This the legendary history of Schefing this establish culture values of heroism.

If this was 2023 I wonder how much it has risen since then.

It's nice to see that students similar to myself were able to give their feedback, and have that feedback addressed in later editions.

The majority of this reading after this point is the author, Robert B. Marks, thanking and giving credit to his colleges and organizations that helped or funded this fifth edition and older editions of this book.

I always wondered why schools never really talked about the great recession. Was it too recent? If so, when do major historical events start being put into general education?

I also found it interesting how fast our focus on past events can change.

This shows how problems in the past relate to today and still effect us now

It’s crazy how much damage humans have done to the climate in such a short amount of time

These are big events in the 21st century but I wouldn’t say that if I would them, about how we changed are relationship with the earths environment

It’s important to remember that the past doesn’t change just how historians write it and how they use issues in there time to influence there writings

Even after retiring from teaching, the author remained an active scholar, and continued researching environmental history, showing that learning and contributing knowledge can continue throughout a person’s life

This means history is not just a collection of past events. It continues to affect the present and studying. It helps us understand today’s problems and how the world keeps changing.

This shows that human activities is not only causing climate change, but is also affecting natural systems on a scale that has never been seen before

This shows why he is a source that we can trust. It also demonstrates that everyone can always keep on learning and improving.

It is important to consider new world events that happen in old history textbooks because history is not just memorizing dates from the past, it is also applies to what is happening today.

This shows that all historians can make mistakes and nobody is perfect in their work.

It is interesting to see how recent world events make an author and historian revise and add new perspective to their work.

I found this interesting because it shows how important accurate financial statements are when evaluating a company and everything should be transparent.  Investors use financial information to determine how a company is performing and whether or not to invest. Kraft Heinz reported inflated adjusted EBITDA because some of its supplier discounts weren't recorded. This could make the company look more profitable than it actually was. This is one reason why investors cannot just look at a company's stock price, but also need to understand the financial records thoroughly.

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【方法】这种设计展示了数据处理的谨慎方法，通过分离素材生成和历史更新，防止了数据不一致问题。这种分阶段处理方法值得其他类似工具借鉴，特别是在处理增量数据时。

【非共识】文档明确标注Windows版本兼容性为实验性，这表明开发者对跨平台支持持谨慎态度。不同于许多工具宣称的全面兼容性，这种诚实的局限性说明有助于用户管理预期，避免在实际使用中遇到挫折。

【局限】这里强调了隐私保护的关键限制，但未明确说明如何确保数据安全。对于处理微信这类敏感数据的工具，应补充说明建议的加密存储方式和访问控制措施，以及如何验证本地环境的安全性。

According to the Rights Lens, the AI companies wouldn't have the right to expose the public to potentially harmful experiments. If at any point one person doesn’t want to participate in this, they are having their own rights violated, and therefore the company shouldn't do it. Through this lens, the line between being a means to an end and not, is drawn almost immediately. This would be especially true if it meant people forfeiting their right for the sake of the company and what they are making. When we look at this through a Utilitarian Lens, things change a little bit. If the work of the AI companies results in more people benefiting than without them developing it, it would be ethical. In the utilitarian lens, they would look to see who and how the companies are affecting the people. If the product that the company would invent would prevent generational hurt and damage, I would argue through this lens, it's worth the potential ‘bad’. It would start treating people as a means rather than an end if the end result was no longer needed, or it was more costly to produce the potential benefits.

Applying Utilitarian lens for pacing AI, the benefit that can be lost is rather a lot. AI is already causing enormous amount of money to be spent in today's time already with more harm being done than good. With pacing AI, it would be an enormous operation, in training, data centers, millions of chips and thousands of people working overtime, not to mention how some AI's are rather invasive to privacy.

According to the article pacing AI, AI focuses more on what could help the majority of people in terms of physical safety and economics. Not what they need or want.

I would need to know a lot. Such as would there start to be credit for the works that artist and writers receive from their works are being stolen? Kinda like a watermark? Would I be able to work freely without the stress that my job is going to get taken simply because I work with technology? Cause I know that still is happening. I would need to know a lot, especially if AI is still going to be around.

I think the quote "Carefully wielded, AI can be the latest in a long line of technological miracles that have uplifted and ennobled humanity." Cause I was assuming if wielded carefully would AI be able to be accessible to the medical field. In diagnosing a patient, in being an alert for major things, in assisting nurses in charting or printing just overall being helpful but not taking their jobs.

Looking at this line through the Utilitarian lens is really interesting. I mean, the sentence itself is exactly that. Where do you cause less harm? Thinking about my previous comments using the care lens and narrowing that focus with the Utilitarian lens, we are weighing the harm done minus the efforts to counterbalance that harm with the possible good and bad for the future. Is it possible that AI could cure more diseases than the destruction it causes to Earth? How do you even weigh these things?

Justice Lens: Amodei's proposal to coordinate pacing to help AI developers keep a commercial advantage and allow the US to keep leading in the AI frontier gives these groups a distinct advantage over workers, citizens of other countries, and future generations. The Justice Lens focuses on making choices that give people fair or equal treatment; however, this is not what this proposal does. AI developers, especially those working on more developed models, are put above smaller developers and workers. They are allowed to keep their commercial standing while other companies may struggle to keep up because the pacing would restrict them more. Also, this could impact workers in this field because they could lose jobs due to the lack of work, also putting strain on other fields with a potential flood of people looking for work. By allowing the US to keep a geopolitical advantage in terms of AI, our citizens would have an unfair advantage in terms of access to AI tools compared to the citizens of other countries. This lack of technology could also impact the ability for other countries to create solutions to problems they face. Going back to the top of the article, AI does have the ability to further medical studies, so other countries won't have access to the technology that could help its people with medical issues.

Common Good Lens: I chose to focus on the Common Good lens to complicate my response because it focuses on some systems I mentioned prior, as well as some I want to mention. I do understand that having pacing good positively impact the AI development community, but the tradeoff is the harm to other communities. As I mentioned earlier, citizens in other countries may not have as much access to AI if the US is able to keep its lead in AI. Their communities are impacted because they will be unable to use this technology to create solutions that could strengthen the systems that uphold their community, such as education or medical care. I also want to touch on future generations now. One thing that is a community-wide (and humanity-wide) need is access to clean water and an overall clean environment. One thing that AI, particularly Generative AI, does is use water to cool down data centers. There have been many people who have shared stories of living near data centers and have their water become dirtier or almost non-existent when they turn on the faucet. This not only affects drinking water, but also these people's ability to cook and clean. If AI continues to grow, even with pacing, with the US at the forefront, it is likely that this issue will become more widespread as we need more data centers to keep up with the demands of AI. This could continue these issues into future generations, potentially even worse. This would go against the common good and could impact the healthcare and other systems communities rely on for health and maintaining a safe standard of living.

By focusing on a utilitarian and rights lens, I believe a lot of people are affected, and more will continue to be affected with the consistent emergence of AI development. The utilitarian lens focuses more on how AI will impact everyone, aiming to cause the least amount of harm, which seems to be ideal. Although AI has benefits, it will harm lots of jobs, potentially being able to replace humans as a whole in certain positions. Or companies opting to use AI to "save money" and time. We have seen this already with AI art being used instead of companies hiring artists. AI is already impacting education and how others learn, but through a utilitarian lens, AI helps with production and accessibility, but it also comes with misinformation and privacy breaches. <- These are some big components when looking into the Rights lens. The effects on employment, education, privacy, and energy consumption must all be evaluated when determining how AI can fit the utilitarian lens. But I feel like that conversation needs to be held on more of a global or government angle. When discussing a product as big as AI, I think a utilitarian approach is a good way to look at it, because making a decision based on making the majority happy seems like it would work. But if we focus on the Rights lens, it's evaluating from a fundamental rights view. There are a lot of ethical questions when it comes to AI, like whether it really respects privacy or is it collecting personal information that we don't know about. Will AI have fair treatment to everyone who uses it? Ai is developing at a scary rate, and needs to be trained and controlled correctly in order for humans to be respected.

I think this shows that the Great Wall was strong enough to stop or slow down the Mongol soldiers, even though they had powerful weapons and military skills. However, I wonder if the wall was always effective against invaders.

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After reading TWL pp 60-76 and looking over the annoations for various lines, this section, and lines like this in particular, stood out to me. Forgive me for not writing ragrding tarot cards, but i am wrapped up with the Christian element of this work from T.S. Eliot. He mentions both Ezekiel and Ecclesiastes in his annotations of lines 20 and 23 of 'The Burial of the Dead", which makes perfect sense as there are stark references to objects and organisms described in both biblical books. However, when he writes heavily about rock and water for 29 lines (330-359) and fails to mention anything regarding the story of Moses, i become confused and curious. Surely it can't be a coincidence. Eliot even mentions a spring just after mentioning water and rock together. In his annotations, these lines fall into the beginning of 'What the Thunder Said'. In his annotation of this section, the only biblical reference he mentions is the road to Emmaus, which does not have a relation to Moses. This section and the lack of clarity around it makes me wonder about what Eliot could have been thinking about whike writing it.

The direct translation of this from German to English is: "The wind blows fresh towards home; my Irish child, where do you live?" There are a couple of things that make this passage intriguing; firstly, Eliot made the conscious choice to continue these German excerpts. This continuous German connects to the past few stanzas that hint towards the archduke and his death as a catalyst for WWI. Additionally, this excerpt is a sailor song from the story of Tristan and Isolde, a story very similar to Romeo and Juliet in the sense that they die together as a result of their potion. The presence of the occult/witch alludes to Madame Sosostris, who appears later in the Burial of the Dead.

However, my main point of focus was how this excerpt connects with the hyacinths. Hyacinthia was loved by Apollo. This is the second (could be third) covert appearance of Apollo within the poem. He appears in the preface, as he is the one who punished Sibyl, and appears in the archduke section, as a prophecy that was told to Marie by a woman whose son died in the Starnbergersee lake. He appears in the myth of Hyacinthia, as he is the one who turned him into hyacinths. Despite that, the second version of the myth details Hyacinthia's death because Zephyrus (the god of the wind) envied the attention that Hyacinthia got from Apollo. Wind in this excerpt foreshadows the meaning of hyacinths as mourning flowers. This ultimately connects back to the death of Tristan and Isolde.

Here we see a reference to a very important and specific tarot card: the Hanged Man. This tarot card traditionally signifies voluntary surrender, a necessary pause, and the possibility of gaining a new perspective by letting go of control. It is therefore significant that Madame Sosostris says, “I do not find / The Hanged Man.” Rather than presenting surrender itself, Eliot presents its absence. Perhaps the missing card symbolizes the speaker’s inability to surrender, pause, or relinquish control within a world consumed by despair and the aftermath of war. Yet it is also significant that the absence is explicitly acknowledged: the speaker recognizes what is missing. This recognition suggests an awareness that surrender or a new perspective may be necessary, even if it remains inaccessible. Sometimes we know, deep down, what must change, yet we wait for some catalytic symbol—like a tarot card or even a war—to force us to confront what we already know.

