This makes me think about how children/students who come from more low-income backgrounds are ultimately affected by certain schooling decisions. During this time, these "charity schools" contained a certain stigma that it was catered towards those who couldn't afford higher education which could be seen as embarrassing to the parents and children who were sent to these schools.

Does intertwining religion with education truly educate people to "choose to do right?" This made me think of how there are people who truly believe this and enroll their children in private, religious schools for this sole purpose. In fact, I know people who have done this as they believe children who attend free, public schools will end up becoming rebellious and defiant. Shows how rooted this stigma has been since the 1800s and how it is still prevalent to this day.

This made me think back to the Educational & Social Change article which pondered the question, "Do schools change society, or does society change schools?" For me, this made it evident that for reformers the answer is that schools have the power to "change" society and "improve" it. "Change" and "improvement" being subject to whatever the societal beliefs of reformers are. Depending on such beliefs, this viewpoint of education as a powerful tool to bring about change can be seen as either a major advantage or disadvantage.

Eidsheim, The Race of Sound - Chapter 4: Race as Zeros and Ones

Noble, Algorithms of Oppression: How Search Engines Reinforce Racism - Chapter 5: The Future of Knowledge in the Public

See page note

https://wptavern.com/matt-mullenweg-identifies-godaddy-as-a-parasitic-company-and-an-existential-threat-to-wordpress-future

WordPress open source or WordPress.com? Recall that Automattic is a VC backed company now and Matt has a big dog in the hunt.

Awkward sentence. Breached sounds severe. Maybe re-phrase to "you may want to consider whether boundaries were crossed or violated."

Velleman believes that love is no different from valuing someone. It has nothing to do with their general qualities and everything do to with their perceived value.

testing uncontrollable tether impacts. Ie not tracked debris or meteoroids

multiple strands help tether livelyhood

further needs for study

quote

good source for the same comment

tethers cross wection vs orbitdal time pros

performance of tethers tethers are meant to be included in spacecraft design?

drawabacks of long tether design

Electrodynamic drag concept explaination.

If we're talking about cultural competence here, some cultures view eye contact as rude or inappropriate and facial expressions vary by culture.

mental health problems or mental illness?

or

This whole section is verbatim from the source- should it be in quotes?

or being overly

hallucinations, i.e. when a person...

Difficulty

delete

delete

they may experience

The writing style is very descriptive. The readers are able to get a clear picture of what each character looked like and how they felt. Oral history was not used, but this method actually allows for more detail through description. With oral history, we did not see the same level of description as we do here in terms of attitude and appearance.

Here is an example of how the objectification of women is a norm. Notice how in this excerpt, there is no mention of whether women want to be with a man based on his appearance. This, in part, speaks to the time period as well as the Jewish culture. Even a feminist like herself, it was typical to talk this way.

This section demonstrates how important preserving Jewish religion was. Rav would check on his daughter just to make sure she was only being taught Jewish ideologies. Additionally, this story shows how close the Jews meant to keep their community. Only other Jewish members were chosen for partners for other Jews. Here, Perele's groom was chosen in advance, a well educated Jewish member.

This shows the instant judgement that women faced by other women. Women were less supportive of each other then, and you had to be a leader to be a feminist. People often like to put all women into the feminine stereotype of fragile, so it's not surprising that other women don't believe that Perele has real health issues.

This is not a decorator; wonder if u can use it with decorator

Beyond COPPA

the limit on history is interesting and feels like a missed opportunity. in a world where storage is so cheap, we should be able to keep our own histories! thinking about sousveillance vs. surveillance and personal panopticons

reminds me of locket but web-based and with full user agency to create what they want. their own little pocket of the internet. Love the no account part and think that's super important..

Elwyck and Darwyn Sure

do

bold

,

helps meet this goal but can't guarantee anything ;)

This sentence doesn't make sense. Should it be "and the" instead of "a"?

4:50 new coordination mechanisms that enable us to solve these challenges at scale

5:30 handle the breadth of challenges

problems billions of people

blockchain to build coordination schools

underestimated potential impact

right size scale

web 2.0 extractive and captured

Cryptographic economic structures

mechanisms we can deploy

Software is eating Mechanism Design

incentive structures

Annotation by Breyer, Sotomayor, and Kagan. </br>“With sorrow—for this Court, but more, for the many millions of American women who have today lost a fundamental constitutional protection—we dissent.” A somber, final comment added in response to today’s opinion. #Annotate22 175/365

Testing this out! It's really cool!

Test

For all the statpearls links it may be helpful to denote the article so people can find the specific one if they are interested in looking into it further.

Same here- should this link to the actual text?

This link does not open to the actual text but instead "libretext landing page", whereas the first two open to the text itself. Not sure if this matters or not, but is not consistent.

Blockchain does not improve monetary systems

With cryptocurrencies—built on blockchain—we still need: centralization, trust, regulation, governance, and a whole host of other things that are already in TradFi.

AD = Conduct disorder

Transaction reversibility requires trusted party

In order to mimic the capabilities in TradFi to make business decisions to reverse transactions, cryptocurrencies rely on smart contract systems and an anointed trusted party to achieve the same thing.

Ah, yes...that thing that was driving up hard drive prices a few years back.

Proof-of-Stake can replace Proof-of-Work

This is a big caveat here...the nature of the tech leads to a centralization of power that means "the rich tend to get richer." For the sake of removing the environmental consequences of consensus building, does it seem worthwhile to anoint a subset of users to get richer from the use of the tech?

digital social clubs

Se compararmos o Alaska, que tem 1.477.953 km 2 de extensão territorial, ao estado de Goiás, com seus 340.086 km 2, segundo o Instituto Brasileiro de Geografia e Estatística IBGE, vemos a grande diferença territorial dos dois. Vale ressaltar que no Alaska os invernos são extremamente frios, longos e o verão é muito curto. As temperaturas na maior parte do ano têm valores abaixo de zero com temperaturas mínimas, que variam entre -29°C no inverno e 2°C nos meses de verão.

A comparação com a temperatura nesses locais mais frios é um bom recurso didático, mas o mapa não parece ter nenhuma função específica: como a diferença de temperatura se relaciona com a diferença de tamanho entre os dois territórios?

Na quinta-feira, 19 de maio, os termômetros registraram 5ºC na capital Goiana -- enquanto no Alasca estava um pouco mais agradável, fazendo 6ºC.

O segundo dia de frio intenso em Goiás também fez o estado registrar temperaturas mais baixas do que no Alasca e outras cidades em que o clima frio é predominante. Sibirskiy, na Sibéria, estava em 11ºC, já em Oslo, capital da Noruega, o termômetro registrou 14ºC.

Para os Goianos acostumados com altas temperaturas, esse climinha frio é uma grande novidade. Durante esses dias em que a temperatura caiu, os Goianos precisaram tirar os casacos do armário e se readaptarem a um frio que não se via há muito tempo por aqui.

A foto ficou cortada. Poderia ter customizado o player ou encontrado uma imagem que não produzisse esse efeito.

Segundo ele, “em poucas horas os goianos têm a experiência de sentir o calor e o frio. Eles saem de casa com blusa para se aquecerem e ao longo do dia vão percebendo que a temperatura esquentou”. Ele explica que a umidade do ar vai na contramão dessa situação, pois cai bastante e chega a colocar o município em situação de alerta.

De acordo com o gerente do Centro de Informações Meteorológicas e Hidrológicas de Goiás (CIMEHGO), André Amorim, estamos passando por um período de estiagem, critico em várias situações que envolvem a questão da amplitude térmica.

Seria importante explicar o que é a amplitude térmica.

uma terra quente. Situada no cerrado, a capital do estado de Goiás registra com frequência temperaturas com mais de 30ºC e, em algumas épocas do ano, os termômetros marcam temperaturas mais amenas.

Neste ano, tivemos a madrugada mais fria em 45 anos da história da cidade: a temperatura chegou a 2,7ºC. Isso causou uma susto nos Goianos, que tiveram dificuldades para lidar com tanto frio.

Não se usa ponto final em título.

I totally agree that the Davidson metadata leaves a lot to be desired. From my experiences at the school, I've seen that the archivists are very focused on alumni as their audience and it reflects how they write their metadata. I too wish that the school would archive under the assumption that people seeing their work are not intimately familiar with the college. Even as a student, I don't know why it's called Quips and Cranks or what the Georgia Club is. If I remember right, that club photo contains racist elements and I think that Davidson often tries to minimize their racist past by not addressing it. I think by expanding the description as you said, the harm the school and white student body had on POC could be better addressed.

Diagrams as Code 2.0 • Simon Brown • GOTO 2021

"long-lived documentation" - "Think about diagrams as 'disposable' artifacts"

https://c4model.com/ C4 Model - model for visualizing software architecture; hierarchal dataset; Diagrams as abstractions, differing levels of technical details; think of it as zooming into maps at different levels * Level 1: Context * Level 2: Containers * Level 3: Components * Level 4: Code

Product Comparisions - Working with the DSL (Lite v. CLI v. DSL Editor): https://structurizr.com/help/dsl - Product offerings (Lite v. Cloud v. On-premise): https://structurizr.com/products

The DSL is rendering tool agnostic

Diagrams as code 1.0 - PlantUML, Mermaild, etc are input formats

Diagrams as code 2.0 - PlantUML, Mermaild, etc are output formats

Checkout: - Online REPL: https://structurizr.com/dsl?example=amazon-web-services - DSL Guidance: https://github.com/structurizr/dsl/tree/master/docs/cookbook - Examples: https://github.com/structurizr/examples

Invitation au design des cookies

Une fonction ?

