- Aug 2024
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Thus, when a family has a child with a disability, theparents may have a range of emotional responses that all family members experience at various time
They suggest that the family's emotions will likely shift as the child is experiencing shifts in their life (developmental shifts) and in smaller transitions like from one program to another.
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They suggestthat feelings of denial and grief are emotions that may disappear and reappear in all families
Turnbull and Turnbull urge professionals to explore beyond grief and denial because those are not the only emotions that families experience when dealing with their child with a disability and even so they will not always be experiencing those emotions anyway so the range of the experiences and feelings still need to be explored.
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Winton, in 1990, remindedprofessionals to define “denial” as an internal coping strategy,which may be useful to some parents, rather than view theconcept as a worrisome stage to be overcome before reaching the stage of acceptance
Winton challenged the term denial to have an alternative meaning amongst professionals, one that is less of a pitiful connotation and instead a term that is associated with coping.
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Featherstone (1980) suggested that some parents might notpass through the stages at all or might experience the stages in differing orders or at varying rates ofintensities.
Despite the four elements not being exactly similar to the stages of grief they are comparable in that they can be experienced out of order and in different intensities.
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The four elements of adaptation she describesinclude surviving, searching, settling in, and separating.
Parents and families of children with a disability adapt to having a child with a disability more than they grieve because the adaption allows them to evolve and are not seen as linear like grief.
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Howard, Williams, Port and Lepper (1997) suggest that it may not be helpful for professionals to viewfamily members as being in particular stages of grief.
Viewing family members as experiencing stages of grief, especially linear stages of grief, may not be beneficial because many families don't not see having a child with a disability as something heavy enough to "grieve" over or they don't not grieve in a linear manner about their child.
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Others have recognized that the presence of achild with a disability in a family can have many positive effects, and can even help to strengthenfamilies
Having a child with a disability in the family is not always a negative impact like it is believed to be by some people (and professionals) it can have a positive impact on the family.
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emmarshall.github.io emmarshall.github.io
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Cognition and Latent Learning
I would like to have a more decent understanding of this topic
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www.youtube.com www.youtube.com
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Fascinating and troublesome is the idea that older books are being edited to be less "offensive" in modern times. This alters the meaning of the past. Do not do this.
Keep it original.
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This person argues that one should cultivate a personal library, the tangibleness of the physical, to safeguard knowledge and prevent information control from falling to a single institution or person, so that they may never control the past.
I think he should go deeper into his argument, I do not fully understand what he means.
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Interesting thought. This guy relates the upcome of AI (non-fiction) writing to the lack of willingness people have to find out what is true and what is false.
Similar to Nas & Damian Marley's line in the Patience song -- "The average man can't prove of most of the things that he chooses to speak of. And still won't research and find the root of the truth that you seek of."
If you want to form an opinion about something, do this educated, not based on a single source--fact-check, do thorough research.
Charlie Munger's principle. "I never allow myself to have [express] an opinion about anything that I don't know the opponent side's argument better than they do."
It all boils down to a critical self-thinking society.
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emmarshall.github.io emmarshall.github.io
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Behaviorism
This added a lot of understanding to what I already had knowledge of for behaviorism. I have more understanding of animal behavior than I do of humans so this aded on a lot that knowledge for me
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emmarshall.github.io emmarshall.github.io
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Dyscalculia
This is an interesting article of a personal story from someone with dyscalculia. She explains how it impacts her life as a mom and what she has learned by researching her learning disorder. https://www.dyscalculia.org/stories/elle
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mitchellcameron123.github.io mitchellcameron123.github.io
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pendi
make sure this is cited as an appendix and finish the explanations of the data. make sure you provide a link to the coffee data.
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www.youtube.com www.youtube.com
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Lol seriously? How serendipitous I came across your channel then. Yes I am in the Scheper tribe, but I am not active as much over there--as well as the fact that I do not pay for the newsletter anymore due to finances.
Reply to LibraryLin
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@productivity6693 Hi There! I don't have a community for myself, but I am in Scott Schepler's Antinet Zettelkasten community. Are you there as well?
Interesting. LibraryLin is a Scheperian as well.
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“We must win back the Freys,” said Robb. “With them, we stillhave some chance of success, however small. Without them, I see nohope. I am willing to give Lord Walder whatever herequires ... apologies, honors, lands, gold ... there must be somethingthat would soothe his pride ...”“Not something,” said Catelyn. “Someone.”
UGH NOOO THE WEDDING
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“The next battle,” Robb said. “Well, that will be soon enough.Once Jo rey is wed, the Lannisters will take the eld against meonce more, I don’t doubt, and this time the Tyrells will march besidethem. And I may need to ght the Freys as well, if Black Walder hashis way ...”“So long as Theon Greyjoy sits in your father’s seat with yourbrothers’ blood on his hands, these other foes must wait,” Catelyntold her son. “Your rst duty is to defend your own people, winback Winterfell, and hang Theon in a crow’s cage to die slowly. Orelse put o that crown for good, Robb, for men will know that youare no true king at all.”
you've lost man you've lost :(
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If you had to fallinto a woman’s arms, my son, why couldn’t they have been MargaeryTyrell’s?
IF ONLYY
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I used to think the same as you, that the wolves were ourguardians, our protectors, until ...”“Until?” she prompted.Robb’s mouth tightened. “....until they told me that Theon hadmurdered Bran and Rickon. Small good their wolves did them.
ughh i can't even be upset with him over that
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I used to think the same as you, that the wolves were ourguardians, our protectors, until ...”“Until?” she prompted.Robb’s mouth tightened. “....until they told me that Theon hadmurdered Bran and Rickon. Small good their wolves did them.
ughh i can't even be upset with him over that
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And there’s the heart of it, Catelyn thought. “He is part of you,Robb. To fear him is to fear you.”“I am not a wolf, no matter what they call me.” Robb soundedcross. “Grey Wind killed a man at the Crag, another at Ashemark,and six or seven at Oxcross. If you had seen—”“I saw Bran’s wolf tear out a man’s throat at Winterfell,” she saidsharply, “and loved him for it.”“That’s di erent. The man at the Crag was a knight Jeyne hadknown all her life. You can’t blame her for being afraid. Grey Winddoesn’t like her uncle either. He bares his teeth every time Ser Rolphcomes near him.”A chill went through her. “Send Ser Rolph away. At once.”
brooo your wolf is your own lie detecter listennn
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and there was another Jeyne Westerling who was queen to KingMaegor three hundred years ago.”
and SHE didn't end well
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“I know,” her son said, stricken. “I’ve made a botch of everythingbut the battles, haven’t I? I thought the battles would be the hardpart, but ... if I had listened to you and kept Theon as my hostage,I’d still rule the north, and Bran and Rickon would be alive and safein Winterfell.”
poor guy
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Jeyne had me taken to her own bed, and she nursed meuntil the fever passed. And she was with me when the Greatjonbrought me the news of ... of Winterfell. Bran and Rickon.” Heseemed to have trouble saying his brothers’ names. “That night,she ... she comforted me, Mother.”Catelyn did not need to be told what sort of comfort JeyneWesterling had o ered her son. “And you wed her the next day.”He looked her in the eyes, proud and miserable all at once. “Itwas the only honorable thing to do. She’s gentle and sweet, Mother,
thats kinda cute ig
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It is swords you need, not gentle hearts. How could you do this, Robb?How could you be so heedless, so stupid? How could you be so ... sovery ... young.
yeah...yeah
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“That would be best.” Robb kissed his Jeyne. “The steward will nd you suitable accommodations.”
oh ok so he does like her
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Jeyne Westerling. Lord Gawen’s elder daughter, and my ... ah ... mylady wife.”The rst thought that ew across Catelyn’s mind was, No, thatcannot be, you are only a child.The second was, And besides, you have pledged another.The third was, Mother have mercy, Robb, what have you done?Only then came her belated remembrance. Follies done for love? Hehas bagged me neat as a hare in a snare. I seem to have already forgivenhim.
LMAOOO ITS SO BAD BRUH
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“The honor is mine,” Catelyn said. Can Robb have won the Crag’sallegiance? If so, it was no wonder the Westerlings were with him.Casterly Rock did not su er such betrayals gently. Not since TywinLannister had been old enough to go to war ...
uhh well no
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Lady Mormont tookher hand and said, “My lady, if Cersei Lannister held two of mydaughters, I would have done the same.” The Greatjon, no respecterof proprieties, lifted her o her feet and squeezed her arms with his
real ones even if greatjon hates women
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Thewolf. The wolf is not here. Where is Grey Wind? She knew the direwolfhad returned with Robb, she had heard the dogs, but he was not inthe hall, not at her son’s side where he belonged.
OH NO
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“I have. I know what it is to love so greatly you can think ofnothing else.”Catelyn bowed her head. “Thank you.” I have not lost this child, atleast.
uhh don't be happy yet
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“Enough.” For just an instant Robb sounded more like Brandonthan his father. “No man calls my lady of Winterfell a traitor in myhearing, Lord Rickard.” When he turned to Catelyn, his voicesoftened. “If I could wish the Kingslayer back in chains I would. Youfreed him without my knowledge or consent ... but what you did, Iknow you did for love. For Arya and Sansa, and out of grief for Branand Rickon. Love’s not always wise, I’ve learned. It can lead us togreat folly, but we follow our hearts ... wherever they take us. Don’twe, Mother?”
would've been sweet if yk
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“It was a mother’s folly. Women are madethat way.”
eugh shut up
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He leapt o thedais and pulled Catelyn into his arms. When he said, “It is good tosee you home, Cat,” she had to struggle to keep her composure.“And you,” she whispered.
the dad that stepped up
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If Robb looks at me as Edmure did, I do notknow what I will do. But it seemed to her that it was not anger shesaw in her son’s eyes, but something else ... apprehension, perhaps?No, that made no sense. What should he fear? He was the YoungWolf, King of the Trident and the North.
haha they've both been traitors
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He is sixteen now, a man grown. Just look at him. War hadmelted all the softness from his face and left him hard and lean. Hehad shaved his beard away, but his auburn hair fell uncut to hisshoulders.
oh my age
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One of Robb’s white banners lay on the ground, andone of the knights turned his horse and trampled over the direwolfas he spurred toward the gate. Several others did the same. Thoseare men who fought with Edmure on the fords, she thought. What couldhave made them so angry? Has my brother slighted them somehow, giventhem some insult? She thought she recognized Ser Perwyn Frey,
ahh jeyne
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His eyes went wide, “Gods be good,” he said in a choked voice.“Arya Underfoot? Lem, let go of her.”“She broke my nose.” Lem dumped her unceremoniously to the oor. “Who in seven hells is she supposed to be?”“The Hand’s daughter.” Harwin went to one knee before her.“Arya Stark, of Winterfell.”
