text: In this brief romantic biography of the last mistress of Louis XV, Haslip (Marie Antoinette, 1988, etc.), now 80, brings a special charm to the story of du Barry (1743-93), whose legendary beauty brought both rewards and penalties: power, wealth, envy, fear, and, ultimately, death. Illegitimate child of a French peasant, Jeanne BeÇu was educated in a convent until, at 15, she emerged a golden-haired, lisping beauty who worked as a hairdresser and a milliner's assistant--but who was, in fact, a prostitute. Discovered by Jean du Barry, a dissolute gambler and pimp to the aristocracy, she was introduced at Versailles, where Louis XV, 58, a widower, became infatuated with her. After her hasty and unconsummated marriage to Jean's brother, Guillaume, comte du Barry, Mme. du Barry became the king's official mistress and one of the wealthiest women in France, a wealth she shared with, among others, her beloved ""niece,"" Betsi, whom Haslip ""suspects"" may have been her illegitimate daughter. Mme. du Barry was also hated, especially by Marie Antoinette, who arranged to have her imprisoned after the death of Louis. Released after one year, Mme. du Barry recovered her jewels, her estate, and a series of lovers, including an English neighbor who ultimately rejected her--a ""little-known"" story that Haslip reveals. In the early stages of the Revolution, Mme. du Barry traveled often to England, possibly as a courier for ÉmigrÉs but ostensibly to try to recover stolen jewels that had been impounded there. She returned to France only to be beheaded for her aristocratic associations. Although more extensive, scholarly, and even popular biographies of du Barry are available, this one has a particular appreciation for the decorative arts that women like du Barry cultivated--arts that included the remaking of themselves--and for the hazards of beauty when it comes close to political power. The lavish language recalls the style of Regency novels, for which this could serve as a reference.


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Red Dead Redemption 2's single-player mode contains numerous ways for Arthur Morgan to obtain quick cash, and now the same can be said for Rockstar Games' Red Dead Online. In fact, Rockstar makes it very easy for players of all types of play styles to make their financial mark in the Old West. Red Dead Redemption 2's online portion, Red Dead Online, can be a little more withholding when it comes to rewarding players financially and some may be confused as to the quickest methods to make some cash.
In a lot of ways, Red Dead Online is very similar to Grand Theft Auto V's online component, Grand Theft Auto Online. This is especially true of the game mode's reliance on not only sharing the entire map from the single player mode with dozens of other players, but competing with (and sometimes against) these players to make cash and progress as well. Naturally, to buy better weapons, clothes, and horses for their customizable characters, Red Dead Redemption 2 players are going to need a lot of money in Red Dead Online. In fact, items actually cost a little more in the online component, and as a whole, it's not always clear how to make money quickly. Luckily, there are already a few different methods for making cash quickly in Red Dead Online.
Related: What You Need to Know Before Playing Red Dead Online
For a method that involves relying strictly on story missions contained within Red Dead Online, players will first have to make sure they've completed the tutorial section of the game. Additionally, this method is more for the type of gamer who doesn't mind playing certain story missions out of order. Players will want to use the Quick Join feature of the game and look for Story Mission On-Call option. This will allow you to play random missions with other players. While players won't be able to pick the mission they participate in, they should keep an eye out for one called "Kill Them, Each and Every One." This particular story mission will reward players with around $250 at the end, depending on how many deaths are accumulated. Keep in mind you will only be able to do this mission once for the cash reward.
If Red Dead Redemption 2 missions aren't really your thing, there are always glitches that can be exploited. For this particular one, players should create a character (don't worry about detail) and complete the first six story missions. Also try and get to level 10 so your Red Dead Online character can get an additional $100. Afterwards, delete your character and start a new character to repeat this process. While your characters will be deleted, the money you've earned will stick with your account. Players who do this three or four times can have upwards of $1000 to play around with when it comes time to create their true characters.
Additionally, players can always utilize the same strategies from the single player mode and rely on hunting/fishing, treasure hunting and looting enemy corpses to make some cash. Of course, these methods aren't nearly as fast as the above strategies, but they also don't require playing story missions out of order or being forced to repeat the same missions over and over again. With that in mind, hunting in Red Dead Online is probably the quickest legitimate way of making quick money, especially if you utilize another glitch involving going to Emerald Ranch in single player mode, hunting and skinning animals, and then accessing online from the menu. Players should find they still have all of the animal skins from the single player portion and can sell these skins for around $15-$20, depending on quality.
Relying on glitches is definitely a sure-fire way to make some quick cash, but they don't often last forever as Rockstar will likely patch them when they become public knowledge. Nowhere is this more true than the animal skinning method, which is arguably the most glitch-like method available right now. Luckily, that method really isn't sustainable and doesn't offer as much money as story missions and the re-create method. Either way, players should now have a good idea of how to make some quick cash in Red Dead Online.

subset: commoncrawl


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Three-time African Footballer of the Year Abedi 'Pele' Ayew has launched an appeal to clear his name of match-fixing.
The former Black Star, who owns and manages Nania FC, was among officials from several clubs who were banned last week for one year after being accused of match-fixing.
Nania's 31-0 win over Okwawu United and Mighty Jets' 28-0 defeat at the hands of Great Mariners prompted the Ghana Football Association to investigate.
"I will go wherever I can to get justice as we have been banned on conjecture," Pele told BBC Sport.
Pele has filed an appeal with the help of a lawyer, who will fight to overturn the verdict against Nania.
Apart from the bans on the club officials, all four clubs were demoted from Ghana's second tier to the fourth tier and fined US$20,000.
Yet Pele is angry with the decision.
"There was no evidence that before, during or after the match, the officials of Okwahu United and myself and/or any of my officials engaged in any form of discussion or even camaraderie - nor did any such thing ever take place," he said in a statement.
"What then did we do wrong?"
"The only possible accusation is that my team scored more goals.
"And if that is the case, my contention is that while the scoreline may raise eyebrows, it does not point to an irrefutable conclusion that the match was fixed.
"At the height of my career and even that of Maradona or the legendary Pele, it is very possible to record that score against a team featuring six players and a seventh in goal who is not a natural goalkeeper."
Okwahu finished the game, in which Nania even missed a penalty, with seven men after a number of injuries, which forced an outfield player into goal.
"Maybe my strikers should be educated that when they are next playing with a superior numerical advantage, they should refrain from scoring more goals to avoid being banned," Abedi stated.
"I have also been advised that my players were not treated fairly but I will leave that to my lawyers to handle."
"Scorelines in football may be baffling but cannot be the basis for judging a match as fixed," the 1982 African Nations Cup winner emphasised.
He used last week's Uefa Champions League game when England's Manchester United thrashed AS Roma of Italy 7-1 to buttress his point.
"The recent score between Manchester United and Roma could have recorded a cricket score if Roma had been reduced to seven men.
"In the statutes of Fifa, it is spelt out clearly that there must be evidence of such impropriety before sanctions can be imposed.
"If there is a video recording of the game, I would love for it to be played so that everybody will be able to judge for themselves."
The GFA is expected to meet soon to fix a date to look into the appeals submitted by Nania and the other three clubs.


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Forcing the Sale of a Home
I have a court hearing scheduled in a few weeks to address what seems to be a common problem: two people own a home and one of them refuses to sell. In the case that I am involved in, I am acting for the owner who wishes to sell.
Whether in the context of an estate dispute or in family law, it is not uncommon for such a problem to arise. Often, the party who refuses to sell seeks to buyout the other's interest (surprisingly, the offers in this regard are often for less than market value). The property in question is often the family home or a cottage.
If no agreement can be reached, the owner who wishes to sell can ultimately make application to the court for an order compelling a sale.
Under the Partition Act, a property owner has a prima facie right to an order for the partition and sale of lands held with another owner. The other owner, in turn, has a corresponding obligation to permit the sale. Pursuant to the Act, the court is required to compel the partition and sale if no sufficient reason can be shown why such an order should not be made.
The case law provides that the court's discretion to refuse an order for sale is limited and is usually reserved for cases which involve malicious, vexatious or oppressive conduct. The party who opposes the sale of course has the onus of demonstrating why the sale should not be ordered.
Thanks for reading,


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While not a nationwide program, a number of AAA clubs offer safe ride services on select dates for members and nonmembers. This service is not available everywhere.
The ACT Raising Safe s Program, developed by the APA Prevention Office, teaches positive parenting skills to parents and caregivers of ren from birth
The Riverside County High college (RCHS) Cal-SAFE (California college Age Families Education) program is a part of the RCOE Community college. RCOE began serving
The CIPA/E-Rate program requires colleges to educate students on the topics of Appropriate Online Behavior, Social Networking and Chat Rooms, and Cyber Bullying
Safe Place is a national youth outreach and prevention program for people in need of immediate help and safety. As a community-based program, Safe Place
Teen Oriented. We have certified and experienced driving instructors that will teach your loved one defensive driving techniques in a safe and positive atmosphere.
Safe Routes Brochure; Register Now to become a Volunteer; Download PDF Registration Form; Detroit Youth Prevention Initiative- Progress Report
Safe Dates – an cent dating prevention program
Monday, April 17, 2017: Teens From Around the State Honored in DMV-Travelers 9th Annual Teen Safe Driving Contest Ceremony : The ninth annual DMV-Travelers Teen Safe
Safe Communities America, an accreditation program that recognizes communities prioritizing safety, gets citizens involved and keeps them engaged in reducing injuries


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On Demand Staffing's West Washington Street office needs day laborers. Work today-pay today.
Employees will go to various job sites throughout Indianapolis and assist clients with general construction clean up and other duties as needed.
Report to the West Washington Street office at 5:30 a.m. with long pants and work boots, ready to go out the door to different job sites throughout the city. Remember this is a work today and get paid today. No background or drug-screens are needed for these assignments.


