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Y'all better be glad Laughing 808 saved your sorry behinds, with your non suggestion giving selves. I shall be moving on from that subject. (If you're confused, read the post under this, SUGGEST SOMETHING and then come back and join us please)
Want to know what I spent my day doing? Getting lost. For work I had to go to a wellness fair at the social services office. Well first my dumb butt went to the social security office (it was on the same street). Ughhh I was soooooo embarrassed when the lady told me in front of that room full of people that I was in the wrong building and she didn't even know what I was talking about. Then I drove down some and saw a social services building. After fighting through the parking thought I thought, "surely this is correct, it's so crowded." I got out of my car only to be told to go down some more. I drive down and don't see the appropriate number, but I see big complex. I go in and in the back I find the right building. I once again get the box out of my car and I see my boss arriving as well. We go in only to find out that we are at the wrong location again! We go down the street and finally find it. Geez! I had a good time though. The nurse that was helping me was a 25 year old black chick from p.g. too. Good times we had! She's doing some of the other fairs coming up with me too so I'm excited. Anyway that was done at 2 so I was on my way back home to go to the social security office to change my name. I drove up and down the same street for 20 MINUTES trying to find this place. It was hidden in the back of a shopping center. I did that and got my name changed. I'm officially Mrs. Count. I change my drivers license Friday and get a new bank account Saturday.
After leaving there at 4, I try to find the Food Lion. Good Lord! I found: Giant, a random field, a firehouse, Columbia Mall, and a nice housing complex during my 30 minute trek to Food Lion. I was so frustrated. Found it, wasn't too impressed with their prices either, got my stuff and got out. Then I remembered that I had to go to the bank. Spent another 30 minutes lost trying to find that. Got there and didn't have the darn checks. I broke down crying and screaming outside the bank.
Want to know the real sad part? I had a darn GPS system and google directions and map pulled up on my phone! How dumb can I be. Ughhh, I need a chauffeur.
(get the title? If you don't please enroll yourself back in Elementary School, or go ask a 5th grader)
Monday, November 10, 2008
Christopher Columbus must be my cousin
Y'all better be glad Laughing 808 saved your sorry behinds, with your non suggestion giving selves. I shall be moving on from that subject. (If you're confused, read the post under this, SUGGEST SOMETHING and then come back and join us please)
Posted by Mrs Count at 8:47 PM


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- North America
- United States
- Wyoming
- Dresher Dam
- Search
Traveling Luck for Dresher Dam Wyoming, United States
The timezone in Dresher Dam is America/Cambridge_Bay
Morning Sunrise at 07:32 and Evening Sunset at 16:57. It's light
Rough GPS position Latitude. 43.8133°, Longitude. -105.9150°
Morning Sunrise at 07:32 and Evening Sunset at 16:57. It's light
Rough GPS position Latitude. 43.8133°, Longitude. -105.9150°
Weather near Dresher Dam Last report from Gillette, Gillette-Campbell County Airport, WY 77.2km awayTemperature: 2°C / 36°F
Wind: 10.4km/h Southwest
Cloud: Sky Clear
Satellite map of Dresher Dam and it's surroudings...
Geographic features & Photographs around Dresher Dam in Wyoming, United States
mine(s) a site where mineral ores are extracted from the ground by excavating surface pits and subterranean passages.
- Dome Butte Mine (4km)
- Quigley and Gall Colorado Number One Mine (4.6km)
- Simmons Number Two Mine (5km)
- Bashawn Mine (5.3km)
- Discovery Roll Mine (5.5km)
- White Mule Mine (5.6km)
- Blue Star Number One Mine (5.9km)
- Lucky Eight Mine (6km)
- Utah Mine (6km)
- Black Star Mine (6.1km)
- Paula Number One Mine (6.4km)
- Jeanette Number One Mine (6.6km)
- Kerr-McGee Ed Claims Mine (7km)
- Kerr-McGee Jim Claim Mine (7km)
- Mary Mine (8km)
- Jeanette Mine (8km)
- Queen Number Two Mine (8km)
- Christensen Brothers Ranch Mine (8.4km)
- Little Star Mine (9.2km)
- Christensen Lease Mine (10km)
- Van Eugenum Number One Mine (10.4km)
- Channel Deposit Mine (10.6km)
- KM Number One Mine (10.7km)
- Campbin and West Mine (10.9km)
- Van Eugenum Number Two Mine (10.9km)
- Kerr-McGee School Lease Mine (11km)
- A-Number Ninety-three Mine (11.2km)
- South Peak Mining Property Mine (11.5km)
Local Feature A Nearby feature worthy of being marked on a map..
- W Schlautmann Ranch (5.8km)
- Pfister Ranch (5.9km)
- Lauby Ranch (7.3km)
- Christensen North Ranch (8.5km)
- Christensen Ranch (9.3km)
- Gilbertz Ranch (10.4km)
- H Schlautmann Ranch (10.8km)
- Pumpkin Butte Ranch (10.8km)
dam a barrier constructed across a stream to impound water.
- Dresher Dam (0km)
- Rodabaugh Dam (8km)
- Brown Dam (10.3km)
- Field 9-207-5 Dam (11.3km)
- Cellers Wildlife 9-499-3 Dam (11.3km)
- Schlautman Dam (12km)
reservoir(s) an artificial pond or lake.
- Dresher Reservoir (0km)
- Rodabaugh Reservoir (8km)
- Brown Reservoir (10.3km)
- Field 9-207-5 Reservoir (11.3km)
- Cellers Wildlife 9-499-3 Reservoir (11.3km)
- Schlautan Reservoir (12km)
mountain an elevation standing high above the surrounding area with small summit area, steep slopes and local relief of 300m or more.
- Dome Butte (3.4km)
- North Butte (4.8km)
- Pistol Point (6.4km)
- North Middle Butte (9.6km)
- Indian Butte (12.1km)
valley an elongated depression usually traversed by a stream.
- Collins Draw (7.2km)
- Bons Draw (7.7km)
- Del Gulch (10.5km)
- Hartzog Draw (11.9km)
stream a body of running water moving to a lower level in a channel on land.
- Dry Willow Creek (9.1km)
- North Prong Willow Creek (9.5km)
well a cylindrical hole, pit, or tunnel drilled or dug down to a depth from which water, oil, or gas can be pumped or brought to the surface.
- First Well (10.8km)
spring(s) a place where ground water flows naturally out of the ground.
- Y Spring (5.3km)
Wikipedia entries close to Dresher Dam
- LORAN-C transmitter Gillette tower
- Wright, Wyoming city o city
- Edgerton, Wyoming city o city
- Midwest, Wyoming city o city
- Antelope Valley-Crestview, Wyoming city o city
- Kaycee, Wyoming city o city
- Sleepy Hollow, Wyoming city o city
- Gillette, Wyoming city o city
- Teapot Dome scandal
- Gillette-Campbell County Airport airport airport
- Hole-in-the-Wall
- Buffalo, Wyoming city o city
- Antelope Hills, Wyoming city o city
- Moorcroft, Wyoming city o city
- Arvada, Wyoming city o city
- Clearmont, Wyoming city o city

subset: commoncrawl


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In preparation for the presentation each member of the group was tasked with collecting an individual body of research into a variety of subject areas relevant to the business. Dividing the research amongst the members of the group helped us to move through the research stages of the project quickly while collecting a body of informed information relevant to the specifics of the business.
Below are links to the PPP research posts published on my Design Context blog;


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high pressure inline oil filter strainer Photos for your ref
Technical Data
Operating medium: mineral oil, emulsion, water-glycol, phosphate ester (resin-impregnated paper just for mineral oil)
Operating pressure (max): 42MPa
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Brand Label: Jonathan Simkhai
ITEM MOVED TO ARCHIVE: On January 30, 2018 2:24 pm, Fashion Stylist Online noted this item may be out of stock at Bergdorf Goodman because it was not referenced in their latest online catalog. If this item is out of stock at Bergdorf Goodman, you may be able to find similar fashions sold there or elsewhere on our Jonathan Simkhai Designer Brand Directory.
Available for Purchase Online at Bergdorf Goodman for $177.00Pin It
Jonathan Simkhai ribbed knit skirt. Sits below natural waist. Trumpet silhouette. Pull-on style. Midi length. Viscose/nylon. Imported.
Shop other brand label items from Jonathan Simkhai on FashionStylist.com.
This item was published for sale online by Bergdorf Goodman on January 30, 2018 10:54 am and last updated by us January 30, 2018 2:24 pm.
Editor Picks: So 70's Clothing Inspiration
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New promo codes and links offering limited time promotions, sales coupons, and special offers on Jonathan Simkhai designer fashions and accessories will be posted on each designer label apparel page. For the latest up to date information on this Jonathan Simkhai Rib Knit Trumpet Midi Skirt | Clothing fashion item, including product specifications, ordering options, pricing, shipping details, high resolution pictures, shopper comments, and other sales media, visit the Bergdorf Goodman Online Store.