The tarot cards, regarded with a high degree of antiquity by many scholars, reflect an unsure ancestral line. Whether or not they are direct predecessors to modern playing cards or of a distinct group remains unclear. However, what they do certainly contain is a connection to Le Morte D'Arture. The Cup, Lance, Sword, and Dish, corresponding images from the aforementioned story, converge to represent the four distinct suits of a tarot deck. Within Le Morte D'Arture, these images can be percieved as harbingers of fertility, of healing to an emaciated landscape left barren by the impotence and perpetual illness of the king. Once they are found, procreation and naturalistic recovery are able to commence once again, cyclically so, providing reprieve from a constant state of waste land. Within the context of tarot, these symbols take a central role in the cards' common divination purposes, one that is relevant to the healing of land. Transcending the commonality of mundane tarot use, these symbols would - within the concept of the Grail - be used to represent a different form of divination, one that foretells the recovery and restoration of the landscape. The symbols thus become not just thematically connected with an evanescent prospect of rebirth, but also with a rich history of fortune telling that reveals their status as prophetically tied to the return of fertility. Critically, this status extends to the cited text. The card, borne aloft on the back, is noted as blank, seemingly absent of either of the four suits - thus non-indicative of restoration. However, the following phrase, describing the forbidden nature of the card's hidden visage, challenges this notion. If the card is indeed blank, what prevents wandering eyes from glancing at its empty surface? Eliot seems to be tantalizing the audience, questioning the candor of his own work. In the balance hangs the prospect of the card's prophetic powers. Obviously, if it is blank, no catalytic change will occur, no prophecy will return fertility to the land. On the off-chance that it contains one of the four suits, restoration seems within reach. In many ways, this ambiguous structure mirrors the poem's internal struggle to reconcile damnation, ruin, and waste, with an indefatigable hope for better things to come. The card may indeed be blank, and indeed, the pursuit of its visage may be in vain. It is in this near futility that the future remains uncertain. The prospect of divination is clear, but the land, and the audience, cannot quite grasp the prophecy.

There are many undertones in this part of Eliot's narrative of the girl who recieves flowers from the man who is courting her. Read after the initial foreign text from a sailor seeking kind winds returning him home to his lover on a distant Irsish shore, the quotation marks around this phrase and the previous 'You gave me hyacinths first a year ago;' could be a distant lover remembering fondly when he was last reunited with his girl uner positive terms. The quotation marks indicate that the words were said by someone else, or something that the speaker has heard before. Hyacinths have a deep purple color, and a sweet smell, and since Hyacinth himself was a close friend to Apolo, there is evidently deep affection beteween the speaker and the person they remember. However the second part from lines 36-39 take a dark turn, which is then foretold after the fact by the clairvoyante, who foretells "fear water" and there is a rememberence of wet hair, as if the person who went to collect flowers came back a changed person, not physically dead, but perhaps emotionally changed in an unalterable way.

Tarot cards depict vague images which may be adapted with symbology to indicate one of a myriad of things in modern life. For instance, one who draws the hanged man may not necessarily walk the gallows the very next day. However, the symbology remains pertinent even to this day. Similarly, the story of star-crossed lovers dying a tragic death together is one that is repeated throughout the ages. Tristan and Isolde, in addition to being a precursor to Le Morte D'Arthur, could also have images that remind the reader of Romeo and Juliet. Belladonna reminds the reader of the Virgin Mary. Any one card can be compared to and contrasted against millions of situations. A tarot card with the Virgin Mary is itself a form of irony, contrasting the occult with the Christian faith. However, that same card can evoke the story of Tristan and Isolde, albeit Tristan is an English sailor and not a Phoenician one, nor was his end one of drowning. However, his untimely and tragic death brings Isolde, seeking to heal him or at least die alongside him. She is the lady of situations, originally due to be wed to Mark and now in love madly with Tristan. Similarly, Mary did not expect to be with the child of God, however, it altered her life. As Isolde says before her death alongside her lover, "How softly and gently he smiles, how sweetly his eyes open - can you see, my friends, do you not see it? How he glows ever brighter, raising himself high amidst the stars? Do you not see it?" (25) Though his eyes may not be pearls, they are the eyes of a dead sailor, seen one last time by a woman who comes to heal him, love him, or die alongside him.

The Hyacinthus article emphasizes Hyacinthus as a beautiful young man who is loved by Apollo but is accidentally killed. Apollo is devastated, so he names a flower after him as a symbol of his love and grief. Eliot uses the "hyacinth garden" as a way to convey heavy emotion if they are "leaving nor dead." This idea directly aligns with the Hyacinthus myth because the beautiful flower connects both the living and the dead. Hyacinthus is dead, but his memory remains intact through the flower that is named after him.

Lines 43-59 explore the theme of prophecy and tarot, but I find it interesting that rather than use the name of a real “fortune-teller,” Eliot refers to Madame Sosostris, a “famous clairvoyant” from Aldous Huxley’s Chrome Yellow. Chrome Yellow is a satirical commentary on the “fads and fashions of the time”. Through his use of Madame Sosostris, who is also referred to as Mr. Scrogan, as a figure representative of prophecy, Eliot acknowledges the tensions between those who do and don’t believe in the truth of fortune telling. At the end of From Ritual to Romance, the author describes how Tarot may have been designed to predict the fertility of the land. The commentary on Mme. Sosostris’ readings could suggest that tarot has lost its original purpose, tainted by human greed for their own futures, or that the fertility of the land is in some way connected to human life. On line 55, Eliot wrote, “Fear death by water,” connecting the human life cycle with the natural life cycle (since tarot could have once been used to tell if there would be an excess of rain that negatively affected the fertility of the land).

looked up an article about the Civil Rights Movement and compared it to the section in my textbook.

Questions I wrote for both:

• What is the main process / event being explained?

• What are the key steps or causes?

• Why is this important?

• Are there any diagrams or data that support it?

Most of the questions were similar. The textbook answers them in a more structured, step-by-step way with clear definitions and diagrams. The online article answered them more casually and skipped some details.

I tried the phrase “I’m so tired I could sleep for a week” in Spanish and then back to English. It came back almost the same. But when I used slang like That test was lowkey brutal it turned into something weird and didn’t make sense.

1 The quiet study floor of the library 2 A study carrel in the student center 3 my desk at home not the bed or couch the library quiet floor is best suited because i’s already designed for focus and has good lighting. To improve it even more I can go during less busy times, bring noise-cancelling headphones just in case, and make sure i have water and snacks so I don’t have to get up and break concentration

my biggest distractions are my phone notifications the urge to check social media and background noise people talking to control them I can put my phone on do not disturb and leave it in another room, use noise-cancelling headphones or go to the library, and set a 50 minute timer so I know I only have to focus for a short block

For my hardest textbook human geo, I can use the chapter learning objectives at the beginning, the bolded key terms, the summary at the end of each chapter, the review questions, and the diagrams tables those help me know what’s important before I even start reading and give me a quick way to check if I actually understood it

The four steps are 1 Preparing 2 Reading 3 Capturing the key ideas 4 Reviewing I think capturing the key ideas will take the most time because that’s when you have to really think about what you just read write good answers to your questions in the cornell notes define the bold terms and carefully highlight/annotate without overdoing it. It forces you to process the material instead of just passively reading.

yes knowing eactly what questions m trying to answer keeps my mind from wandering as much im not just reading words im hunting for specific answers.

definitely breaking it down into sections and knowing which parts look most important took away that this is so much material feeling definitely breking it down into sections and knowing which parts look most important took away that this is so much material feeling

yeah i do, having the questions written down in the left column of my cornell notes makes me feel like Im in control instead of just hoping Ill figure it out as I go

yes after scanning the chapter and turning the section titles into questions I already know what the main ideas are and what I should be looking for it feels less like Im just diving into a bunch of pages and more like I have a clear plan.

The color filter is clearly labelled, so users can easily understand what they can filter. Having the label along with the colour options makes it easier to navigate.

The all filters button has a clear and descriptive label that tells users what the control does. Clearly labeled interactive elements make websites easier to understand and navigate, particularly for users who rely on assistive technologies.

The product name is presented clearly with the product and its price. I think providing clear textual information about products means users do not have to rely solely on visual images to understand what is being offered.

The page has a clear sale heading that identifies the main purpose of the webpage. Descriptive and big headings help users understand the page structure and allow users, including screen-reader users, to identify the main content more easily.

I wonder how statistically accurate this is. Is the bay area in fact the best suited for success in that field?

This is a claim of fact because it is something that can be argued and proven through research or trials. This is a fact because of the research that proved it. I have friends who have claimed that smoking cigarettes isn't as bad as vapes, and that it's more natural. But it is foolish to claim that it isn't cancer causing, as there are mountains of research that prove the opposite.

Although it seems like federal governments, particularly Canada's, loves AI and data centres, I remain hopeful seeing people stand up against unregulated AI and the construction of these data centres. It reinforces that action does matter, it is not futile, and any positive shift can be substantial.

This is kind of terrifying for me in the context of education. With all the information compiled and summarized by an LLM, there is a lot of convenience that comes with that, but the price is that now teaching that skill is going to pose a lot of challenges. Not to mention the countless artists who get their hard work stolen from them to train LLMs, only to have their own work spat out in the form of slop.

I really resonate with the sentiment that actions can make a difference in the course we make in the future. All the tech overlords and Big Capitalists want us to believe that we can't avoid inevitable disaster, we are going to get left behind if we don't embrace technology, and there is nothing we can do as people. but that is simply never true! Standing up and resisting "bad AI" enables the people around to do the same, and every individual action adds up.

Bad: While Zara’s home page is visually appealing, it relies heavily on large images and minimal text to create that experience. This design creates accessibility barriers for users with visual impairments. Without descriptive text or context, screen reader users may struggle to understand the images, identify featured products, and theerefore, navigate the website effectively. This makes important information less accessible to users who rely on assistive technologies.

When Berman speaks in regard to cattle producing methane gas, it is a topic that most nod their heads on and do not give second thoughts about. Cows are natural = they are living beings that are supposed to be on earth. The biggest methane sources in the world are from humans. From a study in 2022, humans account for almost 70% of the world's total methane emission. 55% of methane is our waste and fossil fuel, while only 22% is from a combination of cattle enteric fermentation and manure management.

As great as eco-fonts could be, it is a shame that companies and businesses have not adopted the usage due to the video being 12 years old and this being one of the first times that I have personally heard of/seen eco-fonts. Unfortunately, it seems that companies and businesses are not as green-thinking as we have hoped.

To be accepted into something

Probation is when you get warned or monitored for breaking certain rules.

Enrollment means to be officially added as a student in a college class, or any class in general

This demonstrates egocentrism

Two stages of preoperational period

Definition

In short: help is given to you as a student and it is to your choice that you either can ask for it or ignore it but the sources will always be there.

This is very true, I find that there is lots more help than I recently thought there was when starting, you just have to reach out for it.

this makes sense, I find that figuring it out myself gives me more insight about the given objective or task I need to complee

helping students get the help they need and having the resources given to them

This created a unhealthy habit and teaches the kids to ignore their natural hunger cues

Does this create “picky eaters” due to knowing and eating what they’re used to?

Obesity affects about 40% of U.S. adults and significantly increases the risk of type 2 diabetes and heart diseases

Healthy daily intake of calories for children and teens should start with school meals. Healthier choices can help reduce the lack of balanced diets.

This control makes them feel safe and secure.

Estatuto Nacional da Microempresa e da Empresa de Pequeno Porte.

Art. 42. Nas licitações públicas, a comprovação de regularidade fiscal e trabalhista das microempresas e das empresas de pequeno porte somente será exigida para efeito de assinatura do contrato.

