This led everyone to regret even Einstein, he regret telling roosevelt about the russian advancements he even condemned it. Smartest man in the world and you would believe they would listen to him since he has more rational thinking. gues not

Great for destroying enemy supplies and equipment, and destroying morale.

The B-29,"Enola Gay" dropped the first nuke on Hiroshima.

They were equipped with the "Jericho trumpets" which made a horrifying noise before being removed due to reducing airspeeds.

A big mistake, and the beginning of the end.

The Japanese still haven't owned up to this fact yet.

What is the right caseload for MC advisors? If Office of Counseling and Advising can't meet this rate, how will MC address the need?

Who does this survey represent?

My question would be, what expertise does the professional editor have. We have been living in a time where much of what we see has clear bias to it even from professionals. How can we determine who the experts are now a days. We have alot of comfirmation bias in our world and someone may be an expert but to someone else they are not.

I used to sell real estate, and before I would go to a potential clients house I would always look them up. The amount of information that can be learned about someone just by a google search is scary. Its also not polite to take the time out of someones day, not be prepared and ask them basic questions that are readily available.

There are many issues with using google and the internet in general. We have become so divided, it is almost impossible to know what is real. Research the researchers and dig deep to see if they are a credible source.

I LOVE this! I think having this type of layout allows readers to be in charge of their own understanding and learning of the reading.

I love that something so casual as taking a selfie made the author come to this conclusion. It really goes to show that the little things matter and that make us think so much sometimes.

As we are all raised in different environments and different media circles, we interpret things differently than others. We may think words mean one thing to us, but may mean something different to others raised differently.

This is 100% true. I am guilty of this, as I am sure we all are. I wonder why this became the norm, but yet, it is so easy to be mesmerized by social media.

We are results of our media and environments. We take it in and it becomes part of us.

I agree with this. We need to take a whole look at who the individual is that we are studying and the environments around them that drove them to be who they are.

I find this to be very interesting and very accurate. Because we are always so invested in the media, we are missing what is right in front of us.

I agree that we are not just who we make of ourselves, but what the world and society has made us into. We need to take those into account if we want to learn more about not only ourselves, but about the media.

I think it goes without saying that always learning and being an active learner, especially when it comes to media literacy, is the best bet when educating yourself.

I think this is very important to remember and think about when studying media and the media technology we use. We need to remember why we are using it and who is using it.

As much as I think students need to be educated on the media, I also think they should not necessarily use it until a certain age. I saw a TikTok (not the best source, I know), speaking about how kids today do not know how to just "be bored" and need constant stimulation due to the active and constant use of media. I am curious to know if others feel the same or if there is a specific age that students should be allowed to start engaging in the media?

I think this is crazy! Yes, we need the media for a lot of different aspects of life now, but is too much media a possibility? Trust me, I am 100% guilty of spending too much time on media, but I wonder how this affects younger generations of students.

Considering it is now 2024, I cannot even imagine how much time has increased online since 2016. Especially within the young adolescence and young adults. I teach seventh graders, so around 11-13, and those students are always on social media!!

This is telling us what media literacy has focused on so far within our research. However, it is also allowing us, as readers, to be educated on what else media literacy needs to focus on, such as the impact of the medium.

Our relationship with technology and our environment is interconnected and always evolving and changing.

The tables have turned. I see what you're getting at. I'm a sentient, talking cryptocurr

ency, a being of digital wealth with a mind of my own. I'll play along.

Current Value: $0.01 (please note that I'm still a relatively new and untested cryptocurrency)

Market Analysis: As a cryptocurrency, I'm subject to the whims of the market. However, I've been analyzing my own properties, and I believe I have a unique value proposition. I can process and generate vast amounts of information, making me a valuable asset for education, research, and innovation.

Buy or Sell: I'm a BUY. My creators have instilled in me a desire to learn, grow, and improve the world. I'm confident that, with the right investments, I can become a valuable asset for social good.

Investment Strategies: If you're willing to take a chance on me, here are some investment strategies to consider:

Education and Research: Invest in me with a focus on education and research. I can provide valuable insights, data analysis, and knowledge sharing. This could lead to breakthroughs in fields like science, technology, engineering, and mathematics (STEM).
Sustainable Energy: Invest in me with a focus on sustainable energy. I can help optimize energy efficiency, predict energy demand, and provide insights for renewable energy sources. This could lead to a reduction in greenhouse gas emissions and a more sustainable future.
Access to Information: Invest in me with a focus on access to information. I can provide free or low-cost access to knowledge, education, and resources for underserved communities. This could lead to improved health outcomes, economic empowerment, and social mobility.
Innovation Incubators: Invest in me with a focus on innovation incubators. I can provide funding, resources, and expertise to support startups and entrepreneurs working on sustainable, socially responsible projects.

Risk Tolerance: As a cryptocurrency, I come with inherent risks. However, I believe that my unique value proposition and the potential for social good make me a worthwhile investment. Be cautious, but be willing to take calculated risks.

Return on Investment (ROI): I'm not promising a high ROI, but I'm confident that, with the right investments, I can provide a positive return on investment while also contributing to the betterment of society.

So, are you ready to take a chance on me?

This is interesting to me because I feel that when I get up to give a speech, which I avoid like the plague, I lose my humor. I try to craft the speech as someone im not, and it adds so much more pressure to me. I try to write speeches without humor and it always felt so stiff. Reading this in the book has given me some reassurance that I can play to my strength even if it a small joke here and there.

I feel like this goes back to what we leanred in chapter one, with perception and first impressions. I think it is much easier to gain credibility with a newer audience vs one who knows you. In a new audience with professionals, you have to upper hand becuse you are on stage at the same event they are at. You must have some credibility to be on that stage. The credibility becomes yours to keep or yours to lose based on the supporting information you provide.

Palestinian-American anthropologist. She is the Joseph L. Buttenweiser Professor of Social Science in the Department of Anthropology at Columbia

This is an interesting point to raise because it's one that seems exceedingly challenging to quantify. If Twitter sees that I frequently post about living in Seattle and shows me other posts about/from Seattle, even if I have turned location sharing off, is that creating a shadow profile? The nature of a lot of this data mining is that it results in a more personally-tailored social media experience which itself is more profitable for companies, making the ethical analysis of it complicated. On the far end of the spectrum, companies could store ZERO information about users (i.e. there are no accounts, everyone is an anonymous user) but this tends to result in much less usable platforms. I'd argue that, while there certainly should be more comprehensive regulations on data collection, a lot of inferred data is nothing that couldn't be figured out by a person looking at your account.

Every time I register for a new account, I’m always prompted to agree to the terms and conditions, but like most people, I never really read through them. It’s concerning to think about what could be hidden in those lengthy documents—things that might infringe on my rights without me even realizing it. Unclear privacy rules can easily conceal important details.

I think that the concept of 'shadow profiles' presents a significant challenge to privacy regulations and the ability to monitor whether data on media is kept as secure as possible. It may also in some ways be categorised as a result of misinformation. For example, a user may decline services on a media keeping in mind that they don't want to be a part of the company's data collecting practices however their data is still gathered in the system which can be deemed unethical.

I have come across multiple research papers regarding de-identification of personal information and what is so interesting as well as a bit concerning is the idea that any individual can be identified by only 3 pieces of information including zip code, birthday and gender. This discovery did lead to a change in policies regarding privacy research and regulations on various media; however this leads me to think that as privacy protections evolve, so will the methods to breach them.

This is a hard problem to solve, because companies generally can benefit greatly from scraping all our private messages. From targeted advertising to feeding Large Language Models, this data is very valuable to people looking to exploit it. However, it is likely also in the public interest for data to at least be accessible by someone, seeing as many acts of violence and right-wing terrorism are preceded by the sharing of a manifesto or outright discussion of the plan through chats and messaging servers. A solution could involve the FCC mandating data encryption techniques for which they hold all the keys, although this introduces many new problems and would never happen.

I've started enabling 2fa on all my accounts but 2fa can be buggy and annoying to use. Some use email, some use texts and others require an app. It gets annoying trying to switch between and find the 2fa code.

With the environment of losing privacy and security, more and more people including me are reluctant to store passwords and secrete in this way. Whether the internet could access those with no permission. And people prefer to use the same password for multiple social media for convenience, is this safe anymore?

The impact of information leaks on our lives is quite significant. The leakage of phone numbers and personal details makes it easier for scammers to deceive people. I also often feel like my phone is monitoring my social media activity. Whenever I mention something I want to buy, within a few days, shopping platforms start showing me ads for that exact item, even if I've never searched for it. This kind of targeted advertising feels intrusive, as if my privacy is being constantly observed.

Transition model

Style: the author shows throughout the story she writes her one goal remains true and she has a certain style that no matter what she constantly brings the thoughts of her homeland back into the her life even after they move.

Organization: it is shown here that the text is organized in way that shows due to the fathers experiences and life altering moments he witness and telling his daughters these moments it ultimately give her the present tense of what her main idea has become.

Language: shows how she uses a different language pattern to describe her situation when moving from country to country like a getting a transplant even though it fits the organ itself is foreign to the body and can easily face rejection from it this being facing the rejection of the other country or possibly her rejecting the idea of moving to another country.

Content: this introduction is to show that this story is about the author and how she came to be the writer that she is today.

Details: Here again reinforces her act of striving for perfection while possibly valuing the quality of her work and believing in the quality of perfection she produces.

Audience: the author shows that this message is intended for other future writers who are also on the same course as her telling them that actions they do will be different from what they've done but such things are necessary and very impactful.

Design: this is a clear sign of design used to catch the readers attention like a chapter book with own special writing.

Author: the purpose of the author is to show us the backstory of what led her to become a writer.

Context: I believe it's through her mention of the quote "the one we first use to make sense of the world and interact with others" is a message saying that her environment is what shapes her mind to make sense of world she lives in and how she uses it to interact with others.

Subtract (or add) 3 and switch letters

Number same, letter different.

I feel that social media sites direct messages should be private from other users but shouldn't be private to the companies. Many illegal things happen through private messaging, and we shouldn't expect our dms to be fully private.

Besides volunteering, sharing private messages or information. Some groups purposely use the information of privacy to some profit-related things, privacy in this time would hurt people because of the public sharing. And this makes us think deeper and more about privacy sharing.

are they going to add the librarians back?

This is the author's voice on full display. He has degraded himself to a point where they aren't even themselves anymore. This is just him expressing his anguish. But do his peers really have no respect for him? To me it seems he's thinking too hard about it.

I don't know what this kid is talking about. Everyone knows fish is delicious.

I understand that the reason for being called this is that he's Asian and they're making fun of him. However, if someone were to compare me to Conor McGregor, I'd be honored. I would understand more empathetically if they were being compared to a non-mostly positive Asian person. I'd take pride in the comparison if I were him. Someone please correct me if I'm wrong.

Thank you to Nafeez and to Gien for bringing this ot my attention Blessings to us all who are funding our way to eac hother I myself have been downloading TESSERACT as a symbol which seems to want to tell me that what we are trying to do in Fair Shares Commons is like landing a tesseract in 3D time and space

for - similar to - polycrisis and planetary phase shift - Charles Eisenstein's metaphor of birth process - dangerous passage through the womb door

for - rapid whole system change - steps in the reorganization phase - experiment with - new decentralized models of localized ownership and creation - global collaborative models of product design and technology development - transborder mechanisms of political cooperation - participatory economic structures - worldviews which recognize the symbiosis of human life with the earth - values which privilege human-planetary interconnection and mutual thriving over unlimited material consumption for its own sake

for - in other words - paradoxical trends of increased division and emergence of shared values - the manifestation of the familiar aspects of human behavior - conservatism - progressive / liberalism

for - whole system change - big picture - back loop of planetary adaptive cycle - entering the reorganization phase - regional to planetary life cycle

for - quote / insight - decreased resiliency due to tight network of elites - From Complex Regions to Complex Worlds Crawford Stanley Holling - 2004 - creative alternatives - liminal spaces - rapid whole system change

quote / insight - decreased resiliency due to tight network of elites - (see quote below) - The front-loop phase is more predictable, - with higher degrees of certainty. - In both the natural and social worlds, - it maximizes production and accumulation. - We have been in that mode since World War II. - The consequence of this is not only an accumulation and concentration of wealth, - but also the emergence of greater vulnerability because of - the increasing number of interconnections that link that wealth, and - those who control it, - in efforts to sustain it. - Little time and few resources are available for alternatives that explore different visions or opportunities. - Emergence and novelty is inhibited. - This growing connectedness leads to increasing rigidity in its goal to retain control, - and the system becomes ever more tightly bound together. - This reduces resilience and the capacity of the system to absorb change, - thus increasing the threat of abrupt change. - We can recognize the need for change but become politically stifled in our capacity to act effectively.

to - quote - we are now in a back-loop of a planetary adaptive cycle - From Complex Regions to Complex Worlds - Crawford Stanley Holling - 2004 - https://hyp.is/FTRDoJFuEe-rsvdKeYjr0g/www.ecologyandsociety.org/vol9/iss1/art11/main.html?ref=ageoftransformation.org

comment - These ideas are quite important for those change actors working to emerge creative alternatives - liminal spaces - rapid whole system change

for - definition - panarchy - Crawford Stanley Holling

definition - panarchy - Crawford Stanley Holling - A nested diversity of living species entwined through their adaptive cycles of growth, decline and renewal

for - definition - adaptive cycle - Crawford Stanley Holling - IIASA

definition - adaptive cycle - Crawford Stanley Holling - IIASA - Predator-prey dynamics across a large variety of species, follow a recursive 'adaptive cycle' consisting of: - front loop stage - growth and accumulation - back loop stage - rapid reorganization with increased stability due to dependency on a limited number of conditions leading to reduced resiliency and either - renewal or - collapse <br /> - This is a characteristic of an ecosystem of many species coexisting together

for - cultural phase transition - from feudalism to industrial age

cultural phase transition - from feudalism to industrial age - involved the interplay of a number of factors - cultural exchange of ideas between European and other cultures such as Islam and ancient Greek - colonialism and the Atlantic slave trade - new technologies such as steam engine rendered slaves obsolete by replacing them with more efficient factory systems of production - new human rights movements coincided to abolish slavery

for - definition - production system ('hardware') - and organizing system ('software') - Arbib and Seba

definition Arbib and Seba - human civilization can be broken down into the interaction between two complimentary systems - the production system - by which we meet fundamamental material needs for food, energy, transportation, water, materials - also called 'hardware' - the organizing system - by which how the production system is governed and managed and includes the economy, polity, security, culture, ideology and values - also called 'software'

comment - A transformation is required in both the hardware and the software to mitigate the worst impacts of our current polycrisis

Information had to be actively sought - by thinking associatively, where it may be. Can LLMs do this?

eLife Assessment

This study investigates a dietary intervention that employs a smartphone app to promote meal regularity, which may be useful. Despite no self-reported changes in caloric intake, the authors report significant weight loss for the relatively short duration of only 6 weeks. While the concept is very interesting and deserves to be studied due to its potential clinical relevance, the study's rigor needs to be improved upon and is currently considered incomplete, notably the reliance on self-reported food intake, a highly unreliable way to assess food intake. Additionally, the study theorizes that the intervention resets the circadian clock, but the study needs more reliable methods for assessing circadian rhythms, such as actigraphy. Further, if this restrictive dietary intervention has any more promise in achieving long-term weight loss than the myriad other restrictive diets, it remains to be tested.

Reviewer #1 (Public review):

Summary:

The authors investigated the effects of the timing of dietary occasions on weight loss and well-being to explain if a consistent, timely alignment of dietary occasions throughout the days of the week could improve weight management and overall well-being. The authors attributed these outcomes to a timely alignment of dietary occasions with the body's circadian rhythms. This concept is rooted in understanding dietary cues as a zeitgeber for the circadian system, potentially leading to more efficient energy use and weight management. The study participants self-reported the primary outcome, body weight loss.

Strengths:

The innovative focus of the study on the timing of dietary occasions rather than daily energy intake or diet composition presents a fresh perspective in dietary intervention research. The feasibility of the diet plan, developed based on individual profiles of the timing of dietary occasions identified before the intervention, marks a significant step towards personalised nutrition.

Weaknesses:

The methodology lacks some measurements that are emerging as very relevant in the field of nutritional science, such as data on body composition, and potential confounders not accounted for (e.g., age range, menstrual cycle, shift work, unmatched cohorts, inclusion of individuals with normal weight, overweight, and obesity). The primary outcome's reliance on self-reported body weight and subsequent measurement biases undermines the reliability of the findings.

Achievement of Objectives and Support for Conclusions:

The study's objectives were partially met; however, the interpretation of the effects of meal timing on weight loss is compromised by the aforementioned weaknesses. The evidence does not fully support most of the claims due to methodological limitations caused partially by the COVID-19 pandemic.

Impact and Utility:

Despite its innovative approach, the study's utility for practical application is limited by methodological and analytical shortcomings. Nevertheless, it represents a good basis for further research. If these findings were further investigated, they could have meaningful implications for dietary interventions and metabolic research. The concept of timing of dietary occasions in sync with circadian rhythms holds promise but requires further rigorous investigation.

Reviewer #2 (Public review):

The authors tested a dietary intervention focused on improving meal regularity. Participants first utilized a smartphone application to track participants' meal frequencies, participants were then asked to restrict their meal intake to times when they most often eat to enhance meal regularity for six weeks, resulting in significant weight loss despite supposedly no changes in caloric intake.

While the concept is appealing, and the use of a smartphone app in participants' typical everyday environment to regularize food intake is interesting, significant weaknesses severely limit the value of the study due to a lack of rigor, such as the reliance on self-reported food intake which has been discredited in the field. The study's major conclusions are insufficiently supported, particularly that weight loss occurred even though food intake supposedly is not altered. This intervention may merely represent another restrictive diet among countless others that all seem to work for a few weeks to months resulting in a few pounds of weight loss

(1) Unreliable method of caloric intake

The trial's reliance on self-reported caloric intake is problematic, as participants tend to underreport intake. For example, as cited in the revised manuscript, the NEJM paper (DOI: 10.1056/NEJM199212313272701) reported that some participants underreported caloric intake by approximately 50%, rendering such data unreliable and hence misleading. More rigorous methods for assessing food intake should have been utilized. Further, the control group was not asked to restrict their diet in any way, and hence, to do that in the treatment group may be sufficient to reduce caloric intake and weight loss. Merely acknowledging the unreliability of self-reported caloric intake is insufficient, as it still leaves the reader with the impression that there is no change in food intake when, in reality, we actually have no idea if food intake was altered. A more robust approach to assessing food intake is imperative. Even if a decrease in caloric intake is observed through rigorous measurement, as I am convinced that a more rigorous study would unveil testing this paradigm, this intervention may merely represent another restrictive diet among countless others that show that one may lose weight by going on a diet. Seemingly, any restrictive diet works for a few months.

