text:
Factors to Consider When Buying or Selling a Car at an Auto Auction
If you are looking to buy or sell a car at an auto auction, there are several important factors to consider. These factors can affect prices, availability, and transit times. Generally, auction auto transport is less expensive than standard door-to-door shipping. Car transporters can often pick up multiple loads at an auto auction, which is called co-loading.
Pre-sale inspections are available at some auctions. These inspections are helpful for determining if the vehicle is worth buying. You can also test drive the vehicle before making a purchase. While pre-sale inspections are not required, they can help you avoid purchasing a car that has a mechanical issue or has been resold. This can give the buying dealer peace of mind and give them a better understanding of the car's condition before making a final decision.
Private auctions are a popular option for buying a car. These events are only open to licensed car dealers. These events often feature vehicles that have been impounded or seized by the government. In addition to private auctions, there are also government auctions. Some government agencies hold these auctions to sell off older vehicles and replace their fleets with new ones.
Cars and trucks arrive at auction sites 24 hours a day. They are transported to the auction site by rail or transport trucks. Sometimes, they are driven by dealership representatives or employees. This arrangement is made prior to the auction. When vehicles arrive at the auction site, they are scanned using a Vehicle Identification Number (VIN). Professional auction inspectors are trained to detect problems with the VIN. Once the vehicle has been checked, it is placed into the auction inventory where it will be sold later.
The benefits of bidding at an auction include the large selection of used cars and cheap prices. Many auctions are open to the public, while others are limited to licensed dealers. In the end, an auction is the most convenient option for used car buyers. It's worth the time and effort it takes to find a quality vehicle at an affordable price.
Unlike traditional retail car sales, auto auctions often use a black book, a miniature car pricing guide. Updated every month, the black book lists recommended selling prices for most vehicle models and makes. Dealers and auto auctioneers prefer to pay less than these recommended prices. But they don't always have this luxury.
Auctions can be intimidating. While they may sound like an exciting way to find a bargain, there are several things you must be aware of. First, it's important to understand the auctioneer's language. He or she may talk at an incredibly fast pace that most people can't understand. Secondly, there is always the chance that other potential bidders will raise the price.
Lastly, before you bid, be sure to inspect the car in person. Photos and condition descriptions may be misleading and leave out important details. So, it's important to come early to inspect the car and make sure it is in good shape. Also, look out for rust, blemishes, and oil leaks. It's also a good idea to practice bidding at an auction so that you can make the right decision fast.
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Oil group Cairn Energy (LON:CNE) has set out the details of a planned US$600 million exploration drilling campaign offshore Greenland later this summer. Following an initial three-well operation in 2010, Cairn will drill up to four wells targeting a gross unrisked mean prospective resource potential of 3.2 billion barrels of oil equivalent. It will also shoot around 4,500 sq km of 3D seismic. The group said it had agreed seven exploration targets with the Greenland government, giving it some flexibility in programme planning and possible options for follow up appraisal wells in the event of a discovery.
Cairn currently operates 11 blocks, with a combined area of 102,000 sq km offshore Greenland, which is equivalent to 15 quadrants or 450 blocks in the UK North Sea. Last year it drilled three wells and acquired 15,000km of seismic data. In January 2011 it increased its equity interest to 87.5% and became operator of the sub-Arctic Atammik and Lady Franklin blocks offshore west Greenland. As part of the new programme, one well will be drilled in each of the Atammik and Lady Franklin blocks and one in each of the Napariaq and Eqqua blocks in the southern Baffin Bay west Disko area. The 3D seismic acquisition will comprise three separate surveys; one in the north of Baffin Bay, one in the south of Greenland and one contingent location to be decided dependent on operational results.
Cairn noted that all of the proposed wells were targeting oil but that evidence of both oil and gas generation had been found regionally. In view of the frontier nature of the exploration and paucity of offset well information the individual prospects are estimated to have chances of success ranging from around 10% to 20%. The wells will be drilled in water depths ranging between 288 metres and 1,530 metres.
Mike Watts, Cairn's deputy chief executive, said: "The group's strong financial position and entrepreneurial exploration focus has allowed it to build a strategic and leading early entry position in the frontier offshore basins of Greenland, which Cairn believes has the necessary geological ingredients for exploration success."
In a note to investors, broker Evolution Securities said the operations update left Cairn shareholders standing at a cross roads. It said that the prize was not just the leverage that this summer's four well campaign provides but the wider implication of more barrels in the underexplored Greenland basins. "On the flip side is the high risk nature and if this summer's campaign disappoints Cairn will have spent around a US$1bn for nothing. If the Cairn India deal with Vedanta also falls through then shareholders don't have much to look forward to," it said. On the question of whether investors should take profits now and come back if there is success in Greenland, Evolution noted that past form in Rajasthan suggests there is ample opportunity to make profits by buying on the day major success is announced and unlike Rajasthan the potential for material follow-up success could be much higher. Evolution said its risked valuation for the new prospects was in the 60-70p range while fully de-risked they could be worth nearer £6. "With little for Greenland in the current share price we remain positive," it concluded. The Cairn Energy share price rose by 5.8p to 425p during the morning.
The final selection of prospects for the 2011 exploration campaign is the LF-7 prospect in the Lady Franklin block and the AT-7 prospect in the Atammik block. Both prospects are understood to contain multiple structural targets in the Tertiary and Cretaceous sections, with expected mean prospective resources of 480 and 350 million barrels respectively, with the probability of success estimated to range from around 10% to 20% dependent on the individual target horizons. Follow-up prospects are present in both blocks with an estimated combined gross mean in-place resource of more than eight billion barrels or 14 Tcf.
At the Napariaq block, which was awarded to Cairn in the Baffin Bay Bid round last December, the exploration well will target the Delta prospect, which is a large structural culmination in the Cretaceous section where the overlying Tertiary volcanics are prognosed to be thin or possibly even absent. The Delta Prospect has an expected mean prospective resource of 530 million barrels and probability of success estimated at around 10%. The potential for stacked pay could add further volumes to these estimates. Cairn said the Delta well would be important for understanding the potential of a regional Cretaceous play in the offshore Disko area.
Finally, at the Eqqua block, Cairn will target the Gamma prospect, which is a combination structural-stratigraphic trap in an interpreted Tertiary basin floor fan at 2,500 metres overlying a deeper Mesozoic structural culmination at around 4,500 metres. The fan targets potentially thicker reservoir sands 100 km from the earlier T-8 well but at the same stratigraphic level as the gas seen in thin sands in the T-8 well drilled in 2010. The Tertiary Gamma Prospect has a mean prospective resources of 1,840 million barrels or 7 Tcf and probability of success of around 10%.


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Доверие () — американский фильм 2016 года в жанре «чёрная комедия». Режиссёрами картины стали Алекс и Бен Брюэр, а авторами сценария Бен Брюэр и Адама Хирш. В фильме снимались Николас Кейдж, Элайджа Вуд, Скай Феррейра, Джерри Льюис, Кевин Уайзман и Стивен Уильямс. Премьера фильма сначала состоялась на телеканале DirecTV (14 апреля 2016 года) и лишь затем состоялась кинопремьера (13 мая 2016 года). Прокатом занималась компания . Этот фильм оказался последним, в котором сыграл Джерри Льюис перед смертью в 2017 году (хотя картина «» оказалась выпущена после ленты «Доверие»).

Сюжет
Молодой сержант полиции Дэвид Уотерс (Элайджа Вуд) и его старший коллега и друг лейтенант Джим Стоун (Николас Кейдж) трудятся в отделе хранения улик департамента полиции Лас-Вегаса. Работа не очень интересная, а зарплата еле-еле позволяет сводить концы с концами. Просматривая одно из досье, Джим обнаруживает интересный случай: мелкого наркодилера избавляют от тюремного заключения за внушительную сумму в 200 тысяч долларов наличными. Это говорит о том, что наркоторговец связан с крупными суммами.

В выходные дни Джим начинает вести несанкционированное наблюдение за гостиницей, в которой работает наркоторговец. Полицейский обнаруживает, что все товары, которые перевозит банда дилера, оказываются в одном и том же здании. Более того, после этого из хранилища ничего не вывозится. Дэвид добывает чертежи здания и обнаруживает, что там на первом этаже построен огромный сейф в дальней части промышленного морозильника. Причём круглосуточной охраны у этого хранилища нет.

Джим и Дэвид решают взломать сейф и украсть его содержимое. Они вычисляют точное расположение сейфа и приобретают мощную дрель с алмазным наконечником, чтобы просверлить стенку сейфа. Кроме того, полицейские приобретают на чёрном рынке огнестрельное оружие (вместо служебных пистолетов). По плану следует просверлить железобетонную крышу огромного сейфа из жилой квартиры, расположенной над морозильной камерой. Это позволило бы, видя изнутри замок, вскрыть входную дверь. План продуман до мелочей. Но характер действий в жилой квартире остается неясным.

В день взлома сообщники в масках врываются в квартиру над первым этажом. Там находятся два человека — мужчина и девушка. Полицейские не планировали убийства. Но дерзкое поведение мужчины выводит из себя Джима и он стреляет. Обитатель квартиры убит. Девушку связывают и запирают в одной из комнат.

Грабители используют дрель и специальные свёрла, чтобы проделать отверстие в бетонном полу и получить доступ в нижнее помещение. Далее им приходится пустить в дело сверло с алмазным наконечником. Но из-за обрыва приводного ремня станок ломается. Тогда Джим оперативно мастерит взрывное устройство, чтобы всё-таки проделать дыру. Всё проходит удачно. Наконец, Джим с помощью специального прибора (длинный гибкий цистоскоп) может видеть изнутри замок входной двери в хранилище. По рации он инструктирует Дэвида, как следует поворачивать циферблат с шифром. Дэвид записывает маркером после каждого поворота искомую цифру на поверхности двери. Наконец дверь открывается.

Взломщики проникают внутрь хранилища. Здесь они обнаруживают коробки с большим количеством алмазов. Джим находится в приподнятом настроении и желает поскорее заполнить сокровищами заранее подготовленные баулы. Но Дэвид начинает нервничать. Он догадывается, что кража этих ценностей не останется безнаказанной и бандиты выследят похитителей. Дэвид убеждает Джима пойти наверх, чтобы собрать оборудование, а сам в это время закрывает дверь в хранилище и стирает цифры кода. Дэвид также поднимается на второй этаж, чтобы помочь напарнику. Связанная заложница, рыдая, смогла убедить Дэвида, что ради спасения малолетнего сына ей надо сделать телефонный звонок бывшему мужу. Короткий разговор не вызывает подозрений.

