Operations research never gets respect

If you incentivize the people with resources appropriately, they can make magic happen.

I agree if we are not cautious about the information we receive it could lead to bigger problems and misconceptions.

I believe the united states isn't the only country spreading rumors. I've heard about false accusations coming from china and it spread into the United States.

rumors were a huge problem in my high school they spread on the internet and in person. I definitely feel like social media made this issue worse.

this is very interesting now I have some great tips on how to avoid false information.

I believe this is how fight in schools and even on the streets are caused by fake news and rumors.

If I had to choose between the two I feel I would go into the intuitionist group.

very true people can make up lies and rumors and spread them just as quick as the internet can.

How can the reproducibility of digital media preserve both that ownership/freedom and what's good about curation / editing?

Copies seem good here to me...

https://www.reddit.com/r/typewriters/comments/1muy8n1/an_olympia_sg3_makes_an_appearance_in_mi_the/

source for this?? interesting, if true

https://www.reddit.com/r/typewriters/comments/1mxxqkl/dieter_rams_typewriter_model/

Electrons that spend most of their time between the nuclei of two atoms are placed into the bonding orbitals, and electrons that spend most of their time outside the nuclei of two atoms are placed into antibonding orbitals. This is because there is an increasing in electron density between the nuclei in bonding orbitals, and a decreasing in electron density in antibonding orbitals (Chang 459). Placing an electron in the bonding orbital stabilizes the molecule because it is in between the two nuclei. Conversely, placing electrons into the antibonding orbitals will decrease the stability of the molecule. Electrons will fill according to the energy levels of the orbitals. They will first fill the lower energy orbitals, and then they will fill the higher energy orbitals. If a bond order of zero is obtained, that means that the molecule is too unstable and so it will not existItalic

https://www.scottishpoetrylibrary.org.uk/poem/breathes-there-man/

https://sustainingcommunity.wordpress.com/2019/02/01/4-types-of-power/#comment-122967

Given your area, if you haven't found it yet, you might appreciate going a generation further back in your references with: Mary P. Follett. Dynamic Administration: The Collected Papers of Mary Parker Follett, ed. by E. M. Fox and L. Urwick (London: Pitman Publishing, 1940). She had some interesting work in organization theory you might appreciate. Wikipedia can give you a quick overview. https://en.wikipedia.org/wiki/Mary_Parker_Follett#Organizational_theory

This is the current NPI standard from HIPAA which now explicitly mentions the NPI by name.

Specific language in the law allowing for fees for issuing identifiers.

This is the HIPPA right to identify codesets etc.

This is the "standards for transactions" part of HIPAA. This gives HHS/CMS the right to dictate FHIR or X12, for different transactions.

This is the specific legal basis for the enumeration of NPIs and plans.

This is the place where the constraints for the HIPAA administrative rule is defined.

This is the "trigger clause" in HIPAA that caused HHS to create the Privacy and Security Rules.

English Explanation

The term "midriff" refers to the part of the body between the chest and the waist. In the provided context, there is a discussion about dress codes, particularly criticizing tight-fitting clothing that exposes the midriff, indicating that such attire is not appropriate for the setting. The conversation emphasizes the need for a more professional appearance, as showing one's midriff may be deemed too casual or not aligned with the standards expected in that environment.

Chinese Explanation

“midriff”是指胸部和腰部之间的身体部分。在提供的上下文中，讨论了着装规范，特别是批评那些暴露腹部的紧身衣物，表明这种穿着不适合该场合。对话强调了需要更加专业的外观，因为暴露腹部可能被认为太随便，或者不符合该环境中预期的标准。

but this is the driving range and nobody official from the club has mentioned anything to us.

In this excerpt, the speaker argues that they are at the driving range and emphasizes that no official from the club has raised concerns about their dress code. This suggests they believe their attire is acceptable as it aligns with the informal setting of the driving range, highlighting a disconnect between personal views on dress and perceived official standards.

在这段摘录中，讲述者主张他们身处练习场，并强调俱乐部的任何官方人员都没有对他们的着装提出过担忧。 这表明他们认为自己的着装是可以接受的，因为这符合练习场的非正式氛围，突显了个人对着装的看法与官方标准之间的脱节。

The excerpt "pompous" describes a person's attitude or demeanor that is characterized by an exaggerated sense of self-importance or superiority. In the surrounding context, it appears to refer to someone who is perceived as arrogant or haughty, particularly in their interactions with others. The conversation indicates a sense of disapproval regarding this behavior, highlighting the impact of such attitudes on relationships.

这段摘录“自负”形容人的态度或举止，表现出夸大自我重要性或优越感。在周围的语境中，它似乎指的是一个被认为傲慢或高傲的人，尤其是在与他人的互动中。对这种行为的看法显示出不满，突显了这种态度对人际关系的影响。

English Explanation: The term "topiary" refers to the art of trimming and shaping trees or shrubs into decorative forms, usually as a feature in gardens. In the given excerpt, the speakers discuss a garden where topiary is perhaps not executed perfectly, as one character humorously notes they could "hardly keep a straight line." Despite this, another character appreciates the garden's appearance and notes there is enough space for activities, which shows a playful, lighthearted atmosphere amidst their conversation.

Chinese Explanation: “修剪艺术”指的是将树木或灌木修剪成装饰性形状的艺术，通常用于花园中。在该摘录中，发言者讨论了一个花园，其中的修剪艺术可能没有执行得很好，因为其中一个角色幽默地提到他们“几乎不能保持直线”。尽管如此，另一个角色欣赏花园的外观，并注意到有足够的空间供活动，这表明他们的对话中存在一种轻松愉快的气氛。

But this is not the kind of place where we have fisticuffs and slanging matches. If you don't like it here, then you should toddle off home.

In this excerpt, the speaker emphasizes that the environment does not tolerate physical violence or aggressive exchanges ("fisticuffs and slanging matches"). Instead, it's a place that values civility and decorum. The phrase "if you don't like it here, then you should toddle off home" suggests that those who cannot adhere to these standards should leave rather than disrupt the atmosphere.

在这段摘录中，讲话者强调这个环境不容许身体暴力或侵略性的争吵（“拳脚和辱骂”）。相反，这里是一个重视礼貌和端庄的地方。“如果你不喜欢这里，就应该回家”的说法暗示，那些无法遵守这些标准的人应该离开，而不是扰乱气氛。

good to bear your teeth every so often.

In the excerpt "good to bear your teeth every so often," the speaker emphasizes the importance of standing up for oneself and being assertive, particularly when dealing with difficult individuals. It suggests that occasionally showing one's strength or willingness to confront can be beneficial in navigating social dynamics. This idea is reinforced by advice from the speaker's mother, implying that the speaker has internalized this lesson about confrontation and self-advocacy.

在摘录“每隔一段时间露出牙齿是好的”中，说话者强调了为自己辩护和自信的重要性，尤其是在与困难的人打交道时。这表示在处理社会关系时，偶尔展现自己的力量或愿意对抗是有益的。这个观点由说话者母亲的建议进一步加强，暗示说话者已经内化了关于对抗和自我倡导的这一教训。

cuddle

English Explanation:

The excerpt "cuddle" refers to a warm and affectionate embrace, often indicating comfort and closeness between people. In the context snippet, the conversation revolves around moving, relationships, and shared living space. It suggests feelings of nostalgia and excitement about having family and friends close by, as well as the playful mention of potential chaos brought by a child named Martha. The interaction captures a blend of humor, care, and the complexities of shared living arrangements.

中文解释：

“cuddle”指的是温暖和亲密的拥抱，通常意味着人们之间的安慰和亲近。在上下文中，谈话围绕着搬家、关系和共享生活空间的主题。它暗示着对与家人和朋友亲近的怀旧和兴奋之情，以及对名为玛莎的小孩可能带来的混乱的幽默提及。这段对话捕捉了幽默、关怀和共享居住安排的复杂性。

reproductive burden indeed

There is an explicit requirement to have data that is FOIA available from NPPES publicly available for download.

This is a SORN change notice for NPPES

The research use case in the original NPPES SORN.

The homepage for the NPI standard

NPI can be used to uniquely identify healthcare provider for any purpose

At the level of the rule, HHS is responsible for NPPES

This is a specific mandate to keep NPPES up-to-date technologically.

Providers are required to provide updates to NPPES within 30 days of the change.

This is where the "intelligence-free" mandate comes from

NPI mandated to be a 10 digit code with a check digit as per the ISO standard.

Type 1 (individual) and Type 2 (organizational) are mandated here.

Personal NPIs cannot have "sub" ids. However, they could in the future according to this rule.

Group healthcare providers are organzations for the purpose of the NPI rule.

This is the connection between an organization, legal entity, sub-parts and being a covered entity.

Organization subparts are intended to address specific legal obligations regard organizational provider locations.

But if you move healthcare data around, based on covered transactions, you -do- need an NPI

Having an NPI does not make you HIPAA covered.

All healthcare providers are eligible to receive NPIs assuming they have been identified as a healthcare provider in any HHS regulation

NPS was the original name for NPPES in the regulation.

