Does this hold for other studies too?

Accuracy encompasses both sensitivity and specificity

So the model overall accurate but lacking in specificity (tends to have more false positives)

Text + Demographics + APOE + Health = 78.5/79.9 highest score as expected

Text alone: 77.8/79.4 (so it doesn't look like adding in the other info helped a a lot)

Traditional neuropsychological tests: 71.3/75.5

Demographics alone: 68.8/71.1

so incorporating speech improved the AUC by >10%

Apolipoprotein E, an LDL associated with the formation of amyloid plaques and tau tangles

How much of the predictability is coming from the speech recognition and how much is coming from the other factors it is also accounting for (demographic, health factors, apolipoprotein E)? Now that is a specific question...

used the same population for testing so not really independent testing. Would be interesting to see how it held up with a completely unrelated group of people. Potentially wouldn't hold up as well.

A Transformer is a neural network architecture introduced by Google. It revolutionized how machines understand and generate sequences

Instead of processing words one at a time, transformers look at all words in a sequence simultaneously and use a mechanism called self-attention to understand how each word relates to every other word.

Self-Attention Mechanism Each token “looks” at other tokens in the sentence and assigns attention weights — numbers that represent how important each word is to understanding the current one.

Example: In the sentence “The patient who had pneumonia was discharged.”, the word “was” should pay more attention to “patient” than to “pneumonia.” The self-attention mechanism captures this context automatically.

1. Stacked Layers

Many layers of self-attention and feed-forward networks are stacked.

Each layer learns increasingly abstract relationships — syntax, semantics, and even reasoning patterns.

Okay so it works pretty good...what are some next steps? Application to a different disease that affects speech?

Okay there is value in this because it another way to help differentiate between MCI and Alzhimer's. Risk stratification, get them a MRI, then potentially some of the drugs that can prevent plaques and tangles from forming

The author suggests the addition of a 4th process which consists of forcing fed members to adhere to same norms, standards, and objectives as ones determined by central state in the name of efficiency, performance, nad rationality

The 3rd process of renationalisation regards anti-immigration policy implementations aimed to "protect the national cultures of Euro countries against population flows of global migration". France, Germany, and UK are challenging the principle of deep div by arguing that multicul is detrimental to maintenance of pol and soc cohesion.

There are observed efforts from the minority nations to "get a better deal" with existing pol settings (ex: Meech Lake prop with Quebec and Canada & suspension of pol institutions in Catalonia through application of Article 155). These are situtations where the central state are using the carrot (Canada) or the stick (Spain) to tame/silence opposition.

The 1st is about the recurrence of violent conflicts in Cen and East Euro following collapse of USSR in 1993. Discusses two fallen multinat fed (Yugoslavia and Czechoslovakia) because they were affected by the pol tensions from the collapse. Their dismantlement led experts to say multinat fed could only lead to or feed pol instability. Or the author reframes as the experts are saying multinat fed contained the seeds of their own failure in their ethno-nationalist comp.

Just introducing the 3 key processes of renationalisation that is taking place in Euro identitfied by Joseph Marko

There are countries (Belgium, Canada, Finland, Germany, Switzerland, and UK) that have been willing to provide support to minority nations through dynamic push and pull that was benefitted by new sensitivity to cul pluralism, nat div, and linguistic div. However, in countries like Spain it wasn't going to last the whole time due to impending dom pol forces trying to reverse it back to central state hegemony and they themselves upholf state stability.

Trend of renationalisation (powerful forces bringing together the central state back) in the past 3 decades. This leads to weaker groups from holding onto their own power which lead to distrust and mistrust among leaders and neg impact between pol comms.

This the final definition the author landed on regarding multinat fed. Its a more collaborative and mutual agreement of the diversity of a multinat or multi level fed.

*I'm a little confused about this paragraph but here's my go. The author is engaging with the discussion about how most forms of multinat fed is linked to ethnofed whic is linked to be unstable "as narrow ethnic identities would prevail over broad civic achievement" or to the unraveling of various communist feds. The author's aim is to take rep of pol interests and their dem exp seriously instead of assuming like Bunce and her colleagues that all pol actions are motivated by ethnic aspirations/goals.

This is the author's definition of multinat fed, one about recognising presence of multiple identities and sustaining appropriate institutions capable of empowering pol comm to promote state differentiation and state collab.

another counter arugment which involves students saying that multinat fed need a Staatsvolk to impose pol will to assert more dominance to be stable. Author fights this claim by arguing that we have seen the consequences of this through Spain (jailing pol leaders) and India when they removed the statute of pol autonomy from the Kashmir.

all nat subjects of a sovereign country. or those subjects belonging to the dominant ethnic group of a country, excluding minorities, especially those who belong to nations that have states of their own

The author brings in counter arguments from Christian Joppke (who argues that states are more inclined to secure and fortify majority culture and impose their authority, through adopting measurements) and Brian Barry (who argues that cultural diversity -> state fragmentation that turn into exploitation from ethnocultural pol entrepreneurs). Author concludes they are insensitive to minority claims

The author highlighted the period of advancement of deep diversity from late 1980s - mid 2000s. Within this period sprouted the ideas that diversity is really important to creativity that sustain public life. But after this period liberal Western dems witnessed backlash against this trend.

Mentions Myanmar, India, Malaysia, Sri Lanka, Iraq. Author shares these cases as a way to demonstrate where the n. gative protrayals of multinational federations are coming from + the root of the issue (the imposition of common nationality).

tables are just very helpful way to look at data for machine learning modeling

as everyone has told me, 90% of ML is data

Data is extaordinarily important, literally one of the reasons im planning to go through all this now before i start going through my data, you can have a good loss function, but ultimately its all dependent on your data

Although Eliot, Carpenter, and Spenser all depict the River Thames as a significant reflection of society, they diverge in their characterization of the river and its role in modern sexuality. Carpenter, an early proponent of sexual and relational freedom, describes the Thames as a fluid beauty balancing nature and humanity.Eliot warns against sexual freedom, contradicting Carpenter and inverting Spenser. The line “Sweet Thames, run softly, till I end my song” comes directly from the refrain in Spenser’s “Prothalamion.” Spenser describes the River Thames as a beautiful sight, and an even more beautiful sight being the two swans of Jove and Leda. He describes the swans as so “purely white,” that even the water was impure to them, so much so that the river didn’t wet their feathers to spare them from self-pollution. Here, he paints an idealized image of a society that shines “as heaven’s light,” following religious values.

“So purely white they were, That even the gentle stream, the which them bare, Seemed foul to them, and bade his billows spare To wet their silken feathers, lest they might Soil their fair plumes with water not so fair, And mar their beauties bright, That shone as heaven's light, Against their bridal day, which was not long”

Eliot, on the other hand, paints the Thames as extremely polluted, indicating that while he borrowed the line from Spenser, he views the more popular version of “Prothalamion” as a better reflection of society. In the more known version, Jove disguises as a swan to sexually assault Leda. In this case, the swans are not actors of love, but of sexual violence. Water, often a symbol of holiness and purity, is physically polluted by industrialization and overconsumption, but is also a reflection of how society has lost its holiness with the rise of sexual assault and sexual fluidity. Going deeper into Eliot’s religious allusions, Jesus washes his disciple’s feet as an act of mutual humility.Here, the “unholy” people of the Waste Land’s River Thames wash their own feet in “soda water,” indicating that the water has been polluted by modernity (soda being a “mutated,” modern form of water and the relational modernity rising in the 20th century) and even humility has been lost.

The opening of this section seemed to me to reflect the structure in the beginning of Keats’ “What The Thrush Said.” Keats begins with “O thou whose face hath felt the Winter’s wind” and other such lines describing winter before writing “To thee the spring will be a harvest-time.” This repeats once more with slightly different description around light. “What the Thunder Said” begins with these lines highlighted—the second is the most explicitly wintry image, and the others become as such positioned around it. The “torchlight” fading suggests warmth and light being extinguished, as winter approaches; “frosty” feels an early winter quality; “stony places” are barren and hard, describing the frozen, unyielding quality of winter ground; the sounds of “shouting and crying” break through the wintry, “frosty,” silence as harsh winds or storms; and both “prison” and “palace” feel cold and stone-bound, echoing with emptiness. Then follows the sixth line, “Of thunder of spring over distant mountains.” This feels as/seems to function as the lines “To thee the spring will be a harvest-time” and “To thee the Spring shall be a triple morn” from Keats—presenting the rejuvenation and renewal that is to come, that is arriving. In Harrison’s study, she writes, “‘Thunder,’ said Umbara headman of the Yuin tribe, ‘is the voice of Him (and he pointed upwards to the sky) calling on the rain to fall and everything to grow up new.’” So this lovely spring thunder brings God (is God), and brings restorative rain. Interestingly, this is in opposition to the start of the poem, where April, the beginning of spring, is marked “the cruelest month.” But then, in fact, what follows is a seeming inversion of the story of Lazarus (as noted by William) and actually no water of any form. So what is left—from this dry state, to the expectation of water, to no water—is an intense thirsting. And then this is teased, with the way the second stanza goes round and round seemingly getting closer to reaching water, really—but not. Psalm 63 relates thirst to a thirst for God. I think this is definitely at play here. So again we are left with more paradoxes. God is present in the thunder, but absent with the absence of water. Spring at the beginning of “The Burial of the Dead” stirs life from death; spring here settles death (from life). How can these be reconciled?

Between his textual narrative of Lil and his reference to Ophelia, Eliot examines the contrast and connection between love, virginity, purity, and exploitation. Here, the speakers discuss how Lil is not taking care of her appearance and will not be appealing to her husband, then one says “What you get married for if you don’t want children?” This implies that the sole reasons for marriage are sexuality and having children, while the concept of love is not mentioned. The final line of “The Game of Chess” is “Good night, ladies, good night, sweet ladies, good night, / good night,” which is a reference to Ophelia’s farewell in Hamlet prior to her suicide. By ending the passage with Ophelia’s words of distress, it is implied that Ophelia’s situation is very significant to Eliot’s message. Ophelia says, in the excerpt from Hamlet, that “To-morrow is Saint Valentine's day, All in the morning betime, And I a maid at your window, To be your Valentine. Then up he rose, and donn'd his clothes, And dupp'd the chamber-door; Let in the maid, that out a maid Never departed more.” She mentions arriving at Hamlet’s window as a virgin (a maid – older description for a young unmarried virgin), looking to be his Valentine, or to find love. Ophelia leaves this meeting no longer a maid, or a virgin. Afterwards, Ophelia adds, “Before you tumbled me, You promised me to wed. So would I ha' done, by yonder sun, An thou hadst not come to my bed.” Hamlet promised to marry her, which she was ready for, but instead just slept with her and then shunned her. Ophelia seems to feel used and exploited for her body. Lil, having already lived a life of five children, chooses to resist the need to appease her husband with superficial changes to her appearance. It is almost as if Lil lived Ophelia’s life, but continued living with a different mindset, though she is still subject to the same expectations and judgement. The difference between the two women is that Lil experiences this while married, and Ophelia is a young unmarried woman. Considering the time period of the piece, having lost her virginity to a man who decides not to marry her after all, Ophelia is left “ruined” and “dishonored” in society and in future romantic relationships. Essentially, by taking her virginity without marrying her, Hamlet has sentenced Ophelia to a life without the authentic love she originally desired. Left without clear choices and grieving the loss of her father, Ophelia becomes mentally unstable and feels that she has no other option than suicide by drowning. This is significant, because water is most often viewed as spiritually pure, especially as the medium for baptism. At the start of one’s life, they are baptized, and at the end of Ophelia’s life, she drowns. So, at line 170, when the women in the bar say “goonight” to each other, they are just going home for the night. In the final line, however, when the farewells shift to Ophelia’s voice, she is saying goodbye to the “Game of Chess” – the “game” of a woman experiencing sexual exploitation and a loss of pure connection – and transitioning the reader to the next section where water (the River Thames) becomes polluted and “impure,” as well.

Eliot’s “The Game of Chess" and its referenced sources characterize women (or the queen piece) as the real pawns of society, exploited by men (the king piece) despite their power. Eliot begins the section with “The Chair she sat in, like a burnished throne, / Glowed on the marble…” In older versions of chess, specifically the marble-like Lewis chessmen, the queen piece sits on an elaborate throne, cradling her head in her hand with a tired expression. So, Eliot's description aligns closely with the chess piece of the Queen. At the same time, this description is a direct reference to Antony and Cleopatra: “The Chair she sat in, like a burnished throne, Glowed on the marble.” So, The Game of Chess begins with Cleopatra, the queen of Egypt and one of the most well known women of immense power labeled a seductress. In fact, the six assigned sources all display women used as scapegoats, always described but never given a chance to never given a chance to stand up for themselves. They are used as pawns in literature, society, and history. Further, these women are almost all associated deeply with snakes, or a symbol for the devil in many works. The foundation of this comparison is shown in Paradise Lost, as Eve is tempted by a serpent, or the devil, and then is blamed alongside the serpent for eternity. Notably, Cleopatra kills herself with an asp, or a serpent, to escape a future of humiliation at the expense of being forever silenced. In Ovid, Philomela’s tongue is cut out because the king dislikes her words, and severed tongue is compared to a snake. By taking away her tongue, or her voice, the king seems to believe he has stunted her ability to tempt and manipulate. In Baudelaire, he writes “The haunches slightly sharp, and the waist sinuous / As a snake poised to strike, / That she's still quite young!” Even as the woman is described in an undone state, she is still viewed as “a snake poised to strike.” Tying these references back to the text, Eliot argues through his characterization of “ the nightingale Filled all the desert with inviolable voice And still she cried, and still the world pursues,” that these women are labeled snakes, always poised to strike and poison others with their cunning manipulation, while they are truly nightingales, only afforded a grieving voice in the night. The thread is clear of women being exploited by men then blamed by those same men and the rest of society without a chance to share their voice.

