Lung tissue from infected and control mice was homogenized and frozen in RLT buffer (Qiagen Inc, Valencia CA). Extraction of RNA was done as per protocol, followed by reverse transcription and amplification using FAM-labeled probe and primers on the ABI Prism 7400HT detection system (Applied Biosystems, Foster City CA). Calculation of ΔΔct was done from the fold increase in signal from infected samples over that of uninfected samples. The primer and probes sequences have been previously published [1], [24] or were purchased (Applied Biosystems, Foster City CA).
Isolation of Alveolar Macrophages, Fibroblasts and Tracheal Epithelial Cells
Alveolar Macrophages were obtained from mice by performing bronchoalveolar lavage with 0.2mM EDTA solution. Mice were dissected to expose their trachea and cannulated with a blunt end 18 gauge needle. Six lavages were performed per mouse with 1ml volume per lavage. The cells were all pooled, spun, resuspended in Minimum Essential Media (MEM, Gibco #11095-080) with 1% Penicillin-Streptomycin, and 10% Fetal Bovine Serum, counted and plated in 96 well plates which were incubated for 1 hour at 37°C, following which the wells were washed to remove non-adherent cells. The adherent alveolar macrophages were then rested overnight before being used in experiments.
Mouse tracheal epithelial cells (MTEC) were generated using the air liquid interface as described before [26]. Mouse tracheas were placed in 0.15% Pronase solution, tracheal cells removed with DNase, followed by incubation in MTEC Basic media with 10% FBS. The cells in supernatant were resuspended in MTEC Plus media and then added to collagen coated transwell membranes (Corning Costar #3460), followed by change of media on days 3, 5 and 6 in both apical and basal compartments. The resistance of cells to electric current was checked on days 5, 6 and 7 and cells were switched to air-liquid interface once a resistance of 800–1000 ohms was reached. At this point, media was removed from apical compartment and media from basal compartment was replaced with 2% NuSerum (BD Biosciences #355100) with Retinoic Acid (Sigma R-2625). For generation of lung fibroblast, lungs were perfused with 5 units/ml dispase followed by 1% low melting agarose, washed in PBS and incubated in dispase for 30 minutes at 37oC. Single cell suspensions were then obtained from DNase/Collagenase treated lungs and fibroblasts generated by passaging 2–4 times. Recombinant Lcn2 was made as described [21] and used at a concentration of 10 ug/ml for all in vitro experiments. The endotoxin levels were tested in preparations of recombinant Lcn2 and found to be low (12 ng/ml total protein preparation).
Determination of Protein Levels
Protein levels of cytokines and chemokines were measured in cell culture supernatants or lung homogenates using a mouse Luminex assay (Linco/Millipore). Lipocalin 2 protein was measured as previously described [21]. Briefly, Ab sandwich ELISA was performed per standard protocol by coating 96-well plates with affinity-purified anti-mouse lipocalin 2, application of 100 µl of diluted protein lysate, detection with monoclonal rat anti-mouse lipocalin 2 (R&D Systems), and HRP-conjugated goat anti-rat IgG (Invitrogen), followed by colorimetric development using a 3,3′,5,5′-tetramethylbenzidine substrate reagent set (BD Biosciences).
Statistical Analysis
Comparison of the means between the uninfected and infected sample groups was performed with the two tailed Student's t-test using GraphPad Prism 5 software (La Jolla, CA). Differences were considered significant at p≤0.05. The data from bacterial growth and RT-PCR were log-transformed for statistical analyses.
Lcn2 Expression is Induced in the Lungs in Response to Low Dose Aerosol M.tuberculosis Infection
Lcn2 gene expression is induced in the lung in response to M.bovis BCG acute pulmonary infection [18]. However, M.bovis BCG is an attenuated strain of mycobacterium and it is not known whether Lcn2 is induced in the lung in response to exposure to pathogenic mycobacterium such as M.tuberculosis H37Rv. Therefore, we infected wild type mice with low dose aerosolized M.tuberculosis H37Rv and found that Lcn2 mRNA was induced between day 15 and day 21 post-M.tuberculosis infection and the mRNA levels were maintained at later time points (Figure 1a). Furthermore, the timing of induction of Lcn2 mRNA between days 15–21 coincided with the induction of other protective host immune genes, such as IFNγ and TNFα, in the lungs of M.tuberculosis-infected mice [28]. In addition, we found significant induction of Lcn2 protein in day 30 WT Mtb-infected lungs (Figure 1b).
Wild type (WT) mice were infected with ∼100 CFU M.tuberculosis via the aerosol route and at specific times after infection, lung tissue was harvested and processed to extract RNA and the expression of Lcn2 mRNA was determined by RT-PCR. Log 10 fold induction of Lcn2 gene in the lungs of M.tuberculosis-infected mice over lungs from uninfected controls is shown (a). WT M.tuberculosis-infected lungs (Mtb) on day 30 post infection or uninfected lungs (Un) were assayed for Lcn2 protein by ELISA (b). In situ hybridization with a Lcn2 cRNA probe was carried out in uninfected and M.tuberculosis-infected lung samples at various time points post infection (c). Original magnification. 100X-left panel; 200X-rightpanel). Lung fibroblasts, alveolar macrophages and tracheal epithelial cells were generated from WT lungs and cells were exposed to irradiated M.tuberculosis (100 µg/ml) for 48 hours. The induction of Lcn2 mRNA was measured by RT-PCR and the log 10 fold induction was measured in treated samples relative to untreated controls (d). The data points represent the mean and SD for four mice for each time point and one experiment representative of two shown. ***, p ≤0.0005.
