#Gene Cleaves

[savage-kult-of-gorthaur]

BEFORE HE REALLY WENT "CUCKOO," SONNY WORE A PIN-STRIPED SUIT.

PIC INFO: Spotlight on Sonny the Cuckoo Bird in his earliest cereal box art incarnation, and was designed by Gene Cleaves in 1962. Sonny's first cereal box appearance was made in the mid '60s, a wholenseven years after Cocoa Puff's first hit market shelves.

Source: www.pinterest.com/pin/370913719307824920.

[kurithedweeb]

I know we always talk about Garroth ending up looking exactly like his father, but what about Dante growing up to look eerily like Gene.

When he joins up with Phoenix Drop, he's still young. He's a little on the short side, still a bit too thin from life in the wild and imprisonment, and he's a little anxious and shaky around so many people after having grown unused to living in a village. The smiling faces of the citizens remind you of your old home, of clamoring crowds and standing frozen in the plaza as your brother . . .

Anyway, it's good here. It's easy to fit in. The guards joke around with you and make sure you're healthy. They don't know a thing about dual wielding, but you get plenty of sparring partners out of helping the local baker practice her magick, and you maybe make a friend too. You're not too sure how you feel about the Lord, but she's a kind soul and does her best to make sure you're comfortable here in town, and her kids are great. Babysitting the boys is easily your favorite duty. Yeah, it's good here. For the first time in a long while, you feel like you're doing good.

Then the war comes. The children and non-combatants are sent away. The jovial atmosphere of the guard tower has soured into solemn silence as you make your final preparations. In the morning, you step into the battlefield and you go to war for the first time in your life. You have a horrible feeling in your gut that it won’t be the last.

You, Sir Laurance and Sir Garroth make a good team. It makes you sick. The three of you cross the battlefield at a slow and inevitable pace, cutting down any soldier that dares stray too close, and together you cleave the enemy forces in half, scattering them. The killing comes easy to you. You had hoped that in this peaceful new village, with time, you would become unfamiliar to how easily you were once able to take a life, but right then you’re glad your body never forgot the motions of death. Glad for the blood that stains your hands—how can you be glad?

You can’t remember how long you fought for. Days, weeks? Surely not months, or so you think. Yours is a small force, and though Miss Lucinda is a good healer, she grows tired while the other army’s numbers are replenished time and again. You remember the bags under her eyes as she tipped a potion sip by sip into your mouth the time you were shot through the face.

You remember sneaking into the enemy camp in the dead of night, skirting around the edges of it to the back line where the archers rested. You quietly slit five of their throats before you were noticed, and managed to slash another across the belly before the arrow caught you in the side of the face, in one cheek and out the other. The wood of the shaft cracked when you bit down. It was everything you could do not to scream as you fled. Dale thought you were a fiend when you first stepped out of the shadows, face obscured in blood and cradling your jaw as you cupped a hand beneath your mouth in an effort to catch more blood before it left a trail. Laurance held you while Garroth split the arrowhead from the rest of it with a knife and pulled the shaft out the other side of your face, your jaw gripped tight in one hand to keep you from struggling. It took hours to pull the splinters from your cheeks and tongue before they sent you to wake the healer. The whole ordeal had been excruciating. You might have cried. You remember that a lot more clearly than most other times at war. After a while, it’s hard to tell which side spills more blood when so much is shed that red squishes out of the earth wherever you step.

Every day, you fought dawn to dusk. And then one day you won. By Nicole literally knocking some sense into her father, of all things! You find a quiet corner to throw up in and for a beautiful moment, you think life in this little town you’ve started thinking of as home will go back to being good. Until your Lord tells you to guard the village as she races past the gates, and she doesn’t come back. None who followed her do either.

For days, you stand waiting at the gates. You don’t eat, you don’t sleep. O’khasis is gone, Scaleswind has made a refuge of the plaza, and still there is no sign of your Lord or your brothers-in-arms. You won’t even leave to have your wounds seen to. Nicole has to drag a doctor to the gates to treat you, and the entire time you watch the forest hoping that any moment they will reappear. You only step away when someone brings you news that the ship that took the children away has returned. You should be the one to tell them.

Zoey knows something is wrong the moment she sees you. Levin and Malachi smile and ask where their mother is—they call you ‘uncle’ while they do. You get down on your knees before them, and you gather them close in your arms, and you cry as you tell them their mother has been missing since the day the war ended. You’re still holding them when the exhaustion catches up with you.

Zoey is with you when you wake. She tells you you’ve been out nearly two days. She fusses over you, and you know you’ve worried her because that’s what she does when she’s worried. You’re a mess anyway, so you let her fuss. You drink the broth she makes you, you change into the clothes she provides, you sit still while she cuts the unruly mats of your hair and shaves your face. You used to cut yourself shaving all the time, no one ever taught you how and you were only six or so when Gene was learning to; you don’t remember now how he showed you each step or the laugh in his voice at the face of disgust you made when you slapped a little hand into the lather on his face and left behind a tiny palmprint. Zoey doesn’t cut you once. When she’s done with you, she takes you by the arm and guides you back into civilization, where everyone who remained has decided already on search parties to go out looking for your missing friends.

You head each expedition. Dale brings himself out of retirement to watch over the town while you’re gone, and asks only that you also look for his son. Does he know you used to be a tracker, used to spend days in the woods trailing coyotes and runaways for enough coin to carry you through the cold months? You try for him, but the ground is soft still and every step anyone takes leaves a print, all overlapping and muddled. You keep an eye out as you circle the same stretches of woods for days, but you find nothing. Your group goes further and faster than any other, the first to find and dismantle bandit camps and dens of fiends, but no matter how far you go you find not a sign of anyone who has disappeared that day. It’s as though they vanished into thin air. Every time you return home, Dale looks at you with hopeful eyes, and every time you must take him aside and break his heart a little more. Eventually, he stops asking.

For a year, you search. The area has never been safer. You have never felt so alone as when people start to suggest that a funeral may be in order.

You feel like a monster for the rage in your voice when you denounce these people. You know they aren’t dead—you would have felt such a thing, you know, you would have felt pieces of yourself snapping like wire pulled too taut, you would have felt the sharp edges tangling inside you—it would have felt like it did when the brother you killed rose from the grave to slit your throat and cut your very existence from the memory of Boboros. You hear white noise rumbling in your ears when the first brave soul says Sir Dante, there’s been no sign for a year now, and your blood is boiling when you slap their comforting hand off your shoulder. You spit that you’re not giving up just because everyone else has taken no evidence of life to mean the surety of death, and with their pitying looks burning into your back to return to the woods. You scream into the trees until you can’t anymore. When it doesn’t help, you use your considerable tracking skills to hunt something, anything, until you feel human again.

You crawl back home the day before the funeral with your cape stained with blood; they held it back so you could attend. You polish your armor and swords until they shine, and the warped reflection of your own face makes you feel sick the way waging war did. You stand at attention the entire ceremony without moving a muscle. When Dale reads the names of the deceased at the end, offering their souls into the embrace of the Matron, you salute, and the clatter of your armor silences the crowd.

Everyone who fought in the war salutes with you. So do your Lord’s sons. You’re too tired to cry. You hold your salute long after everyone else has left.

The remaining forces of Scaleswind return home. One by one, family by family, the streets of your home empty. Without your Lord, without your guard, the citizens trickle out the front gates and never turn back. Some apologize to you as they say their goodbyes, and some of them you actually believe. You close the gate behind each of them until all that remains is you, Zoey, and your Lord’s sons. Then Zoey tells you she’s taking the boys to the Yggdrasil Forest. She holds you tight for too long and kisses your brow when you show them to the gate for the last time.

You can’t believe you ever thought you knew what loneliness was before this.

For five years, you are completely and utterly alone. You search and you patrol and you do your best to maintain the village. You don’t believe in Irene, but every day before dawn you stand before her statue and look down down down over the cliff’s edge and pray that this won’t be the rest of your life. That you haven’t deluded yourself into believing a fantasy, that you haven’t made such an incredible fool of yourself that people can’t bear to be around you, that you haven’t been forgotten. For five years, you pray that someone, somewhere, remembers that you exist. You look down down down over the cliff’s edge and have the terrible thought that you don’t know what you’d do if you were forgotten again.

The gate is falling apart. You don’t know how to repair the damage the weather’s done to it, you tried to patch the cracks but it never holds. With each year, you’ve been pushed further and further outtowards the coast. The only places you have the energy to maintain anymore are the guard tower and your Lord’s home. You blockaded the gates when the mechanism broke, you check it on occasion to be sure no bandits get in, and one day you hear voices from the other side. Familiar voices. You scramble up the wall and look over the other side at a boy you don’t recognize looking back up at you. He says, Is that Uncle Dante? and you climb down as fast as you can to embrace Malachi.

He’s nearly the age you were when you first met his mother. He’s grown tall, and strong enough to carry his brother on his back. Levin is fevered when you first see him, flush and hurting even as he dozes, and Malachi tells you he can’t walk from how bad he hurts. You remember how Zoey fretted over him when he was young, how sometimes he’d scream for seemingly no reason, and once you show them to their mother’s home Malachi refuses to leave his bedside.

You sit with them and ask where Zoey is. Malachi tells you of her obsession, and the relief that you are not alone in the belief that those who disappeared are alive feels like a hint of betrayal. You’re relieved that she’s driving herself into a downward spiral because of what? Because it makes you feel like you were reasonable to fight not to let their souls be put to rest?

You wait for her at the gates deep into the night and take her to her boys when she bursts from the woods, frantic that she’d lost them, and safe if your Lord’s home she holds you so tight your ribs hurt from the force of her grip. After so long, you’re not alone anymore.

You wake before dawn and strap your swords to your back. For the first time in a long time, you feel safe enough to go without your armor. You hike up the steep cliff to the Irene statue. You kneel before her to offer your thanks. You look into the pool at her feet and fear grips you by the throat.

Your brother’s face looks back at you.

You wear your swords the way he did. Your hair falls like his, dark in the shadow of Irene. Your face is gaunt and pale from old habits, eating only enough to sustain yourself so rations will stretch long enough for you to find more—do you remember how they starved Gene before they killed him? How they weakened him so he wouldn’t have the energy to fight? How pale and gaunt he was, dirt streaking over the side of his face, blood and grime drying in his hair, shaking and sweaty with how hard he fought back? Do you remember the scar that twisted around his throat when he returned from the dead to get his vengeance? Your collar is open over the scar he left twisting across your own, and it matches his own so very well. In the shadows of your eyes, you see his own staring back.

You think of the war. You think of how easy the killing was. You think of how easily Gene cut through the guards, the Lord, the memories of Boboros. The rage in his voice when he denounced you as his brother, the twist of his smile when he told you he would leave you to rot, Dante. No one will ever remember you. You can see that twist in the corners of your own smile, pushed into shape by the deep scars on your cheeks. You and your brother are the same.

