When I was younger I remember one of my friends mentioning this problem. She would read, but not remember what she just read about and she wasn't applying herself to the reading. I believe, especially in the world we live in today, that many students are not participating in active reading, which hinders their learning and comprehension of subject material.

This information is all very helpful on explaining how to actually annotate. It explains what to look for, how to write it down, and the important things to note.

This is a really helpful note to think about. When reading the first time it is good to jus read through and not worry abut annotating. It helps you focus more on the material and make sense of all the information.

These are great questions to think about before reading something. The can help prepare us to have a better understanding of what we are about to read and give us things to look for and think about as we are reading.

Active reading is a skill. It isn't just something you are easily able to do without understanding it and learning what it means. I am glad it is a skill that we will learn during this semester as it is something that will be useful for a lot of different things!

The passage presents a multifaceted view of work, acknowledging its importance for survival and material well-being, while also highlighting its potential to provide psychological and social benefits. It challenges the purely economic view of work as simply a negative aspect of life.

The consists of a series of questions prompting the reader to observe and reflect on the presence, quality, and accessibility of public services and infrastructure in their neighborhood.

The text describes the approach and supplementary resources for a book aimed at making economics accessible to a general audience. and also the passage emphasizes the book's commitment to making economics understandable and engaging, while also offering additional resources for those interested in deeper study.

The passage introduces the perception of the economy as a complex and volatile entity, often portrayed as such by the media. and it sets a tone of skepticism towards the common media representation of the economy, suggesting it might be overcomplicated and repetitive.

Essentially, this passage outlines the book's approach to explaining economics through visual aids that build in complexity, with the ultimate goal of providing readers with a useful understanding of capitalism.

This part shows the fundamental activities and relationships that constitute capitalism. Work, or human effort, is identified as the driving force of the economy.

This section lays the groundwork by defining what an economy is and pinpointing the specific characteristics of a capitalist economy.

Basically, the author's trying to convince us that the economy isn't just about individual greed and competition. It's about how we're all connected and rely on each other, even in a capitalist system. It's a way of saying that everyone contributes, and nobody's truly "self-made."

The author's basically saying that economists tend to gloss over the big stuff about capitalism – like how some people have way more money and power than others. Instead of calling it capitalism, they call it a 'market economy,' which kind of sweeps the real problems under the rug."

Emphasizes the need to understand capitalism's specific characteristics

Challenges the idea that capitalism is the only or inevitable economic system.

economists are overrepresented in media discussions, and that other professions might offer more valuable insights.

I can tell ya, friend, I never thought I'd have to accidentally come across a little button in the Share Sheet when passing (literally any filetype atm lol) to discover that Apple had finally complied with the creation of custom ringtones.

why is is -2 instead of -1

This is good to note! A lot of my classes require me to submit my assignments in either pdf form or a word document, but I've always loved the commenting and collaborative nature of google docs! I know word has these functions, but I used google docs a lot in high school, and I prefer this program more!

I feel this will push me harder to be comfortable being uncomfortable with my writing. Sometimes, I believe I hold back in what I am writing, and stick within my comfort zone because I don't want to earn a bad grade. Knowing that if I take risks I might end up doing better, I feel like I will be more likely to push myself!

I love this! I feel like this will be a very vulnerable class for me, especially knowing that many of my classmates will see my annotations. However, I am going to try and make it a goal for myself to not only annotate what I'm comfortable with, but push the boundaries of what I'm comfortable annotating!

I feel this sums up most of my writing experience in high school pretty well. I feel like I always stuck to the "correct formula" because that's what I knew would get me the grade that I wanted. I look forward to pushing my comfort zone, and advancing my writing skills!

This statement is something everyone should be aware of going into this course. There are a lot of different platforms to use for writing, so make sure that google docs is the one that you use.

This statement is something everyone should know and think about throughout the semester. AI is not permitted unless otherwise stated.

This information is good to know in case of an emergency or if something comes up and you aren't able to do your work. You must be thinking ahead.

This is very encouraging for me, and good to know that we are able to express our beliefs and share with the class. This is very important and valuable to know.

This sentence is challenging for me because it highlights a key part of scientific research that can be easily missed. It points out that researchers need to know the difference between questions that can be answered through observation and those that involve personal values and opinions, which can't be tested. Why do researchers and people in general often have a hard time understanding this difference? How can making this distinction clearer improve the way research is done and make scientific findings easier to understand?

Should mention in the ScR methodology

Would put the definition of Visual aid as 1.8.2 to enhance flow

Might be useful in the rational

Kindly add abbreviations under the figure

This sentence is important because it points out a key issue with using intuition to make decisions. While our gut feelings can sometimes help us, they can also lead us to make mistakes because they are influenced by biases that affect our judgment. Understanding this helps me appreciate research methods better. It shows that even though instincts can guide us, we shouldn't rely on them alone without any evidence. This awareness encourages me to look for more reliable ways to gain knowledge, like careful observation and logical thinking, rather than just going with how I feel.

Regardless of your position held with your employer, employees should be allowed to seek time off without any issues prohibiting them to do so. Now, bear in mind if they are in Finance or Accounting the middle of the month is typically ideal when considering month end closures. That would indeed be the understanding within this field of work- similar to that of a Tax Accountant during tax season. Work-life balance is just as important as adhering to your employer's deadlines.

When I worked for a manufacturing company at the height of COVID-we as small company were forced to pivot. This company sold dog kennels which by no means made us "essential workers." However we did have connections with our manufacturer in China...thus creating the new business endeavor as a result of the pandemic. The small business decided to purchase medical masks (N95) at wholesale and resale them on our website at a reduced rate if purchased in bulk.This allowed the company to stay open for another year prior to its inevitable closure. Had we not decided to complete, our doors would've closed a lot sooner than a year that was prolonged.

The owner of this company is completely transparent. Transparency is vital although not always given in either a corporation nor a small business. With this information considered, certain exceptions for Senior level managers and other decision makers will want an increase in pay or annual / quarterly bonus. Whereas in retrospect, this could also mean company wide layoffs if the financials are seen in the red. Both situations are crucial when sharing financial reporting. (unless these companies are publicly traded- financials are typically only shared with decision makers and those composed of the finance department).

Revenue is recognized when a product is delivered. Sales are apart of revenue-sales are one of the primary contributors to total revenue.

I enjoy that the book mentioned both the internal and external Demographic influences. Corporations must make discussions based both their own employee demographics and the pressure of consumers. An example of changes for consumers from my personal interests is the fact that in the video game series Kirby, the box art for the american releases of the games has kirby with his brows furrowed. This was to appeal to the interests of teenage boys, who were the primary demographic of american nintendo consoles, while in japan they were marketed towards families.

I would consider labor the most important part of the business. Without labor, neither the commodities would be produced, and the capital wouldn't exist without someone else's labor. I believe that giving the laborers their fairest wage is very important if only on ethical grounds.

This explains the concept that the economy moves in cycles, and the highs and lows of these cycles can have a direct impact on employment, income, and business decisions. This is why economic influences are considered to be a significant external influence on businesses.

This defines how the standard of living is measured, which is not just by wages, but actually by what those wages can purchase. This highlights the difference between income levels vs purchasing power.

One of the main reasons why I have always wanted to start my own business was for this exact reason. Business owners and managers have such a big control over everything and they get to get their hand dirty with everything. They get to be apart of everything when it comes to who their hiring to what their selling.

I have seen this first hand when it comes to my dad's company. He has always thought me that to grow you need to take risks.

This shows the impact of man-made disasters and even natural disasters. One question is, is there a possibility for businesses to prepare for natural disasters that they cannot control or won't be aware of until its too late?

This quote highlights the role that businesses play in everyday life. This quote tells us that we are surrounded by businesses everywhere, not only the things we buy come from businesses, but things such as the medical industry is also considered a business.

I selected this part of the article to highlight as it ties into the main point of the article. Implicit bias is completely normal, but on the other hand it raises moral and societal controversies. The social consequences being discrimination or unfair treatment which are considered problematic.

This statement is interesting. Knowing how much social media/ technology influence us. I do believe that is where most of our generations implicit bias stems from. We scrolled the social media so often. We see that many people live lifestyles that we would like to have. Some individuals become so heavily influenced that they judge other for not having certain items, or looking a certain way. We allow what we see on a day to day basis determine how we view others before meeting that individuals.

This quote supports the main point in many ways. Many individuals say "we don't judge a book by the cover", not realizing they do it every day. Everyday you walk out of your front door individuals will judge you before speaking to you. Many factors stem from their beliefs/ upbringings. They will attempt to paint a picture of you that is not always accurate to your story. You can be the smartest person in the world with many qualifications. But many people will not take a change on you, because of implicit bias.

Excuse me!?

This text is important because it challenges a common belief that women talk more than men. The researchers found that the difference in the number of words spoken each day is very small. This shows that we shouldn't just believe stereotypes without real evidence.Understanding this helps me see how important research methods are in psychology. It demonstrates that scientific studies can reveal truths that change how we think about people. Using tools like audio recorders to collect actual data shows how research can help us understand behavior more accurately and challenge outdated ideas.

This sentence highlights how implicit bias and in-group/out-group thinking can influence how we interpret others’ actions. It suggests that when someone from a different racial group does something negative—like cutting someone off in traffic—we’re more likely to blame their personality or character rather than the situation. In contrast, if someone of our own race did the same thing, we might be more likely to excuse the behavior or assume it was circumstantial.

This sentence emphasizes the importance of first impressions and suggests that being good at reading others quickly is linked to other positive traits, like strong social skills or good judgment. I personally agree—I do believe that first impressions matter. If someone makes a bad first impression, that tends to stick in my mind. Every time I see them after that, the first thing I remember is that initial experience, almost like a mental picture I can't unsee. It’s not that people can’t change or redeem themselves, but that first moment really sets the tone for how I view them going forward.

Focusing on how the mind works and process like problem solving how we remember things notice things and languages .

Self actualization ,self esteem , social, security and physiological are all basic tenets of the humanism

To better understand the health and well being of yourself and others by understanding development

Seeing thing in the whole pattern and not in parts our mind naturally organizes the information

Influencing new ways to understand the mind and therapy

“Breaking down consciousness like feeling & sensation to understand the structure of the mind “

“Wundt studied the mind through experiments making it scientific”

The career option in psychology that is most appealing to me is sports psychology. I run cross country and track, so I know firsthand how it is to be an athlete. The mental challenges are just as hard as the physical challenges; you're not only exerting your body but your mind as well. For running especially, there is a huge mental aspect to it, it's really hard to keep going at times. I think I could relate my experience as an athlete to other athletes as a sports psychologist because I know what it's like. I want to be able to help athletes that are struggling with their mental health and make sure they are at their best mentally and physically. At first, I thought I wanted to be a therapist/counselor for people, but I think I would be better working with athletes and id be able to connect with them on a more personal level.

this experiment demonstrates the effects racism and colorism has on children.

the theory that human and animal behavior can be explained in terms of conditioning, without appeal to thoughts or feelings, and that some mental conditions are best treated by altering behavior patterns.

this can be helpful for psychology students who are looking to become therapists.

