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How parents argue affects kids—sometimes in positive ways
Emily Glover
The nature of any relationship has ups and downs, since it's likely impossible to agree on everything all the time. Sometimes being a partner is more stressful than being a parent, so it's also inevitable for children to sense tension between their parents every now and then. And while conflict isn't inherently bad, how you manage it matters, especially in front of your children.
Research suggests we keep kids out of adult arguments as much as possible, and when an issue does come up, dealing with it in a constructive way can turn a negative into a positive for kids.
"The message is clear: Even low-level adversity like parental conflict isn't good for kids," says Alice Schermerhorn, an assistant professor in the University of Vermont's Department of Psychological Sciences and the lead author of a new study published in the Journal of Social and Personal Relationships.
But the one thing parents can do to avoid those pitfalls? Demonstrate how to reach a positive conclusion to the argument.
For the study, researchers divided a group of 99 pre-teens into two groups based on how much conflict they perceived in their parents' relationships. The kids were then shown pictures of couples having happy, angry or neutral interactions. They found that the children from low-conflict homes were correctly able to interpret the tone of the pictures, but the children from higher-conflict homes had trouble placing the emotions of the people in the neutral photos.
"If their perception of conflict and threat leads children to be vigilant for signs of trouble, that could lead them to interpret neutral expressions as angry ones or may simply present greater processing challenges," says Schermerhorn, adding the results also showed the shy children were more sensitive to these disputes.
However, there is one significant catch: Rather than pretend conflicts don't happen, if parents resolve heated discussions in ways that display positive communication skills, children can benefit.
"No one can eliminate conflict altogether," says Schermerhorn. "But helping children get the message that, even when they argue, parents care about each other and can work things out is important."
Another recent study found this is true even for very young children: According to findings published in the Journal of Family Psychology last year, constructive conflict management demonstrates calmness and respect in ways that toddlers pick up on.
"Not all conflict is bad—it's about how you manage it," says University of Arizona researcher Olena Kopystynska. "Given that children are going to encounter conflict out there in the real world, exposure to some conflict can be beneficial. However, it's really how parents handle that conflict that sets the tone for how safe children feel, and may further promote similar conflict management behaviors for when children are confronted with conflict of their own."
In fact, a 2009 study published in the Journal of Child Psychiatry and Psychology found the 5 to 7-year-old children of parents who argued constructively showed greater empathy toward their classmates during a three-year period.
As for how to achieve those constructive arguments, Ashley Davis Bush, LICSW, previously wrote for Motherly it may help adults to take three steps:
Diffuse your defensiveness: Think to yourself, "I wonder what pain is under their anger?"
Turn the flashlight to yourself: Ask, "I wonder why I'm feeling so agitated?"
Cultivate a sense of curiosity: Think to yourself, "I wonder if we can grow together through this?"
Just as children's spats with each other can actually be good learning examples, so too can be the disputes we parents have. The key is just making sure love and respect are central to the conversation—and if that's too hard, it's best to save the talk for after bedtime.
The science behind play-fighting—and why it's not all bad
Being kind vs. being right: 5 tips to fight fair and know then to surrender
New parents: Stop feeling guilty about fighting—it's normal
Emily is Motherly's Senior News Writer. She lives in Colorado with her husband and children.
This mom's plea for coffee in the hospital goes viral after her internet friends come to the rescue
Zara Hanawalt
Every parent out there knows that caring for a sick child isn't just heartbreaking, it's absolutely exhausting. Jaiden Cowley knows this all too well: The single mama's daughter, Amira, was born with a heart defect and has been waiting more than a year for a heart transplant.
Last week, when Amira ended up in the emergency room late at night for her ongoing heart issues, Jaiden reached out for a helping hand. She desperately needed a coffee to get her through the night, but she also couldn't leave her sweet baby to get one for herself.
So the single mother who moved to Toronto to be closer to its Hospital for Sick Children reached out to her virtual Mom Squad, a Facebook group that allows moms to connect. But she didn't expect the outpouring of generosity she received.
As first reported by TODAY, Jaiden posted the following message to the group, writing: "Is anyone at sickkids right now? I have a huge favor to ask. I'm in the er and I can't leave my daughter alone, but I really need a coffee."
She hoped another mom would coincidentally be in the hospital—she didn't expect a perfect stranger to go out of her way, but that's exactly what happened. A woman named Elizabeth drove to the hospital and presented Jaiden with a coffee half an hour later.
"To some it was just a simple cup of coffee, to me her bringing it to me meant I could stay awake and alert for my daughter. I was able to properly advocate for her," Jaiden tells Motherly.
TODAY on Instagram: ""I'm in the ER and I can't leave my daughter alone, but I really need a coffee." What happened next will make your day. Full story at the…"
Elizabeth wasn't the only amazing mama who came forward to help: Another reportedly sent Jaiden money so she could treat herself to coffee, and several others offered to help in any way possible.
"It meant so much to me," Jaiden tells TODAY. "Going through this as a single mom has been a lot. It's exhausting. But now I don't feel so alone."
She later told Motherly: "No act of kindness goes unnoticed. No matter how small you think it may be to that person it means the world."
We couldn't love this story more! It's such an important reminder that mamas can do amazing things when they help each other out in times of need. We all need a little support sometimes, and if this story is any indication, there are still incredibly kind people out there who are willing to offer it.
It's science: There's a connection between mama's support system and baby's brain development
Moms, we need each other
What went viral this week: A triplet who had triplets, a meteorologist's clap back + an awesome tweet
mom squad mom coffee mom friends moms helping moms mom support support support for moms supporting mothers
Tia Mowry co-slept with her kids for years—because it was the right choice for her family
[Editor's note: This article describes one parent's experience with bed-sharing. To learn more about the American Academy of Pediatrics safe sleep recommendations please visit the AAP.]
Raise your hand if you've ever found yourself asleep with your child next to you in bed. (🙋🏽♀)While the American Academy of Pediatrics recommends room-sharing, they discourage bed-sharing, particularly in the first four months of a baby's life, due to safety concerns.
But the reality is that many parents fall asleep with their babies next to them in bed. Whether it's because your baby won't sleep without those cuddles, because you've drifted off while nursing, because you didn't have the heart to put a sick baby in their crib, or because your doctor has given you the okay to snooze alongside your babe, bed-sharing is very much a thing.
And Tia Mowry is getting real about her experience with it.
When asked about her most "non-traditional" parenting move, Tia shared that she's a big-time bed-sharer. "My 1-year-old [daughter, Cairo] is still in my bed," the actress said during an interview with PEOPLE. "Ever since she was born she was always in our bed." But this isn't her first experience with co-sleeping: Tia also shared that she slept with her son until he was 4 years old.
Tia is hardly alone when it comes to sleeping with her kids. A 2016 study found that only about 44% of survey responders never slept with their babies in bed with them—and that those who slept with their babies were more likely to keep breastfeeding for the recommended six months. Fellow celeb Kourtney Kardashian is a co-sleeper, and many mamas find that while they didn't plan to co-sleep, it is what works for them. That's why there are even special co-sleeping beds big enough for parents and kids.
But as popular as co-sleeping is, it can still be seen as controversial. Even Tia's own mom isn't on board with the Sister Sister star's decision to bed-share with her kids. "[My mom is] like, 'You need to do the cry-out method. Put your baby in the crib. And I'm like, 'No!' I don't want my baby to have any sign of stress whatsoever," Tia explains.
Whichever side of the line you fall on, one thing is clear: Sometimes parents need to do things they never expected to do in the name of more sleep. When it comes to parenting, there's only one absolute: You have to do what keeps your family safe, healthy and happy. And while we'd urge all mamas to familiarize themselves with child safety guidelines, ultimately we all have to make the choices that are best for our families.
We need to stop shaming moms for co-sleeping—and research shows why
True life: I never thought I'd co-sleep, but it's what works for my family
One mama's scientific case for co-sleeping
co-sleeping co-sleeping with newborn co-sleeping with toddler tia mowry bed sharing sleep sleeping with baby celebrity moms celebrity parents
Not all breastfeeding babies with tongue-ties automatically need to get surgery, says new study
Many new parents receive the confusing news that their newborn baby has a tongue-tie. It means the skin attaching their tongues to the bottom of their mouths is longer than normal. And while this condition can cause complications from infancy on, a new study shows that not all newborns need to get surgery to correct it.
The condition is known as ankyloglossia, which occurs in 4-10% of people, usually looks like nothing but an extra strip of skin under the tongue. But because that skin (called the lingual frenulum) acts like a taut rubber band restricting movement, babies with a tongue-tie often have difficulty forming a good latch to nurse. This can mean they don't get enough milk, so they have to nurse for longer. Meanwhile, they're causing their mother a whole lot of pain because their latch is shallower and mostly clamping down on the tip of the nipple.
I speak from experience here: When my son had a tongue-tie, it felt like I was feeding an angry piranha. He was definitely not getting enough to eat, and my milk supply was steadily decreasing.
But this new study published in JAMA Otolaryngology Head & Neck Surgery suggests that not all babies diagnosed with ankyloglossia need to undergo a frenotomy—a simple procedure in which a doctor snips the skin with a pair of surgical scissors.
Infants don't usually need anesthesia, because this tissue doesn't have many nerve endings or blood vessels. Their mother is asked to breastfeed them immediately after the snip, to get the tongue moving properly and reduce the chance of the skin growing back. According to the Mayo Clinic, complications from frenotomies include bleeding, infection and damage to the tongue or salivary glands, but they are rare.
What's not rare is the number of patients getting frenotomies: Referrals for the procedure in the U.S. went up tenfold, from 1,200 in 1997 to 12,400 in 2012.
"We have seen the number of tongue-tie and upper lip tether release surgeries increase dramatically nationwide without any real strong evidence that shows they are effective for breastfeeding," study co-author Christopher J. Hartnick, MD MS, of the Massachusetts Eye and Ear Infirmary, said in a press release.
The researchers looked at 115 infants (between 19-56 days old) who were referred for a frenotomy. Instead of sending them straight into surgery, the babies and their parents met with a pediatric speech-language pathologist for a feeding evaluation. These specialists observed the babies breastfeeding and gave parents feedback and tips to overcome any challenges they were experiencing. After this, 72 (62.6%) patients did not have the frenotomy after all, while 10 (8.7%) had a labial frenotomy (releasing extra tissue from the lips) and 32 (27.8%) had both a labial and lingual frenotomy.
"We don't have a crystal ball that can tell us which infants might benefit most from the surgeries, but this preliminary study provides concrete evidence that this pathway of a multidisciplinary feeding evaluation is helping prevent babies from getting this procedure," Hartnick said.
For now, parents' best bet is to consult more than one specialist to identify the best plan of action. In addition to lactation consultants, children with tongue-ties might need to see speech pathologists later. In some cases, the frenulum loosens over time. In others, they might wind up needing the procedure after all.
Anecdotally, I'll add that I visited with lactation consultants and my son's pediatrician more than once before deciding he should have a frenotomy. It was no fun for me (who wants a strange man sticking scissors in their baby's mouth?), but my kid was fine. It didn't solve all our problems, but feeding was much less painful immediately afterward.
The bottom line here seems to be that not everyone needs to rush into a procedure just because it's easy. Our kiddos deserve more than a one-size-fits-all approach to their health.
What's a tongue tie or lip tie in babies?: Here's what to know—and do
4 experts on the 1 piece of breastfeeding advice new moms need to know
The early weeks of breastfeeding are exhausting—but fleeting
baby tongue tie tongue tie breastfeeding tongue tie ankyloglossia breastfeeding frenotomy