You’re shaking too much to stand. You never go without your armor again.

[whitetaileddeer]

Extracts from Gio’s diary.

a little transcript in case my handwriting is too terrible:

august 2021 (before first breakup with aaron)

you are expanding and constricting in blues and purple, bruised lungs, beaten and deoxygenated above me. me, trapped in your ribcage. i feel like the body who’s spleen i sleep on had swallowed a stone, and i’m just rattling around your torso.

i wish i was buried between someones vertebrate but theres no one around to eat me. i dont think im a very a pretty stone, maybe id be more edible if i were smaller or if i were a kinder stone. i dont think people would care that i was hard to swallow then. But im not a pretty stone and im not a smart stone so they feel every scratch as i drag myself down their oesophagus.

I think it would be better if people got operations and surgeries to remove me anyway. i dont want to be in there anymore, i want to turn faster and farther in between their organs so they take me out. i dont really want them anyway. i want to be a stone on the shore, i dont think id be on a shingle beach. i dont know if there are any stones anymore, i think im the last to be removed. ive been feeling sick a lot more recently, my heart beats so fast and heavy in my chest. it shocks me

i think sometimes its easy to forget im alive, im just going through the motions

october 2021 (just moved schools)

“I am not an earth nor an adjunct of an earth / I am the mate and companion of people, all just as immortal and fathomless as myself”

I cant begin to understand him, his heart is pouring out love for everyone that passes him, how could he feel more connected to uncaring strangers than uncaring nature? the stranger chooses not to care, chooses to loathe and anger, nature has no say in it. It creates storms and tsumanis and fells trees and cleaves land in on itself through no fault of it own, through no choice. Maybe the stranger doesnt have a choice either, maybe that is nature, a passage of self and outward destruction writen into genes and dna and into lines of bark and blades of grass. Maybe its nature not to care, but that makes me too sad to believe. and anyway, if i was to be hurled into an uncaring sea or an uncaring crowd of strangers, id ask to be ploughed against rocks and turn sea foam pink, i dont like strangers.

I need to stop thinking about that stupid passage about his stupid abundance of understanding. I need to read something else, probably do some homework or something.

[shadowqueenjude]

Ok but what if bc Lucien has strong magic genes from his mother and the spell-cleaving genes from his father plus that all-seeing eye he can break mating bonds and he breaks the feysand mating bond and sparks chaos. Should I write feral Lucien?😈

[anulithots]

Random ask, can I ask what are apoptosis and caspases (in simple terms)?

I have been summoned

(my 'noorie rambles. be very afraid' tag will make so much sense now. I tried to talk about it in a way that's a way that's both engaging and simple. Lots of metaphors.)

Caspases destroy the cell if it needs to die. They dismantle the cell parts as the cell membrane turns into bubbles, containing all the damaged pieces within so that a white blood cell might disposes of them. That's apoptosis in a nutshell.

But why must the cell die you ask?

Plenty of reasons. Human embryos have webbed fingers, and apoptosis causes the webbing to dissipate. Tadpoles too, use apoptosis to rid of their tails so they can become frogs. Apoptosis opposes growth - aka mitosis - and it keeps the body in homeostasis - or a balance of sorts. The destruction to mitosis's creation.

However, what I find most interesting, what is the subject of much research nowadays, and what holds the most opportunity for angsty biology fanfics (most important/j) is that apoptosis opposes tumorigenesis. In other words, if something goes wrong, if the cell gets some notion that it might try to cheat death and live forever, if it has the inklings of an idea to hijack the rest of the body through growing its own lump of cells and draining the body of its nutrients... if it decides to throw a mutiny at the expense of peace...

Then the cellular system realizes, and it activates the caspases.

(Usually how a cell 'decides' is when something wrong happens with genetic replication, mutations and such and such. There are two labels of genes relating to this. Proto-oncogenes promote cell growth and avoid apoptosis - these are the creation genes, the ones that wish to achieve the heights of production and throw all caution to the wind. Whereas tumor suppressor genes are... tumor suppressors. They are the little 'angel on the cell's shoulder' that says 'you've done wrong, now commit cell death and accept your fate'. When a proto-oncogene mutates, it becomes an oncogene. Basically, the genes that said to grow for the sake of the body now says to grow at the expense of the body, the 'devil on the cell's shoulder' won... and now it'll try to cheat death)

Caspases are a type of enzyme, which is a subset of proteins. I like thinking about it like this: enzymes are witches, spell crafters. Proteins in general function like worker bees, but enzymes are the ones who actively create and destroy, the ones that change the way the universe manifests so life can exist. They utilize reactants from their environment - materials which they are named after, such as proteases that cleave other proteins - to either create larger, energy storing molecules (endergonic reactions), or break down large, energy storing molecules to release energy (exergonic reactions).

(One type of enzyme that I like is called kinases, they basically initiate things, they tell the cell 'it's okay to do things' or 'this will be dangerous, let's not do that', or 'oof this cell doesn't have the proper genes, that could be a problem, how about we destroy everything so this doesn't become a big issue?')

When the cell needs to die, the caspases activate.

Humans have 14 caspases. I wish I knew what all of them are, but the pub med articles I read do not have a whole list of them like a pokedex from pokemon (at least that I have read so far, after ap testing there's one article I want to read that seems to have ANSWERS to a lot of my questions)

So imagine this, the cell has lots of caspases floating around in the cytoplasm/water jelly environment. These beings of death in huge numbers in a perpetual slumber, just floating around, until the cell decides it needs them to destroy its existence, and it activates them.

(ALTHOUGH some of my questions refer to the "caspase-dependent non-lethal cellular processes", so far what I've been able to find is that if the cell needs some repair to the cytoskeleton/structure of the cell, then it activates a few caspases to destroy parts of it, so that the repair may start.)

There are two types: inflammatory caspases (if I remember correctly, this is caspase 1,4, and... a few others I'll have to check) and apoptotic caspases. What we're interested in are the apoptotic caspases. Initiator caspases get activated first. The way I story-fy them/see them in my head is as the older sibling types who condone violence. If the cell needs to die because of an external signal - a message from far away to destroy itself, to which it must oblige - then caspase 8 activates. If the cell needs to die because of an internal signal - something inside isn't right, and what a havoc it would be to pass that on through replication, so the proteins decide the fate of the world it lives in, and it choses death for the sake of the wider body- then caspase 9 activates.

Both these initiator caspases cleave (meaning 'cut', these inactive beasts are not yet 'complete', so they need further modification after activation to work properly. If the cell were to create caspases fully functional and finished, the large number of caspases would kill the entire cell, so they don't finish, they subdue the ones behind their death, and make sure to regulate their slumber.) their 'younger, violent siblings': caspase 7 and caspase 3. The doll I have is a caspase 3.

(I've found more information on them than caspase 7... although.. the one article I found.... it should have information on them all, the amount of searching I've done for those sorts of answers... but alas... I need to read the material for the ap bio exam... which does not include caspases.... one day...)

Caspase 3, as far as I could gather, destroys the cytoskeleton. The cytoskeleton supports the entire cell, acting as the 'tent poles' that keep the floppy cell membrane from collapsing. Collapsing, however, is exactly what the caspase 3 wants. It dismantles the whole thing, along with activating and inactivating a slew of other proteins. (it's an assassin basically.)

And with this, the caspases bow, for the cell is separated within these small little 'blebs' (they look like bubbles) and the cell is destroyed.

OH AND LAST LITTLE THING!

If something inhibits the caspases, the cell goes through with necrosis, which is basically instead of becoming little bubbles, the cell membrane ruptures and the cell 'guts spill out'

THANK YOU SO MUCH FOR THE ASK!! <3 <3 I got to blabber about caspases and it gave me happy sparkles.

ALSO, here's the doll I made for caspase 3:

[quaft-delicates]

by happenstance I read this poem today and now it's rattling around inside my skull like it has the place to itself

(if you're a cryer like me, be warned)

ALZ Ghazal

By Rebecca Foust

For my sister

It’s the same house, same rugs, same wallpaper, and bedroom repeating;

same dresser; same rocker. Same window and frame, repeating.

Same birds at the pane, same pots and pans, and—on the alarm clock,

the wall clock, the phone clock—the same time, repeating

each hour’s increment in a lived life. But, This is no life, each day like

before and to come, repeating.

The furniture set in a known pattern. The rugs there, like always, inking

the blueprint of home, repeating

jewel tones on the floor, but what was once north–south now seems to lie

east–west—who moved the rugs?—in sum, repeating

the familiar, but sideways. Your inner axis has shifted, the landmarks

somehow changed but the same, you repeating

Why do they keep moving the rugs?  The desk, the chair, your keys?

Home its own balm, repeating

the familiar, but neither keys nor your purse can be found—I know

I just had them—repeating

the questions yields the same, that is, no real answers. Your sense of taste

gone, like eating chum, repeating

the same million small motions: fork to plate then mouth, then back down,

always the same, repeating

the flavor of cardboard. You used to love to cook, that joyous jazz variation-

on-a-theme now a repeating

like pages of musical staffs, xeroxed blank with no notes. Lately, you refuse

to eat anything at all. In a poem, repeating

lines compose a refrain and, echoed again and again, the sum of refrains

is a song. But there is also empty repeating:

zero plus zero plus zero still zero, a void. No accretion, no growth, no life,

no thrum. Then again, birds—some, repeating

one clear note, are said to singing without tune—and, the same set of sounds

from a beaten drum, repeating

means nothing and everything at the same time. The gene runs in families

and can be followed like breadcrumbs, repeating

the precise map for getting lost, down through generations. She took the same

route to work on the town tram, a repeating

my sister relied on. We rely on a plum to taste purple when our teeth break

its skin. Some numbers go on ad infinitum, repeating

that reminds us of architecture and God. Rings of a tree, whorls of a shell,

sections of a lime, repeating

a sequence that re-enacts growth and change. Meiosis, mitosis, Eve cleaved

from Adam, other kinds of repeating

so life can go on. We are “pattern-seeking animals,” Hass says, and even

atheists pray in a jam, repeating

lessons learned as a child: “Now I lay me” like the promise of mercy, like mercy

itself, soothing and warm, you repeating

your talisman against terror. After your nap, I worry you’ll wake confused,

but then you resume, repeating

Where are my keys? Why do they keep moving the rugs? Always theft and loss

the theme, repeating.

