- Oct 2024
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experiments.myhub.ai experiments.myhub.ai
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So while each cozyweb can be a village — “small, most people know each other and you all share a common interest in keeping the sidewalks tidy” — each can build federated roads to many others, including bigger ones “crowded with people, plenty of them sleazy and more than the occasional sidewalk madman… But you’ll always discover something or someone new there. Every second person’s selling something, but one in 20 is selling what you need. Besides, you’re selling too…” (Welcome to the Fediverse, starry-eyed noob).
How does this relate to the Fediverse?
There's a nascent movement within the Fediverse precisely to experiment with 'insularity', building smaller opt-in-federation-based networks.
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Annotators
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academic.oup.com academic.oup.comEmpires2
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As a sop to appease their Spanish allies, the French gave them New Orleans (page 112)p. 112and most of Louisiana
-granted new orleans and looudiana to spain to satisfy spanish allies -wanted to please spain to get their help to fight against the brirish
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Pitt’s policy was financially reckless: by compounding the national debt, Pitt saddled the colonists and Britons with a burden that would later disrupt the empire.
-they were irresponsible and led to national debt -contributed to tensions with the british empire
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www.mdpi.com www.mdpi.com
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“I was taught that the people who hurt you, violently hurt you, every day, for years, are doing it because they love you”.
WHAT!!
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“a baby is a ‘viper in [a] diaper, a ‘depraved’, ‘diseased’ beast” (p. 617). The effect of being raised in this type of environment is evident in my own experience. I remember sermons about the indisputable proof of total depravity being children, who are bad in all natural inclinations and behaviors.
This makes me so sad and angry, not only are these children being brought into environments that preach inherent badness, but the people who are supposed to raise and care for them have had it engrained in them that they are inherent sinners, liars, etc. This fosters a sense of distrust and negative perception of children in adults, including their parents. Both the home and community are infiltrated by the idea that children were born depraved and will act accordingly, giving children no way to separate themselves from that opinion. Because of this, it becomes absorbed into their self-perception.
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“Recent research on shame and trauma has more explicitly linked the two, locating shame at the core of symptomatology of Post-Traumatic Stress Disorder”
Is this true across all cases of PTSD? Has investigation into veterans with PTSD revealed the same root symptom? I have doubts about this, and it's caused a little bit of doubt about the reliability of this source.
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In large measure this is because Christian tradition has not distinguished between guilt and shame, even sometimes using the terms synonymously.
I think even if Christianity were to advocate for a sense of self formed by guilt, this would still result in an equivalently damaging impact. However, it seems that these researchers disagree. Why do they disagree? If shame and guilt are so often intertwined and mistaken for each other in religion (and outside of it), how are the impacts different?
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shame occurs in reflexive bodily processes before language develops; this means it functions in a child’s body before reflective thought about the physiological responses are possible.
The idea that small children are experiencing possibly repeated bodily feelings of shame and are unable to verbalize or truly process that negative experience through communication or thought makes me incredibly sad.
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it may shape a relationship with religion which is opposite to the generally stated goals of spiritual and religious practices. Instead of religious or spiritual practices deepening and developing personal truthfulness,
If I were to write my essay on this topic, I would need to be very mindful of my biases toward this topic and approach it very objectively. I am aware of my biases coming through here and the opinions I have formed impacting my ability to believe the goals of religious practice as stated here.
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At the same time, they often also employ “spiritual practices and beliefs to ‘transcend’ or deny problems rather than understand them”
I'm not sure I understand this fully. I need to do more research on spiritual bypassing.
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As trauma, the harm is not resolved simply by leaving a religious group or avoiding the religious tradition because trauma shapes bodies into a “fight/flight/freeze response”
I think, especially if someone was exposed to such thought processes in their formative years, it becomes almost an instinct to question, doubt, and perceive actions/thoughts as shameful or "wrong," which, even after leaving the practice, still impacts an individual especially when gone untreated/managed.
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One of these messages is that emotions exist in a binary of good and bad, acceptable and unacceptable. In such contexts, bad emotions cannot be acknowledged or managed; they can only be condemned.
This brings me back to my statement about spiritual harm and the possibility that environments in which harm is done may return to being a safe or comfortable space because there is space granted to talking about it to either troubleshoot or soothe/protect an individual. In religious spaces, this is often not the case and may even cause more shaming to take place.
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Spiritual harm may occur in any social context, from a workplace to a hospital to a community center.
True, however, is this type of harm as widely accepted as religious teachings which may result in harm, and is support more accessible within that space which allows the space to feel safe enough to still engage in?
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the modifier religious is less definitionally fraught than the term spiritual, though contestation and overlap is inescapable.
The concept and definition we have so far of "spiritual harm," I would argue, is integral in understanding how religious trauma comes about from the complex and chronic repeat exposure to religious teachings and because we are a spiritual being with psyche vulnerable to harm on a level other than physical.
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“Religious trauma is more prevalent than the research suggests and often is a contributing factor to many of the problems that bring people to therapy, including depression, anxiety, and relationship difficulties. For this reason, religious trauma deserves careful attention”
Do we need to approach treating mental illness in those with religious trauma any differently than those with mental health problems from a different psychological cause? What treatment is shown to be the most effective, CBT, DBT, or other therapies?
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Scholarship investigating how religious teachings and practices may traumatize remains scant. Though the scholarship is sparse, religiously traumatizing experiences are not.
The limited research may be because, historically, this behavior and type of teaching were accepted, and only recently have people started to vocalize the negative aspects of participating in a religion so widely accepted and touted as "the right way."
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In this approach, Christian religious trauma is not an added element to traumas of domestic, physical, or sexual abuse by a religious person or leader. Instead, the source of the trauma is formative experience of participating in Christianity.
I wonder how shame and fear are made more pernicious with an added element of physical and sexual abuse by church leaders. However, it is important to understand how Christianity creates an environment fostering shameful feelings at a very basic level without those added elements.
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academic.oup.com academic.oup.com
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involve no loss
Except the loss incurred by the owner, who has had their rights as the owner abrogated by the trespasser - whilst more of an abstract loss it is a loss nonetheless
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At least in the sense that the defendant is deemed properly answerable to a plaintiff, the defendant is deemed legally responsible for having injured the plaintiff.
So where the defendant is legally responsible for having caused injury to the plaintiff, corrective justice insists that the defendant is answerable to the plaintiff for this result
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unsophisticated Africans.
But they are more sophisticated than the "captain"
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hearse-like roll of the hull.
Its giving ghost ship
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Whether the ship had a figure-head
No captain spells a very bad time
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and, at the least, pilot her in
as in the ship?
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apors partly mantling the hull
From where?
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observing that, the ship, in navigating into the harbor, was drawing too near the land; a sunken reef making out off her bow.
Bad at steering
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the stranger, viewed through the glass, showed no colors
What?
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the former seemed as docile as the latter the contrary? The whites, too, by nature, were the shrewder race. A man with some evil design
Is Herman Melville racist?"
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The alleged Don Benito was in early manhood, about twenty-nine or thirty
rich kid went on an adventure and got into some trouble
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a black man’s slave was Babo, who now is the white’s.
Explains why Babo is so content to be a servant
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Suddenly, one of the black boys, enraged at a word dropped by one of his white companions, seized a knife, and, though called to forbear by one of the oakum-pickers, struck the lad over the head, inflicting a gash from which blood flowed.
that took a turn
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the black upholding the white
Like the early americas
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What the San Dominick wanted
What the ship wanted? Melville has taken personification and really ran with it
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involuntary victim of mental disorder
suffering has made the captain mute
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peculiar natures on whom prolonged physical suffering seems to cancel every social instinct of kindness;
the people ahve kind of turned to anarchy
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Babo.
Is Babo the chained up guy?
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greater energy, misrule would hardly have come to the present pass
loss of supplies=loss of power
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with the peculiar love in negroes of uniting industry with pastime
I think every slave ever would disagree
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the raw aspect of unsophisticated Africans.
woah... racism just jumped a few notches there
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with a sort of stoical self-content, were picking the junk into oakum
religious ritual from the POV of an outsider maybe?
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The whites, too, by nature, were the shrewder race
more racism
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Left to himself, the American, to while away the time till his boat should arrive, would have pleasantly accosted some one of the few Spanish seamen he saw; but recalling something that Don Benito had said touching their ill conduct, he refrained; as a shipmaster indisposed to countenance cowardice or unfaithfulness in seamen.
I want to analyze again how this characterizes Americans
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Ah, ah–if, now, that was, indeed, a secret sign I saw passing between this suspicious fellow and his captain awhile since; if I could only be certain that, in my uneasiness, my senses did not deceive me, then–
The quick pacing makes you almost feel his heart rate accelerating!
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like a doe in the shade of a woodland rock. Sprawling at her lapped breasts, was her wide-awake fawn, stark naked, its black little body half lifted from the deck, crosswise with its dam’s; its hands, like two paws, clambering upon her; its mouth and nose ineffectually rooting to get at the mark; and meantime giving a vexatious half-grunt, blending with the composed snore of the negress.
i hate how hes comparing people to animals.
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Upon this, the servant looked up with a good-natured grin, but the master started as from a venomous bite.
OOP
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Captain Delano thought he observed a lurking significance in it, as if silent signs, of some Freemason sort, had that instant been interchanged.
Ominous! Also the Freemason reference is fascinating
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the silky paw to his fangs
Love the spooky imagery!
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The spars, ropes, and great part of the bulwarks, looked woolly, from long unacquaintance with the scraper, tar, and the brush. Her keel seemed laid, her ribs put together
the boats hasn't been maintained properly the "woolly" look is barnacles and peeling paint
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Deploring this supposed misconception, yet despairing of correcting it, Captain Delano shifted the subject; but finding his companion more than ever withdrawn, as if still sourly digesting the lees of the presumed affront above-mentioned, by-and-by Captain Delano likewise became less talkative, oppressed, against his own will, by what seemed the secret vindictiveness of the morbidly sensitive Spaniard. But the good sailor, himself of a quite contrary disposition, refrained, on his part, alike from the appearance as from the feeling of resentment, and if silent, was only so from contagion.
Hmm there's a lot of characterization to unpack here
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The black was silent.
This line is powerful
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the good captain put several baskets of the fish, for presents
Christmas at his house must suck
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An iron collar was about his neck, from which depended a chain, thrice wound round his body; the terminating links padlocked together at a broad band of iron, his girdle.
Uh oh
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“Yes.” “But died of the fever?” “Died of the fever. Oh, could I but–” Again quivering, the Spaniard paused.
This feels off putting
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showed no colors
The stranger hid their intentions?
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had he not been a person of a singularly undistrustful good-nature, not liable, except on extraordinary and repeated incentives, and hardly then, to indulge in personal alarms, any way involving the imputation of malign evil in man.
I think this means he is ignorant of, and not experienced in the "evil" ways of man.
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Is it, thought Captain Delano, that this hapless man is one of those paper captains I’ve known, who by policy wink at what by power they cannot put down? I know no sadder sight than a commander who has little of command but the name.
Tossing this back to "what is the author saying about being American" because I think you could analyze this in that lens
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Suddenly, one of the black boys, enraged at a word dropped by one of his white companions, seized a knife, and, though called to forbear by one of the oakum-pickers, struck the lad over the head, inflicting a gash from which blood flowed.
Uhh this reminds me of that racist stereotype that black people are violent. Yikes
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When Don Benito returned, the American was pained to observe that his hopefulness, like the sudden kindling in his cheek, was but febrile and transient.
What is he trying to say about being American here?
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Such generosity was not without its effect, even upon the invalid.
Bro leave disabled people alone. And stop being racist. And-
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However unsuitable for the time and place, at least in the blunt-thinking American’s eyes
Again - what is the author trying to say about being American here?
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As master and man stood before him, the black upholding the white, Captain Delano could not but bethink him of the beauty of that relationship which could present such a spectacle of fidelity on the one hand and confidence on the other. The scene was heightened by, the contrast in dress, denoting their relative positions.
Interestingly symbolic...
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a privileged spot
Maybe the author should think more about privilege lol
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Would Don Benito favor him with the whole story.
Style of storytelling reminds me again of Frankenstein
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what the emigrant ship has,
Again, slave ship, no need to "pretty it up" by calling it an emigrant ship
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The San Dominick was in the condition of a transatlantic emigrant ship,
A slave ship you mean
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But this the American in charity ascribed to the harassing effects of sickness, since, in former instances, he had noted that there are peculiar natures on whom prolonged physical suffering seems to cancel every social instinct of kindness; as if, forced to black bread themselves, they deemed it but equity that each person coming nigh them should, indirectly, by some slight or affront, be made to partake of their fare.
Again, what is the author saying about being American here?
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a black man’s slave was Babo, who now is the white’s.
man poor babo
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Marking the noisy indocility of the blacks in general
More racism...ugh
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A prey to settled dejection, as if long mocked with hope he would not now indulge it, even when it had ceased to be a mock, the prospect of that day, or evening at furthest, lying at anchor, with plenty of water for his people, and a brother captain to counsel and befriend, seemed in no perceptible degree to encourage him. His mind appeared unstrung, if not still more seriously affected.
Sounds like depression
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Struggling through the throng, the American advanced to the Spaniard, assuring him of his sympathies, and offering to render whatever assistance might be in his power.
What is the author trying to say about American mentality/culture/demeanor?
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But as if not unwilling to let nature make known her own case among his suffering charge, or else in despair of restraining it for the time, the Spanish captain, a gentlemanly, reserved-looking, and rather young man to a stranger’s eye, dressed with singular richness, but bearing plain traces of recent sleepless cares and disquietudes, stood passively by, leaning against the main-mast, at one moment casting a dreary, spiritless look upon his excited people, at the next an unhappy glance toward his visitor. By his side stood a black of small stature, in whose rude face, as occasionally, like a shepherd’s dog, he mutely turned it up into the Spaniard’s, sorrow and affection were equally blended.
...correct me if I'm wrong, but is the captain here the only white person? If so, ughh
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barbarous din. All six, unlike the generality, had the raw aspect of unsophisticated Africans.
RACISM
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with the peculiar love in negroes of uniting industry with pastime
This feels racist
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They accompanied the task with a continuous, low, monotonous, chant; droning and drilling away like so many gray-headed bag-pipers playing a funeral march.
Ghosts!
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who, in venerable contrast to the tumult below them, were couched, sphynx-like, one on the starboard cat-head, another on the larboard, and the remaining pair face to face on the opposite bulwarks above the main-chains.
In-human, creepy description of black people...yikes
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Marking the noisy indocility of the blacks in general,
more racism
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, had swept off a great part of their number, more especially the Spaniards.
As far as I know, I don't think Spanish people are particularly susceptible to scurvy?? What is the author trying to say with this?
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hearse-like roll of the hull.
Yay death! Yay ghost ship!
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f Castile and Leon, medallioned about by groups of mythological or symbolical devices; uppermost and central of which was a dark satyr in a mask, holding his foot on the prostrate neck of a writhing figure, likewise masked.
Tell me you're scared of paganism without telling me you're scared of paganism
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All six, unlike the generality, had the raw aspect of unsophisticated Africans.
racism
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As the whale-boat drew more and more nigh, the cause of the peculiar pipe-clayed aspect of the stranger was seen in the slovenly neglect pervading her. The spars, ropes, and great part of the bulwarks, looked woolly, from long unacquaintance with the scraper, tar, and the brush. Her keel seemed laid, her ribs put together, and she launched, from Ezekiel’s Valley of Dry Bones.
Personification of the ship - genuinely if I heard this out of context and ship-specific stuff wasn't mentioned, I would've just thought of a woman. It's either really pervading personification or I'm just too sleepy lol
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with the peculiar love in negroes of uniting industry with pastime
this line feels really questionable considering how similar it is to things that have been said to justify slavery
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nd the true character of the vessel was plain–a Spanish merchantman of the first class, carrying negro slaves, amongst other valuable freight, from one colonial port to another.
