#tcr work in progress

[lin-iva]

TCR Birthday Bash 2023 Day 4: Secret Heritage

If any of you want to ask if she's their biological daughter ? For now yes. I'm still trying to put together the story of Dani's relationship and background story, so there probably will be changes in the future.

In here, Dani got stuck while she travels back in time and till now is trying to find a way to go back to her own time and universe. In her efforts of trying to get back, she tends to help them with their cases while trying not to interfere too much in their relationship.

Story Time :

For now, her story goes like this. In the far future, Haru after an accident from one of their adventures becomes immortal and then both Baron and her decides to get married. Also, the Bureau expand and gathers members from all accross the world, universes, etc.

One day, they have a child, through magic from one of their adventures. Sadly because of how she comes to being, her young body is fragile, prone to sickness and unfortunately wouldn't last long. Baron and Haru tries every way they could to perfect her body so that she could live and grow long enough. They finally found a way to do so, that is by drinking some sort of magic embued potion that unbeknownst to them is sealing a dark and powerful entity. Whoever drinks it, will cast demise to a lot of worlds.

Danielle (Dani for short) ever since young have accept that she won't live long, and doesn't want to put pressure to her parents. In Her tenth birthday, they suprise her with the cure. After she drinks it, the party that once brought joy turns into a catastrophe. The entity emerge, thanks her, and proceed to cause chaos.

Dani's appearance changes, the physical traits that she has gotten from her parents suddenly dims and make her appearance seems darker. Once she realized that the situation has gotten people killed, she then blames Baron and Haru for the things that happened that day. Finally putting a strain to their relationship.

Ever since then, Dani became bitter and closed, she still hasn't forgiven her parents despite their efforts. But, deep down she loves them too much and doesn't want them to get hurt. She'll do anything to keep them safe.

Oof this is long, there are still a lotttt of stuff that i want to explain but for now this is enough.

[chialeah]

TCR Episode 4 and 5

I didn’t have a chance to write any notes on last week’s episode (i was very busy lol) so here’s some thoughts about episode 4 and 5!! I’d love to hear your thoughts! Spoilers ahead!

Episode 4

1. I lowkey had the biggest crush on Jason Isaacs as Lucius Malloy when I was a kid so I was so excited to see tom get to work with him! His accent threw me off but it makes sense since he is an alter-it’s legit the most quintessential British accent for a reason!

2. Toms acting! His expression of Danny’s emotions gets better and better every episode!

3. I did take notice of the stock footage of London from that era used to connect the scenes. At first I thought it was weirdly out of place and unbelievable. But now I know it was probably intentional! Danny 100% didn’t actually leave New York! When would he actually have the time between the shooting and getting arrested? Was the stock footage from films Danny’s watched before?

4. Watching Danny realize it has only ever been himself in these situations is gonna hurt so bad!

Episode 5

1. This freakin’ show 😭 Episode 5 almost made me cry multiple times. the london travel guide! danny’s abuse and trauma! the racist attack in front of danny! tom acting with his eyes! adam sacrificing himself for danny! What an emotionally haunting episode.

2. So Adam was Danny’s first alter! So Adam was created before any of the emotional/sexual abuse Danny experienced but was used so danny could disassociate from the trauma and details of it. The moment Rya helps Danny understand what has been going on is going to end me lol!

3. The softness and vulnerability in Tom’s expressions this episode was so so so good! Emmy was also excellent in this episode! Episode 5 was definitely a turning point in establishing what led Danny to where he is now. Every point of this episode leads the audience to be more empathetic towards everything that’s taken place so far.

4. It looks like we’re getting more of Rya’s backstory next episode so yay! This will hopefully help progress the story deeper AND sort of explain the things that we’re missed in the first few episodes. Everyone’s been acting SO WELL so far so no doubt it’s only going to get better with these next five. I’m excited!

My episode ranking so far:

3-5-4-2-1

[candyswift-ny]

CAR-T Cell Therapy & Surgery for Solid Tumor Treatment: The Whole is Greater Than Sum of Parts

The past decade has witnessed ongoing progress in immune therapy to ameliorate human health. As an emerging technique, chimeric antigen receptor (CAR) T-cell therapy has the advantages of specific killing of cancer cells, a high remission rate of cancer-induced symptoms, rapid tumor eradication, and long-lasting tumor immunity, opening a new window for tumor treatment.

CAR T cell therapy works by reprogramming patients' own immune cells to attack their tumor cells. A recent study conducted by researchers in the Perelman School of Medicine at the University of Pennsylvania found that this therapy may also enhance the effectiveness of surgery for solid tumors.

The study is published in Science Advances on Jan. 11, 2023, titled “Chimeric antigen receptor T cells as adjuvant therapy for unresectable adenocarcinoma”, and reports that the research found a method to allow the mice to survive the tumor recurrence.

Surgery is highly effective if the solid tumor has not spread. The main obstacle is that during the surgery, it’s usually hard for surgeons to clearly distinguish a tumor from the surrounding healthy tissues. Thus, post-surgical recurrence due to remaining microscopic tumor cells is common.

The research team tried to find an answer to this obstacle with an eye to applying an anti-tumor treatment to kill any residual tumor cells immediately after tumor removal, and they tested with CAR T cells for two cancer types: triple-negative breast cancer (TNBC), which lacks all of the three major breast cancer markers, and pancreatic ductal adenocarcinoma (PDA), the most common type of pancreatic cancer. Both types are notoriously hard to cure.

The CAR T cells were engineered to home in on the protein mesothelin, a surface marker on both types of tumor cell in the experiments. Without the CAR T cell and fibrin gel, the remaining tumor tissue grew and the mice succumbed within about seven weeks. With the gel, however, residual tumor tissue swiftly disappeared in 19 of 20 mice, and these animals survived without wound-healing complications or other apparent side effects for the remainder of the observation period.

Further experiments showed that CAR T cells targeting mesothelin have the potential to attack healthy cells bearing that protein marker after intravenous injection, and the toxicity was decreased by local injection of the CAR T cells compared to direct injection of the cells into the blood.

“This study demonstrates the promise of CAR T as an add-on to surgery for solid tumors. And this approach can be broadened to deliver other cellular therapies and anticancer agents in addition to CAR T cells, potentially boosting the antitumor effectiveness even further.” commented a scientist at Creative Biolabs, a biotech company providing TCR and CAR T therapy development services as well as ready-to-use TCR and CAR T&NK cell construction products.

Researchers all over the world are trying to enhance the effectiveness and safety of CAR T-cell therapy, and it’s believed that this direction will be a big step toward curing cancer.

[traceemerald]

-January 27th, Factory Tyrant's Journal, Mini Entry 24.1-

This journal entry won't actually have any progress in my survival world, instead I'll note what I've learned from testing IC2 nuclear reactors, as well as what I learned from browsing the Industrial Craft 2 wiki.

This is quite long for a mini journal entry, so feel free to skip this if you don't like walls of text telling you how reactors work.

First off, nuclear reactors are more complicated than just "put in fuel rods and coolant, get power".

Nuclear reactors come in 2 versions, EU reactors and fluid reactors, here's what the different types of reactors look like.

From left to right, there's an EU reactor, and EU reactor with 6 reactor chambers, and a fluid reactor.

Fluid reactors aren't a separate block, to make a fluid reactor you have to cover an EU reactor with a hollow 5x5x5 cube of reactor pressure vessels (or reactor access hatches, reactor fluid ports, and reactor redstone ports).

Both types of reactors require a redstone signal in order to run, otherwise they'll shut off.

Here's what an EU reactor's GUI looks like.

In this screenshot, you can also see some different types of uranium fuel rods, heat vents, heat exchangers, and neutron reflectors in my inventory.

You can't place anything in the X marked slots, but you can turn them into regular slots by placing up to 6 reactor chambers directly adjacent to the main reactor block, each reactor chamber will add 6 slots.

Any fuel rods you place in here will generate power, but will also generate heat that you need to get rid of.