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Like many of the reviews here on OutdoorGearLab, this one is a compilation of years of winter boot testing. This year's analysis includes testing we conducted during mid-winter months in Washington State, Idaho, the San Juans of Colorado, and the Sierra Nevada of California. We took the top picks from our previous reviews, dropped the lowest performing models and replaced them with new ones. Our testing includes standardized, quantifiable tests as well as hours and hours of wear time.
During winter testing, we spent long days stomping around in snowshoes, putting down some miles on the local winter trails, and shoveling heaps of snow from our long driveway, deck, and even the roof to evaluate each boot's hiking abilities and long-term comfort. We also made countless trips to the post office, the grocery store, and around town dog walks during cold and snowy days to test the ease of use and comfort levels in models meant for less rugged activities. Field testing took place primarily in the mountain ski town of Mammoth Lakes, California, and the greater Lake Tahoe area.
Fall testing required a little more creativity to hunt down icy snow patches on high mountain passes. But between those high-altitude, snowy hikes, ice bath tests, and cold water submersion tests, we tested the newest boots in the review to their limits.
We tested warmth in a few ways. First, to objectively measure how well each boot retains heat, we placed the boots side by side in an ice bath and took internal temperature readings with a laser thermometer at three-minute intervals for twelve minutes. This showed us precisely how each of the models tested retained or lost heat without the addition of body heat. At the end of this test, we recorded how much heat was retained or lost, by each model on this level playing field.
We also tested for warmth by putting on a single pair of merino wool socks on with each pair of boots. Then we stepped into a 32-degree slush comprised of ice cubes, snow, and water and stood there for 8 minutes. We allowed our feet to fully rewarm in front of the fire before retesting so that we did not start testing the next model with already cold feet. This test was also performed as a side by side comparison with two different models on at the same time for a direct comparison. We also wore these boots for numerous hours apiece shoveling snow, stacking wood, and while tackling relatively sedentary winter chores out of doors.
Water Resistance
Using both the standardized eight minutes slush bucket test and a ten-minute water submersion test, we dunked our booted feet around 5 inches deep to see if the boots would show any signs of leaking. While this is an easy way to quantify the water resistance of the boots, we also tromped through countless puddles and snow banks, going out of our way to see if we could get them to leak in real-world applications. The submersion test goes above and beyond the demands that most people will put on their winter boots from a water resistance standpoint, but it quickly becomes evident which boots are truly waterproof and those that are simply water and splash resistant.
Fit and Comfort
We tried to get as many people's feet into the boots as possible to reach a consensus on fit and noted whether each pair is available in a wider fit. We assessed comfort by walking around in the boots, spending time in and out of doors, and by polling our various review contributors. Testers took detailed notes on the comfort and support of the insole, the width and length of each model, as well as the stiffness and support offered by the shaft of the boots.
Ease of Use
We tested ease of use by seeing how much time it took to put on and take off each boot. We compared lacing systems and, for the Pac models that include a removable liner, we included how easy or difficult it is to put the liner back in after drying it.
We scored traction after testing each one of the boots on icy pathways and parking lots, firm old snow patches, and freshly fallen snow to see which rubber compounds and lug patterns provide the best grip in a variety of conditions. In some instances, we wore a different boot on each foot to test traction on slick surfaces side by side.


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A young man has incurred the wrath of some social media users after he complained about his girlfriend demanding marriage after dating her for seven years.
The Nigerian man identified as Chisomjoel Owuamanam referred to the years they have dated as "just seven years".
Sharing his frustration in a Facebook group, he claimed the woman was impatient and intolerant for asking what direction the relationship was taking after the seven years of dating.
"Just seven years of dating she is already asking me where the relationship is leading to, Hmhmh women and impatience," said the man.
Chisomjoel concluded his post with a running man emoji, perhaps sensing that his rant was sure to make people angry and come after him.