YIKES...
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Peering over the bulwarks were what really seemed, in the hazy distance, throngs of dark cowls; while, fitfully revealed through the open port-holes, other dark moving figures were dimly descried, as of Black Friars pacing the cloisters.
Ghost ship?
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_saya-y-manta._
Underscores? Also thanks to the person who commented what this is!
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more especially the Spaniards
the spanish once again being brought up in relation to the slave trade
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To Captain Delano’s surprise, the stranger, viewed through the glass, showed no colors;
Why am I reminded of the Creature's appearance on the ship in Frankenstein?
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The morning was one peculiar to that coast. Everything was mute and calm; everything gray
What did I just say about eerie lol?
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On the second day, not long after dawn, while lying in his berth, his mate came below, informing him that a strange sail was coming into the bay. Ships were then not so plenty in those waters as now. He rose, dressed, and went on deck.
I feel like there's an interesting, eerie tone in this.
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the true character of the vessel was plain–a Spanish merchantman of the first class, carrying negro slaves, amongst other valuable freight, from one colonial port to another.
oh...
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as of Black Friars pacing the cloisters
Religious simile!
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singularly undistrustful good-nature, not liable, except on extraordinary and repeated incentives, and hardly then, to indulge in personal alarms, any way involving the imputation of malign evil in man.
character is an unbelievably good person apparently
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with the shreds of fog here and there raggedly furring her, appeared like a white-washed monastery after a thunder-storm, seen perched upon some dun cliff among the Pyrenees
Very descriptive language.
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vapors partly mantling the hull
Super mysterious writing, lots of imagery.
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www.ecologyandsociety.org www.ecologyandsociety.org
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for - planetary adaptive cycle - entering back-loop phase - paper - From Complex Regions to Complex Worlds - Crawford Stanley Holling - 2004 - from - essay - The End of Scarcity? From ‘Polycrisis’ to Planetary Phase Shift - Nafeez Ahmed - 2024
from - essay - The End of Scarcity? From ‘Polycrisis’ to Planetary Phase Shift - Nafeez Ahmed - 2024 - https://hyp.is/okOeDJFqEe-9ZsMEsKWR9w/ageoftransformation.org/the-end-of-scarcity-from-polycrisis-to-planetary-phase-shift/
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academic.oup.com academic.oup.com
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The line of resurrection and heaven is not clear ( the moment when you go to heaven )
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Most of the heresies come from a logical breakdown of a. paradox
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Latin A describes how the crucification was a plat by satin that went wrong
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We are troubled by the description of the cross and crucification
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en.wikipedia.org en.wikipedia.org
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https://tana.inc/docs/supertags
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ageoftransformation.org ageoftransformation.org
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panarchy
for - definition - panarchy - Crawford Stanley Holling
definition - panarchy - Crawford Stanley Holling - A nested diversity of living species entwined through their adaptive cycles of growth, decline and renewal
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adaptive cycle
for - definition - adaptive cycle - Crawford Stanley Holling - IIASA
definition - adaptive cycle - Crawford Stanley Holling - IIASA - Predator-prey dynamics across a large variety of species, follow a recursive 'adaptive cycle' consisting of: - front loop stage - growth and accumulation - back loop stage - rapid reorganization with increased stability due to dependency on a limited number of conditions leading to reduced resiliency and either - renewal or - collapse <br /> - This is a characteristic of an ecosystem of many species coexisting together
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explaining the phase transition from the feudal to the industrial age
for - cultural phase transition - from feudalism to industrial age
cultural phase transition - from feudalism to industrial age - involved the interplay of a number of factors - cultural exchange of ideas between European and other cultures such as Islam and ancient Greek - colonialism and the Atlantic slave trade - new technologies such as steam engine rendered slaves obsolete by replacing them with more efficient factory systems of production - new human rights movements coincided to abolish slavery
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Arbib and Seba explain this by categorising human civilisation into two fundamentally intertwined complexes: the production system, encompassing all the foundational systems by which we meet fundamental material needs across energy, transport, food and materials (corresponding to ‘hardware’); and the organising system, encompassing how the former systems are governed, regulated and managed by society through economic, political, military, cultural and ideological structures and values (corresponding to ‘software’)
for - definition - production system ('hardware') - and organizing system ('software') - Arbib and Seba
definition Arbib and Seba - human civilization can be broken down into the interaction between two complimentary systems - the production system - by which we meet fundamamental material needs for food, energy, transportation, water, materials - also called 'hardware' - the organizing system - by which how the production system is governed and managed and includes the economy, polity, security, culture, ideology and values - also called 'software'
comment - A transformation is required in both the hardware and the software to mitigate the worst impacts of our current polycrisis
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www.fastcompany.com www.fastcompany.com
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arbinger.com arbinger.com
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Carla Debow from CenturyLink talks how transformational it is to truly “understand the person on the other side of the table” here:
Please watch this video.
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outward mindset is focused on others and their needs.
And using this information and insight to help pursue the goals of the organization and the greater good.
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then our relationships will continue to be strained and the results we accomplish together much less than they could be.
Does anyone come to mind for you when you read this paragraph?
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An inward mindset is a way of seeing ourselves and others that keeps us focused on our own needs, wants, and goals, often at the expense of others. When we live and work with an inward mindset we see people as objects rather than as people–as vehicles to be used, irrelevancies to be ignored, or as obstacles to be overcome, rather than as individuals with their own thoughts, feelings, and perspectives. Inward mindset tendencies can show up in various ways such as blaming others, justifying our own mistreatment of others, or avoiding responsibility.
With the way this is phrased it seems obvious that we should have an inward mindset and we may initially balk at the idea that we might be working from an outward mindset. However, this can show up in subtle ways. For example, much of our job is trying to get people to change old habits for new ones. We often lead with processes, rules, and exactly WHAT we want people to do without taking into account why they do what they do and WHY they shoud change.
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Outward mindset examples Now that you know what an outward mindset is, how it impacts employees and organizations, and what benefits it offers, you’re probably wondering what it looks like in practice. Take a look at the impact transforming to an outward mindset has had on these businesses: Raytheon leveraged Arbinger’s outward mindset principles to cut $100M in expenses without laying off a single employee. See how:
The following videos are optional: * Raytheon (video 1) * Tubular Steel (video 2)
This video is recommended: * Kansas City Police Department
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www.biorxiv.org www.biorxiv.org
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Version 2 of this preprint has been peer-reviewed and recommended by Peer Community in Genomics.<br /> See the peer reviews and the recommendation.
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www.cos.io www.cos.io
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Publish-then-Review; Transparent and Open Peer Review
The Unjournal supports this. I think we have done this in more depth and rigor than other initiatives in economics and social science
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social-media-ethics-automation.github.io social-media-ethics-automation.github.io
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Unclear Privacy Rules: Sometimes privacy rules aren’t made clear to the people using a system. For example: If you send “private” messages on a work system, your boss might be able to read them. When Elon Musk purchased Twitter, he also was purchasing access to all Twitter Direct Messages
Every time I register for a new account, I’m always prompted to agree to the terms and conditions, but like most people, I never really read through them. It’s concerning to think about what could be hidden in those lengthy documents—things that might infringe on my rights without me even realizing it. Unclear privacy rules can easily conceal important details.
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This includes the creation of Shadow Profiles, which are information about the user that the user didn’t provide or consent to
I think that the concept of 'shadow profiles' presents a significant challenge to privacy regulations and the ability to monitor whether data on media is kept as secure as possible. It may also in some ways be categorised as a result of misinformation. For example, a user may decline services on a media keeping in mind that they don't want to be a part of the company's data collecting practices however their data is still gathered in the system which can be deemed unethical.
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Sometimes companies or researchers release datasets that have been “anonymized,” meaning that things like names have been removed, so you can’t directly see who the data is about. But sometimes people can still deduce who the anonymized data is about. This happened when Netflix released anonymized movie ratings data sets, but at least some users’ data could be traced back to them.
I have come across multiple research papers regarding de-identification of personal information and what is so interesting as well as a bit concerning is the idea that any individual can be identified by only 3 pieces of information including zip code, birthday and gender. This discovery did lead to a change in policies regarding privacy research and regulations on various media; however this leads me to think that as privacy protections evolve, so will the methods to breach them.
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social-media-ethics-automation.github.io social-media-ethics-automation.github.io
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Hacking attempts can be made on individuals, whether because the individual is the goal target, or because the individual works at a company which is the target. Hackers can target individuals with attacks like: Password reuse attacks, where if they find out your password from one site, they try that password on many other sites Hackers tricking a computer into thinking they are another site, for example: the US NSA impersonated Google
The impact of information leaks on our lives is quite significant. The leakage of phone numbers and personal details makes it easier for scammers to deceive people. I also often feel like my phone is monitoring my social media activity. Whenever I mention something I want to buy, within a few days, shopping platforms start showing me ads for that exact item, even if I've never searched for it. This kind of targeted advertising feels intrusive, as if my privacy is being constantly observed.
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readingpoetryatmit.wordpress.com readingpoetryatmit.wordpress.com
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dread
Do you see the poem as a celebration of creativity or a warning about potential hard truths one might come across during the creative process?
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For he on honey-dew hath fed And drunk the milk of Paradise.
Describes the transcendent experience of being all consumed within your creativity.
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A sunny pleasure-dome with caves of ice!
Another contradiction to illustrate the complex reality of his creativity.
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Floated midway on the waves;
The deep true poetic vision he wants to create will never be perfect and is always trying to run.
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The sha
Shift to more quiet tone
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flung up momently the sacred river.
Coleridge plays between moments of stasis and intense movement. It relates to his creative process by saying that sometimes you don't know what to do, while others, it is clear what direction you should be heading.
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savage place! as holy and enchanted
Contradiction between his words illustrates how his creativity is not straightforward and even he can't fully describe the ways his mind works.
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sunless sea
Represents the mystery of this place and how it can't be fully understood. Could be mapped to the subconscious part of his imagination.
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twice 5 miles of fertile ground
emphasis on the idyllic nature of this place.
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the-orb.arlima.net the-orb.arlima.net
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After lunch all the youth of the city go out into the fields to take part in a ball game.
It's lovely to see how this has not changed so much even today, especially with people in England crazy for sports like soccer/football and rugby.
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it is not productive for urban society to be always serious or practical
Humans have always had the tendency to find ways to kickback and relaxed, and from this phrase we can see the author's view of what the ideal urban society is: serious and practical at times, while recreational at other times. If I were to compare it to modern day society I feel like the desire to seek constant recreation and fun can be seen especially when it comes to overwork and stress found in the expectations of modern society, which seems like an unhealthy balance and maybe, I think, people of the past would have found this type of living quite depressing and scary.
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readingpoetryatmit.wordpress.com readingpoetryatmit.wordpress.com
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But hush! there is a pause of deepest silence! And all that noise, as of a rushing crowd, With groans, and tremulous shudderings — all is over — It tells another tale, with sounds less deep and loud!
The wind changes and so too does the speaker's internal feelings in that it now has a more manageable as if paying attention to the tumultuous sounds of the wind was akin to a release of some sort.
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I may not hope from outward forms to win The passion and the life, whose fountains are within.
Here, the speaker realizes that they must not rely on nature to lift his spirits and instead needs to gain real passion and life from within.
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And I fear, I fear, my Master dear! We shall have a deadly storm.
Serves as foreshadowing for the "deadly storm" about to take place which correlates to the speaker's future explanation of their emotional distress.
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In its own cloudless, starless lake of blue; I see them all so excellently fair, I see, not feel, how beautiful they are!
Emphasizes the speaker's disconnect with nature as the speaker can see the moon, yet can not feel its presence. This contributes to the speakers overwhelming sense of isolation.
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For hope grew round me, like the twining vine, And fruits, and foliage, not my own, seemed mine. But now afflictions bow me down to earth: Nor care I that they rob me of my mirth;
These lines have a contrast in that Coleridge first explains how there were once positive feelings associated with hope and imagination; however, now, these feelings have morphed into despair and a lack of an ability to connect with nature.
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“Dejection: An Ode” by Coleridge
Discussion questions: - What role does nature play in the poem? - What is the significance of the epigraph? - What is the relationship between imagination and emotion?
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www.espn.com www.espn.com
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"We've looked at outside the building, but to get a true No. 1 in here, you got to make some massive trades and give up quite a bit.
Because of the injury the Buccaneers may try to make a trade which would impact many people in both in the NFL, fans, and fantasy football players
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The Buccaneers' Chris Godwin will undergo surgery for a dislocated left ankle and will likely be done for the year
This event just happened on Monday night so it is an example of timeliness
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one day after the star wide receiver was carted off the field in the final minute of a "Monday Night Football" loss to the Baltimore Ravens.
This comment shows prominence as Chris Godwin is a star player for the Tampa Bay Buccaneers.
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rmi.org rmi.org
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The research finds
for - stats - green growth - 2024 - Global South vs Global North
stats - green growth - 2024 - Global South has - 60% of world population - 20% of fossil fuel production - fossil fuel production in decline - 70% of global renewable resource potential - In 2024, 87% of capex of electricity generation is renewable - From 2019 to 2024, renewable energy has grown 23% annually and now supplies 9% of its electricity - 17% of Global South has already overtaken Global North in % of renewable electricty generation
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for - Report - Powering Up the Global South - Rocky Mountain Institute - RMI - 2024 - Vikram Singh - Kingsmill Bond
Summary - This report shows that the Global South is adopting cleantech faster than the Global North
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Local file Local file
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pathology in the radiograph may be seen in due to these factors
Radyografideki patoloji bu faktörlerden dolayı görülebilir
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Inability to eliminate the infection
Enfeksiyonu ortadan kaldırma yeteneğinin olmaması
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Inflammation in PDL with the direct effect of periodontal diseases. Traumas canalso have inflammatory results.
Periodontal hastalıkların doğrudan etkisiyle PDL'de iltihaplanma meydana gelir. Travmalar da iltihabi sonuçlar doğurabilir
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Inflammation in the pulp tissue is transmitted to the PDL via the apical foramenor lateral canals.
Pulp dokusundaki iltihap, apikal foramen veya lateral kanallar aracılığıyla periodontal ligamente (PDL) iletilir
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www.accessengineeringlibrary.com www.accessengineeringlibrary.com
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Steel erection essentially consists of four main tasks:Establishing that the foundations are suitable and safe for an erection to commence.Lifting and placing components into position, generally using cranes but sometimes by jacking. To secure components in place, bolted connections will be made, but will not yet be fully tightened. Bracings may similarly not be fully secured.Aligning the structure, principally by checking that column bases are lined, and level and columns are plumb. Packing in beam-to-column connections may need to be changed to allow column plumb to be adjusted.Bolting-up, which means completing all the bolted connections to secure and impart rigidity to the frame.
main topic
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books.openbookpublishers.com books.openbookpublishers.com
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The posthumanist approach understands the human subject as constantly becoming through the myriad of constituting relations in their life.
Our relationship with technology and our environment is interconnected and always evolving and changing.
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www.medrxiv.org www.medrxiv.org
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eLife Assessment
The authors analyze a comprehensive cohort of human plasma samples to identify an extracellular vesicles protein signature for early diagnosis of pancreatic cancer. The application of liquid biopsies is valuable, and the work addresses a key clinical problem as pancreas cancer is often diagnosed in late stages. The strength of evidence is solid. Altogether, this work supports the potential use of extracellular vesicles in clinical settings, with promising value to scientists and clinicians.
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Reviewer #1 (Public review):
This study presents a large cohort of plasma-derived extracellular vesicle samples from 124 individuals, including patients with PDAC, benign pancreatic diseases and controls. The authors identified a panel of protein markers for the early detection of pancreatic cancer and validated in an external cohort.