Minimalisme

But somehow we can make some places more ordered? Like we are working against disorder? but like the world in general is still increasing in entropy?

More time actually doesn't mean you will do better, it might take away the sense of urgency so you end up doing worse

todo see the over-engineering zettel - add to this to it

Wow that example is horrifying but like people do tend to exploit metrics as much as possible, yet if you don't give them metrics but a vague goal, that doesn't really work as well because they don't know what to improve on.

It is kinda a problem that you need the metric to be very comprehensive - humans aren't like AI that can totally misunderstand... it is more that if you link that metric to some reward then people have to max the metric even if it means they are doing the wrong thing. e.g. cheating on exams (because it will determine your future...)

the problem isn't so much making the metrics as the artificial importance we place on the metrics

Work smart, not hard? Pick an easier solution to execute or problem that you can solve, you don't have to make things hard for yourself on purpose.

Actually if you consider that people can have very simple apps that perform one service and then lots of subs (e.g. readwise) then maybe you really don't have to do something complicated with AI just to get money

Science advances one coffin at a time - this is likely the limitation of the academic system, if one's work is linked to their career then people are encouraged to push agendas that are not necessarily the true best way but just the way they picked in the beginning when they were ignorant and new to the field.

Also like old biases perpetuate - see lack of male birth control pill. When you let elites control the research direction, the problem is that elites may not be representative and unbiased.

Not sure about this? Defining what is acceptable (to society) does actually coz unfair restrictions because it may not be a rule but societal pressure can cause some pretty horrific things. E.g. in ancient china, castration was normalized because you cannot have 'men' other than imperials in the palace (near consorts) (wait they had imperial guards?) but anyways if you say you cannot speak against the emperor as a societal thing... even things related to the emperor get hit with "no debate"

People will get used to whatever new tactics you used to get them to engage yet the software remains the way it was because when first met with that thing, it worked really well. Problem of lock in?

Not sure how wise the past wisdom is... kinda like history right ? we should learn from it yet we are not the people of the past (social science show that new generation vs old is diff) so what we can learn is limited.

I do feel like "The internet forgets" is a thing tho, or maybe when we look back after the emotional haze fades and realize that things weren't how we thought they were. Then again justice delayed is justice denied - people think they will change their perception once given information about that topic yet not really? they subconsciously retain - e.g. jennie lazy scandal

Basically if things don't make sense that is coz model of world is incorrect - not necessarily yours

But you should imitate people who you think are good at things you wanna do? Like that is how to develop style - so while you want to develop in an unknown niche that lacks competition, you also want to model what you do over what people have done and don't reinvent the wheel

Not sure if I agree but - interesting definition of genius and talent? Usually Talent → Genius is the assumption. I do get how society tends to stifle creativity with rigid education so we only really hyper focus on the goals that are given - great worker bee but not actually good at directing society? because the lack of creativity is a problem when you need to make leaps off data in a new way or need to think out of the box. It is just hard without instructions

Not sure I agree with this? Like you can have conflict because you are different too, but we might not choose to interact with people who are too different. Like we fight over our differences but we only interact with people who have some similarity to us (when do we even meet?)

I think this is similar to how I tunnel vision onto a solution for a problem and spend more time making the solution than I would have if I kept doing things the old way - but hey convenience and reducing friction is super important for making me keep doing it.

Ja!

Vielleicht ein komischer Vergleich, aber bspw. sollte auch m.E. Kriegsphilosophie keine eigene phil. Disziplin sein. Hier eignet sich auch ein Phänomen dafür, als phil. Feld erforscht zu werden.

Muss wieder an den Anime Film Belle denken.

Wenn, wie Jörg Noller sagt, die als digital unmündig gelten, die die Digitalisierung bzw. Digitalität als äußerliches Instrument verstehen und sie damit reduzieren - also reduktionistisch sind - dann läuft ein Großteil der Lehrer*innen und des didaktischen Diskurses Gefahr, digital unmündig zu sein bzw. Unmündigkeit zu verbreiten, wenn sie ihren Unterricht als digital gestützten Unterricht (miss-)verstehen. Digitalität ist keine Stütze und kein bloßes technologisches Phänomen, das lästig oder nützlich ist.

Kant

Keine Dualität, keine Dichotomie - Kollektive - sind es virtuelle Kollektive?

Finde in diesem Kontext auch die Betrachtung von click farms interessant

In diesem Sinne ist die Annotierung durch z.B. hypothes.is auch eine Erweiterung des virtuellen Handlungsraums. D.h. hier scheint mir auch eine gewisse Haltung, die die erweiternde Person hat, eine Rolle zu spielen. Ich könnte ja auch trollen oder fake-news verteilen, aber ich will am Inhalt sprechen, ich will die sachliche Diskussion.

Reicht die Verfügbarkeit des Internet für das Bildungsrecht? Ich bezweifle das mindestens im Bezug auf die aktuelle kommerzielle Geschlossenheit des Internets in großen Teilen. Es braucht Mündigkeit im Digitalen - es braucht digital literacies.

Das wirft auch Fragen auf, inwiefern Menschen, die an der Realität abseits von Digitaliät nicht partizipieren können (z.B. ÖPNV nicht leisten können), sich eine Partizipation durch autonomes Handeln in der Digitalität verbessern können? Oder ob hier eins gegen das andere ausgespielt wird

Wenn man dieser These zustimmt - ich stimme zu - dann hat das Konsequenzen für den Bildungsbetrieb und wenn das der Fall ist, hat das Konsequenzen für die Professionalisierung der Lehrkräfte. Und wenn das der Fall ist, kann die Forschung von und über PLN in diesem Bereich als Desiderat bezeichnet werden.

Hier sehe ich eine Stärke in dem philosophischen, begrifflichen Ansatz von Jörg Noller. Er geht auf transsubjektive und transzendente Aspekte ein - so kann das über das Bekannte Gehende begriffen werden, ohne dass man gleich den gesamten Humanismus transzendieren müsste.

Man sollte - denke ich - Qualia hier zwar auch betonen jedoch nicht überbetonen. Der Mensch macht ne Menge unbewusst, wir bestehen nicht nur aus innerlichen Erfahrungsqualitäten und Bewusstsein. Gerade Verhalten ist stark durch habituelle Aspekte geprägt

Ja!

Utilizar el enfoque interseccional no supone encasillar a las personas en categorías rígidas para poder reivindicarlas, sino entrecruzar las categorías de identidad para, a partir de esos cruces, analizar la discriminación que se da a consecuencia de la combinación de aquellas. La interseccionalidad permite entender y establecer el impacto de dicha convergencia en situaciones de oportunidades y de acceso a derechos

Kimberly Crenshaw (1989) presentó el concepto de interseccionalidad y señaló que hay categorías como la raza y el género que se interseccionan e influyen en la vida de las personas. P. ej. las mujeres blancas no experimentan las mismas situaciones de sexismo que las mujeres afrodescendientes. Por ende, la interseccionalidad no se trata de una suma de desigualdades, sino de cómo estas están conectadas (se cruzan) o se interseccionan de forma diferente en cada situación, persona y grupo social, lo que permite mostrar estructuras de poder existentes en el seno de la sociedad para cada caso (Expósito Molina, 2012)

Los autores mencionan que en este estudio quieren evidenciar cómo se manifiesta la doble discriminación (por ser mujer y ser indígena) en el caso de las mujeres indígenas que participan en espacios políticos de gobierno. Para lograr ese objetivo, en este estudio se utilizará el concepto de interseccionalidad, que es una herramienta para entender las manera en que el género se cruza con otras identidades, principalmente la indígena, y cómo estos cruces contribuyen a experiencias de opresión, discriminación y privilegio que forman parte del desenvolvimiento del rol de la mujer indígena en los procesos de toma de decisiones de los distintos niveles de gobierno en el Perú.

Video 5

block 数会大于 processor 数：所以 block 是个逻辑概念，会被调度到 processor上？多个 grid 又是什么概念？

Und hier drängt sich mir die Frage auf: Sind persönliche Lernnetzwerke die Instrumente oder der Lernmodus, der uns dazu bringt, in der Lage zu sein, diese Pflichten zu erfüllen??

Absolut, es ist selbst wiederum nicht einfach ein Annex - es ist mehr, da es im Allgemeinen menschliche Lebenswelt durchringt, bedingt und verändert.

Also auch um die Praxis?

Gut, dass Bildung in diesem Kontext genannt wird. Reicht das? Müssten diese Rechte nicht weitergehen? Sollte nicht die Autonomie oder Mündigkeit als Ziel gesetzt werden? Welche Rolle nimmt eine technische Bildung ein? Was sollte im Sinne der Autonomie in der Lebenswelt, im Handlungsraum der Digitalität an technischen Grundsätzlichkeiten gelernt werden - nicht inhaltlich festgelegt, sondern ein Wort zur Relation von technischem Know-How und anderen.

Evaluation Summary:

This is potentially an interesting paper in which extensive MD simulations are used to probe the effect of phosphorylation of a tyrosine residue on the conformational ensemble of Ras GTPase. The insights form the basis for a screen of small molecule(s) that disrupt interaction with its target Raf kinase, and predictions are tested experimentally. Overall, the integrated approach is of interest to a wide range of biochemist and protein scientists and could potentially be used to modulate the activities of other proteins.