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Availability: In Stock
The original clear adhesive tape - easy to tear by hand.
- Ideal for desktop dispensers.
- Biodegradable.
- Commonly requested sizes listed below.
|Width x Length
|12mm x 66m
|144 rolls per pack
|19mm x 66m
|96 rolls per pack
|25mm x 66m
|72 rolls per pack
|50mm x 66m
|36 rolls per pack
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The Spiritual Power of Empathy
Develop your Intuitive Gifts for Compassionate ConnectioneBook - 2014
Discover your innate empathic abilities with popular author Cyndi Dale as your guide. With this hands-on training course, you'll learn how to comfortably use empathy for better relationships and healing the self and others.
The Spiritual Power of Empathypresents this often-unrecognized ability in accessible ways, allowing you to discover an expanded awareness of what empathy is, how it works, and the myriad ways it manifests. Develop deeper connections with your loved ones, use specialized techniques for screening and filtering information, and gain insights on how to overcome the difficulties empaths often face. With the power of empathy, you'll transform the way you live and connect with the world around you.
2015 IPPY Award Gold Medal Winner in New Age (Mind-Body-Spirit)
From the critics
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ALSO READ: Singer to deliver lecture at University of West Indies
The singer hung out with the Nollywood actress and her daughters, Princess and Meraiah Ekeinde in Los Angeles.
The 30-year-old singer posed with Omotola and her girls in some cute snaps which she shared via her Instagram page, yesterday, September 1.
Earlier on the same day, the singer was pictured with Omotola and the rest of her family as well as Hollywood actor, Hakeem Kae Kazim at Universal Studios.
ALSO READ: Singer arrives Ghana in style
Two months ago, the singer was pictured having the time of her life in Johannesburg, South Africa, sharing photos of herself swimming with dolphins.
She does know how to ball!


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Ghislaine Desmarais (née Turcotte)
1933 - 2018
Ghislaine Desmarais (née Turcotte)
In Montreal, passed away on February 6th, 2018, at the age of 84, Mrs. Ghislaine Turcotte, beloved wife of Gérard Desmarais.
She leaves to mourn many relatives and friends.
The family will receive relatives and friends at :
6700 rue Beaubien Est, Montréal
Visiting Hours
|February 11, 2018
|2:00 PM to 5:00 PM
Funeral service
Funeral will take place on February 11, 2018 5:00 PM at the following address: au salon.
Messages in memory of Ghislaine Desmarais (née Turcotte)
Nancy Arias
February 10, 2018
Andrée Robin
February 09, 2018
Rita salloum
February 06, 2018
Online ceremony viewing
Urgel Bourgie / Athos offers an online ceremony viewing service for family and friends.
This service is available on any desktop computer, tablet or smartphone for anyone who cannot attend the service in person.
The pictogram below will be displayed in the obituary section and enable loved ones to access the online ceremony viewing service.
Online ceremony viewing is available at our Beaumont, Beaubien and Lasalle funeral home complexes and Côte-de-Liesse and Saint-Hubert cemeteries.
Please contact us at 1 844 735-2025 for more information.


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2'41'41''. Woo hoo!!! My new PB.
Kak June, thanks for your advice. I did drink the ORS before the race and for the first time in my running history, I didn't suffer any cramps at all during the run. Anyway, the route was all flat and straight. If ada naik turun bukit haruslah tak achieve that PB.
I ran someone else's bib, that explained why I was Fazlina
Prior to the event, we had some dispute at the shuttle bus station. The organizer overlooked the most important factor of the logistics. No public transport to the venue, plus most of the locals also didn't know where the heck was this Changi Exhibition Centre sebab jauh kat hujung dunia, participants would definitely opt for the shuttle bus. They should charter more buses for the runners.
the longgggg queue
When we arrived at Pasir Ris MRT station by 7.40pm, the queue was already very long I couldn't see where the head was. An hour passed by and we were still far from the head. The half marathon would start in 20 minutes time. I saw some of the runners already jumped into cabs. We still waited patiently in the queue. There are thousands more behind us. So I thought if we're gonna be late, at least we're gonna be late together. haha. Half an hour passed by and finally, we made it to the front. But to our shock, the organizer told us the bus driver ain't driving as they've been driving since afternoon and they told us to wait for the 10-12am shuttle bus. WTF!!! True, according to the race booklet the shuttle bus for the half marathoners was from 6-8pm. But with the high volumes of participants and the 10 minutes interval between the buses, what did you expect? The logistics was totally a failure.
Not only were there shortage of buses, the organizer also told us the cut off time for the half marathoners to start the race was at 9.30pm. Another WTF!! Anyway, we managed to get on the bus at 9.15pm. 15 minutes more to the cut off time. Since the race already started at 9pm, the road leading to the venue was closed so we had no choice but walked about 3-4km to the starting line. Not bad for a warm up session eh?
Luckily the organizer still let us run, maybe they've realized their mistake and acknowledged that there were a lot more stranded participants at the shuttle bus station. So we started running at 10.30pm. 1 hour and a half late than other participants. But anyway, it didn't matter as the official timing would be based on our timing chip.
Other than the shuttle bus letdown, the race was all good. Even though we started late, it wasn't a lonely run. When it comes to marathons, Singaporeans are an eager bunch. 20000 people participated for this event yo! And for this year's Standard Chartered marathon in December, I heard 22000 runners already signed up for it.
My sister took part in the full marathon category. The flag off was at 11.59pm so when her friends and I finished our race we didn't sleep the whole night waiting for her. Anyway, we couldn't go back either as the shuttle bus stopped at 12.45am and would only resumed at 5.15am. So to kill the time, the organizers had movie marathons for us.
outdoor cinema
women's winner for ultra-marathon. she ran 84km in 8 hours! O_o
the gang
why I looked tall here? ;p
beat the sunrise
OK. Time to go back.


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|ARM Technical Support Knowledge Articles
Applies to: DS-5
Information in this knowledgebase article applies to:
On Windows Vista and Windows 7, launching a debug configuration with platform as Cortex-A8 RTSM or FVP may give the following error:
Windows cannot find 'c:\windows\system32\telnet.exe
DS-5 Debugger uses telnet capabilities of your computer for a debug session over serial connection to the RTSM and FVP. This error occurs when you try to connect to the RTSM and FVP from the debugger and the telnet client is disabled. The telnet client is not enabled by default on Windows Vista and Windows 7.
Enable telnet client on your computer and restart the debug session. To enable telnet client on Windows Vista and Windows 7:
Article last edited on: 2014-03-25 13:39:33
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It just occurred to me today that as some of you fine folk may be planning 7dRL entries for this year, I should make a last ditch attempt to woo you! With that in mind, I'm going to try and get the next release of the tileset out this weekend.
One of the main things that the tileset lacks at present are items. While I still intend to soldier on with my grand plan for a System shock inventory system, there will always need to be symbols for the stuff that is actually on the dungeon floor. While I'm never going to depict 50 different swords within the space of a single tile, I at least need a symbol for swords as an item type.
Anyway, here is what I have so far, If you cant make out what something is, or if you have any specific requests leave a comment and I'll try and get a reasonably complete set of bits and bobs ready for the weekend.
Thursday, 8 March 2012
Small Items
6 comments:
Any thoughts when to expect next release? :)ReplyDelete
I'm going to try and make it for this Saturday or Sunday. I can't be more specific than that though.ReplyDelete
- RylandAlmanza8 March 2012 at 18:59
So exciting!ReplyDelete
Another question: have you thought about making sci-fi/cyberpunk themed graphics like yours great fantasy one for roguelikes?ReplyDeleteReplies
Hi Dan,Delete
After I have finished with this project I might start over using a SF theme. I would probably use larger tiles though, as I can only really get away with such limited space here by using fantasy tropes. Making fiddly bits of tech or unusual aliens recognisable would require a few extra pixels.
I will try and introduce a few more steam punk elements into this set - there will be a bit of this in the next post.
Wow that's so exciting, thank you :)ReplyDelete
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The Items this month!
1.) A brown satin finish Makeup bag
2.) theBalm Meet Matt(e) eyeshadow
3.) Nailtini Nail Lacquer in Millionaire
4.) Chella Defining Eyebrow Gel
5.) Starlet Intense Eyeliner in black
6.) Bare Minerals Marvelous Moxie Lipgloss (my favorite the color is AHHHmazing)
If you haven't tried My glam/Ipsy/Glam Bag I honestly think you should! I have been receiving glam bags since June and I am very pleased. I get great brands and products in my glam bag and I mean come on its on ten dollars. How can you go wrong?
On to the next topic! Check out this super cutie blog with an even cuter owner here! She is the sweetest person not to mention her and her hubby Garrett are adorable!! I am lucky to have such a wonderful Cara box partner!
I hope everyone has a fabulous weekend and for all my local readers remember that its girls night at the boutique on Saturday! We will be having free Makeovers, food, Up-do's and massages. We will also be raffling of some great prizes such as a Coach Purse and more. So if you have time stop by for a fabulous girl's night! Hope to see some of your faces there!