Like Nerval’s fragmented account of his dreams, the Waste Land is a sort of dreamscape with a patchwork of seemingly random images, which all share a similar intention and background. “Unreal City” begins line 60 of The Waste Land, and Eliot leaves a footnote in French, nearly translating to “SWARMING city, city full of dreams, Where in a full day the spectre walks and speaks.” That quote in the footnote is from Baudelaire’s “The Seven Old Men,” which explores the shifting definition of beauty in nature, especially while Paris is rapidly industrializing. The city of Paris is labeled “Unreal City,” as a place built up by dreams and romanticized images, while industrialization begins to degrade the city.

The experience of reading the poem is similar to slipping in and out of consciousness, as literary, cultural, and historical references wash over the reader. So, “dreams” serve here as those of sleeping and those of hopes for the future. The concept of dreams as a whole is repeated throughout "The Burial of the Dead,” so ending the section with a direct connection to De Nerval’s account of dreams is symbolic for the poem’s overall structure. So, if we theorize that, here, this constant flow of people crossing the bridge are on a similar hell-like journey as the people who are neither here nor there in Dante (the uncommitted, unclassified , outcasts, etc.) The inscription in Dante on the gates of Hell translates to “"Abandon all hope, ye who enter here,” suggesting that this characterization of The Waste Land is one of abandoned hopes and dreams, leaving people to wade through the dreamscape (or nightmare) that is a fragmented modern society.

TL;DR: Most interesting to me is the mirrored structure which exists both thematically and structurally within this section—and within all its references. At first glance I thought it might indicate a doubling or twinning of sorts; but I’ve now understood this “mirror” to be more of a replication (or regression).

Midway through the stanza comes the line “With a dead sound on the final stroke of nine.” Upon my initial reading two significant features revealed themselves. First, there is numerical weight to the chosen number “nine.” In Baudelaire's poem the unnamed narrator watches seven old men, all identical in misery and form, tread somberly before him, before at last arriving at the terrible understanding that he himself is the “awful eighth.” And so of course the natural progression is to uncover a ninth member in this hellish parade; and obligingly Eliot supplies us with one. The ninth is, of course, himself, or perhaps the reader; either way the mention of the number “nine” suggests that this repetitive pattern has not yet ceased. In the original Les Fleurs Du Mal, Baudelaire plays with the notion of what I initially thought was doubling: there is the “spectre” of self which walks and speaks in our “city full of dreams,” the hypocritical Reader decried as Baudelaire’s “second self—[his] brother,” and, of course, the seven (or eight) old men, whose presence compels the narrator to double back and flee home. But at the very end there is a line which suggests that—as I’ve come to realize—that this doubling is more of a regression: “In vain my reason tried to cross the bar,/ The whirling strom but drove her back again”. And so in this way Baudelaire introduces another creation story for the ‘doubled’ men, in which they are not copies of one another but one and the same, forced back along the same path after having failed to “cross the bar”. So what is this “bar” which cannot be crossed? Eliot appears to suggest it is death itself. It seems to me that “a dead sound on the final stroke of nine” is operating as a threshold of sorts, or the “bar” in question: the line cleaves the stanza into two exact halves, each of which can be considered a “double” of the other. In this way it becomes clear that the “doubles” we are dealing with are the stanza’s halves, each of which depict an eerily similar motif. The first half of the stanza (lines 1-8) pays homage to Dante. The line “undone by death” comes from a section of the Inferno in which Dante meets the Uncommitted, a group of souls who reside neither in hell nor out of it. But interposed over this image of dead souls in limbo is the living, since Eliot roots this classical reference in a visual description of modern London. And so through this overlay Eliot melts any barrier between the living and the dead, perhaps suggesting that they are one and the same (a suggestion, it is worth noting, somewhat loosely put forth in De Nerval’s dream writings). The second half of the stanza is much the same. Eliot introduces the image of the deathly bloom, where the narrator expects mysterious flowers to erupt from planted corpses. This description seems to be most obviously an allegory for the afterlife: flowers growing from corpses are, after all, a continuation of life after death. But interestingly, the afterlife appears to be inaccessible. The lines “'Or has the sudden frost disturbed its bed?/'Oh keep the Dog far hence, that’s friend to men,/'Or with his nails he’ll dig it up again!” suggest that small discrepancies are capable of disrupting our entire future (which, again, is the plot of De Nerval’s writings—he spends the entire excerpt ranting about how our lives are the sum product of infinitely small moments and knick-knacks); and the questions marks suggest that as a result of these discrepancies, the flowers’ ability to bloom, or our ability to access the afterlife, has been compromised. Looping back to Baudelaire’s model of duplication—in which doubles are produced by a ‘doubling back,’ or re-walking of the same path—we can see how the stanza’s halves inform our understanding of the poem as a whole. Both parts reference our inability to move into the afterlife: the first half speaks of life and death as another doubled pair, implying that the dead souls ‘double back’ onto the living (aka they hit a dead end, cannot move past into the afterlife, so turn around and go back into the realm of the living); and the second half’s tie to the continuation of life post-death has already been thoroughly explained. This understanding gives new resonance to “a dead sound on the final stroke of nine”. Yes, the line references death as a threshold; but importantly, it frames death as the “bar” we fail to cross. There exists “a dead sound,” suggesting a sudden impact or some unsuccessful attempt. I think what Eliot is trying to say is that nothing ever changes; having died, we fail over and over again to access the afterlife, instead ‘doubling back’ onto life. As a consequence all future generations become copies of the past; and we get this weird circularity that Eliot flirts with for the rest of the poem.

I think the notion of orientation is really important here. Addie suggests that the "Fishing" metaphor alludes to the Fisher King, positioned so that his back is facing hell. Her analysis sinks into a particularly relevant section from Weston's essay: ‘the Fish was sacred to those deities who were supposed to lead men back from the shadows of death to life,' and is brilliant enough that I was sufficiently convinced that the Fisher King does, indeed, face away from hell. Hurling ourselves back in time to the segment on Death by Water -- where the threshold of death is, thematically, embodied by water itself -- we can thus see that we find ourselves (as MacLeish scholars past have noted, shoutout Jeannie + Addie!) in the exact same position from which we began. We are at the edge of death. Water is before, us, and behind us is hell. And so what is the way forward? If the Waste Land is said to have a central conflict, it would be the struggle to escape the repetition of human life. Life flows into death flows back into the life; and in this way it appears that in Eliot's world, no souls have ever successfully reached the afterlife. And so at the end of the poem we find ourself faced with this exact same dilemma: we have effectively traversed through hell, and are standing before the Death: will we cross the bar into the afterlife? Or will we descend into the fated spiral once more?

Props to Quisha for drawing a connection between these lines and Bradley's Appearance and Reality. In her annotation she isolated a specific line from his essay: “In either case my experience falls within my own circle, a circle closed on the outside; and, with all its elements alike, every sphere is opaque to the others which surround it." I find this quote extremely revealing. In my past annotations I have loosely discussed how TS Eliot appears to be presenting the thematic motif of DOUBLING, and as such, has constructed two separate worlds: the world in reality and the world of perspective. The world in perspective collides resoundingly with the concepts of Tarot and orientation; that is, how our own internal worlds can shape our perception of the environments around us, and as such, potentially imprint upon them. The world in fact, however, is far bleaker - this is the waste land of which Eliot speaks, populated entirely by vast tracts of infertile land and a cacophony of disembodied voices. Upon reading Bradley’s essay, I was most struck by the notion of a soul truly “knowing” another - and the rarity of that occurrence. I think part of the Waste Land’s pessimism is birthed from the notion that while our perceptions can filter the external world in a way which infuses it with hope, our perceptions are exactly that: OURS. There is no guarantee that any one else will ever see into your soul, or has a soul similar to yours; and there is also no way to ensure that a communication is as accurate as you desire it to be. “The key”, then, is turned only when all these factors miraculously unite: when the world of perspective and reality are guaranteed to be identical.

Last year, Addie annotated this exact section and described how Eliot purposefully confuses the reader's sense of right-side-up and upside-down. In an especially insightful section of analysis she claims that if the reader were to orient herself with respect to Dracula (whom "crawled head downward down a blackened wall"), the tower down which he crawls becomes inverted - and the corresponding Tarot Card, the Dark Tower, is similarly flipped. Nested in this idea is a broader understanding: that in the chaos and turbulency of the modern world, the only form of agency we truly have is our perspective. When Dracula is flipped upside down, the world appears to him inverted; and though in fact it remains exactly the same as it always was, in his mind's eye all has been reoriented. That's precisely Eliot's point. Though the world itself may be a wasteland, there exists a copy of this world - a world of shadows, of impressions, of perspectives and opinions - which is completely up to interpretation. I think he invokes Tarot as a way of imbuing this doppelganger realm with purpose and value: Tarot is all about perspective. Your interpretation of the card, and what it tells you about your life in this theoretical duplicate of reality, informs the way you act in the real physical world - and so perhaps our agency, though constrained to our own perspectives, is more powerful than we think. The following two lines are relevant insofar as they condense several central thematic discussions: the voices, time, familiarity and remembrance, and water. All of these strands weave together a picture of reality IN FACT: that is, a world in which people are consigned to make the same mistakes over and over, a world where several voices overlap but never really hear one another, a world analogous to a dry rock. I think Eliot piles up all these images to drive home the fact that though our perspectives may change (though the Dark Tower may become inverted, or vice versa), objective reality is constant. In this way he DOES put a pessimistic constraint on the extent to which our conception of life can actually influence the events occuring around us; but nevertheless I do think there are some shards of positivity embedded in there.

To Olivia: I agree! I think that gender fluidity is a big thematic motif throughout this section. Expanding that a bit further, I think there's a way to rope in Donne into how Eliot plays with male-female binaries. In his Elegy XIX, Donne describes the male desire for possession of the female body, or more specifically, the female soul. Initially it seems that Donne is simply illustrating the basic desire for ownership inherently embedded within sexual relations; but there exists another reading too, in which sexual yearning is a product of Man's intense wanting to be Woman. The elegy begins; "Come, madam, come, all rest my powers defy,/ Until I labor, I in labor lie." Of course there is the most obvious understanding of these lines as "Come to my bed, because I cannot rest until I can make love to you!" - but this is far too tame. I think there is an equally plausible interpretation in which Donne is saying he cannot rest until he not only makes love to Woman, but BECOMES a woman. "Labor" is here taken to mean work (sexual work, presumably), but one cannot help but think of its second meaning; that is, childbirth. In this way labor, being a biological capability that only woman possess, becomes a replacement for womanhood itself; and when Donne writes that he cannot rest until he lies "in labor," he is actually suggesting that he cannot rest before experiencing womanhood. This analysis colors the rest of the elegy in a different shade. The narrator's visceral descriptions of an undressing woman become not a product of sexual desire, but rather a deep-rooted envy of her femininity; and in this way Donne reframes love, lust, and hatred for the female figure as a function of Man's desire to be Woman.

To Lucas - I completely agree, but I also think there's an alternate meaning of the thrush in which the barren wasteland not only becomes hopeful, but grows un-wasted. You are definitely right to say that the thrush preaches a life of simplicity, and of thriving off of scant resources. If you've only ever lived off nothing, than something--even the smallest of crumbs--seems enormous to you. But there is another subtlety to Keats's poem as well: as he writes, "He who saddens/ At thought of idleness cannot be idle,/ And he's awake who thinks himself asleep." I think what he means here is that THINKING itself is something: having the thought of idleness inherently means that you are not idle, and the fact that you are thinking about being asleep proves that you are not, in fact, asleep. Similarly, thinking about the waste-land proves that the wasteland is not, in fact, wasted. Though there may be no water, nor soil, and perhaps just a rock, there nevertheless are THOUGHTS: and these thoughts are material growths in of themselves, proving that something in fact has grown out of this barren world. Thus, the wasteland cannot be wasted - it really just depends on the way you think about it. Perhaps this is Eliot's way of suggesting that all the spectres that he references - shadow selves, twins, the "sound" of water, thoughts - constitute something valuable? Something that, in a world of waste, has worth?

Death by Water represents the convergence of two central motifs: the twins and the voices. In the Inferno, Dante encounters two sinners who are being burned together in a double flame: that is, "who are twinned within a single fire." The identicality of their hellish fate stems from the fact that their sins were wrought together, or similarly; in this way the twins become a generalizable metaphor for the repetitive nature of the human race. Prone to the same errors, we live the same lives. Interestingly, one of these sinners (Ulysses) explains his final journey, in which he hit a underwater whirlwind and died via drowning. The convergence of these images - of fire after death, of water AS death, and of the whirlpool being life itself funneling towards death - mirror Phlebas's narrative structure exactly. How does this relate to Dante, you may ask? I am of the opinion that Eliot roots Phlebas's arc in the "twin" sinners as a way of indicating that Phlebas (much like Sybil with the voices) represents not one person, but many. Having been "a fortnight dead," he is presumably burning in hell; but if his Inferno counterparts are Ulysses and Diomedes, he must have twin counterparts too. Eliot helpfully introduces us to his double: the reader. The final line ("Consider Phlebas, who was once handsome and tall as you") uses a visual comparison to directly frame the reader as Phlebas's twin. In this way it becomes clear what Eliot is really trying to get at: that we as humans are doomed to repeat the same sins over and over, and as a result, will burn together in a "twinned flame." What that flame is we do not yet know.

“Moisture is the essence of wetness, and wetness is the essence of beauty,” says Derek Zoolander, dead serious. Keats would agree. His world glistened—wine, nightingale, sleep, feeling itself was fluid. Eliot’s isn’t. In What the Thunder Said, language cracks open: “Here is no water but only rock.” The Romantic current congeals to dust. Keats seeps into forest; Eliot splinters in desert. The thunder stutters where the nightingale once sang. “If there were water”, the words twitch, dehydrated prayer. Keats drinks. Eliot chokes on sand.