Epithelial cells and macrophages are considered to be one of the major sources of Lcn2 expression [18], [26]. Interestingly, following M.tuberculosis infection, the early induction of Lcn2 mRNA (day 12–15) was detected within the lung parenchyma consistent with type II epithelial cells and macrophages, whereas late expression of Lcn2 mRNA (day 21) was detected both within the lung parenchyma and the airways of the M.tuberculosis-infected lungs (Figure 1c). These data suggest that multiple cell types can likely produce Lcn2 in response to M.tuberculosis infection. Accordingly, we found that following M.tuberculosis exposure, lung fibroblasts and alveolar macrophages produced high levels of Lcn2 mRNA, while Mouse Tracheal Epithelial Cells (MTECs) induced lower levels of Lcn2 mRNA (Figure 1d). IL-23 is a key mediator of IL-17 and IL-22 responses [29], [30], [31], and IL-22 and IL-17 can induce Lcn2 expression in lung epithelial cells and participate in protection against gram-negative bacteria [26]. However, we found that Lcn2 mRNA induction was not significantly different among wild type, IL-23KO, IL-22KO and IL-17AKO M.tuberculosis-infected lungs (data not shown). These data together suggest that following low dose pulmonary infection with M.tuberculosis, Lcn2 mRNA is induced both within the lung parenchyma and epithelium, and that several lung cell types can produce Lcn2 in response to M.tuberculosis exposure.
Lcn2 is not Required for Protective Immunity Against Primary Low Dose M.tuberculosis Aerosol Infection
To determine whether Lcn2 was required for protective immunity during low dose M.tuberculosis infection, we assessed the bacterial burden in M.tuberculosis-infected wild type (WT) and Lcn2 knock out (Lcn2KO) mice and found that the total lung bacterial burdens in Lcn2KO and WT mice were similar (Figure 2a). In addition, dissemination to the peripheral organs was also similar, since bacterial burdens in spleen and lymph node were comparable between WT and Lcn2KO mice on day 50 post infection (Figure 2b). These results were consistent with the comparable accumulation of IFNγ producing CD4+ T cells (Figure 2c) in M.tuberculosis-infected lungs and similar induction of iNOS mRNA (Figure 2d) in WT and Lcn2KO lungs. These data suggest that even in the absence of Lcn2, IFNγ T cell responses are generated effectively to induce macrophage activation that can mediate protective immunity against M.tuberculosis infection.
WT and Lcn2KO mice were infected with low dose aerosolized M.tuberculosis and the bacterial burden determined in the lungs at different time points post infection (a) and spleens and mediastinal lymph nodes (MLN) on day 50 post infection (b). Lymphocytes were isolated from the lung and the number of CD4+ IFNγ+ producing T cells were determined by intra cellular staining and flow cytometry following stimulation with PMA and Ionomycin (c). Lung from uninfected and infected WT and Lcn2KO mice were processed and the Log 10 induction of iNOS mRNA in infected samples relative to uninfected control samples was determined by RT-PCR (d). The data points represent the mean and SD for four mice for each time point and one experiment representative of two shown. ns-not significant.
Lcn2 Regulates Pulmonary Inflammation during TB
TB is an inflammatory disease of the lung and the formation of the granuloma is a hallmark of the disease. Therefore, despite Lcn2 not being required for overall bacterial control against Mtb infection (Figure 2), we next examined if Lcn2 had a role in granuloma formation. We quantified the average size of the lung granulomas in WT and Lcn2KO M.tuberculosis-infected mice by histology. At early stages of the infection, we found that the average size of granulomas were similar in WT and Lcn2KO mice (Figure 3a). However, later during chronic M.tuberculosis infection, we found the average size of granulomas were significantly increased in the Lcn2KO mice when compared to granulomas found in lungs of M.tuberculosis-infected WT mice (Figure 3a). To further validate the role for Lcn2 in inflammation during M.tuberculosis infection, we compared the T cell accumulation in response to pulmonary M.tuberculosis infection in WT and Lcn2KO M.tuberculosis-infected lungs. Despite similar numbers of lung T cells in WT and Lcn2KO mice at early time points, the T cell numbers were significantly increased in day 50 M.tuberculosis-infected Lcn2KO lungs when compared to WT M.tuberculosis-infected lungs (Figure 3b). This was consistent with higher accumulation of T cells histologically within granulomas of Lcn2KO M.tuberculosis-infected lungs (Figure 3c). These data suggest a novel function for Lcn2 in regulating lymphocytic recruitment and granuloma formation during pulmonary TB.
WT and Lcn2KO mice were infected with low dose aerosolized M.tuberculosis and formalin-fixed paraffin-embedded lungs were stained using H&E. Morphometric analysis of the average size of the granuloma in H&E stained sections of M.tuberculosis-infected WT and Lcn2KO lungs was determined and a representative section of the lung granuloma is shown for D20 and D50 post M.tuberculosis infection (a). Original magnification shown-100X. Lymphocytes were isolated from the lungs of WT and Lcn2KO M.tuberculosis-infected mice at day 20 and day 50 post infection and the number of CD3+ T cells determined by surface staining and flow cytometry (b). Lung sections from M.tuberculosis-infected WT and Lcn2KO mice at D50 post infection were stained for B220 (green) and CD3 (red) and shown at 200X magnification (c). Formalin-fixed, paraffin-embedded lung sections from WT and Lcn2KO from day 50 M.tuberculosis-infected mice were assayed for CXCL9 mRNA localization by ISH using a murine CXCL9 mRNA probe or control probe (d) and total lung CXCL9 mRNA levels induced quantitated using RT-PCR in day 20 (D20) or day 50 (D50) M.tuberculosis-infected mice (e). Original magnification-100X (d). The data points represent the mean and SD for four mice for each time point and one experiment representative of two shown. *, p ≤0.05. ***, p ≤0.0005.
