8,429 Matching Annotations
  1. Dec 2021
    1. But ifall we’re doing is cherry-picking, we could just as easily have chosenthe much earlier burial known to archaeologists as Romito 2 (afterthe Calabrian rock-shelter where it was found). Let’s take a momentto consider what it would mean if we did this.

      Keep in mind here that these are only singular examples they're talking about amongst millions of data points that we don't have.

    1. violencia de género

      «Todo acto de violencia sexista que tiene como resultado posible o real un daño físico, sexual o psíquico, incluidas las amenazas, la coerción o la privación arbitraria de libertad, ya sea que ocurra en la vida pública o en la privada».

  2. Nov 2021
    1. El factor tiempo de trabajo está también muy relacionado con la variabilidad de la presión de inyección, ya que a medida que incrementa el tiempo de operación de un motor, se incrementa el desgaste de las piezas, siendo agravado el sistema de alimentación por la variación de la calidad del combustible

      También depende del factor de cuanto uso se le ha dado a ese motor, no solo por el incremento de desgaste significa que va a bajar su calidad.

    1. painted an American flag on his bare chest, but painted it upside down.

      It was over the fact that there was a burning of the Flag not a painting on his chest

    1. Eliminating Covid-19 seemed theoretically possible, because the original 2002 SARS virus ultimately disappeared.

      Eliminating SARS-CoV-2 was deemed plausible, because SARS-CoV-1 had been eliminated.

    1. Epstein and Walker, p194

      This is only a citation from our textbook (which is still wrong, the brief can be found on p. 394). The proper legal citation is 536 U.S. 639 (2002).

  3. Oct 2021
    1. which might disappoint its most important designs

      What is meant by "most important designs"? Is it meant that the state governments are supposed to depend on the federal government and not vice versa? If so, that seems to go against the core contractual aspect of federalism.

    2. every argument which would sustain the right of the General Government to tax banks chartered by the States, will equally sustain the right of the States to tax banks chartered by the General Government.

      Is this relationship between State, banks, and General Government rooted in reciprocity or mutual responsiblity?

    3. CONFIDENCE.

      Is this something that is actively being said? If so, is this something that was yelled a little but louder? Or was this only written and capitalized for effect.

    4. incompatible with the language of the Constitution.

      How exactly is the necessity of resorting to mean which it cannot control not compatible with the language outlined in the Constitution?

    5. It is true, they assembled in their several States—and where else should they have assembled?

      When assembling in their states, did they form permanent locations to meet or was it a random?

    6. The people of all the States have created the General Government,

      A lot of what we've read up to this point make it seem like the Supreme Court acts as a kind of voice for the Constitution, that it would interpret and enforce the objectives of it. It's never felt like a branch that was representative of the people. Does the Supreme Court ever act or treat itself as representative of the people, or does it more view itself as a representation of the Constitution?

    7. The Constitution of our country, in its most interesting and vital parts, is to be considered, the conflicting powers of the Government of the Union and of its members, as marked in that Constitution, are to be discussed, and an opinion given which may essentially influence the great operations of the Government....

      Does Marshall regularly define the scope of the case like this at the start of his decisions? I don't recall anything similar in Marbury v Madison.

    8. the Bank of the United States is a law made in pursuance of the Constitution, and is a part of the supreme law of the land....

      What does this mean? so the bank of the united states is inherently also the supreme law of the land?

    9. may be carried into execution, would partake of the prolixity of a legal code, and could scarcely be embraced by the human mind. It would probably never be understood by the public.

      I am lost here. Is this referring to the idea that if the constitution had every subdivision of rules stated that it would not be embraced by human kind and that it would never be understood by the public? Because wouldn't a constitution with accurate details be the most accepted to the public?

    10. quieting the excessive jealousies

      Is here referring to the jealousies of the states to the federal government or the federal government to the states? Who in this scenario is the jealous party leading to the creation of the 10th amendment?

    11. Its nature, therefore, requires that only its great outlines should be marked, its important objects designated, and the minor ingredients which compose those objects be deduced from the nature of the objects themselves.

      At the end of this where it states 'deduced from the nature of the objects themselves' - the objects themselves are the individual states and people in those states, correct? So if there was no mention of the laws regarding bank taxes of states in the constitution, but the grand power of the law is saved for the people, which influence the government, makes me wonder- was this creation of Maryland's bank and bank tax, at all ever voted on by the people? Or was it 'secretly' created and decided by the government of the state alone?

    12. the power of establishing a branch in the State of Maryland might be properly exercised by the bank itself,

      This section confuses me. Is Marshall saying here that not only can the government create a national bank, but they can delegate powers to this bank for it to exercise? Or is it saying that the State of Maryland, where the bank will be established, is to delegate powers to this national bank?

    13. That the power to tax involves the power to destroy;

      Definitely seems like a major quote. Is he basically saying that this would give state governments the power to destroy the federal government?

    14. burden,

      This word choice seems dangerous in how vague it is. A lot of actions that a state might take could be defined as "burdening" a law enacted by Congress. Who decides what is a burden? I wonder if there have been cases that have happened since that argue over whether a state action is a "burden" to a national government function? Or if this part of Marshall's argument has ever been used as precedent for the federal government to stifle state power?

    15. This, then, is not a case of confidence, and we must consider it is as it really is.

      I feel like I've been lost in this paragraph. I'm not entirely sure what is trying to be said here?

    1. Vietnam War, President Truman issued an executive order commanding the secretary of commerce to seize the nation's steel mills and keep them in operation

      incorrect: the book version states: "President Harry S. Truman was not about to let a strike hit the steel industry. The nation was engaged in a war in Korea, and steel production was necessary to produce weapons and other military equipment."

    1. Barros-Martins, J., Hammerschmidt, S. I., Cossmann, A., Odak, I., Stankov, M. V., Morillas Ramos, G., Dopfer-Jablonka, A., Heidemann, A., Ritter, C., Friedrichsen, M., Schultze-Florey, C., Ravens, I., Willenzon, S., Bubke, A., Ristenpart, J., Janssen, A., Ssebyatika, G., Bernhardt, G., Münch, J., … Behrens, G. M. N. (2021). Immune responses against SARS-CoV-2 variants after heterologous and homologous ChAdOx1 nCoV-19/BNT162b2 vaccination. Nature Medicine, 1–5. https://doi.org/10.1038/s41591-021-01449-9

    1. This is of the very essence of judicial duty.

      When deciding a case, how is being able to disregard the constitution part of judicial duty? Isn't that the backbone for making laws in this country? It the court's duty to determine if the laws are constitutional or not.

  4. Sep 2021
    1. The secretary of state, being a person holding an office under the authority of the United States, is precisely within the letter of the description; and if this court is not authorized to issue a writ of mandamus to such an officer,

      I am a little lost in this area. Why is Madison the name on this case and not Jefferson? Is this sentence explaining that? I don't quite understand they way this is phrased.

    2. appellate jurisdiction may be exercised in a variety of forms

      What does that mean appellate can be exercised in a variety of forms? Is this referring to the different types of lower courts that hears under this type of jurisdiction?

    3. practical and real omnipotence

      As a Federalist, Marshall knows that many of the people reading this opinion will be Anti-Federalists. I'd love if my classmates would weigh in: Do you think he is using the exaggerated language of "omnipotence" to appeal to the Anti-Federalist fear of concentrated power? Do we think Marshall is speaking from a totally neutral judicial stance here, or do aggrandized depictions like these hint that he is letting his political bias shine through?

    4. If then the courts are to regard the constitution; and the constitution is superior to any ordinary act of the legislature; the constitution, and not such ordinary act, must govern the case to which they both apply.

      What would make the constitution not superior if put in the situation of conflict? From my understanding the Constitution is going to be more superior or hold more weight

    5. If the solicitude of the convention, respecting our peace with foreign powers, induced a provision that the supreme court should take original jurisdiction in cases which might be supposed to affect them; yet the clause would have proceeded no further than to provide for such cases, if no further restriction on the powers of congress had been intended.

      What exactly is this stating? I this suggesting that the courts don't have any jurisdiction over foreign powers, and only take cases that are related to their branch?

    6. The principles, therefore, so established, are deemed fundamental. And as the authority, from which they proceed, is supreme, and can seldom act, they are designed to be permanent.

      I know that the peaceful transfer of power between one administration to another is a big deal in the United States and a coveted tradition. Does this case have anything to do with that precedent? Obviously this has to do with judgeship and not the presidency, but refusing to deliver the commissions in the last days of office certainly screams sore loser. So other than establishing judicial review within the courts maybe this case had other effects on the tradition of American politics?

    7. If this obloquy is to be cast on the jurisprudence of our country, it must arise from the peculiar character of the case....

      This whole phrase is just completely in one ear and out the other for me. Would anybody be able to explain what this means? It is just a fancy way for getting into the opinion on the case?

    8. appointment conferred on him a legal right to the office for the space of five years.

      When mentioning Mr. Marbury's commission, they state that he is given a five year term. Were judicial appointments not lifetime appointments within the early years of the court system? Was the term five years for each appointee?

    9. Affirmative words are often, in their operation, negative of other objects than those affirmed; and in this case, a negative or exclusive sense must be given to them or they have no operation at all.

      I am confused on what affirmative words are. What do they have to do with this case? If anything, shouldn't there need to be positive affirmation words towards the case? If it is only negative, then wouldn't that be the opposite of having no operation at all?

    10. That it thus reduces to nothing what we have deemed the greatest improvement on political institutions -- a written constitution -- would of itself be sufficient, in America, where written constitutions have been viewed with so much reverence, for rejecting the construction.

      At this point, Marshall has invoked the idea of and principle behind written constitutions generally about 5 times. I don't take issue with his argument, but is there a reason he acts as if the principle behind a constitution is so well understood but does not invoke specific examples from somewhere else? Did he think this was not helpful for a young United States in defining the scope of its own laws? Or did he not have a useful example of a written constitution from another nation to cite?

