Is consideration in your life, explained as careful thought considering others, do people give you consideration, do you give consideration to others, something that should always be present in decision-making and making evaluation's on facts etc., something that should always be kept in mind and that of others, thoughtful or sympathetic regard or respect for others, and for yourself, an opinion based on reflection,consideration is necessary in our lives everyday, it is as important to give as to receive, we all need to be considered on any scale, the decisions of others affect us in many ways, good and bad whether it be a work or social area, couples also need to consider all points of view, not just from one side, if an important change is made in your working environment directly affecting you, the consideration taken when expressed to you will maybe help you to understand the reasons behind it, although it may not be what you wish to hear, you will always respond better, if you are receiving or giving, if consideration is within the equation. Consideration is also a form of love, therefore a very important thing which should not be missing in your life……… Kindness is the other emotion we should all have in our lives, again it is as important to give as to receive, it is a behaviour marked by characteristics, it shows a pleasant disposition, and shows concern for others, it is known as a virtue and recognized as a value, it is defined as helpfulness towards someone in need,NOT in return for anything OR the advantage of the helper themselves, but simply for that of the person helped. We receive kindness from the day we are born and as we grow we learn to give kindness, some are born with it and will give kindness all there lives, we appreciate kindness in all different walks of our life, when we are served in restaurants, supermarkets,cloths stores etc., if it is done with kindness and consideration we feel good and appreciate it, we return to that place, in the same way that we gravitate to people who are kind to us, we all have our special places to go and to shop, we sometimes go out of our way to obtain something because we know we will be attended with kindness, also a form of love and should be ever-present in our lives.
This entry was posted in Chase your dream, energy dreams success attraction, Informative, inspiration self development personal growth, Motivational, positivity, self help and tagged blog blogger blogging consideration & kindness, consideration kindness love. Bookmark the permalink.
Que bueno ! Me gusta ! cuando tenemos estos valores devemos entendernos ….
Another essential thing, you need in everyday life………………….


After a tragic accident of falling off a cliff whilst on a class trip, Tomie appears dead as a dodo before her teachers and classmates eyes. In a frantic attempt to hide her body her teacher and male classmates begin to dismember her only to find out that she is in fact alive. Following through on the dismemberment and putting her to rest each classmate is asked to get rid of a part of her body. What none of them expected though was her to come back from the grave and seek havoc on anyone and everyone she encounters.
Tomie is one of those iconic manga characters. Whether you have read Ito's work or not you have probably seen manga panels with her face on floating around the internet. I guess you could call her a bit of a horror icon of the manga world.
She is beauty. She is grace. You will totally murder her face.
Tomie has to be one of my favourite collections of Ito's work I have read so far. There is something so striking about her character that really sucked me in as a reader. When picking it up to read it at night I felt eager to read another few chapters and delve further into her sick and twisted world.
Tomie is beautiful on the outside but rotten to the core on the inside. She uses and abuses men making them fall in love with her before breaking down the sweet girl facade and revealing her heartless nature. She would often laugh at them and reject them which would often lead to her demise of being brutally attacked and murdered. I feel she has a siren like quality in the sense of luring men. Women are never apart of her devious deeds and it's only men that she lures with her beauty. Even when she is brutally mutilated or just a head which was grotesque and deformed she was still able to control men and get them to do whatever she asks of.
I find the supernatural element of Tomie fascinating. Although it is never revealed how Tomie continues to come back to life and multiply it's something as a reader that you just accept. As much as I'd love to know the ins and outs of how her regeneration works I never felt I needed to know the formula of how it worked. Her character and the stories were far too engrossing for me to question it. The fact that Tomie also detests other versions of herself and they all want to get rid of each other was an interesting twist. It displayed the idea that each Tomie thought of herself to be more superior than the others and told of her truly vain, self obsessed nature.
I had a constant battle of both loving and hating Tomie as a character. She is narcissistic who will uses anyone who crosses her path. As a reader I also fell for her beauty and found her such a captivating character. I cannot recommend Tomie enough to horror lovers. It's a staple in any manga collection.
It was good! I thought it was interesting how she will always live on.
Yeah I really enjoyed that element of the story!


KRASNODAR (Reuters) - The shadow of the dramatic sacking of coach Julen Lopetegui on the eve of the World Cup still hung over Spain as they set off for their final Group B game against Morocco in Kaliningrad after midfielder Saul Niguez openly criticised the decision.
The common line from Spain's players since Lopetegui was sacked by Spanish football federation president Luis Rubiales for negotiating a move to Real Madrid behind his back was that they have accepted the move, while admitting it was far from ideal for their campaign.
But Saul, who has known Lopetegui since he was his coach in the Spain under-19 team, broke ranks in an interview with Spanish state television on Saturday, saying the players should have been given a say in the seismic decision.
"It was surprising and I think that even though there were motives and reasons behind the decision, the players had no say in it and it was not the right time," Saul said.
Interim coach Fernando Hierro, who before the tournament was working as the team's sporting director, said he had no problem with Saul's comments.
"It's logical, healthy, natural and what everyone is thinking," Hierro said in an interview with Spanish radio station Onda Cero.
Spain will qualify for the last 16 of the World Cup with a draw against already-eliminated Morocco on Monday although in order to guarantee top spot in Group B they will need to outscore Portugal, who will be in action against Iran.


text:
Read Next Purple Mountains' Debut is a Richly Depressive Comeback Album from a Brilliant Songwriter	Send Us a Tip	Subscribe
Home Culture Culture News
'Spider-Man' Musical Delayed Again; U2 Producer to Fine-Tune Songs
Longtime U2 collaborator Steve Lillywhite will also produce the original cast recording
Bruce Glikas/FilmMagic
The opening day for Spider-Man: Turn Off the Dark has been delayed yet again. The show, which is currently in previews and had been scheduled to open on February 7th, will now officially debut on March 15th at the Foxwoods Theater on Broadway.
According to director Julie Taymor, the production is being delayed because she is still fine-tuning the story, music and technical execution. Most significantly, Taymor is in the process of reworking the show's final act.
Photos: U2: Three Decades of the World's Biggest Band, Onstage and Backstage
U2's Bono and the Edge, who wrote the music for the show, have been refining their songs and working on the audio mix in the venue. As of this week, the two have brought in their old collaborator Steve Lillywhite to help them out in working with the actors on the show's music. Lillywhite, who is responsible for producing songs on a majority of U2's albums including War and Achtung Baby, is now set to produce the original cast recording.
Photos: U2 in Italy
This delay probably won't have much impact on audience turnout. The musical, which is the most expensive production in the history of Broadway, is performing very well at the box office in its unusually long preview run. Despite some bad buzz and negative publicity regarding its multiple cast injuries, Spider-Man was the highest-grossing show on Broadway last week.
"Spider-Man" musical delayed again [Reuters]
U2 Album Producer To Work On 'Spider-Man' Music [NY Times]
In This Article: Spider-Man: Turn Off the Dark,	Steve Lillywhite,	U2

redpajama_set_name: RedPajamaCommonCrawl


text:
Australian Militia Forces, New South Wales, Contents
Topic: Militia - Inf - NSW
Australian Militia Forces
Outline and History of Military Structure, 1900
Military Structure - Location of Units, 1900
The 6th Volunteer Infantry Regiment, History
Militia Regiments Mobilised for service in South Africa, 1899 - 1900
New South Wales Lancers
New South Wales Infantry Contingent
New South Wales Mounted Rifles, "A" Squadron
1st Australian Horse
"A" Battery, Royal Australian Artillery, New South Wales
New South Wales Army Medical Corps
New South Wales, Military Structure, 1903
14th (Hunter River) Infantry
16th Infantry (Newcastle Regiment) raised 1912
18th (North Sydney) Infantry
19th (Kuring-Gai) Infantry raised 1913
21st (Woollahra) Infantry
24th Infantry (East Sydney Regiment)
25th Infantry (City of Sydney)
26th Infantry raised 1913
29th Infantry (Australian Rifles)
31st (Leichardt) Infantry
33rd Infantry
37th (Illawarra) Infantry
39th Infantry
41st (Blue Mountains) Infantry
42nd (Lachlan Macquarie) Infantry
43rd (Werriwa) Infantry
Sydney University Scouts
Militia Distribution in Australia, 1914, 2nd Military District, Part 3
New South Wales Militia Unit Location, 1913
New South Wales Infantry
Australian Infantry Militia
Citation: Australian Militia Forces, New South Wales, Contents
Updated: Sunday, 23 October 2011 9:10 AM EADT
12th Light Horse Regiment Routine Order No. 270, 1 January 1917
War Diary, 5 January - 18 January 1917
Routine Order No. 270, 1 January 1917
Routine Order No. 275, 6 January 1917, p. 1
Routine Order No. 280, 11 January 1917
War Diary, 19 January - 7 February 1917
Routine Order No. 295, 26 January 1917, p. 1
Routine Order No. 301, 1 February 1917
War Diary, 9 February - 27 February 1917
Routine Order No. 310, 10 February 1917
Routine Order No. 324, 24 February 1917, p. 1
War Diary, 28 February - 20 March 1917
Routine Order No. 329, 1 March 1917, p. 1
Routine Order No. 332, 5 March 1917
Orders for EEF Canteen, 6 March 1917
Routine Order No. 337, 10 March 1917, p. 1
Routine Order No. 338, 11 March 1917
Gong Calls, 11 March 1917
March Discipline, 18 March 1917
Routine Order No. 356, 1 April 1917
Routine Order No. 358, 3 April 1917, p. 1
Routine Order No. 365, 10 April 1917, p. 1
Routine Order No. 369, 14 April 1917
Routine Order No. 373, 26 April 1917, p.2
War Diary, 1 May - 4 May 1917
Routine Order No. , 1 May 1917
Routine Order No. 378, 2 May 1917, p. 1
Routine Order No. 380, 4 May 1917
War Diary, 6 May - 6 May 1917, p. 1
War Diary, 10 May - 12 May 1917
Routine Order No. 386, 10 May 1917
Routine Order No. 388, 12 May 1917, p. 1
Routine Order No. 390, 14 May 1917, p. 10
Routine Order No. 406, 1 June 1917
Routine Order No. 408, 3 June 1917, p. 1
Routine Order No. 416, 11 June 1917, p. 1
Album - 12th Light Horse Regiment War Diary and Routine Orders (2), 1916 - 1919
War Diary, 30 June - 4 July 1917
Routine Order No. 436, 1 July 1917, p. 1
War Diary, 5 July - 7 July 1917
War Diary, 9 July - 12 July 1917
4th Light Horse Brigade Shoulder Patches
War Diary, 1 August - 4 August 1917
Programme of 5th Mounted Brigade Sports
5th Mounted Brigade Sports Competitions
War Diary, 1 September - 7 September 1917
War Diary, 8 September - 15 September 1917
War Diary, 1 October - 7 October 1917
Routine Order No. 521, 1 October 1917, p. 1
War Diary, 30 October - 2 November 1917
Report of the assault on Beersheba on 31 October 1917
War Diary, 3 November - 3 November 1917
War Diary, 11 November - 12 November 1917
War Diary, 1 December - 1 December 1917
War Diary, 9 December - 10 December 1917
Updated: Sunday, 11 December 2011 2:31 PM EAST


Mine: Fun and enjoyable. Tone is set from the very beginning. Some well delivered, witty dialogue and a good example of the genre.
Leonard Maltin/Plot synopsis: Two corporate executive assistants hatch a plan to match-make their two bosses.