FINALLYYYY
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For a moment she did not know how to answer. She’d had somany names. Had she only dreamed Arya Stark? “I’m a girl,” shesni ed. “I was Lord Bolton’s cupbearer but he was going to leave me
i wish he'd know her
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A moment later a man came swaggeringthrough the open door, a Tyroshi even bigger than Lem with a greatthick beard, bright green at the ends but growing out grey.
OH NOOO
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“There’s no cause for spilling good ale onmy oor,” said Sharna. “Sit back down and calm yourself, boy,there’s rabbit coming. You too, girl. Whatever harm’s been doneyou, it’s over and it’s done and you’re with king’s men now. We’llkeep you safe as best we can.”
they'll be safe? suree
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“Three golden dragons. We need to buy those horses.”
bruh sell horses and then gain them back
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Anyways, you lot were supposed to get them back.”
ahh interesting
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“We were thinking we might eat it,Sharna. With lemons. If you had some.”“Lemons. And where would we get lemons? Does this look likeDorne to you
ahh so dany was kept in dorne
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“A bear there was, a bear, a bear!All black and brown, and covered with hair ...”
that song...
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“Sharna’s husband, and an orphan boy they took in. They won’tharm you. There’s ale, if you think you’re old enough. Fresh bread
ugh they could've met brienne and jaime
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Escaped from Lord Bolton’s kitchen, did you?”“How did you know?” Arya demanded, uneasy.“You bear his sigil on your chest, little one.”
forgot about that
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“Squab, if you want,” she said. “I don’t care.”
lord these names are hideous
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s and a hundreddoves baked into a great pie,” Shae gushed. “When the crust’sopened, they’ll all burst out and y.”“After which they will roost in the rafters and rain down birdshiton the guests.” Tyrion had su ered such wedding pies before. Thedoves liked to shit on him especially, or so he had always suspected.
how does that work??
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Lollys won’t go. I toldher no one’s like to rape her in the king’s own throne room, butshe’s so stupid.”
wtf did i just read
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“My lord.” A woman sidled into the light; plump, soft, matronly,with a round pink moon of a face and heavy dark curls. Tyrionrecoiled. “Is something amiss?” she asked.Varys, he realized with annoyance. “For one horrid moment Ithought you’d brought me Lollys instead of Shae. Where is she?”“Here, m’lord.” She put her hands over his eyes from behind.“Can you guess what I’m wearing?”“Nothing?”“Oh, you’re so smart,” she pouted, snatching her hands away.“How did you know?”“You’re very beautiful in nothing.”“Am I?” she said. “Am I truly?”“Oh yes.”“Then shouldn’t you be fucking me instead of talking?”“We need to rid ourselves of Lady Varys rst. I am not the sort ofdwarf who likes an audience.”“He’s gone,” Shae said.Tyrion turned to look. It was true. The eunuch had vanished,skirts and all. The hidden doors are here somewhere, they have to be.That was as much as he had time to think, before Shae turned his
CRYING WHY IS HE THIRD WHEELING LMAOO
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I had my ownlove once, and we had a song as well.” I loved a maid as fair assummer, with sunlight in her hair.
interesting
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“Prince Aemon the Dragonknight took his vows at seventeen,” SerLoras said, “and your brother Jaime was younger still.”“I know their reasons. What are yours? T
they joined for love and and now loras's lover is dead
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It was strange to see the Knight of Flowers all in whitewhen before he had always been as colorful as a rainbow
its so obvious lmao
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It’s even worse than I feared. “And my father? Who does he havespying on me?”This time the eunuch laughed aloud. “Why, me, my lord.”
lmao
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“And the second Aegon fed GrandMaester Gerardys to his dragon.”
oh!
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“My lord Tyrion,” came out in a squeak,punctuated by a nervous giggle.
you can suprise varys?
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Jaime hauled the last dragonking bodily o the steps, squealing likea pig and smelling like a privy.
thats insaneee
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So spare me your envy. It was the gods who neglectedto give you a cock, not me.”
cersei and brienne both being eveious of him is kinda perfect
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A gift you want desperately, wench, and can never have.
dany make her a kingsguard pleaseee
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Orperhaps your moon’s blood was on you. Never give a wench a swordwhen she’s bleeding.”
i-
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“Yes, and what you did as well. We’re both kingslayers here, ifwhat I’ve heard is true.”
similar yet opposite
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but heresigned the Handship on some thin pretext and returned to CasterlyRock, taking his daughter with him. Instead of being together,Cersei and Jaime just changed places, and he found himself alone atcourt, guarding a mad king
woops
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excited him all the more. Jaime had never seen her more passionate.Every time he went to sleep, she woke him again. By morningCasterly Rock seemed a small price to pay to be near her always. Hegave his consent, and Cersei promised to do the rest.
eughh
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Aerys had Ser Ilyn Payne’s tongue torn out just forboasting that it was the Hand who truly ruled the Seven Kingdoms.The captain of the Hand’s guard, and yet Father dared not try andstop it! He won’t stop this, either.
bruhh
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sleeping lion
sleeping lion? bruh don't steal from the lannisters
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Cersei took him aside andwhispered that Lord Tywin meant to marry him to Lysa Tully, hadgone so far as to invite Lord Hoster to the city to discuss dower. Butif Jaime took the white, he could be near her always.
jaime and LYSA?? NAHH
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Jaime sat against the bole of an oak and wondered what Cerseiand Tyrion were doing just now. “Do you have any siblings, mylady?” he asked.Brienne squinted at him suspiciously. “No. I was my father’s onlys—child.”Jaime chuckled. “Son, you meant to say. Does he think of you as ason? You make a queer sort of daughter, to be sure.”Wordless, she turned away from him, her knuckles tight on hersword hilt. What a wretched creature this one is. She reminded him ofTyrion in some queer way, though at rst blush two people couldscarcely be any more dissimilar. Perhaps it was that thought of hisbrother that made him say, “I did not intend to give o ense,Brienne. Forgive me.
can never doubt that this man loves his siblings lmao
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Brienne considered them brie y, and thenswung her horse onto the southern road. Jaime was pleasantlysurprised; it was the same choice he would have made.
oh yeah
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huge ugly cow ofa woman.
stfu
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Thesaddlecloth had originally been checkered pink and black,
related to arya i just know it
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And the lightning lord’s been seen in these parts as well. He crossesthe river wherever he likes, riding this way and that way, neverstill.”“And who is this lightning lord?” demanded Ser Cleos Frey.“Lord Beric, as it please you, ser. They call him that ’cause hestrikes so sudden, like lightning from a clear sky. It’s said he cannotdie.”They all die when you shove a sword through them, Jaime thought.“Does Thoros of Myr still ride with him?”“Aye. The red wizard. I’ve heard tell he has strange powers.”
yess i wanna know more abt him
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ride by way ofDuskendale
uhh isn't yhe ighting heading there
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“Seize him, and take his dirk. He means to use it onour lady.”
NOOO
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The boy went down as well, but he was up again almost at once.“What are you doing here?” he demanded as he brushed himself o .Jet-black hair fell to his collar, and his eyes were a startling blue.“You shouldn’t get in my way when I’m running.”
oh edric storm!
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hell
they have hell? oh wait i forgot about the seven hells
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Salladhor Saan pushed himself to his feet. “You are no true friend,I am thinking. When you are dead, who will be bringing your ashesand bones back to your lady wife and telling her that she has lost ahusband and four sons? Only sad old Salladhor Saan. But so be it,brave ser knight, go rushing to your grave. I will gather your bonesin a sack and give them to the sons you leave behind, to wear inlittle bags around their necks.” He waved an angry hand, with ringson every nger. “Go, go, go, go, go.”
OH HE ATE HIM UP
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“And this is why,” said Davos. “To do this thing. To make an endof Melisandre of Asshai and all her works. Why else would the seahave spit me out? You know Blackwater Bay as well as I do, Salla.No sensible captain would ever take his ship through the spears ofthe merling king and risk ripping out his bottom. Shayala’s Danceshould never have come near me.”“A wind,” insisted Salladhor Saan loudly, “an ill wind, is all. Awind drove her too far to the south.”“And who sent the wind? Salla, the Mother spoke to me.”The old Lyseni blinked at him. “Your mother is dead ...”“The Mother. She blessed me with seven sons, and yet I let themburn her. She spoke to me. We called the re, she said. We calledthe shadows too. I rowed Melisandre into the bowels of Storm’s Endand watched her birth a horror.” He saw it still in his nightmares,the gaunt black hands pushing against her thighs as it wriggled freeof her swollen womb. “She killed Cressen and Lord Renly and abrave man named Cortnay Penrose, and she killed my sons as well.Now it is time someone killed her.”
davos you are no hero i fear
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Melisandre. Davos shivered. “The red woman did this to him,” hesaid. “She sent the re to consume us, to punish Stannis for settingher aside, to teach him that he could not hope to win without hersorceries.”
ummm
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“Illyrio Mopatis.
so e's trying to smugglethings in hmm
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Salladhor Saan threaded his way between the jars of spice and boltsof cloth that lled the hold of the merchanter, wrapped Davos in a erce embrace, then kissed him once on each cheek and a third timeon his forehead. “You are still warm, ser, and I feel your heartthumpety-thumping. Can it be true? The sea that swallowed you hasspit you up again.”
lmao
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table he spent the rest of the day withone end or the other dangling over the rail
thats how they poop at sea...
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“So wide open that I fear you may fall through it, and live all therest of your days as a wolf of the woods.”
lmao
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He was the only one who liked Bran’s plan, though. Meera justsmiled at him and Jojen frowned. They never listened to what hewanted, even though Bran was a Stark and a prince besides, and theReeds of the Neck were Stark bannermen
bran's a bit annoying
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wo crannogmen a thousand leagues fromthe Neck.
why not ask your dad for help
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jen Reed was thirteen, only fouryears older than Bran.
i thought he was like 9
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But his sister hadleft the wilds, to walk in the halls of man-rock where other huntersruled, and once within those halls it was hard to nd the path backout.
yeah sansa
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.. all but the sister they had lost. His taildrooped when he remembered her. Four now, not ve. Four and onemore, the white who has no voice.
:(( lady killed by lannisters and soon robb too
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Prince. The man-sound came into his head suddenly, yet he couldfeel the rightness of it. Prince of the green, prince of the wolfswood. He
bloodraven?
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. And I tell youtruly, Daenerys, there is no man in all the world who will ever behalf so true to you as me.”