    11. To enable this court then to issue a mandamus, it must be shown to be an exercise of appellate jurisdiction, or to be necessary to enable them to exercise appellate jurisdiction.

      I think I'm lost at this point? I'm not entirely sure what point they are trying to make, and there are far too many large words in the previous paragraph for me to be able to make sense of what is happening in this section. I certainly feel lost.

    1. Ovelha

      Animal adulto, se torna ovelha no momento em que parir. Caso nunca pariu, se torna ovelha depois que trocar dentes, por volta de ansos.

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    1. L’esercizio fisico deve essere raccomandato per il controllo del diabete nelle persone con diabete di tipo 2?

      Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum.

    1. Stephen G. Breyer

      I believe Justice Breyer is part of the majority. Similar to Justice Alito, he gives Layton several hypotheticals. Layton's argument toward these hypotheticals show discrimination by not giving benefits, such as policemen, to a church simply because it is religious. Justice Breyer also sounds annoyed and short with Layton.

    2. Elena Kagan

      I believe Justice Kagan is the second dissenter. She seems to side against Cortman as she argues that the States should have some flexibility in their choices. She questions what would happen if one church received benefits while a different church didn't. Cortman goes on to say that the program is religion blind, but its seemed to me that she was not agreeing with anything he said.

    3. and there is a point where you can accommodate religion,

      I am a little confused with Cortman's argument. If the court favors religion, then theres an Establishment Clause problem, but if the court attacks religion, then theres a Free Exercise Clause problem. How can these things be balanced out? When is it appropriate to accommodate religion? What are other examples of the court accommodating religion?

    4. World Vision brief,

      What is the World Vision Brief? Is this apart of the amicus brief? Im confused and already not familiar about what exactly an amicus brief is.

    5. Neil Gorsuch

      I think justice Gorsuch is another one of the majority voters here. He seems very secure on the basis of religious discrimination as opposed to the questions of sotomayor and kagan who seemed to recognize it as a possible issue with the inclusion of other religions. He already seems convinced that it was a case of discrimination against the church.

    6. For example, in that case, we have independent decisionmaking, which has been key to many of this Court's decisions.

      Layton is in majority rule for the church funding the playground by his statement of independent decision making. He specifically says its in the hands of the state.

    7. But there's -- there's government coercion when you say there's a public benefit, and the only way you could receive that public benefit is if you do not exercise your religion.

      I understand the idea that public benefit from the government and religious states should not mix. Are there other examples of government coercion to public benefit you could provide as examples?

    8. we don't want to, as a country -- well, the vast majority of States, to fund houses of worship.

      I also read this as a sign of a dissenting justice. I believe Sotomayor is a dissenting justice because she strictly says the state does not fund places of worship. She goes on to say that if the state is free from places of worship, then funding it would affect free exercise.

    9. Elena Kagan

      I think this is a great point brought up by justice Kagan. It ties back to one of my questions I left earlier. She seems to think that there may be a risk of favoritism and a violation of that establishment clause that Cortman keeps talking about but going the other way. And again Cortman doesn't do a good job answering that question, in fact in this example he sidesteps it all together and simply says the system is set up to not discriminate and be "religion blind" Although that sounds great, the problem is discriminatory people wont be "religion blind" which is something she seems to realize.

    10. And so on one side you have the Establishment Clause.

      I looked up the establishment clause and it is essentially a clause put within the fifth amendment that prohibits congress from establishing religion. To me it seems like funding a religion is a very good way to establish it so I'm not sure exactly what his point is here. Unless he is suggesting that all religious schools recieve the same public benefits/funding. And unfortunately I have a hard time believing that certain states would ever fund anything other than christianity if it was left to a state level decision.

    11. Okay?

      Based on Justice Breyer's tone in this section, which seems to indicate annoyance with the respondant, I'm going to conclude that he was one of the Justices who voted with the majority. While it is possible that his tone is merely a tool to probe the strength of Layton's legal argument, it sounds more like he has already made up his mind and is growing impatient with having to hear Layton's case.

    12. I know your white light is on.

      Do any of my fellow students know what Justice Kagan is referring to here? Is Cortman's "white light" perhaps a five-minute warning light, since we are reaching the 25-minute mark, and each party gets 30 minutes? Or perhaps it just means "it's time to wrap it up?"

    13. That's a history that's even longer than the Locke history.

      Although I wasn't feeling sure beforehand, this moment from Justice Sotomayor fortified my notion that she was the second dissenting Justice in this case. Just as Ginsberg did, she chose to utilize the Stare Decisis method of decision making, and asserts here that the older a legal precedent that has been set is, the more firmly it holds as an establishment of proper interpretation of law. This solififies her argument that follows, that as a country we should not fund religious places of worship.

    14. Because the kind of examples Your Honor is giving are examples where the -- the benefits are universal. They are not selective, which they are here; they are universal. So we start on the endorsement side.

      This is a very good point that I feel like is ignored. I have to wonder why this was not expanded on further - both by Layton himself or one of the justices.

    15. how Missouri interprets the term "church" in its constitution?

      This question by Justice Alito made me realize that each state may have its own definition of "church". If this is to be one of the deciding factors of this particular case, and a case of the same material opens up in a different state, would the Supreme Court use Trinity Lutheran v. Comer to decide in a similar manner? Or would they have to judge with no stare decisis because of the different terms within the different state constitutions?

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    1. s. This study originated from the desire of tenants to obtain reliable data which they could use for their cause. They did not carry out the study but they were consulted so that its form would serve their needs. They subsequently made use of the findings to argue their case both to the media and to the local authori

      The study had benefit for the communities.

    2. Damp ho

      Damp rental homes creating health issues in Scotland.

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    1. Refusing to stand convicted on the teacher's charges of laziness, I'd spend four hours a night on my homework,working even longer whenever we were assigned an essay. I suppose I could have gotten by with less, but I wasdetermined to create some sort of an identity for myself. We'd have one of those "complete the sentence"exercises, and I'd fool with the thing for hours, invariably settling on something like, "A quick run around thelake? I'd love to. Just give me a minute to strap on my wooden leg." The teacher, through word and action,conveyed the message that, if this was my idea of an identity, she wanted nothing to do with it.

      trying really hard to make himself stand out. spending alot of time on his work maybe going above and beyond. its all for nothing though teacher doesn't seem amused

    2. When called upon, I delivered an effortless list of things I detest: blood sausage, intestinal pâté, brain pudding.I'd learned these words the hard way. Having given it some thought, I then declared my love for IBM typewriters,the French word for "bruise," and my electric oor waxer. It was a short list, but still I managed to mispronounceIBM and afford the wrong gender to both the oor waxer and the typewriter. Her reaction led me to believe thatthese mistakes were capital crimes in the country of France.

      he thought he was doing well until he mispronounced ibm and learns that it was a mistake he shouldn't of made because of how the teacher was ridiculing every single student he was confused as to why the teacher was referring to objects as genders it just didn't make sense to him

    3. While the optimist struggled to defend herself, I scrambled to think of an answer to what had obviously becomea trick question. How often are you asked what you love in this world? More important, how often are you askedand then publicly ridiculed for your answer? I recalled my mother, ushed with wine, pounding the table lateone night, saying, "Love? I love a good steak cooked rare. I love my cat, and I love . . ." My sisters and I leanedforward, waiting to hear our names. "Tums," our mother said. "I love Tums.

      he realized at this moment he was screwed becasue of the teachers attitude towards everyone. he was reminded of his childhood and how he felt towards his mother.

    4. The second Anna learned from the rst and claimed to love sunshine and detest lies. It sounded like a translationof one of those Playmate of the Month data sheets, the answers always written in the same loopy handwriting:"Turn-ons: Mom's famous ve-alarm chili! Turnoffs: Insincerity and guys who come on too strong!!!

      very cliche he probably felt like she was boring

    5. Oh, really," the teacher said. "How very interesting. I thought that everyone loved the mosquito, but here, infront of all the world, you claim to detest him. How is it that we've been blessed with someone as unique andoriginal as you? Tell us, please."

      very sarcastic towards the student

    6. 've moved to Paris in order to learn the language. My school is the Alliance Française, and on the rst day ofclass, I arrived early, watching as the returning students greeted one another in the school lobby. Vacations wererecounted, and questions were raised concerning mutual friends with names like Kang and Vlatnya. Regardlessof their nationalities, everyone spoke what sounded to me like excellent French. Some accents were better thanothers, but the students exhibited an ease and condence I found intimidating. As an added discomfort, theywere all young, attractive, and well dressed, causing me to feel not unlike Pa Kettle trapped backstage after afashion show

      hes feeling nervous apparently and is aware of his surroundings and kind of feels out of place.

    1. Đọc sách nghe tưởng chừng là việc quen thuộc và phức tạp vô cùng đối với nhiều người mà lại trở nên đơn giản đến không tưởng với cách diễn tả sâu sắc qua từng câu chuyện của tác giả Phan Thanh Dũng.

    1. The main idea comes at the beginning, the bodyparagraphs support the main idea, and the conclusion wraps up the whole thing

      This is good to know and remember so you know what to look for when reading something. i have to make sure to have these 3 components when writing any kind of essay.

    1. art. D. 411-2 du code de l’éducation).

      Le conseil d'école vote le règlement intérieur de l'école, établit le projet d’organisation pédagogique de la semaine scolaire, dans le cadre de l'élaboration du projet d'école à laquelle il est associé, donne tous avis et présente toutes suggestions sur le fonctionnement de l'école et sur toutes les questions intéressant la vie de l'école (par exemple, intégration des enfants handicapés, activités périscolaires, restauration scolaire, hygiène scolaire, sécurité des enfants, art. D. 411-2 du code de l’éducation).