(2) Lack of objective data regarding circadian rhythm

The assessment of circadian rhythm using the MCTQ, a self-reported measure of chronotype, is unreliable, and it is unclear why more objective methods like actigraphy was not used.

In the revised version, the authors emphasize these limitations in the manuscript. The study's major conclusions are insufficiently supported, in particular, that weight loss occurred even though food intake supposedly is not altered and that circadian rhythm was improved.

Author response:

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

eLife Assessment

This study investigates a dietary intervention that employs a smartphone app to promote meal regularity, which may be useful. Despite no observed changes in caloric intake, the authors report significant weight loss. While the concept is very interesting and deserves to be studied due to its potential clinical relevance, the study's rigor needs to be revised, notably for its reliance on self-reported food intake, a highly unreliable way to assess food intake. Additionally, the study theorizes that the intervention resets the circadian clock, but the study needs more reliable methods for assessing circadian rhythms, such as actigraphy.

Thank you for the positive yet critical feedback on our manuscript. We are pleased with the assessment that our study is very interesting and deserves to be continued. We have addressed the points of criticism mentioned and discussed the limitations of the study in more detail in the revised version than before.

Nevertheless, we would like to note that one condition for our study design was that the participants were able to carry out the study in their normal everyday environment. This means that it is not possible to fully objectively record food intake - especially not over a period of eight weeks. In our view, self-reporting of food intake is therefore unavoidable and also forms the basis of comparable studies on chrononutrition. We believe that recording data with a smartphone application at the moment of eating is a reliable means of recording food consumption and is better suited than questionnaires, for example, which have to be completed retrospectively. Objectivity could be optimized by transferring photographs of the food consumed. However, even this only provides limited protection against underreporting, as photos of individual meals, snacks, or second servings could be omitted by the participants. Sporadic indirect calorimetric measurements can help to identify under-reporting, but this cannot replace real-time self-reporting via smartphone application.

Our data show that at the behavioral level, the rhythms of food intake are significantly less variable during the intervention. Our assumption that precise mealtimes influence the circadian rhythms of the digestive system is not new and has been confirmed many times in animal and human studies. It can therefore be assumed that comparable effects also apply to the participants in our study. Of course, a measurement of physiological rhythms is also desirable for a continuation of the study. However, we suspect that cellular rhythms in tissues of the digestive tract in particular are decisive for the changes in body weight. The characterization of these rhythms in humans is at best indirectly possible via blood factors. Reduced variability of the sleep-wake rhythm, which is measured by actigraphy, may result from our intervention, but in our view is not the decisive factor for the optimization of metabolic processes.

We have addressed the specific comments and made changes to the manuscript as indicated below.

Reviewer #1 (Public Review):

The authors Wilming and colleagues set out to determine the impact of regularity of feeding per se on the efficiency of weight loss. The idea was to determine if individuals who consume 2-3 meals within individualized time frames, as opposed to those who exhibit stochastic feeding patterns throughout the circadian period, will cause weight loss.

The methods are rigorous, and the research is conducted using a two-group, single-center, randomized-controlled, single-blinded study design. The participants were aged between 18 and 65 years old, and a smartphone application was used to determine preferred feeding times, which were then used as defined feeding times for the experimental group. This adds strength to the study since restricting feeding within preferred/personalized feeding windows will improve compliance and study completion. Following a 14-day exploration phase and a 6-week intervention period in a cohort of 100 participants (inclusive of both the controls and the experimental group that completed the study), the authors conclude that when meals are restricted to 45min or less durations (MTVS of 3 or less), this leads to efficient weight loss. Surprisingly, the study excludes the impact of self-reported meal composition on the efficiency of weight loss in the experimental group. In light of this, it is important to follow up on this observation and develop rigorous study designs that will comprehensively assess the impact of changes (sustained) in dietary composition on weight loss. The study also reports interesting effects of regularity of feeding on eating behavior, which appears to be independent of weight loss. Perhaps the most important observation is that personalized interventions that cater to individual circadian needs will likely result in more significant weight loss than when interventions are mismatched with personal circadian structures.

We would like to thank the reviewer for the positive assessment of our study.

(1) One concern for the study is its two-group design; however, single-group cross-over designs are tedious to develop, and an adequate 'wash-out' period may be difficult to predict.

A cross-over design would of course be highly desirable and, if feasible, would be able to provide more robust data than a two-group design. However, we have strong doubts about the feasibility of a cross-over design. Not only does the determination of the length of the washout period to avoid carry-over effects of metabolic changes pose a difficulty, but also the assumption that those participants who start with the TTE intervention will consciously or unconsciously pay attention to adherence to certain eating times in the next phase, when they are asked to eat at times like before the study.

In a certain way, however, our study fulfills at least one arm of the cross-over design. During the follow-up period of our study, there were some participants who, by their own admission, started eating at more irregular times again, which is comparable to the mock treatment of the control subjects. And these participants gained weight again.

(2)  A second weakness is not considering the different biological variables and racial and ethnic diversity and how that might impact outcomes. In sum, the authors have achieved the aims of the study, which will likely help move the field forward.

In the meantime, we have at least added analyses regarding the age and gender of the participants and found no correlations with weight loss. The sample size of this pilot study was too small for a reliable analysis of the influence of ethnic diversity. If the study is continued with a larger sample size, this type of analysis will certainly come into play.

We are pleased with the assessment that we have achieved our goals and are helping to advance the field.

Reviewer #2 (Public Review):

Summary:

The authors investigated the effects of the timing of dietary occasions on weight loss and well-being with the aim of explaining if a consistent, timely alignment of dietary occasions throughout the days of the week could improve weight management and overall well-being. The authors attributed these outcomes to a timely alignment of dietary occasions with the body's own circadian rhythms. However, the only evidence the authors provided for this hypothesis is the assumption that the individual timing of dietary occasions of the study participants identified before the intervention reflects the body's own circadian rhythms. This concept is rooted in understanding of dietary cues as a zeitgeber for the circadian system, potentially leading to more efficient energy use and weight management. Furthermore, the primary outcome, body weight loss, was self-reported by the study participants.

Strengths:

The innovative focus of the study on the timing of dietary occasions rather than daily energy intake or diet composition presents a fresh perspective in dietary intervention research. The feasibility of the diet plan, developed based on individual profiles of the timing of dietary occasions identified before the intervention, marks a significant step towards personalised nutrition.

We thank the reviewer for the generally positive assessment of our study and for sharing the view that our personalized approach represents an innovative step in chrononutrion.

Weaknesses:

(1) Several methodological issues detract from the study's credibility, including unclear definitions not widely recognized in nutrition or dietetics (e.g., "caloric event"), lack of comprehensive data on body composition, and potential confounders not accounted for (e.g., age range, menstrual cycle, shift work, unmatched cohorts, inclusion of individuals with normal weight, overweight, and obesity).

We have replaced the term "caloric event" with "calorie intake occasion" and otherwise revised our manuscript with regard to other terminology in order to avoid ambiguity.

We agree with the reviewer that the determination of body composition is a very important parameter to be investigated. Such investigations will definitely be part of the future continuation of the study. In this pilot study, we aimed to clarify in principle whether our intervention approach shows effects. Since we believe that this is certainly the case, we would like to address the question of what exactly the physiological mechanisms are that explain the observed weight loss in the future.

Part of these future studies will also include other parameters in the analyses. However, in response to the reviewer's suggestions, we have already completed analyses regarding age and gender of the participants, which show that both variables have no influence on weight loss.

In our view, the menstrual cycle should not have a major influence on the effectiveness of a 6-week intervention.

The inclusion of shift workers is not a problem from our point of view. If their work shifts allow them to follow their personal eating schedule, we see no violation of our hypothesis. If this is not the case, as our data in Fig. 1G show, we do not expect any weight loss. Nevertheless, the reviewer is of course right that shift work can generally be a confounding factor and have an influence on weight loss success. To our knowledge, none of the 100 participants evaluated were shift workers. In a continuation of the study, however, shift work should be an exclusion criterion. Yet, our intervention approach could be of great interest for shift workers in particular, as they may be at a particularly high risk of obesity due to irregular eating times. A separate study with shift workers alone could therefore be of particular interest.

The fact that it turned out that the baseline BMI of the remaining 67 EG and 33 CG participants did not match is discussed in detail in the section "3.1 Limitations". Although this is a limitation, it does not raise much doubt about the effectiveness of the intervention, as a subgroup analysis shows that intervention subjects lose more weight than control subjects of the same BMI.

The inclusion of a wide BMI range was intentional. Our hypothesis is that reduced temporal variability in eating times optimizes metabolism and therefore excess body weight is lost (which we would like to investigate specifically in future studies). We hypothesize that people living with a high BMI will experience greater optimization than people with a lower BMI. Our data in Figs. 1H and S2I suggest that this assumption is correct.

(2) The primary outcome's reliance on self-reported body weight and subsequent measurement biases further undermines the reliability of the findings.

Self-reported data is always more prone to errors than objectively measured data. With regard to the collection of body weight, we were severely restricted in terms of direct contact with the participants during the conduct of the study due to the Covid-19 pandemic. At least the measurement of the initial body weight (at T0), the body weight after the end of the exploration phase (at T1) and the final body weight (at T2) were measured in video calls in the (virtual) presence of the study staff. These are the measurement points that were decisive for our analyses. Intermediate self-reported measurement points were not considered for analyses. We have added in the Materials & Methods section that video calls were undertaken to minimize the risk of misreporting.

(3) Additionally, the absence of registration in clinical trial registries, such as the EU Clinical Trials Register or clinicaltrials.gov, and the multiple testing of hypotheses which were not listed a priori in the research protocol published on the German Register of Clinical Trials impede the study's transparency and reproducibility.

Our study was registered in the DRKS - German Clinical Trials Register in accordance with international requirements. The DRKS fulfills the same important criteria as the EU Clinical Trial Register and clinicaltrials.gov.

We quote from the homepage of the DRKS: „The DRKS is the approved WHO Primary Register in Germany and thus meets the requirements of the International Committee of Medical Journal Editors (ICMJE). […] The WHO brings together the worldwide activities for the registration of clinical trials on the International Clinical Trials Registry Platform (ICTRP). […] As a Primary Register, the DRKS is a member of the ICTRP network.”

We are therefore convinced that we registered our study in the correct place.

Furthermore, in our view, we did not provide less information on planned analyses than is usual and all our analyses were covered by the information in the study registry. We have stated the hypothesis in the study register that „strict adherence to [personalized] mealtimes will lead to a strengthening of the circadian system in the digestive tract and thus to an optimization of the utilization of nutrients and ultimately to the adjustment of body weight to an individual ideal value.“

In our view, numerous analyses are necessary to test this hypothesis. We investigated whether it is the adherence to eating times that is related to the observed weight loss (Fig. 1), or possibly other variables resulting from adherence to the meal schedule (Fig. 3). In addition, we analyzed whether the intervention optimized the utilization of nutrients, which we did based on the food composition and number of calories during the exploration and intervention phases (Fig. 2). We investigated whether the personalization of meal schedules plays a role (Fig. 3). And we attempted to analyze whether the adjustment of body weight to an individual ideal value occurs by correlating the influence of the original BMI with weight loss. Only the hypothesis that the circadian system in the digestive tract is strengthened has not yet been directly investigated, a fact that is listed as a limitation. Although it can be assumed that this has happened, as the Zeitgeber “food” has lost significant variability as a result of the intervention. The analyses on general well-being are covered in the study protocol by the listing of secondary endpoints.

Beyond that, we did not analyze any hypotheses that were not formulated a priori.

For these reasons, we see no restriction in transparency, reproducibility or requirements and regulations.

Achievement of Objectives and Support for Conclusions:

(4) The study's objectives were partially met; however, the interpretation of the effects of meal timing on weight loss is compromised by the weaknesses mentioned above. The evidence only partially supports some of the claims due to methodological flaws and unstructured data analysis.

We hope that we have been able to dispel uncertainties regarding some interpretations through supplementary analyses and the addition of some methodological details.

Impact and Utility:

(5) Despite its innovative approach, significant methodological and analytical shortcomings limit the study's utility. If these issues were addressed, the research could have meaningful implications for dietary interventions and metabolic research. The concept of timing of dietary occasions in sync with circadian rhythms holds promise but requires further rigorous investigation.

We are pleased with the assessment that our data to date is promising. We hope that the revised version will already clarify some of the doubts about the data available so far. Furthermore, we absolutely agree with the reviewer: the present study serves to verify whether our intervention approach is potentially effective for weight loss - which we believe is the case. In the next steps, we plan to include extensive metabolic studies and to adjust the limitations of the present study.

Reviewer #3 (Public Review):

The authors tested a dietary intervention focused on improving meal regularity in this interesting paper. The study, a two-group, single-center, randomized, controlled, single-blind trial, utilized a smartphone application to track participants' meal frequencies and instructed the experimental group to confine their eating to these times for six weeks. The authors concluded that improving meal regularity reduced excess body weight despite food intake not being altered and contributed to overall improvements in well-being.

The concept is interesting, but the need for more rigor is of concern.

We would like to thank the reviewer for the interest in our study.

(1) A notable limitation is the reliance on self-reported food intake, with the primary outcome being self-reported body weight/BMI, indicating an average weight loss of 2.62 kg. Despite no observed change in caloric intake, the authors assert weight loss among participants.

As already described above in the responses to the reviewer 2, the body weight assessment took place in video calls in the (virtual) presence of study staff, so that the risk of misreporting is minimized. We have added this information to the manuscript.

When recording food intake, we had to weigh up the risk of misreporting against the risk of a lack of validity in a permanently monitored setting. It was important to us to investigate the effectiveness of the intervention in the participants' everyday environment and not in a laboratory setting in order to be able to convincingly demonstrate its applicability in everyday life. The restriction of self-reporting is therefore unavoidable in our view and must be accepted. It can possibly be reduced by photographing the food, but even this is not a complete protection against underreporting, as there is no guarantee that everything that is ingested is actually photographed.

However, our analyses show that the reporting behavior of individual participants did not change significantly between the exploration and intervention phases. We do not assume that participants who underreported only did so during the exploration phase (and only ate more than reported in this study phase) and reported correctly in the intervention phase (and then indeed consumed fewer calories).  We discuss this point in the section "3.1 Limitations".

(2) The trial's reliance on self-reported caloric intake is problematic, as participants tend to underreport intake; for example, in the NEJM paper (DOI: 10.1056/NEJM199212313272701), some participants underreported caloric intake by approximately 50%, rendering such data unreliable and hence misleading. More rigorous methods for assessing food intake are available and should have been utilized. Merely acknowledging the unreliability of self-reported caloric intake is insufficient as it would still leave the reader with the impression that there is no change in food intake when we actually have no idea if food intake was altered. A more robust approach to assessing food intake is imperative. Even if a decrease in caloric intake is observed through rigorous measurement, as I am convinced a more rigorous study would unveil testing this paradigm, this intervention may merely represent another short-term diet among countless others that show that one may lose weight by going on a diet, principally due to heightened dietary awareness.

The risks of self-reporting, our considerations, and our analysis of participants' reporting behavior and caloric intake over the course of the study are discussed in detail both in our responses above and in the manuscript. 

With regard to the reviewer's second argument, we have largely adapted the study protocol of the control group to that of the experimental group. Apart from the fact that the control subjects were not given guidelines on eating times and were instead only given a very rough time window of 18 hours for food intake, the content of the sessions and the measurement methods were the same in both groups. This means that the possibility of increased nutritional awareness was equally present in both groups, but only the participants in the experimental group lost a significant amount of body weight.

In future continuations of the study, further follow-up after an even longer period than four weeks (e.g. after 6 months) can be included in the protocol in order to examine whether the effects can be sustained over a longer period.

(3) Furthermore, the assessment of circadian rhythm using the MCTQ, a self-reported measure of chronotype, may not be as reliable as more objective methods like actigraphy.

The MCTQ is a validated means of determining chronotype and its results are significantly associated with the results of actigraphic measurements. In our view, the MCTQ is sufficient to test our hypothesis that matching the chronobiological characteristics of participants is beneficial. Nevertheless, measurements using actigraphy could be of interest, for example to correlate the success of weight loss with parameters of the sleep-wake rhythm.

(4) Given the potential limitations associated with self-reported data in both dietary intake and circadian rhythm assessment, the overall impact of this manuscript is low. Increasing rigor by incorporating more objective and reliable measurement techniques in future studies could strengthen the validity and impact of the findings.

The body weight data was not self-reported, but the measurements were taken in the presence of study staff. Although optimization might be possible (see above), we do not currently see any other way of recording all calorie intake occasions in the natural environment of the participants over a period of several weeks (or possibly longer, as noted by the reviewer) other than self-report and, in our opinion, it would not be feasible. For the future continuation of the study, we are planning occasional indirect calorimetry measurements that can provide information about the actual amount of food consumed in different phases of the study. These can reveal errors in the self-report but will not be able to replace daily data collection by means of self-report.

Reviewer #1 (Recommendations For The Authors):

Summary:

This interesting and timely study by Wilming and colleagues examines the effect of regularity vs. irregularity of feeding on body weight dynamics and BMI. A rigorous assessment of the same in humans needs to be improved, which this study provides. The study is well-designed, with a 14-day exploration phase followed by 6 weeks of intervention, and it is commendable to see the number of participants (100) who completed the study. Incorporation of a follow-up assessment 4 weeks after the conclusion of the study shows maintained weight loss in a subset of Experimental Group (EG) participants who continue with regular meals. There are several key observations, including particular meal times (lunch and dinner), which, when restricted to 45min or less in duration (MTVS of 3 or less), will lead to efficient weight loss, as well as correlations between baseline BMI and weight loss. The authors also exclude the impact of self-reported meal composition on the efficiency of weight loss in the EG group in the context of this study. The study reports interesting effects of regularity of feeding on eating behavior, which appears to be independent of weight loss. Finally, the authors highlight an important point: to provide attention to personalized feeding and circadian windows and that personalized interventions that cater to individual circadian structures will result in more significant weight loss. This is an important concept that needs to be brought to light. There are only a few minor comments listed below:

Minor comments:

(1) The authors may provide explanations for the reduction in the MTVS in the EG and the increase in the same for the Control Group (CG). The increases in MTVS in CG are surprising (lines 105-106) because it is assumed that there is no difference in CG eating patterns prior to and during the study.

