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can you please tell me that when mpsc preliminary exam forms will be out....??what is the submission date for the same one??on which date prelim exam ??
can you please tell me that when mpsc preliminary exam forms will be out....??what is the submission date for the same one??on which date prelim exam ??
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i had done m.com,B.ED, and have 4years experience in teaching junior college.do im eligible for appearing for mpse exam,what this exam for?
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Meg, a Franklin Pierce track recruit, overcame a serious shoulder injury. The 6-2 sophomore also plays basketball and runs cross country. A top student, his GPA is 4.4 and his goal is to attend an.
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Lindsay Little, was named to the chancellor's list with a 4.0 GPA for the spring semester at UMass Dartmouth. Tanya Petronio was named to the dean's list for the spring semester at Franklin Pierce.
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Maintain a minimum cumulative grade point average (GPA) of 3.00. Complete the residency requirement: Master of Business Administration students must earn in residence at Franklin University at least 24 of the 36 required credits.
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A National Honor Society member, she sports a 4.53 GPA. The Patriot MVP. A four time SCC All-Star in tennis, she will attend Franklin Pierce. A two-time Middlesex League All-Star, Nolan has led her.
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subset: commoncrawl


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Nocked's second pillar of revised gameplay is its regional mechanics. While the immediate focus of the story is on Robin and his relationships, those actions have repercussions on the larger world outside of Sherwood Forest, and I needed a way to show it; the challenge lay in giving the player some control over those repercussions.
Fortunately, the previous version of the game already had something of a side quest system known as grace points, which you could spend to influence the church, the nobility, the fairies, and the peasants in order to gain rewards. Unfortunately, the implementation was less than ideal, but the points – as placement of in-game rewards – were perfect. I kept the points, converted them to Power, and rebuilt from the ground up. Story actions and granular resources are both means to gain power, which is also modified by your background.
Power can be used to gain favor with the regional factions (the church, nobility, fairies, and peasants). Gain enough favor (thresholds which are determined by your background), and you'll earn influence which can be spent to increase your standing with a given faction. This, in turn, opens up side quests that may take you far from Sherwood, grant you granular resources, and are directly related with issues concerning each faction. The side quests alone constitute an additional 60,000 words of adventure added to the new version.
Power, favor, and influence are also integrated with third pillar of Nocked's revised gameplay mechanics; join us next time for the examination of structural resources.


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Everything posted by racewayzx
racewayzx replied to Spring2188's topic in OCC Member's Loungerenters insurance isn't very expensive and if you get it through the same company that does your car insurance you usally get a discount.
Contest for Core2Duo donators: Mushkin 650w power supply
racewayzx replied to Angry_Games's topic in OCC Member's Lounge20 congratz Blooz
Random rebooting issue.
racewayzx replied to theVash's topic in Processors, Motherboards and MemoryThere is no master and slave with sata. Run prime 95 and try reinstalling your drivers and anti-virus as those are the most common software to cause this problem. Don't no if its been fixed but running zonealarm and avg can cause it to.
Contest for Core2Duo Donators! (Mushkin 2x1GB DDR2) congrats Blooz
racewayzx replied to Angry_Games's topic in OCC Member's Lounge91 (fingers crossed)
dr_bowties WTB thread... Looking for 754 CPU....3200up
racewayzx replied to dr_bowtie's topic in OCC Market PlaceI could check shipping with a address. I can ship from work so the cost shouldn't be to bad as I get a discount. As far as the leak it isn't very bad but it is under the outer frame so it would be a pain to fix. Cheaper to buy a new one unless you can do it yourself.
dr_bowties WTB thread... Looking for 754 CPU....3200up
racewayzx replied to dr_bowtie's topic in OCC Market PlaceI have a black ice stealth 240 you can have. It needs to be repaired as I used screws that were to long and now leaks. If you want it I will send it to you for the cost of shipping.
Actually after a little investigation it is possible on the nforce chipset as long as you have the sata drivers installed which gives you the safely remove hardware icon.
I think I read somewhere that xp dosen't support hot swapping but vista is supposed to. I would personaly shut it down just to be safe.
DDR memory in short supply
racewayzx replied to ArchVile's topic in OCC Market Placewevsspot: Hopfully you have more luck than me but praz's timmings had me clearing cmos on my ultra-d. If they don't work here is a good starting point straight from muskin http://forums.mushkin.com/phpbb2/viewtopic.php?t=2837 Praz: I was wondering which slots are you running your readlines in? I haven't had time to play lately so I was wondering if you had better luck with the yellow or orange.
20% off at danger den coupon
racewayzx posted a topic in OCC Market PlaceUse coupon code PAGLAN06 and you get appoximately 20% off. I decided to jump on the band wagon and go water cooling. I got: TDX block & D5 pump Stealth gts 240 rad 10 ft of tygon tubing DD filport & 10 clamps reg $196.52 I paid $157.26 Coupon is good till 12/31/06.
19" LCD Monitor
racewayzx replied to a topic in OCC Market PlaceI have the 940bw which is the widescreen version of that monitor and am very happy with it.
wtb: Anybody sick of their Venus yet?
racewayzx posted a topic in OCC Market PlaceAnyone looking to get rid of their venus soon I am looking to purchase one. PM me with what you want for it and I will get back to you. Heat is racewayzx jason in the process of buying one
Who Wants To Join The "Get H_G / Rgone A Conroe Fund"?
racewayzx replied to EllisD's topic in OCC Member's LoungeI'm in for $20. Since RG has an enginering sample already why not get the money together now so HG can get his asap. In the meantime we can finishing collecting donations for RG.
Forgot to update. I found one, and the guy accepted a resonable offer for it. Thanks anyways.
cpu-z 1.36 is out
racewayzx posted a topic in Softwarehttp://www.cpuid.com/cpuz.php * New processor information display. * Intel 5000X/P/Z/V SPD support. * Intel Pentium D 925 & 945. * AMD K8 family HyperTransport link speed.
I am looking for a venus motherboard. The processors are venice. I'm sure someone has one and is planning on going conroe soon.
SVC August Coupon Code Sale!
racewayzx replied to Angry_Games's topic in OCC Market Placehttp://www.petrastechshop.com/evvcvgcodnf4.html I know these guys ship international. They are fairly new but have excelent reseller rating. http://www.resellerratings.com/seller11322.html
Thermaltake Armor/Kandalf question.
racewayzx replied to Nak's topic in Cases, Power Supplies and ModdingI have that panel on the side of my case And personaly love it. As far as temps go it helps keep everything nice and cool at stock clocks. The only problem I am having is keeping the pwmic cooled when overclooking. If your going for the best cooling possible I say start cutting holes and wait for some better 250mm fans to come out. And yes the thing is very quiet, Puts out about the same amount of noise at 7 and 12 volts.
fs:TT BT & Zalman vf900cu led
racewayzx posted a topic in OCC Market PlaceI have a TT Big Typhoon & a Zalman vf900-cu led for sale. I have had the big typhoon for about 4 months and the zalman for 2 months. I just recently got the bugs worked out of my watercooling and as its a shame to let these coolers sit here I am looking to sell them. I will take $35 shipped for each seperately or $60 shipped together.sold I accept non cc paypal and money order. heat is under racewayzx
64 bit burning software
racewayzx replied to svtfast's topic in SoftwareNero 7 works fine. You just need to update it.
5 free days of Red Orcherstra
racewayzx posted a topic in Gamingfound this over on hardocp. They are letting people play the full version for 5 days starting tomarrow the august 2nd. http://www.steampowered.com/index.php?area=news&id=711
Prime95 (32-bit & 64-bit) + Orthos (SP2004 for dual-core/HT)
racewayzx replied to Angry_Games's topic in SoftwareMuch apreciated. Didn't know there was a dualcore version of sp2004.
Not really. It's stable in everything but prime with less volts. The good thing is load is only 45 to 46 with those volts.
I was more worried about happy:)
I just post my OCDB entry here Let me know if I didn't do it right.