The main way to get rid of heat is by using heat vents, which will take heat from adjacent components (or the reactor itself if you have the right kind of heat vent), then expel heat into the air where it can't harm anything.

If a heat vent is taking heat faster than it can expel it, it will start to overheat and eventually break.

Heat exchangers won't get rid of heat, but they can move it between adjacent components to prevent them from breaking.

Heat exchangers will also break if they can't get rid of heat fast enough.

Any generated heat that isn't handled by components goes directly to the reactor's core, causing the reactor itself to eventually overheat and explode, here's what the crater of a reactor explosion looks like.

As you can see, I don't want this to happen right next to my base, luckily this is in a creative mode world I made specifically to test reactor explosions.

One more thing about EU reactors is that fuel rods generate more power (and more heat) when placed next to each other, or when placed next to neutron reflectors.

If you fill every slot with fuel rods, the reactor will explode in less than 5 seconds.

Anyway, onto fluid reactors, which are mostly the same apart from a few key differences, here's the fluid reactor GUI.

Fluid reactors have 2 internal tanks, one for coolant, one for hot coolant.

The functionality of components placed inside a fluid reactor is largely the same as inside an EU reactor, with 2 main differences.

First, fuel rods don't generate power, only heat.

Second, any heat expelled via heat vents is used to convert coolant into hot coolant, which is the only product of a fluid reactor.

If the reactor doesn't have any coolant, heat cannot be expelled, and the reactor will overheat.

In order to get power from a fluid reactor, you need to put hot coolant through a liquid heat exchanger, which will produce regular coolant as well as heat.

Any coolant from the liquid heat exchanger can be sent back to the reactor.

IC2 actually adds 3 different types of energy, electric energy, heat energy, and kinetic (or rotational) energy, although most machines only use electric energy.

Create (not part of TCR) goes a lot more in-depth with kinetic energy/rotational force, in IC2 it's basically just arbitrary game units transferred between adjacent machines.

There are 2 ways to convert heat to electric, either using a stirling generator (simpler, but less efficient), or using a more complicated process which I'll describe below.

Putting heat and water (or distilled water) into a steam boiler (steam generator post 1.12) will cause it to produce steam (or superheated steam), which can then be sent to a steam turbine (kinetic steam generator post 1.12).

Steam turbines turn steam into kinetic energy and distilled water (superheated steam will be turned into kinetic energy and regular steam, which can be passed to a second steam turbine), kinetic energy is passed to a kinetic generator which uses it to generate electric energy.

Any distilled water from the steam turbine can be sent back to the steam boiler.

So that's the extend of my reactor knowledge after looking through the IC2 wiki, but I also did some research on other IC2 things that I'm either using now, or could possibly use in the future.

I think at the point I'm at in my survival world, making a nuclear reactor isn't worth the risk, especially now that I know more about my other options.

First off, I learned the actual numbers behind geothermal generators.

Geothermal generators will generate exactly 20 EU/t (electric units per tick), assuming they're provided with lava, so the 12 geothermal generators I have produce a total of 240 EU/t.

The problem with my setup isn't that I've moved past the point when geothermal generators are viable, it's that I'm still using copper cables for everything, which can only transfer up to 128 EU/t.

The reason my cables haven't burned is because the 240 EU/t of 12 geothermal generators is still tier 1 power, which doesn't burn copper cables, but I still need to upgrade my cables so they can transfer all the power I'm producing.

IC2 cables come in 5 varieties, each one can transfer 4x as much as the last (tin cables transfer 32 EU/t, copper transfers 128 EU/t, gold transfers 512 EU/t, HV (iron) transfers 2048 EU/t, glass fibre transfers 8192 EU/t).

Each cable also loses twice as much EU/block as the last, apart from copper cables (loses 0.2 EU/block, same as tin cables) and glass fibre cables (loses 0.025 EU/block, making them the best cable).

As for my pumping setup in the nether, while I can't easily improve it with Buildcraft, I can improve it with AE2, using fluix cables and ME fluid import/export busses, although this is more expensive than Buildcraft pipes.

I'm probably going to switch over to AE2 for fluid transport next time I play in my survival world.

Anyway, I should probably end this here.

I don't personally think a mini journal entry should be longer than a regular journal entry, but I also don't think I should post a regular journal entry unless I actually play in my survival world.

-End Mini Journal Entry 24.1-

[thecultrevived]

Lichenometry

[blairette-on]

Story: Once upon a broken world

https://www.fanfiction.net/s/13775967

Hello everyone!

Here I am with my new fanfic! It's a TCR one!

I don't know where I'm going with it, but I'll try my best! The summary and the title are bound to change as some point, haha!

I hope you'll like it!

Summary :

"A car passed by her, and the young woman had to move away to avoid being soaked by the water that splashed in her direction.

That’s how she noticed that strange house."

***

"The stranger looked familiar in a way she couldn't pinpoint, and it bothered her. Because she knew deep down she had seen him somewhere before."

[cornerstonc]

1, 3, 10 for the writing asks :D

Fic Meta | Accepting

Tell us about your current project(s)  – what’s it about, how’s progress, what do you love most about it?

oh l o r d i'm working on. a number of different WIPs tbh lies down they're all tcr-related atm tho. but if i narrowed them down to those which i intend to Finish and most likely publish at some point, we end up with uhhh. five?? five fics lies down

this is going to be a long ramble isn't it let me just preemptively throw down a readmore i'm so sorry

a condensed history of natori's career in the kingdom of cats

aka the 'young natori and cat king meet and Hate Each Other' fic jfifeoa honestly just an excuse for me to write out my headcanons on how they met, their early relationship, natori's family, and the cat king's childhood/family/Issues lmao. the third chapter is so close to being Done, but the fourth and fifth are. Not. so once that third chapter is posted, i'm not sure how long the wait for the next two will be rip

my favorite thing about it is probably just. idk, getting to explore those headcanons i'm attached to, aha. also getting to write natori in a lot of different ways i don't normally get to Here is fun

playin possum

a much shorter post-canon fic in which the cat king comes down with a cold and makes it everyone else's problem. it's. well. Not Particularly Close to being finished, but i at least have an idea of where it's going rip

my favorite thing is that it gave me the opportunity to write natori uttering the line 'sire, if you make me hand-feed your own cold medicine, it will not be as sensuous as you're clearly expecting it to be' jfieoa

have courage and be bold

an excuse plot involving yuki's wedding attire and natori's sewing skills which exists only bc i wanted to write them interacting with each other rip

it's somewhat close to being done. there's little patches here and there which need to be filled in, and the ending is still eluding me lmao. my favorite thing tho is honestly just getting to write for yuki and delve into my own ideas about her, aha, especially after canon wraps up. also love the idea of natori (and probably natoru, too) sharing embarrassing baby stories about lune with her

the willow bud processional

or 'the cat king tries to teach natori how to dance while natori tells him a fairy tale and the self-indulgence levels inherent in that plotline alone blew away the ozone layer'

i've actually posted this one before on my fic blog, but it was unfinished. i posted it anyway bc i was So Tired of looking at it, i just wanted to sort of wash my hands of it rip now it's been some time and i've been looking at it again like. 'u know maybe i have the motivation to finish this now that i'm not Sick Of It'

the ruins

wheezes okay last one the fic i keep bringing up which basically amounts to 'i wonder how much suffering i can dump on my favorite character before i feel bad.' the answer is A Fuckn Lot. natori being kept alive after what's for all intents and purposes a coup bc one particular noble took a not easy to define interest in him. it's ended up way frickn longer than i intended it to but i've had a lot of. well. maybe not fun, exactly, but Something Positive while writing it despite its bleak tone oof

it's inching closer to being finished rip my favorite thing about it is without a doubt getting to indulge not only in my love for Despair but also getting to write natori reacting in ways which i hope are maybe. somewhat surprising considering his usual demeanor and such, aha he literally gets into what amounts to a physical fight in this fic ok that's kind of hilariously unfitting