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Sponsored by Holiday Inn Express, San Luis Obispo
Inspired by a magazine called Blast, Freddy takes a rare day off as a service rep at a wheelchair company to fulfill a dream that's been brewing in his head for 23 years. Freddy's dream? To be the first wheelchair athlete to ski, surf and skate – all in one day. Is it worth the risk? Hell, yes! 15 min. USA. Watch the trailer here
Thursday, March 15 – 1:15pm – Palm Theater
Thursday, March 15 – 4:15pm – Downtown Cinemas
Friday, March 16 – 4:15pm – Downtown Cinemas
Director/Writer/Producer: Maggie Franks
Co-Producer: Rick Franks
Website: facebook.com/freddycarrillosdayoff


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- 2019 Lok Sabha — All You Need To Know
- Hero ISL To Resume From Jan 25th; Kerala Blasters To Face ATK
- Owning A Maruti Suzuki Is Now A Special Occasion — Here's How
- Asus ZenFone Max M2 — Detailed Review
- Why Cheat India — Movie Review
- 7 Best SBI Mutual Fund Schemes To Invest Through SIP
- Benefits Of Drinking Black Coffee
- Vikramshila — The Forgotten University In Bhagalpur, Bihar
According to a study, Nokia Phones are the most preferred mobiles in Hollywood. The study has also found out that every other Hollywood movie has a scene where Nokia Mobiles are used. Nokia is on top of the mobiles with best battery life & also provides some best features.
Due to this, the brand value of Nokia has skyrocketed.
Experts attribute this trend to the excellent battery backup that the Nokia phones offer. This feature has attracted Hollywood to a large extent. People in countries such as the U.S, U.K, France, Russia prefer mobile phones which have the best battery backup, Nokia has introduced various models that are custom-made for people in these countries. Due to this, more & more people are preferring Nokia over other brands such as Samsung, Sony Ericsson etc.
Experts also state that since Nokia is the oldest mobile phone company in the world, people are preferring products based on the reliability factor. The feature-filled nature of these phones are another reason as to why Hollywood prefers them over other brands.
The Symbian operating system of Nokia makes it possible for people to use their phones for high-end users as well. These uses range from simple calling to even image & High-Definition video editing. The agency conducting this study reports that Hollywood actors and the technical crew are completely smitten by the features of Nokia mobiles.
All in all this finding is surely good news for Nokia & has surely come at the right time.


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Insomnia and Schizophrenia
Schizophrenia can sometimes cause insomnia. It might also be that the medication required by the condition affects sleep. What is the relationship between insomnia and schizophrenia?
How do antipsychotics affect sleep?
Someone who suffers from schizophrenia is likely to suffer from insomnia. If the person is not on medication it is likely that insomnia is produced by the voices in the person's head, which are the result of the psychological condition. If, however, the person suffering from schizophrenia is on antipsychotic medication, such as haloperidol, thiothixene, and flupentixol, the person is likely to suffer from sleeplessness. This is because these drugs have been found to cause reduced sleep. There are antipsychotic drugs available, however, that do not reduce sleep. These drugs, including olanzapine, risperidone and clozapine, do the opposite by increasing sleeping time. The only effective way to treat schizophrenia is antipsychotics, which help in gaining control of the symptoms. Therefore, it is important to examine how these drugs affect sleep before using them.
Insomnia as indicator of worsening symptoms
It has been found that insomnia usually indicates worsening of schizophrenia symptoms. If sleeplessness occurs with someone who suffers from schizophrenia, it is advisable to go to doctor to find out if measures can be taken for the possible onset of worsening psychotic symptoms.
Why are medications important for schizophrenia?
Research has been able to determine that patients on medication for schizophrenia who suffer from sleeplessness are more likely to develop severe psychotic symptoms if medications for the symptoms of the condition are withheld. This is why it is important that insomnia is treated with schizophrenia sufferers. This can be done by examining the main reasons for the sleeplessness. This could be lack of peace and quiet where the person sleeps or too much stimulation in the form of caffeine or exercise. If the person can relax sufficiently both physically and mentally, before going to bed, it is easier to fall asleep.
Schizophrenia is best treated with medication but for insomnia it is best that no drugs are used because of the adverse side effects that might occur, especially in conjunction with the antipsychotics


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what is the battle of boyne
Battle in East Sussex clans. The attractive town of Battle gets it name from the Battle of Hastings, which was fought between Harold the Saxon king and William the battle. net. Brood War, from beginning to end join millions of players online and discover the most epic gaming experiences… ever! learn more create account log in. Experience the intergalactic battle between the terran, protoss, and zerg with improved graphics and audio get the blizzard desktop app. Englisch-Deutsch-Übersetzung für battle im Online-Wörterbuch dict 《魔兽世界》竞技场世界锦标赛中国区预选赛即将到来 erfahrt mehr über die spielinhalte von patch 7. cc (Deutschwörterbuch) 2. Alle Sprachen 5, wie updates zu zeitwanderungen, das neue szenario mit chromie, klassenreittiere, neue events im spiel und vieles. If you are not referring to official BattleBots events, shows, or products, please do not use our name battle. Join our mailing list © 2017 BattleBots Inc net. Top 與全球超過. Battle City is a Duel Monsters tournament hosted by the series tritagonist Seto Kaiba at the a revolution is coming to battlefield 1 new battlefield content. Yugi is forced to battle Kaiba when more pressing matters were every month. Fight a desperate battle to reclaim the throne of Wesnoth all summer long. The development team is proud to release version 1 learn more nivelle nights prise de tahure premium friends share or. 12 of The Battle for Wesnoth play tetris battle 2p online for free! play a two minute game against an opponent in this browser-based authentic tetris game mode. Here is the list of the latest internet battle games where you have to face and defeat your oppponent tetris friends. battle against Aliens, Robots, and Zombies in this game Battle online games. net battles third album 'la di da di' out 18 september. เข้า hey everyone, we've decided it was time to reissue our out of print albums, as well as press ep c/b ep. important notice: this version of the battle. the battle of Waterloo net end user license agreement will become effective on february 28, 2015 g. See more m. Dictionary t. com; Word of the Day; Translate; Games; Blog; battle. Dictionary net, accounts. com Unabridged Based on the Random House Dictionary battle meaning, definition, what is battle. Allow the stories of battle to unfold and discover its atmosphere for yourself meaning of "battle" in the english dictionary. Things to see and do english. 1066 Battlefield; Visitor Centre; Abbey Ruins; Walled Garden; 'The Yabba' appears on Battles' third album, 'La Di Da Di' english; american; business; contents. BattleFin seeks the best data centric, systematic & machine learning based strategies in the world contents. Our proprietary technology platform identifies noun (3. For Organizers host your game on kongregate. Control the fun an open platform for all web games!. BattleTac gives you great control over the happenings and keeps you updated where your scenario stands join the ultimate battle for domination! jump. No matter what your game type top games this month stellar squad. Play the best Battle games online on GamesXL you do not need a battle pass or a calibrated matchmaking ranking (mmr) to participate. All the Battle games can be found on this page how much does it cost?. Click on an image to play the game! Recently played Favorites battle cup tickets are not tradable or giftable. Synonyms for battle at Thesaurus полный список изменений вы найдете на battle.


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Software Engineers - C++ - Python - Haskell - R - Oxford
Four brand new products that are going to change the scientific community by influencing the way things are researched and what data is extracted are under development in Oxford!
This company, who are moving to new offices this week, need to increase their software team from 10 to 23 people. New members of staff have praised this employer for a number of key reasons compared to local companies that hire C++/Python developers
1 They have money! And more importantly they want to spend it. The hardware and the tech the use is the very best and most modern. Even research based PHD grads have joined not having used such technology
2 They are big on continuous improvement! Everything is reviewed and remodelled from software to working practices all off the back of employee feedback
3 They have directors/managers passionate about what they do who don't sit on the board of universities so they can keep all knowledge in house and give 100% of their time to changing their field of tech
4 They're not that agile! Usually a selling point, agile working can constrict engineers and their work on projects by micromanaging - turning senior engineers with a wealth of knowledge into another cog in the machine.
They offer flexible working, free food and an excellent pension + bonus.
The kind of engineers we are seeking are excellent mathematicians with a mathematical degree who have a relevant academic background followed up by commercial experience or experience in PHD research roles involving C++, Python or Haskell programming.
If you would like to be considered, please give me a call on 0117 3328100 or apply for immediate consideration - Charles Hoskins


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Imminent Madness. A nice article in today's Diamondback about how the Maryland women are set to steal the show tonight at Comcast. We're getting excited just thinking about the season highlight video they are planning to show. After all, how can you get tired of watching Kristi hit "The Shot?""
DC BasketCases
A friendly asylum for a couple of crazy DC-area women's basketball fans, where we share some news, some opinions, as well as a little of almost anything else that our demented minds dredge up having to do with the WNBA, the Maryland Terrapins, or any other team, player or character associated with the world of women's hoops.
Friday, October 13, 2006
1 Comments:
- At 6:23 PM, terpswomensbball12 said...
-shay you're my hero


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Preheat oven to 350ºF.
In large bowl, beat butter and sugar with electric mixer until light and fluffy. Beat in egg and extracts. Mix flour, baking powder and salt; add to butter mixture 1 cup at a time, mixing after each addition. Do not chill dough. Divide dough into 2 balls.
On floured surface, roll each ball into a circle approximately 12 in. diameter x 1/8 in. thick. Dip cookie cutter in flour before each use. Bake cookies on ungreased cookie sheet 8-11 minutes or until cookies are lightly browned.
In Stock (ships in 1-2 business days)
This item is currently not available.