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Reviewer #2 (Public review):
This work investigates the use of extracellular vesicles (EVs) in blood as a noninvasive 'liquid biopsy' to aid in differentiation of patients with pancreatic cancer (PDAC) from those with benign pancreatic disease and healthy controls, an important clinical question where biopsies are frequently non-diagnostic. The use of extracellular vesicles as biomarkers of disease has been gaining interest in recent history, with a variety of published methods and techniques, looking at a variety of different compositions ('the molecular cargo') of EVs particularly in cancer diagnosis (Shah R, et al, N Engl J Med 2018; 379:958-966).
This study adds to the growing body of evidence in using EVs for earlier detection of pancreatic cancer, identifying both new and known proteins of interest. Limitations in studying EVs in general include dealing with low concentrations in circulation and identifying the most relevant molecular cargo. This study provides validation of assaying EVs using the novel EVtrap method (Extracellular Vesicles Total Recovery And Purification), which the authors show to be more efficient than current standard techniques and potentially more scalable for larger clinical studies.
The strength of this study is in its numbers - the authors worked with a cohort of 124 cases, 93 of them which were PDAC samples, which considered large for an EV study (Jia, E et al. BMC Cancer 22, 573 (2022)). The benign disease group (n=20, between chronic pancreatitis and IPMNs) and healthy control groups (n=11) were relatively small, but the authors were not only able to identify candidate biomarkers for diagnosis that clearly stood out in the PDAC cohort, but also validate it in an independent cohort of 36 new subjects. Proteins they've identified as associated with pancreatic cancer over benign disease included PDCD6IP, SERPINA12 and RUVBL2. They were even able to identify a set of EV proteins associated with metastasis and poorer prognosis , which include the proteins PSMB4, RUVBL2 and ANKAR and CRP, RALB and CD55. Their 7-EV protein signature yielded an 89% prediction accuracy for the diagnosis of PDAC against a background of benign pancreatic diseases that is compelling and comparable to other studies in the literature (Jia, E. et al. BMC Cancer 22, 573 (2022)).
The limitations of this study are its containment within a single institution - further studies are warranted to apply the authors' 7-EV protein PRAC panel to multiple other cases at other institutions in a larger cohort.
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Author response:
The following is the authors’ response to the original reviews.
Reviewer #1 (Public Review):
In this manuscript, Bockorny, Muthuswamy, and Huang et al. performed proteomics analysis of plasma extracellular vesicles (EVs) from pancreatic ductal adenocarcinoma (PDAC) patients and patients with benign pancreatic diseases (chronic pancreatitis and intraductal papillary mucinous neoplasm, IPMN) to develop a 7-EV protein signature that predicts PDAC. Moreover, the authors identified PSMB4, RUVBL2, and ANKAR as being associated with metastasis. These studies provide important insight into alterations of EVs during PDAC progression and the data supporting predict PDAC with EV protein signatures are solid. However, there are certain concerns regarding the rigor and novelty of the data analysis and interpretation, as well as the clinical implications, as detailed below.
(1) Plasma EVs were characterized by transmission electron microscopy and nanoparticle tracking analysis to confirm their morphology and size. The authors should also include an analysis of putative EV markers (e.g., tetraspanins, syntenin, ALIX, etc.) to confirm that the analyzed particles are EVs.
We thank the reviewer for this comment. In the previous study from our co-authors who developed EVtrap method (PMID:32396726), they used electron microscopy and NTA , as well as quantification of typical EV protein markers, such as CD9, to confirm that particles isolated using EVtrap had typical characteristics of the extracellular vesicles. As such, these experiments were not replicated here. We added the following statement to the manuscript:
“Previous analyses using electron microscopy and nanoparticle tracking also confirmed that the vast majority of particles isolated by EVtrap had diameters between 100-200 nm, consistent with exosomes (PMID:32396726). In addition, EVtrap isolates demonstrates higher abundance of CD9, a common exosome marker, as compared to isolates from other traditional EV isolation methods such as size exclusion chromatography and ultracentrifugation (PMID:32396726)”
(2) The authors identified multiple over-expressed proteins in PDAC based on their foldchange and p-value; however, due to the heterogeneity of PDAC, it is necessary to show a heatmap displaying their abundance in all samples. High fold change does not necessarily indicate consistently high abundance in all PDAC samples.
We thank the reviewer for this suggestion. We have now included the heatmap in the new Supplementary Figure 3.
(3) PSMB4, RUVBL2, and ANKAR were identified as being associated with metastasis. The authors state that they intended to distinguish early and late-stage cancer samples, but it is unclear why they chose to compare metastatic and non-metastatic samples, as the non-metastatic group also includes late-stage cancer samples. This sentence should be rephrased to more accurately reflect the sample types profiled.
We thank the reviewer for pointing this out. We would like to clarify that this analyses shown in Figures 3B and 3C pertain to patients with Metastatic vs Non-Metastatic disease, not early versus late stage. We edited the text to ensure this information is clear.
(4) Non-metastatic and metastatic patients were separated based on global protein abundance. The samples within each group display significant heterogeneity, with some samples displaying similar patterns although they were classified into different groups (Figure 3A), and the samples within the same group, particularly the metastasis group, did not consistently exhibit similar patterns of protein abundance. The authors should clarify this point.
We thank the reviewer for this comment. The EV proteomic expression is anticipated not to show the exact pattern across of samples of each group. The purpose of this experiment depicted in Figure 3 heatmap is to show the enrichment for pattern of expressions, but we acknowledge that not all samples from the same group have the exact proteome pattern.
We added this statement in the discussion section:
“As expected, the EV proteomic profiles of PDAC patients exhibited significant heterogeneity. While the above mentioned markers exhibited strong association with disease states at population levels, their abundances in individual patients varied significantly. Those observations highlight the need to develop multi-protein panels for pancreatic cancer diagnosis and prognosis.”
(5) The authors performed the survival analysis on a set of EV proteins but did not specify the origin of these markers or how many markers were examined. The authors should show their abundances across different groups, such as different stages and metastasis status.
We thank the reviewer for the comments. The goal of this experiment was not to identify EV proteins that performed similarly well for diagnosing and prognostication. In Figure 3A, 3B and 3C, we identified EV proteins that had better performance for diagnosis of metastatic disease. In these experiments we made comparative analysis between patients with metastasis versus non-metastasis. In the experiment depicted in Figure 3D, the goal was to identify EV markers that had better performance is prognosticating outcomes as measured by overall survival, out of the markers identified in the previous experiments from Figure 3A. We would like to further clarify that based on our observation and others, it has become clear that EV profiles from cancer patients are highly heterogenous and we do not anticipate that a single marker will have sufficient test performance for cancer diagnosis or prognosis assessment when measured isolated. Rather, we anticipate that a panel of markers may yield better performance for diagnosis while a different combination of EV markers may have better performance for prognosis assessment.
(6) The classification model yielded a 100% accuracy, which may refer to AUC, in their discovery cohort, but it decreased to 89% in the independent cohort. This suggests that the authors have encountered overfitting issues with their model, where it performed well on the discovery cohort but did not generalize well to the independent cohort. The authors should clarify this point. The AUC score of the 7-EV signature is 0.89 and is not equivalent to prediction accuracy. In order to demonstrate prediction accuracy, the authors should show the confusion matrix of training and testing data as well as other evaluation metrics, such as accuracy, precision, and recall.
We thank the reviewer for providing these insightful comments. As you noted, the 7-biomarker signature machine learning model attained an impressive 100% accuracy within the internal Discovery Cohort, raising concerns about potential overfitting in the external validation dataset. Acknowledging the noted difference in AUROC of 0.11 in the external validation cohort, which surpasses the typical reported range of ~0.06-0.09, the model demonstrated a commendable AUROC of 0.89 in an independent patient cohort. Moreover, the utilization of an alternate technology to measure protein abundance in the validation dataset, underscores the model’s reproducibility and validity. We have provided the model metrics for both internal- and external-validation cohort. For these, please see updated Supplementary Figure 7, as well as the new Supplementary Figure 6 and Supplementary Figure 8. We also amended the discussion section to acknowledge that the validation cohort had limited sample size and proteins were measured in using a different method. Those factors likely contributed to the lower accuracy of predictions in the validation cohort. We addressed these limitations in the discussion section of the manuscript.
(7) The authors should include more details of their model and the process of selection of signatures to enhance the reproducibility and transparency of their methods.
We thank the reviewer for their valuable comments. To enhance clarity, we have incorporated additional information regarding the method employed for biomarker signature identification into the ‘Methods Section’ in page 23. We note that Supplementary Table 7a provides details on ‘Sensitivity, Specificity, Precision, and AUC’ for the 16 markers included in the external validation study. Additionally, Supplementary Table 7b presents the contingency table for 7-biomarker signature, offering insights into model accuracy for both the Internal-Discovery and External Validation cohorts.
Reviewer #2 (Public Review):
The authors intended to identify a protein signature in extracellular vesicles of serum to distinguish pancreatic ductal adenocarcinoma from benign pancreatic diseases.
A major strength of the work presented is the valuable profiling of a significant number of patient samples, with a rich cohort of patients with pancreatic cancer, benign pancreatic diseases, and healthy controls. However, despite the strong cohorts presented, the numbers of patient samples for benign pancreatic diseases as well as controls were very limited.
Also, the method used to isolate vesicles, EVTrap, recognizes double bilayers, which means that it can detect cellular debris and apoptotic bodies, which are very common in the circulation of patients that are undergoing chemotherapy. It would be important to identify the patients that are therapy naïve and the ones that are not because of this possible bias.
We thank the Reviewer for these comments. We want to point out that the experiments presented in Supplementary Figure 1 (Transmission electron microscopy images and Nanoparticle tracking analysis) confirm that the vesicles isolated with EVTrap are not cellular debris and apoptotic bodies. Rather, these structures are in the nano range expected for exosomes. This is further supported by the additional work from our co-author and collaborator describing the development of EVtrap and its performance in isolating exosomes when compared to other traditional methods such as ultracentrifugation and size exclusion chromatography (PMID:32396726).
As per the Reviewer’s request, we have provided an additional heatmap figure depicting whose patients are treatment naïve to differentiate from those who have received treatment (revised Figure 2C).
Additionally, the transmission electron microscopy data reflect this heterogeneity of the samples, also with little identification of double bilayered vesicles. It would be important to identify some extracellular vesicles markers in those preparations to strengthen the quality of the samples analyzed.
We appreciate the comment from the Reviewer and acknowledge the importance of identifying exosome markers on the isolate from EVtrap. These experiments have already been done and are reported in the original paper describing the development of this method by our co-authors in a separate work. In the manuscript PMID: 30080416, our collaborators demonstrated the detection of CD9, a well-known exosome marker, using Western Blot from isolates using EVtrap or ultra-centrifugation, a traditional technique to isolate exosomes. This work showed that EVtrap yielded much higher recovery rate of exosomes with lower contamination from soluble proteins. We did not repeat these already published experiments, but we amended our manuscript to reference these results.
What is more, previously published work with this same methodology identifies around 2000 proteins per sample. It would be important to explain why in this study there seems to be a reduction in more than 50% of the amount of proteins identified in the vesicles.
We thank the Reviewer for pointing out this important detail. In the previous work in which EVtrap was developed by our co-authors, the blood samples were processed using a different protocol, with shorter centrifugation (2,500g for 10 min) (PMID: 32396726). In the current work, we employed three centrifugation steps. As detailed in the Methods section of the manuscript, blood samples were centrifuged at 1,300g for 15 min. Then plasma was removed from the top carefully avoiding cell pellet; Repeat centrifugation of plasma at 2,500g for 15 min; Again, plasma was removed from the top carefully avoiding cell pellet; Third centrifugation at 2,500g for 15 min. This more extensive centrifugation process was intended to further increase the removal of platelets, apoptotic bodies, and other large particles and aggregates. Accordingly, we anticipate that the additional centrifugation steps decreased the contamination of our isolates but may have also decreased the amount of exosome proteins, hence the lower amount of exosome proteins identified in our study as compared to the original study from our co-authors (PMID: 32396726).
One of the proteins that constantly surges on the analysis is KRT20. It would be important to proceed with the analysis by first filtering out possible contaminants of the proteomics, of which keratins are the most common ones.
We thank the Reviewer for this comment. We would like to point out that we do believe that KRT20 is, in fact, cancer related and a not a contaminant. This is supported by our results presented in this manuscript showing enrichment or KRT20 in PDAC cases, and lower expression in benign samples. If this protein was a contaminant, its expression would be found uniformly in all samples, there would be no apparent reason for different expression between malignant vs benign cases, as all samples were processed following the same procedures. In addition, increased expression of KRT20 in PDAC tissues has also been reported by others. For instance, in a study by Schmiz-Winnthal (PMID: 16364723), the authors showed that Cytokeratin 20 (KRT20) were expressed in 76% of PDAC patients and expression of KRT20 was associated with poor survival after surgical resection. Based on these observations, we believe that the KRT20 identified in our study is indeed a tumor associated EV protein rather than contamination.
Finally, none of the 7-extracellular vesicle protein signatures has been validated by other techniques, such as western blot, in extracellular vesicles isolated by other, standard, methods, such as size exclusion chromatography.
A distinct technique for protein analysis was done but not a different method of isolation of these vesicles. This would strengthen the results and the origin of the proteins.
We appreciate the Reviewer’s comment. We would like to again emphasize that the goal of this manuscript was not to compare the performance of EVtrap with other traditional EV isolation approaches such as ultracentrifugation and size exclusion chromatography. The main goal of study is to determine proteomic profiles of EVs isolated from clinical samples and provide such information to research community for further studies. As the Reviewer points out, proteins in EVs are highly heterogeneous which highlight the complexity of EV biology and interpatient heterogeneity of pancreatic cancer. We do not anticipate the development of EV-based markers for pancreatic diagnosis can be achieved by a single team, but by a community of researchers. We hope information presented in the current study will help other researchers identify additional candidates for validation in future work. Nonetheless, we edited the manuscript to discuss the limitation of not doing cross-validation of protein detection using a different method.
The conclusions that are reached do not fully meet the proposed aims of the identification of a protein signature in circulating extracellular vesicles that could improve early detection of the disease. The authors did not demonstrate the superiority of detection of these proteins in extracellular vesicles versus simply performing an ELISA, nor their superiority with respect to the current standard procedure for diagnosis.
We would like to clarify to the Reviewer that the goal of this manuscript was not to prove superiority of the EV signature biomarker in diagnosing pancreatic cancer as compared to current standard of care (SOC) practice, i.e., CT scans, endoscopic ultrasound and CA19-9. In order to prove such superiority, one would require a large, randomized phase III trial with several hundred patients. This was not the pursue of our discovery EV proteomics study and we double checked our manuscript to ensure no such claim was made. Rather, we aimed at developing a new pipeline for discovery of new EV biomarkers and we believe we were able to prove that this approach was successful in discovering a new class of biomarkers based on proteins expressed on extra-cellular vesicles that have predominant expression on patients with pancreatic cancer. Future studies should continue to advance this field with goals of improving on the current standard of care diagnostic methods.
The authors also suggest that profiling of circulating extracellular vesicles provides unique insights into systemic immune changes during pancreatic cancer development. How is this better than a regular hemogram is not clear.
We would like to clarify that the overall goal of this study is to provide patient-relevant information for the research community to further investigate biology of extracellular vesicles. For the state 'unique insights into systemic immune changes' we referred to the fact that we discovered EVs carrying proteins involved in immune responses. Previous studies have shown that EVs play important roles in cell-cell communication, discoveries from our study provide candidates for future studies on cellular mechanisms underlying immune regulation during pancreatic cancer development.
Finally, it would be important to determine how this signature compares with many others described in the literature that have the exact same aim. Why and how would this one be better?
We would like to again clarify that comparing the diagnostic performance of the EV biomarkers discovered in the study against standard of care methods (CA19-9, ctDNA, CT scan) was beyond the scope of this discovery EV proteomics work. We reviewed the manuscript to ensure that no claims were made as far as superiority against point-of-care tests available in clinic.
