This makes college a step stone, its not the final goal its just the first step you put towards getting towards something thats a big pointer to what you do in the future. That mindset makes some general education classes feel like obstacles instead of learning.

Whether they know exactly what they want or just figuring it out now, they all see college as one thing: A way to get a better job than whatever they got now. Its almost like the economic benefits are the one thing everyone bets on.

Kevin has this whole path mapped out with getting his certification, landing an internship, getting a job, some more certifications, a bachelors degree, then more money! I respect the hustle, i really do, but he's MONEY HUNGRY (can't say I blame him).

This is social pressure, College is just the default after high school. Sebastian isn't even sure what he wants at this point he's just following what "everyone's saying" even if its not really what they're saying.

A fifteen cent raise is garbage. Thats a reinforced concrete reason to go to school, to leave these terrible jobs that pay so little. I can see what sebastian wants something better, I do too!

Miguel had to experience first hand what the real world gives him before he started valuing college. His parents couldn't convince him so only working these terrible jobs did. That shows how experiencing something is the most powerful.

THIS is the core of the economic arguement, because while the jobs are available, they're menial and just pay below whats needed to live comfortably, but enough for you to balance your education with work.

This is the main idea, because every decision is a trade off between a short term cost and the long term benefits of it. It shapes when, how long, and what degree you choose.

When school and work conflict, work usually wins. Thats because bills are due, family issues arise, etc... Its not that school isn't important, it is, its just about survival. This explains why some students drop out

After a year of work, Yolanda realized she was missing one thing... an EDUCATION. Work experience taught her what she needed. A common theme real world experience motivating college.

This is a last attempt type of motivation. She doesn't really care about what job it might be, as long as it pays to get out of debt. It's honest and shows how debt drives decisions, sometimes good, most bad.

Miguel waited until his car was paid off, which showed how financial obligations control when a student can enroll. It's really all about the money, he couldn't focus on school with that debt.

The second cost is interrupted advancement at work, Students will give up promotions or raises while they're in school, which makes college somewhat a gamble since you risk current progress for future gain (which sometimes isn't guranteed).

Kyra feels that time pressure, since she is getting older and as you get older you feel this need to have more security, so she wants to finish fast and this itself connects to the idea of short term vs long term tension mentioned in this chapter.

Hugh knows what he wants, and his plan is very specific especially mentioning LVN then RN. This shows how his career goals shape his educational choices, and its not really about the learning itself, its about getting a real career afterwards.

This is a safety net mentality, she's betting against uncertainty. Getting the associates degree first is practical even if its delaying her bachelors.

She's being strategic, shes not particularly against transfer courses, she just can't afford the extra time and money for it. Efficency is her priority.

One class at a time shows how financial limits can slow down your progress, Grants and aids (FINANCIAL SPECIFICALLY) make full time possible.

Charmaine hit the ceiling, so college was pretty much the only way up from there, thats a real motivator and not learning just for fun.

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In my life I often hear people usually adults use the phrase "social media isn't real life." I totally understand this and think it needs to be articulated more to the younger generation. I believe this because so many people have fallen into the trap of comparing themselves to other individuals online. While I dont think social media is inherently evil I do believe it can cause negative attitudes towards yourself and others when you spend too much time comparing yourself to others lives. You have to remember that those pictures or videos are only a small portion of what we see in their life. People are usually showing the highest highs on social media when they post to boost their presence without showing the lows and negative feelings they have. I would advise every person to make their own conscious decision on how they want to interpret and consume media, but make sure to know they negative implications overconsumption and comparison can lead to.

I think of the relationship between social media and "real life" as social media being a part of real life. Social media is so integrated into our lives that I think recently it has become a part of real life. We all carry phones in our pockets and computers in our backpacks, there isn't really a distinction between being online and offline anymore, I would argue that for the most part we are all online. We check our phones for notifications every couple minutes and sometimes we even have notifications being relayed to us through our headphones when we aren't looking at a screen. The way we receive and send information is for the most part through social media and this has become the new norm. Humans are social creatures, we need connection, attention, and community, and nowadays we are fed all of these things through social media, for that reason I think there is no difference between social media and real life.

In my opinion, in the future, Internet can be the real life. For now, some of the rule of the internet are vague. It means even if someone did impolite or criminal behavior, they would not be punished a lot. So, that makes bad people have low cost to do a crime. However, there would be more and more comprehensive law in the internet. Then, I think internet can be a real life.

This is a very problematic sentence lmaooooo

A histographic reading that presents history as a movement from the oppressed past to the enlightened present and future, with teleological and hero-villain dualism

The reading says that existentialism could be considered a form of nihilism, but I would have to disagree. While I do agree that both agree that there's no form of a built-in morality system, but nihilism's main ideal is that nothing matter. On the other hand existentialism argues that since there's no rulebook for life we are left with a complete responsibility for every choice we make. That is more responsibility onto us and the decisions we make which argues against the idea of nihilism that nothing matters. I think a better wording to this would be that existentialism is a response and action to nihilism and not a form of it.

To add on to this description of Consequentialism ethics, it is also about maximizing the good, meaning that the morally right action would be the one that has the greatest net balance of good over evil. A common criticism of this ethic framework is that the idea of "the ends justify the means" can sometimes hurt minorities if the result is deemed beneficial to the majority.

I don't agree with the condensed summary of Nihilism. Primarily because it doesn't acknowledge that Nihilism was a pessimistic ethical view in history that Friedrich Nietzsche himself considered to be a threat to society and its values. Philosophers worried that nihilism would lead to a global crisis so they downplayed its pessimistic nature. But the interpretation has gone on further. Nihilism is not simply about how nothing matters, but how it is important that one views themself in regard to the world. Nihilism is about acknowledging one's flaws and mistakes and not trying to fix it in order to become an "ideal person," but remembering that people are ultimately flawed and one shouldn't obsess over it.

1. For Taoism, I would like to add more information. In the textbook, it said:" Trying to force something to happen will likely backfire." It's right, but we should know more. In Taoism, spring means growing up, summer means prosperity, autumn means recession, and winter means taking a break. Therefore, the further comprehension of Taoism is understand and control. For example, ancient Chinese water wheel reflects that they can use the natural flow of water to help them cultivate, rather than forcing nature.

Kantianism is my favorite ethical lens because of the infinite number of hypothetical situations it creates for us to analyze. For example, say you come across a problem on whether you should lie or tell the truth in a certain situation. Well Kantianism might prompt you to ask yourself the question "What would life look like if everyone lied?" You would probably come to the conclusion that society would collapse because nobody would be able to trust one another. This would prompt you to then question "What would life look like if everyone told the truth?" and you might discover that these two hypothetical realities look quite similar. From here you would have to ask the more specific question "What would life look like if everyone in my certain situation lied?". This is why I love Kantian ethics, there is an unlimited amount of pondering to be done and hypotheticals to be worked out. I find it fascinating to work out these hypotheticals and see how much the answer depends on how the question is framed.

I believe a key figure of divine command theory could be Pope Francis. As a Catholic, we believe and follow gods and Jesus many teachings. Some include love your neighbor, forgive others and caring for those in need. Overall he guides us in how to treat others in which I believe are morally good.

The Aztec Virtue ethics say that it isn’t the action that causes one to be good or bad (balanced or unbalanced), but that the virtue of an action is more about context and circumstance. For example, public drunkenness for nobility was seen as a crime punishable by death, but the elderly at a wedding were permitted and expected to get drunk. They believed “the middle path” to be the one of most virtue - dressing not too formal and not too drab. But most of all virtue was communicated regularly by community. The King had advisors, before leaving on a perhaps risky journey, merchants would make sacrifices and receive virtue adjustments in the form of a community ritual - a “refresher course” if you will.

I agree with this statement because using an ethics framework doesn't necessarily mean you're going to lead to the right answer. Different frameworks can lead people to completely different decisions because we all have different ethics and we all have different opinions. We all interpret information differently, which is why I believe it is important to consider multiple ethical frameworks instead of just relying on one when making a decision.

I loved reading about Virtue Ethics and thought it was very interesting and I'd love to add more information to the frame work. I feel like acting through your virtue definitely a way to flourish as an individual as stated in the text, but is also impacted as the way you are raised. Living through your virtue is based on actions and believes through your own point of view which can be impacted by social media. But I also feel like when you live through your virtues its based on how you were raised as your parents have an impact on you. As we get older we form our own opinions but no matter what I feel like the way we are raised always has an affect to our virtues.

This actually feels like the opposite of Ethics. Is egoism really 'rational'? I think we could easily argue no - how many happy, healthy, loved people exist that live only for their own self interest?

The concept of considering the balance of all parts of the ecosystem in which we live is especially intriguing to me. I appreciate the idea that Ethics considerations go beyond just person to person. We only exist as a part of a larger system - with air, water, land, plants - all in need of balance. Maximizing for people, can easily mean the deterioration for one of the other resources - considering the balance from the beginning would be wise it seems.

Another ethics framework I researched is feminist ethics. HIstorically, men have been privileged, no matter the social class. This ethic focuses on re-routing the common opressive practices that harm others, focusing on the ethic of "doing things". Feminist ethics can be expressed in multiple different ways and overall universal principles.

As times change, the way modern people view issues has become different from that of people in the past. When modern and ancient ways of thinking clash, should we respect so called cultural heritage or adapt to the times?

I have always found this way of thinking ,very disturbing. At its core, nihilism argues that existence is without objective meaning, purpose, or moral truth, which can lead to extreme detachment or numbness. The only person I've met who thinks this way was utterly depressed and found no meaning in life. They found no joy, sadness, or emotion. It always broke my heart to find more people who related to that on social media. The person I knew had dead eyes, with no soul or emotion behind them.

Another Ethics framework that could be included is Ethics egoism. Whether an action is moral mainly depends on whether it is in line with the interests of the actor. This framwork could be interesting for social media because the company may want more profit and more users online, they may release more atractive video or news, in order to make their user addicted to their apps. So that they can make more profit during this process. If the company can bring greater benefits, then the company might consider this choose as morally justified. But i wonder whether should a compamy's own benefit be enough to make a decision ethical if their users are harmed.

Could Confucianism become a way of appearing respectful without actually feeling respect? If people follow them only to meet social expectations, could they make society seem harmonious even when people don’t really mean it?

I would say that I am the one making the post. Even though I am not the one write the code, or the one actually sending it, but that post is only sent with my consent. Without me, the bot would not be able to send it in the account I created first.

A vague stereotype that changes from subculture to subculture, and person to person. Essentially they are simply expressing their personality and interjecting a stereotype/social role to it that may be offensive and might not fit someone else’s definition of that stereotype.

Example: Bob wears a dress and tells Sally that it means he is a girl because girls wear dresses.

Sally tells Bob that since girls don’t wear dresses to her, he is actually a boy and he would have to wear sandals to be seen as a girl to her because to her girls wear sandals.

This would mean that they are not in fact trans as nobody has to affirm a stereotype (social “transition”) being applied to yourself.

So their biological sex is perceived at the opposite sex of what it actually is. The idea is to relieve symptoms of distress resulting from an intense hatred of one’s sex characteristics due to a mental illness called Gender Dysphoria.

No child is trans. Some children have gender dysphoria. Transgender is when a person medically induces secondary sex characteristics to imitate that of the opposite sex. Transitioning a child is medical malpractice. A person should only be allowed to transition after Gender Dysphoria has persisted well into adulthood proving to be chronic and therapy resistant.

Exploratory therapy (non-affirming, non-directive) is mental health care that some advocate for— it doesn't steer toward transition, but it also doesn't condemn the identity. The UK's Cass Review and several European health agencies have moved in this direction.

The model assumes that the internal stress is externally imposed. It doesn't account for the possibility that some of the social friction comes from the interaction between the person's demands and the environment's response. The research literature on this is mixed — some studies find that social support and identity affirmation improve mental health outcomes, while others find that stronger identification with a marginalized identity can increase sensitivity to rejection and worsen outcomes .

So the causal arrow may run both ways — not just "society rejects → distress," but also "distress → heightened sensitivity to rejection → more perceived rejection."

The sociological literature on "preferred pronouns" and workplace dynamics documents that mandating affirmation can create resentment and backlash, which in turn affects the person demanding it .

Transgender is when one is diagnosed with gender dysphoria and medically induces secondary sex characteristics to present in the image of the sex that is the opposite of what they really are.

Not something anyone has to agree with or validate, and the majority of clinicians and the world population disagree with.

This is highly contested. Many still believe that male and female are still a binary, that the small percentage of people with Disorders of Sex Development still only have male and female traits (not a secret 3rd category), and the .0018% of the population that are truly intersex (fully ambiguous) don’t define the system and again, are still not a secret 3rd category.

