DOI: 10.3390/biomedicines12071612

Resource: (Cell Signaling Technology Cat# 12105, RRID:AB_2716685)

Curator: @scibot

SciCrunch record: RRID:AB_2716685

What is this?

DOI: 10.3390/biomedicines12071612

Resource: (Cell Signaling Technology Cat# 31718, RRID:AB_2687580)

Curator: @scibot

SciCrunch record: RRID:AB_2687580

What is this?

DOI: 10.3389/frdem.2023.1198006

Resource: IMSR_JAX:000664

Curator: @evieth

SciCrunch record: RRID:IMSR_JAX:000664

What is this?

DOI: 10.7554/eLife.96979

Resource: RRID:Addgene_12260

Curator: @evieth

SciCrunch record: RRID:Addgene_12260

What is this?

DOI: 10.1016/j.scr.2024.103507

Resource: (Abcam Cat# ab150121, RRID:AB_2801490)

Curator: @vtello

SciCrunch record: RRID:AB_2801490

What is this?

DOI: 10.1016/j.xpro.2024.103228

Resource: IMSR_JAX:000664

Curator: @vtello

SciCrunch record: RRID:IMSR_JAX:000664

What is this?

DOI: 10.1016/j.celrep.2024.114565

Resource: (Bio X Cell Cat# BE0015-1, RRID:AB_1107624)

Curator: @vtello

SciCrunch record: RRID:AB_1107624

What is this?

DOI: 10.1016/j.celrep.2024.114565

Resource: AB_1107634

Curator: @vtello

SciCrunch record: RRID:AB_1107634

What is this?

DOI: 10.1016/j.cmet.2024.07.005

Resource: AB_2338072

Curator: @vtello

SciCrunch record: RRID:AB_2338072

What is this?

DOI: 10.12688/f1000research.135151.1

Resource: SCR_011958

Curator: @vtello

SciCrunch record: RRID:SCR_011958

What is this?

https://danallosso.substack.com/p/science-of-reading-meeting-2

Images on this page do not open

To be clearer, it is probably better to specify how excavation strategy was changed. What was the old strategy, and what is the new one? Why such changes were made?

When the strategy changed, we decided to ....

https://www.antiquetypewriters.com/

Before making any business transaction or partnership with other companies, it is important to conduct your research or do a background check on potential business partners.

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It is unclear where the vessel is found, what "other elements" are, and what was excavated by A15.

an

"vessels," not "vessel"

Add "and" before "the."

"it seems". Add "it" before "seems"

When a sentence starts with the number of a feature, it is better to capitalize the letter at the beginning of the sentence. Similar situation applies below.

Perhaps it should be "a hearth" instead of "heart"?

"the roof" is better than just "roof."

not commensurable; having no common basis, measure, or standard of comparison.

A parish beadle is a minor official in a parish who has various duties, including keeping order at services and other functions, ushering, and sometimes acting as a messenger. They may also wait on the rector and punish petty offenders

“To wit” is a phrase that means "that is to say" or "namely". It can also be used to introduce examples for a more general statement, or to indicate that something is about to be described more precisely.

equivalent, as in value, force, effect, or signification

Latin phrase that translates to "of his own accord and on his own responsibility". It appears in Fear and Trembling by Søren Kierkegaard, where it is used to describe the infinite movement of resignation:

abbreviation for that is to say; namely (used especially to introduce examples, details, etc.): the two forms of energy that our society demands in enormous quantities, viz. electricity and liquid fuels.

"it concerns you"

embraced or adopted, as an idea, principle, or cause: There was an immediate negative reaction to his clearly espoused beliefs on the subject.

a formal expression of high praise; eulogy: An encomium by the president greeted the returning hero.

If you are reading this article, maybe your company incorporation in Singapore is done.

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Singapore's Companies Regulatory Agency released a new version of Annual Filing for Foreign Companies and Applications for Reliefs Under the Companies Act.

Read this article to guide you about the annual statements’ legal requirements, including the balance sheets’ preparation and lodgement of audited accounts for branches of foreign corporations, as well as requests for reliefs for local and foreign companies under the Companies Act.

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Trump Media Quietly Enters Deal With a Republican Donor Who Could Benefit From a Second Trump Administration by [[Justin Elliott]], [[Robert Faturechi]] and [[Alex Mierjeski]]

When stocks can become called "meme stocks" they cease to have actual value.

https://www.propublica.org/article/trump-media-truth-social-jedtec-james-davison-conflict-of-interest

the whole list needs a little redo

need to define these in the background and ml section

cite something

add dates here?

Should be "we found," instead of "were find."

https://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.memedroid.com%2Fmemes%2Ftag%2Fcognitive%2Btest&psig=AOvVaw06miNleLOFpV4DV_GEKaSq&ust=1722476651782000&source=images&cd=vfe&opi=89978449&ved=0CBEQjRxqFwoTCLjWyd2T0IcDFQAAAAAdAAAAABAR

This is what companies like Turian does as a part of their business model. Teaching the companies in an creative way how to think with design.

• Having said that how, what is so special about DT for business?

Medical Research Council

I found this study very interesting and creative! Fine-tuning the embedding space to account for structural similarity via contrastive learning seems like a wonderful idea and the results are very impressive. Here are some of my thoughts about your paper, presented in no particular order. Please feel free to take or leave any of my suggestions.

One advantage of CLAlign compared to structural aligners is that you don't need to calculate structures. However, the hardware requirements for CLAlign are probably non-trivial, since pLM embeddings have to be calculated. Hardware requirements are missing from the manuscript so it's hard to know. Relatedly, there is no information provided about the speed of CLAlign. I think the manuscript should be expanded to include detailed runtime statistics and hardware requirements so that CLAlign can be better benchmarked against the other tools.

While Table 1 gives us an overall picture of the alignment quality, it would be nice to know the tool's strengths and weaknesses. How does it perform when sequences are distant homologs? Or when there are large length mismatches? Since embedding-based alignments are state-of-the-art, this kind of information would be broadly useful for readers.

Figure 1 looks more like a draft than a complete figure. And without a caption it doesn't make sense.

The performance is very impressive, and it has me curious how much further the performance could be improved simply by increasing the epochs or training dataset. Visualizing the loss curve could help contextualize the performance and help readers understand the extent to which there is room for improvement.

Small notes:

• Throughout the manuscript, consistent reference to pLMs is made without any specificity. But there are many different architectures, e.g. BERT, T5, autoregressive, etc. I found this confusing.

• There are many grammatical mistakes. Consider passing the manuscript through a grammar checker.

Final thoughts:

Great work! I am curious to try CLAlign once it is made available.

Mover a la parte de investigación reproducible abajo.

Sería adecuado considerar...

Se recomienda...

varios

Aclarar o borrar. Hablando de que exporta trinos individuales en lugar de grupales y su estrutura de datos arbórea en lugar de tabular dificulta la exploración y el análisis.

El entorno reproducible permitió efectivamente continuar el trabajo en distintos computadores, con diferentes sistemas operativos y sin permisos especiales sobre las máquinas, incluyendo computadores domésticos, del tutor, del estudiante y del LabCI. En sentido dicho entorno permitió configurar una "nube personal académica" de baja complejidad tecnológica, que permitía mayor autónomia en la escritura y colaboración fluida, más allá de lo que permiten otras formas de escritura en línea más convencionales.

Aclarar o quitar.

Aclarar o quitar

Aclarar o quitar.

mas datos sobre las interacciones de los usuarios con un trino particular. Es decir ...

Aclarar o quitar.

In this case does s denote tuning to how results are classified when calling true and false positives and negatives? Or weight terms in the F-measure score it self?

Is this a vector with only a value at position j? So a vector of size N with only position j having a value set, hence being different than x_{j}(t)?

A simple camera could be positioned (perhaps attached to the head of the guitar) so as to view the hands moving over the fretboard. Perhaps visual methods which use commercial cameras to infer blood pressure could be used to estimate stress being applied by fingers [reference, reference]. Perhaps this would involve looking at changes in skin colour corresponding to blood flow restrictions resulting from stresses being applied. The apparatus described in this paper could be used to validate such an approach. Potentially the installation of a camera could potentially result in an apparatus more easily deployed at scale, and perhaps usable on many different instruments (without special fretboard modifications and such). Potentially it could also enable more dimensions of resolution, extending the one-dimensional limits of the described apparatus.

The term is Eulerian Video Magnification:

• https://www.youtube.com/watch?v=ONZcjs1Pjmk

Perhaps contemporary machine learning visual analysis could be applied also, and it would be potentially straightforward to record video data for training.

A more straightforward extension to the current study could be to apply visual analysis such that merely finger placements and gestures were identified to supplement the stress measurements. Indeed, visual analysis or motion capture approaches could be applied to video of the whole posture of the player for assessment of relationships between stress measurements across the fretboard and posture (such as sitting and standing), which can be significant.

Another complimentary measurement is audio. Stress/force sensor data could be supplemented with audio data analysis such as is employed by the likes of Yousician. A mentioned limitation of the apparatus is the measurement of vibrato effects/techniques. Could audio analysis supplement the existing apparatus's measuring capabilities to include such effects/techniques? Audio analysis can infer keys being typed on a keyboard, could it not also be used to infer frets being played, and with different forces applied?

Another form of sensor which may not have been considered is the Fiber Bragg Grating (FBG). Could this be feasible, perhaps in a specially-constructed fretboard? Would it add a greater resolution? Would it add a greater dimensionality to the stress measurements?

The characteristics of silicon strain gauges in terms of drift and hysteresis effects are well-understood and can be accounted for. Velostat foil can be used, but is perhaps more suited to cheaper, simpler applications like switches. The resistive sensors are dependent on temperature, but so are the light sensors used in the apparatus described.

Capacitive sensors are well-understood and potentially very cheap to make, e.g. they can be made from printed inks, beyond the more usual contemporary approach of Indium Tin Oxide (ITO). However, could the electrical fields of the strings be an issue? Although perhaps this could be understood using the Projected Capacitance Touch (PCT) approach with mutual capacitive sensors (for multi-touch sensors).

2 x ~$7,000

Apparently the smallest commercially-available 6-axis transducer, suited to robotic arms.

Couldn't this be higher? The TI MSP430 microcontroller family being used started in 1992. Still, it has good open source support.

Couldn't simple temperature sensors be added?

Use a non-breaking space.

Finite Element Analysis analysis

DC Comics, Detective Comics Comics

a fixed boundary condition against which the string can vibrate

What skill!

Style: For SI and NIST consistency, there should be a space between the numerical value and the unit symbol, here and in other cases in this document.

Could a clear definition of "correct" for this context be added?

Could more objective measurements of health improvements etc. be added?

Why refer only to careers? Why not refer to all musical instrument usage? The priority should be wellbeing, not economic productivity.

Technically perhaps this could be referred to as impulse-sensing, as impulse is force by time.

It might be good to mention this rationale/justification and thought process earlier in the preprint so people understand why the code isn't available right now.

Not sure if done on purpose, but this figure shows up really light and it's difficult to see some details

When the code is available, it would be good to link it in the abstract since this is a cool tool people will want to use!

https://www.youtube.com/watch?v=RKK7wGAYP6k

I watched this TED talk a while ago, and I thought that it connected well to this concept. It talks about how language shapes the way an individual thinks, and how different languages cause those who speak it to think differently. An example I find interesting is that a certain aboriginal group uses cardinal directions instead of right or left, and this causes them to think completely differently about distance and time than English speakers.

