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Grey's Anatomy recap: It's a slow burn for Meredith and Cormac
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Grey's Anatomy recap: It's a slow burn for Meredith and Cormac - Entertainment Weekly News
Twice a day, meteorologists at the Twin Cities and other National Weather Service offices launch weather balloons into the sky.
The balloons carry an instrument package called a radiosonde several miles up into the atmosphere. The radiosonde beams back real-time data on atmospheric parameters like temperature, wind speed and direction, humidity and pressure.
Michelle Margraf, from NOAA's Twin Cities National Weather Service, launches a weather balloon carrying instruments that measure atmospheric variables.
Courtesy of Anthony Zaleski
Thursdays 6 a.m. Twin Cities NWS weather balloon launch rose nearly straight up into the atmosphere. Light winds in the lowest mile caused the balloon to rise fairly close to the launch point.
As the balloon gained altitude, stronger but still relatively light westerly winds gently pushed the balloon eastward along Interstate 494 in the southern Twin Cities.
The balloon continued to gain altitude over Eden Prairie. By the time it reached Bloomington, it was more than 20 miles above ground level 108,675 feet above the ground to be precise.
Then it burst.
The parachute on the radiosonde slowed carried the unit gently back to earth. The instrument package began to descend as it drifted over Richfield, the Minneapolis-St. Paul International Airport, Mendota Heights and West St. Paul.
Finally, the parachute guided the instrument package to a landing in Woodbury in the eastern Twin Cities.
Heres the map of the flight path from Thursday mornings launch.
Twin Cities NWS 6 a.m. weather balloon flight path on Thursday.
Twin Cities National Weather Service
Weather balloons launched front the Twin Cities NWS office in Chanhassen, Minn., typically travel 80 to 200 miles. But Thursdays morning flight was unusually short.
The light wind profile above the Twin Cities produced a flight of only about 30 miles. The NWS Twin Cities tweeted Thursday about the unusually short flight.
So, what did data the balloon launch gather? This is a little inside weather baseball geeky, but take a look at the sounding below. The left-hand image shows the temperature and dew-point profile with height through the atmosphere.
You can see the wind direction and velocity barbs on the right-hand side of that image at different levels above ground. The right-hand side shows the balloons corkscrew path as it rises.
6 a.m. sounding from the Twin Cities NWS Thursday balloon launch.
NOAA
Thursdays launch occurred under nearly ideal conditions given the light surface wind. Thats not always the case. Check out this launch under extreme conditions during the April blizzard in 2018.
Neither rain nor snow nor raging blizzard.
Continued here:
Anatomy of a very short weather balloon flight over MSP - Minnesota Public Radio News
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MINNEAPOLIS, MINNESOTA NOVEMBER 09: Safety Antoine Winfield Jr. #11 of the Minnesota Golden Gophers intercepts a pass intended for wide receiver KJ Hamler #1 of the Penn State Nittany Lions during the second quarter at TCFBank Stadium on November 09, 2019 in Minneapolis, Minnesota. He is one of the top safeties in the 2020 NFL Draft. (Photo by Hannah Foslien/Getty Images)
The safety position has become a role that is no longer just a role of sitting twelve yards deep. A safety in todays game has to do multiple things if they want to be considered the best. Having a good safety can change the way a defense is played. Safeties cant afford to stand back and watch things happen, they have to make things happen. Good news: there are a handful of great ones in the 2020 NFL Draft.
Your favorite safety now probably looks a little bit different than what your favorite safety was as a kid. The evolution from safety prospect to a starting safety is evolving. They look more versatile, theyre all shaped a little differently, and yet there are so many good ones without there being a true prototype.
Some of the most exciting players in todays game are safeties who roam around the back end while also contributing in every area. Not all safeties are built the same though and that is what makes the game so exciting today. We are seeing different breeds of safeties who are allowing the game to continue to evolve.
No matter how different they are though, the safety position comes with its own core traits. Imagine what your perfect safety would look like if you combined the best trait from each of the top safeties in the game.
Think about the versatility of a Derwin James that made him an All-Pro as a rookie. Maybe you like the versatility of Jamal Adams because after all the two are both versatile yet so different. Okay I see, you love to see a ballhawk like Eddie Jackson on the back end of your defense.
If youre more of an underdog type of guy then maybe you love the tenacity and beautiful recklessness that guys like Tyrann Mathieu and Budda Baker bring to the game in a small frame as the underdog. Wait I know now your style is more of a sure open field tackler like Jamal Adams that will get you off the couch in excitement. Unless you prefer your safeties in the box and delivering big hits like a Landon Collins.