This in the NPI Final Rule, which establishes the details of what NPPES is an how it works.

1. category and tag pages are not working...
2. can we change category url from https://adswom.com/category/the-search-to-sql/ to https://adswom.com/the-search-to-sql/
3. post pages filter as per the category

only the form style like below references

Contact lead on which email ID

ref - https://www.parashifttech.com/contact

1. In footer same the reduce the logo size and align the logo vertical

2. add the social link

3. add privacy and disclaimer page

Make the description word count under two lines

1. Background should be frosted background
2. Make the description word count under two lines.

The Playbook for Winning High-Value Clients Online. Your step-by-step guide to turning search visibility into a steady flow of qualified project inquiries.

Reduce the image size by 30%

Quote

Lessons * It takes great holiness to discover the sweetness of penances. * The "good life" is discovered only in the act of divine union * Greatest impact in the world is made when we completely give ourselves over to service of God so that He works through us.

• Died at the age of thirty-one
• Predicted the day of her own death
• Canonised after around fifty years

• Became saint as she entered divine union with her saviour.
• Had divine experiences and Ecstasies
• Experienced exceptionally painful spiritual dryness
• This interior cross produced good fruit of perseverance and fidelity, consequently deepening her divine union.

• Engaged in charitable works
• Supported her family
• Emulated Saint Catherine of Siena
• (Penances) Slept on hard floor, wore crown of thorns and fasted.
• Went to mass daily and adored blessed sacrament
• Became lay member of the third order of Saint Dominic
• Wore chain of spikes around her waist

• Cut her beautiful hair
• Rubbed pepper on her face and lime juice on hands to make her skin less appealing

• Received sacrament of confirmation at 11years of age
• Took the name of Rose.

• Saint Rose of Lima was born in Peru, governed by the Kingdom of Spain
• Fifty-five years after spanish conquerors arrived and colonized the territory.
• Birth Name: Isabel Flores de Oliva
• Nickname: Rose after family servant says she saw her transformed into Rose

• 1532, covetous spanish explorers arrived in modern day Peru to covet available silver and Gold.
• 1537, Pope Paul III Issued papal bull named Sublimus Deus which Highlighted the Intrinsic dignity of the natives

A humorous celebration of the value of quality blogging within the Hive Blogger Ecosystem. Learn that true merit lies in the art of writing rather than superficial metrics or visual content. Content creation is not merely driven by algorithms or financial incentives. But by the integrity and passion of writers dedicated to sharing insightful narratives.

The fascinating world of Fibonacci numbers. See their mathematical properties and diverse applications in nature, art, and trading. Learn how these numbers not only influence the growth patterns of plants and the structure of galaxies. But they are also utilized in financial analysis through concepts like Fibonacci retracement.

Announcing the winners of a previous giveaway and introducing Giveaway 25. Where participants can win various prizes, including Hive Engine tokens and an NFT.

t5和输出之间应该还有网络？

the act of speaking angrily to someone because you disapprove of what they have said or done, or the things that someone says to show disapproval like this

a short fight between small groups of soldiers, etc., especially one that is not planned

a visit to a place that is considered special, where you go to show your respect

speed and eagerness

a small part on a piece of equipment or furniture

a young person who works for an employer for a fixed period of time in order to learn the particular skills needed in their job

a small town or group of buildings in a lonely part of a country

​unhappy because you are alone and do not want to be or because you have no friends

a flat, wide area of land in North America and Canada, without many trees and originally covered with grass

​the large size of something

remove the animation

Make this as per our styling, not as a folder, and remove my photo, add the gradient image

add the user functionality

Add the colour to the table heading (dark blue)

This whole section should be in the first fold when user scrolls then the second section should appear. Check the user experience in the reference below

Ref - https://www.halo-lab.com/blog/llms-how-they-work-and-how-to-use-them

Jesus' Incarnation is the perfect exemplification of the virtue of humility. It was an act of complete selfless service to humanity

What is humility? * I acknowledge my limitations, weakness and fault without seeking to exalt myself over others. * I act with modesty, simplicity and goodness. Always keeping in mind the good of others before myself.

• The ideal life is the greatest life
• Greatness is found in servant hood/humble service
• What is humble service? It is the act of selflessly serving others without seeking recognition or reward.

1. What is your desire in life; your driving force?
2. It should not be indulgence in pleasures.

Weaving the IndyWeb

♖ HyperPost OrigoWeb folder

getting ready to be ready to launch the IndyWeb

The Permanent, Evergreen, Co-evolving

commons based,Peer produced Autonomous, Person-first Virtual Cloud Services

Control yOur Information flows, Control yOur Destiny

of Autonomous

Inter- - Planetary - Personal - Plays

over the Web

Predictor: I.Pure Substance: Always the same composition II.Mixture: Variable composition IIa.Homogeneous: Uniform IIb. Heterogeneous: Not uniform

Noble Gases

Summarize: Matter is never lost or created; it only changes form.

This sentence points out that mass communication involves a deliberate effort to craft and deliver messages, often requiring additional steps like using technology or media platforms to reach a brough audience. This is especially relevant in today’s world, where media—such as television, social media, and online new—is a major channel for mass communication, allowing messages to reach millions of people quickly and effectively. Mass communication means that the message is often carefully constructed to influence, inform, or entertain a large and diverse audience. As someone who regularly interacts with media, I now realize how much planning goes into the messages I consume every day.

This sentence explains that group-level communication often centers around completing a shared task or objective. It gives an example of a practical, goal-oriented situation, where group members must collaborate and communicate effectively to achieve a common outcome. It emphasizes how communication plays a key role in coordinating efforts and solving problems withing groups. Although, like also stated in the article, “you don’t enjoy it.”.

This sentence highlights two different approaches to teaching or evaluating public speaking. One approach emphasized the content, such as the strength of ideas, arguments, and how well the speech is organized, and overall delivery. While the other, focuses on performance elements, like voice, body language, and overall delivery. It shows that effective public speaking involves both strong content and strong presentation skills.

I found this sentence interesting because it explains the evolution of early record-keeping systems. Initially, physical objects were used to represent quantities in transactions. Over time, these mechanisms were replaced by abstract symbols, marking a shift toward more advanced, symbolic methos of recording information. As of today, there are many more advanced methods for record-keeping such as digital databases, showing how far the practice has progressed.

Earlier in the text, it mentioned how the ability to talk in humans had an evolutionary advantage, which is due to the goal-oriented nature of interpersonal communication. Communication can transform the goals of one individual into a united goal for many. One's intrapersonal goals and communication can only be shared with the individual. Interpersonal communication is the driving factor of society; without it, we may have never been able to create the society we have today.

I think this draws a fairly interesting comparison to the evolution of communication today. For years, our conversation, vocabulary, and dialect have evolved far past anything our prehistoric ancestors could've imagined. But now, to communicate certain ideas, we use words like drip, cap, and fire to convey different ideas than the word suggests. Similar to the onomatopoetic nature of the first words spoken, we have shortened our language in areas and introduced new meanings to words already created to communicate how we best see fit.

This passage shows how committed the south was to restoring the life that the were use to having. The used laws to enact the old was of the south, it was a new era of salvery. If not in name but in action.

Money and wealth was more important than injustice and mistreatment

They were promised land and rightfully so. Instead of being given land they were still offered to work for white men but with wages. The very same men who had tortured and berated them.

Although massive progress was made, Reconstruction was not sustained permanently until later on

After emancipation, many Black people desired legal marriage as a way to gain dignity, family stability, and recognition long denied under slavery. This personal goal aligned with the government’s Reconstruction agenda. Officials, especially through the Freedmen’s Bureau, wanted free Black men to take responsibility as heads of households in line with patriarchal norms. At the same time, marriage was seen as a way to reduce government aid by preventing Black women and children from becoming dependent on welfare.

important

Can I apply this framework for my research workflow to accommodate both "structure" and "flexibility"? I guess the Heilmeier Catechnism will stand on the "structure" part. Iteration, "fail fast" will stand on the "flexibility" part. Can I formulate this sweet combination of "structure" and "flexibility" in research practice? Is there anyone before me who adapt this strategy? How to balance between planning, execution and replanning?

The right question frame might be: "How to navigate uncertainty efficiently?"

Parento 80/20 Need lots of elaboration since it's important

Taxonomy means system of classification per merriamwebster.com. I never knew that word before. annotation can help you expand on ideas for papers by getting you to think outside the box.

以下のようにあるので、小文字の"pip"が正しそうです。

pip is the package installer for Python. https://pip.pypa.io/en/stable/

Students must maintain a 3.0 GPA with a minimum of 30 credit hours earned within the approved program of study.

I think this seems nice so far, not as intimidating as speaking in front of everyone

This is the first class in which Open Pedagogy has been utilized. I'm curious how the content differs compared to those found in other college textbooks. I am hopeful that it will make digesting information easier, as the information within was written from the perspective of students previously in our positions rather than from the perspective of an educator or expert on the subject as is usual in these textbooks.

the forming or devising of a plan or idea.

organize the team into smaller, specialized units focused on specific areas of support, create a shared knowledge base to ensure consistent and accurate information

the general mental capacity for learning from experience, reasoning, and adapting to new or challenging situations

If I were to take another english class I feel like I could improve with these tips.