  5. Aug 2021
    1. Task 2 Briefly not the main topics throughout each section of the text. This section explains the differences in philosophy from other types of science. It shows how they collect knowledge and that there is not a definite answer to philosophical questions. Restate the main point of th opening paragraph in your own words. The opening paragraphs main point is that philosophy is the study of value. Many time it is undermined by other types of science that claim it is pointless. These people do not allow any kind of growth outside of said science. Estate the main point of the closing paragraph in your own words Philosophy’s goal is not to come to a conclusion but instead expand you thoughts. This is the knowledge that makes it possible to grow how we think. Everyone find and define two vocabulary words Unalloyed-complete and unreserved Dogmatism-the tendency to lay down principles as incontrovertibly true.

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    5. ZDB-ALT-141023-2

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    6. ZDB-ALT-060301-2

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    1. Hillus, David, Tatjana Schwarz, Pinkus Tober-Lau, Hana Hastor, Charlotte Thibeault, Stefanie Kasper, Elisa T. Helbig, et al. “Safety, Reactogenicity, and Immunogenicity of Homologous and Heterologous Prime-Boost Immunisation with ChAdOx1-NCoV19 and BNT162b2: A Prospective Cohort Study,” June 2, 2021. https://doi.org/10.1101/2021.05.19.21257334.

    1. ZDB-ALT-080528–2

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    1. Prof. Devi Sridhar. “Rest of the World Watching Closely: ‘In Scotland, Estimated That 92.5% of Adults Would Have Tested Positive for Antibodies against SARS-CoV-2 on a Blood Test in the Week Beginning 12 July 2021’- Is This Enough to Dampen Transmission & Protect under 12s from Infection? Under 18s?” Tweet. @devisridhar (blog), August 4, 2021. https://twitter.com/devisridhar/status/1422852550957617157.

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    1. RRID:ZFIN_ZDB-ALT-060322-2

      DOI: 10.7554/eLife.44431

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    2. RRID:ZFIN_ZDB-GENO-100820-2

      DOI: 10.7554/eLife.44431

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    1. RRID:ZFIN_ZDB-ALT-010919-2

      DOI: 10.1016/j.cub.2018.11.059

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      SciCrunch record: RRID:ZFIN_ZDB-ALT-010919-2


      What is this?

    1. RRID:ZFIN_ZDB-GENO-060623-2

      DOI: 10.1002/cne.24001

      Resource: (ZFIN Cat# ZDB-GENO-060623-2,RRID:ZFIN_ZDB-GENO-060623-2)

      Curator: @scibot

      SciCrunch record: RRID:ZFIN_ZDB-GENO-060623-2


      What is this?

    1. I like the differentiation that Jared has made here on his homepage with categories for "fast" and "slow".

      It's reminiscent of the system 1 (fast) and system2 (slow) ideas behind Kahneman and Tversky's work in behavioral economics. (See Thinking, Fast and Slow)

      It's also interesting in light of this tweet which came up recently:

      I very much miss the back and forth with blog posts responding to blog posts, a slow moving argument where we had time to think.

      — Rachel Andrew (@rachelandrew) August 22, 2017
      <script async src="https://platform.twitter.com/widgets.js" charset="utf-8"></script>

      Because the Tweet was shared out of context several years later, someone (accidentally?) replied to it as if it were contemporaneous. When called out for not watching the date of the post, their reply was "you do slow web your way…" #

      This gets one thinking. Perhaps it would help more people's contextual thinking if more sites specifically labeled their posts as fast and slow (or gave a 1-10 rating?). Sometimes the length of a response is an indicator of the thought put into it, thought not always as there's also the oft-quoted aphorism: "If I Had More Time, I Would Have Written a Shorter Letter".

      The ease of use of the UI on Twitter seems to broadly make it a platform for "fast" posting which can often cause ruffled feathers, sour feelings, anger, and poor communication.

      What if there were posting UIs (or micropub clients) that would hold onto your responses for a few hours, days, or even a week and then remind you about them after that time had past to see if they were still worth posting? This is a feature based on Abraham Lincoln's idea of a "hot letter" or angry letter, which he advised people to write often, but never send.

      Where is the social media service for hot posts that save all your vituperation, but don't show them to anyone? Or which maybe posts them anonymously?

      The opposite of some of this are the partially baked or even fully thought out posts that one hears about anecdotally, but which the authors say they felt weren't finish and thus didn't publish them. Wouldn't it be better to hit publish on these than those nasty quick replies? How can we create UI for this?

      I saw a sitcom a few years ago where a girl admonished her friend (an oblivious boy) for liking really old Instagram posts of a girl he was interested in. She said that deep-liking old photos was an obvious and overt sign of flirting.

      If this is the case then there's obviously a social standard of sorts for this, so why not hold your tongue in the meanwhile, and come up with something more thought out to send your digital love to someone instead of providing a (knee-)jerk reaction?

      Of course now I can't help but think of the annotations I've been making in my copy of Lucretius' On the Nature of Things. Do you suppose that Lucretius knows I'm in love?

    1. RRID:ZFIN_ZDB-GENO-100402-2

      DOI: 10.7554/eLife.37024

      Resource: (ZFIN Cat# ZDB-GENO-100402-2,RRID:ZFIN_ZDB-GENO-100402-2)

      Curator: @scibot

      SciCrunch record: RRID:ZFIN_ZDB-GENO-100402-2


      What is this?

    1. RRID:ZFIN_ZDB-ALT-070118-2

      DOI: 10.7554/eLife.68755

      Resource: (ZFIN Cat# ZDB-ALT-070118-2,RRID:ZFIN_ZDB-ALT-070118-2)

      Curator: @scibot

      SciCrunch record: RRID:ZFIN_ZDB-ALT-070118-2


      What is this?

  6. Jul 2021
    1. Well, no. I oppose capital punishment, just as (in my view) any ethical person should oppose capital punishment. Not because innocent people might be executed (though that is an entirely foreseeable consequence) but because, if we allow for capital punishment, then what makes murder wrong isn't the fact that you killed someone, it's that you killed someone without the proper paperwork. And I refuse to accept that it's morally acceptable to kill someone just because you've been given permission to do so.

      Most murders are system 1-based and spur-of-the-moment.

      System 2-based murders are even more deplorable because in most ethical systems it means the person actively spent time and planning to carry the murder out. The second category includes pre-meditated murder, murder-for-hire as well as all forms of capital punishment.

    1. RRID:ZFIN_ZDB-GENO-060619-2

      DOI: 10.1002/cne.24042

      Resource: (ZFIN Cat# ZDB-GENO-060619-2,RRID:ZFIN_ZDB-GENO-060619-2)

      Curator: @scibot

      SciCrunch record: RRID:ZFIN_ZDB-GENO-060619-2


      What is this?

    1. Zebrafish: Tg(sox10:Gal4-VP16,cmlc2:EGFP)sq9

      DOI: 10.1016/j.cell.2019.08.001

      Resource: (ZFIN Cat# ZDB-ALT-130826-2,RRID:ZFIN_ZDB-ALT-130826-2)

      Curator: @ethanbadger

      SciCrunch record: RRID:ZFIN_ZDB-ALT-130826-2


      What is this?

    2. Zebrafish: Tg(olig2:DsRed)vu19

      DOI: 10.1016/j.cell.2019.08.001

      Resource: (ZFIN Cat# ZDB-ALT-080321-2,RRID:ZFIN_ZDB-ALT-080321-2)

      Curator: @ethanbadger

      SciCrunch record: RRID:ZFIN_ZDB-ALT-080321-2


      What is this?

    3. Zebrafish: Tg(sox10(4.9):nls-Eos)w18

      DOI: 10.1016/j.cell.2019.08.001

      Resource: (ZFIN Cat# ZDB-ALT-110721-2,RRID:ZFIN_ZDB-ALT-110721-2)

      Curator: @ethanbadger

      SciCrunch record: RRID:ZFIN_ZDB-ALT-110721-2


      What is this?

    1. Zebrafish: Tg(mbp:egfp-caax)ue2

      DOI: 10.1016/j.celrep.2019.03.013

      Resource: (ZFIN Cat# ZDB-ALT-120103-2,RRID:ZFIN_ZDB-ALT-120103-2)

      Curator: @ethanbadger

      SciCrunch record: RRID:ZFIN_ZDB-ALT-120103-2


      What is this?

    2. Zebrafish: Tg(olig2:dsred)vu19

      DOI: 10.1016/j.celrep.2019.03.013

      Resource: (ZFIN Cat# ZDB-ALT-080321-2,RRID:ZFIN_ZDB-ALT-080321-2)

      Curator: @ethanbadger

      SciCrunch record: RRID:ZFIN_ZDB-ALT-080321-2


      What is this?

    3. Zebrafish: Tg(sox10(4.9):nls-eos)w18

      DOI: 10.1016/j.celrep.2019.03.013

      Resource: (ZFIN Cat# ZDB-ALT-110721-2,RRID:ZFIN_ZDB-ALT-110721-2)

      Curator: @ethanbadger

      SciCrunch record: RRID:ZFIN_ZDB-ALT-110721-2


      What is this?

    1. ZDB-ALT-170509-2

      DOI: 10.1016/j.neuron.2017.06.001

      Resource: (ZFIN Cat# ZDB-ALT-170509-2,RRID:ZFIN_ZDB-ALT-170509-2)

      Curator: @Zeljana_Babic

      SciCrunch record: RRID:ZFIN_ZDB-ALT-170509-2


      What is this?

    2. ZDB-ALT-131122-2

      DOI: 10.1016/j.neuron.2017.06.001

      Resource: (ZFIN Cat# ZDB-ALT-131122-2,RRID:ZFIN_ZDB-ALT-131122-2)

      Curator: @Zeljana_Babic

      SciCrunch record: RRID:ZFIN_ZDB-ALT-131122-2


      What is this?

    1. ZFIN ID: ZDB-ALT-140424-2

      DOI: 10.7554/eLife.66596

      Resource: (ZFIN Cat# ZDB-ALT-140424-2,RRID:ZFIN_ZDB-ALT-140424-2)

      Curator: @evieth

      SciCrunch record: RRID:ZFIN_ZDB-ALT-140424-2


      What is this?