They say you’ll forget my name, and your own. How to talk, small mercy

then, you no longer doomed to repeating

your aching questions. They say you’ll forget how to swallow, to breathe—

O God, will you forget to breathe?—just succumb, repeating

the pause in place of the heartbeat? The doctor says keep things simple

and flat, no drang or sturm, repeating

“Is it nice there today,” and “Have the tulips bloomed,” conversations dull

with the same datum repeating

catechism and cliché. You no longer work on your dollhouses, potting trees

the size of your thumb, repeating

postage-stamp paintings on walls, building one-inch-to-the-foot the lavish life

you never had or presumed, repeating

a litany of loss enlarged from what-was to what-will-never-be. The Christmas

tree shadowbox in simulacrum repeating

the childhood obsessions. Some of your dollhouses have tiny dollhouses

within them, repeating

themselves in infinite regression. All that helpless sap and surge of creation,

your xylem and phloem repeating

what to make of a diminished thing. You say you’ll burn them come spring,

without reflection or qualm, the future repeating

and eating the past. It’s happening so fast: a page torn, a book lost, then

whole libraries gone dumb, repeating

erasure until only erasure remains. O trochee, lilting your 10-beat line.

O river, O iamb repeating

the tide. Bishop called loss an “art.” I last saw you at home in October,

a glorious autumn repeating

saffron and scarlet against azure sky. Outside now, a robin taps at the pane,

ravished by her own image, an eye-rhyme repeating—

there’s been one bird each spring. Some beat their beaks bloody and die,

some stay for days, numb with repeating

their broken routine. Who moved the keys? Who moved the goddamn keys?

Your patient husband is grim, repeating

they’re-in-a-safe-place-so-we-can-keep-you-safe. We are all of us trapped

under a great dome repeating

our words and the cries of wild birds. This is harder in some ways, he says,

than Vietnam, repeating

his pledge to stay till the end. He tries to give you small pleasures, picking

and handing you the plum repeating

all you can recall of lush purple juice run down your chin, but at best,

it’s a dim meme repeating

as holograph plums eaten before. Soon, where are my keys will dwindle

to where keys, the totem repeating

as keys, then just key—what matters, the way out of labyrinth and code.

A chant or psalm, repeating

open vowels like water or rain. Luck—the river in poker, the silver ball

spun at the wheel’s rim—can be repeating

until it turns, and isn’t it possible to hear a persistent tune as anthem,

not ear-worm? Can’t repeating

strengthen a seam, or knit a long row? After you fell, I imagined you there

on the floor, the refrigerator’s hum repeating

its cold comfort until the ambulance came. Your husband cradling you

on the linoleum, repeating

presence in absence in presence in absence—cognitive dissonance—

or an inscrutable charm repeating

its mysteries. Your hip and arm are mostly healed. The house is still there,

but an interior door slams shut every second, an alarm repeating

its warning: all doors soon will be sealed. Where we stand in an earthquake

is an empty doorframe. Soon, repeating

itself will disappear: all language flown, a slaughtered lamb,

a shattered paradigm. Will your repeating

heart just—miss its cue? Will you forget how to swallow? O Fates, come, come,

/ Cut thread and thrum, repeating

its end-times refrain. Your jacks and pick-up sticks—you still love any game

—played now with your left arm, repeating

that makes of small movements an art, like dance. Your little jokes,

like the “ALZ tip jar”—now crammed—repeating

the ring of each coin dropped in. You pull my leg when I call, pretending

to forget who I am, repeating

Becky who? Becky who? until we both laugh, the sounds that sound so much

like crying but with a whetted edge; in time, repeating

a knife against stone will bring forth a terrible blade. O sweet sad funny sister,

you are the bird who flies away in my dream, repeating

its looping script on the blank page of sky. An unhooded hawk, a wild bird

freed. Calling Key? Key? Key?—repeating, repeating, repeating.

Source: Poetry (May 2021)

by way of: www.poetryfoundation.org/poetrymagazine/poems/155783/alz-ghazal

[worldbuilding-tomfoolery]

Magiautotrophs

When Olua is deep into star-feeding, magic floods the atmosphere, and the air fills with critters who feed on it. It's so thick with life, certain macrofauna swim the sky throughout summernoon. Many microorganisms have developed ways essentially eat magic. These magisynthetic processes are fundamentally different from photo- or chemosynthetic ones, leading to a new set of trophic categories for Oluan microbes: Magiotrophs. An organism may get their energy, their electrons, or both from magic. An organism who does both and obtains carbon via CO2 fixation would be fully termed a magiomagiautotroph -- magiotroph for short. Though the convention is to shorten a -troph to its first (and sometimes third) prefix, a non-magisynthetic organism who nonetheless oxidizes magic for electrons will often be called a magitroph anyway, due to the novelty and contextual significance of the concept.

Side note

Though plenty of species use magic in myriad ways, including various energy-boosting strategies, no known eukaryote has figured out how to use magic directly as food, the way plants use sunlight. Magic, by definition, is poorly understood, so it is yet unknown why this is a power exclusive to the micros.

One popular hypothesis posits the ability originated in domain Alcana and spread via horizontal gene transfer to some other species; among alcans with fully sequenced genomes, over 90% have genes that code for magisynthetic biomachinery. Same and similar genes have been found in several bacteria and archaea (some of which are relative newcomers to Olua). Trace amounts of these genes have even been found in lichens. Though the magisynthetic section of archaea and bacteria is much smaller, that they possess these sequences at all implies remarkable things for interdominal HGT, and the role magic may play in the process.

Second side note

There is emerging evidence certain alcan lineages have evolved ways to avoid cleaving oxygen during carbon fixation, likely due to the sheer abundance of atmospheric oxygen and magiparticles' resemblance to it. Big if true.

(Is it possible some of these microbes have complexes unrelated to Rubisco at all?)

[pheonixdrop]

🔫 hand over the lore toxi

OK SO quick context: I was feverous last night, and when I’m feverous I semi-hallucinate Horrors.

This is gonna be LONG

Shadowknights are born when a mortal dies with a heart full of hate and betrayal. These souls are pulled down into the nether by Shad, and reborn as shadow souls.

shadow souls spend their first century in a converted Nether Fortress called The Nursery.

Gene is the head of the Nursery, in charge of… how should we say… re-educating the shadow souls, and guiding them through their new majick.

When the shadow souls make their first vessel— twist their magic into something tangible— they are graduated to the rank of Fledgeling.

as fledgelings, they receive intensive training, in majicks, combat, deception, etc etc and then sent out into the overworld for their first kill.

The First Kill is a very important milestone for every fledgeling— both culturally and metaphysically.

they are meant to hunt down their old leader, be it a lord/parent/etc. this destroys the ties to their past, permanently cleaving themselves to the ranks of the Shadow-knights.

But in a more real way, their First Kill is also their first true meal.

The Shadow Knights don’t eat human food, obviously, so what powers them?

…why, magic, of course! particularly, soul magic. They eat the dead. any soul will feed adequately, (though the more magic, the better) but the intensity of the relationship is significant.

When they return victorious from the Overworld, they can begin being sent on missions.

Laurence is an interesting case.

Unlike other shadowknights, Laurence doesn’t… die. or rather, he’s never separated from his mortal body. He isn’t alive. He isn’t dead, either.

[sevenrs]

Do you have more iterator immune system headcanons? I would absolutely love to hear them >:) (or just immune system facts in general those are really cool)

inspectors definitely act on a more macro scale when it comes to defenses. there isn't a single cell i can compare them to because the roles of immune cells are very specific and inspectors are pretty general in how they act (poking around crevices, attacking, etc.) i know their design is very hydra-inspired, but they also look like dendritic cells to me.

these cells are best described as the "brains" of the immune system (except they dont have real thoughts and are stupid because they are cells but. moving on). these cells sample the surrounding fluid and if they identify a protein that is foreign, they are the first step in the adaptive immune response. they are technically part of the innate immune system, but they bridge the gap

iterators have a lot of microbes in them that need to stay alive. they probably defend themselves through one of the various immune systems they can have. i think the most interesting is one that a lot of bacteria have, CRISPR-CAS. this defense is primarily used against viruses. when viruses inject their genetic material into the bacteria, the bacteria will take a part of the virus's genome and add it to its own for memory (CRISPR). rna is then transcribed from this dna and cleaved into individual parts, each with one with one piece of rna from one virus. then, with tracer RNA (tracRNA), bind to the CAS9 protein and this complex searches the cell for the matching DNA sequence. when found, it will unwind the viral dna and chop it up, rendering it ineffective as more proteins and enzymes dissolve the DNA. yes this is where gene editing was discovered

this part is speculation, but maybe it helps with iterating. iirc new ideas are thought of when the microbes grow and change :p

[cyanidas]

Let's play a game of how much Lumian lore you can dump on me so I can compile a complete post and use it for world building for a Lumian character of mine (pls give me the knowledge I crave it)

Oh god. Ok you're on

I'll do my best 😵‍💫 if you're looking for anything else / more specific, lmk!

Keep in mind my bs can and will differ from everyone else because everyone is either normal about them or is also weird about them but this time with tails instead, and also in a fundamentally unique way from my shit. And I have a LOT.

So,

Some world setup, because it matters to me; KirinDave as a concept is like 3 characters - Kirin (Craftia), Quetzal (Earth), and Chimera (Twilight). There's other deific rulers of other places but they don't matter rn. Kirin is the god of the Heart, Chimera the Mind, and Quetzal the Form. The 3 have their own backstory but I'll skip it for now.

Quetz came in contact with their people, the humans, in early historic times they were completely terrified of them. Out of shame, Quetz invented different religions to interact with them in a way they could better understand, and chose forms that were more pleasing and comprehensible. Under these guises, these personas, Quetzal fell in love with several humans and they just kinda sired a bunch of demigod children as a result.

Kinda messed up but I'm choosing to cover it up and say the humans they loved knew what they were getting into when they agreed to have sex with a god, including the whole polyamory thing. For all intents and purposes everyone was cool with it except general society who took one look at these "horrific hybrid" half-human half-animalish demigod kids and freaked out, killing nearly half of them and their parents in the process.

This didn't happen like, all at once, just over time for a while until Quetz couldn't take it anymore and decided to relocate the remaining humans, their children, and a good chunk of people who were supportive and capable of running a society successfully, and had an arsenal of angels help relocate them to a planet so far away, Quetzal's Siblings couldn't detect them easily. They kept this planet so secret that they would've loved to take this secret to the ends of the universe and with their deaths if they could've, but you see...

In no time at all, this society came together and grew into one species; The Lumians.

Lumians did not retain their Origin story and have no idea where they came from. The Guardian Angels cleared their ancestor's minds of Earth, and they simply began anew, knowing only their relationships and skills. Lumians became a race of highly scientifically progressed people whose genetics are influenced by the "throw a dart at a board to see what animal you land on" genes of their demigod ancestors, and the naturally adaptive influence of being descended from the god of Life itself.