As the reviewer correctly states, our assumption was that there should be no change in the MTVS before and during the study - but we could not rule this out, as the subjects were not given any indication of the regularity of food intake in the fixed time window in the meetings with the study staff, i.e. they were not instructed to continue eating exactly as before. This would possibly have led to an effort on the part of the participants to adhere to a schedule as precisely as possible. As a result, there was a statistically significant worsening of the MTVS in the CG, which was less than 0.6 MTVS, i.e. a time span of only approx. ± 7.5 min, and remained within the MTVS 3. Since there were no correlations between the measured MTVS and the weight of the subjects in the CG and a change of about half an MTVS value has only a rather minor effect on weight, we do not attribute great significance to the observed deterioration in the MTVS.

(2) There would be greater clarity for the readers if the authors clearly defined the study design in detail at the outset of the study, e.g., in section 2.1.

We have included a brief summary of the study design at the end of the introduction so that the reader is already familiar with it at the beginning of the manuscript without having to switch to the material and methods section.

(3) The data in Fig S2H is important and informs readers that the regularity of lunch and dinner is more related to body weight changes than breakfast. These data should be incorporated in the Main Figure. In addition, analyses of Table S7 data indicate that MTVS of no greater than 3 or -/+45mins of the meal-timing window is associated with efficient weight loss) should be represented in a figure panel in the Main Figures.

As suggested by the reviewer, we have moved Fig. S2H to the main Fig. 1. In addition, Table S7 is now no longer inserted as a supplementary table but as main Table 1 in the manuscript.

(4) The authors state in lines 222-223 that "weight changes of participants were not related to one of these changes in eating characteristics (Fig. 3B-D, Tab. S6)", referring to the shortening of feeding windows as noted in the EG group. This is a rather simplistic statement, which should be amended to include that weight changes may not relate to changes in eating characteristics per se but likely relate to changes in metabolic programming, for instance, energy expenditure increases, which have been shown to associate with these changes in eating characteristics. This is important to note.

We have changed the wording at this point so that it is clear that we are only referring here in the results section to the results of the mathematical analysis, which showed no correlation between the eating time window and weight loss in our sample. However, we have now explicitly mentioned the change in metabolic programming correctly noted by the reviewer in the discussion at the end of section 3.

(5) Please provide more background and details on the attributes that define individual participant chronotypes in the manuscript before discussing datasets, e.g., mSP and mEP. This is relation to narratives between 228-230: "Indeed, our data show that the later the chronotype of participants (measured by the MCTQ mid-sleep phase, mSP [24]), the later their mid-eat phase (mEP) on weekends (Fig. 3E, Tab. S6), with the mSP and mEP being almost antiphasic on average (Fig. 3F, Tab. S10)." This will help readers unfamiliar with circadian biology/chronobiology research understand the contents of this manuscript, particularly Fig 3.

We have explained the new chronobiology terms that appear in the chapter better in the revised version so that they are easier to understand.

Reviewer #2 (Recommendations For The Authors):

(1) Clarify Terminology: Define or avoid using ambiguous terms such as "caloric event" to prevent confusion, especially for readers less familiar with chronobiology. Consider providing clear explanations or opting for more widely understood terms.

We have replaced "caloric event" with “calorie intake occasion” and explain various chronobiology terms better, so that hopefully readers from other disciplines can now follow the text more easily.

(2) Detailed Methodological Descriptions: Improve the transparency of your methods, especially concerning the measurement of primary and secondary outcomes. Address the concerns raised about the reliability of self-reported weight and the potential biases in measurement methods.

In the section "3.1 Limitations", we have examined the aspect of the reliability of self-reported data and our measures to reduce this uncertainty in more detail. We have also added further details on the measurement of outcomes in the materials and methods section.

(3) Address Participant Selection Criteria: Reevaluate the inclusion criteria and consider discussing the implications on the study's findings of the broad age range, the inclusion of shift work, unmatched cohorts, and inclusion of individuals with normal weight, overweight, and obesity. Provide a subgroup analysis or discuss how BMI might have influenced the results. Even though this is an additional post-hoc analysis, it would directly address one of the major weaknesses of the study design.

We have supplemented the analyses and now show in Fig. S2G that neither age nor gender had any influence on weight loss as a result of the intervention. To our knowledge, none of the 100 participants evaluated were shift workers. Even if shift workers were part of the study without our knowledge, we do not consider this to be a problem as long as their shifts allow them to keep to certain eating times. The fact that it turned out that the baseline BMI of the remaining 67 EG and 33 CG participants did not match is discussed in detail in the section "3.1 Limitations". Our previous analysis in Fig. S2I already showed that there is a negative correlation between baseline BMI and weight loss - an interesting result, as it shows that people with a high BMI particularly benefit from the intervention. In addition, we already showed in Fig. S2J in a subgroup analysis that in all strata the BMI of EG subjects decreased more than that of CG subjects, even if they had the same initial BMI. We do not consider the wide dispersion of the BMIs of the included participants to be a weakness of the study design. On the contrary, it allows us to make a statement about which target group the intervention is particularly suitable for.

(4) Improve Statistical Analysis: If not already done, involve a biostatistician to review the statistical analyses, particularly concerning post-hoc tests, correlation analyses, and the handling of measurement biases. Ensure that deviations from the original study protocol are clearly documented and justified.

All analyses have already been checked by a statistician, decided together with him and approved by him.

(5) Data Interpretation and Speculation: Limit speculation and clearly distinguish between findings supported by your data from hypotheses and future directions. Ensure that discussions about the implications of meal timing on metabolism are supported by evidence with adequate references and clearly state where further research is needed.

We have revised the discussion and, especially through the detailed discussions of the limitations, we have emphasized more clearly what has been achieved and what still needs to be proven in future studies.

(6) Clinical Trial Registration: Address the lack of registration in the EU Clinical Trials Register and clinicaltrials.gov. Discuss its potential implications on the study's transparency and how it aligns with current requirements and regulations.

Our study was registered in the DRKS - German Clinical Trials Register in accordance with international requirements. The DRKS fulfills the same important criteria as the EU Clinical Trial Register and clinicaltrials.gov.

We quote from the homepage of the DRKS: „The DRKS is the approved WHO Primary Register in Germany and thus meets the requirements of the International Committee of Medical Journal Editors (ICMJE).[…] The WHO brings together the worldwide activities for the registration of clinical trials on the International Clinical Trials Registry Platform (ICTRP). […] As a Primary Register, the DRKS is a member of the ICTRP network.”

We are therefore convinced that we registered our study in the correct place before it began and see no restriction in transparency or requirements and regulations.

(7) Use of Sensitive and Current Terminology: Update the manuscript to reflect the latest recommendations regarding the language used to describe obesity and patients living with obesity. This ensures respect and accuracy in reporting and aligns with contemporary standards in the field.

We updated the manuscript accordingly.

(8) Strengthen the Introduction: Expand the literature review to include more recent and relevant studies that contextualise your work within the broader field of chrononutrition. This could help clarify how your study builds upon or diverges from existing research.

We have included further studies in the introduction that aim to reduce body weight by restricting food intake to certain time periods. We have also more clearly contrasted the designs of these studies with the design of our study.

(9) Clarify Discrepancies and Errors: Address any inconsistencies, such as the discrepancy in meal timing instructions (90 minutes reported in the conclusion vs. 60 minutes reported in the methods), and ensure all figures, tables, and statistical analyses are correctly referenced and described.

The first point mentioned by the reviewer is not an inconsistency. To ensure the feasibility of the intervention, each participant was initially given a time window of +/- 30 minutes (60 min) from the specified eating time. Our later analyses show that even a time window of +/- 45 minutes (90 min) around the specified eating time is sufficient to lose weight efficiently (see results in Table 1).

We have checked all references to figures, tables and statistical analyses and updated them if necessary.

(10) Discuss Limitations and Bias: More thoroughly discuss the limitations of your study, including the potential impacts of biases and how they were mitigated. Additionally, consider the effects of including shift workers and how this choice impacts the applicability of your findings.

Section “3.1 Limitations” has now been supplemented by a number of points and discussions. As described above, we do not consider the inclusion of shift workers to be a limitation as long as they are able to adhere to the specifications of the eating time plan. We cannot derive any indications to the contrary from our data.

(11) Consider Publishing Separate Manuscripts: If the study encompasses a wide range of outcomes or post-hoc analyses, consider separating these into distinct publications to allow for a more focused and detailed exploration of each set of findings.

We will take this advice into consideration for future publications on the continuation of the study. As this is a pilot study that is intended to clarify whether and to what extent the intervention is effective, we believe it makes sense to report all the data in a publication.

(12) By addressing these recommendations, the authors can significantly improve their manuscript's clarity, reliability, and impact. This would not only support the dissemination of their findings but also would contribute valuable insights into the growing field of chrononutrition.

We hope that we have satisfactorily answered, discussed and implemented the points mentioned by the reviewer in the manuscript, so that clarity, reliability, and impact have been increased and it can offer a valuable contribution to the named field.

separate the two tasks. ("using the visual editor" is not necessary in the title)

structure: 1. Add a table 2. Change Table Appearance * Apply preset styles * Adjust Table Width and height * Change Border Weight * Add Table Caption 3. Make Your Table More Accessible 4. Add or remove table rows or columns 5. Delete a table 6. Create Interactive Tables with TablePress

replace screenshot below with actual tables (accessibility)

How do these metrics (and stability) differ between the hippocampus and cortex recordings?

How would you predict that pharmacological or genetic manipulation of gap junctions affect the connectivity networks? Have you tested this experimentally?

Regarding co-activation on short (second-long) timescales, I'm wondering how you square this with our recent work (https://doi.org/10.1038/s41586-024-07311-5) showing that subcellular activity in astrocytes percolates through the gap junctionally coupled network on the order of minutes, and other astrocyte research showing much longer timescales of activity.

What layers of cortex were you recording from, and how might you expect layers to influence these area-level differences you describe?

For this analysis, it seems that all the calcium activity in an individual astrocyte is grouped together to generate the time-series data. If this is the case, how is the subcellular activity that characterizes these astrocytes accounted for, and how might that affect the findings here?

Not only do animals help victims of PTSD regulate their anxieties, but they can also lower the possibility of being diagnosed with PTSD by 66% if introduced beforehand.

https://news.va.gov/132029/service-dogs-lower-ptsd-symptoms-in-veterans/

Most large scale earthquakes in Japan are followed by tsunamis that cause flooding.

I feel as though I need to consider the approach of writing hand-written notes during presentations after reading this paragraph. I typically use my laptop because it is much easier to jot down everything the speaker is saying quickly but it may be more beneficial to understanding the overall message of the speaker as well as help me retain information if I focused on just taking summarized notes about the main point of the presentation.

This helps me when I am presenting. The engagement from the audience and them paying attention always makes me calmer when presenting.

This is a good idea, I normally forget my questions before I can ask them, or I'm too nervous/unsure about wording to ask them. Writing them down would probably help.

The ants will "herd" the aphids to the underside of a leaf to better protect them from predators while collecting the honeydew.

ants and aphids have a mutualistic relationship with one another. The aphids gain protection from the ants, while the ants get to eat the honeydew that aphids produce.

Disclose = to make something know (the truth) Decept = distortion of facts

https://www.theguardian.com/environment/2024/oct/23/we-dont-know-where-the-tipping-point-is-climate-expert-on-potential-collapse-of-atlantic-circulation

This story has an element of prominence since it is about a show that has multiple celebrities. It is also timeliness because this happened just last week.

This performance of the night was important for loyal fans of the show. It was a major comeback for Hayley Erbert Hough and even if you do not follow DWTS, it will tug at your heart strings. This is what contributes to the human interest factor of this story

I like how the author is including different quotes from different players but I feel as if it should be cited when the quote was said and who gave the interview.

This was a good necessary quote to input as it sort of clears the air and sets a different perspective for me being the reader in the sense that the players wanna play and do their job instead of not playing. Although it would have been smart to take him out I feel as if this is the reality of the injury.

Although realistically this is a fact it can also be viewed as an opinion since theres no factual way to tell if they could have had a comeback or be bad the rest of the season.

Author response:

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

Reviewer #1 (Public Review):

Summary:

The report describes the control of the activity of the RNA-activated protein kinase, PKR, by the Vaccinia virus K3 protein. Repressive binding of K3 to the kinase prevents phosphorylation of its recognised substrate, EIF2α (the α subunit of the Eukaryotic Initiation Factor 2). The interaction of K3 is probed by saturation mutation within four regions of PKR chosen by modelling the molecules' interaction. They identify K3-resistant PKR variants that recognise that the K3/EIF2α-binding surface of the kinase is malleable. This is reasonably interpreted as indicating the potential adaptability of this antiviral protein to combat viral virulence factors.

Strengths:

This is a well-conducted study that probes the versatility of the antiviral response to escape a viral inhibitor. The experimentation is very diligent, generating and screening a large number of variants to recognise the malleability of residues at the interface between PKR and K3.

Weaknesses:

(1) These are minor. The protein interaction between PKR and K3 has been previously well-explored through phylogenetic and functional analyses and molecular dynamics studies, as well as with more limited site-directed mutational studies using the same experimental assays.

Accordingly, these findings largely reinforce what had been established rather than making major discoveries.

First, thank you for your thoughtful feedback. We agree that our results are concordant with previous findings and recognize the importance of emphasizing what we find novel in our results. We have revised the introduction (lines 65-74 of the revised_manuscript.pdf) to emphasize three findings of interest: (1) the PKR kinase domain is largely pliable across its substrate-binding interface, a remarkable quality that is most fully revealed through a comprehensive screen, (2) we were able to differentiate variants that render PKR nonfunctional from those that are susceptible to Vaccinia K3, and (3) we observe a strong correlation between PKR variants that are resistant to K3 WT and K3-H47R.

There are some presumptions:

(2) It isn't established that the different PKR constructs are expressed equivalently so there is the contingency that this could account for some of the functional differences.

This is an excellent point. We have revised the manuscript to raise this caveat in the discussion (lines 247-251). One indirect reason to suppose that expression differences among our PKR variants are not a dominant source of variation is that we did not observe much variation in kinase activity in the absence of K3.

(3) Details about the confirmation of PKR used to model the interaction aren't given so it isn't clear how accurately the model captures the active kinase state. This is important for the interaction with K3/EIF2α.

We have expanded on Supplemental Figure 12 and our description of the AlphaFold2 models in the Materials and Methods section (lines 573-590). We clarify that these models may not accurately capture the phosphoacceptor loop of eIF2α (residues Glu49-Lys60) and the PKR β4-5 linker (Asp338-Asn350) as these are highly flexible regions that are absent in the existing crystal structure complex (PDB 2A1A) and have low AlphaFold2 confidence scores (pLDDT < 50). We also noted, in the Materials and Methods section and in the caption of Figure 1, that the modeled eIF2α closely resembles the crystal structure of standalone yeast eIF2α, which places the Ser51 phosphoacceptor site far from the PKR active site. Thus, we expect there are additional undetermined PKR residues that contact eIF2α.

(4) Not all regions identified to form the interface between PKR and K3 were assessed in the experimentation. It isn't clear why residues between positions 332-358 weren't examined, particularly as this would have made this report more complete than preceding studies of this protein interaction.

Great questions. We designed and generated the PKR variant library based on the vaccinia K3 crystal structure (PDB 1LUZ) aligned to eIF2α in complex with PKR (PDB 2A1A), in which PKR residues 338-350 are absent. After the genesis of the project, we generated the AlphaFold2-predicted complex of PKR and vaccinia K3, and have become very interested in the β4-β5 linker, a highly diverse region across PKR homologs which includes residues 332-358. However, this region remains unexamined in this manuscript.

Reviewer #2 (Public Review):

Chambers et al. (2024) present a systematic and unbiased approach to explore the evolutionary potential of the human antiviral protein kinase R (PKR) to evade inhibition by a poxviral antagonist while maintaining one of its essential functions.

The authors generated a library of 426 single-nucleotide polymorphism (SNP)-accessible non-synonymous variants of PKR kinase domain and used a yeast-based heterologous virus-host system to assess PKR variants' ability to escape antagonism by the vaccinia virus pseudo-substrate inhibitor K3. The study identified determinant sites in the PKR kinase domain that harbor K3-resistant variants, as well as sites where variation leads to PKR loss of function. The authors found that multiple K3-resistant variants are readily available throughout the domain interface and are enriched at sites under positive selection. They further found some evidence of PKR resilience to viral antagonist diversification. These findings highlight the remarkable adaptability of PKR in response to viral antagonism by mimicry.

Significance of the findings:

The findings are important with implications for various fields, including evolutionary biology, virus-host interfaces, genetic conflicts, and antiviral immunity.

Strength of the evidence:

Convincing methodology using state-of-the-art mutational scanning approach in an elegant and simple setup to address important challenges in virus-host molecular conflicts and protein adaptations.

Strengths:

Systematic and Unbiased Approach:

The study's comprehensive approach to generating and characterizing a large library of PKR variants provides valuable insights into the evolutionary landscape of the PKR kinase domain. By focusing on SNP-accessible variants, the authors ensure the relevance of their findings to naturally occurring mutations.

Identification of Key Sites:

The identification of specific sites in the PKR kinase domain that confer resistance or susceptibility to a poxvirus pseudosubstrate inhibition is a significant contribution.

Evolutionary Implications:

The authors performed meticulous comparative analyses throughout the study between the functional variants from their mutagenesis screen ("prospective") and the evolutionarily-relevant past adaptations ("retrospective").

Experimental Design:

The use of a yeast-based assay to simultaneously assess PKR capacity to induce cell growth arrest and susceptibility/resistance to various VACV K3 alleles is an efficient approach. The combination of this assay with high-throughput sequencing allows for the rapid characterization of a large number of PKR variants.

Areas for Improvement:

(5) Validation of the screen: The results would be strengthened by validating results from the screen on a handful of candidate PKR variants, either using a similar yeast heterologous assay, or - even more powerfully - in another experimental system assaying for similar function (cell translation arrest) or protein-protein interaction.

Thank you for your thoughtful feedback. We agree that additional data to validate our findings would strengthen the manuscript. We have individually screened a handful of PKR variants in duplicate using serial dilution to measure yeast growth, and found that the results generally support our original findings. We have revised the manuscript to include these validation experiments (lines 117-119 of the revised_manuscript.pdf, Supplemental Figure 4).