    2. ZFIN ID: ZDB-ALT-070118-2

      DOI: 10.7554/eLife.66596

      Resource: (ZFIN Cat# ZDB-ALT-070118-2,RRID:ZFIN_ZDB-ALT-070118-2)

      Curator: @evieth

      SciCrunch record: RRID:ZFIN_ZDB-ALT-070118-2


      What is this?

    1. A Digital Scholarly Edition: The Willa Cather Archive

      This website is what I kept picturing when I heard the words "digital humanities project". This is like most of the websites I have interacted with, with posts on the main page and categorized tabs at the top for anything else you are looking for. I think I could probably branch out and explore different ones now that I know the wide variety out there.

    2. A searchable map of the addresses contained in the 1956 Negro Travelers’ Green Book, which the user can filter by state or establishment type.

      I think that this idea for a digital project is really interesting. I like that they have mapped it out and you can click on a point to filter by state or establishment type to zero in on what interests you. I never thought that a digital humanities project could look like this. I think it is really cool how different all of these projects are on this site, yet they all fall under the umbrella of digital humanities.

    3. Many  students tell me that in order to get started with digital humanities, they’d like to have some idea of what they might do and what technical skills they might need in order to do it.

      I am usually that person who likes to know what they're getting into before they start. I like to know what skills I need as well so I can see if I will be able to do it easily or if it will be more of a learning curve. I definitely did not know anything about digital humanities before I started this class, but I am learning with each exercise we do. I think a lot of people like to be confident in what they are doing so to have some idea of what they are going to do and what skills they need is reassuring.

    1. Informal and pre-or postpublication communication with fellow scholars to share research questions or results was traditionally carried out through letter-writing, then by phone or fax and in the digi-tal age variably through Gophers, forums, chat rooms, RSS feeds, wikis, listservs and e-mail. Blogging is a way of discussing or sharing informa-tion on the web by uploading posts (discrete, usually brief notices). These are often displayed with the most recent item at the top.

      Blogging is a convenient way to get information out to an audience. Rather than letter-writing or phone, and then into email and chat rooms, blogging allows you to share information and knowledge by posting your thoughts. It can stay up for as long as you like, allowing a variety of people to view it. I think it is a way to share your ideas and get information out faster.

    2. Might we be approaching the time when the distinction created by the term homo Jaber, the human as maker, outside and above the world of her creations, becomes meaning-less in the world of the semantic web and 3D bacterial printing?

      I do not think the term homo faber, or human as maker has become meaningless because of the digital world of the web and 3D printing. The digital is using that term in a different way. Yes, digital things are done online with the help of certain tools and software, but it is still the human behind the screen. It is the human as maker with the ideas and creativity for these new digital concepts and the ability and knowledge to develop them. People can use the digital to enhance their ideas.

    3. Only most recently with the digital has this kit of tools begun to change rapidly and fundamentally. Yet in many ways these new digital tools carry on, in analogous ways, the same functions of the traditional humanities.

      I think it is true that for the most part the environments of the humanities have been things like the scholar's desk, lecture halls, campuses, and convention halls. In the last little bit there has been a shift from these environments in that the digital has now come in to play. I agree that the digital tools carry on the same functions as traditional humanities in comparable ways. We are still learning about the humanities by using a digital form, it is just a newer way of presenting them.

  7. Jun 2021
    1. Oversharing. Crying, disclosing intimate details, and telling long (unrelated and/or unsolicited) stories about one’s personal life may indicate the lack of an essential social work skill: personal boundaries.

      Testing out the annotate feature. Student 1 will highlight sections according to the prompts, as shown HERE.

      For example: "This is me during interviews. I say too much and veer off topic."

    1. ZFIN: ZDB-ALT-061204-2

      DOI: 10.1016/j.cub.2021.05.042

      Resource: (ZFIN Cat# ZDB-ALT-061204-2,RRID:ZFIN_ZDB-ALT-061204-2)

      Curator: @Naa003

      SciCrunch record: RRID:ZFIN_ZDB-ALT-061204-2


      What is this?

    2. ZFIN: ZDB-ALT-150324-2

      DOI: 10.1016/j.cub.2021.05.042

      Resource: (ZFIN Cat# ZDB-ALT-150324-2,RRID:ZFIN_ZDB-ALT-150324-2)

      Curator: @Naa003

      SciCrunch record: RRID:ZFIN_ZDB-ALT-150324-2


      What is this?

  8. May 2021
    1. One solution that fixed this issue with my ISP was that when I went through the first and second line and got in touch with the people that fixed my problem, I asked them if they could give me one of their personal numbers in case the same problem happened again. The problem did occur a couple more times, and I just directly called the same guy.
    1. Reviewer #2 (Public Review):

      This study presents a novel machine learning tool (termed T-REX) for automated analysis of single cell cytometric data that is capable of identifying rare cell populations, such as antigen-specific T cells. This ability to detect low frequency cells is a distinct advantage over existing tools. The demonstration of this ability is appropriately shown by examining antigen-specific CD4+ T cells before and after rhinovirus infection in a challenge study. Useful demonstrations are also included for examining SARS-CoV-2-specific T cells and changes in cellular populations in cancer patients upon treatment. These examples use both mass cytometry and fluorescence-based cytometry. Since both of these are commonly-used single-cell technologies that generate highly complex data sets, new automated analysis methods such as T-REX are needed.

      The first data set examined changes in cell phenotype before and 7 days after rhinovirus infection in healthy adults. The flow cytometric staining panel included markers of T cell differentiation and activation as well as rhinovirus-specific tetramers. The results of T-REX convincingly demonstrate "hotspots" that are expanded at 7 days and enriched for tetramer-staining cells. Thus, this study succeeds at demonstrating the utility of this method for identification of rare cells and the authors use this data set to appropriately determine the model parameters. Combining the results of this algorithm with the "Marker Enrichment Modeling (MEM)" method to characterize the markers expressed on those cell populations identified through T-REX is also very informative since this automates the characterization (that traditionally needs to be done by manual investigation).

      This first data set is relevant for this demonstration, but in some aspects it represents a best case scenario. "Phenotypic" identification of antigen-specific T cells in this way is only possible because the time point was chosen to capture the relatively narrow window when T cells would be activated, and there was access to a baseline sample for comparison. The authors do address the second point, and perform the analysis comparing day 7 to a later time point, day 28, as an appropriate alternative. The first concern limits the generalizability of this approach. In fact, the second example dataset examining mass cytometry data in patients with COVID-19 does in fact demonstrate limited ability to detect change in cell populations for many study participants.

    1. Reviewer #2 (Public Review):

      The use of convalescent plasma (CCP) to treat patients with Covid-19 has changed over the course of the pandemic (from rates as high as 40% of hospitalized patients in October, 2020 to a low of less than 10% by March 2021). To explore the efficacy of CCP therapy and the impact of the drop in CCP use, the authors assess whether there was a link between CCP use and patient mortality rates over time in the U.S. Using information from blood centers to estimate CCP usage and population level information on deaths from public databases, they found a strong inverse correlation between CCP usage per hospital admission and deaths due to Covid-19 after admission. The model estimates that the case fatality rate decreased by 1.8 percentage points for every 10 percentage point increase in the rate of CCP use. The detailed analysis suggests that the observed effect could not be attributed to changes in patient ages over time or the emergence of variant viruses. Other cofounders such as changes in the use of additional therapeutic agents or clinical interventions were not analyzed. The authors acknowledge the main limitation of this type of analysis i.e. that establishing a correlation does not prove a causal role. With that caveat, they conclude that the decline in usage may have resulted in excess deaths, possibly 29,000 to 36,000 over the past year in the U.S. Because the decreased usage of CCP occurred during the time that several randomized clinical trials and some media coverage reported no benefit of CCP, the authors suggest that resultant "plasma hesitancy" may have contributed to increased mortality. These findings add an important perspective to future considerations for clinical care, treatment guidelines and regulatory approvals of CCP. Emphasizing the importance of using high-titer units and administering CCP early in the disease course, the authors urge a more nuanced interpretation of the available evidence and a holistic approach to decisions about the use of CCP in individual patients.

    1. Reviewer #2 (Public Review):

      The authors should be commended on the sharing of their data, the extensive experimental work, the experimental design that allows them to get opposite predictions for both hypotheses, and the detailed of analyses of their results. Yet, the interpretation of the results should be more cautious as some aspects of the experimental design offer some limitations. A thorough sensitivity analysis is missing from experiment 2 as the safety margin seems to be critical to distinguish between both hypotheses. Finally, the readability of the paper could also be improved by limiting the use of abbreviations and motivate some of the analyses further.

      Major:

      1) The text is difficult to read. This is partially due to the fact that the authors used many abbreviations (MA, PO, IMD). I would get rid of those as much as possible. Sometimes, having informative labels could also help FFcentral and FFlateral would be better than FFA and FFB.

      2) The most difficult section to follow is the one at the end of the result sections where Fig.5 is discussed. This section consists of a series of complicated analyses that are weakly motivated and explained. This section (starting on line 506) appears important to me but is extremely difficult to follow. I believe that it is important as it shows that, at the individual level, PO is also superior to MA to predict the behavior but it is poorly written and even the corresponding panels are difficult to understand as points are superimposed on each other (5b and e). In this section, the authors mention correcting for Mu1b and correcting for Sig2i/Sig1Ai but I don't know what such correction means. Furthermore, the authors used some further analyses (Eq. 3 and 4) without providing any graphical support to follow their arguments. The link between these two equations is also unclear. Why did the authors used these equations on the pooled datasets from 2a and 2b ? Is this really valid ? It is also unclear why Mu1Ai can be written as the product of R1Ai and Sig1Ai. Where does this come from ?