They were born and raised on a harsh planet with a huge sun and massive insectoid monsters, so they naturally thrived in caves and darker biomes, where they quickly evolved to glow. Soon, their bodies rapidly changed to include pointed ears with sharper hearing to better listen for specific bugs, sharper teeth to cleave through incredibly tough bug meat, stronger/bigger bodies and longer lifespans to survive with, and colored blood/glow for attraction reasons bc blood color is pretty meaningless to their society and it has little to no effect on their bodies or survival.

Lumians have a superiority complex to humans, and assume that they're the result of ancient Lumians who colonized Earth and "devolved". This is a point of contention in their society and anywhere else in space that Lumians interact with humans. Funny enough, despite being smart with technology and science and weaponry, Lumian society valued art and creativity so poorly that entertainment is virtually nonexistent here. They get their kicks by starting and joining wars across space, and partaking in what human culture they can capture from lightyears away.

Their buildings and cities are very sterile-looking, and what color they do intentionally use are organized in mathematical and artificial/generated ways. Idk how to explain it because I haven't looked for architecture that displays this, but think of pretentious billionaire houses. That's kinda what we're working with. Lumians also don't have a class system, so even a lowly outback-type reclusive farmer or woodsman with a small 2x2 home has access to the same rights, funding, and technology as everyone else.

Their planet, Lumina, has a large variety of biomes with jungles, swamps, deserts, temperate zones, rocky barrens, and so on. They only have one ocean, and it's about the size of South America, and in their southern hemisphere. Everywhere else are lakes, rivers, and an incredible amount of underground pocket water and plants that retain a lot of water (like "cut the root open and drink it like a straw" amounts). They do have some mammal-like creatures, but they are usually very tiny and serve as pets.

Some dogs came with their ancestors on the trip and they've also evolved! Due to the strange food they've fed on, they've gotten sleek coats, bigger and tougher teeth, pointy tongues, bigger/wider ears, and very unusual colors and eyes. I'll have to draw them later!

They have similar careers to us, but their society celebrates joining the armada, to an even bigger extent than the US. To them, it's THE job to have, the most noble position to take, you basically become a hero to your family and town. For all interns and purposes, they absolutely are a military state. The leader of the armada is Xephos's dad and he is basically a rockstar and democratically elected emperor of the planet. Matching that, their language is also monolith, but has distinct accents and dialects. I've written the alphabet somewhere on this blog in the past. They do have actual leaders of society that lead certain areas, very much like our countries do, but they all take a backseat to Xeph's father in the end. Everyone in general follows along with him in a similar fashion to how certain white people obsess over the royal British family.

Religion can be niche and taboo depending on the region, and magic is a marginal subculture in general. Lumians don't care a whole lot about bloodlines, lineages, family cultures, so on; everyone is motivated to become independent of themselves, and visiting family is socially accepted to be a rarity. Sometimes their bodies can have more animal-like features, like colored patterns, patches of floof, different shaped pupils or more "wild" irises, sharper nails, maybe even a tail who knows - but these physical differences are as rare as unique physical differences on Earth (i imagine white and black blood could even be possible if the Lumian were albino/melanistic). They have their own spaceships, news, radio, television, school systems, so on so forth. They just don't have a lot of variety in life and it kinda sucks. they suck lmao

Quetzal's secret is now as thinly veiled as tissue paper, thanks to the Lumians' exploration and interaction with Earth and Craftia. Humanoids are very distinctly localized to our solar system, and at least two of the deities are needed to create a living/sentient creature, so a humanoid race existing outside the solar system is definitely a huge cause for suspicion.

also Xeph's dad is planning a secret invasion of Earth and enslavement of humanity and he's overall a horrible person, but only in secret. to the public he's just the bee's fuckin knees.

I hope u like ;w; search my blog for other Lumian stuff, bc I've posted quite a bit about them! ALSO TAG ME IN ANY OCs.....I LIVE FOR THAT SHIT DUDE

[axgmented]

❛ if you take side with my enemies , i will not hesitate to cleave your head from your shoulders. ❜

@saishuu-heiki

"cleave it then!"

Rem screams, pushing against that initial fear with the only other thing she knew: anger. It floods her veins, boiling through the hollow channels of her body and causing her limbs to shake. The rage courses through her, eyes filling with the cerulean glow of their shared genes, pupils thinning and her vision feels sharper. Her blazer is torn from her body, laying in a crumpled heap behind her as she storms forward, boots thudding against the concrete of the roof; brass knuckles gripped tightly in her right hand to the point of her joints screaming from the pressure of her grasp.

"tear it from my shoulders with your bare hands!"

rem shouts, that churning in her stomach causing waves of nausea to crest and fall like a wave. Her voice breaks, quivering with the unbridled rage she feels towards the image of his figure. He had to be real this time; the thrumming of her heart matched his own-- she felt his breath with every inhale of own lungs and her cells vibrate with intensity: they call to him, cry for a reunion between the two of them. She barely feels the tug at the center of her core: the lead that cinches tighter and tighter is ignored as she stands but a few feet from his towering body.

"They are my family.. they are my home."

she snarls, fangs gleaming as exasperation waivers her voice. She abandons the half-jargon she usually speaks, ire clarifying her words. Rem heaves, panting as she stands before the general in a sullied uniform; red dries to a rusty colour, staining the whites of her shirt and there are patches of crimson that are drying against the black slacks. The blood isn't hers, but of her comrades-- of the other turks and she feels tears prick at the corner of her eyes.

"we.. are not your enemies. you are your biggest adversary. your hatred blinds you and makes you a fool!"

her heart aches, the voices in her head writhing like smoke and screaming out for his touch. Her skin crawls, goosebumps breaking across the surface of her flesh. She's hot to the touch, outrage causing a flush to break across high cheekbones and her lip is tender from a wound that would bruise in the coming days. She feels a hiccup strangle her throat, and Rem tries to speak past it.

"if you are a man of your word then do it! end my suffering and bring this reunion she wants so desperately to happen, but know this, Sephiroth. The planet will not weep for you as it will for me!"

[mariacallous]

Philosophers have grappled with the nature of evil for thousands of years, but these days, immorality can feel like a solved problem. Take the case of Bryan Kohberger, the prime suspect in a quadruple homicide near the University of Idaho whose arrest ignited rampant media speculation about the psyche of a killer, as if properly diagnosing his personality disorder could mitigate the damage already done. His “psychopathic stare” made headlines in UK tabloids, while The New York Times dissected Kohberger’s self-described feelings of remorselessness as an adolescent. Dr. Drew brought on a former FBI agent to discuss Kohberger in the context of the “dark triad”: narcissism, psychopathy, and Machiavellianism. 

Americans understandably want help making sense of the otherwise senseless deaths that populate the front pages of local papers and constitute Netflix’s extensive true-crime back catalog. But attempts to characterize evil remain scientifically dubious, say criminologist Jarkko Jalava and psychologist Stephanie Griffiths, coauthors of The Myth of the Born Criminal. When it comes to crime, psychologists frequently “get really sloppy,” Jalava says, adding, “we’re functioning on this folkloric level.”

The perpetrator of the University of Idaho murders should be condemned, but getting inside the mind of a killer is easier said than done. Prediction and prevention—the supposed end goal of criminal profiling—is even harder. And the proliferation of quasi-scientific terms for jerks, assholes, and even killers has far-reaching consequences. 

The medicalization of evil—that is, the physician-led diagnosis and management of diseases like “moral insanity” and “criminal psychosis”—stretches back to the early 19th century. Where clerics once drew the line between good and evil, psychiatrists began to take people who engaged in impulsive, self-defeating, or otherwise un-Christian acts into their care. 

Early on,  these doctors-cum-criminal-profilers explained bad apples through theories such as atavism. Proponents believed that, over time, bad breeding led to degeneration of the gene pool, and the concentration of poverty, criminality, and other undesirable traits in certain ethnic groups or social classes. While the theory of degeneration was slowly replaced by a strikingly similar notion of “psychopathy” (literally “soul sickness”), many of the concerns remained the same: deviants who showed a lack of remorse or guilt, exhibited sexual promiscuity, and developed a lengthy rap sheet, perhaps from a young age.

New variations on this theme pop up all the time. The “dark triad,” coined in 2002 by Canadian psychologists Delroy Paulhaus and Kevin Williams, aims to describe “offensive but non-pathological personalities,” including CEOs, politicians, and bad boyfriends. There are also labels like antisocial personality disorder, a diagnosis given to individuals with severe impulsivity, aggression, and criminal behaviors—in other words, a DSM-approved twist on the old “psychopathic” standard.

At first glance, these attempts at categorization appear to be trending positive. For one thing, researchers are slowly cleaving obvious wrongdoing from the more inadvertent harms of mental illness. Similarly, it’s a relief to be able to use the dark triad to acknowledge just how commonplace selfishness really is. 

But the shadow of degeneracy still looms large. In addition to further medicalizing everyday discourse (“jerks,” Jalava and Griffiths point out, have become “psychopaths,” with all the attendant baggage), these models uphold the dubious belief that every human has an immutable personality—and that those personalities can be easily classified as good or bad. In reality, recent research shows that many people change—and, in some cases, change dramatically—over the course of their lifespan. At the same, many researchers remain critical of the historic characterization of personality disorders, in part because it is stigmatizing and can obfuscate trauma, and even then it doesn’t lead to clear directions for treatment.

Many popular ideas about evildoers seem to stem from tabloid news, rather than scientific evidence. For example, Jalava and Griffiths have shown that many experiments that make claims of a genetic or neurobiological basis have not been replicated, and those that have been replicated have produced contradictory results. Soon, they will publish a new review detailing similar problems in studies of psychopathy and fMRI brain imaging. Perhaps most importantly, the husband-wife duo have documented how meta-analyses of psychopathy research, ostensibly the gold standard in scientific research, often ignore published results with null findings. 

Even if researchers working on the problem of evil content themselves with the shortcomings of the existing scientific literature on psychopathy, dark triad, and the like, a new issue arises: People don’t just want to describe existing traits. They want to use these scales to predict future behavior. 

In the criminal system, the results of assessments like the Hare Psychopathy Checklist are used to assess an individual’s risk of recidivism, and therefore the possibility or terms of parole. Paulhaus, the creator of the Dark Triad Personality Test, wants prospective employers, including police and the US military, to screen prospective candidates with his scale—and not always for the reasons you’d imagine. “It makes a big difference whether you diagnose someone as a Machiavellian or a sadist or a narcissist,” Paulhaus says. “There may be occasions where that's what you’re looking for.” At the same time, it’s easy to imagine a world in which individuals are screened for psychopathy and, if their score is high enough, closely monitored for potential crimes.