Art. 43. As microempresas e as empresas de pequeno porte, por ocasião da participação em certames licitatórios, deverão apresentar toda a documentação exigida para efeito de comprovação de regularidade fiscal e trabalhista, mesmo que esta apresente alguma restrição.

Art. 44. Nas licitações será assegurada, como critério de desempate, preferência de contratação para as microempresas e empresas de pequeno porte. (Vide Lei nº 14.133, de 2021

§ 1o Entende-se por empate aquelas situações em que as propostas apresentadas pelas microempresas e empresas de pequeno porte sejam iguais ou até 10% (dez por cento) superiores à proposta mais bem classificada.

§ 2o Na modalidade de pregão, o intervalo percentual estabelecido no § 1o deste artigo será de até 5% (cinco por cento) superior ao melhor preço.

Art. 45. Para efeito do disposto no art. 44 desta Lei Complementar, ocorrendo o empate, proceder-se-á da seguinte forma: (Vide Lei nº 14.133, de 2021

I - a microempresa ou empresa de pequeno porte mais bem classificada poderá apresentar proposta de preço inferior àquela considerada vencedora do certame, situação em que será adjudicado em seu favor o objeto licitado;

II - não ocorrendo a contratação da microempresa ou empresa de pequeno porte, na forma do inciso I do caput deste artigo, serão convocadas as remanescentes que porventura se enquadrem na hipótese dos §§ 1o e 2o do art. 44 desta Lei Complementar, na ordem classificatória, para o exercício do mesmo direito;

III - no caso de equivalência dos valores apresentados pelas microempresas e empresas de pequeno porte que se encontrem nos intervalos estabelecidos nos §§ 1o e 2o do art. 44 desta Lei Complementar, será realizado sorteio entre elas para que se identifique aquela que primeiro poderá apresentar melhor oferta.

§ 1o Na hipótese da não-contratação nos termos previstos no caput deste artigo, o objeto licitado será adjudicado em favor da proposta originalmente vencedora do certame.

§ 2o O disposto neste artigo somente se aplicará quando a melhor oferta inicial não tiver sido apresentada por microempresa ou empresa de pequeno porte.

§ 3o No caso de pregão, a microempresa ou empresa de pequeno porte mais bem classificada será convocada para apresentar nova proposta no prazo máximo de 5 (cinco) minutos após o encerramento dos lances, sob pena de preclusão.

Art. 46. A microempresa e a empresa de pequeno porte titular de direitos creditórios decorrentes de empenhos liquidados por órgãos e entidades da União, Estados, Distrito Federal e Município não pagos em até 30 (trinta) dias contados da data de liquidação poderão emitir cédula de crédito microempresarial.

Art. 47. Nas contratações públicas da administração direta e indireta, autárquica e fundacional, federal, estadual e municipal, deverá ser concedido tratamento diferenciado e simplificado para as microempresas e empresas de pequeno porte objetivando a promoção do desenvolvimento econômico e social no âmbito municipal e regional, a ampliação da eficiência das políticas públicas e o incentivo à inovação tecnológica. (Redação dada pela Lei Complementar nº 147, de 2014) (Vide Lei nº 14.133, de 2021

Parágrafo único. No que diz respeito às compras públicas, enquanto não sobrevier legislação estadual, municipal ou regulamento específico de cada órgão mais favorável à microempresa e empresa de pequeno porte, aplica-se a legislação federal.

Art. 48. Para o cumprimento do disposto no art. 47 desta Lei Complementar, a administração pública: (Redação dada pela Lei Complementar nº 147, de 2014) (Vide Lei nº 14.133, de 2021

I - deverá realizar processo licitatório destinado exclusivamente à participação de microempresas e empresas de pequeno porte nos itens de contratação cujo valor seja de até R$ 80.000,00 (oitenta mil reais); (Redação dada pela Lei Complementar nº 147, de 2014)

II - poderá, em relação aos processos licitatórios destinados à aquisição de obras e serviços, exigir dos licitantes a subcontratação de microempresa ou empresa de pequeno porte; (Redação dada pela Lei Complementar nº 147, de 2014)

III - deverá estabelecer, em certames para aquisição de bens de natureza divisível, cota de até 25% (vinte e cinco por cento) do objeto para a contratação de microempresas e empresas de pequeno porte. (Redação dada pela Lei Complementar nº 147, de 2014)

§ 1o O valor licitado por meio do disposto neste artigo não poderá exceder a 25% (vinte e cinco por cento) do total licitado em cada ano civil.

§ 2o Na hipótese do inciso II do caput deste artigo, os empenhos e pagamentos do órgão ou entidade da administração pública poderão ser destinados diretamente às microempresas e empresas de pequeno porte subcontratadas.

§ 3o Os benefícios referidos no caput deste artigo poderão, justificadamente, estabelecer a prioridade de contratação para as microempresas e empresas de pequeno porte sediadas local ou regionalmente, até o limite de 10% (dez por cento) do melhor preço válido. (Incluído pela Lei Complementar nº 147, de 2014)

Art. 49. Não se aplica o disposto nos arts. 47 e 48 desta Lei Complementar quando: (Vide Lei nº 14.133, de 2021

I - os critérios de tratamento diferenciado e simplificado para as microempresas e empresas de pequeno porte não forem expressamente previstos no instrumento convocatório;

II - não houver um mínimo de 3 (três) fornecedores competitivos enquadrados como microempresas ou empresas de pequeno porte sediados local ou regionalmente e capazes de cumprir as exigências estabelecidas no instrumento convocatório;

III - o tratamento diferenciado e simplificado para as microempresas e empresas de pequeno porte não for vantajoso para a administração pública ou representar prejuízo ao conjunto ou complexo do objeto a ser contratado;

IV - a licitação for dispensável ou inexigível, nos termos dos arts. 24 e 25 da Lei nº 8.666, de 21 de junho de 1993, excetuando-se as dispensas tratadas pelos incisos I e II do art. 24 da mesma Lei, nas quais a compra deverá ser feita preferencialmente de microempresas e empresas de pequeno porte, aplicando-se o disposto no inciso I do art. 48.

one leads after the other.

they are all connected.

this has helped me alot.

summarize the source and helps organize.

start off with mind mapping outlining.

everyone can get different results

free online service that provides writing resources.

I should brainstorm before I start researching.

we can use what we already know to find reliable information.

This sets clear boundaries and gives children to openness to be able to talk and express with their parents.

Children’s from10-13 years old are able to have sexual attraction or excitement.

This is borrowed from a passage in The Programming Task Considered as an Intellectual Challenge (EWD273).

What's interesting about the earlier piece is that just a few paragraphs earlier, he discusses abstract data types some 4–5 years before the paper by Liskov and Zilles and then immediately shifts into riffing on something very close to the Liskov Substitution Principle well before the latter was coined.

every time you research you can find new things.

Question: Why’s it more common in males ?

Definition

Definition

Studies of toddler girls having higher fine motor skills, language skills and social maturity.

how and why questions help look deeper into a problem.

asking questions are important in research.

to understand better reading and writing research situations related to you.

learning isn't just about memorizing answers. Its about being curious and aasking questions.

This description/ title for this shirt and many other products use appropriate text alternatives by descripting the products colour, if its embroidered and also what thread colour it may be.

The products are structured to be categorized and filtered for easy access. They have very clear product controls with the select options making it have distinguishable content. Additionally hovering over the product can show the back of the shirt if there is extra text.

I believe the titles are structured logically, the text is easy to read and understand making this a good practice.

The page is perceivable, operable, understandable and robust. The user can understand how to use and navigate the page.

Tadpoles because it represents simplistic human features of a head and limbs

research is about asking questions about real problems.

I can use things I am interested in to turn them into questions that I can research and learn more about.

Eliminação de doenças e infecções

eLife Assessment

This revised submission remains a valuable contribution to influenza surveillance. After further addressing a previously highlighted methodological issue, the claims of providing an early warning tool aligns more with the reported study results and is a solid addition to the evidence base.

Reviewer #2 (Public review):

[Editors' note: The Reviewing Editor has assessed the revised article without further input from the original reviewers. The Reviewing Editor noted the authors further addressed a methodological concern, and eLife's Assessment remains unchanged from the previous review.]

Summary:

The study aimed to assess the associations between meteorological drivers and influenza is important although not new. The authors used 6 years of surveillance data and deep learning models, combining distributed lag non-linear models (DLNM) with Bayesian-optimized LSTM neural networks for predictive modeling. The key interest in this area is to explore the subtropical locations, where influenza is less common and circulates year-round. The authors further claimed that such an association could be able to provide an early warning in the community.

Strengths:

Study design based on a prospective cohort to analyse the data for retrospective outcomes.

Author response:

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

Public Reviews:

Reviewer #2 (Public review):

Summary:

The study aimed to assess the associations between meteorological drivers and influenza is important although not new. The authors used 6 years of surveillance data and deep learning models, combining distributed lag non-linear models (DLNM) with Bayesian-optimized LSTM neural networks for predictive modeling. The key interest in this area is to explore the subtropical locations, where influenza is less common and circulates year-round. The authors further claimed that such an association could be able to provide an early warning in the community.

Strengths:

Study design based on a prospective cohort to analyse the data for retrospective outcomes.

We would like to express our sincere and heartfelt gratitude to all of you for your exceptionally thorough, constructive, and intellectually rigorous evaluation of our manuscript. The breadth and depth of the feedback we have received reflect a high standard of scientific scrutiny that we deeply respect and appreciate.

eLife Assessment

This important study compares how different classes of drugs act on the SARS-CoV-2 main protease, a key antiviral target, and shows that many of them work by controlling whether the enzyme assembles into its active dimeric form. The evidence, based on a range of complementary biophysical methods, is convincing and points to the interface between the two protein protomers, including a newly found binding site, as a promising target for broad-spectrum antiviral drugs. This work will be of interest to biochemists and virologists working on treatments for coronaviruses.

Reviewer #2 (Public review):

Summary:

This manuscript presents a sophisticated investigation into the mechanisms by which different inhibitor classes affect the SARS-CoV-2 main protease (Mpro), a pivotal antiviral drug target. This study reveals that effective inhibition can be achieved by modulating the stabilization of the essential dimeric state. It also indicates the dimer interface could be a druggable allosteric site, which may offer a strategy for developing broad-spectrum anticoronaviral agents.

Strengths:

The identification of dimer interface stabilization/destabilization as distinct inhibitory mechanisms and the discovery of C300 as a potential allosteric site for ebselen are important contributions to the field. The experimental approach is modern, multi-faceted, and generally well-executed.

Comments on latest version:

All of my concerns have been adequately addressed.

Author response:

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

Reviewer #1 (Public review):

Summary:

Since dimerization is essential for SARS-CoV-2 Mpro enzymatic activity, the authors investigated how different classes of inhibitors, including peptidomimetic inhibitors (PF-07321332, PF-00835231, GC376, boceprevir), non-peptidomimetic inhibitors (carmofur, ebselen, and its analog MR6-31-2), and allosteric inhibitors (AT7519 and pelitinib), influence the Mpro monomer-dimer equilibrium using native mass spectrometry. Further analyses with isotope labeling, HDX-MS, and MD simulations examined subunit exchange and conformational dynamics. Distinct inhibitory mechanisms were identified: peptidomimetic inhibitors stabilized dimerization and suppressed subunit exchange and structural flexibility, whereas ebselen covalently bound to a newly identified site at C300, disrupting dimerization and increasing conformational dynamics. This study provides detailed mechanistic evidence of how Mpro inhibitors modulate dimerization and structural dynamics. The newly identified covalently binding site C300 represents novelty as a druggable allosteric hotspot.