9745 Touchton Rd 522 has a Walk Score of 41 out of 100. This location is a Car-Dependent neighborhood so most errands require a car.
Beautiful condo in Il Villagio!!! 2 story condo with 2 master suites upstairs with 2.5 bathrooms with wood floors and private screened patio. Living room upstairs 2 bedroom all hard wood floors. Great Amenities including guarded gate. Strictly no pets per HOA.
Explore how far you can travel by car, bus, bike and foot from 9745 Touchton Rd 522.
9745 Touchton Rd 522 has some transit which means a few nearby public transportation options. Car sharing is available from RelayRides.
9745 Touchton Rd 522 is in the Windy Hill neighborhood. Windy Hill is the 83rd most walkable neighborhood in Jacksonville with a neighborhood Walk Score of 31.


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Le Cycle épique des Taïra et des Minamoto est une collection qui rassemble la traduction de trois œuvres majeures de la littérature japonaise, Le Dit de Hôgen (Hōgen monogatari), Le Dit de Heiji (Heiji monogatari) et enfin Le Dit des Heike (Heike monogatari). Ces traductions sont l'œuvre du célèbre japonologue René Sieffert. Elles ont été éditées en deux ouvrages aux Publications orientalistes de France, plus connues sous le nom de P.O.F.

Le Cycle épique des Taïra et des Minamoto a été réalisé entre 1976 et 1993. Il représente un travail de traduction considérable qui aura duré plus de quinze ans.

René Sieffert offre là au lecteur francophone les œuvres qui sont au fondement de la littérature japonaise et qui ont joué un rôle fondamental dans l'évolution de la langue, des lettres et de la civilisation du Japon dans son ensemble.

Sources
Le Dit de Hōgen, Le Dit de Heiji, trad. René Sieffert, Publications orientalistes de France, rééd. Verdier/poche.
Le Dit des Heike, trad. René Sieffert, Publications orientalistes de France, rééd. Verdier/poche.

Liens externes
Le Dit de Hôgen et Le Dit de Heiji
 Le Dit des Heiké

Littérature japonaise ancienne

redpajama_set_name: RedPajamaWikipedia


I am trying to load the map part of a saved game into memory (SGAME0.OP2). Saved game files contain some header data not present in map files. I don't really need the info in the saved game header, so it could just be ignored, or loaded into a variable.
When I load a map file, the code works fine. When trying to load a save file, it appears the map header data isn't being loaded properly. I'm assuming that my ignore commands are not placing the ifstream at the proper position to start reading the map data, but I'm not very familiar with doing this.
Your math is wrong. Read the documentation again. The structure after the header is an array of 0x74 elements with two structures each of size 0x1E0, not a single structure of size 0x74.
And now, all the map data is reading in properly.
I see my comment in the other thread was superfluous. Glad to see you got this working.
I ended up using HXD to determine the offset of 0x1E025 for loading the map data from the save file. You can basically search for the width and height of the map which appears near the beginning of the map portion.
I cannot get the math to work to get 0x1E025 from the structure sizes listed in the file snippet earlier in the thread.
If someone has time to break it down for me for my own knowledge on the subject, it would be appreciated.
You're right, the math doesn't seem to work out. Looks like an error in the description of the file format.
I've updated the text file describing the saved game and map file formats. It was missing a section in the header of saved game files. I also filled out some details on the meaning of the previously unknown fields.
Note: Technically each object loads data through a virtual function call, so each object could load a different amount of data. However, there are no overrides, so they all call the same base class function, and hence all load the same data.
Since I'm new to HXD and memory offset, I was worried the problem would be that I didn't know how to sum a range of hexadecimal values (at least after sirbomber's first correction).
Good to see someone break my dozen long commit streak into the repo.


We live in crazy, busy times. We're always rushing and we want to do it all. A career and a family need to fit our schedules and we are led to believe that nothing ever suffers as a result. I'm not criticizing anyone for wanting to have a comfortable lifestyle, hey, I like nice things too. But it's hard not to see that for my generation of young parents the upbringing seems to happen somewhere beside and in between the daily things that distract us. The pressure is great and the drive is enormous and sometimes you can't even afford to do it any other way. Kids are raised by video games and TV's. I've met children who played Warcraft before they could read. But they get by. Some parents can't really afford to adopt any different kind of lifestyle and work hard just to make ends meet.
My mom quit her job when I was born, but it was a different time. Things seemed to have been flowing a little slower back then. It was easier to make it, although my father did end up getting a second job. In communist Poland it was pretty common for the mother to become a stay at home parent with a disabled child. Mine decided it she needed to to make sure I'd get all those hours of exercises in a day, extra care and attention and that my brother could also benefit from her presence. My parents gave up, changed their lives and goals for me. Altered where they wanted to be and what they wanted to accomplish. I sometimes wonder if that kind of approach would be possible today. Disability isn't something that you can schedule. That you can pencil in between your tasks. I've met parents who give their kids maybe two, maybe three hours of physical or occupational therapy a week. And even that becomes a problem. Getting there, driving, making it work.Now, I'm not really a critic of these approaches, but as with everything you need to make it a continuous everyday commitment for it all to have any kind of benefit. Doing it twice a week, if that's all you do, probably it is not going to work. I see it with our Conductive Education program too. For some parents, scheduling and transportation becomes a really big issue and they end up attending our Academy sporadically. They can't make it every day. And yes, the children suffer as a result. Perhaps something is better than nothing, because there is no other way, what can you do? Disabilities seem incompatible with modern world.
On the other end our Summer Camp is always busy. Parents want to do something that will help their children, that will perhaps make up for an entire year of doing very little. Because it's easier to commit to something short term. It's doable, workable to do it for six weeks out of a year. Is it the same as doing it every day for the other ten months? Of course not. That is the tragedy of our times. Not to say that these people are bad parents. Some barely make it from day to day. Some are poor and uneducated, some struggle as single parents. As much as you want to help your child you can only work within the means you have. America doesn't have a social and healthcare system developed in a similar fashion we so in Europe. And yes, it's often criticized, may be ineffective and will probably go bankrupt at some point. But right now it could really help those parents. Conductive Education, the method we use is not well recognized by the medical community, it's not covered by insurance and we don't offer transportation. Those parents are on their own.
One thing I will say for mine. We didn't have much, but they always found a way to make it work, be it extra jobs for my dad or some work in the West. I do think part of it was mentality. My rehabilitation was more an adopted lifestyle than just something we did for an X hour a week, which what it seems like today. In, out, you're done and on to the next thing. Somebody else does it for you. The sad thing is, disability needs that extra time, that extra care and some things you neglect you may never make up for.