Gender ideology

🔬 Evidence for Biological Influence

The research literature points to prenatal androgen exposure as a significant factor in shaping sex-typed interests. Studies on girls with Congenital Adrenal Hyperplasia (CAH), who are exposed to high levels of androgens prenatally, consistently show they have more male-typical play preferences and interests in male-typical careers compared to unaffected girls .‌

📊 The "Gender Equality Paradox"

One of the strongest challenges to the purely social explanation is the "gender equality paradox." Research shows that countries with greater gender equality (like Nordic nations) often have a lower percentage of women in STEM fields . The proposed explanation is that when economic security is high, people are freer to follow their intrinsic interests rather than choosing careers for financial stability—and those intrinsic interests tend to be more sex-differentiated .‌‌

⚖️ The Textbook's Framing vs. the Research

The textbook is correct that social and cultural factors matter, but it presents a one-sided view by stating these roles are "derived not from biology." The actual scientific consensus is that it's a complex interaction. Even researchers who emphasize biological factors stress that hormones act "jointly with the social environment".

Notice how it uses the same definition worded differently as “gender (ideology)”

Cohort effect. Gen Z will call Harry Styles in a ball gown incredibly masculine.

Stereotypes/social roles applied to oneself relating to sex.

According to the APA’s position in favor of Gender Ideology but highly contested and ironically socially determined to be false by the majority in both USA and globally.

The belief according to APA.

Gender (ideology): gender roles are gender.

This is highly dissented. Majority of clinicians and society reject gender ideology. The general consensus is that gender is sex.

Gender identity: applying a gender role/stereotype to oneself.

This is not something anyone has to validate or accept. Many believe this is harmful and just reinforces stereotypes for everyone even if unintentional. It also cuts down on women’s rights by stripping down protective spaces separated on the basis of biology when you try to change definitions to fit your narrative.

eLife Assessment

This important study identifies underappreciated experimental factors that influence α-synuclein amyloid polymorphism, with practical implications for the reproducibility of in vitro fibril preparations. Extensive cryo-EM analyses provide convincing evidence that protein purity, monomer preparation, and agitation conditions influence polymorph selection, including substantial effects from small amounts of an N-terminally truncated variant. The study provides a substantial resource for researchers studying amyloid assembly and synucleinopathies, although the proposed nucleation mechanisms need stronger kinetic support and reliable protocols for producing specific polymorphs require further development.

Reviewer #1 (Public review):

In this work, Frey and colleagues have carried out a very large study of α-synuclein polymorphism as a function of aggregation conditions and sample preparation. They provide valuable insight into the many critical factors affecting α-synuclein polymorphism, thereby illuminating the need for detailed reporting in the literature as well as both rigorous and detail-oriented protocols when working with this protein. Indeed, their observations are in line with the difficulties of reproducing structural outcomes across different laboratories and experiments. The authors must be complemented on their openness about the difficulties experienced and the thoroughness of their work. Efforts like these are going to be crucial to achieve an understanding of the unparalleled structural plasticity of α-synuclein amyloid fibrils. It is particularly notable that the authors have managed to optimize protocols to form single-polymorph aggregation reactions with high reproducibility.

In this work, the authors focus on the influence of α-synuclein purity in aggregation reactions. They find that using reverse-phase HPLC to purify the protein significantly alters the aggregation behaviour. It is interesting, and rather uncommon in the field, to use reverse-phase HPLC as a final purification step, rather than SEC, which is commonly used in many laboratories. It would be useful to compare this new protocol even more directly and extensively with the commonly used SEC protocol. When mentioning their previously published work, it should be mentioned explicitly how the protein was purified in these previous studies.

On the point of protein purification, the authors lyophilize their protein prior to storage. In their work, they also find that pre-aggregation oligomer formation alters the aggregation pathway of α-synuclein. While they demonstrate that this can be solved by appropriate filtration, it should be discussed why the lyphilizaiton step was not reconsidered/omitted given that this process is known to facilitate oligomer formation. Do the authors have experience with aggregation studies using α-synuclein that has not undergone lyophilisation and are able to comment on the influence of this step in the protocol?

The authors make note of several degradation products affecting their aggregation reactions, which is why they employ a much more thorough purification protocol. However, they also point out that some of the degradation products found at the end of their reaction could form during the reaction itself. Unfortunately, they never investigate this further. In particular, it would be very useful to know if different aggregation conditions (pH, salt, agitation) lead to different and characteristic degradation patterns. HPLS/mass spec of the supernatant at the end of each aggregation reaction would have been a very insightful thing to do.

With respect to degradation products, in this work a NΔ4-variant is produced to mimic a disease-relevant degradation product and indeed it is found to alter the structural outcome even at low relative concentrations (5%). Have the authors investigated the minimal fraction of the NΔ4 variant necessary to still influence the structural outcome of the predominant WT protein? This type of analysis could have significant relevance to disease-related analysis, where several variants (truncations and PTM variants) are present in trace amounts.

While on this topic, the authors note that in several of their type 5 fibrils, they find unresolved peptide fragments in their cryo-EM structures. Can the authors speculate if these fragments are indeed peptide degradation products or residues wrapping around the fibril originating from the fibril-incorporated protein?

In this work, the authors have performed an extensive study of α-synuclein polymorphism. However, despite generating what is likely the largest single data set of fibril structures, they perform very little quantitative analysis of their data. It would be interesting to analyse the relative abundance of fibril polymorphs produced in each reaction. Perhaps from the particle-picking data it could be estimated the relative abundance of each polymorph as well as non-resolved fibrils to generate a more nuanced view of polymorphism beyond overall classifications of the resolved structures. Indeed, from such data it could also be studied if certain protofilaments are more prone to pair in asymmetric fibril structures over others or if fibril asymmetry can be attributed to random pairing of protofilaments in accord with their abundance. This latter point is particularly interesting for the type 1 fibrils. Perhaps, the propensity of α-synuclein to form specific symmetries could also be estimated.

The authors note that pH is a strong factor in determining polymorph selection. This does indeed appear to be the case, but other parameters do not appear to show any clear trend. Have the authors investigated the influence of aggregation parameters (agitation, duration) on structural outcomes systematically or quantitatively, such as with principal component analysis? Indeed, they also find that some fibril types that otherwise are not compatible at the same pH appear to co-exist when the shaking parameter is modified. Are the authors then confident in the claim that pH is a deterministic parameter?

It is evident from this and other work that amyloid aggregation is highly sensitive to kinetic effects. It is therefore curious that the effect of protein concentration and reaction time has not been systematically investigated. The authors have some data studying dilution series (Figure 6A) and different reaction times (reaction 56 & 57). Could the authors comment on the effects of these two parameters, and might there be more information touching upon this that could be highlighted in this work?

The authors state that this work likely underreports fibril polymorphisms in samples due to population size or data quality challenges. Could the authors, based on their extensive experience, try to quantify this statement?

Additionally, the authors point out that the current framework for classifying α-synuclein fibril polymorphism is not sufficient to describe the real complexity of this protein system. However, they do not seem to address some of the recent literature aiming to solve such issues (see Scheres 2026, Connor et al. 2025, Milchberg et al. 2025 & Price et al. 2025).

Was any biophysical/biochemical analysis performed of the many structures produced here, such as CD spectroscopy, Proteinase K digestion, dye binding or FTIR, which could act as low-resolution structure identifiers and might help to retrospectively explain some findings in the older literature? Such data would be very useful for the vast majority of researchers, who do not have access to cryo-EM.

Reviewer #2 (Public review):

Summary:

This manuscript describes insights gained during efforts to reproduce disease-relevant alpha-Synuclein (aSyn) fibrils using recombinant protein in vitro. It follows up on a similar article from this team published in 2024. Although the authors have not been able to produce fibrils with the structure of ex vivo fibrils isolated from patients, they share insights gained into which factors influence the formation of specific fibril polymorphs.

Strengths:

This is quite an unusual manuscript because it goes into minute detail about sample preparation that are usual just mentioned in the Materials and Methods sections of other manuscripts (if at all). This makes it very valuable for the scientific community working on exactly the problem of reproducing disease-relevant aSyn fibrils in vitro (which will be a major breakthrough in the field). The authors present an impressive array of cryo-EM fibril structures, some of which have not been described before.

Weaknesses:

A major concern with the manuscript is that its story and messaging are a bit murky. The authors describe a few new polymorphs, show that some polymorphs (type 1) have small variations, show that sample purity and fragmentation will influence polymorph formation, and present a helical-symmetry mystery. This all reads like a loose collection of findings without any major takeaway. Looking at Table 1, it still seems that the authors do not have a good control over any of these polymorphs. Are they able to make any of these polymorphs reliably? I think the impact of this work could be strengthened if it ended with a reliable protocol for the production of any of the polymorphs described.

A second major concern is the quality of the aggregation kinetics and their interpretation. Are these kinetics just done once per concentration? The figure caption talks about 'three independent samples' but it is unclear if this refers to the three different concentrations or NΔ4 percentages used, or true repetitions. Looking at the curves themselves, it seems that only one of the conditions was actually done in triplicate, which should be the minimum to draw conclusions. Further, it would have been helpful to characterize the kinetic data quantitatively. Finally, because there are only kinetic data for a fraction of the conditions tested, it is not clear what they add to the overall manuscript. My recommendation is to either remove the kinetics from the manuscript or substantially expand this section.

Author response:

Reviewer #1 (Public review):

In this work, Frey and colleagues have carried out a very large study of α-synuclein polymorphism as a function of aggregation conditions and sample preparation. They provide valuable insight into the many critical factors affecting α-synuclein polymorphism, thereby illuminating the need for detailed reporting in the literature as well as both rigorous and detail-oriented protocols when working with this protein. Indeed, their observations are in line with the difficulties of reproducing structural outcomes across different laboratories and experiments. The authors must be complemented on their openness about the difficulties experienced and the thoroughness of their work. Efforts like these are going to be crucial to achieve an understanding of the unparalleled structural plasticity of α-synuclein amyloid fibrils. It is particularly notable that the authors have managed to optimize protocols to form single-polymorph aggregation reactions with high reproducibility.

In this work, the authors focus on the influence of α-synuclein purity in aggregation reactions. They find that using reverse-phase HPLC to purify the protein significantly alters the aggregation behaviour. It is interesting, and rather uncommon in the field, to use reverse-phase HPLC as a final purification step, rather than SEC, which is commonly used in many laboratories. It would be useful to compare this new protocol even more directly and extensively with the commonly used SEC protocol. When mentioning their previously published work, it should be mentioned explicitly how the protein was purified in these previous studies.

On the point of protein purification, the authors lyophilize their protein prior to storage. In their work, they also find that pre-aggregation oligomer formation alters the aggregation pathway of α-synuclein. While they demonstrate that this can be solved by appropriate filtration, it should be discussed why the lyphilizaiton step was not reconsidered/omitted given that this process is known to facilitate oligomer formation. Do the authors have experience with aggregation studies using α-synuclein that has not undergone lyophilisation and are able to comment on the influence of this step in the protocol?

We will include details in our revised version on how samples were prepared in our previously published work. However we do not plan to do a comparison between SEC and HPLC as final purification steps because these are truly orthogonal separation methods. SEC is the gold standard for oligomer removal but not particularly useful for removing degradation products of similar size (which we believe to affect aggregation outcomes). In our hands, the highest purity and reproducibility come from HPLC-purified material followed by a stringent oligomer removal. Because oligomers in the solubilized sample are a concern, SEC as a final post-solubilization/pre-aggregation step might be ideal. However, as we mentioned in the manuscript, SEC dilutes the sample to the point that much of the sample is too dilute for our purposes (aggregation without seeds) and adding an additional concentration step would risk promoting the formation of new oligomers in the concentration device. That is why we resorted to using a 100 kD MWCO filter to remove oligomeric species.

We considered skipping the lyophilization step and dialyzing the HPLC-purified sample into the buffer of choice but stuck with lyophilization because it provides an easy control over the protein concentration in the solubilized sample.

To clarify the logic in this choice of sample preparation steps, we will add a section to the revised manuscript listing/explaining our suggested protocols for preparing alpha-synuclein samples for reproducible aggregation experiments.

The authors make note of several degradation products affecting their aggregation reactions, which is why they employ a much more thorough purification protocol. However, they also point out that some of the degradation products found at the end of their reaction could form during the reaction itself. Unfortunately, they never investigate this further. In particular, it would be very useful to know if different aggregation conditions (pH, salt, agitation) lead to different and characteristic degradation patterns. HPLS/mass spec of the supernatant at the end of each aggregation reaction would have been a very insightful thing to do.