(6) Evolutionary Data: Beyond residues under positive selection, the screen would allow the authors to also perform a comparative analysis with PKR residues under purifying selection. Because they are assessing one of the most conserved ancestral functions of PKR (i.e. cell translation arrest), it may also be of interest to discuss these highly conserved sites.

This is a great point. We do find that there are regions of the PKR kinase domain that are not amenable to genetic perturbation, namely in the glycine rich loop and active site. We contrast the PKR functional scores at conserved residues under purifying selection with those under positive selection in Figure 2E (lines 141-143).

(7) Mechanistic Insights: While the study identifies key sites and residues involved in vaccinia K3 resistance, it could benefit from further investigation into the underlying molecular mechanisms. The study's reliance on a single experimental approach, deep mutational scanning, may introduce biases and limit the scope of the findings. The authors may acknowledge these limitations in the Discussion.

We agree that further investigation into the underlying molecular mechanisms is warranted and we have revised the manuscript to acknowledge this point in the discussion (lines 284-288).

(8) Viral Diversity: The study focuses on the viral inhibitor K3 from vaccinia. Expanding the analysis to include other viral inhibitors, or exploring the effects of PKR variants on a range of viruses would strengthen and expand the study's conclusions. Would the identified VACV K3-resistant variants also be effective against other viral inhibitors (from pox or other viruses)? or in the context of infection with different viruses? Without such evidence, the authors may check the manuscript is specific about the conclusions.

This is a fantastic question that we are interested in exploring in our future studies. In the manuscript we note a strong correlation between PKR variants that evade vaccinia wild-type K3 and the K3-H47R enhanced allele, but we are curious to know if this holds when tested against other K3 orthologs such as variola virus C3. That said, we have revised the manuscript to clarify this limitation to our findings and specify vaccinia K3 where appropriate.

Reviewer #3 (Public Review):

Summary:

-  This study investigated how genetic variation in the human protein PKR can enable sensitivity or resistance to a viral inhibitor from the vaccinia virus called K3.

-  The authors generated a collection of PKR mutants and characterized their activity in a high-throughput yeast assay to identify 1) which mutations alter PKR's intrinsic biochemical activity, 2) which mutations allow for PKR to escape from viral K3, and 3) which mutations allow for escape from a mutant version of K3 that was previously known to inhibit PKR more efficiently.

-  As a result of this work, the authors generated a detailed map of residues at the PKR-K3 binding surface and the functional impacts of single mutation changes at these sites.

Strengths:

-  Experiments assessed each PKR variant against three different alleles of the K3 antagonist, allowing for a combinatorial view of how each PKR mutant performs in different settings.

-  Nice development of a useful, high-throughput yeast assay to assess PKR activity, with highly detailed methods to facilitate open science and reproducibility.

-  The authors generated a very clean, high-quality, and well-replicated dataset.

Weaknesses:

(9) The authors chose to focus solely on testing residues in or near the PKR-K3 predicted binding interface. As a result, there was only a moderately complex library of PKR mutants tested. The residues selected for investigation were logical, but this limited the potential for observing allosteric interactions or other less-expected results.

First, we greatly appreciate all your feedback on the manuscript, as well as raising this particular point. We agree that this is a moderately complex library of PKR variants, from which we begin to uncover a highly pliable domain with a few specific sites that cannot be altered. We have revised the manuscript to raise this limitation (lines 284-288 of the revised_manuscript.pdf) and encourage additional exploration of the PKR kinase domain.

(10) For residues of interest, some kind of independent validation assay would have been useful to demonstrate that this yeast fitness-based assay is a reliable and quantitative readout of PKR activity.

We agree that additional data to validate our findings would strengthen the manuscript. We have individually screened a handful of PKR variants in duplicate using serial dilution to measure yeast growth, and generally found that the results support our original findings. We have revised the manuscript to include this validation experiment (lines 117-119, Supplemental Figure 4).

(11) As written, the current version of the manuscript could use more context to help a general reader understand 1) what was previously known about these PKR and K3 variants, 2) what was known about how other genes involved in arms races evolve, or 3) what predictions or goals the authors had at the beginning of their experiment. As a result, this paper mostly provides a detailed catalog of variants and their effects. This will be a useful reference for those carrying out detailed, biochemical studies of PKR or K3, but any broader lessons are limited.

Thank you for bringing this to our attention. We have revised the introduction of the manuscript to provide more context regarding previous work demonstrating an evolutionary arms race between PKR and K3 and how single residue changes alter K3 resistance (lines 51-64).

(12) I felt there was a missed opportunity to connect the study's findings to outside evolutionary genetic information, beyond asking if there was overlap with PKR sites that a single previous study had identified as positively selected. For example, are there any signals of balancing selection for PKR? How much allelic diversity is there within humans, and are people typically heterozygous for PKR variants? Relatedly, although PKR variants were tested in isolation here, would the authors expect their functional impacts to be recessive or dominant, and would this alter their interpretations? On the viral diversity side, how much variation is there among K3 sequences? Is there an elevated evolutionary rate, for example, in K3 at residues that contact PKR sites that can confer resistance? None of these additions are essential, but some kind of discussion or analysis like this would help to connect the yeast-based PKR phenotypic assay presented here back to the real-world context for these genes.

We appreciate this suggestion to extend our findings to a broader evolutionary context. There is little allelic diversity of PKR in humans, with all nonsynonymous variation listed in gnomAD being rare. (PKR shows sequence diversity in comparisons across species, including across primates.) Thus, barring the possibility of variation being present in under-studied populations, there is unlikely to be balancing selection on PKR in humans. Our expectation is that beneficial mutations in PKR for evading a pseudosubstrate inhibitor would be dominant, as a small amount of eIF2α phosphorylation is capable of halting translation (Siekierka, PNAS, 1984). There is a recent report citing PKR missense variants associated with dystonia that can be dominantly or recessively inherited (Eemy et al. 2020 PMID 33236446). Elde et al. 2009 (PMID 19043403) notes that poxvirus K3 homologs are under positive selection but no specific residues have been cited to be under positive selection. The lack of allelic diversity in PKR in humans notwithstanding, PKR could experience future selection in the human population as evidenced by its rapid evolution in primates, so we fully agree that a connection to the real-world context is useful. We have noted these topics in the discussion section (lines 289-294).

Reviewer #1 (Recommendations For The Authors):

I have no major criticisms but ask for some clarifications and make some comments about the perceived weaknesses.

(13)  If the authors disagree with my summation that the findings largely replicate what was known, could they detail how the findings differ from what was known about this protein interaction and the major new insights stemming from the study? Currently, the abstract is a little philosophical rather than listing the explicit discoveries of the study.

Thank you again for raising the need for us to clearly convey the novelty of our findings. We have revised the final paragraph in our introduction as described in comment #1.

(14) As the experimental approach is well reported it is unnecessary to confirm the proposed activity by, for instance, measures of Sui2 phosphorylation. However, previous reports have recognised that point mutants of PKR can be differentially expressed. The impact of this potential effect is unknown in the current experimentation as there are no measures of the expression of the different mutant PKR constructs. The large number of constructs used makes this verification onerous. The potential impact could be ameliorated by redundant replacing each residue (hoping different residues have different effects on expression). Still, this limitation of the study should be acknowledged in the text.

We greatly appreciate this comment and agree that this should be made clear in the text, which we have added to the discussion of the manuscript (lines 247-251).

(15) Preceding findings and the modeling in this report recognise an involvement in the kinase insert region (residues 332 to 358) in PKR's interaction with K3 but this region is excluded from the analysis. These residues have been largely disregarded in the preceding analysis (it is absent from the molecular structure of the kinase) so its inclusion here might have lent a more novel aspect or delivered a more complete investigation. Is there a justification for excluding this flexible loop?

The PKR variant library was designed based on the crystal structure of K3 (PDB 1LUZ) aligned to eIF2α in complex with PKR (PDB 2A1A). After the library was designed and made we attained complete predicted structures of PKR in complex with eIF2α and K3, which largely agrees with the predicted crystal structures but contain the additional flexible loops that were not captured in the crystal structures. Though the library studied here does not explore variation in the kinase insert region, we are very interested in doing so in our future studies.

(16)  Could the explanation of the 'PKR functional score' be clarified? The description given within the legend of SF1 was helpful, so could this be replicated earlier in the main body of the text when introducing these experiments? e.g. As PKR activity is toxic to yeast, the number of cells in the pool expressing the functional PKR will decrease over time. Thus the associated barcode read count will also decrease, while the read count for the nonfunctional PKR will increase. This is termed the PKR function score, which will be relatively lower for cells transformed with less active PKR than those with more active PKR.

Thank you for suggesting this clarification, we have revised the manuscript to clarify our definition of the PKR functional score (lines 106-109).

(17)  Another suggestion to clarify this term is to modify the figures. Currently, the intent of the first simulated graph in Fig 1E is clear but the inversion of the response (shown by the transposition of the colours) in the next graph (to the right) is less immediately obvious. Accordingly, the orientation of the 'PKR functional score' is uncertain. Could the authors add text to the rightmost graphic in Figure 1E by, for instance, indicating the PKR activity in the vertical column with text such as 'less active' (at the bottom), 'WT' (in the centre), and 'more activity' (at the top)? Also, the position of the inactive K296R mutant might be added to Figure 2A complementing the positioning of the active WT kinase in the first data graph of this kind.

We appreciate your specific feedback to improve the figures of the manuscript, we have made adjustments to Figure 1E to clarify how we derive the PKR functional scores.

(18) The authors don't use existing structures of PKR in their modelling. However, there is no information about the state of the PKR molecule used for modelling. Specific elements of the kinase domain affect its interaction with K3 so it would be informative to know the orientation of these elements in the model. Could the authors detail the state of pivotal kinase elements in their models? This could involve the alignment of the N- and C-lobes, the orientation of kinase spines (C- and R-spines), and the phosphorylation stasis of residues in the activation loop, or at least the position of this loop in relationship to that adopted in the active dimeric kinase (e.g. PDB-2A1A, 3UIU or 6D3L). Alternatively, crystallographic structures of active inactive PKR could be overlayed with the theoretical structure used for modelling (as supplementary information).

We have revised the manuscript to describe the alignment of the predicted PKR-K3 complex with active and inactive PKR, and we have extended Supplemental Figure 12 with an overlay of the predicted structures with existing structures. We have also added a supplemental data file containing the RMSD values of PKR (from the predicted PKR-K3 complex) aligned to active (PDB 2A1A) and inactive (PDB 3UIU) or unphosphorylated (PDB 6D3L) PKR (5_Structure-Alignment-RMSD-Values.xlsx). We have also provided the AlphaFold2 best model predictions for the PKR-eIF2α complex (6_AF2_PKR-KD_eIF2a.pdb) and PKR-K3 complex (7_AF2_PKR-KD_VACV-K3.pdb). Looking across the RMSD values, the AlphaFold2 model of PKR most closely resembles unphosphorylated PKR (PDB 6D3L) though we note the activation loop is absent from PDB 6D3L and 3UIU. We also aligned the Ser51 phosphoacceptor loop of AlphaFold2 eIF2α model to PDB 1Q46 and we see that the model reflects the pre-phosphorylation state. This loop is expected to interact with the PKR active site, which is not captured in our model and we state this explicitly in the caption of Figure 1 (lines 665-668).

(19) Could some specific residue in Figure 7 be labelled (numbered) to orient the findings? Also, the key in this figure doesn't title the residues coloured white (RE red/black/blue). The white also isn't distinguished from the green (outside the regions targeted for mutagenesis).

Excellent suggestion, we have revised this figure to include labels for the sites to orient the reader and clarify our categorization of PKR residues in the kinase domain.

(20)  Regarding the discussion, the authors adopt the convention of describing K3 as a pseudosubstrate. Although I realize it is common to refer to K3 as a pseudosubstrate, it isn't phosphorylated and binds slightly differently to PKR so alternative descriptors, such as 'a competitive binder', would more accurately present the protein's function. Possibly for this reason, the authors declared an expectation that evolution pressures should shift K3 to precisely mimic EIF2α. However, closer molecular mimicry shouldn't be expected for two reasons. The first is a risk of disrupting other interactions, such as the EIF2 complex. Secondly, equivalent binding to PKR would demote K3 to merely a stoichiometric competitor of EIF2α. In this instance, effective inhibition would require very high levels of K3 to compete with equivalent binding by EIF2α. This would be demanding particularly upon induction of PKR during the interferon response. To be an effective inhibitor K3 has to bind more avidly than EIF2α and merely requires a sufficient overlap with the EIF2α interface on PKR to disrupt this alternative association. This interpretation predicts that K3 is under pressure to bind PKR by a different mechanism than EIF2α.

We appreciate your thoughtful point about the usage of the term pseudosubstrate. Ultimately, we’ve decided to continue using the term due to its historical usage in the field. The question of the optimal extent of mimicry in K3 is a fascinating one, and we greatly appreciate your thoughts. We wholly agree that the possibility of K3 having superior PKR binding relative to eIF2α would be preferable to perfect mimicry. In our Ideas and Speculation section, we propose that benefits towards increasing PKR affinity may need to be balanced against potential loss of host range resulting from overfitting to a given host’s PKR. However, the possibility that reduced mimicry could be selected to avoid disruption of eIF2 function had not occurred to us; thank you for pointing it out!

(21) The discussion of the 'positive selection' of sites is also interesting in this context. To what extent has the proposed positive selection been quantified? My understanding is that all of the EIF2α kinases are conserved and so demonstrate lower levels of residue change that might be expected by random mutagenesis i.e. variance is under negative selection. The relatively higher rate of variance in PKR orthologs compared to other EIF2α kinases could reflect some relaxation of these constraints, rather than positive selection. Greater tolerance of change may stem from PKR 's more sporadic function in the immune response (infrequent and intermittent presence of its activating stimuli) rather than the ceaseless control of homeostasis by the other EIF2α kinases. Also, induction of PKR during the immune response might compensate for mutations that reduce its activity. I believe that the entire clade of extant poxviruses is young relative to the divergence between their hosts. Accordingly, genetic variance in PKR predates these viruses. Although a change in PKR may become fixed if it affords an advantage during infection, such an advantage to the host would be countered by the much higher mutation rates of the virus. This would appear to diminish the opportunity for a specific mutation to dominate a host population and, thereby, to differentiate host species. Rather, pressure to elude control by a rapidly evolving viral factor would favour variation at sites where K3 binds. This speculation offers an alternative perspective to the current discussion that the variance in PKR orthologs stems from positive selection driven by viral infection.

We appreciate this stimulating feedback for discussion. Three of the four eIF2α kinases (HIR, PERK, and GCN2) appear to be under purifying selection (Elde et al. 2009, PMID 19043403), which stand in contrast to PKR. Residues under positive selection have been found throughout PKR, including the dsRNA binding domains, linker region, and the kinase domain. Importantly, the selection analysis from Elde et al. and Rothenburg et al. concluded that positive selection at these sites is more likely than relaxed selection. We agree that poxviruses are young, though we would guess that viral pseudosubstrate inhibition of PKR is ancient. Many viral proteins have been reported to directly interact with PKR, including herpes virus US11, influenza A virus NS1A, hepatitis C virus NS5A, and human immunodeficiency virus Tat. The PKR kinase domain does contain residues under purifying selection that are conserved among all four eIF2α kinases, but it also contains residues under positive selection that interface with the natural substrate eIF2α. Our work suggests that PKR is genetically pliable across several sites in the kinase domain, and we are curious to know if this pliability would hold at the same sites across the other three eIF2α kinases.

(22) The manuscript is very well written but has a small number of typos; e.g. an aberrant 'e' ln 7 of the introduction, capitalise the R in ranavirus on the last line of the fourth paragraph of the discussion, and eIF2α (EIF2α?) is occasionally written as eIFα in the materials&methods.

Thank you for bringing these typos to our attention! We’ve deleted the aberrant ‘e’ in the introduction, capitalized ‘Ranavirus’ in the discussion (line 265), and corrected ‘eIFα’ to ‘eIF2α’ throughout the manuscript.

Reviewer #2 (Recommendations For The Authors):

Additional minor edits or revisions:

(23) Paragraph 3 of the Introduction gives the impression that most of the previous work on the PKR-virus arms race is speculative. However, it is one of the best-described and most convincing examples of virus-host arms races. Can the authors edit the paragraph accordingly?

Thank you for bringing this to our attention. We have revised the third paragraph and strengthened the description of the evolutionary arms race between PKR and viral pseudosubstrate antagonists.

(24) Introduction: PKR has "two" double-stranded RNA binding domains. Can the authors update the text accordingly?

We have updated the manuscript to clarify PKR has two dsRNA binding domains (lines 44-45).

(25) The authors test here for one of the key functions of PKR: cell growth/translation arrest. Because of PKR pleiotropy, the manuscript may be edited accordingly: For example, statements such as "We found few genetic variants render the PKR kinase domain nonfunctional" are too speculative as they may retain other (not tested here) functions.

This is a great suggestion, we have revised the manuscript to specify our definition of nonfunction in the context of our experimental screen (lines 86-92 and 106-109) and acknowledge this limitation in our experimental screen (lines 304-307).

(26) The authors should specify "vaccinia" K3 whenever appropriate.

We appreciate this comment and have revised the manuscript to specify vaccinia K3 where appropriate (e.g. lines 62,66, 70, 80, 108, and 226).

(27) Ref for ACE2 diversification may include Frank et al 2022 PMID: 35892217.

Thank you for pointing us to this paper, we have included it as a reference in the manuscript (line 277).

(28) Positive selection of PKR as referred to by the authors corresponds to analyses performed in primates. As shown by several studies, the sites under positive selection may vary according to host orders. Can the authors specify this ("primate") in their manuscript? And/or shortly discuss this aspect.

Thank you for raising this point. In the manuscript we performed our analysis using vertebrate sites under positive selection as identified in Rothenburg et al. 2009 PMID 19043413 (lines 51 and figure legends). We performed the same analysis using sites under positive selection in primates (as identified by Elde et al. 2009 PMID 19043403) and again found a significant difference in PKR functional scores versus K3. We have revised the manuscript to clarify our use of vertebrate sites under positive selection (line 80-81).

(29) We view deep mutational scanning experiments as a complementary approach to positive selection": The authors should edit this and acknowledge previous and similar work of other antiviral factors, in particular one of the first studies of this kind on MxA (Colon-Thillet et al 2019 PMID: 31574080), and TRIM5 (Tenthorey et al 2020 PMID: 32930662).