ANAHEIM, Calif., March 6, 2019 /PRNewswire/ — Flow Alkaline Spring Water, the premium alkaline wellness water brand, launches unique augmented reality mirrors as part of their innovative in-store demonstration program. The technology, powered by memomi , will be the first of its kind in the grocery space.
The experience will bring to life Flow's summer campaign of the "Superpower of Alkalinity" and help allow consumers tap into their own super powers. The interactive AR experience with customized memomi Memory Mirrors® will track the customer's face to add super power illustrations that coincide with Flow's original alkaline spring water or one of the brand's six organic flavors to create an immersive experience that can then be shared on social media.
At in-store demonstrations, the team on the ground often takes photos and records the customer's thoughts on the product and it's been a source of data and inspiration for Flow. The brand wanted to deepen that experience for our customer and add a new element of excitement into an otherwise routine trip to the grocery store. At the same time, the brand can learn more about our customer, listen to their needs, share offers and invites, and better engage with them.
The technology will be revealed at the 2019 Natural Products Expo West in Anaheim, CA this March and will launch in participating grocery stores with Flow product demonstrations in May 2019.
Flow naturally alkaline spring water is of the highest quality and is sourced from a family-owned artesian spring in South Bruce County, Ontario, Canada. The water comes from a limestone aquifer where it naturally collects healthful, essential minerals like Calcium, Magnesium and Potassium that give the water an alkaline pH of 8.1 to keep the body in optimal hydration. Flow's water comes straight from the earth into the package, no industrial processing necessary.
Flow recently launched the first organic flavored premium alkaline water to the market. The 100% naturally alkaline certified-organic flavor options are ideal for consumers looking for optimal hydration and amazing taste. Six refreshing flavors are now available: Cucumber + Mint, Lemon + Ginger, Strawberry + Rose and Watermelon + Lime, and new for 2019, Grapefruit + Elderflower and Blackberry + Hibiscus.
For more information on Flow visit flowhydration.com.
Interviews and photography available upon request.
Flow is the fastest growing global wellness water, providing naturally alkaline spring water while maintaining responsible business practices including sustainability and social good. The B-Corp Certified brand was founded in 2015 by entrepreneur Nicholas Reichenbach. Flow is mindfully sourced from the founder's family-owned artesian spring in South Bruce County, Ontario; a natural source virtually untouched by man or machine. Flow is uniquely packaged in a Tetra Pak® paper carton made from nearly 70 percent renewable resources. Due to its unique origins, Flow's water is filled with naturally occurring electrolytes, essential minerals, and an alkaline pH of 8.1. Flow is available in over 15,000 retailers across North America and the United Kingdom including Whole Foods Market, Sprouts Farmers Market, Safeway, Bristol Farms, Earth Fare, Central Market, Jewel Osco, Gelson's, and Kowalski's in the US; Whole Foods Market, Loblaws, Sobeys, Metro and Farm Boy in Canada. For more information on Flow Alkaline Spring Water, please visit flowhydration.com, or follow Flow on social media; Instagram and Twitter and facebook.com/FlowHydration.
Memomi is the creator of the Memory Mirror® an award-winning Augmented Reality and Artificial Intelligence platform that transforms the in-store and online experience by enabling customers to try products virtually and get recommendations based on profile, style and preference. HQ in Silicon Valley, R&D in Israel, Offices in Paris and Tokyo. The Memory Mirror® Platform runs on iOS, Web and Android and allows multiple use cases in a single device. Memomi currently supports fashion, beauty, haircare, eyewear, jewelry, accessories brands and more and is trusted and used by LVMH Group, L'Oréal,Estee Lauder, Luxottica , Neiman Marcus, Berluti, Sephora, Tom Ford and more.


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https://www.sfgate.com/politics/article/Dean-leads-Democrats-in-California-Field-Poll-2600801.php
Dean leads Democrats in California Field Poll / President's numbers 'have begun to sag'
Carla Marinucci, Chronicle Political Writer
Published 4:00 am PDT, Tuesday, July 22, 2003
7/22/2003 | B/W | 5star | 4p11x7p6 | A5 | A-Section | Warren, x6169 | DEAN
Photo: KELLY WEST
Presidential candidate Howard Dean -- buoyed by Internet campaigning and strong word-of-mouth among grassroots voters -- has rocketed to the top of the Democratic pack in crucial California, a new Field Poll released Monday showed.
The former Vermont governor has more than doubled his support since April, to 16 percent among likely Democratic voters. Dean is closely followed by Massachusetts Sen. John Kerry with 15 percent, and Connecticut Sen. Joe Lieberman -- touring the Bay Area Tuesday -- with 14 percent. Missouri Rep. Dick Gephardt -- who plans a major foreign policy speech before the Bar Association of San Francisco today, has only 7 percent of Democratic support in the state, while North Carolina Sen. John Edwards posted the lowest showing of any of the major candidates, with just 4 percent of the party's support.
MOSELEY BRAUN AT THE BOTTOM
Among Democrats considered to be second-tier candidates, Ohio Rep. Dennis Kucinich is tied with Florida Sen. Bob Graham and the Rev. Al Sharpton with 3 percent, and former U.S. Sen. Carol Mosley Braun posted 2 percent.
But the latest poll numbers for President Bush suggested he too may have work ahead in the nation's most populous state. Just 46 percent of the voters of California were inclined to re-elect the president, the poll showed -- numbers that appear to mirror those of his father in the months leading up to the 1992 election.
"After six months of campaigning, the Democratic race is more unsettled than it has ever been," said Field Poll Director Mark DiCamillo.
BAD NEWS FOR EDWARDS
But the numbers are "not a good news poll for those who are not in the top tier," particularly Edwards, who "hasn't broken through at all," said DiCamillo.
Dean's rise to the top of the Democratic pack here is due to his position as "the anti-Bush candidate . . . at least in the top tier," said DiCamillo. "Dean has caught on in a way that is more appealing to people . . . but the big tipoff is where his support is coming -- the liberal wing, what he calls the Democratic wing of the Democratic Party. He has pretty much staked out the left side of the party . . . and it's not a bad play to start out from."
Lieberman's decline -- from 22 percent in April to 14 percent in July -- is due to "the function of higher name recognition he brought to the race," said DiCamillo. "Voters were familiar and fairly comfortable with him early on. But as the other candidates get better known, it becomes a more competitive arena."
On the GOP side, Bush's approval ratings "have begun to sag," and his re- election figures are stagnating, said DiCamillo. He said the current numbers show that voters' preferences are "much more wide open" than just three months ago. Then, "Bush looked so strong, and looked like he would carry California," he said. Now "it's a more wide open field on the Democratic side, and the president looks to be more vulnerable here."
The latest Field Poll was conducted July 1-13 statewide by telephone, of 1, 040 California registered voters. The margin of error is plus or minus 3.8 percentage points.