Inflammatory chemokines such as CXCL9,10 and 11 [2] and CCL2 [3], [4], [5] are critical for lymphocytic recruitment and mediating granuloma formation during TB. Logically, mice deficient in these chemokines or their receptors also have decreased lymphocytic recruitment and decreased granulomatous inflammation when compared to WT mice [2], [3], [4], [5]. Therefore, we hypothesized that Lcn2 may limit expression of some CXCR3-ligating chemokines and restrain lymphocytic accumulation and inflammation during TB. Consistent with this hypothesis, we found increased localization of mRNA for one of the CXCR-3 ligating chemokines, namely CXCL9 mRNA, within granulomas of M.tuberculosis-infected Lcn2KO mice when compared to WT M.tuberculosis-infected lungs (Figure 3d). In addition, we found that increased inflammation during chronic infection coincided with increased induction of total CXCL-9 mRNA levels in Lcn2KO M.tuberculosis-infected lungs when compared to WT infected lungs (Figure 3e). However, we did not find any differences in total CXCL10 mRNA or localization within the granulomas in WT and Lcn2KO M.tuberculosis-infected lungs during both early and chronic tuberculosis (data not shown).These novel data suggest that Lcn2 regulates some lymphocyte recruiting chemokines such as CXCL9, to constrain lymphocytic infiltration and granuloma formation during TB.
Lcn2 Promotes Production of Neutrophil-attracting Chemokines and Neutrophil Recruitment during Pulmonary TB
Our data show that Lcn2 has a regulatory role in induction of lymphocyte-attracting chemokines and that absence of Lcn2 during M.tuberculosis infection results in enhanced T cell recruitment and increased granuloma inflammation. Paradoxically, we also found, upon immunofluorescent staining for neutrophilic accumulation in the lungs, that Lcn2KO mice had significantly reduced neutrophilic influx compared to WT mice (Figure 4a, b). This also coincided with decreased total numbers of neutrophils in Lcn2KO M.tuberculosis-infected lungs compared to WT M.tuberculosis-infected lungs (Figure 4c). These data suggest that, apart from its regulatory role in cellular inflammation, Lcn2 may promote neutrophil generation and recruitment. Alveolar macrophages are likely first-responders to infection that would produce these molecules. We show that following exposure to M.tuberculosis, alveolar macrophages produced G-CSF, a key regulator of neutrophil production (Figure 4d) and KC, a neutrophil chemoattractant (Figure 4e). Interestingly, Lcn2 treatment alone is sufficient to induce neutrophil chemoattractant, KC in alveolar macrophages (Figure 4e). Addition of exogenous Lcn2 to M.tuberculosis exposed alveolar macrophages resulted in significantly higher induction of G-CSF and KC than M.tuberculosis alone. Since M.tuberculosis exposure induces Lcn2 expression in alveolar macrophages (Figure 1d), we then tested whether the induction of G-CSF and KC was dependent on endogenous Lcn2 expression in alveolar macrophages. Therefore, we exposed WT or Lcn2KO alveolar macrophages to M.tuberculosis and measured G-CSF and KC levels in culture supernatants. We show that in alveolar macrophages, M.tuberculosis-induced G-CSF and KC production is dependent on endogenous production of Lcn2, since alveolar macrophages derived from Lcn2KO mice induced significantly lower levels of these molecules. In addition, exogenous Lcn2 was able to rescue induction of both these proteins in alveolar macrophages (Figure 4f & 4g). These data suggest that M.tuberculosis-induced G-CSF and KC production in alveolar macrophages is partly dependent on endogenous Lcn2 production, but that Lcn2 made by other cells, such as epithelial cells and fibroblasts (Figure 1d), may amplify the response.
WT and Lcn2KO were infected with low dose aerosolized M.tuberculosis infection as described in Figure 2. Formalin-fixed paraffin-embedded lung sections from infected WT and Lcn2KO mice were stained for neutrophils (Gr1-red) by immunofluorescence. The number of neutrophils per 20X field was estimated at D20 and D50 (a) and a representative image is shown at 200X magnification (b). Lung cells were isolated from WT and Lcn2KO M.tuberculosis-infected lungs at D50 post infection and the total number of neutrophils (Gr1+CD11b+) determined by surface staining and flow cytometry (c). Alveolar macrophages isolated from WT mice were exposed to M.tuberculosis (100 µg/ml) alone, Lcn2 alone (10 µg/ml), or both Lcn2 (10 µg/ml) and M.tuberculosis (100 µg/ml) and levels of G-CSF (d), KC (e) were measured in culture supernatants after 48 hours. Alveolar macrophages were isolated from WT or Lcn2KO mice were exposed to M.tuberculosis (100 µg/ml) and exogenous Lcn2 (10 µg/ml) and the levels of G-CSF (f) and KC (g) determined in culture supernatant after 48 hours. The data points represent the mean and SD for 3–4 samples for each time point and one experiment representative of two shown. *, p ≤0.05. **, p ≤0.005. ***, p ≤0.0005.