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With KPIs, the situation is not so good, there are not many options available, mainly due to the fact that KPIs are usually different and calculated in a different way for each company.
One method of displaying KPIs directly within Redmine's project would be by combining two plugins. The first plugin lets you display custom SQL on the wiki page, and the second plugin lets you apply permissions on this particular page so that no users with a particular role can access this page.
This recipe was tested on the 2.5 version of Redmine. At the time of writing this book, plugins were not upgraded to support 3.x version. Readers are advised to monitor this book's website www.redminecookbook.com because plugin updates used in this ...
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One in five American children today has a mental disorder and the rate is rising, according to a report from the Centers for Disease Control and Prevention (CDC). Affecting 13 to 20 percent of youth under 18, mental disorders impact a child's behavior, ability to learn, and cope with their emotions. Although researchers don't have a definitive explanation for the rise, studies have linked a … [Read more...]
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The pleasure boat was on a tranquil, Sunday afternoon cruise when suddenly half the approximately 20 passengers turned out to be Kurdish activists in disguise.

Some of the group shoved the boat's captain and driver to one side. "They demanded that the crew steer the boat along the bank, and wanted to read out their manifesto through loudspeakers," police spokesman Bruno Ethen told Der Spiegel magazine.

No-one was injured in the incident, and the activists, who were unarmed, have been interviewed and are expected to be released on bail, according to police.

In September last year more than 30 PKK sympathizers briefly occupied the studios of commercial TV channel RTL in Cologne. They tried to get the broadcaster to air a report on captive PKK leader Abdullah Öcalan who was arrested in Germany in 1999 and deported to Turkey.

The PKK is fighting an armed struggle in eastern Turkey in an attempt to establish an autonomous Kurdistan and greater rights for Kurds in Turkey. It is listed as a terrorist organization by the US and the European Union.

The Local/DAPD/bk


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India are going into the series with 127 rating points and a 2-0 win will take it to 130 points while defeats in both the Tests will see it slip to 122.
Australia enters the series in fourth position on 113 points, just one ahead of England, but will move ahead of Sri Lanka if it wins the series. A 1-0 win will put it on 117 ratings points while a 2-0 win will give it 118 ratings points.
On the other side of the coin, Australia will drop behind England if India wins the series.
There is a chance for Sehwag to regain the top spot which he had lost to Sri Lanka captain Kumar Sangakkara last month.
Sehwag trails Sangakkara by just 15 ratings points and a good first Test with the bat will potentially put him ahead of the left-handed middle-order batsman from Matale.
Besides Sehwag, third-ranked Michael Clarke of Australia and India's Sachin Tendulkar also have good opportunities to move up the ladder. Clarke leads Tendulkar by just five ratings points but trails Sehwag by 45 ratings points.
Simon Katich is the only other batsman from either side inside the top 10 in ninth position while the other batsmen inside the top 20 include VVS Laxman (12th), Gautam Gambhir (13th) and Ricky Ponting (17th).
Batsmen sitting outside the top 20 and eyeing an upward movement include Michael Hussey (22nd), Rahul Dravid (23rd), Mahendra Singh Dhoni (32nd), Shane Watson (39th) and Marcus North (47th).
In bowlers rankings, India has Zaheer Khan (seventh) and Harbhajan Singh (10th) inside the top-10 while Australia boasts Mitchell Johnston and Doug Bollinger inside the top 20 in sixth and 14th positions, respectively.
Bowlers aiming to improve their rankings include Ishant Sharma (22nd), Ben Hilfenhaus (23rd), Nathan Hauritz (28th), S.Sreesanth (29th) and Shane Watson (34th).
The bowlers' list is headed by Dale Steyn of South Africa with England's Graeme Swann in second place.
In rankings for Test all-rounders, the highest-ranked player from either side is Sehwag in sixth position while Watson is eighth.
South Africa's Jacques Kallis leads the field with New Zealand captain Daniel Vettori in second spot.
Sehwag to target number 1 rank in the series against Aussies
India are going into the series with 127 rating points and a 2-0 win will take it to 130 points while defeats in both the Tests will see it slip to 122.


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By Michelle Simmons
Debunking some of the most common water survival myths
Water is one of the most important items you will need for survival, but it is often the first thing that everyone runs out of. Drinking water in the wilderness may also be challenging because people continue to follow dangerous water survival myths — and this article aims to debunk them. (h/t to PreppersWill.com) Water […]
By Zoey Sky
What you need to know about water purification so you won't put your life at risk
As a prepper, you can ensure the quality of your water supply by learning about water purification. This knowledge can also help you choose the best water bottles and filters when you're looking for prepping gear. Refer to these five tips about water purification so you can gather potable water when you're out in the […]
By Ethan Huff
The top dangers you will face when you finally bug out
Hollywood movies are notorious for glamorizing the survival lifestyle of a post-apocalyptic world. But the reality is that, in the event of a major societal catastrophe, life is hardly going to be easy – even for those who think they're fully prepared for the worst. The following seven scenarios need to be carefully considered by […]
By JD Heyes
As Hurricane Florence approaches the East Coast, these should be the top preparation priorities for residents
Once again, Mother Nature is about to teach a large segment of the country how important advanced planning and preparation is when it comes to dealing with disaster and emergency situations. Hurricane Florence is on a path to strike North and South Carolina, with the latest weather forecasts claiming that the storm will make landfall […]
By Jhoanna Robinson
Be smart, stay safe: Your primer to shutting off utilities in case of an emergency
Calamities and natural disasters come and go as they please – they don't announce themselves at the door. This is why it pays to prepare for their coming; emergency planning can help you address the challenges that you may encounter when the time comes that you have to endure a crisis situation. One of the ways you can […]
By Jhoanna Robinson
6 Survival skills you need in the wilderness
No one knows what the future may bring, and no one can ever anticipate the occurrence of an emergency situation. However, if you are planning to seek solace for a weekend of want to enjoy the simplicity in the forest, it pays to be prepared before you embark on your journey. Here are some of […]
By Zoey Sky
How to grow food when the lack of water becomes a problem
Gardening itself can be a very arduous task, but it's more difficult if you're facing problems like drought and heat. What do you do when you don't have access to enough water for your crops or if the weather isn't ideal for growing vegetables? (h/t to PreppersWill.com) Even if the weather in your area is perfect […]
By Russel Davis
How to secure water during an emergency disaster
Water is an essential commodity, especially during trying times such as emergencies and natural disasters, due to its many roles in body function. An article featured on the Live Science website notes that the human body is made up of 65 percent water, which explains why it is important for survival. According to the article, water flows […]
By Rhonda Johansson
Do you know how long it's safe to store drinking water?
Yes, water can go bad. Those who have traveled will know that water can taste different depending on where you are. The difference is not bad, but there are nuances in flavor. This is because the water is sourced from various areas so the potential for chemical contaminants or even environmental organisms differs too. Scientists […]
By Zoey Sky
How to store water in your car
If you've ever forgotten plastic water bottles in your car, you might have noticed a strange aftertaste if you drink the water. The strange plastic aftertaste is especially evident during the summer, or whenever you've left a plastic water bottle sitting in the heat of a warm car, even if it's already cooled down. (h/t to ModernSurvivalBlog.com) While […]
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Intelligent Case Management
Work threat-centric cases instead of meaningless alerts
Group related alerts from across your detection tools (SIEM, EDR and more) into a prioritized queue of threat-centric cases. Assign, collaborate and escalate cases to higher tiers, all while capturing and documenting all analyst activity.
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Get to the root cause in seconds, not hours
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Real-time SOC Metrics and KPIs
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Make data-informed decisions, demonstrate the value of security operations to senior management and drive continuous improvement by tracking and analyzing a wide range of SOC key performance indicators across people, process and technology.