Degradation! Slop is now!

I do enjoy the idea of more creative work being rewarded with better grades. I feel some classes are completion based and that stinks when you put in more effort and thought compared to others. I like to focus on making my work high quality often.

I would like to think that we have been doing so because of the evolution of humans we naturally cling to things that describe emotions we feel. I feel that speaking, writing, and singing poems allow us to express emotions. This unlocks connection between those around us.

This is extremely helpful! My outlook gets packed full with emails so this helps me stay organized through the whole semester.

I really like how professor Folasade included this in his syllabus. Because one of my worries is failing my teacher's expectations when I gave an assignment my all. Especially with poetry. Poetry is an art and I believe that if you give your all in an art it should be recognized rather than getting a poor grade on it. In conclusion, it is reassuring to know my professor will be looking at my hard work with my own mind.

These two sentences highlight the importance of individuality of Instructor John.

This is an important thing to note considering how AI has grown even just in the past year. Even 3 years ago you would not have seen this last sentence in a syllabus, and it probably would've seemed outlandish. But here we are.

It’s really not and it would take away the fun out of the assignments in my opinion. Use your own creativity!

This will help a lot because I have a few other assignments that are due at the same time.

This should take a lot of weight off of my shoulders.

This might be the most important thing to talk about because people are afraid to fail, especially in front of others and if we (and when) we create a community where people are comfortable to admit their failures and mistakes, we will be able to flourish throughout the semester!

Thank you for posting the schedule. It makes it easy to plan ahead and compare what I have to do with other classes!

I think that the grace period I nice in case you have a class and you forgot to submit something.

Is this dictated by you (the professor) or by us? And is something like a book in free verse a book that we would be able to pick? It seems extremely interesting if we are all able to pick our own 4 books to help guide us through this class. I am also very excited for the poetry slam, and whatever that entails!

This comment is so joyful and hopeful, as pushing ourselves is so innately human. I love to be pushed and told that I will be growing.

This place has been really helpful in the past! Especially when I had writers block and didn't know how to make my writing even better, or which direction to take it!

I think this will help us break out of our shells and help us get more comfortable with different writing styles in poetry! Sometimes you learn one way to do it and it becomes a default, this will help expand our knowledge and help us be even more creative.

This makes me a little more relaxed about this class so I can focus more on being creative and being open to trying new things, with out the fear of failing.

The method of using multiple senses and experiences in learning makes me think about how effective the experience can really be. When students are only hearing information, it can and is easier for them to forget, but when they see, hear, and physically interact with the lesson and activity, it seems more memorable and as though they absorb the information better.

If teachers are too focused on making sure the final project looks perfect, I feel that takes away from students being able to think creatively and make their own choices. I personally feel like learning happens most when students can take risks, make mistakes, and figure things out amongst themselves.

Observation: This quote is stating her innocence and the fact that she has been held captive for over a week. It shows us the unjust in the trials and what these women went through during this time in history.

In this source, the speaker is pressuring the accused by saying, “They accuse you of hurting them, & if you think it is not unwillingly but by designe, you must look upon them as murderers.” What I see here is an early example of interrogation tactics that are still recognizable today—getting people to confess to things they may not have done. The logic being pushed is that if the accused did not hurt the children by accident, then it had to be intentional, and therefore she is the one guilty. The trap in the statement is that it flips the blame onto the children, making them seem like the true “murderers,” so in the accused’s eyes anything she might have done to them would appear justifiable. It connects to Context, since understanding the fear of witchcraft and the religious panic in 1692 helps explain why this kind of statement would carry so much weight in Salem.

My observation is as follows; That "upon the motion of her body fits followed...abundantly and very frequently" is that movement from her (the nurse), would cause bouts of affliction to the people accusing her of hurting them/witchcraft. Interpretation of this is, how back then, seeing this in person, live, would cause anyone being accused of the occult and power of people to be believed to do so if any sort of movement from them (witches) cause the accused to act that way. A linkage of sorts, construing her movement to their fits.

Observation: People in the room say familiar spirits come near Goody Nurse during questioning. Multiple people repeat the claim. Interpretation: The Assembly treats these reports as real signs of harm.  Context:  Shared belief in familiar spirits sets how people read the scene. Belief explains why these reports count. Why it matters: Shows why faith based claims carry weight during the examination, because of how faith based the people are

Observation: One of the afflicted people reports harm this morning Interpretation: The statement accuses Goody Nurse of beating the speaker. Causality: One accusation leads to harmful judgment.  This is important, as it shows what counts as proof during the examination.

Observation: Mr Harthorn suggests temptation even if Goody Nurse thinks she is not a witch. The question starts soft, then pushes towards her being guilty Interpretation: The wording steers answers toward guilt. Trying to trap the Nurse into a confession  Causality: Questioning attempts to lead, admission, or misstep. This matters as it shows pressure inside the questioning. And how you can guide answers to the path you want them on.

Observation: Mr Tho: Putman’s wife links Goody Nurse to the Devil and to urging her temptation. Interpretation: The Assembly reads harm through a shared religious point of view. Context: Their belief frames how the charge is understood. This matters, as it shows and explains that unseen claims and faith count as evidence.

Observation: Goody Nurse is not feeling well and the judge believes it's witch craft or demons based on what others have been saying about her. She is an old woman and is just sick from age.

Interpretation: Her illness cannot be seen by the naked eye so it is hard for those to understand why she is so sick. She's been standing in front of everyone with no emotions yet she claims to be sick.

Connection: When someone accuses you of witchcraft in Salem, it was extremely hard to convince anyone that you are not. They took even a simple illness that you have and thought it was from a demon. The judge didn't believe Goody Nurse.

Complexity: I think about how hard it must have been to live in Salem during the 1600's. You could be doing anything and all of a sudden you are called a witch. It was much harder for woman than a man. Even being sick people will think you are a witch. Goody Nurse could do nothing but deny everything and keep her faith in God known. The voices of everyone else and the actions of the young girls were more believable to the judge than anything she had to say.

Observation: The accused, Goody Nurse used to be a professor in the community making her well known throughout the village. She isn't showing any emotion during her trial while everyone else is crying.

Interpretation: The way the accusers and the crowd watching are acting is sending a more powerful reaction to the judge. It's hard for him to find her innocent because of her lack in emotion to being convicted of abusing two young girls.

Connection: What one see's with their eyes is more powerful than what one says. if you act like a victim, then the more people are going to believe you. People in Salem only knew the truth in the Bible so if anyone acted out in a way the Lord would condemn evil, they were looked at as witches or being posessed by the devil because if you are living a holy life, how could you commit such an act of violence?

Complexity: When you think of the early 1600's people didn't know much except what they are taught. This document shows us that people only knew how to live their lives as what it say's in the bible. No real evidence is needed as long as enough people agree with you.

Observation: The person in charge of finding Goody Nurse guilty or not guilty is trying to understand how she claims her innocents when two grown people have come forward now.

Interpretation: No matter how innocent you are, the more people that accuse you, the less innocent you look. Not only are the two girls accusing Goody Nurse, two adults have joined them.

Connection: Goody Nurse's trial is based on whose account of a situation is true or false. During this time period of 1691, there is no way to show proof of a crime, only what people have seen or heard. The people in Salem dedicate their lives to their worship of God and live their life by the rules of Christianity. Goody Nurse knows only God can see her innocence.

Complexity: Showing your love and faith in God was a way to prove your innocence when someone accused you of a crime. God cannot save you if many people are accusing you, and all you have on your side is just yourself.

Observation: The person in charge of finding Goody Nurse guilty or not guilty is trying to understand how she claims her innocents when two grown people have come forward now.

Interpretation: No matter how innocent you are, the more people that accuse you, the less innocent you look. Not only are the two girls accusing Goody Nurse, two adults have joined them.

Connection: Goody Nurse's trial is based on whose account of a situation is true or false. During this time period of 1691, there is no way to show proof of a crime, only what people have seen or heard. The people in Salem dedicate their lives to their worship of God and live their life by the rules of Christianity. Goody Nurse knows only God can see her innocence.

Complexity: Showing your love and faith in God was a way to prove your innocence when someone accused you of a crime. God cannot save you if many people are accusing you, and all you have on your side is just yourself.

Observation: Goody nurse is on trial because the two girls are saying she hurt them. The girls cried while accusing Goody nurse.

Interpretation: Regardless if it is true or false, you can take someone to be accused no matter the crime. You have the right to defend yourself in front of your accusers.

Connection: The source tells us Goody Nurse is accused of hurting the girls by means of witchcraft. The only evidence they have is the two girls words against Good Nurse's words. The girls are crying and acting as if they are in such pain from the violence Goody put them through. Goody stands firm she did not touch the girls and her calm demeanor strikes everyone as odd as the girls can't help but cry.

Complexity: During this time in Salem, people lived their lives as Christians according to the bible. It seems the person judging Goody Nurse cares more about her faith in God and if she fell prey to demons which caused her to harm the two young girls. Goody Nurse denied all these claims with a stoic face that was opposite from what the girls portrayed. Your actions as the accuser and the accused seem more validating than your own words.

This sentence is very relatable. It highlights how our brains rely on past experiences and familiar patterns to make sense of what’s around us, sometimes without us even realizing it. The idea of going on “autopilot,” as stated in the text, is something I experience often. For example, there are times when I’m sitting in my living room and I think I see someone walking past my big front window. But when I actually look outside, there’s nobody there. This has happened multiple times, and I’ve always wondered why. Now, I think it’s because the walkway to the front door is right outside that window, so my brain may be expecting someone to come up to the door.

I once got pulled over twice in one week for two different instances, the first was for speeding, and I was on my way to work and got hit by a speed trap on a foggy road, I'd accidentally spilled chili on my backseat on my way to work as well, which didn't help much. I'd been respectful and I think because of the chili situation he felt bad for me.<br /> I think the schemata that contributed to his interpretations of me was I was a younger person, just trying to get to work, who'd spilled a container of chili in her backseat.

This idea is definitely true in my own experience. There were plenty of times when I was younger and riding in the car with my parents, and they would prefer listening to the radio rather than letting me connect my phone to the aux. Because of that, I’d scroll through different radio stations trying to find something we could all enjoy. It was during those moments that I stumbled upon country music—something I probably wouldn't have chosen on my own, but grew to like over time just from those car rides. It really shows how even when we’re making quick decisions, like changing the station, our preferences and environment shape what we stop and listen to.