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

Reviewer #1 (Public Review):

Using molecular dynamic simulations, the authors first explored the impact of phosphorylation on the structure and dynamics of G12D-Ras, a protein of great interest due to its involvement in various cancers. The results then motivated the authors to screen for small molecules that mimic the effect of phosphorylation, which perturbs the conformation of SwI and therefore the interaction with RAF. The prediction was then tested experimentally and shown to be correct. Therefore, the authors have established an approach that is potentially applicable to the modulation of other proteins for biomedical purposes.

Reviewer #2 (Public Review):

In the manuscript, the authors explored RAS-RAF interactions upon phosphorylation of Y32, mutation of G12D, and the ligand binding using molecular dynamics simulations. Their in-silico findings were validated by in-vivo cell assays successfully. The strength of this paper is that from both simulations and experiments, they could show molecular mechanisms underlying RAS-RAF interactions related to the above three factors, which opens the possibility of new anti-cancer drugs. In contrast, the weakness of this paper comes from the technical aspects of their molecular dynamics simulations. 2.5 microsecond simulation is fine as it is a standard simulation length nowadays. However, a few replicated simulations are required to evaluate the statistical significance of the main results. Actually, no statistical error is shown in all the main-text figures. Also, more quantitative analysis is required, if the authors want to discuss PMF (Figure 9, for instance). Not only SMD but also umbrella sampling is recommended to get more reliable PMF for ligand-bound and unbound states.

I consider that the insight from their MD simulations has sufficient impact on the HRAS research as well as anti-cancer drug developments if technically sound methods are applied to their computational studies.

Reviewer #3 (Public Review):

In their manuscript "Inhibition of mutant RAS-RAF interaction by mimicking structural and dynamic properties of phosphorylated RAS", Ilter, Kasmer, et al. search for druggable sites in the RAS mutant G12D in computer calculations, and verify their results by experiments. RAS is a major oncogene for various types of cancer and is notoriously hard to target with drugs. Any significant insight into how to find drugs targeting RAS mutants is therefore of high interest. The present manuscript tries to provide such insight, and the connection between simulation and theory appears sound, as the identified compound cerubidine apparently indeed blocks mutant RAS activity.

As I am an expert in simulations, but not in experiments, I will only focus on the presented computational part. In this function, however, I see some significant problems with the results: The data basis that the authors base their analysis on is quite small (only two simulations of 2.5 µs total simulation time), and from the presented data set I do not see any information on if the results on Y32 dynamics are anecdotal or reproducible. All presented distance distribution plots miss error bars/error ranges, as well as some time course plots that the simulations have indeed converged. So I cannot confirm whether the presented results are valid or if the authors were just lucky in their small data set.<br /> Furthermore, it might be that I have overlooked this information, but this work is not the first finding of druggable sites in RAS (see e.g. review of Moor et al., Nat. Rev. Drug Discov. 2020). The authors should include such a comparison in their manuscript. Especially the PMF presented in Figure 9 is erroneous, and all arguments based on this plot need to be discarded from the manuscript. From the Methods and Eq. (9), I assume the authors indeed use only the first two cumulants to calculate the PMF. The artificially low PMF with a difference of up to ~800 kcal/mol is a well-understood artefact (see Jäger et al., J. Chem. Mol. Model. 2022) that indicates the breakdown of the second-order approximation in Eq. (9) due to the presence of different pathways in the steered MD data set. This artefact overlays the PMF and obfuscates any information on the true free energy profile.

решили изучить

хорошенько изучить этот вопрос, прежде чем сделать выбор

не понимали

Evaluation Summary:

This work describes the function of N6-methyladenosine (m6A) modification in developing retina. Enriched scRNA-seq and MeRIP-seq data will be an excellent resource for neurodevelopmental community.

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

Reviewer #1 (Public Review):

The manuscript by Xin et al. investigated the function of N6-methyladenosine (m6A) modification in the retina. They conditionally deleted Mettl3, which is the key enzyme depositing m6A on mRNAs, in the mouse retina from the beginning of retinogenesis, and studied the consequences of Mettl3 deletion-mediated m6A depletion. They found that Mettl3 deletion led to disorganization of the retina structure at postnatal day 14 (P14), and claimed that Muller glial dysfunction was underlying these morphological abnormalities. They then focused on investigating how Mettl3 deletion affected late retinogenesis after birth and claimed that Mettl3cko retinal progenitor cells (RPCs) withdrew from the cell cycle slower than control RPCs. As a result, there was an increased number of Muller glial cells at P7. They then performed single cell RNA seq and MeRIP-seq to uncover the underlying molecular mechanisms. They claimed that Mettl3-mediated m6A modification promotes the degradation of RPC-related mRNAs, facilitates termination of retinogenesis and fine-tuning the transcriptomic transition from RPCs to Muller glial cells.

The authors performed a significant amount of analyses. But they didn't integrate and explain their data clearly enough for readers to follow. The logic behind the study is not strong. The lack of rigor, data inconsistency and confusing explanations further reduced the weight for this work.

Major concerns:

1. The authors showed disrupted retina structure in Mettl3cko and claimed that dysfunction of Muller glial cells was the underlying cause. However, based on the data presented in the manuscript, it is not clear whether retinal structure abnormalities are direct effects of Muller glial defects or secondary effects induced by defects in RPCs or other cell types.

2. The authors claimed that m6A is important for RPC to Muller glial transition but didn't clearly state whether m6A regulates gliogenesis, which can be easily assessed by quantifying the number of Muller glial cells. The authors showed that the number of Muller glial cells was increased in mettl3cko at P7 based on Sox9 staining, but the number of Muller glial cells in single cell RNA-seq data stayed unchanged between Mettl3cko and controls. In addition, the number of Muller glial cells in adult retinas (for example, at P14) was not examined. Based on images in Fig 1E, the number of Muller glial cells seemed to be slightly decreased compared to controls. Moreover, in line 130, the authors mentioned that Mettl3cko retinas resembled those with loss of Muller glia in the literature. These discrepancies need to be addressed.

3. The authors showed that there was an increased number of RPCs in Mettl3cko at late stages of retinal development compared to controls. However, some of the data do not seem to be consistent with published literature and their own supplementary data. Specifically, at P7, based on their single cell RNA-seq data, 6% of retinal cells were RPCs in control retinas, and 11% of retinal cells were RPCs in Mettl3cko (Fig 3-figure supplement 2). However, the number seems to be very high for control P7 retinas. In published single cell datasets (Clark et al. 2019), the percentage of retinal cells identified as RPCs was close to 0 at around P7. In addition, in Figure 3-Figure supplement 3, the authors stained P7 Mettl3cko and control retinas with Ki67. There was a very limited number of Ki67+ cells in Mettl3cko, which doesn't quite match the 11% found in single cell RNA-seq data.

4. The stage of the retina was not consistent across experiments. The single cell RNA-seq experiment was performed using P7 retinas, whereas MeRIP-seq used P6 retinas. Twenty-four hours could make a big difference in terms of transcription at this stage.

5. Many claims in the manuscript are not fully supported by the data. For example, the authors over-expressed candidate m6A-regulated genes in RPCs and showed that they prevented the RPCs from exiting the cell cycle. However, the data presented in Fig 6 cannot fully support this conclusion. PCNA is not a pan cell cycle marker. Reduction of GFP+PCNA- cells in Mettl3cko doesn't necessarily mean that mutant cells failed to exit the cell cycle.

6. Individual data points and N numbers were not shown in the bar graphs.

Reviewer #2 (Public Review):

In this study, Xin et al. investigated the role of m6A epitranscriptomic modification in the developing mouse retina. They found the structural disorganization and functional deficits of P14 retinae in Mttl3CKO mice. Combined immunohistological and single-cell transcriptome analyses showed that RPCs and Müller glia are subject to Mettl3 deficiency. Further integrative analyses of scRNA-seq and MeRIP-seq suggested the RPC-specific transcript degradation at the late stages of retina development. Finally, misexpression of specific m6A-regulated RPC-enriched transcripts at P1 could partially recapitulate the phenotype as Mettl3-deficient retinae. The finding is potentially interesting, but a direct mechanistic link remains inadequate.

Strength: The study clearly demonstrated the defects of Mettl3CKO retinae mice, including cellular disorganization and abnormal physiological responses. Enriched scRNA-seq and MeRIP-seq data presented here will be an excellent resource to study the function of m6A modification in retinogenesis.

Weakness: While identifying the essential role of m6A modification in gliogenesis by late RPCs is potentially interesting, the direct evidence remains missing mainly. Overall mechanistic exploration was conducted mainly at the populational level such that many results lose cellular specificity, which is critical to uncover the direct link between late RPC-specific m6A-modified transcripts and gliogenesis. Besides, some results seem inconsonant, which needs further clarification.

Findet Carsten Roeger auch, sogar für den Philosophieunterricht - siehe dieses Padlet

Volle Zustimmung - daher finde ich ja PLN so wichtig - unser Lernen nicht der Kommerzlogik zu unterwerfen, sondern selbstbestimmt, autonom, mündig unseren eigenen unvertretbaren Bildungsprozess zu gestalten, dazu möchte ich forschen, meine Gedanken dazu sollen am besten Aufschlüsse für die Professionalisierung von Lehrer*innen bieten - Wir müssen die Schule in der Digitalität aktiv denken.