YEAH NO
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“You ... you should not have ...”“I should not have waited so long,” he nished for her. “I shouldhave kissed you in Qarth, in Vaes Tolorru. I should have kissed youin the red waste, every night and every day. You were made to bekissed, often and well.” His eyes were on her breasts.
ewww
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“And my vest—” shestarted to say, turning.Ser Jorah slid his arms around her.“Oh,” was all Dany had time to say as he pulled her close andpressed his lips down on hers. He smelled of sweat and salt andleather, and the iron studs on his jerkin dug into her naked breastsas he crushed her hard against him. One hand held her by theshoulder while the other slid down her spine to the small of her
STOPPP
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but three living dragons, andthose were hers; they were a wonder, and a terror, and beyondprice.
suprised no one in westeros has mentioned them yet
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Until one day Prince Rhaegar found something in hisscrolls that changed him.
the prophecy
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Young Lord Connington was dear to the prince as well,
oh im sure
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His name wasArstan,
barristan selmy?
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ut the dragons the Seven Kingdoms knew best werethose of House Targaryen. They were bred for war, and in war theydied.
the dance..
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ut when she told her brother, Viserys had twisted her hairuntil she cried. “You are blood of the dragon,” he had screamed ather. “A dragon, not some smelly sh.”
:(
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DaenerysTargaryen was as happy as she could ever remember being.
YESS
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dragons chase each other across a cloudless blue sky,
omg they can fly now
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academic.oup.com academic.oup.com
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What is more, axonal SMN is reported to interact with distinct RNA-binding proteins,36,37,38,39 an indication that it assembles numerous RNP particles.
Lack of specificity to MNs?
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www.biorxiv.org www.biorxiv.org
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Referee #3
Evidence, reproducibility and clarity
The manuscript by Balachandra and Amodeo presents Bellymount-Pulsed Tracking as a technique for continuous long-term imaging of Drosophila oogenesis. This approach modifies the existing Bellymount technique by exposing restrained female flies to pulses of CO2 anesthesia in combination with image acquisition. Flies that survived the restraint were kept alive for many hours by addition of a liquid diet in the restraint apparatus. This allowed for imaging and tracking of egg chamber development over longer time periods than capable with ex vivo culturing methods. However, the authors did report a 40% mortality rate and decreased fecundity compared to unrestrained flies. Using this method the authors were able to image and measure the growth rate of developing egg chambers in living flies, and capture events like vitellogenesis which relies on the interactions of multiple organ systems.
This technique is a notable contribution to the fly community, as it could be useful for studying processes that require interactions between multiple tissues and organs, as well as for long-term imaging of other internal structures in the adult fly. The significance is somewhat reduced due to the relatively high mortality rate and the decreased fecundity and egg chamber growth rate reported. However, the authors should be commended for their diligence in documenting the limitations of the procedure, as this now provides a strong jumping off point to improve the technique if it becomes widely adopted by the fly community. Overall, the experiments appear to have been carefully performed and the manuscript is clearly written. However, there are several issues that should be addressed prior to publication.
Major concerns
- The movies of egg chamber development are challenging to interpret. They could be improved by the addition of timestamps and other annotations. Having multiple example movies of the same process would also be valuable. It could be helpful to potential users of this technique to show the process the authors used for identifying the same egg chamber between such long time points.
- Figure 4 - Given that the Bellymount PT technique slows oogenesis and reduces egg chamber growth in vitellogenic stages (Figure 3E), it is possible that Bellymount PT slows yolk protein uptake. It would be important to establish a baseline for how much to expect yolk protein levels to change across stages to compare to measurements obtained with Bellymount PT. It would be a relatively simple experiment to show the change in yolk protein uptake across stages in fixed samples. This could also be performed for His2Av dynamics during nurse cell dumping.
- Movie 11 - The authors propose that Bellymount-PT can be used to visualize the process of border cell migration. However, there is no obvious movement of the cluster relative to the nurse cell nuclei over the course of the 3 hour long movie. The authors should either show a better movie of border cell migration, or remove this claim from the manuscript.
- Movie 13 - The authors claim that they see egg chamber rotation continue in stage 9 and 10 egg chambers. This movie is not convincing. There is also very strong evidence in the literature that egg chamber rotation ends at stage 8. Chen et al., Cell Reports, 2017 showed using a method that tracks follicle cell migration in vivo that rotational migration ends during stage 8. The only movement of follicle cells after stage 8 is due to the epithelial reorganization that occurs during the posterior movement of the follicle cells as the stretch cells flatten. Additionally, after stage 8 follicle cells lose their circumferentially oriented actin protrusions that drive rotation. This claim should be removed from the manuscript.
Minor comments
- Line 104 - The authors mention that CO2 affects fertility in flies. They should also reference Sustar et al., Genetics, 2023 and Zimmerman and Berg, PLoS One, 2024 for wider ranging effects of CO2 on oogenesis.
- Line 244 - Although it is true that the original paper describing egg chamber rotation reported that it starts at 5, subsequent studies from multiple labs have confirmed that it begins much earlier. First shown by Cetera et al., Nature Communications, 2014 but later confirmed by Bilder, Dahmann, and Mirouse labs. Chen et al., Cell Reports, 2016 has even published a movie of an egg chamber initiating rotation as it buds from the germarium.
- Figures of egg chambers are generally oriented anterior on the left and posterior on the right. Reorienting all the figures would be challenging, so the recommendation is to be clear in the figure legends the orientation of the images. This is important given they are shown in different orientations in Figure 1 than throughout the rest of the paper, and also will be helpful for readers who may not be familiar with the structure of the ovary/egg chambers.
- Figure 1B and Methods line 334 - Should "Rely" be "Relay"?
- Figure 1E - Oocyte nuclei are missing from the diagrams of stage 7, 13 and 14 egg chambers. Also, "G" looks like a figure panel label, could just say Germarium
- Figure 3F-H - "Stagee" should be "Stage"
- Figure 4B - Why is the fluorescence for egg chamber #6 so much higher than the others? It makes the slopes of the other samples hard to see.
- Figure 4D,E,G - For clarity, the labeled boxes should be the same color as the lines on the associated graphs. In line 790 "Note the steady increase of H2Av in all three regions as it exits the nurse cell nuclei" - this is not actually shown without the nurse cell nuclei average intensity being on the graph as well.
- Line 787 - "Note the flow of H2Av" - "flow" is not actually shown in these static images. Consider a more precise description.
Referee Cross-commenting
The other reviewers make several excellent points. We personally feel that it is beyond the scope of this initial report to ask the authors to show that they can see all aspects of oogenesis with this technique. If the method becomes widely adopted by the oogenesis community, individual researchers can optimize it to suit the exact process they want to study. If the authors want to claim they can see a particular process, it needs to be well documented and convincing. For example, we agree that the movies that claim to show egg chamber rotation (both during established stages and later) and border cell migration need to be improved or the claims need to be removed. However, we feel that the authors have documented enough other interesting processes to make the study worthy of publication. Likewise, asking the authors to determine the minimal time window that can be used for imaging could take months of open-ended work and is something that could be better tackled by subsequent users depending on the requirements of the biological process they want to study. It seems better to get the work out into the public sooner rather than later so that improvements can be crowd sourced.
Finally, although Flp-out clones were used for cell tracking in the original Belly mount paper, this technique will be less effective during the first half of oogenesis when the egg chamber is rotating, as the clone is likely to rotate into and out of sight between imaging time points.
Significance
This technique is a notable contribution to the fly community, as it could be useful for studying processes that require interactions between multiple tissues and organs, as well as for long-term imaging of other internal structures in the adult fly. The significance is somewhat reduced due to the relatively high mortality rate and the decreased fecundity and egg chamber growth rate reported. However, the authors should be commended for their diligence in documenting the limitations of the procedure, as this now provides a strong jumping off point to improve the technique if it becomes widely adopted by the fly community. Overall, the experiments appear to have been carefully performed and the manuscript is clearly written. However, there are several issues that should be addressed prior to publication.
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Referee #2
Evidence, reproducibility and clarity
Summary:
The authors describe an improvement of the Bellymount imaging method for internal tissues of the fly's abdomen. They are able to increase the total duration of the imaging by introducing pulsed anesthesia. This allows the immobilized flies to take up food in between the imaging; this increases survival rate and allows for longer total imaging times. The authors illustrate the technique by tracking the development of egg chambers.
Major Points
- The Bellymount PT method results in decreased fecundity, which might affect the processes (oogenesis) the authors looked at. Indeed, the authors conclude that "oogenesis is not completely stalled under the Bellymount-PT protocol" (line 140). The authors do provide some data indicating that egg chambers develop (Fig. 2G,H; Fig. 3F,H), in particular a stage 10 egg chamber proceeding to a stage where dorsal appendages seem to form. However, for early stage egg chambers this is less convincing. The egg chambers show an increase in (cross-sectional) area, however, what is the evidence that they also mature? For example, during egg chamber maturation, the ratio of oocyte/nurse cell volume changes, follicle cells re-arrange, etc. The authors should test whether any of these characteristics can be observed in egg chambers imaged using Bellymount PT. This may include the imaging of egg chambers in which both nuclei and plasma membranes are visualized.
- A potential advantage of the Bellymount PT method is the ability to follow the dynamics of processes. A current drawback, however, is the rather low temporal resolution as the fly needs to wake up between single images. The authors should provide an estimate for the minimal possible cycle time and should test whether flies imaged at 10 minutes interval show lower survival/fecundity than flies imaged at 2 hours interval.
- The authors claim that they can track on a cellular level (based on nuclei), but it is unclear how accurate the tracking is. Especially cell tracking over very long times might be challenging here, as the time delay between two time points is big. The authors should test the accuracy of their tracking, potentially by creating Flip-out clones and using them as a control.
- The authors show that they can visualize cell membranes (Moesin-GFP, Fig. 2C). Tracking cells over time based on their membranes would greatly widen the applicability of the method as it would enable to analyze the complex cellular dynamics during egg chamber maturation. The authors should test whether cells can be tracked over time (e.g. using Moesin-GFP) using their technique.
- Movie 11. The authors claim that they can capture border cell migration. However, it is unclear whether the border cells actually migrate towards posterior. The authors should track and quantitatively analyze the migration path of the border cells in their movies.
- Movie 12. The authors claim that they can observe egg chamber rotation. However, it is unclear whether the egg chambers actually rotate. The authors should track cells and quantify the angular velocity of movement.
Minor Points
- Please move the labels of the scale bars to the legends.
- The figures (especially 2 and 3) would benefit from a clearer structuring. Moving part of them to supplementary figures would also help.
- "stage" typo in figure 3
Significance
The authors describe here an improvement of an existing technique. The advantage of the improved technique is the longer imaging time, which potentially allows users to track cells/organelles/proteins over time. However, tracking requires the user to connect single time points with each other, which is somewhat unclear at this time. Moreover, the potential applicability (and significance) of the technique would be widened if visualization and tracking of cell membranes/organelles/vesicles would be possible. With these further optimizations, the technique would add a useful tool to the Drosophila community.