But Jalava and Griffiths insist that meaningful prediction isn’t possible with existing measures, either. If you remove questions on psychopathy evaluations that ask about previous criminal activity, these scales fail to anticipate what the test-taker does next. It’s an unsexy finding: “Past behavior can predict future behavior” is never going to be an A1 headline. But unlike other, more elaborate theories of pathological personalities, this at least is real. 

The desire for strong language to match heinous acts is only natural. Unfortunately, sadistic bosses, everyday assholes, and even murderers are still human—shaped by and shaping the world. Condemning people as the subhuman (or, paradoxically, superhuman) embodiment of evil isn’t based on “the characteristics of the individual in front of us,” Griffith says, “but our response to them.” Such illusory categories make real understanding almost impossible.

In the context of evil, pursuing understanding is itself controversial. For decades, people have rightly criticized the media for the breathless attention it pays to killers—on the assumption that it feeds the psychopath’s or narcissist’s desire for attention, and may in turn inspire copycats. But there’s a difference between a news consumer’s insatiable desire for serial-killer content, and the responsibility we all have to face the harm in our society—and, perhaps, the capacity for harm in ourselves.

For example, many true-crime aficionados will cite data showing that something like one-third of serial killers (many of whom are presumed to be psychopaths) experienced physical abuse, one-fourth experienced sexual abuse, and half experienced psychological abuse as children. But these statistics don’t explain much at all about these “evil” adults, more than half of whom weren’t abused. Rather, they raise a more interesting question: Why are so many ostensibly non-psychopathic American parents abusive of their children? 

This reformulation isn’t an excuse or an absolution; adults can, by and large, be considered responsible for their own actions. But it makes clear that “evil” doesn’t exist neatly in an individual in the way inherently judgemental labels like “psychopath” might imply. It’s not simply that psychopaths aren’t born but made, either. It’s that, if psychopaths exist at all, the same forces that shape them are at work on the rest of us, likely with similar, if more subtle consequences. 

Flipping cruelty on its head also opens avenues for new solutions. We know, for example, that poverty is the number one cause of child abuse. One might reasonably conclude that the money devoted to studying dark traits might better be used for a universal basic income. Similarly, the notion that past behavior can, in meaningful ways, suggest future behavior is a helpful starting point. For example, if the only part of a psychopathy test that can predict crime are questions about past criminality, we should be able to ditch the armchair psychoanalysis and focus on documented, real-world behavior. Even then, because people can and do change, these rules cannot be hard and fast, and context and compassion will remain essential.

It’s still possible to divest even further from useless labels, by focusing on the idiosyncrasies of troubled—or, more precisely, troubling—individuals, as well as the specific context in which they emerged. Despite all of their scholarship, Jalava and Griffiths say they don’t have a silver bullet for defeating, let alone replacing, the myth of the born criminal. Rather, they advocate for a descriptive (rather than the standard normative) approach. Understanding how a certain person arrived at a certain point in time can’t undo the hurt they’ve caused, but it might help them to chart a new path—perhaps even one that deviates from their dark “personality.”

[helen0086]

Molecular Cloning and Functional Study of the Phycocyanin Cleaving Enzyme CPCS from Spirulina

Abstract: In order to study the catalytic function of algal cyanine lyase CpcS in Spirulina subsalsa, the gene encoding algal cyanine lyase SscpcS was first amplified from the genomic DNA of S. subsalsa FACHB351 by PCR, and then pCDFDuet-SscpcS was constructed as an expression plasmid, which was co-transformed into E. coli BL21 (DE3) together with pETDuet-SscpcB-Ssho1::SspcyA expression plasmid of auxin and pigment synthase. The expression plasmid pCDFDuet-SscpcS was then co-transfected with the cofactor protein and pigment synthase expression plasmid pETDuet-SscpcB-Ssho1::SspcyA into Escherichia coli BL21 (DE3) and recombinantly synthesized into phycocyanin by IPTG (Isopropyl β-D-Thiogalactoside) induction. Sequencing of the PCR products showed that SscpcS was successfully amplified, and the plasmid pCDFDuet-SscpcS was successfully constructed and was able to express the target protein by double digestion and SDS-PAGE.

The cellular product of recombinant phycocyanin PCB-CpcB was dark blue; the purified pigment protein showed a maximum absorption peak at 620 nm and a maximum fluorescence emission peak at 646 nm; the pigment protein was stained with zinc ions, and showed obvious fluorescence under ultraviolet irradiation. In this study, the gene encoding SsCpcS, a cyanobacterial proteolytic cleavage enzyme from Spirulina subsalsa Salina, was successfully cloned, and its product SsCpcS can effectively catalyze the biosynthesis of cyanobacterial proteins. This study lays the foundation for the recombinant synthesis of S. subsalsa cyanobacterial proteins and the development of antioxidant reagents, and also provides a scientific basis for the investigation of the catalytic mechanism of SsCpcS in Spirulina salina.

Spirulina, a cyanobacterium, is widely recognized as an ideal health food. In addition to containing many minerals, carotenoids and other natural pigments, it is also the richest food source of vitamin B12 [1]. Among the pigmented proteins it contains, Phycocyanin (C-PC) has been widely recognized for its strong antioxidant and free radical scavenging ability [2-4], and its significant role in anticancer, antiviral, immunity regulation, and non-toxicity to mammals [5-7]. However, the current extraction process of algal blue protein is complicated, which limits its application. As the biosynthesis mechanism of phycocyanin has been analyzed, the recombinant synthesis of phycocyanin in Escherichia coli using genetic engineering technology is a better choice. It has been demonstrated that this recombinant phycocyanin is physicochemically similar to natural phycocyanin and exhibits good antioxidant activity [8-12]. However, the production of recombinant phycocyanin generally requires the catalysis of lytic enzymes [13-14], so the study of lytic enzymes and their catalytic mechanism is crucial in the heterologous synthesis of phycocyanin.

Studies have shown that phycocyanin has two subunits, α and β [15], and that there are three families of alginate ligases, CpcE/CpcF (E/F type), CpcT (T type) and CpcS (S type) [16-18]. Lysin CpcE/F catalyzes the covalent coupling of the α-subunit (CpcA) of phaeocyanin (PCB)[19] , and lysin CpcS catalyzes the covalent coupling of the β-subunit (CpcB) of PCB[20] , in which PCB is produced by heme oxidase (HO1) and biliverdin reductase (PcyA) using heme as the substrate[21] . It has also been found that some of the S-type ligases need to form a heterodimer with another ligase, CpcU, to perform their catalytic function, e.g., the S-type ligase of Synechocystis sp. PCC6803, which is known as the CpcS-I isoform; however, some of the S-type ligases, such as the S-type ligase of Nostoc sp. PCC7120, which is known as CpcS-III, are able to catalyze alone [22]. However, some CpcS alone can perform the catalytic function, such as the S-type ligase of Nostoc sp. PCC7120, which is called CpcS-III isoform, and CpcS-II, which is only found in marine polychlorophylls, performs the catalytic function together with CpcU-II [22].

It can be seen that the catalytic mechanisms of different types of lytic enzymes are different. Among the commonly cultivated Spirulina, Spirulina salina has the highest total amount of proteins and the widest adaptability to salinity and humidity [23], but there are few studies on the functional mechanism of the cyanobacterial lysin lyase of Spirulina salina. In our previous study, we used CpcS ligase from Cichlidium spp. to catalyze the biosynthesis of the cyanobacterial β-subunit of Leptosphaeria salina [24], and found that, although different sources of cyanobacterial ligases showed similar catalytic functions, their structural differences might lead to different catalytic efficiencies. Therefore, the study of the catalytic function of recombinant Leptospira salina CpcS lysylase may not only improve the efficiency of the catalytic enzyme and increase the production of recombinant algal cyanobacterial proteins, but also help to clarify the taxonomic properties of Leptospira salina CpcS and to investigate the catalytic mechanism of the lysylase CpcS.

Based on the successful construction of the expression plasmid pETDuet-SscpcB-Ssho1::SspcyA for cyanobacterial β-subunit de-coordinating protein and pigment synthase in Spirulina salina [24], the present study was carried out to clone the gene encoding for cyanobacterial proteolytic cleavage enzyme, SscpcS, and co-expressed it with pETDuet-SscpcB-:SspcyA in E. coli by PCR technology. Ssho1::SspcyA in E. coli to synthesize the cyanobacterial protein CpcB-PCB. This study can lay a foundation for the recombinant synthesis of cyanobacterial proteins and the development of antioxidant reagents in Spirulina salina, as well as provide a scientific basis for the investigation of the catalytic mechanism of the cyanobacterial lysylase CpcS in Spirulina salina.

1 Materials and Methods

1.1 Materials and reagents

Spirulina subsalsa FACHB351 was purchased from Institute of Aquatic Biology, CAS. The clone strain Escherichia coli DH5α, expression strain Escherichia coli BL21 (DE3), and expression plasmid pETDuet-SscpcB-Ssho1::SspcyA of phycocyanin β-subunit deacylation and pigment synthase were kept in the laboratory, of which SscpcB, Ssho1, and SspcyA were from Spirulina subsalsa FACHB351, SscpcB, Ssho1, and SspcyA were obtained from Spirulina subsalsa FACHB351. pCDFDuet-1 expression vector was purchased from Novagen; restriction endonucleases Bgl Ⅱ, Xho Ⅰ, and T4 DNA ligase, and the corresponding buffers were from Thermo Fisher Scientific; and the plasmid miniaturization kit, DNA purification and recovery kit, Pfu DNA Polymerase, dNTP, and dNTP were from Thermo Fisher Scientific. DNA  Polymerase、dNTPs ；1000 bp DNA ladder、Protein marker ；The 1000 bp DNA ladder and Protein marker were produced by SMOBIO; Isopropyl β-D- Thiogalactoside (IPTG) was produced by Beijing Boao Toda Technology Company; Ni-NTAAgarose was produced by GE Healthcare. The primer synthesis and gene sequencing were done by Nanjing Kingsley Biotechnology Co.

1.2 Primer design and molecular cloning

The genomic DNA of cyanobacteria was extracted according to the method of Hao Min et al [25]. The polymerase chain reaction (PCR) method was used to amplify the cleavage enzyme encoding gene SscpcS from the genomic DNA of the cyanobacterium Spirulina subsalsa FACHB351, and the primer design was based on the reported sequence of S. subsalsa PCC9445, which was obtained from the NCBI GenBank database (Accession No. WP9445), and the sequence was obtained from the NCBI GenBank database (Accession No. WP9445). Since the CpcS sequence of S. subsalsa FACHB351 has not been reported, the primers were designed based on the reported sequence of S. subsalsa PCC9445, which was obtained from the NCBI GenBank database (Accession No. WP_017305822). The upstream primer for amplification of SscpcS was 5'- GCG AGA TCT GAT GGA CGG TTT AAC ATT TTT TGAG- 3 ', and the downstream primer was 5'-CCTCTC GAGTTACCA ACC ACT AAC AGC ATAAAG- 3 '.