What is that one scene that you’ve always wanted to write but can’t be arsed to write all of the set-up and context it would need? (consider this permission to write it and/or share it anyway)

there’s some ideas i have that fall under this category tbh-- that one idea i’ve brought up involving a groundhog day loop, where haru figures out she’s been living the cat kingdom day over and over again and can’t remember Why. she and the bureau, when she eventually gets them involved again, come to the conclusion it’s the cat king’s doing, and that he’s doing it to get his own way (tho they also find that conclusion kind of odd, considering he didn’t seem particularly broken up about how it all went down by the end of it)

of course. as i’m so. Wont to do rip the ultimate reveal would be that it was natori who had been behind the time loop, bc i have this story idea nicknamed ‘tfw u realize how things worked out in canon was the good end’

the basic idea is that natori was offered the opportunity to redo That Particular Day in order to alleviate some of the damage, but his efforts caused an even worse ending to the fiasco, and so now he’s been redoing it over and over again trying to get it Back On The Right Track. more than likely whatever creature gave him the original opportunity knew this would happen

bc i love the groundhog day loop trope to pieces, and i also love a good skirting too close to the despair event horizon fjioaef

i also have this random desire to write baron interacting with natori in that one ‘organized crime dark comedy’ idea i brought up once, where yuki is the cat king’s daughter and lune is the hapless ditz who fell in love with her, got all mixed up in the family business, and now is in too deep and not digging it lmao mostly bc i just have this vivid Idea of natori being just. profoundly two-faced in a conversation with someone who can send that polite, ambiguous energy right back at him lmao (bc i had the thought that, much like canon, natori is the one technically running everything. but in this case, it’s a good deal more Sinister, and his meticulously crafted bearing is quite brittle and wan in the right light)

How would you describe your writing process?

lies down scattered and capricious are two words which come to mind i'm too laid-back and lack the discipline to write on a legit schedule or when i'm not Feeling It, and my attention can sometimes flit from one WIP to the next in just a matter of half an hour (if not less rip)

it's also frustratingly easy for me to get burned out on an idea or a fic, which is part of the reason i usually have so many different WIPs going on at once orz

[mashasxart]

hi i’ve been working on this the last few. uh. the Last While here. idk, i’ve always wanted to do this kinda meme laughs tho i think there’d be much more noticeable and Cooler progress if i’d started at like. 2007 or something l ma o

i might do that if anyone wants to see my oldest bad art the internet had to suffer through jfjfk;da

this was fun, but admittedly i don’t. see much change in the last seven years now that it’s all here together, ha. i’m also hoping this ends up large enough that u can actually read it rip

also after looking at it myself, i’m not sure the smallest text is readable, so here’s a transcript under the cut

2014-- get into kfp somewhere in the first half of the year, shortly after joining tumblr, too. pretty sure it was all kfp all the time from theni really liked palette challenges

2015-- i had opened mr. ping's blog by now, so some of these are references to AUs and verses on that blog. also randomly using crayons on occasion. used a lot of brush and ink this year thanks to art classes

2016-- still lots of kfp. also a random dip into svtfoe for a couple months, too, at the end of the year when the excitement for kfpIII had worn off and i was feeling restlessi remember figuring out some shading/color theory stuff here

2017-- the invasion of whis. still using some pen and ink, but also running out of steam on the whole coloring thing, apparently

2018-- still in the dbs fandom. inking pictures falls by the wayside bc Too Much Effort. instead i started relying on darkening lines digitally after they were scanned in lmao

2019-- i don't recall when i started using the yellow copier paper proper but this year i'm pretty sure i drew almost exclusively on it. with some new colors occasionally. tcr and natori Invaded in august

2020-- The Year Of The Furry. on a small scale, too. flip flopping between dbs and tcr fandoms and ultimately staying mostly in tcr. slowly starting to get the energy back for coloring like i used to

2021-- floating between various fandoms with a focus still on tcr but. who knows. coloring at least has come back a bit. maybe this will be the year i finally start inking again!

[drama-queen-supreme]

Author Interview Tag

Tagged by @hella1975 , aka the jerk who doesn't realize I haven't written anything besides essays since middle school and thus am freaking out about getting back into fanfic.

Name: Espí, Shamooda

Fandom(s): Cyrano de Bergerac, A:TLA, The Adventure Zone, Hadestown, Newsies, Hedwig and the Angry Inch, Good Omens, Check, Please!, The Cat Returns, Matilda, etc.

Where you post: Here on tumbo

Most popular oneshot: I think it was a songfic for "The Cat Returns" that I posted on FFN.Net? (We don't talk about my middle school fics)

Most popular multichapter fic: Another songfic, we don't talk about my middle school works for a reason, okay?

Favorite story you've written so far: NONE OF THEM, THEY WERE ALL FUCKING CRINGE

Fic you were nervous to post: ALL OF THEM, I WAS A MIDDLE SCHOOLER

How you choose your titles: They were all songfics, take a fucking guess

Do you outline?: I didn't used to, but if there's one thing fandom in general taught me, it's that it sucks when a fic gets abandoned because the writer loses motivation or doesn't know where to take the plot. Plus, I always respected the TCR writers, because they always finished writing their fics before posting.

Complete: All my fics were oneshots, so yes?

In progress: Ghost AU. No I won't elaborate ♡

Coming Soon/Not yet started: Ghost AU. Really nervous since my middle school multichap was a oneshot collection, and this might turn into a multichapter thing? So we'll see how it goes.

Do you accept prompts?: Not really, my writing is crappy and I don't want to disappoint people.

Upcoming work that you're most excited about: The next chapters of The Art of Burning and Fractures, most definitely!

Tagging the following people: @zuko-just-wants-his-honor , @jekkiefan , @joyfulsongbird , @bongripsncoffee , @red-0ak-tree (y'all don't have to do it if you don't want to! ♡)

[massieh]

Various TCR inspiration, part 1 :

SCP FOUNDATION. To absolutely no one’s surprise. It would be an injustice not to credit the entries among the SCP Foundation that helped to inspire the Cage, Appalachian Unit, Divide, Depths, and more. The series’ original form was an AU thread with a roleplay partner of 10+ years, with Andy’s character loosely based on the role their OC took in the thread. Not only do I have permission to use this, but they regularly give feedback on the story’s progress; they may be one of three who have always known the story in full. The original thread barely holds a resemblance to TCR’s final product, but inspiration can still be seen in the SCP Foundation, primarily in the entries SCP-354 and SCP-096. The former, I’m still working toward weening out, as it’s still too similar.

HOZIER’S TAKE ME TO CHURCH. I feel like everyone was inspired by this one at some point. It happened to come out when I was just starting out with the story. The deep tones of his voice, the poetry of the lyrics, the haunting-but-empowering swell of the choir backing him? I want to create ( without forcing it, of course ) that same feeling. 

NEW ORLEANS. Honestly, the whole place, since that’s where Sector 52 was settled. With both its sci fi scope and a post-apocalyptic setting, however, things aren’t wholly recognizable. However, a few places littered throughout the city did supply direct inspiration, as well as landmarks for the narrative. Included are Bourbon Street, Jackson Square, Jazzland / Six Flags New Orleans, the Mercedes-Benz Superdome, Iberville Projects / Bienville Basin Apartments, and the Delta Queen. These locations were used in narrative, but with Six Flags on its way to being demolished and Iberville recently being renovated, a change was both necessary and exciting. The locations will remain, but my approach to portraying them as individual settings in an altered timeframe is changing.

ANGEL LORE AND FICTION. And when I say “angel lore”, I don’t mean flying babies in diapers. I also don’t mean whatever tf came out of spn. I mean those scary motherfuckers with six wings and bodies made of eyes. Utilized throughout the book are Enochian, various references to the angels Azrael and Apollyon, as well as plot devices designed around the various mythologies of angels. Yes, the Bible is one source, but it isn’t the only one. Not by a long shot. I’ll give the full list as I find references during the next edit.