Strengths:

This manuscript investigates how different classes of inhibitors modulate SARS-CoV-2 main protease dimerization and structural dynamics, and identifies a newly observed covalent binding site for ebselen.

Weaknesses:

None. The requested mutagenesis data have been provided in the revised manuscript, and all of my previous concerns have been satisfactorily addressed.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

None. The overall quality of the manuscript has been substantially improved. The authors have added supportive mutagenesis data in the revised manuscript to validate the proposed role of C300. All of my concerns have been adequately addressed.

We appreciate the reviewer’s recognition of the improvements made in the revised manuscript.

Reviewer #2 (Public review):

Summary:

This manuscript presents a sophisticated investigation into the mechanisms by which different inhibitor classes affect the SARS-CoV-2 main protease (Mpro), a pivotal antiviral drug target. This study reveals that effective inhibition can be achieved by modulating the stabilization of the essential dimeric state. It also indicates the dimer interface could be a druggable allosteric site, which may offer a strategy for developing broad-spectrum anticoronaviral agents.

Strengths:

The identification of dimer interface stabilization/destabilization as distinct inhibitory mechanisms and the discovery of C300 as a potential allosteric site for ebselen are important contributions to the field. The experimental approach is modern, multi-faceted, and generally well-executed.

Comments on revised version:

The authors have very nicely addressed most of the previous comments raised. But one comment remains to be clarified relating to original point 5 and the authors' response:

"We agree with the reviewer about the need for quantitative rigor in reporting HDX changes. We have calculated the fractional deuterium uptake difference for each peptide fragment discussed in the text between the inhibitor-bound and unbound states. These values, along with their statistical significance (p-values from a two-tailed t-test), have been provided in the revised manuscript (Legends for Figures 3 and 4). Although the HDX change of residues 296-306 is relatively small (<5%), this region showed a reproducible difference with low experimental variability and statistical significance (p < 0.05). Given its location within the C-terminal dimerization interface and its consistency with native MS, we interpret this change as a subtle local conformational perturbation."

Two questions remain for the statements in line 376-380. First, while it is stated "residues 296-304 in the C-terminal region of Mpro were more flexible upon ebselen binding", the segment of 296-306 is shown Figure 4c. Second, the HDX change for this segment upon ebselen binding is very subtle in the figure (in contrast to the significant HDX change of the same segment in the protein upon PF-07321332 binding), thus making the strong conclusion that "This suggests that ebselen targeting C300 may induce structural changes in the C-terminal helical segment, weakening key hydrogen bonds at the dimer interface and ultimately inhibiting activity" not convincing. The reviewer would suggest the authors either delete this conclusion or largely tone it down.

We thank the reviewer for the recognition of our efforts and agree with the reviewer’s suggestion. We have corrected “residues 296–304” to “residues 296–306” in Line 377 and removed the statement “This suggests that ebselen targeting C300 may induce structural changes in the C-terminal helical segment, weakening key hydrogen bonds at the dimer interface and ultimately inhibiting activity.”, as suggested.

1. Making a good income – 5
2. Having good friends – 4
3. Learning new things about your interests – 5
4. Having a nice car – 5
5. Having intelligent conversations – 5
6. Staying current with the news – 4
7. Playing sports – 2
8. Hanging out with friends – 3
9. Playing computer or video games -2
10. Cooking – 4
11. Online social networking – 2
12. Sleeping – 5
13. Reading a good book – 4
14. Traveling to new places – 5
15. Shopping – 3
16. Being liked by others – 2
17. Studying and reading textbooks – 4
18. Having nice clothing – 3
19. Watching television – 2
20. Enjoying time alone – 5
21. Getting out in nature – 4
22. Working your job – 5
23. Looking good/personal hygiene – 5
24. Meeting new people – 3
25. Going to movies or entertainment – 3
26. Exercising/being physically active – 4
27. Being your own boss – 5
28. Having a positive romantic relationship – 4
29. Engaging in your hobbies – 4
30. Setting your own schedule – 4
31. Volunteering your time for a good cause – 4
32. Cleaning your house – 5
33. Attending class – 5
34. Going to religious services – 3
35. Talking on the phone, texting, emailing, etc. – 3
36. Going to parties – 2
37. Participating in clubs/organized activities – 2
38. Family time – 5
39. Personal time – 5

I think one of the most importnt part of our jobs as mentors is to continue to be refelctive throughout every part of the process. Reflecting on our alignment is just another example!

Why would women be disproportionately hit?

I feel as though this further supports the idea that it is less about the Economic gains and more about an asserting dominance over Canada.

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The headings are compatible with assistive technologies such as VoiceOver that are used to recognize content and navigate between sections. This makes it easier for users who depend on assistive technology to view the website.

The “Build fast on Shopify” section displays how to use Shopify in a clear and simple way using numbered and descriptive sentences. This section makes it easy to understand how Shopify can be used to improve operations in a way that all users can understand.

The “Start for free” clickable link can be reached using the TAB key and clicked on by pressing the ENTER key without using a mouse. This keyboard accessibility allows different users to operate the website without a mouse.

The “Why we build Shopify” video provides closed captions in 18 different languages. This makes the video a lot more perceivable by providing a text alternative for users that cannot hear or understand the audio.

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Interview/Conversation to another person

Outside evidence

Personal Experience

personal experience

personal experience

Personal Experience

Outside Research

Personal Experience

Personal Experience

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This adds another perspective because the problem may involve the lack of non-pornographic content created for and by Black women, not only Google's algorithm. It makes me wonder how Google and content creators can work together to reduce bias and improve representation online.

This passage shows bias in the technology because Google's system connected a racial identity with a large number of sexualized search terms. The issue may also reflect broader cultural bias and stereotypes in the data the algorithm learns from.

This evidence shows that Google's Keyword Planner connected the search term "Black girls" to hundreds of suggestions, many of which were classified as adult content. It provides specific numbers that demonstrate the problem the reporters discovered.

This challenges the assumption that Google search results are always neutral and accurate. It shows that algorithms can reflect the information available online, including harmful stereotypes and unequal representation.

The authors move from their research findings to a larger conclusion about racial bias in Google's systems. The evidence supports this reasoning because the results differed between racial groups, although the findings alone cannot establish every cause of the bias.

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I like how they are promoting their collaboration on their main page. the colours and textures of the logo looks very good. Having the slow-motion dynamic videos below to help display the product is always a good addition.

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It's smart that they have a "Sale" page on the website especially for kids, parents might be trying to find some essentials for a cheaper price. Again, weeding out the expensive stuff, so nobody has to sift threw all the products to find something good on sale.

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This is a good header and display of all the different shoes they carry so people can click basketball or running specifically better for user experience and better for the company to make money as they are weeding out all the products the customer does not want and only having things they are interested in up front.

I feel like this should be smaller, and in a different area of the web page as it is taking up about a 3rd of the screen when it really doesn't need to. It also cuts off the words from the photo slides right below.

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Como parte de los recursos individuales, tras la experiencia de inifdelidad, importará en la experiencia emocional donde se deposita la atención de lo ocurrido, si es en la pérdida del otro prevalece la tristeza, si es en la infidelidad en si misma surge la rabia o dolor

Las emociones que surgen a la par de la anifestación de los celos son el resneitiento, dolor, soledad, rechazo, sospecah y descnifazna y decepción

principal hallazgo

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Every social media button has its own "box" that the user intuitively knows to click on since it functions and looks like a button. It sharply contrasts the background and grabs the user's attention by being distinguishable from the other buttons on this part of the page. Plus, it passes the POUR acronym

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lalala

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Irish playwright, critic, polemicist and political activist

The large pictures and explanatory text close to it appeal to accessibility. It is largely due to how it makes the reader understand. The pictures and text are structured logically and are very distinguishable

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

Learn more at Review Commons

Reply to the reviewers

Response to the Reviewers

Manuscript number: RC-2026-03632

Corresponding authors: Olivier Sperandio and Eugénie Romero

We thank the Editor and the three reviewers for their careful and constructive assessment of our manuscript. We are grateful for the positive feedback on the concept of a pocketome framework for comparing local binding environments across amyloid fibrils. The comments have also helped us clarify an important distinction that was not sufficiently explicit in the original manuscript: our analysis characterizes recurrent structural and physicochemical pocket environments, but does not by itself predict ligand affinity or selectivity. In the revised manuscript, we therefore narrow the interpretation of the pocketome and strengthen the study through experimental ligand-site validation, repeatability and calibration analyses, sensitivity analyses, and an external validation on TDP-43, TMEM106B and transthyretin. We also clarify that the reference pocketome is constructed from amyloid-β, tau and α-synuclein and represents the sampled ordered-core surface environments rather than an exhaustive catalogue of all amyloid ligand-binding modes.

Reviewer #1

1. __ Although this is a computational study, there is a lack of validation. This paper is mainly focused on alpha-synuclein however it would be useful if the authors could validate on tau where ligands such as APN-1607 and MK-6240 have been found to bind to AD PHFs and SFs by cryo-EM. They could also test AV1451 which binds to CTE filaments by cryo-EM. __We thank the reviewer for this helpful and important comment. We have assembled 38 ligand-bound amyloid fibril structures, including tau structures containing APN-1607, MK-6240 and flortaucipir/AV-1451. For each structure, the ligand is removed before pocket detection and the ligand-bound structure is used only afterwards to identify whether a detected pocket corresponds to the experimentally occupied site. We will report site recovery, spatial overlap and filter survival, including which filter removes experimentally occupied sites when they are not retained. This provides a direct validation of the pocket-detection procedure without retuning the detector to individual ligands.
2. __ Robustness of the pocket detection pipeline. If you change the rotamer of a side chain where the ligand has been observed to bind or the charge state, does the pocket remain stable? I suppose one could test this on the high redundancy of the same in vitro structures that reappear in the PDB of alpha-synuclein (as these models have all been built in different cryo-EM maps with different resolutions) - is the same pocket always identified and if not, what is causing that? __We thank the reviewer for this relevant comment. Rather than selecting rotamers or charge states post hoc, we will assess repeatability using corresponding pockets in symmetry-related copies, independent structures belonging to the same structural groups, and repeated experimentally occupied sites. Correspondence will be defined independently of PSI, after which pocket properties and PSI variability will be quantified. We will also examine whether pocket number, volume or similarity depend on structural/model quality.

3. __ The manuscript implies that similar pockets will bind to similar ligands which makes sense. However it would be helpful to describe that with specifics: i.e. electrostatics, solvent accessibility, water molecules nearby, induced fit etc. An overall conclusion Figure that describes pocket architecture would be useful. We will explicitly describe the geometric and physicochemical descriptors used in the pocket representation and distinguish these from factors not currently captured, such as detailed hydration, induced fit and ligand–ligand interactions. We will also add a conceptual figure summarizing the structural features represented by the pocketome and those that remain outside its scope. __Minor comments:

4. __ There is no reference to Figure 1 in the main text. Please include MK6240 as well as a tau pet tracer.__ We will add the missing reference to Figure 1 and incorporate MK-6240 and relevant tau PET tracers into the introductory discussion. We will also correct the terminology concerning the PET tracers, including the spelling of florbetaben and the characterization of ACI-12589.