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Why you must prepare baby's birth passage
There's a joke that giving birth is like sh…ing a watermelon. That's really an exaggeration. Giving birth is like sh…ing a grapefruit. That's big enough! There's an old adage that 'women will stretch' … some will and all will be well. Others will not stretch successfully and be damaged for life. Others will tear and heal fine. Others will tear and be damaged for life. Which one will you be? We have got to get real about the 2nd stage of labor. You don't want your baby to suffer from you having a tight vagina. You don't want to suffer in your own body because your soft tissue of your vagina was so tight that your vagina, possibly bladder or rectum and you were damaged for life.
The birth of your child is one of the most amazing life experiences … both in its positive and negative qualities. We can reduce a great deal of some of the negative by preparing our pregnant body to become a birthing body as part of our childbirth preparation. Prepare our vagina for 2nd stage of labor is essential as part of your birth preparation. Your vagina is your baby's birth canal.
Listen to the Internal Work audio in the Birthing Better Childbirth Preparation Online Course. The audio will instruct your partner how to do the preparation from 32 weeks onward. The audio will alos guide you to do the Internal Work for yourself. It takes between 5-15min/day or two. You can both reduce damage to your birth canal, shorten 2nd stage and protect your baby's well being.
It's more than …
The Internal Work in Birthing Better Childbirth Preparation is not the same as perineal massage. Perineal massage only stretches the small amount of skin between your Vjayjay and your rectum. Instead you have to soften and make pliable all the soft tissue inside your birth canal … and that includes the muscles that you can tighten up right now. Are you familiar with your ability to tighten up inside your vagina? If you're not familiar with how to tighten up (which hinders your baby's efforts to come down, through and out your vagina) then you have no hope to learn how to soften and open this part of your body. Try doing these things:
- Tighten up your rectum … those are some of the muscles inside your baby's birth canal.
- Draw the bones you sit on together … those are other internal muscles
- Pull your butt muscles together … those are others.
Because our baby feels SO BIG when its coming through this soft tissue, it's instinctive and intuitive to tighten up down there and pull back unless you've prepared thoroughly. You must learn to soften and stretch all those muscles and the soft tissue inside that is not 'muscle'.
You've probably heard about the 'ring of fire'. Imagine that this intense stretching and burning sensation can be reduced by your preparation before hand! Preparing your baby's birth canal is also essential in keeping your bony pelvis open, helps your cervix to dilate and keeps your pelvis mobile … but that's another story.


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A Star Is Born
Great book, a remarkable journey through the early days of
Fratelli Nemici – Close Enemies
Starcraft. Frontline
A Star Is Born
Boy Erased
Cold War
Il Professore e il Pazzo
Vilde Rolf smadrer Internettet
Burn the Stage: The Movie
The Mule
Escape Room
Nice Girls Dont Stay for Breakfast
Doon de Maience, Chanson de Geste (Classic Reprint)
LEGO Filmen 2
Alita: Battle Angel
Burn the Stage: The Movie
The Front Runner
A Dog's Way Home
This helps immensely
I have not received the
An Elephant Sitting Still
Jeune bergre
Great, helpful, book. It cost much less on
Boy Erased
Bohemian Rhapsody
Love this! Highly recommended!! d3c7d16
Peppa Celebrates Chinese New Year
The Mule
Már 14
Wish I would have had this
A Star Is Born


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new orleans saints image of superdome events october 12th national2019-04-15 11:07:02
new orleans saints game today youtube clips johane sudithar cutting If you are interested in the history of the Democratic Party of the 1990s then you should read about the Democratic Leadership Council (DLC) this was formed in the '80s, peaked under Bill Clinton (Both the platforms above are from your lifetime Clinton ran for President), Declined this Bush years, And power down in 2011. It moved the Democratic Party to the correct by promoting what used to be Republican positions on balanced budgets, Free trade, Tough on crime strategies, And wellbeing reform. in the meantime, The Republicans moved even further off to the right.The NFL says it tests all players for banned substances before the season starts, And then randomly tests seven players per team every week of the season."within the last five seasons, Just to take one of these, Weu0027ve only had 25 players who have violated our program and been hung," speaks Tagliabue. "may be far below 1 percent."NFL officials declined to give 60 Minutes sunday an on camera interview. new orleans saints score by quarter panthers roster 2020 nba all star But at the NFL team owners meeting yesterday morning, Tagliabue said the league was looking into your situation with the Carolina Panthers. Campy was a success from the day he found its way to Brooklyn in mid 1948. The stocky catcher had a rocket for an arm, formidable bat, And guided a the well-known teen pitching staff to five pennants in 10 years. Campanella was a prime reason the 1950s Dodgers were the brilliant team in the NL. through 1951, Campanella won the first of three most valuable Player Awards, A feat performed by only a tiny group of stars. he or batted.325 in addition to 33 homers and 108 RBI. His 1953 MVP season was a good ever recorded by a catcher, As he led the little league with 142 RBI, Clubbed 41 homers, scored 103 runs, to batted.312.
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Postpartum Soup Use
Mugwort (Koj ntshuab) translate to green boat. pick 4 or 5 leaves of this plant used in the chicken soup.
Medicinal use:
It's good for food poisoning. (Zoo tsuaj piv.) Those of you that know more please let me know, thanks in advance. We can all learn together.
This is information purpose only. Seek professional help. Back to herbs.
More information on this at eurekamag.com/pdf
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|Related Articles
Dysphagia following anterior cervical spinal surgery: A systematic review.
Bone Joint J. 2013 Jul;95-B(7):868-73
Authors: Cho SK, Lu Y, Lee DH
Dysphagia is a common complication of anterior surgery of the cervical spine. The incidence of post-operative dysphagia may be as high as 71% within the first two weeks after surgery, but gradually decreases during the following months. However, 12% to 14% of patients may have some persistent dysphagia one year after the procedure. It has been shown that female gender, advanced age, multilevel surgery, longer operating time and severe pre-operative neck pain may be risk factors. Although the aetiology remains unclear and is probably multifactorial, proposed causes include oesophageal retraction, prominence of the cervical plate and prevertebral swelling. Recently, pre-operative tracheal traction exercises and the use of retropharyngeal steroids have been proposed as methods of reducing post-operative dysphagia. We performed a systematic review to assess the incidence, aetiology, risk factors, methods of assessment and management of dysphagia following anterior cervical spinal surgery. Cite this article: Bone Joint J 2013;95-B:868-73.
PMID: 23814234 [PubMed – in process]