Вскоре полицейские возвращаются на первый этаж. Увидев, что хранилище закрыто, Джим приходит в ярость. Он угрожает Дэвиду расправой и заставляет снова открыть вход. Затем напарники перегружают ценности в свою машину. Напоследок Джим решает убить захваченную в квартире девушку. Дэвид, не желая допустить этого, стреляет в товарища. После короткой перестрелки Джим убит.

Дэвид возвращает все ценности обратно в сейф, а девушку сажает в свою машину. Он обещает ей, что выпустит на свободу, отъехав подальше от города. Когда они едут по безлюдному участку дороги, фургон неожиданно оказывается окружён тремя другими автомобилями. На одном из них Дэвид с ужасом видит рекламную надпись с номером телефона, по которому ранее звонила девушка. Полицейский слишком поздно понял, что попал в ловушку. Из идущей впереди машины стреляют и убивают Дэвида. Заложница успевает перебраться на переднее сиденье и остановить мчащуюся машину.

Фильм заканчивается сценами со склада улик, где работали неудачливые грабители. В том числе на стеллажах можно увидеть оборудование, которым они пользовались.

Актёры
 Николас Кейдж — лейтенант Джим Стоун
 Элайджа Вуд — сержант Дэвида Уотерса
 Скай Феррейра — девушка, оказавшаяся в квартире
 Джерри Льюис — мистер Стоун, отец Джима
 Итан Сапли — детектив
 Кевин Уайзман — Рой
 Стивен Уильямс — Клифф

Съёмки
Работа над картиной началась 14 мая 2014 года. Изначально кроме Николаса Кейжда в роли его напарника должен был сниматься Джек Хьюстон. Но вскоре на съёмочной площадке его сменил Элайджа Вуд. 6 ноября 2014 года права на распространение фильма приобрела кампания Saban Films. Непосредственно съёмки начались 26 января 2015 года. Все сцены снимались в Лас-Вегасе.

Критика
Картина получила противоречивые отзывы критиков. На сайте Rotten Tomatoes фильм имеет рейтинг одобрения 63%, основанный на 46 рецензиях. Средний балл — 5,47 из 10 возможных. Итоговая оценка гласит: «Возможно, «Доверие» не станет классикой кинематографа, но его тщательно продуманный сюжет и актёрская игра Николаса Кейджа и Элайджи Вуда — должны понравиться любителям подобных картин». На сайте Metacritic фильм получил 58 баллов из 100 возможных (по результатам  опроса 12 критиков).

Примечания

Фильмы 2016 года
Фильмы США 2016 года
Фильмы о Лас-Вегасе
Фильмы-ограбления США

redpajama_set_name: RedPajamaWikipedia


Catherine is a senior communications professional with over twenty years experience advising major listed corporations in the telecom, technology and finial services sectors. Most recently she held the position of General Manager, Media Relations for Telstra Corporation leading the organisation's media unit, one of the most demanding and intense communications roles in Australia.
Prior to this Catherine spent ten years in the finance services sector as Head of Corporate Affairs for Credit Union Australia and General Manager, Business Unit Communications for the Commonwealth Bank of Australia. Catherine also spent nearly a decade in London working for mobile telecommunications provider Orange and as an Associate Director for Hill & Knowlton.
Catherine is seasoned in providing guidance at Board and Executive Leadership Team level across the entire spectrum of corporate reputation and issues management fields. She has managed large national and international teams responsible for communications strategy, media relations, social media, internal and external communications. Areas of specific corporate communications expertise include issues management, brand positioning, mergers and acquisitions, divestments, major restructures and downsizing, legislative impacts and crisis management.


3. Turn away three bolts 24 (fig. of the Detail of system of greasing of the engine of 1,4 l, 55 and 74 kw see) fastenings маслоприемника 25 to the block of cylinders and the oil pump. Remove маслоприемник and its lining 23.
4. Remove a belt of a drive of camshafts and a pulley 20 cranked shaft (subsection 2.4.2 see).
5. Turn away bolts 14 and remove the oil pump 13 with a lining 22.
7. Establish the new pump upside-down, having replaced new linings 22 and 23.
3. Remove a forward cover 5 (fig. of the Detail of system of greasing of the diesel engine see) block of cylinders (subsection 2.10 see).
4. Loosen the an inhaling of a bolt of fastening of the tension device 7.
5. Take aside the tension device 7 and remove a chain 4 drives of the oil pump.
6. Turn away bolts 6 and remove a partition 16 oil картера with the oil pump 1 in gathering.
7. Clear of a dirt and wash out uajt-spiritom a grid маслоприемника. If the grid does not manage to be cleared or it is damaged, replace маслоприемник 15. For this purpose turn away two bolts of fastening маслоприемника to the pump. At installation маслоприемника replace a sealing ring 14.
8. Replace a gear wheel 2 with the damaged teeths. For this purpose, keeping a gear wheel from проворачивания, turn away a bolt 3.
9. Establish the new pump upside-down.


Total transtec 5 85W-140 is a new generation rear axle oil manufactured from superior quality base oils and stable additives.
Smooth torque transmission for longer life of gear teeth.
Good thermal stability and wear protection while climbing sleep inclines.
API GL-5 AND MIL-L 2105D.


I cannot believe we have only one week left in this round of Insanity. Yes, I said "this round" because we are going to keep working out. The question is what we should next: Power 90, which I bought years ago; another round of Insanity; or get the CharLEAN DVDs. I know I am not ready to graduate to Asylum, the new Shaun T. program. We definitely want to focus on muscle building. For me, it's all about strengthening my upper body and core. I want to be able to lift and hold up my body weight.
But first we are going to do a 3-day detox before we jump back into another grueling exercise program. I wish we had done this before Insanity, it would have been a nice way to kick start our weight loss by ridding our bodies of water weight, built up toxins, and undissolved waste.
I was planning to have us do the Shakeology detox, but at $120 per kit that is way too expensive. I think a better investment is to buy a juicer. I used to detox regularly as part of my health regime so I know what to do. We are researching juicers and I found several juice cleansing recipes online. The first step is to ease into it by eating light a couple of days before the juice fast (no simple carbs, no meat, no fatty foods or refined sugars...nothing that the digestive system has to work hard to metabolize). Then 3 days of juice, juice, juice! Then we break the fast slowly by incorporating solids back into our diet starting with fruits/veggies, whole grains and leaving meat and dairy for last.
Glad that you've stayed with it. I can see a difference between your day one photo and this one.
First of all - you look fantastic!! What a lovely body you have. And I have wondered about cleansing. Would love a step-by-step post if that's something you'd consider. I'm still nursing so I couldn't really do it now but the idea really intrigues me.
btw I haven't heard back from my "guy"...gonna call him next week.
You look great! Way to go. Keep up the good work.
There is a big difference from Day 1 to Day 60. I hope you are happy with your progress!


Grate the cabbage and collect it in a big bowl.
Add all the spices and flours, oil and mix well. Now add eno and mix again.
Do NOT add any extra water or moisture.
Boil water in a steamer or in the rice cooker.
Grease the steamer plate well with cooking oil.
Now grease your palms and take approx.4 tblsp of mixture and shape into cylinders and place on the steamer plate.
If you are having difficulting in shaping them due to excess moisture then add some more gram flour and semolina.
Leave some gap in between the rolls,so that it cooks properly.
Put the tray in the steamer, close the lid and steam for approx 25-30 minutes on medium heat.
After that open the lid and check if it is done or not. When you touch the roll, the mixture should not stick on your finger or if you insert a tooth pick it should come out clean.
you can steam these muthiya and keep refrigerated for 2- 3 days ,temper when you want to serve them.
Heat 3 tbsp of oil in a pan, add mustard and sesame seeds, and once they start crackling, add slit green chillies, asafoetida and curry leaves.
Now add sliced muthiyas and stir fry for one minute.
Now add lemon juice, fresh coconut and chopped fresh coriander.
Cabbage Kababs – You can deep fry the dumplings after slicing and serve as cabbage kababs.or just make fritters with this mixture without steaming the dumplings.
Cabbage Kofta – Make a tomato or curd based gravy and add these muthiyas in it just before serving.
Cabbage Dahi Vada – Arrange muthiya in a dish and pour whipped salted curd over it, sprinkle red chilli powder, roasted cumin powder, sweet chutney and green chutney and serve as cabbage dahi vada.
Cabbage Appe – Make cabbage appe, in a appe maker with the same mixture.
Low Fat Snack – Slice muthiyas and add lemon juice and fresh coriander and serve with ketchup.
1. You can make muthiyas from cabbage, bottle gourd (Lauki), spinach (palak), fresh fenugreek leaves (methi), grated mix vegetables, left over dry khichdi (an Indian lentil and rice preparation) or even with left over vegetable pulao (mix vegetable rice).
2. If using Sweet Gourd (Lauki) then squeeze extra water from it after grating and if using pulao then mash the rice a little and add 1 tbls extra curd. into it.
I was looking for light dinner ideas n got one..
I ecently came upon your blog and have been trying several recipies. The dumplings came out awesome and everyone in my family loved them. Thanks for posting such wonderful recipies.
Hi, I am so glad to have stumbled upon your wonderful blog.. you are a true true treasure house of pure vegetarian delights.. thanks a ton for all the recipes, I will definitely try out these. And soooo glad to meet you.
Actually I don't have an Idli or Dhokla stand, and no steamer insert either, that's why I had asked the question above.
But still, I liked your recipe & photo so much, that I was determined to try it today (but with spinach)… I somehow steamed them in pressure cooker without the whistle, and then stir-fried, and when I ate it – I was super addicted to it!
And just to let you know, I also really, really liked how you gave other ideas of using these steamed dumplings!
Muthiyas are totally new to me..You have experienced hands in coking and so happy to follow you..
Do drop by my space when you find time ,wishing you will love my space too!!
Wish you and your family a very Happy New Year…super delicious dish….
Wish you and your family a very Happy New Year.
Very innovative and interesting recipe. Cabbage dumplings looks delicious and tempting.
Excitingly new recipe..:)) Loved it..
Super yummy and healthy recipe.
steaming time depends on the thickness of the roll and the quantity placed in the container.if you make thin rolls and put only few then they get cooked in lesser time .
Thank you friends for your lovely comments and suggestions!!!
you can easily steam these muthiya in a idli stand or in a dhokla stand.
Wishing you a very Happy new year !!!
Thanks to you i am trying out all the gujarati recipes I never cooked simply cos always considered them "mommy special"..now i guess can't reply on mom all the time, am a mom myself!
sounds so healthy n yummy one…looks great…bookmarked!!
New recipe to me! Looks delicious..steaming for 40 minutes??!!! Will it take that much time??!
Can we keep 1(or) 2 logs and steam cook for lesser time?!
Muthiya looks delicious, Wishing you and family and happy and prosperous New year.
If I don't have the steamer insert, can you suggest me some other idea on how I can make steamed dishes like these?
yes Balvinder you can make these muthiyas with besan only,wheat flour and semolina is added to give softness and to make them lighter to digest.if you use only besan then add 1 t.s eno and 2 tbls of oil.
I have thought to cook this recipe but not sure what to use in place of wheat and semolina. The muthiyas look so nice. Can I just make with besan?
wow this z really amazing recipe….
Super tempting dumplings, sounds so delicious. Thank you for stopping by my space and leaving such lovely comments and I glad to follow u!!