5. __ Have the authors tried to group filaments based on Scheres amyloid packing algorithm (APD)? __We agree that amyloid packing difference provides a useful orthogonal structural comparison. We will therefore examine the relationship between pocket similarity and APD where technically appropriate, while retaining the existing structural grouping for redundancy control and representative selection rather than replacing the entire classification procedure.
6. __ Figure 5 legend: L298 Please add that this pathological phenotype (i.e. the inclusions formed in cell and mouse models) do not necessarily recapitulate what is observed in disease. This may or may not be due to the structures formed in these model systems. __We will clarify that pathological phenotypes and inclusions observed in cellular or mouse models do not necessarily reproduce the structures observed in human disease and that our structural analysis should not be interpreted as establishing such equivalence.
7. __ L390 - Implications for in vitro models: It should be mentioned that it depends on the question. Distinct structures of alpha-synuclein form in synucleinopathies, as such, studying the disease (e.g. in mouse), it is important to study this within that context. Indeed if a binding site is identical, which they are in many of the greek-key like fold of alpha synculein then yes this can be used in the development of a ligand. However, it should always be validated with brain derived filaments.__ We will revise this section to emphasize that the relevance of an in vitro fibril depends on the biological question. In particular, a conserved local binding environment may support the use of an in vitro fibril as a ligand-development model, but this does not establish that the complete structure reproduces the disease-associated filament. We will therefore emphasize the importance of validation against brain-derived filaments when these are available.
8. __ L408: Missing references to the filament structures. The relevant filament structures and corresponding references will be added at this location and throughout the manuscript where appropriate. __Reviewer #2

__1, A clear definition of a pocket should be included. How deep can it be, what volume, how solvent accessible? This was not at all clear to me and as shown in Figure S15c, if a larger definition of a pocket is used amyloid-specific ligands are found. This might be expected: small pockets are more likely to be shared in common compared with larger ones. Hence, how does the analysis perform if pockets of larger size are considered? Such an analysis would really improve the article and be of immense use for the field. __We agree that the operational definition of a pocket should be made substantially clearer. The revised Methods will provide the VolSite parameters, volume and accessibility criteria, layer and interface filters, symmetry rule, descriptor definitions and complete filter attrition. We will also perform a sensitivity analysis of the pocket-size criterion using the existing detections and examine how pocket counts and similarity patterns change with pocket size. The 38 ligand-bound structures will provide an independent empirical benchmark for determining whether experimentally occupied sites are represented by the retained pockets or by sites excluded by the current filters.

__ Abeta was considered in the work, yet there is little discussion of its pockets in the latter parts of the Results section. This fibril type is especially interesting as it does not have a fuzzy coat. Please add this detail. __We will expand the comparison of amyloid-β pockets and explicitly discuss how its structural organization differs from the other two proteins considered in the reference dataset. __ Regarding the fuzzy coat, this could occlude some of the pockets. Have the authors considered this fact? A pocket may not be solvent exposed in the context of the full fibril and not just the fibril core. __We agree that the fuzzy coat may influence the biological accessibility of pockets. Our analysis is based on the experimentally resolved ordered fibril core and therefore does not reconstruct unresolved or dynamically disordered regions. We will make this limitation explicit and distinguish accessibility in the resolved structural model from accessibility in the complete biological fibril. __ A unique feature of the amyloid fold is its repeating beta strands and the twist. So how does this impact a pocket? Surely many pockets will repeat along the fibril axis creating grooves rather than pockets? Please explain. And regarding the twist, how does this affect the pockets defined? The polymorph analysis in Calypso uses only a very few layers so the twist is not considered in their analysis. __We will clarify that our pocket representation describes local environments within a finite number of fibril layers and does not necessarily capture the complete recognition surface of a ligand spanning several rungs. This limitation will also be discussed in relation to the ligand-bound validation analysis. __ As the field will be especially interested in finding polymorph-specific ligands for disease related amyloids, I would appreciate adding a section that compare the pockets in those fibrils for Abeta, Alpha-synuclein and tau with detailed figures to assist the analysis and clarity. __We will strengthen the cross-protein comparison of amyloid-β, tau and α-synuclein, including representative examples of recurrent and more restricted pocket environments. We will also make the scope explicit throughout the manuscript so that conclusions concerning shared environments are clearly understood as applying to the three-protein reference dataset unless independently supported by the external validation analysis.

Reviewer #3

1. __ The filters may exclude experimentally observed ligand-binding site classes . __We agree that the current filters may exclude experimentally occupied site classes and that this limits the interpretation of the original conclusions. We have therefore initiated an analysis of 38 ligand-bound fibril structures in which ligands are removed before pocket detection, allowing us to determine whether experimentally occupied sites are detected and, if not, which filter excludes them. We will also distinguish surface, enclosed and interface-related site classes in the revised analysis. Importantly, we will revise the conclusions so that the reported recurrence of pockets applies to the sampled ordered-core surface pocket class and is not presented as evidence that selectiv sites are generally rare across all amyloid ligand-binding modes.
2. __ The dataset is restricted to three proteins without justification . __We agree that the restriction to amyloid-β, tau and α-synuclein needed to be stated more explicitly. The revised manuscript will clearly define these three proteins as the reference dataset and will qualify the corresponding conclusions accordingly. To test transferability without compromising the independence of the validation, we have additionally completed representative selection and pocket detection for TDP-43, TMEM106B and transthyretin. These proteins will be treated as an external hold-out set and projected into the frozen three-protein reference space rather than being used to redefine the reference metric.
3. __ The central claim is not yet supported by an explicit test. __We will quantify within- and between-protein similarity using structure-balanced analyses and blocked/hierarchical resampling, accounting for the fact that multiple pockets can originate from the same structure. We will compare the observed mixing with appropriate structure-level null models and report both pocket-weighted and structure-balanced results.
4. __ The metric is unvalidated and the counts behind it are unreported__ __Major comment 4a : __We agree that the PSI scale requires empirical calibration. We will report the full and nearest-neighbour distance/PSI distributions and interpret individual values relative to these empirical distributions rather than treating a fixed PSI threshold as equivalent to uniqueness. We will also explicitly state which dataset defines the reference scale and retain this scale unchanged when projecting the external validation datasets.

Major comment 4b : We will provide the exact mathematical definition of the descriptor vector and distance calculation, including the descriptor index set, units, preprocessing and scaling. We will additionally assess the contribution of descriptor families and test whether the principal conclusions are robust to standardization and feature-family sensitivity analyses.

Major comment 4c : We will quantify similarity and variability for symmetry-related pocket copies, closely related independent structures and repeated experimentally occupied sites, defining correspondence independently of PSI. We will report dispersion and matching failures rather than relying only on average similarity.

Major comment 4d : We will add a complete structure-to-pocket flow showing the number of structures at each stage, the structural groups, representatives, raw cavities, filter attrition and final retained pockets. We will explicitly distinguish the approximately 400 structures used for structural classification from the representative structures subjected to the pocket analysis and from the final pocket dataset.

__Major comment 4e : __We agree that the use of one representative per structural group limits the statistical power to establish how frequent polymorph-specific pockets are. We will therefore qualify the absence of polymorph-specific clusters as an observation within the current representative sampling rather than evidence that such environments are intrinsically rare.

__Major comment 4f : __We agree that model quality is particularly relevant because the pocket descriptors depend on side-chain placement. We will report resolution and available model-quality information for the representatives and test whether pocket properties and similarity correlate with structural quality. We will also perform targeted quality sensitivity analyses and examine the effect of retaining non-identical second protofilaments where appropriate.

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Referee #3

Evidence, reproducibility and clarity

Summary

The authors ask whether amyloid fibril polymorphism produces enough diversity in local surface pockets to allow protein-selective or polymorph-selective ligands. They treat the fibril surface as a catalogue of cavities rather than a set of folds. From 400 cryo-EM structures of α-synuclein, tau and amyloid-β they keep one representative per fold group, detect surface cavities, filter to those judged accessible to a small molecule, encode each as a vector of geometric and physicochemical descriptors, and compare all pairs with a single similarity index. The resulting map is the amyloid pocketome.

They find it largely continuous. Pockets recur across polymorphs and across the three proteins, they do not track fold classification, and the commonest class is a small cavity formed by a few charged or polar side chains. The Concluding perspective states the objective plainly: the work "argues for a shift from a fold-centric to pocket-centric paradigm in amyloid ligand discovery" (l. 480), reports that "many pockets are shared across proteins and polymorphs, whereas truly isolated pockets are rare" (l. 483-485), and holds that this landscape "helps explain why selective amyloid ligands have been difficult to obtain" and "defines the structural limits and opportunities for selective amyloid targeting" (l. 487-492). The study is computational throughout: no new structures and no binding measurements are reported.