Creatures are drawn to bright, eye-catching colors and reflections, just as much as humans are too. I’ve been a fisher since I was a little girl, and I’ve always noticed how the rods, the lures, and even the bait seem to shine brilliantly in the sunlight, especially when they hit the water. The shimmer seems to call out to fish, catching their attention in a way that feels almost magical. It’s fascinating how both underwater and airborne creatures respond so instinctively to light and color.

This goes along with what I said in my previous annotation. Paying attention to the smallest details in what a student does, says, or chooses, can reveal so much in who they are as a person. What they have experienced in life even. There is so much that can be expressed, without even using words to intentionally express them.

This raised an eyebrow for me...and this is something I have thought about. Why do our brains make sense of the world through stories? Something I have always thought about.

Right off the bat, I would totally agree that this statement is true. I think that people resort to storytelling when in doubt, when there is nothing else to say. It's sort of the go to move when you first meet someone, or to break the ice.

This stood out to me because it reminds me that even trained experts can fall into the same traps as anyone else, which is why skepticism, evidence, and peer review are such an important part of psychology.

I think Khartum finds a way of highlighting the agency women navigated despite the misogyny and constraints placed on them by white australian and south asian men.

This is terrible.

Fuck this guy.

That's fucked. Also, they're basically raping her again to confirm whether she was raped... which is so fucked up.

.... A little fuzzy here, but come back to this.

A white woman marrying a south asian. Wow.

Ah, right.

Because diaspora cannot happen without women to go with the men (or, in many cases men to go with the women).

I am interested in if the police officers saw results in the violence they took. As well as whoever through the bomb. If they thought violence was the way to get their voice heard?

Intrapersonal communication takes place before, during, and after an interaction. For example, you wanting to go out with friends is a thought before an interaction. While you’re out you think about what you’re going to eat. After, you reminisce about the day you had with your friends.

Ah. Coexistence, conflict, and entanglement in the margins of a white nation. have we all been there...

This... is wild.

Right.

I believe Khatun is very brave to openly write about this, notwithstanding the backlash she may receive from her own community.

interesting...

!!!

Ah shit...

You know, I think this story complicates the position of South Asian migrants as participants in settler colonialism and displacement of indigenous people.

fucking psychopath. His actions cost someone's life, by the way.

THis is so fucked up...

In my life experience, I have the understanding that we as people grow up in different ways and at different rates. This also means that when we do become adults, everyone has a different meaning of an adult, and that is what the film is doing here.

This challenged my thinking because normally, wouldn't a grandmother be happy that her grandsons were playing with girls?

THe idea of "blank spaces" awaiting imperial penetration deserves some unpacking.

Will points be deducted for work that is submitted late?

Are points deducted for work submitted late?

Are points deducted for work submitted late?

I personally like the idea of consuming ideas; those ideas can nourish your mind and your soul. Once you have glutted yourself on this smorgasbord of ideas, you can at last begin to create new ideas with confidence of a practiced mind through ingestion and reflection.

Being able to humble and modest about a text is not enough; it also takes perseverance and commitment to see a learning journey through the very end. There will be struggles, but no fruit can truly be savored without the struggle to acquire it.

This reminds me of my study of the science of reading. How text levels need to be instructional level and not frustration level. It is always important to challenge yourself when reading, and that is where the tie in for modesty comes into the picture. If we aren't modest about our abilities and our understanding, then we do not take the first step to knowing something truly without the weight of shame anchoring us to the bottom of the ocean floor as our insecurities grind us down into nothingness. It is a pivotal thought to realize that learning should be approached with humility. We are not all knowing and everyone can stand to teach us something if we are modest and willing to learn it.

Many intellectuals also have some degree of neurodivergence, and I think that--in addition to historical-sociological and ideological conditioning--there is also a neurochemical or neurological link to inventive and innovative ideas. There must be more at play than these two concepts to create such a wide array of learning experiences and a varied array of individual schema within our society. Think about how varied people are; we find people who are compatible with us, but we rarely ever find someone who is a copy of us due to permutations.

This is referencing schema creation within learners that helps them form new opinions based on prior experience. I wonder if schema creation is not just a sum of prior experiences, but also a conglomeration of genetic factors as well. I would love to see how neurochemistry and genes affect how we learn, and what chemicals develop the feelings we get when we have those a-ha neuron connecting moments.

I recall writing about this earlier before arriving at this part of the text. Learning is an experiential process and more than rote memorization; it is a deep dive into the meaning of an experience and the reflective process of a domino-flick that begins a cascade.

Many readers a guilty of skimming and scanning to capture contextual meaning; however, this process often leaves out the words found in between that further enrich contents. It is only through a full, careful reading of a text that we can hope to facilitate a proper discourse about its material.

Studying is a quest to remember and understand what we read. The real breakthrough in learning is in applying the material to real-world situations. Employers and teachers don't care that you know something like the back of your hand, they care what you do with that information. Regurgitating information because you are well studied is no longer relevant; experiential learning is the future. People don't have fond memories of that time they studied; fond memories derive from beneficial experiences.

REQUIREMENTS

Soft Skill

Qualifications

Almost every and any surplus of animals or materials was exploited by white newcomers.

U.S. went so far as to oversee their OWN promises of peace to be rid of Native Americans

Is this used in the procedure? I don't see it. Maybe delete.

Materials list mentions a "shape sheet" (which I authored easily in Google Slides). Not sure what a "three shape object" is... maybe just use the shape sheet.

Materials list says "sugar", but the procedure says "salt".

Will we see our grade in brightspace and the modules there also?

Related to the pooling operator, I think large gains may come from the use of 1) edge weights in your GCN layers so that not all neighbors are treated equally by the message passing mechanism, and 2) alternative MPNN layer types, including use of the graph attention mechanism (i.e. GAT) or graph transformers, which use the attention mechanism to learn which neighbors are more "important." I suspect that even with simple mean-pooling, these alternative layer types will be much more performant and generalizable (e.g. from CRBD to BiSSE). In effect the GCN layers (particularly without using edge weights) is more akin to the CRBD in that it assumes uniform, homogeneous contribution by all neighbors to feature updates.

Again, I think this is a case for exploring the use of more general pooling operators, such as EdgePooling, etc, that might capture the relevant signal, but without imposing such rigid inductive biases on the architecture that could prove harmful to more general application.

Have you considered other pooling layers other than mean-pooling? For instance existing pooling operators like EdgePooling might confer similar benefits by retaining the temporal signal through iterative edge contraction. https://pytorch-geometric.readthedocs.io/en/latest/generated/torch_geometric.nn.pool.EdgePooling.html#torch_geometric.nn.pool.EdgePooling

Related to the above (regarding the number of GNN layers used), have you considered/looked into using either gate residual connections, or using something like jumping knowledge (https://arxiv.org/abs/1806.03536) between layers to mitigate oversmoothing in deeper networks which GNNs tend to be prone to? This could be another simple modification with outsized benefits.

I presume you're using the "base" GCN layer here? If so I'd be clear about this, since unlike the CNN and MLP, there is a massive diversity of MPNN layer types - its worth being very explicit about what you're using, since their choice could have massive impacts on the performance of these architectures!

Is there a reason you chose not to include edge weights/attributes corresponding to each respective branch length? I suspect this would be quite useful/informative for providing additional context to the message passing layers and could boost performance further. Otherwise, each neighbor is assumed to contribute equally to feature updates, which is not something we innately suspect to be true.

True, but only necessarily when the prediction task is graph-level. This is not necessary for many other common GNN prediction tasks, including node or link prediction. This is a bit of a nitpick, but I think useful to distinguish since this architecture is so new to the field!

I know these details are present in the supplement, but here and for the other NNs, it could be useful to specify the number of layers used - particularly for the GNN, where this relates to the effective "diameter" of visibility or neighborhood size (i.e. how many hops away the message passing mechanism aggregates neighbor information).

The use of LM has indeed demonstrated a large dependency of getting an answer whether that is writing a paper or trying to solve a math problem. It's clear that the use of LM can cause a lack of brain development and understanding of a topic.

One of the key differences to keep in mind when utilizing AI is that it thinks and operates differently from humans. Intuition and the ability to read between the lines are among the major differences between humans and AI, and it becomes very easy to overlook something obvious for us but might be interpreted differently for AI and vice versa.

I find it interesting that with these systems, the more data collected/the greater the scope and demands are, creates a greater risk of inaccuracy.

People tend to try to find patterns, even when there are none

The impacts of Environmental Racism are very prevalent now. Historically, marginalized communities have been affected by unfair practices of real estate and redlining. The use of AI could be seen as adding fuel to the fire, especially when going outside of the US and looking at the impacts of the rise of AI against third world countries.

When dealing with AI we often forget that the computers does not have immediate access to the meaning of our words, different versions have evolved to reflect specific "meaning" back to us, but to the computer, language is just another form of symbols to communicate something. It is important to think about that as it becomes more present in our life.

If this piece is from 2021 I wonder how the LM training regiment and affects have changed.

It is important to remember that LMs are a reflection of human input, and therefore, human error. Our individual experiences create subconscious biases that make it impossible to deliver an unbiased LM system.

As an ENST major, I have understood the environmental impact of the use of LM and Generative AI models. While it can be impressive and convenient for our day to day use, there could be too much irreversible damage created, which will keep us stagnant in our progress for a more environmentally friendly future

Community accessibility is a huge issue for public goods. Just because a product or service is available and targeted for a specific group doesn't guarantee access and beneficial use.

LMs are indeed impressive, and come through whenever we seek their aid. However, knowing and understanding the implication which the counter factors have on us as humans, and the way the "wins" are even got to, is imperative to protecting our sense of being, and minds.

This is significant as it can cause issues for research. I have experienced this to be the case, where it has made up specific dates or even made up a specific event that never occured.

When I first read this, I started being curious about these Germanic tribes. Who were these people? What was their culture like before they had to flee from Attila the Hun? I also noticed the wording, “The city of Rome itself fell to the barbarians.” Why are these people who destroyed the city of Rome called “barbarians?” Where the “barbarians” the same Germanic tribes that escaped the Huns? I don’t know about others, but I personally find the term “barbarians” both vague and kind of insulting. But that's just a personal bias of mine.

Another thing I found interesting is that this population crisis occurred over 150,000 years ago. Our professor said we don’t fully understand how this population crisis happened, which makes it even more intriguing. Was it a plague that caused the population decline? Was it because of climate conditions or some other environmental impact? I just find this interesting to think about.

I find it interesting how our understanding of the agricultural revolution has changed over the years. We as humans tend to think about history, and really a lot of things, in a chronological order. We’ve learned over the years that it isn’t always the cause, especially in our understanding of pre-written eras.

LLMs themselves are literal plaigiarism generators.

Jim, the machine has no perspective other than the one that is programmed / trained / tuned into it.

WHICH SCHOOLS JIM?! This feels like a broad and sweeping claim with no evidence cited.

What is being analyzed? What is being processed?

I am begging you to stop humanizing the LLM

Already this is a little weird because do calculators even do algebra? At least the ones generally available to students?