The upstream primer was designed with Bgl Ⅱ and the downstream primer was designed with Xho Ⅰ. The PCR reaction system was 50 μL, containing 0.2 μmol/L primer, 10 ng DNA template, 0.25 mmol/L dNTPs, 1×PCR reaction buffer, and 2U of fast Pfu DNA polymerase (Tiangen, Beijing, China). The PCR program was set as follows: pre-denaturation at 94 ℃ for 3 min, denaturation at 94 ℃ for 30 s, annealing at 58 ℃ for 30 s and extension at 72 ℃ for 1 min. The annealing temperature was set from 58 ℃ to 52 ℃, and each temperature was cycled once, and then 52 ℃ was cycled for 30 times, and 72 ℃ was extended for 5 min.

The PCR-amplified target genes were detected by DNA agarose gel electrophoresis and sent to Nanjing Kingsley for sequencing. The amplified SscpcS fragment and expression vector pCDFDuet-1 were double digested by restriction endonuclease Bgl Ⅱ and Xho Ⅰ, then purified and recovered by TAE agarose gel electrophoresis, and then ligated at 16 ℃ overnight under the action of T4 ligase, and then the ligated product, pCDFDuet-SscpcS, was transferred into Escherichia coli DH5α receptor cells and incubated at 37 ℃ in the presence of streptomycin (25 μg/mL). The ligation product pCDFDuet-SscpcS was transferred into Escherichia coli DH5α receptor cells and cultured overnight at 37 ℃ on LB agar plates containing streptomycin (25 μg/mL), and independent single colonies were selected and inoculated into LB liquid medium containing streptomycin (25 μg/mL), and after incubation at 37 ℃, plasmids were extracted by using plasmid mini-extraction kit, and the plasmids were digested by restriction endonuclease Bgl Ⅱ and Xho Ⅰ, and then detected by DNA gel electrophoresis.

1.3 Protein expression and purification

The plasmid pCDFDuet-SscpcS was transferred into E. coli BL21 (DE3) receptor cells by CaCl2 method, and the transformants were screened by streptomycin (25 μg/mL) in LB solid medium. The positive single clones were picked into 5 mL of LB liquid medium containing streptomycin (25 μg/mL), incubated at 37 ℃ until the OD600 was 0.6, and then IPTG was added at a final concentration of 1 mmol/L. The expression was induced by light at 16 ℃ and 150 rpm on a shaking table for 12 h. The supernatant was discarded by centrifugation, and the cells were collected, and then resuspended and washed twice with twice-distilled water, and the cell precipitates were stored at -20 ℃ for spare parts. The cells were collected and resuspended and washed twice with secondary distilled water.

In order to synthesize fluorescent phycocyanin by co-expression of the constructed plasmid pCDFDuet-SscpcS and the expression plasmid pETDuet-SscpcBSp-Ssho1::SspcyA, the two plasmids were co-transfected into E. coli BL21 (DE3) receptor cells by CaCl2 method for in vivo reconstitution, and the transformants were screened with benzylpenicillin (10 μg/mL) and streptomycin (25 μg/mL), and the transformant colonies were picked from solid medium. Ampicillin (10 μg/mL) and streptomycin (25 μg/mL) were used to screen the transformants, and the transformant colonies on solid medium were picked. E. coli BL21 (DE3) containing pETDuet-SscpcB-Ssho1::SspcyA alone was used as the control, and the transformant colonies on solid medium were picked and inoculated into 5 mL of LB liquid medium containing the corresponding antibiotics, and cultured overnight at 37 ℃, and then transferred to 200 mL of TB medium, and then cultured at 37 ℃ until OD600 was 0.6. The cells were collected by centrifugation at 6,500 rpm, resuspended and washed twice with distilled water, and the cells collected by centrifugation were stored for the detection of cytofluorescence or the extraction of recombinant proteins by ultrasonic crushing.

To purify the recombinant protein, 8 mL of pre-cooled Start buffer (20 mmol/L KH2PO4-K2HPO4, 500 mmol/L NaCl, pH 7.2) was added to each of the collected PCB-CpcB cells to resuspend the cell products in the buffer and the cells were broken by sonication in an ice bath. The fractured cells were centrifuged at 12 000 rpm for 40 min at 4 ℃ and the supernatant was collected. The supernatant was purified using a nickel ion affinity chromatography column, and the purified pigmented protein was dialyzed in KPB buffer (50 mmol/L KH2PO4-K2HPO4, 500 mmol/L NaCl, pH 7.2), because the expression vector used was histidine His-tagged, and the recombinant protein was able to bind to the affinity layer medium.

1.4 SDS-PAGE electrophoresis and zinc electrophoresis of pigment proteins

Protein SDS-PAGE samples were prepared by using the pCDFDuet-SscpcS cell product preserved in Experimental Method 1.3. After centrifugation, 45 μL of 1×SDS sample buffer and 5 μL of β-mercaptoethanol were added, and then centrifuged to remove the precipitate in a boiling water bath at 100 ℃ for 5 min. The supernatant obtained was used as the protein samples for SDS-PAGE electrophoresis. After electrophoresis, the sample was stained with Cauloblue and decolorized with decolorizing solution until clear bands appeared on the electrophoresis gel, and then photographed by a gel imager (Geno-Sens 1850, Qinxiang, Shanghai, China) for preservation.

SDS-PAGE electrophoresis of protein samples was prepared by using the recombinant pigment protein PCB-CpcB purified by Experimental Method 1.3, and the purified protein samples of recombinant products without the participation of cleavage enzyme SsCpcS were prepared as the control group at the same time. 50 μL of the purified protein samples were mixed with 45 μL of 2× SDS buffer and 5 μL of β-mercaptoethanol, and the samples were mixed homogeneously, and the supernatant was centrifuged to remove the precipitation for 5 min at 100℃ in a boiling water bath, which was the protein sample for SDS-PAGE electrophoresis. After mixing homogeneously, the samples were subjected to 100 ℃ boiling water bath for 5 min, and the supernatant was centrifuged to remove the precipitation, which was the protein sample, and then subjected to SDS-PAGE electrophoresis. After electrophoresis, the gel was soaked in 1.5 mol/L zinc acetate solution for 15 min, and then put into the gel imager for detection by 280 nm ultraviolet light. After the fluorescent bands were detected, the gel was stained with Cauloblue and decolorized with decolorizing solution, and then imaged by the gel imager and photographed for storage.

1.5 Spectral analysis

The cell products were resuspended in Start buffer, and the fluorescence emission spectra of the expressed E. coli recombinant chromoprotein cell products were examined, and the absorption and fluorescence emission spectra of the purified recombinant chromoprotein were further examined. Absorption spectra were measured by a UV-visible spectrometer (Lambda 325, USA) with a scanning range of 200-800 nm, a scanning speed of 960 nm/min, and a slit width of 1.0 nm; fluorescence emission spectra were analyzed by a fluorescence spectrometer (Perkin Elmer LS55, USA) with a scanning range of 300-800 nm, a scanning speed of 1200 nm/min, and a slit width of 1.0 nm. The fluorescence emission spectra were analyzed by a fluorescence spectrometer (Perkin Elmer LS55, USA).

2 Results and analysis

2.1 PCR amplification and plasmid construction of the gene encoding the lytic enzyme SsCpcS

According to the experimental method 1.2, PCR amplification of SscpcS, the coding gene of lysyl CpcS, was performed, and then the PCR output was inserted into the expression vector pCDFDuet-1 to construct a new plasmid pCDFDuet-SscpcS. The constructed plasmid was double-enzymatically cleaved by Bgl Ⅱ and Xho Ⅰ, and the product of the enzyme cleavage was electrophoresed by DNA agarose gel, and the target gene SscpcS band was found to be clearly visible at 600 bp (Fig. 1), which was basically consistent with the prediction of 588 bp, indicating that the cloning was correct. After DNA agar gel electrophoresis, there was a clear SscpcS band at around 600 bp (Figure 1), which was basically consistent with the predicted 588 bp, indicating that the cloning was correct.

2.2 Sequence analysis of cleavage enzyme SsCpcS

The coding gene of lysin CpcS, SscpcS, was amplified according to Method 1.2, and the sequence of the gene and the amino acid sequence of the protein (Fig. 2) showed that there were 196 amino acids in the protein. Further amino acid sequence clustering of SsCpcS with other cyanobacterial lysin CpcS (Fig. 3) revealed that SsCpcS was distant from NCpcS from Nostoc sp. PCC 7120, and had a high degree of homology with YCpcS from Synechocystis sp. PCC 6803. Since YCpcS has been shown to be a CpcS-I-type cleavage enzyme [26], it was inferred that SsCpcS should also be a CpcS-I-type cleavage enzyme. In order to confirm this assumption, the amino acid sequence of SsCpcS was compared with that of the cyanobacterial lysylase database (http://cyanolyase.genouest.org/), and it was confirmed that SsCpcS is indeed a CpcS-I type lysylase.

2.3 Expression of the cleaving enzyme SsCpcS

To determine the expression product, plasmid pCDFDuet-SscpcS was transferred into Escherichia coli BL21 (DE3). The expression of the ligase SsCpcS was induced by IPTG in E. coli according to Method 1.3, and the expression product was prepared according to Method 1.4, and SDS-PAGE protein gel electrophoresis was carried out. The results showed that there was a clear band around 25 kDa, which was basically consistent with the approximate molecular weight of the protein based on the amino acid sequence (Fig. 2); and there was no band of the same size at the same position in the control group (Fig. 4), which indicated that the constructed plasmid was able to correctly express lysosomal enzyme SsCpcS.

2.4 Function of the cleaving enzyme SsCpcS

In order to study the catalytic function of SsCpcS, the SsCpcS expression plasmid pCDFDuet-SscpcS was co-expressed with the plasmid pETDuet-SscpcB-Ssho1::SspcyA, which is an expression plasmid for the Semi-subunit of Semi-coordinated protein and pigment synthesizing oxidoreductase of Spirulina salina, in Escherichia coli as described in the Experimental Procedure 1.3. The recombinant cells were collected and co-expressed in E. coli. The results showed that the recombinant cells containing pCDFDuet-SscpcS had a dark blue color (Figure 5A inset b), and the fluorescence emission spectra were measured by fluorescence spectrometer, which showed the maximum fluorescence value at 640 nm under the excitation light of 580 nm (Figure 5A), whereas the control without pCDFDuet-SscpcS did not show any obvious color (Figure 5A inset a), and the control without pCDFDuet-SscpcS did not have a clear color (Figure 5A inset b). The control group without pCDFDuet-SscpcS did not show significant color (Figure 5A inset a), and also did not show any fluorescence emission peaks under the excitation light of 580 nm (Figure 5A). These results tentatively suggest that the cleaving enzyme SsCpcS can catalyze the biosynthesis of the fluorescent phycocyanin β subunit (PCB-CpcB).