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That's right folks. The women who gave us "Jesus Take The Wheel" and "Before He Cheats" has given birth yet again to an adorable little boy named Jacob Bryan Fisher.
Since her debut in 2005, Carrie Underwood has done nothing but rise to the top. With her music, her awards such as her seven Grammy awards, her movies and let's not forget her marriage to Hockey hunk Mike Fisher, the birth of her two children and then making a comeback after an accident that she thought would leave her never looking the same, the woman is just an amazing, talented storm that has taken over the country genre.
Now for those who may not know who Carrie Underwood really is, Carrie is a talented country singer, who is known for her albums such as, Some Hearts, Carnival Ride, Play On and Blown Away. According to en.wikipedia.org, she rose to success so quickly in 2005 thanks to her debut album "Some Hearts," which had songs such as, Inside Your Heaven, Jesus, Take The Wheel, and Before He Cheats, on it.
Her album did so well that it became the best-selling solo female debut album in country music history, the fastest-selling debut country album in Nielsen SoundScan history, and the best-selling country album of the last 15 years. It led her to win three Grammy Awards for is, including Best New Artist. Keep scrolling to learn more about the talented young mom of two and her new baby boy that she just gave birth to.
20 She Gave Birth To A Little Boy
It was on Wednesday, January 23, 2019, that Carrie Underwood and her husband Mike Fisher welcomed their second child together, a little boy named Jacob Bryan Fisher.
According to cbsnews.com, the talented country singer announced the birth of her little boy via her social media page with this message: "Jacob Bryan Fisher entered the world in the wee hours of the morning on Monday," Underwood wrote. "His mom, dad and big brother couldn't be happier for God to trust them with taking care of this little miracle! Our hearts are full, our eyes are tired and our lives are forever changed. Life is good..."
She then shared a few pictures of herself, her husband and her three-year-old all holding little Jacob.
19 This Past Year Was The Toughest Year Of Her Life
When a new year begins, typically people love to start fresh and put the previous year behind them. No one was more excited for a new year and a fresh start more than Carrie Underwood due to how bad 2017-2018 was for her.
According to eonline.com, the now mother of two had a tough last two years. Between her struggles to have a second child and the accident she suffered outside her estate, 2018 was defiantly not her year.
She was strong though, and she knew that no matter what, she was going to be ok. She had her career, her little boy and a loving husband. Her luck did come around, however. Near the end of 2018, she found out she was pregnant with another little boy.
18 She Struggled At Getting Pregnant With Baby Number 2
Back in 2017, Carrie Underwood had two goals: work on new music and have a baby. Sadly, one thing was not in the cards for her. According to cbsnews.com, Carrie went through three miscarriages between 2017 and 2018.
"We got pregnant early 2017, and didn't work out," Carrie admitted during an interview with Tracy Smith For The Record. "At first I was like, 'Okay, God, we know this is, just wasn't Your timing. And that is all right. We will bounce back and figure our way through it.' And got pregnant again in the spring, and it didn't work out. Got pregnant again, early 2018. Didn't work out. So, at that point, it was just kind of like, 'Okay, like, what's the deal? What is all of this?'
Luckily for Carrie, her time did come and she is now yet again a mommy to another beautiful little boy.
17 She LOVES Animals
Carrie Underwood is a huge animal lover and because of her love of animals she even became a vegan at the age of thirteen due to not having the heart, nor the stomach, to eat one of her own animals.
According to en.wikipedia.org, Carrie was even voted by PETA as the "World's Sexiest Vegetarian" in 2005 and 2007.
During an interview with PETA in 2007, Carrie had this to say about her love for animals: "Ever since I was little, I loved animals. If you told me I could never sing again, I'd say that was horrible, but it's not my life. If you told me I could never be around animals again, I would just die."
16 She Broke Her Wrist During A Bad Fall
Back in November 2018, just days after having the amazing opportunity to host the CMA Awards, Carrie Underwood fell on the steps outside her home and suffered some serious injuries.
According to cbsnews.com, among all the cuts and abrasions she suffered, Carrie also received a broken wrist from her fall, a break that required her to receive surgery and have a metal plate put in. Her husband, Mike Fisher, travelled overnight back to Nashville to be with her.
A rep for the singer was quoted saying: "Her recovery will take some time and she appreciates all prayers and well wishes," Underwood's rep said.
15 Her Face Will Never Be The Same
In November 2018, Carrie Underwood suffered a fall down her steps located outside her home that left her with a few cuts, abrasions and even a broken wrist.
According to people.com, later it was learned that there was more that happened during her fall than she had originally told her fans. She had also cut her face so badly that she had to get between 40 and 50 stitches.
Seven weeks after the accident, Carrie admitted that her face did not look the same and that she was nowhere near ready to get back in front of a camera. "When I am ready to get in front of a camera, I want you all to understand why I might look a bit different," she explained. Luckily, she got over her fear and is not blessing her fans with her beautiful face again. She will always beautiful to her fans, no matter what happens.
14 She Announced The Gender Of Her Baby During The CMT Awards
It was back in 2014 when Brad Paisley accidentally told the world the gender of Carrie Underwood's first child. The act caused "#BradBlewIt" to blow up. Well, it seems like the tradition continues. Thanks to Brad, Carrie told the world the gender of her second child while hosting the CMA Awards in 2018.
According to cbsnews.com, Brad started the conversation on stage by saying: "Tonight we're going to reveal something even more exciting. Who the father is ... Mike Fisher we're all rooting for you buddy. What's your gut feeling?"
Carrie shook her head laughing in amusement. Brad then continued, "Seriously, Carrie give me a little baby hint. Johnny or June? Keith or Nicole? Garth or Trisha? Tim or Faith? George or Tammy? Waylon or Willie?" Carrie responded, "Oh my gosh, Willie! It's a Willie."
13 She Got Engaged After A Year Of Dating
It was on December 20, 2009, that Mike Fisher popped the question to his girlfriend of a year, Carrie Underwood.
According to people.com, a rep for the singer said: "I'm happy to confirm that Carrie Underwood is engaged to Mike Fisher, and the couple couldn't be happier. No wedding date has been set at this time."
Later the next Day, Mike himself opened up and admitted that it was true: "It's true! We're both obviously excited and very happy." Mike proposed to Carrie in their home Sunday afternoon, and news of their engagement spread later that day after the two sent messages of their news to their friends and family.
12 She Had A Southern Style Wedding
When Carrie Underwood said "I Do" to her now-husband of eight years, Mike Fisher, it took place in a ceremony that her wedding planner called, "a spiritual expression of their love."
According to people.com, the loving couple tied the knot at the Ritz Carlton Reynolds Plantation in Georgia on Saturday, July 10, 2010, in front of 250 close friends and family. The ceremony featured classical music and the couple's favorite Bible verses.
After the ceremony, the couple gave PEOPLE a statement: "We could not feel more blessed to have found each other and to have shared this day with our friends and family that mean so much to us!"
11 She Announced She Was In Love On Her CD Play On
When Carrie Underwood first started dating her now husband Mike Fisher in 2008, the pair kept their relationship off the radar for a while.
According to people.com though, when Carrie was ready to let everyone know she was in love, she did it in the sweetest way ever. The talented country singer announced her love for her boyfriend at the time by writing a sweet message on her CD Play On for all her fans to see for.
"I love you so much!" she wrote. "You make my life better in every way! I thank God for you every day." How adorable is that?
10 She Adopted A Puppy Just Weeks Before Giving Birth
When Carrie Underwood was just a few weeks from giving birth to her second child with husband Mike Fisher, the duo decided to go out and adopt the cutest puppy in the world. According to dailymail.co.uk, the family of three adopted the loveable puppy and their three-year-old son Isaiah named him Zero.
Mike shared the news via social media with the message, "'Meet the newest member of our family! Isaiah affectionately named him Zero. I guess it fits because it's the chance of surviving if you try to break into our home." Along with the message was an adorable picture of the new addition the family.
Mike also tagged the rescue they adopted the puppy from, Cedar Valley Canine, whom also shared a picture of the goofy puppy with the message, "'Little Zero needs those big ears to hear all the new things he's learning!!"
9 She Gave Birth To Her First Child In 2015
When Carrie Underwood first gave birth to her first son, the marvelous event happened on February 27, 2015.
According to rumorfix.com. the beautiful singer took to her social media accounts to announce the event with a picture of her little one's hands and feet and this message: "Tiny hands and tiny feet...God has blessed us with an amazing gift! Isaiah Michael Fisher - born February 27."
When it came to the gender of her baby, the couple played a small game with her OBGYN. The Doctor wrote down the gender of their little bundle of joy and gave it to them in a sealed envelope. The couple were allowed to either look at it to see the gender, or leave it sealed and keep it a surprise.
It was during a romantic dinner that the two lovebirds decided to open the envelope. Thing is, they already had strong suspicions that it was a boy, and the envelope just told the what they already knew.
8 Her Fame Started After Winning American Idol
For those who don't know, Carrie Underwood first came into the spotlight in the summer of 2004 when she appeared on the show American Idol in St. Louis, Missouri.
According to enwikipedia.org, Carrie auditioned for the show with the song "I Can't Make You Love Me," by Bonnie Raitt. During her time on the show, producers admitted that Carrie dominated the voting every week.
During the final episode, Carrie sand "Bless the Broken Road," by Rascal Flatts and won the competition. Along with winning the competition, she also received a recording contract worth almost a million dollars, a private jet for a year and a Ford Mustang convertible. She's been on top ever since.
7 She Has Done A Little Acting
Most fans know Carrie Underwood for her many songs, but singing is not the only thing this little mamma has done. According to en.wikipedia.org, this little mamma has dipped her toes a time or two in the acting pool, and she rocked that too.
In March 2010, the talented singer appeared as Tiffany, a medical sales rep that falls for Ted, in How I Met Your Mother. Her episode she appeared on became the show's second highest rated episode with over 10.48 million viewers.
In April 2011 she went on to debut in her first film. Carrie played the role as Sarah Hill, a church youth leader who helps the young Bethany Hamilton overcome her troubles after the shark attack, in the movie Soul Surfer.
6 Her Music Was "Banned" From Being Played On A Certain Radio Station
Back in 2011, Carrie Underwood's husband, Mike Fisher was traded to the Nashville Predators, an act that made the Ottawa radio station, 105.3 CISS-FM, threaten to ban Underwood's music because of it.
According to en.wikipedia.org, fans of Carrie Underwood pitched a fit and threatened to never tune into the radio station again, which caused some issues with the station. The station later apologized for its threat about banning Carrie's music from their station and said it was all a joke and that they don't even play her music anyway. It was their way of wishing Mike good luck in Nashville... supposedly.
Mike later criticized the station and talked about how it was not right either way how Carrie was blamed for his trade when it's obvious she had nothing to do with the move or the trade in the first place. Joke or not, to imply that was wrong.
5 She Found A Benefit Of Having A Big Pregnant Belly
It was on September 1, 2009, when Carrie Underwood and Mike Fisher announced that they were expecting their first child together, a little boy named Isaiah.
According to rumorfix.com, while pregnant, Carrie found a few joys that went with being pregnant- one being a new place to store snacks. The country singer learned that her ever growing baby belly was the perfect spot to place a bowl of treats. Her own personal table.
She took to social media to share her new love for her pregnant belly with her fans: "Pregnancy perk: using my belly as a tabletop for my snacks! Nom nom nom."
4 Her and Her Husband Fought Over The Name Of Their First Child
Naming a child is a hard decision as the child is going to be stuck with that name for the rest of their life. A parent wants to make sure to give them the best name they can. According to rumorfix.com, when it came to the name of their first child, Carrie Underwood and Mike Fisher had different opinions on what they should name him.
"He keeps trying to push the name 'Bo' for our little guy," Carrie admitted during an interview with Country Countdown. "I just won't have it. Bo Fisher? I don't know. I don't like it. He thinks of it like a bow and arrow. There's kind of a couple of names that we're okay with, but it's hard, labeling your child for the rest of its life. I was like, 'Why don't we come up with a short list we're both agreeable to and then meet the little guy and see what we're feeling?'" That was a great idea.
3 Her Birth Announcements Were Different
When Carrie Underwood announced the birth of her two little boys, each birth was different.
According to tasteofcountry.com, when she gave birth to her first child, Isaiah, she only shared a picture of his foot and hand with a sweet message announcing he had finally arrived. In fact, it was only recently that she has allowed her almost four-year-old to be seen in public.
When she gave birth recently to her new little one, Jacob, she shared a full picture of her the new bundle of joy. It is defiantly obvious that she isn't as protective over this birth as she was her first.
2 Her Second Pregnancy Was A Lot Different
Pregnancy is a hard thing for a woman to go through, besides the birth of course. It takes a toll on one's body. There are some pregnancies that seem to go by a breeze and those women are the luckiest women ever, but then there are some that can make a woman hate being pregnant.
When it comes to Carrie Underwood, she learned really quick that not ever pregnancy is the same. According to tasteofcountry.com, while Carrie seems to be a pro at being pregnant, she has admitted that this time around pregnancy has been a lot harder on her body.
"It's definitely different than the first time," Underwood confessed. "When they say every pregnancy is different, it really is. Just different symptoms. I feel like this one is just a little harder on my body for some reason. But it's been really good."
1 Her Three-Year-Old Is Excited To Become A Big Brother
There are times when a child is being raised alone in a household that they do not like when another sibling is born and that it takes away the attention they are so use to having on them all the time.
When it comes to Carrie Underwood, she got lucky in that area and her almost four-year-old son was ecstatic to learn that he was going to be a big brother soon. According to etonline.com, the little man had one stipulation, though: "He says he won't change any poopy diapers though," Underwood shared. "I understand, maybe I can change his mind."
Now that his little brother is here, the question is will she really be able to change his mind?
References: people.com, etonline.com, en.wikipedia.org, cbsnews.com. rumorfix.com, dailymail.co.uk, tasteofcounrty.com,


text: Screwing my young Labrador in cowgirl pose Your browser does not support the video tag. Download video. Duration: 0:00 Added: 2018-07-19 Tags / Categories: cowgirl labrador screwing animalsexhost.com Related Animal Porn Movies Big angry Labrador and sexy zoophile Inked zoophile fucks his dog in cowgirl pose Sweet Labrador gives a nice cunnilingus Young blonde shows striptease for a Labrador Fat female zoofil got licked by Labrador Screwing my lovely dog in missionary pose Two 3D dogs are screwing a passionate bae Sweet cowgirl is sucking a stallion's boner Trained Labrador is trying to suck himself