Oh, this will be good! Adam has been working on, thinking and writing about forms for quite a while and he has distilled that down into ten patterns. You just know that progressive enhancement will be at the heart of this book.
alphagov/accessible-autocomplete: An autocomplete component, built to be accessible.
If you're looking for an accessible standalone autocomplete script, this one from GDS looks very good (similar to Lea's awesomplete).
Accessible star ratings (progressively enhanced from radio buttons) with lots of animation options. The code is on Github.
An interesting idea from Ruth—using subtle sounds to augment inline form validation.
There aren't any extremely established best practices for this stuff. The best we can do is make tasteful choices and do user research. Which is to say, the examples in this post are ideas, not gospel.
A look at the feedback needed for a slider control that feels "right".
<input type="range" min="0" max="100" value="50"
Here's a CSS file that will give you a bit more control over styling some form elements. The thinking behind the CSS for each element is explained nice and clearly.
A wonderful piece by Ethan taking issue what the criticism that responsive design is over-reliant on screen size. Instead, he says, it begins with screen size, but there's no limit to where we can go from there.
Responsive design might begin with the screen, but it doesn't end there.


text:
Q: Ubuntu 12.04 installation failed - terminated by signal 9 ( killed) I tried to install Ubuntu 12.04 via USB on my Acer TravelMate x483, but the installation stopped and this is what is showing on the screen:
Udevd[261]: '/sbin/blkid -o udev -p /dev/sdb'[3102]terminated by signal 9 ( killed)

How can I fix this?

A: If you were installing via Bootable USB, it would be that which showed that error. Either your USB disconnected accidentally or it is malfunctioned (i,e contains some error) or not made bootable in proper way.
You can try to install with USB also but I suggest you to make bootable USB using Startup Disk Creator which is available in Ubuntu by default. So for that you can refer to any friend who are using Ubuntu. Although it would be better to install using Live DVD.. ;)

redpajama_set_name: RedPajamaStackExchange


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Two Hall of Famers We Are Very Proud Of In Lycoming County
Lycoming NCAA Hall of Fame coach Frank Girardi (L) and 2019 Baseball Hall of Fame pitcher Mike Mussina.
(Photo Courtesy of John Green taken June 2, 2015)
FRANK GIRARDI:Longtime Lycoming head football coach Frank Girardi was inducted into the College Football Hall of Fame on Tuesday. Girardi was the head coach at Lycoming from 1972 to 2007, amassing 257 wins in that time, good for 16th all time in NCAA history. Girardi led the Warriors to the NCAA Division-III tournament 11 times, and reached the national championship game in 1990 and 1997.
MIKE MUSSINA:Legendary Major League Baseball (MLB) pitcher and Montoursville, Pa., native Mike Mussina will earn his iconic bronze plaque this summer as he has officially been named a member of the 2019 National Baseball Hall of Fame Class by the Baseball Writers' Association of America. A dedicated Little League® volunteer and Little League International Board of Directors member, Mr. Mussina will join fellow inductees and former Little Leaguers, Roy Halladay, Edgar Martinez, and Mariano Rivera, as well as Today's Game Era ballot winners, Harold Baines and Lee Smith, in this year's induction.
Labels: 2019, Awards and Recognition

redpajama_set_name: RedPajamaCommonCrawl


"My Deepest Sympathy and Prayers of comfort to his family and friends. Thanks for the music and laughs. Rest In Peace # Fan "
"My grandfather Adrian Michaels and Daryl's father Carmen were good friends and colleagues for many years. I am sorry for Daryl's passing and wish his family love, hope and eternal peace. I was a very young boy but I remember Carmen. I know my..."
"I WAS 16 YEARS OLD WHEN THERE HIT SONGS CAME OUT BEST TIME OF MY LIFE THANK YOU FOR YOUR MUSIC YOUR SONGS BRING GOOD MEMORIES BACK.MAY YOU REST IN PEACE IN HEAVEN WITH THE ANGELS GOD BLESS YOU."
"Condolence to the family on your loss of loved one in his prime of life! Do be comforted Isaiah26:19"
"Thanks for the music and memories. RIP."


Glithero presents a new furniture series titled 'Les French' at gallery Fumi. The work represents an ad-hoc exercise in translation from sketch model to finished piece. A family of unique characters, each piece is named after a french phrase that has been adopted and appropriated by the english language.


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The Eleanor Bradley Fellowship - Alan and Cyril Body Trust
Maternal mortality is a desperate blight on sub-Saharan Africa where the lifetime risk of maternal death is 1 in 16. By contrast the lifetime risk of maternal death for developed nations is only 1 in 2,800. Maternal death is a major cause of health inequality and also has a significant impact on the life chances of surviving children.
Find out about the work of the Eleanor Bradley Fellows
"At the Royal College we are very proud of the Eleanor Bradley Fellowship and use it as a model for other initiatives. The key point is its sustainability, with one Fellow building off the achievements of his or her predecessors. We hope it continues for many years to come. Please support it. "
Professor Anthony Falconer RCOG President
For a link to the
Royal College of Obstetricians and Gynaecologists
Eleanor Bradley (1920 - 1976) was Cyril Body's wife and the mother of David Body, the current Chair of Trustees.
She was born in Llanelli and became an SRN and midwife. She trained at the Royal Gwent Hospital in Newport at the beginning of the Second World War, and became a District Midwife in Cardiff.
The prizes given by the Trust and the Fellowship that bear her name were established in her memory by Cyril and David.
Unfortunately the Trust has not had sufficient funds in recent years to continue with this Fellowship, but is proud of its creation of the project and the change that it brought about. This is a page to commemorate what it achieved.
In conjunction with the Royal College of Obstetricians and Gynaecologists the Trust established the Eleanor Bradley Fellowship, which provided a one-year training experience in Uganda for an obstetric and gynaecological Specialist Registrar. It iwas open to doctors at either Cardiff University or the University of Liverpool.
From 2008, for four years, the Trust was able to send a Fellow each year, whose presence at the Mulago Hospital in Kampala Uganda has made a significant difference to maternal perinatal and neonatal mortality. In the first year, a maternal death audit and a programme for training midwives were established. In the second year, a triage system for the labour ward was introduced, which
supported the development of a High Dependency Unit in the third year. The net result has been that the number of maternal deaths at the Mulago Hospital has fallen substantially over three years. And good practice spreads outward from the hospital.
The Fellowship had two-way benefits. It is also of major educational benefit for the Fellow, who encounters a range of clinical experiences outside the usual UK practice and finds that s/he has to exercise ingenuity and initiative in a way that greatly enhances his or her skills, which s/he then brings back to the UK.
The fellowship cost between £15,000 and £18,000 a year. At its crudest, this amounts to around £400 for a woman's life. The scheme also yields other benefits: children are spared coping with the loss of their mother in an already challenging environment; a significant number of babies' lives have been saved as a direct or indirect result of the work of the Fellows; and a number of women are living healthier and fuller lives as a result of the medical interventions at the Mulago Hospital.


text:
#include "NuiFrameCacheImpl.h"

class NuiFrameBuffer : public NuiFrameCacheImpl
{
public:
	NuiFrameBuffer();
	virtual ~NuiFrameBuffer();

	virtual void								clear() override;

	virtual bool								pushbackFrame(std::shared_ptr<NuiCompositeFrame> pFrame) override;
	virtual std::shared_ptr<NuiCompositeFrame>	getLatestFrame() override;
	virtual std::shared_ptr<NuiCompositeFrame>	getFrame(UINT frameId) override;

private:
	std::shared_ptr<NuiCompositeFrame>			m_pFrame;
};

redpajama_set_name: RedPajamaGithub


Ensuite Tents are small privacy rooms perfect for your camp shower, change room or toilet. They are simple in design so that they can be set up and packed away quickly and easily. Get a double to separate your shower, change room or toilet!
Ensuite's make a perfect camping shower or toilet tent. They can also be used as change rooms for sporting events, markets, camping and anything else you would need a bit of privacy for!
They come in 3 main styles - Pop Up, Instant Frame and Dome.
Pop up Ensuite's are the quickest to set up and pack away but don't offer quite the same stability or strength of the other 2 styles.
The OZtrail Privacy Ensuite is the cheapest and smallest ensuite tents. It is a great budget option but doesn't provide the structural support and stability of the other models.
The OZtrail Privacy Ensuite XL provides a bit more room but again doesn't offer the structural stability of the other models.
The OZtrail Pop Up Ensuite provides generous dimensions with more upright walls than the Privacy Ensuites providing more room for showering in comfort. It also has wall and roof braces for extra stability. Get the Tactix model if you want to dissappear! The OZtrail Pop up Double Ensuite features a shared wall that can be unzipped to create one large room.
The OZtrail Ensuite Instant Frame has a super quick set up frame already attached to the tent so that you have the advantage of a steel stable frame combined with the speed of a "pop-up" design.
The OZtrail Ensuite Dome provides a super stable & sturdy steel frame that is still quick and easy to set up and pack away. The OZtrail Ensuite Duo Dome provides twice the privacy with only a few more poles.
The OZtrail Ensuite Dome Jumbo provides the same strength of the other Domes with larger dimensions for a great feeling of spaciousness.


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Emballonura monticola is een zoogdier uit de familie van de schedestaartvleermuizen (Emballonuridae). De wetenschappelijke naam van de soort werd voor het eerst geldig gepubliceerd door Temminck in 1838.