As customer-brand engagement progressively shifts to digital domains, understanding social media effects in branding has become a vital issue. Social media effectiveness is especially important for the US retail sector due to intense competition among retailers for consumer attention and engagement on digital channels. Yet, the research on the effectiveness of social media in the retail industry remains sparse. Thus, the purpose of this paper is to investigate how social media affects US retailers' customer-based brand equity (CBBE) which is an important indicator of brand success. Using a dataset of 15,717 retailer-day observations, the authors empirically test the dynamics between owned and earned social media and CBBE using panel vector autoregression (PVAR). The authors find strong impacts of owned and earned social media on CBBE across the board. However, they find that owned social media harms CBBE of retailers dealing in hedonic and high involvement products. Whereas owned social media helps general retailers in building CBBE, it reduces CBBE of specialty retailers.


The 2019 OHSAA State Championships was wicked fast: a total of ten Division I state records were broken throughout the course of the meet, one of which was a national high school record. The meet was packed, too – in fact, part of the reason why the state meet is held at Canton is because it has more spectator seating than any other pool in Ohio.
Defending OHSAA Division I state champs Mason High School was challenged from the get-go by North Canton Hoover. Though the lead shifted back and forth between the two across the course of the meet, it was ultimately Mason High School that claimed their second straight state team title – even without winning a single individual event. The Mason Comets barely edged out North Canton Hoover in the final team scores, 204 to 201.
North Canton kickstarted their state meet campaign with an impressive showing in the 200 medley relay. The Vikings relay team of Caitlin Cronin, Maya Lewis, Parker Timken and Caroline Ehlers touched first in a time of 1:41.02, breaking the state record of 1:41.88 set by Upper Arlington in 2017.
In the 50 free, North Canton picked up a ton of points thanks to a 1-2 finish by seniors Parker Timken and Caroline Ehlers, clocking times of 22.61 and 23.03. Timken went on to double up with another victory in the 100 free, claiming the top spot with a 49.65 – the only swimmer under 50 seconds.
Ursuline Academy senior Megan Glass, who in 2017 tied Maddie Martin's now eight year old state record of 53.34 in the 100 fly, has become the sole occupant of that 100 fly record. She won the event in a final time of 53.31, over a second ahead of the rest of the field. Earlier in the meet, Glass was victorious in the 200 free, topping the competition with a 1:47.77.
Junior Paige McCormick of Hudson earned an individual victory in the 500 free, stopping the clock at 4:48.90, which is over a six second drop from her third place finishing time from last year of 4:55.15.
North Canton and Mason again went 1-2 in the 200 freestyle relay – the quartet of Caroline Ehlers, Reagan St Clair, Kate Lochridge and Parker Timken combine for a final time of 1:32.43, touching ahead of the 1:33.21 from Mason's squad.
The last state record of the girls meet was achieved by Brecksville senior Hannah Bach. Though Bach was seeded with a time of 59.54, which is well under the state record of 1:00.75 she established last year; she ended up lowering it even further in finals, taking the top spot with a 59.29.
Winning the final contested event of the meet was New Albany's 400 free relay team consisting of Lexi Elkovitch, Olivia Horovka and sisters Mia and Ava Lachey. They earned a final time of 3:25.92.
North Canton Hoover's 400 free relay ended up placing seventh in the finals – and even though Mason's relay was DQ'd in the morning, and therefore couldn't score points in the final, they still managed to lock in the team title.
Dublin Coffman sophomore Ellie Andrews was the only competitor to break the 2:00 barrier in the 200 IM, narrowly out-touching senior Felicia Pasadyn of Brunswick, 1:59.73 to 2:00.08.
Pasadyn would later emerge victorious in the 100 backstroke, taking the event in a time of 53.31 – identical to the winning 100 fly time.
In the diving, West Claremont senior Morgan Southall tallied a final score of 479.55 to clinch the win over fellow senior Maycey Vieta out of Gahanna Lincoln, who earned a total 474.40.
Saint Xavier has a rich program history of Division I state titles – coming into the meet on a ten year win-streak, as well as 39 total state team championships. Similarly to last year, they are rivaled by a team backed a lot of star power: Sycamore's Carson Foster and Jake Foster. Despite the five collective event victories that the brothers accrued, St Xavier remained relentless; taking home their 11th straight state crown with a final score of 310, well ahead of Sycamore's 240.
Responding to the 200 medley relay state record on the girls side, the Sycamore boys set a 200 medley relay state record of their own. Lead off by the Foster brothers, with teammates Ansel Froass and Elliott Carl holding down the back-end, the team touched in a time of 1:28.48.
The old state record was held by St Xavier at 1:29.66 from 2017 – this year, St Xavier finished second to Sycamore in a time of 1:28.94, also under their old record.
Following up on his morning 200 free, in which his 1:33.38 broke his own state record of 1:34.19, Carson Foster broke the national high school record with a time of 1:32.99. The junior took down Trey Freeman's year old national record of 1:33.09.
That time would currently rank 10th in the NCAA this year, including the times posted at the SEC meet; and is the 3rd fastest time in the USA Swimming SWIMS database for the 17-18 age group, with Foster having an additional year remaining in the age group.
Older brother Jake Foster swam the exact same time in both prelims and finals in the 200 IM – a 1:43.99, which obliterated the previous record of 1:46.58 set by Cooper Hodge in 2016.
Mason junior Adam Chaney rewrote the record books in both the 50 and 100 freestyles during the preliminaries, posting times of 19.69 and 43.27. In finals, he took wins in both of the events, touching slightly slower at 19.79 and 43.67. Leading off Mason's 200 free relay in finals, however, Chaney lowered his 50 free record to a blistering 19.64.
St Xavier collected a relay victory in the men's 200 free relay – JP Khouzam, Owen Taylor, Ian Van Gorp and John-Pierre Khouzam touched the wall first in a time of 1:22.69 over North Canton's 1:23.03.
In his second individual event, the 100 back, Carson Foster annihilated Justin Grender's old state record of 47.39, posting a 46.28 in the preliminaries. He later secured the victory in finals, recording a 46.62 – marking the only two 46s in OHSAA history.
As if the two brothers were destined to follow each other up, Jake Foster took down the 100 breast state record of 54.35 held by Ross Palazzo, registering a scorching 53.31 during the heats. Jake added a bit in finals, but easily managed to net his second state title with a 53.76.
Though Sycamore's 400 free relay – comprised of the Foster brothers, as well as Noah Patterson and Ansel Froass – took the win over St Xavier's, 2:59.97 to 3:01.79, the team championship had long been secured for St Xavier. This marks their 40th title in program history.
St Xavier got an individual title out of junior Jean-Pierre Khouzam in the 100 fly. He touched the wall in a swift 47.21, over a second ahead of the rest of the field.
Jacob McDonald, a junior out of Liberty Township, posted a dominant win in the 500 free; his 4:22.17 was easily ahead of St Xavier senior Grady Wheeler's 4:25.94 for second.
Shaker Heights senior Lyle Yost claimed the top spot in the men's 1m diving event, amassing a final score of 571.20 points.
The results link indicate that the Finals times were: Sycamore 2:59.97 and St Xavier 3:01.79. It appears that Swimswam referenced the Prelims times.
Soooo the Mason Manta Rays won?
We know u rep the fosters. Thought u worked for marlins?
Even with Chaney's points, SycaRays still don't outscore St. Xavier. This is a pointless, fanboi comment.
What about the points from St.X that are from Manta Ray Swimmers?
None of the St. X kids train with Mason during the year…that's what makes St. X a TEAM and their coaches real high school coaches. Your argument is ridiculous, just like you changing user names every post.
You guys need to stop doing this every time Ohio High School swimming comes up. It's not a good look for anybody.
What if you count McDonald's points as well?
Of course, the RAYS would win a High School championship. They would probably beat the Dayton Raiders and Cincinnati Marlins as well. They are top 5 swim club team in the country. But, they have no standing in Ohio high school swimming and are rightly not mentioned in the article above….
It was truly an epic meet. The best part about it from my observation on deck was the interactions between the athletes, be they St. X or Sycamore or Hudson or Mason or Hoover…..they were FOR each other. They were genuinely happy for the record breakers. They were tough competitors but they were not wishing someone else to not do well. I overhead so many conversations. It was encouraging all the way around.


Yesterday was a big day for the broad coalition of employer and labor groups -- including Mercer and The Alliance to Fight the 40 -- that have been advocating for repeal of the Cadillac Tax. The president signed a short-term spending measure that included a two-year delay of the effective date for the 40% excise tax on high-cost medical plans, from 2020 to 2022. The act will also halt the ACA's 2.3% medical device tax for 2018 and 2019 and suspend the health insurance tax (HIT) for 2019. Both of those taxes were reinstated this year. The ACA tax changes approved by the House that repealed the individual mandate penalty remain in effect.
In 2010, when the ACA was signed into law, the so-called Cadillac Tax was the biggest concern for employers that provide health insurance to 178 million Americans. That's not surprising, since nearly a fourth of employers were likely to get tagged by the tax in its first year, even though it was supposedly going to affect only very rich-- "Cadillac" – health plans. The design of the tax is flawed in that it uses cost as a proxy for benefit richness, when in fact there are many factors beyond plan design that can drive up the cost of coverage: geographic location, population age and health risk, and the number of enrolled dependents, to name a few. In addition, the tax could compel employers to stop offering wellness programs and on-site clinics, and to reluctantly ask employees to bear even higher out-of-pocket costs as a way to get the premium cost below the tax threshold.
While we are grateful for the two-year delay, we know there is bipartisan support for a full repeal. Along with other employer coalitions and advocacy groups, we are committed to keeping the pressure on.


In my typical Night Notes, I take pictures of silly things and talk about my daily life. I did not take this photo, even though it's slightly silly.
Japanese underwear maker Triumph recently held a press conference for this "Welcome To Japan bra". It's possible to hear "Welcome to Japan" in Chinese, Korean and English — just by pressing those gold buttons. With the APEC summit in Japan and Tokyo's Haneda Airport pushing international flights, this bra is timely.
But...it is simply a PR stunt. That's fine, really! Every year, Triumph shows off all sorts of crazy bras. These don't actually go on sale, and they're not for public consumption. They are, as I said, PR stunts. There seems to be some confusion in the West about these garments — that these are going on sale. Like I said, they're not.
Here's another bra that Triumph unveiled. Likewise, not a real product! Triumph, of course, makes loads of bras, and women buy them. They do not buy these bras, however. Nor do they buy Triumph's solar-powered bra or its rice-growing bra or whatever. These are like "concept bras" more than anything. Fun to look it, but not ready to drive.
That is all. Be sure to check my Night Note on Monday when I don't post Japanese bras!
Hey, Korea, Why You So Down On Starcraft II?