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Bandung is one of the largest city in Indonesia. There are several international flights from Malaysia and Singapore to Bandung Hussein Sastranegara Airport, in regular (daily) basis. There are also a number of domestic flight connecting Bandung to major cities in Indonesia, e.g. Jakarta, Surabaya and Denpasar.
From Capital City Jakarta, Bandung can also be reached by car, travel bus, and train. With approximate distance of 150 km, trip from Jakarta to Bandung takes 4 hours or more depends on the mode of transportation and traffic condition.
Aryaduta Hotels is located at the very center of Bandung city. It is only 5 kilometers from Hussein Sastranegara Airport and 4 kilometers from Bandung Train Station. Several shopping centers and tourist attractions are located nearby.
International participants are suggested to take connecting flight to Hussein Sastranegara Airport, but another options maybe of interest.
Take airport train from Cengkareng (Soekarno-Hatta) International Airport to Manggarai Station (last stop). Take local train (commuterline) to Bekasi Station. From Bekasi Station, there is Argo Parahyangan Train for Bandung Station. Please consult Railink, Commuterline and KAI for airport train, commuterline, and intercity train schedule and ticketing. From Bandung Station, take taxi to Aryaduta Hotels.
Several travel bus are available every hour at the Cengkareng International Airport. Xtrans, Daytrans, and CitiTrans can be used to reach Bandung. The booth for those travel bus are located at the corner of the terminal.
Bandung has minibus as the main public transportation. However, it is not recommended for tourist/foreigner to take public transportation without any guidance as it is quite challenging as most of the drivers can not speak English. App-based taxi or motor-taxi are more preferable. Smartphone application such as Gojek, Grab, and Uber can be used for local transportation in Bandung.
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Click here to print - Click here to close this windowGardening Know How - https://www.gardeningknowhow.com
Healthy cucumber plants will provide the gardener with a bountiful harvest of the delicious, crisp fruit, sometimes too bountiful. Unfortunately, there are plenty of insect pests that might get to the cucumbers before you do or transmit diseases, rendering plants unable to produce. It isn't just insects that cause cucumber plant damage, however. Sudden cold snaps can kill the plants as well, so protecting cucumber plants is of paramount importance. Read on to find out how to protect cucumber plants and about keeping cucumbers protected from predatory insects.
Cucumbers [1] (Cucumis sativus) are tender annuals that thrive in warm temperatures of between 65-75 degrees F. (18-23 C.). Even prolonged exposure to temperatures below 55 degrees F. (13 C.) can cause decay, pitting and water soaked areas on fruit. Sudden cold snaps can cause cucumber plant damage on leaves, stems and fruit or even kill the plants. Frost damage is seen as shriveled, dark brown to black foliage.
While global warming has been increasing temperatures around the world, it also makes for unpredictable weather such as sudden cold snaps. So, it's important to have a plan and take steps to protect cucumber plants and other warm season annuals at the risk of sudden frost, thereby avoiding damage to cucumbers.
First off, grow cucumbers in sheltered areas of the garden. Avoid open, exposed sites or low spots in the garden where cold air will collect. Grow the fruit along fences [2], boulders or shrubs to provide them with some protection from the cold. If a sudden cold snap is forecast, cover the cucumbers.
The plants can be covered with whatever you have on hand, old bed sheets, plastic, newspaper or other light material. Push some sturdy sticks into the ground around the plants to support the covering and weigh down the corners with stones. You can also use wire (extra wire coat hangers will work) to form a curved arch upon which to lay the covering. Tie the ends of the covering to sticks pushed into the ground. Remember to open the row cover daily to allow condensation to evaporate. Close them again by mid-afternoon to trap heat overnight.
Temperatures inside a row cover will be from 6-20 degrees warmer than outside and soil temps 4-8 degrees warmer down to 3 inches deep.
In lieu of covering the cucumbers with row covers, there are other methods for keeping cucumbers protected from cold. Use a shingle or other broad board stuck into the ground on the windward side of each plant to protect them from cold winds. Place a plastic milk container, bottom cut out, over each plant; large aluminum cans will also work.
There are many insect pests that are more than happy to sample your cucumbers. Some of them even introduce disease into the cucumber patch. Cucumber beetles [3] are guilty of introducing bacterial wilt. They carry the disease in their bodies and it overwinters with them as they hibernate in vegetation left in the garden.
Avoiding damage to cucumbers due to cucumber beetles and the resulting bacterial wilt requires a two part approach. Be sure to clean up detritus, including weeds, in the garden at the end of the growing season to avoid leaving any hidey holes for the beetles to hibernate and overwinter in. Then in the spring after planting, cover the cukes with a light weight floating row cover [4]. Remember to remove the cover after the plants begin to flower so they can be pollinated.
Aphids [5] will also get at cucumbers, actually aphids seem to get at everything. They reproduce rapidly and colonies of them are difficult to control. At the first sign of aphids, treat the plant with an insecticidal soap. Other ideas to combat aphids are planting in an aluminum foil covered bed, and filling yellow pans with water, which will entice the aphids and drown them. Encourage beneficial insects that prey on aphids by planting flowers nearby that attract them. Aphids and leafhoppers also introduce mosaic virus into the garden.
Leafhoppers [6] suck the juice form the leaves and stems of cucumbers. Here again is a situation where the use of row covers can mitigate infestation. Also, spray with insecticidal soap.
Leaf miner [7] larvae tunnel through leaves. Use floating row covers and destroy any infected leaves. Cutworms [8] are another hazard to cucumbers. They chew on stems, roots and leaves. Cutworms live under the surface of the soil so protect the plants by placing a 3-inch paper collar around the stem of the plant or use saved canned food containers with the top and bottom cut out. Also, keep the garden free from weeds and sprinkle wood ash around the base of the plants.
Spider mites [9] also love cucumbers. Spray them with water or insecticidal soap or rotenone. Encourage beneficial predators, such as ladybugs and lacewings. Whiteflies [10] can also be found congregating on the underside of the cucumber leaves. Again, beneficial insects should be encouraged. Also, remove infested leaves.
Other types of insects enjoy munching on cucumbers. Where they can be seen, hand pick them and dump them in a bucket of soapy water. Snails [11] and slugs [12] will snack on cucumbers, especially young plants. Hand pick them as above or if that's too disgusting for you, bait some traps. Pour some beer into a low bowl and place a few around the plants. The slugs will be enticed by the beer and crawl in and drown. Diatomaceous earth [13] sprinkled around the plants will thwart these pests as well.
Article printed from Gardening Know How: https://www.gardeningknowhow.com
URL to article: https://www.gardeningknowhow.com/edible/vegetables/cucumber/protecting-cucumber-plants.htm
URLs in this post:
[1] Cucumbers: https://www.gardeningknowhow.com/edible/vegetables/cucumber/tips-for-growing-cucumbers.htm
[2] Grow the fruit along fences: https://www.gardeningknowhow.com/edible/vegetables/cucumber/planting-cucumber-fence.htm
[3] Cucumber beetles: https://www.gardeningknowhow.com/edible/vegetables/cucumber/cucumber-beetle-control.htm
[4] floating row cover: https://www.gardeningknowhow.com/edible/vegetables/vgen/row-covers-for-garden-plants.htm
[5] Aphids: https://www.gardeningknowhow.com/plant-problems/pests/insects/homemade-aphid-control.htm
[6] Leafhoppers: https://www.gardeningknowhow.com/plant-problems/pests/insects/leafhopper-control.htm
[7] Leaf miner: https://www.gardeningknowhow.com/plant-problems/pests/insects/leaf-miner-control.htm
[8] Cutworms: https://www.gardeningknowhow.com/plant-problems/pests/insects/get-rid-cutworms.htm
[9] Spider mites: https://www.gardeningknowhow.com/plant-problems/pests/insects/spider-mite-treatment.htm
[10] Whiteflies: https://www.gardeningknowhow.com/plant-problems/pests/insects/whitefly-control.htm
[11] Snails: https://www.gardeningknowhow.com/plant-problems/pests/insects/organic-snail-control.htm
[12] slugs: https://www.gardeningknowhow.com/plant-problems/pests/insects/kill-garden-slugs.htm
[13] Diatomaceous earth: https://www.gardeningknowhow.com/plant-problems/pests/pesticides/diatomaceous-earth-insect-control.htm
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text: Add to Cart Compare Quick view Hardcore Energy, Orange Gold Spike MSRP: Now: $43.00 Was: Dietary supplement.The hardcore energy drink. Not to burst anyone's bubble, but there are no such things as monsters-and bulls really can't fly. So if you believe in these fictional characters, you need not apply. Spike Hardcore Energy-it's as real as it... MSRP: Now: $43.00 Was: Add to Cart Compare Quick view


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Content Warnings
Here is a list of content warnings for I Wish You All the Best. I've tried to collect this list to the best of my ability, but if you have read the book and see something I did not include, please don't hesitate to reach out to me with the information.
Content Warnings
Parental Abuse (both emotional and physical)
Misgendering (both accidental and purposeful)
Detailed Anxiety & Panic Attacks
Detailed Depressive Episodes
Suicide Idealization
Underage Drinking


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When Father Jonathan Morris started doing some work on behalf of The Christophers, he noted that he was a longtime fan of our message and mission encouraging individuals to light a candle rather than curse the darkness. That mission, of course, was started by Father James Keller, M.M. in 1945.
Watch the video below for more details and click here to buy "Light in the Darkness" or The Christophers' new "Three Minutes a Day" book (Volume 50).
RELATED: An Ideal Book for the Overly Busy, Those with Spiritual ADD, and Everyone Else, Too


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04 July 2012
Details of Defendant:
McEvoy's Self Service Drapery Limited
Company Number:
Registered Office:
Backhouse Centre, Clanbrassil Street, Dundalk, Co. Louth.
Dundalk Circuit Court
Judge Fulham
The appeal was confined to severity of sentence only. The District Court Orders of 3rd February, 2011 were affirmed save for a reduction of the fines from a total of €3,000 to a total of €2,400. The defendant was directed to pay costs of €1,250 to the prosecution in respect of the appeal proceedings.
District Court Case 3rd Feb 2011
The Director of Public Prosecutions v Mary Donnelly »
The Director of Public Prosecutions v Thomas Clarke »
Disclosure - Non Financial Information
Large - companies, groups and undertakings are required to disclose and publish their policies on non-financial matters each financial year.