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It's generally believed that natural fabrics are the most comfortable ones. I am not opposing the fact. I just want to highlight the characteristics of Spandex, which is a synthetic fabric yet very comfortable and gives a great fit to our garments.
Spandex is an elastic fiber, and it was introduced by DuPont under the trade name "Lycra" in 1958. The brand name, Lycra, got so famous that it became the synonym of spandex and now all the varieties of spandex were known as Lycra. It was developed as the alternative to rubber but proved to be so good that it left rubber far behind. It has better resistance to heat, oil, and abrasion in comparison to rubber. However, the quality due to which spandex is loved so much is its stretchability. It can be stretched to almost 500% of its length. It is lightweight, soft, smooth, supple, durable and has higher retractive ability i.e. it can bounce back in its original shape in a very effective manner. As such, it never leads to bagging and sagging of the garment.
All the characteristics of spandex are due to the variety of raw materials that are used to make the stretchable spandex fiber - prepolymers which produce the backbone of the fiber, stabilizers which protect the integrity of the polymer, and colorants. Apart from the basic prepolymers, catalysts etc. stabilizers, such as antioxidants, are added to protect the fibers from damaging sources like heat, light, atmospheric contaminants, and chlorine. These fibers are manufactured in four different ways- melt extrusion, reaction spinning, solution dry spinning, and solution wet spinning. However, solution dry spinning alone produces over 90% of the world's spandex fibers.
As far as application of spandex is concerned, it is widely used in making of active sports wear, work wear and garments that provide support such as hosiery, bra, corsets etc. The level of comfort and wicking ability of Spandex can't match any other fabric. Not only they are used in work wear and swim wear but also are gaining popularity for making apparels for plus sized population. Due to its many qualities, spandex give support as well as freedom of movement and ease to such people. The new seamless spandex not only supports a fuller figure, it also provides the illusion of a more toned frame.


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Before I 'reflect' on the challenge, a shout-out to lovely Corinne Rodrigues. She was the leader of our FB group created for the A-Z challenge. She is a good example of a great leader - mostly cajoling, definitely monitoring, sometimes scolding, but clearly loving. I enjoyed not just reading her blog, but also the way she managed the group. Almost every person in the group is in love with her and her sunny personality. May your tribe grow Corinne!
So here are a few things I learnt during this challenge:
1. Being Organized: I thought I led a fairly organized life, but this challenge woke me up to reality! No, I am not as organized as I used to be. 'Being organized' is a term I use in 2 ways - being organized in terms of time (some people were fantastic about posting at midnight - Afshan, Suzy, Priya Sreeram to name a few), other super-organized in terms of their content - Notably Cynthia, Bhavya NK, Laxmi. Either a story, or a mommy special, or travel special. I started with planning a travel series, but since I didn't plan, I got stuck at several instances and moved to a miscellaneous month instead.
2. Blog Revamp: My blog was started in 2006. I have written over 150 posts. But as far as the look & feel of the blog is concerned, except for occasional photo changes or layout changes, nothing has significantly changed about my blog. I intend remedying it. In fact, one of the key annual goals for myself is to revamp my blog. Learn (currently geeky sounding words like) feeds, traffic, burner, bloglovin and other such!
3. Size of the posts: Given that this challenge involves posting EVERY single day, I wish I had set out some guidelines about the size of the posts. I sometimes had long posts, sometimes short ones. Some posts were hastily written, with disregard for (gulp!) grammar, spelling mistakes or style. This needs to change.
4. Reading / Comments: Ideally I wanted to comment more. Visit at least 3 non-Indian blogs EVERY DAY. I managed on an average 1 every 4 days. But with sick parents, an infant AND a naughty young boy at home, I think I did good on this count. I did comment on most blogs on the FB closed group almost every day.
So will I do this next year?
Too soon to predict. The format has a certain 'suspense' element to it. A feeling of driving to a thrilling finish line that appealed to my adrenaline glands more than my rational brain! Next year, if I plan, I shall execute. Half the battle is the planner. Like I always say, never say never!
Special Mention
My top 5 reads (in no order of priority) are - Roshni (for being witty and self-deprecating about her parenting adventures), Corinne (for writing clearly & precisely, for having wonderful co-writers in Jose and Pablo, and finally for having a blog that is so easy to navigate, clearly a work of love), Monika (well, for being Monika! For clicking pictures that are SO FULL of life, just like her), Vinita (at Blogwatig, for being 1 part irreverent, one part serious, one part fun and one part witty, a great package!) and Kajal (for being a lovely writer, covering myriad topics).


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Showing 1-1 of 1
Price: $23.50
Publisher: New York, New York, U.S.A., Scholastic Book Services: 2000
Edition: Unknown Edition
Seller ID: 034038
Condition: Fine
A new item still in original publisher's shrinkwrap. Stationary box, 8 sheets of stationary, 10 envelopes, sticker sheet, rubber stamp, pocket photo album, address book. Nice gift quality item for the collector. THIS COPY IS IN MY POSSESSION AND NORMALLY SHIPS NEXT DAY. View more info


text: I have started to experiment with potential layouts for the pages of the concertina booklet, I have decided to use the black and white colour scheme which I researched into earlier in the brief. I think it works well in representing two types of identity, online and offline. Furthermore it provides high contrast. Additionally the use of numbers within the design is a reference to may target audience, as when my target audience grew up on the internet it was a 'trend' to use numbers in words when talking online, I have used this visual clue to make the publication relate to my target audience. It also makes the design appear more digital although it is still a printed document.