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Ideal Fulton Place location, awesome value! This top floor, renovated 820 sqft 1 bedroom condo is the best in it's price range. Featuring a bright and open floor plan with upgraded vinyl plank flooring and fresh paint throughout. Kitchen boasts plenty of counter space and crisp white cabinetry, open to the large living room and dining room areas. Additional highlights are the large bathroom, super sized master bedroom, and the in suite laundry and storage room. Great views from the large balcony. Within walking distance to shops (including Capilano Mall), restaurants, public transportation, a variety of services, and scenic river valley and ravine walking trails. Quick access to Downtown Edmonton, King's University, U of A, etc. Low condo fees of $293 monthly include heat and water. Includes 1 assigned parking stall. Klass Point is a quiet, well managed condominium community in the heart of south/central Edmonton. Move in condition!
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Defense has been a point of emphasis for the University of Dayton basketball team since last season, and coach Archie Miller wanted to check to see if the Flyers had been making any progress by comparing film of their first exhibition in 2011-12 to the one eight days ago against Findlay.
"It was way, way different," Miller said.
That's different in a good sense, which UD fans will be glad to hear.
Miller's first team at UD last season was exceptionally fluid on offense but was gashed frequently on defense. The Flyers' defensive field-goal percentage was 44.8, the highest shooting mark for opponents since the 1994-95 team, which finished 7-20, allowed foes to hit 48.1 percent.
The Flyers cobbled together a 20-13 record and a fifth-straight postseason appearance despite giving up 67.6 points per game, the most they've allowed in 13 years. Nine foes shot 50 percent or better from the field, including Saint Joseph's (52.7), Rhode Island (52.6) and Duquesne (51.7) in consecutive Atlantic 10 losses.
But while the offense may take an early hit because of the turnover in personnel, the Flyers should be much more active on defense with nimble newcomers Jalen Robinson and Devon Scott alternating with senior forward Josh Benson to form an agile inside attack and swift shooting guard Vee Sanford joining veteran point guard Kevin Dillard to apply pressure on the perimeter.
"Our defense last year had holes. I think a lot of those holes are filled up by some new bodies," Miller said. "You throw Jalen and you throw Devon out there with Josh inside — and the athleticism with Devin (Oliver) and Vee combined with Kevin — and your quickness level is much different.
"We're also much more understanding of what we're trying to accomplish off the ball with seasoned returning guys. The younger guys are being taught. We had a much more defensive-minded offseason than we did a year ago. The natural progression of our team has to become that."
The Flyers were 11th in the A-10 last season in field-goal percentage defense. In the three previous years under coach Brian Gregory, they were tied for fourth (41.4), second (39.3) and second (39.7).
They also were eighth in 3-point percentage defense (33.5). They finished fourth (32.3), fifth (30.6) and second (31.2) in that category in the three years before that.
"Those things last year at times were there, and at times they weren't," Miller said. "They have to be consistent this year to give us a chance to win."
Miller saw promise against Findlay even before the video comparison and will be looking for more tonight against Walsh.
"Defensively, we're still a 20- to 25-minute team as far as playing hard. We've got to slowly but surely start creeping into the 30's and, hopefully, by our opener, we're ready to go a full 40 because that's what we're going to need," he said.
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Like most other US cities in the US, cost of living in Sumner has significantly changes in the last 30 years. As somebody who needs or can provide Landscape Design in Sumner, how does this relate to you? Because the more wealthy a city, the higher demand for freelancers , it is the optimal place to find freelancers to do landscape designing .
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Election leaflets from Mansfield Independent Forum
We don't have any leaflets for Mansfield Independent Forum. That won't do. They must have some right?
Your mission, if you choose to accept it, is to find us one and photograph it for posterity.
- Put a call out on Twitter or tweet the party directly
- Ask friends on Facebook
- Go rummaging through the bin