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Referee #1
Evidence, reproducibility and clarity
Summary
The Drosophila ovary is an established model system for many aspects of development and cell biology. In vitro culture of live ovaries has provided valuable insight, yet these methods do not accurately mimic oogenesis in vivo for some stages. Here the authors develop a new method that allows for sustained imaging of ovaries in intact flies, maintaining normal physiology.
The method provides a valuable addition to the field. Processes such as growth, cell migration, egg chamber rotation, yolk uptake and nurse cell dumping can be observed in the intact fly. Time lapse and 3D reconstruction provide valuable tools. While the detail/resolution of the images is not as good as ex vivo or fixed samples, the ability to maintain normal development and homeostasis provides a novel advantage. The figures and movies are well-presented and sufficient detail is provided in the methods.
Major comments
- Why do the authors think that growth is slowed? The imaging process or the trapping/anesthesia of the fly? For example, if the frequency of imaging was varied, it could reveal whether it was the actual imaging that affected development. Did the length of time the fly had been in the trap make a difference? The sentence on lines 190-191 is not clear.
- In Movie 6, the nurse cell nuclear shape does not look normal - more ovoid than round. Perhaps some settings are off in the 3D reconstruction.
- Movie 11 - why do the border cells seem stalled?
- There is no discussion of the earliest stages of oogenesis. Is it possible to see egg chambers forming from the germarium?
Minor comments
- It would be helpful to mention if the egg chambers stay in similar locations or move around - is it challenging to locate the same egg chamber after 2 hours?
- Are any egg chambers degenerating? This could indicate stress in the fly.
- In Figure 4D, release of HisAV into the cytoplasm is described. Similar release of nuclear proteins was described by Cooley et al. 1992 so this paper could be cited.
- At 321 minutes in Figure 4D, a large nucleus is apparent in the oocyte. Is this an oocyte nucleus or evidence for nurse cell translocation to the oocyte as described in Ali-Murthy et al. 2021?
Significance
The technique provides a significant advance to the field, extending the time period currently possible to image ovaries through the Belly Mount method. It will immediately benefit researchers working on the ovary but could be extended to many other tissues in the fly abdomen such as the gut and tumor models.
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platform.openai.com platform.openai.com
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Both input and output tokens count toward these quantities. For example, if your API call used 10 tokens in the message input and you received 20 tokens in the message output, you would be billed for 30 tokens. Note however that for some models the price per token is different for tokens in the input vs. the output (see the pricing page for more information).
输入输出都计价 输入的价格和输出的价格不一样
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www.youtube.com www.youtube.com
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use the Neuroscience principle of education for corporate learning systems so instead of just having a classic a classic lesson to teach people
for - neuroscience and education - problem solving - active learning
neuroscience and education - problem solving - active learning - this is much like Socratic dialogue technique, engaging the learner actively to recreate the problem in their own consciousness - and play an active role in solving it - just like historical innovators did
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is it possible to teach machine values
for - question - AI - can we teach AI values?
question - AI - can we teach AI values? - it's likely not possible because we cannot assign metrics to things like - ethics - kindness - happiness
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Solutions or systems that are created uh to solve problems
for - question - neuroscience - creating neuroscience-based systems for solving problems
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studies that are coming in right now from the last two years where we were forced to work remotely we see a decrease in Innovation and creative potential in in companies
for - neuroscience research - remote intentional working during Covid - showed decreased productivity and innovation
neuroscience research - remote intentional working during Covid - showed decreased productivity and innovation - Due to only creating intentional work times and eliminating the opportunities for informal meeting - When it is purely intentional work contexts created and no relaxing, informal opportunities to meet, innovation suffers
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the first question that came in and as we're embracing remote and hybrid working as The New Normal how do you address this from a neuroscience perspective
for - question - neuroscience - efficacy of hybrid remote and live work environments
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the future future for education and this is a mega Trend that will last in the next decades is that we use artificial intelligence to tailor um educational let's say or didactic Concepts to the specific person so let's say in in the future everybody will have his or her specific let's say training or education profile he or she will run through and artificial intelligence um will will tailor the different educational environments for everybody in the future this is this is a pre this is a pretty clear Trend
for - AI and education - children will have custom tailored education program via AI
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before puberty before let's say 30 and 14 years of age um we know that the Restriction of those devices is beneficial for the development of the brain because children learn to to think in a three-dimensional world
for - neuroscience - education of children - recommend no digital devices before puberty - allows learning in a 3 dimensional world
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children for instance ask 500 2 000 questions a day and as you are grown up it's maybe 10 or 20 Questions per day
for - neuroscience - importance of maintaining curiosity - 1000 questions a day for children - 20 questions a day for adults
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in fact the best ideas happen when you are not planning them when you are just creating an environment where people get together in an informal way this is the reason why um Steve Jobs when he designed the Pixar building um he the initial idea was there's just one bathroom for the whole company
for - neuroscience - building design - common area to converge everyone - creates diverse social meetings - increases work efficacy - example - Steve Jobs - Pixar bathroom
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upport cross-divisional thinking and that the best ideas are already in a company and it's just a matter of sort of um getting people together
for - neuroscience - validation for Stop Reset Go open source participatory system mapping for design innovation
neuroscience - validation for Stop Reset Go open source participatory system mapping for design innovation - bottom-up collective design efficacy - What Henning Beck validates for companies can also apply to using Stop Reset Go participatory system mapping within an open space to de-silo and be as inclusive as possible of many different silo'd transition actors
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we are slower we are irrational we are imperfect we are drifting away we are forgetting stuff we are making mistakes but we are learning from our failures we get support from our from our friends from our from our colleagues and we are understanding and instead of just analyzing the world and this is giving us the ultimate cognitive Edge
for - key insight - human vs artificial intelligence - humans will create the best ideas
key insight - human vs artificial intelligence - humans will create the best ideas - why? - because we are - slower - imperfect - less rational - drifting away - forgetting - and we learn from the mistakes we make and from different perspectives shared with us
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by doubling the size of the tables in the in the eating in the eating areas they increase cross-divisional across talk um in a very informal way they found out that cross-department um Corporation increased after that and the and the code and the code output increased two months later
for - neuroscience - example - informal diversity - increases work efficacy - via sharing diverse and novel perspectives
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all the great ideas come um with a price tag of it's maybe a mistake
for - neuroscience - innovation - great ideas - mistakes and - risk
neuroscience - innovation - great ideas - mistakes and - risk - Any new idea involves taking a risk that it could be wrong - we cannot be innovators if we are not able to risk making mistakes
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when we analyze what is happening in the brain when we are doing a mistake then we we see that a lot of different areas active when one region is missing the region for fear
for - neuroscience - mistakes - and fear
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a good projects always benefit from cross-divisional from cross-divisional cooperation from asking some guys from outside not because they are showing the better um the better solution but usually they they give a good they give a good question they ask questions that nobody ever asked before and thereby giving you some kind of some kind of New Perspective
for - Indyweb - Stop Reset Go participatory system mapping - benefits of open source - Henning Beck - neuroscience support
Indyweb - Stop Reset Go participatory system mapping - benefits of open source - Henning Beck validates the importance of an open source design of the Stop Reset Go participatory system mapping - By developing an open source graph for many silo'd actors to participate, they mutually desilo each other - The sharing of diverse perspectives helps to mitigate progress traps
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here you see a company with three different departments depicted in blue red and green
for - neuroscience - example - diverse and low density connections beats non-diverse and high connections
neuroscience - example diverse and low density connections vs non-diverse high density connections - having access to many diverse perspectives is a key enabler of good problem-solving and innovation
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catching a break is necessary in order to refill your mental capacities and as a rule of thumb you can say that it's it's five to one five parts of work one part of doing a break so 50 minutes working 10 minutes catching a break
for - neuroscience - efficient work - relaxation rule
neuroscience - efficient work - relaxation rule - It is necessary to build NO WORK time into effective work - 5 time units work - 1 time unit relaxation - It is necessary to step back from concentrating on a problem - for the brain to drift away from it and - relax from concentrating on the problem - so that new perspectives can develop that can be brought back to solve the problem
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I have never seen a single project that did not benefit from asking a non-expert
for - quote - neuroscience - perspective shift - benefits
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it's about concentration prioritization and drifting away and doing something different
for - neuroscience - ideation depends on three different brain functions and brain areas - concentration - prioritization - and drifting away
neuroscience - ideation depends on three different brain functions and brain areas - concentration<br /> - frontal area of brain - prioritization and - deep inner part of the brain - drifting away - back part of the brain
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what is the most brain friendly working environment in our digital in our digital working area and interestingly there are as I've shown you before there are different aspects of our way of thinking I mean we are not thinking the same way throughout the day um there are phases at the day
for - neuroscience - optimal working environment - varies with brain state - different phases during the day - engagement - inspiration - concentration - communication - relaxation
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how long did it take you to understand the word brexit
for - neuroscience - human abilities - example Brexit and variations
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we know from Lab studies that children understand the meaning of stuff at first or second or third site you
for - neuroscience - children's understanding - 3 examples is enough to consolidate new concept
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this is the reason why I'm not afraid of artificial intelligence taking over
for - question - AI - can AI learn to be intentionally distracted?
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human beings are good at getting distracted at mentally drifting away doing something else and thereby thereby understanding the world and give meaning to stuff
for - neuroscience - human understanding - what makes us excel? - forgetting and getting distracted!
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usually it sticks you you know that moment you know that aha moment when you say ah I got it I understood it and suddenly from one second to the next your your way of thinking completely changes and this is the main difference in our world
for - human learning - key feature - evolutionary nature - indyweb - key feature - evolutionary nature of learning
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human beings don't do that we understand that the chair is not a specifically shaped object but something you consider and once you understood that concept that principle you see chairs everywhere you can create completely new chairs
for - comparison - human vs artificial intelligence
question - comparison - human vs artificial intelligence - Can't an AI also consider things we sit on to then generalize their classifcation algorithm?
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the brain is Islam Islam is it is lousy and it is selfish and still it is working yeah look around you working brains wherever you look and the reason for this is that we totally think differently than any kind of digital and computer system you know of and many Engineers from the AI field haven't figured out that massive difference that massive difference yet
for - comparison - brain vs machine intelligence
comparison - brain vs machine intelligence - the brain is inferior to machine in many ways - many times slower - much less accurate - network of neurons is mostly isolated in its own local environment, not connected to a global network like the internet - Yet, it is able to perform extraordinary things in spite of that - It is able to create meaning out of sensory inputs - Can we really say that a machine can do this?