This establishes a public service role for government officials compared to the divine right of kings. The citizens of Virginia are responding to the unjust actions committed previously by the King acted as though he was above the law because he was a monarch and divinely appointed so. The VA Declaration of Rights ensures that no governing body that is established in VA will abuse their power in the way the King did.

eLife Assessment

This important study investigates how signals from the nervous system can influence the response to different food sources. To demonstrate the role of specific neuronal and intestinal regulators in sensing food quality and modulating digestion, the authors present evidence through a combination of genetic screening, RNA-seq analysis, and functional studies. These findings shed light on an adaptive strategy to integrate food perception with physiological responses, with a mix of solid and convincing evidence supporting the work.

Reviewer #1 (Public review):

Summary:

In this manuscript, Liu et al have tried to dissect the neural and molecular mechanisms that C. elegans use to avoid the digestion of harmful bacterial food. Liu et al show that C. elegans use ON-OFF state of AWC olfactory neurons to regulate the digestion of harmful gram-positive bacteria S. saprophyticus (SS). Authors show that when C. elegans are fed on SS food, AWC neurons switch to OFF fate, which prevents the digestion of S. saprophyticus, and this helps C. elegans avoid these harmful bacteria. Using genetic and transcriptional analysis as well as making use of previously published findings, Liu et al implicate p38 MAPK pathway (in particular, NSY-1, the C. elegans homolog of MAPKKK ASK1) and insulin signaling in this process.

Strengths:

The revised manuscript has improved significantly. The authors have addressed almost all the comments that I had in my initial review.

Weaknesses:

None.

Reviewer #2 (Public review):

Summary:

Using C. elegans as a model, the authors present an interesting story demonstrating a new regulatory connection between olfactory neurons and the digestive system. Mechanistically, they identified key factors (NSY-1, STR-130 et.al) in neurons, as well as critical 'signaling factors' (INS-23, DAF-2) that bridge different cells/tissues to execute the digestive shutdown induced by poor-quality food (Staphylococcus saprophyticus, SS).

Strengths:

The conclusions of this manuscript are mostly well supported by the experimental results shown.

Weaknesses:

The authors have done a nice job in addressing my comments.

Reviewer #3 (Public review):

Summary:

The study explores a molecular mechanism by which C. elegans detects low-quality food through neuron-digestive crosstalk, offering new insights into food quality control systems. Liu and colleagues demonstrated that NSY-1, expressed in AWC neurons, is a key regulator for sensing Staphylococcus saprophyticus (SS), inducing avoidance behavior and shutting down the digestive system via intestinal BCF-1. They further revealed that INS-23, an insulin peptide, interacts with the DAF-2 receptor in the gut to modulate SS digestion. The study uncovers a food quality control system connecting neural and intestinal responses, enabling C. elegans to adapt to environmental challenges.

Strengths:

The study employs a genetic screening approach to identify nsy-1 as a critical regulator in detecting food quality and initiating adaptive responses in C. elegans. The use of RNA-seq analysis is particularly noteworthy, as it reveals distinct regulatory pathways involved in food sensing (Figure 4) and digestion of Staphylococcus saprophyticus (Figure 5). The strategic application of both positive and negative data mining enhances the depth of analysis. Importantly, the discovery that C. elegans halts digestion in response to harmful food and employs avoidance behavior highlights a physiological adaptation mechanism.

Weaknesses:

Major weaknesses have been addressed.

Author response:

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

Reviewer #1 (Public review):

Summary:

In this manuscript, Liu et al have tried to dissect the neural and molecular mechanisms that C. elegans use to avoid digestion of harmful bacterial food. Liu et al show that C. elegans use the ON-OFF state of AWC olfactory neurons to regulate the digestion of harmful gram-positive bacteria S. saprophyticus (SS). The authors show that when C. elegans are fed on SS food, AWC neurons switch to OFF fate which prevents digestion of S. saprophyticus and this helps C. elegans avoid these harmful bacteria. Using genetic and transcriptional analysis as well as making use of previously published findings, Liu et al implicate the p38 MAPK pathway (in particular, NSY-1, the C. elegans homolog of MAPKKK ASK1) and insulin signaling in this process.

Strengths:

The authors have used multiple approaches to test the hypothesis that they present in this manuscript.

Weaknesses:

Overall, I am not convinced that the authors have provided sufficient evidence to support the various components of their hypothesis. While they present data that loosely align with their hypothesis, they fail to consider alternative explanations and do not use rigorous approaches to strengthen their overall hypothesis. The selective picking of genes from the RNA sequencing data and forcing the data to fit the proposed hypothesis based on previously published findings, without exploring other approaches, indicates a lack of thoroughness and rigor. These critical shortcomings significantly diminish enthusiasm for the manuscript in its totality. In my opinion, this is the biggest weakness in this manuscript.

We appreciate the reviewer’s all the suggestions which help us to improve this paper. We now addressed reviewer’s comments at the section of “Reviewer #1 (Recommendations for the authors)”

Reviewer #2 (Public review):

Summary:

Using C. elegans as a model, the authors present an interesting story demonstrating a new regulatory connection between olfactory neurons and the digestive system.

Mechanistically, they identified key factors (NSY-1, STR-130 et.al) in neurons, as well as critical 'signaling factors' (INS-23, DAF-2) that bridge different cells/tissues to execute the digestive shutdown induced by poor-quality food (Staphylococcus saprophyticus, SS).

Strengths:

The conclusions of this manuscript are mostly well supported by the experimental results shown.

Weaknesses:

Several issues could be addressed and clarified to strengthen their conclusions.

(1) The word "olfactory" should be carefully used and checked in this manuscript. Although AWCs are classic olfactory neurons in C. elegans, no data in this manuscript supports the idea that olfactory signals from SS drive the responses in the digestive system. To validate that it is truly olfaction, the authors may want to check the responses of worms (e.g. AWC, digestive shutdown, INS-23 expression) to odors from SS.

We appreciate the reviewer’s careful attention to terminology. We agree that the term "olfactory" requires direct experimental validation. However, in this paper, we only used "olfactory" to specific define the AWC neurons. As reviewer’s suggestion, we now deleted the word “olfactory”.

(2) In line 113, what does "once the digestive system is activated" mean? The authors need to provide a clearer statement about 'digestive activation' and 'digestive shutdown'.

Previously, we observed that activating larval digestion with heat-killed E. coli or E. coli cell wall peptidoglycan (PGN) enabled the digestion of SS as food (Hao et al., 2024). Additionally, when animals reached the L2 stage by feeding normal OP50 diet, they could utilize SS as a food source to support growth (Figure 1—figure supplement 1D). These findings suggest that once digestion is activated (via E. coli components or L2-stage maturation), worms gain the capacity to process SS as a viable food source, abolishing SS-induced growth impairment (Hao et al., 2024) ( Figure 1—figure supplement 1D).

(3) No control data on OP50. This would affect the conclusions generated from Figures 2A, 2B, 2D, 3B, 3C, 3G, 4D-G, 5D-E, 6B-D.

We appreciate  this point. The central goal of the experiments listed (Figures 2A,B,D; 3B,C,G; 4D-G; 5D-E; 6B-D) was not to compare growth or behavior between SS and OP50 under standard conditions, but rather to understand the genetic basis of the C. elegans response specifically to SS, as identified through our nsy-1 mutant screen.

Our data in Figure 1 clearly establishes the fundamental difference in growth and feeding behavior when larvae encounter SS compared to OP50 (Figures 1A,B). Having established SS as an unfavorable food source that triggers a specific protective response (digestive shutdown), the subsequent experiments focus on deciphering how this response is mediated.

Therefore, within these specific experimental contexts under SS feeding: The primary comparison is between wild-type (N2) and nsy-1 mutant animals. All assays (growth, behavior, survival) are performed under the same SS feeding conditionsfor both genotypes.

This design allows us to directly assess the functional role of NSY-1 in mediating the SS-specific response pathway we are investigating. Including an OP50 control for every figure would not address this core genetic question and could introduce confounding variables given the established difference in how C. elegans treats these two food sources. The critical internal control for these specific experiments is the performance of the wild-type under SS versus the mutant under SS.

(4) Do the authors know which factors are released from AWC neurons to drive the digestive shutdown?

Enrichment analysis revealed that genes related to extracellular functions, such as insulin-related genes, are induced in nsy-1 mutant animals (Figure 5—figure supplement 1A, Supplementary file 4). Further analysis of insulin-related genes from the RNA-seq data showed that ins-23 is predominantly induced in nsy-1 mutant animals (Figure 5—figure supplement 1B), suggesting its potential role in promoting SS digestion. We found that knockdown of ins-23 in nsy-1 mutants inhibited SS digestion (Figure 5D). Given that INS-23 is expressed in AWC neurons (Figure 5—figure supplement 3A, CeNGEN), this suggests increased production and likely enhanced release of INS-23 from AWC neurons in the nsy-1 mutant background, which promotes SS digestion.

The insulin/insulin-like growth factor signaling (IIS) pathway, particularly through the DAF-2 receptor, integrates nutritional signals to regulate various behavioral and physiological responses related to food (Kodama et al., 2006; Ryu et al., 2018). It has been shown that INS-23 acts as an antagonist for the DAF-2 receptor to promote larval diapause (Matsunaga et al., 2018). To test whether ins-23 induction in nsy-1 mutants promotes SS digestion through its receptor, DAF-2, we constructed a nsy-1; daf-2 double mutant. We found that the SS digestion ability of the nsy-1 mutant was inhibited by the daf-2 mutation. This suggests that the nsy-1 mutation induces the insulin peptide ins-23, which promotes SS digestion through its potential receptor, DAF-2.

The data supports a model where AWC neurons regulate digestion via the release of INS-23. Loss of nsy-1 function increases INS-23 release from AWC, activating DAF-2 signaling and promoting digestion. Conversely, in wild-type animals, reduced INS-23 release from AWC contributes to digestive shutdown in response to SS food.

Reviewer #3 (Public review):

Summary:

The study explores a molecular mechanism by which C. elegans detects low-quality food through neuron-digestive crosstalk, offering new insights into food quality control systems. Liu and colleagues demonstrated that NSY-1, expressed in AWC neurons, is a key regulator for sensing Staphylococcus saprophyticus (SS), inducing avoidance behavior and shutting down the digestive system via intestinal BCF-1. They further revealed that INS-23, an insulin peptide, interacts with the DAF-2 receptor in the gut to modulate SS digestion. The study uncovers a food quality control system connecting neural and intestinal responses, enabling C. elegans to adapt to environmental challenges.