This draft class has its own set of unique talents at the safety position despite the feeling it isnt a deep prospect group. An elite safety can be built based on some of the best traits from each safety prospect. The anatomy of an elite safety using this draft class looks a little something like this
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2020 NFL Draft: The anatomy of an elite safety prospect - With the First Pick
Its no secret that most fans are annoyed with Debbie Allens character, Catherine Fox, on this season of Greys Anatomy. However, in her defense, Richard Webber (James Pickens, Jr.) did have some shady business going on with his friend from AA. Lets take a look at why fans think their relationship is about to crumble, and why fans are ecstatic about it.
[Warning: Greys Anatomy Season 16 spoilers ahead]
Although Richard was in a good majority of scenes this week, Catherine only made a quick appearance in the last two minutes. She took that time to grill her husband on where he was all day. The only Catherine we see this season is the jealous one.
The entire episode, Richard was consoling his daughter, Maggie Pierce (Kelly McCreary), and trying to get her to come back to work. However, Catherine doesnt think that should have taken him all day.
Catherine, this isnt going to work if you dont trust me, Richard tells his wife.
Who said this is working? she responds. Richard, I think its time for us to have a much bigger conversation.
By the way she is looking at him, we know that this is not going to go well. Next weeks promo for Greys Anatomy reveals that it is Richard and Catherines anniversary. We are in for a snarky dinner party.
Does anyone really care about Catherine and Richards marriage at this point?one Reddit userposed the question. Out of all the relationships, this one annoys me the most. Its either arguing, or shes out of town. I just felt his relationships with Adele and Ellis were way better and worth being invested in.
After the fan complained about the couple, several other viewers joined in. They feel that Richards relationship with Adele (Loretta Divine) or even Merediths mother, Ellis (Kate Burton), was better than this one. Thats saying a lot since Richard had an affair with Ellis and cheated on Adele.
The last season she has been on the STUPIDEST jealousy-trip, another Redditor added. And she becomes such a bitch. Its really annoying. She always demands to be so POWERFUL. Yes, she is strong and has been through a lot, but that DOES NOT mean she can bulldoze through everyone else just to fulfill her demands.
There are many instances where Catherine dismisses others feelings. She does not seem like the right fit for Richard, with how demanding she is.
Viewers of the show are calling for the couple to break up. Some fans are asking for worse than a breakup.
Well, its been a while since a major character death, her cancer might come back soon, one fan pointed out. Or they might break up, and one or both of them end up retiring. Because yeah, the relationship sucks.
Whether Catherine dies or they break up, it sounds like fans will be happy about it.
I get that shes a powerful, strong woman and is a leader, another fan added. But she acts like she is the end all be all of the relationship. I feel like Richard has consistently lessened himself just to give her her own way. Every once in a while, he stands up for himself, but she basically whaps him with a newspaper. I dont like what their relationship has done to Richards character.
Viewers are ready to see Richard without Catherine. He is an original character to the show and deserves a better storyline without her destructive personality.
Find out what happens with the couple when Greys Anatomy returns on Feb. 6, 2020, at 9 p.m. ET on ABC.
Read more: Greys Anatomy: Fans May Hate Catherine Fox, but They Despise Richard Webber Right Now Even More
Grey's Anatomy said goodbye to Justin Chambers' Alex Karev (sort of), but fans don't have to worry about another exityet.
Jesse Williams, Jackson Avery on the show since season six, will make his Broadway debut in the revival of Take Me Out in the spring of 2020. But that doesn't mean Williams is leaving the show or Jackson is leaving Grey Sloan Memorial.
"I've known since the beginning of the season and I've been able to plan [Jackson]'s storyline [accordingly]," executive producer Krista Vernoff told TVLine about any possible scheduling conflicts. "Jesse is able to fly back one day a week; we're just making it work [because] this was important to him."
Typically, Broadway shows are dark (meaning they don't run) one day out of the week. Rest easy, Grey's Anatomy fans. Jackson is sticking around...for now.
Meanwhile, the show has yet to fully explain Karev's exit aside from the fact that he's still visiting family. His character's last episode aired in November. Chambers has been with the show since the start.