Beautiful, straight legs and feet are indispensable for a good look. But it's also important for your health to take good care of your feet and legs. Parents can save their children a lot, if at a young age the right footwear and a healthy diet is respected.
As long as we still struggle in Mother's stomach, our legs are bent and we are born with O-legs. This is normal and no mother should worry if her baby will walk like John Wayne later. The correct attitude of a newborn is reminiscent of a small frog. This is because the hip joints are not directed to the side as an adult, but more forward.
Ideal for healthy development is therefore a kind of squatting position, in which the legs are kept at 90 degrees. If you look closely, you can see this leg position in babies being carried on their hips by their mother. The femoral heads lie optimally in the joint sockets. A sling can be a reasonable solution. However, it must be adjusted properly and neither mother nor child may be harmed by improper carrying techniques.
To prevent hip joint damage, such as hip dysplasia, the baby's legs should not hang limp down, otherwise the thighs may be forced out of their pans.
The first steps we take on our O-legs. This is advantageous because the gait becomes so stable and we tend to find our balance in the wide gait. But at least three years later, the picture changes. Children of this age have X-legs, but they should have grown to school maturity.
Also, the impression that toddlers have flat feet, deceives quite often. It is rather a fat pad in the midfoot area that gives this impression. But these fat pads disappear soon and leave in almost all cases, a beautiful, slightly curved arch of the foot back.
If parents are unsure whether their child is not suffering from leg malformations that go beyond normal levels, it is often worth having a visit to the pediatrician and going to an orthopedist specializing in children.
Important signs of a malposition requiring treatment may be the distance between the knees or the ankles when the legs are closed. In the first grader should - depending on O-leg or X-leg suspicion - be measured at these locations less than ten inches distance.
Extreme O-legs are very rare in our society. In former times, they were often the result of a lack of vitamin D. Although our organism can produce this vitamin with the help of solar radiation itself to a sufficient extent. However, we live in a relatively sun-poor area, so our babies often come thickly packed in the open and therefore get relatively little sun.
Often, therefore, the missing vitamin is given as a nutritional supplement, usually in conjunction with fluoride, to aid tooth development.
Only 20 percent of all foot deformities are innate. Many forms are the result of incorrect footwear, everyday stress, nervous disorders or arise due to other causes such as rheumatism, arthritis or accidents. The flat foot, in which the arch of the foot has sunken, is caused in many cases by weak connective tissue.
Other foot deformations are for example the buckling, lowering, splayfoot and many more. The range of possible misalignments is great. No wonder that this field could develop into a field of medicine.
Especially women torment their feet and legs with chic, but extremely unhealthy footwear. The big toe is squeezed in by tapering shoe ends inside, high heels prevent the normal rolling of the foot. The foot musculature atrophies and can not support the vault of the foot sufficiently. A healthy foot can get so sick.
Important is the freedom of movement!
Barefoot is healthy, shoes must support the foot, but not constrict. And what is right for children is also true for adults. Sure is against fancy, high heels now and then nothing wrong. But every day and over long distances such shoes damage the feet and can cause diseases such as hallux valgus. Constant pain from bursitis and corneal formation are the result.
Parents should be a role model for their children. Because even for the little ones is: Dear non-slip socks in the apartment as a tight footwear.
Winter depression - what to do?


As a technical partner of many large companies, we are always actively working on the development of our approach. Techniques and frameworks are increasing in number and becoming more complex. It requires a strong vision to stay ahead in the adoption curve and to track the best innovations. Smart application architecture and the right choices for technical platforms are the foundation for a successful digital future.
As a reputable Sitecore partner (Silver) we see our customer's demands changing. "In the past, a customer would ask whether we would like to build them a platform in a predetermined format. Nowadays, the focus is on the digital ambitions. ", Rob Habraken, Sitecore MVP at We are you. "We therefore start by defining a concept and strategy. Only then do we select the appropriate technology with the customer."
Looking at the technology we see a shift in the application architecture. In the past, the monolith was the standard: horizontally stacked layers of functionality in one big application. Nowadays we select standalone applications (microservices) and APIs much more often. The independent components allow you ensure that the maintenance, replacement or expansion of specific functionality is a lot easier.
The future-proof flexible architecture today consists of a fusion of best-of-breed technologies. The adoption of external frameworks for certain functionalities is, according to Rob, not only powerful because of their flexibility: "I am thinking of the application of Identity Providers, DAM tooling or, more technically, GraphQL for more efficient communication between different application layers. Such frameworks reduce customization and enable our focus to be primarily on adding value to our customer's business."
"By embracing this technique, we have a shorter time to market for our customer with less effort required. We contribute to our customer's business using technology. "
The same trend in components can also be seen in modern Cloud hosting architecture. You increasingly only host the application that is required for a specific function, such as Platform-as-a-Service (PaaS). Sometimes you even host only specific functions or application components, such as the serverless approach, of which Functions-as-a-Service (FaaS) is a known component.
According to Rob, the gains in the shift to an application architecture based on components are on three levels: "The user experience improves. You remain relevant because of your flexibility. The time to market is shorter and we can pay more attention to adding real business value for our clients. Everyone wins."


With its warm, grainy finish and cool asymmetry, the Tonnari TV stand with fireplace option sure takes center stage. Its two-tone treatment merges a lightly finished butcher block design with sleek black for a fresh, modern twist. Compatible with not only the W100-22 large fireplace insert but also our W100-32 split integrated audio insert for superior sound quality, this TV stand that fits a 75″ television puts on a show in more ways than one.
Made with Mango and Acacia veneers and select hardwood solids in a two-tone, dark brown and light brown finish scheme that highlights the textural and natural grain characteristics. Asymmetrical design with a butcher block veneer layup on the door. Thick tops with waterfall veneer look on edge. Cord management openings and vent holes throughout. Large center opening that is compatible with our W100-22 large fireplace units or use the shelving insert. Fits a 75″ TV.


text:
'Made in Chelsea' Has a New York Moment
The hit British reality show is debuting its New York episodes. What happens when the young, rich characters bring their hard-partying, bed-hopping antics stateside?
Tim Teeman
Senior Editor and Writer
It was only a matter of time before the hedonistic yet anguished Sloane Rangers of Made in Chelsea, the hit British reality show, came to spread their well-spoken dysfunction to Manhattan. Not content to bitch and glare at each other on the King's Road, the rich and entitled young people are now in New York, braying about "paaaaarties" and petty betrayals above the honking of taxis.
The transatlantic transfer is shameless. Nothing, absolutely nothing—bar the introduction of a few sundry American characters—has been changed for the show's American incarnation. And so, just as implausibly as the characters once bumped into each other—"Oh, hi, amazing!"—on the streets of London, do they now in New York. Sure, coincidences happen, but these guys make big cities look like one-horse villages.
And still, according to the MiC vision, does New York for Brits mean pictures of the skyline and Empire State Building, and melting shots of 34th Street and 5th Avenue road signs. It is New York for visual dummies. There's a little too much amazement among the British cast at meeting actual Americans, even though they're in America, but perhaps their TV fishbowl is so small and incestuous New York might seem a little more intimidating to populate.
Of course, they are not cowed. The partying starts immediately with a group of extras, with the men whooping they are going to take the city, and fist-pumping at the skyline. They are embarrassing fools—and really, don't be misled by their cut-glass accents. This may be futile advice: One of the points a male cast member makes is that the American accent goes by unnoted in London, whereas—mysteriously—the British accent in New York is still prized and delighted over. It means, they say, they can have sex with any American girl they want. Charmers, all.
It may have all the trappings of luxe and money, but the Made in Chelsea world is, like Jersey Shore, about a group of young people falling in and out of bed with each other, drinking, partying, and feuding—all dizzyingly quickly. The show is as precisely plotted as a soap opera and beautifully lit; entirely, fatally addictive. The difference with MiC is that its participants are incredibly rich and posh, and the show, shot as if through gossamer Instagram filters, is the TV equivalent of soft cashmere.
The show is six seasons old in Britain. But the young hoorays have run out of streets in London SW3 to bump into each other, hence the trip to New York. In their own steam-rolling way, the Made in Chelsea cast achieve the impossible and make New York look small. "I'll be in Central Park," the philanderer Alex tells his much-wronged girlfriend Binky, and somehow those words enshrine precise geographical co-ordinates, because Binky finds Alex to tell him what a shit he is, and why she has come to New York to escape him and get over him.
And then Jamie, a happy-go-lucky blond candy-maker, finds Alex in Central Park to tell him how awful he is for allegedly hooking up with his ex-girlfriend Tara. Jamie bursts into the most embarrassing set of man-tears ever to flow forth on television.
Minutes before, cameras in perfect position, two other characters met, supposedly randomly on a park bench. They flirt, swap numbers, next scene. The girls typically bitch about the boys in tea shops, the boys play poker ("Fucckiing aces") and exchange boy-talk about girls that—sweetly—is every bit as gossipy and shrill as the girls'. "No wayyyyy, I don't believe it" is the show's cross-gender, most over-used phrase.
In the New York episodes, there is one new American female character, Billie, and there are two New York guys joining the British guys ("What do you have to do to pick up a British chick?"); one a roughish, raffish douche with a beard called Alik, the other a creepy, smoother, Ken Doll-type called Jules. Their presence makes you think all the better (worryingly) of all the arrested development-struck British men.
The key plot device of the Americans so far is to underscore how differently both countries perceive the notion of dating: in the U.S. you can date many simultaneously, in Britain, it is done one at a time, or you are immediately an adulterous wretch.
The irony that Made in Chelsea seeks to make this point is that none of the sexually carnivorous characters have much time for either notion in either country: They hop in and out of bed with each other as if these were involuntary body reflexes.
If they missed its quiet debut ages ago on the Style channel, American viewers can pick up the Made in Chelsea basics via the New York episodes, helpfully uploaded to YouTube.
Spencer is the resident man-slut who sees himself as a one-man axe in a forest of gratefully swaying female trees. He dumps or cheats on his girlfriends. His cheek is very used to being slapped. Jamie is his best friend, sweet and luckless, who pines after Lucy, the female approximation of Spencer—oh, hang on, she and Spencer were together once—who none of the girls like, and then they do like her, even though she will casually walk into a party as she does in the first New York episode and call someone a bitch and smile thinly.
The men are as catty as the women in MiC. In order to defend himself against Jamie, the shabby Alex, says: "You're no angel, mate. You chased Phoebe. You ratted me out to Lucy, you told Lucy about the orgy." And so it goes on. We rarely see these over-subsidized reprobates engaged in any kind of meaningful employment.
One of the guys, Proudlock, seems to do something with T-shirts. Cheska—blonde, tall, fretful, a human being with thoughts and sensitivities and therefore an alien in MiC—does something in public relations. The most outrageous non-workers are Victoria and Mark Francis, a bitching tag team, their noses permanently turned up at the world. A bad day for Mark Francis would involve having to take public transport and having the crusts left on his bread. They are mostly separate from the main group and their most memorable scene in New York so far saw them taking sanctuary in a church for the peace and quiet, although I think Mark was probably after some stained-glass window design inspiration.
It is never too late to catch you up on all six seasons of this phenomenal show. But soon enough, watching "New York," you'll wonder, a little too obsessively, if creepy Ken-toy Jules will romance Rosie or Lucy; how Binky will exorcise the ghost of the no-longer bearded Alex; whether Louise, long recovered from being Spencer's most grievously injured relationship roadkill, will get it on with Alik; and whether Billie, the new American female character, will ultimately choose master-player Spencer or the seemingly sweeter Stevie.
Rosie having a romance storyline is, for a fan, radical in itself. She usually restricts herself to judging and bitching; but this awakening has been hastened in New York by feeling a yoga teacher's cock imprinted on her back as he rearranged her pose. As she relayed the anecdote the normally unshockable group was—as the viewer at home—open-mouthed at the sudden emergence of potty-mouthed, horny Rosie. More, please.
In between the romances and confrontations, you will be seduced by MiC's ridiculous shots of champagne glasses being filled, with its soft, fizzy sound; the trays of canapés and cakes that no one eats; the whooping rooftop parties illuminated by twinkly lights; sun-kissed days by the pool, the boys as smooth of body and toned as the girls.
You'll wonder why the characters stare at each other for a second too long, without anyone saying, "Why are we not saying anything to each other?" There is ridiculous, swelling music as people walk out on each other's lives, car doors slammed, yearning looks to the right of the camera, lives torn asunder…until next week's episode when everyone reconfigures again.
The show seeks to suggest, as Dynasty and Dallas did, that rich people suffer too. But, just as Dallas and Dynasty did, it also supposes that these agonies are best recovered from with day spas and endless champagne. You become used to the quick reconciliations after volcanic confrontations, the tossing of glossy hair, and of course the promises that "Tonight's going to be great, we're going to have so much fun."
And you know, just as they would say the same thing every week in The Hills, that no one, ever, ever, ever, has an evening out on MiC (or The Hills) without a bitch-fight, flung glass of wine, and an evening culminating in tears on the curb.
These young British poshos are every bit as uncouth as Snookie and the gang. Bad behavior, especially on scripted reality shows, is universal. That Made in Chelsea has a story editor listed on its end credits is no surprise; it is blatantly set up. That shouldn't lessen the pleasure. Indeed, see it as a starter kit. The British channel E4, which shows Made in Chelsea in the UK, is currently making another reality show featuring a bunch of good-looking Brits trying to make it in Manhattan, and drinking and bed-hopping as they go. The early word is that Taking New York may be just as irresistible as Made in Chelsea. And if their horny hijinx leave you cold, well...there's always Dame Maggie and Downton Abbey.