I always try to double check my grammar in my writing

I always try my best to write in academic format and more clearly so that my professor can give me full credit for the long hours I put into my work. I think citation are very beneficial

Clarify how these tests contributed to the project's reliability or ease of future updates.

Specify the impact of bug elimination—did it improve performance, user satisfaction, or reduce crashes?

Explain the significance of this feature—how does it enhance the application's functionality or user experience?

Quantify the impact of this training—did it lead to increased project success rates or skills assessments?

Explain how reduced costs translated to benefits for the company (e.g., increased revenue, efficiency).

Clarify the context of 'user workload' reduction—what tasks were simplified or eliminated?

Specify how much student understanding improved (e.g., grades, feedback) to quantify impact.

I honestly thought apple would have been the highest ranking instead of elsevier i'm surprised.

I believe this even my school pushes for open access to the library online and in person we even have tutoring that is held in the library.

This is very interesting to me because i never would have thought california would have been the largest for academic publishers I thought it would have been a bigger univeroty maybe even Harvard or Yale.

I always ask my children "do you understand why you are in trouble" and I make sure that they can repeat back to me the reason. I feel like it really helps gauge their understanding and whether I'm wasting my time and energy.

I want to have my class make their classroom rules and sign it like a contract. I loved when my cooperating teacher had her 8th grade class do that.

One thing that my husband and I do with our kids: we give them two or three options and sometimes one of the options would be something clearly negative. It has helped because my daughter knows that she doesn't want that option. Therefore, she usually gravitates towards other options. Meanwhile, I feel like it's helping her critical thinking. Does anyone have any experience with this type of reinforcement for behaviors? Do you think it would work in a classroom setting?

I have noticed when managing my own children that they thrive in an environment where I am consistent. I do notice that they start to act up more and more whenever my husband and I aren't on the same page about the expectations we have regarding their behavior.

However, we give varying reasons to not accept His invitations

God constantly invites us to deeper union with Him through participation in the Church

Mode of rejection Business Smartphones Computers and tablets Workaholics

Ideal christian attitude to the invitation

Hostility mode of rejection

The Church is the Bride of Christ

Earlier in the chapter, I made an annotation on the point made that interpersonal communication is more goal-oriented than intrapersonal communication. However, I would like to challenge that thought. In every discipline of life, nobody is ever perfect. If we were all perfect, there would be no meaning to trying and striving forward. So, when we mess up, we set up goals for ourselves intrapersonally to help us grow in the areas we recognize we need growth. Thus, it can be noted that intrapersonal communication could very well be just as or more goal-oriented than interpersonal communication.

Humans are relational beings. We are capable of feeling complex emotions like love, and we spend time with loved ones and friends to communicate such emotions with them. We have many aspects of our lives in which we depend upon communication and relationships to survive. When these relationships are stripped away from us, it can severely damage any communication skills we have acquired, not to mention the emotional struggle loneliness can put a person through.

By this the author could mean that the man is dark and mean, but he could also mean that he is smart and strategic.

This line repeats in every stanza, showing the author's hope of "this" passing over.

Physical context does indeed play a massive role in communication. I have gone through a handful of interviews, and I have had very diverse experiences. I have had an interview at a table in a crowded restaurant, one in a quiet enclosed room, but most recently, I had my first online interview. Online interviewing was a completely different ballpark from what I was used to. Not being able to see their physical self in front of me caught me off guard, and so did the slow internet speed with lag that followed it. The physical changes led to adaptations in my communication style that I was not prepared to make.

I really enjoy how the textbook offers real world applications. Antibiotic resistance is a big issue especially with pandemics and insecticides is another great real world issue to think about when applying this knowledge.

We discussed this in PB 360 but the genetic mutations must cause more benefit than harm to the species in order to be passed on. Some genetic mutations can be harmful for species but somehow increase their fitness and ability to reproduce however the pros must outlay the cons.

Reading this reminded me of when wolves were re-introduced into Yellowstone national park. This could have also happened during their reintroduction but what's so cool about their reintroduction is how they changed the environment due to their presence in the park. I wonder if this could connect to possible changes in phenotypes and genotypes (as discussed later on in this chapter) of other species due to the wolves effect of changing the environment.

This reminded me of Gregor Mendel pea plant experiments and how certain traits are inherited.

The DOI is new to me

Where would you find the name of a compiler of a website, i did not know that you supposed to do that.

Is the compiler name the same as the publisher?

Is this the same as a permalink

Thomas & Friends: The Great Bubbly Build Bath Time Book

Finding a character named just after me in The Worst Witch books

The Many Adventures Of Winnie The Pooh

This is referring to the death and diseases that the Europeans endured when travelling from Europe to the Americas. The Natives are defending themselves that they are keeping themselves safe and growing their lives and tribes perfectly here without have to go and find new foreign areas to start over.

Again, the Natives are so happy in the way that they live in their simple lives that they see no reason on why they should completely change the way they live for these Europeans that are trying to come in and take over their spaces.

The Patriarch essentially means that the Natives just want to live their simple lives and don't want to change to fit into the Europeans standards. They like their traditions and don't see it as necessary to change to do things that they don't want to be apart of.

Can we separate the art from the artist? What are the moral implications of enjoying The Cosby Show? In light of the serious allegations against Bill Cosby.

This shows that the Natives were actually really thoughtful people and liked to share their food between the different members of their tribe and their family. This completely goes against all of the negativity that was being spread to them in the quotes prior.

The Natives are being compared to beggars which gives them another untrue negative attribute. The Natives had their own establishments with a stable way to get food and live peacefully.

The word "Savages" was used to describe the natives. This is such a negative connotation of the Natives making them seem evil or not established, even thought they have their own societies and were open to making treaties and peace with the Europeans. Also referring the Natives to the devil gives another negative untrue attribute about them. This writing will make those back in Europe fear for when coming to America and thing of the Natives poorly.

A step-by-step guide on creating a high-quality AI-generated video. It discusses the tools and processes involved in video production. Emphasizing the importance of quality over free options. Also, the challenges of sourcing appropriate sound.

Knowing that the Natives were actually very peaceful people and were looking for allies with the Europeans this makes sense. They are open to trying new things while creating new relations.

This shows that not everyone in Spain, and Europe in general, agreed with the exploitation of the Native Americans. Some believed that it wasn't a just thing to do and that awareness should be spread. This writing was a great way to do it with it being seen all around Europe.

The academic community is unable to live up to it's fullest potential if members within the community do not create their own perspectives and ideas.

and what good that did!

if sacrifices should not be made solely for the sake of the economy- given that the economy is supposed to support people and not the other way around then this raises important questions about which policies are truly for the people and which ones are just for economic growth.

I think this could be much shorter. A description of your writing style is a bit overkill or? But go off Mr. Hemingway

Also deine Motivation für deine Arbeit ist es die Arbeit in nem geilen Format zu schrieben so dass es jeder versteht?:D Ich würd das vielleicht eher auf das Framework verallgemeinern? Also deine Motivation war es ja nen Framework zu bauen was self-documenting workflow hat.

Sowas ist doch eher Abstract statt Motivation? Würde ich woanders hinpacken. Und man hat mir mal gesagt, dass in den Abstract keine Referenzen (zu Kapiteln oder so) sollen.

Gehört sowas in die Motivation? Ist das nicht eher Zusammenfassung?

But doesn’t Hadronspectroscopy then reflect the underlying quark/gluon (strongly interacting components) configs? Vorher hast du aber gesagt, dass die innere Zusammensetzung nicht beachtet wird.

Boah I guess the term unify could be a bit missleading here. Because Grand Unified Theory of forces is something of its own.

Sounds like a pretty Country

It seems like these are similar weather variations in countries nowadays.

The rat trap ski binding was developed in Norway in 1927. See its impact on cross-country skiing. Learn the versatility of adjustable ski bindings.

12 years old gathering their own food shows how much society has changed over time.

It is crazy how barbarious people were in this time period and the extent they were willing to go to.

I find this very intriguing that a 3 sylable word can have so much meaning.

Nigeria gained independence on October 1st. See the significance of the New Yam Festival in Igbo culture. Which involves rituals and community gatherings.

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

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Reply to the reviewers

Manuscript number: RC- 2025-03073

Corresponding author(s): Shaul Yogev

1. General Statements [optional]

We kindly thank our reviewers for their enthusiasm, thoughtful feedback, and constructive suggestions on how to strengthen our manuscript. Below, we provide a point-by-point response to reviewer comments and outline the experiments we will do to address every concern that has been raised.