In order to further verify the catalytic function of the cleaving enzyme SsCpcS and to determine the spectral characteristics of the fluorescent phycocyanin β subunit (PCB-CpcB) synthesized in Escherichia coli, the recombinant product was purified according to the experimental method 1.3, and the absorption and fluorescence emission spectra were examined. The results showed that the maximum absorption and fluorescence emission values of the purified pigment protein PCB-CpcB occurred at 620 nm and 646 nm, respectively, while no absorption and fluorescence peaks appeared in the control group without cleavage enzyme (Figs. 5C and 5D). These characteristics were similar to those reported in a study [24].

In addition, fluorescence quantum yield and molar extinction coefficient are also important parameters for testing biosynthesized pigment proteins [27]. Taking the absorption spectra of natural C-PC proteins and the fluorescence integrals of the corresponding fluorescence spectra of different absorption values as a reference, the fluorescence quantum yield of the recombinant pigment protein PCB-CpcB was calculated to be 0.26 by using the measured absorption and fluorescence emission data; the molar extinction coefficient was calculated to be 5.63×104/(mol-cm) from the ratio of absorbance before and after the denaturation of the pigment protein, based on the denaturation experiment. The molar extinction coefficient of the recombinant pigment protein PCB-CpcB was calculated from the ratio of absorbance before and after denaturation as 5.63×104/(mol-cm). These results indicate that the cleaving enzyme SsCpcS catalyzes the synthesis of the β-subunit of fluorescent phycocyanin in E. coli.

In order to further confirm the covalent coupling between CpcB, the β-subunit deacylated protein of phycocyanin, and PCB, SDS-PAGE protein samples were prepared according to the method 1.4, and protein electrophoresis was carried out. After electrophoresis (Figure 5B), the protein samples were stained with Cauloblue, and it was found that PCB-CpcB and the control without SsCpcS had obvious bands around 20 kDa, which was consistent with the theoretical value of CpcB [28], whereas the cell samples with induced expression of E. coli BL21 did not have bands at the corresponding positions, which indicated that the exogenous product CpcB was obtained in this experiment; After staining with zinc acetate and irradiation with ultraviolet light, a fluorescent band was found in the PCB-CpcB lane, indicating that CpcB covalently binds the phycobiliprotein PCB, which is catalyzed by the cleavage enzyme SsCpcS.

3 Discussion

Spirulina is a widely used health food, and its alginate has significant antioxidant and free radical scavenging properties [29]. Since the direct extraction of phycocyanin from cyanobacteria is a complex and costly process, the production of phycocyanin by genetically engineered E. coli reorganization has a greater advantage [30]. Studies have shown that the synthesis of cyanobacterial proteins in E. coli requires the catalytic action of ligases, such as CpcS, a ligase derived from Arthrospira plantensis FACHB314, which covalently binds phycocyanin to the β-subunit of phycocyanin and synthesizes fluorescent cyanobacterial proteins [20]. In a previous study by this group [24], the de-coordinating protein of the β-subunit of Spirulina salina was successfully cloned and showed some antioxidant potential. Although the pigment-binding fluorescent β-subunit was successfully synthesized by heterologous phycocyanin ligases, homologous ligases may have stronger catalytic ability and enhance the pigment-binding rate.

A number of studies have shown that there is a strong correlation between the pigment binding rate and the antioxidant activity of phycocyanin [24], and the strength of antioxidant capacity of recombinant phycocyanin not only depends on its de-coordinating protein, but also has a strong relationship with its co-chromophore, and the higher the content of the co-chromophore, the higher the antioxidant capacity of the recombinant phycocyanin is [28]. Therefore, in order to understand the function of CpcS, a homologous cyanobacterial proteolytic cleavage enzyme of Spirulina salina, the present study cloned its coding gene SscpcS from Spirulina salina, and used it to catalyze the recombinant pigment fluorescent protein to show its enzyme function, i.e., it successfully catalyzed the covalent coupling of the β-subunit of Spirulina salina cyanobacteria algins and phycobiliproteins in Escherichia coli. The resulting pigmented fluorescent protein PCB-CpcB exhibited maximum fluorescence emission and absorption at 646 nm and 620 nm, which were similar to the spectral characterization of the pigmented fluorescent protein catalyzed by NCpcS cleavage enzyme from Nostoc sp. PCC 7120 that has been reported [26]. This also suggests that, like NCpcS from Nostoc sp. PCC 7120, SsCpcS ligase alone in Spirulina salina is capable of catalyzing the binding of phycocyanin to phycocyanin by the de-coordinating protein of phycocyanin β-subunit.

On the other hand, by analyzing the evolutionary tree of amino acid sequences of the ligase SsCpcS from Spirulina salina and other cyanobacterial ligases, it was found that SsCpcS is a CpcS-I-type ligase. It is generally believed that the participation of CpcU ligases in cyanobacteria is required to form a heterodimer to co-catalyze the binding of CpcB to PCBs, but SsCpcS alone has shown catalytic activity in in vivo recombination, which is similar to that reported for YCpcS and SpCpcS (Spirulina platensis NIES-39) [31,20]. This further suggests that cleavage enzyme CpcS-I alone is also catalytically active, but in the absence of cleavage enzyme CpcU, its catalytic ability may be inferior to that of CpcS-III (e.g., NCpcS), i.e., it may not be able to further increase the degree of pigmentation of phycocyanin, which needs to be further confirmed.

The next step is to clone CpcU, a ligase from Spirulina salina, and to study the pigmentation of cyanobacterial proteins in E. coli through synergistic interaction with SsCpcS, so as to lay the foundation for the development of recombinant recombinant Spirulina salina cyanobacterial proteins with higher antioxidant potentials and more efficient antioxidant agents. In addition, by comparing the catalytic efficiency of SsCpcS with that of other ligases, such as NCpcS, in the biosynthesis of the same algal cyanobacterial protein, we can provide a reference for further understanding of the catalytic mechanism of CpcS, and for the selection of ligases with higher catalytic ability to improve the yield.

4 Conclusion

SscpcS was amplified from the genome of Spirulina salina S. subsalsa FACHB351 by PCR, and according to the amino acid sequence clustering analysis, SscpcS belongs to the CpcS-I type of ligase, which usually forms a heterodimer with the ligase CpcU to perform catalytic action, but also has catalytic function when it exists alone, i.e., it can catalyze the covalent coupling of the β-subunit of the cyanoprotein and phaeocyanin in E. coli, and shows 620 nm covalent coupling between PCB-CpcB and alginin. It can effectively catalyze the covalent coupling of phycocyanin β-subunit and phaeocyanin in Spirulina salina, and it can recombine into the phycocyanin PCB-CpcB in E. coli, and it exhibits the maximum absorbance value of 620 nm and the maximum fluorescence emission value of 646 nm, which indicate that the gene encoding for the phycocyanin lysyl enzyme SsCpcS derived from Spiroplasma salina is cloned successfully in the present study and the product SsCpcS can be expressed in the following ways SsCpcS can effectively catalyze the biosynthesis of cyanobacterial proteins. Therefore, this study can lay a foundation for the recombinant synthesis of S. subsalsa and the development of antioxidant reagents, as well as provide a reference for the understanding of the catalytic mechanism of CpcS in Spirulina salina.

References :

[1] Ravi M, De S L, Azharuddin S, et al. The beneficial effects of Spirulina focusing on its immunomodulatory and antioxidant properties[J]. Nutrition & Dietary Supplements, 2012, 2: 73-83.

[2] Mitra S, Siddiqui W A, Khandelwal S. C-Phycocyanin protects against acute tributyltin chloride neurotoxicity by modulating glial cell activity along with its anti-oxidant and anti-inflammatory property: a comparative efficacy evaluation with N-acetyl cysteine in adult rat brain[J]. Chemico-Biological Interactions, 2015, 238(3): 138-150.

[3] Nagaoka S, Shimizu K, Kaneko H, et al. A novel protein C-Phycocyanin plays a crucial role in the hypocholesterolemic action of Spirulina platensis concentrate in rats[J]. The Journal of Nutrition, 2005, 135(10): 2425-2430.

[4] Liao G, Gao B, Gao Y, et al. Phycocyanin inhibits tumorigenic potential of pancreatic cancer cells: role of apoptosis and autophagy[J]. Scientific Reports, 2016, 6: 345-357.

[5] Kuddus M, Singh P, Thomas G, et al. Recent developments in production and biotechnological applications of C-Phycocyanin[J]. Biomed Research International, 2013, 3: 1-9.

[6] Shishido T K, Humisto A, Jokela J, et al. Antifungal compounds from cyanobacteria[J]. Marine Drugs, 2015, 13(4): 2124-2140.

[7] Sigamani S. Antibacterial activity of extracted phycocyanin from Oscillatoria[J]. Journal of Applied Pharmaceutical Science, 2017, 7(3): 62-67.

[8] Yu P, Li P, Chen X, et al. Combinatorial biosynthesis of Synechocystis PCC6803 phycocyanin holo-α-subunit (CpcA) in Escherichia coli, and its activities[J]. Applied Microbiology & Biotechnology, 2016, 100(12): 1-14.

[9] Zhang W, Guan X, Yang Y, et al. Biosynthesis of fluorescent allophycocyanin alpha-subunits by autocatalysis in Escherichia coli[J]. Biotechnology and Applied Biochemistry, 2008, 52(2): 135-140.

[10] Hao S, Qin Y, Wang C T. Research progress of the physiological activity of functional food phycocyanin[J] . Journal of Food Science & Biotechnology, 2017, 36(12): 1233-1240.

[11] Pleonsil P, Soogarun S, Suwanwong Y. Anti-oxidant activity of holo-and apo-c-phycocyanin and their protective effects on human erythrocytes[J] . International Journal of Biological Macromolecules, 2013, 60: 393-398.

[12] Chen M, Wang N, Yang D L, et al. The structure and energy transfer of phycobiliproteins in Cryptophytic algae[J] . Plant Physiology Journal, 2015, 51(12): 2070-2082.

[13] Zhao K H, Wu D, Wang L, et al. Characterization of phycoviolobilin phycoerythrocyanin-α 84-cystein- lya se- (isomerizing) from Mastigocladus laminosus[J]. European Journal of Biochemistry, 2002, 269(18): 4542-4550.

[14] Wu X J, Chang K, Luo J, et al. Modular generation of fluorescent phycobiliproteins[J]. Photochemical & Photobiological Sciences, 2013, 12(6): 1036-1040.