Schedestaartvleermuizen
IUCN-status niet bedreigd


For the 10th Anniversary of Project: Mooncircle, a 46 tracks-compilation released the 16 March 2012, with artists such as Sweatson Klank aka Take, Robot Koch, fLako, Jehst, Beat Butcha, Long Arm, Kelpe, Pavel Dovgal, Dday One, Rain Dog, Kidkanevil, Sorrow, 1000Names, DZA, Sun Glitters and many more...!
Listen and download the track for free on Soundcloud.
Full EP "Wooden Love" available on bandcamp here.


text:
Rey Carabeo
Washington State University, United States
George Liechti
Uniformed Services University of the Health Sciences, United States
Ashok Aiyar
LSU Health Sciences Center New Orleans, Louisiana State University, United States
Maria T. Damiani
Instituto de Histología y Embriología Dr. Mario H. Burgos (IHEM CONICET), Argentina
Front. Cell. Infect. Microbiol., 30 November 2018 | https://doi.org/10.3389/fcimb.2018.00415
Genetic Inactivation of Chlamydia trachomatis Inclusion Membrane Protein CT228 Alters MYPT1 Recruitment, Extrusion Production, and Longevity of Infection
Jennifer H. Shaw1*, Charlotte E. Key1, Timothy A. Snider2, Prakash Sah3, Edward I. Shaw3, Derek J. Fisher4 and Erika I. Lutter3*
1Department of Integrative Biology, Oklahoma State University, Stillwater, OK, United States
2Department of Veterinary Pathobiology, Oklahoma State University, Stillwater, OK, United States
3Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK, United States
4Department of Microbiology, Southern Illinois University, Carbondale, IL, United States
Chlamydia trachomatis is an obligate intracellular pathogen with global health and economic impact. Upon infection, C. trachomatis resides within a protective niche, the inclusion, wherein it replicates and usurps host cell machinery and resources. The inclusion membrane is the key host-pathogen interface that governs specific protein-protein interactions to manipulate host signaling pathways. At the conclusion of the infection cycle, C. trachomatis exits the host cell via lysis or extrusion. Extrusion depends on the phosphorylation state of myosin light chain 2 (MLC2); the extent of phosphorylation is determined by the ongoing opposing activities of myosin phosphatase (MYPT1) and myosin kinase (MLCK). Previously, it was shown that MYPT1 is recruited to the inclusion and interacts with CT228 for regulation of host cell egress. In this study, we generated a targeted chromosomal mutation of CT228 (L2-ΔCT228) using the TargeTron system and demonstrate a loss of MYPT1 recruitment and increase in extrusion production in vitro. Mutation of CT228 did not affect chlamydial growth in cell culture or recruitment of MLC2. Moreover, we document a delay in clearance of L2-ΔCT228 during murine intravaginal infection as well as a reduction in systemic humoral response, relative to L2-wild type. Taken together, the data suggest that loss of MYPT1 recruitment (as a result of CT228 disruption) regulates the degree of host cell exit via extrusion and affects the longevity of infection in vivo.
Chlamydia trachomatis (serovars D-K, L1-L3) is the most commonly reported bacterial sexually transmitted infection (STI) in the United States and worldwide with over 130 million new cases each year (Newman et al., 2015). While antibiotic therapy is efficacious, re-infection is common and many chlamydial infections are asymptomatic thereby left untreated. Ongoing infection in women leads to scarring within the reproductive tract, which may cause pelvic inflammatory disease, ectopic pregnancy and infertility. Likewise, extensive scarring in response to ocular infection (serovars A–C) is the leading cause of preventable blindness in underdeveloped countries (Bourne et al., 2013). The global impact on health and economic burden necessitates the development of a vaccine. Vaccine strategies were initially investigated in the 1960s wherein short term immunity was produced against ocular trachoma in children vaccinated using live or fixed whole bacteria (Grayston et al., 1963; Dhir et al., 1967) and more recently, a live-attenuated vaccine protected non-human primates against trachoma (Kari et al., 2011). Currently, there are five vaccine candidates in preclinical trials and one (MOMP-VD4) is in Phase I (Poston et al., 2017). While significant progress has been achieved, a major limitation to elucidating the drivers of pathogenesis and long-term immunity has been the prior lack of genetic tools.
The technical challenge in studying chlamydial pathogenesis is largely due to the complex nature of this obligate intracellular pathogen which exhibits a biphasic development cycle involving an infectious form, the elementary body (EB), and a non-infectious replicative form termed a reticulate body (RB) (Moulder, 1991). Upon infection, Chlamydia directs the formation of a protective niche within the host cell called an inclusion, which becomes decorated with Chlamydia-derived inclusion membrane proteins (Incs) that interface with the host cytosol (Hackstadt et al., 1997) as well as kinase laden microdomain regions where inclusion membrane proteins interact with the host cytoskeleton and signaling pathways (Mital et al., 2010; Mital and Hackstadt, 2011). Within this specialized vacuole, EBs differentiate into RBs followed by abundant replication of RBs causing the inclusion to enlarge. Within 24–48 h the RBs undergo asynchronous differentiation back to EBs in preparation for dissemination while avoiding detection by the host innate immune response. Host cell egress occurs either by lysis of the infected cell releasing EBs or extrusion of EB-filled inclusions wrapped in host membrane leaving behind an intact host cell (Doughri et al., 1972; Hybiske and Stephens, 2007, 2008).
The extrusion exit mechanism may provide a means to temporarily evade the host immune response via enclosure within host membrane and maximize dissemination by delivery of Chlamydia-rich vesicles to neighboring cells rather than individual EBs. Uptake of extrusions by macrophages and survival within has been documented in vitro (Zuck et al., 2016a), however whether this occurs in vivo remains unknown. While there may be benefits to the pathogen for this exit strategy, unbridled extrusion would likely be accompanied by a substantial metabolic cost to its host. Studies show that extrusion is negatively regulated by the interaction between C. trachomatis inclusion membrane protein CT228 and host myosin phosphatase target subunit 1 (MYPT1). Whether Chlamydia exit via lysis or extrusion is controlled by the phosphorylation state of myosin light chain 2 (MLC2): dephosphorylated MLC2 favors lysis while phosphorylated MLC2 enhances extrusion (Lutter et al., 2013). Therefore, recruitment of host MYPT1 to the inclusion by CT228 serves to regulate the manner and degree of host cell egress. Although the extrusion process has been observed in vitro for many years (Todd and Caldwell, 1985; Hybiske and Stephens, 2007, 2008), it was only recently documented to occur in vivo across multiple serovars (Shaw et al., 2017).
In the last 8 years genetic approaches have proven feasible across chlamydial species and serovars enabling the generation of targeted chlamydial mutants and expression of recombinant genes (Hooppaw and Fisher, 2015). These advances have been crucial in confirming the secretion and localization of C. trachomatis Incs (Weber et al., 2015) and open the path to discovery of virulence factors. Herein, we utilized the TargeTronTM method (Johnson and Fisher, 2013) to produce a targeted chromosomal mutation in C. trachomatis serovar L2 inclusion membrane protein CT228 (L2-ΔCT228). We report that the resultant loss of CT228 did not affect chlamydial growth in cell culture. However, recruitment of MYPT1 to the inclusion membrane failed to occur and a significant increase in extrusions was observed in vitro. Moreover, we documented a delay in clearance of L2-ΔCT228 during murine intravaginal infection, suggesting that enhanced host cell exit of EBs within the protective enclosure of an extrusion may reduce antigen presentation to the host immune system. This is further supported by a significant reduction in mean systemic anti-Chlamydia IgG2a titers following infection with L2-ΔCT228 compared to the L2-wild type.
Chlamydial Strains and Cell Culture
Chlamydia trachomatis serovar L2-wild type and L2-ΔCT228 (LGV 434/Bu, originating from the Hackstadt Lab at Rocky Mountain Laboratories, Hamilton, MT) were propagated in HeLa 229 cells and purified by Renografin density gradient centrifugation as previously described (Caldwell et al., 1981). HeLa were grown in RPMI 1640 + 5% fetal bovine serum (FBS) at 37°C with 5% CO2 and 1 μg/mL of cycloheximide added as needed.
Construction of pDFTT295 and Selection of a C. trachomatis L2-ΔCT228
The chlamydial TargeTron vector pDFTT3(bla) was modified to allow for spectinomycin selection by replacing the bla cassette with the aadA cassette (Lowden et al., 2015). The TargeTron algorithm from Sigma-Aldrich was used to predict CT228 insertion sites and primers were chosen that would target intron insertion at bp 295 (A in ATG used as position 1) in an antisense orientation (E-value of 0.200). The vector was then retargeted for insertion into CT228 using PCR with Phusion High-Fidelity PCR Master Mix (Thermo-Fisher) and primers 228A, 228B, 228C, and EBS universal (Integrated DNA Technologies, Coralville, IA; Table S1) followed by DNA restriction digestion as previously described (Johnson and Fisher, 2013). The pDFTT295 vector was propagated in Escherichia coli DH5α grown at 30°C in LB supplemented with 20 μg/ml chloramphenicol. Chlamydia trachomatis transformation and mutant selection was carried out as described in Lowden et al. using 500 μg/ml spectinomycin (Lowden et al., 2015). The passage 4 mutants were plaque purified to obtain a clonal L2-ΔCT228 strain. The plaque isolate was serially expanded in cell culture using HeLa cells and stored in sucrose-phosphate-glutamic acid (SPG) buffer at −80°C. Stocks were titered using the infectious progeny quantitation assay.
PCR Validation of L2-ΔCT228
Genomic DNA was harvested from L2-wild type and L2-ΔCT228 EB stocks using the DNeasy Blood and Tissue Kit from Qiagen. PCR reactions were performed with 50 ng of genomic DNA or 5 ng of plasmid DNA and Phusion High-Fidelity PCR Master Mix. The primers used are provided in Table S1. PCR products were run on 1% agarose gels, stained with ethidium bromide, and viewed using UV transillumination. To confirm the location of the GII insertion in CT228, the PCR product generated from primers CT228-2F and CT228-2R was extracted from the agarose gel using the GeneJET gel extraction kit (Thermo-Fisher) and ligated into pJET (Thermo-Fisher) to generate pJET::CT228GIIaadA. Ligation products were transformed into E. coli DH5α and transformants were selected at 37°C on Luria-Bertani (LB) agar plates supplemented with 100 μg/ml of ampicillin. A colony was then selected for overnight growth in LB with ampicillin and the plasmid was extracted using the GeneJET Plasmid Miniprep kit (Thermo-Fisher). The gene insert in pJET::CT228GIIaadA was sequenced using the vector specific primers JETF and JETR via Sanger sequencing performed by Macrogen USA. Sequencing results were analyzed using BioEdit (TA Hall 1999) and Clone Manager (Sci-Ed Software).
Transcript Analysis of the L2-Wild Type and L2-ΔCT228 CT229-CT224 Gene Linkage Group