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May 19, 2019 we had our first training game. I said it wasn't a real battle but rather a training exercise so there wouldn't be too much embarrassment when things went terribly wrong or to be too worried about rules perfection. We started out with a 1940 First Wave German platoon and 1939 British Platoon… Continue reading Chain Of Command Training Game
Category: Chain of Command
Chain of Command
I am learning Chain of Command (click rules name for its website) in my seemingly never-ending search for an elegantly simple but somewhat realistic "skirmish" ruleset. We started with Tractics (or really its predecessor, GRT) in 1970 but noted its flaws of morale and turn sequence. Then after years of D&D, I gained a bit of… Continue reading Chain of Command


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Brittany is an ABA Therapist and student of the Disability Studies program at Ryerson University.
With a Disability Studies background, Brittany brings a unique perspective to her work, which she finds challenging but believes is important. The idea for Brittany's video was inspired by her experiences working with students with ASD in the school environment. Please note that the video is not based on any one particular experience and the purpose was not to showcase any one person's experience. Her hope is that the video will highlight that, while the word 'inclusion' gets thrown around a lot, it is not necessarily being practiced to the full extent.


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Shaker Country Club and St. Dominick's Church
Caroline said one of her favorite moments was when the Navy Pilots sang you've lost that lovin feeling so be sure to scroll through this blog! Daughter's Florist did a great job on the Decor, making the church and reception absolutely beautiful! I really enjoyed shooting this couple and incorporated several different lighting styles and techniques


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Nintendo officially confirms that patch 3.1.0 for Super Smash Bros. Ultimate will feature fighter adjustments
Earlier today, SSBUBot prophesied that Super Smash Bros. Ultimate users will be prompted with an in-game notification that patch 3.1.0 would be releasing within the next seven days. That declaration ended up being accurate.
Seeing as how replays from the previous version would become unwatchable, it was speculated that this upcoming patch will feature balance adjustments. Well, we no longer have to speculate about this. Nintendo has officially confirmed that version 3.1.0 will host adjustments for the game's roster.
"Version 3.1.0 of Super #SmashBrosUltimate is coming this week," said Nintendo Versus. "This update will include fighter adjustments, meaning replay data from previous versions will be incompatible."
Of course, it was not revealed exactly what those adjustments are. If you have any replays you want to keep, you'd best make the preparations to convert them into a video fairly soon.
Thus far, version 2.0.0 and 3.0.0 held the highest number of balance adjustments for the game's lifespan so far. While they were initially criticized for the lack of notes, Nintendo has done a good job of releasing information about what exactly was changed when these two versions were released.
It's worth mentioning that the Piranha Plant was added to the roster when the game went from version 1.x.x to version 2.0.0 and Joker was made playable when the game updated from version 2.x.x to 3.0.0. Since the version is changing from 3.0.1 to 3.1.0, we probably shouldn't expect a new fighter to suddenly appear.
So which combatants are considered to be strong right now? Characters like Olimar, Snake, Peach, Daisy, Wolf, Joker, and Pichu are considered to be very strong in the tournament scene right now.
Meanwhile, fighters like Little Mac, Bowser Jr., King K. Rool, and Kirby are struggling a bit right now. There's no telling if Nintendo plans on balancing based on these perceptions.
Click images for larger versions


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Single Family Residence
- 3 beds
- 2.5 baths
- $212,900 price
- 1638544 mls id
- Half Baths: 1
- Neighborhood: Hamilton West
- Days on Market: 32
- Status: Active
New 1970 sq ft 2 story home open floor plan. 3 bd 2 1/2 baths, HUGE MBR. 2 car attached garage. Alum Cliff Builders quality construction with warranties. Home delivery date approx mid Nov.
- Appliances Dishwasher, Garbage Disposal, Microwave, Other
- Area Butler-W15
- Cooling Ceiling Fans, Central Air
- Directions West On Millville Rd Out Of Downtown Hamilton, N On Smalley To Subject
- Exterior Features Deck, Porch
- Heating Electric
- Interior Features Cathedral Ceiling
- Parking Driveway, On Street
- Roofing Shingle
- Rooms 7
- School District Hamilton City SD
- View City, Park
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Welcome Home! Shows like a model. 3BR 2.5 bath, 2nd floor laundry, ,new appliances, cathedral ceilings, eat in kitchen-large fenced yard -great for entertaining, sprinkler system, HWH 10/18, extra parking pad. Updated windows. Close to Badin and Ridgeway Schools.Agent related to seller
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Contemporary tri-level w/mod touches. Desirable location. Open concept space opens to private yard. Backs up to park. Perfect for entertaining. Nat. light every room. Granite counters, ss app, gas FP, new roof, new tankless WH, NEST therm, new driveway.
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The Marion design by Cristo Homes. Ranch living with 3 bds, 2 full bath.The kitchen has a lg island perfect for quick meals and meal preparation. The Breakfast Room is large enough for a dining room table and leads to the deck.
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Over 3100 sq ft in this spacious Condo! Handicap accessible shower. Study or Craft Rm in Lower Level could be used for extra Bedroom. All appliances stay in this Gated Community! HOA incl: Water/Sewer, Trash, Snow Removal, Security Lighting & Gate, Termite Prevention, Maint, Landscaping, Roads...
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Completely renovated, this condo is move in ready! Gated community close to restaurants & shopping, condo features updated eat-in gourmet kitchen, all new SS appliances & granite countertops. New hardwood floors, carpet and freshly painted throughout; Master Bdrm with bay window & walk...
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Over 1800Sq. Ft. 3 Bedroom open Foyer Plan. Generous Bedroom Sizes. 2nd. Floor Laundry. Luxury Vinyl Plank on 1st. Floor. Large eat-in Kitchen with Stainless Appl. and Pantry. Cul-De-Sac Street.
105 Oxford Circle Hamilton, OH
3 beds, 2 baths, $223,000
Courtesy of: Paul Howe Realty, Inc.
Office Phone: 143
Listing Agent: Gregg Russell
Updated: 21st October, 2019 5:34 PM.
451 Smalley Hamilton, OH