text:
H.S. baseball: Waldwick's Mike Salmonese on a hot streak
by NJIC Web Master | May 3, 2015
By MARK J. CZERWINSKI
STAFF WRITER | The Record
WALDWICK – The streak began almost a year ago.
Mike Salmonese had settled nicely into a personal comfort zone.
And the Waldwick senior only has enhanced his reputation as one of North Jersey's most productive hitters, swinging the bat at a .556 clip, while riding a 21-game hitting streak stretching back to last May 19.
"It just started to click for him," said Waldwick coach Frank Clark, whose team is ranked No. 13 in The Record baseball Top 25. "Now, everything is on a line, gap to gap."
But it is interesting to note that Salmonese's comfort zone has expanded to include center field. A top shelf shortstop who likely will play second base for Division II Pace University, Salmonese moved to the outfield this season in an effort to sure up the Waldwick defense.
"In Group 1, you've got to be creative," Clark said. "He's our best overall athlete. He's been getting to balls he has no business getting to, covering everything gap to gap."
The 5-foot-6, 145-pound senior said center field was "a little weird at first," especially since he had to change his throwing motion. He hasn't made an error in 16 games at a new position, handling 33 chances flawlessly and picking up five assists.
"Don't get me wrong, I love the infield," Salmonese said. "It's my thing. But center field has been fun, even though I don't get as many chances as I did at shortstop."
Salmonese said before the season that he had to work on his mental approach to the game, and so far, he's pleased with the results. It took a while for him to get comfortable as the focal point in a good offensive lineup, but now Salmonese is just churning out hits.
Salmonese has two or more in 11 straight games, including 24 hits in his last 38 at-bats (.632). He had a four-hit game Thursday against Leonia and has scored at least one run in 15 games with a .581 on base percentage.
"He's also not afraid to take a walk," Clark said. "When his game is right, a walk could be the same as a triple."
For the season, Salmonese has 30 hits, including six doubles, three triples and 12 RBI. He's stolen 20 bases, scored 27 runs and struck out five times in 62 plate appearances.
http://www.northjersey.com/sports/h-s-baseball-waldwick-s-mike-salmonese-on-a-hot-streak-1.1323950


text:
Le District de Cachar (en assamais : কাছাড় Kasaŗ, en bengali : কাছাড় Kachhaŗ, Sylheti: কাছাড় জেলা)  est un district de l'état d'Assam en Inde.

Géographie
Il couvre une surface de , et sa population est de  habitants en 2011.

Son chef-lieu est Silchar, l'ancienne Khaspour. C'est aujourd'hui une des plus importantes villes d'Assam et l'une des six à disposer d'un aéroport.
La langue officielle au Cachar est le bengali.
Curieusement, bien que les États indiens soient constitués sur une base linguistique, le Cachar reste partie de l'Assam et n'a pas été rattaché au Tripura voisin, qui partage avec lui le bengali.

Liens internes
Liste des districts de l'Assam

Liens externes

Références

District en Assam

redpajama_set_name: RedPajamaWikipedia


text:
Kid's Page
Welcome Kids..
Free Tutorials!
This week's special...
Story Time:
Ducky duck's Mischief
He could not sleep at night, his heart was heavy and he was cussing himself for his bad habits. But he had no idea how to come out of his bad habits. He spent whole night crying. He was afraid to tell anybody about his mischief. He prayed to God and said, "I will not commit this type of mischief anymore..........
For full detail click here....
This Week's Riddle: #48
God promised to whom saying, "I will make you father of many nations"?
Send your answers
Find out details....
Answer of Riddle: #47 and Winner
Find out....
Home | Games | Stories | Historical Events | Riddle Archive
World Travel | Quiz and Riddles | Rack 1 | Email


Toronto (Canada) From 24 to 27 January 2019, IOM's General Director Tomasz Abramowski payed a technical visit at the CVMR company. Engaged in mining and metallurgical processing of metals and metal ores, the CVMR company is the owner of the carbonyl technology (http://www.cvmr.ca). The patented technology is used for metal extraction, especially of high purity metal powders, e.g. nickel.
The meeting was held with the view of CVMR cooperation with Interoceanmetal in the scope of ore extraction for metallurgical processing and employment of carbonyl technology in metallurgical processing of polymetallic nodules. Attended by Kamran Khozan, CEO of CVMR, and Tomasz Abramowski, IOM's General Director, the meeting discussed possibilities for further cooperation within which the carbonyl technology would be included in the IOM's activity plans and economical models. CVMR declared cooperation for providing the relevant data. CVMR has already tested, at its own cost, processing of nodules delivered by IOM. Possible fields of cooperation within the framework of Horizon 2020 programme were also considered, those included the possibility of performing metallurgical processing on board of an mining support vessel.


Trevor Pateman's Philately Blog: Soviet and Post Soviet Covers ... What Do You Think of These?
Soviet and Post Soviet Covers ... What Do You Think of These?
(1) On this first card, the bisected stamp does contribute the necessary 5 kopecks to make up the 10 kopeck postcard rate of 1 June 1931. The card was posted at ALEXANDROV VOKSAL 1 8 31 and arrived at BERDYANSK 3 8 31. There is a long family message in German on the back but at the end the sender writes, " Diese Karte bitte züruck" - Please send this card back (and underlined). So the sender knew that the card was philatelically interesting and maybe was responsible for the bisect. However, when this card was illustrated in The Post Rider in November 1998, Andrew Cronin commented that another bisect is known from the same region [ Added: See now below]. If the sender is not the same person, then the bisect may be non-philatelic but recognised by the sender as philatelically important. The fact that the post office clerk has cancelled the bisect carelessly does suggest that the clerk did not think of this card as philatelic. And another things I have just noticed: the pencil used for the "Please send this card back" is not the same pencil as was used to write the long message! The sender added it later ... So is this card worth 10 € or 1000 € ? Comments?
Added: Vasilis Opsimos has copied me the Andrew Cronin article from The Post Rider 1998 which gives the date of first known use as 17 7 31 from Khortytsa when a card with a bisect was sent to the German stamp dealers and catalogue makers, Senf, in Leipzig. They announced the local bisect in the German philatelic press. We do not have an illustration of this card. Cronin does illustrate a card from KHORTYTSA 3 2 32 to Kiev, and translates the Russian message which makes reference to the bisect. Opsimos copied me the following images of a card sold on ebay (for $355) and going from ALEXANDROVSK VOKSAL 18 11 31 to Vienna. This card is also from a philatelist.
So in total we now have 5 recorded cards, all with bisects of the 1905 Revolution commemoration 10 kopeck. Two are internal uses, two are used to Germany and one to Austria (inland and foreign Tariffs were the same). Three are written in German and one in Russian. Three are from Khortytsa and two from Alexandrov Voksal. The period of use is just over a year. The message on the Raritan card with the use of the word "Gelegenheit" [Opportunity]strongly suggests these cards were linked to the German colony of Khortytsa and may have had their origins as a local post office provisional in July 1931, even if they were then sought out by philatelists. It may be relevant that at this time travelling in Ukraine would not have been a straightforward matter.
(3) Finally, a post-Soviet example of an interesting but frustrating 1992 Belarus cover. Here the address and the sender's address suggest that this is non-philatelic (no Box Numbers, as one so often sees on philatelic confections of this period). But are these Manuscript revaluations the work of the NOVO POLOTSK VITEB[sk] 22 12 92 post office? That question can only really be answered YES if you can find other examples of these revaluations going to other addresses from other senders. Does anyone have an example?


text:
<!-- Check the boxes with 'x' like '[x]' -->
- [ ] A new feature/enhancement.
- [ ] Fix an issue/feature-request.
- [ ] An improvement to existing modules.
- [ ] Other (Please mention below).

#### Where has this been tested?
<!-- Check the boxes with 'x' like '[x]' -->
- Python Version
    - [ ] v3.x
    - [ ] v2.x
- Operating System:
    - [ ] Linux (Please specify distro)
    - [ ] Windows
    - [ ] MacOS

#### Does this close any currently open issues?
[Mention any issue which this PR closes]

#### Does this add any new dependency?
[Mention if this PR includes any new library]

#### Does this add any new command line switch/argument?
[Mention if the changes add any new arguments like `--arg`]

#### Any other comments you would like to make?
[Anything more you'd want the reviewer to know]

redpajama_set_name: RedPajamaGithub


This entry was posted on Monday, August 15th, 2011 at 10:10 am	and is filed under General bike news. You can follow any responses to this entry through the RSS 2.0 feed. You can leave a response, or trackback from your own site.
Thank you for you alert! I contacted my commissioner and he is a Republican. Hopefully he listens to my comments and votes to support this resolution. It would be a great first step for the county.
I contacted my (R) commissioner as well. Thanks for the alert!
That's great — and the result is the resolution passed this morning. I'll post more about it soon.