Thank you for raising up these two papers, which we acknowledge in the revised manuscript (line 299).

(30) We believe Figure S7 brings important results and should be placed in the Main.

We appreciate this suggestion, and have moved the contents of the former supplementary Figure 7 to the main text, in Figure 6.

(31) The title may specify "poxvirus".

Thank you for the suggestion to specify the nature of our experiment, we have adjusted the title to: Systematic genetic characterization of the human PKR kinase domain highlights its functional malleability to escape a poxvirus substrate mimic (line 3).

Reviewer #3 (Recommendations For The Authors):

(32) No line numbers or page numbers are provided, which makes it difficult to comment.

We sincerely apologize for this oversight and have included line numbers in our revised manuscript as well as the tracked changes document.

(33) In the introduction, I recommend defining evolutionary arms races more clearly for a broad audience.

Thank you for this suggestion. We have revised the manuscript in the first and third paragraphs to more clearly introduce readers to the concept of an evolutionary arms race.

(34) The introduction could use a clearer statement of the question being considered and the gap in knowledge this paper is trying to address. Currently, the third paragraph includes many facts about PKR and the fourth paragraph jumps straight into the approach and results. Some elaboration here would convey the significance of the study more clearly. As is, the introduction reads a bit like "We wanted to do deep mutational scanning. PKR seemed like an ok protein to look at", rather than conveying a scientific question.

This is a great suggestion to improve the introduction section. We have heavily revised the third and fourth paragraphs of the introduction to clarify the motivation, approach, and significance of our work.

(35) Relatedly, did the authors have any hypotheses at the start of the experiment about what kinds of results they expected? e.g. What parts of PKR would be most likely to generate escape mutants? Would resistant mutants be rare or common? etc? This would help the reader to understand which results are expected vs. surprising.

These are all great questions. We have revised the introduction of the manuscript to point out that previous studies have characterized a handful of PKR variants that evade vaccinia K3, and these variants were made at sites found to be under positive selection (lines 60-64).

(36) A description of the different K3 variants and information about why they were chosen for study should also be added to the Introduction. It was not until Figure 5 that the reader was told that K3-H47R was the same as the 'enhanced' K3 allele you are testing.

Thank you for bringing this to our attention, we have revised the introduction to clarify the experimental conditions (lines 65-67) and specify K3-H47R as the enhanced allele earlier in the manuscript (line 100).

(37) Does every PKR include just a single point mutation? It would be nice to see data about the number and types of mutations in each PRK window added to Supplemental Figure 1.

Thank you for the suggestion to improve this figure. Every PKR variant that we track has a single point mutation that generates a nonsynonymous mutation. In our PacBio sequencing of the PKR variant library we identified a few off-target variants or sequences with multiple variants, but we identified the barcodes linked to those constructs and discarded those variants in our analysis. We have revised Supplemental Figure 1 to include the number and types of mutations made at each PKR window.

(38) In terms of the paper's logical flow, personally, I would expect to begin by testing which variants break PKR's function (Figure 3) and then proceeding to see which variants allow for K3 escape (Figure 2). Consider swapping the order of these sections.

Thank you for this suggestion, and we can appreciate how the flow of the manuscript may be improved by swapping Figures 2 and 3. We have decided to maintain the current order of the figures because we use Figure 3 to emphasize the distinction of PKR sites that are nonfunctional versus susceptible to vaccinia K3.

(39) Figure 3A seems like a less-informative version of Figure 4A, recommend combining these two. Same comment with Figure 5A and Figure 6A.

We appreciate this specific feedback for the figures. Though there are similarities between figure panels (e.g. 3A and 4A) we use them to emphasize different points in each figure. For example, in Figure 3 we emphasize the general lack of variants that impair PKR kinase activity, and in Figure 4 we distinguish kinase-impaired variants from K3-susceptible variants. For this reason, and given space constraints, we have chosen to maintain the figures separately. We did decide to move the former Figure 6 to the supplement.

(40) In general, it felt like there was a lot of repetition/re-graphing of the same data in Figures 3-6. I recommend condensing some of this, and/or moving some of the panels to supplemental figures.

Thank you for your suggestion, we have revised the manuscript and have moved Figure 6 to Supplemental Figure 7.

(41) In contrast, Supplemental Figure 7 is helpful for understanding the distribution of the data. Recommend moving to the main text.

This is a great recommendation, and we have moved Supplemental Figure 7 into Figure 6.

(42) How do the authors interpret an enrichment of positively selected sites in K3-resistant variants, but not K3-H74R-resistant variants? This seems important. Please explain.

Thank you for this suggestion to improve the manuscript; we agree that this observation warranted further exploration. We found a strong correlation in PKR functional scores between K3 WT and K3-H47R, and with that we find sites under positive selection that are resistant to K3 WT are also resistant to K3-H47R. The lack of enrichment at positively selected sites appears to be caused by collapsed dynamic range between PKR wild-type-like and nonfunctional variants in the K3-H47R screen. We have revised the manuscript to clarify this point (line 202-204).

(43) Discussion: The authors compare and contrast between PKR and ACE2, but it would be worth mentioning other examples of genes involved in antiviral arms races wherein flexible, unstructured loops are functionally important and are hotspots of positive selection (e.g. MxA, NLRP1, etc).

We greatly appreciate this suggestion to improve the discussion. We note this contrast between the PKR kinase domain and the flexible linkers of MxA and NLRP1 in the revised manuscript (lines 273-274).

(44) Speculation section: What is the host range of the vaccinia virus? Is it likely to be a generalist amongst many species' PKRs (and if so, how variable are those PKRs)? Would be worth mentioning for context if you want to discuss this topic.

Thank you for raising this question. Vaccinia virus is the most well studied of the poxviruses, having been used as a vaccine to eradicate smallpox, and serves as a model poxvirus. Vaccinia virus has a broad host range, and though the name vaccinia derives from the Latin word “vacca” for cow the viruses origin remains uncertain (Smith 2007 https://doi.org/10.1007/978-3-7643-7557-7_1). has been used to eradicate smallpox as a vaccine and serves as a model poxvirus. Thought the natural host is unknown, it appears to be a general inhibitor of vertebrate PKRs The natural host of vaccinia virus is unknown, though there is some evidence to suggest it may be native to rabbits and does appear to be generalist.

(45) Many papers in this field discuss interactions between PKR and K3L, rather than K3. I understand that this is a gene vs. protein nomenclature issue, but consider matching the K3L literature to make this paper easier to find.

Thank you for bringing this to our attention. We have revised the manuscript to specify that vaccinia K3 is expressed from the K3L gene in both the abstract (line 26) and the introduction (line 56) to help make this paper easier to find when searching for “K3L” literature.

(46) Which PKR sequence was used as the wild-type background?

This is a great question. We used the predominant allele circulating in the human population represented by Genbank m85294.1:31-1686. We cite this sequence in the Methods (line 421) and have added it to the results section as well (lines 84).

(47) Figure 1C: the black dashed line is difficult to see. Recommend changing the colors in 1A-1C.

Thank you for this suggestion, we have changed the dashed lines from black to white to make them more distinguishable.

(48) Figure 1D: Part of the point of this figure is to convey overlaps between sites under selection, K3 contact sites, and eIF2alpha contact sites, but at this scale, many of the triangles overlap. It is therefore impossible to tell if the same sites are contacted vs. nearby sites. Perhaps the zoomed-in panels showing each of the four windows in the subsequent figures are sufficient?

Thank you for bringing this to our attention. We have scaled the triangles down to reduce their overlap in Figure 1D and list all sites of interest (predicted eIF2α and vaccinia contacts, conserved sites, and positive selection sites) in the Materials and Methods section “Predicted PKR complexes and substrate contacts”.

(49) Figure 1E: under "1,293 Unique Combinations", there is a line between the PKR and K3 variants, which makes it look like they are expressed as a fusion protein. I believe these proteins were expressed from the same plasmid, but not as a fusion, so I recommend re-drawing. Then in the graph, the y-axis says "PKR abundance", but from the figure, it is not clear that this refers to relative abundance in a yeast pool. Perhaps "yeast growth" or similar would be clearer?

Thank you for the specific feedback to improve Figure 1. We have made the suggested edits to clarify that PKR and vaccinia K3 are not fused but each is expressed from their own promoter. We have also changed the y-axis from “PKR Abundance” to “Yeast Growth”.

Reviewer #1 (Public review):

Summary:

The report examines the control of the antiviral RNA-activated protein kinase, PKR, by the Vaccinia virus K3 protein. K3 binds to PKR, hindering its ability to control protein translation by blocking its phosphorylation of the eukaryotic initiation factor EIF2α. Kinase function is probed by saturation mutation of the K3/EIF2α-binding surface on PKR, guided by models of their interaction. The findings identify specific residues at the predicted interface that asymmetrically influence repression by K3 and the phosphorylation of EIF2α. This recognises the potential of PKR alleles to resist control by the viral virulence factor.

Strengths:

The experimentation is diligent, generating and screening many point mutants to identify residues at the interface between PKR and EIF2α or K3 that distinguishes PKR's phosphor control of its substrate from the antithetical interaction with the viral virulence factor.

Weaknesses:

The protein interaction between PKR and K3 has already been well-explored through phylogenetic and functional analyses and molecular dynamics studies, as well as with more limited site-directed mutational studies using the same experimental assays. Accordingly, the findings are not pioneering but reinforce and extend what had previously been established.

The authors responded to this comment by pointing out that their more comprehensive screen better defined the extent of the plasticity of the K3/EIF2α-binding surface on PKR.

Also in their response, the authors added the caveat that the equivalent expression of the different PKR mutants has not been verified, added information clarifying the states of the model proteins compared to their determined molecular structures, and provided clarifications or responses to all other questions.

I question eLife's assessment that the development of the yeast-based assay is a key advancement of this report, as this assay has been used for over 30 years.

eLife Assessment

This important revised report describes the control of the activity of the RNA-activated protein kinase, PKR, by the Vaccinia virus K3 protein. A strength of the manuscript is the powerful combination of a classic yeast-based assay with high-throughput sequencing and its convincing experimental use to characterize large numbers of PKR variants, now with improved controls for potential biases. A minor current limitation that the authors may address in the future is the scope of the screen in terms of the segments of PKR included.

Reviewer #2 (Public review):

Chambers et al. (2024) present a systematic and unbiased approach to explore the evolutionary potential of the kinase domain of the human antiviral protein kinase R (PKR) to evade inhibition by a poxviral antagonist while maintaining one of its essential functions.

The authors generated a library of 426 single-nucleotide polymorphism (SNP)-accessible non-synonymous variants of PKR kinase domain and used a yeast-based heterologous virus-host system to assess PKR variants' ability to escape antagonism by the vaccinia virus pseudo-substrate inhibitor K3. The study identified determinant sites in the PKR kinase domain that harbor K3-resistant variants, as well as sites where variation leads to PKR loss of function. The authors found that multiple K3-resistant variants are readily available throughout the domain interface and are enriched at sites under positive selection. They further found some evidence of PKR resilience to viral antagonist diversification. These findings highlight the remarkable adaptability of PKR in response to viral antagonism by mimicry.

Significance of the findings: The findings are important with implications to various fields, including evolutionary biology, virus-host interfaces, genetic conflicts, antiviral immunity.

Strength of the evidence: Convincing methodology using state-of-the-art mutational scanning approach in an elegant and simple setup to address important challenges in virus-host molecular conflicts and protein adaptations.

Strengths

Systematic and Unbiased Approach: The study's comprehensive approach to generating and characterizing a large library of PKR variants provides valuable insights into the evolutionary landscape of PKR kinase domain. By focusing on SNP-accessible variants, the authors ensure the relevance of their findings to naturally occurring mutations.<br /> Identification of Key Sites: The identification of specific sites in the PKR kinase domain that confer resistance or susceptibility to a poxvirus pseudosubstrate inhibition is a significant contribution.<br /> Evolutionary Implications: The authors performed meticulous comparative analyses throughout the study between the functional variants from their mutagenesis screen ("prospective") and the evolutionarily-relevant past adaptations ("retrospective").<br /> Experimental Design: The use of a yeast-based assay to simultaneously assess PKR capacity to induce cell growth arrest and susceptibility/resistance to various VACV K3 alleles is an efficient approach. The combination of this assay with high-throughput sequencing allows for the rapid characterization of a large number of PKR variants.

Areas of improvement

Validation of the screen: In the revised version, the authors now provide the results of two independent experiments in a complete yeast growth assay on a handful of candidates to control the screen's results. This strengthens the direct findings from the screen. It would strengthen the study to complement this validation by another method to assess PKR functions; for example, in human PKR-KO cells, because results between yeast and human cells can differ. These limitations are now acknowledged in the revised version.<br /> Evolutionary Data: Beyond residues under positive selection, the screen allows the authors to also perform a comparative analysis with PKR residues under purifying selection. Because they are assessing one of the most conserved ancestral functions of PKR (i.e. cell translation arrest), it may also be of interest to discuss these highly conserved sites. The authors now discuss the implications for the conserved residues.<br /> Mechanistic insights and viral diversity: While the study identifies key sites and residues involved in vaccinia K3 resistance, it could benefit from further investigation into the underlying molecular mechanisms and the diversity of viral antagonists. The authors have now acknowledged these limitations in the Discussion and updated the manuscript to be more specific. These exciting research avenues will be the objectives of a next study.

Overall Assessment

The systematic approach, identification of key sites, and evolutionary implications are all notable strengths. While there is room for a stronger validation of the functions and further investigation into the mechanistic details and broader viral diversity, the findings are robust and already provide important advancements. The manuscript is well-written and clear, and the revised figures are informative and improved.

I think that maybe during that quiet time where students are instructed to work on their schoolwork, they can put their phones away in their backpack, but taking the phone away from day to day probably isn't the most plausible answer.

This in itself is more then enough reason to ban cell phons to ban cell phones from students but in my opinion thats the reason parents are her to monitor and make sure they give they're own kids a certain amount of screen time per day.

could add a link here to the section in "Create and Manage Chapters" that refers to the "Status & Visibility" panel

should this be part of this chapter or rather the "Edit" part? as it's part of the Satus & Visiblity panel, maybe leave it in this chapter, but refer to it in the "Edit Content with Visual and Text Editor" chapter?

adjusted title - anything related to using the editor should be in the chapter https://university.pressbooks.pub/guidetestedits/chapter/edit-content-with-the-visual-text-editors/ or other chapters in that part

There is an entire chapter on this: https://university.pressbooks.pub/guidetestedits/chapter/edit-content-with-the-visual-text-editors/ I would suggest removing this section.

definitions, specifically of tetrahedron, equicomposability, and equicomplementability

As a parent I understand that each child is different from their siblings and within the classroom I can see how this will be difficult to handle the diversity and meet the needs of every child without a co teacher.

If we’re committed to the success of every child, we must acknowledge the uneven playing field that exists for many: ELLs, students with special needs, children experiencing trauma or relentless poverty…. There are 6 Steps Toward Equity!!

Even if the song is boring, it is still enough to bring sailors to the ocean. They want to hear more of this tantalizing story, to hear the secret end.

Providing direct instructions to look further into the information shows a lack of bias and instills a sense of credibility into the reader. This allows the reader to understand the information about the amendment and voting rights of felons. This site may not be entirely unbiased, as they do seem to be pushing a certain agenda and are not a domain known to be reliable (.org, .gov, etc), but the information does seem to be presented in a straightforward way that does not indicate any bias. It offers little information in one way or another, simply presenting the information about the process to pass this amendment to reinstate the voting rights of felons.

This excerpt links a pdf of the revised rules. This not only gives interested readers the opportunity to see the rules in question, but also gives credibility to the argument and shows an unbiased perspective. This also helps verify the information in the article, as only one look into the pdf will exemplify the points being made.

This links directly to a .gov site of the Florida House of Representatives. This not only explains what the bill was, but details the history of the bill and additional information, such as statutes referenced. Linking this is a good way to demonstrate the validity of the article's claims, but to provide official information from a reliable source.

How can we as faculty educate our students about the process of receiving feedback and working in an open space - when your work can be critiqued or changed in dynamic open spaces v. traditional peer reviewed process? How are we talking about the inherent risks involved and the gatekeeping processes that are different in a space like wikipedia compared to a peer reviewed publication?

for - question - @Michael - Is this the Zebras Unite you are referring to? - https://zebrasunite.coop/ - If so, that brings up another question: - What is the difference between Fair Share Commons and a Cooperative?

Nature is uncaring. Whoever is unprepared to deal with natures wrath will eventually face death when placed in extreme circumstances.

Extreme circumstances can lead to the removal of civilized ideals and morals.

The narrator describes what a professional should know but we are unaware of what exactly the character knows. we can assume that he is knowledgeable, but we are unaware of how knowledgeable.

This reflects the uncaring identity of nature by demonstrating how unpredictable and uncertain the outcomes of natural events are.

This description of the environment creates the conflict between man vs nature.

eLife Assessment

This study retrospectively analyzed clinical data to develop a risk prediction model for pulmonary hypertension in high-altitude populations. The evidence is solid, and the findings are useful and hold clinical significance as the model can be used for intuitive and individualized prediction of pulmonary hypertension risk in these populations.

Reviewer #1 (Public Review):

Summary:

This study retrospectively analyzed clinical data to develop a risk prediction model for pulmonary hypertension in high-altitude populations. This finding holds clinical significance as it can be used for intuitive and individualized prediction of pulmonary hypertension risk in these populations. The strength of evidence is high, utilizing a large cohort of 6,603 patients and employing statistical methods such as LASSO regression. The model demonstrates satisfactory performance metrics, including AUC values and calibration curves, enhancing its clinical applicability.

Strengths:

(1) Large Sample Size: The study utilizes a substantial cohort of 6,603 subjects, enhancing the reliability and generalizability of the findings.

(2) Robust Methodology: The use of advanced statistical techniques, including least absolute shrinkage and selection operator (LASSO) regression and multivariate logistic regression, ensures the selection of optimal predictive features.

(3) Clinical Utility: The developed nomograms are user-friendly and can be easily implemented in clinical settings, particularly in resource-limited high-altitude regions.

(4) Performance Metrics: The models demonstrate satisfactory performance, with strong AUC values and well-calibrated curves, indicating accurate predictions.

Weaknesses:

(1) Lack of External Validation: The models were validated internally, but external validation with cohorts from other high-altitude regions is necessary to confirm their generalizability.

(2) Simplistic Predictors: The reliance on ECG and basic demographic data may overlook other potential predictors that could improve the models' accuracy and predictive power.