2. Description of the planned revisions

• *

Reviewer #1 (Evidence, reproducibility and clarity (Required)):

This interesting study uses an unbiased genetic screen in C. elegans to identify SAX-1/NDR kinase as a regulator of dendritic branch elimination. Loss of SAX-1 results in an excess branching phenotype that is striking and highly penetrant. The authors identify several additional regulators of branch elimination (SAX-2, MOB-1, RABI-1, RAB-11.2) by using a candidate genetic screen aimed at factors that interact physically or genetically with SAX-1. They propose that SAX-1 acts by promoting membrane retrieval based on the nature of these interactors and the results of an imaging-based in vivo assay for endocytic puncta.

Major comments.

1. My biggest concern is that the phenotypes are only observed in temperature-sensitive dauer-constitutive mutant backgrounds, and not in wild-type dauers. That is, wild-type animals exiting dauer do not require SAX-1 for dendrite elimination. While this does not undermine the importance of the results, it does require more explanation. The authors write that "the requirement for sax-1... relies on specific physiological states of the dauer stage," but I do not understand what this means. Are they saying that daf-7 and daf-2 dauers are in a different "physiological state" than wild-type dauers? In what way? What is the evidence for this? A more rigorous explanation is needed. We agree that this is puzzling, and we thank the reviewer for recognizing that this does not undermine the importance of the results. There is ample evidence that daf-2 and daf-7 differ from starvation-induced dauers. For example, a recent preprint finds that the transcriptomes of these two mutants at dauer cluster much closer to each other than to starvation-induced dauers (Corchado et al. 2024). Older work has noted other differences, such as the time the dauer entry decision is made (Swanson and Riddle 1981), the synchronicity of dauer exit, the ability to force dauer entry in daf-d mutants, as well as additional dauer-unrelated phenotypes (reviewed in Karp 2018). We agree with the reviewer that this merits further clarifications and will perform the experiments suggested by the reviewer below:

To me, the simplest genetic explanation is that daf-7 and daf-2 are partially required for branch retraction in a manner redundant with sax-1, and the ts mutants are not fully wild-type at 15C. Thus, the sax-1 requirement is revealed only in these mutant backgrounds. Can the authors examine starvation-induced dauers of daf-7 or daf-2 raised continuously at 15C?

We will do this experiment.

daf-7 and daf-2 ts strains can form "partial dauers" that have a dauer-like appearance but are not SDS resistant. Could the difference between partial dauers and full dauers account for the difference in sax-1-dependence? The authors could use SDS selection of the daf-7 strain at 25C to ensure they are examining full dauers.

We tested daf-7 mutants with 1% SDS when we set up the system – they are fully dauer at 25°C and are SDS sensitive after exit. We will repeat this important control with daf-7; sax-1 double mutants.

The Bargmann lab has created a daf-2 FLP-OUT strain (ky1095ky1087) that allows cell-type-specific removal of daf-2. Could this be used to test for a cell-autonomous role of daf-2 in IL2Q related to branch elimination?

We can attempt this experiment. However, since IL2 promoters turn on prior to dauer, the interpretation would not be straightforward – it would be hard to exclude that a cell autonomous defect in dauer entry does not account for the IL2 dauer exit phenotype, even if branching appears normal.

These ideas are not a list of specific experiments the authors need to complete, rather they are meant to illustrate some possible approaches to the question. Whatever approach they use, it is important for them to more rigorously explain why SAX-1 is not required for branch removal in wild-type animals.

We completely agree. We will carry out the 15°C experiment, examine morphological characteristics and test SDS resistance. In addition, we will test neuronal markers that differ between dauers and non-dauers to determine whether the mutants are full or partial dauers at the relevant timepoints.

The SAX-2 localization (Fig. 4) and endocytosis assay (Fig. 6) results were not clear to me from the data shown. Overall a more rigorous analysis and presentation of the data would be important to make these conclusions convincing. This may involve refining the data presentation in the figures, modifying the claims (e.g., "we propose" vs "we find"), or saving some of the data to be more fully explored in a future paper. In my view, these figures are the biggest weak point of the manuscript and also are not important for the central conclusions (which are well supported and convincing), indeed these results are barely mentioned in the Abstract or last paragraph of Introduction.

We agree that the analysis and presentation of Figures 4 and 6 need to be improved. The presentation has already been updated, and the figures are clearer now. In the revision, we will increase sample size to provide stronger conclusions, consolidate some of the analysis and further improve presentation. While we agree with the reviewer that conclusions from these figures are not as strong as those drawn from genetic experiments, they do complement and support the conclusions of those other figures.

• In Fig. 4D, why is SAX-2 visible throughout the entire neuron and why is the "punctum" marked with an arrow also seen in the tagRFP channel? One gets the impression that some of the puncta may be background, bleed-through, or artifacts due to cell varicosities.

There is no bleed-through: this is most evident by looking at the brightest signals in the cell body (now labelled with an asterisk in a zoomed-out image) and noting that they do not bleed between channels. In sax-1 mutants, the SAX-2::GFP puncta are very obvious and distinguishable from the tagRFP channel. In control, SAX-2::GFP is very faint in the dendrite, so we increased the contrast to allow visualization. The reviewer is correct that under these conditions, some puncta look like the cytosolic fill. In the revision, we will re-analyze the data and will not consider these as bona-fide SAX-2 puncta, but rather cytosolic SAX-2 that accumulates due to constrictions and varicosities in the dendrite.

• Related to both Fig. 4 and Fig. 6, where does SAX-1 localize in IL2Q in dauer and post-dauer? Does its expression or localization change during branch retraction? Does it co-localize with SAX-2 or endocytic puncta?

We generated an endogenously tagged sax-1 with a 7xspGFP11 tag; however, this was below detection in the IL2s. For the revisions, we can test an overexpressed cDNA construct.

**Referee cross-commenting**

I think we all touched on similar points. I wanted to follow up on Reviewer 3's comment, "Is the failure to eliminate branches an indication of incomplete dauer recovery? Do sax-1 mutants retain additional characteristics of dauer morphology in post dauer adults." I thought this was an excellent point. It made me wonder if that might explain why the defect is only seen in daf-7 and daf-2 mutant backgrounds - maybe these strains retain partial dauer traits even after exit. Is there a specific experiment that they could do? Did you have specific characteristics of dauer morphology in mind for them to check? (Ideally something in the nervous system that can be scored quantitatively.)

Please see response to point #1 regarding experiments we will do to confirm the “dauer state” of daf-7 and daf-7; sax-1 double mutants.

Reviewer #1 (Significance (Required)):

A major strength of this work is the pioneering use of a novel system to study neuronal branch retraction. C. elegans has provided a powerful model for studying how dendrite branches form, but much less attention has been paid to how excess neuronal branches are removed. The post-dauer remodeling of IL2Q neurons provides an exciting and dramatic physiological example to explore this question.

This paper is notable for taking the first steps towards developing this innovative model. It does exactly what is needed at the outset of a new exploration - a forward genetic screen to discover the main regulators of the process. Using a combination of classical and modern genetic approaches, the authors bootstrap their way to a sizeable list of factors and a solid understanding of the properties of this system, for example that retraction of higher vs lower order dendrites show different genetic requirements.

We thank the reviewer for recognizing the novelty and significance of our work.

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

In this manuscript, the authors establish C. elegans IL2 neurons as a system in which to study dendrite pruning. They use the system to perform a genetic screen for pruning regulators and find an allele of sax-1. Unexpectedly sax-1 is only required for post-dauer pruning in two different genetic backgrounds that induce dauer formation, but not starvation-induced dauer formation. Sax-1/NDR kinase reduction has previously been associated with increased outgrowth and branching in other systems, so this is a new role for this protein. However, the authors show that proteins that work with Sax-1 in other systems, like sax-2/fry, also play a role in this pathway. The genetic experiments are beautiful and the findings are all clearly explained and strongly supported. The authors also examine sax-2 localization, which localizes sax-1 in other systems, and show it in puncta in dendrites that increase with dauer exit, consistent with function at the time of pruning. They also show that membrane trafficking regulators associated with NDR kinases function in the same pathway here, hinting that endocytosis may play a role during pruning as in Drosophila. The link to endocytosis was a little weak (see Major point below). Overall, this study describes a new system to study pruning and identifies NDR/fry/Rabs as regulators of pruning during dauer exit. The work is very high quality and both the imaging and genetics are extremely well done.

We thank the reviewer for their positive assessment of the manuscript.

Major points

1. The only place where there were any questions about the data was the last figure (6G and I). Here they use uptake of GFP secreted from muscle as a readout of endocytosis in IL2 neurons. They nicely show that more internalized puncta accumulate as animals exit dauer. The claim that this is reduced in sax-1 mutants doesn't seem to match the images shown well. In the image there are many more puncta in the GFP channel and much more accumulation of the RFP-tagged receptor everywhere. It seems like some additional analysis of this data is important to fully capture what is going on and whether this really represents an endocytic defect. We agree and will provide additional data in Figure 6. The specific discrepancy between the image and the quantification is because we showed a single focal plane rather than a projection. This does not capture all the puncta in a neurite. The current version shows a projection, making it evident that the mutants has fewer puncta compared to the control.