“Why is the rum gone?” Jack Sparrow asks, staring out at an empty sea that feels both comic and mournful. That’s how Mr. Apollinax reads to me, a poem laughing at its own drowning. His laughter is submarine and profound, already half below the surface, intellect bubbling through the drawing room air like something that shouldn’t survive there. Beneath the teacups and lemon slices, coral and green silence are already rising. The scene is civil, but the tablecloth is soaked through. Years later, in The Waste Land, Eliot returns to that same sea, but the laughter has gone still. The descent is no longer metaphorical. What was once a philosopher’s joke about depth becomes Phlebas’s silence. The current that once worried the drowned now picks his bones clean. The wit has been replaced by ritual. In Mr. Apollinax, the drowning is intellectual—a mind so deep in abstraction it begins to suffocate itself. In The Waste Land, the body follows. Phlebas is the same figure, just further down. The philosopher’s laughter becomes a whisper, the thought turned to tide. Both poems trace the same descent. One plays with it, the other completes it. The drawing room and the sea are the same room, seen at two depths. The shift from irony to elegy, from chatter to current, is the sound of Eliot’s tone sinking. Phlebas is Apollinax returned to the water, his laughter dissolved into the sea that always waited beneath the surface.

What interests me most here is the question of power. It seems to be the fundamental resource beneath all male-female relations: does man overpower woman, or vice versa? In this instance the answer seems to be neither; for the first time we see a productive partnership between Man and Woman - Elizabeth and Leicester - in which they are united against the court of public opinion. The following line ("beating oars") reveals another facet to this partnership. This reference is preceded by a section on the Thames, and so visually, we can imagine Elizabeth and Leicester trapped together on a tiny rowboat, working furiously to fight against - or even, perhaps, to escape - the dirty Thames. This metaphor fits neatly into historical records: Elizabeth and Leicester were partners in crime when it came to escaping public scolding, which in some sense can be viewed as a dirty contaminant (like the Thames) threatening to lay waste to their reputations. Importantly, both of them were unsuccessful in escaping the waste. Despite their "beating oars," they never did escape the Thames; the two were never married, and public speculation concerning their relationship--and the foul play which it may have catalyzed--never ceased. Perhaps a moral generalizable to the larger poem: that it is impossible to flee the waste.

“Are we there yet?” Donkey whines in Shrek, somewhere between tedium and revelation. The question feels small, but it’s also about arrival—what it means to get somewhere, or never really arrive at all. Augustine writes, “To Carthage I came, where there sang all around me in my ears a cauldron of unholy loves.” The line is clean and chronological, a simple confession of arrival. Eliot adds one word: then. “To Carthage then I came.” That small word changes everything. In Augustine, the journey is complete; in Eliot, it feels ongoing. Then marks not only sequence but recurrence, as if the speaker is caught in the act of remembering and re-experiencing at once.The addition of then places the line in the voice of Tiresias, who exists across genders, times, and stories. For Tiresias, to see is always to relive; his vision folds every moment into one. The then mirrors that condition. He cannot witness without returning, cannot perceive without repeating. Augustine’s journey to Carthage happens once in time; Tiresias’s happens perpetually, in vision. Eliot’s then transforms a historical confession into an eternal one. It turns Augustine’s movement through sin into Tiresias’s endless cycle of seeing. “To Carthage then I came” is no longer an arrival in place, but a return in consciousness—a moment that Tiresias, who can never stop seeing, must witness forever.

This line is a modified version of Psalm 137: "By the rivers of Babylon, there we sat down, yea, we wept, when we remembered Zion." The general plot of Psalm 137 revolves around the Babylonian exiles, who have been sent away from Zion (their home) and made to live, against their will, in a foreign land. The captors demand of them a happy song from Zion, and describing this demand, the captives write: "they that wasted us required of us mirth." In the Eliot-Psalm parallel, the Thames supplants Zion. This replacement is peculiar, since the Thames is neither traditionally "sweet" nor a homeland to be longed after, as Zion is for the exiles. Perhaps Eliot means this tongue-in-check reference to be a nod to the industrialization of the modern world: those who "wasted" society--that is, big business, factories, etc.--expect the public to sing mirthfully of the disgusting Thames, polluted and chemical-filled as it is. Interestingly, Eliot obliges: over the course of the next two lines it is revealed that he does in fact sing his song. In Psalm 137, the exiles refuse to sing a song of mirth out of principle: "How shall we sing the Lord's song in a strange land?" The fact that Eliot has no such hesitations, and sings willfully, suggests that perhaps this land is not so strange - he feels perfectly at home in this foreign land (and no sorrow at his home having been "wasted") simply because he comes from a wasteland also. And so perhaps the true optimism in the poem does not come from broad generalizations of "beauty in the waste" or promises that "we're all in this together"...rather, Eliot seems to take the most comfort in the understanding that we are all at home, always. No matter how bad the world gets or how wasted we become, we can still sing our songs of mirth - for after all, we come from the wasteland.

I suspect the "Goonight" is a nod back to one of Eliot's previous subtextual motifs; that is, the idea that life and death exist in an endless loop of repetition. Sofia theorized (in a fantastic annotation of her own) that Eliot believed small changes---aka waste (the tiny objects or moments which comprise our entire lives)---to be the reason behind massive changes in our destinies; in one of my own earlier annotations, I spoke very briefly about how these small changes are responsible for the cyclical nature of life and death (in which dead souls are repurposed into new ones, thus condemning the human race to an eternity of the same lives lived over and over again). Zooming into this section specifically, the dropping of the "d" in "Goodnight" represents one of these many "small changes." Perhaps Eliot means to parody the decay of great literature by directly contrasting the "Goonight" with the Shakespearian original below; but either way such a decay represents only one of the many ways in which human society signs its own death sentence.

What struck me most as I was reading through Baudelaire's rather grotesque image of "A Martyred Woman" was the way in which the physical image of the woman blends seamlessly into the environment. It makes me think all the way back to De Nerval's dream essays, where he ponders the "eternal distinction between good and evil. Is my soul this indestructible molecule, this tiny bubble of breath which plays its part in nature none the less? Or is it instead merely this void, this image of nothingness receding into infinite space?" If we take his understanding- that to be good is to be active, to have a part in society, to play one's part in nature; and that to be bad is to be nothing, to blend into the void of life itself - then is there no clearer distinction between good and evil than man and woman as presented by Eliot? Baudelaire begins the trend. His woman, who, being dead, is quite literally "an image of nothingness," recedes quietly into the background of the poem; in fact, the linen literally "drinks up" her blood. Stylistically, there is no distinction between his extravagant descriptions of furniture (the external world) and his reverential (and objectifying) analysis of her body. This blurring of a woman and her environment extends to Eliot's poem, too, where this section on Woman is cloaked in references to sensory experience and classical literature; she is a function of the world, not the other way around. In this way it becomes clear which of the two camps Woman falls into: "this image of nothingness receding into infinite space," or, as Baudelaire calls it, evil. And yet to Eliot, Man is the "indestructible molecule"; throughout the entire poem, men are the actors, the changers, the doers. They form the very fibers of life, and as such, meet Baudelaire's criteria for "good" - and Eliot's criteria to be represented explicitly.

“Violet! You’re turning violet, Violet!” Wonka shouts as the girl swells into blue, her face and hands and body shading into purple—it’s almost a violent process. I am certain Eliot was not thinking of Charlie and the Chocolate Factory as he wrote of this violet, just as he was not Tiresias, gifted with foresight. In Ovid, Tiresias is blinded after striking two snakes, only to regain vision through prophecy. To perceive the present, Tiresias must see the future in the past. In this story, past, present, and future exist as articulated, distinct definitions that blend into a single dissonant moment of perception, one where time collapses into color. In Eliot, as in Ovid, sight is never linear; to see is to blur. Tiresias’s vision moves backward and forward, a violet haze where beginnings and endings share the same hue. The present becomes residue, what has already happened and what is still to come– a constant turning, violet to violent, prophecy to memory. That blur extends beyond time into the human. The women of The Waste Land are not distinct but refracted images of one another. They fold into Tiresias’s field of vision until they become the same woman, seen again and again under different light. Each one repeats the same gestures: speaking into silence, waiting for a knock, cleaning up the fragments of her life. The plural dissolves into the singular, but not individuality; rather, a collective exhaustion. The private and public collapse too. The boudoir bleeds into the barroom, the domestic into the civic, until all speech feels communal—shared, overheard, half-remembered. The commons replaces the person; intimacy becomes collective. Tiresias watches as individuality gives way to type, as woman becomes women becomes one.

“RON: Once I make my move, the queen will take me. Then you’re free to check the king. HARRY: No. Ron, no! HERMIONE: He’s going to sacrifice himself. RON: Do you want to stop Snape from getting that stone or not? Harry, it’s you that has to go on. I know it. Not me. Not Hermione. You. Knight to H3.” The scene feels like the final game, the sacrifice, the victory THE GAME. Pound’s The Game of Chess works that way too. The definite article locks the world in structure: “Red knights, brown bishops, bright queens.” Everything burns with precision, every piece belongs to the pattern. “The” implies consequence. Each move means something, each color holds.<br /> But Eliot’s A Game of Chess loosens that grip. A, not the. Suddenly the game isn’t singular or grand but one of many, maybe endless. The definite becomes indefinite, the sacrifice hollow. “‘My nerves are bad tonight. Yes, bad. Stay with me.’” The moves don’t land, “‘What shall I do now? What shall I do?’” There’s no check, no king, just exhaustion masquerading as strategy. The board gleams under purpose; a board flickers under repetition. Pound’s definite article closes the frame; Eliot’s indefinite article opens it until it collapses. THE Game demands sacrifice. A Game doesn’t even notice one’s been made. You don’t know A game is over, until THE game starts, and it's time to say goodnight.

“Mirror Mirror on the wall, who is the fairest of them all?” (Snow White), the evil queen utters, jealous of the pure perfect young girl. While Eliot does not write of a pristine princess (rather a queen), there is much regality in A Game of Chess, seeming to draw from Baudelaire’s opulent descriptions in A Martyred Woman. What fascinated me the most in both pieces is their olfactory descriptions, more specifically their representation of perfume, and its reflection or rather refraction. These two lines seem to reflect one another visually, both beginning with Un–; however, that is really the extent of their mirroring. “Unstoppered, lurked her strange synthetic perfumes, Unguent, powdered, or liquid, troubled, confused.” The prefix un- does more than repeat; it undoes. To unstopper a bottle is to release what was meant to remain contained, while unguent evokes the oily, ceremonial luxury of queens and corpses alike. The language itself opens and unravels, performing the act of unsealing that the scene describes. Yet what spills out is not clarity but confusion. The perfumes, once symbols of beauty and refinement, have become dense, chemical, and suffocating. In Baudelaire’s A Martyred Woman, perfume occupies the same paradox. The room is decadent yet dying, filled with “perfume flasks” and “bouquets exhaling their final breath.” The air is both intoxicating and fatal, heavy with sweetness that edges toward rot. Eliot refracts this atmosphere into the modern world; his perfumes are “synthetic,” their allure artificial. Where Baudelaire’s fragrance veils decay in beauty, Eliot’s amplifies decay through imitation. Perfume, then, becomes a mirrored contradiction, both attraction and repulsion, luxury and poison. Its scent seduces even as it suffocates. In both poets, the air itself becomes a reflection of moral and physical decay, a beautiful corruption, a sweetness turned stale, lingering long after life has left the room.

“The beginning of the end” is a term typically associated with gradual endings—the kind that are drawn out, painful, sometimes bittersweet. Think graduations, moving away from home, drifting from friends, or summer turning into a wall of autumn. But what does it mean to end at something’s beginning? The last few lines of The Burial of the Dead are borrowed directly from Baudelaire’s preface to Les Fleurs du Mal. A preface traditionally begins a work and offers the reader a lens through which to read what follows. Eliot’s decision to close his first section with another writer’s beginning is not accidental; it creates a deliberate tension between origin and conclusion. To end with a preface is to deny resolution, to suggest that endings are never final but cyclical, that one artist’s conclusion depends on another’s start. The moment that should signify closure instead opens outward, invoking a different text, a different time, and a different artistic vision. This reversal also reflects the poem’s modernist structure. The Waste Land builds itself out of fragments– pieces of prior works, cultures, and languages. By ending with Baudelaire’s opening, Eliot acknowledges his dependence on the past while reanimating it in a new context. It becomes an act of literary resurrection: the “burial” of the dead poets whose voices still speak through his own. What appears to be an ending is actually a beginning disguised as decay, echoing the poem’s central paradox of death and rebirth. In this way, Eliot transforms the very concept of closure. His ending refuses to end, it begins again, looping the reader back into the lineage of the poem, where every beginning is reminiscent of what came before and every ending is a begging in some sort of way.

Benvolio returns and tells Romeo that Mercutio is dead. He mentions he was a good man went up to heaven. Romeo says bad days just began

Benvolio, concerned about fighting out in public but Mercutio isn’t worried about fighting in front of people

Hyperbole

Make sure that you have the right information.

I think this sentence is important because I have gathered sources I enjoy in the past and tried really hard to make one work when I could have just set it aside and picked a different source.

This can inform wether the author is bias

online wikis are not reliable to get good quality and the information could be false

Find the sources that are most relevant and reliable.

I need to make sure to ask the professor if i am doing things the right way.

Some other examples upon a google search include: -Orginal documents and records. Ex: Birth Certificate, Marriage Record, Census Data.

Seccondary sourses refer to the author, but are written and analized by other people talking about their point of view or perspective

primary sources are official documents that come from the original author, or are part of history

Looking at multiple sources ensures your reliability and cancels out any false information

Secondary source examples

Primary source examples

Potential angle: improving the differential diagnoses between AD and non AD

follows a step by step predictable pathway that yields the same result each time (no randomness involved)

Lack of a diverse data set again a common theme.

Is there any potential research question here? What should the standard definitions be? What details are needed?

type of AI method where humans create categories and the AI sorts data points into them

you need to have confident words included in the thesis.

the thesis has to contain opinions of your own

a thesis has to be precise

a thesis has to be specific.

A thesis is not the topic of your essay instead it is the interpretation of the essay like what are the key points.

A thesis statement is very useful and needed for your essay

It’s really shocking to realize how much personal information can be inferred from simple online behavior. The idea that AI or data mining can guess someone’s sexual orientation or addiction tendency just from their friend list or social activity feels invasive and unethical. I personally think it crosses a line between public and private life.

At the same time, I understand why companies want to use data to “predict” users—it’s part of how social media algorithms work. But when this data is used to judge people’s race or personality through pseudo-scientific facial recognition, it becomes a form of digital discrimination. It makes me wonder if we are gradually losing control of our identities online.

Wow, the bare faced lie on it, too.