Major comments

1. The filters may exclude experimentally observed ligand-binding site classes Ligand-bound amyloid structures include at least two recurrent geometries, defined by the orientation of the ligand relative to the helical axis. The present filters appear to retain exposed surface-groove sites while removing several enclosed or interface-bound sites. Perpendicular (columnar). The ligand lies across the fibril and copies stack into a column along the axis. MK-6240 adopts "a stacked arrangement perpendicular to the fibril axis" and spans about two tau rungs [R2]; GTP-1 stacks across three rungs [R1]; flortaucipir sits at about 46{degree sign} to the helical axis [R3] and F0502B at about 50{degree sign} [R6]. Burial is substantially ligand-ligand: 243 Ų against 208 Ų of protein contact for MK-6240 [R2]. EGCG is the flat limit of this mode, in register with the 4.7 to 4.8 Šrise at 1:1 stoichiometry [R5]; separating flat from tilted cases is secondary to the point made here. Parallel. The ligand's long axis runs along the fibril axis, lying lengthwise in a surface groove and spanning several rungs. APN-1607 binds tau this way at sites 1, 2a and 2b, "parallel to the long helical axis", in both paired helical and straight filaments [R4]. PM-PBB3 is modelled the same way on TMEM106B, "paralleling to fibril axis and spanning four rungs" [R12]. Filter 3 removes cavities at the protofilament interface, and filter 4, together with the criterion illustrated in Fig. S9E for pockets "localized within the adopted monomeric fold", removes cavities buried within the fibril or enclosed by a single fold. Several perpendicular or cleft-bound ligands occupy these site classes. MK-6240 and APN-1607 site 3 are resolved within the C-shaped cavity of the tau fold [R2, R4]; EGCG binds tau at "the polar cleft at the intersection of the two protofilaments" [R5]; F0502B occupies a site at "the protofilamental interface of WT polymorph 5a" on 8ZMY [R7], one of the manuscript's own representatives. What survives filtering appears dominated by shallow surface grooves and by the recurrent 100 to 150 ų cavities defined by two or three residues that the paper calls non-discriminatory (l. 216-222; l. 269-271). State whether the pipeline detects and retains the C-shaped cavity of the Alzheimer tau fold and, if not, which filter removes it. Under either objective stated in the Concluding perspective, this materially limits the claimed scope. If the aim is to describe the amyloid pocketome, a map that omits empirically occupied site classes is incomplete. If the aim is to guide selective ligand design, excluding sites used by several of the best structurally characterised tracers leaves the central question only partially addressed. Retain the excluded classes as separate strata and analyse them alongside the retained surface grooves rather than discarding them before analysis. Isolated pockets are then counted in what remains. Concluding from that count that selective sites are rare in general (l. 385), and that this explains why selective ligands have been hard to obtain (l. 487-488; also l. 355-360, l. 414-419), selects on the predictor. Fold-restricted sites do exist: flortaucipir gives clear density on chronic traumatic encephalopathy Type I filaments while the same study was "unable to visualize additional cryo-EM density for flortaucipir for AD paired helical or straight filaments" [R3]. Fig. S10 does not settle this. VolSite finds a 511 ų cavity coincident with F0502B in 7WMM, so detection works. But 7WMM is holo, so a cavity recovered after deleting the ligand is a cast of it; the test is whether it appears in the apo form, since the representatives are largely apo. And 511 ų is set partly by the four-layer model and the tip filter, so comparing it with a 100 to 150 ų per-rung trough in one unscaled Euclidean space compares different objects. Requested (essential; existing data). Perform a sensitivity analysis of the pocket definition rather than treating the 80 ų minimum (l. 524) as sufficient evidence of ligand relevance. The one cavity shown here to accommodate a real fibril ligand is 511 ų (Fig. S10), whereas the recurrent class called non-discriminatory is 100 to 150 ų (l. 217-218) and cross-amyloid cluster 4 is "approximately 150 ų or less" (l. 266-267). Because a ligand may engage a shallow subpocket or extend beyond the detected volume, this example does not establish one correct cutoff. Re-run the analysis across a justified range anchored to the dimensions and contact footprints of published ligand sites; report how the pocketome, intermingling and isolated-pocket counts change; and report buried contact area per pocket alongside volume. Also compare apo and holo forms of the same polymorph. Separately, recast the conclusion as an explanation of ligand promiscuity rather than of the absence of selectivity, scope the negative-design proposal to the site classes actually sampled, and qualify or remove l. 355-360, l. 385 and l. 487-488. Report how many cavities each filter removes, per protein, and reinstate interface and enclosed classes as separate strata so that empirically occupied binding-site classes are represented. The expected outcome is itself the interesting result. A site able to host a ligand across several rungs is an extended groove or cleft, and not every fold presents one: the C-shaped cleft of the Alzheimer paired helical filament does, whereas flatter folds such as the Pick's disease filament (6GX5, a representative here) are unlikely to. If raising the threshold leaves pockets concentrated on a subset of folds, that is fold-level discrimination emerging from the authors' own data, and it bears directly on the conclusion drawn at l. 385.
2. The dataset is restricted to three proteins without justification The Methods inclusion criteria (l. 497-501) specify cryo-EM structures from the Amyloid Atlas and exclude ssNMR assemblies, monomers and peptides. They never state a restriction to amyloid-β, tau and α-synuclein. The three proteins first appear at the classification step (l. 504-506) as an assumption, so the criteria as written do not reproduce the dataset, and the restriction is absent from the limitations at l. 441-470. The Atlas already holds cryo-EM fibril structures for TDP-43, TMEM106B, transthyretin, IAPP, serum amyloid A, β2-microglobulin, immunoglobulin light chain and prion protein. Extending the map would be a direct test of the central claim, although it would require additional curation, representative selection and filtering rather than being purely mechanical. If convergence reflects backbone-lined grooves that any cross-β spine presents, these folds should intermingle with the three already included; if the map instead separates once more proteins are present, the claim changes. The restriction also sets the scale of the whole metric, since σ is the standard deviation of distances within this three-protein set (l. 543-544). "Isolated in the amyloid pocketome" therefore currently means "isolated among these three proteins", while the title, the term "cross-amyloid pockets" (l. 265) and the Concluding perspective generalise to amyloid as such. TMEM106B is the most pointed omission. It forms amyloid filaments in aged and diseased human brain [R9], and it is a documented off-target of tau PET tracers: both [¹⁸F]PM-PBB3 and [¹⁸F]flortaucipir bind TMEM106B-containing choroid plexus homogenate with high affinity, and PM-PBB3 co-localises with TMEM106B-immunoreactive Biondi ring structures in the choroid plexus epithelium [R10]. Cross-protein binding by clinical tracers is the phenomenon this manuscript sets out to explain, in a protein it excludes. The comparative literature the manuscript relies on is in any case already broader than three proteins: ref. 78 compares disease-associated folds of prion protein, tau, α-synuclein, TDP-43 and TAF15 [R8]. Requested (essential scope correction; expansion strongly recommended). State and justify the three-protein restriction in Methods and add it to the limitations. Revise the title, Abstract and conclusions so that the present map is explicitly a pocketome of the sampled site classes in amyloid-β, tau and α-synuclein, or broaden the analysis sufficiently to support the general terminology. A strongly recommended extension is to add other Atlas proteins, at minimum TDP-43, TMEM106B and transthyretin, and report whether intermingling, the σ-normalised outlier set and the clusters change. If distinguishing sites emerge, describe them and outline how they might be targeted.
3. The central claim is not yet supported by an explicit test "Extensively intermingled" (l. 160-162) and "did not systematically co-localize" (l. 183-184) are inferred from the colours in Figs. 5, 6 and S11 to S13; no test is reported. A minimum spanning tree connects every node by construction (l. 549-551), so connectivity itself cannot establish mixing or non-isolation. A terminal node joined by a long edge can still represent geometric isolation, but the figures provide no interpretable PSI or edge-length scale. Fig. 5 also shows long single-colour runs, which the authors concede as "local enrichments" (l. 162-163). Requested. Compare PSI within proteins against PSI between proteins using structure-balanced summaries. Count how often neighbouring pockets share a label and compare the result with a null obtained by permuting labels at the representative-structure or fold level, preserving all pockets from the same structure. Several pockets are nested within one model, and every pocket contributes to many pairwise PSI values; pocket-level shuffling or treating all PSI pairs as independent would inflate the effective sample size and create impossible mixed labels within a structure. Use blocked permutations or a hierarchical bootstrap with uncertainty intervals, report both pocket-weighted and structure-balanced estimates, and show that the result is not driven by structures yielding unusually many cavities. Repeat for polymorph and for fibril source, which Fig. 2B records but no analysis uses. The second claim, that pockets are decoupled from fold, compares two rulers that measure different things. Pockets are defined by side chains (l. 565-566); polymorph groups come from a Cα RMSD (l. 506-508), which is blind to side-chain packing, since Cα RMSDs "can lead to relatively low values for structures that share similar backbone conformations but differ in their side-chain packing interactions" [R8]. Two structures can therefore share a group and still present different pockets, which is precisely the reported observation (l. 185-186). Requested. Drop the hand-cut groups and correlate PSI against pairwise fold distance directly, computed both as Cα RMSD and as the amyloid packing difference [R8], which counts differing side-chain packing contacts instead of superposing coordinates. If PSI tracks the packing difference but not Cα RMSD, the decoupling is an artefact of the comparator. RMSD also assumes a common superposition, and the α-synuclein models do not share one: ordered spans run 42 to 102 residues and about a quarter of entries contain internal chain breaks (Fig. S1), so equal RMSD values do not describe equal differences. Tau is largely exempt (Fig. S2); amyloid-β mixes Aβ40 and Aβ42 constructs on one axis (Fig. S3). Requested. State the superposed residue range for every comparison, and report whether the α-synuclein and amyloid-β groupings survive restriction to a common core.
4. The metric is unvalidated and the counts behind it are unreported (a) No scale. PSI = exp(−d²/2σ²) restates distance in units of σ: 0.624 is 0.97σ, 0.148 is 1.95σ, and 0.005 is 3.3σ. No distribution is shown, so no value can be judged typical or extreme. The kernel strongly compresses the tail beyond about 3σ, and the "PSI below 0.005" rule operationally groups a broad range of large distances into the same outlier category. It therefore does not by itself separate unusual from unique, yet it defines the selective-targeting opportunities (l. 277-281; l. 320-325). σ is computed within the dataset analysed (l. 543-544), so global and protein-specific values sit on different scales while being quoted together, and every added structure changes every PSI, complicating the claim that new structures can be placed in an unchanged existing map (l. 464-470). Requested. Publish the full and nearest-neighbour PSI distributions and define "similar" and "isolated" against them; rank outliers on raw distance in σ; state which matrix each quoted value comes from; and either give a dataset-independent normalisation or drop the extensibility claim. (b) Ambiguous distance, unscaled descriptors. "Non-zero descriptor values only" (l. 540-541) names no index set. Dropping globally zero columns, or keeping descriptors non-zero in either pocket, equals the full-vector distance; keeping only those non-zero in both compares each pair in a subspace of different size and discards the largest differences, since a descriptor present in one pocket and absent in the other is deleted rather than counted (Fig. 4D: OD1 = 32.2 against 0). Small simple cavities carry the most zeros, so this rule could bias the analysis toward the reported convergence. No scaling is specified, so volume in ų shares a sum with bounded percentages and may dominate. Requested. Give the rule as one equation, recompute under the standardized full-vector alternative and report the correlation between PSI and volume difference. With 109 descriptors and only on the order of 10² retained pockets, correlated descriptor families can overweight one physical property and Euclidean distances can concentrate. Report descriptor correlations or effective rank and the nearest-to-farthest distance contrast, and show that nearest neighbours and DBSCAN assignments are stable after standardization, correlation pruning or PCA, and feature-family ablation. (c) No repeatability benchmark. Two controls sit in the authors' own data. Symmetry-related copies of one pocket are detected twice and the smaller discarded (l. 532-534); their similarity distribution would provide an empirical repeatability envelope or expected upper range, and Fig. S9F already shows five clouds against four on supposedly identical protofilaments. Second, structures sharing a fold group at different resolutions can be run against each other to test whether the same surface yields the same pockets. Until one is reported, observed PSI differences cannot be separated from detection and model variability. Requested. Report both controls, including matching failures and dispersion rather than only a mean PSI. (d) Counts. The Abstract and Significance statement attribute the analysis to 400 structures (l. 5-6; l. 21-22), but pocket detection is performed on roughly 50 representatives selected after classification of those structures. No pocket total is given, in any breakdown, so the DBSCAN result and the claim that "most pockets were not assigned to any cluster" (l. 249-251) cannot be checked. The two headline families are seven pockets from six tau structures and only three pockets spanning two proteins, so the cross-protein example is very small. Requested. Add a flow table from retrieved structures through grouping, representative selection and each filter to retained pockets, and distinguish clearly between the approximately 400 structures used for classification and the approximately 50 representatives subjected to pocket analysis. (e) Limited power for polymorph-specific clusters. No cluster composed only of pockets from a single polymorph was found (l. 275-276), but only one representative structure per group entered the analysis. Several pockets from that representative could in principle form a cluster, but the design supplies no independent within-polymorph replication and has little power to establish that polymorph-specific environments are rare. Requested. Qualify the conclusion as a limitation of the sampling, or test several members of each group. (f) Model quality. Selecting the best-resolved member of each group (l. 125) removes redundancy and is sensible, but it is a relative criterion with no quality floor: a group whose best member is poor still contributes a poor model. The α-synuclein representatives span 1.93 Å (9EUU) to 4.8 Å (9D5C), with 7L7H at 4.0 Å. Global resolution is not identical to local side-chain certainty, but these values raise a material concern for a side-chain-based comparison. One such model carries a reported result: the Lewy-fold match at PSI 0.307 (Fig. 7B) compares a pocket from 9D5C at 4.8 Å against one from 8A9L at 2.2 Å. Separately, five of the 18 α-synuclein representatives contain a second non-identical protofilament, including 6XYO, 8ZMY and 9OBP, so keeping unit 1 only discards a different surface rather than a redundant copy. Requested. Report the global resolution and, where available, local map and model-validation measures for every representative; test whether pocket count, volume and PSI correlate with model quality; apply a justified quality sensitivity analysis; and include non-identical second protofilaments where they were dropped. Bearing on the Concluding perspective (l. 478-492) Read against the analysis, one of the claims made there survives intact.
5. "Ligand selectivity is ultimately governed by the local binding environments exposed on fibril surfaces" (l. 482-483) is the premise rather than a result, and it sits awkwardly with the paper's own material: for a stacked ligand most of the buried surface is ligand-ligand rather than protein, 243 Ų against 208 Ų for MK-6240 [R2], so selectivity there is not governed by the local protein environment alone.
6. "Many pockets are shared across proteins and polymorphs, whereas truly isolated pockets are rare" (l. 483-485) rests on an untested visual reading (point 3), a rarity criterion that saturates and so cannot separate unusual from unique (point 4a), a σ fixed by the three-protein dataset (point 2), and a filtered set that excludes the enclosed sites where ligands are actually observed (point 1).
7. "This constrained pocket landscape helps explain why selective amyloid ligands have been difficult to obtain" (l. 487-488) is not supported. No binding or selectivity measurement enters the analysis at any point, and the ligands being explained bind by a mode the Limitations concede the descriptors do not capture (l. 449-454).
8. "Avoid recurrent cross-amyloid pockets that are structurally predisposed to off-target recognition" (l. 490-491) rests on a cross-amyloid cluster of only three pockets spanning two proteins. Those cavities are 100 to 150 ų, well below the 511 ų of the one site shown here to accommodate a ligand, and whether they constitute complete ligand-binding sites has not been established.
9. "Use in vitro fibrils when they reproduce the relevant local binding environment" (l. 489-490) is the recommendation the analysis does support, and the refinements listed under Patient-derived pockets would strengthen it further. Accordingly, "defines the structural limits and opportunities for selective amyloid targeting" (l. 492) overstates what was done. "Maps the cavity landscape of the sampled site class" is defensible on the present analysis, and would still be a useful contribution. A constructive extension (OPTIONAL): two catalogues this analysis could deliver As run, the analysis chiefly produces a negative result: shared pockets are common and isolated pockets are rare. Two concise catalogues would make the framework more useful and give each result a testable prediction. Broadening them beyond the present three proteins would strengthen their scope but is an optional extension rather than a prerequisite for a properly scoped paper. A catalogue of genuinely selective sites. Rank pockets by similarity to the nearest pocket from another fold. Candidates whose nearest out-of-fold neighbour is distant should be reported with lining residues, volume, accessibility, putative binding mode and the folds in which no counterpart was found. The prediction is direct: ligands designed against these sites should discriminate among a defined fibril panel. A catalogue of minimally selective sites. The converse list is also useful and is closer to what the present data support. Pockets recurring across many folds or proteins are candidate targets for pan-amyloid, pan-tauopathy or other coverage-oriented applications. Report each recurrent class and the proteins and folds in which it occurs; here recurrence is a design specification rather than only a liability. Anchoring makes both lists interpretable. Run published amyloid-ligand cryo-EM complexes through the same pipeline after ligand removal and use the recovered sites as empirical anchors. These include enclosed tau-fold cavities (MK-6240 [R2], flortaucipir [R3]), extended grooves (APN-1607 [R4], F0502B [R6]) and a polar protofilament cleft (EGCG [R5]). If sites occupied by related chemotypes are neighbours, the map gains predictive content; if not, the descriptor set requires revision. A pass/fail test is available now. GTP-1 and MK-6240 independently occupy the same Alzheimer tau site involving Gln351, Lys353, Asp358 and Ile360 [R1, R2]. This provides two checks with known structural answers: whether the pipeline detects and retains that site, and whether it separates the site from tau folds that do not present the cavity, including Pick's disease (6GX5), corticobasal degeneration (6TJX) and progressive supranuclear palsy (7P65). Recovery and discrimination on this case would be a much stronger validation than the single holo example in Fig. S10. One list says where to aim for specificity, the other where to aim for coverage. Both follow from work already done, and together they would convert the pocketome from a catalogue of cavities into an instrument for choosing targets, which is closer to what the Concluding perspective claims.