Reviewer #3 (Public Review):
This work investigates the use of extracellular vesicles (EVs) in blood as a noninvasive 'liquid biopsy' to aid in the differentiation of patients with pancreatic cancer (PDAC) from those with benign pancreatic disease and healthy controls, an important clinical question where biopsies are frequently non-diagnostic. The use of extracellular vesicles as biomarkers of disease has been gaining interest in recent history, with a variety of published methods and techniques, looking at a variety of different compositions ('the molecular cargo') of EVs particularly in cancer diagnosis (Shah R, et al, N Engl J Med 2018; 379:958-966).
This study adds to the growing body of evidence in using EVs for earlier detection of pancreatic cancer, identifying both new and known proteins of interest. Limitations in studying EVs, in general, include dealing with low concentrations in circulation and identifying the most relevant molecular cargo. This study provides validation of assaying EVs using the novel EVtrap method (Extracellular Vesicles Total Recovery And Purification),which the authors show to be more efficient than current standard techniques and potentially more scalable for larger clinical studies.
The strength of this study is in its numbers - the authors worked with a cohort of 124 cases,93 of them which were PDAC samples, which are considered large for an EV study (Jia, E etal. BMC Cancer 22, 573 (2022)). The benign disease group (n=20, between chronic pancreatitis and IPMNs) and healthy control groups (n=11) were relatively small, but the authors were not only able to identify candidate biomarkers for diagnosis that clearly stood out in the PDAC cohort, but also validate it in an independent cohort of 36 new subjects.
Proteins they have identified as associated with pancreatic cancer over benign disease included PDCD6IP, SERPINA12, and RUVBL2. They were even able to identify a set of EV proteins associated with metastasis and poorer prognosis, which include the proteins PSMB4, RUVBL2 and ANKAR and CRP, RALB and CD55. Their 7-EV protein signature yielded an 89% prediction accuracy for the diagnosis of PDAC against a background of benign pancreatic diseases that is compelling and comparable to other studies in the literature (Jia,E. et al. BMC Cancer 22, 573 (2022)).
The limitations of this study are its containment within a single institution - further studies are warranted to apply the authors' 7-EV protein PRAC panel to multiple other cases at other institutions in a larger cohort.
We are very thankful to the Reviewer for the positive feedback. We are similarly optimistic that EV-based biomarkers will assist future researchers to develop better diagnostic assays for patients with pancreatic cancer, as well as other tumor types lacking accurate blood-based tests.
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www.biorxiv.org www.biorxiv.org
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Author response:
The following is the authors’ response to the current reviews.
Reviewer #1 (Public review):
Summary:
In this manuscript, Herrmannova et al explore changes in translation upon individual depletion of three subunits of the eIF3 complex (d, e and h) in mammalian cells. The authors provide a detailed analysis of regulated transcripts, followed by validation by RT-qPCR and/or Western blot of targets of interest, as well as GO and KKEG pathway analysis. The authors confirm prior observations that eIF3, despite being a general translation initiation factor, functions in mRNA-specific regulation, and that eIF3 is important for translation re-initiation. They show that global effects of eIF3e and eIF3d depletion on translation and cell growth are concordant. Their results support and extend previous reports suggesting that both factors control translation of 5'TOP mRNAs. Interestingly, they identify MAPK pathway components as a group of targets coordinately regulated by eIF3 d/e. The authors also discuss discrepancies with other reports analyzing eIF3e function.
Strengths:
Altogether, a solid analysis of eIF3 d/e/h-mediated translation regulation of specific transcripts. The data will be useful for scientists working in the Translation field.
Weaknesses:
The authors could have explored in more detail some of their novel observations, as well as their impact on cell behavior.
The manuscript has improved with the new corrections. I appreciate the authors' attention to the minor comments, which have been fully solved. The authors have not, however, provided additional experimental evidence that uORF-mediated translation of Raf-1 mRNA depends on an intact eIF3 complex, nor have they addressed the consequences of such regulation for cell physiology. While I understand that this is a subject of follow-up research, the authors could have at least included their explanations/ speculations regarding major comments 2-4, which in my opinion could have been useful for the reader.
Our explanations/speculations regarding major comments 2 and 3 were included in the Discussion. We apologize for this misunderstanding as we thought that we were supposed to explain our ideas only in the responses. We did not discuss the comment 4, however, as we are really not sure what is the true effect and did not want to go into wild speculations in our manuscript. We thank this reviewer for his insightful comments and understanding.
The following is the authors’ response to the original reviews.
Reviewer #1 (Recommendations For The Authors):
Major comments:
(1) The authors report the potential translational regulation of Raf kinase by re-initiation. It would be interesting to show that Raf is indeed regulated by uORF-mediated translation, and that this is dependent on an intact eIF3 complex. Analyzing the potential consequences of Raf1 regulation for cancer cell proliferation or apoptosis would be a plus.
We agree that this is an interesting and likely possibility. In fact, another clue that translation of Raf1 is regulated by uORFs comes from Bohlen et al. 2023 (PMID: 36869665) where they showed that RAF1 translation is dependent on PRRC2 proteins (that promote leaky scanning through these uORFs). We noted in the discussion that our results from eIF3d/e/hKD and the PRRC2A/B/CKD partly overlap. It is a subject of our follow-up research to investigate whether eIF3 and PRRC2 co-operate together to regulate translation of this important mRNA.
(2) The authors show that eIF3 d/e -but not 3h- has an effect on cell proliferation. First, this indicates that proliferation does not fully correlate with eIF3 integrity. Depletion of eIF3d does not affect the integrity of eIF3, yet the effects on proliferation are similar to those of eIF3e. What is the possibility that changes in proliferation reflect functions of eIF3d outside the eIF3 complex? What could be the real consequences of disturbing eIF3 integrity for the mammalian cell? Please, discuss.
Yes, proliferation does not fully correlate with eIF3 integrity. Downregulation of eIF3 subunits that lead to disintegration of eIF3 YLC core (a, b, c, g, i) have more detrimental effect on growth and translation than downregulation of the peripheral subunits (e, k, l, f, h, m). Our previous studies (Wagner et al. 2016, PMID: 27924037 and Herrmannová et al. 2020, PMID: 31863585) indicate that the YLC core of eIF3 can partially support translation even without its peripheral subunits. In this respect eIF3d (as a peripheral subunit) is an amazing exception, suggesting it may have some specialized function(s). Whether this function resides outside of the eIF3 complex or not we do not know, but do not think so. Mainly because in the absence of eIF3e – its interaction partner, eIF3d gets rapidly degraded. Therefore, it is not very likely that eIF3d exists alone outside of eIF3 complex with moonlighting functions elsewhere. We think that eIF3d, as a head-interacting subunit close to an important head ribosomal protein RACK1 (a landing pad for regulatory proteins), is a target of signaling pathways, which may make it important for translation of specific mRNAs. In support is these thoughts, eIF3d (in the context of entire eIF3) together with DAP5 were shown to promote translation by an alternate capdependent (eIF4F-independent) mechanism (Lee et al. 2016, PMID: 27462815; de la Parra et al. 2018, PMID:30076308). In addition, the eIF3d function (also in the context of entire eIF3) was proved to be regulated by stress-triggered phosphorylation (Lamper et al. 2020, PMID: 33184215).
(3) Figure 6D: Surprisingly, reduced levels of ERK1/2 upon eIF3d/e-KD are compensated by increased phosphorylation of ERK1/2 and net activation of c-Jun. Please comment on the functional consequences of buffering mechanisms that the cell deploys in order to counteract compromised eIF3 function. Why would the cell activate precisely the MAPK pathway to compensate for a compromised eIF3 function?
This we do not know. We can only speculate that when translation is compromised, cells try to counteract it in two ways: 1) they produce more ribosomes to increase translational rates and 2) activate MAPK signaling to send pro-growth signals, which can in the end further boost ribosome biogenesis.
(4) Regarding DAP-sensitive transcripts, can the authors discuss in more detail the role of eIF3d in alternative cap-dependent translation versus re-initiation? Are these transcripts being translated by a canonical cap- and uORF-dependent mechanism or by an alternative capdependent mechanism?
This is indeed not an easy question. On one hand, it was shown that DAP5 facilitates translation re-initiation after uORF translation in a canonical cap-dependent manner. This mechanism is essential for translation of the main coding sequence (CDS) in mRNAs with structured 5' leaders and multiple uORFs. (Weber et al. 2022, PMID: 36473845; David et al., 2022, PMID: 35961752). On the other hand, DAP5 was proposed to promote alternative, eIF4F-independent but cap-dependent translation, as it can substitute the function of the eIF4F complex in cooperation with eIF3d (de la Parra et al., 2018, PMID: 30076308; Volta et al., 2021 34848685). Overall, these observations paint a very complex picture for us to propose a clear scenario of what is going on between these two proteins on individual mRNAs. We speculate that both mechanisms are taking place and that the specific mechanism of translation initiation differs for differently arranged mRNAs.
Minor comments:
(5) Figure S2C: why is there a strong reduction of the stop codon peak for 3d and 3h KDs?
We have checked the Ribowaltz profiles of all replicates (in the Supplementary data we are showing only a representative replicate I) and the stop codon peak differs a lot among the replicates. We think that this way of plotting was optimized for calculation and visualization of P-sites and triplet periodicity and thus is not suitable for this type of comparison among samples. Therefore, we have performed our own analysis where the 5’ ends of reads are used instead of P-sites and triplicates are averaged and normalized to CDS (see below please), so that all samples can be compared directly in one plot (same as Fig. S13A but for stop codon). We can see that the stop codon peak really differs and is the smallest for eIF3hKD. However, these changes are in the range of 20% and we are not sure about their biological significance. We therefore refrain from drawing any conclusions. In general, reduced stop codon peak may signal faster termination or increased stop codon readthrough, but the latter should be accompanied by an increased ribosome density in the 3’UTR, which is not the case. A defect in termination efficiency would be manifested by an increased stop codon peak, instead.
Author response image 1.
(6) Figures 5 and S8: Adding a vertical line at 'zero' in all cumulative plots will help the reader understand the author's interpretation of the data.
We have added a dashed grey vertical line at zero as requested. However, for interpretation of these plots, the reader should focus on the colored curve and whether it is shifted in respect to the grey curve (background) or not. Shift to the right indicates increased expression, while shift to the left indicates decreased expression. The reported p-value then indicates the statistical significance of the shift.
(7) The entire Figure 2 are controls that can go to Supplementary Material. The clustering of Figure S3B could be shown in the main Figure, as it is a very easy read-out of the consistent effects of the KDs of the different eIF3 subunits under analysis.
We have moved the entire Figure 2 to Supplementary Material as suggested (the original panels can be found as Supplementary Figures 1B, 1C and 3A). Figure S3B is now the main Figure 2E.
(8) There are 3 replicates for Ribo-Seq and four for RNA-Seq. Were these not carried out in parallel, as it is usually done in Ribo-seq experiments? Why is there an extra replicate for RNASeq?
Yes, the three replicates were carried out in parallel. We have decided to add the fourth replicate in RNA-Seq to increase the data robustness as the RNA-Seq is used for normalization of FP to calculate the TE, which was our main analyzed metrics in this article. We had the option to add the fourth replicate as we originally prepared five biological replicates for all samples, but after performing the control experiments, we selected only the 3 best replicates for the Ribo-Seq library preparation and sequencing.
(9) Please, add another sheet in Table S2 with the names of all genes that change only at the translation (RPF) levels.
As requested, we have added three extra sheets (one for each downregulation) for differential FP with Padjusted <0.05 in the Spreadsheet S2. We also provide a complete unfiltered differential expression data (sheet named “all data”), so that readers can filter out any relevant data based on their interest.
(10) Page 5, bottom: ' ...we showed that the expression of all 12 eIF3 subunits is interconnected such that perturbance of the expression of one subunit results in the down-regulation of entire modules...'. This is not true for eIF3d, as shown in Fig1B and mentioned in Results.
This reviewer is correct. By this generalized statement, we were trying to summarize our previous results from Wagner et al., 2014, PMID: 24912683; Wagner et al.,2016, PMID: 27924037 and Herrmannova et al.,2020, PMID: 31863585. The eIF3d downregulation is the only exception that does not affect expression of any other eIF3 subunit. Therefore, we have rewritten this paragraph accordingly: “We recently reported a comprehensive in vivo analysis of the modular dynamics of the human eIF3 complex (Wagner et al, 2020; Wagner et al, 2014; Wagner et al., 2016). Using a systematic individual downregulation strategy, we showed that the expression of all 12 eIF3 subunits is interconnected such that perturbance of the expression of one subunit results in the down-regulation of entire modules leading to the formation of partial eIF3 subcomplexes with limited functionality (Herrmannova et al, 2020). eIF3d is the only exception in this respect, as its downregulation does not influence expression of any other eIF3 subunit.”
(11) Page 10, bottom: ' The PCA plot and hierarchical clustering... These results suggest that eIF3h depletion impacts the translatome differentially than depletion of eIF3e or eIF3d.' This is already obvious in the polysome profiles of Figure S2C.
We agree that this result is surely not surprising given the polysome profile and growth phenotype analyses of eIF3hKD. But still, we think that the PCA plot and hierarchical clustering results represent valuable controls. Nonetheless, we rephrased this section to note that this result agrees with the polysome profiles analysis: “The PCA plot and hierarchical clustering (Figure 2A and Supplementary Figure 4A) showed clustering of the samples into two main groups: Ribo-Seq and RNA-seq, and also into two subgroups; NT and eIF3hKD samples clustered on one side and eIF3eKD and eIF3dKD samples on the other. These results suggest that the eIF3h depletion has a much milder impact on the translatome than depletion of eIF3e or eIF3d, which agrees with the growth phenotype and polysome profile analyses (Supplementary Figure 1A and 1D).”
(12) Page 12: ' As for the eIF3dKD "unique upregulated" DTEGs, we identified one interesting and unique KEGG pathway, the ABC transporters (Supplementary Figure 5A, in green).' This sentence is confusing, as there are more pathways that are significant in this group, so it is unclear why the authors consider it 'unique'.
The eIF3dKD “unique upregulated” group comprises genes with increased TE only in eIF3dKD but not in eIF3eKD or eIF3hKD (500 genes, Fig 2G). All these 500 genes were examined for enrichment in the KEGG pathways, and the top 10 significant pathways were reported (Fig S6A). However, 8 out of these 10 pathways were also significantly enriched in other gene groups examined (e.g. eIF3d/eIF3e common). Therefore, the two remaining pathways (“ABC transporters” and “Other types of O-glycan biosynthesis”) are truly unique for eIF3dKD. We wanted to highlight the ABC transporters group in particular because we find it rather interesting (for the reasons mentioned in the article). We have corrected the sentence in question to avoid confusion: “Among the eIF3dKD “unique upregulated” DTEGs, we identified one interesting KEGG pathway, the ABC transporters, which did not show up in other gene groups (Supplementary Figure 6A, in green). A total of 12 different ABC transporters had elevated TE (9 of them are unique to eIF3dKD, while 3 were also found in eIF3eKD), 6 of which (ABCC1-5, ABCC10) belong to the C subfamily, known to confer multidrug resistance with alternative designation as multidrug resistance protein (MRP1-5, MRP7) (Sodani et al, 2012).
Interestingly, all six of these ABCC transporters were upregulated solely at the translational level (Supplementary Spreadsheet S2).”
(13) Note typo ('Various') in Figure 4A.
Corrected
(14) The introduction could be shortened.
This is a very subjective requirement. In fact, when this manuscript was reviewed in NAR, we were asked by two reviewers to expand it substantially. Because a number of various research topics come together in this work, e.g. translational regulation, the eIF3 structure and function, MAPK/ERK signaling, we are convinced that all of them demand a comprehensive introduction for non-experts in each of these topics. Therefore, with all due respect to this reviewer, we did not ultimately shorten it.