Kritik an Nida-Rümelin: reduktionistische Tendenz - sehe ich auch so, ich vermute, dass es eine Überbetonung des Menschlichen als ursprünglich Menschliches ist, das zu dieser Tendenz führt und ich glaube, dass Latour hier wirklich helfen kann, indem er sagt: Technische Entwicklung funktioniert im Laufe der Menschheitsgeschichte spiralförmig (Technik X hilft bei Problem X, sorgt aber für Problem Y, dabei hilft die Technik Y, sorgt aber für Problem X1, dabei hilft Technik X1 usw usf. es sind die Übersetzungsprogramme der Techniken, die Inskriptionen und Präskriptionen an Entitäten vermitteln, die sie in Kontexte betten, in denen sie wiederum ihr nun verändertes Programm auf andere richten, wodurch sie Inskriptionen und Präskriptionen fordern, die wiederum den Prozess beeinflussen usw.) Wir lösen die prinzipiellen Probleme der Transhumanisten und die ihrer Fronten mit den Humanisten, indem wir zeigen: Der Mensch lebt immer schon mit anderen Menschen UND Dingen. Es ist immer ein Kollektiv. Ergänzend zu dem Kollektivgedanken von Latour finde ich den Gedanken von Jörg Noller, dass virtuelle Realität etwas Menschliches ist, hilfreich. Der Mensch lebt immer schon in Kollektiven UND in physischer SOWIE virtueller Realität - beides sind grundsätzliche menschliche Eigenschaften, die die Digitalität nicht neu entwirft, sondern in ihrer Rupturhaftigkeit nur mit Schlaglichtern versieht.

Ich glaube, dass ich einen ähnlichen Gedanken bereits als Notiz bei der Lektüre von Nida-Rümelins Text in Hauck-Thums und Nollers Was ist Digitalität notiert habe.

Stichwort Transsubjektivität

Wenn die Vergegenständlichung von Menschen ein Problem ist, dann sind auch die transsubjektiven und interobjektiven Tendenzen der Digitalität problematisch. Bzw. je nach unserem Umgang mit diesen Tendenzen kann es problematisch sein.

Lässt mich an Zimmerli denken - komplette Transparenz bedeutet ja Ununterscheidbarkeit und das kann nicht das sein, was uns interessiert

Lebensweltdurchdringung der Digitalisierung nennt man auch Digitalität

Dieser Diskussionspunkt ist interessant für die Klärung des Akteurstatus. Muss ein Akteur denken können, um Akteur zu sein? Latour würde nein sagen. Gegen die Kritik von Nida-Rümelin am Transhumanismus könnte man den Akteurbegriff der ANT - so denke ich gerade - ganz gut abgrenzen.

Digitaler Humanismus - der Versuch eines Mittelwegs zwischen Apokalyptik und Heilsversprechung

Acces la documentații

PT nu poate modifica DT

what does this mental health care capacity look like? How do we address the white supremacy that's the main drive for this

to keep guns away from people most likely to kill and not to regulate guns amongst all. what do potential killers look like?

UPDATED

UPDATED According to ECOI was published 3 March 2022. ECOI link: https://www.ecoi.net/en/document/2046539.html

UPDATED Not available as this is a constantly-updated page and no archived version of 10 May 2022 seems to exist - 24 May 2022 is closest: https://web.archive.org/web/20220526114553/https://www.cia.gov/the-world-factbook/countries/sri-lanka/

Nihiliste, va !

In two months we designed the lab, planned all constructional changes, briefed the construction workers and zoned all labs according to Formo's lab workflows.

remove this section and the image OR show a screenshot on a laptop frame of our Lab Selector

Add section here on everything that we did. Its a checklist for each sprint phase similar to what we have on the website and it will show everything we did here

What means combed? Dont know that word. Write instead "...searched the real estate market", found 80 locations and evaluated them on XXX criteria. A week later the favorite was Frankfurt, so the lease was signed a month later".

Build

Discover

Completed in under 6 month [from first contact to operational]

350m² Bioprocess Up- & Downstreaming

Version 4 of this preprint has been peer-reviewed and recommended by Peer Community in Zoology.

See the peer reviews and the recommendation.

This may be useful but need to know what the control group was and the method of non-operative management

Knowing know that the city was divided into 3 municipalities almost 200 years ago, gives insight into the wards and how they can be drastically different.

(en complément) Le démarrage de la prochaine session est prévue en janvier 2023

La photo ci-dessous ne convient pas car le CHESP se fait hors les murs, jamais en salle de classe. Changer cette photo

I think that this section sheds minimal light into what's going on today after each Hurricane. Many citizens say that New Orleans never recovered from Katrina and that the people who were most effected are not getting any help. Instead, they are building around it and not fixing the issue at hand.

The cookie _gc_lang must be "eng" or "fra"

The cookie _gc_lang must have a single text value.

The cookie _gc_lang must exist.

w

This is a roughly worded statement indeed - but I can see it as a way that people may talk. They are expressing concern & care - just not in so many words. It is possible more words were said - but from the angle of most children, often only parts of adult conversation would be taken note of.

also, calling him "old man" was a colloquial way people would call someone's father, they did not literally mean a person is old.

I saw this as the father's maladaptive grieving behavior. It did not reflect on the rest of the community as drunks though! Just this father.

I personally didn't read it as Natasha being seen as a feared voodoo person - rather I did see it as respect. It gave me the impression that Natasha was a forceful, no-nonsense woman who became an unofficial community leader due to the way she interacted with others. & sometimes this means children can indeed say they are a little afraid of someone.

I see this, interestingly, as how the figure of adulthood has become simplified in modern narrative. In Western culture today, an adult in a story who isn't always kind & solicitous would have to be seen as abusive... whereas in many cultures, including Western culture in the past (see for example Tom Sawyer & Anne of Green Gables), adults could be more complicated. Setting limits on others could be a harsh scolding. We all know that a completely rational, loving approach to discipline takes much more time and involvement - which is not a luxury most human societies had in their daily lives when it came to curbing the more destructive tendencies of their fellow community members. Throughout history, communities have had to police themselves in many different ways in order to maintain quality of life, not to mention survive.

In this story it appears to me that Natasha's husband was less perceptive to helping people by setting limits, as he took the children's father out to drink, when he knew that the father had a drinking a problem & was trying to be sober. And you could also see that Natasha was still respectful towards her husband enough that she did not impose her opinion of this on him - & the effect was that 1 person died, & his 2 children very nearly did.

It's a good point here that this writing can be interpreted as people drinking alcohol!

I wasn't thinking about it either way when I first read it, but I can see it interpreted both ways, so it is written a bit ambiguously. I can understand that this was written 'from a child's perspective' & thus would be more of a generalized impression, rather than being accurate or specific. I can definitely get the sense that the child felt as if the adults didn't care so much...

In fact, I got that impression myself as a child attending a grandparents' traditional funeral in rural Taiwan. Now I see it as what happens when traditional rituals are still upheld, but performed by a generation of adults who are a bit removed from the cultural context (having been somewhat "modernized"). So we see towers of fruits & canned foods wrapped in plastic & gaudy plastic ribbons, we see adults who are uncomfortable talking about emotions (though this is probably accurate throughout generations - people in many old cultures were generally more taciturn & pragmatic in their communication than people today) & in a performative setting (a ceremony with a lot of people attending, crowded & busy & full of music & incense & decor...etc) where they are also trying to be comfortable... in a time when funerals happen relatively infrequently as well...

I appreciate the reviewer bringing up the point that drinking alcohol in the community hall was never allowed. This info would be something useful to include in a reading guide for the book.

would this have been the practice back then? or was it a practice among some families?

Because these were people who were living in more settled lives, with western amenities, I saw this as a time period where the culture was changing & a lot of the things that used to be done were not being continued or passed down necessarily. It would be interesting to see if the homemade casket was more universal.

Moreover, a word can be adopted differently by peoples whose native language was not English, & then evolve later on - the difference between a casket & a coffin, for example, is not so clear to me as a non-native English speaker. This would be interesting to see how the English introduced to this region has evolved ... I would imagine generations of children quickly become more literate in English, with greater diversity of vocabulary that matches that of the English speaking areas of the country as more resources become available.

this told me that the father was more sensitive to crowds.

Code duplication

Is this import really needed?

It's not a nice message indeed if it were interpreted that way. But from understanding the diversities of families & individuals I thought it was a valuable example. In this same family in the book, the grandparents were mentioned as very supportive of the children going to school. It's quite useful for children to see how adults can be different & have their individual, sometimes complicated & unoptimized motivations.

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Reply to the reviewers

Reviewer #1 (Evidence, reproducibility and clarity (Required)): **Summary:** Techniques to probe the local environment of membrane proteins are sparse, although the influence of lipids on the membrane protein's function are known since many years. Therefore, the paper by Umebayashi et al. is important. The environment-sensitive dye Nile red (NR) coupled to a membrane protein is an appropriate sensor for monitoring the local membrane fluidity. Linking of Nile red to the receptor via a flexible tether was achieved with the acyl carrier protein (ACP)-tag method. Experiments showed that depending on the ACP site a certain linker length is required to have NR inserted in the membrane and thus be an effective sensor for lipid disorder. This technology could be of general usability to study the environment of membrane proteins in the context of their function. As an example, the technique allowed insulin induced membrane disorder in the close insulin receptor vicinity to be observed. Further, results suggested that tyrosine activity is required for this disorder to happen. The experimental results appear to be complete and controls were made.