      3) In experiment 1, does the presence of a central target not cue the participants to plan a first movement towards the center while such a central target was never present in other motor averaging experiment. In the adaptation domain, people complain that asking where people are aiming would induce a larger explicit component. Similarly, one could wonder whether training the participants to a middle target would not induce a bias towards that target under uncertainty

      4) The predictions linked to experiment 2 are highly dependent on the amount of safety margin that is considered. While the authors mention these limitations in their paper, I think that it is not presented with enough details. For instance, I would like to see a figure similar to Fig.4B when the safety margin is varied.

      The sensitivity analysis is very difficult to follow and does not provide the right information. First, this is only done for exp2 and not exp1. For exp1, it would be good to demonstrate that, even when varying the weight of the two one-target profiles for motor averaging, one never gets a prediction that is close to what is observed. It is unclear in the text that the performance optimization prediction simply consists of the force-profile for the center target. The authors should motivate this choice. For the second experiment 2, the authors do not present a systematic sensitivity analysis. Fig. 5a and d is a good first step but they should also fit the data on exp2b and see how this could explain the behavior in exp 2a. Second, the authors should present the results of the sensitivity analysis like they did for the main predictions in Fig.4b. While I understand where the computation of the safety margin in eq.2 comes from, reducing the safety margin would make the predictions linked to the performance optimization look more and more towards the motor averaging predictions. How bad becomes the fit of the data then ? How does the predictions look like if the motor costs are unbalanced (66 vs. 33%, 50 vs. 50% (current prediction), 33 vs. 66% ). What if, in Eq.2 the slope of the relationship was twice larger, twice smaller, etc. The safety margin is the crucial element here. If it gets smaller and smaller, the PO prediction would look more and more like the MA predictions. This needs to be discussed in details. I also have the impression that the safety margin measured in exp 2a (single target trials) could be used for the PO predictions as they are both on the right side of the obstacle.

      5) On several occasions (e.g. line 131), the authors mention that their result prove that humans form a single motor plan. They don't have any evidence for this specific aspect as they can only see the plan that is expressed. They can prove that the latter is linked to performance optimization and not to the motor averaging one. But the absence of motor averaging does not preclude the existence of other motor plans.... Line 325 is the right interpretation.

      6) Line 228: the authors mention that there is no difference in adaptation between training and test periods but this does not seem to be true for the central target. How does that affect the interpretation of the 2-target trials data ? Would that explain the remaining small discrepancy between the refined PO prediction and the data (Fig.2f) ?

    1. Reviewer #2 (Public Review):

      The authors established a comprehensive map of neurogenetic sites with evolutionary conserved neurogenic and postmitotic gene expression in a common octopus, Octopus vulgaris that has been a historically important species in comparative neuroscience and behavioral studies. The selected molecules include representative regulatory genes such as achaete-scute, neurogenin, and neuroD, and also proliferating cell markers such as elav and PCNA. In subsequent experiments by using a fluorescent dye, the authors carefully traced the migratory pathways from the target ectodermal sites surrounding eyes to many developing brain lobes of clearing staged embryos with light sheet microscopy for 3D reconstruction.

      They found that the special regions called lateral lip and other special ectodermal areas produced a pool of migratory postmitotic neurons that might contribute a novelty for developing octopus large and complex brains as in mammals, in contrast to those of other invertebrates such as flies or worms.

      I find the bodies of evidence convincing. A good study usually opens many new questions. Before publication, I found two major points that may enhance the author's conclusions.

      1) Are the migratory cells only neurons, or could they also be glia, neurosecretory, blood, or immune cells? The enlarged views of the migratory cells and the cytological features must be clarified.

      2) The expression of canonical neurogenic genes disappears during middle and late stages, meaning that octopus has very unique neurogenic mechanisms compared to mammals? Consider the octopus-specific novelty.

    1. Reviewer #2 (Public Review):

      In this work, Corbett and colleagues investigate how value influences speeded decisions. In a random dot motion task with speed pressure, shortly before motion onset it is indicated which of both choices has a higher value if answered correctly. EEG recordings show a buildup of motor beta in response to the cue (earlier for high value choices, steeper for low value choices) and a dip in LRPs for low value choices in response to stimulus onset. A computational model constructed based on these findings provides a good account of the data. The EEG informed modeling is impressive and deserves merit. The paper is well-written, but rather dense.

      • I am struggling with the idea that cue-evoked motor beta reflects urgency. As it currently reads, this is more taken as a given than actually demonstrated. Could this claim be corroborated by e.g. showing that response deadlines modulate this signal? Related to that, how can we be sure that the pre-stimulus patterns seen in motor beta feed into the decision making process itself? It is not hard to imagine why left and right pointing arrows directly trigger motor activity (i.e. simple priming), but does that also imply that such activity leaks into the decision process?

      • I had a hard time understanding the choice for this specific design. As the authors write they "primarily focused on the value biasing dynamics in common across these challenging regimes" so I wonder whether conditions with different value differences could have been more instructive (e.g., according to the author's hypothesis different levels of value should parametrically affect motor beta, whereas if this reflect a simple priming process value itself should not matter). Alternatively, it should be better explained why these conditions where crucial for the current findings.

      • One of the main selling points of the paper is that we currently lack a model that can explain fast value-based decisions, mostly because the constant drift rate assumption in evidence accumulation models seems invalid. This conjecture is very similar to literature on response conflict, where performance in conflict tasks (such as Stroop, Flanker, etc.) is best modelled using a time-varying drift rate. I wonder to what extent current data reflect the same process, i.e. the value cue "primes" a response, which then has to be suppressed in favor of the correct response. A clear difference is that the value remains relevant here, but could e.g. the motor beta effect just reflect priming?

      • If I understand correctly the model was fit to all data effectively ignoring between-participant differences. It is unclear why this was this done (rather than fitting data separately per participant or fitting the data using a hierarchical model), because it induces substantial variance in the fits caused by between-participant differences.

    1. Reviewer #2 (Public Review):

      In this work, Sobczak et al. suggest that correlations between fMRI and pupil diameter vary over time, and propose an approach to identify distinct clusters of such correlation patterns. The proposed methods are applied to data acquired from anesthetized rats. Based on the clusters obtained, the authors conclude that pupil dynamics are linked with different neuromodulatory centers over different intervals of time.

      Overall, I believe that the study is novel and uncovers potential new modes of coupling between neuromodulatory nuclei and pupil diameter. However, additional analysis may be needed to fully support the validity of the derived clusters, and the decoding methods may need some modification before the accuracy values can be properly interpreted. The mechanisms behind the time-varying fMRI-pupil coupling exhibited under anesthesia could also be further clarified. Specifically:

      • The clusters appear to involve interpretable brain regions. However, a more formal analysis of reproducibility of these clusters, and statistical testing against an appropriate null model, are not present. Such tests would be useful for establishing the validity of the derived clusters, ensuring that the conclusions are strongly supported. Similarly, the differentiation between power spectral density of each cluster is not yet supported by statistical testing.

      • With regard to the decoding models, it appears there could be interdependence between the training and testing data (the PCA step seems to include all scans, and it was not clear if the training/testing sets contained data drawn from the same animal).

      • While the paper is motivated by discussion that pupil diameter changes are complex and related to rich behaviors (mental effort, decision making, etc.), this paper examines data from anesthetized rats. The mechanisms behind the time-varying changes in fMRI-pupil coupling in the current data, and the potential impact of anesthesia, were not clear and could be elaborated upon.

    1. Reviewer #2 (Public Review):

      The various pathogenic, parasitic, symbiotic, and mutualistic interactions between insects and the microbes they interact with represents a rich area of research. This study by Xiao et al. represents a very interesting example of such a relationship. Overall the study is well designed and executed. The approach they utilize to test their hypothesis is valid and they combined both laboratory and field collected insects to address the question. The RNAseq analysis also provides potential insights into possible mechanisms by which the virus HaDV2 enables enhanced resistance to Bt Cry1Ac. The RNAseq data also represent one of the minor issues. The authors focused on analyzing only development and immune systems, however, they do not report on any other significantly different changes in gene expression other than reporting that there were 1573 significant differences. The authors should at least provide some holistic analysis and report the data in the supplemental results. Focusing on development and immune systems is valid and rationally supported but a complete analysis should be presented. The relationship between H. armigera and HaDV2 is more a mutualistic relationship, thus, the authors should consider changing the titles of the manuscript and the supplementary data. This is an exciting study and is well written and will be of general interest to the field.

    1. Reviewer #2 (Public Review):

      Transposable elements (TEs) have been shown to play an important role in genome evolution, in shaping genomic organization, structure, and genome size. The importance of TEs in evolutionary processes such as adaptation, has been rather limited so far. Here, Oggenfuss et al. used intraspecies data from six global populations of the wheat pathogen Zymoseptoria tritici to study the process of TE insertion dynamics, to detect candidate adaptive regions associated with TE insertions, and to show that TE expansion has driven to genome size of some populations in about 25 years. Using publicly available short read sequencing, as well as newly sequenced populations, they created a pipeline to specifically identify TE insertions, then used TE frequency insertions to infer patterns of selection, which they hypothesize is under strong purifying selection for regions into genes. They further tried to detect evidence for positive selection at loci associated with adaptation to new environment or resistance to fungicide, and finally contrasted the TE expansion in a window of 25 years to show that two populations have increased their genome size due to TE expansion.

      Strengths:

      The dataset used in this study with different global populations at different time makes the authors in an ideal position to detect TE expansion in a short timeframe of 25 years. While it has been shown in multiple eukaryotic genomes that TEs contribute substantially to variation in genome size, the evidence provided in this paper is compelling and shows that it is unlikely due to genome duplication events.

      The pipeline to identify TE insertions from short read sequencing provides a well detailed path to apply in other genomes, and provide logical reasoning detecting TEs absent from the reference or absent from the isolates but present in the reference. Providing validation for some of the steps would be desired if well-known regions can be used.

      The authors explored the interesting perspective of identifying TEs under positive selection by focusing on loci with increased frequency in specific populations. They also added a level of functional validation to better understand specific TE insertions under selection, which is often not included. They identified three loci that did confer resistance to fungicide according to their assay. These results would benefit from additional key details in the methods and the rationale behind the choice of loci in order to fully measure the impact of this finding.