JOB 41. “Can you pull in Leviathan with a fishhook or tie down its tongue with a rope? Can you put a cord through its nose or pierce its jaw with a hook? Will it keep begging you for mercy? Will it speak to you with gentle words?” The entirety of the chapter can be read here. Job questions God’s competence on the grounds that Job himself is suffering, so God is clearly doing something wrong. Chapters 40-41 are God’s response, asking if Job can do the work of a god, since he’s so goshdang smart. Personally, I’ve always loved the description of the Leviathan and wanted to show that love in narrative.

[catsafarithewriter]

From the resent promt list (if you feel up to it): 15 (“I’ve come to the conclusion that I can no longer rely on you to make any rational decisions.") for Baron and Haru. Have fun! :)

Day 4: Prank War

A/N: So I am definitely cheating and re-purposing a half-written prompt I got sent ages ago (sorry, nalua!) for Day 4: Prank War of the TCR Birthday Bash. This is a human AU ficlet. 

Based on this post.

x

Having Louise for a sister meant that Baron wasn’t entirely unaccustomed to strange messages at stranger times of day. 

Like the time she had texted that there was an intruder making noises in her bathroom, and only later sent a photo of a ROOMBA hover trapped behind the door that her landlord had set to automatic. 

(Baron had been halfway to her house armed with a crowbar before the situation had been resolved.)

Or the week that she communicated solely in memes.

(She sent him the entirety of the Bee movie. Twice.)

Or the 11pm text demanded to know which Greek philosopher followed Plato.

(Aristotle. It was Aristotle. And she was cheating in a pub quiz.)

(She won.) 

So he wasn’t completely shocked when he received the frustratingly contextless text stipulating the urgent need for him to send a photo of a shaved leg.

He scanned the message for signs of any obvious typos and, when he came to the reluctant conclusion that Louise had meant what she’d said, sent back, “Why?”

“it’ll be funny”

“You already have shaved legs. Take your own picture”

“a guy accidentally messaged me and he wants a pic”

“There are easier ways to pick up guys. Also. You already have a girlfriend??”

“I KNOW. god. okay but he’s being creepy and needs to be taken down like a 100 pegs you’re my big brother aren’t you meant to protect me?”

“We’re twins, Louise”

“an even better reason why you should be helping me twinkie”

“*twinnie damn autocorrect”

Baron ignored the mistake. “You were born first”

“taller = big brother”

“You’ve been calling yourself the ‘alpha twin’ for years”

“your point is?”

Baron didn’t reply. 

Eventually, Louise sent, “Humbeeeeeeerrt”

“Do you know how much effort it takes to shave a leg?”

“try shaving two on a regular basis. it sucks. okay but seriously H this guy has obviously pressured some girl into giving him her number and the fake one she gave just happens to be mine I’m not going to throw this chance away come on he’s an absolute Creep he asked for a shower pic I NEED to troll him”

Baron stared at the almost incomprehensible mess of run-on sentences, weighing up the options as if he wasn’t already mentally commited to the cause. 

“How much leg do you need?”

“!!! Thank you!! also as much as you can convincingly pass for a lady”

“How long is this going to take?”

“lol like no time at all”

x

There was no answer at Baron’s door when Haru knocked, so she let herself in with her spare key and found the beginnings of dinner in the works. It looked like Baron had at least selected out the food - pots and pans arranged and the vegetables ready to be prepared - but that was where it ended. Which was a pretty long way off completion, considering that she had been promised a meal. The radio was faintly playing and the lights were still on. 

So. A tad horror movie-esque. 

She knocked hesitantly on a wall and called up, “Uh, is anyone home?”

There was a noise from the floor above, and she cautiously took to the stairs. If this was a horror movie, she considered, she’d probably be the first to die. Curiosity and the cat and all. 

The sounds of life - or, at least, movement - lead her to the bathroom door and she gently nudged it open. 

She stared. 

Baron stared back. 

He flashed a nervous grin. “Hello, Haru.”

“We were going to have dinner, remember?” she asked, latching onto the sole remnant of logic she had left. “You invited me over, remember?”

Baron looked at her, and then at the single, hairless leg sticking out of the bathtub. “Ah.”

“Yes,” Haru agreed. “Ah.” 

She swept her gaze over the scene before her: her boyfriend predominantly hidden beneath a layer of bubbles save for his head, his phone in one hand, and that immaculately shaved leg tipped with hot pink nail polish. 

“I like the toes,” she eventually settled on.

Baron wiggled his foot. “A bit brighter than my usual shade, I’ll admit, but I think it suits me.”

“Is it mine?”

“To be fair, I didn’t have any other to use.”

A beat went by. Haru had exhausted all other possible lines of conversation and Baron seemed unusually taciturn. She inhaled and then sighed heavily into her facepalming hand. “Alright. Alright. Now I’ve come to the conclusion that I can no longer rely on you to make any rational decisions, can you tell me what the hell is going on?”

He looked at her, and then his phone, his leg, and then back to her. “A favour for my sister?” he offered.

“Oh god, is she blackmailing you again?”

“No!” Baron dropped his gaze. “Anyway, she doesn’t have anything to blackmail me with since I asked you out.”

“Sure. And what kind of favour-not-blackmail requires you to pose so prettily for the world’s most misleading selfie when we’re meant to be having dinner?”

“She’s trolling a guy.”

A flicker of understanding - far faster than Baron had pegged Louise’s plan - flashed across Haru’s face. “Oh. Ohhh.”

Baron raised an eyebrow. “The speed at which you accepted that alarms me.”

“Then you’ve never been chatted up by a guy who won’t leave you alone,” Haru answered and, fair enough, Baron conceded, she had a point. “Have you sent Louise the picture?”

He nodded, and she motioned for him to hand her the phone. “Now, dry yourself off and get some clothes on so we can have dinner. I’m starving.”

x

It was just as they were moving onto pudding that there was a ring at the door and Louise arrived in a flurry of chaos and dramatics. She flounced to the spare seat at the table with, “I got a reply from leg guy!”

Baron paused in sharing out the tiramisu to give his sister a withering look. “Hello to you too, Louise.”

“Leg guy?” Haru echoed. “Is that what we’re calling him now?”

“Well, there are other things I could call him that would be far less suitable for the dinner table. Hi Haru.”

Haru couldn’t help it. She grinned. “Heya, Louise. Baron’s told me all about your trolling plan.” 

“I take it you approve?”

“I love it. So, what did he say?”

“He wants to see the rest of me.”

Both ladies looked to Baron. 

“I’m not shaving my other leg,” he immediately said. 

Louise’s phone beeped, and she pulled it up. “Oh wow, I leave him on ‘read’ for five minutes and he’s already getting lewd. Trust me, guys, I’m saving you by not showing that pic. Whoever Yuki is, she dodged a bullet.”

Haru suddenly went very quiet. “He thinks he’s talking to a Yuki?” she eventually asked. 

“Yeah… why…?”

“I told a guy who was hitting on me last night that my name was Yuki,” Haru said, her voice getting progressively lowering with every admission. “And gave him a fake number.”

“You gave him my number,” Louise corrected.

“I thought it was random!” Haru cried. “I was picking numbers out of my head, I didn’t mean to pick an actual number I knew!”

“You told him your name was Yuki?”

“It was the name of the cat I had as a kid,” Haru admitted miserably. “I did tell him I was taken. And, even if I wasn’t, I wouldn’t have been interested. He was a creep.”

Louise elbowed her brother. “See? I told you he was a creep.”

“Yes, yes, your intuition is very smart,” Baron dutifully replied. “Now, hand me this phone so I can teach this creep a lesson.”

Louise hesitated. “Baron, I love you, but I am not burying a body for you.” 

“The only thing I’m going to be killing today is this snivelling brat’s ego.” 

Louise raised an eyebrow and, after sharing a shrug with Haru, passed across her phone. Baron set to taking a selfie. 

“What if he’s bi?” Haru asked suddenly.