Total RNA was harvested from C. trachomatis serovar L2-wild type and L2-ΔCT228 (LGV 434) infected Hela cells at 24 h post-infection using Tri Reagent (Ambion, Austin, TX). To remove contaminating DNA, all RNA samples were treated with RQ1 DNase (Promega, Madison, WI). Removal of contaminating DNA was confirmed using PCR. Reverse Transcriptase (RT)-PCR analysis was carried out using the Access Quick RT-PCR Kit (Promega, Madison, WI) following the manufacturer's instructions. All oligonucleotide primers used in this study are shown in Table S1. RT-PCR analysis of groEL (control), CT229, CT228, CT227, CT226, CT225, and CT224 open reading frames (ORFs) was performed using oligonucleotide primer pairs within each ORF, respectively. For CT228, primers pairs designed upstream (5′), downstream (3′), or spanning the TargeTron insert were employed. Amplicons were separated by 1.5% agarose gel electrophoresis. Ethidium bromide stained gels were visualized on a BioRad Imager (BioRad, Hercules, CA). Genomic DNA samples were analyzed in parallel during each assay as a PCR positive control.
Growth and Infectious Progeny Quantitation
Chlamydia trachomatis L2-wild type and L2-ΔCT228 were used to infect HeLa cell monolayers at 90% confluency in 24 well plates at a MOI of ~0.5; inoculated plates were centrifuged for 1 h at 700 × g then any free EBs were washed away prior to feeding cells. At 0, 6, 12, 24, and 48 h post-infection cells were lysed with water, lysates were serially diluted in Hank's Balanced Salt Solution and used to infect HeLa cells in 96 well plates. At 24 h post-infection, monolayers were fixed in methanol and stained with rabbit anti-EB antibody followed by anti-rabbit DyLight 488 (Jackson ImmunoResearch Laboratories, Westgrove PA). The number of inclusions per field of view were counted in 20 fields for each sample using a Leica DMI600B fluorescent microscope. Total Infectious Forming Units (IFUs)/ml were calculated for each sample. Titers for both the C. trachomatis L2-wild type and L2-ΔCT228 were repeated 3 times to ensure 5 μL contained 1 × 106 EBs.
Enumeration of Extrusions
Extrusions were enumerated by a method similar to that described by Lutter et al. (2013). HeLa cell monolayers (50% confluence) were plated into 24-well plates and incubated overnight with MYPT1 or Scramble Targetplus Smartpool siRNA (Dharmacon, Lafayette, CO.), complexed with DharmaFECT1 in RPMI with 5% FBS. At 48 h post-transfection, cells were infected in triplicate with C. trachomatis L2-wild type and L2-ΔCT228 at a MOI of ~0.5. At 48 h post-infection, the supernatants were removed and gently pelleted by centrifugation at 300 x g with the resulting pellet gently resuspended in 50 μL of media. Trypan blue (ThermoFisher Scientific) and NucBlue Hoechst live-cell stain (ThermoFisher Scientific) were added and 10 μL samples were loaded onto a Hausser Bright-line hemacytometer and a total of 16 grids were counted for each condition. Extrusions free of host cell nuclei (to distinguish from an infected cell) were enumerated on a Leica DMI6000B fluorescent microscope. The entire experiment was repeated on 3 separate days to ensure consistent data across multiple trials.
Immunofluorescence to Measure MYPT1 and Myosin Recruitment
C. trachomatis L2-wild type and L2-ΔCT228 were used to infect HeLa cell monolayers on glass coverslips in 24 well plates (CellTreat Scientific, Pepperell MA) at a MOI of ~0.5 (performed in technical triplicates). At 18 h post-infection supernatants were removed and cells were fixed in cold methanol. Recruitment of host proteins was tested with primary antibody staining to MYPT1 (United States Biological Life Sciences, Salem, MA), phospho (pSer-19)-MLC2 (Abcam, Cambridge, MA), phospho (pTyr419) Src (Millipore Sigma, Burlington, MA), Myosin IIa (ThermoFisher Scientific), Myosin IIb (ThermoFisher Scientific), and phospho (pTyr 471) Myosin Light Chain Kinase (Santa Cruz). Chlamydiae were detected with anti-Chlamydia LPS (ThermoFisher Scientific). Fluorescent secondary antibodies, anti-mouse or anti-rabbit DyLight 594 and DyLight 488 (Jackson ImmunoResearch Laboratories, West Grove, PA) were used for indirect immunofluorescence. The entire experiment was repeated on three separate occasions (n = 3 biological replicates). Twelve images were captured per condition using a Leica DMI600B fluorescent microscope and a representative image was selected for presentation.
HeLa cells were infected with C. trachomatis L2-wild type and L2-ΔCT228; at different time points during infection, cells were lysed with 2X Laemmli Sample Buffer (Biorad, Hercules, CA). Protein samples were separated by SDS-PAGE electrophoresis (12 or 15% gel) and transferred to 0.2 um nitrocellulose membrane (Bio-Rad, Hercules, CA). Membranes were blocked with 5% non-fat dry milk/5% BSA in 1X Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at room temperature. After blocking, membranes were incubated with primary antibodies diluted in 5% non-fat milk/5% BSA in 1X TBST at 4°C overnight followed by incubation with horseradish peroxidase conjugated anti-rabbit/mouse antibodies (Cell Signaling, Danvers, MA) for 1 h at room temperature. Detection was performed by enhanced chemiluminescence using SignalFire ECL reagents (Cell Signaling Technology, Danvers, MA). Immunoblot images were acquired using the Fluorchem E FE0622 system (ProteinSimple, San Jose, CA). Primary antibodies to MLC2 (Millipore Sigma, Burlington, MA), p-MLC2 (S19) (ThermoFisher Scientific), HsP60 (ThermoFisher Scientific), GAPDH (Santa Cruz), and MYTP1 (Cell Signaling, Danvers, MA) were used herein.
Intravaginal Infection of Mice
Six-week old female inbred C3H/HeJ mice were purchased from Jackson Laboratories (Bar Harbor, Maine). All animal work was performed according to The Animal Care and Use Guidelines and approved by the Institution of Animal Care and Use Committee at Oklahoma State University. At 7 and 3 days prior to infection, all mice were subcutaneously injected with a depot formulation of 2.5 mg of medroxyprogesterone acetate (Upjohn, Kalamazoo, MI) to synchronize estrous prior to infection. Mice were infected intravaginally with 1 × 106 C. trachomatis L2-wild type EBs or L2-ΔCT228 EBs in 5 μL sucrose-phosphate-glutamate (SPG) to monitor both the course of infection and immune response (n = 8 infected/colonized with L2-wild type; n = 9 infected/colonized with L2-ΔCT228) and histopathology (n = 5 for L2-wild type and L2-ΔCT228). Due to the inherent technical imperfection of the intra vaginal route of infection using small volumes (e.g., 5 μL) and that the human L2 serovar produces less robust colonization relative to other strains, any mice swabbed early (Day 7) post-infection who shed 0 IFU (100% clear of infection) were not processed any further.
Quantification of Recoverable IFUs From the Cervicovaginal Tract
At days 7, 14, 21, 28, and 42 days post-infection, cervicovaginal tracts were swabbed (Puritan Diagnostics, HydraFlock 6″ 15 cm swabs; Guilford, ME) and each swab was added to tubes containing 600 μL SPG and two glass beads on ice, as adopted from previously described studies (Shaw et al., 2001, 2017). Swab samples were vortexed vigorously to liberate EBs from the swab followed by serial dilution and inoculation of confluent HeLa cell monolayers in 96 well plates (CellTreat Scientific, Pepperell MA). Plates were centrifuged at 700 × g for 1 h to promote EB entry, then incubated at 37°C for 30 min. Inoculated HeLa monolayers were washed to remove any extracellular EBs then incubated in RPMI 1640 + 5% FBS + gentamicin (10 μg/mL) for 24 h at 37°C with 5% CO2. Plates were fixed with methanol and stained with anti-MOMP (courtesy of Dr. Harlan Caldwell) followed by anti-mouse DyLight 488 (Jackson ImmunoResearch). Twenty fields of view were counted using a Leica MI6000B fluorescent microscope. Total recoverable IFUs per mouse were calculated for each sample. While variation ranging across 2 logs of recoverable IFU exists throughout the Chlamydia murine model literature and periodically within the same study, replicated studies using this current L2 stock at 1 × 106 per mouse consistently produces 104-105 recoverable IFU at early time points post-murine infection and is therefore used for internal comparison to mice infected with 1 × 106 L2-ΔCT228 EBs herein.
Quantification of Systemic and Mucosal Antibody Response
To measure systemic and mucosal immune responses to infection, sera and vaginal lavages were collected 31 days post-infection for ELISA. Vaginal lavages were obtained by gently washing the vaginal vault with 60 μL 0.5% BSA-PBS twice and stored at −20°C until the ELISA was performed. Briefly, 96-well polystyrene plates (Immulon 2HB; Thermo, Milford, MA) were coated with 1 μg of formalin fixed EBs (serovar L2) per well in 100 μL TBS (50 mM Tris buffer pH 7.5, 0.15 M NaCl) overnight at 4°C. Following adsorption of fixed EBs, wells were washed to remove unbound EBs then blocked with 200 μL 2% BSA in 0.012 M Tris pH 7.4, 0.14 M NaCl, 3.0 mM KCl, 0.05% Tween 20 for 90 min at 37°C. Blocking solution was removed and wells were washed, then serial dilutions of sera and vaginal washes were added and incubated for 90 min at 37°C. Chlamydia-specific antibody titers were measured using alkaline phosphatase-conjugated anti-mouse IgG2a (sera samples) and anti-mouse IgG and IgA antibodies (vaginal lavage samples) (Southern Biotech Associates, Birmingham, AL). P-nitrophenyl phosphate (pNPP) was used as a substrate and resulting absorbance was read at 405 nm (BioTek Synergy, Winooski, VT). Antibody titers were considered positive at the highest sample dilution that displayed an absorbance that was ≥3X the absorbance of concentrated sham (uninfected) samples.
Reproductive Tract Histology
Mouse uteri were excised and immersion fixed in 10% buffered neutral formalin on days 23 and 64 post-infection for scoring inflammatory damage. Following fixation, they were processed in whole and embedded en bloc into paraffin followed by sections cut at 4 μm and stained with hematoxylin and eosin (H&E). Sections were examined and scored via light microscopy by an American College of Veterinary Pathologists (ACVP) certified veterinary pathologist with the following numerical designations: 0, normal; 1, minimal change; 2, mild change; 3, moderate change; 4, severe change. Scored parameters (mean ± SE) included an overall impression, periglandular mucinous change, hydrosalpinx, uterine tubal dilation, and luminal PMN. Morphometric analyses of endometrial luminal epithelial height were determined using calibrated measurements via Olympus CellSens software coupled to an Olympus DP70 microscope camera. Each reproductive tract was evaluated regionally and morphometrically at six locations (proximal, mid-, and distal locations of the two uterine horns) determined to be uniform and free of tissue bends and curves.
Student's unpaired two-tailed t-test with equal variance was used to compare mean ± SE growth (IFUs) and extrusion numbers between L2-wild type and L2-ΔCT228. One-way ANOVA repeated measures was used to analyze the recoverable log IFU data across four time points following murine infection with L2-wild type or L2-ΔCT228. Student's unpaired two-tailed t-test was used to analyze systemic (IgG2a) and mucosal (IgA, IgG) mean ± SE antibody titers in mice infected with L2-wild type vs. L2-ΔCT228 and performed on clinical scores for histopathology between L2-wild type and L2-ΔCT228 infected mice. Statistical analyses were performed using Prism 5.0 and data were considered significant at p< 0.05.
C. trachomatis CT228 Deletion and Resulting Effects