In retrospect, we agree that this could have been important from the standpoint of understanding how in situ degradation during the aggregation at 37º C could also play a role in polymorph selection. We have begun to save frozen aliquots of our aggregation samples for subsequent MS analyses of the interesting samples in order to be able to address this in the future. However, it was outside the scope of our original search for the PD polymorph.

With respect to degradation products, in this work a NΔ4-variant is produced to mimic a disease-relevant degradation product and indeed it is found to alter the structural outcome even at low relative concentrations (5%). Have the authors investigated the minimal fraction of the NΔ4 variant necessary to still influence the structural outcome of the predominant WT protein? This type of analysis could have significant relevance to disease-related analysis, where several variants (truncations and PTM variants) are present in trace amounts.

We did not try lower than 5% because it was our goal to test if impurities at this level (which usually go undetected) could influence the aggregation outcomes. We think that directly relating the precise impurity level in these in vitro experiments to in vivo aggregation would be difficult due to the many factors we do not yet understand that appear to guide in vivo polymorph selection.

While on this topic, the authors note that in several of their type 5 fibrils, they find unresolved peptide fragments in their cryo-EM structures. Can the authors speculate if these fragments are indeed peptide degradation products or residues wrapping around the fibril originating from the fibril-incorporated protein?

We will add this speculation to the revised manuscript. The unassigned peptide density most likely originates from the C-terminal residues of the intact chains rather than from a degradation product. The levels of degradation products observed by MS in our other samples were far too low to account for the amount of peptide that would be required for >50% of the fibrils in sample 23 to contain this extra density. Furthermore, in the 5A polymorph the extra peptide density is sometimes present (e.g. sample 52) and sometimes absent (e.g. sample 3), despite the fact that in both samples the coexisting type 5 polymorphs (5m and 5B, respectively) have the peptide bound. Therefore, it appears that subtle differences between 5A polymorphs determine the presence or absence of this density, whereas for 5m and 5B it is consistently present.

In this work, the authors have performed an extensive study of α-synuclein polymorphism. However, despite generating what is likely the largest single data set of fibril structures, they perform very little quantitative analysis of their data. It would be interesting to analyse the relative abundance of fibril polymorphs produced in each reaction. Perhaps from the particle-picking data it could be estimated the relative abundance of each polymorph as well as non-resolved fibrils to generate a more nuanced view of polymorphism beyond overall classifications of the resolved structures. Indeed, from such data it could also be studied if certain protofilaments are more prone to pair in asymmetric fibril structures over others or if fibril asymmetry can be attributed to random pairing of protofilaments in accord with their abundance. This latter point is particularly interesting for the type 1 fibrils. Perhaps, the propensity of α-synuclein to form specific symmetries could also be estimated.

We agree that Cryo-EM datasets contain considerable information that could potentially be used to better understand polymorph populations. We have previously used particle counts as an approximate measure of polymorph abundance; however, the biases introduced during particle picking and subsequent curation are substantial, and we therefore do not consider these counts sufficiently reliable for quantitative comparison of polymorph populations.

Regarding the symmetry of paired filaments, it is clear that the overwhelming preference of all filaments is to pair as symmetric dimers. Among the in vitro polymorphs, type 1 is the most commonly observed to form an asymmetric dimer, either with itself or with the new type 7. However, the number of observations is too small to establish that this represents a statistically meaningful preference. Types 2 and 3 have also been observed to pair in asymmetric fibrils. Overall, we do not think that our dataset is large enough to add statistical weight to previous observations.

The authors note that pH is a strong factor in determining polymorph selection. This does indeed appear to be the case, but other parameters do not appear to show any clear trend. Have the authors investigated the influence of aggregation parameters (agitation, duration) on structural outcomes systematically or quantitatively, such as with principal component analysis? Indeed, they also find that some fibril types that otherwise are not compatible at the same pH appear to co-exist when the shaking parameter is modified. Are the authors then confident in the claim that pH is a deterministic parameter?

We did not systematically vary the agitation but in two cases where it was either intentionally or accidentally varied, we found surprising polymorph outcomes. We felt that these observations were worth reporting, but on their own they do not constitute a thorough study. We would rather conclude that pH is a strong selector and can be deterministic under certain conditions: pure sample, no seeds, continuous or intermittent moderate agitation. We will revise the manuscript to make this clearer.

It is evident from this and other work that amyloid aggregation is highly sensitive to kinetic effects. It is therefore curious that the effect of protein concentration and reaction time has not been systematically investigated. The authors have some data studying dilution series (Figure 6A) and different reaction times (reaction 56&57). Could the authors comment on the effects of these two parameters, and might there be more information touching upon this that could be highlighted in this work?

We did not collect sufficient data to draw conclusions about the effects of protein concentration or aggregation time, and therefore do not think that a quantitative analysis of these parameters is justified by the present dataset.

The authors state that this work likely underreports fibril polymorphisms in samples due to population size or data quality challenges. Could the authors, based on their extensive experience, try to quantify this statement?

Precise quantification would be difficult. The literature has many mentions of amyloids that could not be solved by Cryo-EM due to a lack of twist. In our hands it is very common to have a small subset of non-twisted filaments in a sample and some samples appear to be exclusively non-twisted. Low-abundance or low-quality fibrils are also rather common in our data but also difficult to quantify. Nevertheless, we agree that it would be useful to place a lower bound on this estimate, and we will re-examine our datasets to determine whether this can be quantified in the revised manuscript.

Additionally, the authors point out that the current framework for classifying α-synuclein fibril polymorphism is not sufficient to describe the real complexity of this protein system. However, they do not seem to address some of the recent literature aiming to solve such issues (see Scheres 2026, Connor et al. 2025, Milchberg et al. 2025 & Price et al. 2025).

We agree that we should have discussed this literature in greater detail and will do so in the revised manuscript.

Was any biophysical/biochemical analysis performed of the many structures produced here, such as CD spectroscopy, Proteinase K digestion, dye binding or FTIR, which could act as low-resolution structure identifiers and might help to retrospectively explain some findings in the older literature? Such data would be very useful for the vast majority of researchers, who do not have access to cryo-EM.

We did not perform these analyses precisely because they are low resolution. Retrospectively, such analyses might have been useful for interpreting past data, but most of these methods (particularly Proteinase K resistance) are difficult to compare between laboratories and work best with side-by-side controls. This is why developing a facile method for polymorph identification is one of our main future research goals.

Reviewer #2 (Public review):

Summary:

This manuscript describes insights gained during efforts to reproduce disease-relevant alpha-Synuclein (aSyn) fibrils using recombinant protein in vitro. It follows up on a similar article from this team published in 2024. Although the authors have not been able to produce fibrils with the structure of ex vivo fibrils isolated from patients, they share insights gained into which factors influence the formation of specific fibril polymorphs.

Strengths:

This is quite an unusual manuscript because it goes into minute detail about sample preparation that are usual just mentioned in the Materials and Methods sections of other manuscripts (if at all). This makes it very valuable for the scientific community working on exactly the problem of reproducing disease-relevant aSyn fibrils in vitro (which will be a major breakthrough in the field). The authors present an impressive array of cryo-EM fibril structures, some of which have not been described before.

Weaknesses:

A major concern with the manuscript is that its story and messaging are a bit murky. The authors describe a few new polymorphs, show that some polymorphs (type 1) have small variations, show that sample purity and fragmentation will influence polymorph formation, and present a helical-symmetry mystery. This all reads like a loose collection of findings without any major takeaway. Looking at Table 1, it still seems that the authors do not have a good control over any of these polymorphs. Are they able to make any of these polymorphs reliably? I think the impact of this work could be strengthened if it ended with a reliable protocol for the production of any of the polymorphs described.

It is true that the initial results were a collection of findings compiled while searching for the PD polymorph. However, the trends that we saw in these data inspired us to pursue a more systematic approach which was used to show how impurities play a crucial role in polymorph selection even at low levels. We agree that the impact of the work will be enhanced by summarizing the protocols that can be used to obtain the types 1, 2, 3 and 5 polymorphs and will add this to the revised manuscript.

A second major concern is the quality of the aggregation kinetics and their interpretation. Are these kinetics just done once per concentration? The figure caption talks about 'three independent samples' but it is unclear if this refers to the three different concentrations or NΔ4 percentages used, or true repetitions. Looking at the curves themselves, it seems that only one of the conditions was actually done in triplicate, which should be the minimum to draw conclusions. Further, it would have been helpful to characterize the kinetic data quantitatively. Finally, because there are only kinetic data for a fraction of the conditions tested, it is not clear what they add to the overall manuscript. My recommendation is to either remove the kinetics from the manuscript or substantially expand this section.

In Figure 6A, the three curves represent three independent samples; in panels B–D, each curve similarly represents one independent sample. We will try to clear up the ambiguity in the revised version. We agree that the kinetic data have limited utility for determining kinetic parameters of the aggregation. The data were collected primarily to determine when the aggregation reactions were complete so that samples could be prepared for cryo-EM. Nevertheless, we think that the kinetic traces provide two qualitative observations that are relevant to the structural results: 1) In panels A and C of figure 6, the type 5 polymorphs are associated with shorter lag times, suggesting that they either nucleate faster, or as we propose, arise from a small amount of oligomeric protein in the original sample. 2) The longer lag phase associated with the NΔ4 construct was reproducible (panels B, C and D), suggesting that its intramolecular self-chaperoning effect is enhanced due to the increased positive charge in its N-terminal region. Future studies will determine whether this same electrostatic change also gives rise to enhanced secondary nucleation.

12張5000台幣的話可以

Interesting that the textbook spelled the platform wrong.

I think "social media" is no longer limited to these platforms. More and more people use different methods to express their opinions, such as songs. So, for me, Spotify is also a form of social media because singers can express their ideas through their songs, and audiences can post comments about them. A lot of people also try to make friends through games. They can become online friends by playing games together.

l

If this is not the case, the perceptron algorithm will not terminate, but surprisingly it will stay bounded. See Block and Levin, "On the Boundedness of an Iterative Procedure for Solving a System of Linear Inequalities", Proc. Amer. Math. Soc., Vo. 26., No. 2, (1970), 229-245.

There exists \(M\) only dependent on the vectors \(v_1, \dots, v_m\), such that

$$ |\alpha_i| \leq |\alpha_1| + M $$

for every \(i = 1, 2, \dots\). In particular, if the vectors have rational coordinates, the algorithm will eventually cycle.

ok put it doesn't look like a map, and white boxes on beige backround... loets design it a bit better with gaming ux and ui, for all the stuff right?

my desk vs the board, whats the diff, also the design is different. some elements of each are good how about we use the best of both for this, so its readable, accessible,a dn easy to know what im supposed to be looking for

what is this used for?

when do i do this? or need it?

why not out that on the bard on the right, marked as a phon instead of a initial?

if laude or somethign is disconnectde, how do we reconnect? through game or claude chat?

? what is this?

whats the difference between this and control room or workfloor?

this is part of the sun, that i can decide ohw dim i want the sreen, no the actual time i see in game lol

whats i more?

what the limit? active, non active, just queue/log style thing? whats it for vs the rest?

Learn essential sales communication phrases to build trust, handle customer conversations, address concerns, and improve engagement throughout the sales process.

queire quieren gustan gusta quiere gusta quiero

gusta te les le nos gusta gustan gustan

I appreciate this footnote, as computers truly would not exist if it weren't for the minds of many amazing women. Hidden Figures, to me, was not simply a movie about brilliant female African American scientists, but about how women truly work wonders in the shadows of progress. If anything, the entire foundation of computers, from NASA to creating a language of code, is ethically fascinating because a majority of people surrounded by computers nowadays are men. The 21st century preaches technological progress, but none of it would be possible without computers. And computers wouldn't be possible without women. It makes me wonder if the further we dive into ethics on social media, the more of this patriarchal censorship is present.

me gusta les gusta les gusta nos gusta le gusta os gusta le gusta les gusta

開兩萬日幣, 若兩萬二日幣可下單

四片2000可下單

四片2200可直接下單

看能否爭取8片或多片合售

satou議價留言: コメント失礼いたします。挙げていただいた8本（マリオパーティ、スーパースターズ、ポケモン 剣、盾、イーブイ、ピカチュウ、ポッ拳、マリオ3D）をまとめて20,000円でお譲りいただくことは可能でしょうか？

もしお値段を変更していただければ、すぐに購入させていただきます。ご検討のほどよろしくお願いいたします。

兩片瑪莉歐1000 加DQ 三片1500可直接下單

diciembre de siete el febrero diecinueve abril mayo

febrero septiembre noviembre mayo Julio agosto agosto y septiembre agosto y mayo

enero

octubre

deciembre

febrero

I think that one feature on Twitter that allowed these events to happen is that you are able to retweet the post and share it to your followers. Even if Justine only had a few followers, one of them could have thousands and when the post was retweeted, more and more people saw the post and most likely also retweeted it which continued the cycle. Another feature of the app that enabled the events is how public her information was. People were able to see exactly where she was and could figure out where she was going which lead to photos of her coming off the plane to e taken and spread online.