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Living cells accumulate potassium (K+) to fulfil multiple functions. It is well documented that the model yeast Saccharomyces cerevisiae grows at very different concentrations of external alkali cations and keeps high and low intracellular concentrations of K+ and sodium (Na+) respectively. However less attention has been paid to the study of the intracellular distribution of these cations. The most widely used experimental approach, plasma membrane permeabilization, produces incomplete results, since it usually considers only cytoplasm and vacuoles as compartments where the cations are present in significant amounts. By isolating and analysing the main yeast organelles, we have determined the subcellular location of K+ and Na+ in S. cerevisiae. We show that while vacuoles accumulate most of the intracellular K+ and Na+, the cytosol contains relatively low amounts, which is especially relevant in the case of Na+. However K+ concentrations in the cytosol are kept rather constant during the K+-starvation process and we conclude that, for that purpose, vacuolar K+ has to be rapidly mobilized. We also show that this intracellular distribution is altered in four different mutants with impaired vacuolar physiology. Finally, we show that both in wild-type and vacuolar mutants, nuclei contain and keep a relatively constant and important percentage of total intracellular K+ and Na+, which most probably is involved in the neutralization of negative charges.
Potassium (K+) is a fundamental element for living cells. It is usually scarce in the majority of ecosystems and it is well known that it is required to fulfil multiple cellular functions, such as regulation of intracellular pH, compensation of negative charges in many macromolecules, or maintenance of cell volume, turgor or plasma membrane electrochemical potential [1–3]. In contrast, sodium (Na+) is the most abundant cation in natural environments, but its accumulation is toxic for most organisms. It has been reported by several groups that the model yeast Saccharomyces cerevisiae is able to grow in a broad range of external [K+], that the intracellular concentration usually ranges from 200 to 300 mM under normal growth conditions and that it decreases at limiting K+ or in the presence of high Na+ [2,3]. Therefore S. cerevisiae cells tend to accumulate K+ against high concentration gradients, and the main plasma membrane potassium transporter Trk1 plays a crucial role in the process [2,3]. Two recent genome-wide studies identified multiple genes from different functional categories involved in K+ homoeostasis highlighting the complexity of the process [4,5]. In addition, little attention has been paid to the detailed analysis of K+ and Na+ intracellular distribution in yeast. The subcellular localization of cations under different external conditions has not been definitively clarified, despite the fact that subcellular compartmentalization is critical for eukaryotic cellular physiology and, for example, it is generally accepted that vacuolar Na+ sequestration plays an important role in both in yeast and plant cells [6,7]. The existence of K+/Na+ transporters in organelles such as vacuoles and endosome/prevacuolar compartments [8–10] or mitochondria [11] reinforces the possible physiological importance of cation compartmentalization.
By using energy-dispersive X-ray microanalysis, it was reported more than three decades ago that the intracellular distributions of K+, rubidium (Rb+) and caesium (Cs+) were very similar, and that the concentrations of these ions in the cytoplasm were approximately equal to those in the nucleus and twice those found in the vacuole [12]. However, possibly due to the fact that the technique was not readily available for many groups or maybe because those experiments were performed under non-physiological conditions (prior to analysis, yeast cells were suspended in water for 1 day), the fact is that this work did not have the impact and significance that it may have had. The only relatively widely used approach to investigate the question of subcellular cation localization in yeast is the use of substances such as cytochrome c [13–15], DEAE dextran [16] or digitonin [17] to permeabilize the plasma membrane. This is a convenient and rapid method, but the main limitation of the procedure is that the results are too simplistic, since it usually considers only two important compartments where the cations are present in significant amounts; K+ extracted after permeabilization is considered the cytoplasmic fraction, whereas the remaining amount is ascribed to the vacuole. After using these procedures, most of the investigations conclude that cations accumulate in the vacuolar fraction to significantly higher amounts than in the cytoplasmic fraction [13,15,16], which is in contradiction with what was previously published by Roomans and Sevéus [12].
On the basis of the isolation of the main yeast organelles, we have optimized a procedure to determine the subcellular location of K+ and Na+ in S. cerevisiae wild-type cells harvested under several conditions. Moreover, we have applied the same procedure to four different vacuolar mutants. We conclude that while vacuoles accumulate most intracellular K+ and Na+, the cytosol contains relatively low amounts of theses cations. However, the concentrations of K+ in the cytosol are kept rather constant during the K+-starvation process and for that purpose vacuolar K+ has to be rapidly mobilized. We also found that this intracellular distribution is altered in four different mutants with impaired vacuolar physiology. Finally, we show that in all strains we have studied, the nuclei have and maintain a relatively constant and important percentage of the total intracellular K+ and Na+.
Yeast strains and culture conditions
The S. cerevisiae wild-type and isogenic mutant strains used in the present study and their genotypes are listed in Table 1. Yeast cells were cultured with shaking at 28°C. 'Normal K+ cells' were grown in YPD medium [1% (w/v) yeast extract and 2% (w/v) glucose], 'Na+-grown cells' were grown in YPD supplemented with 0.8 M NaCl, and 'K+-starved cells' were obtained by growing cells in synthetic K+-free medium [YNB-F (0.17% yeast nitrogen base without amino acids, ammonium sulfate and K+ (pH 5.8), Formedium™] supplemented with 50 mM KCl and incubating them for an additional 4 h in the same medium lacking added K+ [18]. Cells were generally harvested during the exponential growth phase at D600 values close to 1.0. In the case of nhx1 mutant grown in Na+, cells were collected at D600 values of 0.2–0.3.
|Gene function
|MATa his3Δ1 leuΔ0 met15Δ0 ura3Δ0
|PP2C type protein phosphatase
|vam6:: kan MX4
|Vacuolar morphogenesis protein
|Vacuolar Na+ (K+)/H+exchanger
|Prevacuolar–vacuolar Na+(K+)/H+exchanger
|Gene function
|MATa his3Δ1 leuΔ0 met15Δ0 ura3Δ0
|PP2C type protein phosphatase
|vam6:: kan MX4
|Vacuolar morphogenesis protein
|Vacuolar Na+ (K+)/H+exchanger
|Prevacuolar–vacuolar Na+(K+)/H+exchanger
Growth curves
To estimate the growth of the different strains, liquid YPD, supplemented or not with 0.8 M NaCl, was inoculated with yeast cells (D600=0.05) and growth was monitored for 25 h.
Isolation of main organelles from yeast
All centrifugation steps were carried out at 4°C in plastic tubes and all buffers were kept on ice. Protocols were adapted in order to use K+- and Na+-free buffers. Figure 1 shows the general workflow followed in these experiments.
Steps in the workflow for the determination of intracellular cation distribution
Isolation of vacuoles
Vacuoles were isolated as described previously [19,20] with minor modifications. Briefly, cells were grown, washed and treated with lyticase-100KU (2 mg/g of cells for 55 min). The spheroplasts were resuspended in 10 mM Tris/Mes (pH 6.9), 0.1 mM MgCl2 and 12% Ficoll, homogenized using a Dounce homogenizer and centrifuged at 26600 g for 60 min. The white layer at the top of the tubes, which contained most of the vacuoles, was collected and resuspended in the same buffer.
The vacuole protein concentration was determined using the Bradford protein assay [21]. For measuring V-ATPase (vacuolar ATPase) activity, we followed the procedure described by Cagnac et al. [9] with minor modifications. Vacuole protein (25 μg) was added to a buffer containing 50 mM tetramethyl ammonium chloride, 5 μM Acridine Orange, 5 mM Tris/Mes (pH 7.5), 3.125 mM MgSO4, 1 mM sodium azide (to inhibit F1F0-ATPase) and 1 mM sodium vanadate (to inhibit P-type ATPases) [22]. The V-ATPase activity was activated by the addition of 5 mM Tris-ATP, and time-dependent fluorescence changes were monitored using a fluorometer (SPECTRAFluor Plus, Tecan), with excitation and emission wavelengths at 485 and 535 nm respectively. The vacuolar activity was inhibited with the addition of 1 μM bafilomycin A. The V-ATPase activity was defined as the difference between the activities obtained with and without 1 μM bafilomycin A in the total homogenate and in the vacuole fraction.
Isolation of nuclei
The isolation of nuclei from yeasts was performed following the procedure described by Zhang and Reese [23]. Basically, the cells were grown, washed and treated with lyticase-100KU. The spheroplasts obtained were resuspended in sorbitol digestion buffer [1.4 M sorbitol, 40 mM Hepes-(NH4)2SO4 (pH 7.5) and 0.5 mM MgCl2], supplemented with 1 mM PMSF and 2 mM 2-mercaptoethanol, homogenized using a Dounce homogenizer and centrifugated at 21500 g for 30 min. The pellet was resuspended in digestion buffer.
To estimate the proportion of nuclei recovered, 100 μl of the nuclei suspension was diluted in 900 μl of digestion buffer and D600 was measured. The reading should be approximately 0.2 for 1 g of cells [23]. In addition, the number of nuclei was determined in spheroplasts and the nuclear fraction using minor modifications of the Prescott–Breed method [24]. Basically, the spheroplasts and the nuclei fraction was thinly smeared on microscope slides and equilibrated with PBS. DAPI staining solution was added and the slides were incubated for 5 min at room temperature (25°C). Background fluorescence was eliminated adding antifade reagent (Slowfade® Gold antifade reagent, Life Technologies). Samples were observed and nuclei counted using a fluorescence microscope with a Mercury-arc lamp (the excitation maximum of DAPI bound to dsDNA is 358 nm and the emission maximum is 461 nm).
Isolation of mitochondria
Isolation of mitochondria was performed following the procedure described by Zotova et al. [25] with some modifications. After lyticase-100KU treatment, the spheroplasts were centrifuged, washed and resuspended in 2.4 M sorbitol and 0.2 M NH4PO3 (pH 7.4). The spheroplasts were homogenized in a Dounce homogenizer. Mitochondria were recovered by centrifugation at 11000 g and the pellet was resuspended in the same buffer.
Mitocondrial protein concentration was determinated by the Bradford protein assay [21]. Cytochrome c oxidase activity was measured as described previously [26,27]. The oxidation of cytochrome c by cytochrome c oxidase was followed spectrophotometrically at 550 nm for 30 s, assuming ϵ1 cm=19600 per mol for horse heart cytochrome c [28]. The mitochondrial fraction cytochrome c oxidase activity was expressed as a percentage of the total homogenate activity.
Isolation of ER (endoplasmic reticulum)