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this blue ball with three stumps a chair or this strange design object here because you can sit on it and what you see here is the difference the main difference between the computer world and the brainworld
for - comparison - brain vs machine intelligence - comparison - human intelligence vs artificial intelligence
comparison - human intelligence vs artificial intelligence - AI depends on feeding the AI system with huge datasets that it can - analyze and make correlations and - perform big data analysis - Humans don't operate the same way
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you can Google data if you're good you can Google information but you cannot Google an idea you cannot Google Knowledge because having an idea acquiring knowledge this is what is happening on your mind when you change the way you think and I'm going to prove that in the next yeah 20 or so minutes that this will stay analog in our closed future because this is what makes us human beings so unique and so Superior to any kind of algorithm
for - key insight - claim - humans can generate new ideas by changing the way we think - AI cannot do this
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you can measure data but you cannot measure having an idea you cannot measure Innovation you cannot measure knowledge there's no metric there is no quantifiable scale for knowledge or having an idea you cannot say one meter of knowledge one kilogram of idea
for - comparison - data vs ideas - no metric for ideas
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for - Henning Beck - neuroscientist - video - youtube - The Brain vs Artificial Intelligence
Tags
- neuroscience - example - informal diversity - increases work efficacy - via sharing diverse and novel perspectives
- human learning - key feature - evolutionary nature
- neuroscience - ideation depends on three different brain functions and brain areas - concentration - prioritization - and drifting away
- question - neuroscience - efficacy of hybrid remote and live work environments
- key insight - claim - humans can generate new ideas by changing the way we think - AI cannot do this
- neuroscience - importance of maintaining curiosity - 1000 questions a day for children - 20 questions a day for adults
- Indyweb - Stop Reset Go participatory system mapping - benefits of open source - Henning Beck - neuroscience support
- Indyweb - key feature - evolutionary nature of learning
- comparison - human intelligence vs artificial intelligence
- neuroscience - human abilities - example Brexit and variations
- question - AI - can we teach AI values?
- quote - neuroscience - perspective shift - benefits
- question - comparison - human vs artificial intelligence - Can't an AI also consider things we sit on to then generalize their classifcation algorithm?
- neuroscience - children's understanding - 3 examples is enough to consolidate new concept
- Henning Beck - neuroscientist
- question - AI - can AI learn to be intentionally distracted?
- neuroscience - problem solving efficacy - no work periods are required
- neuroscience - human understanding - what makes us excel? - forgetting and getting distracted!
- comparison - data vs ideas - no metric for ideas
- neuroscience - mistakes - and fear
- neuroscience - validation for Stop Reset Go open source participatory system mapping for design innovation - bottom-up collective design efficacy
- comparison - brain vs machine intelligence - what brains and consciousness can do but AI cannot
- video - youtube - The Brain vs Artificial Intelligence
- neuroscience - innovation - great ideas - mistakes and - risk
- neuroscience - building design - common area to converge everyone - creates diverse social meetings - increases work efficacy - example - Steve Jobs - Pixar bathroom
- neuroscience and education - problem solving - active learning
- neuroscience - efficient work - relaxation rule
- question - neuroscience - creating neuroscience-based systems for solving problems
- neuroscience research - remote intentional working during Covid - showed decreased productivity and innovation
- AI and education - children will have custom tailored education program via AI
- neuroscience - education of children - recommend no digital devices before puberty - allows learning in a 3 dimensional world
- neuroscience - example - diverse and low density connections beats non-diverse and high connections
- neuroscience - optimal working environment - varies with brain state - different phases during the day
- key insight - human vs artificial intelligence - humans will create the best ideas
Annotators
URL
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drive.google.com drive.google.com
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Case: Patient Proband SS, Female, Caucasian
DiseaseAssertion: UCD/OTCD
FamilyInfo: De novo inheritance, no family history of disease
CasePresentingHPOs: Hyperammonemia (HP:0001987), oroticaciduria (HP:0003218), Childhood onset (HP:0011463)
CaseHPOFreeText:
CaseNOTHPOs: Positive allopurinol test
CaseNOTHPOFreeText:
CasePreviousTesting: Genomic DNA isolated from peripheral blood leukocytes or cultured skin fibroblasts. Amplification by PCR used. SSCP analysis performed. Sequencing of both free and immobilized single strands carried out by dideoxy chain termination method.
SupplementalData: Table 1: Mutations in the Ornithine Transcarbamylase Gene of 17 Females
Variant: NM_000531.6:c.77+1G>A
ClinVarID: 97313
CAID: CA224773
gnomAD:
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drive.google.com drive.google.com
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Case: Female Patient #1, Female, Japanese
DiseaseAssertion: UCD/OTCD
FamilyInfo: N/A
CasePresentingHPOs: Childhood onset (HP:0011463)
CaseHPOFreeText: Onset at 2 years
CaseNOTHPOs:
CaseNOTHPOFreeText: 16% OTC activity
CasePreviousTesting:
Variant: NM_000531.5:c.67C>T (p.Arg23*)
ClinVarID: 97292
CAID: CA224742
gnomAD:
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www.newyorker.com www.newyorker.com
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read in a less linear, more selective fashion.
this is happening to me right now, as I keep getting side tracked with all the other notes and highlights previously made on this article using this app.
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drive.google.com drive.google.com
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Case: Patient #38, Female, Chinese
DiseaseAssertion: UCD/OTCD
FamilyInfo: No family history of disease, mutation is inherited
CasePresentingHPOs: Hyperammonemia (HP:0001987), oroticaciduria (HP:0003218), vomiting (HP:0002013), coma (HP:0001259), lethargy (HP:0001254), seizures (HP:0001250), childhood onset (HP:0011463)
CaseHPOFreeText: Elevated plasma ammonia at 190 umol/L (Normal: 9-30 umol/L), Elevated urinary orotate at 202 mmol/mmol creatinine (Normal: 0-1.5 mmol/mmol creatinine)
CaseNOTHPOs:
CaseNOTHPOFreeText: Normal plasma glutamine at 13.5 umol/L (Normal: 6-30 umol/L), Normal plasma citrulline at 15.75 umol/L (Normal: 7-35 umol/L)
CasePreviousTesting: gDNA extracted from peripheral blood leukocytes. PCR all coding exons and exon–intron boundaries of the OTC gene using 9 pairs of synthetic oligonucleotide primers, and the primer sequences and annealing temperature. PCR products were then purified and bidirectionally sequenced. The library was sequenced using Illumina HiSeq4000 and generated 150 bp paired-end reads. Data analysis was performed as previously described [Sun Y, Hu G, Liu H, Zhang X, Huang Z, Yan H, et al. Further delineation of the phenotype of truncating KMT2A mutations: the extended Wiedemann–Steiner syndrome. Am J Med Genet A. 2017;173:510–4.]. Multiplex ligation-dependent probe amplification analysis was performed for samples in which Sanger sequencing or WES failed to detect any disease-causing mutation.
Variant: NM_000531.6:c.67C>T (p.Arg23*)
ClinVarID: 97292
CAID: CA224742
gnomAD:
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drive.google.com drive.google.com
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Case: Patient #4, Female, Caucasian
DiseaseAssertion: UCD/OTCD
FamilyInfo: N/A
CasePresentingHPOs: Hyperammonemia (HP:0001987),
CaseHPOFreeText: Elevated plasma ammonia concentration at 123 uM/L
CaseNOTHPOs:
CaseNOTHPOFreeText: Anxiety, plasma citrulline at 2 uM/L
CasePreviousTesting: N/A
Variant: NM_000531.5:c.67C>T (p.Arg23*)
ClinVarID: 97292
CAID: CA224742
gnomAD:
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pressbooks.montgomerycollege.edu pressbooks.montgomerycollege.edu
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A climatologist wants to check the claim on a Wikipedia article that San Diego averages a maximum of 66 days of measurable precipitation per year.
H₀: μ less than or equal to 66. Hₐ: μ > 66.
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www.biorxiv.org www.biorxiv.org
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Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.
Learn more at Review Commons
Reply to the reviewers
Reviewer #1
__Evidence, reproducibility and clarity __
The work by Przanowska et al., sought to understand the role of ORC2 in murine development and further wanted to discover its role in liver endo-reduplication. The overall methods used is sufficient enough to address its role but is not very conclusive based on their overall results and data provided as elaborated in below comments.
Major Comments:
- The major issue of the paper is how well is ORC2 depleted in perinatal liver (Fig. 2C) and is not very clear from the data as all the western blots are at very low exposure levels and bands are very weak (still weak bands seen). There are good antibodies of ORC2 which can be used for IHC staining and can be used to address the extent of ORC2 depletion.
We have now shown that ORC2 protein is significantly decreased in the hepatocytes of the Orc2 KO and DKO livers (New Fig. 2C and 6D). The decrease is consistent, with 4-5 mice examined, and all showing the depletion. We have been unable to do immunohistochemistry on tissue sections of the mouse livers with the anti-ORC antibodies we have tried, and this could be a reflection of the low level of the proteins. On hepatocytes in culture we have obtained faint signal with the anti-ORC2 antibody in WT cells, and this is clearly absent in 100% of the hepatocytes. See Fig. R1 below.
__Reviewer Fig R1: __
A) Immunofluorescence of hepatocytes in culture from livers of WT and two DKO mice.
B) Quantitation of A) from counting 70-100 cells from each specimen.
However, the calculations in the methods and the discussion are very compelling that at least the last 6-9 cell divisions in normal development start with 2n nuclei in the livers at baseline (Fig. 3B-G and 6I).
Why in Fig 2C, the M2 mice is showing an equivalent level of ORC2 protein compared to mice M1 with NO CRE expression (compare lane1 and lane5). So, the results are based on one mouse which I do not think is significant enough to come to the conclusion. The authors need to add more data from different mice for statistical significance. Please use IHC to show the depletion of ORC2 protein in the liver sections.
We had used total liver and had pointed out that residual ORC2 protein will be seen from stromal cells (endothelia, blood vessels and blood cells). We have therefore removed the figure which measured ORC2 levels in total liver and have now shown that when hepatocytes are isolated from five animals there was a massive depletion of ORC2 in all five animals (new Fig. 3C).
As nicely demonstrated in the previous paper by Okano-Uchida et al., 2018 that ORC1 depletion in the liver shows an DNA ploidy effect from 6-week onwards. The authors need to demonstrate in this paper also when the 16N phenotype is observed starting from week1 to 12 months.
Based on the results from our previous paper (Okano-Uchida et al., 2018) we decided to measure 16N phenotype at 6 weeks of age. The endoreduplication occurs at a stage when ORC2 protein is undetectable during normal development or during regeneration.
In the double knockout experiments (ORC1 and ORC2) the authors are not even bothered to demonstrate that how much are both the proteins are actually depleted from the cells, so on the results obtained from these mice experiments are not conclusive or explanatory.