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Rift Valley fever virus (RVFV) is a member of the genus Phlebovirus within the family Bunyaviridae. It can cause severe epidemics among ruminants and fever, myalgia, a hemorrhagic syndrome, and/or encephalitis in humans. The RVFV M segment encodes the NSm and 78-kDa proteins and two major envelope proteins, Gn and Gc. The biological functions of the NSm and 78-kDa proteins are unknown; both proteins are dispensable for viral replication in cell cultures. To determine the biological functions of the NSm and 78-kDa proteins, we generated the mutant virus arMP-12-del21/384, carrying a large deletion in the pre-Gn region of the M segment. Neither NSm nor the 78-kDa protein was synthesized in arMP-12-del21/384-infected cells. Although arMP-12-del21/384 and its parental virus, arMP-12, showed similar growth kinetics and viral RNA and protein accumulation in infected cells, arMP-12-del21/384-infected cells induced extensive cell death and produced larger plaques than did arMP-12-infected cells. arMP-12-del21/384 replication triggered apoptosis, including the cleavage of caspase-3, the cleavage of its downstream substrate, poly(ADP-ribose) polymerase, and activation of the initiator caspases, caspase-8 and -9, earlier in infection than arMP-12. NSm expression in arMP-12-del21/384-infected cells suppressed the severity of caspase-3 activation. Further, NSm protein expression inhibited the staurosporine-induced activation of caspase-8 and -9, demonstrating that other viral proteins were dispensable for NSm's function in inhibiting apoptosis. RVFV NSm protein is the first identified Phlebovirus protein that has an antiapoptotic function.
Rift Valley fever virus (RVFV), a member of the genus Phlebovirus within the family Bunyaviridae, causes periodic outbreaks among livestock and humans in sub-Saharan African countries (42). RVFV infection results in severe hepatic diseases, with high mortality and abortion rates, in domestic ruminants and also causes an acute febrile myalgic syndrome, a hemorrhagic syndrome, ocular disease, and encephalitis in humans (3, 39). The most recent outbreak was reported in Kenya and resulted in a high reported case-fatality ratio for infected humans (12). RVFV is transmitted primarily by mosquitoes or through direct contact with infected animal blood. Even though the natural infectious cycle of RVFV has been linked to mosquitoes of Aedes species, many other mosquito species can be infected and subsequently transmit the virus (16), implying that these mosquito species can increase the possibility of RVFV becoming epidemic and perhaps endemic outside of its traditional area.
RVFV has a single-stranded, tripartite RNA genome composed of the L, M, and S segments. The L segment is of negative polarity and encodes the RNA-dependent RNA polymerase (L). The S segment uses an ambisense strategy for gene expression; a nonstructural protein, NSs, is translated from viral-sense mRNA, while the nucleocapsid (N) protein is expressed from anti-viral-sense mRNA (42). The NSs protein has been known as an interferon antagonist due to its shutting off of host transcription (5, 34). The anti-viral-sense M segment encodes two envelope glycoproteins, Gn and Gc, and two accessory proteins, the 14-kDa NSm protein and the 78-kDa protein. The M gene open reading frame (ORF) in M mRNA contains five in-frame translation initiation codons within the preglycoprotein (pre-Gn) region, which is located upstream of the Gn and Gc genes (see Fig. 1A) (20, 26, 48). The 78-kDa protein is translated from the first AUG codon of the ORF, and its coding sequence includes the entire NSm and Gn coding sequences. NSm is translated from the region from the second AUG codon to the end of the pre-Gn region (see Fig. 1). The NSm and 78-kDa proteins are not essential for RVFV replication in cell culture (18, 51), whereas all RVFVs thus far sequenced carry the pre-Gn region (7), strongly suggesting that there is selection pressure(s) to retain an RNA element(s) in the pre-Gn region and/or proteins encoded (fully or partially) by the pre-Gn region, i.e., NSm and the 78-kDa protein. Currently, the biological functions of the NSm and 78-kDa proteins of RVFV and proteins encoded by the pre-Gn regions of other phleboviruses are totally unclear, though a mutant RVFV lacking both NSm and 78-kDa protein expression showed attenuated virulence in rats (6), implying a possible role of the RVFV NSm and/or 78-kDa protein in viral pathogenesis.
In the present study, we have generated a deletion mutant of an attenuated MP-12 strain of RVFV, which expresses neither the NSm protein nor the 78-kDa protein, due to a large deletion in the pre-Gn region of the M segment. We found that cells infected with the deletion mutant underwent apoptotic cell death earlier than cells infected with the parental virus and that NSm expression inhibited the rapid apoptotic cell death in mutant virus-infected cells. NSm expression also suppressed staurosporine (STP)-induced apoptosis, demonstrating that NSm performed its antiapoptotic function in the absence of other viral proteins. This is the first demonstration of the biological function of the NSm protein of any phlebovirus.
Cells and viruses.Vero E6 cells, human embryonic kidney 293 cells, and cells of the murine macrophage-like line J774.1 were maintained in Dulbecco's modified minimal essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100 μg/ml). BHK/T7-9 cells, which express T7 RNA polymerase (25), were grown in MEM-alpha containing 10% FBS. arMP-12, a recombinant MP-12 vaccine candidate strain of RVFV, and arMP-12-del21/384, carrying a deletion in the pre-Gn region of arMP-12, were generated using an RVFV reverse-genetics system (22, 51). Both viruses were grown, and their titers were determined by a plaque assay, in Vero E6 cells.
Plasmids and rescue of mutant virus.The entire NSm, 78-kDa, and 73-kDa ORFs (nucleotides [nt] 135 to 479, 21 to 2090, and 174 to 2090, respectively, of the anti-viral-sense M segment) were independently cloned into a pCAGGS plasmid (28), resulting in pCAGGS-NSm, pCAGGS-78, and pCAGGS-73, respectively. For the generation of pCAGGS-78, the second and third AUG codons of the 78-kDa ORF were replaced with GCC to eliminate expression of the NSm and 73-kDa proteins. For the generation of arMP-12-del21/384, two EcoRI sites were created at nt 21 and 384 of the M gene ORF of a plasmid expressing an anti-viral-sense M segment. EcoRI digestion of this plasmid and subsequent self-ligation resulted in pPro-T7-avM(+)-del21/384. arMP-12-del21/384 was recovered from BHK/T7-9 cells that were cotransfected with pPro-T7-avM(+)-del21/384, pPro-T7-avS(+), and pPro-T7-avL(+), all of which express anti-viral-sense RNA segments, pT7-IRES-vN, expressing N protein, pT7-IRES-vL, expressing L protein, and pCAGGS-vG, expressing proteins encoded in the M gene ORF, as described previously (22, 51). The rescued virus was amplified once in Vero E6 cells and used in the experiments.
Plaque assay.After adsorption of virus to Vero E6 cells at 37°C for 1 h, the inoculum was removed, and the cells were washed three times with phosphate-buffered saline and overlaid with modified Eagle's medium containing 0.6% tragacanth gum (MP Biomedicals, Inc.), 2.5% FBS, and 5% tryptose phosphate broth. At 4 days postinfection (p.i.), the overlay was removed, and cells were fixed and stained with 0.75% crystal violet, 10% formaldehyde, and 5% ethanol.
Northern blot analysis.Intracellular RNAs were extracted from virus-infected cells using TRIzol reagent (Invitrogen). Approximately 100 ng of RNA was denatured and separated on 1% denaturing agarose-formaldehyde gels and transferred to a nylon membrane (Roche Applied Science). Northern blot analysis was performed with strand-specific RNA probes as described previously (23).
Antibodies.An anti-NSm rabbit polyclonal antibody and an anti-RVFV mouse polyclonal antibody were used to detect viral proteins (51). A rabbit polyclonal antibody against an L protein peptide was prepared by immunizing rabbits with the synthetic peptide N-RDRSKQPFSPDHD, corresponding to amino acids 434 to 446 of the L protein.
Western blot analysis.Virus-infected and mock-infected cells were lysed with sample buffer and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The separated proteins were transferred to a polyvinylidene difluoride (Bio-Rad) membrane. After being blocked for 1 h, the membranes were incubated with a primary antibody overnight at 4°C and with a secondary antibody for 1 h at room temperature. The membrane was developed with an enhanced chemiluminescence (ECL) kit (Amersham Biosciences).
Radioimmunoprecipitation.Virus-infected and mock-infected cells were radiolabeled with 100 μCi/ml of Tran35S-label (MP Biomedicals) at 8 h p.i. for 30 min. Cytoplasmic extracts were prepared as described previously (51) and incubated with an anti-NSm antibody or preimmune serum. Radioimmunoprecipitation was performed as described previously (51).
Cell viability assay.Confluent Vero E6 cells grown in 24-well plates were either mock infected or independently infected with arMP-12 or arMP-12-del21/384 at a multiplicity of infection (MOI) of 10. Cell viability was determined by using an MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide)-based in vitro toxicology assay kit (Sigma), which measures the activity of mitochondrial dehydrogenases in living cells. At various times p.i., MTT (0.5 mg/ml) was added to each well for 4 h, and the reaction was terminated by adding an MTT solubilization solution. After MTT formazan crystal was completely dissolved, the absorbance of samples was measured at a wavelength of 570 nm and the background absorbance at 690 nm was subtracted. The viability of the infected cells was determined as a percentage of the viability of mock-infected cells at the corresponding time p.i.