Lcn2 Regulates Inflammation and Contributes to Protective Immunity Against Acute M.bovis BCG Infection
Lcn2 is induced early in the lung in response to M.bovis BCG infection from day 2 post infection [18]. Consistent with this data, we found induction of Lcn2 mRNA (Figure 5a) and protein (Figure 5b) in the lungs on day 30 post M.bovis BCG pulmonary infection. In addition, we found that Lcn2 mRNA induction in the M.bovis BCG-infected lungs were not dependent on IL-23, IL-17 or IL-22 (data not shown). Furthermore, we could detect Lcn2 mRNA within both the lung parenchyma and airways of M.bovis BCG infected WT mice (Figure 5a). Although Lcn2 is shown to inhibit intercellular M.bovis BCG growth in vitro in alveolar epithelial cells [18], it is not known if absence of Lcn2 results in increased susceptibility in vivo following pulmonary infection. We found that following pulmonary infection with M.bovis BCG, Lcn2KO mice showed increased early bacterial burden that was maintained at later time points (Figure 5c). Interestingly, increased bacterial burden was observed in the spleen at early time points, but was controlled by day 50 post infection (Figure 5c). Importantly, we found that increased lung bacterial burden coincided with increased lung inflammation (Figure 5d). Consistent with our observations in the low dose aerosol model of M.tuberculosis infection, we found that lungs of M.bovis BCG-infected Lcn2KO mice exhibited increased induction of CXCL9 mRNA when compared to WT M.bovis BCG-infected lungs (Figure 5e,f). These data for the first time show that Lcn2 restrains inflammation and is required to mediate immunity to pulmonary acute M.bovis BCG infection in vivo.
Wild type (WT) mice were infected with 5×106 CFU of M.bovis BCG by the intratracheal route and day 30 post-infection, formalin-fixed, paraffin-embedded lung sections were assayed for Lcn2 mRNA localization by ISH using a murine Lcn2 mRNA probe (a) and Lcn2 protein levels determined by ELISA (b). Bacterial burden was determined in the lungs and spleen of M.bovis BCG infected WT and Lcn2KO mice at the described time points (c). Formalin-fixed paraffin-embedded lungs were stained using H&E and morphometric analysis of the area occupied by the granuloma in H&E stained sections of M.bovis BCG infected WT and Lcn2KO lungs was determined (d). A representative section of the lung granuloma is shown at 100X. Formalin-fixed, paraffin-embedded lung sections from infected WT and Lcn2KO were assayed for CXCL9 mRNA localization by ISH using a murine CXCL9 mRNA probe or control probe (e) and total lung CXCL9 mRNA levels induced quantitated using RT-PCR in day 30 infected lungs (f). Original magnification 40X-left panel, middle panel; 100X-right panel (e).The data points represent the mean and SD for four mice for each time point and one experiment representative of two shown. *, p ≤0.05. **, p ≤0.005. ns-not significant.
A protective role for Lcn2 in acute high dose M.tuberculosis infection has been recently reported [18]. However, given that the natural course of M.tuberculosis infection occurs as a result of inhalation through aerosol droplets harboring the bacteria, it is critical to test the protective role of Lcn2 in the low dose aerosol model of M.tuberculosis infection. Our results using a low dose aerosol M.tuberculosis infection model show that Lcn2 is dispensable for overall protective immunity against primary M.tuberculosis infection. Despite a number of studies showing in vitro inhibition of M.tuberculosis by Lcn2 [17], [18], [19], our data suggests that absence of Lcn2 does not impact overall control of bacteria in the lung or dissemination to other organs following low dose pulmonary M.tuberculosis infection in mice. This could be due to a limited role of Lcn2 in inhibiting M.tuberculosis growth in some cell types, but not others, and therefore not having an effect on overall bacterial control in the lung in response to chronic M.tuberculosis infection. Importantly, we document a new role for Lcn2 in constraining and limiting lymphocytic inflammation and promoting neutrophilic accumulation during mycobacterial infections.
We recently showed that key inflammatory immune mediators following low dose M.tuberculosis infection are upregulated in the lung between D12 and D20 post infection [28]. Consistent with this finding, we show here that induction of Lcn2 mRNA also takes place within this crucial time period. Strikingly, our data suggests that early in vivo cellular sources of Lcn2 following low dose M.tuberculosis infection are within the lung parenchyma, while later sources of Lcn2 are likely both cells within the lung parenchyma and airways. Furthermore, other studies have shown that exposure to M.tuberculosis in macrophage cell lines [19], M.avium in bone marrow derived macrophages [32] and M.bovis BCG in alveolar macrophages [18] induces Lcn2 mRNA, suggesting that pathogenic, non-pathogenic and attenuated strains of mycobacteria can all induce Lcn2 in the host. Furthermore, cytokines such as IL-17 [26], [33] and IL-22 [26] can drive the induction of Lcn2 in epithelial cells. While they are sufficient, they do not appear to be necessary, suggesting that other cytokines can also likely induce Lcn2 during M.tuberculosis infection. Furthermore, since IL-23KO M.tuberculosis-infected lungs have reduced levels of both IL-17 and IL-22 mRNA [25], this suggests that Lcn2 can be induced even in the absence of both IL-17 and IL-22. Accordingly, other studies have shown that IL-1βinduced in response to extracellular bacterial pneumonia [21], plays a prominent role in infection-induced Lcn2 production, suggesting that it is likely that multiple cytokines can induce Lcn2 expression in response to inflammation.