Reviewer #2 (Recommendations For The Authors):
- In Figure 2, it would be useful to know why eIF3d is destabilized by eIF3e knockdown - is it protein degradation and why do the eIF3d/e knockdowns not more completely phenocopy each other when there is the same reduction to eIF3d as in the eIF3d knockdown sample?
Yes, we do think that protein degradation lies behind the eIF3d destabilization in the eIF3eKD, but we have not yet directly demonstrated this. However, we have shown that eIF3d mRNA levels are not altered in eIF3eKD and that Ribo-Seq data indicate no change in TE or FP for eIF3d-encoding mRNA in eIF3eKD. Nonetheless, it is important to note (and we discuss it in the article) that eIF3d levels in eIF3dKD are lower than eIF3d levels in eIF3eKD (please see Supplementary Figure 1C). In fact, we believe that this is one of the main reasons for the eIF3d/e knockdowns differences.
- The western blots in Figures 4 and 6 show modest changes to target protein levels and would be strengthened by quantification.
We have added the quantifications as requested by this reviewer and the reviewer 3.
- For Figure 4, this figure would be strengthened by experiments showing if the increase in ribosomal protein levels is correlated with actual changes to ribosome biogenesis.
As suggested, we performed polysome profiling in the presence of EDTA to monitor changes in the 60S/40S ratio, indicating a potential imbalance in the biogenesis of individual ribosome subunits. We found that it was not affected (Figure 3G). In addition, we performed the same experiment, normalizing all samples to the same number of cells (cells were carefully counted before lysis). In this way, we confirmed that eIF3dKD and eIF3eKD cells indeed contain a significantly increased number of ribosomes, in agreement with the western blot analysis (Figure 3H).
- In Figure 6, there needs to be a nuclear loading control.
This experiment was repeated with Lamin B1 used as a nuclear loading control – it is now shown as Fig. 5F.
- For Figure 8, these findings would be strengthened using luciferase reporter assays where the various RNA determinants are experimentally tested. Similarly, 5′ TOP RNA reporters would have been appreciated in Figure 4.
This is indeed a logical continuation of our work, which represents the current work in progress of one of the PhD students. We apologize, but we consider this time- and resource-demanding analysis out of scope of this article.
Reviewer #3 (Recommendations For The Authors):
(1) Within the many effects observed, it is mentioned that eIF3d is known to be overexpressed while eIF3e is underexpressed in many cancers, but knockdown of either subunit decreases MDM2 levels, which would be expected to increase P53 activity and decrease tumor cell transformation. In contrast, they also report that 3e/3d knockdown dramatically increases levels of cJUN, presumably due to increased MAPK activity, and is expected to increase protumor gene expression. Additional discussion is needed to clarify the significance of the findings, which are a bit confusing.
This is indeed true. However, considering the complexity of eIF3, the largest initiation factor among all, as well as the broad portfolio of its functions, it is perhaps not so surprising that the observed effects are complex and may seem even contradictory in respect to cancer. To acknowledge that, we expanded the corresponding part of discussion as follows: “Here, we demonstrate that alterations in the eIF3 subunit stoichiometry and/or eIF3 subcomplexes have distinct effects on the translatome; for example, they affect factors that play a prominent (either positive or negative) role in cancer biology (e.g., MDM2 and cJUN), but the resulting impact is unclear so far. Considering the complex interactions between these factors as well as the complexity of the eIF3 complex per se, future studies are required to delineate the specific oncogenic and tumor suppressive pathways that play a predominant role in mediating the effects of perturbations in the eIF3 complex in the context of neoplasia.”
(2) There are places in the text where the authors refer to changes in transcriptional control when RNA levels differ, but transcription versus RNA turnover wasn't tested, e.g. page 16 and Figure S10, qPCR does not confirm "transcriptional upregulation in all three knockdowns" and page 19 "despite apparent compensatory mechanisms that increase their transcription."
This is indeed true, the sentences in question were corrected. The term “increased mRNA levels” was used instead of transcriptional upregulation (increased mRNA stabilization is also possible).
(3) Similarly, the authors suggest that steady-state LARP1 protein levels are unaffected based on ribosome footprint counts (page 21). It is incorrect to assume this, because ribosome footprints can be elevated due to stalling on RNA that isn't being translated and doesn't yield more protein, and because levels of translated RNA/synthesized proteins do not always reflect steady-state protein levels, especially in mutants that could affect lysosome levels and protein turnover. Also page 12, 1st paragraph suggests protein production is down when ribosome footprints are changed.
Yes, we are well-aware of this known limitation of Ribo-seq analysis. Therefore, the steadystate protein levels of our key hits were verified by western blotting. In addition, we have removed the sentence about LARP1 because it was based on Ribo-Seq data only without experimental evaluation of the steady-state LARP1 protein levels.
(4) The translation buffering effect is not clear in some Figures, e.g. S6, S8, 8A, and B. The authors show a scheme for translationally buffered RNAs being clustered in the upper right and lower left quadrants in S4H (translation up with transcript level down and v.v.), but in the FP versus RNA plots, the non-TOP RNAs and 4E-P-regulated RNAs don't show this behavior, and appear to show a similar distribution to the global changes. Some of the right panels in these figures show modest shifts, but it's not clear how these were determined to be significant. More information is needed to clarify, or a different presentation, such as displaying the RNA subsets in the left panels with heat map coloring to reveal whether RNAs show the buffered translation pattern defined in purple in Figure S4H, or by reporting a statistical parameter or number of RNAs that show behavior out of total for significance. Currently the conclusion that these RNAs are translationally buffered seems subjective since there are clearly many RNAs that don't show changes, or show translation-only or RNA-only changes.
We would like to clarify that S4H does not indicate a necessity for changes in FPs in the buffered subsets. Although opposing changes in total mRNA and FPs are classified as buffering, often we also consider the scenario where there are changes to the total mRNA levels not accompanied by changes in ribosome association.
In figure S6, the scatterplots indicate a high density of genes shifted towards negative fold changes on the x-axis (total mRNA). This is also reflected in the empirical cumulative distribution functions (ecdfs) for the log2 fold changes in total mRNA in the far right panels of A and B, and the lack of changes in log2 fold change for FPs (middle panels). Similarly, in figure S8, the scatterplots indicate a density of genes shifted towards positive fold changes on the x-axis for total mRNA. The ecdfs also demonstrate that there is a significant directional shift in log2 fold changes in the total mRNA that is not present to a similar degree in the FPs, consistent with translational offsetting. It is rightly pointed out that not all genes in these sets follow the same pattern of regulation. We have revised the title of Supplementary Figure S6 (now S7) to reflect this. However, we would like to emphasize that these figures are not intended to communicate that all genes within these sets of interest are regulated in the same manner, but rather that when considered as a whole, the predominant effect seen is that of translational offsetting (directional shifts in the log2 fold change distribution of total mRNA that are not accompanied by similar shifts in FP mRNA log2 fold changes).
The significance of these differences was determined by comparing the ecdfs of the log2 fold changes for the genes belonging to a particular set (e.g. non-TOP mTOR-sensitive, p-eIF4E-sensitive) against all other expressed genes (background) using a Wilcoxan rank sum test. This allows identification of significant shifts in the distributions that have a clear directionality (if there is an overall increase, or decrease in fold changes of FPs or total mRNA compared to background). If log2 fold changes are different from background, but without a clear directionality (equally likely to be increased or decreased), the test will not yield a significant result. This approach allows assessment of the overall behavior of gene signatures within a given dataset in a manner that is completely threshold-independent, such that it does not rely on classification of genes into different regulatory categories (translation only, buffering, etc.) based on significance or fold-change cut-offs (as in S4H). Therefore, we believe that this unbiased approach is well-suited for identifying cases when there are many genes that follow similar patterns of regulation within a given dataset.
(5) Page 10-"These results suggest that eIF3h depletion impacts the translatome differentially than depletion of eIF3e or eIF3d" ...These results suggest that eIF3h has less impact on the translatome, not that it does so differently. If it were changing translation by a different mechanism, I would not expect it to cluster with control.
This sentence was rewritten as follows: “The PCA plot and hierarchical clustering (Figure 2A and Supplementary Figure 4A) showed clustering of the samples into two main groups: RiboSeq and RNA-seq, and also into two subgroups; NT and eIF3hKD samples clustered on one side and eIF3eKD and eIF3dKD samples on the other. These results suggest that the eIF3h depletion has a much milder impact on the translatome than depletion of eIF3e or eIF3d, which agrees with the growth phenotype and polysome profile analyses (Supplementary Figure 1A and 1D).”
Other minor issues:
(1) There are some typos: Figure 2 leves, Figure 4 variou,
Corrected.
(2) Figure 3, font for genes on volcano plot too small
Yes, maybe, however the resolution of this image is high enough to enlarge a certain part of it at will. In our opinion, a larger font would take up too much space, which would reduce the informativeness of this graph.
(3) Figure S5, highlighting isn't defined.
The figure legend for S5A (now S6A) states: “Less significant terms ranking 11 and below are in grey. Terms specifically discussed in the main text are highlighted in green.” Perhaps it was overlooked by this reviewer.
(4) At several points the authors refer to "the MAPK signaling pathway", suggesting there is a single MAPK that is affected, e.g in the title, page 3, and other places when it seems they mean "MAPK signaling pathways" since several MAPK pathways appear to be affected.
We apologize for any terminological inaccuracies. There are indeed several MAPK pathways operating in cells. In our study, we focused mainly on the MAPK/ERK pathway. The confusion probably stems from the fact that the corresponding term in the KEGG pathway database is labeled "MAPK signaling pathway" and this term, although singular, includes all MAPK pathways. We have carefully reviewed the entire article and have corrected the term used accordingly to either: 1) MAPK pathways in general, 2) the MAPK/ERK pathway for this particular pathway, or 3) "MAPK signaling pathway", where the KEGG term is meant.
(5) Some eIF3 subunit RNAs have TOP motifs. One might expect 3e and 3h levels to change as a function of 3d knockdown due to TOP motifs but this is not observed. Can the authors speculate why the eIF3 subunit levels don't change but other TOP RNAs show TE changes? Is this true for other translation factors, or just for eIF3, or just for these subunits? Could the Western blot be out of linear range for the antibody or is there feedback affecting eIF3 levels differently than the other TOP RNAs, or a protein turnover mechanism to maintain eIF3 levels?
This is indeed a very interesting question. In addition to the mRNAs encoding ribosomal proteins, we examined all TOP mRNAs and added an additional sheet to the S2 supplemental spreadsheet with all TOP RNAs listed in (Philippe et al., 2020, PMID: 32094190). According to our Ribo-Seq data, we could expect to see increased protein levels of eIF3a and eIF3f in eIF3dKD and eIF3eKD, but this is not the case, as judged from extensive western blot analysis performed in (Wagner et. al 2016, PMID: 27924037). Indeed, we cannot rule out the involvement of a compensatory mechanism monitoring and maintaining the levels of eIF3 subunits at steady-state – increasing or decreasing them if necessary, which could depend on the TOP motif-mediated regulation. However, we think that in our KDs, all non-targeted subunits that lose their direct binding partner in eIF3 due to siRNA treatment become rapidly degraded. For example, co-downregulation of subunits d, k and l in eIF3eKD is very likely caused by protein degradation as a result of a loss of their direct binding partner – eIF3e. Since we showed that the yeast eIF3 complex assembles co-translationally (Wagner et. al 2020, PMID: 32589964), and there is no reason to think that mammalian eIF3 differs in this regard, our working hypothesis is that free subunits that are not promptly incorporated into the eIF3 complex are rapidly degraded, and the presence or absence of the TOP motif in the 5’ UTR of their mRNAs has no effect. As for the other TOP mRNAs, translation factors eEF1B2, eEF1D, eEF1G, eEF2 have significantly increased FPs in both eIF3dKD and eIF3eKD, but we did not check their protein levels by western blotting to conclude anything specific.
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The Master never reaches for the great;thus she achieves greatness.
Maybe by this the author implies that the process is more important than the goal, so by following the process "the master" automatically reaches grateness.
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Confront the difficultwhile it is still easy;accomplish the great taskby a series of small acts.
This means that one seemingly very hard or impossible task/goal still can be reached if it is divided into smaller; hence easier tasks.
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Think of the small as largeand the few as many.
By saying this the author means that we should treat even small things as something important, because it can grow into something bigger over time.
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www.embopress.org www.embopress.org
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Figure EV2
The authors wanted to start by directly impairing ubiquitylation of Mcm7. However this was not simple, because Mcm7 has multiple sites for possible ubiquitylation.
Figure 2EV shows the process of broadly finding regions of ubiquitylation sites on Mcm7.
Pannel A:
Their previous findings from 2020 show that the first lysine residue #29 on Mcm7 was the sole residue for ubiquitylation by SCF-Dia2 in vitro, even with Mrc1 present, which stimulates the formation of long ubiquitin chains on Mcm7.
However,
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52.2.80.92:1336 52.2.80.92:1336Edm8ker2
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Repair Cafe (Ages 13-16 OR 17-18)
The other programs (3) are missing from the carousel. Also, the title should be one line (if possible).
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+1 (646) 335 3297 (US)
X (twitter) icon to the footer besides the LinkedIn icon.
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www.poetryfoundation.org www.poetryfoundation.org
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Only you, only you can, you are unique
the song is made for the individual person
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it is a boring song but it works every time.
Bro this is the ol' reliable SpongeBob meme. But also, the idea of a cliche song that still works every time is not very common.
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Shall I tell you the secret and if I do, will you get me out of this bird suit?
This basically saying more about how important the song is to them and the spirit and the hope of the less doubt.
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the song that is irresistible:
This annotation is referring to the serin song that everybody is willing to do anything to hear it
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the
The author introduces this song as irresistible to give an information about the poem.
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This song is a cry for help: Help me! Only you, only you can, you are unique
This could be saying that the song is so beautiful that all the sailors try to help the beautiful voice.
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FF6 binds to and activates μ-opioid receptors (MOR) at both low and physiological pH
It only activates in the targetted area. This makes it less addictive. The words used in this source is also more complex. There are terms in the study that I do not entirely understand. I had to look up some meanings of them.
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Chemical structures of fentanyl, N-{1-[2-(2,6-difluorphenyl)ethyl]piperidine-
example of how the atom was replaced
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two hydrogens were replaced by two fluorine atoms at the phenyl ring in the fentanyl structure
the changing of the atoms makes the opioid less addictive. The new opioid works in the targetted area instead of like a traditional opioid. The traditional opioid just releases everywhere, giving the brain that feeling of dopamine.
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strategy to preclude side effects.
to stop the harmful side effects of opioids
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S1, S2, S3, S4. Explain the absence of an S4 in Celia Jeffers’ examination
S1 = ventricles contract; SL valves open, AV valves close S2 = atrial contract; AV valves open, SL valves close S3 = dilated cardiomyopathy (rapid filling of excess volume of blood into dilated ventricles; overfilled water balloon) S4 = hypertrophied cardiomyopathy (ventricle is stiff, so atria has to push extra hard to get blood into stiffed ventricles)
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techwontsave.us techwontsave.us
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Japan and Germany are on one side of this and want one type of standard because they are really advanced in robotics, and whatnot, and want to keep control of that market. Then on the other hand, you have China and South Korea
All of these countries are currently experiencing population crisis, so it makes sense that they are competing for influence on how the future of care will look
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Innovation 25, which imagined what Japan would look like in the year 2025, with all of these high tech devices, including robots
It feels like this did not happen, at least not the the extent they expected. 18 years also seems a bit unrealistic for this change, especially considering that the first iphone came out the same year this was created.
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www.theguardian.com www.theguardian.com
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With Japan’s ageing society facing a predicted shortfall of 370,000 caregivers by 2025, the government wants to increase community acceptance of technology that could help fill the gap in the nursing workforce.
I wonder if something similar will happen in the US eventually if birth rates drop lower than the sustainability rate of 2.1.
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technology that guides people to the toilet at what it predicts is the right time.
This part implies that the future aid devices will need some level of advanced AI, as it "predicts" timings.