I think people need to spread more awareness about how easily social media users and companies forget or forgive someone who did something wrong. They act like nothing ever happened. I don't think it's right that she was rehired. She should have known better, especially since she works in public relations.

I think the Sacco case raises an interesting question about the boundaries of public rights. They have the right to critique her remarks. However, i think this does not necessarily mean that thay have the right to further pry into her private life. Does the public information disclosed on social media imply that the public can freely search for person's private informations?

I think it is the responsibility of anyone who works for any company to be aware of the ethics and values that the company holds. Is this benevolent non-profit with only good intentions and a transparent approach that keeps everything on the up and up? Or is this a giant tech conglomerate that has its hands in many pies and seeks making money most of all, no matter who they support and no matter how it gets done? It is the responsibility of the worker, I believe, to work for companies that match their own moral beliefs. I couldn’t work for a place that was poisoning the Earth, for example. I think the lead tech workers, the ones actually steering the ship, have a huge responsibility to ask themselves is this technology 1. Helpful 2. Harmful to anybody Along side making tech should be a failsafe, something to combat any negative effects any piece of tech might hold.

I believe tech workers should take on the same responsibility that Justine Sacco took on by acknowledging the issues that society can face with the evolution of technology. Even though many platforms have faced backlash over these issues, society does not seem to do much to address them. The movie The Social Reckoning, even though it has not been released yet, presents the idea that everything has consequences, including technology. The bigger question, however, is how these large tech companies can be held accountable for the consequences of the platforms they create

eLife Assessment

This useful study reports potential loss-of-function variants, pseudogenes and gene presence-absence variation across multiple chicken genomes, with potential implications for understanding genome evolution and domestication. The evidence for the central claims is unfortunately incomplete, as the inferences of gene loss are not sufficiently robust to account for assembly and annotation artifacts, and, in addition, the analyses can not distinguish between positive selection and relaxed constraint. The overall claim of large-scale gene loss being adaptive and thus being a major driver of chicken evolution and domestication is therefore not sufficiently supported. The area of the study is of interest to colleagues in evolutionary and comparative genomics as well as animal domestication.

Reviewer #1 (Public review):

Summary:

The authors have assembled the genome of four local chicken breeds from China and analysed their gene content. They come to the conclusion that thousands of genes present in the current reference genome of chicken have become pseudogenized during chicken evolution and domestication. They argue that their study provides strong support for the importance of the "less-is-more" hypothesis for adaptive evolution.

Strengths:

The paper provides medium-quality genome assemblies for four individuals representing four local populations of chickens and analyses their gene content.

Weaknesses:

They have not excluded the possibility that the high rate of putative pseudogenes reflects the presence of errors in gene models, in particular in GC-rich microchromosomes that are challenging to assemble correctly. The paper contains no genotype-phenotype analysis, which means that the adaptive significance of a high rate of pseudogenization, if it exists, is unknown.

Reviewer #2 (Public review):

Summary:

The authors set out to investigate the evolutionary role of gene presence-absence variation and pseudogenization in chicken evolution and domestication. By comparing draft genome assemblies of four indigenous Chinese chicken breeds against the red junglefowl reference genome (GRCg6a) and four PacBio HiFi assemblies, the study proposes that the common ancestor possessed nearly 22,000 genes, and that each domestic lineage independently lost thousands of genes (identifying ~8,000 dispensable genes). The authors conclude that massive loss of function and pseudogenization represent major drivers of chicken evolution under the "less-is-more" hypothesis.

While the concept that gene loss can drive phenotypic diversification during domestication is compelling, the results do not convincingly support the central conclusions. The scale of reported gene loss and the specific patterns of pseudogenization appear to be potentially driven by well-known genome assembly gaps, annotation errors, and sequencing dropouts rather than genuine evolutionary events, and the authors do not provide enough convincing evidence that this is not the case.

Strengths:

(1) The study addresses an important and timely evolutionary question regarding the role of gene loss and loss-of-function variation in animal domestication.

(2) The inclusion of multiple indigenous Chinese chicken breeds alongside high-accuracy PacBio HiFi assemblies provides a valuable comparative genomic dataset.

(3) The authors attempt to evaluate pseudogene transcription using RNA-seq data and check for transcript isoforms that bypass candidate loss-of-function mutations.

Weaknesses:

(1) Unvalidated gene and pseudogene annotations: Long-read and consensus genome assemblies are known to suffer from residual indel errors that create artificial frameshifts and premature stop codons. The frequency of predicted pseudogenes in this study (~3.5%-4.5%) aligns closely with expected baseline annotation error rates. Although the authors state in their Methods that mutations were validated using short reads, this validation is never quantitatively demonstrated or shown in the results. The fact that most proposed pseudogenes are actively transcribed and lack a paralog strongly suggests that many are intact, functional genes affected by sequencing or annotation artifacts.

(2) Assembly gaps and GC-bias mistaken for gene loss: The claim that ancestral chickens possessed ~22,000 protein-coding genes and lost thousands of genes in only ~10,000-50,000 years is inconsistent with the evolutionary conservation of avian genomes. The missing genes are enriched for high GC content and preferentially located on microchromosomes. Avian microchromosomes and dot chromosomes are notoriously GC-rich, repeat-dense, and prone to severe assembly gaps in non-telomere-to-telomere assemblies. The reported gene absences reflect assembly fragmentation and coverage dropouts rather than evolutionary deletions.

(3) Lack of synteny validation: Genuine gene absence requires demonstrating conserved collinear synteny of flanking orthologous genes with an unambiguous sequence deletion at the locus. Relying on sequence alignment or short-read mapping failures across fragmented scaffolds substantially inflates false-positive gene loss calls.

(4) Positional bias of pseudogenization mutations: The observed concentration of pseudogenization mutations in the terminal 10% of coding sequences (the "bathtub" distribution) is characteristic of alignment boundary artifacts and non-canonical translation start/stop annotations, rather than positive selection to disrupt gene ends. Mutations in the terminal 3' region often produce functional proteins with slightly altered C-termini rather than complete loss of function.

(5) Inconsistent terminology: The manuscript alternates between identifying pseudogenes as unitary (lacking a functional paralog in the same genome) and evaluating sequence identity against "parental genes," creating substantial confusion regarding whether loci are duplicated paralogs or orthologous reference genes.

Reviewer #3 (Public review):

Summary:

The authors reanalyze genome assemblies of four indigenous chicken breeds from Yunnan Province together with the red jungle fowl reference (GRCg6a), and search for genes with disrupted protein-coding sequences. They catalog candidate pseudogenes and missing genes to estimate that 7,993 of the ancestral genes are dispensable. They characterize the positional distribution of pseudogenization mutations along coding sequences, their fixation in breeds, their estimated ages, and their pathway enrichments. Most results are replicated in four independent PacBio HiFi-based chicken assemblies. From the biased position of pseudogenization mutations toward CDS ends, their frequent fixation, and the phylogenetic signal in gene-loss patterns, the authors conclude that large-scale loss of function is a major driver of chicken evolution and domestication, consistent with the "less-is-more" hypothesis.

Strengths:

The catalog itself is a substantial resource: the comparison spans multiple closely related genomes, the main patterns are checked in a second, independently assembled set of HiFi genomes, and population resequencing data are used to ask whether the pseudogenization mutations are fixed rather than segregating. The finding that candidate loss-of-function genes are under relaxed purifying selection is well supported. The question of what gene loss contributes to domestication is worth asking, and this is a useful dataset for asking it.

Weaknesses:

The finding that genes carrying disruptive mutations are under relaxed selection is not particularly surprising, and the more interesting claim, that the observed patterns reflect positive selection for gene loss, is less certain in my opinion.

(1) Relaxed purifying selection versus positive selection. The bias of pseudogenization mutations toward the two ends of coding sequences is interpreted as positive or artificial selection, along with elevated dN/dS. But the alternative, that disruptive mutations at gene ends are simply better tolerated, is equally consistent with the results. Several mechanisms would produce this pattern under relaxed constraint without positive selection per se: alternative downstream start sites that rescue 5-prime disruptions; the small fraction of protein truncated by 3-prime disruptions; and the enrichment of disordered regions at protein termini.

(2) The functional status of the pseudogenes is assumed, not demonstrated. Genome-scale work cannot be expected to validate individual genes, but the language of the paper should reflect the candidate status of these calls. Nearly all predicted pseudogenes (~95%) were reported as transcribed in multiple tissues. It is possible for a nonfunctional coding sequence to retain intact regulatory sequences, but the observation deserves more attention in the paper, particularly because transcripts carrying premature termination codons can be the targets of nonsense-mediated decay, which is not discussed. These remain candidate pseudogenes defined by the presence of a putatively large-effect mutation (e.g. premature stop or frameshift).

(3) What "missing" means. For a gene to be scored as completely absent, it could be genuinely deleted, or its allele could be diverged enough that annotation and orthology/mapping no longer detect it. These are different phenomena. Related, since most pseudogenization mutations are reported as fixed or nearly fixed in their populations, the history of alleles matters: it is not clear how the authors established which state is derived, and whether the reference sequence assumed to be functional is in fact the "functional" version.

(4) Limited biological insight into domestication. The main biological interpretation rests on hierarchical clustering of dispensable genes followed by ontology enrichment within clusters (Figure 7a), with narrative connections to breed phenotypes. Only 19.6% of dispensable genes have Gene Ontology assignments, and the phenotype links are speculative. The section on the subspecies origin of the GRCg6a reference is only loosely connected to the loss-of-function story, and the population-genetic analysis supporting it is thin as described.

eLife Assessment

This study provides a valuable and comprehensive in vivo map of proteins in proximity to three vesicle-tethering complexes. The evidence supporting the overall proximity mapping is convincing. However, some aspects of the functional characterization of selected candidates remain incomplete.

Reviewer #1 (Public review):

Summary:

The CATCHR complexes are a family of five multisubunit complexes that act in vesicle tethering in several key transport steps. COG and GARP act at the Golgi, EARP acts on early endosomes, EXOCYST acts in transport to the cell surface, and DSL1 acts on the endoplasmic reticulum (ER). The authors have used in vivo proximity biotinylation and mass spectrometry to look for new neighbours of three of these complexes, COG, GARP, and EARP (despite the title, only two of the three are on the Golgi). The authors then follow up two of the hits, CCDC186 and WWOX, with more directed experiments.

Strengths:

The strength of the paper is that proximity biotinylation is of a high standard. To avoid overexpression, the authors express TurboID-tagged CATCHR subunits in cell lines from which the genes have been deleted. This allows them to confirm that the tagged proteins are functional and correctly located. Mass spectrometry is used to identify the proteins biotinylated in each cell line with four replicates, and the data are clearly presented in figures and supplementary tables. The authors make a good choice of proteins to follow up, as both CCDC186 and WWOX appear potentially interesting.

Weaknesses:

Overall, although the paper is based on a high-quality initial set of data, it seems somewhat incomplete and preliminary. There is undoubted value in presenting a through if descriptive set of in vivo proximity labelling data for a comprehensive overview of proteins or complexes. However, in this case the authors have only addressed three of the five CATCHR complexes, and so it is not a complete overview. It was also somewhat unclear why they examined VPS52 and VPS53, as these are present in both GARP and EARP, which adds some ambiguity, even if they did at least provide useful confirmation of some of the hits with EARP. Of course, a proximity biotinylation analysis does not need to cover all members of a family if it generates substantial biological insight.

However, the investigation of CCDC186 and WWOX does not provide significant insight into either function or mechanism. CCDC186 has already been identified in C. elegans as a protein involved in dense core vesicle biogenesis (CCCP-1, as the authors acknowledge), and work in C. elegans and mammalian cells has already linked it to EARP function. Less has been published on WWOX, but all that is found here are some small changes in Golgi appearance and glycosylation when it is reduced by RNAi. Thus, the paper falls between two stools: it is neither a comprehensive application of proximity biotinylation to the CATCHR family, nor is it the application of proximity biotinylation to reveal new insight into membrane traffic. I feel that for a broad-interest journal such, it should be one or the other of these.