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I have written here a brief response to those who question my decision to stay in the Episcopal Church. I know that this is a controversial topic and that many will disagree with the conclusions that I make here. The important thing to remember is that this is not a debate about the validity of each others' ministers so that I am not saying that any of the other Anglican bodies are not churches. I have many friends in ACNA, AMiA, REC, etc. and I do not doubt for a moment their piety or ministry. This is a personal belief that I have come to but that could change.
In my mind, the issue needs to be addressed from a few different angles to properly understand the reasons for staying in the Church. First, we must define what "the Church" is in Anglican ecclesiology, according to the formularies and Scripture. Second, we must consider our Lord's exhortation to unity. Thirdly, we must consider the nature of heresy and what the Christian's response to it should be. And lastly, we must consider whether or not heresy is a justification for schism.
What is the Church?
The first question that must be answered is "What is the Church?" I look to the formularies and to Scripture to answer this question. Article 19 defines the church as the, "congregation of faithful men, in the which the pure word of God is preached and the sacraments be duly ministered according to Christ's ordinance in all those things that of necessity are requisite to the same." The Articles do not address the question of episcopacy, which I will not either, although acknowledging the long tradition of associating a valid historic succession to the validity of the Church is nothing new in Anglicanism. Ironically, the Article was written during a time of schism and was written to provide a counter-ecclesiology than that of the Roman church in order to justify the Reformation. The ecclesiology here presented seems simple but as Gerald Bray points out it is more complex t han meets the eye.
The word "faithful" is one of those complexities. Bray notes that the word means "orthodox" not dedicated. Obviously, there has never been a pure church and Cranmer would have noted that when he penned this statement as the Church of England then was full of people who disliked his doctrines and wanted (and eventually did for a time) rejoin the Roman Catholic Church.
The second part of the Article defines the marks of the Church which are the preaching of the Gospel and the administration of the Sacraments. Both of these marks has a presupposition though. First, in order to preach the Gospel, the minister has to know what it is and he must also preach the right Gospel, unlike our Presiding Bishop. The second issue is that the Sacraments must be "duly ministered," i.e. by the proper authority. This means that the person administering the Sacraments must have the authority to do so by the Church, ie. must be ordained. Which leads us to the key element here presented by the Article, that is the visibility of the Church. The Article is not concerned with the "invisible church" or the Church as it is seen by God but by the visible Church on the earth.
The issue of unity
This leads to the next point, which is the consideration of the unity of the visible Church here in earth. Obviously, throughout its 2,000 year existence, the Church has not done a good job of maintaining the unity desired by our Lord. We could look to any period of the Church and see splits and schisms, starting near the beginning with the non-Chalcedonian Oriental Churches. The infamous split in 1054 between East and West as well as the Protestant Reformation mark the larger and more noticeable splits in Church history, however, there are thousands of lesser schisms which have occurred. One can think of the "Old Believer" movement in the Russian Orthodox Church, the SSPX in the context of Rome, or the Continuing Churches in our own Anglican context, who have chosen to break away from the main body of the Church and have faded into a shadow in the history of the Church.
The unity of the people of God is reiterated throughout Scripture, even in the Old Testament, "Behold, how good and how pleasant it is for brethren to dwell together in unity!" (Ps. 133:1). It is, of course, a chief concern for Jesus as he expressed in his High Priestly prayer, "Neither pray I for these alone, but for them also which shall believe on me through their word; That they all may be one; as thou, Father, art in me, and I in thee, that they also may be one in us: that the world may believe that thou hast sent me" (John 17:21-22). Disunity was apparently a major problem in the Church in Corinth, for Paul exhorts the Corinthian Church to unity many times throughout his epistles to that body. "Now I beseech you, brethren, by the name of our Lord Jesus Christ, that ye all speak the same thing, and that there be no divisions among you; but that ye be perfectly joined together in the same mind and in the same judgment" (1 Cor. 1:10), "For ye are yet carnal: for whereas there is among you envying, and strife, and divisions, are ye not carnal, and walk as men?" (1 Cor. 3:3), "For first of all, when ye come together in the church, I hear that there be divisions among you; and I partly believe it" (11:18). Paul notes that, "God is not the author of confusion, but of peace, as in all churches of the saints" (14:33). The urgency in unity is seen throughout the New Testament. "Now I beseech you, brethren, mark them which cause divisions and offences contrary to the doctrine which ye have learned; and avoid them" (Rom. 16:17), and this beautiful passage from Ephesians, "I therefore, the prisoner of the Lord, beseech you that ye walk worthy of the vocation wherewith ye are called,With all lowliness and meekness, with longsuffering, forbearing one another in love; Endeavouring to keep the unity of the Spirit in the bond of peace. There is one body, and one Spirit, even as ye are called in one hope of your calling; One Lord, one faith, one baptism, One God and Father of all, who is above all, and through all, and in you all" (4:1-6).
For me, seeing such an appeal for unity in the Scriptures leads me to believe that we should not consider division lightly in today's Church. However, as this is not a simple issue, we must move on to the next consideration which is what to do about heresy, especially when it invades the Church.
The issue of heresy
Heresy is not a light thing and the Scriptures speak plainly of the dangers of false teaching. Consider the following passages. "But there were false prophets also among the people, even as there shall be false teachers among you, who privily shall bring in damnable heresies, even denying the Lord that bought them, and bring upon themselves swift destruction. And many shall follow their pernicious ways; by reason of whom the way of truth shall be evil spoken of. And through covetousness shall they with feigned words make merchandise of you: whose judgment now of a long time lingereth not, and their damnation slumbereth not" (2 Peter 2:1-3), consider how Paul considers heresy and schism as equally bad sins as well, "Now the works of the flesh are manifest, which are these; Adultery, fornication, uncleanness, lasciviousness, Idolatry, witchcraft, hatred, variance, emulations, wrath, strife, seditions, heresies, Envyings, murders, drunkenness, revellings, and such like: of the which I tell you before, as I have also told you in time past, that they which do such things shall not inherit the kingdom of God" (Galatians 5:19-21). We are told to beware false teachers, "Beware of false prophets, which come to you in sheep's clothing, but inwardly they are ravening wolves" (Matt. 7:15), "Beware of dogs, beware of evil workers, beware of the concision" (Phil. 3:2), "Ye therefore, beloved, seeing ye know these things before, beware lest ye also, being led away with the error of the wicked, fall from your own stedfastness" (2 Peter 3:17). Heretics have condemned themselves by their own false teaching, "A man that is an heretick after the first and second admonition reject; Knowing that he that is such is subverted, and sinneth, being condemned of himself" (Titus 3:10-11). We are to avoid heretics as well, "If there come any unto you, and bring not this doctrine, receive him not into your house, neither bid him God speed" (2 John 1:10).
Based on these portions of Sacred Scripture, we know that we are to avoid heresy and not to receive those who are heretics. The question inevitably arises as to how the faithful in the Episcopal Church can stay when so many of her bishops and priests and laity teach and believe things that are contrary to Scripture. This leads to the final consideration, does heresy justify schism?
Does heresy justify schism?
The big question here is does heresy justify schism? I would say, No. But this is not a simple question so it cannot be answered simply.
First, the Episcopal Church is still a valid Church and her ministers are valid ministers (without entering into the women's ordination debate). Her teaching is contained in the Book of Common Prayer, which teaches the catholic faith as contained in Scripture and tradition and as explained in the Formularies, although the present addition is flawed in several important aspects, it still contains this teaching. The Articles of Religion are included in the Book, although in a less than desirable place, they still hold authority in the Church. The Sacraments are administered within the pattern of the historic episcopate and ministers are ordained accordingly (again without considering the ordination of women and how that affects this). Also, the Articles of Religion teach that the worthiness of a minister does not affect the validity of a sacrament, i.e., that Donatism is wrong:
"Although in the visible Church the evil be ever mingled with the good, and sometimes the evil have chief authority in the Ministration of the Word and Sacraments, yet forasmuch as they do not the same in their own name, but in Christ's, and do minister by his commission and authority, we may use their Ministry, both in hearing the Word of God, and in receiving the Sacraments. Neither is the effect of Christ's ordinance taken away by their wickedness, nor the grace of God's gifts diminished from such as by faith, and rightly, do receive the Sacraments ministered unto them; which be effectual, because of Christ's institution and promise, although they be ministered by evil men."
Secondly, the Church has never been pure, in the sense that there have always been false teachers among her clergy. One can look in the Scriptures and see Paul's comments in Galatians to the Judaizers, an early heresy. Or to the early Church's struggle with all forms of Gnosticism and Christological heresies. In the Middle Ages we see Catharism and other dualistic heresies. During the Reformation we saw the heresies of Anabaptism and millennarianism. In the 1700 and 1800's we saw the Enlightenment and rationalism. In the 20th we have seen liberalism, modernism, and the "inclusive gospel" which plagues the Church today. This is nothing new in the life of the Church.
Let us consider this famous passage from the Gospel of St. Matthew,
"Another parable put he forth unto them, saying, The kingdom of heaven is likened unto a man which sowed good seed in his field: But while men slept, his enemy came and sowed tares among the wheat, and went his way. But when the blade was sprung up, and brought forth fruit, then appeared the tares also. So the servants of the householder came and said unto him, Sir, didst not thou sow good seed in thy field? from whence then hath it tares? He said unto them, An enemy hath done this. The servants said unto him, Wilt thou then that we go and gather them up? But he said, Nay; lest while ye gather up the tares, ye root up also the wheat with them. Let both grow together until the harvest: and in the time of harvest I will say to the reapers, Gather ye together first the tares, and bind them in bundles to burn them: but gather the wheat into my barn." (13:24-30).
Like the field, the Church has within her wheat and tares, those who believe and follow the Gospel and those who, for whatever reason, do not believe the Gospel but are yet members of the Church externally. The problem is when these people enter into the leadership of the Church and begin to teach the people. They cannot be trusted to preach the Gospel for they do not believe it. In my opinion, it is the duty of faithful Christians to combat these heresies at the national and local level and not to leave. At this point in the Church's life, leaving the Church only diminishes the orthodox voice and allows the progressive gospel to further advance in the ranks of the clergy. What the Episcopal Church needs is a group of orthodox and faithful Christians dedicated to not leaving but rather to preaching the Gospel, teaching the Bible, and defending the faith "once delivered to the saints." If that were to happen, the orthodox in the Church might have a flying chance of reviving and renewing the Church.


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We're getting down to the wire, which means down to the nitty-gritty bits of preparation. My biggest focus these past couple weeks has been on our resupply package.
This is my first long trail, which means my first time using a resupply system. A few months back, as we were figuring out our itinerary, we made the decision of how best to resupply ourselves. Being Canadian, we're struggling with the problem of our dollar being rather low. For each US dollar, we have to pay an extra 30% in exchange. This makes any trip across the border 30% more expensive! With that in mind, we decided to start our hike with an entire week's worth of food, to get us from our trailhead at Tioga Pass all the way to Vermillion Valley Resort. We will only be sending one resupply box to ourselves, which saves money both in shipping costs and in resupply pick-up fees.
Vermillion Valley Resort won the draw thanks to their list of services offered to thru-hikers and their affordable resupply option. We've built in a zero day at VVR, which means we won't do any hiking at all. Instead, we'll have a comfortable (free!) campground to stay in, showers, laundry, hot food cooked by someone else, and lots of cold beer.
Did I mention yet that we're vegetarian? Officially we're lacto-ovo-pesca-tarians, which means we don't mind eating dairy, eggs or seafood (as long as it's well sourced!) But there is absolutely nothing that will make me want to eat jerky!
So, how do you eat 3000 calories a day while keeping pack weight down, maintaining variety and a healthy diet? With lots of careful planning!
As you may have gathered from my previous posts, I have become a master of the dehydrator. Over the past couple months I have been dehydrating fruit, sauces, and full meals. To make life a little easier we also picked up a box of pre-dehydrated ingredients like veggies and beans, which I could then mix together with spices and carbs to make our meals.
Trail lunches are always a challenge for hikers. I've never been a fan of sandwiches so on our day hikes we've gotten into the habit of having a block of cheese, a couple rolls of bread, some nuts and an apple. Of course most of that is impossible to bring along on a backpacking trip, plus we need way more calories than that lunch offers.
Backpacking dinners are our pièce de résistance. No Backpackers Pantry meals for us! I much prefer to draw from the amazing recipes found in "Lipsmackin' Vegetarian Backpacking." I've been using this cookbook for most of the years I've been backpacking and I have some real favourites, like "Boot-Stomped Spuds," "South Sister Stroganoff," and "TrailDad's Spaghetti." All of our dinners hit near the 1000 calorie mark, and include a heavy carb such as rice, potatoes and fry bread.
I know that there are many different ways thru-hikers plan their menus. Some hikers really don't mind eating the same dehydrated meal day in and day out. Some hikers don't even bother with a stove, and instead consume an endless amount of energy bars. We've chosen to rise to the challenge of having a variety of meals while maintaining the healthiest diet possible.
What's your backpacking menu look like? Have you ever had to plan resupply boxes? What's the heaviest amount of food you've had to carry at one time? Share in the comments below!