text:
Rapid generation of a mouse model for Middle East respiratory syndrome
Jincun Zhao, Kun Li, Christine Wohlford-Lenane, Sudhakar S. Agnihothram, Craig Fett, Jingxian Zhao, Michael J. Gale Jr., Ralph S. Baric, Luis Enjuanes, Tom Gallagher, Paul B. McCray Jr., and Stanley Perlman
PNAS April 1, 2014 111 (13) 4970-4975; https://doi.org/10.1073/pnas.1323279111
Jincun Zhao
Departments of aMicrobiology and
Kun Li
bPediatrics, University of Iowa, Iowa City, IA 52240;
Christine Wohlford-Lenane
Sudhakar S. Agnihothram
cDepartments of Microbiology and Immunology and of Epidemiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599;
Craig Fett
Jingxian Zhao
Michael J. Gale
dDepartment of Immunology, University of Washington School of Medicine, Seattle, WA 98109;
Ralph S. Baric
Luis Enjuanes
eDepartment of Molecular and Cell Biology, Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas, Campus Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain; and
Tom Gallagher
fDepartment of Microbiology and Immunology, Loyola University Medical Center, Maywood, IL 60153
Paul B. McCray
Stanley Perlman
For correspondence: stanley-perlman@uiowa.edu
Edited by Michael B. A. Oldstone, The Scripps Research Institute, La Jolla, CA, and approved February 21, 2014 (received for review December 16, 2013)
The Middle East respiratory syndrome (MERS)-coronavirus, a newly identified pathogen, causes severe pneumonia in humans, with a mortality of nearly 44%. Human-to-human spread has been demonstrated, raising the possibility that the infection could become pandemic. Mice and other small laboratory animals are not susceptible to infection. Here, we describe the development of a small-animal model for MERS, in which we use an adenovirus expressing the human host-cell receptor to sensitize mice for infection. We show that these mice are useful for determining immune responses and for evaluation of an anti-MERS vaccine and an antiviral therapy. This approach will be generally useful for the rapid (2–3 wk) development of relevant mouse and other animal models for emerging viral infections.
In this era of continued emergence of zoonotic virus infections, the rapid development of rodent models represents a critical barrier to public health preparedness, including the testing of antivirus therapy and vaccines. The Middle East respiratory syndrome coronavirus (MERS-CoV) was recently identified as the causative agent of a severe pneumonia. Given the ability of coronavirus to rapidly adapt to new hosts, a major public health concern is that MERS-CoV will further adapt to replication in humans, triggering a pandemic. No small-animal model for this infection is currently available, but studies suggest that virus entry factors can confer virus susceptibility. Here, we show that mice were sensitized to MERS-CoV infection by prior transduction with adenoviral vectors expressing the human host-cell receptor dipeptidyl peptidase 4. Mice developed a pneumonia characterized by extensive inflammatory-cell infiltration with virus clearance occurring 6–8 d after infection. Clinical disease and histopathological changes were more severe in the absence of type-I IFN signaling whereas the T-cell response was required for virus clearance. Using these mice, we demonstrated the efficacy of a therapeutic intervention (poly I:C) and a potential vaccine [Venezuelan equine encephalitis replicon particles expressing MERS-CoV spike protein]. We also found little protective cross-reactivity between MERS-CoV and the severe acute respiratory syndrome-CoV. Our results demonstrate that this system will be useful for MERS-CoV studies and for the rapid development of relevant animal models for emerging respiratory viral infections.
emerging pathogen
interferon
The spread of the severe acute respiratory syndrome (SARS)-coronavirus (CoV) in 2002/2003 and of the Middle East respiratory syndrome (MERS)-CoV in 2012 indicated that coronaviruses could cause severe pneumonia in humans (1, 2). As of February 7, 2014, 182 patients had been infected with MERS-CoV, with a 43.4% mortality rate. Human-to-human spread has been documented (3). A majority of patients with severe disease were elderly and had preexisting illnesses such as diabetes or renal failure whereas immunocompetent patients mostly developed mild disease (4). The pathogenesis of the infection is not well understood, in part because no autopsy information is available. Experimental infection has been demonstrated only in macaques (5⇓–7). The expense and limited availability of these animals makes it imperative to generate a small-animal model for MERS for development of vaccines and antiviral therapies. Mice and hamsters are not infectable, and, because virus entry factors can confer virus susceptibility (7, 8), we postulated that exogenous expression of human host-cell receptor dipeptidyl peptidase 4 (hDPP4) would render mice susceptible (9).
Here, we describe a novel approach to developing a mouse model for MERS by transducing mice with a recombinant, nonreplicating adenovirus expressing the hDPP4 receptor. After infection with MERS-CoV, mice develop an interstitial pneumonia. We show that these transduced, infected mice can be used to determine antivirus immune responses and to evaluate anti–MERS-CoV vaccines and therapies.
Development of Mice Susceptible to MERS-CoV Infection.
Adenoviral vectors have been used for gene therapy and to sensitize mice to systemic infection (10⇓⇓⇓–14). However, their ability to render mice susceptible to mucosal infections, including those of the respiratory tract, has not been examined previously. Second-generation E1/E3-deleted Ad5 vectors are configured to minimize outgrowth of wild-type (WT) virus and to reduce immunogenicity (15). To develop adenoviruses for expressing hDPP4 in mouse lungs, we cloned a FLAG and myc-tagged cDNA into a replication-deficient adenovirus (Ad5-hDPP4). hDPP4 expression was validated by transducing MLE15 cells, a mouse alveolar type-II cell line, with Ad5-hDPP4 and analyzing cell lysates for hDPP4 expression. hDDP4 was detected using anti-hDPP4 or anti-FLAG antibodies (Fig. 1A). Surface expression of hDPP4, required to enable virus entry, was demonstrated by flow cytometry (Fig. 1B). To determine whether hDPP4 rendered cells susceptible to MERS-CoV, we infected Ad5 control (Ad5-Empty) and Ad5-hDPP4–transduced cells with MERS-CoV. Control cells were resistant to MERS-CoV whereas MERS-CoV replicated to high titers in Ad5-hDPP4–transduced cells (Fig. 1C). To identify hDPP4 expression in mice, we transduced BALB/c mice with 2.5 × 108 pfu of Ad5-hDPP4 or Ad5-Empty and detected DPP4 with an antibody that recognized human and mouse DPP4. After Ad5-Empty transduction, only endogenous mouse DPP4 expression was detected whereas, after Ad5-hDPP4 transduction, there was widespread hDPP4 expression in airway and alveolar epithelial cells (Fig. 1D). Next, we infected 6- to 12-wk-old and 18- to 22-mo-old C57BL/6 and BALB/c mice with MERS-CoV 5 d after Ad5-hDPP4 transduction (Fig. 1 E and F). We expected that innate responses triggered by the Ad vector would be largely dissipated by 5 d after transduction (16). Consistent with this, histological examination revealed no evidence for inflammatory-cell infiltration after Ad5-hDPP4 transduction but before MERS-CoV infection (Fig. 1H and Fig. S1, day 0). After MERS-CoV infection, there was no mortality, but young BALB/c mice failed to gain weight and aged mice of both strains lost weight. Transduced hDPP4 was required for virus replication (Fig. 1D), which reached ∼107 pfu/g lung tissue by 2–3 days postinfection (d.p.i.), (Fig. 1 E and F). After MERS-CoV infection, virus was then cleared by days 6–8 in young mice and days 10–14 in 18- to 22-mo-old mice. Clearance did not reflect loss of hDPP4 expression because mice could be infected as late as days 17–22 posttransduction, dependent upon strain (Fig. 1G). As expected, viral antigen was detected in the lungs of mice transduced with Ad5-hDPP4 but not control vector (Fig. 1D). After MERS-CoV infection, we identified perivascular and peribronchial lymphoid infiltration initially, with progression to an interstitial pneumonia at later times postinfection (p.i.) (Fig. 1H and Fig. S1).
Development of mice susceptible to MERS-CoV infection. To assess hDPP4 expression (A) and surface localization (B), MLE15 cells were transduced with Ad5-hDPP4 or Ad5-Empty at an MOI of 20 at 37 °C for 4 h. hDDP4 expression was monitored by Western blot assay (A) or flow cytometry (B). (C) Ad5-hDPP4–transduced cells were infected with MERS-CoV at an MOI of 1 at 48 h posttransduction, and virus titers were determined by plaque assay. Five days after transduction with 2.5 × 108 pfu of Ad5-hDPP4 or Ad5-Empty in 75 μL of DMEM intranasally, mice were intranasally infected with 1 × 105 pfu of MERS-CoV in 50 μL of DMEM. (D) Lungs were harvested from BALB/c mice at day 3 after MERS-CoV infection, fixed in zinc formalin, and embedded in paraffin. Sections were stained with an anti-hDPP4 or with an anti–MERS-CoV nucleocapsid antibody (blue signal). (Original magnification, 20×.) (E and F) Weight changes in 6- to 12-wk-old (young) and 18- to 22-mo-old (aged) B6 (E) and BALB/c (F) mice were monitored daily. For B6 mice, n = 8 in Ad5-Empty group; 12 in Ad5-hDPP4 group; 8 in Ad5-hDPP4 aged group. For BALB/c mice, n = 8 in Ad5-Empty group; 12 in Ad5-hDPP4 group; 8 in Ad5-hDPP4 aged group. To obtain virus titers, lungs were homogenized at the indicated time points and titered on Vero 81 cells. Titers are expressed as pfu/g tissue (n = 4–8 mice per group per time point). Data are representative of two independent experiments. Δ, P < 0.05 when Ad5-hDPP4 aged were compared with Ad5-hDPP4 and Ad4-Empty. (G) To evaluate the length of time that Ad5-hDPP4–transduced mice could be infected with MERS-CoV, Ad5-hDPP4–transduced mice were infected with MERS-CoV at the indicated times. Lungs were harvested for titers at 2 d.p.i. (n = 4 mice per group per time point. (H) Lungs from B6 mice were removed at the indicated time points p.i., fixed in zinc formalin, and embedded in paraffin. Sections were stained with hematoxylin/eosin.
Requirements for Type-I IFN Induction and Signaling in MERS-CoV Clearance.
There are roles for both the innate and adaptive immune responses for protection from coronavirus infection (17). Initially we showed that depletion of natural killer (NK) cells, a cellular component of the innate immune response, did not change clinical disease or the kinetics of virus clearance (Fig. S2). MERS-CoV does not induce significant amounts of IFNα/β expression in vitro (18⇓–20), but the role of type-I IFN induction and signaling in vivo is unknown. IFN is induced via RIG-I–like receptors (RLRs) and Toll-like receptors (TLRs) in coronavirus infections (21). To determine the role of each in MERS-CoV–infected mice, Ad5-hDPP4–transduced mice impaired in RLR [mitochondrial antiviral signaling protein−/− (MAVS−/−)] or TLR [myeloid differentiation primary response gene 88−/− (MyD88−/−)] signaling were infected with MERS-CoV (Fig. 2A). Infection of MyD88−/− but not MAVS−/− mice resulted in up to 20% weight loss. Without type-I IFN signaling (IFNAR−/−), infection was even more severe than in MyD88−/− mice, with weight loss beginning 2 d earlier p.i. Virus was cleared with the same kinetics in MAVS−/− and WT mice, but clearance was delayed in both MyD88−/− and IFNAR−/− mice (Fig. 2B), suggesting that TLR-dependent and IFN signaling pathways were required for MERS-CoV control. Consistent with these results, we observed increased vascular congestion and inflammation on gross pathological lung specimens from infected MyD88−/− and IFNAR−/− mice (Fig. 2C). Histological examination of Ad5-hDPP4–transduced, MERS-CoV–infected IFNAR−/− compared with B6 mice revealed earlier onset of peribronchial, perivascular, and interstitial infiltrates, relative to B6 mice (Figs. 1H and 2D). Alveolar thickening and edema and increased infiltration of granulocytes, especially eosinophils, were observed only in infected IFNAR−/− lungs (Fig. 2E). To further address the role of IFN induction and signaling in MERS-CoV protection, we transduced B6 mice with Ad5-hDPP4 and, 5 d later, treated them with poly I:C, a TLR3 agonist that signals through the MyD88-dependent pathway, or with IFN-β or IFN-γ. We then infected mice 6 h later with MERS-CoV. The treatments, particularly poly I:C and IFN-β, accelerated virus clearance (Fig. 2F) without appreciably affecting weight or extent of inflammatory-cell infiltration (Fig. S3). Poly I:C delivered 6 h after infection also accelerated virus clearance, although to a lesser extent than if delivered before infection (Fig. S4). Of note, administration of IFN-α2b (with ribavirin) to MERS-CoV–infected macaques improved clinical, radiological, and virological parameters of infection (22).