ER was obtained by using the Endoplasmic Reticulum Isolation kit (Sigma) with some minor modifications. Spheroplasts were prepared and homogenized as described above [19,20]. The homogenate was centrifuged at 1000 g for 10 min. Then, the corresponding supernatant was centrifuged at 15000 g for 15 min. The resulting supernatant was the so-called post-mitochondrial fraction. This fraction was then precipitated with 7 volumes of 8 mM CaCl2 and centrifuged at 8000 g for 10 min. The ER partitioned to the insoluble fraction, which was resuspended in 10 mM Tris/Mes (pH 6.9), 0.1 mM MgCl2 and 12% Ficoll buffer.
The ER protein concentration was determined using the Bradford protein assay [21]. The ER NADPH-cytochrome c activity was determined using the Cytochrome c Reductase (NADPH) Assay kit (Sigma) and was expressed as a percentage of the total homogenate activity.
Isolation of the Golgi apparatus
The Golgi apparatus was isolated as described previously [29] with some minor modifications. Spheroplasts were homogenized and centrifuged, and the post-mitochondrial fraction was obtained as described for ER. A 1 ml aliquot of that fraction was placed on the top of a gradient containing 1 ml steps of 22, 26, 30, 34, 38, 42, 46, 50 and 54% (w/w) sucrose in 10 mM Hepes (pH 7.5) and 1 mM MgCl2. Gradients were centrifuged at 30000 rev./min for 150 min in a Beckman SW41 rotor. Fractions of 660 μl (12-drop) were collected from the top to the bottom. The sucrose density was measured with a refractometer (Atago). Protease inhibitors were added to the fractions.
Step 26 was enriched with Golgi apparatus protein. The Golgi GDPase activity was measured in vesicle fractions of the different steps in buffer containing 0.2 M imidazole (pH 7.5), 10 mM CaCl2, 0.1% Triton X-100 and 2 mM GDP; 100 μl of this solution, containing 20 μg of sample protein, was incubated at 30°C for 30 min. The reaction was stopped by transferring the tubes to ice and adding 10 μl of 10% SDS. To determine the amount of phosphate released during hydrolysis, 200 μl of ultra-pure water and 700 μl of Ames reagent (1:6 mixture of 10% ascorbic acid and 0.42% ammonium molybdate in 0.5 M sulfuric acid) were added to each tube; following incubation at 45°C for 20 min, absorbance was measured at 660 nm [30]. The Golgi apparatus fraction GDPase activity was expressed as a percentage of the total homogenate activity.
Cytosolic fraction
In order to obtain the cytosol, spheroplasts were homogenized and centrifuged, and the post-mitochondrial fraction was isolated as described for the ER. That fraction was centrifuged at 35000 rev./min for 60 min and then the supernatant was again centrifuged at 45000 rev./min for 150 min (in a Beckman 50.2 TI rotor). The resulting supernatant corresponded to the soluble fraction of the cytoplasm [31].
Cross-contamination activity
The vacuolar cross-contamination in the nuclei, and mitochondrial and ER fractions was determined fluorimetrically by measuring the V-ATPase activity [19,20]. The nuclei cross-contamination was estimated by using a fluorescence microscope to observe nuclei [24,32] and spectrophotometrically to determine the presence of nucleic acids [23] in the vacuolar, ER and mitochondrial fractions. The degree of mitochondrial cross-contamination in the vacuolar, ER and nuclear fractions was determined spectrophotometrically measuring cytochrome c oxidase activity [26,27]. The Golgi apparatus cross-contamination was determined spectrophotometrically measuring Golgi GDPase activity in vacuolar, mitochondrial, nuclei and ER fractions.
Plasma membrane permeabilization procedure
In order to permeabilize yeast plasma membranes, the procedure described by Okorokov et al. [13] was slightly modified. Cells were washed with 0.9 M mannitol, 1 mM Hepes, 5 mM MgSO4 and 10 mM CaCl2. To make the plasmalemma readily permeable to cations, the cells were treated for 15 min with 2% cytochrome c solution (0.9 M mannitol, 2% cytochrome c, 18 μg/ml antimycin, 1 mM Hepes, 5 mM MgSO4, 10 mM CaCl2 and 5 mM 2-deoxy-D-glucose). Permeabilized cells were recovered by centrifugation and the supernatant was kept (the 'cytoplasmic fraction'). Then vacuoles in the pellet were lysed by osmotic shock by adding 5 ml of water (the'vacuolar fraction').
K+ and Na+ measurements
To estimate internal K+ and Na+ content, cell samples were collected on Millipore filters, which were rapidly washed with 20 mM MgCl2. Whole cells [33] and subcellular fractions were then extracted with acid and K+ and Na+ were analysed by atomic emission spectrophotometry. Cation content in the different organelles was calculated taking into consideration the percentage of nuclei, vacuoles, mitochondria, Golgi apparatus and ER isolated. In all cases values are expressed as nmol of K+/Na+ per mg of dry weight cells.
Statistical analyses of the results
In all cases three to four independent experiments were used for every condition. Values shown are means±S.E.M. In some experiments, the significance of differences between mean values was determined by ANOVA using Tukey's test. A P value<0.05 was considered significant. Statistical processing was carried out using SPSS software.
Efficiency of the isolation procedure
Yeast cells obtained under several conditions were treated as described in the Experimental section in order to determine the intracellular cation distribution, efficiency of the isolation procedures and cross contaminations. As a first step, we checked that intracellular K+ was not lost during the spheroplasting procedure both in wild-type and vacuolar mutants (less than 5% of total K+ was lost in all cases). After the different organelle isolation steps, 91% (±8%), 40% (±8%), 27% (±3%), 71% (±6%) and 87% (±3%) of the nuclei, vacuoles, mitochondria, ER and Golgi apparatus were isolated and used to determine their cation content. Those percentages are comparable with what has been previously described in the literature when these organelles are isolated for different purposes [34–41]. Moreover our control tests to determine possible cross-contaminations showed that the mitochondria, vacuoles, Golgi apparatus or ER contaminating other fractions were usually below 5%. Owing to the isolation procedure, the Golgi apparatus fraction is practically free of other organelles [29]. Vacuolar, ER and mitochondrial fractions were virtually free from nuclei contamination since on the one hand, no nuclei were detected by microscopy and, on the other hand, spectrophotometric reading of nucleic acids gave values of approximately 0.001 for 1 g of cells compared with 0.2 in the control [23].
Taken together, these results indicate a low degree of cross-contamination in the different fractionation procedures.
Subcellular K+ distribution
Intracellular cation distribution was studied in wild-type yeast and vacuolar mutants. Our objective was to analyse a set of different mutants affected in vacuolar physiology. For that purpose, we selected two mutants lacking a cation transporter (vnx1 and nhx1) and, to determine whether possible changes observed were due to the ion transport activity of these proteins or to general defects in vacuolar morphology, we included two other mutants for comparison: ptc1 and vam6. These mutants also display alterations in vacuolar morphology, although these mutants are not expected to affect ion transport, the fact is that they are Na+/lithium (Li+)-sensitive [42–44]. All strains grew similarly in rich medium without potassium limitation (normal K+ cells) (Figure 2A) and although nhx1 mutants, lacking a K+(Na+)/H+ antiporter located in prevacuolar and vacuolar membranes [45–47], showed a slight delay in growth, both the growth rate constant and maximum biomass yield were similar to those obtained for the wild-type, vam6, ptc1 and the vnx1 mutant, lacking the K+(Na+)/H+ transporter in the tonoplast [9]. Under these growth conditions, total intracellular K+ content was determined in all of the strains. It is worth highlighting that the vam6 strain accumulated approximately 20% more K+ than the wild-type with highly significant differences (Tukey's test, P<0.001) (Table 2). Intracellular distribution of the cation in wild-type cells showed that the vacuole was the intracellular compartment containing more K+ (approximately 45% of the total). It is relevant that nuclei contained 29% of the total K+ and only a minor amount of this cation was present in the mitochondria, Golgi apparatus and ER. (Figure 2B). Finally, it is important to mention that when complex YPD or synthetic YNB-F plus 50 mM KCl were used, growth rates were comparable (0.27 and 0.24 h−1 respectively) (Figure 2A and [18]), as were intracellular K+ concentrations (571±38 and 564±47 nmol/mg respectively) and K+ distribution (results not shown).
Subcellular K+ localization in wild-type and vacuolar mutants
|Total intracellular K+ (nmol/mg of cells)
|Normal K+ cells
|K+-starved cells
|Total intracellular K+ (nmol/mg of cells)
|Normal K+ cells
|K+-starved cells
We applied the same isolation procedure in four different vacuolar mutants. We first checked that the alterations in vacuoles of the mutants did not lead to different organellar yields. In general, the total protein yield was 38–42 mg/g of cells in all strains, and in all cases 15–17% corresponded to vacuolar proteins. The fact that, for example, ER proteins were 11–13% of total protein in all strains reinforced the idea that cell organelle isolation outcomes in all of the mutants was sufficiently similar to wild-type to ensure that the comparisons were sufficiently robust. Although affected in very different processes, all of them showed altered distribution of K+ with decreased amounts of this cation in the vacuole and increased amounts in the cytosol. This redistribution was more relevant in the case of the two mutants affected in transport in which the cytosol accumulated even more K+ than the vacuole. For example, in the vnx1 mutant, we measured 37% of internal K+ in the cytosol and only 21% in the vacuole. Moreover, K+ in the nuclei, mitochondria, Golgi apparatus and ER was not greatly affected by these mutations, although we observed an increase in K+ in the mitochondrial fraction of the ptc1 mutant (Figure 2B). Importantly, we did not observe increased amounts of vacuolar cross-contamination of other fractions in these mutants, suggesting that vacuolar integrity was not compromised during the extraction process. Moreover, we examined the isolated vacuoles from each of these strains microscopically and did not observe significant differences in the morphology of the purified vacuoles, thus discarding an artefact or vacuolar fragility.
In a subsequent experiment, we prepared K+-starved cells. Cells were grown in YNB-F supplemented with 50 mM KCl. After 4 h of incubation in the same medium without added K+, all strains lost more than 50% of their intracellular K+ content (Table 2). Determination of the intracellular distribution of K+ revealed that vacuolar K+ spectacularly decreased in wild-type cells and that the percentage in the cytosol was 3.3-fold higher than in normal K+ cells, which means higher cytosolic K+ amounts under starvation conditions. Therefore it is conceivable that K+ was transported from the vacuole to the cytosol in order to fulfil cytosolic requirements. Finally, it is worth mentioning that the nuclei kept a similar percentage of K+ after starvation (Figure 2C).
The general behaviour described above was also observed in all of the mutant strains. However, the changes in the vacuolar and cytosolic distribution of K+ during starvation were much less relevant in the mutants, probably because of the impaired vacuolar physiology which probably leads to the observed decreased vacuolar and increased cytosolic K+ concentrations before the starvation process (Figure 2C). Figure 2(D) shows radar graphs illustrating the quantitative changes in K+ distribution after the K+-starvation process in the wild-type and the vacuolar mutants.