Reviewer #2 (Public review):

In this manuscript, Aragon-Ramirez et al. present the first systematic proximity-interaction map of the three Golgi/endosomal CATCHR tethering complexes - COG, GARP, and EARP. They generated hTERT-RPE1 knockout lines rescued with C-terminally TurboID-tagged subunits (COG4, COG6, VPS50, VPS52, VPS53, VPS54) expressed from the COG4 promoter at near-endogenous levels, verified that each construct rescues its KO phenotype, and confirmed expected localization by immunofluorescence. Fifteen-minute biotin pulses followed by streptavidin capture and label-free DIA mass spectrometry, benchmarked against GFP-TurboID, yielded compartment-resolved neighbor lists. The central claim of this work is that each complex sits within a distinct "trafficking module" of coiled-coil tethers (CCTs), SNAREs, SM proteins, Rab GTPases, coats, and homeostasis regulators. COG neighbors nearly all Golgi golgins plus the STX5-SCFD1 fusion machinery, with COG4 and COG6 lobes showing overlapping but non-identical hierarchies - this is offered as in vivo support for a two-lobe model. GARP associates with TGN golgins GOLGA1/GOLGA4, STX16 and partners, TBC1D23, and CLINT1. EARP associates with GRIPAP1, RELCH, RAB11FIP5, and the VPS33B-VIPAS39 (CHEVI) SM complex; a VPS53 MUN-domain mutant loses VPS33B proximity while retaining VPS50 labeling. Two hits are followed up in this study. CCDC186, a poorly characterized coiled-coil protein, localizes to the TGN and, when ectopically anchored to mitochondria, captures ~60 nm vesicles - presented as direct evidence of tethering activity. WWOX, a tumor suppressor with no prior trafficking role, colocalizes with COG8 in the medial Golgi; siRNA knockdown reduces Golgi area, increases HPA and GNL lectin binding (O- and N-glycosylation defects), and displaces COPB2 from the Golgi. Based on these findings, the authors conclude that CATCHRs are not isolated tethers but organizing hubs that assemble compartment-specific tethering-and-fusion modules. Overall, this is a solid piece of work that will be of interest to cell biologists.

(1) The CCDC186 mitochondrial assay demonstrates sufficiency in a non-native context but includes no loss-of-function work, and the cargo-specificity result carrying most of the interpretation is "data not shown." At a minimum, the authors should show KO/KD and rescue data.

(2) The WWOX section rests on a single siRNA with no second oligo, no rescue, knockdown shown only by RT-PCR, and localization based entirely on overexpressed myc-tagged protein.

(3) Proximity of labeling does not distinguish direct from indirect interactions. When referring to "interactions", the authors either need to show recombinant protein binding data, or soften the tone to acknowledge potential indirect interactions.

(4) The VPS53 MUN-domain mutant design rests on a "manuscript in preparation," and the mutant's localization isn't shown (VPS50 labeling establishes complex incorporation, not correct targeting). The HEK293T WWOX replication is "data not shown." The authors should either show the data or delete these claims.

(5) References 73 and 78 are duplicates.

(6) "NZR" is used in the introduction. Should this be NRZ?

Reviewer #3 (Public review):

Summary:

This manuscript presents a comprehensive proximity proteomics analysis of Golgi- and endosome-associated CATCHR complexes, including COG, GARP, and EARP, using near-endogenously expressed TurboID-tagged subunits. The study aims to define how these tethering complexes organize distinct trafficking modules involving coiled-coil tethers, Rab-associated proteins, SNAREs, SM proteins, and other trafficking regulators. In addition to validating known associations, the authors identify CCDC186 as a candidate vesicle tether and WWOX as a potential regulator of Golgi homeostasis and glycosylation. The work provides a broad resource for understanding the spatial organization of CATCHR-associated trafficking networks. However, several conclusions require further experimental and statistical support, particularly those interpreting proximity-labeling data as physical interactions or evidence of discrete functional complexes.

Strengths:

The manuscript addresses an important question in membrane trafficking and provides a systematic comparison of the proximity interactomes of COG, GARP, and EARP complexes. The use of near-endogenously expressed TurboID-tagged subunits is a strength, as it may reduce artifacts associated with protein overexpression. The dataset is comprehensive and has the potential to serve as a valuable resource for investigators studying Golgi and endosomal trafficking. The comparative analysis identifies both known trafficking factors and potentially novel regulators, including CCDC186 and WWOX. The functional follow-up experiments add biological relevance to the proteomic findings and extend the study beyond descriptive mapping. Overall, the scope is well defined, and the manuscript proposes an interesting model in which CATCHR complexes act as organizing hubs for vesicle tethering and fusion.

Weaknesses:

A major limitation is that TurboID proximity labeling is repeatedly interpreted as evidence of physical interaction or assembly into discrete complexes, although the method primarily reports spatial proximity. The soluble GFP-TurboID control may not adequately account for enrichment caused by membrane confinement and high local protein concentration in Golgi/endosomal microdomains. Some spatial enrichment analyses do not reach statistical significance, weakening claims of compartment-specific labeling. Several low-fold-change SNARE hits are emphasized despite limited enrichment. Additional quantitative imaging is needed, including colocalization analysis of streptavidin labeling with Golgi/endosomal markers and localization of GFP-TurboID. The WWOX knockdown phenotype requires validation by rescue, independent siRNA, or CRISPR-based approaches. Several supporting data, quantifications, figure corrections, and nomenclature revisions are also needed.

all the sources deal with some version of loss at sea which points to the title of Death by water. specifically the Dante's excerpt deals with the specific instance of celebrating because they sea the land, but immediately after they get swallowed up by the sea. This idea relates back to the poem as a whole because the people are celebrating that the war is over, but not realizing that they are being swallowed up by the destroyed land and environment.

This line reminds me of lines 23-24: "And the dead tree gives no shelter, the cricket no relief, / And the dry stone no sound of water." An obvious parallel emerges between the cricket and the cicada, and their connection to the absence of water. In the first reference, Eliot responds to the fifth verse of Ecclesiastes 12, transforming the biblical verse to reflect a darker understanding of mortality in the wake of World War I. Similarly, in the later reference, Eliot engages with Psalm 63, which conveys a message of devotion to God, particularly in the context of death. I see these two references as reflecting on one another: the first emphasizes the literal reality that mortality is cyclical and inevitable, while the second prompts us to seek God as we face our departure.

However, Eliot may also be rejecting the message of Psalm 63 with his wish for "the sound of water." Psalm 63 states that the body seeks God "where no water is." Therefore, if Eliot longs for water, it suggests that he does not desire to seek God in the absence of it.

"What the Thunder Said," a barren, corrupt mountainous landscape, seems to be a land of human detritus. Not only is it physically barren, with "sterile thunder without rain" but seems volatile and plagued by human vice and aggression. Line 343 conveys this most explicitly: "There is not even solitude in the mountains/ But red sullen faces sneer and snarl/ From doors of mud-cracked houses." Opening lines 321 to 324 introduce Section V.'s landscape. The line, "After the torchlight red on sweaty faces," implies that the reader is temporally after a creation event and is reminiscent of mortal warfare. Firstly, consistent use of anaphora in the three lines, repeating "After," transforms the world into the product of a state of desolence. The natural world is what's left of a distant past. Secondly, the diction (red torch light and sweaty faces) connote a barbarity and archaism innate to the human. Further bolstering this observation, the line, "There is not even solitude in the mountains" suggests that even in a world of waste, there is intricacy in ‘the human’ to plant to the divine’ relationship.

part 2 (10/5/26)

While textual symbols relating to the hermit-thrush are sparse, and the provided source only describes the bird's behavior, its presence is significant. The Handbook of Birds of Eastern North America highlights the birds insular living habits and ability to modulate tone in voice. It's described to prefer dense shrubbery and forest rather than open space; Eliot's final section, "What the Thunder Said," conversely, is a barren vast plain (which, arguably, contains little to no vegetation). Thus, the hermit-thrush is a moment of antithesis and respite from the sections overall tone and setting; the bird is only mentioned in reference to a land with water. Eliot’s relation to the hermit-thrush, nevertheless, is obscure.

Post 1 (10/5/26)

Stone and rock differ distinctly in “The Rite of the ‘Thunders’ in regards to the "Wasteland" and John 11. A stone is a smaller, manageable piece of rock that has either been weathered or shaped for human use, whereas rocks are large, raw, naturally occurring masses of material.

These two different factors make up the Rites of Thunder, which is typically performed with a thunder-stone, as well as thunderbolts. Thunder in general is the oldest tool for purification. The fact that there is no water and there are no stones, only rocks, connects the "Rites of Thunder" and John 11.

When Jesus brings Lazarus back from the dead, he is doing so while weeping. Thunder-stones, as well as tears, which contain salt, are ultimate forms of purification. There is no water, and there are no stones, only rocks. This is the exact reason why the location is filled with death and suffering, as the absence of those things (stones and water/tears) results in no purification.

Phlebas is dead, and he maintains no control over his corporeal movement. He moves like a jellyfish, wherever the current takes him. The section immediately following him portrays not Phlebas being a victim of the tides, but rather, a man denying the will of the tides. "The sailor, attentive to the chart and to the sheets. A concentrated will against the tempest and the tide..." Phlebas is likely the drowned Phoenician sailor alluded to by Madame Sosostris, even if it is not explicitly stated. Pound, in fact, puts this beyond any doubt. In stating Phlebas is the only part worth keeping, Pound seems to express a desire to showcase a sense of helplessness or lack of control. Phlebas was once as handsome and tall as you, but what is he now? Even those who are strong may die, drown, and lose agency. Sosostris, in telling her fortune, states that one must be so careful these days and to fear death by water. Even those who were once strong may become weak. My mom always tells me not to mess with tides, and that in the battle with nature I will never win. Corinthians refers to the same concept as well, "[22] Shall we provoke the Lord to jealousy? Are we stronger than he?" Nature and the elements are eternal and indefatigable. In a sense, the seas and oceans are treated as sentient and all powerful objects in and of themselves. They moved Phlebas, and in De Quincey, "The sea was rocking, and shaken with gathering wrath." The sea is increasingly personified as a vengeful and powerful being that we struggle to defy. Pound, in shortening the original, emphasizes this fact.

Eliot’s description of Phlebas’s death, “a current under sea”(IV, 315), “picked his bones in whispers”(IV, 316), presents water as a physical force that gradually destroys the human body. Caroline Block pointed out on October 18th 2024 that this use of water contrasts with 1 Corinthians 10, where the Israelites are “baptized into Moses in the cloud and in the sea.” The verb “picked” is especially significant because it makes the current seem almost like an animal or scavenger feeding on Phlebas. Rather than presenting water as peaceful or cleansing, Eliot emphasizes its ability to take apart what remains of a human being. The wording “in whispers” also makes the destruction strangely quiet and ominous . There is no dramatic description of Phlebas’s death, the water silently erodes his body, making death seem inevitable and natural.

Caroline’s comparison with 1 Corinthians 10 highlights the different functions of water in the two works. In Corinthians, the sea is not primarily important because of what it physically does to the body. It is the passing through the sea that becomes a spiritual transformation, connecting the Israelites to Moses and to God. Water represents religious belonging and salvation rather than physical destruction. Although the Israelites are still punished by the wilderness. So, water doesn’t guarantee salvation, but it is a step in that direction.

Shakespeare’s “The Tempest” offers another way of understanding water as a force of renewal. At first, the sea seems destructive because Prospero’s storm separates the characters and creates the appearance of death, but Ariel’s song transforms this destruction into something “rich and strange.” The drowned father becomes part of the sea, suggesting that death does not necessarily mean an absolute end, but can become the beginning of something new and beautiful. This connects to Eliot’s question, “Has it begun to sprout? Will it bloom this year?” because the image of something dead beginning to grow suggests that water can exist between death and resurrection.

Caroline Block’s comparison shows that, while Eliot presents water as something that can erase and destroy the body, the Corinthians (and now with my addition of The Tempest) suggests that water can also transform death into renewal and help people, be it before or after death.

So, it is interesting, what’s the role of water? Eliot mentions water a lot. Perhaps when people took care of water it helped, but when they started polluting it, it became a symbol of death?

eLife Assessment

This important study investigates how GDNF treatment restores nerve cells in the bowel of a mouse model of Hirschsprung disease, and proposes that this regeneration occurs through diverse resident precursor cells and a non-canonical signaling pathway. The evidence is solid for the ability of the treatment to induce new bowel nerve cells and for a contribution from multiple precursor populations, supported by extensive cell tracing and imaging experiments. This work will be of interest to researchers in developmental biology and peripheral neurobiology.

Reviewer #1 (Public review):

Gary, Soret et al., present an interesting and ambitious study investigating the cellular and signaling mechanisms by which GDNF induces enteric neurogenesis in a disease model of HSCR. The manuscript combines scRNA-Seq, pharmacological inhibition, and several lineage-tracing strategies. The lineage tracing experiments in particular are technically impressive and clearly represent a substantial amount of work. The figures are generally beautiful and high quality, and the biological question is an important one.