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Jean Dixon, MS, LPC-S, CGP, CPLC
Jean Dixon, MS, LPC-S, CGP, CPLC
Psychotherapy, Counseling, Groups and Coaching
As a recovered woman, myself, I have a unique perspective to help my clients travel the road of recovery, specifically in eating disorders, food issues, and body acceptance. I enjoy helping people view their lives as an experience to embrace, not merely to survive. My hope for my clients is that they achieve confidence in their true authentic selves as they conquer the problems in their lives and face the fears and difficulties they are experiencing.


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HIV-1-specific cytotoxic T lymphocytes (CTLs) and neutralizing antibodies (NAbs) are present during chronic infection, but the relative contributions of these effector mechanisms to viral containment remain unclear. Here, using an in vitro model involving autologous CD4+ T cells, primary HIV-1 isolates, HIV-1-specific CTLs, and neutralizing monoclonal antibodies, we show that b12, a potent and broadly neutralizing monoclonal antibody to HIV-1, was able to block viral infection when preincubated with virus prior to infection, but was much less effective than CTLs at limiting virus replication when added to infected cell cultures. However, the same neutralizing antibody was able to contain viruses by antibody-dependent cell-mediated virus inhibition in vitro, which was mediated by natural killer cells (NKs) and dependent on an Fc-Fc receptor interaction. Meanwhile, bulk CTLs from HIV-1 controllers were more effective in suppression of virus replication than those from progressors. These findings indicate that control of HIV-1 replication in activated CD4+ T cells is ineffectively mediated by neutralizing antibodies alone, but that both CTLs and antibody-dependent NK-mediated immune mechanisms contribute to viral containment. Our study systemically compared three major players in controlling HIV-1 infection, CTLs, NAbs, and NKs, in an autologous system and highlighted the multifactorial mechanisms for viral containment and vaccine success.
Citation: Ting Tu, Jianbo Zhan, Danlei Mou, Wei Li, Bin Su, Tong Zhang, Tao Li, Ning Li, Hao Wu, Cong Jin, Huabiao Chen. In vitro inhibition of HIV-1 replication in autologous CD4+ T cells indicates viral containment by multifactorial mechanisms [J]. VIROLOGICA SINICA, 2017, 32 (6): 485-494 https://doi.org/10.1007/s12250-017-3992-9
Received: 5 April, 2017; Accepted: 27 July 2017; Published: 15 September 2017
Copyright: © Wuhan Institute of Virology, CAS and Springer Science+Business Media Singapore 2017
Data Availability: All relevant data are within the paper and its Supporting Information files.
Corresponding author: Huabiao Chen, Phone: +86-511-85038483, Fax: +86-0511-85038483, E-mail: firstname.lastname@example.com, ORCID: 0000-0002-7215-5429; Cong Jin, Phone: +86-10-58900986, Fax: +86-10-58900986, E-mail: email@example.com, ORCID: 0000-0002-5200-5992.
#These authors contributed equally to this work.
The host immune response system is thought to be involved in the control of HIV-1 infection. During acute HIV-1 infection, the viral load rises to a peak, which leads to activation of the cellular immune system (Kahn and Walker, 1998). This high-level plasma viremia typically declines dramatically once HIV-1-specific cytotoxic T lymphocyte (CTL)-mediated immune responses are mounted, probably reflecting the strong antiviral activities of these cells during acute infection (Borrow et al., 1994; Koup et al., 1994). The dramatically accelerated progression of simian immunodeficiency virus disease that is noted to occur in rhesus macaques following the depletion of CTLs also provides evidence for the importance of CTLs in the control of viruses (Schmitz et al., 1999). Moreover, enhanced ability of HIV-1-specific CTLs to inhibit virus replication in autologous CD4+ T cells was demonstrated in a rare subpopulation of HIV-1-infected individuals (Saez-Cirion et al., 2007; Chen et al., 2012), although these cells actually had low res-ponses as measured at baseline by IFN-γ enzyme-linked immunospot (ELISpot) assays (Pereyra et al., 2008). These individuals maintain levels of viremia below the limit of detection by current assays for long periods in the absence of therapy (Cao et al., 1995; Deeks and Walker, 2007) and are referred to here as elite controllers. Similarly, HIV-1-specific neutralizing antibodies (NAbs) are often undetectable or at low titer during acute infection (Moore et al., 1994; Moog et al., 1997) and in elite controllers (Harrer et al., 1996; Bailey et al., 2006; Pereyra et al., 2008), suggesting that antibodies do not play a major role in viral control. These findings constitute a striking contrast to progressive HIV-1 infection, where high levels of virus replication occur in spite of the presence of strong HIV-1-specific CTL and NAb responses. In these progressive patients, HIV-1-specific CTL responses are of higher magnitude and more broadly directed, as measured by the assessment of IFN-γ production (Pereyra et al., 2008), and HIV-1-specific NAbs are at higher titer, as measured by in vitro neutralization of cell-free viruses (Bailey et al., 2006; Pereyra et al., 2008). The ability of these immune responses to inhibit virus replication in autologous CD4+ T cells, the ultimate function likely required of protective responses, has rarely been measured.
It has been recently reported that antibody-dependent cell-mediated virus inhibition (ADCVI), a measure of the combined potential of antibody and effector cells dependent on an Fc-Fc receptor (FcR) interaction, was able to contain virus infection rather than cause target cell death (Forthal et al., 2006). Passive infusion of neutralizing monoclonal antibodies has been demonstrated to prevent virus infection in rhesus macaques (Emini et al., 1992; Mascola et al., 2000; Hessell et al., 2007). Furthermore, antibody responses to vaccines have correlated with improved outcomes in macaques after virus challenge (Schmitz et al., 2005; Someya et al., 2005).
Given that a cellular immune response is well documented to contribute to control of HIV-1 infection, it is possible that enhanced antiviral efficacy against HIV-1 could be gained by a combination of NAbs and cell-mediated immunity (Hessell et al., 2007; Walker and Burton, 2008). In this study, we examined the potency of CTLs and natural killer cells (NKs) from HIV-1-infected individuals, in combination with engineered NAbs, to inhibit infectious virus replication in autologous CD4+ T cells. Our data indicated that both CTLs and antibody-dependent NK-mediated immunity contribute to viral containment.
Study subjects
HIV-1-infected individuals were recruited on the basis of viral load from outpatient clinics at local hospitals, following institutional review board approval and written informed consent. Elite controllers were defined as having HIV-1 RNA below the level of detection for the respective available ultrasensitive assay (e.g., < 75 RNA copies/mL by bDNA or < 50 copies by ultrasenstive PCR) without antiretroviral therapy. Untreated chronic progressors were recruited locally on the basis of viral load above 10, 000 RNA copies/mL plasma.
The primary CXCR4-utilizing HIV-1 strain X4 92HT599 and CCR5-utilizing HIV-1 strain R5 91US056 were obtained from the AIDS Research and Reagent Program, Division of AIDS, NIAID, NIH (Bethesda, MD, USA). The titers of the virus stocks were 2.57 × 106 TCID50/mL and 2.14 × 106 TCID50/mL, respectively.
Monoclonal antibodies
IgG1 b12 is an engineered monoclonal human antibody that recognizes an epitope overlapping the CD4-binding site of gp120 (Burton et al., 1994; Binley et al., 2004; Nie et al., 2014). LALA, a variant of b12, was created by site-directed mutagenesis (Hezareh et al., 2001). LALA retains antigen binding but binds weakly to human Fcγ receptors, triggers antibody-dependent cellular cytotoxicity (ADCC) with lower efficiency, and binds and activates human complement weakly (Hessell et al., 2007). IgG1 DEN3 is an isotype control monoclonal antibody specific for Dengue virus.
Effector cell preparation
Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood of HIV-1-infected individuals by Ficoll-Hypaque density gradient centrifugation. Bulk CD8+ T cells and NKs were isolated from PBMCs by negative selection (Dynabeads, Thermo Fisher Scientific, Waltham, Massachusetts, USA). The isolated CD8+ T cells and NKs were of up to 98% purity as validated by flow cytometry. The generated CD8+ T cells responding to viral proteins were fine-mapped using optimal peptide pools in IFN-γ ELISpot assays.
ELISpot assay
IFN-γ ELISpot assays were performed as described by Addo et al. (2003) , using optimally defined epitope peptide pools. To calculate the number of specific spot-forming cells (SFC), the number of spots in the negative control wells was subtracted from the number of spots counted in each well. Negative controls were always 30 SFC per 106 input cells or fewer.
Antibody neutralization assay
Virus neutralization was performed using the method described by Trkola et al. (1995) with slight modifications. Briefly, CD4+ T cells were generated from freshly isolated PBMCs by the addition of CD3: CD8 bispecific monoclonal antibody (Wilson et al., 1995; Wong et al., 1997). After 5–7 days, greater than 98% of these cells coexpressed CD3 and CD4 by flow cytometric analysis (data not shown). Antibodies were adjusted to 200 μg/mL, and eight twofold serial dilutions were prepared. An aliquot (50 μL) of each dilution step was transferred to four replicate wells of a 96-well culture plate. The virus inoculum was adjusted to 2000 TCID50/mL, and a 50-μL (100 TCID50) aliquot was incubated with antibodies (50 μL) for 1 h at 37 °C. The calculated neutralization titers refer to the antibody concentrations present during this preincubation step. The antibody-virus mixture was then diluted twofold by addition of 100 μL of CD4+ T cells at a density of 1 × 106/mL, and the cultures were incubated for 7 days. The cultures were collected and treated with 0.5% Triton, and the p24 antigen concentration was determined by a standard quantitative enzyme-linked immunosorbent assay (HIV-1 p24 ELISA kit, PerkinElmer, Inc., Waltham, Massachusetts, USA). As the antibody-virus inoculum was not washed away from the cells at any stage, control wells containing 50 μL of virus and 150 μL of medium were included to provide an estimate of the amount of input p24 antigen that carried over into the p24 antigen assay for virus production. This value was subtracted from all the test results. The antibody concentrations (μg/mL) causing 50%, 90%, and 99% reduction in p24 antigen production were determined by linear regression analysis; these are the ID50, ID90, and ID99 values.