redpajama_set_name: RedPajamaCommonCrawl


text:
Home Technology Honor 10 is official and it also has a notch
Honor 10 is official and it also has a notch
Weeks ahead from its launch in the UK and other global markets, Huawei has made its upcoming midranger, Honor 10, available in China.
The newly unveiled Honor 10 smartphone from China seems to be a less-costly version of Huawei P20 which is the most recent flagship smartphones from the company. The first thing that strikes the user is definitely the design of Honor 10 which closely resembles to that of P20. The smartphone comes with the same shiny color gradient design which has already earned the interests of the smartphone experts and critics from around the globe.
Also Read: Huawei sold $15 million worth of P20 series smartphones in just 10 seconds
The fingerprint scanner, however, has been moved to the front of the smartphone and now sits at the top of a home button. On the front, the smartphone packs an iPhone X like notch to house important sensors. There are several similarities under the hood too. Honor 10 comes with Kirin 970 chip and 6GB of RAM. As far as the camera section on this smartphone is concerned, the specifications look promising. The smartphone has a dual-camera setup at the back with 24-megapixel and 16-megapixel lenses. The smartphone also has a 24-megapixel front-facing camera for selfies.
As far as the pricing of this smartphone is concerned, Huawei Honor 10 (with 64GB of internal storage) has been made available for 2,599 yuan in China which translates to $414 (Pakistani Rupees 48,000). The price increases for the 128GB version which can be bought for 2,999 yuan (Pakistani Rupees 55,000). The smartphone is expected to release in the UK and elsewhere around 15 of May.
Image — TR
Huawei International News
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Julia Park Tracey
Writer | Editor | Poet
Amaryllis: Collected Poems
I've Got Some Lovin' To Do: The Diaries of a Roaring Twenties Teen
Reaching for the Moon: More Diaries of a Roaring Twenties Teen (1927 – 1929)
Tongues of Angels
Veronica Layne Gets the Scoop (Hot Off the Press, #1)
Veronica Layne Has a Nose for News (Hot Off the Press, #2)
The Cause of Conscience: Go Set a Watchman (Book Review, Part 1)
(WARNING: Spoilers)
I have never read To Kill a Mockingbird. This is cause for alarm among you many literate people, but you needn't think me unlettered. In high school I read Poe, Shakespeare, JD Salinger and Carson McCullers; lots of plays, many short stories, and certainly some fat summer beach reads, too (The Thorn Birds, Jaws, Roots, Shogun and Hotel, to name a few). In college I majored in journalism and had no cause for deep American reading, and no affinity for Southern lit, but I did read Flannery O'Connor and William Faulkner. I went on to study early 20th-century British literature for my master's degree, and drank my fill of Woolf, Joyce, and Orwell. I still read scads of early and mid-century Brit lit. It's my crack. But I didn't have Harper Lee on my radar for pleasure reading until now.
I'll add a caveat to my preamble that I realize there is an issue with the release of Lee's first draft/second book. I have read her biography and understand the nature of her friendship with Truman Capote, her anxiety about meeting people and the "sophomore slump" that often hits young, successful authors who feel unequal to producing as fine a work again. I also recognize that there are claims of elder abuse and mixed opinions about the role of Lee's sister as protector or as censor. History will judge those who abused Lee's trust, I'm sure.
With all that in mind, I set out to read Go Set a Watchman before reading TKAM, in hopes of seeing which felt the more true. GSAW was written first, as the earlier draft of TKAM, so they say, but her agent and editor did not think it would sell. Lee rewrote the story from the young Scout's viewpoint and had a bestseller on her hands. She eventually won the Pulitzer Prize for her work. That's pretty heady stuff.
"For thus the Lord said to me: "Go, set a watchman; let him announce what he sees." Isaiah 21:6
It is but a quirk of fate that in the past month, there was an assassination of nine African-Americans in Charleston, SC, and the Confederate flag has at last begun to come down. The #BlackLivesMatter and #Occupy campaigns have raised the African American issue in the media — and a new generation of activists are tired of waiting for justice and equality. As well, in the past few months I have been engaged in family history research and have been grappling with my own Alabama roots and heritage of slave-owning in my DNA. White privilege? Yes. So much yes.
I opened Go Set a Watchman with virgin eyes and no idea other than generalities on what to expect. And was — riveted.
Scout as a young woman — Jean Louise — is New York City-cynical, streetwise, on vacation to Maycomb, Alabama. Her brother is dead. Her beau, childhood friend Hank, is on hand to woo her back to small-town life. Attorney Atticus Finch, whom she calls by his first name, is her father, crippled by arthritis, a longtime widower living with his pushy Southern matron sister, Alexandra. Adult Jean Louise is a sexual being, pushing the boundaries of Southern small-town mores by behaving like the child Scout; a late-night swim with her beau creates a furor. She's also enlightened, having crossed the border into Yankee territory and lived and worked alongside a rainbow of skin-tones. She returns to find Maycomb charmingly simple, the same as always, but smaller in size and in their way of thinking. The problem isn't that home has changed; it's that she has changed.
The era is the 1950s and the Civil Rights movement is underway, with integration and the like on the agenda. The whites of Maycomb don't like it, and their "citizens' committee" looks a lot like the KKK. The N-word is thrown around with vitriol and Jean Louise is alarmed — she thinks of herself as color-blind. There is an incident whereby a white neighbor is accidentally killed by the grandson of Calpurnia, Jean Louise's African American surrogate mother — their housekeeper as she and Jem were growing up. When Jean Louise comes face to face with a grieving Calpurnia, the young woman knows everything is different, that black and white are not the same, at least not down in Maycomb. And when she discovers her father and fiance in a citizens' committee meeting, she realizes her whole life has been a lie.
I will say no more about the plot — too much already, perhaps. But to turn to the Atticus problem: I am familiar with the figure of righteous Atticus waving his figurative sword at prejudice and racism, despite not having read TKAM or seeing the entire movie. He shows up as a cultural touchstone in films, books, essays and news articles. The Atticus I find in GSAW is an aging man of his era; he was steeped in racism in small-town Alabama from his youth, and whatever his personal beliefs, the tint of the standard philosophy is going to seep in. I read his protests to adult Scout as plausible — he's at the meeting to keep an eye on things. He's in the group but not of the group. Hank is there because he wants to succeed in small town life; it would be social suicide not to participate — plausible today but frankly, reprehensible. Atticus is a character with his own moral compass who's dragging his feet into the new era; he is less obstructionist and certainly less vocal and violent about it than his sister or the neighbors. Is he a good guy? A bad guy? He's a racist — but they all are. What I don't follow is Dr. Jack Finch, Scout's uncle (Atticus's brother), teaching a brutal lesson fraught with bizarre classical literary references, to Jean Louise on hero-worship. That final episode seems overwrought and histrionic in the extreme. Dr. Finch is the oddball in the bunch, acting as Greek chorus to show Jean Louise her errors.
What is hard to read, especially in light of current events — and as I said above, I read a lot of novels from the early 20th century, weekly, in fact; I am used to seeing non-PC expressions like "nigger in the woodpile" or "worked like a nigger" in those dated texts — what strikes me as much harder to swallow is the infantilization of the African American race as a whole by the characters, including Jean Louise, that continues today. "The Negroes down here are still in their childhood as a people…They're [the NAACP are] trying to wreck us," Atticus says. While Jean Louise argues for a race's humanity, all the characters accept the belief that the Negroes have miles to go before they sleep, even as the population is being oppressed and prevented from enjoying their civil rights (no wonder they have so far to go). I see this attitude still alive in the world today; African Americans are "that way," are "like that," fill in the blank as you wish. They are Different from white people, and don't know better, and have to be protected from themselves (cue talking heads nattering about black-on-black violence instead of the centuries of causes for violence in a shell-shocked community). And the stratification endures.
As a text, as fiction, Lee's work is stellar. Jean Louise is a thoughtful character, sympathetic and well drawn. The scenes where African American characters have agency are few and far between, despite a story that purports to have their interest at heart, but what is there is sharp and very keenly felt. Calpurnia calling Jean Louise "Miss Jean" for the first time upon the girl's menarche, or Calpurnia's silence when Jean Louise arrives to commiserate over the tragedy, speaks volumes about the strict racial divide. Hank's unsuitability as a mate for Jean Louise, the class division of this same society, is portrayed in the subplot. Setting, language, dialogue, plot and characters are beautifully crafted for a first novel, and I am in awe of Lee's youthful skills.
Where the book fails for me is the denouement, the confrontation between Scout and her father. I don't think it works. The strange tutelage of Uncle Jack feels dropped in from another planet. I have trouble following his Red Queen logic, and so does Scout, for that matter. And I certainly don't buy the neat reconciliation that ends with Jean Louise remembering not to bump her head on anything difficult in life, like racism in her town, or her beau's KKK connection.
Ophelia has thoughts on being black and white, but prefers to leave it to the reader. #catsofwordpress
I have not yet cracked the spine of TKAM. I look forward to reviewing that story, and then, comparing and contrasting the two, side by side, as an author's oeuvre and as documents of our national racist heritage.
Your thoughts? Tell me.
Because I Haven't Known What to Say
Because the events of the past week — the horrific shooting deaths of nine African-Americans in a Charleston church by a young white racist, and the — maybe — final straw that will bring down the Confederate battle flag, and bring the longed-for change, I am trying to say —
Because when we were children, in the extremely white liberal suburbs of Marin County in the late 1960s, we used to say, "Eenie meenie miney mo, catch a nigger by the toe," called Brazil nuts "nigger toes," and when someone asked, "Where'd you get that?," the response was, "Stole it off a dead nigger."
Because the one African-American girl in
my elementary was so beautiful, but so different from me, and the time she invited me to sleep over, I felt so strange at being the only white person in the house that I never slept over again.
Because when I was in junior high, we watched "Roots" on TV and saw the story of slavery in America, and then named our black cat Kunta Kinte and my sister's sheep Kizzy.
Because the most strikingly odd groups at my white high school were the exchange students from Germany and Norway, or the handful of punk rockers dyeing their hair blue or green in 1979.
Because I never talked to the one African-American boy in our class, and to this day I still don't know his name.
Because my father still says things like "black as the ace of spades."
Because as a young adult, although I was beginning to meet people of color, of all colors, I still used to say that Richmond (CA) was where all the black people lived and was careful never to go there.
Because I married into a Nicaraguan family, I got to hear skin-toned racism as my then-husband swore at African-Americans and Afro-Hispanics.
Because I learned from them that being "pure Spanish" (white and cultured) was better than being "puro jincho" (a peasant, a country hick).
Because when I was suddenly a single mother in 1986 and went down to the welfare office to see about getting help, and was one of few white women there, in shame, I never returned.
Because when I married again, into a Portuguese family, I found myself sitting in a relative's trailer home watching the Super Bowl in 1991, when Whitney Houston sang the most beautiful rendition of "The Star Spangled Banner," and listening to a spew of racist slurs from the man of the house. He said he'd rather kill himself than ever hear the song sung by a black person, and I did not speak up. (But I told my then-husband I'd never go back, which was something.)
In between then and now, I grew, I learned, I opened up and am continuing to ruthlessly self-examine my words and my actions as a citizen of the human race. I don't always succeed.
Because as I now examine my family's American history, I find slaveholders among them, as well as casual racism in every generation (the vintage Valentine above is from my grandmother's childhood scrapbook, circa 1910).
Because it took so long for consciousness to dawn, and for me to understand and own my own racism, I offer this apology to my African-American brothers and sisters for not speaking up before, and my pledge to be an ally going forward.
As we all are, I am a work in progress. May my movement be forward, never backward. It's not about guilt. It's about being accountable and owning our history.
May I be as brave, some day, as Bree Newsome, who climbed the flagpole in Charleston and pulled down the Confederate flag, in seeking to change the world.
Three Writing Lessons from Life
I'm in the midst of promoting one book and writing another, but, as is the way with life, other projects and adventures get in the way. And yet, they all lead back to writing, somehow.
Take our grand-dog, for example. I'm not a dog person; I am most definitely a cat lady. But the more time I spend babysitting Peanut, who's a puggle (pug and beagle mix — who looks like a boxer!), the more dog-person-y I become. I take him for walks, I chat with him, I use a special "Peanut voice" when in conversation with him, I make sure he has water and a snuggly place to sleep, and so on.
I think I'm "not a dog-person" but, according to my Facebook friends, I am. I'm doggier by the day.
When I was writing Veronika Layne Gets the Scoop, I was pretty sure I was writing a chick-lit romance. Sex and the City! Hot sweet love! I like romance, but not so much on reading mysteries and suspense. I like a good love story. Who doesn't?
But when the publisher set the BISAC codes for the book, I found that I had written a mystery-suspense-thriller.
I don't read mysteries. I don't write them. I don't even like them. Except sometimes (Dick Francis horse racing thrillers). I filled in my love story with the adventures of a young reporter. That part kind of took over, until the romance was just about ten percent of the total story. The publisher said it no longer counted as a romance.
So apparently I wrote a thriller. And apparently I like dogs. One dog. Just this one. There's a surprise.
In another example, we bought a crack house last summer. Not that we wanted a crack house, but it was a troublesome entity in the neighborhood, and out of frustration over tenants who trashed the place, we offered to buy it from the landlord, and, to our shock, he agreed. We suddenly found ourselves with another fixer-upper (koff, gasp, wheeze). We have been working our way through permit-red tape all through fall and have been having a survey done of the property.
We just found out a few days ago that the house is actually partially built on the neighbor's land. Since 1940, that is (koff, gasp, wheeze). We kind of eminent domained, unintentionally. Once again, we thought we had one thing, and we ended up with another. We had plans. Big plans. And the map has changed. If that doesn't sound like your latest writing project, then you must have a better plan than I do.
I've got a third example, and it isn't pretty. I've been watching the events of Ferguson and New York, and everywhere else where there is police brutality, gun violence, and racism. A lot of arguments have played out in the streets, on Facebook, in the news, between friends and among family members. I've blocked people and rebutted trolls, tried reason and logic, written passionate replies and stood my ground. And I fear this is not going to end soon, though perhaps it will end well.
I am trying to have faith about progress of the human race.
Where this leads me is to truth. Truth can be ugly, but it's better to see it than to hide it. It's better to speak it than to lie.
Ernest Hemingway said
of his work that as long as he could write one true thing every day, he felt that he was progressing. I am well aware of my privilege and intend to make use of whatever bully pulpit comes of it. It is my intention to write truly. The mot juste. The truth about whatever story or lesson comes my way.
Follow these examples in your writing:
Dig deeper and find out who you really are.
Let go of the road map and deal with what is.
Look at — and tell — the truth, however painful.
And let that be a lesson to you.
2020 Vision December 20, 2019
A Whole New World September 10, 2017
Slingshot: This Is Not Where I'm Supposed to Be November 16, 2015
Behind Closed Doors October 26, 2015
Birthrights and Wrongs October 12, 2015
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Slingshot: This Is Not Where I'm Supposed to Be
Birthrights and Wrongs