Don't capitalize

The final scene showing Draupadi as the rightful queen shows that good has beat evil in which justice has been restored. Even after many long years, the story shows that justice will always be reached and that cruel acts will face consequences. Draupadi's adversity is highlighted her as it is a personal victory for her and shows how her resilience and strength was able to prevail. The difference between "us" and "them" can be seen through the Pandavas and Kauravas. In which the Pandavas are seen as the good forces while the Kauravas are the embodiment of evil creating a good framework for the narrative as a result. This is undoubtedly the highest point in the narrative as the Pandavas are able to live out their former glory and are once again all-powerful and live in their kingdom. Not only does the ending of the story give closure, but readers gain a better understanding of morality and how human conflict is such a big factor in all of this. Because of the rivalry between the Pandavas and Kauravas, it led to lots of pain and suffering for the Pandavas until they finally waged war and were victorious. It serves as a lesson for readers to not be afraid of facing conflict as it might be the only and best way of handling it altogether. Draupadi as a person shows why resilience and strength are important qualities to have as they can help put conflicts to an end and not accept defeat. CC BY Ajey Sasimugunthan (contact)

This is turning point in the story because her husband is finally taking some action to enact revenge on the Kauravas for their cruel acts over the years. The image of her husband holding the weapon with so much anger can be felt by the reader and shows how much of the anger he has held over the years and is finally able to take it on the same guy who disrespected and humiliated her wife many years ago. Bhima's vow to break Duryodhana's thigh as a response to the disrobing of Draupadi is admirable and shows his determination and motivation behind the war that the Pandavas agreed to do. Not only does it show his honor, but serves as a huge sign of his loyalty to Draupadi and commitment to protecting her which propels the narrative forward. The collective effort of the Pandavas marks the unity of the group and how an attack on one individual is an attack on all of them and this is highlighted in the manner in which they fight the Kauravas with so much anger and anguish. CC BY Ajey Sasimugunthan (contact)

A key moment happens here where Draupadi once again questions the purpose of her husbands if they offer her no sense of security. In a day and age where people are much safer, women still expect security from men through money and even protection which shows that it is innate. Even with this being the case, her husbands have not shown the same loyalty to her showing that in their time period, husbands can do the bare minimum and can still expect the most amount of love and loyalty from their wife representing an unfair balance. Keechaka's attempts to force Draupadi into being his wife represents a systematic oppression towards women as there is no sense of decency and manners. By refusing to let Keechaka get what he wants, it shows the strength that she has and how she will not let men take away her dignity and respect. This moment not only shows how men in a patriarchal society failed to be protectors, but also highlights the strong spirit of Draupadi which makes her an admirable character. CC BY Ajey Sasimugunthan (contact)

Driven by envy and hate towards the Pandavas, Duryodhana devises a plan to make them suffer by utilizing the short temper of Durvasa who is also known for his curses. Because the Pandavas have a reputation for being hospitable, the plan was to ruin their reputation and have a curse placed on them as a result which results in more suffering and pain inflicted upon them. Even though the Pandavas had their backs against the wall, Krishna came to their aid and made Durvasa and his people feel full so that they would no longer need food from them. This marks another divine intervention in the story coming to the rescue of the Pandavas. The concept of dharma is on full display as the Pandavas' reputation for being hospitable and righteous still remain. Not only does their reputation remain intact, Krishna's loyalty to the Pandavas can be highlighted and shows that he protected the right people and that divine figures will intervene in human affairs to uphold righteousness in certain scenarios. CC BY Ajey Sasimugunthan (contact)

Filled with anger and anguish, the Pandavas are forced to live away from the comforts that they are used to and must face the harsh realities of existence. Krishna's presence after they have been exiled and gives a sense of assurance for them and makes them feel secure as they are in new territory that they are not accustomed to. He is a symbol of hope for their people. In a way, the exile helps bring the Pandavas closer as they have a common enemy that they all hate and want to avenge creating an even strong unity among them. While the exile does not compare to the moment when Draupadi was gambled away, this marks another low point in the story as the moral is still low for the Pandavas and they know it will be many years before they can inflict any type of pain or suffering onto the Kauravas. CC BY Ajey Sasimugunthan (contact)

This moment marks a pivotal moment in the story where the narrative is set to change significantly from this point and the fate of the characters are bound to change. For one thing, the husband using his wife as a wager shows how women were viewed at the time. The status of women at the time was merely property and eye candy to some extent. Because of her husband's actions, Draupadi is humiliated in front of a large group of people and has a lot of anger and resent towards the Pandavas for letting this moment occur. The gambling done by Yudhishtra puts his dharma into question especially as a king because he did not consider how his actions would affect his wife and showed his lack of compassion. Not to mention, this scene highlights the cruelty of the Kauravas to inflict the most amount of humiliation upon Draupadi and the Pandavas. There must be a lot of anger within them that will eventually lead to a war for revenge. Even though Draupadi as an individual being is receiving the worst amount of embarassment, the Pandavas see it as an attack on them as well showing the bond between their group and how they always stand up for one another. The vivid imagery through the art and diction creates empathy for Draupadi as she is helpless and her cries for help cannot do anything. This is the lowest point in the narrative and reader can tell without even reading the rest of the text because of the emotional intensity of the moment. Moreover, the moral implication of this incident will be massive and is very disturbing for the audience. CC BY Ajey Sasimugunthan (contact)

An interesting point has been reached in the story where they learn that Draupadi cannot marry them and belongs to the Pandava brothers. Arjuna's victory in using the bow and arrow symbolizes the difficulty in a challenge in which completion rewarded him with Draupadi as a wife. The garland symbolizes this victory and her acceptance of Arjuna as a husband. In their culture, garlands symbolize winners in a contest to show triumph and honor which is why Draupadi offered a garland to Arjuna after he won. It also shows the exceptional skill and courage and becomes a medal of honor while Arjuna is wearing it. In addition, Draupadi's willingness to be in a relationship with Arjuna is shown here and carries a sense of commitment and dedication. Arjuna's victory does not only help him win over Draupadi, but is also elevates his status within the Pandavas showing that they are a unified unit in which people can raise the ranks through accomplishments and skill. Individual goals in the Pandava group are aligned with their group's goal so it is a win-win situation for everyone. CC BY Ajey Sasimugunthan (contact)

The phrase "be careful for what you wish for" perfectly applies in this situation and becomes the first challenge and humiliation that she faces in this story. Because Shiva intervened as a higher power in her affairs, it continues to show that their society believed gods ultimately affected the daily lives of mere mortals and the best thing that they can do is to remain good people that remained faithful. Despite being innocent and pure, Draupadi's wishes were misinterpreted and shows why wishing for something is not always the best thing. It is all too common in shows, movies, and books to see a character wish for something but not receive what they want because of the wording behind that wish. This situation relates to Draupadi and will now affect the way everyone will view her and will be humiliating. Not to mention, the standards and views relating to love and marriage will be challenged when Draupadi has five husbands because the common belief is that people commit to only one person. Even though this is the case, the concept of arranged marriage comes into play as Draupadi must respect the decision made by Shiva putting into perspective why arranged marriages are still acceptable in India as parents and divine beings alike know what is best for their children. The idea of marrying five men challenges the western notion of monogamy and shows what practices were acceptable during that time period. CC BY Ajey Sasimugunthan (contact)

Draupadi's birth is nothing short of a miracle suggesting that a higher power had some influence on this event. It also alludes to the fact that their society believes that their gods effect their daily lives and can affect whether certain events happen or not at a moment's notice. The audience up to this point learns about the rivalry between the Kauravas and Pandavas which is foreshadowing. In addition, the Pandavas being disguised as Brahmins show the spot they are in and how they have been exiled up to this point. They are seeking a powerful son for that reason to help them be in a better situation. This highlights how important leadership is in society and can be the difference between whether a society lives in harmony or constant suffering. CC BY Ajey Sasimugunthan (contact)

Draupadi is immediately introduced as a character of importance especially in Hinduism and shows that she carried a unique position as a woman during her time. Despite having some privileges that made life easier for her, the contrast to the challenges she faced being in a patriarchal society balances it out and highlights how Draupadi was resilient during those times. The passage as a whole allows the reader to understand that she carries many characteristics such as wisdom and strength that women of her time are not accustomed to which will make this story more compelling as a result. Compared to other women of her time, she was embarrassed much more and was put in an interesting situation being married to multiple men but still remained strong for her people and was determined. Draupadi as a character also breaks a lot of social norms of India at the time and proves that women are much more capable than what their society believes. CC BY Ajey Sasimugunthan (contact)

This passage in particular highlights the finality of death and how Gilgamesh ultimately faces the same ending as other ordinary people. Despite the wisdom and courage that he had, it was not enough to overcome the fragility of human life. The imagery behind Gilgamesh "[going] into the mountain" suggests that he might be going to the underworld and shows that there is an afterlife after death. Another theme that a reader would think about is the meaning of life as Gilgamesh was unable to complete his main goal despite his accolades and characteristics. It does raise questions about the purpose of human existence and how people should view life and death. With this being the case, the ending and epic in general does a good job highlighting the fact that our time and existence is very limited which is why it is important to cherish every moment and make the most of it. Moreover, the ending of the epic does a good relaying the main message of the epic which is that human connection and contributions to society can provide a sense of purpose and fulfillment which makes life worth living despite everyone knowing that their time will eventually come to an end. CC BY Ajey Sasimugunthan (contact)

This quote really highlights how Gilgamesh's effort and quest in general for immortality ended up being a failure as he was not able to achieve this goal. It shows how people can put their best effort into accomplishing a goal and still not be able to achieve it which is seen through Gilgamesh. Many people that have succeeded talk about how hard work is not enough to actually reach success. There is some luck involved in being successful especially with ambitious goals and we can see that luck was missing in the case for Gilgamesh which led him to falling short of his goal. He is now at a position where he must reconsider his goals and priorities since the goal was the main focus in his life for a long stretch of time so it will be interesting to see how that perspective shapes as the story goes on. Not only must he reconsider his priorities, but he must evaluate his life since immortality is no longer a guarantee for him so that anxiousness exists once again and he must learn to accept that his time alive will be limited. The epic seems to suggest that our time being alive is better to be limited since the quality of life is more important the quantity or else life itself would no longer be enjoyable. CC BY Ajey Sasimugunthan (contact)

The catastrophic flood symbolizes a punishment for the people as they have committed a lot of sins and this is their way of making it up to the gods. With the phrase "smashed the land like a cup", it truly encapsulates the destructive nature of the flood and how the humans were so helplessness in that moment. We also learn that the gods are in fear of the flood because of how powerful it is highlighting the vulnerability that exists between both humans and divine beings. Water is always known to have significant meanings in many cultures which means that the flood is supposed to be a punishment for humans for their wrongdoings. There is a story in the Bible where God punished humans as he sees a lot of corruption and does this through a flood. It is interesting to see that parallel between both texts and shows that floods are universally seen as punishments for humans. CC BY Ajey Sasimugunthan (contact)

Gilgamesh's impulse and actions have made it much more challenging for him to be able to reach immortality. It shows how there are limitations in humans that leads to mistakes which is seen through Gilgamesh. He has been creating his own challenges up to this point and becomes even more apparent in this moment. This is also another important or even turning point in this epic as Gilgamesh is forces to realize that he depends a lot on others and this has been one of the biggest reasons for the consequences he has been facing. While this may be the case, it also highlights his resilience and persistence in achieving immortality as he remains strong willed despite the challenges he faced so far. This is similar to how bad habits tend to ruin some of the goals people have because it distracts them and makes them lose focus on what they want to achieve. CC BY Ajey Sasimugunthan (contact)