“Or pan?” Louise added.

Baron sent the photo and hesitated. “Then I guess I have a date.”

Louise flicked a piece of tiramisu at him. “You also have a girlfriend.”

“The offer of a date,” Baron quickly amended. There was a ping from Louise’s phone, and the two ladies watched as his eyebrows rose. “Or not.” The eyebrows rose a little further. “I’m vaguely impressed by how many times he manages to repeat the same word in various forms. That is a one-word vocabulary and no mistake.”

“Give,” Louise commanded.

“Hang on, I have an idea.” He quickly took another photo, this one highlighting the stark comparison between the shaved and non-shaved parts of his leg, his face visible just to make his identity abundantly clear. “I don’t want him getting any ideas about my first photo being anything but mine.” 

Following that, Louise’s phone beeped several more times in rapid succession, each one shorter than the previous. Louise finally managed to snatch back her phone. “Are you quite done?”

“It looks like he’s had enough for today.”

“Yeah, no shit.” Louise’s eyes widened as she scrolled through the waves of swearing she’d received. Haru leant over her shoulder and gave an appreciative whistle. 

“You weren’t kidding about the vocab thing,” Haru said. “Oh, wait, he got inventive - there’s a ‘mother’ in front of that one. And... he’s finished.” She gave an impish grin. “Well. On to dessert.” 

x

Baron had almost forgotten about the incident until a week later, when Louise texted him again. 

“message from leg guy’s number again” she sent.

Baron paused in his book to answer back. “Is it an apology?”

“not from him”

“?”

“from his flatmate. he apologises for his ex-friend and wants to know if you’re single. name’s Toto”

“You know I’m taken??”

“that’ll be a no then”

[adalhied-prime]

Author Meme

Stealing from @ao3commentoftheday​ cause I wanna and they said we could

Author name: Rowena Bensel on FFN, Rowena_Bensel on AO3

Fandoms you write for: My main fandoms are Transformers (Bayverse and Prime) and The Cat Returns, though I also have an old HP fic, and some Mass Effect, Dragon Age and Game of Thrones WIPs. 

Where you post: On AO3 and FFN, Cat Returns fics will also be posed on my TCR sideblog (@ro-visiting-the-bureau​), and I try to post or reblog to my writing blog (@writing-ro​). 

Most popular one shot: As Beautiful - a MegOP rewrite of the Stoik/Valka scene from HTTYD 2

Most popular multi-chapter: The AllSpark Changed My Life - my current WIP where a self-insert oc wakes up in the place of Sam at the start of the Transformers movies and decides to change things in an attempt to prevent the events of Dark of the Moon and Age of Extinction.

Favourite story you wrote: Tuck Me In! - a one-shot of Optimus tucking his daughter into bed. I just love parent-child dynamics and cute kids, as well as the country song “It Won’t Be Like This For Long” by Darius Rucker. 

Story you were nervous to post: You’re All Mine Now - It was my first completed smut fic and I never posted anything smutty outside of a few tumblr rps, so I didn’t know how the response would be. It’s been good so far, and now all I need to worry about is my mom finding out about it.

How do you choose your titles: Sometimes it’s a line in the fic, like Tuck Me In or As Beautiful, and other times it just summarizes the theme or feel of the fic, like AllSpark was originally “The Transformers Screw Up My Life”, before some narrative alterations made me go for the current title.

Do you outline: Vaguely, though generally only my multi-chapter WIPs, and they function more as a timeline of events than a specific outline 

Complete: Most of my fics

In progress: AllSpark is my only published current WIP, but I also have a Miko/Megatron fantasy AU, a Mass Effect time-travel fix-it AU I’m working on with my brother, a Mass Effect/TFP crossover, a TFP/Naruto AU, and a few various AllSpark AUs/X-overs that can’t be published until after the main story finishes.

Coming soon: I post new chapters of AllSpark on Fridays, and the Miko/Megatron fantasy AU is the closest of the other WIPs to publication. 

Do you accept prompts: No, I’ve tried but aside from something short like a “i’ll write three or five sentences for __ prompt” meme, I can’t get ideas to flow with random prompts.

Upcoming story you are most excited to write: Probably any of the Mass Effect WIPs, just cause ME is very prevalent in my mind right now. 

[lupine-publishers-lojpcr]

Lupine Publishers | A Hmsh2 c.2332 T>A Mutation and Membrane-hMSH2/ TCRγδ/NKG2D-Mediated Cytotoxicity of Human Vγ9vδ2 T Cells in Ovarian Serous Cystadenocarcinoma

Lupine Publishers | LOJ Pharmacology & Clinical Research

Abstract

Membrane human MutS homologue 2 (mhMSH2) is a well-characterized endogenous ligand for human Vγ9Vδ2 T cells, but its germline gene expression in mhMSH2-overexpressing ovarian serous cystadenocarcinoma (OSC) cells and tissues is unclear. Herein, we discovered a silent hmsh2 c.2332 T>A mutation in the OSC HO8910 cell line (GenBank accession no.MG674653) and identified serval novel soluble forms of hMSH2 protein from its whole cell protein extracts and culture supernatant. The mhMSH2/ TCRγδ/NKG2D-mediated recognition and cytotoxicity of Vγ9Vδ2 T cells were validated in mhMSH2/6-overexpressing SKOV3 cells. Positive expression of cytoplasmic and/or membrane hMSH2/6, cytoplasmic and/or nuclear hMSH3 and complete loss of nuclear expression of hMSH2 was observed in all examined ovarian cancer tissues. The clinical significance of the novel hmsh2 c.2332 T>A mutation, the soluble full-length and truncated forms of hMSH2 proteins, and the complete loss of nuclear hMSH2 protein expression in OSC development and human Vγ9Vδ2 T cell-mediated anti-OSC immunity remain to be further elucidated.

Keywords: Hmsh2; Mutation; Membrane hMSH2; Vγ9Vδ2 T cells; Ovarian serous cystadenocarcinoma.

Abbreviations: hMSH2: Human MutS Homologue 2; MMR: Mismatch Repair; M: membrane; EBV: Epstein-Barr virus; OSC: Ovarian Serous Cystadenocarcinoma; ATCC: American Type Culture Collection; IHC: Immunochemistry; mAb: monoclonal Antibody; FCM: Flow Cytometry; PFA: Paraformaldehyde; FITC: Fluorescein Isothiocyanate; DAPI: 4’,6-diamidino-2-phenylindole; LS: Lynch Syndrome.

Introduction

Human MutS homologue 2 (hMSH2), a critical element of the DNA mismatch repair (MMR) system, is normally localized in the nucleus of host cells and dimerizes with hMSH3 or hMSH6 (hMSH3/6) to form complexes that participate in DNA damage and cell apoptotic signaling. Inherited or acquired defects in the hmsh2 gene or protein lead to dysfunctional correction of errors in DNA synthesis and duplication, the cell cycle and Ig isotype switching of B cells and thus have a close association with the genesis of many types of tumors [1,2]. In our published study, we reported a broad overexpression of membrane hMSH2 (mhMSH2) in a series of epithelial tumor cell lines and Epstein-Barr virus (EBV)- transformed B lymphoid cells [3]. mhMSH2 overexpressed on malignant cells was subsequent characterized as a stress-inducible endogenous protein ligand for human Vγ9Vδ2 T cells, a subset of innate immune cells that play a crucial role in antitumor/antiviral immunity and autoimmunity [3-5]. The induction of ectopic mhMSH2 expression on renal carcinoma cells by oxidative stress via the p38 mitogen-activated protein kinase and c-Jun N-terminal kinase pathways with interleukin-18 promotion was subsequently revealed, but the systemic expression of hmsh2 gene in mhMSH2- overexpressing carcinomas is still unknown [6]. There are 238,700 estimated new cases of ovarian cancer and 151,900 deaths per year worldwide, and thus, this disease is the fifth most common cause of female cancer-related death in the United States in 2018 [7,8]. Among the 4 subtypes of ovarian cancer (high-grade serous, endometrioid, clear cell ovarian carcinomas and non-epithelial subtypes), Ovarian Serous Cystadenocarcinoma (OSC) is the most common and lethal type in women [9]. Previously, we reported the unusual ectopic mhMSH2 expression on the human OSC cell lines HO8910 and SKOV3 and the specific binding of mutated mhMSH2 on SKOV3 cells to synthesized OT3 peptides of human Vδ2 TCR [3,10]. In this work, we further elucidated the hmsh2 gene mutation and ectopic subcellular protein expression in HO8910 cells and the mhMSH2-mediated recognition and cytolytic effects of Vγ9Vδ2 T cells towards SKOV3 cells. The abnormal subcellular distribution of hMSH2/3/6 in different subtypes of ovarian cancer tissues was demonstrated by immunochemistry (IHC).