Strengths:

The study employs a genetic screening approach to identify nsy-1 as a critical regulator in detecting food quality and initiating adaptive responses in C. elegans. The use of RNA-seq analysis is particularly noteworthy, as it reveals distinct regulatory pathways involved in food sensing (Figure 4) and digestion of Staphylococcus saprophyticus (Figure 5). The strategic application of both positive and negative data mining enhances the depth of analysis. Importantly, the discovery that C. elegans halts digestion in response to harmful food and employs avoidance behavior highlights a physiological adaptation mechanism.

Weaknesses:

Major points:

(1) While NSY-1 positively regulates str-130 expression in AWC neurons and is critical for SS avoidance and survival, the authors should examine whether similar phenotypes are observed in str-130 mutants.

In this study, we mainly focused on how worms sense adverse food sources (SS food) and shutdown digestion (not growth as digestion shutdown readout). We found that nsy-1 in AWC play key roles in response SS food, once nsy-1 mutation, mutant animals cannot detect SS food and digest it, therefore growth under SS food. From RNA-seq, we found that nsy-1 positively regulates several sensory perception related genes (sra-32, str-87, str-112, str-130, str-160, str-230) (Figure 4—figure supplement 1A, Supplementary file 2). After screen, we found that we found that knockdown of str-130 in wild-type animals promoted SS digestion, thereby supporting animal growth (Figure 4D), and the proportion of animals with two AWC^OFF neurons decreased (Figure 4E). Secondly, we found that overexpression of str-130 in nsy-1 mutant animals inhibited SS digestion, thereby slowing animal growth (Figure 4F), and the proportion of animals with two AWC^OFF neurons increased (Figure 4G). These results demonstrate that NSY-1 promotes the AWC^OFF state by inducing str-130 expression, which in turn inhibits SS digestion in C. elegans.

(2) NSY-1 promotes the AWC-OFF state through str-130, inhibiting SS digestion. The authors should investigate whether STR-130 in AWC neurons regulates bcf-1 expression levels in the intestine.

We agree with the reviewer's suggestion regarding the potential role of STR-130 in AWC neurons regulating intestinal bcf-1 expression. To address this, we generated transgenic worms with AWC-specific knockdown of str-130, achieved by rescuing sid-1 cDNA expression under the ceh-36 promoter (AWC-specific) in sid-1(qt9);BCF-1::GFP background worms.

We observed that AWC neuron-specific RNAi of str-130 elevated intestinal BCF-1::GFP expression (Figure 6—figure supplement 1B). This demonstrates that STR-130 functions cell-non-autonomously in AWC neurons to repress BCF-1 expression in the intestine.

(3) The current results rely on str-2 expression levels to indicate the AWC state. Ablating AWC neurons and testing the effects on digestion would provide stronger evidence for their role in digestive regulation.

To confirm the important of AWC state in SS digestion, we performed AWC-specific neuron ablation experiments using previously validated transgenic strain that expresses cleaved caspase under the AWC-specific promoter, ceh-36 (ceh-36p::caspase). Critically, worms with ablated AWC neurons completely failed to digest SS food (Figure 3—figure supplement 4), phenocopying the non-digesting state of wild-type worms on SS when AWC-OFF signaling is impaired. This result directly confirms that functional AWC neurons are essential for initiating SS digestion, aligning with our model where the AWC-OFF state (induced by SS) inhibits digestion while the AWC-ON state promotes it.

Furthermore, we previously study discovered that AWC ablation activates the intestinal mitochondrial unfolded protein response and inhibits food digestion, mechanistically linking neuronal integrity to gut stress responses and digestive inhibition.

Together, these functional ablation studies provide compelling physiological evidence that AWC neurons act as central regulators of food-state sensing and gut function.

(4) The claim that NSY-1 inhibits INS-23 and that INS-23 interacts with DAF-2 to regulate bcf-1 expression (Line 339-340) requires further validation. Neuron-specific disruption of INS-23 and gut-specific rescue of DAF-2 should be tested.

We agree with the reviewer that the proposed NSY-1 ⊣ INS-23 → DAF-2 → BCF-1 signaling axis requires tissue-specific validation. To address this, we conducted compartment-specific functional dissection of INS-23 and DAF-2:

AWC neuronal role of INS-23:

To test whether INS-23 acts in AWC neurons to regulate intestinal BCF-1, we generated AWC-specific knockdown strains which was achieved by rescuing sid-1 cDNA expression under the ceh-36 promoter in a sid-1(qt9);BCF-1::GFP background. We found that AWC-restricted ins-23 knockdown significantly reduced intestinal BCF-1::GFP expression (Figure 6—figure supplement 1A). This confirms that INS-23 functions cell-non-autonomously within AWC sensory neurons to activate intestinal BCF-1, consistent with NSY-1’s upstream inhibition of INS-23 in this neuronal  subtype

Intestinal role of DAF-2 as INS-23 receptor:

To investigate weather DAF-2 acts as the gut-localized receptor for neuronal INS-23 signaling, we performed tissue-specific rescue experiments in the nsy-1(ag3);daf-2(e1370) double mutant. When DAF-2 was re-introduced specifically in the intestine (using the ges-1 promoter), we observed a significant suppression of SS digestion (Figure 5—figure supplement 3B), but not rescue digestive defect. This indicates that INS-23 induction in nsy-1 mutants promotes digestion independently of intestinal DAF-2 function.

(5) Figure Reference Errors: Lines 296-297 mention Figure 6E, which does not exist in the main text. This appears to refer to Figure 5E, which has not been described.

We corrected this.

Reviewer #1 (Recommendations for the authors):

I would like the authors to address the following comments in a resubmission.

(1) The hallmark of the activated p38 MAPK pathway is the phosphorylation of most downstream kinase p38 (PMK-1/PMK2 in C. elegans) of this kinase cascade. Previous work from Bergmann lab showed that the most downstream kinase of this pathway, PMK-1/PMK-2, is not required for AWC asymmetry. I wonder whether that is the case also for the model that Liu et al have presented in this manuscript. Since p38/PMK-1 undergoes activation (phosphorylation) in response to pathogenic bacteria like P. aeruginosa, it is worth testing whether PMK-1 plays a role downstream of NSY-1 in the model that Liu et al present in this manuscript. It would be worth testing whether there is increased phosphorylation of p38 when C. elegans are fed SS and whether that phosphorylation regulates downstream components that Liu et al have identified in this manuscript.

We thank the reviewer for raising this important point regarding PMK-1/p38 MAPK signaling. As established in our prior work (Reference 1), SS exposure triggers phosphorylation of PMK-1 (P-PMK-1) in C. elegans, and pmk-1 mutants exhibit enhanced growth on SS (Figure-1, Figure-2). This confirms that PMK-1-mediated innate immune signaling actively regulates SS responsiveness and digestion.

To address whether PMK-1 functions downstream of NSY-1 within our proposed model, we performed critical epistasis analyses. While we observed that nsy-1 mutation elevates ins-23 (indicating NSY-1 suppression of ins-23), knockdown of pmk-1 did not alter ins-23 expression levels (Figure 5-figure supplement 3C). This demonstrates that PMK-1 does not operate through the ins-23 pathway to regulate SS digestion. Thus, although both pathways respond to SS, the PMK-1-mediated innate immune response and the NSY-1/INS-23 axis constitute distinct regulatory mechanisms governing digestive adaptation.

Reference 1: Geng, S., Li, Q., Zhou, X., Zheng, J., Liu, H., Zeng, J., Yang, R., Fu, H., Hao, F., Feng, Q., & Qi, B. (2022). Gut commensal E. coli outer membrane proteins activate the host food digestive system through neural-immune communication. Cell host & microbe, 30(10), 1401–1416.e8. https://doi.org/10.1016/j.chom.2022.08.004

(2) Since p38 MAPK pathway has a well-established role in host defense in the C. elegans intestine, it is important to show that NSY-1 does not function in the intestine in the model that Liu et al present. I would like the authors to reintroduce nsy-1 in C. elegans intestine in nsy-1 mutant animals and then test whether it has any effect on worm length on SS food (similar to what is done in Figure 3 for AWC-specific nsy-1).

Beyond its  established  role  in  AWC  neurons,  we  detected  NSY-1 expression in the intestine (Figure 3-figure supplement 2A). To assess intestinal NSY-1 function, we performed tissue-specific rescue experiments in nsy-1 mutants using the intestinal-specific vha-1 promoter. Intestinal expression of NSY-1 significantly suppressed the enhanced SS digestion phenotype in nsy-1 mutants (Figure 3-figure supplement 2B), demonstrating functional involvement of gut-localized NSY-1 in regulating digestive responses. We propose intestinal NSY-1 mediates this effect through innate immune signaling, consistent with its known pathway components. As previously established (Reference 1), the canonical PMK-1/p38 MAPK pathway functions downstream of NSY-1, with both sek-1 and pmk-1 knockdown enhancing SS digestion through immune modulation. This indicates intestinal NSY-1 suppresses digestion may act through PMK-1-mediated immune responses. Since neuronal NSY-1's role in digestive control was previously undefined, we prioritized mechanistic analysis of its neuronal function in digestion regulation.

Notably, this immune-mediated mechanism operates independently of NSY-1's neuronal regulation pathway. In AWC neurons, NSY-1 controls digestion exclusively through the neuropeptide signaling axis (INS-23/DAF-2/BCF-1) without engaging innate immune components.

Reference 1: Geng, S., Li, Q., Zhou, X., Zheng, J., Liu, H., Zeng, J., Yang, R., Fu, H., Hao, F., Feng, Q., & Qi, B. (2022). Gut commensal E. coli outer membrane proteins activate the host food digestive system through neural-immune communication. Cell host & microbe, 30(10), 1401–1416.e8. https://doi.org/10.1016/j.chom.2022.08.004

(3) At multiple places, wild-type (WT) controls have been labeled as N2. It is better to label all controls as WT (and not as N2).

Corrected.

(4) In Figure 2B, the aversion response should be scored at multiple time points, like Figure 1C, rather than at just one timepoint.

We thank the reviewer for suggesting multi-timepoint analysis of aversion behavior. In accordance with this recommendation, we have now quantified SS avoidance at multi-timepoint. As shown in the revised Figure 2B, nsy-1 mutants exhibited significantly impaired avoidance responses at both 4h and 6h but not at 8h, confirming that NSY-1 is essential for sustained aversion to SS food in the early response. This data demonstrates that the critical role of NSY-1 in food discrimination at initial sensory responses.

(5) Does the re-introduction of nsy-1 in AWC neurons in nsy-1 mutant background help animals avoid SS in dwelling and food-choice assays? Along the same lines, does the CRISPR-generated AWC-specific mutant of NSY-1 fail to avoid SS in dwelling and food-choice assays similar to the whole-animal mutant? These behavioral data are missing in Figure 3.