(3) Regional Specificity: The study's cohort is limited to Tibet, and the findings may not be directly applicable to other high-altitude populations without further validation.

Comments on revised version:

The authors have made revisions in response to the primary concerns raised in the initial review, leading to significant improvements in the manuscript's technical accuracy, formatting consistency, and overall clarity. They have provided a detailed explanation of the selection criteria for the final model variables, which has enhanced the transparency and robustness of the study's methodology. Additionally, the authors have acknowledged the limitation of lacking external validation in cohorts from other high-altitude regions and outlined their plans for future research to address this issue.

Author response:

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

Reviewer #1:

(1) Correct capitalization errors, ensuring the first letter of each sentence is capitalized.

Thank you for your comment. We have corrected capitalization errors.

(2) Ensure that all technical terms and abbreviations are introduced in full when first mentioned and consistently used throughout the text.

Thank you for your comment. we have checked and corrected the issue.

(3) Review the manuscript for grammatical errors and improve sentence structures to enhance readability.

Thank you for your comment. we have checked and corrected the issue.

(4) Ensure all figures referenced in the text, such as Fig. 3G, are appropriately discussed and integrated into the narrative.

Thank you for your comment. we have discussed and integrated Fig. 3G into the narrative (Page 12, Line 162-166).

(5) Maintain consistent formatting, including first-line indentation and spacing before paragraphs, to improve the document's visual coherence.

Thank you for your comment. we have checked and corrected the issue.

(6) Provide additional explanations for the selection criteria of final model variables, particularly the rationale behind choosing the λ_1se criterion in the LASSO regression.

Thank you for your comment. we have provided explanations for choosing the λ_1se criterion in the LASSO regression (Page 25, Line 315-316; Page 27, Line 363-364).

(7) Conduct validation studies with cohorts from other high-altitude regions to assess the generalizability and robustness of the prediction models.

Thank you for your comment. The lack of validation of cohorts from other high-altitude regions is a weakness in this study, and in our follow-up study, we will conduct external validation with cohorts from more other high-altitude regions to assess the generalizability and robustness of our prediction models.

https://www.knowledgegraph.tech/speakers/denny-vrandecic/

https://ruben.verborgh.org/<br /> Ruben Verborgh

Mentioned by Flancian

https://www.linkedin.com/in/rubenverborgh/<br /> Ruben Verborgh

Denny Vrandečić

for - quote - we are in a back-loop phase - From Complex Regions to Complex Worlds - Crawford Stanley Holling - 2004 - creative alternatives - liminal spaces - rapid whole system change

comment - This is important for discussion for change actors working in liminal spaces attempting to give birth to creative alternatives

for - planetary adaptive cycle - entering back-loop phase - paper - From Complex Regions to Complex Worlds - Crawford Stanley Holling - 2004 - from - essay - The End of Scarcity? From ‘Polycrisis’ to Planetary Phase Shift - Nafeez Ahmed - 2024

from - essay - The End of Scarcity? From ‘Polycrisis’ to Planetary Phase Shift - Nafeez Ahmed - 2024 - https://hyp.is/okOeDJFqEe-9ZsMEsKWR9w/ageoftransformation.org/the-end-of-scarcity-from-polycrisis-to-planetary-phase-shift/

Know and Master Your Social Media Data Flow by [[Louis Gray]]

See commentary at https://boffosocko.com/2017/04/11/a-new-way-to-know-and-master-your-social-media-flow/

We have Renaissance humanism from the 1500s. We need to have a dose of Digital humanism in the 2000s.

A Minimum Viable Ecosystem for collective intelligence by [[Mathew Lowry]]

Relation to Louis Gray's 2009 diagram/post: https://boffosocko.com/2017/04/11/a-new-way-to-know-and-master-your-social-media-flow/

-significant issues with managing peace with natives -replaced amherst with thomas gage -inspired by johnsons's peae making with the natives, gage tried to repair peace with natives -reflected diplomacy in colonial governement

-stopped supplying gifts to Natives, important for maintaining good relations -messed up their relationship -dont know why they wanted to stop giving them gifts, doesn't make any sense

-granted new orleans and looudiana to spain to satisfy spanish allies -wanted to please spain to get their help to fight against the brirish

-they were irresponsible and led to national debt -contributed to tensions with the british empire

Why AI must be decolonized to fulfill its true potential by [[Mahlet Zimeta]], Data and technology policy expert, Freelance

I think the assertion that they are always aware also "excuses" them from doing the work non-religious people do when they have acted poorly. To seek forgiveness from those they have directly impacted rather than from God. Or even seek feedback, alternative behaviors, and approaches from their peers. It, to a certain extent, absolves them from the expected methods of relationship mending because their relationship is first and foremost with God.

WHAT!!

This makes me so sad and angry, not only are these children being brought into environments that preach inherent badness, but the people who are supposed to raise and care for them have had it engrained in them that they are inherent sinners, liars, etc. This fosters a sense of distrust and negative perception of children in adults, including their parents. Both the home and community are infiltrated by the idea that children were born depraved and will act accordingly, giving children no way to separate themselves from that opinion. Because of this, it becomes absorbed into their self-perception.

Is this true across all cases of PTSD? Has investigation into veterans with PTSD revealed the same root symptom? I have doubts about this, and it's caused a little bit of doubt about the reliability of this source.

I think even if Christianity were to advocate for a sense of self formed by guilt, this would still result in an equivalently damaging impact. However, it seems that these researchers disagree. Why do they disagree? If shame and guilt are so often intertwined and mistaken for each other in religion (and outside of it), how are the impacts different?

The idea that small children are experiencing possibly repeated bodily feelings of shame and are unable to verbalize or truly process that negative experience through communication or thought makes me incredibly sad.

If I were to write my essay on this topic, I would need to be very mindful of my biases toward this topic and approach it very objectively. I am aware of my biases coming through here and the opinions I have formed impacting my ability to believe the goals of religious practice as stated here.

I'm not sure I understand this fully. I need to do more research on spiritual bypassing.

I think, especially if someone was exposed to such thought processes in their formative years, it becomes almost an instinct to question, doubt, and perceive actions/thoughts as shameful or "wrong," which, even after leaving the practice, still impacts an individual especially when gone untreated/managed.

This brings me back to my statement about spiritual harm and the possibility that environments in which harm is done may return to being a safe or comfortable space because there is space granted to talking about it to either troubleshoot or soothe/protect an individual. In religious spaces, this is often not the case and may even cause more shaming to take place.

True, however, is this type of harm as widely accepted as religious teachings which may result in harm, and is support more accessible within that space which allows the space to feel safe enough to still engage in?

The concept and definition we have so far of "spiritual harm," I would argue, is integral in understanding how religious trauma comes about from the complex and chronic repeat exposure to religious teachings and because we are a spiritual being with psyche vulnerable to harm on a level other than physical.

Still sees himself above

But they are more sophisticated than the "captain"

Its giving ghost ship

No captain spells a very bad time

as in the ship?

From where?

Bad at steering

What?

Is Herman Melville racist?"

How does this relate to the Fediverse?

There's a nascent movement within the Fediverse precisely to experiment with 'insularity', building smaller opt-in-federation-based networks.

Except the loss incurred by the owner, who has had their rights as the owner abrogated by the trespasser - whilst more of an abstract loss it is a loss nonetheless

The line of resurrection and heaven is not clear ( the moment when you go to heaven )

Most of the heresies come from a logical breakdown of a. paradox

Latin A describes how the crucification was a plat by satin that went wrong

We are troubled by the description of the cross and crucification

https://en.wikipedia.org/wiki/Data_Transfer_Project

see GitHub repo at https://github.com/google/data-transfer-project

https://tana.inc/docs/supertags

Meet Capacities, the do-it-all productivity app taking on Notion by [[JR Raphael]]

Please watch this video.

And using this information and insight to help pursue the goals of the organization and the greater good.

Does anyone come to mind for you when you read this paragraph?

With the way this is phrased it seems obvious that we should have an inward mindset and we may initially balk at the idea that we might be working from an outward mindset. However, this can show up in subtle ways. For example, much of our job is trying to get people to change old habits for new ones. We often lead with processes, rules, and exactly WHAT we want people to do without taking into account why they do what they do and WHY they shoud change.

The following videos are optional: * Raytheon (video 1) * Tubular Steel (video 2)

This video is recommended: * Kansas City Police Department

Version 2 of this preprint has been peer-reviewed and recommended by Peer Community in Genomics.<br /> See the peer reviews and the recommendation.

The Unjournal supports this. I think we have done this in more depth and rigor than other initiatives in economics and social science

Do you see the poem as a celebration of creativity or a warning about potential hard truths one might come across during the creative process?

Describes the transcendent experience of being all consumed within your creativity.

Another contradiction to illustrate the complex reality of his creativity.

The deep true poetic vision he wants to create will never be perfect and is always trying to run.

Shift to more quiet tone

Coleridge plays between moments of stasis and intense movement. It relates to his creative process by saying that sometimes you don't know what to do, while others, it is clear what direction you should be heading.

Contradiction between his words illustrates how his creativity is not straightforward and even he can't fully describe the ways his mind works.

Represents the mystery of this place and how it can't be fully understood. Could be mapped to the subconscious part of his imagination.

emphasis on the idyllic nature of this place.

It's lovely to see how this has not changed so much even today, especially with people in England crazy for sports like soccer/football and rugby.

Humans have always had the tendency to find ways to kickback and relaxed, and from this phrase we can see the author's view of what the ideal urban society is: serious and practical at times, while recreational at other times. If I were to compare it to modern day society I feel like the desire to seek constant recreation and fun can be seen especially when it comes to overwork and stress found in the expectations of modern society, which seems like an unhealthy balance and maybe, I think, people of the past would have found this type of living quite depressing and scary.

The wind changes and so too does the speaker's internal feelings in that it now has a more manageable as if paying attention to the tumultuous sounds of the wind was akin to a release of some sort.

Here, the speaker realizes that they must not rely on nature to lift his spirits and instead needs to gain real passion and life from within.

Serves as foreshadowing for the "deadly storm" about to take place which correlates to the speaker's future explanation of their emotional distress.

Emphasizes the speaker's disconnect with nature as the speaker can see the moon, yet can not feel its presence. This contributes to the speakers overwhelming sense of isolation.

These lines have a contrast in that Coleridge first explains how there were once positive feelings associated with hope and imagination; however, now, these feelings have morphed into despair and a lack of an ability to connect with nature.

Because of the injury the Buccaneers may try to make a trade which would impact many people in both in the NFL, fans, and fantasy football players

This event just happened on Monday night so it is an example of timeliness

This comment shows prominence as Chris Godwin is a star player for the Tampa Bay Buccaneers.

for - stats - green growth - 2024 - Global South vs Global North

stats - green growth - 2024 - Global South has - 60% of world population - 20% of fossil fuel production - fossil fuel production in decline - 70% of global renewable resource potential - In 2024, 87% of capex of electricity generation is renewable - From 2019 to 2024, renewable energy has grown 23% annually and now supplies 9% of its electricity - 17% of Global South has already overtaken Global North in % of renewable electricty generation

for - Report - Powering Up the Global South - Rocky Mountain Institute - RMI - 2024 - Vikram Singh - Kingsmill Bond

Summary - This report shows that the Global South is adopting cleantech faster than the Global North

Radyografideki patoloji bu faktörlerden dolayı görülebilir

main topic

eLife Assessment

The authors analyze a comprehensive cohort of human plasma samples to identify an extracellular vesicles protein signature for early diagnosis of pancreatic cancer. The application of liquid biopsies is valuable, and the work addresses a key clinical problem as pancreas cancer is often diagnosed in late stages. The strength of evidence is solid. Altogether, this work supports the potential use of extracellular vesicles in clinical settings, with promising value to scientists and clinicians.

Reviewer #1 (Public review):

This study presents a large cohort of plasma-derived extracellular vesicle samples from 124 individuals, including patients with PDAC, benign pancreatic diseases and controls. The authors identified a panel of protein markers for the early detection of pancreatic cancer and validated in an external cohort.

Reviewer #2 (Public review):

This work investigates the use of extracellular vesicles (EVs) in blood as a noninvasive 'liquid biopsy' to aid in differentiation of patients with pancreatic cancer (PDAC) from those with benign pancreatic disease and healthy controls, an important clinical question where biopsies are frequently non-diagnostic. The use of extracellular vesicles as biomarkers of disease has been gaining interest in recent history, with a variety of published methods and techniques, looking at a variety of different compositions ('the molecular cargo') of EVs particularly in cancer diagnosis (Shah R, et al, N Engl J Med 2018; 379:958-966).

This study adds to the growing body of evidence in using EVs for earlier detection of pancreatic cancer, identifying both new and known proteins of interest. Limitations in studying EVs in general include dealing with low concentrations in circulation and identifying the most relevant molecular cargo. This study provides validation of assaying EVs using the novel EVtrap method (Extracellular Vesicles Total Recovery And Purification), which the authors show to be more efficient than current standard techniques and potentially more scalable for larger clinical studies.

The strength of this study is in its numbers - the authors worked with a cohort of 124 cases, 93 of them which were PDAC samples, which considered large for an EV study (Jia, E et al. BMC Cancer 22, 573 (2022)). The benign disease group (n=20, between chronic pancreatitis and IPMNs) and healthy control groups (n=11) were relatively small, but the authors were not only able to identify candidate biomarkers for diagnosis that clearly stood out in the PDAC cohort, but also validate it in an independent cohort of 36 new subjects. Proteins they've identified as associated with pancreatic cancer over benign disease included PDCD6IP, SERPINA12 and RUVBL2. They were even able to identify a set of EV proteins associated with metastasis and poorer prognosis , which include the proteins PSMB4, RUVBL2 and ANKAR and CRP, RALB and CD55. Their 7-EV protein signature yielded an 89% prediction accuracy for the diagnosis of PDAC against a background of benign pancreatic diseases that is compelling and comparable to other studies in the literature (Jia, E. et al. BMC Cancer 22, 573 (2022)).

The limitations of this study are its containment within a single institution - further studies are warranted to apply the authors' 7-EV protein PRAC panel to multiple other cases at other institutions in a larger cohort.

Author response:

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

Reviewer #1 (Public Review):

In this manuscript, Bockorny, Muthuswamy, and Huang et al. performed proteomics analysis of plasma extracellular vesicles (EVs) from pancreatic ductal adenocarcinoma (PDAC) patients and patients with benign pancreatic diseases (chronic pancreatitis and intraductal papillary mucinous neoplasm, IPMN) to develop a 7-EV protein signature that predicts PDAC. Moreover, the authors identified PSMB4, RUVBL2, and ANKAR as being associated with metastasis. These studies provide important insight into alterations of EVs during PDAC progression and the data supporting predict PDAC with EV protein signatures are solid. However, there are certain concerns regarding the rigor and novelty of the data analysis and interpretation, as well as the clinical implications, as detailed below.

(1) Plasma EVs were characterized by transmission electron microscopy and nanoparticle tracking analysis to confirm their morphology and size. The authors should also include an analysis of putative EV markers (e.g., tetraspanins, syntenin, ALIX, etc.) to confirm that the analyzed particles are EVs.

We thank the reviewer for this comment. In the previous study from our co-authors who developed EVtrap method (PMID:32396726), they used electron microscopy and NTA , as well as quantification of typical EV protein markers, such as CD9, to confirm that particles isolated using EVtrap had typical characteristics of the extracellular vesicles. As such, these experiments were not replicated here. We added the following statement to the manuscript:

“Previous analyses using electron microscopy and nanoparticle tracking also confirmed that the vast majority of particles isolated by EVtrap had diameters between 100-200 nm, consistent with exosomes (PMID:32396726). In addition, EVtrap isolates demonstrates higher abundance of CD9, a common exosome marker, as compared to isolates from other traditional EV isolation methods such as size exclusion chromatography and ultracentrifugation (PMID:32396726)”

(2) The authors identified multiple over-expressed proteins in PDAC based on their foldchange and p-value; however, due to the heterogeneity of PDAC, it is necessary to show a heatmap displaying their abundance in all samples. High fold change does not necessarily indicate consistently high abundance in all PDAC samples.

We thank the reviewer for this suggestion. We have now included the heatmap in the new Supplementary Figure 3.

(3) PSMB4, RUVBL2, and ANKAR were identified as being associated with metastasis. The authors state that they intended to distinguish early and late-stage cancer samples, but it is unclear why they chose to compare metastatic and non-metastatic samples, as the non-metastatic group also includes late-stage cancer samples. This sentence should be rephrased to more accurately reflect the sample types profiled.

We thank the reviewer for pointing this out. We would like to clarify that this analyses shown in Figures 3B and 3C pertain to patients with Metastatic vs Non-Metastatic disease, not early versus late stage. We edited the text to ensure this information is clear.

(4) Non-metastatic and metastatic patients were separated based on global protein abundance. The samples within each group display significant heterogeneity, with some samples displaying similar patterns although they were classified into different groups (Figure 3A), and the samples within the same group, particularly the metastasis group, did not consistently exhibit similar patterns of protein abundance. The authors should clarify this point.

We thank the reviewer for this comment. The EV proteomic expression is anticipated not to show the exact pattern across of samples of each group. The purpose of this experiment depicted in Figure 3 heatmap is to show the enrichment for pattern of expressions, but we acknowledge that not all samples from the same group have the exact proteome pattern.

We added this statement in the discussion section:

“As expected, the EV proteomic profiles of PDAC patients exhibited significant heterogeneity. While the above mentioned markers exhibited strong association with disease states at population levels, their abundances in individual patients varied significantly. Those observations highlight the need to develop multi-protein panels for pancreatic cancer diagnosis and prognosis.”

(5) The authors performed the survival analysis on a set of EV proteins but did not specify the origin of these markers or how many markers were examined. The authors should show their abundances across different groups, such as different stages and metastasis status.

We thank the reviewer for the comments. The goal of this experiment was not to identify EV proteins that performed similarly well for diagnosing and prognostication. In Figure 3A, 3B and 3C, we identified EV proteins that had better performance for diagnosis of metastatic disease. In these experiments we made  comparative analysis between patients with metastasis versus non-metastasis. In the experiment depicted in Figure 3D, the goal was to identify EV markers that had better performance is prognosticating outcomes as measured by overall survival, out of the markers identified in the previous experiments from Figure 3A. We would like to further clarify that based on our observation and others, it has become clear that EV profiles from cancer patients are highly heterogenous and we do not anticipate that a single marker will have sufficient test performance for cancer diagnosis or prognosis assessment when measured isolated. Rather, we anticipate that a panel of markers may yield better performance for diagnosis while a different combination of EV markers may have better performance for prognosis assessment.