Gilgamesh's journey to the underworld highlights a common experience people face when loved ones die and shows a common archetype that can be seen in a lot of heroes between movies and books. He will set out to become a better person that lives up to his potential after a loved one dies which is seen throughout many movies, shows, books, epics, and other pieces of writing. As a result, he seeks to become immortal since he has a new fear of death. He has come to the realization that his life can perish at anytime and wants to have a lasting legacy and he believes this can be done through immortality. In addition, this desire allows him to not be forgotten and allows him to not worry about his legacy being forgotten. It is interesting that he feels this way as a lot of people usually face the idea of disbelief when someone dies and have a hard time coming to accept that. On the other hand, Gilgamesh has a hard time accepting death itself which is now causing him a lot of pain and suffering. CC BY Ajey Sasimugunthan (contact)

Gilgamesh's words for Enkidu shows his love for Enkidu and the deep bond between the two of them and how he was devastated by his loss. The repetition of "weep" highlights how deeply Gilgamesh is mourning for him and creates a haunting yet melancholic tone at the same time to enhance the meaning behind his message. Vivid imagery plays a role in enhancing the emotions that are felt and showing the impact of Enkidu's death. His death also seems to suggest that there is a interconnectedness between humans, animals, and the environment as it is common between all three groups and something that haunts all of us. Something interesting to think about is how people show a lot of appreciation for another person after they have died. It is similar to the phrase that people will not be fully appreciated until they have died. While Gilgamesh appreciated having Enkidu as a companion and said nice things to him, it was not on this level and shows an inherent human flaw which is the inability to fully appreciate and recognize someone's efforts and qualities until they are gone. CC BY Ajey Sasimugunthan (contact)

Enkidu's death carries a lot of significance and shows the consequences of the decisions that both Gilgamesh and Enkidu made. For one thing, the death shows how fragile human life is and how it can easily be taken away at a moment's notice. The grief and sadness that Gilgamesh holds as a result of the death shows the effect that death can have on humans and relationships alike. We lost a part of ourselves when close ones die and the emotional toll is highlighted through Gilgamesh. It is similar to how people wonder why they are okay with putting so much time, resources, and love for pets when you know that they will die before you and that will cause a lot of pain and emotional suffering. The text itself seems to teach us that it more about the quality spent rather than the amount of time spent between loved ones. Not to mention, the power of words can be highlighted in this section as Enkidu's curses show the destruction that words hold and the reasons behind the current consequences up to this point. CC BY Ajey Sasimugunthan (contact)

The relationship between Gilgamesh and Ishtar is very compelling and challenges many ideas that we have seen before in other pieces. Gilgamesh was the one to reject Ishtar after she proposed her love for him especially being a powerful goddess which may have come as a big surprise and slap in the face for her. It challenges the gender norm that men are usually the ones to propose their love and the women are able to reject the man if they choose to. This is a very interesting take on that idea and shows that men should also be respected if they choose to reject a woman and that women can also step up and propose their love if that is how they feel. Not to mention, the idea of love is viewed differently between both people as Gilgamesh seeks more of an equal and Ishtar views it as possession over the other person. This is why the recounting of the previous lovers she has can tell a lot about Ishtar and why she is not a suitable partner for Gilgamesh. In a day and age where people are constantly looking for something better especially with dating apps, the epic reminds the audience to reject someone because of who they are as a person and if the qualities suggest that they might be a bad partner. CC BY Ajey Sasimugunthan (contact)

At this point in the story, Enkidu and Gilgamesh come across their first challenge together and it was facing off against Humbaba. Their teamwork and trust in each other is fully evident and shows how much they rely on one another leading to their eventual victory. An interesting symbol is the cedar in this moment. The falling of the cedars can be seen as a symbolic victory over nature and Humbaba who is protecting the forest. This can be seen that way because they gain resources but also disturb the natural order that existed before them. Enil's anger is another interesting point to mention because it shows that the victory should not have happened and that possibly the gods had other plans in mind. People talk about the idea of destiny and whether their lives have already been planned out or if people have full control over their destiny. The take here appears to argue that humans can control their destiny but the gods typically have a plan in place that is usually followed in most circumstances. CC BY Ajey Sasimugunthan (contact)

Gilgamesh and Enkidu learn more about the Cedar Forest and the use of imagery helps the reader understand that is a sacred place and has lots of reverence. We can see some of the fear in the fact that they are in an unknown place and learning as they go along their journey. The mix of courage and fear from both characters show the bond that the two of them has created. Gilgamesh, the person who was complained about frequently has become better and has respect for Enkidu as he trusts him and relies on him throughout this journye and also listens to what he has to say when navigating through the forest. This shows why it is important for people to have friends or mentors that they respect because those are the people who can sway their opinion or change them for the better. Respect is crucial in any relationship and it can be seen here between Enkidu and Gilgamesh. CC BY Ajey Sasimugunthan (contact)

Gilgamesh is undergoing an interesting point in the story where he is starting to figure out whether he wants to have a legacy that will be remembered or just die altogether. It shows the theme of human mortality because people at some point eventually think about whether their life had a positive impact on others leading them to feel panicked. While Gilgamesh is contemplating and thinking about this, Enkidu serves as a good friend by his side offering support and advice for him. He serves as a good example as to why it is important to have the right people close to us as they can make challenging times feel a lot better and provide crucial support when it is needed the most. CC BY Ajey Sasimugunthan (contact)

Before Enkidu can face Gilgamesh and present himself as an equal, the reader can see how Enkidu must transform and changes himself in order to meet certain criteria in order to accomplish this goal. The harlot is responsible for changing Enkidu from being a wild being to someone who can be civilized and proper in their society. In a similar way, children grow up learning societal expectations and what is normal so that they can easily adapt and be accepted by others. It shows the importance of having a teacher or someone like our parents to guide us so that young children can be good members of society and we can see this through Enkidu. CC BY Ajey Sasimugunthan (contact)

The reader immediately figures out that Gilgamesh is an oppressive leader and tends to abuse his power. There are many rumors and people often talk about his poor leadership and how he takes advantage of others. As a result, people call for the gods to create a change in hopes of changing his behavior. Enkidu is created as a result to be an equal to Gilgamesh and keep his power in check. It reminds me of why checks and balances exist within the US government. If each of the three branches of government were not able to keep each other in check, then the President might be able to get away with some things that may result in bad consequences as a result. In a similar fashion, Gilgamesh does not have any checks and balances in which someone keeps him in check resulting in the people suffering as a result and why Enkidu is also created as a result. CC BY Ajey Sasimugunthan (contact)

The tone of this prologue suggests that Gilgamesh is a very powerful and important figure and places him at a high stature. Using a word like "perfect" to describe Gilgamesh further asserts the fact that Gilgamesh is highly respected and the rest of the story will highlight these strong qualities and present him favorably. It shows the divine nature of the hero of this epic and serves as a testament to Gilgamesh as a figure. In addition, the reader learns a lot about the storyteller and understands the weight that their tone carries. CC BY Ajey Sasimugunthan (contact)

Separate into 2 sentences.

hello

A major theme is that of the shifting of identities and disguises in society.

Peevishly means "with irritable discent or petty bad temper." It can allude to the perception against Orsino's intentions towards Olivia, and how Olivia's household sees an insistence which bothers her. It can also allude to a sense of competition as Malvolio will later find himself competing for Olivia's attention.

real

i am a fan of billie's music

39:39 ... damit hast du dein eigenes System zerstört,<br /> das kannst du nicht mit Dummheit irgendwie rechtfertigen, das ist Vorsatz.

ideologische subversion. psychokrieg. vorstufe zum heissen krieg (november 2024?)

der ewige kampf zwischen gnostik und mystik, wissen und ignoranz, wachsein und schlafen...

cool; i lived in houston

i watched this with music in the background nice

I enjoyed reading this chapter. I think it's a good addition to our OER.

I have attributed that quotation to Karl Weick (retrospective sense making).

reply to u/DinoPup87 at https://new.reddit.com/r/typewriters/comments/1efzeor/adler_tippa_id/

It's a common misconception that the database lists all serial numbers.

You'll need to identify the make (and preferably the model) to search the database. Then you'll want to look at the serial numbers which your serial number appears between to be able to identify the year (or month if the data is granular enough) your machine was made. Reading the notes at the header of each page will give you details for how best to read and interpret the charts for each manufacturer. Notes and footnotes will provide you with additional details when available.

You can then compare your machine against others which individuals have photographed and uploaded to the database. Feel free to add your typewriter as an example by making an account of your own. Doing this is sure to help researches and other enthusiasts in the future. Don't forget photos of your manual, tools which came with your machine, your case, and original dated purchase receipts if you have them.

This resource is interesting, long (69 slides), and technical. I have used h5p in my large-lecture human communication class, but I have access to the JMU Libraries' license, so I don't have to pay for it. Faculty who don't have access to that license may not want to pay for their own license.

Is this the name of a textbook that Long, Minervini, and Gladd wrote?

This is a really helpful prompt for students to use.

These prompts would be very helpful in Communication departments that offer Business and Professional/Technical Writing.

syntel. synthetic telepathy. i suspect they can read and write our thoughts and feelings.<br /> it would explain why so many people are so stupid and self-harming. its assisted suicide.<br /> wifi (2 to 5 GHz) is bad, but the power grid is the worst, because the low frequencies (50 or 60 Hz with harmonic overtones up to 20 KHz) are impossible to shield, so really the only solution is to get off grid, as far away as possible from their "civilization".

Chances are that you've got your original metal spools, and if so, definitely keep them. You can make a quick measurement, but I'm guessing you're going to want 1/2" or 13mm wide universal ribbon.

You can buy this in many places and in various color combinations (if you have a bichrome machine—look for a black/white/red switch which can usually be found on the front of your machine) for just a few dollars for 16 yards or about 14 meters to fill up a 2 inch diameter spool. Often it will come on cheap universal plastic spools which you can use to wind onto your own original metal spools if necessary.

Some machines often make use of proprietary mechanisms or geometry on their spools to effectuate the auto-reverse mechanism of the machine (though you'd have to check on your particular unit). Many machines after the 40s used small grommets on the ribbon itself to trigger the auto-reverse mechanism. If yours doesn't, you can trim these off with scissors as you spool the ribbon onto your machine if you're worried they'll get in the way.

Some smaller ultra-portables can and often do use smaller diameter spools which only fit 12 yards of ribbon, but you can always cut your ribbon down from bigger spools if necessary.

A few good sources of ribbon can be found at https://site.xavier.edu/polt/typewriters/tw-faq.html#q1.

If you don't have the original spools and the cheap plastic universal ones don't work on yours, you can find replacements via https://www.ribbonsunlimited.com/ or by calling around to repair shops which may have extras https://site.xavier.edu/polt/typewriters/tw-manuals.html

Incidentally, having your typewriter make and model as well as serial number can be helpful. You can often identify the model via https://typewriterdatabase.com/ if it's not on your typewriter directly. I'm guessing from the 2Y5852 that you've got a Good Companion No. 2 circa 1942, but you can track that down by looking at the database and individual galleries with photos.

If you don't have one already, you might find a manual for your machine (or one very similar to it) at https://site.xavier.edu/polt/typewriters/tw-manuals.html

reply to u/Fancy_Temporary_5902 at https://new.reddit.com/r/typewriters/comments/1eg176q/im_trying_to_id_a_typewriter_of_my_dads_as_it/

Several summers ago, one of my students turned in a paper about John Bowlby's attachment theory. The student "cited" sources from the North Dakota Journal of Speech and Theatre. I was dubious, but I did check the archives and those citations were fake.

This section seems to be aimed at students, not faculty members. The information is useful, but let's aim it at our audience (faculty members).

developed provisional guidelines (caps unnecessary).

I don't know what this is. We need some kind of preamble or explanatory paragraph to tell readers what the two links are for.