We thank the reviewer for prompting behavioral validation of AWC-specific nsy-1 functions. To determine whether NSY-1 in AWC neurons mediates SS sensory perception, we performed dwelling (avoidance) and food-choice assays using AWC-specific nsy-1 knockout and AWC-rescued strains (nsy-1(ag3); Podr-1::nsy-1). In dwelling assays, AWC-specific nsy-1 KO mutants exhibited significantly impaired SS avoidance at 6h (Figure 3-figure supplement 3A), while AWC-rescued strains restored avoidance capacity at 2-6h (Figure 3-figure supplement 3B). Food-choice assays further revealed that AWC nsy-1 KO mutants preferentially migrated toward SS (Figure 3-figure supplement 3C), whereas AWC-rescued showed no preference between SS and HK-E. coli (Figure 3-figure supplement 3D). These data conclusively demonstrate that NSY-1 acts in AWC neurons to mediate SS recognition and aversion behaviors.

(6) In Figure 3E and F, the number of animals that were used for scoring AWC str-2p::GFP expression should be specified.

we added the number of animals in the figure.

(7)  RNA seq analysis identified multiple GPCRs (including STR-130) that are upregulated in an NSY-1-dependent manner when animals are fed with SS bacteria. However, the authors decided to only characterize STR-130 because of previously published findings. It is important to rule out the role of other GPCRs since all are upregulated on SS food as shown in Figure S4 B. I would like the authors to knock down other GPCRs in the same manner as they did for STR-130 and demonstrate that only str-130 knockdown behaves similarly to the nsy-1 mutant (if that is the case) using the assay presented in Figure 4 D.

We appreciate the reviewer’s suggestion to comprehensively evaluate NSY-1-regulated GPCRs. In response, we extended our functional analysis to all six GPCRs (str-130, str-230, str-87, str-112, str-160, and sra-32) identified as NSY-1-dependent and SS-induced in RNA-seq (Figure 4—figure supplement 1).

Using RNAi knockdown and the SS growth assay, we observed that RNAi of str-130, str-230, str-87, or str-112 significantly enhanced SS growth (Figure 4—figure supplement 2A), with str-130 RNAi exhibiting the most robust phenotype—phenocopying nsy-1 mutants. Crucially, none of these GPCR knockdowns further enhanced growth in nsy-1(ag3) mutants (Figure 4—figure supplement 2B), confirming their position downstream of NSY-1. These data establish str-130 as the dominant effector of NSY-1-mediated SS response regulation, while suggesting minor contributions from other GPCRs (str-230, str-87, str-112).

(8) In Figure 4E and G, the number of animals that were used for scoring GFP expression should be specified.

we added the number of animals in the figure.

(9) When comparing Figure 3E and Figure 4E, it appears that the loss of str-130 RNAi does not phenocopy nsy-1 mutant. This raises the question of whether the inefficiency of RNAi targeting str-130 is the cause, or if STR-130 is not the only GPCR regulated by NSY-1 on SS food. I would like the authors to address this discrepancy. If RNAi inefficiency is indeed the cause, using an RNAi-sensitive background, such as an eri- 1 mutant, could help strengthen the data presented in Figure 4E. Conversely, if RNAi inefficiency is not responsible for the discrepancy, I suggest that the authors investigate the roles of other GPCRs that were identified by RNA sequencing.

We appreciate the reviewer’s observation regarding the phenotypic difference between nsy-1 mutants and str-130 (RNAi) animals on SS food (Fig. 3E vs Fig. 4E).

While both genetic perturbations significantly enhance SS growth and increase the proportion of animals exhibiting AWC^ON states compared to wild type (indicating enhanced digestion), the specific AWC^ON neuron configurations differ: nsy-1 mutants predominantly show 2 AWC^ON animals, whereas str-130(RNAi) animals primarily exhibit the 1 AWC^ON /1 AWC^OFF configuration (Fig. 3E vs Fig. 4E).

This difference likely arises because STR-130 is the key GPCR mediating NSY-1's inhibitory effect on SS digestion, but it is not the sole GPCR involved, as evidenced by our RNAi screen identifying several additional NSY-1-regulated GPCRs (str-230, str-87, str-112) whose depletion also enhanced SS growth (Fig. 4A-D).

The robust SS growth enhancement and AWC^ON state increase caused by str-130 (RNAi) (phenocopying the nsy-1 mutant’s functional outcome of enhanced digestion) (Figure 4D, 4E) indicate effective RNAi knockdown for this specific assay. Therefore, the distinct neural configurations reflect the partial redundancy among GPCRs downstream of NSY-1, rather than an inherent inefficiency of the str-130 RNAi.

The nsy-1 mutant phenotype represents the complete loss of all inhibitory GPCR signaling coordinated by NSY-1, while str-130(RNAi) represents the loss of its major component. Investigating the roles of other identified GPCRs (str-230, str-87, str-112) in modulating AWC^ON neuron states is an important direction for future research.

(10) In Figure 4 F and 4 G, the authors show that the overexpression of STR-130 rescues the nsy-1 mutant phenotype suggesting that NSY-1 might function through STR-130 to control digestion on SS food. These data place STR-130 downstream of NSY-1. To further strengthen these epistasis data, authors should knock down str-130 in nsy-1 mutant animals and show that the combined loss of both genes produces the same effect as the loss of either gene alone.

We thank the reviewer for the insightful suggestion to further define the genetic relationship between nsy-1 and str-130. To strengthen our epistasis analysis, we performed RNAi knockdown of str-130 in the nsy-1(ag3) mutant background and assessed development on SS food. Consistent with STR-130 acting downstream of NSY-1, the loss of str-130 via RNAi did not further enhance the developmental capacity (i.e., growth phenotype) of nsy-1(ag3) mutant animals on SS. This lack of enhancement indicates that str-130 and nsy-1 function within the same genetic pathway, with str-130 acting epistatically downstream of nsy-1 (Figure 4—figure supplement 3). This finding reinforces the model proposed from our overexpression data (Fig. 4F-G) – that NSY-1 primarily exerts its inhibitory effect on SS digestion by inducing the expression GPCR STR-130.

(11) In Figure 5C, please mention "ins-23 transcript levels" on the top of the graph so that it is clear what these data represent.

We appreciate the reviewer’s suggestion.

(12) Since all ins genes were upregulated in nsy-1 mutants (though ins-23 was indeed the most highly upregulated gene) on SS food from RNA seq analysis (Figure S5 B), it is important to first phenotypically characterize all of them using "worm length assay". If this analysis shows that ins-23 has the most robust phenotype, it would make more sense to just focus on ins-23.

We agree with the reviewer that initial phenotypic characterization of candidate genes identified through transcriptomic analysis is valuable.Our RNA-seq data revealed that several insulin-like peptide genes, including ins-22, ins-23, ins-24, and ins-27, were significantly upregulated in the nsy-1 mutant on SS food (Figure 5—figure supplement 1B). We prioritized these insulin-like peptide genes for functional validation because they are known to act as neuropeptides capable of mediating non-cell autonomous signaling in previous studies (Shao et al 2016).

To determine if any were functionally responsible for the enhanced SS growth observed in nsy-1 mutants, we performed functional phenotypic screening using the SS growth assay (worm length assay). We individually knocked down each of these candidates (ins-22, ins-23, ins-24, ins-27) in the nsy-1(ag3) mutant background. Among these, only RNAi targeting ins-23 significantly attenuated (i.e., suppressed) the enhanced development of the nsy-1(ag3) mutant on SS (Figure 5—figure supplement 2). This targeted functional screening revealed that ins-23 has the most robust and specific role in mediating the enhanced digestion phenotype downstream of NSY-1 loss, providing the critical justification for our subsequent focus on this particular insulin-like peptide.

Ref:

Shao, L. W., Niu, R., & Liu, Y. (2016). Neuropeptide signals cell non-autonomous mitochondrial unfolded protein response. Cell research, 26(11), 1182–1196. https://doi.org/10.1038/cr.2016.118

Reviewer #2 (Recommendations for the authors):

There are several minor errors and typos in the manuscript

(1) A number of typos in the figures, like "length".

Corrected.

(2) The 'axis labels' are inconsistent from panel to panel, like "relative body length" and "relative worm length".

Corrected.

(3) The fonts are inconsistent from panel to panel.

Corrected.

(4) There is no Ex unique number for transgenic lines.

Corrected.

Reviewer #3 (Recommendations for the authors):

Minor points:

(1)  Figure 3B, 3C, 3G, 4D, 4F, 5D, 5E, and 6C: Replace "lenth" with "length" (consistent with Figure 2A).

Corrected.

(2) Figure 4D: Correct "ctontrol" to "control."

Corrected.

(3) Figure 4G: Update the co-injection marker to Podr-1::GFP instead of Pstr-2::GFP.

Corrected.

(4) Figure 5C: This figure is missing from the Results section.

Corrected.

(5) Figure 6A: Label the graph with Pbcf-1::bcf-1::GFP, as in Figure 6D.

Corrected.

(6) Italicization: Lines 588 and 603-italicize nsy-1.

Corrected.

(7) Supplementary Figure S2A: Correct "Screeng" to "Screening."

Corrected.

(8) Spelling/Proofreading: Ensure consistent spelling and grammar, such as correcting "mutan" to "mutant" in Figure 4A.

Corrected.

Testing

very important aspect

This is new for me to learn, but I'm looking forward to an interesting class! Glad to be here.

Testing

Done

t_man_linux

The links aren't working on my end.

It seems like this grading would be aligned well with standards, but I wonder how well this grading would be interpreted. It also seems a little confusing for the teacher to keep up with different skill levels.

I don't think I would be confident about my grades if I didn't experience a little bit of anxiety about it. If the worry aspect was taken away from grading, I don't think I'd care much about my grades. Granted, I care a lot about my grades and I take great pride in my accomplishments. It hasn't been easy for me, in fact, I dropped out of college when I was 18. So the fact that I am doing well has been a driving factor in my success this time. However, I strive hard to make good grades and I push myself to go above and beyond on assignments.

But then the work the students who did the assignment would be in vain right? Am I understanding this correctly? Because surely teachers aren't just overlooking missed assignments on purpose and not assigning a zero.

Something that I seen in my Pre-Internship I is that parent's seem to not really care that their students are getting bad grades. I seen several instances where this was the case and it seems like there is a lack of engagment from some students to even complete simple assignments. Is that a phenomenon everywhere?

must be present and attentive to be able to participate in class discussions.

What do the shapes and sizes in these networks tell us about potential outcomes?

How are these changes created? How are the outcomes and shapes different?

Can we put a mathematical "measure" on them? What do the (topological) "neighborhoods" look like before and after?

I feel that this shows how some teachers are willing to cross certain lines for their school/students to receive more funding that could benefit them academically. However, educators must keep in mind that with standardized tests you can not cheat for your students, because they are gaining no knowledge of the content and it is illegal.

eLife Assessment

In this valuable manuscript, Rao and colleagues investigate the UFD-1/NPL-4 complex, which is involved in extracting misfolded proteins in the plasma membrane and the accumulation of pathogenic bacteria in the intestine. Using convincing methods, the authors find that knockdown of the ufd-1 and npl-4 genes leads to shortened lifespan of the nematode C. elegans and reduced accumulation of the bacterial pathogen P. aeruginosa in the intestine.