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98 manufacturers test nursing-care robotic devices over the past five years, 15 of which have been developed into commercial products.
This feels like a lot compared to how little I had heard about this previously.
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www.sciencedirect.com www.sciencedirect.com
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Likewise, listening to music might be considered a distraction, but Fife's students reported “using music to increase their enjoyment and energy for the task of writing.
uses music to enjoy doing work and finish work more efficiently
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metacognition
the ability to be aware of one's own thought processes and to regulate them
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but also that writing activity is much more complex and multifaceted than the developers of such tools seem to acknowledge.
This sentence reminds me of "You Can Learn to Write In General" and how it talks about how unalike different kinds of writings / writing styles can be
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she discovered that some students use procrastination to generate “time pressure in order to start writing and maintain focus.
What i'm interpreting from this is that students delay their time with work by procrastinating. When they see that they're running out of time, it motivates them to get the work done (?).
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pressbooks.library.torontomu.ca pressbooks.library.torontomu.ca
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We lose our health in a love of color,
"Lose our health" could be referring to the passage of time and how we slowly lose our health over time. "love of colour" evokes a positive feeling around the idea of love, maybe suggesting that as we grow older we experience many different types of love, in people, things, activities.
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let
formatting allows for emphasis on emotions, impacts and even the consequences
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fraternity ghost
A literal understanding of this term could be someone who doesn't belong within a fraternity because of their background but still participates in the activities behind the scenes.
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soul
could it relate to the soul of both the children and the mother? it is healthy for the children to leave the house, go to the movies, but it can also be for the mother to get the children out her space for a period of time
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young
Almost feels like it could be either an internal rant or someone stressing to a new mother how they should allow their children to experience life in a certain way as to ensure they will not ruin the family
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hating you
separating this line from the previous adds intense dramatic emphasis
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first name Linda I once heard
It almost feels like the poet is having a conversation with themselves, is an observer of the environment around them, invites the reader into the mind of the poet
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I stopped breathing
O’Hara runs through his whole day, mundane points and all, before his day ends with the shock of finding out Billie Holiday has died. I don’t know if the details surrounding her death were that public back then, but it’s ironic that he goes through his routine, start to finish, accomplishing all these tasks, mean while Holiday was locked in a hotel room dying. There’s a pointed contrast there. His day was full of events (monotonous ones) while hers was spent essentially in a prison before it ends with her death.
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days I
unpunctuated poem, this line implies a breath being taken between the sentences and a separation of ideas but is still unpunctuated - partially but not entirely separated, interconnectivity of passages and words
-immersion in the environment, people, places, art, culture, life around the speaker goes on continuously and is not able to be viewed in a vacuum despite the attempts to separate
-throughout there is constant reference to things happening <-- jam-packed with action, but none of the actions try to introduce this separation either; thematically life goes on despite the trauma of the speaker
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the probability of theexposion of the lateral canalsincreases
yan kanalların açığa çıkma olasılığı artar.
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the pocket epitheliumproliferates in the apicaldirection
cep epitelinin apikal yönde çoğaldığında
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Absence of periodontal problems outside the area of thepatient's problematic tooth
Hastanın problemli dişi dışında periodontal sorunların olmaması.
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www.biorxiv.org www.biorxiv.org
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Author response:
The following is the authors’ response to the original reviews.
Public Reviews:
Reviewer #1 (Public Review):
This study delineates an important set of uninjured and injured periosteal snRNAseq data that provides an overview of periosteal cell responses to fracture healing. The authors also took additional steps to validate some of the findings using immunohistochemistry and transplantation assays. This study will provide a valuable publicly accessible dataset to reexamine the expression of the reported periosteal stem and progenitor cell markers.
Strengths:
(1) This is the first single-nuclei atlas of periosteal cells that are obtained without enzymatic cell dissociation or targeted cell purification by FACS. This integrated snRNAseq dataset will provide additional opportunities for the community to revisit the expression of many periosteal cell markers that have been reported to date.
(2) The authors delved further into the dataset using cutting-edge algorithms, including CytoTrace, SCENIC, Monocle, STRING, and CellChat, to define the potential roles of identified cell populations in the context of fracture healing. These additional computation analyses generate many new hypotheses regarding periosteal cell reactions.
(3) The authors also sought to validate some of the computational findings using immunohistochemistry and transplantation assays to support the conclusion.
Weaknesses:
(1) The current snRNAseq datasets contain only a small number of nuclei (1,189 nuclei at day 0, 6,213 nuclei on day 0-7 combined). It is unclear if the number is sufficient to discern subtle biological processes such as stem cell differentiation.
We analyzed a total of 6,213 nuclei from uninjured periosteum and fracture calluses at 3 stages of bone healing. We were able to describe 11 distinct cell populations, revealing the diversity of cell populations in uninjured periosteum and post-injury, including rare cell types in the fracture environment such Schwann cells, adipocytes and pericytes. The number of nuclei was sufficient to perform extensive analysis using a combination of cutting-edge algorithms. We agree that more nuclei would allow more in-depth analyses of cell fate transitions and rare populations, such as pericytes and Schwann cells. However, we concentrated here on SSPC/fibrogenic cells that are well represented in our dataset. Our study robustness is also reinforced by the analysis of 4 successive time points to define the SSPC/fibrogenic cell trajectories. Our validations using immunohistochemistry and transplantation assays also confirmed that our dataset is sufficient to define cell trajectories. There is no clear consensus on the number of cells needed to perform sc/snRNAseq analyses, as it depends on the cell types analyzed and the fold changes in gene expression. Previously reported single cell datasets containing a lower number of cells reached major conclusions including SSPC identification, cell differentiation trajectories and differential gene expression (658 cells in (Debnath et al. 2018), 300 in (Ambrosi et al. 2021), around 175 in (Remark et al. 2023).)
(2) The authors' designation of Sca1+CD34+ cells as SSPCs is not sufficiently supported by experimental evidence. It will be essential to demonstrate stem/progenitor properties of Sca1+CD34+ cells using independent biological approaches such as CFU-F assays. In addition, the putative lineage trajectory of SSPCs toward IIFCs, osteoblasts, and chondrocytes remains highly speculative without concrete supporting data.
We performed additional analyses to further support that Sca1+ SSPCs display stem/progenitor properties. We performed CFU assays with Prx1-GFP+ SCA1+ and Prx1-GFP+ SCA1- periosteal cells (Figure 2F-G). We showed that Prx1-GFP+ SCA1+ display significant increased CFU potential compared to Prx1-GFP+ SCA1- cells. In addition, we isolated and transplanted Prx1-GFP+ Sca1+ and Prx1-GFP+ Sca1- periosteal cells at the fracture site of wild-type mice (Figure 2H). Only Sca1+ cells contributed to the callus formation, reinforcing that Sca1+ cells are the SSPC population mediating bone repair.
The differentiation trajectory of SSPCs presented in our study is supported by a combination of bioinformatic analyses and in vivo validation:
- snRNAseq allowed us to identify the different populations in the uninjured periosteum. In silico, in vitro and in vivo analyses all point to Sca1+ cells as the SSPC population (Fig 2EG).
- At day 3 post-fracture, we did not detect Sca1+ cells in the callus (Fig 4 – Supplementary figure 2). Instead, we observed the appearance of a new population, IIFCs. This population clustered along SSPCs and pseudotime analyses indicate that SSPCs can differentiate into IIFCs (Fig 5B). We confirmed the ability of Sca1+ pSSPCs to form IIFCs, by grafting them in the fracture callus and assessing their fibrogenic fate at day 5 post-fracture (Fig 6B).
- In silico, we observed that IIFCs clustered along osteogenic and chondrogenic cells. The pseudotime trajectory suggests that IIFCs can differentiate into both lineages (Fig 5B-C). This is coherent with the progressive expression of osteochondrogenic genes observed in IIFCs (Fig 5C, Fig 8A, C, E). In vivo, we observed the progressive expression of Runx2 and Sox9 by IIFCs undergoing differentiation (Fig 6A). We now show that IIFCs are not undergoing apoptosis, indicating that these cells further differentiate (Fig 7 – Supplementary figure 2). To functionally assess the osteochondrogenic potential of IIFCs, we used transplantation assay and showed that Prx1-GFP+ IIFCs isolated from day 3 post-fracture form cartilage and bone when transplanted at the fracture site of wild-type mice (Fig 6C).
We would like to insist on the robustness of the bioinformatic analyses performed in our study. First, we used datasets from different time points post-fracture to capture the true temporal progression of cell populations in the fracture callus. We used a large combination of tools shown to be reliable in many studies (Julien et al. 2021; Matsushita et al. 2020; Debnath et al. 2018; Baccin et al. 2020; Junyue Cao et al. 2019; Zhong et al. 2020), and all tools converge in the same trajectory. To further show the relevance of pseudotime in our model, we illustrated the distribution of the cell populations by time point (Fig. 5D). We can observe a parallel between the time points and the pseudotime, reinforcing that the pseudotime trajectory reflects the timing of SSPC differentiation. Overall, the combined in silico, in vitro and in vivo analyses support that Sca1+ Pi16+ cells are the periosteal SSPC population, specifically represented in the uninjured dataset. In response to bone fracture, these SSPCs give rise to IIFCs that are specifically represented in the intermediate stages (days 3 and 5) prior to osteochondrogenic differentiation.
(3) The designation of POSTN+ clusters as injury-induced fibrogenic cells (IIFCs) is not fully supported by the presented data. The authors' snRNAseq datasets (Figure 1d) demonstrate that there are many POSTN+ cells prior to injury, indicating that POSTN+ cells are not specifically induced in response to injury. It has been widely recognized that POSTN is expressed in the periosteum without fracture. This raises a possibility that the main responder of fracture healing is POSTN+ cells, not SSPCs as they postulate. The authors cannot exclude the possibility that Sca1+CD34+ cells are mere bystanders and do not participate in fracture healing.
IIFCs are a population of cells that express high levels of ECM related genes, including Postn, Aspn and collagens. We did not claim that Postn expression is specific to IIFCs. While Postn is detected in the uninjured periosteum, snRNAseq analyses and RNAscope experiments showed that the expression of Postn is limited to a small number of cells in the cambium layer of the periosteum (Fig 4B , Figure 4 – Supplementary figure 1B). These Postn-expressing cells in the uninjured periosteum are not SSPCs, as they do not co-express/co-localize with Pi16+ and Sca1+ cells detected in the fibrous layer (Fig4, Figure 4– Supplementary figure 1A, Figure 6-Supplementary figure 1). These Postn-expressing cells are undergoing osteogenic differentiation as shown by the correlation between Runx2 and Postn expression (Fig. 4 – Supplementary Figure 1C). After fracture, we observed a strong increase in ECM-related gene expression and specifically in the IIFC population. We now show the strong increase of Postn expression after injury (Fig. 4 – Supplementary Figure 1D-E, Figure 6-Supplementary figure 1E).
As mentioned in our response above, we now show that SCA1+ cells form cartilage and bone after fracture, while SCA1- cells (including the POSTN+ population) from the uninjured periosteum did not contribute. These data reveal that Sca1+ CD34+ cells are the main SSPC population mediating bone healing and that POSTN+ IIFCs are a transient stage of SSPC differentiation. We added the following text to the result section: “Pi16-expressing SSPCs are located within the fibrous layer, while we observed few POSTN+ cells in the cambium layer (Fig. 4 – Supplementary Fig. 1A). Postn expression is weak in uninjured periosteum and is limited to differentiating cells. Postn expression is strongly increased in response to fracture, specifically in IIFCs (Fig. 4 – Supplementary Fig. 1B-E). “
(4) Detailed spatial organization of Sca1+CD34+ cells and POSTN+ cells in the uninjured periosteum with respect to the cambium layer and the fibrous layer is not demonstrated.
We performed RNAscope experiments to locate Pi16-expressing and Postn-expressing cells in the uninjured periosteum. We observed that Pi16-expressing cells are in the external fibrous layer of the periosteum while Postn-expressing cells are located along the cortex in the cambium layer. The data are added in Fig 4B and Fig. 4- Supplementary Figure 1 and mentioned in the result section “Pi16-expressing SSPCs were located within the fibrous layer, while Postn-expressing cells were found in the cambium layer and corresponded to Runx2-expressing osteogenic cells (Fig. 4 – Supplementary Fig. 1A-C).”.
(5) Interpretation of transplantation experiments in Figure 5 is not straightforward, as the authors did not demonstrate the purity of Prx1Cre-GFP+SCA1+ cells and Prx1Cre-GFP+CD146- cells to pSSPCs and IIFCs, respectively. It is possible that these populations contain much broader cell types beyond SSPCs or IIFCs.
We agree with the reviewer that our methodology for cell transplantation required more justification and validation. We decided to use a transgenic mouse line to be able to trace the cells in vivo after grafting. Prx1 marks limb mesenchyme during development and the Prx1Cre mouse model allows to label all SSPCs contributing to callus formation. Therefore, we used Prx1Cre, R26mTmG mice as donors for SSPCs and IIFCs isolation (Duchamp de Lageneste et al. 2018; Logan et al. 2002). Prx1 does not mark immune and endothelial cells but can label pericytes and fibroblastic populations (Duchamp de Lageneste et al. 2018; Logan et al. 2002; Julien et al. 2021). In the uninjured periosteum, Sca1 (Ly6a) is only expressed by SSPCs and endothelial cells (Fig 3-Supplementary figure 2, Fig 6-Supplementary figure 1). We sorted GFP+ Sca1+ cells from uninjured periosteum of Prx1Cre, R26mTmG mice to isolate only SSPCs and excluding endothelial cells and pericytes. For IIFCs, we isolated cells at day 3 post-fracture, as in our snRNAseq data, we detected IIFCs but no SSPCs, chondrocytes or osteoblasts at this stage of repair. To eliminate Prx1-derived pericytes, we sorted GFP+CD146- cells, as CD146 is specifically expressed by pericytes. We added Figure 6-supplementary Figure 1 to better illustrate the expression of Prx1, SCA1 (Ly6a) and CD146 (Mcam) in the uninjured and day 3 post-fracture datasets. We further demonstrate the purity of SSPCs and IIFCs isolation by qPCR on sorted GFP+ Sca1+ cells from uninjured periosteum and GFP+ CD146- cells from day 3 post-fracture periosteum and hematoma and confirmed the absence of contamination by other cell populations (Figure 6-Supplementary figure 1E). We made the following changes in the text: “To functionally validate the steps of pSSPC activation, we isolated SCA1+ GFP+ pSSPCs from Prx1Cre; R26mTmG mice, excluding endothelial cells, and grafted them at the fracture site of wild-type hosts” and “we isolated GFP+ CD146- from the fracture callus of Prx1Cre; R26mTmG mice at day 3 post fracture, that correspond to IIFCs without contamination by pericytes (CD146+ cells) (Fig. 6C, Figure 6 – Supplementary Fig.1).
Reviewer #2 (Public Review):
Summary:
The authors described cell type mapping was conducted for both WT and fracture types. Through this, unique cell populations specific to fracture conditions were identified. To determine these, the most undifferentiated cells were initially targeted using stemness-related markers and CytoTrace scoring. This led to the identification of SSPC differentiating into fibroblasts. It was observed that the fibroblast cell type significantly increased under fracture conditions, followed by subsequent increases in chondrocytes and osteoblasts.
Strengths:
This study presented the injury-induced fibrogenic cell (IIFC) as a characteristic cell type appearing in the bone regeneration process and proposed that the IIFC is a progenitor undergoing osteochondrogenic differentiation.
Weaknesses:
This study endeavored to elucidate the role of IIFC through snRNAseq analysis and in vivo observation. However, such validation alone is insufficient to confirm that IIFC is an osteochondrogenic progenitor, and additional data presentation is required.