Case: Korean, Male

DiseaseAssertion: OTCD

FamilyInfo: n/a

CasePresentingHPOs: HP:0003623, HP:0001987, HP:0003218 (Neonatal onset, Hyperammonemia, Oroticaciduria)

CaseHPOFreeText: Elevated plasma ammonia concentration of 856 umol/L (Normal range 10-35 umol/L), Normal plasma citrulline concentration of 18.9 umol/L (Normal range 10-45 umol/L), Elevated urinary orotate of 3677.4 mmol/mmol creatinine (Normal range 0.3-6 mmol/mmol creatinine)

CasePreviousTesting: OTC Mutation Analysis, In Vitro Expression Study

Variant: c.853del (p.(Gln285ArgfsTer4))

ClinVarID: n/a

CAID: n/a

gnomAD: n/a

I think this will be a very useful resource for faculty members.

I learned the meaning of a new word today. According to the Merriam-Webster dictionary, this word may be more familiar to British readers than American readers.

and what makes these ideas a priori valid? from a historicist point of view, these are nothing but conditioned prejudices.

Claro, porque a galera que é contratada para ser atendente tem preparação pra atuar como segurança.

Sem contar que eu duvido que tenha adicional de periculosidade (ou insalubridade). Já me surpreende ser regime CLT.

Os caras não meteram essa. Não é possível.

Porque uma portinhola de vidro cumpre sim o papel de um segurança. Confia.

https://economia.uol.com.br/noticias/redacao/2022/07/06/oxxo-mercado-de-proximidade-memes-expansao-sao-paulo.htm

https://record.r7.com/hoje-em-dia/videos/funcionario-de-mercado-e-baleado-durante-assalto-na-zona-sul-de-sao-paulo-11012023/

Me lembra o episódio de Last Week Tonight sobre Subway.

This chapter seemed . . . truncated. I don't know what it's missing, but I would have difficulty understanding the purpose of this chapter.

I highlighted Khan, but I did so because I wanted to note that this will be a very useful chapter in our OER.

I am not sure what CW stands for.

Do we want to use APA style (7th edition) here? If so, the citations would need to be tweaked.

• ok
• [7] !!! to be published !!! (search for it)

• Hardy's

• Hardy's

-

-

hello

Professor Mendell taught digitial humanities, an english credit, at TAMU. I enjoyed the course and she introduced me to this extension.

Enough with the exclamation marks. Use a period instead here.

Use "analyze" instead of "analyse."

Again, do we want to use the original spelling (endeavour) or the American spelling (endeavor)?

Do we want to use the English spelling of this word or the American spelling (center instead of centre)?

https://imgflip.com/meme/533936279/Bell-Curve Iq often times is used to say whether or not someone is intelligent and how they view something, which does not always work since people with high or low iq can still be diverse in how they view things, besides iq does not always mean that someone is “smart” or “dumb”

Key take-aways: scalability of instruction maintained by outlining a curriculum that can be used department-wide, democratic assessment processes, and anti-deficit-thinking. Inspiring article, but intentionally left open-ended so libraries do their own work on this.

100 bullen im einsatz gegen eine "antidemokratische" eismaschine,<br /> damit "demokratische" messerstecher im hintergrund ihre arbeit machen können.

人权不能作为干预南方国家发展的借口

人权的追求是在建立我们想要的社会

权利与政治之间的关系，人权可以保护尊严

人权运动的核心：同情+对侵犯个人权利的不公正感

用自然自由获得公民自由，并拥有权利，这是卢梭rousseau说的

二战后人权概念建立，人们不止可以通过law来judge，还可以通过social norm等来judge，变成了一个可以审查的标准

人权的两面性：好的——保护每个人的尊严；坏的——保护罪犯的利益

政治中的人权牌

unclear on what this means in this context

1:13 die Menschen die den Weg nach Deutschland finden, kommen meistens aus Ländern in denen die Bevölkerung nichts zu melden hat, wo die Bevölkerung leidet, hungert, keine Perspektive hat, Arbeitslosigkeit, und alles Negative was man sich so vorstellen kann, was in so einem Land passieren kann.

naja, der einzige unterschied zu deutschland ist, deutschland hat ein sozialsystem, finanziert von geraubtem "steuer" geld, das menschen belohnt fürs nichtstun. dieses sozialsystem MUSS sterben, also importiert man millionenfach sozialfälle, bis das sozialsystem endlich kollabiert, und endich wieder ein freier markt herrscht. sozialsystem produziert immer parasiten, die keiner braucht.

Referenced by Tim

https://www.youtube.com/watch?v=SQAidyYqfnI

https://www.youtube.com/watch?v=uf8zQmqe97g

for

بدوي

Does it matter where the database is created?

It might be helpful to have a brief explanation of what Password Depot is.

Use Password Depot to record all of the passwords you use for your Lexsys and SAP accounts.

Deactivate multi-factor authentication on your old device (the user isn't deactivating the device itself)

change the value of variable

Is there any way to get screenshots of these instructions from SAP? (There might not be).

It might be helpful to have a list of the everyday programs the user will need to install, along with links to their corresponding pages in the help portal.

The virtual desktop's toolbar?

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

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Reply to the reviewers

The work would have significant impact in the cilia community, if the conclusion is correct. This reviewer, however, has a concern about the authors concluding the presence/absence of TZ, based on only B9D1 and the H-shaped body among nine doublet microtubules. First, is it really established how the structure of Xenopus embryo TZ is? While Chlamydomonas is well known to have a H-shaped TZ, other species have different form inside the 9+0 doublet, or no feature (Comparison of TZ from various species in Dennis Diener https://doi.org/10.1016/B978-0-12-822508-0.00007-1). Fig.2B of this manuscript shows visible densities in the panel "Pre", but it does not look like an H-shape. The tomogram of TZ before deciliation seems clearer (but judging from wavy MTs and membrane in this tomogram, there could be unevenness of embedding and staining), while the tomogram after deciliation is thin and does not cover the entire width. Therefore it is not sure that absence of TZ can be concluded. If the author claims Xenopus embryo cilia have a H-shaped TZ, they have to provide multiple micrographs (ideally tomogram or serial section TEM to cover the entire TZ structure) and/or past literature on Xenopus embryo TZ. B9D1 is likely a membrane associated protein (according to their deciliation by detergent and mechanical force). This may mean B9D1 is located on or near the membrane, in vicinity to TZ, and thus binds to TZ after the main part of TZ is built. In this case, it is risky to judge presence of TZ based on B9D1. Also in this point, TEM imaging will be helpful to confirm the authors' conclusion.

RESPONSE: We appreciate the reviewer’s thoughtful comments on the loss of TZ upon deciliation and its absence during the initial regeneration period. The reviewer is right in their assessment that the TZ of Xenopus cilia has not been well defined before in any manuscript. We want the reviewer to consider that our goal was not to define the TZ in Xenopus but to study deciliation and how cilia regenerate in a vertebrate model system for the first time. We unexpectedly discovered that cilia are deciliated distal to the basal body at the plasma membrane, and the “H-shaped structure,” similar to TZ, was also removed and did not come back for first hour during regeneration. Given this surprising observation, we felt obliged to study and explain our results. To that end, we explored different resources (antibodies and markers of TZ) and different methods over 6 years trying to define TZ in Xenopus.

Our conclusion about the TZ structure came from multiple lines of evidence from our experiments and published literature, including the similarity in structure compared to other organisms and its physical location in the cilium. Specifically, 1) In a review of the basal bodies, Mitchell indirectly suggested that the electron-dense “H-shaped” structure could be a TZ in Xenopus. 2) The electron-dense “H” shaped structure in Chlamydomonas is similar, if not identical, to that shown in Xenopus cilia. 3) The physical location of TZ is always shown to be distal to the basal body and transition fibers (except in clubmoss Phylloglosum) while proximal to the central pair. The electron-dense “H-shaped” structure in Xenopus fulfills these criteria, suggesting that this structure is the TZ in Xenopus. 4) The TZ bonafide protein B9D1 is localized distal to Chibby, which labels the distal end of the basal body, suggesting that the TZ is localized distal to the basal body. Moreover, the loss of an “H-shaped” structure determined using TEM and tomograms corresponds to the loss of the B9D1 signal, further strengthening the conclusion that the H-shaped structure is the TZ.

We will include serial sectioning and imaging of multiple Xenopus cilia in control and 0hr (after deciliation) to address this reviewer's concerns further. Our preliminary data has suggested that the ciliary membrane is tightened around this electron-dense structure, similar to what has been shown before for other organisms like Chlamydomonas. and thus boosts our confidence that this structure likely corresponds to the TZ in Xenopus.

The reviewer has raised a concern that “the tomogram after deciliation is thin and does not cover the entire width. Therefore, it is not sure that absence of TZ can be concluded”. We note that even if the tomograms do not go through the entire cilium (supplementary videos 2 and 3), it does go through more than the center of cilium as seen by the presence of central pair microtubules and we can observe that the electron-dense “H-shaped” structure is not present in these cilia. Further, in the supplementary videos 5 and 6, even if the tomogram again only covers half of the cilia, we can see the presence of the structure, confirming that our tomograms can demonstrate the presence or absence of the H-shaped structure confidently. We have also provided TEM sections in addition to the tomograms to show the same result.

The Reviewer has commented that “B9D1 is located on or near the membrane, in vicinity to TZ, and thus binds to TZ after the main part of TZ is built”. This reviewer is correct in their assessment. This is why we argue that the presence or absence of B9D1 may be a good marker for understanding the presence or absence of TZ assembly.

TIMELINE: We are performing additional serial TEM in the control and deciliated (0hr.) embryos to address the reviewer’s concern. We will need 1 month to finish these experiments.

Their discussion about length/number of cilia and force generated by cilia is interesting, but in the context of this research, this reviewer is skeptical about its value. The calcium induced deciliation is not a physiological phenomenon, but an artificial event (please correct if I am wrong). The argument how length and number of cilia are regulated upon deciliation makes sense only in case deciliation happens regularly and the species must optimize themselves to survive. The argument about possible passway of protein transport to control ciliary number and length (Line408-) seems, although it is an interesting topic in general, not suitable in this manuscript. For this reviewer's view, it is relatively straightforward to interpret the result of cilia number/length under normal growth, without new protein expression (CHX), with protein degradation blocked. Cilia will extend when components are provided. Growth will slow down when it is exhausted. Existing cilia start degrading, when they lack proteins, which are necessary for turn-over. With the current experimental output, there is no point to describe redistribution of proteins.

RESPONSE: We appreciate the reviewer’s comment; however, we would like to argue that different methods of deciliation have been used in different model systems, such as Chlamydomonas, to study cilia regeneration. Although this reviewer may not find some of the experiments and conclusions appropriate for this manuscript, other research groups have found these results interesting. For example, reviewer 2 states, “To support their observations that cilia length is favored over cilia number under conditions of limiting ciliary precursor availability, the authors use a mathematical model that leads to the conclusion that force generation is optimized by increasing cilia length. This is a convincing conclusion and in agreement with other comparable modeling studies performed in the field.” We have already had great discussions about these results with many cilia researchers at multiple conferences. Therefore, we prefer to keep these experiments and results in the manuscript and let readers come to their own conclusions about their importance.

Minor points:

Line65: do they mean "selected few basal bodies"? – we have removed the word “select”

Line73: extracellular flow is not limited to developmental system. – we have altered the statement to add “growth, development and homeostasis”

Line124: alpha-tubulin signal and SEM image – we have added “and scanning electron microscopy (SEM)”

Line139: Could you define explicitly the two hypotheses? – Now, we have reworded the sentences to clarify the two hypotheses. “Therefore, we considered two hypotheses: First, Xenopus MCCs regenerate cilia or second, Xenopus depend on stem cell-based replacement of damaged MCCs.”