text:
The expression of B23 and EGR1 proteins is functionally linked in tumor cells under stress conditions
Donatella Ponti1,
Daniela Bastianelli2,
Paolo Rosa1,
Luca Pacini1,
Mohsen Ibrahim2,
Erino Angelo Rendina2,
Giuseppe Ragona1 &
Antonella Calogero1
The nucleolus is a multi-domain enriched with proteins involved in ribosome biogenesis, cell cycle and apoptosis control, viral replication and differentiation of stem cells. Several authors have suggested a role for the nucleolus also in malignant transformation. We have recently demonstrated that under specific circumstances the transcriptional factor EGR1 is shuttled to the nucleolus where it functions as a negative regulator of RNA polymerase I. Since this activity is hampered in ARF −/− cells, and ARF transcription is regulated by EGR1 while the turnover of ARF protein is under the control of B23, we speculated that some sort of cooperation between EGR1 and B23 might also exist.
In this work we identified a canonical EGR1 binding site on the B23 promoter through experiments of transactivation and in vitro DNA binding assay. We then found that the levels of B23 expression are directly correlated with those of EGR1, and that this correlation applies to several cellular types and to different stress conditions. Furthermore, we showed that EGR1 stability and accumulation within the nucleolus is in turn regulated by B23 through proteasome involvement, similarly to ARF turnover.
Our results highlight EGR1 as a regulator of B23 expression actively playing within the newly discovered nucleolar B23-ARF-EGR1 network.
During tumorigenesis, cancer cells increase the production of ribosomes to support the higher rate of protein synthesis associated with cancer growth. This is supported by the increase in rRNA synthesis transcribed in the nucleolus by RNA polymerase I, and correlates with adverse prognosis [1–3]. To contrast this mechanism, the products of Rb and p53, two important tumour suppressor genes, negatively interfere with the RNA polymerase I and the assembly of the transcriptional machinery on the rDNA promoter [4, 5]. p53 activation is under direct control of the negative regulator MDM2, which in turn is inactivated by ARF. This explains why ARF is pivotal for triggering the cell-cycle arrest and the apoptotic programme after oncogenic cues [6]. The stability of ARF is significantly increased in cells that overexpress exogenous B23 (known also as nucleophosmin, numatrim, and NOR38). B23 associates with ARF within the nucleolus, delaying its turnover. As a consequence, the inhibition of B23 by shRNA has destabilizing effects over ARF [7]. ARF mutants unable to bind B23 are unstable and functionally impaired [8]. In mouse embryonic fibroblasts (MEFs) lacking both B23 and p53, ARF is mainly found outside the nucleolus and with a shortened half-life, conferring the cells with higher proliferation rates [9]. B23 is an abundant, multifunctional protein present at high amount in the granular region of nucleoli [10]. It is involved in the regulation of ribosome biogenesis, and the control of genome stability and survival in response to a variety of stress stimuli [11–13]. In fact, B23 −/− fibroblasts become aneuploid and have increased levels of P53 in a stable form [7]. Conversely, B23 has been shown to induce senescence in normal primary fibroblasts [14] and its overexpression in tumors lacking p53 is known to promote proliferation [15]. The transcription factor EGR1 (early growth response protein 1) is involved in the transcriptional regulation of responses to a wide number of proliferative, differentiation and stress stimuli [16, 17]. In particular, EGR1 regulates the expression of key genes, including p53, TGFB, CDKN1A/p21 and PTEN, involved in the growth and division of cancer cells, [18–20]. Egr-1 +/− and Egr-1 −/− MEFs bypass senescence and grow as typically immortalized cells. Besides to cover the role as "gatekeeper" of p53-dependent growth regulatory mechanisms [18], EGR1 has been suggested to play a role as tumour suppressor in several tumor types [21–23]. We have recently demonstrated that EGR1 behaves as a negative regulator of RNA polymerase I [24]. EGR1 localizes to the nucleolus and the more it is expressed the less is the 47S pre-rRNA synthesized. ARF is required for the nucleolar localization of EGR1. Indeed, in cells ARF −/− such as the NIH-3 T3 Egr1 does not keep the nucleolar localization and the ability to suppress the synthesis of the 47S ribosomal precursor [24]. In view of the functional interaction of ARF with B23, and the fact that EGR1 regulates the transcription of ARF [25] we have hypothesized that EGR1 might extend its control also on B23 expression. Here, we show that EGR1 promotes its stability and the transcription of B23 gene.
The expression of nucleolar B23 correlates with the levels of EGR1
To verify our hypothesis we first monitored the effects on the expression of B23 in HeLa cells by either increasing or reducing the levels of EGR1. After transient transfection of the pEGFP-EGR1 expression vector, showed a six-fold increase of B23 mRNA in low serum conditions (Fig. 1a). No changes were observed under culture conditions with 10 % FBS. Accordingly, lowering the EGR1 mRNA levels with siRNA specific to EGR1 sequence, we observed a significant reduction of B23 mRNA levels compared to cells treated with non-specific (scrambled) siRNA (Fig. 1d upper panel). Here again, the inhibitory effect on B23 mRNA synthesis was observed only in cells cultured in low serum conditions, and not in presence of 10 % FBS. Similar changes were observed with the B23 protein synthesis under the same experimental conditions. In summary, in condition of serum deprivation the B23 mRNA and protein levels increased after EGR1 overexpression and decreased after EGR1 silencing.
B23 levels are directly correlated to EGR1 under serum deprivation. EGR1 and B23 expression in HeLa cells transfected with a GFP fused full length EGR1 (named EGR1-GFP) or incubated with siRNA specific for EGR1 (named siRNA EGR1). A control empty vector for GFP (named GFP) or a scrambled sequence of the siRNA EGR1 (named siRNA SCRAMBLED) were used for comparison, respectively. Experiments were carried out in medium with either 0.2 % or 10 % FCS. Relative levels of B23 and EGR1 mRNA were quantitated by RT-qPCR in cells transfected with EGR1-GFP or control GFP (Panel a), and in cells incubated with siRNA EGR1 or control siRNA scrambled (Panel d). Protein signal ratios of EGR1 and B23 to GAPDH were quantitated by densitometry in cells transfected with EGR1-GFP or control GFP (Panel c), and in cells incubated with siRNA EGR1 or control siRNA scrambled (Panel f), limitedly to serum deprived conditions. Immunoblot of EGR1 and B23 proteins from a representative experiment (Panel b and e). Data are the mean +/− s.e. of three independent experiments. Comparisons were performed by t-test. Significant results are highlighted with asterisks (*p < 0.05; **p < 0.01; ***p < 0.001)
To verify that the above results in HeLa cells are not dependent upon the specific cellular context, we replicated the experiments with a primary culture of human lung tumor and a glioblastoma derived established cell line, U87MG and 293 T (Additional file 1: Figure S1). In all these cultures the B23 mRNA and protein underwent variations in their levels similar to those observed in HeLa under the same experimental conditions. We conclude that the levels of B23 expression are strongly influenced by EGR1 under lower FBS concentration, a known stress inducing condition.
To investigate whether EGR1 can influence the expression of B23 under different stress conditions, we sought to examine their response to actinomycin D, a genotoxic agent known to inhibit the RNA Polymerase I and II activity and to induce DNA damage [26]. The levels of EGR1 and B23 mRNA and protein showed a step-wise increase when cells were treated with increasing concentrations (0.04, 0.5, and 1 μg/ml) of actinomycin D (Fig. 2a-b-e-f-g). Also the expression of p300, a specific EGR1 target gene, increases, whereas the synthesis of 47S and 45S is strongly inhibited, as shown in Fig. 2c-d. Pre-treatment with EGR1 silencing siRNA abolished the response of B23 and p300 genes and restored the production of 47S rRNA (Fig. 2c-d). Finally, we asked the question of whether the B23 protein would translocate to the nucleoplasm in response to Pol I-induced transcription repression, as suggested by Yao Z. et al. [27]. We confirm that in our experimental conditions actinomycin D causes the translocation of B23 to the nucleoplasm (Additional file 2: Figure S2).
Actinomycin D induces EGR1 and B23. Relative mRNA levels of EGR1, B23, 47S,45S pre-rRNA and p300 (the last two are known gene targets of EGR1) expressed in HeLa cells treated with Actinomycin D for 1 h at 37 ° C (Panel a to d). The smaller inserts in the panels compare the effect of siRNA EGR1 plus Actinomycin D to that of Actinomycin D alone. Quantitative evaluation of EGR1 and B23 proteins under the effect of Actinomycin D, together with a representative immunoblot, are shown in Panel e to g. Data are the mean +/− s.e. of three independent experiments. Comparisons were performed by one way ANOVA. Significant results are highlighted with asterisks (*p < 0.05; **p < 0.01; ***p < 0.001)
EGR1 binds to B23 promoter
Since the levels of B23 expression vary in response to EGR1, we hypothesized that as a transcriptional regulator it might control the B23 expression by interacting with the promoter. To verify this hypothesis, we first searched the B23 promoter (NCBI, Accession: NG_016018) for the presence of canonical sequences that were recognized and bound by-EGR1.
By-bioinformatics-approach (http://bip.weizmann.ac.il/index.html) one of such elements was found at −172 bp to the TATA box (Fig. 3a). To establish the function of the putative EGR1 binding site, we cloned a partial sequence of the B23 promoter (4328 to 5240 bp) from HeLa DNA and inserted into a luciferase reporter gene to assay the responsiveness to the activity of EGR1. As above, EGR1 was exogenously expressed following transient transfection of the pEGFP-EGR1 expression vector. All experiments were carried out in HeLa cells cultured in low serum conditions (0.2 % FBS). The B23 minimal promoter carrying the sequence of the EGR1 binding site found in HeLa was successfully transactivated by the exogenously expressed EGR1, providing high levels of luciferase activity (Fig. 3b). No luciferase activity was detected when the B23 minimal promoter was deleted of the sequence or carried a mutated version of the EGR1 binding site and tested in the same conditions as above (Fig. 3a-b).