The chlamydial TargeTron vector pDFTT3(bla) was modified to allow for spectinomycin selection by replacing the bla cassette with the aadA cassette. The vector was then retargeted to insert into CT228 as previously described (Johnson and Fisher, 2013). The predicted insertion site was at bp 295 within CT228 in an antisense orientation. Mutants were selected using 500 μg/ml spectinomycin and plaque purified to obtain a clonal L2-ΔCT228 strain. The CT228 locus map, TargeTron insertion site, and PCR validation of the intron insertion are shown in Figures 1A–C. Sequencing of the L2-ΔCT228 mutant CT228::GII(aadA) locus confirmed intron insertion at the predicted location. HeLa cell monolayers were infected with both L2-wild type and L2-ΔCT228 for 18 h and processed for immunofluorescence using anti-CT228 and anti-LPS antibodies. CT228 is detected surrounding the chlamydial inclusion in L2-wildtype, however is absent in the L2-ΔCT228 inclusion membrane confirming the inactivation of CT228 (Figure 1D). Southern blotting confirmed a single TargeTron insertion in the L2-ΔCT228 mutant (Figure S1).
Figure 1. TargeTron inactivation of CT228. (A) TargeTron insertion site in CT228. (B) Schematic of CT228 containing GII(aadA). (C) PCR verification of TargeTron insertion in L2-ΔCT228. PCRs were used to amplify incA, CT228 and the Intron in the L2-wild type, L2-ΔCT228 (Mut), and TargeTron vector (pDFTT295). (D) Immunofluorescence images of L2-wild type and L2-ΔCT228 at 18 h post-infection stained with anti-CT228 and anti-Chlamydia LPS followed by fluorescent secondary antibodies. Scale bar, 10 μm.
The CT229 through CT224 gene linkage map suggests that these genes are transcriptionally linked as an operon (Stephens et al., 1998). In effort to determine whether the TargeTron insertion in CT228 may exert polar effects on the transcription of other genes within the putative operon, RT-PCR analysis was performed using oligonucleotide primers (Table S1) designed within each ORF as well as spanning the TargeTron insertion site (Figure 2A). The green and red arrows in Figure 2A indicate the position of forward and reverse primers, respectively. Using total RNA harvested from Hela cells infected with C. trachomatis L2-wild type as template, amplification products were observed (Figure 2B) for each ORF (CT229, CT228, CT227, CT226, CT225, and CT224). In total RNA from C. trachomatis L2-ΔCT228 infected cells, robust amplification products were observed (Figure 2B) for CT229, CT227, CT226, CT225, and CT224. Of particular note is the observation that the region of CT228 that lies upstream of the TargeTron insertion also produced a robust amplification product, while primers spanning the insertion site do not. It was also observed that RT-PCR using primers situated downstream of the TargeTron insertion site, yet within the CT228 ORF, produce a far less robust amplification product (Figure 2B). Taken together, the data indicate that the TargeTron insertion within CT228 does not cause a polar elimination of CT227, CT226, CT225, and CT224 transcripts while it does interrupt the production of a complete CT228 transcript.
Figure 2. TargetTron inactivation of Chlamydia trachomatis CT228 and subsequent CT229-CT224 gene linkage group transcription. (A) Physical map of C. trachomatis CT229-CT224 gene linkage group. Solid arrows represent CT229-CT224 ORFs. Gene designations are indicated above each ORF. Green and red arrows indicate forward and reverse primers, respectively, used for RT-PCR analysis. Red arrowhead indicates the point of TargeTron insertion within CT228. (B) Agarose gel image(s) show RT-PCR products corresponding to groEL (control), and CT229-CT224 gene linkage group expression (correlating to the indicated primers above). Total RNA (L2-wild type or L2-ΔCT228) is indicated above corresponding gel lanes. + indicates RT added to reaction. – Indicates RT not added to reaction. L2-wild type and L2-ΔCT228 DNA act as PCR controls. (C) Growth curves of L2-wild type and L2-ΔCT228 were performed in triplicate, the entire experiment was repeated on three separate occasions and a representative growth curve was selected. Error bars represent standard deviation.
Comparison of Bacterial Growth, Recruitment of MYPT1, and Extrusion Production in C. trachomatis L2-Wild Type vs. L2-ΔCT228
Bacterial growth was measured at 0, 6, 12, 24, and 48 h post-infection of HeLa cell monolayers with C. trachomatis L2-wild type and L2-ΔCT228. There were no statistically significant differences between growth curves of the different strains and the curves followed the expected pattern for growth (Figure 2C). L2-wild type and L2-ΔCT228 were assessed for recruitment of MYPT1 and myosin phosphatase pathway host proteins at 18 h post-infection in HeLa cell monolayers. While the L2-wild type strain recruited MYPT1 to the periphery of the inclusion (Figure 3A), the L2-ΔCT228 failed to do so (Figure 3B). However, both strains demonstrate recruitment of MLCK (pY474), MLC2 (pS19), and Myosin IIa and Myosin IIb to the inclusion co-localizing with active Src Y474 in the microdomains at 18 h post-infection (Figures 3A,B). Recruitment of MLCK (pY474), MLC2 (pS19), and Myosin IIa and Myosin IIb at 48 h post-infection was also visualized confirming recruitment for the duration of the infection (Figure S2). Western blot analysis of MLC2 (pS19) and endogenous MLC2 levels did not show any differences during the course of infection at 24 and 48 h post-infection (Figure 3D). The number of extrusions produced by both L2-wild type and L2-ΔCT228 was quantified at 48 h post-infection in Scramble or MYPT1 siRNA depleted HeLa cells [successful MYPT1 knockdown was verified by western blot (Figure 3E)]. In the Scramble treated cells, L2-ΔCT228 produced significantly more extrusions than the L2-wild type (Figure 3E, p < 0.0001) however, there was no difference in extrusion production between the L2-wild type and L2-ΔCT228 in the MYTP1 siRNA treated cells (Figure 3E). Analysis of total Chlamydia IFUs was comparable between strains and conditions (Figure 3F).
Figure 3. Recruitment of MYPT1 and Myosin phosphatase pathway components and extrusion production by C. trachomatis L2-wild type and L2-ΔCT228. HeLa cell monolayers were infected at a MOI of ~0.5 with L2-wild type and L2-ΔCT228 for 18 h (in technical triplicate). Cells were fixed and stained with primary antibodies to MYPT1, Chlamydia LPS, MLC2 (pS19), Src Y474, MLCK (pY471), non-muscle Myosin IIa and IIb followed by fluorescent secondary antibodies. Experiments were repeated on three separate occasions and representative images were selected. (A,B) Top panel shows individual and merged images of MYPT1 recruitment (green) and Chlamydia LPS staining (red) in both the L2-wild type and L2-ΔCT228. Lower panel of individual and merged images show MLC2 (pS19), MLCK (pY471), and Mysoin IIa and IIb (green) co-localizing with active Src Y474 kinase (red) in microdomains at the periphery of inclusions in both L2-wild type and L2-ΔCT228. Scale bar, 10 μm. (C) Total protein from L2-wild type and L2-ΔCT228 infected HeLa cells at 24 and 48 h post-infection were assessed for MLC2, MLC2 (pS19), HsP60, and GAPDH levels by western blot analysis. (D) HeLa cells were treated with either Scramble (Scr) or MYPT1 siRNA for 48 h prior to infection with L2-wild type and L2-ΔCT228. Protein samples were assessed for MYPT1 and GAPDH levels by western blot. (E) Extrusions collected and (F) IFUs were assessed for L2 wild-type and L2-ΔCT228 at 48 h post-infection in either Scramble (symbols and solid bars) or MYPT1 (open symbols and white bars) siRNA treated HeLa cells. *p < 0.0001.
Course of C. trachomatis L2-Wild Type vs. L2-ΔCT228 Infection in vivo
Bacterial burden in mice infected intravaginally with either 1 × 106 L2-wild type EBs (n = 8) or L2-ΔCT228 EBs (n = 9) was quantitated at days 7, 14, 21, and 28 via swabbing the cervicovaginal tract and culturing on HeLa cell monolayers. The calculated numbers of recoverable log IFUs are shown in Figure 4. Early L2-wild type (Figure 4A) and L2-ΔCT228 (Figure 4B) infection in mice at day 7 produced an infectious burden that was not statistically different from one another. However, there was a statistically significant effect of time on the clearance of L2-wild type (p = 0.006) that was not present in response to L2-ΔCT228 (p = 0.371).
Figure 4. Recoverable IFUs shed by mice infected with C. trachomatis L2-wild type and L2-ΔCT228. Female C3H/HeJ mice were intravaginally infected with 1 × 106 EBs of either L2-wild type or L2-ΔCT228. Recoverable IFUs were obtained by swabbing vaginal tracts and enumerating on HeLa cell monolayers. Recoverable IFU data are expressed for (A) L2-wild type (n = 8) and (B) L2-ΔCT228 (n = 9) on a logarithmic scale from Day 7 to 42 post-infection. Effect of time (*p = 0.006) was observed in mice infected with L2-wild type (repeated measures one-way ANOVA). For day 28, 6/8 L2-wild type infected mice were clear compared to 3/9 L2- ΔCT228. Triangles represent individual mice, bars represent mean of group for each time point.
Systemic and Mucosal Antibody Response to C. trachomatis L2-Wild Type vs. L2-ΔCT228
Mean systemic anti-chlamydia IgG2a antibody titers were significantly higher (p = 0.041) in mouse sera at day 31 post-infection with L2-wild type (n = 8) relative to L2-ΔCT228 (n = 9) as shown in Figure 5A. As expected for serovar L2, mucosal anti-Chlamydia antibody responses were low overall. There were negligible mucosal anti-Chlamydia IgG and IgA titers in mice 31 days post-infection with L2-ΔCT228 relative to L2-wild type (Figures 5B,C).
Figure 5. Systemic and mucosal antibody titers following infection with C. trachomatis L2-wild type and L2-ΔCT228. (A) Sera were collected from mice 31 days post-infection with L2-wild type (n = 8) or L2-ΔCT228 (n = 9) and assayed for the presence of anti-chlamydial IgG2a. (B,C) Vaginal lavages were collected 31 days post-infection and assayed for the presence of anti-chlamydial IgA and IgG. Antibody titers are expressed for individual mice as the highest dilution tested that produced >3-fold the absorbance as control/uninfected mice. Bars represent mean antibody titer per group. *p = 0.0412, unpaired two-tailed Student t-test.
Histopathology in C. trachomatis L2-Wild Type vs. L2-ΔCT228 Infected Mice
Whole reproductive tracts were collected 23 and 64 days post-infection (L2-wild type vs. L2-ΔCT228) and formalin fixed for histological analyses. Sections of uterine tissue were examined and the scores assigned by an AVCP certified veterinary pathologist via light microscopy. Individual clinical scores ranged from normal to moderate pathology for the infected mice (Table 1, raw scores provided in Table S2). As previously shown, intravaginal infection with serovar L2 overall produces less histopathology relative to other human serovars (e.g., serovar D-LC) and the mouse-specific pathogen C. muridarum (MoPn) (Shaw et al., 2017). A single infection with serovar L2 produces only minimal to mild histopathology, however it was important to assess any inflammatory damage following infection with L2-ΔCT228 since this strain had never previously been tested in vivo and studies support the concept that chlamydial genotypes may alter the capacity for ascension and survival within the upper genital tract (Menon et al., 2015). Histological parameters compared between L2-wild type and L2-ΔCT228 revealed no statistical differences with the exception of fewer mucinous changes observed in the L2-ΔCT228 infected mice (Figures 6A–D and Table 1, p = 0.02).
Table 1. Pathological scoring of murine reproductive tracts.
Figure 6. Histopathological assessment of reproductive tracts post-infection. Mice (n = 5 per group) were euthanized at 23 and 64 days post-infection (dpi) and entire reproductive tracts were removed and formalin fixed for histology. Representative images of H&E stained sections of uterine tissue were captured using an Olympus DP70 for mice infected with (A) C. trachomatis L2-wild type, 23 dpi, (B) C. trachomatis L2-ΔCT228, 23 dpi, (C) C. trachomatis L2-wild type, 64 dpi, (D) C. trachomatis L2-ΔCT228, 64 dpi. See Table 1 for pathological clinical scoring of reproductive tracts at 64 dpi.