Materials and Methods

Cell lines, culture medium and tumor tissues

HO8910 cells (OSC) were maintained in RPMI-1640 complete medium (Invitrogen, Shanghai, China). SKOV3 (OSC) and HK-2 (proximal tubular cell line derived from normal kidney) cells were cultured in 10% FBS DMEM/F12 medium (Niuyin, Beijing, China). All cell lines were purchased from the Cell Culture Center of the Institute of Basic Medicine, Chinese Academy of Medical Sciences and confirmed to have no mycoplasma contamination before use. Expansion and subset separation of effector human Vγ9Vδ2 T cells were carried out as we previously described [3]. Ovarian cancer tissues belonging to different histotype (OV-1, serous adenocarcinoma; OV-2, embryo sinus carcinoma; OV-3, cyst-myxomatous adenoma) were freshly obtained from Peking Union Hospital with informed consent provided by the patients. The use of human tissues for research was also approved by the ethics committee of the Guangzhou Women and Children’s Medical Centre, Guangzhou Medical University, Guangzhou Institute of Pediatrics (2015020917).

Gene sequencing of hmsh2 in HO8910 cells

Appropriate numbers of HO8910 cells in the logarithmic growth phase were collected for total mRNA extraction and cDNA reverse transcription. The target hmsh2 gene was amplified using fragment PCR as we described in Subcellular hMSH2 expression is aberrant in membrane-hMSH2-overexpressing cervical, lung and gastric cancer cell lines and tissues (article in progression). Three PCR products with predicted lengths (990, 1250 and 1549bp) were purified and introduced into the pGEM-T easy vector (Promega, USA). Recombinant plasmids were positively selected and characterized by Not I digestion before gene sequencing. Human msh2 variants in HO8910 cells were screened by alignment with the full-length hmsh2 sequence (NM_000251) using Laser gene 7 software.

Soluble full-length and truncated forms of hMSH2 in the whole cell extracts and culture supernatant of HO8910 cells

Soluble forms of hMSH2 protein in the complete protein extracts and the cell culture supernatant of HO8910 cells were simultaneously separated by SDS-PAGE and blotted with antihMSH2 McAb (65021-1 Ig, 1:500, Proteintech Group, USA) and with goat anti-mouse IgG/HRP secondary antibody (1:5000, Zhongshan Jinqiao Company, China). 𝛽-actin was blotted as endogenous reference control (MW 43 kDa).

Growth curves of HO8910 cells

For growth curves of both OSC cell lines, appropriate numbers of trypsin-digested HO8910, SKOV3 and HK-2 cells were planted in 24-well plates in triplicate and cultured for 8 consecutive days in a 5% CO2 atmosphere at 37°C. Growth curves of the examined cell lines were obtained by using the cell counting method. The expansion rates of target OSC cells were compared with that of HK-2 control cells.

Quantitative real-time PCR for hmsh2 mRNA expression in HO8910 cells

The human msh2 gene transcription levels in HO8910, SKOV3 and HK-2 cells were comparatively analyzed by qRT-PCR. The results were processed with Sequence Detector Version 1.2 (Applied Biosystems, USA) and Sigma Plot 11.0 as we previously described [3].

Ectopic membrane, cytoplasmic and nuclear expression of hMSH2 in HO8910 cells

The membrane, cytoplasmic and nuclear hMSH2 expression in HO8910, SKOV3 and HK-2 cells were further investigated by laser confocal microscopy with different fixation reagents. The target cells (2-3×105) were cultured overnight on pre-autoclaved glass cover slips in a 24-well format, properly fixed with 4% paraformaldehyde (PFA) or ice-cold methanol for 10-15 min and blocked with 0.5% BSA for 30 min at 4°C before labeling with specific anti-hMSH2 (N- 20, Santa Cruz Biotechnology, USA) polyclonal antibody or rabbit IgG and fluorescein isothiocyanate (FITC)-conjugated goat antirabbit secondary antibody (Zhongshan Jinqiao Company, Beijing, China). The nuclei of the examined tumor cells were stained with 4’,6-diamidino-2-phenylindole (DAPI) (1:1000, Sigma, USA) before being mounted on a Leica DMIRE2 inverted microscope (objective, 40×; numerical aperture, 1.25).

Participation of mhMSH2/TCRγδ/NKG2D in Vγ9Vδ2 T cell-mediated anti-OSC immunity

For further analysis of the role of mhMSH2 in human Vγ9Vδ2 T cell-mediated anti-OSC immunity, effector Vγ9Vδ2 T cells were incubated with SKOV3 cells at different ratios (effector: target [E: T] 20:1, 10:1, 5:1 and 2.5:1). Cytotoxicity was measured with the LDH method, as we previously described [3]. For cytotoxicity blockade assays, target OSC cells were pretreated with anti-hMSH2 (N20, Santa Cruz Biotechnology), anti-NKG2D (149810, R&D Systems) and anti-TCRγδ (B1.1, Immunotech, France) antibodies before incubation with effector Vγ9Vδ2 T cells at different E:T ratios (20:1, 10:1, 5:1 and 2.5:1). The blockade cytotoxicity was measured and compared to that of the rabbit IgG blockade control group. Target SKOV3 cells were stained with anti-hMSH2, anti-hMSH3 and anti-hMSH6 antibodies to confirm the cell surface expression of hMSH2/3/6 antigens before cytotoxicity and antibody-blocking cytotoxicity assays.

Subcellular hMSH2/3/6 Distribution In OSC Tissues

For IHC analyses, freshly collected ovarian cancer tissues were classically made into paraffin sections (3-4 μm in thickness) after proper neutral formalin fixation. The biopsies were heated at 60°C overnight and gradient dehydrated with methanol before antigen retrieval in boiled citrate buffer, followed by overnight incubation with purified mouse anti-hMSH2 monoclonal antibody (mAb) (clone G219-1129,1:200), mouse anti-hMSH3 mAb (clone 52, 1:20), mouse anti-hMSH6 mAb (clone 44, 1:30) (BD Pharmingen, USA) or isotype-matched mIgG1 at 4°C in a moist environment after 3% H2O2 treatment. The coloration was developed with PV9000 reagents (Zhongshan Jinqiao Company, Beijing, China) and captured with a Leica DM3000 imaging system.

Statistical Analysis

GraphPad Prism 7.2 (GraphPad Software, Inc., La Jolla, CA, USA) was used for statistical analysis and data plotting. Data are presented as the mean ± SD. Differences between/among groups were compared with Student’s t-test (two-tailed) or one-way ANOVA, P<0.05 was considered statistically significant.

Results

Hmsh2 gene sequencing and soluble full-length and truncated hMSH2 protein blotting

 The PCR products of hmsh2 in HO8910 cells were 990, 1250 and 1549 bp in length (Figure 1A). By aligning the spliced full gene sequence with hmsh2 (NM_000251), we observed a point mutation (hmsh2 c.2332 T>A) in the HO8910 cell line (Figure 1B). It was a silent mutation that did not alter the primary structure of hMSH2 protein. The gene sequencing data of mutated hmsh2 in HO8910 cells were submitted to GenBank (Accession number: MG674653) for release. No additional gene mutation of hmsh2 was found in other mhMSH2-overexpressing epithelial tumor cell lines (data not shown here). By SDS-PAGE separation and western blots, several soluble forms of hMSH2 proteins were identified from the whole HO8910 cell protein extract [including the full-length form (MW ~105 kDa) and four truncated variants (MW~70, ~62, ~55 and ~40 kDa)] and the cell culture supernatant (MW~88, ~68 kDa) (Figure 1C).