Line164: 10,31-33 are not suitable citation for the location of calcium induced deciliation in Chlamydomonas. cite Sanders and Salisbury JCB 108, 1751 – We have changed the citation.

Line181: Later -> latter – We have changed the text.

Line195: by mechanical shearing, B9D1 remained with cilia. They concluded that TZ stays with the axoneme by deciliation. How can they exclude the possibility that mechanical separation works differently from calcium shock? – We do not intend to claim that both calcium-based and mechanical ripping of cilia from cells adopt the same deciliation mechanism, and we have mentioned in line 193 that ‘we adopted an alternative approach of mechanical deciliation’. Using these two methods as complimentary to each other, our aim was to show that TZ is lost by both ciliation methods. For the calcium method, because the membrane is ripped with detergent, we show the loss of TZ by examining the MCCs devoid of cilia. In the mechanical deciliation protocol, since no detergent is involved, we can examine cilia that are likely to have intact membranes and thus maintain a B9D1 signal.

Line214: 1.33uM -> 1.33um - We have made these changes to the text.

__RESPONSE: __All the minor points in the manuscript are addressed.

Overall, the results are well presented and allow strong conclusions to be drawn. The results are based on both immunofluorescence studies and EM analysis. To support their observations that cilia length is favored over cilia number under conditions of limiting ciliary precursor availability, the authors use a mathematical model that leads to the conclusion that force generation is optimized by increasing cilia length. This is a convincing conclusion, and in agreement with other comparable modeling studies performed in the field. It would be fascinating to be able to measure the flow parameters at the cell surface during cilia regeneration to see whether this regeneration actually leads to an increase in the overall flow or force generated by the cilia. But as the authors explain, this is probably a difficult experiment to carry out and appears to be optional in the context of this study.

__RESPONSE: __We thank the reviewer for recognizing and stating that “the results are well presented and allow strong conclusions to be drawn”. We also want to sincerely thank the reviewer for understanding the technical difficulties in performing these experiments.

The authors are apparently only able to detect a single TZ protein, B9D1, to follow the fate of the TZ during the deciliation and reciliation process. In some ways, this provides an incomplete demonstration that all the TZ is indeed removed during deciliation, although this is supported by EM observations. It also provides a limited understanding of the time course of TZ re-formation during reciliation. Given the limitations of antibody availability, could it be possible to express tagged proteins in the animal cap system to track more TZ proteins? In particular, would it be possible to track for example Cby and NPHP proteins. What is the behavior of Cep290? This would greatly reinforce the conclusions on the molecular reorganisation of the TZ after deciliation and during cilia regeneration.

__RESPONSE: __We appreciate this reviewer’s brilliant questions on understanding the time course of TZ re-formation during reciliation. When we started this project and observed that TZ was lost upon deciliation in our preliminary TEM experiment, our first goal was to confirm this outstanding result. Thus, we did more TEMs and EM tomography, used bonafide TZ protein B9D1 to label the structure, and observed its loss upon deciliation. Taken together, we feel highly confident that TZ is lost upon deciliation. To address this reviewer’s concerns, we will performing additional serial TEMs to confirm the loss of TZ after deciliation.

Our next goal was to understand what the reviewer has mentioned, the TZ assembly time course. We started with TEMs at different time points and again saw a surprising result: TZ assembly was delayed compared to cilia axoneme. We were driven by this question of understanding how cilia “put together” the complex structure of TZ structurally and molecularly using EM and fluorescence data. We first attempted a few antibodies, including B9D1, CEP290, MKS5, and NPHP4, to localize to the TZ in the Xenopus cilia. Despite our efforts with different fixation strategies, only B9D1 appeared to localize to the TZ, whereas others did not give any signal or localized at the basal body. Next, we tried localizing TMEM216, TMEM67, and NPHP4 using fluorescent tags, but we again found the same result: they localized to the basal body but not at the TZ. We are perplexed by this result and are pursuing the reasons behind them. However, these experiments are out of the scope of this paper. We want to note that we have used Chibby in our experiments and that it is not lost upon deciliation (Fig S1). This is because Chibby is a distal transition fiber protein (distal end of basal body) and does not extend up to the transition zone.

TIMELINE: To address the reviewer's concern, we are performing additional serial TEM in the control and deciliated (0hr.) embryos. We will attempt to localize CEP290-GFP, requiring approximately 1 month to finish the experiment. However, we would like to note that we cannot guarantee that this experiment will work, as similar experiments with other TZ markers have failed before.

Minor comments

1. Figure 4: The images are poorly defined, and it is difficult to distinguish individual basal bodies and cilia. Therefore, it is not clear how the authors can confidently quantify the number of basal bodies in each condition to construct the graph at the bottom of the figure. In addition, it would be interesting to label the basal body with a centriolar marker to better define it. - Figure 4 labels the Transition Zone protein B9D1 and cilia marker acetylated tubulin and not basal bodies. The graph represents the number of cells with the presence or absence of elongated B9d1 signal.

2. Figure 5: not clear why the graph on the lower right does not include the control at 3 and 6 hrs? Is it because the number is too high and difficult to quantify? – Yes, the reviewer is right. Cilia become too long and too many to quantify their number reliably.

3. References: I would like to draw the authors' attention to studies of deciliation in Paramecia that could be cited in the introduction or discussion of the conservation of this pathway through evolution. – We have added multiple references to paramecia throughout the manuscript. Specifically, we mention that deciliation and regeneration in unicellular models like paramecia have added to our understanding of ciliogenesis. Line 102 “While it is important to remember that regeneration of cilia may not be identical to de novo assembly, cilia regeneration studies in Chlamydomonas reinhardtii, Paramecium and Tetrahymena etc., have provided significant insights into ciliogenesis, g., cargo transport, the presence of precursor pool, regulation of ciliary gene expression.18,23–26”. Further, we also added the reference to paramecia in results, line164 “Next, we determined the location where the deciliation treatment severed cilia. Unicellular models such as Chlamydomonas, Paramecium and Tetrahymena lose cilia distal to the TZ and below the central pair (CP) microtubules33.”. We also add discussion on the importance of TZ in paramecia, line 203 “Interestingly in Paramecium also a unicellular multiciliated cell, displays constant shedding of cilia when TZ proteins are depleted.25”. These statements have been supported by the following studies that are now cited in the manuscript: Machemer and Ogura 1979 Journal of Cell Physiology (10.1113/jphysiol.1979.sp012990) and Gogenddeau et al., Plos Biology (10.1371/journal.pbio.3000640).

RESPONSE: All the minor points in the manuscript are addressed.

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Referee #3

Evidence, reproducibility and clarity

The manuscript by Rao et al. focuses on determining the mechanism of cilia regeneration using Xenopus mucociliary epithelium. The authors employ a simple yet powerful approach to trigger deciliation of multiciliated cells, enabling them to study the mechanism of cilia regeneration. This research has a significant impact on the field of cilia biology and enhances our understanding of ciliopathies. Through detailed cell biological methodologies, the authors obtained intriguing results, including the finding that deciliation removes the transition zone and that cilia repair precedes the transition zone assembly. Additionally, the authors demonstrate that IFT proteins involved in cilia construction concentrate at selected basal bodies. Although there are open questions that the authors also highlight, this manuscript provides solid, pioneering insights into the process of cilia regeneration in vivo.

Significance

The manuscript characterizes the mechanism of cilia regeneration, providing new insights into processes that could be harnessed to restore ciliary function in patients suffering from chronic respiratory diseases.

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Referee #2

Evidence, reproducibility and clarity

Summary

This manuscript investigates how cilia regenerate in multi-ciliated cells. The authors have exploited an original multi-ciliated cell system derived from the Xenopus embryonic cap and use chemical and mechanical deciliation to understand the different steps of cilia regeneration. In this model, they show that cilia are excised just above the BB and below the ciliary transition zone. Their results indicate that during ciliary regeneration, axoneme reassembly precedes TZ formation and that ciliary reassembly relies on de novo protein synthesis. In the context of limited protein synthesis, cells regenerate fewer cilia, but of almost the same size as control cells, suggesting the existence of a cell control system to maximise force generation. Mathematical modelling of the forces exerted by defined numbers of cilia of different lengths supports this hypothesis.

Major comments

Overall, the results are well presented and allow strong conclusions to be drawn. The results are based on both immunofluorescence studies and EM analysis. To support their observations that cilia length is favored over cilia number under conditions of limiting ciliary precursor availability, the authors use a mathematical model that leads to the conclusion that force generation is optimized by increasing cilia length. This is a convincing conclusion, and in agreement with other comparable modeling studies performed in the field. It would be fascinating to be able to measure the flow parameters at the cell surface during cilia regeneration to see whether this regeneration actually leads to an increase in the overall flow or force generated by the cilia. But as the authors explain, this is probably a difficult experiment to carry out and appears to be optional in the context of this study.

The authors are apparently only able to detect a single TZ protein, B9D1, to follow the fate of the TZ during the deciliation and reciliation process. In some ways, this provides an incomplete demonstration that all the TZ is indeed removed during deciliation, although this is supported by EM observations. It also provides a limited understanding of the time course of TZ re-formation during reciliation. Given the limitations of antibody availability, could it be possible to express tagged proteins in the animal cap system to track more TZ proteins? In particular, would it be possible to track for example Cby and NPHP proteins. What is the behavior of Cep290? This would greatly reinforce the conclusions on the molecular reorganisation of the TZ after deciliation and during cilia regeneration.

Minor comments

Figure 4: The images are poorly defined and it is difficult to distinguish individual basal bodies and cilia. It is therefore not clear how the authors can confidently quantify the number of basal bodies in each condition to construct the graph at the bottom of the figure. In addition, it would be interesting to label the basal body with a centriolar marker to better define the basal body.

Figure 5: not clear why the graph on the lower right does not include the control at 3 and 6 hrs? Is it because the number is too high and difficult to quantify?

References: I would like to draw the authors' attention to studies of deciliation in Paramecia that could be cited in the introduction or discussion of the conservation of this pathway through evolution.

Significance

The mechanisms of deciliation and re-ciliation have mostly been studied in protozoa (Chlamydomonas, Paramecia) or in primary ciliated cell cultures. Only a few studies have described deciliation in multiciliated cells, such as sea urchins, or physiological deciliation in the oviduct. The Xenopus deciliation system described here has already been used to determine the dynamics of IFT proteins during ciliogenesis or to define the ciliary proteome. In this study, the authors go one step further by describing more precisely which part of the cilium is shed upon induction of deciliation and the dynamics of the recruitment of the Tip and of the TZ proteins.

This study provides a completely new perspective on the deciliation process:

1. the authors show that, contrary to what is generally accepted from protozoan studies, the deciliation process, in Xenopus multiciliated cells, expels the TZ, leaving only the basal body in the cell;
2. While ciliogenesis is described in various models to begin with the formation of the TZ, in this Xenopus system the TZ maturates after the onset of axonemal elongation, calling into question the precise function of the TZ in axonemal elongation. The observations could be further strengthened by analyzing more TZ proteins to better understand the time course of events involved in the deciliation-reciliation program.

The protocol used to deciliate Xenopus multiciliated cells has been described in previous manuscripts. Its use here reveals striking differences in the deciliation-reconciliation pathways from what is known in the field. It provides new conceptual perspectives for researchers working on the basic mechanisms of ciliogenesis. Note that, as a geneticist and specialist in ciliogenesis using various model organisms, I am not fully competent to critically evaluate the mathematical models developed in this study.