Requirements for type-I IFN induction and signaling in MERS-CoV clearance. (A) Five days after transduction with 2.5 × 108 pfu of Ad5-hDPP4, mice were intranasally infected with 1 × 105 pfu of MERS-CoV. Weight changes were monitored daily (n = 8 in B6 group; 14 in IFNAR−/− group; and 13 in MyD88−/− group; n= 9 in MAVS−/− group). (B) Virus titers in the lungs were measured at the indicated time points. Titers are expressed as pfu/g tissue (n = 3–4 mice per group per time point). Data are representative of two independent experiments. *, P < 0.05 compared with B6 group; Δ, P values of <0.05 compared with IFNAR−/− group; #, P values of <0.05 compared with MyD88−/− group. (C) Photographs of gross pathological lung specimens isolated from infected mice at day 7 p.i. (D) Sections of paraffin-embedded lungs from Ad5-hDPP4–transduced, infected IFNAR−/− mice were stained with hematoxylin/eosin. (E) Edema (asterisks) and infiltrating eosinophils (arrows) are indicated. (F) Ad5-hDPP4-–transduced mice were treated with 20 μg of poly I:C, 2,000 units of IFN-β, 200 ng of IFN-γ, or PBS in 50 μL of DMEM 6 h before intranasal infection with 1 × 105 pfu of MERS-CoV. Viral titers in lungs were measured at the indicated time points (n = 4 mice per group per time point. Data are representative of three independent experiments. *, P < 0.05 compared with PBS group; Δ, P values of <0.05 compared with poly I:C group; #, P values of <0.05 compared with IFN-β group.
Requirements for CD8 T Cells and Antibodies for MERS-CoV Clearance and Protection from Subsequent Challenge.
To examine the role of T- and B-cell responses in protection against MERS-CoV, we infected Ad5-hDPP4–transduced mice deficient in T cells [T-cell receptor α−/− (TCRα−/−)], B cells (μMT), or T and B cells [recombination activating gene 1−/− (RAG1−/−) severe combined immunodeficiency (SCID)] and their corresponding controls (Fig. 3 A and B). Virus was not cleared in mice lacking T cells (TCRα−/−, RAG1−/−), or in SCID mice but was cleared in μMT mice. Despite the persistent infections, none of these mice lost weight (Fig. S5). Although T cells are important for acute virus clearance, protection against subsequent challenge is generally antibody-mediated. To generate a protective antibody response, we engineered Venezuelan equine encephalitis replicon particles (VRPs) expressing the MERS-CoV spike protein as previously described (VRP-S) (6). Immunization with VRP-S using a prime-boost regimen reduced MERS-CoV titers to nearly undetectable levels by day 1 p.i. Anti-S antibody, which blocked virus attachment, was largely sufficient for this effect because transfer of sera from VRP-S–immunized mice also accelerated the kinetics of virus clearance (Fig. 3C). To analyze CD8 T-cell activity in vivo, we identified several epitopes using peptides selected for consensus binding to the MHC class I antigen (Table S1) and used them in intracellular IFN-γ–staining assays (Fig. 3D). The immunodominant epitopes recognized in both B6 and BALB/c mice were located in the S protein. The CD8 T-cell response to these epitopes in Ad5-hDPP4–transduced B6 and BALB/c mice peaked at days 7–10 p.i. (Fig. 3E). Specific killing was confirmed in vivo because target cells coated with virus-specific CD8 T-cell peptides were efficiently lysed (Fig. 3 F and G).
Requirements for CD8 T cells and antibodies for MERS-CoV clearance and protection from subsequent challenge. (A and B) Ad5-hDPP4–transduced mice were infected with 1 × 105 pfu of MERS-CoV. Virus titers in the lungs were measured at the indicated time points. Titers are expressed as pfu/g tissue (n = 3–4 mice per group per time point. Data are representative of two independent experiments. *, P values of <0.05 compared with WT group; Δ, P values of <0.05 compared with RAG1−/− group; #, P values of <0.05 compared with TCRα−/− group. (C) BALB/c mice were immunized with 1 × 105 infectious units (IU) of VRP-GFP or VRP-S in the footpad in 20 μL of PBS and boosted with the same dose 4 wk later. Mice were transduced and infected with 1 × 105 pfu of MERS-CoV 2–4 wk after the booster. For adoptive transfer of serum, sera were obtained 2–4 wk after booster. Then, 300 μL of serum was transferred into transduced mice intraperitoneally 1 d before MERS-CoV infection. *, P values of <0.05 compared with VRP-GFP group; #, P values of <0.05 compared with VRP-GFP serum group. (D) To identify MERS-CoV–specific CD8 T-cell epitopes, single-cell suspensions were prepared from the lungs of transduced/infected mice and stimulated with 1 µM peptides for 5–6 h in the presence of brefeldin A. Frequencies of MERS-CoV–specific T cells (determined by IFN-γ intracellular staining) are shown. Kinetics of immune responses to dominant CD8 T-cell epitopes in Ad5-hDPP4–transduced B6 and BALB/c mice are summarized in E. Data are representative of five independent experiments. (F and G) In vivo cytotoxicity assays were performed on day 7 (BALB/c mice; peptides S395, S434, and S1165 combined) or day 8 (B6 mice; peptides S291, S823, and N214 combined) p.i. as described in Materials and Methods (F) and summarized (G) (n = 4 mice per group). Data are representative of two independent experiments.
Low Level of Cross-Reactivity Between MERS-CoV and SARS-CoV.
A critical question is whether SARS-CoV and MERS-CoV, which both likely originate from bat sources (23, 24), elicit cross-reactive, protective immune responses. To address this question, we infected young BALB/c mice (after Ad5-hDPP4 transduction) with either MERS-CoV (Fig. 4 A–C) or a sublethal dose of SARS-CoV (Fig. 4 D–F). BALB/c mice were used in these experiments because young B6 mice are resistant to SARS-CoV (25). After 5 wk, mice were challenged with SARS-CoV or (after Ad5-hDPP4 transduction) with MERS-CoV. Mice initially infected with MERS-CoV were as fully susceptible to subsequent challenge with SARS-CoV as DMEM-treated mice (Fig. 4 A–C). Regardless of the MERS-CoV immunization regimen (no Ad5, Ad5-empty, or Ad5-hDPP4), mortality and weight loss were equivalent (Fig. 4 A and B and Fig. S6 A and B). The reciprocal initial infection with SARS-CoV resulted in a statistically significant but minor decrease in MERS-CoV titers at day 5 p.i. after challenge (Fig. 4F). This decrease in titer may reflect a cross-reactive T-cell response because no differences were detected at early times p.i. Again, the immunization regimen (no Ad5 or Ad5-empty before SARS-CoV infection) did not effect any weight changes after MERS-CoV challenge (Fig. S6 C and D). As controls, mice were treated before infection with serum obtained from SARS-CoV– or MERS-CoV–infected mice, which conferred protection (Fig. 4 C and F).
Low level of cross-reactivity between MERS-CoV and SARS-CoV. (A–C) Ad5-hDPP4–transduced BALB/c mice were inoculated with 1 × 105 pfu of MERS-CoV or DMEM and rested for 5 wk before infection with 1 × 104 pfu of SARS-CoV in 50 μL of DMEM. Control mice received 300 μL of immune serum from mice previously (5 wk) infected with a sublethal dose of SARS-CoV (500 pfu). Mortality (A) and weight (B) were monitored daily (n = 12 in all groups). (C) To obtain virus titers, lungs were homogenized at the indicated time points and titered on Vero E6 cells. Titers are expressed as pfu/g tissue (n = 4 mice per group per time point). Data are representative of two independent experiments. *, P values of <0.05 compared with DMEM group; Δ, P values of <0.05 compared with MERS-CoV–immunized group. (D–F) BALB/c mice were infected with 500 pfu of SARS-CoV or DMEM and rested for 5 wk before Ad5-hDPP4 transduction and infection with 1 × 105 pfu of MERS-CoV. Control mice received 300 μL of immune serum from mice previously (5 wk) infected with 1 × 105 pfu of MERS-CoV. Mortality (D) and weight (E) were monitored daily (n = 12 in all groups). (F) Virus titers in the lungs were measured at the indicated time points (n = 4 mice per group per time point). Data are representative of two independent experiments. *, P values of <0.05 compared with DMEM group; Δ, P values of <0.05 compared with SARS-CoV–immunized group.
Here, we developed a novel platform strategy for sensitizing mice to MERS-CoV infection. We demonstrated that both innate, antibody, and T-cell responses are important for protection from MERS-CoV. Similar to infected patients, Ad5-hDPP4–transduced mice with normal immune systems developed mild disease whereas immunocompromised mice, like patients with underlying diseases, were more profoundly affected. MERS-CoV–infected mice were successfully used to evaluate an antiviral drug and a vaccine. Of note, poly I:C is inexpensive and has been approved for use in humans (26, 27). VRP-S induced a protective immune response (Fig. 3C); VRPs are excellent subunit vaccine candidates (28).
Although there are a few limitations of the Ad-hDPP4 transduction system (level of expression, tissue distribution), development of adenovirus vectors expressing the MERS-CoV receptor was efficient and rapid, resulting in the generation of an easily reproducible murine model for MERS-CoV within 2–3 wk. This short time course compares favorably with the much longer time (several months to years) required to develop transgenic or knock-in mice expressing the human receptor. Further, hDPP4 expression after AD5-hDDP4 intranasal inoculation is restricted to the lungs whereas transgenic expression may not be targeted to the correct organ (29, 30). Although hDPP4 expression may be more physiological in knock-in mice, the low level of native DPP4 expression in the mouse lung presents limitations (8). Another advantage of the Ad5-hDPP4 transduction strategy is that it can be used in genetically deficient mice, facilitating rapid identification of host genes and pathways that play protective or pathogenic roles in disease. Because Ad vectors are polytropic, this approach will have broad utility in rendering animals from multiple species susceptible to infection with emerging respiratory viruses, where speed of development is often critical to enable drug screening and vaccine validation.
Mice, Virus, and Cells.
Specific pathogen-free 6- to 12-wk-old and 18- to 22-mo-old C57BL/6 and BALB/c mice were purchased from the National Cancer Institute. RAG1−/−, μMT, TCR−/−, and IFNAR−/− mice were purchased from The Jackson Laboratory. MyD88−/− mice were obtained from Dr. S. Akira (Osaka University, Osaka, Japan) (31). MAVS−/− mice were obtained from Dr. S. Akira and developed as previously described (32). Mice were maintained in the animal care facility at the University of Iowa. All protocols were approved by the University of Iowa Institutional Animal Care and Use Committee. The EMC2012 strain of MERS-CoV (passage 8, designated MERS-CoV) was provided by Drs. Bart Haagmans and Ron Fouchier (Erasmus Medical Center). Mouse-adapted SARS-CoV (MA15) was a kind gift from Dr. Kanta Subbarao (National Institutes of Health, Bethesda, MD) (33). A human DPP4 ORF clone was purchase from Origene. Recombinant adenoviral vectors expressing-hDPP4 (Ad5-hDPP4) were prepared as previously described (15) by the University of Iowa Gene Transfer Vector Core at titers of 1010 ∼1011 pfu/ml in A195 buffer (29.5 mM histidine, 54 mM Tris⋅HCl, 10.8 mM MgCl2, 108 μM EDTA, 0.0216% Tween 80, 0.54% ethanol, 5% sucrose in PBS). African Green monkey kidney-derived Vero E6 cells and Vero 81 cells (CCL81; ATCC) were grown in DMEM (GIBCO) supplemented with 10% (vol/vol) FBS. MERS-CoV and SARS-CoV were passaged once on Vero 81 or Vero E6 cells, respectively, and titered on the same cell line, as described (34). MLE15 cells, a mouse type-II pneumocyte cell line, were cultured in 2% FBS RPMI 1640 supplemented with 5 μg/mL insulin, 5 μg/mL selenium, 10 μg/mL transferrin, 10 nM hydrocortisone, and 10 nM β-estradiol, all from Sigma and 2% FBS (35).
Chemicals, Cytokines, and Peptides.
Poly I:C (Sigma), IFN-γ (R&D Systems), and IFN-β (PBL) were purchased. MERS-CoV–specific peptides, predicted using online programs (RANKpep, SYFPEITHI, NetMHC 3.4) (36⇓–38), were synthesized by BioSynthesis Inc.
Transduction and Infection of MLE15 Cells and Western Blot Analysis.
MLE15 cells were transduced with Ad5-hDPP4 or Ad5-Empty at multiplicity of infection (MOI) = 20 for 4 h at 37 °C. Extracts were prepared 48 h posttransduction. Identical amounts of protein were separated on a 4–20% SDS/PAGE gel and transferred to PVDF membranes. Membranes were stained with a mouse anti-human DPP4 antibody (clone 11D7; Origene), a rabbit anti-FLAG antibody (Sigma), or a mouse anti–α-tubulin (clone DM1A; Sigma). Proteins were detected using a SuperSignal West Pico Trial Kit (Thermo Scientific). For infection, MLE15 cells were transduced with AD5-hDPP4 or Ad5-Empty for 48 h before infection with MERS-CoV (MOI = 1) at 37 °C for 1 h. Supernatants were collected at the indicated time points and analyzed for infectious virus by plaque assay.
Transduction and Infection of Mice.
Mice were lightly anesthetized with isoflurane and transduced intranasally with 2.5 × 108 pfu of Ad5-hDPP4 or Ad5-Empty in 75 μL of DMEM. Five days posttransduction or at the indicated time points, mice were infected intranasally with MERS-CoV (1 × 105 pfu), or SARS-CoV (500 pfu, sublethal, or 1 × 104 pfu, 1 LD50 dose) in a total volume of 50 μL of DMEM. Mice were monitored daily for morbidity and mortality. All work with MERS-CoV and SARS-CoV was conducted in the University of Iowa Biosafety Level 3 (BSL3) Laboratory.