We next decided to use the wild-type strain to compare our results with those obtained with the most widely used procedure to determine intracellular cation distribution: plasma membrane permeabilization. This procedure is straightforward and less time consuming, but too simplistic since it focuses almost exclusively on vacuolar and cytoplasmic compartmentalization and tends to ignore the nuclear K+ content contribution to total intracellular K+. Our parallel experiments, in which the plasma membrane was permeabilized with cytochrome c, on the one hand fit with previous observations showing the higher content of alkali cations in vacuoles of cells grown without K+ limitation [13,16], since we measured 55/45% in 'vacuolar' and 'cytoplasmic' fractions of normal K+ cells and, on the other hand, confirmed the drastic mobilization of vacuolar K+ to the cytoplasm during the starvation process (28/72%) (results not shown).
Subcellular Na+ distribution
When the YPD medium was supplemented with 0.8 M NaCl ('Na+ grown cells') we observed that while the wild-type and vnx1 mutant grew similarly, vam6 and ptc1 were slightly more affected and nhx1 mutants lacking the vacuolar K+(Na+)/H+ antiporter presented a severe growth impairment (Figure 3A). Under these conditions, all strains accumulated significant amounts of Na+, close to the K+ values (Table 3). It is worth highlighting that the nhx1 mutant, the strain showing poorer growth in the presence of Na+, was the only one in which total intracellular amounts of Na+ were higher than those of K+. The next step was to analyse the intracellular cation distribution. Results in Figures 3(B) and 3(C) show that, also under these growth conditions, wild-type cells accumulated more cations in the vacuole than in the cytosol, and that this was much more significant in the case of Na+, which was kept at very low levels in the cytosol, suggesting a Na+-sequestering function for the vacuole. This function was also confirmed by parallel plasma membrane permeabilization experiments which indicated that approximately 60% of the total Na+ was kept in the 'vacuolar fraction' (results not shown). Very interestingly, nuclei contained both Na+ and K+ in not very different amounts, which was not the case for wild-type mitochondria, Golgi apparatus or ER, which contained lower percentages of Na+ than of K+. Once again, the four vacuolar mutants showed altered distribution of cations, containing less K+ and Na+ in the vacuoles and higher amounts of both cations in the cytosol. The ratio of vacuolar compared with cytosolic Na+ clearly illustrates the defective cation distribution in these mutants: while this ratio was 13.7 for the wild-type, in the different mutants it reached values from 2.2 (ptc1) to 0.7 (vnx1). Radar graphs of the Na+ content in the wild-type and in the vacuolar mutants are shown in Figure 3(D). These results show that while the wild-type maintains a virtually Na+-free cytosol, all of the mutants contain higher amounts of Na+ in the cytosol, being especially relevant in the case of the two mutants affected in transport (vnx1 and nhx1).
Subcellular K+ and Na+ localization in wild-type and vacuolar mutants
|Total intracellular K+ and Na+ in Na+-grown cells
|K+ (nmol/mg of cells)
|Na+ (nmol/mg of cells)
|Total intracellular K+ and Na+ in Na+-grown cells
|K+ (nmol/mg of cells)
|Na+ (nmol/mg of cells)
K+ and Na+ homoeostasis are well-studied processes in yeast and intracellular values of these cations in S. cerevisiae wild-type and mutants grown under different conditions have been frequently reported [3]. However, less attention has been paid to the subcellular localization of these ions. The most widely used approach to this subject is the use of several substances that specifically permeabilize the plasma membrane. As mentioned above, although this procedure seems to be too simplistic, several groups have reported higher concentrations of K+ in the vacuole than in the cytoplasm [13–15]. The results of the present study with wild-type cells grown under non-limiting K+ conditions fit with this idea. Moreover, our results show that cytoplasmic values obtained by using cytochrome c were significantly higher than cytosolic values obtained by isolating organelles in both normal K+ cells and K+-starved cells. These results suggest much higher contamination of the cytoplasmic fraction coming from other organelles when the cytochorome c permeabilization process was used. We also demonstrate that the presence of significant amounts of K+ in the nucleus cannot be ignored, as reported by Roomans and Sevéus [12]. Vacuoles are the organelles which accumulate higher amounts of K+ and, in contrast, cytosolic K+ is relatively low. However, the amounts in the cytosol seem to be very important for cell physiology, i.e. regulation of enzymatic activities [48], as suggested by the extraordinary mobilization of vacuolar K+ to the cytosol during the starvation process in order to keep the levels constant. An open question that remains is the substrate accumulated by the vacuole in K+-starved cells in order to keep osmotic potential since, in addition to the decrease in K+ content, cells lose high amounts of polyphosphates during the K+-starvation process (D. Canadell and J. Ariño, personal communication).
In the case of Na+-grown cells, the subcellular distribution of this cation was similar to what was observed for K+, that is, higher amounts in the vacuole than in the cytosolic fraction. However, in this case, Na+ levels in the cytosol were kept especially low, reinforcing the idea of Na+ as a toxic element.
The four mutants used in the present study lack very different genes, but all of them have impaired vacuolar physiology. It is of note that all of them showed altered intracellular cation distribution. The four vacuolar mutants contained higher amounts of K+ in the cytosol and lower amounts in the vacuole. Specifically, both nhx1 and vnx1 were less effective in accumulating K+ in the vacuole, and ptc1 and vam6 showed a less-severe phenotype, supporting a specific role for the cation transport activity of Nhx1 and Vnx1 in intracellular cation distribution and validating our experimental procedure. These results indicate defective vacuolar accumulation and the absence of a possible sensing mechanism which would regulate K+ transport from the external medium to the cytoplasm. In addition, the four mutant strains showed a different response to K+ starvation than the wild-type strain. Moreover, none of these strains were able to maintain the very low cytosolic amounts of Na+ observed in the wild-type, although their Na+ sensitivity greatly varied from one mutant to another. This behaviour illustrates the complexity of the targets of Na+ sensitivity. In our hands, the vnx1 mutant was not Na+-sensitive, which is in agreement with previous reports [9], and the ptc1 and vam6 showed moderate sensitivity. In this respect, it is worth mentioning that in two genome-wide studies, the growth of vam6 mutants was proposed to be unaffected [49] or to be very slightly inhibited [50] by NaCl. Finally, the most susceptible strain, the nhx1 mutant, accumulated more intracellular Na+ than the wild-type, the K+/Na+ ratio was the lowest and, in addition, an important percentage of this Na+ was in the cytosol, which most probably is linked to its sensitive character. At this point it is worth commenting that Nhx1 regulates membrane fusion of endosomal vesicles and acidification of cytosol and vacuole lumen [46]. In summary, although the underlying reason may be different depending on the mutation, the fact is that all of the mutants showed altered vacuolar cation content and problems to adapt to changes in external medium.
From a general point of view, the cation content in other organelles such as mitochondria, Golgi apparatus and ER was relatively low and constant under the different conditions used in the present study. We cannot completely rule out the possibility of some K+ loss during the cell homogenization in the case of the ER or Golgi; however, it seems to be reasonable to expect low amounts of K+ in these organelles and the results of the present study appear coherent with this idea and with the whole set of data obtained. The possibility of significant amounts of K+ in yeast nuclei has usually been overlooked, although Roomans and Sevéus [12] did mentioned it in their pioneering paper in 1976. We now show that nuclei from wild-type cells contain approximately one-third of the intracellular K+ and Na+ and that this amount is kept quite constant in all of the vacuolar mutants studied and is independent of the external conditions (K+ starvation or the presence of Na+). Apparently, it does not matter if the cell uses K+ or Na+, since in Na+-grown cells, both K+ and Na+ are present. Nuclear pores are big enough to allow K+ and Na+ leak; however, our measurements suggest that important amounts of these cations are found in nuclear fractions and it is tempting to propose that they can be used to neutralize part of the negative charges in this organelle, as has been previously proposed in mammalian cells where Na+ and K+ have been directly implicated in chromosome structure through electrostatic neutralization and a functional interaction with non-histone proteins [51].
Finally we would like to highlight that we have established a robust and optimized procedure to determine subcellular cation localization in yeast that can be used to analyse wild-type cells under several external conditions and many different mutants affected in cation homoeostasis that are not sufficiently understood at present.
Rito Herrera contributed to the experimental design, carried out the experiments and analysed the results. María Álvarez contributed to the design of the research, helped with the organelle isolation procedure and analysed the results. Samuel Gelis helped with the interpretation of the data, scientific discussion and writing the paper. José Ramos conceived and designed the study, supervised the project and wrote the paper with input from all of the authors.
We thank L. Yenush and J. Ariño for critical comments, D. Canadell and J. Ariño for sharing information on intracellular polyphosphates, and the Translucent Consortium for many useful discussions. We also thank S. Demyda, F. Calahorro, and the groups of M.M. Malagón and J.M. Villalba for technical advice.
This work is a part of TRANSLUCENT-2, a SysMo ERA-NET funded Research project and was supported by the Ministry of Science and Innovation, Spain [grant numbers BFU2008-04188-C03-03 and EUI 2009-04153 (to J.R.)]. R.H. is a recipient of a Panama Government fellowship (SENACYT-IFARHU).


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February 10, 2013, Nanaimo, BC (ISN) – Two members of British Columbia's Mid Island Velo Association were honoured at the 2012 Nanaimo Athletic Achievement Awards this Saturday.
National master women cyclo-cross champion, Janna Gillick was recognised as the city's outstanding master athlete and club president Peter McCaffery received the award for outstanding sports' administrator. A third cyclist who won the best male athlete plaque was international downhill champion, Steve Smith.


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Sid decides to sit and watch television all weekend long. There's just one problem with his plan: sitting around and not moving would make his body feel terrible. At school Sid and his friends learn that exercising is a really fun way to stay healthy! And it makes your body feel great! (Health/Exercise and Movement)
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