Drug ionization in a weak acid or base if dependent on pH.

Check the model in the lecture slides about this!!

You become better at detecting broad, low-detail (“coarse”) features — like shapes, movements, or sudden changes in the environment. This comes from increased activity in subcortical visual pathways (like the superior colliculus and amygdala) that specialize in quick, rough visual analysis. You become less focused on fine details (like facial features or small text), but more able to spot threat-relevant cues (like movement in your periphery, or something large approaching you).

research-driven digital derive enabled by the internet

between organization and human nature or to put the matter in another way the

divorce of the - economic motive from the - impulses of creation and possession

Title

Authority and the in-divi-dual 1 I

From the projects, there will be dramatic changes in the chemistry and biology of the coming decades, even if its not as dramatic as reefs and shells actually dissolving. This is why teh planetary ounday is set at up to 80% of preindustrail average of 3.44 We're currently aruond 2.8, at 81% of preindustrail levels, 2.75 is the lowest it can go

When argonite saturation is greater than 1, aragonite favours precipation, less than favours dissolution Some parts of the ocean have historically low argonite saturdation levels, but most temperate and tropical oceans havevalues greater than 3. This can change over time, but much of the oceans are forecast to be below 3 by the end of 2000s. This will make the production of aragonite energetically more expensive.

Global picture of HP matches the patterns evident in GoM and Oregon Coastal zones which drain large areas of croplands into shallow seas are more prone

used to be episodic but human activity make HP worse. nurtirent offrun from land causes by this is polluting the ocean The mississippi runs into the ocean, through 40% of the USA's crops, over nurtirents in this water stimulates algal blooms - nurtrient loading Algal blooms then degrade, are consumed and low oxygen water is consumed - which creates an oxygen dead zone near the sea floor Stratification of water means the water layers don't mix (fresh and salty sit ontop of each other) oxygen is less soulble in temps - marine animals consume more oxygen in warm temps - leading to more stratifcation with global warming (open ocean) coastal areas is similar and nutrient loading is expected to rise with increased storms To manage this we need to reduce nurtrient loading in the large water sheds (gulf of mexico) narrow ganitz bay regulated sewage treatmnet plant water - HP reduced

Hypoxia is caused by a lack of oxygen Shellfish & worms get trapped and suffocate and die Brown shrimp in mexico was a big fisherie - optimal habitat reduced by 25% Hypoxia takes away a food source (veg) which has a chain reaction HP can effect the growth and reproductive potiental of some bottle dwelling fish, even with intermient exposure it's more sub-leathal affects which is an issue rather than death, as they cascade through the food chain Looks like it's causing a reduction in shrimp growth Fish & shirmp tend to stay on the ages when there's HP so fisherman might be taking the shrimp when they're young it has an adverse affect on the economy

I think that socialization for students with differences and disabilities can make a whole world of a difference. It will allow them to learn from their peers academically and behaviorally but also allow them to feel more included which is the whole point!

Researching has been critical in learning about how to interact with special populations. This also helps educators be prepared and have an idea of what to expect in order to be better prepared in interacting with the children.

I think this also shows to have grace for yourself. No one is perfect, and at the end of the day we’re all humans trying our best.

It’s always important to include parents and share progress/expectations of the child. Especially in considering how home-life can often bleed into school-life and affect the child’s behavior and results.

It also shows how we as educators need to retrain our way of speech and thinking, if this is something that you usually do. I think another important factor is to see how you speak to yourself. That can be a tell-tell sign of how you normally speak to others.

This part serves as a reminder for all educators that the environmental setting, and set up of a classroom is a very critical aspect for a child’s learning experience.

Love that the classroom was inclusive and the teacher was able to help other students interact and understand. That is the ultimate goal!

I think it’s very important to showcase that instead of removing the object forcibly, they decided to replace the item with a similar shape and feel as the original object. Also, noting how they “eased” his mind.

This is true, while observing my group mate actually got bit buy a special needs little girl. I know she was probably upset and didn't mean it in that way so once everything was calmed we didn't freak out, however, it just goes to show how you have to be ready for the unexpected.

As I've stated previously, I 100% agree that parents should be involved in one way shape or form. I understand that every parents relationship is different but I think the parent knows their kid the best as well as the kid knowing themselves so its important for parents to have a say.

I agree, I think it is important for teachers to be able to adapt and mold to student(s) needs such as this instance.

I agree with this statement, I think the students AND parents should be involved with any district or rule changes as they best know what is write or wrong and what best suits themselves/ their child.

It is interesting to see how Carson uses and utilizes his surroundings. He tends to explore and Id assume he does so to clear his mind or see new things.

This can be good, as long as the "model student" does not feel uncomfortable about being an example for their classmate, but this most likely would not be an issue. I do agree that learning by observing ones peers is a great method.

I think this is something that educators forget about sometimes. Students just want to be known and seen by their teachers. If they feel that way, they will be more open to participating and overall more engaged in the classroom.

I can connect this to my own placement. When I have taught in self-contained autism classes, students can respond to the music by singing and dancing to the movements (oral and kinesthetic), as well as choose what songs they want by pointing to a choice board I hand them. The choice board encourages them to use their words (Ex. "I want..") or use their communication device.

In the special education classes I've observed and taught, silent gestures and signals have been ineffective. Perhaps this could work in an inclusive classroom rather than a self-contained one? In my placement, when a student is displaying inappropriate behavior, it's accepted schoolwide for teachers to say "No thank you" and give the student a simple and quick redirection. This saying and redirection is used by all teachers consistently, so it has been effective.

This is so smart. Kids love to be creative and show off what they can do, so this was a great way of getting Carson's mood back up.

I witnessed how effective student-to-teacher proximity is today during my placement. In a self-contained autism classroom, all of the paraprofessionals, as well as their teacher, were absent. One substitute para and one substitute teacher were placed there instead for the day. This caused the students to display more class-disruptive behaviors than normal. With a lack of their familiar paras and a lack of adults, the classroom management was lower than usual.

These are common traits in people with special needs, especially those on the autism spectrum. It is incredibly important to know this about your students with special needs, and it seems like the teacher in this vignette was doing an excellent job!

I really like this strategy because while it encourages visual and tactical learning, it also serves as a gentle reminder to follow the rules established in the classroom, rather than a punishment

I really appreciate this. This is deeply shows how easily behavior can be misunderstood when communication is difficult. Growing up I've lived through situations where I acted out in class, not because I felt the urge to misbehave, but because I didn't have the words to express my feelings. It's important for teachers to be patient and have empathy toward students instead of labeling students "bad kids".

It's important to understand that students deserve to have a voice in their own behavioral plans. It'll help them feel more respected and responsible for their own actions, while ensuring they feel heard.

This is very important in not only the classroom setting, but also in the workplace. This ensures that people don't feel isolated, and frustrated. It's important to know that active engagement promotes social integration, acceptance and shared learning experiences.

This is an ongoing issue in today's society, as many schools are short in the music department. In my experience, there was only one music staff in my High School building. This in turn, can make professional support and creative idea sharing, a lot difficult for music educators.

This chapter emphasizes proactive student-centered strategies for inclusive music classrooms, from physical setup and behavior plans to socialization and ethical care. How can music educators balance the need for structure with the flexibility required to support disabled learners in high-performance settings?

This passage reinforces how music educators can contribute to meaningful data intervention plans, even in non-core subjects.

How can music educators ensure paraprofessionals feel empowered to participate in teaching, especially if they don't know a lot about music?

a strategy reframed by thinking about classroom incentives. Instead of external reward, the teachers use musical expression and leadership roles to motivate Carson.

Labeling as "good" or "bad" can harm a student's esteem and identity. Describing actions neutrally to avoid reinforcement of negative self-concepts.

DOI: 10.1145/3332186.3332229

Resource: None

Curator: @bandrow

SciCrunch record: RRID:SCR_019299

What is this?

DOI: 10.1016/j.celrep.2025.116428

Resource: None

Curator: @areedewitt04

SciCrunch record: RRID:MMRRC_034840-JAX

What is this?

下面的图中没有关于不同的Head的信

本项目任务：预测 start and end positions

I suppose this is what I discovered. What I wasn't doing was the thing I should have been doing. Even though that thing was only touching base and not actually doing anything major it was still what I should have been doing.

See this note front he beginning of the article. and this one

Its really sad to think how schools gave better opportunities to white students and worse ones to students of color. That made it nearly impossible for everyone to have the same opportunities.

This article talks about how Facebook limits its users from opting out their phone number for the "look up" feature. This makes the user's number visible to users searching, even though the intention of inputting the number was for extra security. Facebook has viewed this more as a tool rather than a threat, so it doesn't look like they will offer a solution to users' concerns.

This is interesting, and I have personally experienced this firsthand with one of my friends searching up merch from a local brand near me. In about the next 5 scrolls on social media, that brand pops up as an ad for me, which exemplifies the algorithm working with people nearby.

The idea that we perform different roles depending on the context really makes sense it’s almost like we’re all actors managing impressions.

It’s sweet how personal idioms or inside jokes make relationships feel unique it’s like having your own private language.

The section about negative routines made me think — we can fall into toxic communication habits just as easily as healthy ones.

I hadn’t thought about how often we communicate just to ‘get things done,’ like asking for help or giving instructions it’s so routine we don’t even notice.

I liked the comparison between maintaining relationships and car maintenance, it’s a good reminder that relationships need consistent care

This article talks about the people who are affected by societal barriers and how there is more work to be done to include people. However, there are innovations to be talked about, like the new traffic lights to help people who are color blind and can't distinguish the various lights. There is still a lot of work to be done, but there is an effort being made to include everyone.

Ability-based design is very crucial today when it comes to new products. The way your device can adapt to your personalizations and environment is a big draw for consumers and helpful for everyone. An ability-based design that comes to mind for me is Apple CarPlay, adjusting the map brightness dependent on your surroundings and time of day.

French states that there are two types of mysteries in this article. Ones that solve the crime head-on and bring order throughout the mystery. (She put Christie and Holmes as examples). The other is that they don't give clear answers and show that truth and evil aren't always that simple. This shows how mysteries can make us feel safe or make us think deeply about life.

The General Data Protection Regulation is in place to create a bigger barrier for data privacy within the EU. This is very different from the United States, since user privacy is more lenient here. The EU had this regulation in place to restrain illegal data sharing.

Metadata is so tiny yet contains so much information that it can expose privacy. The CEO and murder case is an example of how dangerous it can be. If people were able to find him in Guatemala, it just shows that anyone can be found, which can be kind of scary.

Spurious relationships are two variables that seem to be correlated with each other, but in the end, it turns out they have no effect on one another. This can be challenging for data scientists to decipher, as the human brain is prone to making connections.

This is very interesting to think about in the grand scheme of things. On our phones, all of these social media apps/sites are free to the public, which brings up the question of how these platforms have the money/capital to support the development. This brings up a thought of mine about how much these platforms make for each client/user and how it is measured.

DOME annotations are also available in the DOME registry here https://registry.dome-ml.org/review/futlrtl5w4

I believe this is trying to demonstrate how black women writers are not very represented, even with all the contributions they have done. I think it just points out sexism and racism due to the invisibility black women writers go through.

this basically highlights how girls, especially black girls in this case, and are often punished for speaking out. It points out that the struggle isn't really to speak but to be heard and share opinions.

Important

Important

This line shows how myths explain natural and emotional realities — in this case, grief and the unknown after death. The story uses the wandering spirits to make sense of human sorrow and the mystery of where souls go. It’s both poetic and tragic.

This moment echoes Lewis Hyde’s idea of the trickster as a boundary-breaker. Like Coyote, he challenges the community’s decision and insists that death should be permanent. In doing so, he breaks the social and moral order that values life and rebirth. Although his actions appear selfish and cruel, they ultimately reshape the natural balance, showing how tricksters bring transformation through chaos and contradiction.

It'd be cool to show some examples of this!

What is your intuition for the origin of this capability? e.g. to what extent is it likely due to the size of the SAM encoder, its architecture, the size of the pretraining dataset, etc.

Does this later the number of parameters in the pre-trained SAM model? And would it be expected to degrade performance? (of course the tradeoff could still be worthwhile, but it would be nice to know if this downsampling comes with a cost)

This is a bit of a nit, but I think it would be worth clarifying what is meant by "works well" as it could be interpreted to mean "easy to use", which feels much less important than that biological software produce correct and reproducible results.

This could be prohibitively expensive, but a more robust test of generalization would be some kind of leave-one-dataset-out cross-validation (e.g. calculate a dataset-dataset similarity and hold out the dataset least similar to all other datasets)

what was the motivation for making this change?

Reddy distinguishes two different relationships/bindings in the community: the guru-cela relationship (focus on lineage and hierarchy and entails obligations), and the sister and mother-daughter relationship (motivated by mutual affection, focus on love and caring). I note that she didn't discuss how the sister relationship is organized for hijras, instead comparing the guru-cela and mother-daughter as parallel. As she argues that this latter form were not restricted by houses or lineages, will it unsettle the Hijra's rigid kinship structure by houses? How does the kinship incorporate this form of relationality, or even use it to recuperate the need for care, which is sometimes absent in the obligations-bound guru-cela relationship?

Hooray, at the final bit of the argument in this chapter. Reddy employed the notion of the architecture of conflicting desires from Trawick. It is an architecture, a system for organizing and managing the ambiguous forces in "various polysemic" desires and relations. The kinship system here is the way to make such ambiguity the meaning-maker. This goes back to her critiques of psychoanalytical, relational, and family explanations. The architecture helps us to think about how ambiguity manifested in these practices of desires, and how in turn it is managed in this way.

Reddy shows her concerns with the "resistant" discourse that reads hijras' kinship model as an opposition against the traditional heteronormative one. Reddy pays attention to the fact that hijras also internalize and adopt many ideas and terminologies from others (guru-cela is a clear example of how toxic it can be sometimes). And this understanding somehow feeds into the heteronormative hegemony (as if all other alternatives have to have something to do with it). I love how she used the word "polysemic," which really allows us to see the possibilities in the hijras' community, which does not exclude the role of power relations. It is not the dichotomy of conforming/resisting. Instead, the hijras create a space in the ambiguity in between.