We have performed immunoblotting of isolated hepatocytes and immunohistochemistry of livers for ORC1 and ORC2. Our data shows that both proteins are depleted in all four mice tested (New Fig. 6D).
Minor points:
- Why are scale bars missing in right panel of Fig. 2G, Fig. 6D Supp Fig. 2B KO studies. The authors need to confirm that that all the large nuclei have NO or less significant ORC2 protein through IHC H&E staining.
The scale bars are missing from the right panels to avoid redundancy. We have added “Both panels are at the same scale.” in the figure legend, according to https://doi.org/10.1371/journal.pbio.3001161.
- Please explain why is EYFP in Fig. 5G is cytoplasmic compared to Fig 4C (nuclear). We consistently see this variability and it was there in our previous results (Okano-Uchida et al., 2018), where EYFP was cytoplasmic in tissues, but was nuclear (and some cytoplasmic) in hepatocytes in culture.
We do not know the reason for this difference but consistently see this difference. We now say in the text: “We did not explore why the EYFP protein is mostly nuclear in hepatocytes in culture (Fig. 4C) and mostly cytoplasmic in hepatocytes in the liver tissue (Fig. 5G, 7G), but speculate that differences in signaling pathways or fixation techniques between the two conditions contribute to this difference.”
Are authors using the same genotype of Alb-Cre mice as shown by Okano-Uchida et al., 2018 as I do not find the reference of Schuler et. al., 2004 (PMID:15282742).
We have been using two independent Alb-Cre animals. This is now described in the Methods.
Significance
The article is exactly based on their previous published paper but instead of ORC1, they were interested in dissecting the role of ORC2. Although they have discussed that CDC6 may be involved in replacing ORC1 KO mice to rescue the extensive DNA replication in endoreduplication, but instead of going to hunt the role of CDC6 in endoreduplication they checked the effect of ORC2 which actually lower the overall impact of the paper.
We studied ORC2 conditional KO mice in a similar manner to the previously published ORC1 conditional KO in order to ensure (1) that the lack of effect in the Orc1 KO was not because ORC1 can theoretically be substituted for by CDC6 and (2) to establish the double KO of Orc1 and Orc2. To the best of our knowledge this is the first description of removal of two subunits of ORC complex at once in a mouse model. Moreover, in the light of rising recognition of sex as biological variable, we report sex-dependent effects which are very intriguing.
We have not attempted knocking out CDC6 to uncover novel mechanisms of DNA replication, because we first needed to make sure that the mice can truly endo-reduplicate without two of the six subunits of ORC. Note that our published results in cancer cell lines (Shibata, 2016) show that CDC6 is still essential in the ORC KO cell lines, so a future experiment will likely reveal that CDC6 is still essential for endoreduplication in the ORC KO mice in vivo.
Reviewer #2
__Evidence, reproducibility and clarity __
It has been reported that in the absence of ORC1, liver cells can still endoreduplicate and it has been speculated that this might occur if CDC6 can replace, at least partially, the function of ORC1. Here, authors evaluate if this is also true in the absence of ORC2 and found that ORC2 is required for cell proliferation in mouse hepatocytes but not for endoreduplication. This is also the case after combining the conditional mutations of ORC1 and ORC2. They propose that a mechanism must exist to load sufficient MCM2-7 to support DNA replication in the absence of these two ORC subunits. Some of the conclusions need further experimental support. The rationale for testing the requirement of ORC2, with or without ORC1, for endoreduplication is valid. However, a key point is that the endoreduplication level seems to be higher in the absence of ORC2 or both ORC1 and ORC2, and this is not properly addressed. Also, mechanistic details on how this could be triggered are absent from this study. As indicated below almost every figure in this manuscript contains weak points (see below).
We now discuss the following: “One possible explanation of the greater endoreduplication in both our papers is that mitosis may be arrested earlier in development by G2 DNA damage checkpoints activated by incomplete licensing and replication of the genome in the absence of ORC. As a result, endoreduplication cycles could begin earlier in development resulting in greater endoreduplication.”
Major 1. Fig 1G, needs a detailed comment and justification.
We have added the following to the text: “The proliferation rate of the MEF were measured by MTT assays. Even in the Orc2+/+ MEF, the infection with adeno-Cre decreased proliferation a little (the orange line compared to the blue line in Fig. 1G). However, for Orc2f/f MEF infection with adeno-Cre impairs proliferation even further (yellow line compared to black line in Fig. 1G)..
Note that Adeno-Cre has been reported to be toxic for cell proliferation (citations 1, 2, 3), and so we included Adeno-Cre expression in ORC2+/+ (WT) as a background control.
Citation:
- Pfeifer A, Brandon EP, Kootstra N, Gage FH, Verma IM: Delivery of the Cre recombinase by a self deleting lentiviral vector: Efficient gene targeting in vivo. Proc Natl Acad Sci USA. 2001, 98: 11450-11455. 10.1073/pnas.201415498.
- Loonstra A, Vooijs M, Beverloo HB, Allak BA, Drunen EV, Kanaar R, Berns A, Jonkers J: Growth inhibition and DNA damage induced by Cre recombinase in mammalian cells. Proc Natl Acad Sci USA. 2001, 98: 9209-9214. 10.1073/pnas.161269798.
- Schmidt EE, Taylor DS, Prigge JR, Barnet S, Capecchi R: Illegitimate Cre-dependent chromosome rearrangements in transgenic mouse spermatids. Proc Natl Acad Sci USA. 2000, 97: 13702-13707. 10.1073/pnas.240471297.
- Fig 2D-F. Is this conclusion applicable to other endoreplicating tissues? Have authors consider to analyze body weight and liver weight measurements after normalization with similar data from a non-affected organ? The conditional KO was performed specifically in the liver. ORC is intact in other tissues in these animals. As a future direction our lab plans to study cardiac-specific conditional KO of ORC subunits to test whether other endo-reduplicating tissues can also synthesize DNA in the absence of ORC subunits.
Fig 3 shows inconsistent results or results that lack proper justification in the text. The 2C peak is missing in Fig 3E (yellow line, positive control). However, 2n nuclei appear in Fig 3F-H. Also, the blue and yellow peaks do not coincide in the flow cytometry profiles, in particular for 8C and 16C.
There was an error in the plotting of the former Fig. 3E. The information is better presented in the former Fig. 3F-H (now Fig. 3E-G) and so have removed the former Fig. 3E from the paper.
Fig 4. Shorter EdU pulses could be more informative of the actual amount of S-phase cells. Thus, the use of a 2h EdU pulse needs a clear justification.
The half-life of EDU incorporation differs slightly between in vivo and in vitro conditions. In vivo, slower cell proliferation requires a longer time, approximately 4 hours. However, in vitro, liver cells grow faster, and a 2-hour EDU pulse with 20 µM is sufficient for detection compared to a 3-hour pulse with 10 µM BrdU (Okano-Uchida et al., 2018). Several publications also use a 2-hour EDU incubation time (https://doi.org/10.1098/rsob.150172).
Fig 5. EYFP is cytoplasmic, in contrast with results shown in Fig 4C
We consistently see this variability and it was there in our previous results (Okano-Uchida et al., 2018), where EYFP was cytoplasmic in tissues, but was nuclear (and some cytoplasmic) in hepatocytes in culture.
We do not know the reason for this difference but consistently see this difference. We now say in the text: “We did not explore why the EYFP protein is mostly nuclear in hepatocytes in culture (Fig. 4C) and mostly cytoplasmic in hepatocytes in the liver tissue (Fig. 5G, 7G), but speculate that differences in signaling pathways or fixation techniques between the two conditions contribute to this difference.”
Fig 6. Results obtained with the double mutant are poorly described.
We have split the figure into two figures (New Fig. 6 and 7) edited the results section to ensure that they are easily comprehended by the readers. We have also included Westerns from hepatocyte cell lysates of four DKO mice to show that ORC1 and ORC2 proteins are reproducible decreased (New Fig. 6D).
What are the level of other pre-RC components in the mutants used in this study. This could be easily evaluated by Western blotting
Despite the technical difficulty of not having antibodies that recognize all the mouse initiation proteins, we have now measured mouse ORC1, ORC2, ORC3, ORC5, ORC6, CDC6 and the MCM2 and MCM3 subunits of MCM2-7. The results do not show a consistent decrease or increase of any of these proteins in individual mice of the two genotypes, Orc2-/- or DKO (New Fig. 2D and 6E)
How do authors justify their claim that a very limited amount of ORC are sufficient to load a vast excess of MCM2-7 hexamers?
The rationale is stated in the introduction from data from cancer cell lines: “Given that WT cells have about 150,000 molecules of ORC2, even if this truncated protein is functional ORC2, ~150 molecules of the protein would be expected to load MCM2-7 double hexamers on at least 50,000 origins of replication. Experimentally, we show in Shibata, 2020 (Fig. 7C), that although ORC subunits are undetectable on Westerns, MCM2-7 association with the chromatin is unchanged. By the way, we do not say “vast excess” of MCM2-7, just sufficient MCM2-7 to fire 50,000 origins.
Minor 1. The titles of the Results section could be more informative of the main conclusion rather than simply descriptive
We updated our Results titles to be more informative.
The Discussion is too long
We have shortened the discussion by removing our calculations to the Results section and abbreviating some of the discussion on endoreduplication. However we had to insert new items brough forth by the reviewers. Due to the controversy of this topic in our field, we had to include extensive discussion of current literature and put our results in their proper context.
Significance
The topic is relevant and the hypothesis tested is reasonable, although the conceptual advance is limited (see also below). The major limitation is the absence of mechanistic details addressing the occurrence of extra endoreduplication cycles (compared to controls) in the ORC1 and ORC2 mutants.
Reviewer #3
__Evidence, reproducibility and clarity: __
The origin recognition complex (ORC) is an essential loading factor for the replicative Mcm2-7 helicase complex. Despite ORC's critical role in DNA replication, there have been instances where the loss of specific ORC subunits has still seemingly supported DNA replication in cancer cells, endocycling hepatocytes, and Drosophila polyploid cells. Critically, all tested ORC subunits are essential for development and proliferation in normal cells. This presents a challenge, as conditional knockouts need to be generated, and a skeptic can always claim that there were limiting but sufficient ORC levels for helicase loading and replication in polyploid or transformed cells. That being said, the authors have consistently pushed the system to demonstrate replication in the absence or extreme depletion of ORC subunits.