Hmsh2 gene transcription and ectopic cytoplasm/ membrane expression

The growth and proliferation of both OSC cell lines were depicted with growth curves determined by cell counting assays. Both HO8910 cells and SKOV3 cells achieved rapid growth on day 5 and expanded much faster than the normal control HK-2 cells (Figure 2A). The mRNA expression of hmsh2 was slightly decreased in SKOV3 cells but substantially elevated in HO8910 cells compared with HK-2 cells (Figure 2B). Further detection of the ectopic membrane overexpression of hMSH2 on target OSC cells with laser confocal microscopy showed that both HO8910 and SKOV3 displayed different degrees of green fluorescence on the cell surface after labelling with a specific anti-hMSH2 antibody. The cell surface green fluorescence was much stronger on SKOV3 cells than on HO8910 cells. No green fluorescence was observed on normal control HK-2 cells (Figure 2C, the upper panels of pictures). The subcellular distribution of hMSH2 in HO8910 and SKOV3 cells was further determined by confocal microscopy with ice-cold methanol fixation. The membranes of both cells displayed different degrees of green fluorescence. The density of green fluorescence in the cytoplasm and the nuclei of SKOV3 cells was much stronger than that of HO8910 cells. There was no green fluorescence observed in HK-2 cells (Figure 2C, the lower panels of pictures).

 Previously, we identified the membrane-overexpressed hMSH2 on several epithelial tumor cell lines as a stress-inducible endogenous protein ligand for human Vγ9Vδ2 T cells [3,6]. Herein, mhMSH2-mediated recognition and cytotoxicity of Vγ9Vδ2 T cells towards target OSC cells were confirmed by cytotoxicity and independent specific antibody blockade cytotoxicity assays. mhMSH2 overexpression on OSC targets was validated with flow cytometry (FCM) before cytotoxicity and cytotoxicity blockade assays (Figure 3A). At E:T ratios of 20:1, 10:1, 5:1 and 2.5:1, the cytotoxic efficiency of effector human Vγ9Vδ2 T cells against target SKOV3 cells was 53%, 26%, 23% and 12%, respectively (Figure 3B). The ectopic mhMSH2-mediated recognition and cytotoxicity could be strongly blocked by specific anti-hMSH2, anti-NKG2D or anti-TCRγδ antibodies at different E:T ratios, indicating the participation of mhMSH2 in Vγ9Vδ2 T cell-mediated anti-OSC immunity by NKG2D/γδ TCR recognition (Figure 3C).

 Subcellular distribution of hMSH2/3/6 in OSC tissues

As shown in Table 1, all ovarian cancer cells displayed positive ectopic cytoplasmic and/or membrane hMSH2/6 expression and cytoplasmic and/or nuclear hMSH3 expression, while loss of nuclear expression of hMSH2 was observed in all 3 categories of ovarian cancer tissues. Strong and clear cytoplasmic and/or membrane hMSH2/3/6 expression was strikingly observed in ovary cyst-myxomatous adenoma nests (Figure 4A). Total loss of nuclear expression of hMSH6 was found in 66.67% (2/3) of the examined ovarian cancer tissues (Figure 4A). hMSH2/3/6 expression showed substantial heterogeneity among individual ovarian cancer patients (Figure 4B).

 Discussion

Human msh2 is one of the most crucial genes involved in the DNA MMR pathway and was strongly associated with increased tumor mutational burden in a multivariate analysis [11]. Recent studies have shown that high hMSH2 expression is significantly associated with smoking, while low hMSH2 expression is an indicator of MMR deficiency in lung adenocarcinoma [11]. The high expression of hMSH2 accompanied by increased PD-L1 expression and CD8+ T cell infiltration therefore leads to the development of a prominent immunotherapy-responsive microenvironment for lung adenocarcinoma and acts as a potential surrogate biomarker of tumor mutational burden to identify immune checkpoint blockade responders in this disease [11]. Moreover, recent studies revealed that MSH2-MSH6 played a crucial role in activation-induced deaminase-initiated antibody diversity by recognizing uracil(s) in the Ig gene loci to generate DNA breaks [12]. Many studies have identified mutations in hmsh2 as diagnostic and/or prognostic factors in carcinomas not only in the US and Canada but also in the Middle East and China [13-15]. Human msh gene mutations also have strong potential as novel candidate triple-negative breast cancer predisposition genes and are closely associated with acute adverse events and survival in rectal cancer patients receiving postoperative chemoradiotherapy [16-19]. Pathogenic or likely pathogenic germline mutations in pms2, msh2 or msh6 were detected in 0.5% (6/1,179) of lung cancer patients [18]. In this study, we screened a novel mutation in the gene sequence of hmsh2 at c.2332 T>A in the OSC cell line HO8910. This is a silent mutation that theoretically does not result in a change to the amino acid sequence of hMSH2 protein or to the phenotype of HO8910 cells. However, a rapid expansion and a strong increase in hmsh2 mRNA expression were observed in this cell line compared with the control cell line. Moreover, several soluble full-length or truncated variants of hMSH2 proteins were observed in the whole cell protein extract and the culture supernatant of HO8910. We deduce that these altered biological characteristics of HO8910 cells and the genesis of OSC might be linked to the hmsh2 c.2332 T>A variation, as evidence showed that germline mutations of hmsh2 and its family members were closely associated with the pathogenesis of Lynch syndrome (LS). For example, hmsh2 c.2152 C>T alteration has been recently reported as a founder mutation in Portugal; its high proportion implies combined screening for this mutation and some other previously reported founder mutations will be helpful in the genetic testing of Portuguese families with suspected LS [19]. The occurrence and clinical significance of hmsh2 c.2332 T>A and soluble full-length and truncated forms of hMSH2 proteins in clinical OSC patients remain to be clarified in the future.

Compared with HO8910 cells, SKOV3 cells displayed stronger ectopic membrane and nuclear expression of hMSH2 in laser confocal microscopy and FCM analyses, suggesting better membrane anchoring and a more efficient nuclear importing system in SKOV3 cells. The ability of the notably overexpressed mhMSH2 on SKOV3 cells to promote human Vγ9Vδ2 T cell-mediated recognition and cytotoxicity via TCRγδ/NKG2D receptors towards target OSC cells was later validated by independent cytotoxicity and specific antibody blockade cytotoxicity assays, providing further evidence for mhMSH2 functioning as an OSC-associated self-antigen (ligand) for human Vγ9Vδ2 T cells in anti-ovarian cancer immunity [10]. In the absence of information/data demonstrating that the observed mutation impacts function, one cannot determine if the mutation is physiologically relevant. In further investigations on the subcellular distribution of hMSH2 and its companion proteins in ovarian cancer tissues, we found that all examined ovarian cancer specimens (serous adenocarcinoma, embryo sinus carcinoma, cystmyxomatous adenoma) displayed positive ectopic cytoplasmic and/or membrane hMSH2/6 expression and cytoplasmic and/or nuclear hMSH3 expression, and total loss of nuclear expression of hMSH2/6 commonly occurred in almost all of the ovarian cancer tissues. Considering the high hmsh2 gene transcription and the loss of nuclear distribution of hMSH2 protein in HO8910 cells, we hypothesized that the nuclear importing system of hMSH2 and/ or hMSH6 protein was dysfunctional. By contrast, recently, it has been reported that a high overall rate (16.2%) of MMR deficiency was surprisingly observed in ovarian endometrioid carcinoma and was significantly associated with increased IFOG (International Federation of Obstetrics and Gynecology) grade and CD8+ intraepithelial lymphocyte infiltration but not with cancer-specific death [19]. These findings suggest a promising future in which loss of nuclear expression of hMSH2 and/or hMSH6 protein may function as effective screening/diagnostic markers or therapeutic targets for different subtypes of ovarian cancers and as endogenous immune ligands not only for Vγ9Vδ2 T cells [20]. We expected that a human cell-based assay system for functional testing of hMSH2 and its family members will facilitate the identification of highrisk ovarian carcinoma patients and the generation of individual autoantigen-targeted immunotherapies for OSC.