Identification of the EGR1-binding sequence required for B23 promoter induction. a The human B23 promoter sequence, partial. An EGR1 binding motif is underlined at position −172 from the TATA box. A schematic representation of the WT sequence of B23 promoter and a copy with a mutated EGR1 binding site cloned upstream of Renilla luciferase gene (pB23-Luc, pB23-mut-Luc). b The luciferase assay in HeLa cells transfected with EGR1-GFP or the control GFP in combination with either of the two luciferase vectors (pB23-Luc or pB23-mut-Luc) plus a beta-galactosidase vector for normalization of transfection efficiency. All experiments were done under serum deprivation conditions. c Real time PCR of HeLa cells extracts either transfected with full length EGR1 or control GFP were incubated with DNA fragments amplified from the promoter sequence of B23 gene, in presence or absence of anti-EGR1 antibody
In vitro DNA binding assays were performed to establish whether EGR1 specifically associated with the minimal B23 promoter. We incubated DNA fragments of the promoter sequence with anti-EGR1 antibody in presence of extracts of HeLa cells either transfected with full length EGR1 or EGFP [24]. Cells were cultured either in low serum conditions or at 10 % FBS. An abundant (about twenty-fold) enrichment of promoter sequences was obtained by RT-qPCR (Fig. 3c-d) and PCR (Additional file 3: Figure S3) only with extracts of cells transfected with EGR1 and cultured in low serum. No statistically significant differences were detected in 10 % FBS. Furthermore, when the PCR was performed with oligonucleotides specific for a region located upstream the minimal B23 promoter no amplification products were obtained (data not shown).
B23 expression is downregulated in the brain of Egr1 −/− mice
In order to gain further evidence about the role of EGR1 as a regulator of B23 expression, we compared the levels of B23 mRNA and protein from brain of Egr1 −/− with Egr1 +/+ mice. Since EGR1 is present at high levels in mouse and human brain [28] but not, the levels of B23 expression should likely be different in the brain of wild-type compared to Egr1 −/− mice, in case B23 were regulated by EGR1. A significant reduction of B23 mRNA and protein was detected in the brain of Egr1 −/− mice (Fig. 4).
B23 is downregulated in EGR1 −/− mouse brain. a B23 mRNA and b-c protein levels in EGR1 +/+ and EGR1 −/− mice. Whole brain extracts (80 μg) were obtained from nine wt and EGR1 −/− female three month old mice. The EGR1 protein was quantified as relative to that of GAPDH taken as control. Comparisons were performed by t-test. Significant results are highlighted with asterisks (**p < 0.001)
B23 increases the EGR1 steady-state level of expression
B23 plays an important role in cell growth by regulating the function of proteins such as ARF and p53 [29]. This is accomplished by delaying the turnover of these proteins, thus contributing to the activation of suppressor activities aimed to the control of cell cycle. In the case of ARF, a physical interaction with B23 has been demonstrated which protected ARF from proteasomal degradation [29]. We asked the question of whether a similar interaction might exist also between EGR1 and B23. If B23 stabilizes EGR1, then a fall in the B23 synthesis would likely translate into a change in the detectable levels of EGR1, due to a loss of stability. To test this hypothesis, we treated the HeLa cells with 30nM B23 specific siRNA, and monitored the expression of both genes EGR1 and B23. We show that following a decrease of B23 mRNA and protein, the levels of EGR1 mRNA remained unchanged (Fig. 5c). However, the levels of EGR1 protein detected by immunoblotting were significantly lower (Fig. 5a-b). We also confirm that under the same experimental conditions the level of 47S pre-rRNA increased whereas the level of p300 diminished, both significantly (Fig. 5d-e). To investigate whether the lower levels of EGR1 protein following B23 silencing were the result of a proteasome-dependent degradation, we tested in HeLa cells the effect of the proteasome inhibitor MG-132 (at the concentration of 10 μM) on the stability of EGR1 during B23 silencing after 16, 20, and 24 h. Only after 16 h from the treatment the levels of EGR1 did actually increase (Fig. 5f-g). These experiments clearly suggest the partial involvement of proteasome machinery on EGR1 turnover, and that, EGR1 stability and accumulation within the nucleolus is likely regulated by B23.
EGR1 protein level is linked to B23. Analysis of EGR1 after B23 siRNA in HeLa cells grown at 0.2 % FBS. a Immunoblotting of EGR1 and B23 in whole extracts of HeLa cells after B23 siRNA. EGR1 protein significantly decreases following endogenous B23 silencing at 30 nM. b-c-d-e Real time PCR analysis of B23, EGR1, p300 mRNA and 47S pre-rRNA under B23 silencing. The level of expression of EGR1 remained unchanged (c). As expected p300 mRNA decrease (d) while the 47S rRNA levels increase levels (e). f-g Immunoblotting of EGR1 under MG-132 treatment after B23 siRNA for 16, 20 and 24 h. The level of EGR1 protein is restored during B23 silencing after treatment with proteosome inhibitor at 10 μM for 16 h at 37 ° C. Comparison tests were performed by one way ANOVA and significant results are highlighted with asterisks (*p < 0.05; **p < 0.01; ***p < 0.001)
B23 (also known as NPM1, nucleophosmin, nucleoplasmin, NO38, and numatrin) is the best studied member of the NPM family of chaperones, a highly conserved protein found in humans, rodents, chicken and fish. Human B23 is mapped to chromosome 5q35 and is made up of 12 exons. B23, first isolated from Xenopus laevis oocyte extracts [30] is a histone storage protein affecting many intracellular processes such as: i) the rate of ribosome synthesis by binding to pre-ribosomal complexes; ii) DNA duplication and transcriptional regulation; iii) nucleo-cytoplasmic shuttling and assistance to small basic proteins such as REV, REX, TAT and p120 for transport to the nucleolus [31–34]. Within the nucleolus, B23 binds to ARF. The interaction with B23 increases ARF stability by interfering with the action of the ubiquitin-proteasome system.
In a recent publication, we have shown that the transcriptional factor EGR1 also localizes to the nucleolus and acts as negative regulator of RNA polymerase I activity, similarly to p53 [24]. Both B23 and EGR1 are activated and expressed under at least two different stress stimuli, i.e., after UV exposure and H2O2 treatment [35, 36]. We do not know whether the two genes are independently. To investigate this hypothesis, we conducted a series of experiments where HeLa cells grown in serum deprived conditions and treated either to express high levels of exogenous EGR1 or to silence the expression of the endogenous EGR1 gene, were examined for changes in the localization and the expression of B23 mRNA and protein, and in the regulation of 47S and 45S pre-rRNA synthesis. The above results show that both pre-rRNAs (47S and 45S) have a rise in their levels when EGR1 is silenced. Though very preliminar this observation leading us to hypothesize that the regulation of 47S could be not an abortive transcription. However, further investigations will help us to clarify this point. Here we show that in HeLa cells the levels of B23 mRNA and protein increase after EGR1 overexpression, and decrease after EGR1 silencing. We know that this correlation is not cell specific. In fact, very similar results were obtained also with established glioma cell line and a primary culture of lung adenocarcinoma. In addition, the same effect can be assumed in vivo for the brain of B23 by comparing the expression levels of B23 in the brain of EGR1 −/− with that from EGR1 +/+ mice. An investigation of the human and mouse B23 promoter nucleotide sequence allowed the recognition of a potential EGR1 binding site, which proved to be effective in a reporter gene transactivation assay. The association of EGR1 to the B23 promoter was further demonstrated by chromatin immunoprecipitation (ChIP). It has been shown that following DNA damage B23 binds to chromatin [37] and to other proteins as well, such as GADD45 [38], the retinoblastoma protein pRB [39], PARP1 and PARP2 [40]. They are all intimately linked to the maintenance of DNA structure, replication or repair. Following treatment with actinomycin D, nucleolar B23 protein undergoes a rapid translocation to the nucleoplasm [41]. Here we confirm these data and find that the levels of both EGR1 and B23 increase with different concentrations of the drug. However, if cells are treated under conditions inhibiting the expression of EGR1, then the levels of B23 remain constant. One of the mechanism of actinomycin D is inhibit the transcription by intercalating with DNA, in particular at the GC-rich RNA Polymerase I genes. In our experiments EGR1 seem to facilitate the action of genotoxic drug. We have already demonstrate in a previous paper that EGR1 bind UBF protein and the formation of this proteins complex could help the action of Actinomycin D on the inhibition of RNA Polymerase I gene. This is only one of the possible scenario further experiments will help to clarify the role of EGR1 under actinomycin action. These data further suggest a direct role for EGR1 in regulating the expression of B23, and that the correlation between EGR1 and B23 is maintained under several conditions of cellular stress. Several studies have demonstrated the involvement of B23 in the p53 tumor suppressor pathway. Colombo et al. found that B23 when overexpressed promotes cellular senescence in fibroblasts and regulates the stability of p53 through direct interaction [14]. Further, in response to nucleolar stress, B23 does promote the stabilization of ARF protein [25]. In view of the multiple interactions that link B23 to the above molecules and likely to EGR1, we finally studied the effects of B23 over EGR1. Indeed, by inhibiting the expression of B23 we observed a reduction of EGR1 protein levels but not of EGR1 mRNA. Since MG-132, a proteosomal inhibitor, can reduce this effect we speculate that B23 might have a role for keeping adequate levels of the intracellular concentration of EGR1 following stress conditions.
In summary, these findings provide the evidence that B23 is regulated by EGR1 under stress conditions, and highlights EGR1 as an important link of the ARF-B23-rRNA nucleolar network [42], regulating ribosome biogenesis and promoting growth arrest.
Cell lines, primary tumour cell and mice