"There's no good time to say goodbye to a show and character that's defined so much of my life for the past 15 years," Chambers said in a statement. "For some time now, however, I have hoped to diversify my acting roles and career choices. And, as I turn 50 and am blessed with my remarkable, supportive wife and five wonderful children, now is that time. As I move on from Grey's Anatomy, I want to thank the ABC family, Shonda Rimes, original cast members Ellen Pompeo, Chandra Wilson and James Pickens, and the rest of the amazing cast and crew, both past and present, and, of course, the fans for an extraordinary ride."
What happens to Karev remains to be seen. As of now, his wife, Jo (Camilla Luddington) is still at work in Seattle and he's still visiting his mom out of state.
Grey's Anatomy airs Thursdays, 9 p.m. on ABC.
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Should Grey's Anatomy Fans Be Worried About Jackson Avery? - E! NEWS
The special two-hour Station 19/Grey's Anatomy crossover took place, but all of our favorites survived the ordeal.
On Grey's Anatomy Season 16 Episode 10, there was an engagement, another lawsuit, and a rite of passage for the latest residents.
Join TV Fanatics Meaghan Frey, Paul Dailly, Jasmine Blu, and Grey's Fanatic Berea Orange.
Did the crossover event live up to the hype, and would you consider it a success?
Berea: It didnt live up to the hype. It was okay, sure, but not as epic as they advertised it to be. I think it was a success in that it boosted Station 19s ratings.
Meaghan: I'm going to be honest; I didn't watch the first half of the crossover event. I have never been able to get into Station 19, and it felt like I could get the gist of what happened by just watching Grey's.
However, even just looking at it from a purely Grey's perspective, it was a little bit of a letdown. It was a good episode, but not over the top amazing.
I am worried that with Station 19 being pushed into the 8 pm time slot, they are going to push more of these events on us to try to get more viewers to Station 19.
I understand that the One Chicago block has successfully been able to do this, but Grey's was an established show on its own for far too long to be able to try to switch up the approach now.
Paul: It was a failure. The Grey's Anatomy cast is being used to prop up Station 19, and I'm tired of it. Let Station 19 stand on its own two feet. If it can't, it shouldn't be an ongoing series.
Jasmine: It did not live up to the hype at all. And Meaghan, you didn't need to watch the Station 19 part to understand the Grey's part of everything. I found the first half very disappointing.
They are trying to make it a thing, but it's not working. They can't pull off the Chicago Universe crossovers.
It was designed to help Station 19 only, but it backfired, since the Grey's characters outshined on Station 19, and then none of Station 19 showed up in the Grey's part, so it wasn't a full-blown crossover.
Of all of GSM's own in peril, who were you most concerned about? Which person/case were/are you invested in the most?
Berea: I was most concerned about Helm. They made it seem like there would be a big death, and I thought it would be someone we cared about. To be honest, I was kind of disappointed there wasnt a big death.
I was very interested in Parkers PTSD episode. Ive been dying for some good Parker content.
Brody: I'm okay. I want to help.Bailey: Brody, those are your friends.Brody: That's why I want to help.
Meaghan: I too thought that we were going to get a death. The interns haven't suffered a major blow yet like the past intern groups. I feel like they are due for something, so while they might have all made it out of this one alive, I think something is coming soon for them.
In general, I was most concerned about Parker because of the PTSD aspect of things. A catastrophic event like this is a huge trigger, and I wasn't sure if Parker would recover as easily as he did.
I also really enjoyed all the newer cast coming together at the end. The new cast have all felt very expendable up until this point. It is nice for them to start developing them as more than just the annoying interns that are a thorn in the sides of the core cast.
Paul: I cared for Helm the most. There was a lot of foreshadowing, and her admission of love certainly made it seem like she was going to die.
I felt like all of the cases were interesting. However, I think I would have liked a death. We need some conflict, you guys!
Jasmine: I was expecting someone to die, and we didn't get it. I mean, you don't wish death on a character, but it was set up to kill someone off, and they didn't do it. Why tease it that way at all?
I also think it's unfortunate that all the characters in peril were those who were shoved to the background for so long. Did anyone remember or care about Blake?
I was most concerned about and interested in Parker. He was always one of my favorite of the new interns, and I loved him getting more exploration, especially with his PTSD.
Owen finally proposed to Teddy. React.
Berea: I. Do. Not. Care. This is so stupid to me. Sure theyve known each other forever, and shes pined for him forever, but I dont think they should rush into marriage.
If I were Teddy, Id want to wait and make sure hes not just dating her because she gave him the child hes been so desperate for, for years. I will say I hope she goes back to Tom because I just think he deserves everything. And Jackson is the last person to give someone advice on committing.