Reviewer #2 (Significance (Required)):

Neurite pruning is important in all animals with neurons. Genetic approaches have primarily been applied to the problem using Drosophila, so identifying a new model system in which to study it is an important step. Using this system, a pathway known to function in a different context is linked to pruning. Thus the study provides new insights into both pruning and this pathway.

We thank the reviewer for the positive assessment of our study’s significance.

__Reviewer #3 (Evidence, reproducibility and clarity (Required)): __

Summary: Figueroa-Delgado et al. use a C. elegans neuro plasticity model to examine how dendrites are eliminated upon recovery from the stress induced larval stage, dauer. The authors performed a mutagenesis screen to identify novel regulators of dendrite elimination and revealed some surprising results. Branch elimination mechanism varies between 2{degree sign}, 3{degree sign}, and 4{degree sign} branches. The NDR kinase, SAX-1 and it's interactors (SAX-2 and MOB-2) are required for elimination of second and third order branches but not fourth order branches. Interestingly they showed that branch elimination varies depending on the stimulus of dendrite outgrowth such that the NDR kinase is required for branch elimination after genetically inducing the dauer stage but is not required if dauers are produced through food deprivation. The authors go a step further to include a small candidate screen looking at various pathways of membrane remodeling and identify additional regulators of dendrite elimination related to membrane trafficking including RABI-1, RAB-8, RAB-10, and RAB-11.2.

We thank the reviewer for their time and suggestions below

Major comments:

• While I find the data promising and exciting, several of the experiments have concerningly low sample sizes. Fig 3G, Fig 4G, Fig 5J and L, and Fig 6I all contain data sets that are fewer than 10 animals. Sample sizes should be stated specifically in the figure legends for all data represented in the graphs. We thank the reviewer for finding the data exciting. We agree that the sample sizes in some panels is low and will increase it in the revised version. Sample sizes are now specifically listed in the figure legends.

• All statements based on data not shown should be amended to include the data as a supplemental figure or edited to omit the statement based on withheld data. We agree. Some “not shown” data are already added to the current version of the manuscript and the rest will be added to the fully revised version, or the statements will be omitted.

• Rescue experiments (Fig 2J) should demonstrate failure to rescue from neighboring tissue types (hypodermis and muscle) to conclude cell autonomous rescue rather than a broadly acting factor. Thank you for the suggestion. We will use a hypodermal promoter and a muscle promoter driving SAX-1 cDNA expression to strengthen the claim of cell autonomy.

• Fig 4 needs quantification of higher order branches and SAX-2 proximity to branch nodes as these are discussed in the text. We will add this quantification.

Minor comments:

• Fig 1C-F, It appears like the shy87 allele produces animals of significantly different body sizes. It would improve rigor to normalize the dendrite coverage to body size in the quantification. We do not see a biologically meaningful size difference between shy87 and control, it may be the specific image shown. We will confirm this by measuring animal size for the final revision.

• Is the failure to eliminate branches an indication of incomplete dauer recovery? Do sax-1 mutants retain additional characteristics of dauer morphology in post dauer adults. This important point was also raised by Reviewer 1. We will test SDS sensitivity, morphological markers, and molecular markers to determine the dauer “state” of the mutants used in this study. The results will be included in the final revision.

• The text references multiple transgenic lines tested in Fig 2I-J but only one line is shown. Additional lines were visually examined under a fluorescent compound microscope but not imaged or quantified. We will add this quantification to the final revision.

• Fig 4F, Additional timepoints would enhance the sax-1 localization result and might provide insight into mechanism of action for sax-1. We will add the localization in post-dauer adults.

• Fig 6I Control and sax-1(ky491) example images should be provided in the supplement. We will add these images to the final revision.

**Referee cross-commenting**

I agree that we shared many of the same concerns.

There are several general assays for dauer characteristics that could be used here to determine if the post-dauer animals retain other characteristics of the dauer stage in addition to IL2 branches (SDS resistance, alae remodeling, pharyngeal bulb morphology, nictation behavior). The nictation behavior has been connected very nicely with IL2 neurons (Junho Lee's group). Additionally, FLP dendrites occupy the same space as the IL2 branches and outgrowth in post-dauers occurs in coordination with IL2 branch elimination - this might be another optional experiment, to check if FLP growth is impeded by persistent IL2 branches. All of these could be quantified similar to how the authors have already established with their IL2 model (FLP dendrite branches) or with a binary statistic.

Please see responses to Reviewer 1 and 3 above for the list of experiments to determine whether the animals fail to completely enter or exit dauer.

Reviewer #3 (Significance (Required)):

SIGNIFICANCE ============ These results describe a new role for the NDR kinase complex in dendrite pruning that has clinical significance to our understanding of human brain development and human health concerns in which pruning is dysregulated, such as observed in the case of autism. The authors use an established neuro-plasticity, C. elegans model (Schroeder et al. 2013) which provides a tractable and reproduceable platform for discovering the mechanism of dendrite pruning. These results would influence future work in the fields of cell biology of the neuron and disease models of brain development.

My expertise is in the field of C. elegans neuroscience and stress biology and have sufficient expertise to evaluate all aspects of this work.

3. Description of the revisions that have already been incorporated in the transferred manuscript

Reviewer #1

• In Fig. 4C, the distinction between puncta in the primary or higher-order dendrites is not clear to me, and several puncta that I would have scored as primary are marked as higher-order.

We apologize for a mistake in the arrowhead color and overall presentation of this figure. It has been fixed in the current version.

• Related to this, in Fig. 4B are the two arrows meant to be white as in the top panel, or yellow as in the bottom panel?

We thank Reviewer #1 for their observation, and we apologize for our oversight. We fixed this in the current version.

• In Fig. 4, where in the head are we looking? It would help to show a more low-magnification view of the entire cell.

We added zoomed-out images and indicated where the zoomed in insets are taken from. We thank the reviewer for helping us improve the clarity of the data.

• The main sax-1 phenotype is increased SAX-2 puncta in dauer, but the branch retraction defect is in post-dauers. How is this relevant to the phenotype?

This is a very good point. The increase in SAX-2 puncta in sax-1 mutants is stronger during dauer-exit than in dauer, consistent with this being the time when SAX-1 functions. We agree that some earlier activity of SAX-1 cannot be excluded, and we do not assume that the effect on SAX-2 completely accounts for the pruning defects. This is now acknowledged in the text. However, given that both proteins function together in pruning, and given that the effect is strongest during dauer exit, we do believe that this data is informative and worth showing.

• The number of SAX-2 puncta in sax-1 mutants decreases almost to normal in post dauers. Is there a correlation between the number of remaining branches and the number of SAX-2 puncta? That is, do the many wild-type animals with "excess" SAX-2 puncta also fail to retract branches?

There is no correlation. In other words, the number of SAX-2 puncta does not instruct the extent of pruning. Please note the quantifications underestimate the number of SAX-2 puncta in the mutants, since they were only done on the primary dendrite. This is necessary because the mutant and control have different arbor size, so only branch order that can be appropriately compared are primary dendrites.

• The control post-dauer data in Fig. 4F and 4H are identical (re-used data) but the corresponding control dauer data in Fig. 4F and 4G are different. What is going on here?

We thank the reviewer for raising this point and apologize for the oversight in data presentation. In the revised manuscript, we now show all control and experimental data integrated into a single graph, ensuring that each dataset is represented accurately to provide a comparison between dauer and post dauer recovery conditions.

• Why are sample sizes so small for both strains in Fig. 4G compared to Fig. 4F and 4H?

We sincerely apologize for this mistake, some of the data was erroneously grouped in the original submission. The revised version contains an updated number of neurons, presented on the same graph, and in the final revision we will further increase sample size. We apologize again for this error.

• In Fig. 6C, why are the tagRFP (blue) puncta larger than the neurite? Aren't these meant to represent vesicles inside the surrounding neurite? One gets the impression that this is bleed-through from the GFP channel.

Based on EM, both an endocytic punctum and the diameter of the neuron are smaller than a single pixel. The apparent difference in size in fluorescence microscopy is because the puncta are brighter (they contain more membrane) and thus appear larger. In the current version, the improved presentation of the figure contains zoomed out images that clearly show that there is no bleed-through.

• In Fig. 6E and 6F, why are there no tagRFP (blue) puncta? Is CD8 not endocytosed at all if it lacks the nanobody sequence? One would expect the tagRFP (blue) signal to be the same in both strains and simply to lack yellow if the nanobody is not present.

CD8 lacks clear endocytosis motifs, which is why it is advantageous for labelling neurites and testing endocytosis when paired with an endocytic signal (Lee and Luo 1999; Kozik et al. 2010). Conversely, extracellular GFP binding to a membrane GFP antibody can induce endocytosis (for example, see (Tang et al., 2020)), likely by inducing clustering, although we are not familiar with work that explored the mechanism. In the updated version we included a rare example of an mCD8 punctum.

• The authors report a decrease in endocytic events in sax-1, but qualitatively it looks like there are vastly more puncta inside the neuron in Fig. 6H than in 6G.