The new role for Lcn2 presented in this paper is to constrain CXCL9 induction and lymphocytic accumulation during mycobacterial pulmonary infections. Accordingly, Lcn2KO mice exhibit increased granulomatous inflammation, increased lymphocytic accumulation and enhanced CXCL9 mRNA induction in mycobacteria infected lungs. In contrast, Lcn2 also has a pro-inflammatory role by its ability to promote G-CSF and KC production in alveolar macrophages and neutrophil recruitment to the mycobacterial-infected lung. There is precedence for this idea, as a recent study also showed that Lcn2KO mice have reduced neutrophilic accumulation in response to intranasal Klebsiella infection [34]. However, despite the dysregulated inflammation found in Lcn2KO M.tuberculosis-infected mice, these mice are still able to mediate effective Th1 mediated immunity, induction of anti-mycobacterial killing mechanisms such as induction of iNOS and mediate control of M.tuberculosis. We [28], and others [2], have shown that depletion of neutrophils does not impact overall protective outcomes during TB, suggesting that in the presence of an effective Th1 immune responses, efficient neutrophil recruitment is not necessary to mediate immunity against TB. In contrast, a high dose acute M.tuberculosis infection model showed that Lcn2KO mice exhibited increased bacterial burden and succumbed to M.tuberculosis infection between 6–8 weeks post infection [18]. It is possible that in the high dose model, delivery of high numbers of M.tuberculosis by intratracheal route induces more robust production of Lcn2 and that the Lcn2KO mice die due to pathological responses mediated by the dysregulated inflammation, rather than due to just the higher bacterial burden in the lungs of Lcn2KO mice [18]. Consistent with this hypothesis, Lcn2KO mice infected with high dose M.tuberculosis intratracheally also showed higher inflammation, which was speculated to be due to the higher bacterial burden [18]. However, since we report increased inflammation and T cell accumulation even under conditions of similar bacterial burden seen in the lungs of Lcn2KO mice in the low dose M.tuberculosis infection model, we propose a new physiological role for Lcn2 in regulating inflammation during mycobacterial infections. Based on our data, we suggest that Lcn2 will play a crucial protective role in models of mucosal extracellular bacterial infection where neutrophils recruitment is important for clearance of the pathogen. Accordingly, it has been shown that Lcn2KO mice are more susceptible to mucosal infection with Klebsiella pneumonia [21], [34] and E. coli pneumonia [8]. In these models it is likely that the Lcn2KO mice are more susceptible not only due to the lack of host iron sequestering by Lcn2, but also because Lcn2 mediates cellular recruitment and regulates inflammation. Lcn2 produced by alveolar epithelial cells has been shown to be involved in intracellular M.bovis BCG control [18]. Consistent with this role for Lcn2, using an acute pulmonary M.bovis BCG infection model, we show that Lcn2KO mice show increased mycobacterial burden in the lung and peripheral organs. The likely reasons why Lcn2 is required to mediate protective immunity in the M.bovis BCG acute pulmonary model but not the low dose M.tuberculosis infection model are twofold. Firstly, although Lcn2 has been shown to inhibit both M.tuberculosis and M.bovis BCG growth in vitro and in cultured cells [17], [18] [18], [19], it is possible that this function of Lcn2 is redundant in vivo during M.tuberculosis infection, but is critical and non-redundant during in vivo M.bovis BCG infection. The second, more likely reason for a protective role for Lcn2 in acute pulmonary M.bovis BCG infection may reflect its role in chemokine regulation and neutrophil recruitment. This is supported by studies showing that depletion of neutrophils does not impact overall protective immune responses to M.tuberculosis infection [28], [35], but results in increased susceptibility to M.bovis BCG acute infection [36]. These data together suggest that in acute mycobacterial infection models, Lcn2 regulates inflammation and controls bacterial growth likely through a role in neutrophil recruitment, while in chronic mycobacterial infection models although Lcn2 regulates inflammation, it does not confer protective immunity.
In summary, we project a new role for Lcn2 in pulmonary infections where apart from its role in sequestering iron and inhibiting growth of bacteria, infection-induced Lcn2 can also act on mucosal cells to regulate chemokines such as CXCL9 and restrain inflammation at mucosal sites.
We thank Dr Anuradha Ray and Dr Jay Kolls for critical feedback and guidance regarding this manuscript and Samantha Slight and Brendon Wahlberg for technical help.
Author Contributions
Conceived and designed the experiments: LG RG YRC SAK. Performed the experiments: LG RG JRM BFJ YL SAK. Analyzed the data: LG RG JRM YL. Contributed reagents/materials/analysis tools: TB TWM JFA TDR TAR YRC. Wrote the paper: LG RG SAK.
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Good Morning to all. Hope your Easter weekend was a great one. It's always nice to spend it with family and friends.
Our challenge this week was a cute one. Who doesn't like furry friends. I know I do. I have two dogs and three cats. I would have a lot more if I lived in the country. So the sponsor this week for that lucky winner is The Paper Shelter.
They will be giving away as the prize a $20.00 Gift Certificate. That is a great prize to try and win.
I was able to used this cute little fuzzy dog and I made it into a Birthday card. It's a Birthday card for a little card. So let me show you the card I made with the dog.
I hope you like it and the image that The Paper Shelter let me use. Let me tell you all the items that I used to make this card.
Cardstock -- Close To My Heart
Paper -- The Paper Studio
Ribbon -- Close To My Heart (Designer Ribbon, Pink Collection)
Gems -- The Paper Studio
Font -- Cool Dots
Colored Markers
Colored Pencils
Well I hope you enjoyed the post today and I will see you next week. Don't forget to submit your entries at Incy Wincy Designs and check out what the other design team members made with their furry friends. have a Wonderful Week.