However, while I found the study interesting, I also think that several of the major conclusions are substantially stronger than what the current data support. In particular, the scRNA-Seq dataset that the study is founded on is difficult to interpret because of the RFP-based enrichment strategy, the lack of WT reference controls, and the very non-stringent FACS gating. Several mechanistic claims are also based primarily on expression patterns or sequencing results.

Major concerns:

(1) The G4-RFP based enrichment strategy raises some concerns. The authors note that 88-90% of SOX10+ glial cells (where did this number come from, it doesn't seem to match the range in the bar graph?) and only 70% of GDNF-induced neurons. They then sort in "yield" mode using a non-stringent gating strategy, but do not provide the flow graphs in the supplements for interpretation. Consistent with this, the initial dataset contains multiple non-ENS populations, including lymphoid, myeloid cells etc, suggesting that their strategy, in addition to missing cell populations, additionally includes potential negative cells based on background fluorescence.

(2) It's not clear to me why the authors did not include a wild-type control for comparison. Due to the RFP-enrichment strategy, it is additionally difficult to compare to integrated with scRNA-seq datasets. The comparison seems important because the central question is not only whether GDNF induces neurons in the mutant colon, but whether the induced cells and associated populations resemble those found in a normal ENS.

(3) The proportional changes in Figure 2 are hard to interpret. The authors write that GDNF treatment leads to an enrichment in enteric neurons, "at the expense of" SCPs. This seems to be a strange conclusion or writing, unless the authors are suggesting that certain cell populations are additionally depleted by GDNF treatment. I think the authors should provide stronger support, or dial down on conclusions based on cluster proportions, given the sorting strategy.

(4) Some of the mechanistic conclusions are far too strong for the evidence. For instance, the claim that GDNF signals through GFRa1/2 based on only expression UMAPs is not justified. The authors should at least confirm some of the major findings via smFISH (HCR or RNA-Scope), and dial down the mechanistic claims. The pharmacological data is more supportive but still indirect. This section would be stronger if the authors validated NCAM1/FAK activation in the specific progenitor populations proposed to respond to GDNF.

(5) The inhibitors are administered during the same P4-8 window as GDNF. Given that the authors report cellular-level responses as early as 6 hours post GDNF treatment, it is important to know whether the inhibitors were already active at the time of GDNF administration. Why were the inhibitors not administered prior to the start time of GDNF treatment?

(6) The authors draw inferences and conclusions based on IHC images, but do not provide any details on the analysis procedures. For example, in Figure 3, the authors interpret changes in NCAM and FAK expression level. However, it does not appear that there are notable differences in intensity between the two timepoints. There are however, differences in signal coverage. Without knowing what was actually measured and quantified, it is not possible to interpret the results. Please provide the necessary information for all experiments in the methods section.

(7) The final results section introduces one of the more surprising claims in the paper, that a non-neural crest-derived progenitor contributes to regeneration, but the section ends quite quickly and abruptly after introducing the observation. The authors should provide additional validation or interpretations of the data.

Reviewer #2 (Public review):

Summary:

Previous work from this group demonstrated regeneration of the ENS following exogenous GDNF treatment within the aganglionic portion of the bowel in several Hirschsprung (HSCR) murine models as well as colon from HSCR patients (Soret, et. al., Gastro. 2020). Focusing on the Holstein (HolTg/Tg) HSCR mouse model, the authors build upon their prior work utilizing lineage tracing, immunohistochemistry, scRNA sequencing, and pharmacological methods to further delineate the mechanisms and cell types contributing to ENS regeneration in this context. This work adds to the knowledge base regarding natural ENS development as well as ENS generation outside of natural ENS development. Furthermore, this work provides important clinical implications for potential curatives treatments for HSCR disease.

The conclusions in the paper are overall well supported by the data with the majority of technical and model limitations openly acknowledged. Strengths include the use of scRNA sequencing, several genetic mouse models with ample, well-planned time points within experiments. However, additional analysis, clarity or more detail around experimental protocols, and broader discussion of prior studies would strengthen the manuscript by improving interpretation of the findings and placing them more clearly within the context of existing work in the field.

First, utilizing the G4-RFP transgene for ENS cell selection and subsequent scRNA sequencing of RFP+ cells, the authors nicely demonstrate the presence of known cell clusters (based on markers and analysis from prior sequencing studies) including Schwann cell precursors (SCPs), enteric glial cells (EGCs) and neuron subtypes in the aganglionic colon of HolTg/Tg GDNF-treated and untreated mice. Further use of this dataset led to the discovery of Ncam1 expression, a GDNF receptor, within all ENS cell clusters, suggesting importance of this protein in the GDNF-induced ENS regeneration phenotype. Interestingly, Ret was limited to some neuron types. On the protein level, temporal and GDNF-treatment changes in NCAM1 expression and downstream phospho-FAK(Y397) were nicely demonstrated via Western blot. Mechanistically, the importance of NCAM1 in ENS regeneration was demonstrated given pharmacologic blockade of downstream FAK signaling via PF-562271 resulted in significantly fewer GDNF-induced neurons. The paper would be strengthened here, however, if the following areas were addressed:

1) Although the GDNF-induced regeneration appears likely through an NCAM1 signaling mechanism, inhibition of FAK phosphorylation by PF-562271 could affect more than NCAM1 signaling. Blocking NCAM signaling through another method (such as through NCAM1 targeted monoclonal antibodies or small molecules) would support the authors' interpretations further.

2) A point of ambiguity is the exact cells included in the final scRNAseq analysis. The authors noted they flowed and gated broadly, including RFP negative cells, in the initial set of cells selected for sequencing (given RFP expression is not visualized within all neurons and glia in GDNF-treated HolTg/Tg mice.) Pan-neuronal (Elavl4, Tubb3) and pan-glial (Sox10) marker expression were utilized to select cells for final analysis (unsupervised analysis, pseudotime trajectories, etc.) Here, it is unclear if RFP expression was examined in these cells, either by looking at unmapped reads or if included in the initial alignment. If this is discernable within the authors' data set, this provides an opportunity for an interesting additional analysis to determine if progenitors or neuronal or glia populations differ in the RFP+ and RFP- groups.

3) The authors presumably sequenced both the submucosal and myenteric plexus ENS components within their scRNAseq analysis. Given the relatively sparse numbers of ENS cells in the submucosal plexus compared to myenteric plexus in the distal colon, this is unlikely to change their analysis or conclusions. However, given their tissue analysis focuses on the myenteric plexus, this difference should be acknowledged.

The authors go on to further to utilize their scRNA sequencing data set with pseudotime trajectory analysis to determine that SCPs appear to go through an EGC-like state prior to GDNF-induced neurogenesis. This was followed nicely by use of transgenic mice (Dhh-Cre) and immunohistochemistry within their HolTg/Tg model to capture the SCP population and demonstrate in vivo this progression. Additionally, they were able to demonstrate that temporally, SCPs generated neurons much sooner (within 6 hours of GDNF-treatment) compared to neural crest derived EGCs (labeled by Slc18a2-Cre and GFAP-CreERT2 lines).

Fascinating here as well is that the authors unearthed that most generated neurons following GDNF-treatment appeared to derive from direct cell transdifferentiation (suggested by EdU incorporation investigation at various time points) and that these neurons did not seem to originate from SCPs or EGCs (i.e. a non-neural crest origin.) Acknowledging that their mouse genetic tools and sequencing could have missed a NCC-derived population, the authors go on to label neural crest derivates utilizing the Wnt1-Cre2 transgene in their wildtype and HolTg/Tg mouse model and find that while Wnt1 labels the vast majority of neurons in wildtype mice, nearly 25% of neurons in P20 HolTg/Tg mice appear non-NCC derived. Finally, once again utilizing various genetic mouse lines for lineage tracing and immunohistochemistry, the authors demonstrate NCC-derived neural progenitors (SCPs, EGCs) appear to preferentially give rise to cholinergic neurons where non-NCC derived neurons tend to be more nitrergic.

4) The high magnification of the majority of images provides the reader with extremely compelling evidence in regard to visible overlap or lack thereof across various reporter lines, antibody markers, and EdU labeling. Unfortunately, this can somewhat create a trade off with the area or number of cells examined which appears somewhat less than the number of cells and/or area examined by many prior studies in the field. The near equal averages across the three mice in each group in many of their experiments makes this less of a concern, but the paper may be strengthened by noting exactly how the 3-11 images per mouse were selected. Were these randomly selected across the tissue, moving from mesenteric to anti-mesenteric? Or caudally to distally?

5) In line with this, the authors report a near 100% of Wnt1-Cre2 driven reporter expression in wildtype mice but a large portion (~25%) of neurons in HolTg/Tg model appear non-NCC derived (i.e. not Wnt1-Cre2 labeled.) The original Wnt1Cre line (Danielian, et.al., Curr Biol. 1998) has been reported to incompletely label all neural crest derivatives/ENS (Hari, et. al., Development. 2012.; Deal, et. al., Dev Bio. 2021). To my knowledge, the same type of analysis has not been carried out with the same rigor in the ENS with the Wnt1-Cre2 line. It could be -- depending on tissue sampling, area covered via imaging, and if there is any patchy or unique patterning to non-NCC derived neuro regeneration -- any incomplete labeling of neural crest by Wnt1-Cre2 could be missed. Of course, alternative interpretations are that the non-labeled cells are truly non-NCC derived. Utilizing other lineage tracing models for other non-NCC derived lineages may help address this issue, but would require many mouse crosses and additional experiments, and thus likely outside the scope of this manuscript. However, this caveat should be addressed.

6) A very important and fascinating find by the authors is that the NCC versus non-NCC derivatives appear to preferentially give rise to cholinergic or nitrergic neurons respectively. This will have important implications in treatment options for ENS regeneration not only within HSCR disease, but other ENS disorders as well. A point of discussion the authors missed out on is several previous reports regarding skewing of ENS neurons (typically toward higher nitrergic numbers) in ganglionic and/or hypoganglionic segments of HSCR bowel in murine models and patients (Zaitoun, et.al., Neurogastroenterol. Motil. 2013.; Musser, et.al, CMGH. 2015.; Cheng, et.al., J Pediatr Surg, 2017.; Sukhada, et. al., Front. Cell Dev. Biol. 2022.) Could ganglionic portions of the bowel in HSCR disease contain more of the non-Wnt1 labeled population? Why do proportions in the aganglionic region generate correct proportions following GDNF-induced treatment compared to ganglionic bowel? This does not need to be experimentally addressed in the manuscript, but including in the discussion impresses upon the audience the importance of the authors findings and that these factors need to be further experimentally delineated and considered with any therapeutic interventions.

Reviewer #3 (Public review):

Summary:

In this work, the authors aim to build on their previous work to further define the mechanism by which GDNF can induce enteric nervous system migration and differentiation in the Holstein mouse model (Hol Tg/Tg) of Hirschsprung's disease (HSCR). They show that GDNF can cause neuronal differentiation from either Schwann cell precursors (SCP) that migrate from the periphery and/or enteric glia (or neural crest precursors located in the intestine). Surprisingly, the rescue effect of GDNF is mediated through signaling with NCAM1 rather than the canonical receptor RET, with blockade of NCAM1 significantly reducing the number of GDNF-treatment induced neurons in the distal colon of Holstein mice. The authors then perform scRNA-seq data of the colonic ENS from Holstein mice +/- GDNF, and pseudotime analysis of this data indicates the SCP downregulate SCP genes and upregulate classical enteric glial genes on their transition to becoming enteric neurons. Following this, the authors employ Dhh-Cre mice (to label SCP), Slc18a2Cre and GFAPCreERT2 (to label enteric glia) and finally Wnt1Cre2 mice (to label neural crest derived cells) to assess the kinetics and end identity of enteric neurons driven by GDNF treatment. Using these animal models, the authors propose a model by which transdifferentiation of SCP gives rise to enteric neurons first, followed by transdifferentiation of enteric glia; the resulting enteric neurons labelled by Dhh, Slc18a2 or GFAP Cres are all biased to a cholinergic lineage, raising a questions about the source of nitrergic neurons. Finally, the authors postulate that at least some nitrergic neurons come from a non-neural crest lineage in GDNF treated Hol Tg/Tg mice as they are not labelled by a Wnt1Cre2 model, while all ENS cells are labelled in three wildtype Wnt1Cre2Tg/+;R26YFP/+ mice.