**Major comments:** 1) Sometimes technical terms are used without explanation: What is the GP value? What is ACP-IR? The spectrum was measured in number of rois? The reader can find those abbreveations out, but it would be nice to have them defined.

We have made a list of abbreviations.

2) Fig. 1d) is confusing. The ACP-IR labelling is evident in 3 panels, but there is no difference in the color (emission spectra of 1992-ACP-IR vs 2031-ACP-IR should be visible??). The DAPI staining is very different. When doing the latter, how difficult is it to get the staining equal?

The differences in spectra cannot be seen because we used pseudo colors for display of the DAPI and CoA-PEG-NR staining. The reviewer’s comments about the unequal DAPI staining is correct. The reason for this is most likely that the cell membrane is unequally permeabilized by PFA treatment. As the point of this figure is just to show that the plasma membrane is labeled, dependent upon the expression of the ACP-tagged insulin receptor, we don’t think that the variable intensities of the DAPI staining is important. DAPI is simply used to indicate the position of the cells.

3) How can one interpret Fig. 4: a) Control goes over 4 frames, at 240" insulin is added, and 10 frames should show a fluctuation difference?

We showed 4 frames after control treatment that showed no significant change was observed by control treatment. We expected that clear changes would be invoked by insulin treatment in GP images, however these changes, while visible in the GP images, are difficult to see for the untrained observer. This is the reason why we used the ZNCC method in the subsequent figures to better visualize the changes.

1. b) A color shift from blue to green is visible after insulin addition. But it is faint - difficult to assess from the pseudo color scheme. What does 1000 pixel top/1000 pixel bottom mean in c). Is it an attempt to better visualize the fluctuation? It is difficult to recognize a difference before and after adding insulin. d) It seems that the kymograph set should show this. What is the color scale? Why is 3 so untypical, i.e., no change? Box 6 is also peculiar: the left side does not show a strong change upon insulin administration, the right side does. Why? We appreciate the helpful comments for improving our manuscript.

As pointed out, the change of GP value is extremely small before and after insulin addition, so it is difficult to fully visualize the change with normal pseudo-color expression. To deal with this, we adopted the following two methods to visualize minute changes.

1) Visualization of local changes of the statistical GP value showed by ZNCC throughout the time-lapse images (Fig. 6 and Fig. S2B).

2) Visualization of the top/bottom 1000 pixels of the sorting ZNCC value in each image (Fig. 7 and Fig. S2C). The top 1000 pixels are the ones that showed the largest changes. The bottom 1000 pixels are the ones that showed the smallest changes.

Owing to these expressions, we found out that the level of the response against the insulin signal was spatially and temporally heterogeneous in the membrane.

As for the color scale, in order to clarify the meaning of the difference of color, we have added the description about the relationship between the color and the ZNCC value in the results section.

4) How is the kymogram calculated? The legend says 'The horizontal dimension represents the averaged ZNCC inside the rectangular area, and the vertical dimension represents time'. The averaged ZNCC is a single value, so it is not clear why the kymogram shows a variation from left to right. May it be the ZNCC was averaged just vertically?

We apologize that we did not provide information regarding making the kymograph.

In the yellow rectangular area (Fig. 6B), the ZNCC values of the pixels with the same x coordinate value were vertically averaged, which were represented as the horizontal direction of the kymograph. That is, one horizontal line of the kymograph holds the spatial distribution of the ZNCC value along the horizontal direction of the membrane, and the vertical direction shows their time changes. To make it easier to understand, we refined the description about the kymograph in the legend of Fig. 6.

5) When calculating cross-correlation values on images, they need to be aligned. What fraction of the total image does the selected 19x19 box represent? As described, I imagine that a rolling CC over 19x19 pixels is calculated over an image from the time lapse series comparing it with the reference Iave(x,y). Compared to the 3x3 median filtered CP image, the ZNCC image should then be much more blurred??

Below we provide more information regarding the calculation of ZNCC.

Each local window for ZNCC calculation is set to a 19x19 pixels centered on every single pixel excluding the edges of an image. The ZNCC value calculated in that window is set to a center pixel of that area. After that, a new window centered on the adjacent pixel is set and calculate the new ZNCC. That is, the calculation window is slid throughout the image. Also, the calculated ZNCC value is not set to all the pixels of the window, but is set to only the center pixel of the window, so there is no blur effect like median filtering.

The figure below shows a schematic view of our ZNCC calculation.

Schematic view of our ZNCC calculation

**Minor comment:** On page 16 supplementary is not spelled properly.

corrected

Reviewer #1 (Significance (Required)):

The key point of this paper is convincing and the new technology appears to have a lot of potential. It can be applied to study membrane protein function in the context of its environment, the lipid bilayer.

Membrane fluidity measurements have been developed (e.g., using fluorescent probes like laurdan). However, the trick to link a probe like nile red by ACP technology to the insulin receptor and to observe its activity is quite new.

A most recent description of such a technology is in TrAC Trends in Analytical Chemistry Volume 133, December 2020, 116092.

This is an interesting review, but not directly impacting on our work.

**Referees cross-commenting**

All comments are constructive and important. The paper is important but needs to be amended as proposed.

Reviewer #2 (Evidence, reproducibility and clarity (Required)): **Summary:** In this manuscript, authors generated an ACP-attached Nile Red probe in order to specifically label Insulin receptor in the membrane. Owing to this specificity, one can measure the lipid membrane properties around a specific protein in the membrane. **Major comments:**

For the conclusions in the manuscript to be convincing, in my opinion, these additional data need to be added. Some of these are new experiments, and some are detailed analysis of existing data. The new experiments are not for new line of investigation, instead it is to confirm their statements and conclusions. The major point is the reliability of spectral shift. In usual environment sensitive probes, it is certain that they are in the membrane whatever is done to the membrane. However, when the probe is attached to a protein, it is not trivial to have the same confidence that the probe is always inside the membrane, and it is in the same plane of the membrane. 1992-ACP-IR is a good example; authors state that it binds to the protein outside the membrane, but when there is cholesterol addition and -maybe more interestingly- cholesterol removal, the dye still reacts and changes its emission (even PreCT changes its emission quite a bit at the 570 nm region). This is a clear indication of a change in localization of the probe upon some changes in the membrane. This implies that observed spectral shifts may not be due to lipid packing differences, but due to localization of the probes. For this reason, it is crucial to know where any environment sensitive probe localize in the membrane with respect to membrane normal, and this knowledge is more important for this probe. Related to this, the spectral difference upon insulin treatment and activation of insulin receptor could be due to changes in probe's localization in the membrane. Especially because authors show in Fig1e, the spectra can change depending on the probe localization. Relatedly, quantum yield of NR should be significantly different when it is inside vs outside membrane. Authors should show QY for 1992-ACP-NR and 2031-ACP-NR with different PEG lengths and upon insulin treatment.

We understand the logic of the request to measure the QY, since the QY of Nile red is much higher in organic solvents than in aqueous solutions, so it might be predicted that the QY of Nile red is higher in a lipid bilayer than when covalently bound to the protein in an aqueous environment. However, this argument depends upon the mechanism for the increase in quantum yield when going from aqueous to a non-polar solution. One possible explanation is based on the intrinsic properties of the dye under the two conditions. The alternative explanation would be that the dye would aggregate (be insoluble) in aqueous solution and therefore either not fluoresce or self-quench. In this case, we believe that the latter is the explanation because we and others have previously shown the turn-on properties of the probe when binding to proteins (SNAP-tag and others). It is not simple to measure QY in the cell under a microscope, but we have done something similar shown in supplementary figure 4. We labeled the three ACP-receptor complexes with PEG11-Nile red and co-stained with antibody to the Insulin Receptor. We then calculated a relative quantum yield. There were very little differences at all between the relative quantum yields, so we conclude that it is not the environment of the probe, which affects the quantum yield under these conditions, but the fact that it is covalently attached to a protein and incapable of forming aggregates. What distinguishes these constructs is the emission spectrum, not the quantum yield. In supplementary Table 2 we also did QY measurements in vitro and we could reproduce the increase of quantum yield by association with liposomes or in organic solvents. We tested whether non-covalent association with a protein would increase the QY by incubation with the lipid binding protein, BSA, in PBS. This was not the case, strongly pointing to the conclusion that it is the covalent association with the protein that increases the QY, not association with a protein. We believe that our demonstration of changes in fluorescent spectra with changes in cholesterol, large changes in fluorescent spectra with linker length for the 1992 construct and voltage sensitivity using patch-clamp prove that the Nile red is reporting on the membrane environment under the conditions we propose.

**Minor comments:** - Fig 1d requires quantification We do not agree on this. This is simply to show that the labeling is dependent upon expression of the relevant ACP-IR constructs. There is no detectable labeling of the control.

• Voltage sensitivity of different PEG length of 2031-ACP probe should be added. We have added this data in figure 2 panel E.

• Fig 3a graph should show all data points, not only bar graphs. Also, the band in 3a for +CoA-PEG-NR is dimmer than other bands, is it specific to this particular gel since quantification does not show any difference?

There is no significant difference- Fig 4d, colour code is needed.

Done

• Fig 5b and Fig3d are basically the same experiments in terms of control measurement, why is the difference in 3b is 0.04 GP unit while it is 0.007 GP unit?