Here, the authors generate conditional ORC2 mutants to counter a potential argument with prior conditional ORC1 mutants that Cdc6 may substitute for ORC1 function based on homology. They also generate a double ORC1 and ORC2 mutant, which is still capable of DNA replication in polyploid hepatocytes. While this manuscript provides significantly more support for the ability of select cells to replicate in the absence or near absence of select ORC subunits, it does not shed light on a potential mechanism. While a mechanistic understanding of how these cells proliferate in the absence or extreme depletion of ORC subunits is outside the scope of the current manuscript, it would have been beneficial to see more functional analyses to help guide the field. For example, is there a delay or impairment in Mcm2-7 loading in G1 (FACs-based loading assay from the Cook Lab (Matson et al., eLife. 2017)) in primary hepatocytes with the ORC2 conditional deletion? Is copy number maintained as cells increase polyploidy in the absence of ORC subunits, or are some regions of the genome more sensitive to ORC depletion (CGH arrays or sequencing of the flow-sorted polyploid cells)?
We thank the reviewer for recognizing the main point of these experiments: to dispel the argument that CDC6 can substitute for ORC1 in the six-subunit ORC (although no one has demonstrated this, the argument is made on the basis of close sequence homology between CDC6 and ORC1). The second point, also appreciated by the reviewer is to show that it is possible to find cells that replicate in the absence or near absence of two ORC subunits.
The mechanistic questions raised are important, and we will address them here:
Is there a delay or impairment of MCM2-7 loading in G1? The hepatocytes in culture are fragile and not immortalized and thus, this issue can be much more easily addressed in the cancer cell lines we have made that are missing several ORC subunits and will do that in a later paper. Note however, the surprising lack of change in MCM2-7 association in cell lines where both ORC2 and ORC5 are deleted (Shibata, 2020, Fig. 7C).
Are some regions of the genome more sensitive to ORC deletion during the polyploidization? We could not find any paper where people have investigated whether the whole genome is uniformly polyploidized in livers. In other words, the baseline conditions in WT livers have not been established. We therefore have postponed experiments to answer this question for a later paper. Note that in unpublished data from mapping SNS-seq origins in WT and ORC deletion cell lines there does not appear to be selective firing of certain origins over others in the deletion cell lines.
Additional points: I didn't understand how the numbers were derived in Table 2. Was there really a 20-fold decrease in nuclear density for female ORC1 and ORC2 double-deletion hepatocytes? The differences in Figure S2 are dramatic, but not 20-fold dramatic.
We measure the relative nuclear density by counting the number of plump nuclei (hepatocytes) per field as described for Fig. 5F and 7F now in the Methods section. The reviewer is correct in that we overestimated the decrease of nuclear density in the female DKO mice by two-fold. The revised calculations suggest that 6 cell divisions occur in the female DKO mice after the ORC proteins have decreased to at least __Significance: __
The strengths of this manuscript are the mouse genetics and the generation of conditional alleles of Orc2 and the rigorous assessment of phenotypes resulting from limiting amounts of specific ORC subunits. It also builds on prior work with ORC1 to rule out Cdc6 complementing the loss of ORC1. The weakness is that it is a very hard task to resolve the fundamental question of how much ORC is enough for replication in cancer cells or hepatocytes. Clearly, there is a marked reduction in specific ORC subunits that is sufficient to impact replication during development and in fibroblasts, but the devil's advocate can always claim limiting levels of ORC remaining in these specialized cells. The significance of the work is that the authors keep improving their conditional alleles (and combining them), thus making it harder and harder (but not impossible) to invoke limiting but sufficient levels of ORC. At this point, the investigators and the field are well-positioned to attempt future functional CRISPR screens to identify other factors that may modulate the response to the loss of ORC subunits. This work will be of interest to the DNA replication, polyploidy, and genome stability communities.
We thank the reviewer for getting the important point of this paper: “making it harder and harder (but not impossible) to invoke limiting but sufficient levels of ORC….” In other words, either ORC is completely dispensable for loading MCM2-7 in certain cancer cell lines and hepatocytes or it is highly catalytic and one molecule of ORC can load a few hundred MCM2-7 doublets so that most origins in the genome are licensed and capable of firing. We are trying the CRISPR screens in cancer cell lines that the reviewer envisages
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Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #3
Evidence, reproducibility and clarity
The origin recognition complex (ORC) is an essential loading factor for the replicative Mcm2-7 helicase complex. Despite ORC's critical role in DNA replication, there have been instances where the loss of specific ORC subunits has still seemingly supported DNA replication in cancer cells, endocycling hepatocytes, and Drosophila polyploid cells. Critically, all tested ORC subunits are essential for development and proliferation in normal cells. This presents a challenge, as conditional knockouts need to be generated, and a skeptic can always claim that there were limiting but sufficient ORC levels for helicase loading and replication in polyploid or transformed cells. That being said, the authors have consistently pushed the system to demonstrate replication in the absence or extreme depletion of ORC subunits.
Here, the authors generate conditional ORC2 mutants to counter a potential argument with prior conditional ORC1 mutants that Cdc6 may substitute for ORC1 function based on homology. They also generate a double ORC1 and ORC2 mutant, which is still capable of DNA replication in polyploid hepatocytes. While this manuscript provides significantly more support for the ability of select cells to replicate in the absence or near absence of select ORC subunits, it does not shed light on a potential mechanism. While a mechanistic understanding of how these cells proliferate in the absence or extreme depletion of ORC subunits is outside the scope of the current manuscript, it would have been beneficial to see more functional analyses to help guide the field. For example, is there a delay or impairment in Mcm2-7 loading in G1 (FACs-based loading assay from the Cook Lab (Matson et al., eLife. 2017)) in primary hepatocytes with the ORC2 conditional deletion? Is copy number maintained as cells increase polyploidy in the absence of ORC subunits, or are some regions of the genome more sensitive to ORC depletion (CGH arrays or sequencing of the flow-sorted polyploid cells)?
Additional points: I didn't understand how the numbers were derived in Table 2. Was there really a 20-fold decrease in nuclear density for female ORC1 and ORC2 double-deletion hepatocytes? The differences in Figure S2 are dramatic, but not 20-fold dramatic.
Significance
The strengths of this manuscript are the mouse genetics and the generation of conditional alleles of Orc2 and the rigorous assessment of phenotypes resulting from limiting amounts of specific ORC subunits. It also builds on prior work with ORC1 to rule out Cdc6 complementing the loss of ORC1. The weakness is that it is a very hard task to resolve the fundamental question of how much ORC is enough for replication in cancer cells or hepatocytes. Clearly, there is a marked reduction in specific ORC subunits that is sufficient to impact replication during development and in fibroblasts, but the devil's advocate can always claim limiting levels of ORC remaining in these specialized cells. The significance of the work is that the authors keep improving their conditional alleles (and combining them), thus making it harder and harder (but not impossible) to invoke limiting but sufficient levels of ORC. At this point, the investigators and the field are well-positioned to attempt future functional CRISPR screens to identify other factors that may modulate the response to the loss of ORC subunits. This work will be of interest to the DNA replication, polyploidy, and genome stability communities.
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Learn more at Review Commons
Referee #2
Evidence, reproducibility and clarity
It has been reported that in the absence of ORC1, liver cells can still endoreduplicate and it has been speculated that this might occur if CDC6 can replace, at least partially, the function of ORC1. Here, authors evaluate if this is also true in the absence of ORC2 and found that ORC2 is required for cell proliferation in mouse hepatocytes but not for endoreduplication. This is also the case after combining the conditional mutations of ORC1 and ORC2. They propose that a mechanism must exist to load sufficient MCM2-7 to support DNA replication in the absence of these two ORC subunits.
Some of the conclusions need further experimental support. The rationale for testing the requirement of ORC2, with or without ORC1, for endoreduplication is valid. However, a key point is that the endoreduplication level seems to be higher in the absence of ORC2 or both ORC1 and ORC2, and this is not properly addressed. Also, mechanistic details on how this could be triggered are absent from this study. As indicated below almost every figure in this manuscript contains weak points (see below).
Major
- Fig 1G, needs a detailed comment and justification.
- Fig 2D-F. Is this conclusion applicable to other endoreplicating tissues? Have authors consider to analyze body weight and liver weight measurements after normalization with similar data from a non-affected organ?
- Fig 3 shows inconsistent results or results that lack proper justification in the text. The 2C peak is missing in Fig 3E (yellow line, positive control). However, 2n nuclei appear in Fig 3F-H. Also, the blue and yellow peaks do not coincide in the flow cytometry profiles, in particular for 8C and 16C.
- Fig 4. Shorter EdU pulses could be more informative of the actual amount of S-phase cells. Thus, the use of a 2h EdU pulse needs a clear justification.
- Fig 5. EYFP is cytoplasmic, in contrast with results shown in Fig 4C
- Fig 6. Results obtained with the double mutant are poorly described.
- What are the level of other pre-RC components in the mutants used in this study. This could be easily evaluated by Western blotting
- How do authors justify their claim that a very limited amount of ORC are sufficient to load a vast excess of MCM2-7 hexamers?
Minor
- The titles of the Results section could be more informative of the main conclusion rather than simply descriptive
- The Discussion is too long
Significance
The topic is relevant and the hypothesis tested is reasonable, although the conceptual advance is limited (see also below). The major limitation is the absence of mechanistic details addressing the occurrence of extra endoreduplication cycles (compared to controls) in the ORC1 and ORC2 mutants
-
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
The work by Przanowska et al., sought to understand the role of ORC2 in murine development and further wanted to discover its role in liver endo-reduplication. The overall methods used is sufficient enough to address its role but is not very conclusive based on their overall results and data provided as elaborated in below comments.
Major Comments:
- The major issue of the paper is how well is ORC2 depleted in perinatal liver (Fig. 2C) and is not very clear from the data as all the western blots are at very low exposure levels and bands are very weak (still weak bands seen). There are good antibodies of ORC2 which can be used for IHC staining and can be used to address the extent of ORC2 depletion.
- Why in Fig 2C, the M2 mice is showing an equivalent level of ORC2 protein compared to mice M1 with NO CRE expression (compare lane1 and lane5). So, the results are based on one mouse which I do not think is significant enough to come to the conclusion. The authors need to add more data from different mice for statistical significance. Please use IHC to show the depletion of ORC2 protein in the liver sections.
- As nicely demonstrated in the previous paper by Okano-Uchida et al., 2018 that ORC1 depletion in the liver shows an DNA ploidy effect from 6-week onwards. The authors need to demonstrate in this paper also when the 16N phenotype is observed starting from week1 to 12 months.
- In the double knockout experiments (ORC1 and ORC2) the authors are not even bothered to demonstrate that how much are both the proteins are actually depleted from the cells, so on the results obtained from these mice experiments are not conclusive or explanatory.
Minor points:
- Why are scale bars missing in right panel of Fig. 2G, Fig. 6D Supp Fig. 2B KO studies. The authors need to confirm that that all the large nuclei have NO or less significant ORC2 protein through IHC H&E staining.