Many vignettes above called back here! Reddy criticized the recent scholarship that resolves the theoretical problem raised by hijra with their investment in the family. Reddy has shown that this family is not what we usually conceive: multidimensional, inclusive, and elastic. The family structure of hijra does not oppose the mainstream family value of India, but precisely in the space of ambiguity, it finds its legitimacy and possibility to sustain.

The paragraphs above criticized the psychoanalysis method; here, Reddy also argues that the relational framework is unsatisfying. Indeed, identities are all relational, but it seems to be an easier option not to obscure the specificity of practices and rituals - why in this way? As other relations also provide identifications, why do this and why insist on becoming hijra despite all the hardship and the sacrifices? Reddy further pushes Don Kulick's idea of relational femininity, which glosses many difficulties between the ideal and practices for travestis (maybe they do love their boyfriends!).

This passage delivered the punch of the argument: hijra is a relational identity. This echoes the idea of relational femininity of Travestis in Kulick's ethnography. An identity of self can only emerge in a relational network with others (whether the in-groups or out-groups). Then Reddy criticized Western scholarship that views identity as a matter of individuals, neglecting the significance of specific contexts that condition the manifestation of such identity.

Reddy tries to build dialogue with the Indian psychoanalysis literature that seems to insist on an essentialist approach. This view naturalizes the hijra identity as something intact and given, as simply a desire for fusion with the world. ijras' desires were essentialized as the desires for fusion. Therefore, the intricated innovation of hijra kinship structure can be reduced to their "desire for an idealized relationship" with their maternal mother. Nevertheless, as Reddy also already shown, many of the hijras did maintain good relationship with their biological mothers, most often as their only connection with their natal family. Reddy wages a critique of the psychoanalytic approach and argues for a more in-depth examination of the hijra's kinship to understand their desires not as a taken-for-granted fact but as a mediation (ambiguity_ of the negotiation of their status quo in the society and their own agency.

Hijras identify all Pantis as the othered, denying their membership to join the kinship network of Kotis. Therefore, sexual penetration (penetrator/penetratee) serves as a principal distinguisher for the hijras' relationality. However, at the same time, the desires for panti/coexist with this exclusive concept of family. For example, in previous chapters, Reddy introduced a vignette of a panti who is viewed as a member of the family, who basically lives at the waterbank with the hijras. Many other instances illustrate this physical proximity with their husbands. It once again demonstrated the elastic application of the family system. It is a kind of family that does not exclude other forms of kinship but allows them to coexist. Moreover, it also relates ot Kulick's account of travestis wherein they could reverse the othering, reiterating their subjectivity.

Reddy adopted a semiotic analytical framework here to understand the ambiguity/ and the flexibility of the kinship conception. The subtle difference between family and manollu is interesting, as it shows how they relate to the wider Koti community and other communities they deem with less izzat. It also makes me wonder if it is possible to extend such kin identification to their biological mothers, as Reddy states, women can also be included in. It also differs from the biological family, which is rigid and passes down through vertical lineage only. Perhaps the kinship system of hijra elucidates on how the a system of relationality can be organized multidimensionally.

Another paradox arises: the complicated presence of multiple kinship relations, the hijra kinship and the natal kinship. While it is ideal for hijra to completely renunciate their natal family as the hijra family is supposed to replace the latter, there is a lot more elasticity in practices. It is also interesting to see that the natal family sometimes recovers the individuals when the hijra kinship fails to extend the support and care. The lasting relationship with their natal mothers also points to the differences from the hijra mother-daughter relationship. Do hijras understand them as two fundamentally different relationships?

This section of this chapter provides really good topics to annotate on. I believe that especially in generation Z, value lacks effectiveness and importance because of how everyone in my generation care more about perception and reward rather than living up to values I believe that not many people my age even care to live up to their values and beliefs because of the fact that everyone is so engulfed in social media and disregard how dangerous social media's influence can truly be

This is a very interesting take on values and beliefs in the US. This section of the article in particular makes so much sense, as I can relate to this to my everyday life. It's no question how a youthful adult appearance signifies sexuality, and It can be a very frightening and unsafe thing because of the dangers that goes on with sexual harrasment in the world going on today. This section states that children represent innocence and purity, but in all honstly and unfortunately, it feels as though there's not much innocence and purity left in children anymore because of the rise in child trafficking and sexual abuse either.

High, Low and Popular Culture summary: This section explains the different types of culture mentioned in the subheading being, high, low and popular culture. High culture is often associated with intellectualism, political power and prestige. Low culture is often associated with experiences and attitudes that exist in the lowest segments of society. Popular culture refers to cultural experiences and attitudes that exist in mainstream society. A few examples of this are, baseball games, music, anime and cosplay.

physical properties that are of some culture that can be touched are tangible whereas, nonmaterial culture are just mainly ideas of a society. Ideas of a society are not physical properties that you can touch, therefore, they are intangible.

Its interesting to me how in other nations and in different times, marriages are arranged due to culture and without individual choice of the person. Even if you hadn't gotten to know your wife or husband, that youre inevtiably bound to them for life without really knowing what theyre capable of doing to you. That is a very scary thought.

Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

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Reply to the reviewers

Manuscript number: RC-2024-02830

Corresponding author(s): Julien, Sage

1. General Statements

We thank the Reviewers for a fair review of our work and helpful suggestions. We have significantly revised the manuscript in response to these suggestions. We provide a point-by-point response to the Reviewers below but wanted to highlight in our response a recurring concern related to the strong cell cycle arrest observed upon the acute FAM53C knock-down being different than the limited phenotypes in other contexts, including the knockout mice and DepMap data.

First, we now show that we can recapitulate the strong G1 arrest resulting from the FAM53C knock-down using two independent siRNAs in RPE-1 cells, supporting the specificity of the effects.

Second, the G1 arrest that results from the FAM53C knock-down is also observed in cells with inactive p53, suggesting it is not due to a non-specific stress response due to “toxic” siRNAs. In addition, the arrest is dependent on RB, which fits with the genetic and biochemical data placing FAM53C upstream of RB, further supporting a specific phenotype.

Third, we have performed experiments in other human cells, including cancer cell lines. As would be expected for cancer cells, the G1 arrest is less pronounced but is still significant, indicating that the G1 arrest is not unique to RPE-1 cells.

Fourth, it is not unexpected that compensatory mechanisms would be activated upon loss of FAM53C during development or in cancer – which may explain the lack of phenotypes in vivo or upon long-term knockout. This has been true for many cell cycle regulators, either because of compensation by other family members that have overlapping functions, or by a larger scale rewiring of signaling pathways.

2. Point-by-point description of the revisions

__Reviewer #1 (Evidence, reproducibility and clarity (Required)): __

Summary:

Taylar Hammond and colleagues identified new regulators of the G1/S transition of the cell cycle. They did so by screening public available data from the Cancer Dependency Map, and identified FAM53C as a positive regulator of the G1/S transition. Using biochemical assays they then show that FAM53 interacts with the DYRK1A kinase to inhibit its function. DYRK1A in its is known to induce degradation of cyclin D, leading the authors to propose a model in which DYRK1A-dependent cyclin D degradation is inhibited by FAM53C to permit S-phase entry. Finally the authors assess the effect of FAM53C deletion in a cortical organoid model, and in Fam53c knockout mice. Whereas proliferation of the organoids is indeed inhibited, mice show virtually no phenotype.

Major comments:

The authors show convincing evidence that FAM53C loss can reduce S-phase entry in cell cultures, and that it can bind to DYRK1A. However, FAM53 has multiple other binding partners and I am not entirely convinced that negative regulation of DYRK1A is the predominant mechanism to explain its effects on S-phase entry. Some of the claims that are made based on the biochemical assays, and on the physiological effects of FAM53C are overstated. In addition, some choices made methodology and data representation need further attention.

1. The authors do note that P21 levels increase upon FAM53C. They show convincing evidence that this is not a P53-dependent response. But the claim that " p21 upregulation alone cannot explain the G1 arrest in FAM53C-deficient cells (line 138-139) is misleading. A p53-independent p21 response could still be highly relevant. The authors could test if FAM53C knockdown inhibits proliferation after p21 knockdown or p21 deletion in RPE1 cells. The Reviewer raises a great point. Our initial statement needed to be clarified and also need more experimental support. We have performed experiments where we knocked down FAM53C and p21 individually, as well as in combination, in RPE-1 cells. These experiment show that p21 knock-down is not sufficient to negate the cell cycle arrest resulting from the FAM53C knock-down in RPE-1 cells (Figure 4B,C and Figure S4C,D).

We now extended these experiments to conditions where we inhibited DYRK1A, and we also compared these data to experiments in p53-null RPE-1 cells. Altogether, these experiments point to activation of p53 downstream of DYRK1A activation upon FAM53C knock-down, and indicate that p21 is not the only critical p53 target in the cell cycle arrest observed in FAM53C knock-down cells (Figure 4 and Figure S4).

The authors do not convincingly show that FAM53C acts as a DYRK1A inhibitor in cells. Figures 4B+C and S4B+C show extremely faint P-CycD1 bands, and tiny differences in ratios. The P values are hovering around the 0.05, so n=3 is clearly underpowered here. Total CycD1 levels also correlate with FAM53C levels, which seems to affect the ratios more than the tiny pCycD1 bands. Why is there still a pCycD1 band visible in 4B in the GFP + BTZ + DYRK1Ai condition? And if I look at the data points I honestly don't understand how the authors can conclude from S4C that knockdown of siFAM53C increases (DYRK1A dependent) increases in pCycD1 (relative to total CycD1). In figure 5C, no blot scans are even shown, and again the differences look tiny. So the authors should either find a way to make these assays more robust, or alter their claims appropriately.

We appreciate these comments from the Reviewer and have significantly revised the manuscript to address them.

The analysis of Cyclin D phosphorylation and stability are complicated by the upregulation of p21 upon FAM53C knock-down, in particular because p21 can be part of Cyclin D complexes, which may affect its protein levels in cells (as was nicely showed in a previous study from the lab of Tobias Meyer – Chen et al., Mol Cell, 2013). Instead of focusing on Cyclin D levels and stability, we refocused the manuscript on RB and p53 downstream of FAM53C loss.

We removed previous panel 4B from the revised manuscript. For panels 4E and S4B (now panels S3J and S3K)), we used a true “immunoassay” (as indicated in the legend – not an immunoblot), which is much more quantitative and avoids error-prone steps in standard immunoblots (“Western blots”). Briefly, this system was developed by ProteinSimple. It uses capillary transfer of proteins and ELISA-like quantification with up to 6 logs of dynamic range (see their web site https://www.proteinsimple.com/wes.html). The “bands” we show are just a representation of the luminescence signals in capillaries. We made sure to further clarify the figure legends in the revised manuscript.

The representative Western blot images for 5C-D (now 5F-G) in the original submission are shown in Figure 5E, we apologize if this was not clear. The differences are small, which we acknowledge in the revised manuscript. Note that several factors can affect Cyclin D levels in cells, including the growth rate and the stage of the cell cycle. Our FACS analysis shows that normal organoids have ~63% of cells in G1 and ~13% in S phase; the overall lower proportion of S-phase cells in organoids may make the immunoblot difference appear smaller, with fewer cycling cells resulting in decreased Cyclin D phosphorylation.

Nevertheless, the Reviewer brings up a good point and comments from this Reviewer and the others made us re-think how to best interpret our results. As discussed above, we re-read carefully the Meyer paper and think that FAM53C’s role and DYRK1A activity in cells may be understood when considering levels of both CycD and p21 at the same time in a continuum. While our genetic and biochemical data support a role for FAM53C in DYRK1A inhibition, it is likely that the regulation of cell cycle progression by FAM53C is not exclusively due to this inhibition. As discussed above and below, we noted an upregulation of p21 upon FAM53C knock-down, and activation of p53 and its targets likely contributes significantly to the phenotypes observed. We added new experiments to support this more complex model (Figure 4 and Figure S4, with new model in S4L).

The experiments to test if DYRK1A inhibition could rescue the G1 arrest observed upon FAM53C knockdown are not entirely convincing either. It would be much more convincing if they also perform cell counting experiments as they have done in Figures 1F and 1G, to complement the flow cytometry assays. I suggest that the authors do these cell counting experiments in RPE1 +/- P53 cells as well as HCT116 cells. In addition, did the authors test if P21 is induced by DYRK1Ai in HCT116 cells?

We repeated the experiments with the DYRK1A inhibitor and counted the cells. In p53-null RPE-1 cells, we found that cell numbers do not increase in these conditions where we had observed a cell cycle re-entry (Fig. 4E), which was accompanied by apoptotic cell death (Fig. S4I). Thus, cells re-enter the cell cycle but die as they progress through S-phase and G2/M. We note that inhibition of DYRK1A has been shown to decrease expression of G2/M regulators (PMID: 38839871), which may contribute to the inability of cells treated to DYRK1Ai to divide. Because our data in RPE-1 cells showed that p21 knock-down was not sufficient to allow the FAM53C knock-down cells to re-enter the cell cycle, we did not further analyze p21 in HCT-116 cells.

The data in Figure 5C and 5D are identical, although they are supposed to represent either pCycD1 ratios or p21 levels. This is a problem because at least one of the two cannot be true. Please provide the proper data and show (representative) images of both data types.

We apologize for these duplicated panels in the original submission. We now replaced the wrong panel with the correct data (Fig. 5F,G).

Line 246: "Fam53c knockout mice display developmental and behavioral defects." I don't agree with this claim. The mutant mice are born at almost the expected Mendelian ratios, the body weight development is not consistently altered. But more importantly, no differences in adult survival or microscopic pathology were seen. The authors put strong emphasis on the IMPC behavioral analysis, but they should be more cautious. The IMPC mouse cohorts are tested for many other phenotypes related to behavior and neurological symptoms and apparently none of these other traits were changed in the IMPC Famc53c-/- cohort. Thus, the decreased exploration in a new environment could very well be a chance finding. The authors need to take away claims about developmental and behavioral defects from the abstract, results and discussion sections; the data are just too weak to justify this.

We agree with the Reviewer that, although we observed significant p-values, this original statement may not be appropriate in the biological sense. We made sure in the revised manuscript to carefully present these data.