The cell lines, uterine cervix cancer HeLa (ATCC CCL2), human embryonic kidney 293 T (ATCC CRL-1573) and glioma U87MG (ATCC HTB-14) were grown in DMEM supplemented with 1 % nonessential amino acids, 1 % L-glutamine, 100 IU/ml penicillin, 100 IU/ml streptomycin and FBS (Sigma-Aldrich St. Louis, Mo, USA) 10 % or 0.2 % (serum starvation) at 37 °C in 5 % CO2 humidified atmosphere in air. The cell lines used were obtained from the American Type Culture Collection, Rockville MD, USA. The primary lung tumour cells were grown in DMEM-F12 [43]. The research protocol was approved by ethic committee of S. Andrea Hospital, University of Roma Sapienza. The study was conducted using C57BL/6 wild-type and Egr1 deficient mice (Egr-1−/−) [44]. Animals were housed four per cage. All the experiments were performed in accordance with the EU Directive 2010/63/EU for animal experiments and approved by ethic committee of Department of Medico-Surgical Sciences and Biotechnologies, University of Roma Sapienza.
RT-qPCR
RNA extraction was performed after EGR1 silencing, B23 silencing and EGR1 overexpression into HeLa cells. For silencing, EGR1 and B23 pre-designed siRNA (Invitrogen, Grand Island, NY USA, 4390822) or scrambled sequence RNA oligonucleotide (Negative Control siRNA 4390846) were transiently transfected at 20, 30 nM or 60 nM using High-Perfect Transfecting Agent (as indicated in the figures) (Qiagen, Valencia, CA) following the Qiagen protocol. The treatment with actinomycin D (Sigma) was performed for 1 h at 37 ° C at the three concentrations indicated in Fig. 2. The treatment with the proteasome inhibitor MG-132 (Sigma) was performed at 37 °C for 16, 20 and 24 h during B23 silencing. For EGR1 overexpression pGFP-EGR1 expression vector [24] was transfected using lipofectamin (Invitrogen, Grand Island, NY USA). To analyse the levels of B23 in vivo, total RNA was extracted from female mice of wild type and Egr1 −/− mice. Total RNA extraction and cDNA preparation were accomplished using RNeasy (Qiagen, Valencia, CA) and Superscript III RT (Invitrogen, Grand Island, NY USA) according to the manufacturer's recommendations. For the silencing and overexpression experiments we used the protocols described previously [24]. Quantitative RT-PCR was performed using Fast SYBR Green Master mix and the StepOnePlus real-time PCR system (both from Applied Biosystems). Each experiment was performed in triplicate. Experiments were independently repeated three times. Gene expression levels were quantified from real-time PCR data by the comparative threshold cycle (CT) method using 18S as an internal control gene. The following gene-specific primers were used: B23: FW 5'-AGAAAAAGCGCCAGTGAAGA-3', REV 5'-TGGTGTTGATGATTGGTTTTGA-3' (for human gene), FW 5'-GGGGGTACCGCTTTCTTTCAGGAGG-3', REV 5'-CCGCTCGAGGGCACGCACTTAGGTA-3' (form mouse gene); EGR1: FW 5'- AGCCCTACGAGCACCTGAC-3' and REV 5'-GGTTTGGCTGGGGTAACTG-3'; 47S: FW 5'-TGTCAGGCGTTCTCGTCTC-3' and REV 5'-GAGAGCACGACGTCACCAC-3'. 18S: FW 5'-GCAATTATTCCCCATGAACG-3' and REV 5'-GGGACTTAATCAACGCAAGC-3'; GAPDH: FW 5'-AGCCACATCGCTCAGACAC-3' REV 5'-GCCCAATACGACCAAATCC-3'; p300: FW 5'-GGTCAAGCTCCAGTGTCTCAA-3' REV: 5'-GGGACTTAATCAACGCAAGC-3'. Each experiment of transfection, immunoblotting and qPCR were repeated three times in triplicate.
Immunoblotting
Immunoblotting analysis was performed using whole extracts (40 μg) from HeLa cells after silencing and overexpression of EGR1 [23], or B23 silencing. Total extract (40 μg) were prepared from subconfluent cultures by resuspending cells in RIPA-Buffer (20 mM Hepes, pH 6.8, 5 mM KCl, 5 mM MgCl2, 0.5 % NP-40, 0.1 % sodium. Furthermore whole extracts (80 μg) were obtained from brain of nine of wild type and Egr1 −/− female mice of three months using RIPA buffer (20 mM Hepes pH 6.8, 5 mM KCl, 5 mM MgCl2, 0,5 % NP-40, 0,1 % sodium deoxycholate, protease inhibitor (Sigma), 0.1 mM phenylmethylsulfonyl fluoride) using a Dounce homogenizer. After incubation for 30 min at 0 ° C the lysate was centrifuged at 10000 rpm × 15 min 4 ° C. The samples were loaded on SDS-PAGE gel at 8 % or 10 % of acrylamide (Sigma 29:1) and blotted on PVDF (Biorad) using a semidry apparatus (Biorad). After blocking in 5 % dry milk the membrane was incubated with the appropriate antibody. The primary antibodies used are: rabbit polyclonal anti-EGR1 (sc-101, Santa Cruz Biotechnology), mouse monoclonal antibody anti-B23 (ab10530, Abcam), rabbit polyclonal antibody anti-GAPDH (2118, Cell Signaling Technology Biotechnology) and monoclonal antibody anti-actin (sc-47778, Santa Cruz). The secondary antibodies used for western blot are anti-mouse and anti-rabbit (GE Healthcare Bio-Sciences, Piscataway, NJ, USA). After ECL assay (GE Healthcare) the membrane was incubated with film specific for protein detection (Kodak).
Luciferase and β–galactosidase activity assay
Cloning of the B23 minimal promoter
To amplify the proximal B23 promoter region, genomic DNA from HeLa cells was used as template. The primers used are: FW 5'-GGGGGTACCGCTTTCTTTCAGGAGGAAT-3' REV 5'-CCGCTCGAGGGCACGCACTTAGGTAG-3'. To verify the specificity of the PCR amplification, the fragment obtained was excised from agarose gel and isolated using DNA gel extraction kit (Millipore). The PCR product (region 4328 bp to 5240 bp of B23, NPM1, numatrin sequence; Accession: NG_016018) was KpnI/XhoI digested and cloned into the pGL3 basic luciferase vector (Promega). The correct sequence was checked by direct sequence (Applied Biosystem). Site-specific mutations in the EGR1-binding sites within the B23 promoter were made with the QuickChangeII Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). Briefly, primer sequences used to generate point mutations are: FW 5'- GGAAGGAGGCTTAAAGGAGGTAGAAAGGAGTG-3 REV 5'-CACTCCTTTCTACCTCCTTTAAGCCTCCTTCC-3'. PCR reaction conditions were: 95 °C for 1 min followed by 18 cycles at 95 °C for 50 s, 60 °C for 50 s, 68 °C for 5 min, then 68 °C for 7 min. Following the PCR reaction, the mix was digested by incubating with Dpn I for 1 h at 37 °C. Clones of mutated plasmids were replicated in bacteria and screened for the correct mutation by DNA sequencing (Applied Biosystem). Luciferase assay. HeLa cells were seeded into 6-well plates and transfected with 0.3 μg trans-activator plasmid pEgr1-GFP, 0.3 μg pB23-Luc or 0.3 μg pB23-mut-Luc, plus 0.5 μg of β-galactosidase vector. At 24 h post-transfection, the levels of luciferase activity were measured sequentially from a single sample using the Luciferase reporter assay system (Promega) with a luminometer (TD-20/20 Turner Design). Measurement of luciferase enzyme activity was assayed on whole-cell extract performed using 20 μl of lysate cleared and 100 μl of Luciferase assay Reagent. The luciferase values were normalized to β-galactosidase activity and protein content. The experiments of luciferase assay were repeated three times in triplicate.
Chromatin immunoprecipitation (ChIP)
HeLa cells were transfected with full length EGR1 expression vector pEGFEGR1 or pEGFP empty vector using lipofectamine 2000, fixed 48 h after transfection with 1 % formaldehyde for 15 min at room temperature and the reaction stopped by addition of 125 mM glycine for 5 min. The other steps of the ChIP experiments were performed according to the manufacturer's instructions (Magna ChIP, Millipore). The extracts were immunoprecipitated with anti-EGR1 (4153, Cell Signaling Technology) (24). The samples obtained were analysed by RT-qPCR (Applied Biosystem) and standard PCR (Additional file 4: Material and Methods) using specific couples of oligonucleotides for B23 promoter. Primers used for PCR and real time analysis are: FW 5'-TCGAGGTGCTCTCTGGCTCAT-3', REV 5'-TGCATAATGGCGTCGGCAG-3' REV 5'-TGCATAATGGCGTCGGCAG-3'.
Each experiment was repeated at least three times. Statistical comparisons were performed using Student's t-test and a one-way analysis of variance (ANOVA).
This research was supported by MIUR (PRIN 2011 Prot. 2010 ZEJWN_007) and University of Rome Sapienza Ricerche Ateneo 2011–2014 to AC; Ateneo 2014 Prot. N. 0067282 to DP.
EGR1:
early growth response protein 1
ARF:
alternative ready frame
ChIP:
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We gratefully thank Prof. J. Milbrandt for the generous gift of the Egr1 −/− mouse strain used in this work.
Department of Medico-Surgical Sciences and Biotechnologies, University of Rome Sapienza, Corso della Repubblica 79, 04100, Latina, Italy
Donatella Ponti, Paolo Rosa, Luca Pacini, Giuseppe Ragona & Antonella Calogero
Division of Thoracic Surgery, Department of Medical-Surgical Science and Translational Medicine, University Sapienza, S. Andrea Hospital, via di Grottarossa 1035, 00189, Rome, Italy
Daniela Bastianelli, Mohsen Ibrahim & Erino Angelo Rendina
Donatella Ponti
Daniela Bastianelli
Paolo Rosa
Luca Pacini
Mohsen Ibrahim
Erino Angelo Rendina
Giuseppe Ragona
Antonella Calogero
Correspondence to Antonella Calogero.
Conceived and designed the experiments: DP AC DB. Performed the experiments: DP DB PR LP. Analysed the data: DP AC DB GR. Performed statistical analysis: DB, MI EAR. Wrote the paper: DP AC GR. All the authors have read and approved the final manuscript.
EGR1 levels are directly correlated to B23 expression in primary culture of lung adenocarcinoma tumor, kidney and glioma cell lines under serum deprivation. In all these cultures the B23 mRNA underwent variations in their levels similar to those observed in HeLa under the same experimental conditions. B23 synthesis at 0.2 % FBS is significantly depressed following endogenous EGR1 silencing with 60 mM specific siRNA for lung adenocarcinoma (panel A), 30 nM specific siRNA for U87MG (panel B) and 20 nM specific siRNA for 293 T (panel C). The B23 expression does not change in scramble siRNA control. The data shown are the mean of three independent experiments. Comparison tests were performed by one way ANOVA. (** < p 0.01 *** < p 0.001). (PSD 3175 kb)
B23 nucleoplasm translocation in HeLa cells after Actinomycin D treatment. Immunofluorescence analysis of B23 expression after actinomycin D treatment. (A) Control and (B) HeLa cells treated (0,4 μg/ml) of HeLa cells for 1 h at 37 ° C. The images were taken by under a 40X objective with immunofluorescence microscropy (LEICADM4000B). (PSD 5029 kb)
PCR analysis of ChIP samples. Analysis on agarose gel of PCR amplification products of B23 promoter immunoprecipitated with antibody to EGR1 from extracts of transfected HeLa cells at 0.2 % FBS transfected with full length EGR1. (PSD 10140 kb)
Materials and Methods: Immunofluorescence and Standard PCR. (DOCX 16 kb)
Ponti, D., Bastianelli, D., Rosa, P. et al. The expression of B23 and EGR1 proteins is functionally linked in tumor cells under stress conditions. BMC Cell Biol 16, 27 (2015). https://doi.org/10.1186/s12860-015-0073-5
EGR1
NPM1
Egr-1 −/− mice
Nuclear function and transport