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Referee #1

Evidence, reproducibility and clarity

In this manuscript entitled "Machanisms of cilia regeneration in Xenopus multiciliated epithelium in vivo", the authors mostly focus on the question, whether TZ (transition zone of cilia) plays an essential role for ciliogenesis during cilia regeneration in multiciliated cells. They used Xenopus embryo as a system to examine this question. While cilia regeneration has been actively studies in unicellular green algae, Chlamydomonas reinhardtii, the mechanism of cilia regeneration is not known yet. Their approach is to investigate cells after deciliation by calcium shock, based on a TZ protein B9D1, as well as ultrastructure observation using conventional electron microscopy.

The authors observed loss of signal from B9D1 and H-shaped objects, which is typical for TZ, upon deciliation induced by calcium and also during the following re-growth of cilia. Based on these experiments they concluded that TZ formation is not necessary for cilia regeneration in multiciliated cells, differently from Chlamydomonas. They further conducted experiments to pursue source of component proteins for re-generation. They compared CHX-treated cells (lacking new protein production) and CHX/MG132 (reduced protein degradation) treated cells to find how the massive amount of protein components upon re-ciliation for multiple cilia will be supplied and regulated. This reviewer found the results of the experiments clearly presented and conducted properly.

The work would have significant impact in the cilia community, if the conclusion is correct. This reviewer, however, has a concern about the authors concluding the presence/absence of TZ, based on only B9D1 and the H-shaped body among nine doublet microtubules. First, is it really established how the structure of Xenopus embryo TZ is? While Chlamydomonas is well known to have a H-shaped TZ, other species have different form inside the 9+0 doublet, or no feature (Comparison of TZ from various species in Dennis Diener https://doi.org/10.1016/B978-0-12-822508-0.00007-1). Fig.2B of this manuscript shows visible densities in the panel "Pre", but it does not look like an H-shape. The tomogram of TZ before deciliation seems clearer (but judging from wavy MTs and membrane in this tomogram, there could be unevenness of embedding and staining), while the tomogram after deciliation is thin and does not cover the entire width. Therefore it is not sure that absence of TZ can be concluded. If the author claims Xenopus embryo cilia have a H-shaped TZ, they have to provide multiple micrographs (ideally tomogram or serial section TEM to cover the entire TZ structure) and/or past literature on Xenopus embryo TZ. B9D1 is likely a membrane associated protein (according to their deciliation by detergent and mechanical force). This may mean B9D1 is located on or near the membrane, in vicinity to TZ, and thus binds to TZ after the main part of TZ is built. In this case, it is risky to judge presence of TZ based on B9D1. Also in this point, TEM imaging will be helpful to confirm the authors' conclusion.

Their discussion about length/number of cilia and force generated by cilia is interesting, but in the context of this research, this reviewer is skeptical about its value. The calcium induced deciliation is not a physiological phenomena, but an artificial event (please correct if I am wrong). The argument how length and number of cilia are regulated upon deciliation makes sense only in case deciliation happens regularly and the species must optimize themselves to survive. The argument about possible passway of protein transport to control ciliary number and length (Line408-) seems, although it is an interesting topic in general, not suitable in this manuscript. For this reviewer's view, it is relatively straightforward to interpret the result of cilia number/length under normal growth, without new protein expression (CHX), with protein degradation blocked. Cilia will extend when components are provided. Growth will slow down when it is exhausted. Existing cilia start degrading, when they lack proteins, which are necessary for turn-over. With the current experimental output, there is no point to describe redistribution of proteins.

Minor points:

Line65: do they mean "selected few basal bodies"?

Line73: extracellular flow is not limited to developmental system.

Line124: alpha-tubulin signal and SEM image

Line139: Could you define explicitly the two hypotheses?

Line164: 10,31-33 are not suitable citation for the location of calcium induced deciliation in Chlamydomonas. cite Sanders and Salisbury JCB 108, 1751

Line181: Later -> latter

Line195: by mechanical shearing, B9D1 remained with cilia. They concluded that TZ stays with the axoneme by deciliation. How can they exclude the possibility that mechanical separation works differently from calcium shock?

Line214: 1.33uM -> 1.33um

Significance

The work would have significant impact in the cilia community, if the conclusion is correct. Their discussion about length/number of cilia and force generated by cilia is interesting, but in the context of this research, this reviewer is skeptical about its value.

Would it be correct to say "you will need to repeat these steps every time you reset your Office365 password?"

This could also make for a good entry in the troubleshooting field. What happens if the user doesn’t re-configure these settings after changing their password?)

Is there a reason this isn't in the "before you begin" section?

This might be a good place to explain the "comment" function in Plunet that lets users explain any discrepancies in their hours.

We cannot explain our own actions faithfully.

We're going to understand and be able to explain the actions of a super intelligence?

To me, this is the biggest issue. In the face of deception, behavior tests fail.

• variable names only starts with alphabetic character
• keywords or reserved words cannot be used as the name of variable

easy to use

'=' means set up variable

represents either 0 or 1

creating a vairable

represent names by characters

represent true or false

non digit number. i.e.) 6.3, 60293.93032

represent integer

reference to an object of a class

primitive types of variable

saving through memory of computer

進化ゲーム理論は均衡に到達する過程に興味がある

（無作為に選ばれた）新たな対戦相手

生物学的解釈の包含： 社会的解釈（個人の戦略変更）は、生物学的解釈（死亡と誕生）を包含できます。つまり、個体の「戦略の変更」を「その個体の死亡と新しい戦略を持つ個体の誕生」と見なすことができます。

test

my first annotation.

Author response:

The following is the authors’ response to the current reviews.

(1) Though we cannot survey all mutants, our observation that 774 genetically diverse adaptive mutants converge at the level of phenotype is important. It adds to growing evidence (see PMID33263280, PMID37437111, PMID22282810, PMID25806684) that the genetic basis of adaptation is not as diverse as the phenotypic basis. This convergence could make evolution more predictable.

(2) Previous fitness competitions using this specific barcode system have been run for greater than 25 generations (PMID33263280, PMID27594428, PMID37861305, PMID27594428). We measure fitness per cycle, rather than per generation, so our fitness advantages are comparable to those in the aforementioned studies, including Venkataram and Dunn et al. (PMID27594428).

(3) Our results remain the same upon removing the ~150 lineages with the noisiest fitness inferences, including those the reviewer mentions (see Figure S7).

(4) We agree that there are likely more than the 6 clusters that we validated with follow-up studies (see Discussion). The important point is that we see a great deal of convergence in the behavior of diverse adaptive mutants.

(5) The growth curves requested by the reviewer were included in our original manuscript; several more were added in the revision (see Figures 5D, 5E, 7D, S11B, S11C).

The following is the authors’ response to the original reviews.

Public Reviews.

Reviewer #1 (Public Review): 

Summary: 

In their manuscript, Schmidlin, Apodaca, et al try to answer fundamental questions about the evolution of new phenotypes and the trade-offs associated with this process. As a model, they use yeast resistance to two drugs, fluconazole and radicicol. They use barcoded libraries of isogenic yeasts to evolve thousands of strains in 12 different environments. They then measure the fitness of evolved strains in all environments and use these measurements to examine patterns in fitness trade-offs. They identify only six major clusters corresponding to different trade-off profiles, suggesting the vast genotypic landscape of evolved mutants translates to a highly constrained phenotypic space. They sequence over a hundred evolved strains and find that mutations in the same gene can result in different phenotypic profiles.  

Overall, the authors deploy innovative methods to scale up experimental evolution experiments, and in many aspects of their approach tried to minimize experimental variation. 

We thank the reviewer for this positive assessment of our work. We are happy that the reviewer noted what we feel is a unique strength of our approach: we scaled up experimental evolution by using DNA barcodes and by exploring 12 related selection pressures.  Despite this scaling up, we still see phenotypic convergence among the 744 adaptive mutants we study. 

Weaknesses: 

(1) One of the objectives of the authors is to characterize the extent of phenotypic diversity in terms of resistance trade-offs between fluconazole and radicicol. To minimize noise in the measurement of relative fitness, the authors only included strains with at least 500 barcode counts across all time points in all 12 experimental conditions, resulting in a set of 774 lineages passing this threshold. This corresponds to a very small fraction of the starting set of ~21 000 lineages that were combined after experimental evolution for fitness measurements. 

This is a misunderstanding that we clarified in this revision. Our starting set did not include 21,000 adaptive lineages. The total number of unique adaptive lineages in this starting set is much lower than 21,000 for two reasons. 

First, ~21,000 represents the number of single colonies we isolated in total from our evolution experiments. Many of these isolates possess the same barcode, meaning they are duplicates. Second, and perhaps more importantly, most evolved lineages do not acquire adaptive mutations, meaning that many of the 21,000 isolates are genetically identical to their ancestor. In our revised manuscript, we explicitly stated that these 21,000 isolated lineages do not all represent unique, adaptive lineages. We changed the word “lineages” to “isolates” where relevant in Figure 2 and the accompanying legend. And we have added the following sentence to the figure 2 legend (line 212), “These ~21,000 isolates do not represent as many unique, adaptive lineages because many either have the same barcode or do not possess adaptive mutations.”

More broadly speaking, several previous studies have demonstrated that diverse genetic mutations converge at the level of phenotype and have suggested that this convergence makes adaptation more predictable (PMID33263280, PMID37437111, PMID22282810, PMID25806684). Most of these studies survey fewer than 774 mutants. Further, our study captures mutants that are overlooked in previous studies, such as those that emerge across subtly different selection pressures (e.g., 4 𝜇g/ml vs. 8 𝜇g/ml flu) and those that are undetectable in evolutions lacking DNA barcodes. Thus, while our experimental design misses some mutants (see next comment), it captures many others. Thus, we feel that “our work – showing that 774 mutants fall into a much smaller number of groups” is important because it “contributes to growing literature suggesting that the phenotypic basis of adaptation is not as diverse as the genetic basis (lines 176 - 178).”

As the authors briefly remark, this will bias their datasets for lineages with high fitness in all 12 environments, as all these strains must be fit enough to maintain a high abundance. 

We now devote 19 lines of text to discussing this bias (on lines 160 - 162, 278-284, and in more detail on 758 - 767).

We walk through an example of a class of mutants that our study misses. One lines 759 - 763, we say, “our study is underpowered to detect adaptive lineages that have low fitness in any of the 12 environments. This is bound to exclude large numbers of adaptive mutants. For example, previous work has shown some FLU resistant mutants have strong tradeoffs in RAD (Cowen and Lindquist 2005). Perhaps we are unable to detect these mutants because their barcodes are at too low a frequency in RAD environments, thus they are excluded from our collection of 774.”

In our revised version, we added more text earlier in the manuscript that explicitly discusses this bias. Lines 278 – 283 now read, “The 774 lineages we focus on are biased towards those that are reproducibly adaptive in multiple environments we study. This is because lineages that have low fitness in a particular environment are rarely observed >500 times in that environment (Figure S4). By requiring lineages to have high-coverage fitness measurements in all 12 conditions, we may be excluding adaptive mutants that have severe tradeoffs in one or more environments, consequently blinding ourselves to mutants that act via unique underlying mechanisms.”

Note that while we “miss” some classes of mutants, we “catch” other classes that may have been missed in previous studies of convergence. For example, we observe a unique class of FLU-resistant mutants that primarily emerged in evolution experiments that lack FLU (Figure 3). Thus, we think that the unique design of our study, surveying 12 environments, allows us to make a novel contribution to the study of phenotypic convergence.