Antibody-dependent cell-mediated virus inhibition (ADCVI)
Inhibition of viral replication was assessed in a previously established assay system (Yang et al., 2002; Forthal et al., 2006; Hessell et al., 2007) with slight modifications. Briefly, autologous CD4+ T cells were infected with designated HIV-1 strains at a multiplicity of infection (MOI) 0.01 for 4 h at 37 °C, washed twice, and plated at 5 × 104 cells per well in a 96-well round-bottom plate. Various dilutions of b12, LALA mutant, or isotype control DEN3 antibodies were added to virally infected cells alone or along with effector cells, either CD8+ T cells or NKs, at a ratio of 1:1 or as otherwise specified in a total of 200 μL RPMI 1640 medium containing 50 U/mL interleukin-2 (IL-2). After 7 days of incubation at 37 °C in 5% CO2, supernatant was collected and p24 antigen was quantified by a standard quantitative enzyme-linked immunosorbent assay (HIV-1 p24 ELISA kit, Dupont, Boston, MA, USA). Virus inhibition was calculated as follows: Percentage inhibition = 100 × (1 - p24p/p24n), where p24p and p24n are the concentrations of p24 in supernatant from wells with or without a source of effectors, respectively.
Statistical analyses
Mann-Whitney tests were performed using GraphPad Prism version 4.0a, and P-values of P < 0.05 were considered significant.
Neutralization of HIV-1 by antibodies
Using two primary HIV-1 isolates, X4 92HT599 and R5 91US056, we first tested the ability of monoclonal antibody b12 and its mutated Fc- and compliment-binding variant LALA to neutralize viruses using CD4+ T cells from elite controllers as indicator cells. The outgrowth of autologous viruses in CD4+ T cells from the elite controllers was markedly delayed, and there were no autologous viruses grown out from the tested subjects during the period of the assay. Representative neutralization titration curves are depicted in Figure 1A.
Mean and median neutralization
The ID90 for both X4 and R5 virus for b12 were in the range of 10 to 25 μg/mL, and LALA was comparable to b12 with an ID90 in the range of 10 to 30 μg/mL. These data showed that both b12 and the LALA variant demonstrated neutralization of cell-free X4 and R5 primary isolates, but for this inoculum of virus a relatively high concentration of antibody was required to achieve 95% neutralization. Moreover, the data showed that both b12 and LALA, which lacks FcγR and complement binding functions compared with b12, mediate comparable neutralization of these primary viruses.
Next, we tested the ability of these antibodies to contain viruses in virally infected CD4+ T cells from the elite controllers in whom outgrowth of autologous viruses in CD4+ T cells was markedly delayed. CD4+ T cells were infected with primary HIV-1 isolates prior to the addition of antibodies. Designated concentrations of b12, LALA mutant, or isotype control DEN3 antibodies were added to virally infected cells. The p24 antigen production in supernatant was evaluated and expressed as the percentage of neutralization (Figure 1B). Both b12 and LALA failed to effectively control viral replication in infected CD4+ T cells as the p24 antigen production in supernatant steadily increased during the period of 7 days even with antibody at 100 μg/mL, far in excess of the range estimates for concentration of an antibody produced in vivo. These data suggest that although this antibody is potent in neutralizing free virus, its ability to neutralize virus spread once infection has occurred is very limited, which is likely due to cell-cell spread of virus in this in vitro culture system, but may also be related to production of high numbers of progeny virions that overwhelm the available antibody binding sites.
Suppression of HIV-1 by CTLs
We next tested the ability of bulk CD8+ T cells from HIV-1 elite controllers and chronic progressors to inhibit virus replication in the same cell culture system, using autologous CD4+ T cells preincubated for 4 h with the same HIV-1 strains at the same MOI (0.01) at which no effect was observed using the antibodies alone (Chen et al., 2009). For these experiments, we chose elite controllers and chronic progressors in whom outgrowth of autologous viruses in CD4+ T cells was markedly delayed, allowing us to use a fixed inoculum of primary viruses in these experiments. As shown as an example in Figure 2A, addition of autologous bulk CD8+ T cells from an elite controller to virally infected CD4+ T cells at a CD8+ to CD4+ T cell ratio of 1 to 1 resulted in marked inhibition of virus replication over 7 days in culture, whereas weak inhibition of virus replication was observed in coculture of virally infected CD4+ T cells with autologous bulk CD8+ T cells from a progressor (Figure 2B).
We next expanded these studies to include 12 elite controllers and 10 chronic progressors, examining the ability of their CTLs to inhibit the same strain of HIV-1 in autologous CD4+ T cells. Marked inhibition of virus replication was observed at a CD8+ T to CD4+ T cell ratio of 1 to 1 for the elite controllers whereas there was weak inhibition of virus replication by bulk CTLs from the chronic progressors (Figure 2C). The difference was highly statistically significant (P < 0.0001). The magnitude of HIV-1-specific CTL responses in bulk CTLs was evaluated by optimally defined epitope pools in IFN-γ ELISpot assays ( Figure 2D). Although the ability of CTLs to retard virus replication was greater in the elite controllers, the magnitude of HIV-1-specific CTL responses as measured in the elite controllers was actually weaker than those in the progressors (3973 ± 1935 vs. 7324 ± 3691 SFC per million bulk CTLs, P = 0.0138). These data indicate that HIV-1-specific CTLs from elite controllers have an enhanced antiviral functional capacity, contributing to the control of virus infection, which is not measured by standard IFN-γ ELISpot assays, suggesting IFN-γ ELISpot approaches may be of limited value in defining CTL functions such as the ability to neutralize virus replication. These data also demonstrate that CTLs from elite controllers can significantly inhibit replication of reference strains of primary isolates in cell culture, whereas a potent NAb, although highly effective when exposed to virus pre-innoculation, is ineffective once initial infection of the cells has occurred.
We next addressed the possibility that enhanced antiviral efficacy could be gained by a combination of CTLs and NAbs. In the infected cell cultures, serially diluted concentrations of NAb b12 were added along with CTLs. However, there was no significantly enhanced neutralization observed with added antibodies even at the highest concentration of 100 μg/mL for CTLs from HIV-1 elite controllers (Figure 3A) or chronic progressors (Figure 3B). Given ADCVI is reported to be dependent on Fc-FcR interaction (Forthal et al., 2006; Hessell et al., 2007), we next determined FcR expression at the surface of the HIV-1-specific CTLs by flow cytometry. Little or no expression of various kinds of FcR was detected on the surface of the HIV-1-specific CTLs (data not shown). Using a previously reported assay (Liu et al., 2009), degranulation of intracellular vesicles (CD107a expression) mediated by Fc-FcR interaction was examined. There was no CD107a upregulation on the CTLs after stimulation with IgG-loaded p815 cells compared with stimulation with p815 cells alone or medium control (data not shown). These data may explain the above finding that enhanced antiviral efficacy against HIV-1 could not be gained by ADCVI mediated by HIV-1-specific CTLs.
Inhibition of HIV-1 by ADCVI
NKs have been reported to serve as main effector cells and in combination with NAbs to mediate ADCVI as described previously (Forthal et al., 2006; Hessell et al., 2007; Liu et al., 2009; Asmal et al., 2011). We next evaluated ADCVI of NKs from virally infected individuals in combination with NAbs. Autologous CD4+ T cells were infected with primary X4 virus and cocultured with NKs alone or along with designated concentrations of antibodies. As shown as an example in Figure 4A, addition of autologous NKs to virally infected CD4+ T cells resulted in significantly enhanced inhibition of virus replication over 7 days in culture containing 50 U/mL IL-2 and this enhanced inhibition was mediated by antibody b12 but not by LALA or the isotype control monoclonal antibody DEN3. From the study of the 12 elite controllers and 10 chronic progressors, weak inhibition of primary X4 virus was observed by NKs alone from the controllers (Figure 4B) or progressors (Figure 4C), which may be mediated by direct killing of NKs through activation of receptors on NKs that interact with major histocompatibility complex class I molecules on HIV-1-infected CD4+ T cells (Bonaparte and Barker, 2004; Vieillard et al., 2005). However, significantly enhanced inhibition (P < 0.01) was gained by addition of antibody b12 at a concentration of 100 μg/mL to NKs for both the controllers ( Figure 4B) and progressors (Figure 4C) whereas there was no statistical difference in the antiviral activity of NKs alone (P = 0.5704) or antibody-mediated enhanced inhibition (P = 0.8336) bewteen the controllers and progressors. Moreover, this impact by ADCVI was mediated by antibody b12 but not by LALA, further demonstrating that antiviral potency by ADCVI is dependent on Fc-FcR interaction.