We explain in the MS, but have improved the title of Y-axis in Fig.5 b graph so that the difference in what is plotted is clear. - Why is inhibitor data so noisy? We should discuss.

We don’t know the exact reason why inhibitor data is noisy, but we speculate that the actin cytoskeleton and phosphoinositide-dependent signaling could affect the membrane stability, and the membrane environment would be fluctuated in the presence of latrunculin B or PI3K inhibitor.

Reviewer #2 (Significance (Required)): Overall, this is a very useful approach, and this line of research will yield very useful tools to shed light on how lipids surrounding proteins can change their function. Major advance of the paper is the new chemical biology tool. There is also biological data on how insulin can change the insulin receptor's membrane environment which is contradictory to some old literature claiming that InsR becomes more "rafty" upon insulin treatment (e.g., PMID: 11751579).

If this type of tagging proves robust and reproducible (limitations and concerns listed above and below), it could be used by other researchers to tag their protein of interest and investigate the lipid environment around those proteins.

The downside of this method is that the probe requires ACP tag, a relatively less used tag than others in biology, therefore researchers interested in using this probe should have their proteins with ACP tag. Moreover, the linker length and ACP-tag position are quite crucial parameters (and probably should be optimized for each protein). Longer PEG lengths cannot report on changes efficiently (Fig3b), while shorter lengths are prone to artefacts as they can go out of membrane (Fig1 and Fig2). This might limit its widespread use.

The reason for using the ACP tag is that neither the SNAP tap nor the HALO tag working. The tethered Nile Red preferred to bind to the tqg rather than inserting into the membrane.

**Referees cross-commenting** I agree with all comments and concerns of other reviewers. I see the usability and potential of this new technology along with its limitations as all three reviewers pointed out.

Reviewer #3 (Evidence, reproducibility and clarity (Required)): See below. No concerns on any of these issues.

Reviewer #3 (Significance (Required)): **Critique:** This MS reports a proof-of-principle for using site-directed environmentally sensitive probe technology to assess the local membrane environment of a receptor tyrosine kinase (IR) upon activation. This technology addresses a major gap in our arsenal of tools to study the mechanisms of membrane signaling as the parameters of interest are biophysical parameters rather than purely biochemical ones. How to do this with spatial and temporal resolution is a major challenge. This study builds on previous work by the Riezman group that develops an extrinsic labeling system to tether Nile Red to specific sites on the ectodomain of a signaling receptor and then probe local membrane environments as a function of receptor activity. This is a carefully done study is well-controlled, is clever in design and is well-described. Although the major issues to which such a general technology could contribute involve intracellular (and not extracellular) event, the advances described will be of general interest -- particularly that local membrane order decreases when IR becomes activated. Specific comments for the authors' consideration follow:

**Specific Comments:** (i) As a general comment, the authors are measuring extracellular plasma membrane leaflet properties that may or may not translate to what is happening in the local inner leaflet environment. A general reader may well miss the significance of this. This point needs to be more explicitly emphasized in the Discussion.

This has been discussed in the revised version.

(ii) Why not treat cells with a PLC inhibitor to block PIP2 hydrolysis and ask if that inhibits membrane disorder. It is PIP2 hydrolysis/resynthesis that regulates the actin cytoskeleton at signaling receptors and this seems an attractive candidate for study.

There is a long list of attractive post-signaling events of the insulin receptor and how this works in different cell types that could be tested. We believe that this is beyond the scope of this study and we encourage others to do this.

(iii) The data acquisition time is at least 4 min which is long enough for activated receptors to be recruited to sites of endocytosis. Can the authors exclude the possibility that what they are measuring isn't reflective of such spatial reorganization? Does a clathrin inhibitor block the observed change in local membrane order for activated IR? We determined localization to AP2 adaptor containing clathrin coated pits at the cell surface and showed that during the time-course of the experiment that there is no significant change in co-localization or evidence for endocytosis (new figure 9). Therefore, we decided not to do the clathrin inhibitor blocking experiment because we believe that it could only lead to indirect effects.

(iv) Receptor activation is accompanied by other transitions such as dimerization, etc. Can the authors exclude the possibility that what they are measuring is related to changes in depth of insertion of the NR probe into the plasma membrane outer leaflet that is a consequence of IR conformational transitions associated with activation? This is highly unlikely given the fact that fluidification of the membrane environment is found with all length linkers. Given the intervals in increases in linker length on the 2031 construct, which is the closest to the membrane, it is very difficult to conceive that any of the ones larger than 5 PEGs restrict significantly the membrane insertion of the dye. **Referees cross-commenting**

I think we have a consensus opinion

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Referee #3

Evidence, reproducibility and clarity

See below. No concerns on any of these issues.

Significance

Critique:

This MS reports a proof-of-principle for using site-directed environmentally sensitive probe technology to assess the local membrane environment of a receptor tyrosine kinase (IR) upon activation. This technology addresses a major gap in our arsenal of tools to study the mechanisms of membrane signaling as the parameters of interest are biophysical parameters rather than purely biochemical ones. How to do this with spatial and temporal resolution is a major challenge. This study builds on previous work by the Riezman group that develops an extrinsic labeling system to tether Nile Red to specific sites on the ectodomain of a signaling receptor and then probe local membrane environments as a function of receptor activity.

This is a carefully done study is well-controlled, is clever in design and is well-described. Although the major issues to which such a general technology could contribute involve intracellular (and not extracellular) event, the advances described will be of general interest -- particularly that local membrane order decreases when IR becomes activated. Specific comments for the authors' consideration follow:

Specific Comments:

(i) As a general comment, the authors are measuring extracellular plasma membrane leaflet properties that may or may not translate to what is happening in the local inner leaflet environment. A general reader may well miss the significance of this. This point needs to be more explicitly emphasized in the Discussion.

(ii) Why not treat cells with a PLC inhibitor to block PIP2 hydrolysis and ask if that inhibits membrane disorder. It is PIP2 hydrolysis/resynthesis that regulates the actin cytoskeleton at signaling receptors and this seems an attractive candidate for study.

(iii) The data acquisition time is at least 4 min which is long enough for activated receptors to be recruited to sites of endocytosis. Can the authors exclude the possibility that what they are measuring isn't reflective of such spatial reorganization? Does a clathrin inhibitor block the observed change in local membrane order for activated IR?

(iv) Receptor activation is accompanied by other transitions such as dimerization, etc. Can the authors exclude the possibility that what they are measuring is related to changes in depth of insertion of the NR probe into the plasma membrane outer leaflet that is a consequence of IR conformational transitions associated with activation?

Referees cross-commenting

I think we have a consensus opinion

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Referee #2

Evidence, reproducibility and clarity

Summary:

In this manuscript, authors generated an ACP-attached Nile Red probe in order to specifically label Insulin receptor in the membrane. Owing to this specificity, one can measure the lipid membrane properties around a specific protein in the membrane.

Major comments:

For the conclusions in the manuscript to be convincing, in my opinion, these additional data need to be added. Some of these are new experiments, and some are detailed analysis of existing data. The new experiments are not for new line of investigation, instead it is to confirm their statements and conclusions. The major point is the reliability of spectral shift. In usual environment sensitive probes, it is certain that they are in the membrane whatever is done to the membrane. However, when the probe is attached to a protein, it is not trivial to have the same confidence that the probe is always inside the membrane, and it is in the same plane of the membrane. 1992-ACP-IR is a good example; authors state that it binds to the protein outside the membrane, but when there is cholesterol addition and -maybe more interestingly- cholesterol removal, the dye still reacts and changes its emission (even PreCT changes its emission quite a bit at the 570 nm region). This is a clear indication of a change in localization of the probe upon some changes in the membrane. This implies that observed spectral shifts may not be due to lipid packing differences, but due to localization of the probes. For this reason, it is crucial to know where any environment sensitive probe localize in the membrane with respect to membrane normal, and this knowledge is more important for this probe. Related to this, the spectral difference upon insulin treatment and activation of insulin receptor could be due to changes in probe's localization in the membrane. Especially because authors show in Fig1e, the spectra can change depending on the probe localization. Relatedly, quantum yield of NR should be significantly different when it is inside vs outside membrane. Authors should show QY for 1992-ACP-NR and 2031-ACP-NR with different PEG lengths and upon insulin treatment.

Minor comments:

• Fig 1d requires quantification
• Voltage sensitivity of different PEG length of 2031-ACP probe should be added.
• Fig 3a graph should show all data points, not only bar graphs. Also, the band in 3a for +CoA-PEG-NR is dimmer than other bands, is it specific to this particular gel since quantification does not show any difference?
• Fig 4d, colour code is needed.
• Fig 5b and Fig3d are basically the same experiments in terms of control measurement, why is the difference in 3b is 0.04 GP unit while it is 0.007 GP unit?
• Why is inhibitor data so noisy?

Significance

Overall, this is a very useful approach, and this line of research will yield very useful tools to shed light on how lipids surrounding proteins can change their function. Major advance of the paper is the new chemical biology tool. There is also biological data on how insulin can change the insulin receptor's membrane environment which is contradictory to some old literature claiming that InsR becomes more "rafty" upon insulin treatment (e.g., PMID: 11751579).

If this type of tagging proves robust and reproducible (limitations and concerns listed above and below), it could be used by other researchers to tag their protein of interest and investigate the lipid environment around those proteins.