One of the main observations of the authors is phenotypic space is constrained to a few clusters of roughly similar relative fitness patterns, giving hope that such clusters could be enumerated and considered to design antimicrobial treatment strategies. However, by excluding all lineages that fit in only one or a few environments, they conceal much of the diversity that might exist in terms of trade-offs and set up an inclusion threshold that might present only a small fraction of phenotypic space with characteristics consistent with generalist resistance mechanisms or broadly increased fitness. This has important implications regarding the general conclusions of the authors regarding the evolution of trade-offs. 

We agree and discussed exactly the reviewer’s point about our inclusion threshold in the 19 lines of text mentioned previously (lines 160 - 162, 278-284, and 758 - 767). To add to this discussion, and avoid the misunderstanding the reviewer mentions, we added the following strongly-worded sentence to the end of the paragraph on lines 749 – 767 in our revised manuscript: “This could complicate (or even make impossible) endeavors to design antimicrobial treatment strategies that thwart resistance”. 

More generally speaking, we set up our study around Figure 1, which depicts a treatment strategy that works best if there exists but a single type of adaptive mutant. Despite our inclusion threshold, we find there are at least 6 types of mutants. This diminishes hopes of designing simple multidrug strategies like Figure 1. Our goal is to present a tempered and nuanced discussion of whether and how to move forward with designing multidrug strategies, given our observations. On one hand, we point out how the phenotypic convergence we observe is promising. But on the other hand, we also point out how there may be less convergence than meets the eye for various reasons including the inclusion threshold the reviewer mentions (lines 749 - 767).

We have made several minor edits to the text with the goal of providing a more balanced discussion of both sides. For example, we added the words, “may yet” to the following sentences on lines 32 – 36 of the abstract: “These findings, on one hand, demonstrate the difficulty in relying on consistent or intuitive tradeoffs when designing multidrug treatments. On the other hand, by demonstrating that hundreds of adaptive mutations can be reduced to a few groups with characteristic tradeoffs, our findings may yet empower multidrug strategies that leverage tradeoffs to combat resistance.”

(2) Most large-scale pooled competition assays using barcodes are usually stopped after ~25 to avoid noise due to the emergence of secondary mutations. 

The rate at which new mutations enter a population is driven by various factors such as the mutation rate and population size, so choosing an arbitrary threshold like 25 generations is difficult. 

We conducted our fitness competition following previous work using the Levy/Blundell yeast barcode system, in which the number of generations reported varies from 32 to 40 (PMID33263280, PMID27594428, PMID37861305, see PMID27594428 for detailed calculation of the fraction of lineages biased by secondary mutations in this system). 

The authors measure fitness across ~40 generations, which is almost the same number of generations as in the evolution experiment. This raises the possibility of secondary mutations biasing abundance values, which would not have been detected by the whole genome sequencing as it was performed before the competition assay. 

Previous work has demonstrated that in this evolution platform, most mutations occur during the transformation that introduces the DNA barcodes (Levy et al. 2015). In other words, these mutations are already present and do not accumulate during the 40 generations of evolution. Therefore, the observation that we collect a genetically diverse pool of adaptive mutants after 40 generations of evolution is not evidence that 40 generations is enough time for secondary mutations to bias abundance values.

We have added the following sentence to the main text to highlight this issue (lines 247 - 249): “This happens because the barcoding process is slightly mutagenic, thus there is less need to wait for DNA replication errors to introduce mutations (Levy et al. 2015; Venkataram et al. 2016).”

We also elaborate on this in the method section entitled, “Performing barcoded fitness competition experiments,” where we added a full paragraph to clarify this issue (lines 972 - 980).

(3) The approach used by the authors to identify and visualize clusters of phenotypes among lineages does not seem to consider the uncertainty in the measurement of their relative fitness. As can be seen from Figure S4, the inter-replicate difference in measured fitness can often be quite large. From these graphs, it is also possible to see that some of the fitness measurements do not correlate linearly (ex.: Med Flu, Hi Rad Low Flu), meaning that taking the average of both replicates might not be the best approach.  Because the clustering approach used does not seem to take this variability into account, it becomes difficult to evaluate the strength of the clustering, especially because the UMAP projection does not include any representation of uncertainty around the position of lineages. This might paint a misleading picture where clusters appear well separate and well defined but are in fact much fuzzier, which would impact the conclusion that the phenotypic space is constricted. 

Our noisiest fitness measurements correspond to barcodes that are the least abundant and thus suffer the most from stochastic sampling noise. These are also the barcodes that introduce the nonlinearity the reviewer mentions. We removed these from our dataset by increasing our coverage threshold from 500 reads to 5,000 reads. The clusters did not collapse, which suggests that they were not capturing this noise (Figure S7B).

More importantly, we devoted 4 figures and 200 lines of text to demonstrating that the clusters we identified capture biologically meaningful differences between mutants (and not noise). We have modified the main text to point readers to figures 5 through 8 earlier, such that it is more apparent that the clustering analysis is just the first piece of our data demonstrating convergence at the level of phenotype.

(4) The authors make the decision to use UMAP and a gaussian mixed model to cluster and represent the different fitness landscapes of their lineages of interest. Their approach has many caveats. First, compared to PCA, the axis does not provide any information about the actual dissimilarities between clusters. Using PCA would have allowed a better understanding of the amount of variance explained by components that separate clusters, as well as more interpretable components. 

The components derived from PCA are often not interpretable. It’s not obvious that each one, or even the first one, will represent an intuitive phenotype, like resistance to fluconazole.  Moreover, we see many non-linearities in our data. For example, fitness in a double drug environment is not predicted by adding up fitness in the relevant single drug environments. Also, there are mutants that have high fitness when fluconazole is absent or abundant, but low fitness when mild concentrations are present. These types of nonlinearities can make the axes in PCA very difficult to interpret, plus these nonlinearities can be missed by PCA, thus we prefer other clustering methods. 

Still, we agree that confirming our clusters are robust to different clustering methods is helpful. We have included PCA in the revised manuscript, plotting PC1 vs PC2 as Figure S9 with points colored according to the cluster assignment in figure 4 (i.e. using a gaussian mixture model). It appears the clusters are largely preserved.

Second, the advantages of dimensional reduction are not clear. In the competition experiment, 11/12 conditions (all but the no drug, no DMSO conditions) can be mapped to only three dimensions: concentration of fluconazole, concentration of radicicol, and relative fitness. Each lineage would have its own fitness landscape as defined by the plane formed by relative fitness values in this space, which can then be examined and compared between lineages. 

We worry that the idea stems from apriori notions of what the important dimensions should be. The biology of our system is unfortunately not intuitive. For example, it seems like this idea would miss important nonlinearities such as our observation that low fluconazole behaves more like a novel selection pressure than a dialed down version of high fluconazole. 

Third, the choice of 7 clusters as the cutoff for the multiple Gaussian model is not well explained. Based on Figure S6A, BIC starts leveling off at 6 clusters, not 7, and going to 8 clusters would provide the same reduction as going from 6 to 7. This choice also appears arbitrary in Figure S6B, where BIC levels off at 9 clusters when only highly abundant lineages are considered. 

We agree. We did not rely on the results of BIC alone to make final decisions about how many clusters to include. Another factor we considered were follow-up genotyping and phenotyping studies that confirm biologically meaningful differences between the mutants in each cluster (Figures 5 – 8). We now state this explicitly. Here is the modified paragraph where we describe how we chose a model with 7 clusters, from lines 436 – 446 of the revised manuscript:

“Beyond the obvious divide between the top and bottom clusters of mutants on the UMAP, we used a gaussian mixture model (GMM) (Fraley and Raftery, 2003) to identify clusters. A common problem in this type of analysis is the risk of dividing the data into clusters based on variation that represents measurement noise rather than reproducible differences between mutants (Mirkin, 2011; Zhao et al., 2008). One way we avoided this was by using a GMM quality control metric (BIC score) to establish how splitting out additional clusters affected model performance (Figure S6). Another factor we considered were follow-up genotyping and phenotyping studies that demonstrate biologically meaningful differences between mutants in different clusters (Figures 5 – 8). Using this information, we identified seven clusters of distinct mutants, including one pertaining to the control strains, and six others pertaining to presumed different classes of adaptive mutant (Figure 4D). It is possible that there exist additional clusters, beyond those we are able to tease apart in this study.”

This directly contradicts the statement in the main text that clusters are robust to noise, as more a stringent inclusion threshold appears to increase and not decrease the optimal number of clusters. Additional criteria to BIC could have been used to help choose the optimal number of clusters or even if mixed Gaussian modeling is appropriate for this dataset. 

We are under the following impression: If our clustering method was overfitting, i.e. capturing noise, the optimal number of clusters should decrease when we eliminate noise. It increased. In other words, the observation that our clusters did not collapse (i.e.

merge) when we removed noise suggests these clusters were not capturing noise. 

Most importantly, our validation experiments, described below, provide additional evidence that our clusters capture meaningful differences between mutants (and not noise).  

(5) Large-scale barcode sequencing assays can often be noisy and are generally validated using growth curves or competition assays. 

Some types of bar-seq methods, in particular those that look at fold change across two time points, are noisier than others that look at how frequency changes across multiple timepoints (PMID30391162). Here, we use the less noisy method. We also reduce noise by using a stricter coverage threshold than previous work (e.g., PMID33263280), and by excluding batch effects by performing all experiments simultaneously, since we found this to be effective in our previous work (PMID37237236). 

Perhaps also relevant is that the main assay we use to measure fitness has been previously validated (PMID27594428) and no subsequent study using this assay validates using the methods suggested above (see PMID37861305, PMID33263280, PMID31611676, PMID29429618, PMID37192196, PMID34465770, PMID33493203). Similarly, bar-seq has been used, without the suggested validation, to demonstrate that the way some mutant’s fitness changes across environments is different from other mutants (PMID33263280, PMID37861305, PMID31611676, PMID33493203, PMID34596043). This is the same thing that we use bar-seq to demonstrate. 

For all of these reasons above, we are hesitant to confirm bar-seq itself as a valid way to infer fitness. It seems this is already accepted as a standard in our field. However, please see below.

Having these types of results would help support the accuracy of the main assay in the manuscript and thus better support the claims of the authors. 

While we don’t agree that fitness measurements obtained from this bar-seq assay generally require validation, we do agree that it is important to validate whether the mutants in each of our 6 clusters indeed are different from one another in meaningful ways.

Our manuscript has 4 figures (5 - 8) and over 200 lines of text dedicated to validating whether our clusters capture reproducible and biologically meaningful differences between mutants. In the revised manuscript, we added additional validation experiments, such that three figures (Figures 5, 7 and S11) now involve growth curves, as the reviewer requested. 

Below, we walk through the different types of validation experiments that are present in our manuscript, including those that were added in this revision.

(1) Mutants from different clusters have different growth curves: In our original manuscript, we measured growth curves corresponding to a fitness tradeoff that we thought was surprising. Mutants in clusters 4 and 5 both have fitness advantages in single drug conditions. While mutants from cluster 4 also are advantageous in the relevant double drug conditions, mutants from cluster 5 are not! We validated these different behaviors by studying growth curves for a mutant from each cluster (Figures 7 and S11), finding that mutants from different clusters have different growth curves. In the revised manuscript, we added growth curves for 6 additional mutants (3 from cluster 1 and 3 from cluster 3), demonstrating that only the cluster 1 mutants have a tradeoff in high concentrations of fluconazole (see Figure 5D & 5E). In sum, this work demonstrates that mutants from different clusters have predictable differences in their growth phenotypes.