[15] Glazer A N. Light harvesting by phycobilisomes[J] . Annual Review of Biophysics and Biophysical Chemistry, 1985, 14(1): 47-77.

[16] Zhou W, Ding W L, Zeng X L, et al. Structure and mechanism of the phycobiliprotein lyase CpcT[J] . Journal of Biological Chemistry, 2014, 289(39): 26677-26689.

[17] Shen G, Saunee N A, Gallo E, et al. Identification of novel phycobiliprotein lyases in cyanobacteria[J]. International Society of Photosynthesis Research, 2004, 21(13): 14-15.

[18] Yi J, Xu D, Zang X, et al. Lyase activities of heterologous CpcS and CpcT for phycocyanin holo-β-subunit from Arthrospira platensis in Escherichia coli[J]. Journal of Ocean University of China, 2014, 13(3): 497-502.

[19] Fairchild C D, Zhao J, Zhou J, et al. Phycocyanin alpha-subunit phycocyanobilin lyase[J]. Proceedings of the National Academy of Sciences, 1992, 89(15): 7017-7021.

[20] Wu F, Zang X, Zhang X, et al. Molecular cloning of cpcU and heterodimeric bilin lyase activity analysis of CpcU and CpcS for attachment of phycocyanobilin to Cys-82 on the β-subunit of phycocyanin in Arthrospira platensis FACHB314[J]. Molecules,2016, 21(3): 357-366.

[21] Beale, Samuel I. Biosynthesis of phycobilins[J] . Chemical Reviews, 1993, 93(2): 785-802.

[22] Kronfel C M, Kuzin A P, Forouhar F, et al. Structural and biochemical characterization of the bilin lyase CpcS from Thermosynechococcus elongatus [J ]. Biochemistry, 2013, 52(48): 8663-8676.

[23] Lin H M. Comparison of spirulina subsalsa with other spirulina species[J]. Journal of Aquatic Biology, 1991, 15(1): 27-34.

[24] Wu X J, Yang H, Chen Y T, et al. Biosynthesis of fluorescent β subunits of C-Phycocyanin from Spirulina subsalsa in Escherichia coli, and their antioxidant properties[J]. Molecules, 2018, 23(6):1369-1379.

[25] Hao M, Liu Z Y, Yang T S. Summary of bacterial genomic DNA extraction methods[J]. Bulletin of Biology, 2014, 49(3): 4-6.

[26] Shen G Z, Schluchter W M, Bryant D A .  Biogenesis of phycobiliproteins: I CpcS-I and CpcU mutants of the cyanobacterium Synechococcus sp .  PCC 7002 define a heterodimeric phycocyanobilin lyase specific for β-phycocyanin and allophycocyanin subunits[J] . The Journal of Biological Chemistry, 2008, 283(12): 7503-7512.

[27] Sun Y F, Xu J G, Tang K, et al. Orange fluorescent proteins constructed from cyanobacteriochromeschromophorylated with phycoerythrobilin[J]. Photochemical & Photobiological Sciences, 2014, 13(5): 757.

[28] Wu X J, Yang H, Sheng Y, et al. Cloning, expression and antioxidant activities of apo-c-phycocyanin β-subunits from three cyanobacteria species[J]. . Jiangsu Journal of Agricultural Sciences, 2018, 34(5): 998-1004.

[29] Bermejo P, Piñero E, ángel Ma Villar. Iron-chelating ability and antioxidant properties of phycocyanin isolated from a protean extract of Spirulina platensis[J]. Food Chemistry, 2008, 110(2): 436-445.

[30] Cherdkiatikul T, SuwanwongY. Production of the α and β subunits of Spirulina allophycocyanin and C-Phycocyanin in Escherichia coli: A comparative production of the α and β subunits of Spirulina allophycocyanin and C-Phycocyanin in Escherichia coli: A comparative study of their antioxidant activities[J]. Journal of Biomolecular Screening, 2014, 19(6): 959-965.

[31] Liu B, Chen S, Zhang L .  Functional studies of the gene slr2049 from Synechocystis sp. PCC6803* and its site-directed mutation[J]. Gene, 2015, 563(2): 196-202.

# Phycocyanin #Phycocyaninpowder #cPhycocyanin #Spirulina

[bisexualmangoeswhere]

knowing ke jin was affected by the gene modification drug thing did not help soften the blow at all when you eventually read the explicit confirmation that he was affected. my heart has been cleaved in two

[traaansfem]

@butterfly-sapphire @witchestower @catboybiologist (please tag some people who know gastroenterology sierra that is NOT my zone of expertise)

Alright, so I found some actual papers on this, and my current suspicion about the function of the appendix is linked to this (assuming a bacterial strain not associated with pathogenicity suddenly becoming malevolent and colonizing areas generally held by other gut flora and causing sickness is a common occurrence in times of famine historically.). ANYWAY. The appendix is known to serve as a reservoir of gut flora, but it has weird stuff going on that didn't fit other models of predicted functions- so I suspect that a good hypothesis to test if these viroid adjacent loose "RNA obelisk" plasmid lookin fuckers would be as follows.

Test for presence of obelisk RNA in mouth/saliva samples, check stool samples, and check other tissues if there are volunteers and people willing to have things stuck up their butt or down their throat for science. Determine if there is a correlation between their presence or absence and gut health (I would assume you want a little- enough to trigger some immune response and keep goblet cells.. well, not sharp per se. But alert.). Lots of confounding variables to control for here, you'd need a broad data set.

Secondarily, if there is some negative health outcome, check against appendicitis and appendectomy tissue samples- that is to say, cross reference appendix samples for the presence of obelisks.

This would serve a preliminary purpose of figuring out if another, more ethically dubious test would be necessary at all.

Because I suspect that these obelisks can be corruptive to the proper function of an individual body's gut microbiome, like virions can be to plant vasculature and development. So if they interfere with the health of a gut microbiome, then it stands to reason that an appendix, which serves as a natural reserve for healthy digestive bacteria would have SOME way to keep them out. My suspicion is that the environment of a healthy appendix is designed to be hostile, even inimical to these obelisks. Thus, I would suspect that RNA detecting macrophages and phagocytic cells would be busying themselves in the appendix, cleaving and destroying invading obelisks to maintain a healthy reserve of gut bacteria. Or perhaps an elevated presence of an extracellular RNA destroying enzyme.

So, assuming that the appendix has some cellular or humoral guard dog mechanism, then loss of function in some phagocytic immune cells, loss of function or lack of function in whichever extracellular RNA cleavage and disintegration enzyme you care to name, temporary or permanent immune system collapse, autoimmune disorders, food poisoning (acute OR chronic), starvation, famine conditions, long term nutrient depletion or deficiencies, mutations in the genes coding for the enzymes that up or downregulate them in ways that result in a depletion of them as the body clears them or overproduces them... All of those could contribute to sudden bacterial or viral infections of the appendix, and particularly if the bacteria was already present and considered trustworthy and non hazardous by pathogen identification pathways. And the swelling and subsequent degeneration of the appendix itself could take days, weeks or even months to be apparent after whatever illness(es) or health issue(s) actually caused the antecedent issue.

The main issue I have with this is that you can establish correlation pretty easily here, but establishing causation would almost certainly necessitate invasive tests, removal of a healthy individual's appendix and immediate study thereof, testing the proteins and enzymes present, and genetic testing to determine if some pathways associated with those enzymes are not present in people who have needed appendectomies.

While you could make an argument that this could be done with organ donors who had given consent to have their bodies used for science, the loss of those organs for harvesting and subsequent transplant would result in disruption of the pathways for the appendix, which would skew data immensely. And an issue with the inverse of that, where you purposefully attempt to disrupt the function of the suspected mechanisms used for keeping the appendix healthy by disrupting the pathways for removal of those suspected causative agents for pathogenicity (RNA obelisks), is that if you get the data you needed, you just gave a coms patient appendicitis and may have ruined their other organs for transplant into other people. Whoopsie.

Anyway, there's a whole medical ethics conundrum and a bioethical tarpit to try to avoid here, so I think it might be best to wait until other people specifically studying the human gut weigh in on safer methodologies.

And that's not even touching on the likelihood of preliminary tests with other animals known to have structures that are both homologous AND analogous to the human appendix (start as low on the phylogenetic tree as you can is the usual rule), before working up to mammals and MAYBE lower primates before even getting CLOSE to humans.

Anyway, that's my 10:50PM to 11:29PM rambles. I'll schedule this one for later tomorrow, and I'll pester some microbiologists and immunologists I know about this.

Of course, this assumes the preprint isn't full of shit, and almost all of the papers promising a dramatic upheaval in biology are. The only reason I'm holding out any hope here is that RNA detection got overhauled a lot for covid detection and subsequent attempts at mitigation via biotechnology, so it MIGHT be possible that we missed this. So far. The specifics are dubious though. I hope they pan out though, this could be monumental! Monolithic, even! Why, you might as well carve it into an obelisk :3

They just discovered a new kind of organism that isn't a virus or bacterium

[rnomics]

Critical factors for precise and efficient #RNA cleavage by RNase Y in Staphylococcus aureus

by Alexandre Le Scornet, Ambre Jousselin, Kamila Baumas, Gergana Kostova, Sylvain Durand, Leonora Poljak, Roland Barriot, Eve Coutant, Romain Pigearias, Gabriel Tejero, Jonas Lootvoet, Céline Péllisier, Gladys Munoz, Ciarán Condon, Peter Redder Cellular processes require precise and specific gene regulation, in which continuous #mRNA degradation is a major element. The #mRNA degradation mechanisms should be able to degrade a wide range of different #RNA substrates with high efficiency, but should at the same time be limited, to avoid killing the cell by elimination of all cellular #RNA. RNase Y is a major endoribonuclease found in most Firmicutes, including Bacillus subtilis and Staphylococcus aureus. However, the molecular interactions that direct RNase Y to cleave the correct #RNA molecules at the correct position remain unknown. In this work we have identified transcripts that are homologs in S. aureus and B. subtilis, and are RNase Y targets in both bacteria. Two such transcript pairs were used as models to show a functional overlap between the S. aureus and the B. subtilis RNase Y, which highlighted the importance of the nucleotide sequence of the #RNA molecule itself in the RNase Y targeting process. Cleavage efficiency is driven by the primary nucleotide sequence immediately downstream of the cleavage site and base-pairing in a secondary structure a few nucleotides downstream. Cleavage positioning is roughly localised by the downstream secondary structure and fine-tuned by the nucleotide immediately upstream of the cleavage. The identified elements were sufficient for RNase Y-dependent cleavage, since the sequence elements from one of the model transcripts were able to convert an exogenous non-target transcript into a target for RNase Y. https://journals.plos.org/plosgenetics/article?id=10.1371%2Fjournal.pgen.1011349&utm_source=dlvr.it&utm_medium=tumblr