Minor comments:

Can the authors provide a rationale for each of the proteins they chose to generate the list of the 38 proteins in the DepMap analysis? I looked at the list and it seems to me that they do not all have described functions in the G1/S transition. The analysis may thus be biased.

To address this point, we updated Table S1 (2nd tab) to provide a better rationale for the 38 factors chosen. Our focus was on the canonical RB pathway and we included RB binding proteins whose function had suggested they may also be playing a role in the G1/S transition. We do agree that there is some bias in this selection (e.g., there are more RB binding factors described) but we hope the Reviewer will agree with us that this list and the subsequent analysis identified expected factors, including FAM53C. Future studies using this approach and others will certainly identify new regulators of cell cycle progression.

Figure 1B is confusing to me. Are these just some (arbitrarily) chosen examples? Consider leaving this heatmap out altogether, of explain in more detail.

We agree with the Reviewer that this panel was not necessarily useful and possibly in the wrong place, and we removed it from the manuscript. We replaced it with a cartoon of top hits in the screen.

The y-axes in Figures 2C, 2D, 2E, and 4D are misleading because they do not start at 0. Please let the axis start at 0, or make axis breaks.

We re-graphed these panels.

Line 229: " Consequences ... brain development." This subheader is misleading, because the in vitro cortical organoid system is a rather simplistic model for brain development, and far away from physiological brain development. Please alter the header.

We changed the header to “Consequences of FAM53C inactivation in human cortical organoids in culture”.

Figure S5F: the gating strategy is not clear to me. In particular, how do the authors know the difference between subG1 and G1 DAPI signals? Do they interpret the subG1 as apoptotic cells? If yes, why are there so many? Are the culturing or harvesting conditions of these organoids suboptimal? Perhaps the authors could consider doing IF stainings on EdU or BrdU on paraffin sections of organoids to obtain cleaner data?

Thank you for your feedback. The subG1 population in the original Figure S5F represents cells that died during the dissociation step of the organoids for FACS analysis. To address this point, we performed live & dead staining to exclude dead cells and provide clearer data. We refined gating strategy for better clarity in the new S5F panel.

Figure S6A; the labeling seems incorrect. I would think that red is heterozygous here, and grey mutant.

We fixed this mistake, thank you.

__Reviewer #1 (Significance (Required)): __

The finding that the poorly studied gene FAM53C controls the G1/S transition in cell lines is novel and interesting for the cell cycle field. However, the lack of phenotypes in Famc53-/- mice makes this finding less interesting for a broader audience. Furthermore, the mechanisms are incompletely dissected. The importance of a p53-indepent induction of p21 is not ruled out. And while the direct inhibitory interaction between FAM53C and DYRK1A is convincing (and also reported by others; PMID: 37802655), the authors do not (yet) convincingly show that DYRK1A inhibition can rescue a cell proliferation defect in FAM53C-deficient cells.

Altogether, this study can be of interest to basic researchers in the cell cycle field.

I am a cell biologist studying cell cycle fate decisions, and adaptation of cancer cells & stem cells to (drug-induced) stress. My technical expertise aligns well with the work presented throughout this paper, although I am not familiar with biolayer interferometry.

__Reviewer #2 (Evidence, reproducibility and clarity (Required)): __

Summary

In this study Hammond et al. investigated the role of Dual-specificity Tyrosine Phosphorylation regulated Kinase 1A (DYRK1) in G1/S transition. By exploiting Dependency Map portal, they identified a previously unexplored protein FAM53C as potential regulator of G1/S transition. Using RNAi, they confirmed that depletion of FAM53C suppressed proliferation of human RPE1 cells and that this phenotype was dependent on the presence protein RB. In addition, they noted increased level of CDKN1A transcript and p21 protein that could explain G1 arrest of FAM53C-depleted cells but surprisingly, they did not observe activation of other p53 target genes. Proteomic analysis identified DYRK1 as one of the main interactors of FAM53C and the interaction was confirmed in vitro. Further, they showed that purified FAM53C blocked the ability of DYRK1 to phosphorylate cyclin D in vitro although the activity of DYRK1 was likely not inhibited (judging from the modification of FAM53C itself). Instead, it seems more likely that FAM53C competes with cyclin D in this assay. Authors claim that the G1 arrest caused by depletion of FAM53C was rescued by inhibition of DYRK1 but this was true only in cells lacking functional p53. This is quite confusing as DYRK1 inhibition reduced the fraction of G1 cells in p53 wild type cells as well as in p53 knock-outs, suggesting that FAM53C may not be required for regulation of DYRK1 function. Instead of focusing on the impact of FAM53C on cell cycle progression, authors moved towards investigating its potential (and perhaps more complex) roles in differentiation of IPSCs into cortical organoids and in mice. They observed a lower level of proliferating cells in the organoids but if that reflects an increased activity of DYRK1 or if it is just an off target effect of the genetic manipulation remains unclear. Even less clear is the phenotype in FAM53C knock-out mice. Authors did not observe any significant changes in survival nor in organ development but they noted some behavioral differences. Weather and how these are connected to the rate of cellular proliferation was not explored. In the summary, the study identified previously unknown role of FAM53C in proliferation but failed to explain the mechanism and its physiological relevance at the level of tissues and organism. Although some of the data might be of interest, in current form the data is too preliminary to justify publication.

Major points

1. Whole study is based on one siRNA to Fam53C and its specificity was not validated. Level of the knock down was shown only in the first figure and not in the other experiments. The observed phenotypes in the cell cycle progression may be affected by variable knock-down efficiency and/or potential off target effects. We thank the Reviewer for raising this important point. First, we need to clarify that our experiments were performed with a pool of siRNAs (not one siRNA). Second, commercial antibodies against FAM53C are not of the best quality and it has been challenging to detect FAM53C using these antibodies in our hands – the results are often variable. In addition, to better address the Reviewer’s point and control for the phenotypes we have observed, we performed two additional series of experiments: first, we have confirmed G1 arrest in RPE-1 cells with individual siRNAs, providing more confidence for the specificity of this arrest (Fig. S1B); second, we have new data indicating that other cell lines arrest in G1 upon FAM53C knock-down (Fig. S1E,F and Fig. 4F).

Experiments focusing on the cell cycle progression were done in a single cell line RPE1 that showed a strong sensitivity to FAM53C depletion. In contrast, phenotypes in IPSCs and in mice were only mild suggesting that there might be large differences across various cell types in the expression and function of FAM53C. Therefore, it is important to reproduce the observations in other cell types.

As mentioned above, we have new data indicating that other cell lines arrest in G1 upon FAM53C knock-down (three cancer cell lines) (Fig. S1E,F and Fig. 4F).

Authors state that FAM53C is a direct inhibitor of DYRK1A kinase activity (Line 203), however this model is not supported by the data in Fig 4A. FAM53C seems to be a good substrate of DYRK1 even at high concentrations when phosphorylations of cyclin D is reduced. It rather suggests that DYRK1 is not inhibited by FAM53C but perhaps FAM53C competes with cyclin D. Further, authors should address if the phosphorylation of cyclin D is responsible for the observed cell cycle phenotype. Is this Cyclin D-Thr286 phosphorylation, or are there other sites involved?

We revised the text of the manuscript to include the possibility that FAM53C could act as a competitive substrate and/or an inhibitor.

We removed most of the Cyclin D phosphorylation/stability data from the revised manuscript. As the Reviewers pointed out, some of these data were statistically significant but the biological effects were small. As discussed above in our response to Reviewer #1, the analysis of Cyclin D phosphorylation and stability are complicated by the upregulation of p21 upon FAM53C knock-down, in particular because p21 can be part of Cyclin D complexes, which may affect its protein levels in cells (as was nicely showed in a previous study from the lab of Tobias Meyer – Chen et al., Mol Cell, 2013). Instead of focusing on Cyclin D levels and stability, we refocused the manuscript on RB and p53 downstream of FAM53C loss.

We note, however, that we used specific Thr286 phospho-antibodies, which have been used extensively in the field. Our data in Figure 1 with palbociclib place FAM53C upstream of Cyclin D/CDK4,6. We performed Cyclin D overexpression experiments but RPE-1 cells did not tolerate high expression of Cyclin D1 (T286A mutant) and we have not been able to conduct more ‘genetic’ studies.

At many places, information on statistical tests is missing and SDs are not shown in the plots. For instance, what statistics was used in Fig 4C? Impact of FAM53C on cyclin D phosphorylation does not seem to be significant. In the same experiment, does DYRK1 inhibitor prevent modification of cyclin D?

As discussed above, we removed some of these data and re-focused the manuscript on p53-p21 as a second pathway activated by loss of FAM53C.

Validation of SM13797 compound in terms of specificity to DYRK1 was not performed.

This is an important point. We had cited an abstract from the company (Biosplice) but we agree that providing data is critical. We have now revised the manuscript with a new analysis of the compound’s specificity using kinase assays. These data are shown in Fig. S3F-H.

A fraction of cells in G1 is a very easy readout but it does not measure progression through the G1 phase. Extension of the S phase or G2 delay would indirectly also result in reduction of the G1 fraction. Instead, authors could measure the dynamics of entry to S phase in cells released from a G1 block or from mitotic shake off.

The Reviewer made a good point. As discussed in our response to Reviewer #1, with p53-null RPE-1 cells, we found that cell numbers do not increase in these conditions where we had observed a cell cycle re-entry (Fig. 4E), which was accompanied by apoptotic cell death (Fig. S4I). Thus, cells re-enter the cell cycle but die as they progress through S-phase and G2/M. We note that inhibition of DYRK1A has been shown to decrease expression of G2/M regulators (PMID: 38839871), which may contribute to the inability of cells treated to DYRK1Ai to divide. Because our data in RPE-1 cells showed that p21 knock-down was not sufficient to allow the FAM53C knock-down cells to re-enter the cell cycle, we did not further analyze p21 in HCT-116 cells. These data indicate that G1 entry by flow cytometry will not always translate into proliferation.

Other points:

Fig. 2C, 2D, 2E graphs should begin with 0

We remade these graphs.

Fig. 5D shows that the difference in p21 levels is not significant in FAM53C-KO cells but difference is mentioned in the text.

We replaced the panel by the correct panel; we apologize for this error.

Fig. 6D comparison of datasets of extremely different sizes does not seem to be appropriate

We agree and revised the text. We hope that the Reviewer will agree with us that it is worth showing these data, which are clearly preliminary but provide evidence of a possible role for FAM53C in the brain.

Could there be alternative splicing in mice generating a partially functional protein without exon 4? Did authors confirm that the animal model does not express FAM53C?

We performed RNA sequencing of mouse embryonic fibroblasts derived from control and mutant mice. We clearly identified fewer reads in exon 4 in the knockout cells, and no other obvious change in the transcript (data not shown). However, immunoblot with mouse cells for FAM53C never worked well in our hands. We made sure to add this caveat to the revised manuscript.

__Reviewer #2 (Significance (Required)): __

Main problem of this study is that the advanced experimental models in IPSCs and mice did not confirm the observations in the cell lines and thus the whole manuscript does not hold together. Although I acknowledge the effort the authors invested in these experiments, the data do not contribute to the main conclusion of the paper that FAM53C/DYRK1 regulates G1/S transition.

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

This paper identifies FAM53C as a novel regulator of cell cycle progression, particularly at the G1/S transition, by inhibiting DYRK1A. Using data from the Cancer Dependency Map, the authors suggest that FAM53C acts upstream of the Cyclin D-CDK4/6-RB axis by inhibiting DYRK1A.

Specifically, their experiments suggest that FAM53C Knockdown induces G1 arrest in cells, reducing proliferation without triggering apoptosis. DYRK1A Inhibition rescues G1 arrest in P53KO cells, suggesting FAM53C normally suppresses DYRK1A activity. Mass Spectrometry and biochemical assays confirm that FAM53C directly interacts with and inhibits DYRK1A. FAM53C Knockout in Human Cortical Organoids and Mice leads to cell cycle defects, growth impairments, and behavioral changes, reinforcing its biological importance.

Strength of the paper:

The study introduces a novel cell cycle control signalling module upstream of CDK4/6 in G1/S regulation which could have significant impact. The identification of FAM53C using a depmap correlation analysis is a nice example of the power of this dataset. The experiments are carried out mostly in a convincing manner and support the conclusions of the manuscript.

Critique:

1) The experiments rely heavily on siRNA transfections without the appropriate controls. There are so many cases of off-target effects of siRNA in the literature, and specifically for a strong phenotype on S-phase as described here, I would expect to see solid results by additional experiments. This is especially important since the ko mice do not show any significant developmental cell cycle phenotypes. Moreover, FAM53C does not show a strong fitness effect in the depmap dataset, suggesting that it is largely non-essential in most cancer cell lines. For this paper to reach publication in a high-standard journal, I would expect that the authors show a rescue of the S-phase phenotype using an siRNA-resistant cDNA, and show similar S-phase defects using an acute knock out approach with lentiviral gRNA/Cas9 delivery.

We thank the Reviewer for this comment. Please refer to the initial response to the three Reviewers, where we discuss our use of single siRNAs and our results in multiple cell lines. Briefly, we can recapitulate the G1 arrest upon FAM53C knock-down using two independent siRNAs in RPE-1 cells. We also observe the same G1 arrest in p53 knockout cells, suggesting it is not due to a non-specific stress response. In addition, the arrest is dependent on RB, which fits with the genetic and biochemical data placing FAM53C upstream of RB, further supporting a specific phenotype. Human cancer cell lines also arrest in G1 upon FAM53C knock-down, not just RPE-1 cells. Finally, we hope the Reviewer will agree with us that compensatory mechanisms are very common in the cell cycle – which may explain the lack of phenotypes in vivo or upon long-term knockout of FAM53C.

2) The S-phase phenotype following FAM53C should be demonstrated in a larger variety of TP53WT and mutant cell lines. Given that this paper introduces a new G1/S control element, I think this is important for credibility. Ideally, this should be done with acute gRNA/Cas9 gene deletion using a lentiviral delivery system; but if the siRNA rescue experiments work and validate an on-target effect, siRNA would be an appropriate alternative.