      Weaknesses:

      While the paper does have strengths in principle to study the evolution of TE dynamics, the weaknesses of the paper reside in the fact that the manuscript in its current form does not directly support the key conclusions presented here. Additional analyses would be required to support them. Such as :

      The presence of a large percentage insertions being singleton TEs coupled with low frequency (based on an arbitrary cutoff) is used to conclude strong purifying selection is acting on these new insertions. The authors should have included evidence to convince the reader the presence of a TE in one isolate is not a case of false positive. Additional analyses such as a subset of the singleton TEs to corroborate these single loci or plotting the relationship between how many isolate with one insertion were found and read depth could be informative. Also, understanding the singleton in the context of chromosome locations (core and accessory) could help reinforce the evidence of purifying selection.

      The author's conclusion of relaxed selection in accessory chromosomes and between populations are mainly based on TE density. This conclusion may be better supported by adding quantified information of the relationship between the numbers and the type of TE insertions and genomic features such as recombination, which is often found to be negatively associated with TE content. Showing the recombination rate between the chromosomes (core, accessory) would help strengthen the argument that selection acts more strongly in regions of high recombination. To make their conclusion more robust, the authors could have included information about the recombination landscape.

      Overall, the authors should have provided the adequate statistical analyses to support many of the insertion frequencies that are often only mentioned qualitatively (e.g. "less than expected") without having the quantitative test. Also, the contrast between low TE insertion frequencies versus high frequency is used without providing details about what is expected, either supported by the literature or by more detailed analyses. Arbitrary threshold can be prone to give artifact results.

      Context : This article comes with a number of recent papers exploiting the population genomics dataset of the major wheat pathogen Zymoseptoria tritici (Fouché et al 2020, Krishnan et al. 2018) to show that TE-mediated insertions have in part helped to colonize host plants and tolerate environmental stress. Here, the authors build upon this knowledge to attempt at characterizing the process of TE insertion dynamics, detect signatures of adaptive evolution and changes in genome size at the population level.

    1. Reviewer #2 (Public Review):

      The current study nicely demonstrates that high-order assembly of SMN protein oligomerization is necessary for animal survival and is dependent on a motif exposed to YG zipper dimers. Mutations in the human SMN1 gene have been shown to cause a neurodegenerative disease named Spinal Muscular Atrophy (SMA). About 50% of the SMA-causing mutations are located in the YG zipper domain. The authors used multi-disciplinary approaches such as biophysical, bioinformatic, computational and genetic approaches to demonstrate that a set of YG box amino acids in SMN protein are not involved in dimerization process and formation high-order oligomers is dependent on these residues. Importantly, mutating key residues within this new structural domain impairs SMN dimerization and causes motor dysfunction as well as viability defects in Drosophila. Overall, this is a well-written paper that offers new insights into the structural and functional aspects of SMN protein. The authors should consider addressing the following issues:

      1) The authors should discuss the impact of the YG zipper domain mutations on snRNP biogenesis. SMN protein is a master regulator of snRNP biogenesis. It is a little surprising that the authors did not mention snRNP biogenesis in the whole manuscript.

      2) The authors should provide evidence that their transgenic lines express the desired transgene. A WB or qPCR would be great (even as supplementary data).

      3) Page 12: The authors stated "Both missense mutations display early onset SMA-like phenotypes". Was it age-dependent phenotype? Did adult animals show a more severe motor dysfunction?

      4) There are few statements that the authors should consider making clear. Here is an example "Presumably, the structural changes associated with Cys and Val substitutions do interfere with some aspect of SMN biology, leading to the intermediate and severe SMA phenotypes observed". What do you mean by some aspects? Oligomerization, stability or anything else?

      5) There are few typos throughout the manuscript that the authors should correct (western should be written as Western).

    1. Reviewer #2 (Public Review):

      The article by Breska and Ivry provides a nice, timely, and relevant continuation of their previous recent work on the role of the cerebellum in interval-based (but not rhythm-based) anticipation in time. While in their related prior work (in particular their recent articles in PNAS and Science Advances) the authors used simple reaction time tasks that made it difficult to attribute the observed effects to visual vs. motor anticipatory mechanisms, in the current work they used a perceptual discrimination task with a delayed response to focus on potential contributions of the cerebellum to temporal anticipation specifically for perceptual sensitivity (where the role of the cerebellum is less obvious, given it has traditionally been implicated more in motor control than in perception). They do so by comparing individuals with cerebellar degeneration to controls, and finding a selective impairment of the individuals with cerebellar degeneration to use interval-based temporal predictions to facilitate visual discrimination, while rhythm-based performance benefits are spared (providing a neat comparison and control).

      I have no major comments to detail. The short report is well written, complements related work by the authors nicely, and makes an important and novel contribution to the literature on temporal anticipation (while also having relevant implications more generally for views on the role of the cerebellum in cognition).

    1. Reviewer #2 (Public Review):

      The manuscript describes a tool to independently tune mean protein expression levels and noise. Light induces dimerization and subsequent activation of transcriptional activator GAVPO. By introducing 5xUAS (a target sequence for dimerized GAVPO) upstream a mRuby reporter gene, the effect of light can be measured on mRuby mean and noise.

      By pulsing light at different periods (from 100-400 minutes), the authors reduce the mRuby noise for intermediate average light intensities. Notably, the pulses are all applied at an absolute light intensity of 100 uW/cm2, with the average light intensity being modulated through the light-off time-periods. Therefore, as all periods tend towards 100 uW/cm2 average light intensity, the PWM duty cycles becomes more similar to the 100 uW/cm2 AM case.

      Strengths:

      The proposed method is an elegant way to independently tune protein mean and noise. This would have a broad application in the field and is much needed to be able to study the consequence of protein expression noise, independently of mean. In addition, the authors use multiple powerful single-cell techniques to try and determine the mechanism underpinning the light-induced noise modulation.

      During constant exposure to light, increased light intensity increases the mean expression of mRuby, while decreasing the noise. This high noise is mostly due to observed bimodality in mRuby expression. Through ODEs and by using small molecule inhibitors, the authors show that this bimodality is caused by some cells being stably off, while other cells enter an on state. In this on state a positive feedback can occur where initial binding of dimerized GAVPO induces histone acetylation and chromatin accessibility, and thus stimulates further GAVPO binding. Bistability induced by constant light exposure is disrupted using small molecule inhibitors of CBP/p300 HAT activity, indicating that histone regulation is a cause for this observed bistability. The stable on state is demonstrated to be more active and accessible through ChIP-seq and ATAC-seq respectively.

      Weakness:

      The single-cell ATAC-seq data indicate that pulsing light induces switching from an accessible (light on) to inaccessible (light off) chromatin state. The authors argue that the switching back into a chromatin inaccessible state prevents the positive feedback to occur and thus reduces noise. However, there are weaknesses in the description of the mechanism by which the pulses modulate (i.e., reduce) noise. Overall, since these sections in the manuscript are not easy to understand, it is difficult to parse what mechanism the authors attributed to the observed noise reduction and to assess if the data supports the conclusions.

      The data from the single-mRNA live-cell imaging experiments are somewhat ambiguous and do not necessarily support some of the arguments. The conclusion that transcription, nuclear export, and mRNA degradation flatten the pulsatile chromatin caused by the PWM is not clear from the data. Especially, since most cells do not show any pulsatile behavior both in the single-cell ATAC-seq and the live-cell imaging data.

    1. Reviewer #2 (Public Review):

      Motivated behaviors, such as food seeking when hungry, can also occur spontaneously at irregular intervals. Understanding how this irregular expression arises is important for understanding behavior and is relatively little investigated. The present work thus addresses an important and under-investigated area in neurobiology. Its demonstration of a potential cellular mechanism for irregular behavioral production has wide relevance, ranging from how cells make "decisions" to how whole organisms do so.

      Intact Aplysia occasionally produce bites even in the absence of food, and isolated buccal ganglia (which contain the biting central generator circuit) will occasionally spontaneously produce fictive bite motor patterns. The activity of central pattern generator networks has almost exclusively been ascribed to the actions of the voltage-gated channels in the network neuron cell membranes and the synaptic connectivity among the network's neurons. Bédécarrats et al. show that a small, highly regular cell membrane voltage oscillation occurs in a neuron (B63) in the biting neural network, and that occasionally this oscillation becomes large enough to trigger a plateau potential in B63 and a single fictive bite from the entire circuit. They show that this oscillation is not due to cell membrane voltage dependent conductances, but instead from process involving the endoplasmic reticulum, mitochondria, or both. Although organelle-driven changes in cellular or tissue activity have been observed in other cell types, this is, to my knowledge, its first observation in a neural network. These data thus are potentially of great importance in understanding how neural networks function, most of which do not show the great regularity of central pattern generated behaviors.

      The presented data seem, to me, strong with respect to the small potential oscillations not being generated by voltage-dependent cell membrane conductances, and somehow involving the intracellular organelles. What is less clear to me is how local release of Ca from endoplasmic reticulum or mitochondria would result in changes in ion composition under the cell membrane, which is what gives rise to the cell membrane potential. Ca is highly buffered in the cytoplasm. It is thus unclear to me that free Ca would remain so for any length of time after release. It does, of course, in muscles, but these are evolved for this to occur. The authors themselves raise a variant of these concerns in the Discussion when considering how the B63 cell membrane voltage oscillations are transmitted to neurons electrically coupled to B63, invoking as a possibility Ca activation of second messengers, which would then themselves be responsible for the cell to cell communication. It seems to me that the same concerns arise with respect to how Ca release at sites distant from the cell membrane could charge the membrane's capacitance.