Meaghan: I'm happy for them. The other day I was looking over old reviews I wrote for my blog and found one from when Teddy made an appearance when Megan Hunt came back.
That storyline was when I finally was able to see Teddy and Owen together and really root for them, so watching them finally take that next step was nice to see.
The problem is that it is tainted a little bit by the looming possibility of Owen being the father of Amelia's baby. For the sake of all couples involved, I hope it's Link's baby.
Owen: Teddy, this ring is my mom's. She gave it to me the day Alison was born, and I've been carrying it around in my pocket ever since. And I love you, and there is no perfect moment, and I am not a perfect man, but you are the perfect woman for me. So, Theodore Grace Altman, will you --Teddy: Owen, no.Owen: No? Is it Tom?Teddy: Owen, stand up. Listen to me. You are not obligated to marry me. You do not have to do this. I want you to break the patterns not reinforce them. Owen: I'm not repeating a pattern. I'm staring a new one. Loving you. Loving our family, with my whole heart every day. That's my new pattern. I love you. I love you. Tedd. Marry me. Marry me. Marry me, Teddy.
Paul: It was about time. The showrunner wanted them together from the get-go. That much was clear, but I am glad there is a progression for Owen. This proposal felt different.
Jasmine: Meh. I mean, I remember feeling that way about them too, Meaghan, back when Megan returned. I was rooting for them in Germany too.
Now, I just don't care anymore. Good for them, though. Hopefully, it's drama-free, so please, end this baby daddy thing.
On a scale of 1-10, how emotional was Bailey's breakdown with Richard?
Berea: A solid 10 across the board. Chandra Wilson is so good. This brought me back to the hospital shooting when she broke down at the elevators. She is so good at those scenes. Im glad we got to see her and Ben feel and cope with this loss.
Meaghan: 10. Chandra is an incredible actress, and I love getting to see her shine. The moment of her, Meredith and Richard coming together was also a moment I loved.
They are officially the final three, so it is more important than ever for them to support each other.
I am hoping now that Bailey has opened up about her loss, that we have avoided the downward spiral that I feared she was heading into.
Richard: Bailey, everyone is fine.Bailey: Everyone. Everyone. Everyone I touched today. Everyone I held in my hands, I gave to another surgeon to put back together again. Fine. To lose you, and Grey, Hunt, and Karev. Fine. I made that work. But this. This I --I am not fine. She isn't fine. And I can't even hold her in my hands or put her in someone else's hands or put her back together again, and she just was, and now she just isn't, and I can't do anything but stand here. Stand here and lose her.
Paul: 10. Chandra delivered the best performance I have ever seen, and I could feel the emotion there. It was heartbreaking, and I want her to get awards for that scene.
Jasmine: 10. It was the best moment of the entire two hours. It made the entire crossover worth it. Chandra Wilson is a powerhouse, and I hope she gets the recognition she deserves for her performance. I got choked up.
Should Amelia have told Link about the baby? How are you feeling about this "baby daddy'" arc?
Berea: Yes, she should. But Im sure she will soon enough. This paternity issue is actually pretty annoying for me. Amelink was the only bright spot in this season so far, and they have to ruin it with Owen.
Link: So you had something you want to tell me?Amelia:Uh, I didn't want to know, but, um, it's a boy.
Meaghan: She really needs to. Of course, it is going to be a major blow to him now that he has come to terms with the idea of being a father, but he is a great guy, and he will be understanding about this.
It isn't like Amelia cheated on him. It was just potentially awkward timing. The longer Amelia waits to tell him, the worse it is going to be.
I need this storyline to be over and have a resolution one way or the other, hopefully with Link being the father.
Paul: Yes! That was such an annoying scene. She clearly changed focus when she heard about Teddy and Owen. I dont actually mind the plot, but we need some movement in it.
Jasmine: I'm in denial, so I would rather pretend it isn't happening, and that includes telling my precious Link that the baby he's looking forward to may not be his.
I'm so sick of this contrived, redundant, cheap storyline. It's stupid.
Amelia and Link are what I have consistently loved about the season, and they won't let them be happy. If they already gave Bailey a miscarriage, why couldn't they leave Amelia's pregnancy complication-free?
Burn it with fire.
Do you think Jo will want to pursue motherhood after her time spent with the baby? How do you think they'll carry on with her storyline in Alex's absence?
Berea: Im sure Jo has some baby fever, so Im very interested to see how Alex will get written off, and what theyll do. They might kill him offscreen and send her back into a depression.