We now show data with three cancer cell lines (U2OS, A549, and HCT-116 – Fig. S1E,F and Fig. 4F), in addition to our results in RPE-1 cells and in human cortical organoids. We note that the knock-down experiments are complemented by overexpression data (Fig. 1G-I), by genetic data (our original DepMap screen), and our biochemical data (showing direct binding of FAM53C to DYRK1A).

3) The western blot images shown in the MS appear heavily over-processed and saturated (See for example S4B, 4A, B, and E). Perhaps the authors should provide the original un-processed data of the entire gels?

For several of our panels (e.g., 4E and S4B, now panels S3J and S3K)), we used a true “immunoassay” (as indicated in the legend – not an immunoblot), which is much more quantitative and avoids error-prone steps in standard immunoblots (“Western blots”). Briefly, this system was developed by ProteinSimple. It uses capillary transfer of proteins and ELISA-like quantification with up to 6 logs of dynamic range (see their web site https://www.proteinsimple.com/wes.html). The “bands” we show are just a representation of the luminescence signals in capillaries. We made sure to further clarify the figure legends in the revised manuscript.

Data in 4A are also not a western blot but a radiograph.

For immunoblots, we will provide all the source data with uncropped blots with the final submission.

4) A critical experiment for the proposed mechanism is the rescue of the FAM53C S-phase reduction using DYRK1A inhibition shown in Figure 4. The legend here states that the data were extracted from BrdU incorporation assays, but in Figure S4D only the PI histograms are shown, and the S-phase population is not quantified. The authors should show the BrdU scatterplot and quantify the phenotype using the S-phase population in these plots. G1 measurements from PI histograms are not precise enough to allow for conclusions. Also, why are the intensities of the PI peaks so variable in these plots? Compare, for example, the HCT116 upper and lower panels where the siRNA appears to have caused an increase in ploidy.

We apologize for the confusion and we fixed these errors, for most of the analyses, we used PI to measure G1 and S-phase entry. We added relevant flow cytometry plots to supplemental figures (Fig. S1G, H, I, as well as Fig. S4E and S4K, and Fig. S5F).

5) There's an apparent contradiction in how RB deletion rescues the G1 arrest (Figure 2) while p21 seems to maintain the arrest even when DYRK1A is inhibited. Is p21 not induced when FAM53C is depleted in RB ko cells? This should be measured and discussed.

This comment and comments from the two other Reviewers made us reconsider our model. We re-read carefully the Meyer paper and think that DYRK1A activity may be understood when considering levels of both CycD and p21 at the same time in a continuum (as was nicely showed in a previous study from the lab of Tobias Meyer – Chen et al., Mol Cell, 2013). While our genetic and biochemical data support a role for FAM53C in DYRK1A inhibition, it is obvious that the regulation of cell cycle progression by FAM53C is not exclusively due to this inhibition. As discussed above and below, we noted an upregulation of p21 upon FAM53C knock-down, and activation of p53 and its targets likely contributes significantly to the phenotypes observed. We added new experiments to support this more complex model (Figure 4 and Figure S4, with new model in S4L).

__Reviewer #3 (Significance (Required)): __

In conclusion, I believe that this MS could potentially be important for the cell cycle field and also provide a new target pathway that could be relevant for cancer therapy. However, the paper has quite a few gaps and inconsistencies that need to be addressed with further experiments. My main worry is that the acute depletion phenotypes appear so strong, while the gene is non-essential in mice and shows only a minor fitness effect in the depmap screens. More convincing controls are necessary to rule out experimental artefacts that misguide the interpretation of the results.

We appreciate this comment and hope that the Reviewer will agree it is still important to share our data with the field, even if the phenotypes in mice are modest.

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Referee #3

Evidence, reproducibility and clarity

This paper identifies FAM53C as a novel regulator of cell cycle progression, particularly at the G1/S transition, by inhibiting DYRK1A. Using data from the Cancer Dependency Map, the authors suggest that FAM53C acts upstream of the Cyclin D-CDK4/6-RB axis by inhibiting DYRK1A.

Specifically, their experiments suggest that FAM53C Knockdown induces G1 arrest in cells, reducing proliferation without triggering apoptosis. DYRK1A Inhibition rescues G1 arrest in P53KO cells, suggesting FAM53C normally suppresses DYRK1A activity. Mass Spectrometry and biochemical assays confirm that FAM53C directly interacts with and inhibits DYRK1A. FAM53C Knockout in Human Cortical Organoids and Mice leads to cell cycle defects, growth impairments, and behavioral changes, reinforcing its biological importance.

Strength of the paper:

The study introduces a novel cell cycle control signalling module upstream of CDK4/6 in G1/S regulation which could have significant impact. The identification of FAM53C using a depmap correlation analysis is a nice example of the power of this dataset. The experiments are carried out mostly in a convincing manner and support the conclusions of the manuscript.

Critique:

1. The experiments rely heavily on siRNA transfections without the appropriate controls. There are so many cases of off-target effects of siRNA in the literature, and specifically for a strong phenotype on S-phase as described here, I would expect to see solid results by additional experiments. This is especially important since the ko mice do not show any significant developmental cell cycle phenotypes. Moreover, FAM53C does not show a strong fitness effect in the depmap dataset, suggesting that it is largely non-essential in most cancer cell lines. For this paper to reach publication in a high-standard journal, I would expect that the authors show a rescue of the S-phase phenotype using an siRNA-resistant cDNA, and show similar S-phase defects using an acute knock out approach with lentiviral gRNA/Cas9 delivery.
2. The S-phase phenotype following FAM53C should be demonstrated in a larger variety of TP53WT and mutant cell lines. Given that this paper introduces a new G1/S control element, I think this is important for credibility. Ideally, this should be done with acute gRNA/Cas9 gene deletion using a lentiviral delivery system; but if the siRNA rescue experiments work and validate an on-target effect, siRNA would be an appropriate alternative.
3. The western blot images shown in the MS appear heavily over-processed and saturated (See for example S4B, 4A, B, and E). Perhaps the authors should provide the original un-processed data of the entire gels?
4. A critical experiment for the proposed mechanism is the rescue of the FAM53C S-phase reduction using DYRK1A inhibition shown in Figure 4. The legend here states that the data were extracted from Brad incorporation assays, but in Figure S4D only the PI histograms are shown, and the S-phase population is not quantified. The authors should show the Brad scatterplot and quantify the phenotype using the S-phase population in these plots. G1 measurements from PI histograms are not precise enough to allow for conclusions. Also, why are the intensities of the PI peaks so variable in these plots? Compare, for example, the HCT116 upper and lower panels where the siRNA appears to have caused an increase in ploidy.
5. There's an apparent contradiction in how RB deletion rescues the G1 arrest (Figure 2) while p21 seems to maintain the arrest even when DYRK1A is inhibited. Is p21 not induced when FAM53C is depleted in RB ko cells? This should be measured and discussed.

Significance

In conclusion, I believe that this MS could potentially be important for the cell cycle field and also provide a new target pathway that could be relevant for cancer therapy. However, the paper has quite a few gaps and inconsistencies that need to be addressed with further experiments. My main worry is that the acute depletion phenotypes appear so strong, while the gene is non-essential in mice and shows only a minor fitness effect in the depmap screens. More convincing controls are necessary to rukle out experimental artefacts that misguide the interpretation of the results.

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Referee #2

Evidence, reproducibility and clarity

Summary

In this study Hammond et al. investigated the role of Dual-specificity Tyrosine Phosphorylation regulated Kinase 1A (DYRK1) in G1/S transition. By exploiting Dependency Map portal, they identified a previously unexplored protein FAM53C as potential regulator of G1/S transition. Using RNAi, they confirmed that depletion of FAM53C suppressed proliferation of human RPE1 cells and that this phenotype was dependent on the presence protein RB. In addition, they noted increased level of CDKN1A transcript and p21 protein that could explain G1 arrest of FAM53C-depleted cells but surprisingly, they did not observe activation of other p53 target genes. Proteomic analysis identified DYRK1 as one of the main interactors of FAM53C and the interaction was confirmed in vitro. Further, they showed that purified FAM53C blocked the ability of DYRK1 to phosphorylate cyclin D in vitro although the activity of DYRK1 was likely not inhibited (judging from the modification of FAM53C itself). Instead, it seems more likely that FAM53C competes with cyclin D in this assay. Authors claim that the G1 arrest caused by depletion of FAM53C was rescued by inhibition of DYRK1 but this was true only in cells lacking functional p53. This is quite confusing as DYRK1 inhibition reduced the fraction of G1 cells in p53 wild type cells as well as in p53 knock-outs, suggesting that FAM53C may not be required for regulation of DYRK1 function. Instead of focusing on the impact of FAM53C on cell cycle progression, authors moved towards investigating its potential (and perhaps more complex) roles in differentiation of IPSCs into cortical organoids and in mice. They observed a lower level of proliferating cells in the organoids but if that reflects an increased activity of DYRK1 or if it is just an off target effect of the genetic manipulation remains unclear. Even less clear is the phenotype in FAM53C knock-out mice. Authors did not observe any significant changes in survival nor in organ development but they noted some behavioral differences. Weather and how these are connected to the rate of cellular proliferation was not explored. In the summary, the study identified previously unknown role of FAM53C in proliferation but failed to explain the mechanism and its physiological relevance at the level of tissues and organism. Although some of the data might be of interest, in current form the data is too preliminary to justify publication.

Major points

1. Whole study is based on one siRNA to Fam53C and its specificity was not validated. Level of the knock down was shown only in the first figure and not in the other experiments. The observed phenotypes in the cell cycle progression may be affected by variable knock-down efficiency and/or potential off target effects.
2. Experiments focusing on the cell cycle progression were done in a single cell line RPE1 that showed a strong sensitivity to FAM53C depletion. In contrast, phenotypes in IPSCs and in mice were only mild suggesting that there might be large differences across various cell types in the expression and function of FAM53C. Therefore, it is important to reproduce the observations in other cell types.
3. Authors state that FAM53C is a direct inhibitor of DYRK1A kinase activity (Line 203), however this model is not supported by the data in Fig 4A. FAM53C seems to be a good substrate of DYRK1 even at high concentrations when phosphorylations of cyclin D is reduced. It rather suggests that DYRK1 is not inhibited by FAM53C but perhaps FAM53C competes with cyclin D. Further, authors should address if the phosphorylation of cyclin D is responsible for the observed cell cycle phenotype. Is this Cyclin D-Thr286 phosphorylation, or are there other sites involved?
4. At many places, information on statistical tests is missing and SDs are not shown in the plots. For instance, what statistics was used in Fig 4C? Impact of FAM53C on cyclin D phosphorylation does not seem to be significant. IN the same experiment, does DYRK1 inhibitor prevent modification of cyclin D?
5. Validation of SM13797 compound in terms of specificity to DYRK1 was not performed.
6. A fraction of cells in G1 is a very easy readout but it does not measure progression through the G1 phase. Extension of the S phase or G2 delay would indirectly also result in reduction of the G1 fraction. Instead, authors could measure the dynamics of entry to S phase in cells released from a G1 block or from mitotic shake off.

Other points

1. Fig. 2C, 2D, 2E graphs should begin with 0
2. Fig. 5D shows that the difference in p21 levels is not significant in FAM53C-KO cells but difference is mentioned in the text.
3. Fig. 6D comparison of datasets of extremely different sizes does not seem to be appropriate
4. Could there be alternative splicing in mice generating a partially functional protein without exon 4? Did authors confirm that the animal model does not express FAM53C?

Significance

Main problem of this study is that the advanced experimental models in IPSCs and mice did not confirm the observations in the cell lines and thus the whole manuscript does not hold together. Although I acknowledge the effort the authors invested in these experiments, the data do not contribute to the main conclusion of the paper that FAM53C/DYRK1 regulates G1/S transition.

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Referee #1

Evidence, reproducibility and clarity

Summary:

Taylar Hammond and colleagues identified new regulators of the G1/S transition of the cell cycle. They did so by screening public available data from the Cancer Dependency Map, and identified FAM53C as a positive regulator of the G1/S transition. Using biochemical assays they then show that FAM53 interacts with the DYRK1A kinase to inhibit its function. DYRK1A in its is known to induce degradation of cyclin D, leading the authors to propose a model in which DYRK1A-dependent cyclin D degradation is inhibited by FAM53C to permit S-phase entry. Finally the authors assess the effect of FAM53C deletion in a cortical organoid model, and in Fam53c knockout mice. Whereas proliferation of the organoids is indeed inhibited, mice show virtually no phenotype.

Major comments:

The authors show convincing evidence that FAM53C loss can reduce S-phase entry in cell cultures, and that it can bind to DYRK1A. However, FAM53 has multiple other binding partners and I am not entirely convinced that negative regulation of DYRK1A is the predominant mechanism to explain its effects on S-phase entry. Some of the claims that are made based on the biochemical assays, and on the physiological effects of FAM53C are overstated. IN addition, some choices made methodology and data representation need further attention.