Virus Titers.
To obtain virus titers, lungs were removed into PBS and homogenized using a manual homogenizer. Virus was titered on Vero 81 cells or Vero E6 cells. Cells were fixed with 10% formaldehyde and stained with crystal violet 3 d.p.i.. Viral titers are expressed as pfu/g tissue for MERS-CoV and SARS-CoV.
Histology and Immunohistochemistry.
Animals were anesthetized and transcardially perfused with PBS followed by zinc formalin. Lungs were removed, fixed in zinc formalin, and paraffin-embedded. Sections were stained with hematoxylin/eosin for histological analysis. A MERS-CoV–specific antibody was developed by immunizing rabbits with a peptide encompassing residues 244–257 of the N protein (AAAKNKMRHKRTST) per company protocol (BioGenes). For hDPP4 staining, high-pH microwave antigen retrieval was performed on sections using Antigen Unmasking Solution (Vector Laboratories) and a Mouse-on-Mouse (M.O.M.) kit (BMK-2202; Vector Labs) to reduce endogenous mouse IgG staining. Sections were then blocked using goat serum (MERS-CoV) or M.O.M.-blocking protein (DDP4) and incubated overnight with rabbit anti-hDPP4 antibody (TA500733, 1:800; Origene) or rabbit anti-N antibody (1:600). Sections were incubated with a biotinylated goat anti-rabbit secondary antibody (Vector Laboratories). Binding of the secondary antibody was detected with an alkaline phosphatase–biotin–avidin mixture. Sections were then visualized with Vector Blue (Vector Laboratories) and counterstained with Nuclear Fast Red.
Preparation of Cells from Lungs.
Mice were killed at the indicated time points. Lungs were removed, cut into small pieces, and digested in HBSS buffer containing 2% FCS, 25 mM Hepes, 1 mg/mL Collagenase D (Roche), and 0.1 mg/mL DNase (Roche) for 30 min at room temperature. Tissues were dispersed using a 70-μM cell strainer, and single-cell suspensions were prepared. Live cells were enumerated by 0.2% trypan blue exclusion.
In Vivo Cytotoxicity Assay.
In vivo cytotoxicity assays were performed on day 7–8 after MERS-CoV infection, as previously described (34, 39). Briefly, splenocytes from CD45.1 congenic naive mice were stained with either 2 μM or 100 nM carboxyfluorescein succinimidyl ester (CFSE) (Molecular Probes) and then pulsed with the indicated peptides (3 μM each) at 37 °C for 1 h. Then, 5 × 105 cells from each group were mixed together (1 × 106 cells in total) and transferred intranasally into mice. At 12 h after transfer, total lung cells were isolated. Target cells were identified on the basis of CD45.1 staining and were distinguished from each other by differential CFSE staining. After gating on CD45.1+ cells, the percentage lysis was calculated as previously described (34).
Flow Cytometry.
The following monoclonal antibodies were used: rat anti-mouse CD8α (53-6.7), rat anti-mouse CD45.1 (A20), rat anti-mouse NKG2D (CX5), all from BD Bioscience; rat anti-mouse IFN-γ (XMG1.2) from eBioscience; and mouse anti-hDPP4 (BA5b) from Biolegend. For surface staining, 106 cells were blocked with 1 μg of anti-CD16/32 antibody and 1% rat serum and stained with the indicated antibodies at 4 °C. For intracellular cytokine staining (ICS), 1 × 106 cells per well were cultured in 96-well dishes at 37 °C for 5–6 h in the presence of 1 μM peptide and brefeldin A (BD Biosciences). Cells were then labeled for cell-surface markers, fixed/permeabilized with Cytofix/Cytoperm Solution (BD Biosciences), and labeled with anti–IFN-γ antibody. All flow-cytometry data were acquired on a BD FACSCalibur or BD FACSVerse and were analyzed using FlowJo software (Tree Star, Inc.).
Venezuelan Equine Encephalitis Replicon Particles.
Venezuelan equine encephalitis replicon particles (VRPs) expressing the MERS-CoV spike glycoprotein were constructed as previously described (6, 40). Briefly, a VRP construct expressing MERS-CoV S glycoprotein was generated using overlap PCR by fusing an amplicon containing the S gene in frame with an amplicon containing sequences from the Venezuelan equine encephalitis (VEE) replicon. The primers for VEE replicon have been described previously (1), and the primers used for generating the S gene amplicon are available upon request. Ligated DNA was digested with ApaI and PacI and inserted into the pVR21 plasmid. VRPs were packaged using helper RNAs encoding structural proteins, as described before (2). A hemagglutinin (HA) tag was added to the C terminus of S protein for titering in BHK21 cells as described previously (41, 42).
Statistical Analysis.
A Student t test was used to analyze differences in mean values between groups. All results are expressed as means ± SEs of the means (SEM). P values of <0.05 were considered statistically significant.
We thank Drs. Bart Haagmans and Ron Fouchier (Erasmus Medical Center) for providing MERS-CoV (isolate HCoV-EMC/2012) and Dr. David Meyerholz for analysis of lung sections. We thank Beverly Davidson for helpful discussions. We acknowledge the excellent technical support of the University of Iowa Gene Transfer Vector Core. This research was supported in part by National Institutes of Health Grants RO1AI091322 (to S.P.), PO106099 (to S.P., P.B.M., T.G., and L.E.), AI074973 and AI083019 (to M.J.G.), and AI057157 (to R.S.B.).
↵1To whom correspondence should be addressed. E-mail: stanley-perlman{at}uiowa.edu.
Author contributions: Jincun Zhao, T.G., P.B.M., and S.P. designed research; Jincun Zhao, K.L., C.W.-L., C.F., and Jingxian Zhao performed research; S.S.A., M.J.G., R.S.B., and L.E. contributed new reagents/analytic tools; Jincun Zhao, K.L., C.W.-L., Jingxian Zhao, P.B.M., and S.P. analyzed data; and Jincun Zhao, T.G., P.B.M., and S.P. wrote the paper.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1323279111/-/DCSupplemental.
Zaki AM,
van Boheemen S,
Bestebroer TM,
Osterhaus AD,
Fouchier RA
(2012) Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia. N Engl J Med 367(19):1814–1820.
Peiris JS,
Guan Y,
Yuen KY
(2004) Severe acute respiratory syndrome. Nat Med 10(Suppl 12):S88–S97.
Assiri A,
et al.,
KSA MERS-CoV Investigation Team
(2013) Hospital outbreak of Middle East respiratory syndrome coronavirus. N Engl J Med 369(5):407–416.
(2013) Epidemiological, demographic, and clinical characteristics of 47 cases of Middle East respiratory syndrome coronavirus disease from Saudi Arabia: A descriptive study. Lancet Infect Dis 13(9):752–761.
Munster VJ,
de Wit E,
Feldmann H
(2013) Pneumonia from human coronavirus in a macaque model. N Engl J Med 368(16):1560–1562.
Scobey T,
(2013) Reverse genetics with a full-length infectious cDNA of the Middle East respiratory syndrome coronavirus. Proc Natl Acad Sci USA 110(40):16157–16162.
(2013) The Middle East respiratory syndrome coronavirus (MERS-CoV) does not replicate in Syrian hamsters. PLoS ONE 8(7):e69127.
Coleman CM,
Matthews KL,
Goicochea L,
Frieman MB
(2014) Wild-type and innate immune-deficient mice are not susceptible to the Middle East respiratory syndrome coronavirus. J Gen Virol 95(Pt 2):408–412.
Raj VS,
(2013) Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC. Nature 495(7440):251–254.
Pietzsch J,
(2012) A mouse model for HIV-1 entry. Proc Natl Acad Sci USA 109(39):15859–15864.
Dorner M,
(2011) A genetically humanized mouse model for hepatitis C virus infection. Nature 474(7350):208–211.
Crystal RG,
(1994) Administration of an adenovirus containing the human CFTR cDNA to the respiratory tract of individuals with cystic fibrosis. Nat Genet 8(1):42–51.
Knowles MR,
(1995) A controlled study of adenoviral-vector-mediated gene transfer in the nasal epithelium of patients with cystic fibrosis. N Engl J Med 333(13):823–831.
Nabel GJ
(2004) Genetic, cellular and immune approaches to disease therapy: past and future. Nat Med 10(2):135–141.
Anderson RD,
Haskell RE,
Xia H,
Roessler BJ,
Davidson BL
(2000) A simple method for the rapid generation of recombinant adenovirus vectors. Gene Ther 7(12):1034–1038.
Liu Q,
Muruve DA
(2003) Molecular basis of the inflammatory response to adenovirus vectors. Gene Ther 10(11):935–940.
Perlman S,
Netland J
(2009) Coronaviruses post-SARS: update on replication and pathogenesis. Nat Rev Microbiol 7(6):439–450.
Kindler E,
(2013) Efficient replication of the novel human betacoronavirus EMC on primary human epithelium highlights its zoonotic potential. MBio 4(1):e00611–e00612.
Zielecki F,
(2013) Human cell tropism and innate immune system interactions of human respiratory coronavirus EMC compared to those of severe acute respiratory syndrome coronavirus. J Virol 87(9):5300–5304.
Chan RW,
(2013) Tropism of and innate immune responses to the novel human betacoronavirus lineage C virus in human ex vivo respiratory organ cultures. J Virol 87(12):6604–6614.
Totura AL,
Baric RS
(2012) SARS coronavirus pathogenesis: Host innate immune responses and viral antagonism of interferon. Curr Opin Virol 2(3):264–275.
Falzarano D,
(2013) Treatment with interferon-α2b and ribavirin improves outcome in MERS-CoV-infected rhesus macaques. Nat Med 19(10):1313–1317.
Li W,
(2005) Bats are natural reservoirs of SARS-like coronaviruses. Science 310(5748):676–679.
(2012) Genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans. MBio 3(6):e00473.
Zhao J,
Legge K,
Perlman S
(2011) Age-related increases in PGD(2) expression impair respiratory DC migration, resulting in diminished T cell responses upon respiratory virus infection in mice. J Clin Invest 121(12):4921–4930.
Okada H,
(2011) Induction of CD8+ T-cell responses against novel glioma-associated antigen peptides and clinical activity by vaccinations with alpha-type 1 polarized dendritic cells and polyinosinic-polycytidylic acid stabilized by lysine and carboxymethylcellulose in patients with recurrent malignant glioma. J Clin Oncol 29(3):330–336.
Butowski N,
(2009) A phase II clinical trial of poly-ICLC with radiation for adult patients with newly diagnosed supratentorial glioblastoma: A North American Brain Tumor Consortium (NABTC01-05) J Neurooncol 91(2):175–182.
Davis NL,
(2002) Alphavirus replicon particles as candidate HIV vaccines. IUBMB Life 53(4-5):209–211.
McCray PB Jr.,
(2007) Lethal infection of K18-hACE2 mice infected with severe acute respiratory syndrome coronavirus. J Virol 81(2):813–821.
Tseng CT,
(2007) Severe acute respiratory syndrome coronavirus infection of mice transgenic for the human Angiotensin-converting enzyme 2 virus receptor. J Virol 81(3):1162–1173.
Adachi O,
(1998) Targeted disruption of the MyD88 gene results in loss of IL-1- and IL-18-mediated function. Immunity 9(1):143–150.
Suthar MS,
(2010) IPS-1 is essential for the control of West Nile virus infection and immunity. PLoS Pathog 6(2):e1000757.
Roberts A,
(2007) A mouse-adapted SARS-coronavirus causes disease and mortality in BALB/c mice. PLoS Pathog 3(1):e5.
Van Rooijen N,
(2009) Evasion by stealth: Inefficient immune activation underlies poor T cell response and severe disease in SARS-CoV-infected mice. PLoS Pathog 5(10):e1000636.
Wikenheiser KA,
(1993) Production of immortalized distal respiratory epithelial cell lines from surfactant protein C/simian virus 40 large tumor antigen transgenic mice. Proc Natl Acad Sci USA 90(23):11029–11033.
Lundegaard C,
(2008) NetMHC-3.0: Accurate web accessible predictions of human, mouse and monkey MHC class I affinities for peptides of length 8-11. Nucleic Acids Res 36(Web Server issue):W509-12.
Reche PA,
Glutting JP,
Zhang H,
Reinherz EL
(2004) Enhancement to the RANKPEP resource for the prediction of peptide binding to MHC molecules using profiles. Immunogenetics 56(6):405–419.
Rammensee H,
Bachmann J,
Emmerich NP,
Bachor OA,
Stevanović S
(1999) SYFPEITHI: Database for MHC ligands and peptide motifs. Immunogenetics 50(3-4):213–219.
Barber DL,
Wherry EJ,
Ahmed R
(2003) Cutting edge: Rapid in vivo killing by memory CD8 T cells. J Immunol 171(1):27–31.
Deming D,
(2006) Vaccine efficacy in senescent mice challenged with recombinant SARS-CoV bearing epidemic and zoonotic spike variants. PLoS Med 3(12):e525.
Pushko P,
(1997) Replicon-helper systems from attenuated Venezuelan equine encephalitis virus: Expression of heterologous genes in vitro and immunization against heterologous pathogens in vivo. Virology 239(2):389–401.
Heise MT,
Simpson DA,
Johnston RE
(2000) A single amino acid change in nsP1 attenuates neurovirulence of the Sindbis-group alphavirus S.A.AR86. J Virol 74(9):4207–4213.
You are going to email the following Rapid generation of a mouse model for Middle East respiratory syndrome
Generation of MERS-CoV mouse model
Jincun Zhao, Kun Li, Christine Wohlford-Lenane, Sudhakar S. Agnihothram, Craig Fett, Jingxian Zhao, Michael J. Gale, Ralph S. Baric, Luis Enjuanes, Tom Gallagher, Paul B. McCray, Stanley Perlman
Proceedings of the National Academy of Sciences Apr 2014, 111 (13) 4970-4975; DOI: 10.1073/pnas.1323279111
Article Classifications