      A second remarkable observation is that B63 depolarization and firing does not reset the organelle-derived slow oscillation. B63 firing should result in substantial Ca concentration changes, at least in a shell under the cell membrane, so a possible feedback mechanism can be imagined. Most biological processes contain multiple feedback process that link cause and effect (e.g., the sequential current activations that return a cell to rest after an action potential, the interactions between sympathetic and parasympathetic system activity that maintain functionally proper body activation, the interactions that regulate hormonal levels). One possibility the authors mention is that the organelle-derived oscillation is used only for intermittent bite activities, and in feeding bites are instead generated solely by standard cell-membrane voltage-dependent processes. Regardless, it is a striking observation that merits additional investigation.

      These issues, however, do not change the data, which show a clear association of disruption of endoplasmic reticulum and mitochondrial function and cessation of the cell membrane voltage oscillation. Nor is it reasonable to expect an article like this, showing an organelle-driven cell membrane potential oscillation for the first time in a neuron, to describe every aspect of the mechanism by which it occurs. Indeed, it is a measure of the article's interest that it prompts such thinking. It will be very interesting to see the effects of similar organelle-disrupting treatment on the activity of other well-defined neural networks.

    1. Reviewer #2 (Public Review):

      Wellington Miranda et al. investigated how acyl-homoserine lactone autoinducer mediated quorum sensing systems evolve. The authors used the statistical covariation method GREMLIN to identify key amino acids that have coevolved in the well-studied LasI/LasR quorum sensing AHL synthase/receptor pair from Pseudomonas aeruginosa PAO1. LasI produces and LasR detects 3OC12-HSL. The authors identify some new and some previously reported residues using the GREMLIN tool; they focus on L157 in LasI and G38, R61, A127, S129, and L130 in LasR as residues that determine selectivity of the acyl-homoserine lactone that is produced and detected, respectively. Quite expectedly, these residues are in or near the ligand-binding pocket of LasI and LasR. The authors further engineer the LasI/R system to produce and detect the non-native 3OC10-HSL autoinducer in addition to 3OC12-HSL, thereby broadening the specificity of LasI/R.

      P. aeruginosa is an important pathogen and a powerful system for the study of quorum sensing. The use of GREMLIN to study how autoinducer synthase and receptor pairs coevolve in terms of sensitivity and specificity for a particular autoinducer is impressive. This paper adds an exciting approach to the growing literature on the evolution of sensitivity and promiscuity in quorum-sensing systems. Further, the authors have developed a thin layer chromatography based approach to separate and detect AHLs from nine samples simultaneously. This methodology should be widely useful to researchers.

      The current manuscript does not provide any data about the solubitlity and/or stability of the LasI and LasR mutant proteins being studied. For instance, biochemical analyses would be needed to evaluate if the increased sensitivity of LasRA127L compared to wildtype is due to higher affinity for the autoinducer or because the variant is more stable. Further, this work relies solely on reporter assays and does not address the consequences of these LasI and LasR variants to quorum-sensing dependent P. aeruginosa group behaviors such as pyocyanin production and virulence.

    1. Reviewer #2 (Public Review):

      The manuscript entitled "The Shu Complex Prevents Mutagenesis and Cytotoxicity of Single-Strand Specific Alkylation Lesions" by Bonilla and colleagues reports that the yeast Shu complex promotes repair of 3meC in single-stranded DNA during S phase. Specifically, the authors show that mutations and cell lethality induced by MMS in csm2∆ cells are suppressed by overexpression of the human ALKBH2. Further, the authors find that the Csm2-Psy3 module of the Shu complex has increased affinity for 3meC-containing DNA relative to unmodified DNA. The authors propose a model, where the Shu complex binds to 3meC-containing DNA to facilitate HR-dependent post-replicative gap-filling.

    1. Reviewer #2 (Public Review):

      Mattis et al have used a hemizygous mutant of the gene Scn1a to study changes underlying the severe epilepsy disorder Dravet syndrome. They describe a change in activation of the dentate gyrus in this mouse model, due to altered excitatory synaptic input. They show that this occurs in the age range after normalization of early inhibitory interneuron dysfunction. This provides an interesting potential mechanism by which neural circuit function is altered even after deficits in inhibition are seemingly corrected. They also report that stimulation of inputs to the dentate gyrus increase seizure susceptibility when body temperature is elevated. Overall these findings indicate a new form of circuit dysfunction that may underlie the etiology of this severe genetic epilepsy disorder.

      These findings are not fully complete, and the manuscript suffers from some flaws in experimental design.

      The most pressing issue is the lack of a counter-balanced design in experiments testing the ictogenicity of DG stimulation. The authors attempt to justify this stating "there is a theoretical concern that seizure threshold on Day 2 (the second consecutive day of stimulation) could be lowered by a seizure 24 hours prior (a "kindling"-like phenomenon)". In the very next sentence, they cite a study in which this phenomenon has been shown (thus the concern is not theoretical). That said, this is not a semantic argument, but a flaw in experimental design. On day 1, the authors perform experiment A. On day 2, they perform experiment A+B. In an attempt to show that performing experiment A on day 1 does not by itself lead to changes in experiment A+B, they use a separate cohort and show that experiment A does not lead to changes in a repetition of experiment A. Unfortunately, this is not an adequate control. Experiment A+B involves a different set of stimuli, to which the response could very well be altered by the day 1 experiment, but this change would not be revealed with the described experimental design. To determine whether the effect shown in experiment A+B requires a more rigorous, counter-balanced experimental design where one group undergoes experiment A followed by experiment A+B, and a second group undergoes experiment A+B followed by experiment A.

      The second major issue is a lack of wild type control groups for several experiments. The experiments presented in Figures 4, 6C and F, and 7 all lack the necessary wild type control measures. Wild type controls were done for Figure 6E, but the data are not presented in the figure.

      Some of the cell physiology experiments presented were not optimally designed to provide a relevant mechanistic follow-up to the major findings. For the first major finding of the paper, Figure 2 shows clear and interesting changes in DG activation in the mouse model, and Figure 5 reveals changes to synaptic excitation and inhibition in these neurons. Figure 3 and 4 present data showing changes to PV-interneuron intrinsic properties that only reveal themselves under very intense stimulation. While these findings are interesting and worthy of follow-up, the changes aren't relevant to the synaptic stimulation used in Figure 2.

      Finally, Figure 2 has missing data points, seemingly due to cropping of panels. Data visualization is problematic for this vital figure. The fit lines for individual experiments overwhelm the color-filled variance of the mean. Thus, the data in this figure are very difficult to read and interpret. The figure would benefit from including all the individual data points and summary data, but removing the individual fits or putting them into a supplement.

    1. Reviewer #2 (Public Review):

      In the manuscript entitled "The Crystal Structure of Bromide-bound GtACR1 Reveals a Pre-activated State in the Transmembrane Anion Tunnel", Li et al. analyzed the effect of bromide binding to GtACR1 by X-ray crystallography and electrophysiology. The authors propose that a bromide ion is bound to the intracellular pocket in the dark, inactivated state and induces a structural transition from an inactivated to a pre-activated state.

      I agree that some of the amino acid residues in the current crystal structure change their conformations compared to the previous one reported in 2019 (Li et al., 2019), and it is very impressive that the authors determined the structure using state-of-the-art crystallography technique, ISIMX. However, unfortunately, most of the conclusions and claims described in the manuscript are not well supported by the authors' data.

      1) The most serious problem is that the evidence of bromide binding is too weak. The authors showed the composite omit map in Supplementary Figure 1A, but they should present an anomalous difference Fourier map to validate the bromide binding. The authors also claim that they replaced the bromide ion to the water, run the PHENIX refinement, and observed a strong positive electron density at the bromide position in the Fo-Fc difference map (Supplementary Figure 1B). However, when I do the same thing using the provided coordinate and map (I really appreciate the honesty and transparency of the authors), I could not reproduce their result; a weak positive electron density is observed between the bromide position and Pro58 in chain A and there is no positive peak at the position in chain B (Fo-Fc, contoured at 3σ). I am wondering the occupancy and B-factor of the water molecule they show in Supplementary Figure 1B.

      In addition to the insufficient evidence, the current models of bromide ions have significant steric clashes. The PDB validation report shows that the top 5 serious steric clashes observed in the coordinate are the contacts between the bromide ions and surrounding residues (PDB validation report, Page 10). I analyzed them and found that the distance between the bromide ion and CG and CD atoms of Pro58 in chain A are only 2.43Å and 2.36Å, respectively. The authors claim that such a close proline-halide interaction has also been observed in the structure of the chloride-pump rhodopsin CIR, but in the structure (PDB ID: 5G28), the distances between the chloride ion and CD and CG atoms of Pro45 are much larger (3.43 and 3.91Å, respectively) and there is no steric clash. Moreover, the authors claim that Pro58 changes its conformation by bromide binding, but it is very possible that the PHENIX program just displaces Pro58 to alleviate the steric clash between the proline and the bromide ion, so the authors should carefully check the possibility.

      Overall, the authors should analyze the density again, provide more solid evidence for the bromide binding such as anomalous difference Fourier map, and if they could, they should correct the current significant steric clashes in their models.

      2) To analyze the functional importance of putative bromide binding, the authors prepared W246E and W250E mutants and analyzed their electrophysiological properties. Because tryptophan and glutamate are so different in terms of volume and charge, they should analyze other mutants as well. The authors claim that bromide is stabilized by a hydrogen bond interaction formed by the indole NH group of W246, so they should at least test the W246F mutant.

      3) The authors claim that the bromide binding in the intracellular pocket induces the conformational change of R94, but the causal relationship is doubtful. As mentioned in the manuscript, R94 forms a salt-bridge with D234 in chain A. However, the arginine has a completely different conformation and does not have any interaction with D234 in chain B. If the bromide binds both in chain A and B and induces the conformational change of R94, why only R94 in chain A interacts with D234? The authors change the pH in the crystallization condition compared to their 2019 study (Li et al., 2019), so the pH may affect the protonation state of D223 and/or other titratable residues and induces the conformational change of R94. The authors should provide more solid evidence for the causal relationship between the bromide binding and the conformational change of R94.