As mentioned in the response to Reviewer 1, the differentiation trajectory of SSPCs presented in our study is supported by a combination of bioinformatic analyses and in vivo validation:
- snRNAseq allowed us to identify the different populations in the uninjured periosteum. In silico, in vitro and in vivo analyses altogether showed that Sca1+ cells are the SSPC population (Fig 2E-G).
- At day 3 post-fracture, we did not detect Sca1+ cells in the callus (Fig 4 – Supplementary figure 2). Instead, we observed the appearance of a new population, IIFCs. This population clustered along SSPCs and pseudotime analyses indicate that SSPCs can differentiate into IIFCs (Fig 5B). We confirmed the ability of Sca1+ SSPCs to form IIFCs, by grafting them in the fracture callus and assessing their fate at day 5 post-fracture (Fig 6B).
- In silico, we observed that IIFCs clustered along osteogenic and chondrogenic cells. The pseudotime trajectory suggests that IIFCs can differentiate into both lineages (Fig 5B-C). This is coherent with the progressive expression of osteochondrogenic genes observed in IIFCs (Fig 5C, Fig 8A, C, E). In vivo, we observed the progressive expression of Runx2 and Sox9 by IIFCs undergoing differentiation (Fig 6A). We now show that IIFCs are not undergoing apoptosis, indicating that these cells further differentiate (Fig 7 – Supp 2). To functionally assess the osteochondrogenic potential of IIFCs, we used transplantation assay and showed that Prx1-GFP+ IIFCs from day 3 post-fracture form cartilage and bone when transplanted at the fracture site of wild-type mice (Fig 6C).
We would like to insist on the robustness of the bioinformatic analyses performed in our study. First, we used datasets from different time points post-fracture to capture the true temporal progression of cell populations in the fracture callus. We used a large combination of tools shown to be reliable in many studies (Julien et al. 2021; Matsushita et al. 2020; Debnath et al. 2018; Baccin et al. 2020; Junyue Cao et al. 2019; Zhong et al. 2020), and all tools converge in the same trajectory. To further show the relevance of pseudotime in our model, we illustrate the distribution of the cell populations by time point (Fig. 5D). We can observe a parallel between the time points and the pseudotime, reinforcing that the pseudotime trajectory reflects the timing of SSPC differentiation. Overall, the combined in silico, in vitro and in vivo analyses strongly support that Sca1+ Pi16+ cells are the periosteal SSPC population, specifically represented in the uninjured dataset. In response to bone fracture, these SSPCs give rise to IIFCs that are specifically represented in the intermediate stages (days 3 and 5) prior to osteochondrogenic differentiation.
We made the following changes in the text:
- Line 81-87: “We performed in vitro CFU assays with sorted GFP+SCA1+ and GFP+SCA1- cells isolated from the periosteum of Prx1Cre; R26mTmG mice, as Prx1 labels all SSPCs contributing to the callus formation1. Prx1-GFP+ SCA1+ showed increased CFU potential, confirming their stem/progenitor property (Fig 2F-G). Then, we grafted Prx1GFP+ SCA1+ et Prx1-GFP+ SCA1- periosteal cells at the fracture site of wild-type mice. Only SCA1+ cells formed cartilage and bone after fracture indicating that SCA1+ cells correspond to periosteal SSPCs with osteochondrogenic potential (Fig 2H).”
- Line 120-122: “We did not detect Pi16-expressing SPPCs, consistent with the absence of cells expressing SSPC markers in day 3 snRNAseq dataset compared to uninjured periosteum (Fig. 4 – Supplementary Figure 2).”
- Line 170-172: “Only a small subset of IIFCs undergo apoptosis, further supporting that IIFCs are maintained in the fracture environment giving rise to osteoblasts and chondrocytes (Fig. 7 – Supplementary Figure 2).”
- Line 277-278: “Following this unique fibrogenic step, IIFCs do not undergo cell death but undergo either osteogenesis or chondrogenesis”
- Line 281-283: “During bone repair, this initial fibrogenic process is an integral part of the SSPC differentiation process, and a transitional step prior to osteogenesis and chondrogenesis.”
Reviewer #3 (Public Review):
In this manuscript, the authors explored the transcriptional heterogeneity of the periosteum with single nuclei RNA sequencing. Without prior enrichment of specific populations, this dataset serves as an unbiased representation of the cellular components potentially relevant to bone regeneration. By describing single-cell cluster profiles, the authors characterized over 10 different populations in combined steady state and post-fracture periosteum, including stem cells (SSPC), fibroblast, osteoblast, chondrocyte, immune cells, and so on. Specifically, a developmental trajectory was computationally inferred using the continuum of gene expression to connect SSPC, injury-induced fibrogenic cells (IIFC), chondrocyte, and osteoblast, showcasing the bipotentials of periosteal SSPCs during injury repair. Additional computational pipelines were performed to describe the possible gene regulatory network and the expected pathways involved in bone regeneration. Overall, the authors provided valuable insights into the cell state transitions during bone repair and proposed sets of genes with possible involvements in injury response.
While the highlights of the manuscript are the unbiased characterization of periosteal composition, and the trajectory of SSPC response in bone fracture response, many of the conclusions can be more strongly supported with additional clarifications or extensions of the analysis.
(1) As described in the method section, both the steady-state data and full dataset underwent integration before dimensional reduction and clustering. It would be appreciated if the authors could compare the post-integration landscapes of uninjured cells between steady state and full dataset analysis. Specifically, fibroblasts were shown in Figure 1C and 1E, and such annotations did not exist in Figure 2B. Will it be possible that the original 'fibroblasts' were part of the IIFC population?
As suggested, we now identified the fibroblast population from the uninjured periosteum in the integration of datasets from all time points (Figure 5B and Fig. 5 – Supplementary Figure 2). We identified 4 fibroblast populations in the uninjured periosteum: Luzp2+, Cldn1+, Hsd11b1+ and Csmd1+ fibroblasts. Luzp2+ and Cldn1+ fibroblasts are clustering distinctly from the other populations in the integrated dataset. Hsd11b1+ fibroblasts blend with SSPCs and IIFCs in the integrated dataset probably due to the low cell number. Finally, Csmd1+ fibroblasts are clustering at the interface between SSPCs and IIFCs likely because they correspond to differentiating cells both in the uninjured periosteum and in response to fracture. We modified the resolution of clustering in our subset dataset, in order to represent Luzp2+ and Cldn1+ fibroblasts as an isolated cluster (Figure 5B, cluster 10). In addition, both pseudotime (Fig. 5B) and gene regulatory network analyses (Fig. 7D), show that the fibroblast populations are distinct from the activation trajectory of SSPCs. We added the following sentence to the text “Fibroblasts from uninjured periosteum (Hsd11b1+, Cldn1+ and Luzp2+ cells corresponding to cluster 10 of Fig. 5B) clustered separately from the other populations, suggesting the absence of their contribution to bone healing.”
(2) According to Figure 2, immune cells were taking a significant abundance within the dataset, specifically during days 3 & 5 post-fracture. It will be interesting to see the potential roles that immune cells play during bone repair. For example, what are the biological annotations of the immune clusters (B, T, NK, myeloid cells)? Are there any inflammatory genes or related signals unregulated in these immune cells? Do they interact with SSPC or IIFC during the transition?
In this manuscript, we report the overall dataset and focused our analyses on the response of SSPCs to injury and their differentiation trajectories. We did not include detailed analyses of the immune cell populations, that are out of scope of this manuscript and are part of another study (Hachemi et al, biorxiv, 2024)
(3) The conclusion of Notch and Wnt signaling in IIFC transition was not sufficiently supported by the analysis presented in the manuscript, which was based on computational inferences. It will be great to add in references supporting these claims or provide experimental validations examining selected members of these pathways.
The role of Wnt and Notch in bone repair has been widely studied and both signaling pathways are known to be regulators of SSPCs differentiation (Lee et al. 2021; Matthews et al. 2014; Novak et al. 2020; Wang et al. 2016; Kraus et al. 2022; Dishowitz et al. 2012; Junjie Cao et al. 2017; Matsushita et al. 2020; Steven Minear et al. 2010; Steve Minear et al. 2010; Kang et al. 2007; Komatsu et al. 2010). It was previously shown that Notch inactivation at early stages of repair leads to bone non-union while Notch inactivation in chondrocytes and osteoblasts does not significantly affect healing, confirming its role in SSPC differentiation before osteochondral commitment (Wang et al. 2016). Wnt was shown to be a critical driver of osteogenesis (Matsushita et al. 2020; Steve Minear et al. 2010; Steven Minear et al. 2010; Kang et al. 2007; Komatsu et al. 2010), as Wnt inhibition alters bone formation and Wnt overactivation increases bone formation (Pinzone et al. 2009; Balemans et Van Hul 2007). The role of Wnt is specific to osteogenic engagement as Wnt inhibition promotes chondrogenesis (Hsieh et al. 2023; C.-L. Wu et al. 2021; Ruscitto et al. 2023). A study by Lee et al. recently confirmed the successive activation and crosstalk of Notch and Wnt pathways during osteogenic differentiation of SSPCs during bone healing (Lee et al. 2021). They showed a peak of Notch activation at day 3 post-injury followed by a progressive decrease that parallels an increase of Wnt signaling inducing osteogenic differentiation. These studies correlate with the sequential activation of Notch and Wnt observed in our snRNAseq analyses. Our analyses now reveal how this sequential activation of Notch and Wnt relates to the fibrogenic and osteogenic phase of SSPC differentiation respectively. We clarified this in the discussion and added the references above to support our claims.
Recommendations for the authors:
Reviewer #1 (Recommendations For The Authors):
(1) The manuscript is well-written overall. However, the authors often oversimplify outcomes and overstate the results. Some of the statements (delineated below) need to be recalibrated to be in line with the presented data.
In addition to the suggested conclusions, we also toned down the following ones to avoid overstating our results :
Line 24: suggesting a crucial paracrine role of this transient IIFC population
Line 227: suggesting their central role in mediating cell interactions after fracture
line 243: IIFCs produce paracrine factors that can regulate SSPCs
- Line 77 (86): The authors should add "might" before "correspond to".
We provided new sets of data including CFU experiments and transplantation assay to reinforce our conclusion. We replaced “correspond to” by “encompass”
- Line 102: SSPCs are obviously not "absent" in day 3 snRNAseq (Figure 2d). The percentage dropped (only) 75%, according to Figure 2e, which is far from disappearance. Overall, immunohistochemical staining is often dichotomous with snRNAseq designations. The authors should more carefully describe the results.
We agree that this comment may not reflect the data shown as we observe a strong decrease in the percentage of cells in SSPC clusters, but still detect few cells in the SSPC clusters. However, when we looked at the presence of Sca1+ Pi16+ cells at different time points, we confirmed the absence of cells expressing SSPC signature genes (Sca1, Pi16, Cd34) at day 3 injury. Due to the clustering resolution of the combined integration, some cells in the SSPC clusters might not be Sca1+ Pi16+. We now show these results in Fig. 4 – Supplementary Figure 2. We changed the text accordingly (line 120): “We did not detect Pi16-expressing SPPCs, consistent with the absence of cells expressing SSPC markers in the day 3 snRNAseq dataset compared to uninjured periosteum (Fig. 4 – Supplementary Figure 2)”.
- Line 134: The authors need to clearly state that GFP+IIFCs were isolated based on Prx1CreGFP+CD146-. The authors did not clearly demonstrate the relationship between POSTN+ cells and CD146- cells, which poses concerns about the interpretation of transplantation experiments.
As mentioned above in response to reviewer 1-public review, we have clarified and provided additional information on our strategy to isolate SSPCs and IIFCs. We used the Prx1Cre; R26mTmG mice to mark all SSPCs and their derivatives with the GFP reporter in order to trace these populations after cell grafting. In the uninjured periosteum, Sca1 (Ly6a) is only expressed by SSPCs and endothelial cells. We sorted GFP+Sca1+ cells to exclude endothelial cells. For IIFCs, we isolated cells at day 3 post-fracture, as in our snRNAseq data, we detect IIFCs but no SSPCs, chondrocytes or osteoblasts at this time point. However, we also detected pericytes that can be Prx1-derived. To eliminate potential pericyte contamination, we sorted GFP+ CD146- cells, as CD146 is specifically expressed by pericytes. We added Figure 6-supplementary Figure 1 to better illustrate the expression of Prx1, SCA1 (Ly6a) and CD146 (Mcam) in the uninjured and day 3 post-fracture datasets. We further demonstrate the purity of SSPCs and IIFCs isolation by qPCR on sorted GFP+ Sca1+ cells from uninjured periosteum and GFP+ CD146- cells from day 3 postfracture periosteum and hematoma and confirmed the absence of contamination by other cell populations (Figure 6-Supplementary figure 1E). We made the following changes in the text (line 153): “To functionally validate the steps of pSSPC activation, we isolated SCA1+ GFP+ pSSPCs from Prx1Cre; R26mTmG mice, excluding endothelial cells, and grafted them at the fracture site of wild-type hosts” and “we isolated GFP+ CD146- from the fracture callus of Prx1Cre; R26mTmG mice at day 3 post fracture, that correspond to IIFCs without contamination by pericytes (CD146+ cells) (Fig. 6C, Figure 6 – Supplementary Fig.1).
- Line 211: It is obvious from Figure 8F that ligand expression was not "specific" to the IIFC phase.
The data only shows a slight enrichment of ligand score.
We corrected the text by “ligand expression was increased during the IIFC phase”.
(2) Some of the computational predictions are incongruent with the known lineage trajectory. For example, in vivo lineage tracing experiments, including but not limited to, PLoS Genet. 2014. 10:e1004820, demonstrate that some of the chondrocytes within fracture callus can differentiate into osteoblasts. This is incompatible with the authors' conclusion that osteoblasts and chondrocytes represent two different terminal stages of cell differentiation in fracture healing. How do the authors reconcile this apparent inconsistency?
In this manuscript, we generated datasets corresponding to the initial stages of bone repair until day 7 post-injury. Therefore, our analyses encompass SSPC activation stages and engagement into osteogenesis and chondrogenesis. The results show that a portion of osteoblasts in the fracture callus are differentiating directly from IIFC via intramembranous ossification. The reviewer is correct to mention that osteoblasts have also been shown to derive from transdifferentiation of chondrocytes, which occurs at later stages of repair during the active phase of endochondral ossification (Julien et al. 2020; Aghajanian et Mohan 2018; Zhou et al. 2014; Hu et al. 2017). This process of chondrocyte to osteoblast transdifferentiation is not represented in our integrated dataset and may require adding later time points. However, when we analyzed the days 5 and 7 datasets independent of days 0 and 3, we were able to identify a cluster of hypertrophic chondrocytes (expressing Col10a1) connecting the clusters of chondrocytes and osteoblasts. This suggests that in this cluster, hypertrophic chondrocytes are undergoing transdifferentiation into osteoblasts as shown in the Author response image 1. Additional time points are needed in a future study to perform in depth analyses of chondrocyte transdifferentiation.
Author response image 1.
Periosteum-derived chondrocytes undergo cartilage to bone transformation. A. UMAP projection of the subset of SSPCs, IIFCs, osteoblasts and chondrocytes in the integration of days 5 and 7 post-fracture datasets. B. Feature plots of Acan, Col10a1 and Ibsp expression. C. UMAP projection separated by time points. D. Percentage of cells in the hypertrophic/differentiating chondrocyte cluster.
(3) The authors did not cite some of the studies that described the roles of Notch signaling in fracture healing, for example, J Bone Miner Res. 2014. 29:1283-94. The authors should test the specificity of Notch signaling activities to IIFCs (POSTN+ cells) in vivo.
The role of Notch in the activation of SSPCs during bone repair has been investigated in several studies (Lee et al. 2021; Matthews et al. 2014; Novak et al. 2020; Wang et al. 2016; Kraus et al. 2022; Dishowitz et al. 2012; Junjie Cao et al. 2017). Notch dynamic was previously described with a peak at day 3 post-injury before a reduction when cells engage in osteogenesis and chondrogenesis (Lee et al. 2021; Dishowitz et al. 2012; Matthews et al. 2014). Notch plays a role in the early steps of SSPC activation prior to osteochondral differentiation as Notch inactivation in chondrocytes and osteoblasts does not affect bone repair (Wang et al. 2016). We added the references listed above to emphasize the correlation between our results and previous reports on the role of Notch and made changes in the discussion.