(6) The classification model yielded a 100% accuracy, which may refer to AUC, in their discovery cohort, but it decreased to 89% in the independent cohort. This suggests that the authors have encountered overfitting issues with their model, where it performed well on the discovery cohort but did not generalize well to the independent cohort. The authors should clarify this point. The AUC score of the 7-EV signature is 0.89 and is not equivalent to prediction accuracy. In order to demonstrate prediction accuracy, the authors should show the confusion matrix of training and testing data as well as other evaluation metrics, such as accuracy, precision, and recall.

We thank the reviewer for providing these insightful comments. As you noted, the 7-biomarker signature machine learning model attained an impressive 100% accuracy within the internal Discovery Cohort, raising concerns about potential overfitting in the external validation dataset. Acknowledging the noted difference in AUROC of 0.11 in the external validation cohort, which surpasses the typical reported range of ~0.06-0.09, the model demonstrated a commendable AUROC of 0.89 in an independent patient cohort. Moreover, the utilization of an alternate technology to measure protein abundance in the validation dataset, underscores the model’s reproducibility and validity. We have provided the model metrics for both internal- and external-validation cohort. For these, please see updated Supplementary Figure 7, as well as the new Supplementary Figure 6 and Supplementary Figure 8. We also amended the discussion section to acknowledge that the validation cohort had limited sample size and proteins were measured in using a different method. Those factors likely contributed to the lower accuracy of predictions in the validation cohort. We addressed these limitations in the discussion section of the manuscript.

(7) The authors should include more details of their model and the process of selection of signatures to enhance the reproducibility and transparency of their methods.

We thank the reviewer for their valuable comments. To enhance clarity, we have incorporated additional information regarding the method employed for biomarker signature identification into the ‘Methods Section’ in page 23.  We note that Supplementary Table 7a provides details on ‘Sensitivity, Specificity, Precision, and AUC’ for the 16 markers included in the external validation study. Additionally, Supplementary Table 7b presents the contingency table for 7-biomarker signature, offering insights into model accuracy for both the Internal-Discovery and External Validation cohorts.  

Reviewer #2 (Public Review):

The authors intended to identify a protein signature in extracellular vesicles of serum to distinguish pancreatic ductal adenocarcinoma from benign pancreatic diseases.

A major strength of the work presented is the valuable profiling of a significant number of patient samples, with a rich cohort of patients with pancreatic cancer, benign pancreatic diseases, and healthy controls. However, despite the strong cohorts presented, the numbers of patient samples for benign pancreatic diseases as well as controls were very limited.

Also, the method used to isolate vesicles, EVTrap, recognizes double bilayers, which means that it can detect cellular debris and apoptotic bodies, which are very common in the circulation of patients that are undergoing chemotherapy. It would be important to identify the patients that are therapy naïve and the ones that are not because of this possible bias.

We thank the Reviewer for these comments. We want to point out that the experiments presented in Supplementary Figure 1 (Transmission electron microscopy images and Nanoparticle tracking analysis) confirm that the vesicles isolated with EVTrap are not cellular debris and apoptotic bodies. Rather, these structures are in the nano range expected for exosomes. This is further supported by the additional work from our co-author and collaborator describing the development of EVtrap and its performance in isolating exosomes when compared to other traditional methods such as ultracentrifugation and size exclusion chromatography (PMID:32396726).

As per the Reviewer’s request, we have provided an additional heatmap figure depicting whose patients are treatment naïve to differentiate from those who have received treatment (revised Figure 2C).

Additionally, the transmission electron microscopy data reflect this heterogeneity of the samples, also with little identification of double bilayered vesicles. It would be important to identify some extracellular vesicles markers in those preparations to strengthen the quality of the samples analyzed.

We appreciate the comment from the Reviewer and acknowledge the importance of identifying exosome markers on the isolate from EVtrap. These experiments have already been done and are reported in the original paper describing the development of this method by our co-authors in a separate work. In the manuscript PMID: 30080416, our collaborators demonstrated the detection of CD9, a well-known exosome marker, using Western Blot from isolates using EVtrap or ultra-centrifugation, a traditional technique to isolate exosomes. This work showed that EVtrap yielded much higher recovery rate of exosomes with lower contamination from soluble proteins. We did not repeat these already published experiments, but we amended our manuscript to reference these results.

What is more, previously published work with this same methodology identifies around 2000 proteins per sample. It would be important to explain why in this study there seems to be a reduction in more than 50% of the amount of proteins identified in the vesicles.

We thank the Reviewer for pointing out this important detail. In the previous work in which EVtrap was developed by our co-authors, the blood samples were processed using a different protocol, with shorter centrifugation (2,500g for 10 min) (PMID: 32396726). In the current work, we employed three centrifugation steps. As detailed in the Methods section of the manuscript, blood samples were centrifuged at 1,300g for 15 min. Then  plasma was removed from the top carefully avoiding cell pellet;  Repeat centrifugation of plasma at 2,500g for 15 min;  Again, plasma was removed from the top carefully avoiding cell pellet;  Third centrifugation at 2,500g for 15 min. This more extensive centrifugation process was intended to further increase the removal of platelets, apoptotic bodies, and other large particles and aggregates. Accordingly, we anticipate that the additional centrifugation steps decreased the contamination of our isolates but may have also decreased the amount of exosome proteins, hence the lower amount of exosome proteins identified in our study as compared to the original study from our co-authors (PMID: 32396726).

One of the proteins that constantly surges on the analysis is KRT20. It would be important to proceed with the analysis by first filtering out possible contaminants of the proteomics, of which keratins are the most common ones.

We thank the Reviewer for this comment. We would like to point out that we do believe that KRT20 is, in fact, cancer related and a not a contaminant. This is supported by our results presented in this manuscript showing enrichment or KRT20 in PDAC cases, and lower expression in benign samples. If this protein was a contaminant, its expression would be found uniformly in all samples, there would be no apparent reason for different expression between malignant vs benign cases, as all samples were processed following the same procedures. In addition, increased expression of KRT20 in PDAC tissues has also been reported by others. For instance, in a study by Schmiz-Winnthal  (PMID: 16364723), the authors showed that Cytokeratin 20 (KRT20) were expressed in 76% of PDAC patients and expression of KRT20 was associated with poor survival after surgical resection. Based on these observations, we believe that the KRT20 identified in our study is indeed a tumor associated EV protein rather than contamination.

Finally, none of the 7-extracellular vesicle protein signatures has been validated by other techniques, such as western blot, in extracellular vesicles isolated by other, standard, methods, such as size exclusion chromatography.

A distinct technique for protein analysis was done but not a different method of isolation of these vesicles. This would strengthen the results and the origin of the proteins.

We appreciate the Reviewer’s comment. We would like to again emphasize that the goal of this manuscript was not to compare the performance of EVtrap with other traditional EV isolation approaches such as ultracentrifugation and size exclusion chromatography.  The main goal of study is to determine proteomic profiles of EVs isolated from clinical samples and provide such information to research community for further studies. As the Reviewer points out, proteins in EVs are highly heterogeneous which highlight the complexity of EV biology and interpatient heterogeneity of pancreatic cancer.  We do not anticipate the development of EV-based markers for pancreatic diagnosis can be achieved by a single team, but by a community of researchers. We hope information presented in the current study will help other researchers identify additional candidates for validation in future work. Nonetheless, we edited the manuscript to discuss the limitation of not doing cross-validation of protein detection using a different method.

The conclusions that are reached do not fully meet the proposed aims of the identification of a protein signature in circulating extracellular vesicles that could improve early detection of the disease. The authors did not demonstrate the superiority of detection of these proteins in extracellular vesicles versus simply performing an ELISA, nor their superiority with respect to the current standard procedure for diagnosis.

We would like to clarify to the Reviewer that the goal of this manuscript was not to prove superiority of the EV signature biomarker in diagnosing pancreatic cancer as compared to current standard of care (SOC) practice, i.e., CT scans, endoscopic ultrasound and CA19-9. In order to prove such superiority, one would require a large, randomized phase III trial with several hundred patients. This was not the pursue of our discovery EV proteomics study and we double checked our manuscript to ensure no such claim was made. Rather, we aimed at developing a new pipeline for discovery of new EV biomarkers and we believe we were able to prove that this approach was successful in discovering a new class of biomarkers based on proteins expressed on extra-cellular vesicles that have predominant expression on patients with pancreatic cancer. Future studies should continue to advance this field with goals of improving on the current standard of care diagnostic methods.

The authors also suggest that profiling of circulating extracellular vesicles provides unique insights into systemic immune changes during pancreatic cancer development. How is this better than a regular hemogram is not clear.

We would like to clarify that the overall goal of this study is to provide patient-relevant information for the research community to further investigate biology of extracellular vesicles. For the state 'unique insights into systemic immune changes' we referred to the fact that we discovered EVs carrying proteins involved in immune responses. Previous studies have shown that EVs play important roles in cell-cell communication, discoveries from our study provide candidates for future studies on cellular mechanisms underlying immune regulation during pancreatic cancer development.

Finally, it would be important to determine how this signature compares with many others described in the literature that have the exact same aim. Why and how would this one be better?

We would like to again clarify that comparing the diagnostic performance of the EV biomarkers discovered in the study against standard of care methods (CA19-9, ctDNA, CT scan) was beyond the scope of this discovery EV proteomics work. We reviewed the manuscript to ensure that no claims were made as far as superiority against point-of-care tests available in clinic.

Reviewer #3 (Public Review):

This work investigates the use of extracellular vesicles (EVs) in blood as a noninvasive 'liquid biopsy' to aid in the differentiation of patients with pancreatic cancer (PDAC) from those with benign pancreatic disease and healthy controls, an important clinical question where biopsies are frequently non-diagnostic. The use of extracellular vesicles as biomarkers of disease has been gaining interest in recent history, with a variety of published methods and techniques, looking at a variety of different compositions ('the molecular cargo') of EVs particularly in cancer diagnosis (Shah R, et al, N Engl J Med 2018; 379:958-966).

This study adds to the growing body of evidence in using EVs for earlier detection of pancreatic cancer, identifying both new and known proteins of interest. Limitations in studying EVs, in general, include dealing with low concentrations in circulation and identifying the most relevant molecular cargo. This study provides validation of assaying EVs using the novel EVtrap method (Extracellular Vesicles Total Recovery And Purification),which the authors show to be more efficient than current standard techniques and potentially more scalable for larger clinical studies.

The strength of this study is in its numbers - the authors worked with a cohort of 124 cases,93 of them which were PDAC samples, which are considered large for an EV study (Jia, E etal. BMC Cancer 22, 573 (2022)). The benign disease group (n=20, between chronic pancreatitis and IPMNs) and healthy control groups (n=11) were relatively small, but the authors were not only able to identify candidate biomarkers for diagnosis that clearly stood out in the PDAC cohort, but also validate it in an independent cohort of 36 new subjects.

Proteins they have identified as associated with pancreatic cancer over benign disease included PDCD6IP, SERPINA12, and RUVBL2. They were even able to identify a set of EV proteins associated with metastasis and poorer prognosis, which include the proteins PSMB4, RUVBL2 and ANKAR and CRP, RALB and CD55. Their 7-EV protein signature yielded an 89% prediction accuracy for the diagnosis of PDAC against a background of benign pancreatic diseases that is compelling and comparable to other studies in the literature (Jia,E. et al. BMC Cancer 22, 573 (2022)).

The limitations of this study are its containment within a single institution - further studies are warranted to apply the authors' 7-EV protein PRAC panel to multiple other cases at other institutions in a larger cohort.

We are very thankful to the Reviewer for the positive feedback. We are similarly optimistic that EV-based biomarkers will assist future researchers to develop better diagnostic assays for patients with pancreatic cancer, as well as other tumor types lacking accurate blood-based tests.

Author response:

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

Reviewer #1 (Public review):

Summary:

In this manuscript, Herrmannova et al explore changes in translation upon individual depletion of three subunits of the eIF3 complex (d, e and h) in mammalian cells. The authors provide a detailed analysis of regulated transcripts, followed by validation by RT-qPCR and/or Western blot of targets of interest, as well as GO and KKEG pathway analysis. The authors confirm prior observations that eIF3, despite being a general translation initiation factor, functions in mRNA-specific regulation, and that eIF3 is important for translation re-initiation. They show that global effects of eIF3e and eIF3d depletion on translation and cell growth are concordant. Their results support and extend previous reports suggesting that both factors control translation of 5'TOP mRNAs. Interestingly, they identify MAPK pathway components as a group of targets coordinately regulated by eIF3 d/e. The authors also discuss discrepancies with other reports analyzing eIF3e function.

Strengths:

Altogether, a solid analysis of eIF3 d/e/h-mediated translation regulation of specific transcripts. The data will be useful for scientists working in the Translation field.

Weaknesses:

The authors could have explored in more detail some of their novel observations, as well as their impact on cell behavior.

The manuscript has improved with the new corrections. I appreciate the authors' attention to the minor comments, which have been fully solved. The authors have not, however, provided additional experimental evidence that uORF-mediated translation of Raf-1 mRNA depends on an intact eIF3 complex, nor have they addressed the consequences of such regulation for cell physiology. While I understand that this is a subject of follow-up research, the authors could have at least included their explanations/ speculations regarding major comments 2-4, which in my opinion could have been useful for the reader.

Our explanations/speculations regarding major comments 2 and 3 were included in the Discussion. We apologize for this misunderstanding as we thought that we were supposed to explain our ideas only in the responses. We did not discuss the comment 4, however, as we are really not sure what is the true effect and did not want to go into wild speculations in our manuscript. We thank this reviewer for his insightful comments and understanding.

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

Reviewer #1 (Recommendations For The Authors):

Major comments:

(1) The authors report the potential translational regulation of Raf kinase by re-initiation. It would be interesting to show that Raf is indeed regulated by uORF-mediated translation, and that this is dependent on an intact eIF3 complex. Analyzing the potential consequences of Raf1 regulation for cancer cell proliferation or apoptosis would be a plus.

We agree that this is an interesting and likely possibility. In fact, another clue that translation of Raf1 is regulated by uORFs comes from Bohlen et al. 2023 (PMID: 36869665) where they showed that RAF1 translation is dependent on PRRC2 proteins (that promote leaky scanning through these uORFs). We noted in the discussion that our results from eIF3d/e/hKD and the PRRC2A/B/CKD partly overlap. It is a subject of our follow-up research to investigate whether eIF3 and PRRC2 co-operate together to regulate translation of this important mRNA. 

(2) The authors show that eIF3 d/e -but not 3h- has an effect on cell proliferation. First, this indicates that proliferation does not fully correlate with eIF3 integrity. Depletion of eIF3d does not affect the integrity of eIF3, yet the effects on proliferation are similar to those of eIF3e. What is the possibility that changes in proliferation reflect functions of eIF3d outside the eIF3 complex? What could be the real consequences of disturbing eIF3 integrity for the mammalian cell? Please, discuss.

Yes, proliferation does not fully correlate with eIF3 integrity. Downregulation of eIF3 subunits that lead to disintegration of eIF3 YLC core (a, b, c, g, i) have more detrimental effect on growth and translation than downregulation of the peripheral subunits (e, k, l, f, h, m). Our previous studies (Wagner et al. 2016, PMID: 27924037 and Herrmannová et al. 2020, PMID: 31863585) indicate that the YLC core of eIF3 can partially support translation even without its peripheral subunits. In this respect eIF3d (as a peripheral subunit) is an amazing exception, suggesting it may have some specialized function(s). Whether this function resides outside of the eIF3 complex or not we do not know, but do not think so. Mainly because in the absence of eIF3e – its interaction partner, eIF3d gets rapidly degraded. Therefore, it is not very likely that eIF3d exists alone outside of eIF3 complex with moonlighting functions elsewhere. We think that eIF3d, as a head-interacting subunit close to an important head ribosomal protein RACK1 (a landing pad for regulatory proteins), is a target of signaling pathways, which may make it important for translation of specific mRNAs. In support is these thoughts, eIF3d (in the context of entire eIF3) together with DAP5 were shown to promote translation by an alternate capdependent (eIF4F-independent) mechanism (Lee et al. 2016, PMID: 27462815; de la Parra et al. 2018, PMID:30076308). In addition, the eIF3d function (also in the context of entire eIF3) was proved to be regulated by stress-triggered phosphorylation (Lamper et al. 2020, PMID: 33184215). 

(3) Figure 6D: Surprisingly, reduced levels of ERK1/2 upon eIF3d/e-KD are compensated by increased phosphorylation of ERK1/2 and net activation of c-Jun. Please comment on the functional consequences of buffering mechanisms that the cell deploys in order to counteract compromised eIF3 function. Why would the cell activate precisely the MAPK pathway to compensate for a compromised eIF3 function?

This we do not know. We can only speculate that when translation is compromised, cells try to counteract it in two ways: 1) they produce more ribosomes to increase translational rates and 2) activate MAPK signaling to send pro-growth signals, which can in the end further boost ribosome biogenesis.

(4) Regarding DAP-sensitive transcripts, can the authors discuss in more detail the role of eIF3d in alternative cap-dependent translation versus re-initiation? Are these transcripts being translated by a canonical cap- and uORF-dependent mechanism or by an alternative capdependent mechanism?

This is indeed not an easy question. On one hand, it was shown that DAP5 facilitates translation re-initiation after uORF translation in a canonical cap-dependent manner. This mechanism is essential for translation of the main coding sequence (CDS) in mRNAs with structured 5' leaders and multiple uORFs. (Weber et al. 2022, PMID: 36473845; David et al., 2022, PMID: 35961752). On the other hand, DAP5 was proposed to promote alternative, eIF4F-independent but cap-dependent translation, as it can substitute the function of the eIF4F complex in cooperation with eIF3d (de la Parra et al., 2018, PMID: 30076308; Volta et al., 2021 34848685). Overall, these observations paint a very complex picture for us to propose a clear scenario of what is going on between these two proteins on individual mRNAs. We speculate that both mechanisms are taking place and that the specific mechanism of translation initiation differs for differently arranged mRNAs.

Minor comments:

(5) Figure S2C: why is there a strong reduction of the stop codon peak for 3d and 3h KDs?