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Three members of the Louisiana congressional delegation are furious with U.S. Attorney General Eric Holder, calling for his resignation over a federal gun running program that resulted in a U.S. Customs and Border Protection agent being shot to death.
Read Original at: The Baton Rouge Advocate (La.)
Read the complete article: The Baton Rouge Advocate


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Sunday, 2 November 2014
october 2014 in pictures - instagram
Have you been getting your camera out lately? What's been your focus?
6 comments:
Beautiful - We'll ring later to say a belated happy birthday to Sophie! xxReplyDeleteReplies
Thank you! Look forward to a chat xoxDelete
Gorgeous pictures LucyReplyDeleteReplies
Thank you Jodi, have so enjoyed taking them!Delete
I love the colour of those roses. You are clearly in a different season to us in the UK. I'm no great photographer but I like the idea of taking a picture a day to record my life, something I'm planning to start in the new year.ReplyDeleteReplies
It was a bit of a gamble when I bought the roses, not sure how they would all fit in together but I'm absolutely delighted with them! And yes, a very different season, everything coming back to life here.Delete
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Friends of mine have been inquiring as to when and how many times I'll be screening Star Wars VII when it debuts this December in multiplexes across the world. None have actually asked if I actually wanted to see it, essentially assuming I was going to, except one person and that came about due to my hesitation on giving an answer. That answer is no, I don't want to see Episode VII: The Force Awakens.
Frankly nothing up to this point has stoked my fires and has worked me up into the required fan frenzy needed to throw money and time at the object of said fandom. The trailers have mostly left me cold and that isn't a good sign. And after the "Force Friday" merchandise nonsense ended I have a great opportunity to check out the goods as that might clue in more about the film which might pique my interests.
When I stopped by my local Target to do some basic needs shopping and decided to walk past the toys and seasonal garbage section to check out the Star Wars goods. I spent close to thirty minutes going up and down the aisles, checking out every bit of product, and then I reached a point where I was standing in front of the LEGO displays for J.J.'s space opera odyssey where I said something out loud that took me by surprise.
"....I don't want any of this."
It wasn't spoken in a loud or condescending manner but one of quiet disappointment. If you saw my face when I spoke those words it may have looked like I lost my best friend; a defining part of who I was that up and left. This was the realization of something coming for a long time: I don't need Star Wars any longer.
I have my memories of the original films and those have had a lovely rose colored gloss that has lasted for years yet as I have aged and become more critical of the media I consume as well as how I spend my time I become thankful that I watched Lucas' original films as a child because if I went in today I'm certain I wouldn't have devoted so much energy to this brand. And this isn't anything sudden because when I look back and really think about it all of the signs were present in the Kubler-Ross model for coming to terms with an emotional experience.
Denial appears early and for me that came about during the prequel films which released back in the late 1990's/early 2000's. This was especially true with The Phantom Menace. I kept denying how terrible the movie really was, denying the possiblity that the other films to follow would be equally as bad because George was just getting his movie making legs back with this one so it could be a dog in the manger, and outright denying that I was no longer the target audience for this series; I will return to that later.
Anger manifested a few months after Phantom Menace left the theaters as I had time to digest just what I watched and realized just how bad it was. Not only that but while being caught up in the fandom I went multiple times, gleefully I might add, while also spending my hard earned money on the relevant merchandise on something that I actually hated. I felt bamboozled and, more importantly, wasted my time with this which really made me angry. I also was angry when it was even more painfully obvious that this was and will always be a kids film and the brand didn't mature with its original audiences. I was angry that I didn't get something more developed and evolved but was given 2+ hours of Jar Jar and Jake Lloyd delivering such Shakespearean lines like "Woo-hooo!".
Bargaining hit right around the release of Attack of the Clones with the "if/then" deals I made with myself to justify keeping up with brand such as "If the movie gets ok reviews then I'll check it out a few weeks after release" and "If the movie is ok then I'll consider checking out the toy lines, etc." It wasn't good and I didn't follow through with the second statement. Anger and denial reared back up as well during this phase too. Bargaining also creeped in regarding making silent deals internally as to what merchandise I would consume from the brand to keep up so to speak. My fanboy "drug" of choice was Star Wars branded LEGO model kits; eventually I realized I just enjoyed putting together LEGO and I didn't need the Star Wars branded ones for that amusement.
Depression crept in post prequel trilogy and well before Disney got its white gloved talons into it. Lucas was doing jack shit with his brand except re-release after re-release of the films in various formats, games that were on the horizon were promoted then cancelled, and nothing new was coming out except TV shows such as The Clone Wars but that was aimed again at 1) children and 2) it was set during that prequel era which was horrible. My object of fandom was falling by the wayside in a very slow, methodical march; I was feeling left out of the thing that I adored. I was no longer feeling included for wanting more out of this brand and it really bummed me out that others my age could still find that same bliss which has now escaped me. Anger creeps in here to as I began to resent others for their fandom which is an interesting concept; I was pissed off and jealous that I didn't have another's level of obsession.