- Please explain why is EYFP in Fig. 5G is cytoplasmic compared to Fig 4C (nuclear).
- Are authors using the same genotype of Alb-Cre mice as shown by Okano-Uchida et al., 2018 as I do not find the reference of Schuler et. al., 2004 (PMID:15282742).
Significance
The article is exactly based on their previous published paper but instead of ORC1, they were interested in dissecting the role of ORC2. Although they have discussed that CDC6 may be involved in replacing ORC1 KO mice to rescue the extensive DNA replication in endoreduplication, but instead of going to hunt the role of CDC6 in endoreduplication they checked the effect of ORC2 which actually lower the overall impact of the paper.
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chatgpt.com chatgpt.comChatGPT5
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de la photographie
de cet art
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photographiés
capturé à l'improviste
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lés qui semblaient suinter l'angoisse de ses rêves
par l'horreur
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ses cauchemars
terreurs nocturnes
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Les joues rouges et trempées d’un mélange de sueur et de larmes, Émilien s’éveilla dans un hurlement
garder
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distribution
a way to summarise quantitative data;
describes what values are present in the data, and how often those values appear
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wtcs.pressbooks.pub wtcs.pressbooks.pub
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Read
Red arrow icon is needed
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www.youtube.com www.youtube.com
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curiosity trap
for - new term - curiosity trap - When distractions take us out of the concentration and focusing zone
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the more stuff happened I'm going to think retrospectively oh this was a very long time because there were so so many new things and so much experience in retrospectively
for - time sense - more new events gives a longer sense of time
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this is one reason why we forget stuff it is not a like like something that that is telling us that our brains bad but on the other hand the brain is using active forgetting in order to make the most important information the more precise and more pronounced
for - neuroscience - why brains forget - active forgetting
neuroscience -active forgetting - leaves behind a small set of salient ideas
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we forget stuff yeah and it is even more it is not precise and accurate we invent stuff retrospectively
for - neuroscience - memories - reconstructed in the present - with new information - Indyweb - talking to our old selves - memories
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Avram Lincoln said I don't like this man I have to get to know him better because getting other people into your perspective
for - neuroscience - perspectival knowing - why it's important to know other perspectives - perspectival knowing - Abraham Lincoln quote - I don't know that man - I better get to know his perspective
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a very good advice in order to calculate or estimate the duration of the project is that you ask non-experts
for - neuroscience - time estimation - non-experts are better at providing time budgets - neuroscience - non-experts give better time estimates than consultants
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for - Henning Beck - neuroscience
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the best way to have a very long life is that you have a lot of new stuff around you
for - neuroscience - how to - create perception of a long life - increase new activities
Tags
- neuroscience - time estimation - non-experts are better at providing time budgets
- neuroscience - why brains forget - active forgetting
- new term - curiosity trap
- Indyweb - talking to our old selves - memories
- time sense - more new events gives a longer sense of time
- When distractions take us out of the concentration and focusing zone
- perspectival knowing - Abraham Lincoln quote - I don't know that man - I better get to know his perspective
- neuroscience - perspectival knowing - why it's important to know other perspectives
- neuroscience - non-experts give better time estimates than consultants
- Henning Beck - neuroscientist
- neuroscience - memories - reconstructed in the present - with new information
- neuroscience - how to - create perception of a long life - increase new activities
Annotators
URL
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python-crash-course.pages.dev python-crash-course.pages.dev
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好みであれば、この手法を好むと思います
好み~好む の重複が気になりました。
代案
好みであれば、この手法が気に入るかもしれません
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コマンド
ちなみに command と入力しても同じ画面になります
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。
Click New to add a new path to the current list of paths. の訳が抜けてます。
訳案
続いて新規ボタンを押して現在のパスに新しいパスを追加します。
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XAMPPインストーラーを実行したときのデフォルト設定のままだと仮定し、表示されるボックスに C:\xampp\php を追加して OK をクリックします。
わかりにくいです。
代案
XAMPPインストーラーを実行したときにデフォルト設定のままにしていたのであれば、表示されるボックスにC:\xamp\phpを追加してOKをクリックします。
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変数
typo 編集
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セットアップはここで説明する以上の情報があります
訳が違うかも。
代案
WSL環境のセットアップについてはここでは説明しきれません。
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好きなOSで開発でき、好きなホストにプッシュできます。 プロジェクトをプッシュするためのツールは十分に開発されており、リモートシステムで正確に実行できるように必要に応じてプロジェクトを修正します。
訳は合っていると思うのですがややぶつ切り感があります。
代案
好きなOSで開発でき、好きなホストにプッシュできます。また、プロジェクトをプッシュするツールは十分に開発されており、リモートシステムで正しく稼働するように必要に応じてプロジェクトを修正してくれます。
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が
typo? を
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リソース
リソースにはいろんな意味があるので、ここでは日本語に訳したほうがよいのでは
代案
情報源
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Platform.shの
Platform.sh上の としたほうがPaaSの一種なんだな、と伝わりやすいと思います
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アプリケーションのデプロイがうまくいくと非常に満足します。一度もデプロイしたことがない場合はなおさらです。
日本語としてやや不自然に感じました。
代案
アプリケーションのデプロイがうまくいくというのはとても満足感が高いものです。一度もデプロイしたことがなければなおさらです。
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www.theguardian.com www.theguardian.com
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combiliftpro.com combiliftpro.com
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Pict. 11: Additional functionsFrom top to bottom: The meanings of the five icons are▪ Information on your unit (model, current software version running, serial number)▪ Font selection – only for writing and naming cooking programmes▪ Left: ESG = Empty steam generator | Right: Service menu▪ Return
*See Video Library "Additional Functions screen functions"
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▪ Network settings (but not active in this screenshot)▪ Left: Change from Celsius to Fahrenheit and back | Right: Date and time settings▪ Left: Additional Functions | Right: Return
See Video Library "iCombi Classic Settings screen functions"
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From top to bottom: The meanings of the six icons are▪ Data up-/and download
See Video Library "iCombi Classic Settings screen functions"
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Pict. 9: First screenFrom top to bottom: The meanings of the four icons are▪ Programming mode▪ Automatic cleaning▪ Settings▪ Return
See Video Library "iCombi Classic Initial screen functions"
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Pict. 8: First screen after pressing menu: Six buttons for operat-ing the iCombi Classic. See highlighted difference between but-tons 3 and 5.The screen is not a touch screen. Instead you operate with the four buttons to the left and two buttons on the right ofthe display. Only if an icon has a small white vertical bar on the outside, the corresponding button can be pressed.In the example above you can see that buttons 1, 2, 3 and 6 have a content and lead to an action, buttons 4 and 5 donot.With the return key (button 6) you go back one level and with a green tick you confirm a setting made.
See Video Library "iCombi Classic First Screen after pressing "menu" button demo"
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Pict. 6: iCombi Classic: start screen with button “menu”
See Video Library "iCombi Classic "Start Screen with Menu Button"
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To switch on the unit, press the on/off button for about three seconds. The unit will start. The start screen will appear
See Video Libray "iCombi Classic On/Off switching procedure"
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To switch off the unit, press and hold the on/off button until the blue progress bar at the top of the display is com-pletely filled. A “goodbye” message is displayed
See Video Libray "iCombi Classic On/Off switching procedure"
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The password to enter the service menu for technicians is “TECLEVEL”
It is no longer possible to enter the iCombi Pro Service menu unless you have downloaded the Rational PIN Creator App and have a valid log in and password.
Attempts to enter the Service Menu will generate a QR code on the screen.
Scanning the QR code with the Rational PIN Creator app will generate a PIN which if used on the screen will enable entry to the Service Menu for technicians.
See Video Library "Access to Service menu for Technicians"
If you have undergone technical training by Rational UK or its approved training partners you can obtain a log in and password from Rational UK.
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You can operate the iCombi Pro with a few simple moves on the touch panel and/or turning and – for confirmation -pressing the central dial.
See Video Library " iCombi Pro Start screen function"
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To switch off the unit, press and hold the on/off button until the LED lights orange. You will be asked whether youreally want to switch off the unit or not. Pressing “yes” will trigger the switch off process
Please note: When the on/off switch LED illuminates amber, immediately release the on/off switch.
Failure to do so will result in the LED blinking amber/green/amber/green continuously and the machine will not switch off.
See Video Library "iComb Pro switch off procedure"
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To switch on the unit, press and hold the on/off button until the LED lights green. If all starting conditions are met thestart screen will appear
Please note: When the on/off switch LED illuminates green, immediately release the on/off switch.
Failure to do so will result in the LED blinking green/amber/green/amber continuously and the machine will not switch on.
See Video Library "iCombi Pro switch on precedure"
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The unit features an integrated p-trap (syphon). In case the drain connec-ted to the unit is also having a p-trap (this might be invisible in the floor), theconnected drain must be vented via a T-piece. Failing to do so will result in anoverflow from the emergency drain
See video Library "Drain Connection iCombi"
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Inhaltsverzeichnis
Table of Contents.
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X51: 12 pol connector: 5 V stand-by voltage to A12, 12 V to A12 if unit is on and bus signal from A12
X51 is a 12 pole female connector from which a 12 core cable carries data to and from A10 and the CPU (A12).
The 12 wire cable also caries a 5V "stand by" voltage from I/O board A10 to on/off switch on CPU (A12) .
The "stand by" voltage enables operation of the on/off switch and the CPU as soon as power is supplied to the oven (as the isolator is switched on).
When the on/off switch is operated, ESTL completes its safety checks and providing no high temperature errors or component errors exist, contactor K1 energises and a 12 V supply is also sent to the CPU via this connector and its associated cable.
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drillere-report.pages.dev drillere-report.pages.dev
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次図は、基準価額の推移である。
過去データの期間を変更できるようにする * 1年 * 3年 * 5年 * 10年
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journals.plos.org journals.plos.org
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Local file Local file
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Robert C. Berwick
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www.youtube.com www.youtube.com
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for - climate crisis - psychology - wrong approach
summary - Climate scientist professor Mojib Latif explores why our best efforts at rapid intervention to deal with the climate crisis are failing - Near the end of the program, he interviews professor Henning Beck, a neuroscientist who suggests that human brains have evolved to be rewarded for securing more. - Dopamine is released when we get more and we have not designed our intervention strategies aligned with this basic property of our brains
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www.linkedin.com www.linkedin.com
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for - neuroscience - Henning Beck - more education - can lead to - more stubbornness
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openclassrooms.com openclassrooms.com
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Tirez un maximum de ce cours
Test
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www.youtube.com www.youtube.com
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( ~19:15 )
Johannes Schmidt calls Luhmann: "Without a doubt the most important German sociologist of the 20th century."
Tags
Annotators
URL
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www.theguardian.com www.theguardian.com
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