Minor comments

Patient-derived pockets

• The comparison of patient-derived and in vitro pockets (l. 304-335; Fig. 7) already reports both matches and non-matches, which the surrounding text undersells. Two MSA pockets are matched to in vitro fibrils at PSI 0.624 and 0.335, three Lewy-fold pockets to other polymorphs at 0.154, 0.148 and 0.307, and two Lewy-fold cavities are reported as having no close neighbour at 0.005 or below, with the buried and unassigned-density caveats stated by the authors. The metric therefore produces non-matches as well as matches.
• Three refinements would let the section carry the weight the Discussion places on it. Report all pockets of every ex vivo structure with ranked nearest in vitro matches rather than a selection. Interpret the values against the background distribution requested in point 4a, without which 0.624 and 0.148 cannot be ranked against one another. And use F0502B as a positive control: its site is in the dataset twice (7WMM, Fig. S10; 8ZMY, a representative) and it is roughly tenfold selective for α-synuclein over tau and amyloid-β by direct affinity measurement [R6], so recovering it as selective by PSI would validate the framework on a ligand of known behaviour.
• State how MSA's two protofilaments were handled. The text discusses only "protofilament IA" (l. 310) while Figs. S6 and S8 state the representative unit "were always number 1". Published packing differences for this comparison are 8 to 11% for one protofilament against 60% for the other [R8], so the answer depends on which is used.
• Pocket detection is validated on one example, with no overlap metric and no denominator (l. 141-143; Fig. S10). Tabulate the published amyloid-ligand cryo-EM complexes: cavity detected at the ligand site, numerical overlap, and survival of each filter. The set covers tau [R1-R5], α-synuclein [R6, R7] and TMEM106B [R12]. Internal consistency
• Fig. 6 caption says the legend shows pocket volume; the in-figure legend reads "Polymorph" with entries "-" and 1.0 to 11.0.
• Text gives "7V4C pocket 7" at PSI 0.624 (l. 313); the Fig. 7 legend gives pocket 9. The second MSA pocket at l. 314-316 does not appear in Fig. 7A.
• "Approximately 15 groups per amyloid family" (l. 123) against 18 (Fig. 3), 14 (Fig. S4) and 20 (Fig. S5).
• The dashed cut in Fig. 3 measures 6.72 {plus minus} 0.02 Å against the stated 5.3 Å (l. 507), with 19 to 20 branches crossing rather than 18. The lines in Figs. S4 and S5 measure 4.80 and 3.31 Å and match their legends. Regenerate Fig. 3 or correct the threshold, and confirm which value the 18-group set used.
• Fig. S7 shows 13 tau representatives against 14 groups in Fig. S4; the missing one is explained only in the S4 legend, where 9GG6 is merged with 9GG0 and 6GX5.
• Polymorph labels reach 11 for α-synuclein and 9 for tau, with gaps at amyloid-β 3 and 6, against 18/14/20 groups, plus an unexplained "-" category. Explain the mapping and what "-" denotes.
• Fig. 4C says six filters; Methods number five plus a symmetry rule (l. 523-534). Fig. S15C is titled "Polymorph-specific pockets" although none were found. Typographic: duplicate titles on the title page, "polymorphs.." (l. 329), "Tha alpha-synuclein pocketome" (Fig. S14), "fibil" (Fig. S10).

Figures

• The main and supplementary split does not match where the information is. Fig. 1 contains no data. Fig. 3 shows only the α-synuclein dendrogram, so 55% of structures and 65% of groups are classified in the supplement, and the one dendrogram in the main text is the one that disagrees with its legend. Three supplementary figures are load-bearing: S9, the only place the six filters are shown; S14, on which the isolated-pockets claim rests; S15, the only view of the pocket families. Move Fig. 1 to the supplement, combine the three dendrograms into one main figure, and promote condensed forms of S9, S14 and S15. Fig. S15's image and legend are on separate pages.
• Figs. 5, 6 and S11 to S14 are screenshots of the interactive TMAP page, with an HTML dropdown visible in the legend box, and Fig. 5 labels proteins by PFAM name ("Tubulin-binding"). Regenerate as vector figures with a PSI scale. The 29-category palette of Fig. S11 is unreadable. A volume-coloured pocketome should be shown, given point 4b.
• The most useful missing figure: a PSI distribution with internal anchors on one axis (symmetry-mate duplicates, within-structure pairs, cross-polymorph pairs, cross-protein pairs, permuted null), beside the mixing statistic against that null. If two copies of the same pocket do not score near 1, the labels in Fig. 7 cannot be interpreted.
• Fig. 1: "[¹⁸F]Florbetapen" should read florbetaben. "Clinically-validated" overstates [¹⁸F]ACI-12589, which separates MSA from controls, PD and DLB but shows no increased retention in sporadic PD or DLB [R11]. Second-generation tau tracers are absent although MK-6240 and APN-1607 are central to point 1.

Methods and reproducibility

Not reproducible as written. Absent: VolSite parameters (given as values "previously validated for protein-protein interaction cavities", l. 521-522); DBSCAN eps and min_samples, the definition of the PSI-derived distance matrix, and cluster and noise counts, noting that Fig. S14A shows one chained cluster holding roughly 54 of about 75 assigned pockets across every arm of the tree; the 109 descriptors with units and scaling; MOE version and QuickPrep settings, including pH and whether the default minimisation was disabled, since l. 515 says none was applied; how the four layers were chosen; whether ligands, ions and waters were removed before detection and how many holo structures contribute, since 8ZMY and 9QYL are ligand- or cofactor-bound; TMAP parameters and software versions; the 400 PDB IDs with retrieval date; and analysis code. Supplementary Files 1 to 3 are announced on SI p. 1 but are absent from the supplied review packet; provide them and describe their contents. Only two of the six filters are stated operationally, volume and layer position; give a numeric criterion and per-filter attrition count for each. Manual interventions appear only in SI legends. "Repeated along the fibril axis" (l. 134-135; l. 384; l. 445) is never measured. Useful sensitivity analyses include the RMSD cut, layer count, representative choice, and inclusion versus exclusion of interface and buried pockets. Prior literature Ref. 78 is cited as a preprint but is published [R8]; check refs. 77 and 80 likewise. The ligand-bound amyloid literature is otherwise absent although it bears on point 1: the published classification of binding geometries on α-synuclein polymorphs [R7] is uncited although one of its structures is used as a representative, and no ligand-bound tau or amyloid-β structure is discussed. The FibrilSite preprint (ref. 80) reaches the opposite conclusion on cross-protein site sharing and is answered at l. 421-427 only by appeal to observed ligand non-selectivity, which this manuscript does not test; compare the two methods on a shared subset of α-synuclein structures.

References

[R1] Merz GE, et al. Nat Commun 14:3048 (2023). GTP-1; PDB 8FUG.

[R2] Kunach P, et al. Nat Commun 15:8497 (2024). MK-6240; PDB 8UQ7.

[R3] Shi Y, et al. J Mol Biol 435:168025 (2023). Flortaucipir, CTE filaments; PDB 8BYN.

[R4] Shi Y, et al. Acta Neuropathol 141:697-708 (2021). APN-1607; PDB 7NRV, 7NRX.