Finally Acceptance happened in that toy aisle in Target where I admitted that I didn't want Star Wars any more. More specifically I admitted that I wasn't a child any longer and this brand didn't grow as I did which then lost its value to me and my resources (time, money, etc). I have to say just because I reached this stage does not mean I'm content. In all honesty anger emerged again because of this revelation yet I will move forward and leave this in my past. Interestingly these stages do not fall in a strict pattern and will bounce around depending on the level of the emotional investment towards the object in question. In a bizarre twist when I look back at this process it feels like it is part of a maturation ritual for the mass produced age. There is a journey involved where on an emotional level I am questing towards adulthood and in the process I must kill my childhood to move forward. The destruction of my fandom of a product that is for children is just another step on the path I am on which has taken years.
Side note: when thinking about writing this I went poking around and found an article from 2011 via Slate.com which gives a humorous look at this process that was similar to mine minus watching 150 episodes of Pokemon.
Another side note: Back in 2012 I wrote a post about changes Lucas made regarding Star Wars and I was horribly wrong about it but totally fanboyish.


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This section contains 756 words
(approx. 2 pages at 400 words per page)
Part 1, Chapter 7 Summary
In the final chapter of this portion of the book, we find Alex being dragged into a whitewashed room at the police station. The room smells like vomit, excrement, and disinfectant. Alex can hear the other inmates singing and cursing, and he also hears the police telling the inmates to shut up. Finally, he hears an inmate being beaten and wailing in pain.
Alex watches as the officers drink some tea, and catches his own reflection in the mirror. To his horror, Alex sees how terrible he looks. His mouth and nose are swollen, and his eyes are red. The police laugh.
Alex demands an attorney, telling the officers that he knows the law and his right to counsel. The officers laugh again. They tell Alex that they know the law, too, and one of the officers punches Alex in the...
(read more from the Part 1, Chapter 7 Summary)
This section contains 756 words
(approx. 2 pages at 400 words per page)


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The morning Inb now ox demands the right to be lazy with difficultly levels, as one reader recalls the day he was robbed by a very discerning burglar. To add your own comments to the Inbox email
United frontYou know, having watched a great deal of Japanese animation and played a great many Japanese games, I have come across a common motif in many an end sequence or finale. The 'we all stand together' ending as I like to call it. Okami, Shaman King, Princess Mononoke, Paper Mario: The Thousand Year Door, Skies Of Arcadia and even Dragon Quest VIII to some extent all have this kind of resolution.Only when everybody stands united can evil be overcome. And they're usually quite good at this ending, not one of those examples is too corny or unsatisfying. My question is: where does this come from? Do the Japanese have more of a community spirit than us selfish Westerners?DMRGC:EarthBound was the best at this, one of the best endings ever actually. We wish Nintendo would release it in Europe (even better some sort of Mother compilation).Xbox Live introI'm submitting a post in a similar vein to one also posted last week with the exception that mine concerns Xbox rather than PlayStation.I recently decided to pay for an Xbox Live Gold account and was wondering what GC and the readers regarded as the best online multiplayer experiences on the service. I would tend to favour co-op over competitive play and my skills are average at best, but all suggestions would be considered (although at the moment it is probably too hot to even think about gaming).StanleyScratch (gamertag)Retro discoveriesJust a quick letter of thanks for the suggestions and advice from GC and readers regarding the Japanese shooter Gaiares I picked up from a car boot sale a couple of weeks ago. I managed to get hold of a Mega Drive Game Genie from a friend who had one buried away in their loft. Gaiares works perfectly and it's a pretty decent shooter. Nice bold graphics with loads of layers of parallax, huge bosses and really great music.The opening track for the first level where your ships blasts off from base is especially good. I don't know if it's my old hands slowing me down but the game is rock hard, I can't get past the third stage at the moment. Gun Lord looks fantastic, watching the video brings back misty eyed Amiga memories. It's a shame the obviously talented team behind it decided to make the game for the Dreamcast rather than going for the much larger audience of releasing it as a download game for one of the current consoles or the PC.PE
Catch up on every previous Games Inbox here
The right to be lazyAfter reading Matt Kirk saying that games should be played at higher difficulty levels with the statement 'no one on these pages should be playing a game on the standard default of medium from the start. You won't play the game on this setting and you will ruin the first run through,' I'd like to respectfully disagree. Firstly it presupposes we are all going to play through games more than once – which is not always true – and secondly not everyone has the same ability level and therefore variable difficulty is mostly appropriate.From a personal perspective, for many years I'd play every game on its highest difficulty including the likes of Devil May Cry, Viewtiful Joe, God Hand and Ninja Gaiden. I'm sure most would agree these games are not easy even at the normal level. The reason being that I so thoroughly enjoyed the gameplay that as the credits rolled I just wanted to carry on playing them, the higher difficulties made this possible and worthwhile. It's now quite rare for me to do it, partly because of time constraints but also because of the plethora of other games to play make it impractical. Mainly though, it just stopped being enjoyable to do so, It all became a bit of a slog and some sort of self-imposed obligation to prove that no level of difficulty was beyond my reach.Laziness is now also a consideration and it takes a special game for me to want to bother to do it – Bayonetta being the only one this generation which has qualified as such. Now I'll just meander through games and enjoy playing them that way. I'll sometimes stand around admiring the scenery and artistry on display, absorbing it all in some kind of Zen like reverie, it can be quite cathartic after a long day (I did this a lot in Dead Space 2 but I'm weird that way).Surely though, it's the prerogative of the player to decide to play a game on easy, medium or hard. So Matt, if you enjoy the challenge of greater difficulty power to you mate, It all seems like far too much hard work to me now.Mr Verbosity.PS: Don't call me Shirley.