Srprs.me: I took a trip to a mystery destination!
Key West Helicopter Tour: coolest cruise excursion you can find!
Hotel de la Ville, a superb small luxury hotel in Monza, was my home for a few days in November when I went exploring Lake Como and Lombardy. Read along to learn why I loved it so much.
Last weekend I visited the beautiful coastal town of Oostende in Belgium. I opted to stay in Hotel 't Botteltje ***, a hotel close to the beach that entirely revolves around beer!
I recently got to stay a few days on Ameland, one of the Dutch Wadden Islands. During my time there, I stayed in Hotel Nobel. Hotel Nobel is located right in the centre of the tiny little town of Ballum.


On this episode of Beyond the Tide, Coyote sets off on an another aquatic adventure with Tide Pool expert Aron Sanchez in search of some monster sized Slugs! ...seriously, these giant black slugs are HUGE!! Weighing up to 30lbs the Black Sea Hare is the largest slug in the world and is found in abundance along the Pacific Coast of the United States. Aptly named Sea Hare due to their rabbit ear looking appendages, these creatures are truly a primordial wonder of the ocean. Who would have ever thought a Sea Slug could grow to the size of a small dog!? Get ready to get up close with one BIG, BLACK, SLIMY, SLUG!


In a breast biopsy, cells are removed from a suspicious mass in the breast. These cells are then examined to find breast cancer cells. Biospies can determine if a mass is cancerous or benign. Based on the type of mass, you may have a fine needle aspiration, core biopsy, sentinel node biopsy, or another type. Follow the links below to find WebMD's comprehensive coverage about how breast biopsies are done, what to expect, what the results mean, and more.
Get an overview of various methods of breast biopsy that doctors use to spot breast cancer.
A sentinel lymph node is the first lymph node or nodes to which cancer cells are most likely to spread. WebMD explains how sentinel node biopsy helps treat breast cancer.
WebMD explains minimally invasive breast biopsy, a procedure to help diagnose breast cancer.
Lymph node biopsy and node dissection are common procedures during breast cancer surgery to determine if the cancer has spread.


To answer your good question, let's read Luke 12:37-40, "Blessed are those servants, whom the lord when he cometh shall find WATCHING: verily I say unto you, that he shall gird himself, and make them to sit down to meat, and will come forth and serve them. And if he shall come in the second watch, or come in the third watch, and find them so, blessed are those servants. And this know, that if the goodman of the house had known what hour the thief would come, he would have watched, and not have suffered his house to be broken through. Be ye therefore READY also: for the Son of man cometh at an hour when ye think not." I encourage you to read Matthew 24. This is a 'parallel' chapter to what we have before us and verses 42-44 are almost identical to Luke 12:37-40.
The Lord Jesus is talking to His disciples about His return to the earth at the end of the Tribulation Period. This is made very clear when reading Matthew 24. When the Lord does return physically to the earth, He will establish His Millennial Kingdom on the earth. When will this occur? Matthew 24:36 tells us, "But of that day and hour knoweth no man, no, not the angels of heaven, but my Father only." Luke 12:40 says that, "…the Son of man cometh at an hour when ye think not." When man's heart is turned away from the Lord, when the inhabitants of the earth have completely ignored the Lord's warnings, when He is least expected…the Lord will return to execute judgment and to set up His kingdom on the earth. The Apostle Peter describes it like this: "But the day of the Lord will come as A THIEF IN THE NIGHT; in the which the heavens shall pass away with a great noise, and the elements shall melt with fervent heat, the earth also and the works that are therein shall be burned up." (2 Peter 3:10).
Peter goes on to say in 2 Peter 3:11, "Seeing then that all these things shall be dissolved, what manner of persons ought ye to be in all holy conversation and godliness." Realizing that all the things of the earth will be done away with, how should that impact the way we live? When we consider that everything that is in the world is temporary, we should not live for those things. We should live Godly lives that honor the Lord that has saved us from judgment and destruction.
Let's consider 1 Corinthians 11:31-32, "For if we would judge ourselves, we should not be judged. But when we are judged, we are chastened of the Lord, that we should not be condemned with the world." This is speaking of self-judgment before partaking of the Lord's Supper. Yet, we see that the chastening hand of the Lord on His children is contrasted with His condemnation of the word. This shows us that this world is condemned! All these things shall be dissolved! In view of that, may we Christians live with eternity before us.


One thing that makes me hopeful about the fairness of life is that the pursuit of happiness isn't made more accessible by power or wealth or following. You can't strong-arm it through an iron will or inflammatory rhetoric or hype. You can't beg it, bargain with it, manipulate your way to it, fake it till you make it. You deal with it through your naked soul, stripped bare down to what your heart holds dear. Regardless of your socioeconomic standing, fear appears equally as menacing and debilitating to us all and hope is what we all seek. If anything, what we use to flex and perform with for others become liabilities and hinders in the things that really matter in life. No matter how you try, you can't make yourself feel as good as you make your life look. Your soul can't be bribed because it only answers to the divine.


As a business executive, whether you have visited London before or it is your first stay in this large metropolis, it's important that you choose the perfect accommodation so you feel as settled as possible. Likewise, if you are a PA booking a place for your executive to stay during their trip to London, it's imperative to thoroughly research the options available to ensure a comfortable trip.
Read on to see how a serviced apartment can provide a home away from home during a business trip to London.
Partnering superior convenience with professional service, a serviced apartment can provide you with everything you need for an enjoyable, productive stay. Whether you have scheduled a short-term stay in London or you plan to visit the capital for a prolonged period of time, the facilities and amenities available to you can create a more comfortable stay.
Along with restaurants and bars, local amenities such as supermarkets and shopping centres can make for an all-round enjoyable and pleasant experience, giving you the chance to settle into your surroundings, no matter the length of the stay.
Located in Vauxhall and Stratford, Staybridge Suites provide three socials per week, with complimentary drinks and snacks in the evening for residents to mingle with other guests. Breakfast is included in the nightly rate and there is a 'chilling' area for guests on the ground floor, complete with a television, computer area and games.
This accommodation has a dedicated space where guests can relax with a drink in their modern chill out space; the perfect place to plug in your laptop, relax in front of the TV or unwind with friends.
People staying here will benefit from The Meeting Centre which features a contemporary, spacious break-out area and pantry, along with multiple comfortable seating areas. This provides the ideal place to greet guests or refuel with a cup of coffee.
From PAs to business executives, we provide each and every client with a reliable, unbiased service, ensuring they find the most suitable accommodation for a business trip to the capital. To find out more or book an appointment, don't hesitate to get in touch today.