Strengths and Weaknesses:

This work convincingly shows that NCAM1 is required for GDNF-induced rescue of the Holstein mouse model of HSCR and, along with the NCAM1 staining in human HSCR tissue, highlights the potential of this treatment even in HSCR patients with loss of function RET mutations. The major weakness of this study is in the interpretation of the mouse models; many of these models are not fully penetrant/show mosaicism in other studies (like the DhhCre and Wnt1Cre) or may not only label the specific subpopulation they present (in this case, the GfapCreERT2 and Slc18a2Cre; these genes have been shown in Schwann cells/SCP in other studies, and the expression pattern of these two genes outside of the gut at the relevant developmental timepoints are not shown). While these are unavoidable limitations with our current mouse models, the authors need to clearly acknowledge these limitations in the interpretation of their data (with the DhhCre and Wnt1Cre) or more definitively show the models are only labelling enteric glia (in the case of the Gfap and Slc18a2Cre). In addition, in some figures the authors should add untreated Hol Tg/Tg and ideally WT mice so readers can contextualise the effect of GDNF on Hol Tg/Tg mice. Finally, it appears the statistics throughout the paper are performed on using each ROI taken from n=3-5 animals - this is inappropriate, as images from the same animal are not independent samples. The statistical unit in question for every analysis performed should be 1 animal, thus the statistics should be done on the n=3-5 animals, not the n=3-11 images from 3-5 animals (which leads to an n of 6-55).

Specific comments:

In general I find the author's conclusions are relatively well supported, albeit a little overstated for the evidence presented in the later figures and with the need to correct the statistical analysis. For Figures 1-5, I have only minor comments, as follows.

In Figure 1-2, the Gata4-RFP reporter is used to sort cells for scRNA-seq, and the authors get a large number of non-ENS cells so utilise Elavl4 and Sox10 expression to pull out neurons and glia to analyse. Do other putative neuronal/glial genes pull out the same cells, and can the authors find evidence of non-neural crest derived glia/neurons in this dataset? This would ensure there isn't a bias in this first analysis and strengthen the later claims respectively.

In Figure 3, the interpretation of increased NCAM1 and pFAK would be aided by adding the MFI for the untreated Hol Tg/Tg and ideally WT mice at p10 to assess if this is a normal developmental increase or induced by GDNF treatment - I would ask the authors add at least untreated Hol Tg/Tg at p10 as authors already present this data as images in figure 3D. Similarly, it would be nice to have neuronal counts of untreated Hol Tg/Tg and WT mice presented in Figure 4c to assess if NCAM1 inhibition completely or only partially prevents the effect of GDNF.

In the text accompanying Figure 2 and 5, the authors use a variety of markers of assign SCP or enteric glia identity to the clusters, but some of the enteric glia genes can be expressed by Schwann cells/SCP in other contexts - it is worth adding references to the genes chosen to define SCP and enteric glia so readers can understand why those were chosen and the strength of evidence for each marker. For me, with the exception of Dhh I do not find any of these genes definitive as, at least on the SCP/Schwann cell side, they have been shown to express Gfap, Slc18a2, Sox2 and single cell analysis indicates they also express Cpe. The ability for SCP/Schwann cells to express both Gfap and Slc18a2 is important for the interpretation of the upcoming experiments.

In Figure 6, the authors state in line 213 that "quantitative analysis as a function of time revealed that GFAP+ cells in the distal colon are not all derived from Dhh-expressing SCPs on the first day of treatment"; however, the DhhCre is restricted to specific SCP subsets (Xie, Meng, et al. "Schwann cell precursors contribute to skeletal formation during embryonic development in mice and zebrafish." Proceedings of the National Academy of Sciences 116.30 (2019): 15068-15073) and thus may not be labelling all SCP coming into the intestine. As far as I know there are no models that would allow labelling of all SCP without labelling enteric glia so would require the generation of a new models or techniques that is out of scope for this work - however, the authors should acknowledge the possibility of Dhh negative SCP contributing to the ENS.

Figure 7 and 8 focuses on the contribution of Dhh+ cells, Slc18a2+ cells and cells expressing GFAP between p3-p8 to the ENS of GDNF treated Hol Tg/Tg mice. The authors first show that by p20 the number of neurons per mm2 is equivalent to those at p60, indicating p20 as an appropriate timepoint to assess the relative contribution of each pool of glia to the resulting neurons. The authors show that neurons derived from a DhhCre are detectable first, then Slc18a2, followed finally by GFAP. This data supports the authors claims that neurons first arise from Dhh+ SCP, but as I raised earlier, both Slc18a2 and GFAP can be expressed by schwann cells later in development - the authors in fact show that GFAP is in fact expressed by Dhh+ cells in figure 6, and showed with pseudotime analysis that SCP undergo a transition to Gfap+ Slc18a2+ glial cells before becoming neurons. The data presented here negates that analysis, or at least suggests some Dhh+ cells skip this step, otherwise the kinetics of neuronal differentiation would align across the 3 lines used. Could the authors address this discrepancy? For example, one addition could be to show that SCP/schwann cells on extrinsic gut innervating nerve fibres do not express Gfap or Slc18a2 at the timepoints used. In addition, their scRNA-seq analysis suggests that Gfap & Slc18a2 are expressed in the same cells, so one would expect similar kinetics in both populations - what is the overlap of GFAP staining with Slc18a2-YFP expression at the earlier stages (say p6 and p10?). Alternatively, the inducible nature of the GFAPCreERT2 means some bona fide GFAP expressing cells may be missed if the tamoxifen dose is not saturating, thus underestimating the contribution of this population to the total neuron count. As such, could the authors please show co-staining with GFAP in some induced GFAPCreERT2 samples to assess the effectiveness of this Cre. As with other figures, it would also be nice to add Holstein negative reporters at p20 as a control to assess the level of contribution each one of these cell lineages has in a normally developing animal compared to GDNF treated Hol Tg/Tg mice.

In Figure 9, the authors show that neurons derived from labelled cells in the DhhCre-YFP or Slc18a2Cre-YFP skew strongly cholinergic (81%) compared to nitrergic (13%), but only 52% of the total neurons in GDNF treated Hol Tg/Tg mice are cholinergic, while 42% are NOS1+. The authors say that a similar trend is seen in Slc18a2Cre-YFP mice with a 'slightly higher percentage' reflecting Slc18a2 expression in neurons. However, this 'slightly higher' percentage looks to be about 40%, which is the amount expected in GDNF treated Hol Tg/Tg. I think the authors underplay the level of nitrergic neurons that are labelled in the Slc18a2Cre mice and should consider the possibility that Slc18a2+ glia can give rise to the nitrergic neurons seen in GDNF treated Holstein mice. This data also contrasts with prior reports that indicate SCP tend to become NOS1 expressing in a Ret or Ednrb loss of function HSCR model (Uesaka, Toshihiro, et al. "Enhanced enteric neurogenesis by Schwann cell precursors in mouse models of Hirschsprung disease." Glia 69.11 (2021): 2575-2590), a discrepancy worth discussing.

In Figure 10, the authors assess the contribution of neural crest to the ENS of WT and Hol Tg/Tg mice using a Wnt1Cre2 line, and conclude there is non-neural crest contribution to the ENS of GDNF treated Hol Tg/Tg mice. This is an interesting idea and supported by a few studies the authors mention in the discussion; however, the labelling of neural crest derived cells in Wnt1Cre2 may not be fully penetrant. While this line has a well established leaky phenotype, others have reported it can have patchy recombination in the neural crest too (potentially in a background dependent manner, according to the line information on Jackson laboratories). For example, recent works shows significant mosaicism in the embryonic phase (Gandhi, S., Du, E. J., Pangilinan, E. S., & Harland, R. M. (2024). The Wnt1-Cre2 transgene causes aberrant recombination in non-neural crest cell types. bioRxiv, 2024-11.) This may not be the case in the authors home institution in a FVB background, as they show all HuCD+ and Sox+ cells overlap with the Wnt1Cre2 reporter, but the Hol Tg/Tg mice have aberrant neural crest development - this could interfere Wnt1 expression thus the effectiveness of the Wnt1Cre2, thus leading to non-labelled, but actually neural crest derived cells. It is also possible that those neurons are truly from a non-neural crest background, and the neural crest labelling tools are all flawed in one way or another - as such, I think the authors just need to discuss this alternative possibility in the discussion.

Finally, Figure 11 shows NCAM1 expression in human tissue resections from HSCR patients. This data bolsters the idea that GDNF treatment could be an effective treatment for HSCR. The methods indicate that 3 tissue samples from HSCR patients were obtained, but staining is only shown for one - could all 3 be included, along with secondary only controls? This is important as human tissue resections are often processed many hours after removal, so are prone to higher background. It would also be interesting to include tissue from non-HSCR patients (the controls used most often in other studies are resections from anorectal malformations which are performed at approximately the same age as HSCR resections) to see if NCAM1 expression is influenced by the absence of an ENS in human patients, but I understand these samples are not always possible to obtain. If not possible, the authors could consider analysing publicly available RNA sequencing datasets to assess if RET is solely expressed in mature neuronal populations while NCAM1 is expressed by glial and neuronal populations like in their mice data.

Overall, the authors clearly show that GDNF can drive differentiation of neurons from different glial populations through NCAM1 not RET, and supports continued effort to translate this work to the clinic as a potential therapy for HSCR patients. I am not convinced that the relative contributions of each glial subpopulation can be extrapolated from the mouse models used - this is a problem across the field, and will only be rectified with the development of new models, which is out of scope for this work. This work complements recent work showing NCAM1-GDNF signaling promotes neurogenesis in glia in Ednrb-/- mice (Mueller, Jessica L., et al. "Intramuscular enteric glia persist in Hirschsprung disease and undergo neurogenesis in response to GDNF-NCAM1 signaling." Scientific Reports 15.1 (2025): 33200), and will add to our understanding of neural development.

Reviewer #4 (Public review):

Summary: The manuscript by Gary et al works to reveal processes that generate enteric neurons in the distal colon of Holstein mutant mice that experience aganglionosis and are an established model of Hirschsprung disease (HSCR) following GDNF enema infusion. The primary goal of the study is to determine the how rectal infusion (enema) of GDNF drives neurogenesis in these mutants. The studies focus on determining the molecules that mediate this GDNF effect and identifying the cellular origins of the newly formed neurons. Given that prior work from this group and others has indicated formation of postnatal enteric neurons is feasible, understanding this process is an important step for the field. The data presented partially support the claims of the study. The analysis described do not support unequivocable conclusion that "transdifferentiation" is occurring. To instill confidence in the stated conclusions and make the text appropriately clear, clarifications of the methods and additional information is needed.

Strengths:

(1) The authors use in vivo experimentation with multiple mouse lines including Dhh-cre, Slc18A2-cre, and GFAP-creERT2 to trace the production of enteric neurons in the context of GDNF enema in the established Holstein mouse model of Hirschsprung disease over a time line of postnatal days 4 (P4) to P10.

(2) The authors generate novel molecular profiles of enteric neurons induced in the distal colons of Holstein HSCR mice after GDNF enema induction by flow sorting for fluorescently labeled and adjacent populations with novel gating parameters followed by single cell RNA-sequencing.

(3) The authors recognize broad expression of NCAM1 amongst the induced neurons in scRNA-seq data and apply experimental approaches to further investigate the role NCAM1 in generation of GDNF-induced ENS neurons.

(4) The authors apply a widely utilized experimental approach, EdU-labeling, to assess proliferation in the immediate timeframe of GDNF-inducation in an effort to discern the timing of when the newly produced neurons exit the cell cycle.

Weaknesses:

(1) The article lacks sufficient information justifying use of the Holstein HSCR mouse model for the study. Authors need be more thorough when introducing the readers to the various HSCR mouse models they consider analyzing and provide an expanded justification for use of the Holstein model in the introduction so the audience can appreciate the rationale behind the analysis presented.

(2) The text minimally describes the G4-RFP reporter that is a critical element of the analysis and upon which the scRNA-seq profiling hinges. Given the data presented in this submission it's not clear how much of the ENS this G4-RFP line labels.

(3) The authors state that "NCAM1 is already present in SOX10+ cells before GDNF treatment begins at P4, both within and outside extrinsic nerve fibers (Fig.3b,c)." However, the data shown in Figure 3 panels B and D do not allow one to conclude co-localization of Sox10 with NCAM1 or FAK. Because ENS cells are so closely positioned with one another the signal the authors present could be due to adjacent cells or processes of cells above and below the plane of Sox10+ nuclei.