Conclusion

In this study, we identified a silent hmsh2 c.2332 T>A mutation (GenBank accession no. MG674653) along with serval novel soluble truncated forms of hMSH2 variants in HO8910 cells and validated the mhMSH2/TCRγδ/NKG2D-mediated recognition and cytotoxicity of Vγ9Vδ2 T cells against mhMSH2/6-overexpressing SKOV3 cells. We also demonstrated the abnormal subcellular distribution of hMSH2/3/6 in the examined ovarian cancer tissues. The clinical significance of the critical findings in OSC genesis and human Vγ9Vδ2 T cell-mediated anti-OSC immunity remain to be further investigated.

 https://lupinepublishers.com/pharmacology-clinical-research-journal/pdf/LOJPCR.MS.ID.000142.pdf

https://lupinepublishers.com/pharmacology-clinical-research-journal/fulltext/a-hmsh2-c2332-ta-mutation-and-membrane-hmsh2-tcr%CE%B3%CE%B4-nkg2d-mediated-cytotoxicity-of-human-v%CE%B39v%CE%B42-t-cells-in-ovarian-serous-cystadenocarcinoma.ID.000142.php

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[pharmaphorumuk]

6th CAR-TCR Summit 2021

Calling all CAR & TCR Drug Developers – Welcome to the CAR-TCR Summit

This August, the 6th CAR-TCR Summit returns; bigger and better than ever! This is the premier industry forum to advance the development of CAR and TCR technologies from bench to bedside, emphasizing progress in novel constructs, solid tumors, allogeneic development, and automation in 2021.

Access the program for a first-look preview of the draft agenda and speakers.

As the industry matures, and leaders prepare for commercial readiness, this year we are introducing 7-tracks of content to the agenda bringing everything from discovery to market access, to ensure success on the global stage.

We are excited to announce some HUGE additions to CAR-TCR Summit, including:

New Regulatory Bootcamp – gain end-to-end visibility of the regulatory challenges from IND to BLA approval with seasoned experts from Novartis, Kite, BMS, Celyad

NEW Track Announced – Entering phase trials? Waiting for clinical readouts? Wondering how to scale up? Access case studies and thought leadership to maximize development in the Early Phase Development track – Immatics, Aleta Biotherapeutics, Poseida Therapeutics

Focus on Cell Characterization & Beyond Oncology – join a dedicated post-event day to explore preclinical characterization to CMC gold standards and debate the best indications for CAR-TCR technologies with the pioneers pushing the bounds of therapeutic application.

See how the summit will respond to the industry’s questions and expectations by downloading the agenda.

Don’t forget: the dates for your diary are August 30 – September 2, 2021

This will be an absolute must-attend event for anyone working on CAR and TCR technologies, looking to engineer a disease-free world. Make the most of this opportunity to connect, collaborate and catalyze your candidate’s success.

The post 6th CAR-TCR Summit 2021 appeared first on .

from https://pharmaphorum.com/events/6th-car-tcr-summit-2021/

[kashmirspeaks]

Immune cell which kills most cancers discovered by accident by British scientists in major breakthrough,

A new type of immune cell which kills most cancers has been discovered by accident by British scientists, in a finding which could herald a major breakthrough in treatment.

Researchers at Cardiff University were analysing blood from a bank in Wales, looking for immune cells that could fight bacteria, when they found an entirely new type of T-cell.

That new immune cell carries a never-before-seen receptor which acts like a grappling hook, latching on to most human cancers, while ignoring healthy cells.

In laboratory studies, immune cells equipped with the new receptor were shown to kill lung, skin, blood, colon, breast, bone, prostate, ovarian, kidney and cervical cancer.

Professor Andrew Sewell, lead author on the study and an expert in T-cells from Cardiff University’s School of Medicine, said it was “highly unusual” to find a cell that had broad cancer-fighting therapies, and raised the prospect of a universal therapy.

“This was a serendipitous finding, nobody knew this cell existed,” Prof Sewell told The Telegraph.

“Our finding raises the prospect of a ‘one-size-fits-all’ cancer treatment, a single type of T-cell that could be capable of destroying many different types of cancers across the population. Previously nobody believed this could be possible.”

Asked if it meant that someone in Wales was walking around completely immune to cancer, Prof Sewell said: “Possibly. This immune cell could be quite rare, or it could be that lots of people have this receptor but for some reason it is not activated. We just don't know yet.”

Therapies which engineer immune cells to fight specific types of cancer already exist, but they are currently only useful for some forms of leukaemia, and do not work for solid tumours, which account for most cancers.

Those treatments - known as CAR-T and TCR-T therapies - involve taking immune cells from a patient which are then altered so they can lock onto molecules which sit on the surface of cancer cells.

The cells are then grown in huge numbers and injected back into the patient’s bloodstream.

CAR-T therapy is now given for certain forms of leukaemia but does not work for solid tumours, the vast majority of cancers. TCR-T therapies can work in some other cancers but they need to lock onto molecules called HLA, which vary widely in the population.

In contrast, the new cell attaches to a molecule on cancer cells called MR1, which does not vary in humans.

The GLOBOCAN project, under the World Health Organization, ranks nations around the world based on the frequency of cancer occurrence among citizens. The ranking rates include all forms of cancer, except non-melanoma skin cancer. As per the study released by World Cancer Research Fund Organization, here are the top 49 countries with the highest occurrences of the disease.

All rates are age-standardized and per 100,000 people.

It means that not only would the treatment work for most cancers, but it could be shared between people, raising the possibility that banks of the special immune cells could be created for instant ‘off-the-shelf’ treatment in future.

When researchers injected the new immune cells into mice bearing human cancer and with a human immune system, they found ‘encouraging’ cancer-clearing results.

And they showed that T-cells of skin cancer patients, which were modified to express the new receptor, could destroy not only the patient’s own cancer cells, but also other patients’ cancer cells in the laboratory.

Prof Sewell said the ‘right people’ are now interested in developing the potential new therapy and said progress could now move ‘quite fast’. The team says human trials on terminally ill patients could begin as early as November if the new treatment passes further laboratory safety testing.

Professor Oliver Ottmann, Cardiff University’s Head of Haematology, whose department delivers CAR-T therapy, said: “This new type of T-cell therapy has enormous potential to overcome current limitations of CAR-T, which has been struggling to identify suitable and safe targets for more than a few cancer types.”

Professor Awen Gallimore, of the University’s division of infection and immunity and cancer immunology lead for the Wales Cancer Research Centre, added: “If this transformative new finding holds up, it will lay the foundation for a ‘universal’ T-cell medicine, mitigating against the tremendous costs associated with the identification, generation and manufacture of personalised T-cells.

“This is truly exciting and potentially a great step forward for the accessibility of cancer immunotherapy.”

Commenting on the study, Daniel Davis, Professor of Immunology at the University of Manchester, said it was an exciting discovery which opened the door to cellular therapies being used for more people.

“We are in the midst of a medical revolution harnessing the power of the immune system to tackle cancer. But not everyone responds to the current therapies and there can be harmful side-effects.

“The team have convincingly shown that, in a lab dish, this type of immune cell reacts against a range of different cancer cells.

"We still need to understand exactly how it recognises and kills cancer cells, while not responding to normal healthy cells."

The research was published in the journal Nature Immunology.

#Courtesy #The_Telegraph

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