Reviewer #2 (Recommendations For The Authors):
Suggestions
(1) This research utilized snRNA seq for the basic hypothesis formation; however, the number of nuclei acquired was quite limited. Therefore, please explain the rationale for employing snRNA seq instead of scRNA seq, which includes cytoplasm, and additionally provide the markers used for cell type mapping in the scRNA analysis.
As mentioned in our response to reviewer #1 above, we analyzed a total of 6,213 nuclei from uninjured periosteum and fracture calluses at 3 stages of bone healing. We were able to describe 11 distinct cell populations including rare cell types in the fracture environment such Schwann cells, adipocytes and pericytes. The number of nuclei was sufficient to perform extensive analysis using a combination of cutting-edge algorithms. We agree that more nuclei would allow more indepth analyses of cell fate transitions and rare populations, such as pericytes and Schwann cells. However, we concentrated here on SSPC/fibrogenic cell that are well represented in our dataset. Our study robustness is also reinforced by the analysis of 4 successive time points to define the SSPC/fibrogenic cell trajectories. Our validations using immunohistochemistry and transplantation assays also confirmed that our dataset is sufficient to define cell trajectories. There is no clear consensus on the number of cells needed to perform scRNAseq analyses, as it depends on the cell types analyzed and the fold changes in gene expression. Previously reported single cell datasets containing a lower number of cells reached major conclusions including SSPC identification, cell differentiation trajectories and differential gene expression (658 cells in(Debnath et al. 2018), 300 in (Ambrosi et al. 2021) around 175 in(Remark et al. 2023))
Several studies have shown that snRNAseq provide data quality equivalent to scRNAseq in terms of cell type identification, number of detected genes and downstream analyses (Selewa et al. 2020; Wen et al. 2022; Ding et al. 2020; H. Wu et al. 2019; Machado et al. 2021). While, snRNAseq do not allow the detection of cytoplasm RNA, there is several advantages in using this technique:
(1) better representation of the cell types. To perform scRNAseq, a step of enzymatic digestion is needed. This usually leads to an overrepresentation of some cell types loosely attached to the ECM (immune cells, endothelial cells) and a reduced representation of cell types strongly attached to the ECM, such as chondrocytes and osteoblasts. In addition, large or multinucleated cells like hypertrophic chondrocytes and osteoclasts are too big to be sorted and encapsidated using 10X technology. Here, we optimized a protocol to mechanically isolate nuclei from dissected tissues that allows us to capture the diversity of cell types in periosteum and fracture callus.
(2) higher recovery of nuclei. We performed both isolation of cells and nuclei from periosteum in our study and observed that nuclei extraction is the most efficient way to isolate cells from the periosteum and the fracture callus.
(3) reduction of isolation time and cell stress. Previous studies showed that enzymatic digestion causes cell stress and induces stem cell activation (Machado et al. 2021; van den Brink et al. 2017). Therefore, we decided to perform snRNAseq to analyze the transcriptome of the intact periosteum without digestion induced-biais.
We added this sentence in the result section: “Single nuclei transcriptomics was shown to provide results equivalent to single cell transcriptomics, but with better cell type representation and reduced digestion-induced stress response (Selewa et al. 2020; Wen et al. 2022; Ding et al. 2020; H. Wu et al. 2019; Machado et al. 2021)”.
The list of genes used for cell type mapping are presented in Figure 3 – Supplementary figure 1. We added a detailed dot plot as Figure 3 – Supplementary figure 2.
(2) During the fracture healing process of long bones, the influx of fibroblasts is a relatively common occurrence, and the fibrous callus that forms during bone repair and regeneration is reported to disappear over time. Therefore, inferring that IIFC differentiates into osteo- and chondrogenic cells based solely on their simultaneous appearance in the same time and space is challenging. More detailed validation is necessary, beyond what is supported by bioinformatics analysis.
The first step of bone repair is the formation of a fibrous callus, before cartilage and bone formation. There are no data in the literature demonstrating that an influx of fibroblasts occurs at the fracture site. Several studies now show that cells involved in callus formation are recruited locally (i.e. from the bone marrow, the periosteum and the skeletal muscle surrounding the fracture site) (Duchamp de Lageneste et al. 2018; Julien et al. 2021; Colnot 2009; Jeffery et al. 2022; Debnath et al. 2018; Matsushita et al. 2020; Julien et al. 2022; Matthews et al. 2021). The contribution of locally activated SSPCs to the fibrous callus is less well understood. Lineage tracing shows that GFP+ cell populations traced in Prx1Cre-GFP mice include SSPCs, IIFCs, chondrocytes and osteoblasts.
The timing of the cell trajectories observed in our dataset correlates with the timing of callus formation previously described in the literature as the day 3 post-fracture mostly contains IIFCs while chondrocytes and osteoblasts appear from day 5 post-fracture. We conclude that IIFCs differentiate into osteochondrogenic cells based on multiple evidence beside the simultaneous appearance in time and space:
- In silico trajectory analyses identify a trajectory from SSPCs to osteochondrogenic cells via IIFCs. We added an analysis to show that our pseudotime trajectory parallels the timepoints of the dataset, confirming that the differentiation trajectory follows the timing of cell differentiation (Figure 5D).
- We show that IIFCs start to express chondrogenic and osteogenic genes prior to engaging into chondrogenesis and osteogenesis. In addition, we detected activation of osteo- and chondrogenic specific transcription factors in IIFCs. This shows a differentiation continuum between SSPCs, IIFCS, and osteochondrogenic cells (Figures 6-8).
- Using transplantation assay, we showed that IIFCs form cartilage and bone, therefore reinforcing the osteochondrogenic potential of this population (Figure 6B).
- IIFCs do not undergo apoptosis. We assessed the expression of apoptosis-related genes by IIFCs and did not detect expression. This was confirmed by cleaved caspase 3 immunostaining showing that a very low percentage of cells in the early fibrotic tissue undergo apoptosis.
Therefore, the idea that the initial fibrous callus is replaced by a new influx of SSPCs or committed progenitors is not supported by recent literature and is not observed in our dataset containing all cell types from the periosteum and fracture site. Overall, our bioinformatic analyses combined with our in vivo validation strongly support that IIFCs are differentiating into chondrocytes and osteoblasts during bone repair. Additional in vivo functional studies will aim to further validate the trajectory and investigate the critical factors regulating this process.
(3) The influx of most osteogenic progenitors to the bone fracture site typically appears after postfracture day 7. It's essential to ascertain whether the osteogenic cells observed at the time of this study differentiated from IIFC or migrated from surrounding mesenchymal stem cells.
As mentioned above, there is not clear evidence in the literature indicating an influx of osteoprogenitors. Cells involved in callus formation are recruited locally and predominantly from the periosteum (Duchamp de Lageneste et al. 2018; Julien et al. 2021; Colnot 2009; Jeffery et al. 2022; Debnath et al. 2018; Matsushita et al. 2020; Matthews et al. 2021; Julien et al. 2022). Our datasets therefore include all cell populations that form the callus. Other sources of SSPCs include the surrounding muscle that contributes mostly to cartilage, and bone marrow that contributes to a low percentage of the callus osteoblasts in the medullary cavity (Julien et al. 2021; Jeffery et al. 2022). We provide evidence that IIFCs give rise to osteogenic cells using our bioinformatic analyses and in vivo transplantation assay (listed in the response above). As indicated in our response to reviewer #1, the steps leading to osteogenic differentiation observed in our dataset reflect the first step of callus ossification and correspond to the process of intramembranous ossification (up to day 7 post-injury). Endochondral ossification also contributes to osteoblasts including the transdifferentiation of chondrocytes into osteoblasts (Julien et al. 2020; Zhou et al. 2014; Hu et al. 2017). While this process mostly occurs around day 14 postfracture, we begin to detect this transition in our integrated day 5-day 7 dataset as shown in Author response image 1.
(4) It's crucial to determine whether the IIFC appearing at the fracture site contributes to the formation of the callus matrix or undergoes apoptosis during the fracture healing process. In the early steps of bone repair, the callus is mostly composed of an extracellular matrix (ECM). IIFCs are expressing high levels of ECM genes, including Postn, Aspn and collagens (Col3a1, Col5a1, Col8a1, Col12a1) (Figure 3 – Supplementary Figures 1-2 and Fig. 7 – Supplementary Figure 1B). IIFCs are the cells expressing the highest levels of matrix-related genes compared to the other cell types in the fracture environment (i.e. immune cells, endothelial cells, Schwann cells, pericytes, …) as shown now in Fig. 7 – Supplementary Figure 1A. Therefore, IIFCs are the main contributors to the callus matrix.
We investigated if IIFCs undergo apoptosis. We observed that only a low percentage of IIFCs express apoptosis-related genes and are positive for cleaved caspase 3 immunostaining at days 3, 5 and 7 of bone repair. This shows that IIFCs do not undergo apoptosis and reinforces our model in which IIFCs further differentiate into osteoblasts and chondrocytes. We added these data in Fig. 7 – Supplementary Figure 2 and added the sentence in the results section “Only a small subset of IIFCs undergo apoptosis, further supporting that IIFCs are maintained in the fracture environment giving rise to osteoblasts and chondrocytes (Fig. 7 – Supplementary Figure 2).”
(5) Results from the snRNA seq highlight the paracrine role of IIFC, and verification is needed to ensure that the effect this has on surrounding osteogenic lineages is not misinterpreted.
To assess cell-cell interactions, we used tools such as Connectome and CellChat to infer and quantify intercellular communication networks between cell types. Studies showed the robustness of these tools combined with in vivo validation (Sinha et al. 2022; Alečković et al. 2022; Li et al. 2023). Here we used these tools to illustrate the paracrine profile of IIFCs, but in vivo validation would be required using gene inactivation to assess the requirement of individual paracrine factors. We performed extensive analyses of the crosstalk between immune cells and SSPCs using our dataset in another study combined with in vivo validation, showing the robustness of the tool and the dataset (Hachemi et al. 2024). We adjusted our conclusions to reflect our analyses: “suggesting a crucial paracrine role of this transient IIFC population during fracture healing”, “suggesting their central role in mediating cell interactions after fracture”, “suggesting that SSPCs can receive signals from IIFC”.
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pressbooks.library.torontomu.ca pressbooks.library.torontomu.ca
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’t You Rich?
form: short, scattered, variable lengths of stanzas with visual cues cutting itself off at varied points
form gives a sense of exaggerated speech ('uncontrollable!' 'what good? what worth? dying!') and sets a strong tone for the speaker
stanzas like to use repetition, alliteration, common themes in the stanzas to accentuate the tone and importance; sounds very realistic and grounded in how we speak and over-emphasize ideas or questions or words that matter most to us (stanza one: everything else, anything else, i have nothing, shall have nothing, immediate, inescapable, invaluable) (stanza 2/3/7 refer to pink/orange/green) (stanza 5/7 use multiple questions)
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fathom.video fathom.video
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Why should we have philanthropy?The reason that we have charities and NGOs and all of this is to fix the problems of corporations.
for - meme - abolish philanthropy - to - critique - Andrew Carnegie essay - The Gospel of Wealth
meme - abolish philanthropy - Agree. Corporations, through externalizing social and ecological impacts, have created a majority of the problems of the polycrisis, that non-profits are created to solve - It would be far more efficient to NOT create those problems to begin with - see my annotations on Andrew Carnegie's "Gospel of Wealth" - where I critique Carnegie's philosophy
to - critique - Andrew Carnegie - essay - The Gospel of Wealth - https://hyp.is/go?url=https%3A%2F%2Fwww.carnegie.org%2Fabout%2Four-history%2Fgospelofwealth%2F&group=world
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what is the nature of the invitation.
for - group dynamics of expanding and converging groups
group dynamics of expanding and converging groups - It is natural for groups to expand and grow and when they do, it changes the dynamics of the social interactions - Effort is required to know each other. It requires time to share and absorb what is shared - That legacy knowledge becomes the unspoken and implicit ground for future discourse - When new people are introduced to a group, or new groups are introduced to each other, - a minimum amount of sharing is required to establish common ground, common understanding - When members of a group have unique ideas to share, - a standardized, shareable documentation may become necessary for greater efficacy of sharing - the constitutions that are often at the heart of institutions became necessary for the same reasons
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but people wanting to take projects on that can produce things in the world that get things done.
for - similarity - not just talk, make an impact
similarity - not just talk, make an impact - I think many of us are of like-mind. Surveying the precarity of the current polycrisis, there is immense complexity and very little time - Given these challenging circumstances, it behooves us to perform very careful sense-making to identify both the individual and the collective leverage points that will have the greatest impact in the shortest time - This also means we have to be careful of which groups we choose to work with as an optimal set of synergies is required if the group is to have possibility of reaching the greatest impact collectively
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pressbooks.library.torontomu.ca pressbooks.library.torontomu.ca
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There were very many who were wanting to be ones doing what he was doing that is to be ones clearly expressing something and then very many of them were not wanting to be being ones doing that thing, that is clearly expressing something, they wanted to be ones expressing something being struggling, something being going to be some other thing, something being going to be something some one sometime would be clearly expressing and that would be something that would be a thing then that would then be greatly expressing some other thing than that thing, certainly very many were then not wanting to be doing what this one was doing clearly expressing something and some of them had been ones wanting to be doing that thing wanting to be ones clearly expressing something.
the entire piece, specifically this sentence, is an example of the overall theme of the piece, which is also the repeated refrain of "clearly expressing something" and the consideration of the content expressed. The structure of this sentence presents concrete ideas but in a difficult-to-digest form (long run-on sentence, basic/filler words repeated often) Is the content about clearly expressing something being clearly expressed? if someone is clearly expressing something in a way that is not clear, does that take away the something of the thing expressed?
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Some said he was not clearly expressing what he was expressing and some of such of them said that the greatness of struggling which was not clear expression made of him one being a completely great one.
the humanity/the struggle makes him more human, more convincingly great
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Nearer in fairy sea, nearer and farther, show white has lime in sight, show a stitch of ten. Count, count more so that thicker and thicker is leaning.
very abstract imagery, very intuitively written. Reminds me of poetry I've written that made sense to me but I've scrapped because it would be gibberish to others, but it's interesting to be a reader and to try and unpack the speaker's garbled thoughts.
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saalck.pressbooks.pub saalck.pressbooks.pub
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The Google Safety Center claims it is committed to responsible advertising and never sells your personal information, but still employs other methods to share and monetize upon it
Google is able to track your phone and see where you have been. By seeing what stores you visit, they are able to send ads by places you visit.
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Local file Local file
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By reason of today's decision, I anticipate that Congress will find delegation of itslawmaking powers much more attractive in the future
This case ruled that the Sentencing Reform Act of 1984 and the U.S. Sentencing Commission were constitutional. The court's decision was based on delegation of powers. Congress can delegate authority to other branches of government under broad guidelines.
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conbio.onlinelibrary.wiley.com conbio.onlinelibrary.wiley.com
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Patterns of parasitism are described in terms of prevalence of infection, taxon richness, and the magnitude of multiple infections, fecal egg count and, in the case of protozoa, the intensity of infection.
All of these together provide a comprehensive picture of parasitism in a population.
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Individuals seen on the ground more frequently tended to have both more ciliate and nematode infections
This would make sense since they most likely had increased exposure.
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Researchers may exert subtle effects on wildlife by altering habitat. The creation of trails is common at long-term research sites (Strier 2010). The impact of these activities on animal behavior or disease ecology is unknown. Habitat alteration can affect patterns of parasitic and bacterial infections within primate populations
I never thought about how something so subtle could have such a significant effect on bacterial infections.
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