Annexin V/PI staining and flow cytometry.Vero E6 cells were either mock infected or independently infected with arMP-12 or arMP-12-del21/384 at an MOI of 3. All cells in the cultures, including floating cells, were collected at 20 and 40 h p.i. The harvested cells were washed three times and then stained with annexin V-fluorescein isothiocyate and propidium iodide (PI) for 15 min at room temperature using the Annexin V-FITC apoptosis detection kit I (BD Pharmingen). We then analyzed 1 × 104 stained cells by flow cytometry (BD FACSCanto flow cytometer; Becton Dickinson).
Caspase enzymatic activity assays.The caspase enzymatic activities of virus-infected cells were evaluated via caspase colorimetric assay kits (BioVision, Mountain View, CA). In this assay, approximately 1 × 106 cells were washed with phosphate-buffered saline and lysed with 50 μl of chilled cell lysis buffer for 10 min on ice. After centrifugation for 1 min at 10,000 × g, supernatants (cytosolic extracts) were collected and protein concentration measured by the Bradford assay. Fifty or 100 μg of proteins was diluted in 50 μl of cell lysis buffer and 50 μl of 2× reaction buffer for each assay. The activity of each caspase was determined with colorimetric peptide substrates: DEVD-pNA (chromophore p-nitroanilide) for caspases 3 and 7 (caspase-3/7), IETD-pNA for caspase-8, and LEHD-pNA for caspase-9. Each sample was incubated with the peptide substrate for 2 h, and color changes were measured at 405 nm in a spectrophotomer (Bio-Rad) using a quartz microcuvette. The background reading from cell lysates and buffers was subtracted from the sample reading.
Reagents.An anti-cleaved caspase-3 (Asp175) antibody and an anti-poly(ADP-ribose) polymerase (PARP) antibody were purchased from Cell Signaling Technology (Danvers, MA). STP and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (St. Louis, MO), and Z-IETD-FMK (a caspase-8 inhibitor) and Z-LEHD-FMK (a caspase-9 inhibitor) were purchased from BD Biosciences (San Jose, CA).
Statistical analysis.A paired Student t test was used to compare significance. A P value of <0.05 was considered statistically significant.
Generation of arMP-12-del21/384 carrying a large deletion in the pre-Gn region of the M segment.The pre-Gn region of the RVFV M gene ORF contains five in-frame start codons, and the 78-kDa and NSm proteins are translated from the first and second AUG codons, respectively (Fig. 1A) (26, 48, 51). We previously reported that neither the 78-kDa protein nor the NSm protein is detected in cells infected with arMP-12-delNSm/78, which carries substitution mutations in the first and second AUG codons. This mutant virus and the parental virus, arMP-12, form plaques similar in size and morphology and have comparable growth kinetics in several cell lines (51). However, we unexpectedly detected an additional protein(s) with a molecular mass of 73 to 75 kDa, which is recognized by the anti-NSm antibody, in arMP-12-delNSm/78- and arMP-12-infected cells (51). To completely eliminate the accumulation of detectable viral proteins whose coding sequences contain the pre-Gn region in virus-infected cells, we generated another mutant virus, arMP-12-del21/384, with a deletion from nt 21 to 384 in the pre-Gn region, by using a reverse-genetics system (22, 51) (Fig. 1A). Sequence analysis showed that the rescued arMP-12-del21/384 virus retained the introduced deletion in the pre-Gn region and had no other mutations in the M segment. In contrast to arMP-12-delNSm/78, which produces plaques indistinguishable from those made by arMP-12, arMP-12-del21/384 produced plaques that were larger than those produced by arMP-12 in Vero E6 cells (Fig. 1B). We have successfully recovered arMP-12-del21/384 in three independent rescue experiments, and all rescued viruses produced plaques that were similar in size and morphology; however, they were larger than those produced by arMP-12 (data not shown), suggesting that the larger-plaque phenotype was caused by the deletion introduced within the pre-Gn region.
Analysis of the accumulation of viral RNAs and proteins and the growth kinetics of arMP-12-del21/384.Northern blot analysis revealed the accumulation of similar levels of viral sense RNAs in arMP-12-infected and arMP-12-del21/384-infected Vero E6 cells (Fig. 2A). The two viruses also accumulated similar amounts of anti-viral-sense RNAs (data not shown). Western blot analyses showed no substantial differences in the amounts of L, Gn/Gc, NSs, and N proteins between arMP-12-infected and arMP-12-del21/384-infected Vero E6 cells (Fig. 2B); a modest increase in the accumulation of Gn/Gc, N, and NSs proteins in arMP-12-del21/384-infected cells relative to that in arMP-12-infected cells, shown in Fig. 2B, was not reproducible. Radioimmunoprecipitation analysis of infected-cell lysates with an anti-NSm antibody showed accumulations of the 78-kDa protein, NSm protein, and a viral protein with a molecular mass of 75 kDa (the 75-kDa protein) in arMP-12-infected cells, whereas these proteins did not accumulate in arMP-12-del21/384-infected cells (Fig. 2B and C). A similar radioimmunoprecipitation analysis of arMP-12-delNSm/78-infected cells demonstrated selective immunoprecipitation of a viral protein with a molecular mass of 73 kDa (the 73-kDa protein). arMP-12 and arMP-12-del21/384 showed similar growth kinetics in Vero E6 cells after infection at an MOI of 1 (Fig. 2D). They also replicated with similar kinetics in 293 cells, MRC-5 cells, and cells of the murine macrophage-like line J774.1 (4) after infection at an MOI of 1 or 0.01 (Fig. 2D and data not shown). These data are consistent with previous findings that the lack of NSm and 78-kDa protein accumulation and the presence of a large deletion at the pre-Gn region do not affect virus replication efficiency in cell cultures (18, 51).
arMP-12-del21/384 replication induces more-extensive apoptotic cell death than arMP-12 replication.arMP-12-del21/384 and arMP-12 replicated with similar kinetics in cell cultures, yet the former produced larger plaques than the latter (Fig. 1B). These data led us to hypothesize that arMP-12-del21/384 replication may induce more-extensive cell death than arMP-12 replication. To test this hypothesis, Vero E6 cells were independently infected with arMP-12, arMP-12-delNSm/78, or arMP-12-del21/384 at an MOI of 10, and cell viabilities were determined by an MTT-based cytotoxicity assay, which measures the mitochondrial dehydrogenase activities of live cells. Cell viabilities of infected cell cultures were expressed as percentages of the absorbance of a mock-infected cell culture at the corresponding time p.i. (Fig. 3A). All infected cell cultures showed similar levels of cell viability during the first 24 h p.i., whereas the viabilities of arMP-12-del21/384-infected cells were significantly lower than those of arMP-12-infected cells from 48 h p.i. to 72 h p.i.; at 60 h p.i., the cell viabilities of arMP-12-del21/384-infected cultures were about 40% less than those of arMP-12-infected cell cultures. The kinetics of cell viability for arMP-12-delNSm/78-infected and arMP-12-infected cells were similar throughout infection, except at 24 h p.i., when marginally lower cell viabilities were observed for the former.
To determine whether arMP-12-del21/384 replication induced more-extensive apoptotic cell death than arMP-12 replication, annexin V/PI staining and a subsequent flow cytometry analysis were performed. Annexin V is a Ca2+-dependent phospholipid-binding protein with a high affinity for phosphatidylserine, which is translocated from the inner to the outer leaflet of the plasma membrane in the early stage of apoptosis (38). PI, which intercalates into double-stranded DNAs, is excluded by viable cells but can penetrate the membranes of dying or dead cells (50). Accordingly, annexin V-positive and PI-negative cells are those undergoing early apoptosis. Vero E6 cells were either mock infected or independently infected with arMP-12 or arMP-12-del21/384 at an MOI of 3. An immunofluorescence assay that detected viral proteins showed that nearly 100% of Vero E6 cells and 293 cells were infected with viruses at an MOI of 3 (data not shown). At 20 h and 40 h p.i., all cells, including floating cells, were collected from the cultures and stained with annexin V/PI; then they were analyzed by flow cytometry (Fig. 3B). The lower-right quadrant (Q4) of Fig. 3B represents early-apoptotic cells, i.e., annexin V-positive, PI-negative cells. At 20 h p.i., mock-infected cells and arMP-12-infected cells had 5.9% and 8.4% apoptotic cells, respectively, whereas 20.7% of arMP-12-del21/384-infected cells underwent apoptosis, demonstrating that the number of cells at the early state of apoptosis was substantially higher for arMP-12-del21/384-infected cells than for arMP-12-infected cells. By 40 h p.i., the apoptotic cell populations had increased substantially in infected cells, and the apoptotic cell population in arMP-12-del21/384-infected cells (35.6%) was higher than that in arMP-12-infected cells (22.3%). Furthermore, the population of cells at the late stages of apoptosis and/or necrosis, shown in the upper-right quadrant (Q2), had increased in infected cells by 40 h p.i., and this population was larger for arMP-12-del21/384-infected cells (24.2%) than for arMP-12-infected cells (14.8%). These data suggested that the NSm and/or 78-kDa protein suppressed virus-induced apoptotic cell death.