The nexus of host-pathogen interaction during chlamydial infection resides at the inclusion membrane. It is at this interface that Chlamydia exploit host cellular machinery to scavenge materials necessary to build and maintain their intracellular niche. Inc proteins are characterized by a bilobed hydrophobic domain of ~40 amino acids and are well conserved between C. trachomatis serovars implying a potential role in pathogenesis (Weber et al., 2015). While there are as many as 59 predicted Incs (Bannantine et al., 2000; Toh et al., 2003; Dehoux et al., 2011; Lutter et al., 2012), the host-pathogen functional interaction of relatively few is currently understood. The most extensively studied is IncA (CT119), which encodes SNARE-like motifs and is required for the homotypic fusion of inclusions that occurs late in infection (Hackstadt et al., 1999; Delevoye et al., 2008). IncB (CT232) is localized to microdomain regions saturated with Src kinases that participate in pathways governing inclusion trafficking and intracellular growth (Mital et al., 2010; Mital and Hackstadt, 2011). IncD (CT115) recruits ceramide endoplasmic reticulum transferase (CERT) (Agaisse and Derre, 2014), a lipid transfer protein, while IncG (CT118) recruits adaptor protein 14-3-3β to enhance host cell viability (Scidmore and Hackstadt, 2001). Endosomal trafficking is regulated by interaction between CT229 and GTPase Rab4 (Rzomp et al., 2006), CT850 interaction with dynein drives migration to the MTOC (Mital et al., 2015), IncV (CT005) interacts with the endoplasmic reticulum (ER) integral membrane protein VAP securing the chlamydial inclusion to the ER (Stanhope et al., 2017)and CT813 binding with ARF1 and ARF4 bring inclusions in close proximity to Golgi mini-stacks (Wesolowski et al., 2017). Later during infection, both CT101 and CT228 regulate host cell exit via extrusion; CT101 (also known as MrcA) interacts with host inositol tri-phosphate (IP3) for calcium management (Nguyen et al., 2018) and, as previously mentioned, CT228 recruits myosin phosphatase (MYPT1) to the inclusion membrane (Lutter et al., 2013; Mirrashidi et al., 2015).
Whether Chlamydia exit host cells via lysis or extrusion is controlled by the phosphorylation state of MLC2. Dephosphorylated MLC2 favors lysis while phosphorylated MLC2 enhances extrusion (Lutter et al., 2013); the extent of phosphorylation is determined by the ongoing opposing activities of myosin phosphatase (MYPT1) and myosin kinase (MLCK). In this study we generated a targeted chromosomal mutation of CT228 using the TargeTron system (Johnson and Fisher, 2013) that no longer produced CT228 (Figure 1D). Gene interruption by insertion mutagenesis introduces the possibility of generating polar effects on downstream gene expression. This can confound subsequent findings as to which genes are specifically responsible for an observed effect. We used RT-PCR to analyze total RNA from L2-wild type and L2-ΔCT228 infected Hela cells. The data confirms that L2-ΔCT228 does not produce a full-length CT228 transcript during infection (Figure 2B). However, it does produce transcripts for CT227, CT226, CT225, and CT224, all of which lie downstream of CT228 and the TargeTron insertion (Figures 2A,B). Analysis of the intervening sequence (282 bp) upstream of the CT227 start site revealed −35 and −10 sigma factor binding sequences (TTGTGT-17bp-TATTAT) with strong homology to the Chlamydial σ66 (Yu et al., 2006) consensus binding sequences (TTGACA-17bp-TATTAT), suggesting that a promotor exists for transcription of CT227-CT224 which may be in addition to upstream promotors. These findings indicate that for this putative operon, the insertion does not eliminate the production of downstream gene products, focusing our findings on CT228 cellular interactions.
A direct comparison of the L2-wild type and L2-ΔCT228 in vitro demonstrated that the only identified alteration in protein localization exhibited by cells infected with the CT228 mutant was in the recruitment of MYPT1 to the inclusion (Figure 3B), confirming the role of CT228. The remaining components of the myosin pathway (MLCK, MLC2, and heavy chains Myosin IIa and IIb) showed identical recruitment by L2-wild type and L2-ΔCT228 (Figures 3A,B; Figure S2) suggesting that myosin regulated events such as extrusion ought to be intact and theoretically enhanced in the mutant. If the recruitment of MYPT1 to the chlamydial inclusion is removed (thus loss of phosphatase activity) and MLCK, which phosphorylates and activates MLC2, continues to be recruited then we would predict a two-pronged increase in phosphorylated MLC2 that is localized to the inclusion and subsequent increase in extrusion production. Our observation that L2-ΔCT228 produced significantly more extrusions compared to the L2-wild type supports this hypothesis (Figure 3). This further suggests that while CT228 is not required for extrusion production in Chlamydia, it likely influences the rate of extrusions and may have implications in vivo. The notion that CT228 is non-essential for extrusion production is not novel. Previous studies utilizing the ocular isolate B/Jali20/OT, which contains a natural truncation mutation of CT228 among other SNPs in its genome, have reported a lack of MYPT1 recruitment (Lutter et al., 2013) and, interestingly, a decrease in extrusion production compared to serovar L2 (Zuck et al., 2016b). Although the extrusion mechanism is conserved among Chlamydiae, a comparison of extrusion production by different species or non-isogenic strains carrying multiple mutations such as B/Jali20/OT may confound efforts to verify roles for specific genes. The cross serovar comparison of extrusion production could account for the reported decrease in extrusions observed in B/Jali20/OT, whereas in our study using isogenic strains we observe a significant increase in extrusions produced by L2-ΔCT228 when compared to the L2-wild type.
Considering our finding that in vitro growth of L2-ΔCT228 produced significantly more extrusions than L2-wild type, we hypothesized that this may provide a means to safeguard Chlamydia from the host immune response in vivo. While both extrusion and lysis occur late infection, the favoring toward even a minor increase in extrusion may dampen the ability of the host immune response to recognize EBs due to enclosure within extrusions, which are surrounded by host membrane. Enhancement of host egress via extrusion would theoretically reduce the number of opportunities for presentation of EB antigens and may explain the modest decrease in clearance of mice infected with L2-ΔCT228. Our data show that murine intravaginal infection with L2-ΔCT228 cleared more slowly; only 3/9 L2-ΔCT228 infected mice were clear by day 28 compared to 6/8 L2-wild type infected mice (Figures 4A,B). Likewise, significantly lower systemic anti-Chlamydia IgG2a titers were produced by L2-ΔCT228 infected mice relative to L2-wild type at day 31 post-infection (Figure 5A). As expected in response to serovar L2 infection, there were negligible mucosal IgG and IgA titers in both groups 31 days post-infection (Figures 5B,C). Moreover, histological analyses of whole murine reproductive tissue show a significant, albeit minor, decrease in mucinous changes following infection with L2-ΔCT228 compared to L2-wild type within this current study (Figures 6A–D). It is important to note that since serovar L2 is well known to elicit only minimal histopathological changes, interpretation of these findings is restricted by the compression of the 0–4 clinical scoring scale to 0–2. Clinical scores of mucinous changes from our previous studies in L2-infected mice displayed a somewhat lower but overlapping numerical range for mucinous changes despite higher recoverable IFU (Shaw et al., 2017) than that which was observed in this current study confounding any definitive conclusions as to the physiological relevance of and reliance on this particular parameter between separate studies. Overall, our in vivo findings suggest a delay in clearance following intravaginal infection with L2-ΔCT228 with a concomitant reduction in anti-Chlamydia humoral responses. Taken together with evidence that the mutant and wild-type strains exhibit similar growth, we speculate that the difference in EB release (via lysis or extrusion) may alter the degree of recognition by macrophages and dendritic cells thereby affecting the ability of host immune clearance mechanisms. While there are multiple players involved in clearance, the data suggest that loss of MYPT1 recruitment (as a result of CT228 disruption) affects the longevity of infection in vivo, which may be related to the degree of host cell exit via extrusion.
It will be important to elucidate the mechanism whereby this chlamydial mutant evades and/or alters the immune response. Future studies on how CT228 may specifically affect the required events for antigen presentation and development of protective immunity, such as dendritic cell activation, IFN-γ production, CD4 T cells mucosal imprinting as well as antibody-mediated cytotoxic (neutrophil) events, are warranted and may reveal survival strategies available to Chlamydia that could also serve as targets for new therapies. Generating a CT228 mutant in serovars that are more robust in driving histopathology to better model human disease than L2 is crucial. Likewise, it would be of interest to investigate whether the loss of CT228 protein affects the localization and function of other Incs.
EL: project conceptualization, data acquisition, data analysis, data interpretation, revision of manuscript, principle investigator; JS: project conceptualization, data acquisition, data analysis, data interpretation, revision of manuscript, co-principal investigator; DF: construction of L2-ΔCT228, revision of manuscript; TS: data acquisition, data analysis; CK: data acquisition, data interpretation, revision of manuscript; ES: data acquisition, data interpretation, revision of manuscript; PS: performed all protein and western blotting experiments.
This study was supported by a grant from the US National Institutes of Health (1R15AI119906-01) to EL and JS and R21AI115238 to DF.
The authors would like to thank Dr. Harlan Caldwell for generously providing the C. trachomatis anti-MOMP (L2-I-5) antibody and Amanda R. Behar for help with preliminary experiments.
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcimb.2018.00415/full#supplementary-materia
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Keywords: Chlamydia, extrusion, lymphogranuloma venereum, L2 serovar, sexually transmitted infection, urogenital infection, CT228, mouse infection
Citation: Shaw JH, Key CE, Snider TA, Sah P, Shaw EI, Fisher DJ and Lutter EI (2018) Genetic Inactivation of Chlamydia trachomatis Inclusion Membrane Protein CT228 Alters MYPT1 Recruitment, Extrusion Production, and Longevity of Infection. Front. Cell. Infect. Microbiol. 8:415. doi: 10.3389/fcimb.2018.00415
Received: 30 July 2018; Accepted: 08 November 2018;
Rey Carabeo, Washington State University, United States
Ashok Aiyar, LSU Health Sciences Center New Orleans, United States
George Liechti, Uniformed Services University of the Health Sciences, United States
Maria Teresa Damiani, Instituto de Histología y Embriología Dr. Mario H. Burgos (IHEM CONICET), Argentina
Copyright © 2018 Shaw, Key, Snider, Sah, Shaw, Fisher and Lutter. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Jennifer H. Shaw, jennifer.h.shaw@okstate.edu
Erika I. Lutter, erika.lutter@okstate.edu