We apologize for the presentation in the original version of Figure 6. This impression was because we showed single focal planes that only captured some of the signal. In the revised version we show projections, which makes it evident that there are fewer endocytic events in the mutant.

• In Fig. 6E and 6H, why are there so many GFP (yellow) puncta outside the neuron? What are these structures and why are they absent in the strain with the nanobody?

These puncta are secreted or muscle-associated GFP that has not been internalized by IL2Q neurons. They are present in all strains in this figure, this can be clearly seen in the zoomed-out images that have been added to the updated figure.

• What is the large central blue structure in Fig. 6H - is this the soma? - and why are puncta in this region not counted?

This is indeed the soma. In the updated version this can be clearly seen in the zoom-out. The large puncta in the soma were not counted because they may arise from the fusion of an unknown number of smaller puncta, and their precise number cannot be determined at the resolution of fluorescence microscopy.

• minor: there is text reading "40-" in the bottom panel of Fig. 6H. It is visible when printed but not on screen - adjust levels in Photoshop to reveal it.

We thank the reviewer for catching this oversight, it is now fixed.

Minor points:

1. At several points the authors emphasize the relationship of neurite remodeling to stress, e.g. Abstract and Discussion: "we adapted C. elegans IL2 sensory dendrites as a model [of...] stress-mediated dendrite pruning". It seems unnecessary and potentially misleading to treat this as a neuronal stress response. First, it conflates organismal and cellular stress - there is no reason to think that IL2 neurons are under cellular stress in dauer. In fact parasitic nematodes go through dauer-like stages as part of healthy development and probably have similar remodeling of IL2. Second, dendrite pruning occurs during dauer exit, which is the opposite of a stress response - it reflects a return to favorable conditions. We agree. We modified the abstract and discussion to avoid conflating organismal stress (the alleviation of which is relevant for triggering pruning) and cellular stress. Thank you for pointing this out.

In Fig. 1A, C. elegans is shown going directly from L1 to dauer in response to unfavorable conditions, which is incorrect. Animals proceed through L2 (in many cases actually an alternative L2d pre-dauer) and then molt into dauer (an alternative L3 stage) after completing L2.

We updated the schematic to include the L2d stage where commitment to dauer entry or resumption to reproductive development is made.

In Fig. 1B, please check if it is correct that hypodermis contacts the pharynx basement membrane as drawn. The schematic in the top panel makes it look like there is a single secondary branch and the quaternary branches are similar in length to the primary dendrite. The schematic in the bottom panel makes it look like the entire neuron is a small fraction of the length of the pharynx. Could these be drawn closer to scale?

The hypodermis does contact the pharynx basement membrane. We redrew the schematic for clarity.

Reviewer #2

For context, it might be helpful to know whether branching of other dendrites is increased in sax-1 mutants (as expected based on phenotypes in other animals) or decreased like IL2 neurons.

We examined the branching pattern of PVD, a polymodal nociceptive neuron (new Supplemental Figure 3). We find no significant difference between control and sax-1 or sax-2 mutants, suggesting that these genes function in the context of pruning. Recent work (Zhao et al. 2022) confirms that sax-1 is not required for PVD branching.

Minor:

"shy87 mutant dauers showed a minor reduction in secondary and tertiary branches compared to control (Figure 1G). These results indicate that shy87 is specifically required for the elimination of dauer-generated dendrite branches." Maybe temper the specificity claim some as the reduction in branches is definitely there.

We agree, the claim was tempered.

"three complimentary approaches" should be complementary

Thank you for noticing. We fixed this.

"In control animals, SAX-2 was mostly concentrated in the cell body (data not shown)" It might be nice to include some overview images that show the cell body for completeness.

We added zoomed-out images to the revised figure, thank you for the suggestion.

Reviewer #3

Minor comments:

• Fig 1G-H, are shy87 second and third order branch counts statistically different between dauer and post dauer adults? This comparison would strengthen the claim that these order branches fail to eliminate all together rather than undergo a partial elimination. We added this to Figure S2. The shy87 mutants show a complete failure in eliminating secondary branches (i.e. no difference between dauer and post-dauer) and a strong but incomplete defect in eliminating tertiary branches.

• Fig 4B-E Indicate branch order in the images, this is unclear and a point that is focused on in the text. Done.

• Discussion of Fig 1G from the text claims that shy87 is specifically required for branch elimination yet the data shows significant defects in branch outgrowth as well. This raises the question, are the branches abnormally stabilized that results in early underdevelopment and late atrophy? Authors should acknowledge alternative hypotheses. We agree and will revise the text accordingly. The difference between shy87 and control dauers, while statistically significant, is relatively minor and can only be detected by careful quantification, it is not apparent from looking at the images (in contrast for example to rab-8 and rab-10 mutants, where we acknowledge in the text that their branching defects might affect subsequent pruning.

• Authors reference a branch elimination process but don't outline what this would entail and where their results fit in. We apologize for being unclear. Given that sax-1 and sax-2 function together, one would intuitively expect to see SAX-2 being reduced in sax-1 mutants, yet the opposite is observed. On potential explanation is that SAX-1 does not directly control SAX-2 abundance, but that clearance of SAX-2 is part of the pruning process that both proteins regulate. This would explain the enrichment of SAX-2 in sax-1 mutants. However, additional models cannot be excluded, and we acknowledge this in the revised text.

References:

Corchado, Johnny Cruz, Abhishiktha Godthi, Kavinila Selvarasu, and Veena Prahlad. 2024. “Robustness and Variability in Caenorhabditis Elegans Dauer Gene Expression.” Preprint, bioRxiv, August 26. https://doi.org/10.1101/2024.08.15.608164.

Karp, Xantha. 2018. “Working with Dauer Larvae.” WormBook, August 9, 1–19. https://doi.org/10.1895/wormbook.1.180.1.

Kozik, Patrycja, Richard W Francis, Matthew N J Seaman, and Margaret S Robinson. 2010. “A Screen for Endocytic Motifs.” Traffic (Copenhagen, Denmark) 11 (6): 843–55. https://doi.org/10.1111/j.1600-0854.2010.01056.x.

Lee, T., and L. Luo. 1999. “Mosaic Analysis with a Repressible Cell Marker for Studies of Gene Function in Neuronal Morphogenesis.” Neuron 22 (3): 451–61.

Swanson, M. M., and D. L. Riddle. 1981. “Critical Periods in the Development of the Caenorhabditis Elegans Dauer Larva.” Developmental Biology 84 (1): 27–40. https://doi.org/10.1016/0012-1606(81)90367-5.

Tang, Rui, Christopher W Murray, Ian L Linde, et al. n.d. “A Versatile System to Record Cell-Cell Interactions.” eLife 9: e61080. https://doi.org/10.7554/eLife.61080.

Zhao, Ting, Liying Guan, Xuehua Ma, Baohui Chen, Mei Ding, and Wei Zou. 2022. “The Cell Cortex-Localized Protein CHDP-1 Is Required for Dendritic Development and Transport in C. Elegans Neurons.” PLOS Genetics 18 (9): e1010381. https://doi.org/10.1371/journal.pgen.1010381.

4. Description of analyses that authors prefer not to carry out

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

Learn more at Review Commons

Referee #3

Evidence, reproducibility and clarity

Summary:

Figueroa-Delgado et al. use a C. elegans neuro plasticity model to examine how dendrites are eliminated upon recovery from the stress induced larval stage, dauer. The authors performed a mutagenesis screen to identify novel regulators of dendrite elimination and revealed some surprising results. Branch elimination mechanism varies between 2{degree sign}, 3{degree sign}, and 4{degree sign} branches. The NDR kinase, SAX-1 and it's interactors (SAX-2 and MOB-2) are required for elimination of second and third order branches but not fourth order branches. Interestingly they showed that branch elimination varies depending on the stimulus of dendrite outgrowth such that the NDR kinase is required for branch elimination after genetically inducing the dauer stage but is not required if dauers are produced through food deprivation. The authors go a step further to include a small candidate screen looking at various pathways of membrane remodeling and identify additional regulators of dendrite elimination related to membrane trafficking including RABI-1, RAB-8, RAB-10, and RAB-11.2.

Major comments:

• While I find the data promising and exciting, several of the experiments have concerningly low sample sizes. Fig 3G, Fig 4G, Fig 5J and L, and Fig 6I all contain data sets that are fewer than 10 animals. Sample sizes should be stated specifically in the figure legends for all data represented in the graphs.
• All statements based on data not shown should be amended to include the data as a supplemental figure or edited to omit the statement based on withheld data.
• Rescue experiments (Fig 2J) should demonstrate failure to rescue from neighboring tissue types (hypodermis and muscle) to conclude cell autonomous rescue rather than a broadly acting factor.
• Fig 4 needs quantification of higher order branches and SAX-2 proximity to branch nodes as these are discussed in the text.