Meaghan: You would think the answer will be yes, but with the uncertainty surrounding Alex and how he will get written out, I'm not sure where this storyline is going.
I need them to let us in on how Alex is going to be "officially" written out. Right now we are stuck in limbo, and it doesn't feel right.
Paul: She wants a child, for sure, but Im not sure where her head will be at. Alex is out of town, and shes going to feel like shes alone if he doesnt arrive off-screen to, you know, leave for good.
Jasmine: I have no idea what's going on here anymore. What will they scramble to sort out? I don't know how they plan to resolve Alex's disappearance, but I can envision an adoption arc for Jo.
It's something about it coming full-circle that makes it potentially interesting, even if she does it alone.
Who was the MVP of the hour?
Berea: Miranda Bailey easily. End of discussion.
Meaghan: I'm with Berea, Miranda. She was incredible.
Paul: Its a full house, er, table. Miranda Bailey was outstanding.
Jasmine: Miranda motherf*cking Bailey.
I'll give an honorary mention to Teddy for how she handled the Casey situation, too.
What was your favorite scene/moment/quote?
Berea: Richard: Are you okay?
Warren: No. But Miranda comes first.
Warren: Thanks for coming.Richard: Thanks for calling. Well, she seems okay.Warren: She's not. She says she is, but she's working through a miscarriage. She hasn't had time to break yet. I can't be there in that OR with her, so someone else who loves her needs to be there. In case she breaks.Richard: Of course.Warren: Thank you.Richard: Warren are you okay?Warren: No. But Miranda comes first.
Ben Warren is the best man who has ever graced the halls of Seattle Grace Mercy West Grey-Sloan, and Ill stand by this statement until my dying day. Benley was the only inter show romance we ever needed.
Meaghan: I loved that moment too, Berea! It made me so mad that they took him away from Grey's. I have always loved Ben. Can they please cancel Station 19 and bring him back, or just have him be written out and go back to starring on Grey's?
I also loved Helm declaring her love for Mer.
Paul: The interns chatting in the aftermath of the accident. They all survived the night. It was very reminiscent of early Greys.
Jasmine: Yes, Meaghan and Berea! I got a little emotional when Ben said that. Honestly, for as much as we raved about Miranda, I have to acknowledge that Ben Warren was the MVP of the Station 19 hour, and Jason George was amazing too.
But yes, aside from that, and Bailey's breakdown, I agree with Paul, I loved the final moments with the interns/residents. It did have the old-school Grey's Anatomy vibe, I love.
Over to you, Grey's Fanatics.
Do you agree with us, disagree? Hit the comments below and let us know!
Grey's Anatomy airs Thursdays on ABC at 9/8c.
And we sure would appreciate a follow of our new Twitter account as we work to rebuild our audience!
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Jasmine Blu is a senior staff writer for TV Fanatic. Follow her on Twitter.
Original post:
Grey's Anatomy Round Table: The Crossover Flopped, But Chandra Wilson Delivered! - TV Fanatic
While last week's crossover with Grey's Anatomy was very well-hyped, the most recent episode of Station 19 may have included a plot twist no one saw coming. Tonight's installment, which ended on a cliffhanger. As Us Weekly noted, the whole thing could mean curtains for one fan-favorite character.
Spoilers for the most recent episode of Station 19 below.
During the episode, Ryan (Alberto Frezza) returns to Seattle and ends up involved in an emergency situation and was left to babysit to young kids with Andy (Jaina Lee Ortiz). After a personal, surprisingly laid-back conversation between the two, one of the kids wanders in with a gun before shooting Ryan in the chest as he lunged forward to retrieve the firearm.
While Andy pleads with Ryan to stay with her as he bleeds out on the floor, the episode ended with the sound of a flat line, following by a message from Miguel Sandoval, who plays Pruitt.
"Consider this: 4.6 million children in the United States live in a household with at least one loaded unsecured gun. If you have a gun at home, store it, unload it, locked and separate from ammunition," the actor said. "No matter what, make sure you always ask about how guns are stored in other homes. You could save a life."
The clip also included a link to EndFamilyFire.org.
Last week, the drama had a crossover special with the long-running Grey's Anatomy, which made no secret before it aired that it would lead with the unexpected death of another beloved character. Still, things ended on a sentimental note, as a proposal between Owen and Teddy in the closing minutes left fans a little weepy-eyed.
Prior to the crossover's airing, Jason George, who plays Ben, told Entertainment Weekly that his character will be taking on a leadership role, which could factor into what happens to his character the remainder of the season.