The downside of this method is that the probe requires ACP tag, a relatively less used tag than others in biology, therefore researchers interested in using this probe should have their proteins with ACP tag. Moreover, the linker length and ACP-tag position are quite crucial parameters (and probably should be optimized for each protein). Longer PEG lengths cannot report on changes efficiently (Fig3b), while shorter lengths are prone to artefacts as they can go out of membrane (Fig1 and Fig2). This might limit its widespread use.

Referees cross-commenting

I agree with all comments and concerns of other reviewers. I see the usability and potential of this new technology along with its limitations as all three reviewers pointed out.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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Referee #1

Evidence, reproducibility and clarity

Summary:

Techniques to probe the local environment of membrane proteins are sparse, although the influence of lipids on the membrane protein's function are known since many years. Therefore, the paper by Umebayashi et al. is important. The environment-sensitive dye Nile red (NR) coupled to a membrane protein is an appropriate sensor for monitoring the local membrane fluidity. Linking of Nile red to the receptor via a flexible tether was achieved with the acyl carrier protein (ACP)-tag method. Experiments showed that depending on the ACP site a certain linker length is required to have NR inserted in the membrane and thus be an effective sensor for lipid disorder. This technology could be of general usability to study the environment of membrane proteins in the context of their function. As an example, the technique allowed insulin induced membrane disorder in the close insulin receptor vicinity to be observed. Further, results suggested that tyrosine activity is required for this disorder to happen. The experimental results appear to be complete and controls were made.

Major comments:

1) Sometimes technical terms are used without explanation: What is the GP value? What is ACP-IR? The spectrum was measured in number of rois? The reader can find those abbreveations out, but it would be nice to have them defined.

2) Fig. 1d) is confusing. The ACP-IR labelling is evident in 3 panels, but there is no difference in the color (emission spectra of 1992-ACP-IR vs 2031-ACP-IR should be visible??). The DAPI staining is very different. When doing the latter, how difficult is it to get the staining equal?

3) How can one interpret Fig. 4: a) Control goes over 4 frames, at 240" insulin is added, and 10 frames should show a fluctuation difference? b) A color shift from blue to green is visible after insulin addition. But it is faint - difficult to assess from the pseudo color scheme. What does 1000 pixel top/1000 pixel bottom mean in c). Is it an attempt to better visualize the fluctuation? It is difficult to recognize a difference before and after adding insulin. d) It seems that the kymograph set should show this. What is the color scale? Why is 3 so untypical, i.e., no change? Box 6 is also peculiar: the left side does not show a strong change upon insulin administration, the right side does. Why?

4) How is the kymogram calculated? The legend says 'The horizontal dimension represents the averaged ZNCC inside the rectangular area, and the vertical dimension represents time'. The averaged ZNCC is a single value, so it is not clear why the kymogram shows a variation from left to right. May it be the ZNCC was averaged just vertically?

5) When calculating cross-correlation values on images, they need to be aligned. What fraction of the total image does the selected 19x19 box represent? As described, I imagine that a rolling CC over 19x19 pixels is calculated over an image from the time lapse series comparing it with the reference Iave(x,y). Compared to the 3x3 median filtered CP image, the ZNCC image should then be much more blurred??

Minor comment:

On page 16 supplementary is not spelled properly.

Significance

The key point of this paper is convincing and the new technology appears to have a lot of potential. It can be applied to study membrane protein function in the context of its environment, the lipid bilayer.

Membrane fluidity measurements have been developed (e.g., using fluorescent probes like laurdan). However, the trick to link a probe like nile red by ACP technology to the insulin receptor and to observe its activity is quite new.

A most recent description of such a technology is in TrAC Trends in Analytical Chemistry Volume 133, December 2020, 116092.

Referees cross-commenting

All comments are constructive and important. The paper is important but needs to be amended as proposed.

Evaluation Summary:

This manuscript provides a comprehensive analysis of expression patterns and genomic features of long non-coding RNAs (lncRNAs) in the human developing gonad, using available single-cell RNA-seq datasets from both somatic and germ cells. Using multiple genetic strategies in an in vitro system of female germ cell differentiation, the study further shows a positive regulatory function of the LNC1845 lncRNA on its protein-coding neighbor LHX8, known to have a role in ovarian follicle development. This study has potential interest for reproductive biologists and for the non-coding RNA community.

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

Reviewer #1 (Public Review):

Using available single-cell transcriptomic data, Wang et al., annotated thousands of long noncoding RNAs (lncRNAs) in human germ cells. From their lncRNA data set, the authors focused on one particular lncRNA, lnc1845 located in the genomic proximity to the LHX8 gene that encodes a transcription factor essential for ovarian follicle development. Applying a set of different genetic approaches, the authors report that lnc1845 regulates LHX8 transcript in cis by modulating chromatin modifications.<br /> The manuscript in its current form consists of three parts: (1) analyses of the available single-cell RNA seq data to identify and catalog lncRNAs expressed in human gonads; (2) dissection of the molecular mechanism of action of one of the lncRNAs named lnc1845 (3) identification of a transcription factor that regulates expression of many of the identified gonad-specific lncRNAs including lnc1845.

Strengths of the study:<br /> - The study provides a useful and novel data set of lncRNAs expressed in human germ cells and can be a valuable resource for the community.<br /> - To dissect the regulatory molecular interplay between lnc1845 and its protein-coding neighbor LHX8, the authors applied high-end genome editing strategies using multiple complementary approaches to inactivate or overexpress lnc1845.

Weaknesses:<br /> - In a current form, the three parts of the manuscript look like independent studies put together in one paper, with the last part being the least developed.<br /> - The data sets of the lnc1845 functional part sometimes lack consistency i.e. looking at one but not another mutant allele by given molecular approaches.

Reviewer #2 (Public Review):

Wang et al. elegantly exploit single-cell RNA-seq datasets to question the putative involvement of lncRNAs in human germ cell development. In the first part of the study, the authors use computational approaches to identify and characterize, from existing data, lncRNAs expressed in the germline. Of note, the scRNA-seq data used were generated from polyA+ RNAs, and thus non-polyadenylated lncRNAs could not be retrieved. Most of the lncRNAs identified in the germ cells and in the somatic cells of the gonads were previously unannotated. While this increases the catalog of lncRNA genes in the human genome, further characterization is needed to determine which fraction of these newly identified lncRNAs represent bona fide transcripts or transcriptional noise.

Differential expression analysis between developmental stages, sexes, or cell types led to several observations: (i) whatever the stage of development, the number of expressed lncRNAs is higher in fetal germ cells compared to gonadal somatic cells; (ii) there is a continuous increase in the number of expressed lncRNA during the development of the germline; of note, a similar, although the more subtle trend is observed for protein-coding genes; (iii) the developmental stage at which there is the highest number of lncRNA expressed differs between male and female germ cells. While convincing, the significance of these observations is difficult to assess. However, the authors remain prudent with their conclusion and are not over-interpreting their findings.

Interestingly, integrating lncRNA expression to classify cell types led to the identification of a novel population of cells in the female germline that had not been revealed by protein-coding gene only-based classification. The biological relevance of this population, which cluster with mitotic populations, remains to be demonstrated. Finally, by examining lncRNA biotype, the authors could demonstrate an enrichment, in the germ cells, of the antisense head-to-head organization (in relation to the nearby protein-coding gene) compared to other biotypes. Whether this is different from the general distribution of lncRNA should be discussed.

In the second part of the manuscript, Wang et al focus on one pair of divergent lncRNA-protein coding genes (LNC1845-LHX8). To document the choice of this particular pair, it would be informative to have its correlation score indicated in Figure 3C. The existence of this transcript was validated using female fetal ovaries, and its function was addressed in late primordial germ cells like cells (PGCLC) derived from human embryonic stem cells (hESCs). The authors have used an admirable set of orthogonal approaches that led them to conclude as to a role for LNC1845 in regulating in cis the nearby gene LHX8. They further went on to identify the underlying mechanisms, which involve modification of the chromatin landscape through direct interaction of LNC1845 with a histone modifier. Among the different strategies used (KO, stop transcription, overexpression), the shRNA-mediated knock-down is the only one to specifically address the function of the transcript itself, as opposed to the active transcription. The result of this experiment led the authors to conclude that the LNC1845 RNA is functional, a conclusion that is reinforced by the demonstration of physical interaction between the LNC1845 RNA and WDR5, a component of MLL methyltransferase complexes. The result of the KD experiment is however puzzling as RNAi has been shown not to be the method of choice for targeting nuclear lncRNAs (Lennox et al. NAR 2016).

Overall the functional investigation is convincing and strengthened by the inclusion of multiple clones for each approach, and by the convergence in the outcome of each individual approach. The depth of characterization is also remarkable. The analyses of the mechanisms at stake are somehow less solid, as there is less evidence demonstrating the involvement of the LNC1845 RNA and its interaction with WDR5.

Altogether, this study provides a convincing demonstration of the role of a lncRNA on the regulation of a nearby gene in the context of the germline. However, to have a better understanding of the functionality of lncRNA genes in general, it would be interesting to know whether other pairs of lncRNA-PC genes have been functionally investigated in this context, where no function for the lncRNA gene could be demonstrated. Negative results are highly informative and if so, these could be included in the manuscript.