(2) Mutants from different clusters have different evolutionary origins: In our original manuscript, we came up with a novel way to ask whether the clusters capture different types of adaptive mutants. We asked whether the mutants in each cluster originate from different evolution experiments. They often do (see pie charts in Figures 5, 6, 7, 8). In the revised manuscript, we extended this analysis to include mutants from cluster 1. Cluster 1 is defined by high fitness in low fluconazole that declines with increasing fluconazole. In our revised manuscript, we show that cluster 1 lineages were overwhelmingly sampled from evolutions conducted in our lowest concentration of fluconazole (see pie chart in new Figure 5A). No other cluster’s evolutionary history shows this pattern (compare to pie charts in figures 6, 7, and 8).

**These pie charts also provide independent confirmation supporting the fitness tradeoffs observed for each cluster in figure 4E. For example, mutants in cluster 5 appear to have a tradeoff in a particular double drug condition (HRLF), and the pie charts confirm that they rarely originate from that evolution condition. This differs from cluster 4 mutants, which do not have a fitness tradeoff in HRLF, and are more likely to originate from that environment (see purple pie slice in figure 7). Additional cases where results of evolution experiments (pie charts) confirm observed fitness tradeoffs are discussed in the manuscript on lines 320 – 326, 594 – 598, 681 – 685.

(3) Mutants from each cluster often fall into different genes: We sequenced many of these mutants and show that mutants in the same gene are often found in the same cluster. For example, all 3 IRA1 mutants are in cluster 6 (Fig 8), both GPB2 mutants are in cluster 4 (Figs 7 & 8), and 35/36 PDR mutants are in either cluster 2 or 3 (Figs 5 & 6). 

(4) Mutants from each cluster have behaviors previously observed in the literature: We compared our sequencing results to the literature and found congruence. For example, PDR mutants are known to provide a fitness benefit in fluconazole and are found in clusters that have high fitness in fluconazole (lines 485 - 491). Previous work suggests that some mutations to PDR have different tradeoffs than others, which corresponds to our finding that PDR mutants fall into two separate clusters (lines 610 - 612). IRA1 mutants were previously observed to have high fitness in our “no drug” condition and are found in the cluster that has the highest fitness in the “no drug” condition (lines 691 - 696). Previous work even confirms the unusual fitness tradeoff we observe where IRA1 and other cluster 6 mutants have low fitness only in low concentrations of fluconazole (lines 702 - 704).

(5) Mutants largely remain in their clusters when we use alternate clustering methods:  In our original manuscript, we performed various different re-clustering and/or normalization approaches on our data (Fig 6, S5, S7, S8, S10). The clusters of mutants that we observe in figure 4 do not change substantially when we re-cluster the data. In our revised manuscript, we added another clustering method: principal component analysis (PCA) (Fig S9).  Again, we found that our clusters are largely preserved.

While these experiments demonstrate meaningful differences between the mutants in each cluster, important questions remain. For example, a long-standing question in biology centers on the extent to which every mutation has unique phenotypic effects versus the extent to which scientists can predict the effects of some mutations from other similar mutations. Additional studies on the clusters of mutants discovered here will be useful in deepening our understanding of this topic and more generally of the degree of pleiotropy in the genotype-phenotype map.

Reviewer #2 (Public Review): 

Summary: 

Schmidlin & Apodaca et al. aim to distinguish mutants that resist drugs via different mechanisms by examining fitness tradeoffs across hundreds of fluconazole-resistant yeast strains. They barcoded a collection of fluconazole-resistant isolates and evolved them in different environments with a view to having relevance for evolutionary theory, medicine, and genotypephenotype mapping. 

Strengths: 

There are multiple strengths to this paper, the first of which is pointing out how much work has gone into it; the quality of the experiments (the thought process, the data, the figures) is excellent. Here, the authors seek to induce mutations in multiple environments, which is a really large-scale task. I particularly like the attention paid to isolates with are resistant to low concentrations of FLU. So often these are overlooked in favour of those conferring MIC values >64/128 etc. What was seen is different genotype and fitness profiles. I think there's a wealth of information here that will actually be of interest to more than just the fields mentioned (evolutionary medicine/theory). 

We are grateful for this positive review. This was indeed a lot of work! We are happy that the reviewer noted what we feel is a unique strength of our manuscript: that we survey adaptive isolates across multiple environments, including low drug concentrations.  

Weaknesses: 

Not picking up low fitness lineages - which the authors discuss and provide a rationale as to why. I can completely see how this has occurred during this research, and whilst it is a shame I do not think this takes away from the findings of this paper. Maybe in the next one! 

We thank the reviewer for these words of encouragement and will work towards catching more low fitness lineages in our next project.

In the abstract the authors focus on 'tradeoffs' yet in the discussion they say the purpose of the study is to see how many different mechanisms of FLU resistance may exist (lines 679-680), followed up by "We distinguish mutants that likely act via different mechanisms by identifying those with different fitness tradeoffs across 12 environments". Whilst I do see their point, and this is entirely feasible, I would like a bit more explanation around this (perhaps in the intro) to help lay-readers make this jump. The remainder of my comments on 'weaknesses' are relatively fixable, I think: 

We have expanded the introduction, in particular lines 129 – 157 of the revised manuscript, to walk readers through the connection between fitness tradeoffs and molecular mechanisms. For example, here is one relevant section of new text from lines 131 - 136: “The intuition here is as follows. If two groups of drug resistant mutants have different fitness tradeoffs, it could mean that they provide resistance through different underlying mechanisms. Alternatively, both could provide drug resistance via the same mechanism, but some mutations might also affect fitness via additional mechanisms (i.e. they might have unique “side-effects” at the molecular level) resulting in unique fitness tradeoffs in some environments.”

In the introduction I struggle to see how this body of research fits in with the current literature, as the literature cited is a hodge-podge of bacterial and fungal evolution studies, which are very different! So example, the authors state "previous work suggests that mutants with different fitness tradeoffs may affect fitness through different molecular mechanisms" (lines 129-131) and then cite three papers, only one of which is a fungal research output. However, the next sentence focuses solely on literature from fungal research. Citing bacterial work as a foundation is fine, but as you're using yeast for this I think tailoring the introduction more to what is and isn't known in fungi would be more appropriate. It would also be great to then circle back around and mention monotherapy vs combination drug therapy for fungal infections as a rationale for this study. The study seems to be focused on FLU-resistant mutants, which is the first-line drug of choice, but many (yeast) infections have acquired resistance to this and combination therapy is the norm. 

We ourselves are broadly interested in the structure of the genotype-phenotype-fitness map (PMID33263280, PMID32804946). For example, we are interested in whether diverse mutations converge at the level of phenotype and fitness. Figure 1A depicts a scenario with a lot of convergence in that all adaptive mutations have the same fitness tradeoffs.

The reason we cite papers from yeast, as well as bacteria and cancer, is that we believe general conclusions about the structure of the genotype-phenotype-fitness map apply broadly. For example, the sentence the reviewer highlights, “previous work suggests that mutants with different fitness tradeoffs may affect fitness through different molecular mechanisms” is a general observation about the way genotype maps to fitness. So, we cited papers from across the tree of life to support this sentence.  And in the next sentence, where we cite 3 papers focusing solely on fungal research, we cite them because they are studies about the complexity of this map. Their conclusions, in theory, should also apply broadly, beyond yeast.

On the other hand, because we study drug resistant mutations, we hope that our dataset and observations are of use to scientists studying the evolution of resistance. We use our introduction to explain how the structure of the genotype-phenotype-fitness map might influence whether a multidrug strategy is successful (Figure 1).

We are hesitant to rework our introduction to focus more specifically on fungal infections as this is not our primary area of expertise.

Methods: Line 769 - which yeast? I haven't even seen mention of which species is being used in this study; different yeast employ different mechanisms of adaptation for resistance, so could greatly impact the results seen. This could help with some background context if the species is mentioned (although I assume S. cerevisiae). 

In the revised manuscript, we have edited several lines (line 95, 186, 822) to state the organism this work was done with is Saccharomyces cerevisiae. 

In which case, should aneuploidy be considered as a mechanism? This is mentioned briefly on line 556, but with all the sequencing data acquired this could be checked quickly? 

We like this idea and we are working on it, but it is not straightforward. The reviewer is correct in that we can use the sequencing data that we already have. But calling aneuploidy with certainty is tough because its signal can be masked by noise. In other words, some regions of the genome may be sequenced more than others by chance.

Given this is not straightforward, at least not for us, this analysis will likely have to wait for a subsequent paper. 

I think the authors could be bolder and try and link this to other (pathogenic) yeasts. What are the implications of this work on say, Candida infections? 

Perhaps because our background lies in general study of the genotype-phenotype map, we are hesitant about making bold assertions about how our work might apply to pathogenic yeasts. We are hopeful that our work will serve as a stepping-stone such that scientists from that community can perhaps make (and test) such statements.   

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors): 

I found the ideas and the questions asked in this manuscript to be interesting and ambitious. The setup of the evolution and fitness competition experiments was well poised to answer them, but the analysis of the data is not currently enough to properly support the claims made. I would suggest revising the analysis to address the weaknesses raised in the public review and if possible, adding some more experimental validations. As you already have genome sequencing data showing the causal mutation for many mutants across the different clusters, it should be possible for you to reconstruct some of the strains and test validate their phenotypes and cluster identity. 

Yes, this is possible. We added more validation experiments (see figure 5). We already had quite a few validation experiments (figures 5 - 8 and lines 479 - 718), but we did not clearly highlight the significance of these analyses in our original manuscript. Therefore, we modified the text in our revised manuscript in various places to do so. For example, we now make clearer that we jointly use BIC scores as well as validation experiments to decide how many clusters to describe (lines 436 - 446). We also make clearer that our clustering analysis is only the first step towards identifying groups of mutants with similar tradeoffs by using words and phrases like, “we start by” (line 411) and “preliminarily” (line 448) when discussing the clustering analysis.  We also point readers to all the figures describing our validation experiments earlier (line 443), and list these experiments out in the discussion (lines 738 - 741).

Also, please deposit your genome sequencing data in a public database (I am not sure I saw it mentioned anywhere). 

We have updated line 1088 of the methods section to include this sentence: “Whole genome sequences were deposited in GenBank under SRA reference PRJNA1023288.”

Reviewer #2 (Recommendations For The Authors):

I don't think the figures or experiments can be improved upon, they are excellent. There are a few times I feel things are written in a rather confusing way and could be explained better, but also I feel there are places the authors jump from one thing to another really quickly and the reader (who might not be an expert in this area) will struggle to keep up. For example: 

Explaining what RAD is - it is introduced in the methods, but what it is, is not really explained. 

Since the introduction is already very long, we chose not to explain radicicol’s mechanism of action here. Instead, we bring this up later on lines 614 – 621 when it becomes relevant.

More generally, in response to this advice and that from reviewer 1, we also added text to various places in the manuscript to help explain our work more clearly. In particular, we clarified the significance of our validation experiments and various important methodological details (see above). We also better explained the connection between fitness tradeoffs and mechanisms (see above) and added more details about the potential use cases of our approach (lines 142 – 150).

The abstract states "some of the groupings we find are surprising. For example, we find some mutants that resist single drugs do not resist their combination, and some mutants to the same gene have different tradeoffs than others". Firstly, this sentence is a bit confusing to read but if I've read it as intended, then is it really surprising? It's difficult for organisms (bacteria and fungi) to develop multiple beneficial mutations conferring drug resistance on the same background, hence why combination antifungal drug therapy is often used to treat infections. 

This is a place where brevity got in the way of clarity. We added a bit of text to make clear why we were surprised. Specifically, we were surprised because not all mutants behave the same. Some resist single drugs AND their combination. Some resist single drugs but not their combination. The sentence in the abstract now reads, “For example, we find some mutants that resist single drugs do not resist their combination, while others do. And some mutants to the same gene have different tradeoffs than others.”