Reviewer #1 (Public review):

The authors adequately addressed the concerns I raised in my initial review, which are noted below.

(1) I suggest that the authors choose a different term in their title, abstract and manuscript to describe the phenotypes associated with ufd-1 and npl-4 knockdown other than an "inflammation-like response." Inflammation is a pathological term with four cardinal signs: redness (rubor), swelling (tumor), warmth (calor) and pain (dolor). These are not symptoms known to occur in C. elegans. The authors could consider using "inappropriate," "aberrant" or "toxic" immune activation in the title and abstract.

(2) I think it is important to point out in the context of the authors novelty claim in the abstract and manuscript that the toxic effects of inappropriate immune activation in C. elegans has been widely catalogued. For example: doi.org/10.1371/journal.ppat.1011120 (2023); doi:10.1186/s12915-016-0320-z (2016).; doi:10.1126/science.1203411 (2011); doi:10.1534/g3.115.025650 (2016). In addition, doi:10.7554/eLife.74206 (2022) previously described a mutation that caused innate immune activation that reduced accumulation of P. aeruginosa in the intestine, but also caused animals to have a shortened lifespan.

Thus, I do not think this study reveals the existence of inflammatory-like responses in C. elegans, as stated by the authors. Indeed, I think it is important for the authors to remove this novelty claim from their paper and discuss their work in the context of these studies in a paragraph in the introduction.

(3) The authors rely on the use of RNAi of ufd-1 and npl-4 to study their effect on P. aeruginosa colonization and pathogen resistance throughout the manuscript. To address the possibility of off-target effects of the RNAi, the authors should consider both (i) showing with qRT-PCR that these genes are indeed targeted during RNAi, and (ii) confirming their phenotypes with an orthologous technique, preferably by studying ufd-1 and npl-4 loss-of-function mutants [both in the wild-type and sek-1(km4) backgrounds]. If mutation of these genes is lethal, the authors could use Auxin Inducible Degron (AID) technology to induce the degradation of these proteins in post-developmental animals.

(4) I am confused about the author's explanation regarding their observation that inhibition of the UFD-1/ NPL-4 complex extends the lifespan of sek-1(km25) animals, but not pmk-1(km25) animals, as SEK-1 is the MAPKK that functions immediately upstream of the p38 MAPK PMK-1 to promote pathogen resistance.

I am also confused why their RNA-seq experiment revealed a signature of intracellular pathogen response genes and not PMK-1 targets, which the authors propose is accounting for toxic immune activation. Activation of which immune response leads to toxicity?

(5) The authors did not test alternative explanations for why UFD-1/ NPL-4 complex inhibition compromises survival during pathogen infection, other than exuberant immune activation. For example, it is possible that inhibition of this proteosome complex shortens lifespan by compromising the general health/ normal physiology of nematodes. Immune responses could be activated as a secondary consequence of this stress, and not be a direct cause of early mortality. Does sek-1(km4) mutant suppress the lifespan shortened lifespan of ufd-1 and npl-4 knockdown? This experiment should also be done with loss-of-function mutants, as noted in point 3.

(6) The conclusion of Figure 6 hinges on an experiment that uses double RNAi to knockdown two genes at the same time (Fig. 6D and 6G), an approach that is inherently fraught in C. elegans biology owing to the likelihood that the efficiency of RNAi-mediated gene knockdown is compromised and may account for the observed phenotypes. The proper control for double RNAi is not empty vector + ufd-1(RNAi), but rather gfp(RNAi) + ufd-1(RNAi), as the introduction of a second hairpin RNA is what may compromise knockdown efficiency. In this context, it is important to confirm that knockdown of both genes occurs as expected (with qRT-PCR) and to confirm this phenotype using available elt-2 loss-of-function mutants.

(7) A supplementary table with the source data for at least three replications (mean lifespan, n, statistical comparison) for each pathogenesis assay should be included in this manuscript.

Comments on revisions:

The authors adequately addressed the concerns I raised.

Reviewer #2 (Public review):

Summary:

The authors aimed to uncover what role, if any, the UFD1/NPL4 complex might play in innate immune responses of the nematode C. elegans. The authors find that loss of the complex renders animals more sensitive to both pathogenic and non-pathogenic bacteria. However, there appears to be a complex interplay with known innate immune pathways since loss of UFD1/NPL4 actually results in increased survival of animals lacking the canonical innate immune pathways.

Strengths:

The authors perform robust genetic analysis to exclude and include possible mechanisms by which the UFD1/NPL4 pathway acts in the innate immune response.

Weaknesses:

The argument that the loss of the UFD1/NPL4 complex triggers a response that mimics that of an intracellular pathogen is not thoroughly investigated. Additionally, the finding of a role of the GATA transcription factor, ELT-2, in this response is suggestive, but experiments showing sufficiency in the context of loss of the UFD1/NPL4 complex need to be explored.

Comments on revisions:

The authors have performed several control experiments for their RNAi based experiments and also tested the requirement for xbp-1s in their paradigm. The findings and their interpretations are acceptable.

Author response:

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

Reviewer #1 (Public Review):

(1) I suggest that the author's choose a different term in their title, abstract and manuscript to describe the phenotypes associated with ufd-1 and npl-4 knockdown other than an "inflammation-like response." Inflammation is a pathological term with four cardinal signs: redness (rubor), swelling (tumor), warmth (calor) and pain (dolor). These are not symptoms know to occur in C. elegans. The authors could consider using "tolerance" instead, as this term may better describe their findings.

We have changed “inflammation-like response” to “aberrant immune response” throughout the manuscript.

(2) It would help the reader to better understand the novelty of the findings in this study if the authors include a paragraph in their introduction to put their results in context of the published literature that has examined the relationship between immune activation and nematode health and survival. In particular, I suggest that the authors discuss doi:10.7554/eLife.74206 (2022), a study that charcterized a similar observation to what the authors are reporting. This study found that low cholesterol reduces pathogen tolerance and host survival during pathogen infection. Cholesterol scarcity increases p38 PMK-1 phosphorylation, priming immune effector induction in a manner that reduces pathogen accumulation in the intestine during a subsequent infection. I also suggest that the authors highlight in this introductory paragraph that the toxic effects of inappropriate immune activation in C. elegans has been widely catalogued. For example: doi.org/10.1371/journal.ppat.1011120 (2023); doi:10.1186/s12915-016-0320-z (2016).; doi:10.1126/science.1203411 (2011); doi:10.1534/g3.115.025650 (2016).

In this context, the authors could consider re-wording their novelty claim in the abstract and introduction to take into account this previous body of work.

We have added a paragraph to the Discussion section to place our findings in the context of previous research. The revised manuscript now includes the following text (page 11, lines 336–344): “Previous studies have shown that hyperactivation of immune pathways can negatively affect organismal development. For example, sustained activation of the p38 MAPK pathway impairs development in C. elegans (Cheesman et al., 2016; Kim et al., 2016), and excessive activation of the IPR also leads to developmental defects (Lažetić et al., 2023). Similar to our current study, recent work has demonstrated that heightened immune responses can reduce gut pathogen load while paradoxically decreasing host survival during infection (Ghosh and Singh, 2024; Peterson et al., 2022). However, our study uniquely shows that while such heightened immune responses are detrimental to immunocompetent animals, they can be beneficial in the context of immunodeficiency.”

(3) The authors rely on the use of RNAi of ufd-1 and npl-4 to study their effect on P. aeruginosa colonization and pathogen resistance throughout the manuscript. To address the possibility of off-target effects of the RNAi, the authors should consider both (i) showing with qRT-PCR that these genes are indeed targeted during RNAi, and (ii) confirming their phenotypes with an orthologous technique, preferably by studying ufd-1 and npl-4 loss-offunction mutants [both in the wild-type and sek-1(km4) backgrounds]. If mutation of these genes is lethal, the authors could use Auxin Inducible Degron (AID) technology to induce the degradation of these proteins in post-developmental animals.

We attempted several protocols of CRISPR in our laboratory to generate ufd-1 loss-of-function mutants; however, these efforts were unsuccessful. While this does not rule out the possibility of generating ufd-1 mutants, the failure is likely due to technical limitations on our part rather than an inherent inability to disrupt the gene. Nevertheless, to confirm the specificity of our RNAi-based approach, we quantified ufd-1 and npl-4 mRNA levels following RNAi treatment and found that each gene was specifically and effectively downregulated by its respective RNAi. 

Importantly, ufd-1 and npl-4 RNA sequences do not share significant homology, yet knockdown of either gene results in nearly identical phenotypes, including reduced survival on P. aeruginosa, diminished intestinal colonization, and shortened lifespan. These consistent outcomes strongly support the conclusion that the phenotypes are attributable to the disruption of the functional UFD-1-NPL-4 complex. We have added these results in the revised manuscript (pages 4-5, lines 114-125): “To confirm the specificity of the RNAi knockdowns and rule out potential off-target effects, we examined transcript levels of ufd-1 and npl-4 following RNAi treatment. RNAi against ufd-1 significantly reduced ufd-1 mRNA levels without reducing npl-4 expression, while npl-4 RNAi specifically downregulated npl-4 transcripts with no impact on ufd-1 mRNA levels (Figure 1—figure supplement 1A and B). Additionally, alignment of ufd-1 and npl-4 mRNA sequences against the C. elegans transcriptome revealed no significant similarity to other genes, supporting the specificity of the RNAi constructs. Moreover, the ufd-1 and npl-4 RNA sequences do not share significant sequence similarity. Therefore, the highly similar phenotypes observed in ufd-1 and npl-4 knockdown animals, including shortened lifespan, reduced survival on P. aeruginosa, and decreased intestinal colonization with P. aeruginosa, strongly suggest that these outcomes result from the disruption of the functional UFD-1-NPL-4 complex.”

(4) I am confused about the authors explanation regarding their observation that inhibition of the UFD-1/ NPL-4 complex extends the lifespan of sek-1(km25) animals, but not pmk-1(km25) animals, as SEK-1 is the MAPKK that functions immediately upstream of the p38 MAPK PMK-1 to promote pathogen resistance.

I am also confused why their RNA-seq experiment revealed a signature of intracellular pathogen response genes and not PMK-1 targets, which the authors propose is accounting for toxic immune activation. Activation of which immune response leads to toxicity?

We consistently observe that sek-1(km4) mutants are more sensitive to P. aeruginosa infection than pmk-1(km25) mutants, a finding also reported in previous studies (for example, PMID: 33658510). Given that SEK-1 functions upstream of PMK-1 in the MAPK signaling cascade, it is plausible that SEK-1 also regulates additional MAP kinases, such as PMK-2 (PMID: 25671546), which could contribute to the enhanced susceptibility observed in sek-1 mutants.