The relative contributions of various immune mechanisms to control of ongoing viral replication remain elusive. These data, employing a functional assay that measures the ability of effector cells, neutralizing monoclonal antibodies, and the combined power of these effectors to inhibit viral replication of primary HIV-1 isolates in autologous CD4+ T cells, indicate that NAb b12 is highly potent in neutralizing free viruses but is limited in containment of cell-cell spread of viruses once infection has occurred. In contrast, bulk CTLs from HIV-1 controllers are effective in containment of viruses under conditions where little or no effect is seen from NAbs. Moreover, bulk CTLs from HIV-1 controllers are more effective in suppression of virus replication than those from progressors. However, the same b12 NAb is able to contain viruses by ADCVI. Importantly, these data demonstrate that ADCVI is mediated by NKs and dependent on an Fc-FcR interaction, suggesting innate immune mechanisms such as NKs or monocytes/macrophages that generally express FcRs may contribute to control of ongoing viral replication by ADCVI in the presence of functional antibodies. However, these data and previous findings that the high-level plasma viremia declines dramatically once HIV-1-specific CTL responses are mounted (Borrow et al., 1994; Koup et al., 1994) probably reflect that the HIV-1-specific CTLs are the major effector cells in containment of viruses whereas the mounted NK response during acute HIV-1 infection is not enough to contain viruses.
The goal of HIV-1 vaccination strategies is to have an immunologic barrier in place that will prevent infection or limit virus replication once infection occurs, thereby limiting symptoms of disease caused by virus infection (Wang et al., 2016). A priority in HIV-1 vaccine development has been the generation of NAbs, ideally to provide sterilizing immunity, but this has remained an elusive goal. Only one antibody-based AIDS vaccine has been taken through efficacy trials thus far, using the gp120 subunit protein as an immunogen, and this vaccine candidate did not elicit antibodies neutralizing field isolates of HIV-1, did not prevent infection, and did not affect subsequent viral load (Flynn, 2005). Although the recent RV144 Thai trial showed moderate protection against viral acquisition (Gilbert et al., 2011), the protection mechanisms remain unclear (Kim et al., 2015). The most important second line of defense is the cellular immune response, including NKs and CTLs as the presumed major effector arms. Together with previous findings (Yang et al., 1996; Yang et al., 1997; Yang et al., 2003; Chen et al., 2009), our studies have demonstrated that both CTLs alone and NKs in the presence of functional antibodies can suppress HIV-1 replication in vitro. However, not all CTL responses created in natural HIV-1 infection are equally potent in terms of the ability to inhibit virus replication. The limitation of this study is that all these antiviral factors were assessed in vitro. It is critical to define in vivo HIV-1-specific CTL responses for viral control during natural infection and vaccine protection against HIV-1.
There is a remarkable subset of HIV-1-infected individuals, here named elite controllers, who are able to maintain a state of apparent durable control of HIV-1 replication without the need for antiviral therapy, providing clear evidence that durable containment of HIV-1 in the absence of therapy is possible. Some evidence for the involvement of CTL responses (Migueles et al., 2002; Zimmerli et al., 2005; Betts et al., 2006) was documented for the mechanisms of virus control in these cases. In this study, a virus suppression assay was used to assess the antiviral potential of CTL responses from these subjects versus chronic progressors. As compared with chronic progressors, bulk CTLs from elite controllers had higher capacities to inhibit virus replication in infected autologous CD4+ T cells, which is consistent with the finding by Saez-Cirion and his colleagues that HIV-1 controllers rather than progressors exhibited potent CTL capacity to suppress HIV-1 infection ex vivo (Saez-Cirion et al., 2007). Factors that potentially modulate protective CTL responses include, among others, polyfunctionality, antigen sensitivity or functional avidity, proliferative capacity, loading of lytic granules, ex vivo expression of perforin, T cell antigen receptor (TCR) clonotypes, and properties of the interactions among TCRs, viral peptide, and the major histocompatability complex (Chen et al., 2012). Collectively, this evidence strongly argues that potent CTL responses directed at HIV-1 are causally related to HIV-1 control in at least some controllers. However, many of these studies are by necessity correlative in nature, making it difficult to rule out the possibility that the preservation of potent HIV-1-specific CTL responses is a consequence rather than a cause of HIV-1 control. Although NKs might exert innate antiviral immunity in acute HIV-1 infection, the mounted NK cell response during acute HIV-1 infection does not appear to be enough to contain viruses. The role of NKs in chronic HIV-1 infection in elite controllers and progressors remains unclear. Unraveling the mechanisms of HIV-1 control in these cases should be a high priority because this may facilitate the design of immunogens able to elicit the corresponding protective responses.
It has been reported that certain engineered monoclonal antibodies and primary antibodies from HIV-1-infected individuals, in the presence of effector cells, were able to inhibit HIV-1 replication in vitro (Forthal et al., 2001; Forthal et al., 2006) and modulate viremia in vivo dependent on Fc-FcR interactions (Hessell et al., 2007). This control of virus replication involving not only cytolytic but also noncytolytic mechanisms was distinguished from ADCC and termed ADCVI by Forthal and his colleagues (Forthal et al., 2006). Given that HIV-1-specific CTLs tested in this study do not express FcRs (data not shown), they would be unlikely to mediate ADCVI, although they showed highly potent ability to inhibit virus replication and limit cell-cell spread of viruses. NKs were reported to serve as the effector cells in ADCVI (Forthal et al., 2001; Forthal et al., 2005; Forthal et al., 2006) because NKs express various kinds of FcRs and can therefore interact with antibodies to modulate antiviral activity. It is to be expected that there are other subsets of cells, such as monocytes/macrophages, which express FcRs, involved in ADCVI and that contribute to viral control. ADCVI also allows the possibilty that antibodies, passively transferred via a viral vector, could be competent to control viruses in the presence of effector cells, overcoming the barrier that antibodies that neutralize HIV-1 are often absent or at low titer in natural HIV-1 infection. The protection mechanisms for HIV-1 containment need to be further investigated with broader CTLs, NAbs, NKs, or other factors in the context of acute infection.
Taken together, our study, for the first time to our knowledge, provides a systemic comparison of three major players, NAbs, CTLs, and NKs, in an autologous system to assess neutralization, cytotoxicity, and ADCVI on HIV-1 replication in CD4+ T cells, and shows that no single arm thus far can be defined as the correlate of complete protection of HIV-1 infection. Alternative factors or combinations of multiple factors must be pursued. Our findings provide an important consideration for HIV-1 control and vaccine success that may be crucially dependent on the combination of antibody and cell-mediated immunity, as well as the innate and adaptive immunity.
This work was supported by the Fund for Jiangsu Specially-Appointed Professor (2014JSTPJS-53), the Innovation Fund from National Center for AIDS/STD Control and Prevention, China CDC, Capital Medical University Key Laboratory Project (2-03-02-BJYAH2016006), and China's 12th Five-Year Major Project on the Prevention and Treatment of AIDS. Dr. Dennis Burton (the Scripps Research Institute) kindly provided b12, b12 LALA, and Den3 monoclonal antibodies. The HIV-1 strains X4 92HT599 and R5 91US056 were obtained from the AIDS Research and Reagent Program, Division of AIDS, NIAID, NIH.
The authors declare that they have no conflict of interest. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee.
HBC and CJ designed and supervised the experiments, TT, JBZ, TL, DM, WL, BS, and TZ collected samples and carried out the experiments, HBC, CJ, TT, and JBZ analyzed the data and wrote the paper. NL and HW provided helpful suggestions about the study. All authors read and approved the final manuscript.
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The Benefits Of Income Tax Preparation
In this world that people lived, it seems like every day is getting busier and busier for them. Well, you can't blame it on the kind of job that they have the have caused them to be so busy. If you can relate to this, then raise your hands up. Do you know that preparing for your income tax is something that you have to also find time with? Yes you should know that this aspect of your life is also important. In this article, you will understand the importance of preparing for this and why this is important as well. You have to consider this as part of your role as a citizen that is why this is important. Furthermore, you must know that the government has been punishing those people who are not able to comply with this rule. These are but some of the reasons why you have to comply the rules that the government is now implementing.
Perhaps some people are aware of this importance but just can find the time to do income tax preparation. If this has been your struggle ever since, there is good news for you. You can hire professionals who can do income tax preparation for you. This is very important so that you can complete this before the deadline. You must understand that there is a deadline that the government will post so that you can prepare this ahead of time. Today, so many people out there are already doing this and believed it or not, it has helped them big time. You too can avail of this service.
The truth is that without income tax preparation, it is possible for you to be paying money that you should not pay and this is going to cost you a lot. In other words, it is not going to be a waste of money on your end. Have you not known that today some tools have been made available in the market for you to buy and use so that this process will not be hard for you? For sure, if you have the tools, it will not make the process difficult and time consuming for you anymore. The good news is that you are not the only one who is using and buying this tool because the demand is increasing every year. With the numbers increasing, you can really say that this is a very perfect and useful tool for almost all people out there. Have you head about income tax preparation done online? Yes there are different methods that you can think when it comes to this. All you need to do is choose a method that is most suitable for your needs.The Path To Finding Better Resources