These all natural frames are indivdually crafted. No two frames will be exactly the same.Expect variation in color and appearance.
Note: 1. Each frame will have 1/4'' added to the length and width when you order.
2. The size you select below is the inside dimension of the frame and the molding size. The molding size is the width of the planks on each of the four frame sides. To find the outside dimension based on the selected molding size and inside dimension, double your selected molding size and add that number to each inside dimension.
Example: You select an inside dimension of 15'' W x 20'' L, with a 3'' molding size. 3 *(2) = 6 and (6 + 15) = 21'' W x (6 + 20) = 26'' L. So the outside dimension of your frame is 21'' wide x 26'' long.


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Q: CMAC-AES on iOS I'm converting an Android app to iOS. The Android version uses CMAC-AES cryptography, but I couldn't find CMAC for iOS. Does anyone know a library or could give a tip? I am not familiar with cryptography, so I am little messy.

A: CMAC stands for Cipher-based Message Authentication Code, that means it's a message authentication code (it is used to authenticate data and avoid any corruption of the data being authenticated) that uses ciphers to achieve its goals, in your particular case it uses the AES cipher to do so.
There are indeed some libraries that you could use, you may use OpenSSL (you may try others tutorials that links OpenSSL to your project) which is a well adopted library and heavily maintened.


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Q: Classifying Space of a Group Extension Consider a short exact sequence of Abelian groups -- I'm happy to assume they're finite as a toy example:
$$
0 \to H \to G \to G/H \to 0\ .
$$
I want to understand the classifying space of $G$. Since $BH \cong EG/H$, $G/H$ acts on $BH$ and we can write $BG \cong E(G/H) \times_{G/H} EG/H$. Thus, we have a fiber bundle (which I'll write horizontally)
$$
BH \to BG \to B(G/H)
$$
On the other hand, the central extension is classified by an element of the group cohomology $H^2(G/H,H)$ which is the same as $H^2(B(G/H),H)$. The latter is an element in the homotopy class of maps $[B(G/H),K(H,2)]$ and $K(H,2)\cong BBH$. This map looks like it classifies a principal $BH$ bundle over $B(G/H)$. I find it hard to imagine that this 'principal' $BH$ bundle is not 'the same' as the bundle above, so the question is, how do you see that? From this construction, it's not even obvious to me that the above bundle is a principal bundle.
I would guess (and being a poor physicist, I'm not so up on my homotopy theory), there's a sense that the classifying space of an Abelian group is an 'Abelian group', and taking classifying spaces of an exact sequence gives you back an 'exact sequence'. That gets you a 'principal bundle' (aren't quotation marks fun?), but even then I'm not sure how to see that the classifying map of this bundle is the same as the class in group cohomology.
Any references to the needed background would also be greatly appreciated.

A: Your picture is essentially correct, except you need to specify that the maps take basepoints to basepoints (i.e., they are pointed maps).
BG is given as the homotopy fiber of a pointed map from B(G/H) to BBH.  Applying the based loop space functor $\Omega$ to the pointed maps yields the sequence of group homomorphisms.  This does not require G to be abelian.  There is a subclass of abelian central extensions, which are those that can be delooped again to maps from BB(G/H) to BBBH.
In general, group homomorphisms can be delooped once to maps of pointed spaces, while abelian group homomorphisms can be delooped arbitrarily many times, to infinite loop maps of infinite loop spaces.  There is an equivalence between grouplike homotopy commutative spaces and double loop spaces, but since G is discrete, the double loop space property is strictified for free.
Edit: A standard reference for this material is the second chapter in Adams, Infinite Loop Spaces.

A: @There is an equivalence between grouplike homotopy commutative spaces and double loop spaces
No, grouplike homotopy commutative is not enough. Double loop spaces have much more in thee way of higher homotopies, even if the space were to have a strictly associative homotopy commutative structure. That was one of the motivations for operads.  Also see JF Adams: 10 types of H-spaces.

A: A quick and dirty answer is that if we regard the groups as one-object groupoids, they again form a short exact sequence of groupoids, and the geometric realisation of this sequence is the $BH$ bundle you mention. $BH$ is a (normal) closed subgroup of $BG$ by a result of Segal, so all that needs to be checked for this to be a principal bundle is that $BG \to B(G/H)$ admits local sections. I believe this is true because the groups involved are discrete hence well-pointed - the inclusion of the identity is a closed cofibration (a paper by Baez and Stevenson has relevant calculations for topological groups in a different context).

A: Yes. The principal bundles are the same and your guess that $BA$ is an abelian group is exactly right. A good reference for this story, and of Segal's result that David Roberts quotes, is Segal's paper:
G. Segal. Cohomology of topological groups, Symposia Mathematica IV (1970), 377- 387.
The functors $E$ and $B$ can be described in two steps. First you form a simplicial topological space, and then you realize this space. It is easy to see directly that $EG$ is always a group and that there is an inclusion $G \rightarrow EG$, which induces the action. The quotient is $BG$. Under suitable conditions, for example if $G$ is locally contractible (which includes the discrete case), the map $EG\rightarrow BG$ will admit local sections and so $EG$ will be a $G$-principal bundle over $BG$. This is proven in the appendix of Segal's paper, above. There are other conditions (well pointedness) which will do a similar thing.
The inclusion of $G$ into $EG$ is a normal subgroup precisely when $G$ is abelian, and so in this case $BG$ is again an abelian group.
I believe your question was implicitly in the discrete setting, but the non-discrete setting is relevant and is the subject of Segal's paper. Roughly here is the answer: Given an abelian (topological) group $H$, the $BH$-princical bundles over a space $X$ are classified by the homotopy classes of maps $[X, BBH]$. When $H$ is discrete, $BBH = K(H,2)$. If $X = K(G,1)$ for a discrete group $G$, these correspond to (central) group extensions:
$$H \rightarrow E \rightarrow G$$
If $G$ has topology, then the group extensions can be more interesting. For example there can be non-trivial group extensions which are trivial as principal bundles. Easy example exist when H is a contractible group. However Segal developed a cohomology theory which classifies all these extensions. That is the subject of his paper.


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Looperskapelle is een buurtschap in de gemeente Schouwen-Duiveland, in de Nederlandse provincie Zeeland. De naam heeft betrekking op een kapel van de familie Loopers. De oudste vermelding van de naam stamt uit 1324, als Lopers Capelle, hoewel het in 1280 nog werd vermeld als Capella.

De kapel werd uitgebreid tot kerk, gewijd aan Sint-Antonius. Deze kerk werd in 1590 afgebroken ten behoeve van de fortificaties van Brouwershaven. De begraafplaats is thans een cultuurhistorisch monument.

Looperskapelle was een oude heerlijkheid en een zelfstandige gemeente tot 1813, toen het met Brijdorpe en Klaaskinderkerke werd toegevoegd aan de gemeente Duivendijke. Van 1961 tot 1997 behoorde het tot de gemeente Middenschouwen.

Looperskapelle telt 1 rijksmonument.

Geografie van Schouwen-Duiveland
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