Status of caspase activation in arMP-12- and arMP-12-del21/384-infected cells.It is well established that once apoptosis is triggered, upstream caspases, e.g., caspase-8 or -9, induce the cleavage of caspase-3, yielding active cleaved forms of caspase-3 (p17/p19), which in turn cleave the downstream cellular substrates, e.g., PARP, to execute apoptosis (33). To further establish that arMP-12-del21/384 replication induced a level of apoptotic cell death more extensive than that induced during arMP-12 replication, we examined the activation status of caspase-3 in arMP-12- and arMP-12-del21/384-infected cells. Vero E6 cells, 293 cells, and J774.1 cells were either mock infected or independently infected with arMP-12 or arMP-12-del21/384 at an MOI of 3, and cell lysates were collected at 8, 16, and 24 h p.i. for Vero E6 and 293 cells; the majority of cells in infected Vero E6 and 293 cell cultures were attached to flasks at 24 h p.i. In contrast, a majority of cells in infected J774.1 cell cultures were detached from the flasks at 24 h p.i., suggesting that most of the infected cells were dead by that time. Hence, extracts from infected J774.1 cells were prepared at 2, 4, 6, and 8 h p.i. Cell lysates were subjected to Western blot analysis to determine the levels of cleavage of caspase-3 and its downstream substrate, PARP (Fig. 4). STP, a broad-spectrum protein kinase inhibitor that has been used to induce apoptosis in various cell lines (21, 32, 45), served as a positive control for apoptosis induction. As shown in Fig. 4, STP treatment induced accumulation of cleaved caspase-3 and PARP, while neither was accumulated in cells treated with DMSO alone. In both 293 and Vero E6 cells, throughout infection, cleaved caspase-3 was more abundant in arMP-12-del21/384-infected cells than in arMP-12-infected cells. For 293 cells, caspase-3 cleavage was detected as early as 8 h p.i. in arMP-12-del21/384-infected cells, whereas only a low level of cleaved caspase-3 was detectable at 16 h p.i. in arMP-12-infected cells. Likewise, the levels of PARP cleavage were higher in mutant virus-infected cells than in cells infected with the parental virus, arMP-12. We noted efficient accumulation of cleaved caspase-3 and PARP at 6 h p.i. in arMP-12-del21/384-infected J774.1 cells but not in arMP-12-infected J774.1 cells, whereas these differences were no longer obvious at 8 h p.i. Western blotting using an anti-RVFV antibody showed that accumulation of N and NSs proteins was similar for arMP-12-infected and arMP-12-del21/384-infected cells, a finding that, together with the identical growth curves (Fig. 2D), suggested that increased caspase-3 activation in arMP-12-del21/384-infected cells was not due to the efficient replication of this virus.
To further confirm that the cleaved caspase-3 in arMP-12-del21/384-infected cells was biologically active, caspase-3/7 activities in arMP-12- and arMP-12-del21/384-infected cells were measured by using their colorimetric substrate, DEVD-pNA. The levels of caspase-3/7 activities in mutant virus-infected cells were substantially higher than those in parental-virus-infected cells at 16 h p.i. for both Vero E6 and 293 cells (Fig. 5A) (P < 0.001). In contrast, only an insignificant difference in caspase-3/7 activities was detected between mock-infected cells and arMP-12-infected cells.
Next, we determined the enzymatic activities of the upstream caspases, i.e., caspase-8 (Fig. 5B) and caspase-9 (Fig. 5C), at 16 h p.i. in infected Vero E6 and 293 cells. In both cell lines, caspase-8 activities were higher in arMP-12-del21/384-infected cells than in arMP-12-infected cells; these differences were statistically significant (P < 0.001). Caspase-8 activities in arMP-12-infected cells were also statistically significantly higher than those in mock-infected cells (P < 0.001 for Vero E6 cells and P < 0.005 for 293 cells). Caspase-9 activities were statistically significantly higher in arMP-12-del21/384-infected cells than in arMP-12-infected cells (P < 0.05 for both cell lines), whereas the differences between mock-infected and arMP-12-infected cells, and those between mock-infected and arMP-12-del21/384-infected cells, were not statistically significant in either cell line. These data suggested that activated caspase-8 primarily induced caspase-3 cleavage in arMP-12-del21/384-infected cells. Overall, our data were consistent with the notion that the viral proteins encoded by the pre-Gn region, including the NSm and/or 78-kDa protein, suppress the activities of caspase-8 and its downstream caspase, caspase-3, causing a delay in apoptotic cell death in arMP-12-infected cells.
Expression of the NSm and 73-kDa proteins suppresses caspase-3 cleavage and activation in arMP-12-del21/384-infected cells.To investigate whether the viral proteins whose coding sequences contain the pre-Gn region, i.e., NSm, the 73-kDa protein, and the 78-kDa protein, could inhibit apoptotic cell death in RVFV-infected cells, 293 cells were independently transfected with a eukaryotic expression plasmid carrying the NSm protein ORF (pCAGGS-NSm), the 78-kDa protein ORF (pCAGGS-78), or the 73-kDa protein ORF (pCAGGS-73), or with a control plasmid, pCAGGS. In our hands, the transfection efficiencies achieved in 293 cells were about 70 to 80% (data not shown). At 48 h posttransfection, cells were either mock infected or independently infected with arMP-12 or arMP-12-del21/384 at an MOI of 3. Total-cell lysates were collected at 16 h p.i. and subjected to Western blot analysis in order to compare the levels of caspase-3 cleavage (Fig. 6A). It was evident that transfection of pCAGGS-NSm and pCAGGS-78 resulted in efficient accumulation of NSm protein and the 78-kDa protein, respectively; the abundance of NSm protein in transfected cells was higher than that in arMP-12-infected cells. Transfection of pCAGGS-73 resulted in a modest amount of 73-kDa protein accumulation. In arMP-12-infected cells (Fig. 6, lanes 5 to 8), the expression of all three proteins had little effect on the abundance of cleaved caspase-3 (p17), whereas expression of the NSm and 73-kDa proteins clearly suppressed p17 accumulation in arMP-12-del21/384-infected cells (Fig. 6, lanes 10 and 12). In contrast, expression of the 78-kDa protein had little effect on p17 accumulation in arMP-12-del21/384-infected cells (Fig. 6, lane 11). Consistent with those data, caspase-3/7 activities in arMP-12-del21/384-infected cells that expressed NSm protein or the 73-kDa protein, but not the 78-kDa protein, were statistically significantly lower than those in arMP-12-del21/384-infected cells transfected with pCAGGS (empty vector [EV]) (P < 0.001 for EV versus NSm; P < 0.05 for EV versus pCAGGS-73) (Fig. 6B). These experiments demonstrated that the independently expressed NSm and 73-kDa proteins, but not the 78-kDa protein, from the transfected plasmids worked in trans to suppress caspase-3 activation in arMP-12-del21/384-infected cells; the NSm and 73-kDa proteins indeed inhibited apoptotic cell death in RVFV-infected cells.
NSm protein expression suppresses STP-induced apoptosis.To further establish an antiapoptotic function of the NSm protein, we examined whether NSm protein expression suppressed STP-induced apoptosis. STP activates both caspase-8 and caspase-9 (45), while the exact mechanism by which STP induces apoptosis is unclear. 293 cells were independently transfected with pCAGGS or pCAGGS-NSm, followed by treatment with 3 μM STP at 48 h posttransfection. Cell extracts were prepared at 3 h after STP treatment, and the enzymatic activities of caspase-8 and caspase-9 were measured (Fig. 7). Consistent with our expectation, STP treatment induced activation of both caspases. It was evident that NSm protein expression efficiently suppressed STP-induced activation of both caspase-8 and caspase-9. NSm protein was quite competent to suppress STP-induced caspase-8 and -9 activities, as evidenced by the fact that its inhibitory effects were comparable to those of the synthetic inhibitors of caspase-8 (Z-IETD) and caspase-9 (Z-LEHD) (Fig. 7A and B). These data clearly demonstrated that the NSm protein alone inhibited STP-mediated apoptosis at the levels of activation of the initiator caspases, caspase-8 and -9.
The RVFV NSs protein, 78-kDa protein, and NSm protein are viral accessory proteins, because they are dispensable for virus replication in cell cultures (22, 51). A biological function of NSs, which suppresses host mRNA transcription, has been described (5, 34), whereas the biological functions of the NSm and 78-kDa proteins were unclear. By using arMP-12-del21/384, carrying a deletion in the pre-Gn region of the M gene ORF, and its parental virus, the present study identified RVFV NSm protein as an inhibitor of apoptosis.