"Ben's hit a spot now where he is, at any moment in time, kind of the guy who knows what he's doing," George explained. "If he's not in charge officially, he's capable of being in charge and will push the guy in charge. So in the bar, he definitely takes on a bit of that role, especially with some of the interns where hes barking orders at folks to get stuff done and they leap into action. But Ben is at that crossroads between firefighter and a doctor where he can bark orders at everybody."
New episodes of Station 19 air on Thursdays on ABC at 8 p.m. ET.
Doctors check in to Grey Sloan Memorial on Greys Anatomy both as doctors and as patients as we saw in last weeks back-to-back episodes of the hit ABC series and its sister show Station 19. But they also check out, too. Is that what is happening now that one of the shows actors is headed for Broadway?
Jesse Williams, who plays Dr. Jackson Avery, is making his Broadway debut in April in a revival of Richard Greenbergs Tony-winning play, Take Me Out. Does this mean hell have to temporarily hang up his stethoscope? Fortunately, no!
Ive known since the beginning of the season [about Williams play] and Ive been able to plan [Jackson]s storyline [accordingly], Greys executive producer Krista Vernoff recently told TV Line. Jesse is able to fly back one day a week; were just making it work [because] this was important to him.
The actors fans were thrilled when they learned he signed a new two-year deal with the ABC medical drama that will keep him on the show through Season 17. He joined the cast of Greys Anatomy in Season 6.
Many doctors have come and gone from Greys over the years. Last November, original cast member Justin Chambers suddenly exited as Dr. Alex Karev. Alexs absence has been explained as saying he left town to visit his mother.
There have been no announced plans for any specific returns but just because a doctor checks out of Greys doesnt mean theyre never coming back. Kim Raver, who originated the role of Dr. Teddy Altman in Season 6, left Greys in Season 8, popped up for a few episodes in Season 14, and then returned as a regular in Season 15.
Vernoff is both executive producer and head writer of Greys Anatomy. She also serves as the showrunner for Station 19, which airs Thursdays at 8 p.m on ABC. Greys Anatomy airs Thursdays at 9 p.m. on ABC.
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Is Jesse Williams Checking Out of Greys Anatomy? - Soap Hub
Abstract
Reassessment of citrullinome cargo in neutrophil extracellular traps confirms the presence of citrullinated peptides.
In an article previously published in Science Immunology (1), we reported a previously unidentified mechanism of induction of pathogenic adaptive immunity against citrullinated antigens in rheumatoid arthritis (RA). This mechanism was mediated by an interaction between neutrophil extracellular traps (NETs) and synovial fibroblasts (FLS) that promote the presentation of citrullinated peptides identified in NETs by the FLS to the adaptive immune system. The identification of citrullinated peptides in NETs was performed by a variety of methods described in this and previous manuscripts and included the use of citrulline-specific probes, antibodies, and mass spectrometry (MS) (1, 2).
Following this publication, we were contacted by an investigator with concerns about our MS analysis. Given the magnitude of the mass shift expected between a citrulline and an arginine residue (+0.984 Da), misannotation of citrulline-containing peptides can occur because of fragmentation of misassigned monoisotopic mass peaks. In addition, the +0.984-Da mass increase upon citrullination is identical to the mass increase observed upon protein deamination of asparagine and glutamine residues within a peptide, thereby confounding the correct assignment of citrullinated species. Last, most of the previously identified peptides contained a C-terminal citrulline. Whereas several endogenous proteases, including cathepsin B, cleave after citrulline, trypsin, the protease used in sample preparation, does not (3). These discrepancies prompted us to reevaluate the initial proteomic data more stringently. Upon reevaluation, the spectra were either not consistent with citrullination or not definitive enough to be consistent with a citrullinated residue. Therefore, we generated new samples for analysis. Herein, we report the identification of 58 sites of citrullination on 32 proteins that are present in NETs using a highly stringent data analysis workflow.
In the analysis, healthy neutrophils were purified as described (1), resuspended in RPMI, seeded in 12-well plates, and incubated with 2.5 M calcium ionophore (A23187, Sigma-Aldrich) or 100 g of rheumatoid factor for 4 hours at 37C. Supernatants were carefully removed, and NETs were harvested with micrococcal nuclease (MNase) (10 U/ml) in RPMI for 15 min at 37C. Supernatant-containing NETs were isolated via centrifugation and stored at 80C until analysis. Note that the proteomic analysis was performed on NETs and not from cells treated with ionophore or rheumatoid factor; hence, it is not possible to generate control proteomes.