What does this AWS Solution do?
Amazon Web Services (AWS) offers infrastructure on demand so that customers can control their resource capacity and pay only for what they consume. One simple method to reduce costs is to stop resources that are not in use, and then start those resources again when their capacity is needed.
The AWS Instance Scheduler is a simple AWS-provided solution that enables customers to easily configure custom start and stop schedules for their Amazon Elastic Compute Cloud (Amazon EC2) and Amazon Relational Database Service (Amazon RDS) instances. The solution is easy to deploy and can help reduce operational costs for both development and production environments. Customers who use this solution to run instances during regular business hours can save up to 70% compared to running those instances 24 hours a day.
This webpage provides an overview of the Instance Scheduler design and functionality.
AWS offers a simple solution that allows you to create automatic start and stop schedules for your Amazon EC2 and Amazon RDS instances. The diagram below presents the components and functionality you can build using the Instance Scheduler implementation guide and accompanying AWS CloudFormation template.
An Amazon CloudWatch event triggers an AWS Lambda function that checks the current state of each appropriately tagged instance against the targeted state defined in an associated schedule in Amazon DynamoDB. Then, the Lambda function applies the applicable start or stop action to the instance.
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After thousands of hours in research and testing, Roush Performance has released their new supercharger kit available for the 2018 Ford F-150 equipped with the 5.0L Coyote V8. This whopping 2.65L supercharger designed for performance and dependability takes the new F-150 to a whole new level, and new levels of supercharger whine.
The 2018 Phase 1 Supercharger Kit is also a jump in power from the previous iteration for the 2015-2017 F-150 V8, which came in at 600 hp. For the F-150 refresh, Roush uncorked an additional 255 hp and 210 lb-ft of torque from stock, boosting the truck to 650hp and 610 lb-ft of torque. The output mirrors what was effectively a Phase 2 upgrade from before, but as the entry level augmentation.
Unlike turbocharging, this Phase 1 Supercharger gives drivers instant throttle response and increased power through the engine's entire power band. A part of the TVS (Twin Vortices Series), Roush has been creating this kit for years, integrating it with both the Mustang and F-150 models with the 5.0L V8.
The fully integrated kit for the 2018 Ford F-150 can be purchased on the Roush Performance website for a hefty $7,800. Lots of power means lots of money, but does come with a three-year, 36,000-mile warranty. Is the extra 255 hp worth the price? That is up for you to decide.
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Righteous And Ratchet - Is Drake The New R. Kelly?
Is Drake The New R. Kelly?
In today's episode Kev & Doboy talk about R. Kelly and question whether Drake is getting the same preferential treatment with his questionable relationships with underage women.
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