[R5] Seidler PM, et al. Nat Commun 13:5451 (2022). EGCG on tau; PDB 7UPG.

[R6] Xiang J, et al. Cell 186:3350-3367 (2023). F0502B; PDB 7WMM.

[R7] Liu K, et al. Proc Natl Acad Sci USA 121:e2321633121 (2024). Binding geometries on α-synuclein polymorphs; PDB 8ZMY, 8X7B, 8X7O, 8X7L, 8X7Q, 8ZLI.

[R8] Scheres SHW. Structure 34:1061-1071 (2026). Amyloid packing difference.

[R9] Schweighauser M, et al. Nature 605:310-314 (2022). TMEM106B filaments.

[R10] Yokoyama Y, Harada R, Kudo K, et al. Transmembrane protein 106B amyloid is a potential off-target molecule of tau PET tracers in the choroid plexus. Nucl Med Biol 142-143:108986 (2025). Postmortem autoradiography and binding assays; PM-PBB3 co-localises with TMEM106B-immunoreactive Biondi ring structures, and both PM-PBB3 and flortaucipir bind TMEM106B-containing choroid plexus homogenate with high affinity.

[R11] Smith R, et al. Nat Commun 14:6750 (2023). [¹⁸F]ACI-12589.

[R12] Zhao Q, et al. Cell Discov 10:50 (2024). PM-PBB3 on TMEM106B; PDB 8J7N. Density weak and also present in apo and PiB maps; pose low-confidence. APN-1607, PM-PBB3 and florzolotau are one compound; flortaucipir is AV-1451 or T807.

Significance

General assessment

The framing is the strongest aspect. The gap between the growth of amyloid cryo-EM structures and the absence of selective ligands is real, and locating the bottleneck at pocket discriminability rather than fold classification is productive. The distinction between a pocket being ligandable and being discriminable is useful, and using a pocketome as a negative filter, to exclude sites where selectivity is implausible before chemistry is invested, is the most valuable idea here. The filtering logic reflects real thought about what a fibril-bound ligand can reach, the limitations section is candid, and the data are deposited. The principal weakness is conceptual. The pocketome pools site classes that the ligand-bound literature treats separately, and because the filters remove interface and enclosed cavities, the reported continuum may be a property of the sampled subpopulation rather than of amyloid surfaces. The work supports the claim that certain ligand classes are promiscuous. It does not establish how rare selective sites are across amyloids, and published fold-discriminating and protein-selective examples constrain any broader negative inference. Beyond that, no hierarchy-aware statistic or repeatability control is reported; the similarity index is under-specified and uncalibrated; and the scope wording blurs the distinction between approximately 400 structures used for classification and approximately 50 representatives used for pocket analysis. Most requested corrections are re-analyses or clearer reporting of data already in hand. One structural feature of the manuscript deserves comment. The Limitations section (l. 441-470) already concedes several of the points on which the central conclusions depend: that detecting a cavity does not demonstrate binding, affinity or selectivity (l. 443-447); that ligands binding "in repeated arrays along the fibril axis" and stabilised by "both fibril-ligand and ligand-ligand interactions" are "not fully captured by pocket descriptors based on local cavity geometry" (l. 449-454); and that the analysis is bounded by the structures available. Each is accurate. The difficulty is that they are offered as caveats on precision while the Discussion draws conclusions that require them to be false. A catalogue of detected cavities is not undermined by the fact that stacking is unrepresented; an explanation of why amyloid ligands are non-selective is, because the ligands in question bind by stacking. Likewise, a map of cavities is unaffected by the caveat that detection is not binding, whereas the claim that isolated pockets are rare, and that selective targeting is therefore structurally constrained, requires detected cavities to stand in for targetable sites. As written, the paper is a legitimate descriptive resource carrying an interpretive layer its own Methods disclaims. Either scope the conclusions to what the analysis supports, or address the limitations rather than acknowledge them.

Advance

Conceptual and technical rather than mechanistic or clinical. It reframes amyloid ligand design from fold-centric to pocket-centric and ports pocketome methodology to filamentous assemblies, which required non-trivial decisions about what counts as a pocket on a helical polymer. The closest work is the FibrilSite preprint (ref. 80), which reaches the opposite conclusion on cross-protein site sharing; the framework is complementary to the packing-difference metric [R8]. Against a ligand-bound literature that has characterised sites one structure at a time [R1-R7], a population-level view is new and is the right instrument for the question. The most useful specific contributions are the identification of recurrent Lys/Arg/Gln/Tyr cavities as poorly discriminating, and the demonstration that some patient-derived α-synuclein pockets have in vitro counterparts.

Audience

Specialised: amyloid structural biologists working on cryo-EM of tau, α-synuclein and amyloid-β filaments; computational and structure-based design groups working on shallow or interface-like surfaces; and PET tracer programmes in neurodegeneration, for whom the negative-design idea and the in vitro model-selection argument are directly relevant. With the statistics and the geometric stratification in place, the general message, that polymorphism at the fold level need not imply polymorphism at the pocket level, would reach a broader biophysics and drug-discovery readership. The framework transfers to TDP-43, TMEM106B, hnRNPA1/A2 and transthyretin.

Reviewer expertise

Cryo-EM of amyloid fibrils and helical reconstruction; structural biology of tau and α-synuclein polymorphs; ligand-bound filament structures and PET tracer binding modes; computational structure-based design; clustering methodology on structural datasets.

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Referee #2

Evidence, reproducibility and clarity

This manuscript describes an interesting analysis of the presence of absence of binding pockets displayed on the surface of all know amyloid fibril structures of Abeta, tau and alpha-synuclein. Using their definition of a 'pocket' the authors show that fibrils of the same protein type of different polymorph type (defined by the Calypso algorithm developed by Connor et al) or those from different proteins can share common pockets with a few examples of fibril type specific pockets.

The work will be of broad interest to those seeking to develop amyloid-specific binders for discovery research and for its translation into the clinic in neurodegenerative disorders. I have comments that I hope will improve the understanding and impact of this analysis:

1. A clear definition of a pocket should be included. How deep can it be, what volume, how solvent accessible? This was not at all clear to me and as shown in Figure S15c, if a larger definition of a pocket is used amyloid-specific ligands are found. This might be expected: small pockets are more likely to be shared in common compared with larger ones. Hence, how does the analysis perform if pockets of larger size are considered? Such an analysis would really improve the article and be of immense use for the field.
2. Abeta was considered in the work, yet there is little discussion of its pockets in the latter parts of the Results section. This fibril type is especially interesting as it does not have a fuzzy coat. Please add this detail.
3. Regarding the fuzzy coat, this could occlude some of the pockets. Have the authors considered this fact? A pocket may not be solvent exposed in the context of the full fibril and not just the fibril core.
4. A unique feature of the amyloid fold is its repeating beta strands and the twist. So how does this impact a pocket? Surely many pockets will repeat along the fibril axis creating grooves rather than pockets? Please explain. And regarding the twist, how does this affect the pockets defined? The polymorph analysis in Calypso uses only a very few layers so the twist is not considered in their analysis.
5. As the field will be especially interested in finding polymorph-specific ligands for disease related amyloids, I would appreciate adding a section that compare the pockets in those fibrils for Abeta, Alpha-synuclein and tau with detailed figures to assist the analysis and clarity.

Referee cross-commenting

All three referees appeared to like the concept of the manuscript and all three ask for more detail and to include more proteins to test the generality of the claims made. I agree with all the suggestions and I hope the authors can address the comments with the requested details and analyses added. If so, this would then make an excellent reference for those working in the amyloid field.

Significance

This manuscript describes an interesting analysis of the presence of absence of binding pockets displayed on the surface of all know amyloid fibril structures of Abeta, tau and alpha-synuclein. Using their definition of a 'pocket' the authors show that fibrils of the same protein type of different polymorph type (defined by the Calypso algorithm developed by Connor et al) or those from different proteins can share common pockets with a few examples of fibril type specific pockets.

The work will be of broad interest to those seeking to develop amyloid-specific binders for discovery research and for its translation into the clinic in neurodegenerative disorders.

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

Learn more at Review Commons

Referee #1

Evidence, reproducibility and clarity

This manuscript presents a computational framework for characterizing ligand binding pockets for amyloid filaments. They construct a "pocketome" and the authors describe binding site similarities across different amyloid proteins. They identify conserved and polymorph-specific pockets (which is important for specificity of ligands) and discuss implications for selective ligand design.

The work does address an important problem in that amyloid filaments typically have limited sites in which ligands can bind to and these can be shared across different amyloid polymorphs which is a strength and conceptually interesting. This will be of specialized interest to the ligand-amyloid field. I have a few concerns:

Major comments:

1. Although this is a computational study, there is a lack of validation. This paper is mainly focused on alpha-synuclein however it would be useful if the authors could validate on tau where ligands such as APN-1607 and MK-6240 have been found to bind to AD PHFs and SFs by cryo-EM. They could also test AV1451 which binds to CTE filaments by cryo-EM.
2. Robustness of the pocket detection pipeline. If you change the rotamer of a side chain where the ligand has been observed to bind or the charge state, does the pocket remain stable? I suppose one could test this on the high redundancy of the same in vitro structures that reappear in the PDB of alpha-synuclein (as these models have all been built in different cryo-EM maps with different resolutions) - is the same pocket always identified and if not, what is causing that?
3. The manuscript implies that similar pockets will bind to similar ligands which makes sense. However it would be helpful to describe that with specifics: i.e. electrostatics, solvent accessibility, water molecules nearby, induced fit etc. An overall conclusion Figure that describes pocket architecture would be useful.

Minor comments:

1. There is no reference to Figure 1 in the main text. Please include MK6240 as well as a tau pet tracer.
2. Have the authors tried to group filaments based on Scheres amyloid packing algorithm (APD)?
3. Figure 5 legend: L298 Please add that this pathological phenotype (i.e. the inclusions formed in cell and mouse models) do not necessarily recapitulate what is observed in disease. This may or may not be due to the structures formed in these model systems.
4. L329: typo error with two ".."
5. L390 - Implications for in vitro models: It should be mentioned that it depends on the question. Distinct structures of alpha-synuclein form in synucleinopathies, as such, studying the disease (e.g. in mouse), it is important to study this within that context. Indeed if a binding site is identical, which they are in many of the greek-key like fold of alpha synculein then yes this can be used in the development of a ligand. However, it should always be validated with brain derived filaments.
6. L408: Missing references to the filament structures.

Significance

This manuscript presents a computational framework for characterizing ligand binding pockets for amyloid filaments. They construct a "pocketome" and the authors describe binding site similarities across different amyloid proteins. They identify conserved and polymorph-specific pockets (which is important for specificity of ligands) and discuss implications for selective ligand design.

The work does address an important problem in that amyloid filaments typically have limited sites in which ligands can bind to and these can be shared across different amyloid polymorphs which is a strength and conceptually interesting. This will be of specialized interest to the ligand-amyloid field.

https://i.imx.to/i/2026/09/17/75r8km.jpg

https://youtu.be/NWnX3ZkL_eA?si=mUD52esB34cLxhbJ&t=353

Suggestion to use index card or pocket notebook as a viewfinder for narrowing down one's view of the world.

https://i.imx.to/i/2026/09/17/75r8kl.jpg

https://i.imx.to/i/2026/09/17/75r8kk.jpg