Bargain and a wishFirst of all just a heads-up on some bargain prices that may be of interest: Dark Souls day one edition (with artbook and soundtrack CD) £16.85 from ShopTo.Net. Rage (standard edition) £7.85, Binary Domain (ditto) £9.95 and finally DiRT 3 (complete edition) £14.95, all from Zavvi.com.Now can I be a little bit selfish and invoke some of that Inbox hoodoo and ask DICE for a HD update of the great Rallisport Challenge 2. Used to love that game, one of the most fun and content rich (all on the disk, as it should be) rally games from the last gen.presidentweevil (gamertag)A discerning burglarWith recent discussion I thought I'd share a story of mine with equal laughter and devastation. It was around the time that Super Mario Galaxy 2 had been released. I'd hit around the 100 stars and came home from work determined to collect all the stars that evening. As I came home my housemate was upstairs on Xbox so I thought, 'Great my Wii is on main TV so I can crack on'. I switched on the TV, sat down and picked up the Wiimote.After pressing the on button on the remote nothing happened, just a blank screen. I checked the batteries, everything was fine – so tried again and nothing. I then thought that maybe I should check the wires at the back so pulled the TV out and everything was there plugged in as normal. Getting frustrated now I sat down again and tried the remote again and still nothing. This time I thought I'll just switch it on at the console and as went under the TV to my surprise there was no Wii.I shouted up to my housemate, 'Where's my Wii?' to which he came down wondering what on earth I was on about. It turned out that during the time my housemate was up stairs someone had come in and just taken my Wii console and Mario Galaxy 2 which was inside and must have been disturbed as my pile of games and controllers were all still there.The incident was so ridiculous I had to laugh about it but was devastated I lost all progress on Mario and my Virtual Console games. All was put right though after my girlfriend and guilt ridden housemate helped replace them and it was pure joy to enjoy that gem of a game again.Terror4mer
Teething problemsWith the new consoles from Sony and Microsoft expected to be shown off at some point next year is anyone else thinking that the first models will probably have a fairly high failure rate? Microsoft never really solved the problem with the Xbox 360 overheating and with considerably improved graphics promised by both companies that's going to be a lot of extra strain on the hardware.In this respect Nintendo might have got it right with the Wii U, making sure that the new consoles are stable. Ideally the majority of gamers would just like to buy a new console based solely on the games available for that system but as more advanced technology unfortunately brings more problems I think the potential longevity of a consoles life may have a greater impact on picking one than ever before. Does anyone else agree with this?Mr BumpPS: Just got Deadly Premonition – York may be the best video game character ever written, right Zach?100% successfulVery rarely has a game compelled me to strive for a 100% completion rate like Batman: Arkham City has, it's so ingratiating and one of the most balanced and refined games I've ever played. From the intuitive and dynamic combat, the euphoric sense of mobility and incredibly sophisticated detective and Zelda/Metroid-esque puzzle elements. And the eponymous super open prison is so dense and brimming with character, a great example of a sandbox environment.With such bravura, Rocksteady have come leaps and bounds since the ambitious yet slightly stilted execution of Urban Chaos. Arkham City and its predecessor are the best use of a licence since Astro Boy :Omega Factor and Chronicles Of Riddick: Butcher Bay in my opinion. So what's next on their plate? How about Bat's DC comics stablemates, especially Superman. The poor fella, I don't recall a single decent game starring him since the 8- bit era, unlike Spider-Man, the Bats or even the Hulk. In fact, Superman 64 is one of the worst games ever let alone comic-to-game adaptations. It was so bad that I could of labelled it a survival horror game, it still haunts me till this day.But if any developer could do him justice, Rocksteady would be the obvious candidate surely. Their unprecedented understanding of a comic's source material and ethos would lend them an auspicious effort, and mind you Superman is a particularly difficult superhero to tackle because of his near invincibility. Bat's is far from insuperable and that's what makes his application to gaming so interesting because you have to think carefully about the consequences of being mortal.Alas, I fear that Arkham City has raised the bar for superhero games to such stratospherical heights that other than RockSteady's own work its contemporaries won't match its near perfected remit for some time to come. Galvanized GamerPS: Have not seen it yet but it's a real shame that The Dark Knight Rises is making headlines for the wrong reasons, my heart and condolences go out to those in bereavement of losing loved ones in the shootings.
Inbox also-ransI wonder if any reader can advise on how to access my game saves that I've stored on a USB and am now trying to use via a new PlayStation 3 that I've just purchased. I've installed it onto the hard drive but when loading up any game I simply get the message 'you cannot load save data that you do not own'! All my hours of work gone up in smoke!SamGreenManGaming.com have a 20% off code for PC downloads at the moment which will expire at midnight on the 2nd of August. The code is GMG20-CVIDE-OGAMETrailbreaker83 (PSN ID)PS: I can't find your review for the first Darksiders game, is it worth twelve quid? GC:It's not on the website but we gave it a solid 7/10 at the time.I found this video really interesting . I think it speaks for itself.Rak2012
Every time I hear that David Braben is still in the games industry but not working on Elite IV it breaks my heart.LyleThis week's Hot TopicThe subject for this weekend's Inbox was suggested by reader LeighDappa (PSN ID), who asks if you could live in a video game world which one would it be?Even if it was just for a holiday why would you want to visit there and is it just for the scenery or because of the excitement and danger? What would you do in the game world and what kind of life would you want to lead?Are there any special powers or other perks that your choice would grant you, and are there any downsides to gaining them? Which game world would you absolutely not want to live in and do you think enough is done to make game universes seems like living, breathing places – rather than just vaguely glimpsed backdrops.
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