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Evaluation Summary:

This manuscript will interest a large community of molecular biologists studying translation and mRNA decay. The study provides a large-scale comparison of the roles of protein factors in No-Go Decay (NGD) and Codon-Optimality-Mediated Decay (COMD) in the yeast S. cerevisiae. A major strength of the manuscript is the direct comparison between one mRNA with a single strong translational stall and another similar mRNA with many slow translation sites (caused by changes in the genetic code). The analysis of both the factors that cause decay of these mRNAs as well as the ribosome states on the different mRNAs has the potential to reveal the molecular basis for the different mechanisms of mRNA quality control.

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

Reviewer #1 (Public Review):

In this study, the yeast haploid deletion library was screened for altered expression of a GFP reporter containing an array of rare Arg codons known to induce No-Go-Decay (NGD). Mutants lacking Slh1 or components of the RQT machinery (Hel2, Cue3, Rqt3) were found to decrease expression of this "CGA" reporter relative to an OPT reporter lacking the Arg codons, consistent with the prevailing view that increased collided ribosomes in these mutants will accelerate NGD. In contrast, mutants lacking Syh1, Smy2 (yeast orthologs of mammalian NGD factors GIGYF1/2) or Asc1 show elevated CGA/OPT ratios, indicating reduced NGD. Most, if not all of these factors were implicated previously in yeast NGD. After verifying the effects of the deletions of these genes, I believe by re-creating the deletions de novo, and examining reporter expression by flow cytometry and Northern analysis, they move on to examine double or triple mutants of some of these genes, as well as deletion of the CUE2 endonuclease implicated previously in NGD as a backup to the primary Xrn1-mediated pathway that comes into play when Slh1 is missing, using a different CGA-repeat reporter based on the HIS3 gene. These results support a role for Syh1 in NGD, and for Slh1 in reducing collided ribosomes in a manner that diminishes NGD. The most dramatic results involve a strong synthetic effect of deleting SYH1 together with either CUE2 or HEL2. This leads them to propose that Cue2/Hel2 and Syh1/Xrn1 represent two independent pathways for NGD, although they wish to conclude that Syh1 represents the major pathway in WT cells, and that Cue2 functions only when the ability of Slh1 to diminish collided ribosomes is overwhelmed. They go on to show that deleting MBF1 or EAP1 has no detectable effect on the NGD reporter, indicating that these yeast orthologs of EDF1 and (possibly) 4EHP, respectively, do not function in NGD in yeast in the manner observed for the corresponding mammalian factors. The next notable finding is that deletions of HEL2, SYH1, or both genes, which impair NGD, have no effect on a separate reporter (NONOPT), whose rapid turnover in WT cells is driven by a high level of non-optimal codons. Deletion of NOT5 strongly stabilizes the NONOPT promoter, as shown previously, but also stabilizes the CGA reporter to a degree comparable to deleting SYH1, providing the first evidence that Not5 contributes to NGD as well. Finally, they perform 80S and disome ribosome profiling on the two different reporters. In WT cells, they see a large accumulation of both monosomes and disomes at the CGA codons of the CGA reporter, and very low levels of either species downstream of the CGA block, and both monosomes and disomes are tightly packed and thus appear to be in collisions. In agreement with previous results, 80S monosomes escape the CGA block in cells lacking Hel2, and they see a loss of close-packing upstream of the block, from which they conclude that Hel2 helps to stabilize disomes and facilitate their removal from the stall site. On the NONOPT reporter they do not appear to observe a higher than average disome occupancy; however, they see an increased ratio of ribosomes with 21nt vs 28 nt footprints, indicating accumulation of ribosomes with empty A sites. Interestingly, they also observe a very high 21/28 nt ratio within the CGA repeats of the CGA reporter. From these results they conclude that ribosomes collided at a strong barrier to elongation (eg. CGA repeats) trigger both NGD and mRNA turnover elicited by nonoptimal codons (COMD), whereas COMD is triggered by slowly elongating ribosomes with empty A sites. The Not5 contribution to the turnover of the CGA reporter can be rationalized by the high-level of ribosomes with empty A sites that are arrested in the CGA array.

Reviewer #2 (Public Review):

Veltri and colleagues manuscript, "Distinct ribosome states trigger diverse mRNA quality control pathways" reports on a study designed to compare and contrast the proteins required for NGD and COMD and the ribosomal conformations associated with each pathway in yeast.

The study has many notable strengths, including a large-scale screen to quantitate the effects of deleting yeast genes on NGD and COMD. These screens use fluorescent reporters designed to trigger NGD (due to CGA and AAA repeat stall sequences) and COMD (due to a high frequency of rare codons). Positive hits from this screen were validated independently. In addition, the authors used careful Northern blot analysis to quantitate reporter RNA levels and turnover rates. Finally, the use of high-resolution, conformation-specific ribosome profiling on reporters correlated ribosome configurations to specific decay pathways. These powerful methods were combined with thorough and careful analyses. There are also weaknesses to the manuscript, including some sections that are not completely clear. There are also limitations in the genetic screen, which covered ~80% of viable gene deletions, and failed to identify many previously characterized COMD factors or any novel factors in either NGD or COMD.

i. Overall, the experiments described do provide a broad comparison of the proteins required for NGD and COMD and correlate ribosomal conformations with these decay pathways on specific reporter mRNAs. The authors conclude that the two pathways are largely independent, at least in yeast, and find that some of the components of human NGD do not have functional orthologs in yeast. These conclusions are well supported by their results.

i. The experiments are well executed and I expect this manuscript should have a moderate to strong impact on the fields of translation and mRNA decay. On the one hand, the conclusions are largely consistent with recent work. For example, Not5 has been reported to be the key link between rare codons and mRNA turnover in yeast COMD (Buschauer et al., 2020). Buschauer et al. also revealed that ribosome structures consistent with open A and E sites create the Not5 binding site. Similarly, the importance of Syh1 in NGD was reported by Hickey et al., by some of the authors of this manuscript, and previous work has shown the structures formed by ribosome collisions which induce NGD. The main impact of this manuscript is that it suggests the NGD and COMD pathways are largely separate, at least in yeast under rich media growth conditions, and identifies Syhl as a primary effector of NGD. This is important because both NGD and COMD are involved in coupling translation to mRNA decay. The authors also report that some components that contribute to human NGD are not involved in yeast NGD. The data used in the study also have the potential to be useful for future work, as they include high-resolution ribosome profiling data (21-mer and 28-mer monsomes and disome footprints) from wildtype and mutant yeast.

Reviewer #3 (Public Review):

The molecular basis for distinct mechanisms of mRNA quality control is poorly understood and an important problem in gene expression. In particular, the molecular mechanisms that act in response to a strong translation stall appear to differ from the mechanisms that act in response to continued slow translation. In this manuscript, a genetic screen revealed a quantitatively significant role for Syh1 in decay of an mRNA with a strong translation stall; similar effects of Syh1 on the same reporter had been reported in a 2020 publication from this laboratory. Here, they find that Syh1 function in mRNA decay does not require function of the major NGD regulator Hel2, of the NGD endonuclease Cue2 or of the ribosome disassociation factor Slh1. By contrast, none of these factors affect mRNA stability of a reporter in which translation elongation is likely uniformly slowed by suboptimal codons, but this reporter is a target of COMD (codon optimality mediated decay) and is stabilized by deletion of NOT5. The surprising result is that the strong stalling reporter is also regulated by Not5, in a manner quantitatively similar that of Syh1. Thus, mRNA stability of the strong stalling reporter is regulated by both NGD and COMD. To understand the molecular basis for recruitment of distinct decay factors, the authors investigate the ribosome states on these reporters using ribosome profiling. In the reporters without a strong stall site, single ribosomes and collided ribosomes are uniformly positioned across the coding sequence, while in the reporter with strong stall (due to CGA codons), ribosomes are absent from the region downstream of the CGA repeats and collided ribosome are substantially increased and stacked at the CGA repeat (compared to the OPT reporter). In addition, ribosomes lacking tRNA from the A site are enriched on the slowly translated reporter. The authors infer that the distinct ribosome signals of collided ribosomes or ribosomes with empty A sites are strong determinants of the factors that lead to mRNA decay.

A major strength of the manuscript is the direct comparison between one mRNA with a single strong translational stall and another similar mRNA with many slow translation sites (caused by changes in the genetic code). The analysis of both the factors that cause decay of these mRNAs as well as the ribosome states on the different mRNAs has the potential to reveal the molecular basis for the different mechanisms of mRNA quality control.

The genetic analysis is complicated and not completely consistent with the claim in the abstract that "Syh1 acts as the primary link to mRNA decay in NGD". While deletion of SYH1 does stabilize mRNA with a strong stalling site, the deletion of both SYH1 and genes encoding other factors known to be in the NGD pathway results in much greater stabilization of the mRNA with the strong stalling site. In the discussion, the authors correctly interpret this result as evidence of two independent mechanisms by which NGD is triggered. This is one of the most novel results presented in this manuscript, but is not followed up and leaves some questions about the primary role of Syh1 in NGD. Subsequent findings that neither Syh1 nor Hel2 are involved in decay of an mRNA encoded with non-optimal codons, that Not5 is involved in decay of mRNAs with non-optimal codons or strong stalls are convincing. The analysis of ribosome states on the same reporters in wild type and mutant strains provides clear and convincing differences in the ribosome states and distribution across the different reporters, which are likely to provide mechanistic insights into the distinct pathways. However, in the absence of any evidence of a causal relationship between these differences in ribosomal state and the difference in mRNA decay, the paper lacks sufficient support for the title "distinct ribosome states trigger diverse mRNA quality control pathways."

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