(4) The authors use the inhibitor PF-562271 in an attempt to specifically inhibit phospho-FAK[Y397] shown in Figure 4. This particular inhibitor has the known side effect of causing apoptosis in phospho-FAK[Y397]+ cells as shown by Hu et al 2017 Cancer Sci (DOI: 10.1111/cas.13256). Because this compound causes apoptosis in phospho-FAK[Y397]+ cells, the data presented do not prove that the lack of neurons produced in this condition is due to inhibition of phospho-FAK[Y397]+ cells making the neurons versus those phospho-FAK[Y397]+ cells simply dying very early in the process.

(5) In the text describing the results of Figure 1A compared to Figure 1B the authors have missed an opportunity to elaborate on the spatial distribution of what appear to be small ganglia in the GDNF treated colon of Holstein mutant mice. The schematic shown is rather simple and it's unclear from the text where these ganglia are distributed circumferentially around the gut wall.

(6) The immunohistochemical labeling for Phox2b shown in Figure 6 is odd. Phox2b is expressed in neuronal progenitors, enteric glia, and ALL enteric neurons as shown in multiple publications. The images shown in Figure 6 offer an outlined region that appears to be a ganglion; however, within that encircled area fewer than half of the cells are labeling with Phox2b by this study.

(7) The authors conclude that "transdifferentiation" is the origin of the ENS neurons that appear in GDNF-treated Holstein mice. However, the data show simply that most of the newly produced neurons have not recently gone through cell division based on lack of EdU incorporation. Given the data presented, other mechanisms may be occurring and should be considered as possibilities.

(8) The authors utilized a Wnt1-cre2 transgenic line that has known issues with expression in the male germline and ectopic expression in cells that are dependent upon the reporter line utilized, like the Rosa26-YFP of this study. The methods lack information on whether crosses were performed in such a manner as to avoid issues with male germline activation of this reporter and the potential for ectopic expression cannot be excluded based on the information provided in the study.

(9) The study lacks data on how GDNF enemas affect enteric neuron density and ganglia distribution in wildtype animals. If the signaling mechanism that produces new neurons in the Holstein model is also operating in wildtype animals, this could be crucial information for investigators interested in neuronal replacement to treat ENS damage resulting from environmental damage, disease, or age.

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We as readers may disregard these things, but truth be told they are very important.

on the desktop version there s the secret deval and then it shws app pages 0 of 12 done, why not put that in the title or under or on the door rotating liek the stocakmarket screen of pricing, instead of a sticker outside etc etc

also check now and connect claude? wher is settings? or what does find do? anything in repo, or docs, or somethign else that claude grabs?

why the menu at the bottom, if the mneu on the right isde with peopl epills or start new chat, why coworkers at the bottom, wjats the difference between the notification and top menu and option B and and questions what does more do I guess I have a lot of questions unlike if I have a more things we haven't decided on? feel like this is ujust high level design right, not like per page? or office? do we need to do that ? finish each room? or?

cause im looking at things and im not sure what diff betwen desk, wh is next questions, notifications is?

this

this one, but instead of day and night, option its just a time of the day. so we can have it real time non stop going or just choose a time timght you prefer :)

how aabou a thin rail of cases, that then docked option b style

this but tell us what to do is missing in this version and shoul dnot be in details., so maybe column a is better? But if it's not a vision or I'm looking at question or something like that maybe there's no

oh these looks like tickets.... i donT' know what on top,

this looks mor elike the board?

THIS SEEM TO BE THE EASIEST ONE TO READ. Lts jit make sure it is actually working correctly and like fot is n't too small or thin?

too close to claude orange, how about yellow yellow? or make claude orange a bit more brown or dark?

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There are three parts to reading. Experience, interpretation, and evalutaion.

would we be in a more socialized medicine state had it not been for World War 1? If this bill was enacted, would it have changed health care policy today?

This statement dates this article for me. I know that it was written later because Trump has dismatled this system.

Why is end stage renal care so specifically included on this list? is it not a long-term disability? why is it different?

Irony is used to make a point and is left up to the readers interpretation.

Multiple genres. Each genre's theme is different. And this defines a novel.

Reasons why we read fiction.

China's obesity rate was about 6.89% of its population.‌ The U.S. obesity rate was about 27.4%.‌

This means a much higher percentage of Americans are obese, but because China's total population is so much larger, even a small percentage translates into a massive absolute number.

I thought the “equal sewer” problem was interesting because it shows that creating equal funding for education also raises larger questions about fairness in other public services too. I think it's important to recognize that equal funding doesn't mean equal outcomes, especially when some communities have greater needs than others. this brings up the question whether school funding should focus more on giving every student the same amount of money or giving students the resources they need to have equal opportunities.

I think this is interesting because you would think the funding system is designed to reduce differences between wealthy and poorer school districts, but in reality, it doesn't create equal funding. If the foundation amount is set too low, wealthier districts can still spend more because they have higher property values and can raise more money through local taxes. This makes me wonder then how much a student’s access to educational resources should depend on where they live.

When our only focus became just about money and instead of integration, we forgot why schools exist in the first place. If schools just ignore citizenship and empathy, it becomes a place where it only care about test scores and money. I agree with Justice Marshall, because we cannot build an equal, united society unless kids from different backgrounds actually go to school together. While sending money to struggling schools is necessary, it’s also important to teach students from different backgrounds how to coexist.

It’s just not right that the government usually focuses on small details rather than fixing the real systemic issues that really hurt schools. When rich suburban keep all their tax revenue, it traps schools in a cycle where wealthy kids get great resources, and everyone else gets left behind. This shows us that unfair school funding and property taxes segregate people just as powerfully. In the end, we can't achieve real fairness until we agree that a child’s wealth shouldn't decide how good an education they get, they should give every child from any background a chance.

Here, I think about all those years that the elderly spent paying into social security. Is this statement making an assumption that those would be okay with all the sudden having to use an HSA? Would they get money automatically added to it? I also think that this statement has a notion that people recklessly spend on health care.

I disagree with this claim. I think that society as a whole cannot function if there is no health care for all. I think about Maslow's hierarchy of needs. That bottom necessary layer includes a lot of things that health care provides us.

The author is taking for granted high deductible plans. Some plans now have deductibles that are $10k. How are low income and middle income families supposed to pay for this?

Is socialized medicine the answer? Would we have better satisfaction if we were in socialized medicine? Do people have favorable thoughts about socialized medicine? I am curious if this is a question that we can really answer in this article.

It’s interesting knowing that scholars want their work to become obsolete, not famous. I feel that the difference between Ferguson and Irvine shows that the word "suburb" have different daily realities. While we connect places like McKinney, Staten Island, and Ferguson proves that suburbs struggle with racism just as much as other big cities. This is important to know this because it tells us that suburbs are not safe from racism, forcing us to face the deep inequalities hidden right in our own neighborhoods.

How does a computational approach work in terms of the dual vision system theory?

While the GPS program primarily focuses on thought processes, I wonder how it would hold up when focusing specifically on memory.

When it comes to the GPS program and comparing the human mind to computers, there will almost always be a gap between processing times and margin of error that comes with being human. People can't always be as efficient as computer programs.

Even with different things being studied, both seem to be trying to show that The brain may go through a set of steps to get to an answer and the process is kind of computer like

When I was first doing the reading, I was thinking this the GPS can only really problem solve like a human to very structured problems but more complex problems such as things that involve social situations or being creative or having to create a decision that doesn’t have clear steps to a correct answer like using your morals or emotions. GPS wouldn’t be able to answer like a person would.

I was a little confused when I was first reading this part, but this helped me understand the comparison between human mind and computer a little more. I think it’s saying just like words or numbers can only be combined in specific ways according to rules the theory is suggesting our brain also processes and manipulates symbols that we know of according to rules that we have kind of like a computer

This was interesting to me because GPS was meant to replicate the stages humans use when they problem solve not just producing the same final answer. It made me think a lot recent AI how rapidly it has progressed in the past few years and also whether producing human like results would mean a computer is thinking the same way humans do. I don’t personally think it does because human thinking is a lot more complex and involves things beyond just problem solving and pattern recognition.

I didn’t know this about the GPS and I find it very interesting. It reminded me of the idea from the lecture last week that computer equals mind because the GPS was trying to copy not just human answers but to process things and solve things the way that humans do

This shows that school segregation and inequality affects people differently, even within the same racial group. High income families had the leisure to move to certain neighborhoods or send their children to private schools, while lower income families had fewer choices. Access to education is not only connected to race, but also socioeconomic status and the resources a family has. This also connects to how giving families more choices in education doesn't create equal opportunities if some families can afford more choices than others, which is a common theme we've been visiting.

This is a good example of how stereotypes can affect students even when they don't actually know anything about each other’s backgrounds. Where the student lived immediately shaped how the other student viewed her. This highlights how placing students from different racial or socioeconomic backgrounds in the same school does not automatically mean prejudice will be eliminated. Students and teachers have to be willing to recognize and change the assumptions they make about others.

This is actually a debate we have had in IT, many times, as we are implementing systems changes to ensure we comply with the Digital Accessibility requirements by the DOJ (https://www.ada.gov/resources/2024-03-08-web-rule/). Finding the 'right' balance between the extra work/burden and the rights of the disabled, is more difficult than it may seem, especially when there is limited time, money and other resources involved - so perhaps the use of the American Indigenous Ethics framework would balance better.

Publication update: peer-reviewed version now available

This preprint represents an earlier version of a study that was subsequently expanded and revised, and has now been published as:

Tei, S., Bando, M., Sakata, T. et al. Cohesin acts as a transcriptional gatekeeper by restraining pause–release to promote processive elongation. Nature Communications 17, 8487 (2026). https://doi.org/10.1038/s41467-026-76738-3

An expanded version of the study was also posted as a later preprint: https://doi.org/10.1101/2025.09.30.679672

Although the two preprints have different titles and DOIs, they represent earlier and later stages of the same study. Readers are encouraged to consult the peer-reviewed article for the final results and conclusions, and to cite the published version where appropriate.

Katsuhiko Shirahige & Shoin Tei Corresponding authors

Not sure why I can't use cos(theta)=x instead of x/r seeing as how the equation is multiplied by the radius and will cancel it out.

Not sure why you calculated r, it was given in the problem. And although the 122 is wrong, the answer is right.

Ok, so 2 cos (pi/6) is a number, why aren't we writing it that way?

This notation is not an identity. Please stop. I find it preposterous that we use a cartesian coordinate system for imaginary numbers at all. 3+4i could well be (3,4i), but the point (5,6) would only be 5x+6y, which has never been taught. There's a lot here that isn't being discussed.

I'm not using this cis notation. You can't use it to solve anything since it's a mixture of sin and cos and isn't an identity.

cis? Never heard of it before.

there's a |z| with no value in the graph.

I'm not supposed to be learning formulas in math. I'm supposed to be learning derivation and modeling.

Fill in your own math! I don't see ANYTHING about the complex conjugate in here! I don't see anything about z^2-=(x+iy)(x-iy) making |z|=sqrt(x^2+y^2)

test

The significance behind this question is the overall idea of putting others first. When contemplating a course of action I always think to myself if it would only benefit me or others as well. It is important to put others before yourself in certain situations. In the context that a big number of people will be hurt you must think to yourself, "what is the next best approach?". It is vital you look at the greater scheme of things, as long as you stay loyal to your beliefs.

heyhey

Should be: 1<a/b<2

missing graphs

missing graphs

This whole section would be better if you included math with the +-(pi)(k)

your first zero shows up as \(0\0

Wouldn't this be more useful if we used sin(pi)=0 to show that theta=(pi/5)k?

5pi8 and 3pi4??

r does not equal +-4 if r^2=4

Is an inner-loop limacon considererd a one-loop or two-loop limacon?

Why are the axes labeled with negative numbers if the concentric circles are positive valued?

Attempt #1 using hypothesis

Sample means always wobble a big by chance and the standard error measures how much.

To me when I read this did I think about how sometimes doctors only focus on the sickness, but holistic medicine looks at the whole person. It made me think about how things like stress, family, and your everyday life can also affect your health.

when I read this I thought about how couples today sometimes have to decide where they are going to live after getting married. Some couples live with the husband or wife’s family for help, but a lot of couples want their own place. I think this can be hard because they have to think about money, jobs, and being close to family. It made me think about how different families can have different expectations about where a married couple should live.

Partisan politics does have a major effect on macroeconomic variables

Importance of partisan elements

What is strategic disadvantage?

Central point of the exclusivity of power

Players do not seek merely to promote ideas but to scale politics toward their advantage

Promotion of policy (first face) and then an arrangement of strategic advantage (second face)

bristling of hairs due to involuntary contraction of hair follicles

Does someone with hairless skin experience music slightly differently in this case?

olfactory senses play key role

Would a postiive olfactory experience be rated higher than excellent results?

temp impacts somatosensory perception --> diff temps result in diff valenced emotions

somatosensory perception is influenced by our experiences and psyche