NETs (50 g) were precipitated with 20% trichloroacetic acid. The pellets were washed with acetone and then resuspended in urea (8 M). Ammonium bicarbonate (100 mM) was then added to the solution. Samples were reduced with dithiothreitol (15 mM) and alkylated with iodoacetamide (12.5 mM). Next, samples were diluted to 1.8 M urea with phosphate buffer (pH 7.4). Samples were then digested overnight at 37C with Trypsin Gold (2 g, Promega) and 1 mM CaCl2. Tryptic peptides were separated on a ZORBAX extended C18 column (Agilent) over a 1-hour, biphasic gradient from 0% buffer A (10 mM ammonium bicarbonate plus 5% acetonitrile) to 100% buffer B (10 mM ammonium bicarbonate and 90% acetonitrile). The resulting 96 fractions were pooled to yield 6 fractions for liquid chromatographytandem MS (MS/MS) analysis.
Fractions were dried and resuspended in 25 l of 5% acetonitrile/0.1% trifluoroacetic acid. Peptides were eluted from a gravity-pulled analytical column packed with 3-m (100 ) Magic C18AQ particles using a biphasic linear gradient from 5 to 60% of B (acetonitrile and 0.1% formic acid) in mobile phase A (water and 0.1% formic acid) over 120 min. Ions were introduced by positive electrospray ionization at 1.4 kV into a Thermo Scientific Q Exactive hybrid mass spectrometer. Raw data files were peak processed by MaxQuant 1.6.3.4, and identifications were searched by Andromeda against the Human SwissProt database. First-search mass tolerance was 20 parts per million (ppm) for precursors and 20 ppm for fragment ions, and main search tolerance after precursor mass recalibration was 4.5 ppm for precursors and 20 ppm for fragments. All positive citrullination sites were verified manually.
To unambiguously detect and verify the presence of citrullinated arginines, we followed a recently proposed workflow (4). First, we filtered the data to remove C-terminal citrullines. Next, we compared the observed MS isotopic envelopes to predicted envelopes for arginine- and citrulline-containing (+0.984 Da) peptides. If the MS spectra demonstrated a mass shift correlating with citrulline, then we searched for a neutral loss of isocyanic acid (CNOH, 43.0058 Da) in the MS/MS fragmentation spectra. Neutral loss of isocyanic acid, which is unique to citrulline-containing ion fragments, is frequently observed during high-energy collision dissociation (4). It is important to note that, although deamination of glutamine and asparagine results in a similar mass shift, it does not yield the neutral loss. Using this algorithm, we identified 58 sites of citrullination on 32 proteins (Table 1; spectra data available upon request).
r, citrulline; c, alkylated cysteine; m, oxidized methionine; n or q, deamination; m/z, mass/charge ratio; IO, ionophore; RF, rheumatoid factor.
Notably, three of the proteins we found as being citrullinated, actin, heat shock protein 90 (HSP90), and plastin-2 (PLSL), were also identified as being citrullinated in RA patient samples (5). In addition, over half of the proteins found in the NETs were also previously found in the proteome of RA synovial fluid and neutrophils treated with ionophores (6). Together, our new data uncovered 15 previously unreported citrullinated proteins present in NETs.
The identified proteins include vimentin and myeloperoxidase (MPO), the latter also identified in the original report (1). Notably, citrullinated vimentin, /-tubulin, -actin, and NCF1 are known RA antigens (1, 4, 7, 8). In addition, we identified S100-A8, plastin, coronin, moesin, and erzin, which are all linked to RA (9). Several inflammation-associated proteins were also observed (S100A8, MPO, LKHA, and HMGB1). Furthermore, our list of citrullinated proteins includes numerous cytoskeletal and cell motility proteins such as PLSL, myosin, vimentin, tubulin, and actin. S100A8 is interesting because several targets of the iNOS-S100A8 transnitrosylase complex (i.e., EZR1, MOES, and VIME) are also present as citrullinated proteins in these NETs. Last, linking our analysis to NETosis is our identification of several citrullinated chromatin binding proteins (HMGB1, HP1B3, and HNRPU) and NCF1, which is a component of the nicotinamide adenine dinucleotide phosphate oxidase complex.