Minor comments:

• Fig 1C-F, It appears like the shy87 allele produces animals of significantly different body sizes. It would improve rigor to normalize the dendrite coverage to body size in the quantification.
• Fig 1G-H, are shy87 second and third order branch counts statistically different between dauer and post dauer adults? This comparison would strengthen the claim that these order branches fail to eliminate all together rather than undergo a partial elimination.
• Discussion of Fig 1G from the text claims that shy87 is specifically required for branch elimination yet the data shows significant defects in branch outgrowth as well. This raises the question, are the branches abnormally stabilized that results in early underdevelopment and late atrophy? Authors should acknowledge alternative hypotheses.
• Is the failure to eliminate branches an indication of incomplete dauer recovery? Do sax-1 mutants retain additional characteristics of dauer morphology in post dauer adults.
• The text references multiple transgenic lines tested in Fig 2I-J but only one line is shown.
• Fig 4B-E Indicate branch order in the images, this is unclear and a point that is focused on in the text.
• Fig 4F, Additional timepoints would enhance the sax-1 localization result and might provide insight into mechanism of action for sax-1.
• Authors reference a branch elimination process but don't outline what this would entail and where their results fit in.
• Fig 6I Control and sax-1(ky491) example images should be provided in the supplement.

Referee cross-commenting

I agree that we shared many of the same concerns.

There are several general assays for dauer characteristics that could be used here to determine if the post-dauer animals retain other characteristics of the dauer stage in addition to IL2 branches (SDS resistance, alae remodeling, pharyngeal bulb morphology, nictation behavior). The nictation behavior has been connected very nicely with IL2 neurons (Junho Lee's group). Additionally, FLP dendrites occupy the same space as the IL2 branches and outgrowth in post-dauers occurs in coordination with IL2 branch elimination - this might be another optional experiment, to check if FLP growth is impeded by persistent IL2 branches. All of these could be quantified similar to how the authors have already established with their IL2 model (FLP dendrite branches) or with a binary statistic.

Significance

These results describe a new role for the NDR kinase complex in dendrite pruning that has clinical significance to our understanding of human brain development and human health concerns in which pruning is dysregulated, such as observed in the case of autism. The authors use an established neuro-plasticity, C. elegans model (Schroeder et al. 2013) which provides a tractable and reproduceable platform for discovering the mechanism of dendrite pruning. These results would influence future work in the fields of cell biology of the neuron and disease models of brain development.

My expertise is in the field of C. elegans neuroscience and stress biology and have sufficient expertise to evaluate all aspects of this work.

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

Learn more at Review Commons

Referee #2

Evidence, reproducibility and clarity

In this manuscript, the authors establish C. elegans IL2 neurons as a system in which to study dendrite pruning. They use the system to perform a genetic screen for pruning regulators and find an allele of sax-1. Unexpectedly sax-1 is only required for post-dauer pruning in two different genetic backgrounds that induce dauer formation, but not starvation-induced dauer formation. Sax-1/NDR kinase reduction has previously been associated with increased outgrowth and branching in other systems, so this is a new role for this protein. However, the authors show that proteins that work with Sax-1 in other systems, like sax-2/fry, also play a role in this pathway. The genetic experiments are beautiful and the findings are all clearly explained and strongly supported. The authors also examine sax-2 localization, which localizes sax-1 in other systems, and show it in puncta in dendrites that increase with dauer exit, consistent with function at the time of pruning. They also show that membrane trafficking regulators associated with NDR kinases function in the same pathway here, hinting that endocytosis may play a role during pruning as in Drosophila. The link to endocytosis was a little weak (see Major point below). Overall, this study describes a new system to study pruning and identifies NDR/fry/Rabs as regulators of pruning during dauer exit. The work is very high quality and both the imaging and genetics are extremely well done.

Major points

1. The only place where there were any questions about the data was the last figure (6G and I). Here they use uptake of GFP secreted from muscle as a readout of endocytosis in IL2 neurons. They nicely show that more internalized puncta accumulate as animals exit dauer. The claim that this is reduced in sax-1 mutants doesn't seem to match the images shown well. In the image there are many more puncta in the GFP channel and much more accumulation of the RFP-tagged receptor everywhere. It seems like some additional analysis of this data is important to fully capture what is going on and whether this really represents an endocytic defect.
2. For context, it might be helpful to know whether branching of other dendrites is increased in sax-1 mutants (as expected based on phenotypes in other animals) or decreased like IL@ neurons.

Minor:

"shy87 mutant dauers showed a minor reduction in secondary and tertiary branches compared to control (Figure 1G). These results indicate that shy87 is specifically required for the elimination of dauer-generated dendrite branches." Maybe temper the specificity claim some as the reduction in branches is definitely there.

"three complimentary approaches" should be complementary

"In control animals, SAX-2 was mostly concentrated in the cell body (data not shown)" It might be nice to include some overview images that show the cell body for completeness.

Significance

Neurite pruning is important in all animals with neurons. Genetic approaches have primarily been applied to the problem using Drosophila, so identifying a new model system in which to study it is an important step. Using this system, a pathway known to function in a different context is linked to pruning. Thus the study provides new insights into both pruning and this pathway.

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

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Referee #1

Evidence, reproducibility and clarity

This interesting study uses an unbiased genetic screen in C. elegans to identify SAX-1/NDR kinase as a regulator of dendritic branch elimination. Loss of SAX-1 results in an excess branching phenotype that is striking and highly penetrant. The authors identify several additional regulators of branch elimination (SAX-2, MOB-1, RABI-1, RAB-11.2) by using a candidate genetic screen aimed at factors that interact physically or genetically with SAX-1. They propose that SAX-1 acts by promoting membrane retrieval based on the nature of these interactors and the results of an imaging-based in vivo assay for endocytic puncta.

Major comments.

1. My biggest concern is that the phenotypes are only observed in temperature-sensitive dauer-constitutive mutant backgrounds, and not in wild-type dauers. That is, wild-type animals exiting dauer do not require SAX-1 for dendrite elimination.

While this does not undermine the importance of the results, it does require more explanation. The authors write that "the requirement for sax-1... relies on specific physiological states of the dauer stage," but I do not understand what this means. Are they saying that daf-7 and daf-2 dauers are in a different "physiological state" than wild-type dauers? In what way? What is the evidence for this? A more rigorous explanation is needed.

To me, the simplest genetic explanation is that daf-7 and daf-2 are partially required for branch retraction in a manner redundant with sax-1, and the ts mutants are not fully wild-type at 15C. Thus, the sax-1 requirement is revealed only in these mutant backgrounds. Can the authors examine starvation-induced dauers of daf-7 or daf-2 raised continuously at 15C?

daf-7 and daf-2 ts strains can form "partial dauers" that have a dauer-like appearance but are not SDS resistant. Could the difference between partial dauers and full dauers account for the difference in sax-1-dependence? The authors could use SDS selection of the daf-7 strain at 25C to ensure they are examining full dauers.

The Bargmann lab has created a daf-2 FLP-OUT strain (ky1095ky1087) that allows cell-type-specific removal of daf-2. Could this be used to test for a cell-autonomous role of daf-2 in IL2Q related to branch elimination?

These ideas are not a list of specific experiments the authors need to complete, rather they are meant to illustrate some possible approaches to the question. Whatever approach they use, it is important for them to more rigorously explain why SAX-1 is not required for branch removal in wild-type animals. 2. The SAX-2 localization (Fig. 4) and endocytosis assay (Fig. 6) results were not clear to me from the data shown. Overall a more rigorous analysis and presentation of the data would be important to make these conclusions convincing. This may involve refining the data presentation in the figures, modifying the claims (e.g., "we propose" vs "we find"), or saving some of the data to be more fully explored in a future paper. In my view, these figures are the biggest weak point of the manuscript and also are not important for the central conclusions (which are well supported and convincing), indeed these results are barely mentioned in the Abstract or last paragraph of Introduction.