1. The authors do note that P21 levels increase upon FAM53C. They show convincing evidence that this is not a P53-dependent response. But the claim that " p21 upregulation alone cannot explain the G1 arrest in FAM53C-deficient cells (line 138-139) is misleading. A p53-independent p21 response could still be highly relevant. The authors could test if FAM53C knockdown inhibits proliferation after p21 knockdown or p21 deletion in RPE1 cells.
2. The authors do not convincingly show that FAM53C acts a DYRK1A inhibitor in cells. Figures 4B+C and S4B+C show extremely faint P-CycD1 bands, and tiny differences in ratios. The P values are hovering around the 0.05, so n=3 is clearly underpowered here. Total CycD1 levels also correlate with FAM53C levels, which seems to affect the ratios more than the tiny pCycD1 bands. Why is there still a pCycD1 band visible in 4B in the GFP + BTZ + DYRK1Ai condition? And if I look at the data points I honestly don't understand how the authors can conclude from S4C that knockdown of siFAM53C increases (DYRK1A dependent) increases in pCycD1 (relative to total CycD1). In figure 5C, no blot scans are even shown, and again the differences look tiny. So the authors should either find a way to make these assays more robust, or alter their claims appropriately.
3. The experiments to test if DYRK1A inhibition could rescue the G1 arrest observed upon FAM53C knockdown are not entirely convincing either. It would be much more convincing if they also perform cell counting experiments as they have done in Figures 1F and 1G, to complement the flow cytometry assays. I suggest that the authors do these cell counting experiments in RPE1 +/- P53 cells as well as HCT116 cells. In addition, did the authors test if P21 is induced by DYRK1Ai in HCT116 cells?
4. The data in Figure 5C and 5D are identical, although they are supposed to represent either pCycD1 ratios or p21 levels. This is a problem because at least one of the two cannot be true. Please provide the proper data and show (representative) images of both data types.
5. Line 246: "Fam53c knockout mice display developmental and behavioral defects." I don't agree with this claim. The mutant mice are born at almost the expected Mendelian ratios, the body weight development is not consistently altered. But more importantly, no differences in adult survival or microscopic pathology were seen. The authors put strong emphasis on the IMPC behavioral analysis, but they should be more cautious. The IMPC mouse cohorts are tested for many other phenotypes related to behavior and neurological symptoms and apparently none of these other traits were changed in the IMPC Famc53c-/- cohort. Thus, the decreased exploration in a new environment could very well be a chance finding. The authors need to take away claims about developmental and behavioral defects from the abstract, results and discussion sections; the data are just too weak to justify this.

Minor comments:

1. Can the authors provide a rationale for each of the proteins they chose to generate the list of the 38 proteins in the DepMap analysis? I looked at the list and it seems to me that they do not all have described functions in the G1/S transition. The analysis may thus be biased.
2. Figure 1B is confusing to me. Are these just some (arbitrarily) chosen examples? Consider leaving this heatmap out altogether, of explain in more detail.
3. The y-axes in Figures 2C, 2D, 2E, and 4D are misleading because they do not start at 0. Please let the axis start at 0, or make axis breaks.
4. Line 229: " Consequences ... brain development." This subheader is misleading, because the in vitro cortical organoid system is a rather simplistic model for brain development, and far away from physiological brain development. Please alter the header.
5. Figure S5F: the gating strategy is not clear to me. In particular, how do the authors know the difference between subG1 and G1 DAPI signals? Do they interpret the subG1 as apoptotic cells? If yes, why are there so many? Are the culturing or harvesting conditions of these organoids suboptimal? Perhaps the authors could consider doing IF stainings on EdU or BrdU on paraffin sections of organoids to obtain cleaner data?
6. Figure S6A; the labeling seems incorrect. I would think that red is heterozygous here, and grey mutant.

Significance

The finding that the poorly studied gene FAM53C controls the G1/S transition in cell lines is novel and interesting for the cell cycle field. However, the lack of phenotypes in Famc53-/- mice makes this finding less interesting for a broader audience. Furthermore, the mechanisms are incompletely dissected. The importance of a p53-indepent induction of p21 is not ruled out. And while the direct inhibitory interaction between FAM53C and DYRK1A is convincing (and also reported by others; PMID: 37802655), the authors do not (yet) convincingly show that DYRK1A inhibition can rescue a cell proliferation defect in FAM53C-deficient cells.

Altogether, this study can be of interest to basic researchers in the cell cycle field.

I am a cell biologist studying cell cycle fate decisions, and adaptation of cancer cells & stem cells to (drug-induced) stress. My technical expertise aligns well with the work presented throughout this paper, although I am not familiar with biolayer interferometry.

Sweet spot

Goldilocks

Sweet spot not sweat spot

Never mind the bad consewuences

And missing out on empowering people that could create exponentially more value at scale

Sharinh

X

Spell out better

X

Men behind the machines

Not the other way round primmarily

InterExtellect

intent

Commitment to work on its articulation explicit formulation leading to its emergent coevolutiinary through co-lab-oration to its Realization in the medium of the working software

Structure and INTERPERSONAL Auto-poietic/nomous co-lab-oration

Riffing on the annotation margins

Say the what you feel and mean what you say

Don't prepare the words prepare the feelings

Transmute the caterpillars of verbal narrative trails flights insights as butterflies in flight

General comment. We need to guide/limit the number of H2s in a lesson. These are all H2s, and there are 6, which is a lot for one lesson.

Simplify titles when possible. Ive been seeing some very long and verbose titles.

Let's talk about closing thoughts, summaries, etc. and ways to close lessons. Do we need a closing thought? AI does this, but it rarely feels valuable.

This feels fluffy, but maybe it's because of the "It's not just X, it's X." structure.

eLife Assessment

This work reveals metabolic pathways and molecular events mechanistically linked to B cell activation. Using an unbiased, comprehensive proteome profiling method and various functional validation approaches, this study generated convincing evidence suggesting a role for amino acid uptake, cholesterol accumulation, and protein prenylation in the proliferation, survival, and biogenesis of B cells stimulated with LPS and other activating stimuli. The significance of the findings is considered to be fundamental, in that they will advance our understanding of cell metabolism during B cell activation.

Reviewer #1 (Public review):

The work presented by Cheung et al. used a quantitative proteomics method to capture molecular changes in B cells exposed to LPS and IL-4, a combination of stimuli activating naive B cells. Amino acid transporters, cholesterol biosynthetic enzymes, ribosomal components, and other proteins involved in cell proliferation were found to increase in stimulated B cells. Experiments involving genetic loss-of-function (SLC7A5), pharmacological inhibition (HMGCR, SQLE, prenylation), and functional rescue by metabolites (mevalonate, GGPP) validated the proteomics data and revealed that amino acid uptake, cholesterol/mevalonate biosynthesis, and cholesterol uptake played a crucial role in B cell proliferation, survival, biogenesis, and immunoglobulin class switching. Experiments involving cholesterol-free medium showed that both biosynthesis and LDLR-mediated uptake catered to the cholesterol demand of LPS/IL-4-stimulated B cells. A role for protein prenylation in LDLR-mediated cholesterol uptake was postulated and backed by divergent effects of GGPP rescue in the presence and absence of cholesterol in culture medium.

Strengths:

The discovery was made by proteome-wide profiling and unbiased computational analysis. The discovered proteins were functionally validated using appropriate tools and approaches. The metabolic processes identified and prioritized from this comprehensive survey and systematic validation are highly likely to represent mechanisms of high importance and influence. Analysis of immune cell metabolism at the protein level is relatively compared to transcriptomic and metabolomic analysis.

The conclusions from functional validation experiments were supported by clear data and based on rational interpretations. This was enabled by well-established readouts/analytical methods used to analyze cell proliferation, viability, size, cholesterol content, and transporter/enzyme function. The data generated from these experiments strongly support the conclusions.

This work reveals a complex, yet intriguing, relationship between cholesterol metabolism and protein prenylation as they serve to promote B cell activation. The effects of pharmacological inhibition and metabolite replenishment on the cholesterol content and activation of B cells were precisely determined and logically interpreted.

Weaknesses:

The findings of this study were obtained almost exclusively from ex vivo B cell stimulation experiments. Their contribution to B cell state and B-cell-mediated immune responses in vivo was not explored. Without in vivo data, the study still provides valuable mechanistic information and insights, but it remains unknown, and there is no discussion about how the identified mechanisms may play out in B cell immunity.

The role of HMGCR, SQLE, and prenylation in B cell activation was assessed using pharmacological inhibitors. Evidence from other loss-of-function approaches, which could strengthen the conclusions, does not exist. This is a moderate weakness.

Reviewer #2 (Public review):

This study uses mass spectrometry to quantify how LPS and IL-4 modify the mouse B cell proteome as naïve cells undergo blastogenesis and enter the cell cycle. This analysis revealed changes in key proteins involved in amino acid transport and cholesterol biosynthesis. Genetic and pharmacological experiments indicated important roles for these metabolic processes in B cell proliferation.

This work provides new information about the regulation of TI B cell responses by changes in cell metabolism and also a comprehensive mass spectrometry dataset, which will be an important general resource for future studies. The experiments are thorough and carefully carried out. The majority of conclusions are backed up by data that is shown to be highly significant statistically.

The study would be strengthened by additional experiments to determine whether the detected changes are unique to stimulation with LPS + IL-4 or more generic responses of resting B cells to mitogenic agonists.

Author response:

Reviewer #1:

We agree with the reviewer that a limitation of our study is its focus on cell-based assays rather than in vivo experiments. We did consider evaluating the effects of statins on B cell responses in vivo; however, this approach is complicated by findings that statins can influence antigen presentation by dendritic cells, thereby impacting antibody responses (Xia et al, 2018). One possible solution would be to use B cell-specific conditional knockout models to study the roles of the identified proteins in an in vivo context. However, we currently do not have access to these models and were therefore unable to include such experiments within a feasible timeframe. We will revise the discussion section to acknowledge these points.

The reviewer also noted that our study assessed the roles of HMGCR, SQLE, and prenylation in B cell activation using pharmacological inhibitors and genetic knockdown/out approaches. Loss-of-function techniques such as RNAi, siRNA, and CRISPR can be challenging to apply to primary B cells, but we are exploring their feasibility for future revisions. While we acknowledge the limitations of using pharmacological inhibitors, we have taken several steps to mitigate these, including targeting multiple steps in the cholesterol biosynthetic pathway using structurally distinct inhibitors and conducting rescue experiments by supplementing downstream metabolites. To further investigate potential off-target effects of statins, we have recently performed proteomic analysis of B cells treated with and without fluvastatin. The data suggest that fluvastatin primarily affects cholesterol metabolism and does not cause widespread off-target effects. We will include this new data in the revised manuscript.

Reviewer #2:

The reviewer suggested that the study would be strengthened by determining whether the observed changes are specific to LPS + IL-4 stimulation or represent a more general B cell response to mitogenic signals.

A complementary study by James et al. (James et al, 2024) investigated murine B cells stimulated via the B cell receptor (BCR) and CD40, using anti-IgM and anti-CD40 antibodies alongside IL-4. Their proteomic analysis showed that such co-stimulation induces a fivefold increase in total cellular protein mass within 24 hours, mirroring our findings with LPS + IL-4. They also reported upregulation of proteins associated with cell cycle progression, ribosome biogenesis, and amino acid transport. Furthermore, by using SLC7A5 knockout mice, they demonstrated that this transporter is required for B cell activation. We will expand our discussion to include and these findings.  We will also expand on the final figure in our paper showing that the effects of statins are not limited to LPS.

References:

James O, Sinclair LV, Lefter N, Salerno F, Brenes A & Howden AJM (2024) A proteomic map of B cell activation and its shaping by mTORC1, MYC and iron. bioRxiv 2024.12.19.629506 doi:10.1101/2024.12.19.629506 [PREPRINT]

Xia Y, Xie Y, Yu Z, Xiao H, Jiang G, Zhou X, Yang Y, Li X, Zhao M, Li L, et al (2018) The Mevalonate Pathway Is a Druggable Target for Vaccine Adjuvant Discovery. Cell 175: 1059-1073.e21

• Enhanced Productivity
• Enhanced Reputation
• Economic benefits
• No loss time
• Safe workplace
• Competent personnels

• Enhanced Productivity
• Enhanced Reputation
• Economic benefits
• No loss time
• Safe workplace
• Competent personnels

It is machinery, equipment, materials, facilities and activities in the workplace

Occupation health and safety is a fundamental topic in every productive organization as health and safety are not negotiable in any activity. It aims to adapt work to people such that the working conditions does not negatively impact the physical, mental or social well being of workers.

In the context of occupational health and safety is the protection of the mind and body of workers from illness resulting fom materials, processes and procedures in the workplace.

The Marriage of Medieval Metaphysics and Modern Personalism

Word on Fire Institute 174K subscribers

reply to u/warriorkitten18 at https://reddit.com/r/adhdwomen/comments/1mqzm12/index_card_system_please_discuss/

Apologies for the delay in reply. I've read extensively about card indexes for productivity and the variety of systems and uses over the past couple of centuries.

Given your context, I'd recommend reading one book which describes an index card system in full, walks you through it card by card, helps you make it, and describes how it's used. It's thorough, but fully adaptable to your particular needs. Best, it's written by two women in the early 1980s and though there wasn't much in the culture about ADHD at the time, I suspect that one or both of these women were coping with nearby neurodivergent issues (not to mention the eternal brain fog forced by pregnancy, lack of sleep, and early childhood woes.)

Young, Pam, and Peggy Jones. 1981. Sidetracked Home Executives: From Pigpen to Paradise. ed. Sydney Craft Rozen. New York: Warner Books. http://archive.org/details/sidetrackedhomee00youn (November 3, 2023).

If you need other perspectives, there are also areas with potential solutions like the Bullet Journal (notebooks), Getting Things Done (GTD), Hipster PDA, and a variety of others. Almost all of these were built on the ideas behind the early 1900s version of the Memindex, which I've written about here: https://boffosocko.com/2023/03/09/the-memindex-method-an-early-precursor-of-the-memex-hipster-pda-43-folders-gtd-basb-and-bullet-journal-systems/. They're all roughly the same in shape, practice, and philosophy, but the Young/Jones SHE version uses index cards, speaks directly to your use case, and suggests an approach for the ADHD set. Assuredly a nearby library can get you a copy, you can find them used, or read the linked online version which you can check out.

If you'd like to see portions of my personal system, I've written a bit about it along with lots of other resources at https://boffosocko.com/research/zettelkasten-commonplace-books-and-note-taking-collection/#Productivity

It's worked well for me for many years. The secret is to read the basics and then adapt the pieces of the system to suit your own needs and methods of working. For example, I love crossing things off of lists, which my index cards didn't really encourage because you do them and move them to the next day/week/month/year forward, but I bought a rotating date stamp that allows me to stamp each card with the date as "done" before re-filing it. For me, the haptic feedback of the "thonk" of the stamp is even better than crossing things off and gives me a sense of accomplishment when I see them and finish filling an entire card up with dates.

A decade on, the best part of my collection are the separate index cards I had laying around while using the rest of the system and on which I wrote down quotes from my daughter, new words as she learned them, words she made up, goofy jokes, etc.

Enough for now, the card for the dog groomer came up yesterday, so I'm off to take our dog to the appointment I made when I saw it. Thonk! Refiled for next month's reminder.

Good luck.