In summary, these findings continue to support the conclusions of our previous publications (1, 2), mainly, that NETs are a source of citrullinated autoantigens. We have identified 58 sites of citrullination in 32 proteins from purified NET samples induced via rheumatoid factor or calcium ionophore. Because citrullination is a modification in such low abundance, generating a comprehensive database of citrulline-containing peptides found in any complex proteome is challenging (10). In addition, the need to verify each identified peptide via manual inspection of MS and MS/MS spectra has hampered the growing field exploring this specific modification. Methods to enrich for citrulline-containing peptides and/or computationally automate the process of picking out true citrulline peptides by neutral loss MS/MS analysis would further facilitate the process of decoding the citrullinome.
Funding: This work was supported by the Intramural Research Program at the National Institute of Arthritis and Musculoskeletal and Skin Disease/NIH (ZIAAR041199). This work was also supported in part by NIH grants R35GM118112 (P.R.T.), F32GM128231 (A.J.S.), 1R01GM117004 (E.W.), and 1R01GM118431-01A1 (E.W.).
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Technical comment on Synovial fibroblast-neutrophil interactions promote pathogenic adaptive immunity in rheumatoid arthritis - Science
Chimeric antigen receptor T-cell (CAR-T) therapies that are made from a patients own T cells haveproven effective in blood cancers, and they are showingpromise for treating solid tumors, too. But scientists are still looking for ways to fine-tune this form of immunotherapy.
Now, scientists at the University of North Carolina (UNC) at Chapel Hill havefound new methods for either dialing up CAR-T cells cancer-killing effects when needed or dampening their activity to avoid severe side effects. The technique involvestwo proteins that are responsible for stimulating the cells. The team published the results in Cancer Cell.
In immunology, its always about balance; you don't want to have too much T-cell activation, and you don't want T-cell activation to be too low, the studys co-first author Peishun Shou, Ph.D., said in a statement. We wanted to keep the T-cell activation and tumor killing at a suitable or sustainable level.
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Shou worked in a team led by UNC Lineberger Comprehensive Cancer Centers Gianpietro Dotti, M.D. In 2018, Dottis team developed CARs that can target a protein called CSPG4, and they showed the treatment worked in mice with glioblastoma.
For the current study, the researchers zeroed in on two different types of CARs, whichare differentiated by the signals that activate them. CAR-T cells tumor-regression ability is first triggered by a signal called CD3-zeta, which is commonly used as the main CAR component to recognize markers on tumors. But the cellsalso require co-stimulatory moleculeseither CD28 or 4-1BB proteinin addition to CD3-zeta to further boosttheir activation.
The UNC team found that they could improvethe activityof T cellsthat areco-stimulated by 4-1BBby increasing the expression of a tyrosine kinase called LCK.
What we found is that the LCK molecule can bind to the CAR, enhancing the CAR-T cell activation and signaling transduction, which therefore will help CAR-T cells get a better tumor-killing effect, Shou said.
RELATED:Redesigned CAR-T eliminates dangerous cytokine release syndrome in lymphoma trial
Use of CAR-T therapy has been linked to a dangerous side effect called cytokine release syndrome, so scientists have also been working on methods to rein in CAR-T activitywhen necessary.
Shou and colleagues found such a safety switch in CAR-T cells that are co-stimulated by CD28. They showed that an enzyme called SHP1 attenuates T-cell activity. They discovered that adding a drug named AP21967which is an analog to Pfizers immunosuppressive drug Sirolimus (rapamycin)can pull SHP1 to the CAR to reduce cytokine release. Whats more, the transient recruitment of SHP1 didnt kill the CAR-T cells or impair their antitumor effects, the researchers demonstrated in a mouse model.
Many other efforts have been made to reduce CAR-Ts toxic side effects. For example, a research team led by the University of Southern California Norris Comprehensive Cancer Center developed a CAR variant called CD19-BBz(86) that didnt trigger any serious side effect in 25 lymphoma patients.
As for4-1BB, it's a target that has already attracted some interest among drug developers. Pfizer is developing utomilumab (PF-05082566), a 4-1BB agonist, to leverage the receptors T-cell stimulation effect. The drug is being paired with Pfizer and Merck KGaAs anti-PD-L1 immunotherapy Bavencio in a phase 2 study in solid tumors.
The UNC scientists want to use their findings on CD28 and 4-1BB to improve CAR-T treatments against blood cancers and to potentially make them viable for solid tumors.
Researchers in the CAR-T immunotherapy field now want to solve the solid tumor problem, Shou said. Solid tumors have an immunosuppressive microenvironment, so you need stronger CAR-T activation.
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Improving CAR-T therapy for cancer by regulating 2 proteins - FierceBiotech