• In Fig. 4, where in the head are we looking? It would help to show a more low-magnification view of the entire cell.
• In Fig. 4D, why is SAX-2 visible throughout the entire neuron and why is the "punctum" marked with an arrow also seen in the tagRFP channel? One gets the impression that some of the puncta may be background, bleed-through, or artifacts due to cell varicosities.
• In Fig. 4C, the distinction between puncta in the primary or higher-order dendrites is not clear to me, and several puncta that I would have scored as primary are marked as higher-order.
• Related to this, in Fig. 4B are the two arrows meant to be white as in the top panel, or yellow as in the bottom panel?
• The main sax-1 phenotype is increased SAX-2 puncta in dauer, but the branch retraction defect is in post-dauers. How is this relevant to the phenotype?
• The number of SAX-2 puncta in sax-1 mutants decreases almost to normal in post dauers. Is there a correlation between the number of remaining branches and the number of SAX-2 puncta? That is, do the many wild-type animals with "excess" SAX-2 puncta also fail to retract branches?
• The control post-dauer data in Fig. 4F and 4H are identical (re-used data) but the corresponding control dauer data in Fig. 4F and 4G are different. What is going on here?
• Why are sample sizes so small for both strains in Fig. 4G compared to Fig. 4F and 4H?
• In Fig. 6C, why are the tagRFP (blue) puncta larger than the neurite? Aren't these meant to represent vesicles inside the surrounding neurite? One gets the impression that this is bleed-through from the GFP channel.
• In Fig. 6E and 6F, why are there no tagRFP (blue) puncta? Is CD8 not endocytosed at all if it lacks the nanobody sequence? One would expect the tagRFP (blue) signal to be the same in both strains and simply to lack yellow if the nanobody is not present.
• In Fig. 6E and 6H, why are there so many GFP (yellow) puncta outside the neuron? What are these structures and why are they absent in the strain with the nanobody?
• What is the large central blue structure in Fig. 6H - is this the soma? - and why are puncta in this region not counted?
• The authors report a decrease in endocytic events in sax-1, but qualitatively it looks like there are vastly more puncta inside the neuron in Fig. 6H than in 6G.
• minor: there is text reading "40-" in the bottom panel of Fig. 6H. It is visible when printed but not on screen - adjust levels in Photoshop to reveal it.
• Related to both Fig. 4 and Fig. 6, where does SAX-1 localize in IL2Q in dauer and post-dauer? Does its expression or localization change during branch retraction? Does it co-localize with SAX-2 or endocytic puncta?

Minor points:

1. At several points the authors emphasize the relationship of neurite remodeling to stress, e.g. Abstract and Discussion: "we adapted C. elegans IL2 sensory dendrites as a model [of...] stress-mediated dendrite pruning". It seems unnecessary and potentially misleading to treat this as a neuronal stress response. First, it conflates organismal and cellular stress - there is no reason to think that IL2 neurons are under cellular stress in dauer. In fact parasitic nematodes go through dauer-like stages as part of healthy development and probably have similar remodeling of IL2. Second, dendrite pruning occurs during dauer exit, which is the opposite of a stress response - it reflects a return to favorable conditions.
2. In Fig. 1A, C. elegans is shown going directly from L1 to dauer in response to unfavorable conditions, which is incorrect. Animals proceed through L2 (in many cases actually an alternative L2d pre-dauer) and then molt into dauer (an alternative L3 stage) after completing L2.
3. In Fig. 1B, please check if it is correct that hypodermis contacts the pharynx basement membrane as drawn. The schematic in the top panel makes it look like there is a single secondary branch and the quartenary branches are similar in length to the primary dendrite. The schematic in the bottom panel makes it look like the entire neuron is a small fraction of the length of the pharynx. Could these be drawn closer to scale?

Referee cross-commenting

I think we all touched on similar points. I wanted to follow up on Reviewer 3's comment, "Is the failure to eliminate branches an indication of incomplete dauer recovery? Do sax-1 mutants retain additional characteristics of dauer morphology in post dauer adults." I thought this was an excellent point. It made me wonder if that might explain why the defect is only seen in daf-7 and daf-2 mutant backgrounds - maybe these strains retain partial dauer traits even after exit. Is there a specific experiment that they could do? Did you have specific characteristics of dauer morphology in mind for them to check? (Ideally something in the nervous system that can be scored quantitatively.)

Significance

A major strength of this work is the pioneering use of a novel system to study neuronal branch retraction. C. elegans has provided a powerful model for studying how dendrite branches form, but much less attention has been paid to how excess neuronal branches are removed. The post-dauer remodeling of IL2Q neurons provides an exciting and dramatic physiological example to explore this question.

This paper is notable for taking the first steps towards developing this innovative model. It does exactly what is needed at the outset of a new exploration - a forward genetic screen to discover the main regulators of the process. Using a combination of classical and modern genetic approaches, the authors bootstrap their way to a sizeable list of factors and a solid understanding of the properties of this system, for example that retraction of higher vs lower order dendrites show different genetic requirements.

https://bytes.vadeai.com/how-one-clojure-function-destroyed-agent-framework-completely/

How Clojure's iteration Function Disrupts Agent Frameworks

Agent Framework Pitfalls

Traditional agent frameworks like CrewAI introduce significant complexity with configuration files, rigid agent definitions, and orchestration mechanics. They require developers to manage state, orchestrate tasks, handle errors, and manage resources, but still abstract away critical decisions, making debugging and customization difficult[1].

The Simplicity of iteration

Clojure 1.11 introduced the iteration function, which models any sequential, stateful process — including agentic workflows — far more simply than the framework approach. Its key parameters:

• step: Does the work (e.g., LLM call, tool execution)
• initk: Starting state (prompt, initial data)
• vf: Extracts the meaningful result from each step
• kf: Determines the next state for the following iteration
• somef: Decides if the workflow continues or stops

This aligns perfectly with agentic workflows: - step: agent action - initk: initial task/state - vf: extract agent output - kf: update agent context/state - somef: goal/termination checker[1]

Real World Example

A basic agent workflow with iteration in Clojure:

clojure (defn simple-agent-workflow [initial-prompt max-iterations] (let [llm-instance (create-llm-instance) step-fn (fn [{:keys [iteration prompt response]}] (when (< iteration max-iterations) (let [messages [(create-message :user prompt)] new-response (generate llm-instance messages) next-prompt (extract-next-task new-response)] {:iteration iteration :prompt prompt :response new-response :next-token {:iteration (inc iteration) :prompt next-prompt :response new-response}})))] (iteration step-fn :somef (fn [res] (some? res)) :vf identity :kf :next-token :initk {:iteration 0 :prompt initial-prompt :response {}})))

Production variants in Vade AI simply expand this pattern for live API streaming, logging, and complex branching, without introducing unnecessary abstraction or opaque state[1].

Benefits Over Frameworks

• Complete control: Every workflow step and state transition is transparent and customizable.
• Easy debugging: Print or inspect state at any moment; no special debugging tools needed.
• Flexible termination: Workflow can halt based on any custom logic, not just predefined callbacks.
• Resource efficiency: No framework overhead, predictable and low memory footprint.
• Streaming and real-time: Can process operations incrementally as LLM responses stream in, with immediate visibility for the user[1].
• Composability: Integrates natively with the rest of Clojure — no framework lock-in.

When to Use This Approach

The iteration pattern is ideal when you need:

• Custom agent behaviors
• Transparent workflows
• Performance optimization
• Complex branching or termination logic
• Deep integration with Clojure systems

Especially powerful for research and analysis, planning systems, validation pipelines, and unique business logic that standard frameworks struggle to express[1].

Key Takeaways

Frameworks often create more complexity than they solve. By embracing Clojure’s iteration, you implement agentic workflows with less code, greater clarity, and full control. This enables adaptive, resource-aware, and highly debuggable systems — proven at scale inside Vade AI[1].

Citations: [1] How One Clojure Function Destroyed Agent Framework Completely https://bytes.vadeai.com/how-one-clojure-function-destroyed-agent-framework-completely/

Tina Arena's achievements as an acclaimed Australian singer and songwriter. Her significant record sales. And her influence in the music industry.

I don’t know but should you write the word we? Doesn’t it imply that didn’t do the work on you own?

I don‘t know if I would mention excited here in particular. Would start with the things you mention later: the framework is tested on excited light baryon resonances… Else it kind of implies that excited hardon state are more important.

I wont call Hadron spectroscopy a systematic framework. Or? It is more of an umbrella term for a multitude of methods (PWA, Lattice …) with the goal to classify hadrons into spectra. These methods are then realised many different computer frameworks?

JP opens door for rate cut next month.

JP first time suggested that the effects of higher goods prices due to tarrifs will be relatively short-lived.

JP has opened the door for rate cut next month.

JP believes tarffis on having effect on consumer prices and will further accumulate in the months ahead but he doesnt know if it will materially raise the risk of inflation.

Throughout the year, the Fed has kept rates steady due to strong labor market and uncertainity over the tarrifs impact on inflation.

JP opened the door for possible rate cuts next month because the possiblity of a sharper slodwn down reduces concerns of tarrifs causing inflation.

Preferred to be dead than live an inhumane life with no rights or freedom

Envied the white men who had freedom

the dipole charges symbol is determined by the ends of a dipole molecule for example H2O has two hydrogens and one oxygen. oxygen is taking the electrons because of its higher electronegativity giving it a negative partial charge, and that leaves the two hydrogens with a positive end and those positive ends are attracted to the negative oxygen in other molecules.

In practice, however, I found that many so-called digital gardens lack this important feature: Recent changes/changelog. Many of them don't even have a web feed or newsletter to subscribe to.

This piece of history I found really interesting, because I didn't make the connection between physics and studying the natural world. It might seem silly not to make this connection, but when I imagine studying physics, my mind goes to studying acceleration, how fast planets are moving, and understanding the laws of gravity. These are obviously connected to nature, but not in the way my mind would associate it to be. It is also intriguing that physics was originally called "natural philosophy," which, for some reason, is such a pleasant and poetic way to describe the type of science. It also has a personifying effect on nature itself, like nature is a being that has ideas and philosophies about how the world works and revolves. I will, from now on, think of physics as the study of nature's own philosophy.