We have checked the Ribowaltz profiles of all replicates (in the Supplementary data we are showing only a representative replicate I) and the stop codon peak differs a lot among the replicates. We think that this way of plotting was optimized for calculation and visualization of P-sites and triplet periodicity and thus is not suitable for this type of comparison among samples. Therefore, we have performed our own analysis where the 5’ ends of reads are used instead of P-sites and triplicates are averaged and normalized to CDS (see below please), so that all samples can be compared directly in one plot (same as Fig. S13A but for stop codon). We can see that the stop codon peak really differs and is the smallest for eIF3hKD. However, these changes are in the range of 20% and we are not sure about their biological significance. We therefore refrain from drawing any conclusions. In general, reduced stop codon peak may signal faster termination or increased stop codon readthrough, but the latter should be accompanied by an increased ribosome density in the 3’UTR, which is not the case. A defect in termination efficiency would be manifested by an increased stop codon peak, instead.

Author response image 1.

(6) Figures 5 and S8: Adding a vertical line at 'zero' in all cumulative plots will help the reader understand the author's interpretation of the data. 

We have added a dashed grey vertical line at zero as requested. However, for interpretation of these plots, the reader should focus on the colored curve and whether it is shifted in respect to the grey curve (background) or not. Shift to the right indicates increased expression, while shift to the left indicates decreased expression. The reported p-value then indicates the statistical significance of the shift.

(7) The entire Figure 2 are controls that can go to Supplementary Material. The clustering of Figure S3B could be shown in the main Figure, as it is a very easy read-out of the consistent effects of the KDs of the different eIF3 subunits under analysis.

We have moved the entire Figure 2 to Supplementary Material as suggested (the original panels can be found as Supplementary Figures 1B, 1C and 3A). Figure S3B is now the main Figure 2E. 

(8) There are 3 replicates for Ribo-Seq and four for RNA-Seq. Were these not carried out in parallel, as it is usually done in Ribo-seq experiments? Why is there an extra replicate for RNASeq?

Yes, the three replicates were carried out in parallel. We have decided to add the fourth replicate in RNA-Seq to increase the data robustness as the RNA-Seq is used for normalization of FP to calculate the TE, which was our main analyzed metrics in this article. We had the option to add the fourth replicate as we originally prepared five biological replicates for all samples, but after performing the control experiments, we selected only the 3 best replicates for the Ribo-Seq library preparation and sequencing.  

(9) Please, add another sheet in Table S2 with the names of all genes that change only at the translation (RPF) levels.

As requested, we have added three extra sheets (one for each downregulation) for differential FP with Padjusted <0.05 in the Spreadsheet S2. We also provide a complete unfiltered differential expression data (sheet named “all data”), so that readers can filter out any relevant data based on their interest.

(10) Page 5, bottom: ' ...we showed that the expression of all 12 eIF3 subunits is interconnected such that perturbance of the expression of one subunit results in the down-regulation of entire modules...'. This is not true for eIF3d, as shown in Fig1B and mentioned in Results.

This reviewer is correct. By this generalized statement, we were trying to summarize our previous results from Wagner et al., 2014, PMID: 24912683; Wagner et al.,2016, PMID: 27924037 and Herrmannova et al.,2020, PMID: 31863585. The eIF3d downregulation is the only exception that does not affect expression of any other eIF3 subunit. Therefore, we have rewritten this paragraph accordingly: “We recently reported a comprehensive in vivo analysis of the modular dynamics of the human eIF3 complex (Wagner et al, 2020; Wagner et al, 2014; Wagner et al., 2016). Using a systematic individual downregulation strategy, we showed that the expression of all 12 eIF3 subunits is interconnected such that perturbance of the expression of one subunit results in the down-regulation of entire modules leading to the formation of partial eIF3 subcomplexes with limited functionality (Herrmannova et al, 2020). eIF3d is the only exception in this respect, as its downregulation does not influence expression of any other eIF3 subunit.”

(11) Page 10, bottom: ' The PCA plot and hierarchical clustering... These results suggest that eIF3h depletion impacts the translatome differentially than depletion of eIF3e or eIF3d.' This is already obvious in the polysome profiles of Figure S2C.

We agree that this result is surely not surprising given the polysome profile and growth phenotype analyses of eIF3hKD. But still, we think that the PCA plot and hierarchical clustering results represent valuable controls. Nonetheless, we rephrased this section to note that this result agrees with the polysome profiles analysis: “The PCA plot and hierarchical clustering (Figure 2A and Supplementary Figure 4A) showed clustering of the samples into two main groups: Ribo-Seq and RNA-seq, and also into two subgroups; NT and eIF3hKD samples clustered on one side and eIF3eKD and eIF3dKD samples on the other. These results suggest that the eIF3h depletion has a much milder impact on the translatome than depletion of eIF3e or eIF3d, which agrees with the growth phenotype and polysome profile analyses (Supplementary Figure 1A and 1D).”

(12) Page 12: ' As for the eIF3dKD "unique upregulated" DTEGs, we identified one interesting and unique KEGG pathway, the ABC transporters (Supplementary Figure 5A, in green).' This sentence is confusing, as there are more pathways that are significant in this group, so it is unclear why the authors consider it 'unique'.

The eIF3dKD “unique upregulated” group comprises genes with increased TE only in eIF3dKD but not in eIF3eKD or eIF3hKD (500 genes, Fig 2G). All these 500 genes were examined for enrichment in the KEGG pathways, and the top 10 significant pathways were reported (Fig S6A). However, 8 out of these 10 pathways were also significantly enriched in other gene groups examined (e.g. eIF3d/eIF3e common). Therefore, the two remaining pathways (“ABC transporters” and “Other types of O-glycan biosynthesis”) are truly unique for eIF3dKD. We wanted to highlight the ABC transporters group in particular because we find it rather interesting (for the reasons mentioned in the article). We have corrected the sentence in question to avoid confusion: “Among the eIF3dKD “unique upregulated” DTEGs, we identified one interesting KEGG pathway, the ABC transporters, which did not show up in other gene groups (Supplementary Figure 6A, in green). A total of 12 different ABC transporters had elevated TE (9 of them are unique to eIF3dKD, while 3 were also found in eIF3eKD), 6 of which (ABCC1-5, ABCC10) belong to the C subfamily, known to confer multidrug resistance with alternative designation as multidrug resistance protein (MRP1-5, MRP7) (Sodani et al, 2012).

Interestingly, all six of these ABCC transporters were upregulated solely at the translational level (Supplementary Spreadsheet S2).”    

(13) Note typo ('Various') in Figure 4A.

Corrected

(14) The introduction could be shortened.

This is a very subjective requirement. In fact, when this manuscript was reviewed in NAR, we were asked by two reviewers to expand it substantially. Because a number of various research topics come together in this work, e.g. translational regulation, the eIF3 structure and function, MAPK/ERK signaling, we are convinced that all of them demand a comprehensive introduction for non-experts in each of these topics. Therefore, with all due respect to this reviewer, we did not ultimately shorten it.

Reviewer #2 (Recommendations For The Authors):

- In Figure 2, it would be useful to know why eIF3d is destabilized by eIF3e knockdown - is it protein degradation and why do the eIF3d/e knockdowns not more completely phenocopy each other when there is the same reduction to eIF3d as in the eIF3d knockdown sample?

Yes, we do think that protein degradation lies behind the eIF3d destabilization in the eIF3eKD, but we have not yet directly demonstrated this. However, we have shown that eIF3d mRNA levels are not altered in eIF3eKD and that Ribo-Seq data indicate no change in TE or FP for eIF3d-encoding mRNA in eIF3eKD. Nonetheless, it is important to note (and we discuss it in the article) that eIF3d levels in eIF3dKD are lower than eIF3d levels in eIF3eKD (please see Supplementary Figure 1C). In fact, we believe that this is one of the main reasons for the eIF3d/e knockdowns differences.

- The western blots in Figures 4 and 6 show modest changes to target protein levels and would be strengthened by quantification.

We have added the quantifications as requested by this reviewer and the reviewer 3.

- For Figure 4, this figure would be strengthened by experiments showing if the increase in ribosomal protein levels is correlated with actual changes to ribosome biogenesis.

As suggested, we performed polysome profiling in the presence of EDTA to monitor changes in the 60S/40S ratio, indicating a potential imbalance in the biogenesis of individual ribosome subunits. We found that it was not affected (Figure 3G). In addition, we performed the same experiment, normalizing all samples to the same number of cells (cells were carefully counted before lysis). In this way, we confirmed that eIF3dKD and eIF3eKD cells indeed contain a significantly increased number of ribosomes, in agreement with the western blot analysis (Figure 3H).

- In Figure 6, there needs to be a nuclear loading control.

This experiment was repeated with Lamin B1 used as a nuclear loading control – it is now shown as Fig. 5F.

- For Figure 8, these findings would be strengthened using luciferase reporter assays where the various RNA determinants are experimentally tested. Similarly, 5′ TOP RNA reporters would have been appreciated in Figure 4.

This is indeed a logical continuation of our work, which represents the current work in progress of one of the PhD students. We apologize, but we consider this time- and resource-demanding analysis out of scope of this article.

Reviewer #3 (Recommendations For The Authors):

(1) Within the many effects observed, it is mentioned that eIF3d is known to be overexpressed while eIF3e is underexpressed in many cancers, but knockdown of either subunit decreases MDM2 levels, which would be expected to increase P53 activity and decrease tumor cell transformation. In contrast, they also report that 3e/3d knockdown dramatically increases levels of cJUN, presumably due to increased MAPK activity, and is expected to increase protumor gene expression. Additional discussion is needed to clarify the significance of the findings, which are a bit confusing.

This is indeed true. However, considering the complexity of eIF3, the largest initiation factor among all, as well as the broad portfolio of its functions, it is perhaps not so surprising that the observed effects are complex and may seem even contradictory in respect to cancer. To acknowledge that, we expanded the corresponding part of discussion as follows: “Here, we demonstrate that alterations in the eIF3 subunit stoichiometry and/or eIF3 subcomplexes have distinct effects on the translatome; for example, they affect factors that play a prominent (either positive or negative) role in cancer biology (e.g., MDM2 and cJUN), but the resulting impact is unclear so far. Considering the complex interactions between these factors as well as the complexity of the eIF3 complex per se, future studies are required to delineate the specific oncogenic and tumor suppressive pathways that play a predominant role in mediating the effects of perturbations in the eIF3 complex in the context of neoplasia.”

(2) There are places in the text where the authors refer to changes in transcriptional control when RNA levels differ, but transcription versus RNA turnover wasn't tested, e.g. page 16 and Figure S10, qPCR does not confirm "transcriptional upregulation in all three knockdowns" and page 19 "despite apparent compensatory mechanisms that increase their transcription."

This is indeed true, the sentences in question were corrected. The term “increased mRNA levels” was used instead of transcriptional upregulation (increased mRNA stabilization is also possible).

(3) Similarly, the authors suggest that steady-state LARP1 protein levels are unaffected based on ribosome footprint counts (page 21). It is incorrect to assume this, because ribosome footprints can be elevated due to stalling on RNA that isn't being translated and doesn't yield more protein, and because levels of translated RNA/synthesized proteins do not always reflect steady-state protein levels, especially in mutants that could affect lysosome levels and protein turnover. Also page 12, 1st paragraph suggests protein production is down when ribosome footprints are changed.

Yes, we are well-aware of this known limitation of Ribo-seq analysis. Therefore, the steadystate protein levels of our key hits were verified by western blotting. In addition, we have removed the sentence about LARP1 because it was based on Ribo-Seq data only without experimental evaluation of the steady-state LARP1 protein levels.

(4) The translation buffering effect is not clear in some Figures, e.g. S6, S8, 8A, and B. The authors show a scheme for translationally buffered RNAs being clustered in the upper right and lower left quadrants in S4H (translation up with transcript level down and v.v.), but in the FP versus RNA plots, the non-TOP RNAs and 4E-P-regulated RNAs don't show this behavior, and appear to show a similar distribution to the global changes. Some of the right panels in these figures show modest shifts, but it's not clear how these were determined to be significant. More information is needed to clarify, or a different presentation, such as displaying the RNA subsets in the left panels with heat map coloring to reveal whether RNAs show the buffered translation pattern defined in purple in Figure S4H, or by reporting a statistical parameter or number of RNAs that show behavior out of total for significance. Currently the conclusion that these RNAs are translationally buffered seems subjective since there are clearly many RNAs that don't show changes, or show translation-only or RNA-only changes.

We would like to clarify that S4H does not indicate a necessity for changes in FPs in the buffered subsets. Although opposing changes in total mRNA and FPs are classified as buffering, often we also consider the scenario where there are changes to the total mRNA levels not accompanied by changes in ribosome association.

In figure S6, the scatterplots indicate a high density of genes shifted towards negative fold changes on the x-axis (total mRNA). This is also reflected in the empirical cumulative distribution functions (ecdfs) for the log2 fold changes in total mRNA in the far right panels of A and B, and the lack of changes in log2 fold change for FPs (middle panels). Similarly, in figure S8, the scatterplots indicate a density of genes shifted towards positive fold changes on the x-axis for total mRNA. The ecdfs also demonstrate that there is a significant directional shift in log2 fold changes in the total mRNA that is not present to a similar degree in the FPs, consistent with translational offsetting. It is rightly pointed out that not all genes in these sets follow the same pattern of regulation. We have revised the title of Supplementary Figure S6 (now S7) to reflect this. However, we would like to emphasize that these figures are not intended to communicate that all genes within these sets of interest are regulated in the same manner, but rather that when considered as a whole, the predominant effect seen is that of translational offsetting (directional shifts in the log2 fold change distribution of total mRNA that are not accompanied by similar shifts in FP mRNA log2 fold changes).

The significance of these differences was determined by comparing the ecdfs of the log2 fold changes for the genes belonging to a particular set (e.g. non-TOP mTOR-sensitive, p-eIF4E-sensitive) against all other expressed genes (background) using a Wilcoxan rank sum test. This allows identification of significant shifts in the distributions that have a clear directionality (if there is an overall increase, or decrease in fold changes of FPs or total mRNA compared to background). If log2 fold changes are different from background, but without a clear directionality (equally likely to be increased or decreased), the test will not yield a significant result. This approach allows assessment of the overall behavior of gene signatures within a given dataset in a manner that is completely threshold-independent, such that it does not rely on classification of genes into different regulatory categories (translation only, buffering, etc.) based on significance or fold-change cut-offs (as in S4H). Therefore, we believe that this unbiased approach is well-suited for identifying cases when there are many genes that follow similar patterns of regulation within a given dataset.

(5) Page 10-"These results suggest that eIF3h depletion impacts the translatome differentially than depletion of eIF3e or eIF3d" ...These results suggest that eIF3h has less impact on the translatome, not that it does so differently. If it were changing translation by a different mechanism, I would not expect it to cluster with control.

This sentence was rewritten as follows: “The PCA plot and hierarchical clustering (Figure 2A and Supplementary Figure 4A) showed clustering of the samples into two main groups: RiboSeq and RNA-seq, and also into two subgroups; NT and eIF3hKD samples clustered on one side and eIF3eKD and eIF3dKD samples on the other. These results suggest that the eIF3h depletion has a much milder impact on the translatome than depletion of eIF3e or eIF3d, which agrees with the growth phenotype and polysome profile analyses (Supplementary Figure 1A and 1D).”

Other minor issues:

(1) There are some typos: Figure 2 leves, Figure 4 variou,

Corrected.

(2) Figure 3, font for genes on volcano plot too small

Yes, maybe, however the resolution of this image is high enough to enlarge a certain part of it at will. In our opinion, a larger font would take up too much space, which would reduce the informativeness of this graph.

(3) Figure S5, highlighting isn't defined.

The figure legend for S5A (now S6A) states: “Less significant terms ranking 11 and below are in grey. Terms specifically discussed in the main text are highlighted in green.” Perhaps it was overlooked by this reviewer.

(4) At several points the authors refer to "the MAPK signaling pathway", suggesting there is a single MAPK that is affected, e.g in the title, page 3, and other places when it seems they mean "MAPK signaling pathways" since several MAPK pathways appear to be affected.

We apologize for any terminological inaccuracies. There are indeed several MAPK pathways operating in cells. In our study, we focused mainly on the MAPK/ERK pathway. The confusion probably stems from the fact that the corresponding term in the KEGG pathway database is labeled "MAPK signaling pathway" and this term, although singular, includes all MAPK pathways. We have carefully reviewed the entire article and have corrected the term used accordingly to either: 1) MAPK pathways in general, 2) the MAPK/ERK pathway for this particular pathway, or 3) "MAPK signaling pathway", where the KEGG term is meant.

(5) Some eIF3 subunit RNAs have TOP motifs. One might expect 3e and 3h levels to change as a function of 3d knockdown due to TOP motifs but this is not observed. Can the authors speculate why the eIF3 subunit levels don't change but other TOP RNAs show TE changes? Is this true for other translation factors, or just for eIF3, or just for these subunits? Could the Western blot be out of linear range for the antibody or is there feedback affecting eIF3 levels differently than the other TOP RNAs, or a protein turnover mechanism to maintain eIF3 levels?

This is indeed a very interesting question. In addition to the mRNAs encoding ribosomal proteins, we examined all TOP mRNAs and added an additional sheet to the S2 supplemental spreadsheet with all TOP RNAs listed in (Philippe et al., 2020, PMID: 32094190). According to our Ribo-Seq data, we could expect to see increased protein levels of eIF3a and eIF3f in eIF3dKD and eIF3eKD, but this is not the case, as judged from extensive western blot analysis performed in (Wagner et. al 2016, PMID: 27924037). Indeed, we cannot rule out the involvement of a compensatory mechanism monitoring and maintaining the levels of eIF3 subunits at steady-state – increasing or decreasing them if necessary, which could depend on the TOP motif-mediated regulation. However, we think that in our KDs, all non-targeted subunits that lose their direct binding partner in eIF3 due to siRNA treatment become rapidly degraded. For example, co-downregulation of subunits d, k and l in eIF3eKD is very likely caused by protein degradation as a result of a loss of their direct binding partner – eIF3e. Since we showed that the yeast eIF3 complex assembles co-translationally (Wagner et. al 2020, PMID: 32589964), and there is no reason to think that mammalian eIF3 differs in this regard, our working hypothesis is that free subunits that are not promptly incorporated into the eIF3 complex are rapidly degraded, and the presence or absence of the TOP motif in the 5’ UTR of their mRNAs has no effect. As for the other TOP mRNAs, translation factors eEF1B2, eEF1D, eEF1G, eEF2 have significantly increased FPs in both eIF3dKD and eIF3eKD, but we did not check their protein levels by western blotting to conclude anything specific.