Our results show that inhibition of the UFD-1-NPL-4 complex improves survival specifically in severely immunocompromised animals, such as sek-1(km4) mutants, but not in pmk1(km25) mutants. To further validate this, we generated the double mutant dbl-1(nk3);pmk1(km25), which exhibits reduced survival on P. aeruginosa compared to either single mutant.

Notably, inhibition of the UFD-1-NPL-4 complex also enhances survival in the dbl1(nk3);pmk-1(km25) background, reinforcing the observation that this response is specific to severely compromised immune states.

We would also like to clarify that the observed phenotypes are independent of the SEK1/PMK-1 pathway, as shown in Figure 3A-3C, Figure 3—figure supplement 1, and Figure 4A-4C. The IPR seems to play a role in the observed phenotypes, as inhibition of some of the protease and pals genes (IPR genes) leads to increased P. aeruginosa colonization in ufd-1 knockdown animals (Figure 6—figure supplement 1). The other immune response pathway that leads to the observed phenotypes is ELT-2, as explained in Figure 6. Finally, we have included in the revised manuscript a note that, in addition, as-yet unidentified pathways are also likely contributing to the phenotypes triggered by disruption of the UFD-1-NPL-4 complex.

(5) The authors did not test alternative explanations for why UFD-1/ NPL-4 complex inhibition compromises survival during pathogen infection, other than exuberant immune activation. For example, it is possible that inhibition of this proteosome complex shortens lifespan by compromising the general health/ normal physiology of nematodes. Immune responses could be activated as a secondary consequence of this stress, and not be a direct cause of early morality. Does sek-1(km4) mutant suppress the lifespan shortened lifespan of ufd-1 and npl-4 knockdown? This experiment should also be done with loss-offunction mutants, as noted in point 3.

We have already included this data in Figure 4D, where we observed that ufd-1 and npl-4 knockdown reduce the lifespan of sek-1(km4) animals. It is possible that immune activation is a secondary consequence of cellular stress induced by inhibition of the UFD-1NPL-4 complex. However, our data strongly suggest that the observed phenotypes, including reduced gut pathogen load and decreased survival on the pathogen, are due to the aberrant immune response activated by the inhibition of the UFD-1-NPL-4 complex. Evidence from sek-1(km4) mutants particularly underscores the role of this dysregulated immune activation. While this aberrant immune response is detrimental to wild-type animals under pathogenic conditions, it appears to be beneficial in severely immunocompromised backgrounds. Specifically, in sek-1(km4) mutants, inhibition of the UFD-1-NPL-4 complex enhances survival during P. aeruginosa infection (Figure 4A). However, under non-infectious conditions, where sek-1(km4) mutants exhibit a normal lifespan, the same immune activation becomes harmful (Figure 4D). Together, these findings demonstrate that the aberrant immune response induced by UFD-1–NPL-4 inhibition is context-dependent: it is advantageous only for immunocompromised animals under infection, but deleterious to healthy animals under infection and to both healthy and immunocompromised animals under non-infectious conditions.

(6) The conclusion of Figure 6 hinges on an experiments that uses double RNAi to knockdown two genes at the same time (Fig. 6D and 6G), an approach that is inherently fraught in C. elegans biology owing the likelihood that the efficiency of RNAi-mediated gene knockdown is compromised and may account for the observed phenotypes. The proper control for double RNAi is not empty vector + ufd-1(RNAi), but rather gfp(RNAi) + ufd1(RNAi), as the introduction of a second hairpin RNA is what may compromise knockdown efficiency. In this context, it is important to confirm that knockdown of both genes occurs as expected (with qRT-PCR) and to confirm this phenotype using available elt-2 loss-of-function mutants.

We thank the reviewer for this helpful suggestion. We have repeated all double

RNAi experiments using gfp RNAi as a control instead of the empty vector (Figure 6 and Figure 6—figure supplement 1). Additionally, we assessed the efficiency of gene knockdown in the double RNAi conditions (Figure 6—figure supplement 2) and found that RNAi efficacy was not compromised by the double RNAi treatment.

(7) A supplementary table with the source data for at least three replications (mean lifespan, n, statistical comparison) for each pathogenesis assay should be included in this manuscript.

The source data is provided for all the data presented in the manuscript.

Reviewer #2 (Public Review):

Summary:

The authors aimed to uncover what role, if any, the UFD1/NPL4 complex might play in the innate immune responses of the nematode C. elegans. The authors find that loss of the complex renders animals more sensitive to both pathogenic and non-pathogenic bacteria. However, there appears to be a complex interplay with known innate immune pathways since the loss of UFD1/NPL4 actually results in increased survival of animals lacking the canonical innate immune pathways.

We thank the reviewer for providing an excellent summary of our work.

Strengths:

The authors perform robust genetic analysis to exclude and include possible mechanisms by which the UFD1/NPL4 pathway acts in the innate immune response.

We thank the reviewer for highlighting the strengths of our work.

Weaknesses:

The argument that the loss of the UFD1/NPL4 complex triggers a response that mimics that of an intracellular pathogen has not been thoroughly investigated. Additionally, the finding of a role of the GATA transcription factor, ELT-2, in this response is suggestive, but experiments showing sufficiency in the context of loss of the UFD1/NPL4 complex need to be explored.

We have investigated the role of IPR genes in the phenotypes observed upon ufd1 knockdown (Figure 6—figure supplement 1), and our results suggest that the IPR may contribute, at least in part, to the phenotypic outcomes of ufd-1 RNAi. In the Discussion section (pages 11–12, lines 345–356), we have included a detailed discussion on the possible mechanisms underlying IPR activation upon inhibition of the UFD-1–NPL-4 complex. We agree that the interaction between the UFD-1–NPL-4 complex and the IPR is intriguing and warrants further investigation. However, we believe that an in-depth exploration of this interaction lies beyond the scope of the current study.

We have incorporated new data on ELT-2 overexpression in the revised manuscript. Overexpression of ELT-2 partially phenocopies the effects of ufd-1 knockdown, supporting the idea that other pathways likely contribute to the full spectrum of phenotypes observed upon UFD-1-NPL-4 complex inhibition. The revised manuscript reads (page 10, lines 311319): “To determine whether ELT-2 activation alone is sufficient to recapitulate the phenotypes observed upon UFD-1-NPL-4 complex inhibition, we analyzed animals overexpressing ELT-2. Similar to ufd-1 knockdown, ELT-2 overexpression led to a significant reduction in the colonization of the gut by P. aeruginosa (Figure 6—figure supplement 3A and 3B). However, overexpression of ELT-2 did not alter the survival of worms on P. aeruginosa (Figure 6—figure supplement 3C). Taken together, these findings suggest that the phenotypes triggered by disruption of the UFD-1-NPL-4 complex are partially mediated by ELT-2. However, additional pathways, yet to be identified, likely cooperate with ELT-2 to regulate both pathogen resistance and host survival.”

Reviewer #1 (Recommendations For The Authors):

The authors could consider avoiding the use of descriptors (e.g., "drastic") when presenting their data.

We have removed the descriptors.

Reviewer #2 (Recommendations For The Authors):

What happens with overexpression of ELT2?

Overexpression of ELT-2 partially recapitulates the phenotypes of ufd-1 knockdowns, indicating that additional pathways are likely involved in controlling the phenotypes observed upon inhibition of the UFD-1-NPL-4 complex. The revised manuscript reads (page 10, lines 311-319): “To determine whether ELT-2 activation alone is sufficient to recapitulate the phenotypes observed upon UFD-1-NPL-4 complex inhibition, we analyzed animals overexpressing ELT-2. Similar to ufd-1 knockdown, ELT-2 overexpression led to a significant reduction in the colonization of the gut by P. aeruginosa (Figure 6—figure supplement 3A and 3B). However, overexpression of ELT-2 did not alter the survival of worms on P. aeruginosa (Figure 6—figure supplement 3C). Taken together, these findings suggest that the phenotypes triggered by disruption of the UFD-1-NPL-4 complex are partially mediated by ELT-2. However, additional pathways, yet to be identified, likely cooperate with ELT-2 to regulate both pathogen resistance and host survival.”

The data with xbp-1 loss of function is very different than that of pek1 and atf-6. Does loss of ufd1/npl4 suppress the increased pathogen survival of xbp-1s overexpressing animals?

We have examined worms overexpressing XBP-1s and found that overexpression of XBP-1s does not rescue the phenotypes caused by ufd-1 knockdown. The revised manuscript reads (page 6, lines 167-174): “To further examine the role of XBP-1 in this context, we assessed the effect of ufd-1 knockdown in animals neuronally overexpressing the constitutively active spliced form of XBP-1 (XBP-1s), which has been previously associated with enhanced longevity (Taylor and Dillin, 2013). Knockdown of ufd-1 resulted in the reduced survival of XBP-1s-overexpressing animals on P. aeruginosa, despite a concurrent decrease in bacterial colonization of the gut (Figure 2—figure supplement 1A-C). This indicated that the XBP-1 pathway was not required for the reduced P. aeruginosa colonization of ufd-1 knockdown animals.” 

Lastly, while the pathogen burden is reduced in ufd1/npl4 loss and pumping rates are marginally affected, have you checked defecation rates? Could they be increased?

We thank the reviewer for this valuable suggestion. We measured defecation rates following ufd-1 and npl-4 knockdown and, unexpectedly, found that inhibition of ufd-1/npl-4 leads to a reduction in defecation frequency. These findings clearly indicate that altered defecation cannot explain the observed decrease in gut colonization. The revised manuscript reads (page 5, lines 138-148): “The clearance of intestinal contents through the defecation motor program (DMP) is known to influence gut colonization by P. aeruginosa in C. elegans (Das et al., 2023). It is therefore conceivable that knockdown of the UFD-1-NPL-4 complex might increase defecation frequency, thereby promoting the physical expulsion of bacteria and resulting in reduced gut colonization. To test this possibility, we measured DMP rates in animals subjected to ufd-1 and npl-4 RNAi. Contrary to this hypothesis, both ufd-1 and npl-4 knockdown animals exhibited a significant reduction in defecation frequency compared to control RNAi-treated animals (Figure 1—figure supplement 2C). This reduction in DMP rate persisted even after 12 hours of exposure to P. aeruginosa (Figure 1—figure supplement 2D). Thus, the change in the DMP rate in ufd-1 and npl-4 knockdown animals is unlikely to be the reason for the reduced gut colonization by P. aeruginosa.”

In summary, we would like to thank the reviewers again for providing constructive and thoughtful feedback. We believe we have fully addressed all the concerns of the reviewers by carrying out several new experiments and modifying the text. The manuscript has undergone substantial revision and has thereby improved significantly. We do hope that the evidence in support of the conclusions is found to be complete in the revised manuscript.