Among five in-frame AUG codons in the pre-Gn region of RVFV M mRNA, the fourth AUG codon is used mainly for translation of the Gn and Gc proteins, while the 78-kDa and NSm proteins are translated from the first and second AUG codons, respectively (27, 47). In addition to these proteins, the present study and our previous studies (51) showed an accumulation of the 75-kDa protein, which reacted with an anti-NSm antibody, in MP-12-infected cells. Synthesis of a viral protein from the second AUG codon that corresponds to the 75-kDa protein was also reported for cells expressing RVFV M segment RNA (20). We also detected an accumulation of the 73-kDa protein, which also reacted with the anti-NSm antibody, in cells supporting the replication of arMP-12-delNSm/78; the NSm, 78-kDa, and 75-kDa proteins were not synthesized in arMP-12-delNSm/78-infected cells (Fig. 2C). Our unpublished studies showed that replication of the MP-12 mutant carrying a substitution mutation at the third AUG codon resulted in accumulation of the NSm, 78-kDa, and 75-kDa proteins but not of the 73-kDa protein. These data suggested that the 73-kDa and 75-kDa proteins were translated from the third and second AUG codons, respectively, and that the 73-kDa protein accumulation occurred when the first and/or second AUG codon was abolished. Among those proteins whose coding sequences contain the pre-Gn region, the NSm and 73-kDa proteins, but not the 78-kDa protein, suppressed caspase-3 cleavage and activation in the trans-complementation experiments (Fig. 6). Consistent with these data, the kinetics of cell viability were similar for arMP-12-infected cells and cells infected with arMP-12-delNSm/78, which expressed the 73-kDa protein but not the 78-kDa, 75-kDa, and NSm proteins (Fig. 3A). These data revealed that the NSm and 73-kDa proteins, but not the 78-kDa protein, had an antiapoptosis function. Furthermore, our data suggested that a low abundance of the 73-kDa protein could exert the antiapoptotic activity (see Fig. 2 and 6). Then why did the 78-kDa protein, which includes the sequences that are present in NSm and the 73-kDa protein, not perform an antiapoptotic function? It is possible to speculate that the 78-kDa protein translocates into the lumen of the endoplasmic reticulum through its N-terminal signal peptide, whereas the NSm, 75-kDa, and 73-kDa proteins lack an N-terminal signal peptide, and hence, the portions of these proteins encoded by the pre-Gn region are probably localized within the cytoplasm (19, 20, 26). If the NSm and 73-kDa proteins exert their antiapoptotic activities in the cytoplasm, then it is not surprising that the 78-kDa protein could not perform an antiapoptotic function, since its pre-Gn region may be located within the lumen.
We reported that arMP-12 and its three different mutant viruses, one lacking NSm protein expression, the second lacking 78-kDa protein expression, and the third lacking the expression of both the NSm and 78-kDa proteins, produce plaques with the same sizes and morphologies (51). Because the 73-kDa and/or 75-kDa protein accumulates in cells infected with these mutant viruses (51), we suspect that expression of the 73-kDa and/or 75-kDa protein in the cells infected with these mutant viruses exerted antiapoptotic activities and contributed to the formation of plaques indistinguishable from those produced by arMP-12. Gerrard et al. reported the generation of the ZH501 strain of RVFV lacking most of the pre-Gn region (18). Like arMP-12-del21/384, this mutant virus most probably does not express any viral proteins whose coding sequences carry the pre-Gn region, yet Gerrard et al. reported that this mutant produced plaques of the same size and morphology as those of its parental wild-type RVFV. It should be noted that 0.6% tragacanth gum was used as an overlay in our plaque assay, while Gerrard et al. used 1% agarose for the overlay. It is possible that the tragacanth gum overlay was more sensitive at detecting minute differences in RVFV plaque sizes and morphologies.
There are ample examples of apoptotic cell death occurring in virus infection; apoptosis is induced by host immune responses, virus-encoded proapoptotic proteins, or double-stranded RNA-mediated host cell responses (2, 24, 40). Induction of apoptosis in virus infection is considered to be one of the host defense mechanisms eliminating infected cells to limit viral replication and spread. Several bunyaviruses induce apoptosis in cell cultures and in vivo (9, 13, 14, 36). To secure efficient production of progeny virus, viruses have developed various strategies to suppress apoptosis by expressing viral antiapoptotic proteins (40, 43). The virus-mediated inhibition of apoptosis also contributes to the establishment of latent infections or viral oncogenesis. Large DNA viruses developing low levels of cytotoxicity, including adenoviruses, papovaviruses, herpesviruses, poxviruses, and baculoviruses (11, 17, 40, 43, 44, 49), have been shown to carry more than one antiapoptotic gene. In contrast to the work on large DNA viruses, studies of antiapoptotic proteins in RNA viruses are rather limited; several past studies have suggested the presence of antiapoptotic proteins in several picornaviruses (10), hepatitis C virus (41), respiratory syncytial virus (8),Bunyamwera virus (29), and influenza A virus (15). RVFV NSm protein is the first viral protein in phleboviruses to show an antiapoptosis function. Further studies are required to assess whether other bunyaviruses also encode antiapoptotic functions.
The number of dead cells among arMP-12-del21/384-infected cells was substantially higher than that among arMP-12-infected cells after 48 h p.i. (Fig. 3A), while the two viruses showed similar virus growth kinetics (Fig. 2D). Because efficient release of progeny RVFV occurs within the first 24 h after infection at an MOI of 1 or higher in cell cultures (Fig. 2D) (22, 51), the majority of progeny viruses have been released prior to virus-induced apoptosis. Rapid virus replication prior to induction of apoptotic cell death is one of the strategies that some RNA viruses have employed to secure efficient progeny production (30, 31), and it appears that RVFV also uses this strategy to circumvent the negative effects of virus-induced apoptosis for virus production. If RVFV always replicates quickly prior to the execution of apoptotic cell death, then why is the pre-Gn region in the RVFV M segment strictly retained in all RVFVs thus far sequenced (7)? A virus such as RVFV with such a rapid progression to cell death in virtually all vertebrate cell types infected might require prolongation of the time of productive infection. We also know that when cells that are undergoing transcriptional and/or translational suppression are exposed to tumor necrosis factor alpha, they quickly die by apoptosis (1, 46). It is established that RVFV replication efficiently suppresses host transcription and translation (22, 34). Accordingly, we suspect that a combination of virus replication and exposure of the infected cells to some extracellular factors, e.g., tumor necrosis factor alpha, may induce rapid apoptosis prior to the completion of maximum progeny virus production in RVFV-infected mammals; without NSm, RVFV may not be able to amplify efficiently in an infected host. Consistent with this notion, a recombinant wt RVFV lacking most of the pre-Gn region has attenuated virulence in rats (6), suggesting that proteins encoded by the pre-Gn region have some role in RVFV virulence.
Our data suggested that RVFV infection triggered apoptosis mainly through activation of caspase-8, which is a death receptor-mediated, extrinsic, apoptotic pathway, and that NSm expression suppressed caspase-8 activation (Fig. 5B). We also observed that NSm efficiently inhibited STP-induced caspase-8 and caspase-9 activities (Fig. 7). Because it is well established that activated caspase-8 can activate caspase-9 by provoking the release of cytochrome c from mitochondria via the cleavage of Bid (35, 37), it is conceivable that NSm protein inhibited either activated caspase-8 activity or an apoptotic pathway that activates caspase-8, resulting in the suppression of caspase-9 activation as well. However, we cannot exclude the possibility that the NSm protein also directly inhibits the activation of downstream caspases, including caspase-9 and caspase-3.
This work was supported by grants to S.M. and C.J.P. from the Western Regional Center of Excellence for Biodefense and Emerging Infectious Diseases, NIH grant U54 AI057156, NIH-NIAID-DMID-02-24 Collaborative Grant on Emerging Viral Diseases, and DHS grant NOOO14-04-1-0660, U.S. Department of Homeland Security, National Center for Foreign Animal and Zoonotic Disease Defense. S.W. and T.I. were supported by the James W. McLaughlin Fellowship Fund.
- Received 6 June 2007.
- Accepted 22 September 2007.
- ↵*Corresponding author. Mailing address: Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555-1019. Phone: (409) 772-2323. Fax: (409) 772-5065. E-mail: shmakino{at}utmb.edu
↵▿ Published ahead of print on 3 October 2007.
- American Society for Microbiology


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Red Family (Korean: 붉은 가족) is a 2013 South Korean drama film directed by Lee Ju-hyoung. It was written, produced and co-edited by the renowned art house moviemaker Kim Ki-duk.[1][2]
Plot summary[edit]
Undercover North Korean agents are planted in South Korean society with the mission to take out defectors.[3]
External links[edit]
- Red Family on IMDb
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LAHORE: On Monday Pakistan batsman Umar Akmal was fined 20 per cent of his match fee and given an official reprimand for a late night-out ahead of Pakistan's fifth ODI against Australia in Dubai, the Pakistan Cricket Board (PCB).
The PCB in its statement said that Akmal pleaded guilty to the charge, apologised for his actions and accepted the sanction proposed by the team manager.
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