      4) The authors assume that the conformational change of R94 creates a functional anion binding site with the Schiff base in GtACR1, but it is too speculative. If the anomalous difference Fourier map does not support the idea, they should delete it.

    1. Reviewer #2 (Public Review):

      This study aims at elucidating the substrate-dependent conformational dynamics of TonB-dependent transporter BtuB, which is responsible for vitamin B12 transport in the outer membrane of E. coli. Following the pioneering studies from the same lab, the study employs an innovative approach of in-situ site-directed spin labeling for CW EPR spectroscopy and double electron-electron resonance (DEER) distance measurements in intact E. coli. Despite the intricacy of spin labeling and performing DEER measurements in intact cells, the majority of the obtained DEER spectra are of high quality with impressively long dipolar evolution times and signal-to-noise ratio, enhancing the reliability and accuracy of the data. Despite the limited number of distance constraints on one side of the core domain, experiments are well designed to address the relevant questions and the conclusions are justified by the data. The results fully support the main conclusion that the large substrate-induced structural change on the C-terminal side of the core in the presence of the mutations that mimic the breakage of the R14-D316 ionic lock (i.e., R14A, D316A, D316A/R14A), indicates the shift of the substrate-binding loop 3 towards the periplasm and reproduces the state when the transporter is bound to both substrate and TonB. The authors have utilized the deduced information to assess the currently proposed transport mechanisms. This study provides evidence for a transport mechanism that does not require a mechanical pulling or rotation by TonB as previously proposed. This model is also compatible with the structure of BtuB in complex with TonB that indicates the TonB-dependent release of the ionic lock. It is notable that these results are not seen in reconstituted membranes further highlighting the significance of in-situ structural dynamics studies in general and specifically for the field of EPR spectroscopy. I see substantial advance with respect to, both the mechanism of membrane transport by the TonB-dependent transporters and the application of this innovative approach.

    1. Reviewer #2 (Public Review):

      This short report by Yeh et al. reveals the presence of sfRNA in mosquito saliva and that it might enhance DENV infection in human Huh7 cells. By referring to literature, the authors propose that salivary sfRNA is secreted by EVs, and is immunosuppressive. The salivary sfRNA might facilitate DENV transmission and disease prevalence in nature.

      Strength: The methods are rigorous, results are clearly presented and the manuscript is well written.

      Weakness: sfRNA has long been recognized to interfere with the immune system in the flavivirus field. This study represents a modest advance. Additionally, even as a short report, the study fails to provide sufficient self-standing evidence to support its key claims. The study depends heavily on published literature to support its key conclusions.

    1. Reviewer #2 (Public Review):

      Inamdar and colleagues present a convincing manuscript identifying the unique role of IRSp53 in the successful assembly of HIV-1 particles. The study provides insight into the molecular mechanisms underlying the membrane curvature generation associated with virion budding from infected cells. Notably, the authors postulate a model in which the HIV-1 machinery "hijacks" host functions to generate fully-assembled viral particles by recruiting a central virion-assembly factor, HIV-1 Gag, to the luminal extremities of nascent extracellular vesicles generated by endogenous IRSp53. This is achieved through interactions between the two proteins, resulting in supramolecular complexes containing host and pathogen factors.

      A significant positive aspect of this study is the implementation of an experimental approach that encompasses complementary techniques, namely biochemistry, super-resolution microscopy, and advanced computational analysis and modelling. This is particularly relevant because several critical studies in the field of HIV-1 often rely on either one or the other set of methods and consequently lack the depth and cross-validation power achieved here. Also, the authors take advantage of experimental models that fall into opposite sides of the natural-artificial spectrum and use them adequately to test hypotheses and make conclusions.

    1. Reviewer #2 (Public Review):

      An intact myocardium is essential for cardiac function, yet much remains unknown regarding the cell biological mechanisms maintaining this specialized epithelium during embryogenesis. In this manuscript, Gentile and colleagues discover a novel role for the repressive transcription factor Snai1b in supporting myocardial integrity. In the absence of Snai1b, cardiomyocytes exhibit an enrichment of intermediate filament genes, including desmin b. In addition, the authors detect mislocalization of Desmin, along with adherens junction and actomyosin components, to the basal membrane in snai1b mutant cardiomyocytes, and these mutant cells exhibit an increased likelihood of extrusion from the myocardium. Ultimately, the authors put forward a model wherein Snai1b protects cardiomyocytes from extrusion at least in part by regulating the amount and organization of Desmin in the cell, thereby supporting myocardial integrity.

      Overall, the authors highlight an important aspect of epithelial maintenance in an environment that experiences significant biomechanical stress due to cardiac function. By generating a promoter-less allele of snai1b, the authors have created a clean genetic model in which to work. Coupled with beautiful microscopy and transcriptomics, this story has the potential to enlighten both cell biologists and cardiovascular biologists on the underpinnings of myocardial integrity. However, clarifications regarding the overall model would be particularly beneficial for the reader.

      1) A clearer discussion of the proposed molecular mechanism for Snai1b function would aid a reader's overall contextualization of this work. At one point, the authors suggest that Snai1b regulates N-cadherin localization to adherens junctions, thereby stabilizing actomyosin tension at cell junctions. Later, it is suggested that Desmin activates the actomyosin contractile network at the basal membrane. It is unclear whether the authors believe that these are separate events or whether they may be coupled, perhaps through Desmin disruption at the lateral membranes, leading to modifications in nearby adherens junctions. A more thorough investigation of the phenotype resulting from desmin b overexpression may clarify this relationship.

      2) It appears that extruded cells do not bud off from the myocardium, but rather remain on the apical surface of the existing myocardium. However, it is unclear whether this change in tissue architecture affects cardiac function or the overall morphology of the chamber. A brief discussion of these possibilities would have helped to contextualize the significance of this phenotype.

      3) The authors show that cardiomyocyte extrusion is most prevalent near the atrioventricular canal, and they suggest that this regionalized effect is due to the different types of extrinsic factors, like biomechanical forces, that this region experiences. However, it is also possible that regional differences in certain intrinsic factors are involved, such as junctional plasticity, actomyosin activity at the basal membrane, etc. To distinguish between these possibilities, it would have been informative to know whether the extent of N-cadherin/α-18/p-Myosin/Desmin mislocalization varies depending on the regional location of cardiomyocytes within the snai1b mutant heart. For example, do cardiomyocytes near the atrioventricular canal exhibit more extreme effects on N-cadherin/α-18/p-Myosin/Desmin localization than cardiomyocytes in further away portions of the ventricle? Or, do these cells exhibit similar degrees of protein mislocalization, but cells near the atrioventricular canal have a lower threshold for extrusion?

    1. Reviewer #2 (Public Review):

      In the manuscript 'A novel mechanosensitive channel controls osmoregulation, differentiation and infectivity in Trypanosoma cruzi' the authors show conclusive evidence that TcMscS is a mechanosensitive channel. They also show that TcMscS has additional roles outside of mechanosensation, likely playing a role in the infectivity of T. cruzi. This manuscript is well written with data that clearly supports the authors hypothesis. The evidence provided in the manuscript clearly shows that TcMscS gates in response to tension and that when knocked out of the genome there is a reduction in infectivity. This work will be impactful to both all researchers studying mechanosensation as it shows that mechanosensitive channels have roles outside of tension sensation.

      Recommendations:

      A) In the section 'Electrophysiological characterization of TcMscS', the authors present compelling evidence that TcMscS gates in response to tension in the membrane. However, it is unclear, both in the text and the caption, if the trace shown in Figure 2 panel C was collected under tension. If it was, please include the applied pressure value in either the text or caption. Additionally, within this section the applied pressure to the patch is frequently unclear. One way to clear this up would be to 1- add the applied pressure to each trace or to 2- add the applied pressure for each patch to the figure caption. -In Panel E: can you comment on the conductance of the channels in the three traces? Why do you see channels that are approximately 1/2 the size of the first trace in the second two traces?

      B) In the section 'TcMscS gene targeting by CRISPR-Cas9' the authors utilized CRISPR to KO TcMscS to determine its function, based on the immunofluorescence and qPCR TcMscS has been successfully knocked out. In lines 251-264, the authors complemented the KO with an overexpression vector in an attempt to confirm the role of TcMscS. In this section, it is very unclear what strains C1 and C2 are and how they are different from one another. Neither of these constructs successfully restores the growth rate. The authors can clarify the differences between the two constructs or they can remove this section from the manuscript, particularly Figure 5 supplement 3. The manuscript is strong and compelling without this panel.

    1. .

      Since publication, the FDA has rescinded its authorisation of bamlanivimab (LY-CoV555), due to its lack of efficacy against circulating variants of concern, particularly B.1.351 (South African), as a result of E484K substitution A,B. Eli Lilly are now pursuing the use of their combination therapy of bamlanivimab with etesevimab (LY-CoV016).

      The antibody cocktail REGN-CoV2 showed sustained efficacy against tested variant strains and thus remains a viable treatment option. However, a mutational library scan by Starr et al. revealed that a single amino acid change (E406W) is all that is required for a future variant to escape this therapy C.

      Circulating variants highlight the limited efficacy of monoclonal antibodies to an evolving virus, particularly in those which are restricted to the RBD. A diverse panel of monoclonal antibodies, which bind subdominant epitopes may be a more sustainable approach.


      A – Wang, P et al. Antibody resistance of SARS-CoV-2 variants B.1.351 and B.1.1.7. 2021. Nature. https://doi.org/10.1038/s41586-021-03398-2

      B – Starr, T.N.et al. Complete map of SARS-CoV-2 RBD mutations that escape the monoclonal LY-CoV555 and its cocktail with LY-CoV016. 2021. Cell Reports Medicine. https://doi.org/10.1016/j.xcrm.2021.100255

      C – Starr, T.N.et al. Prospective mapping of viral mutations that escape antibodies used to treat COVID-19. 2021. Science. https://doi.org/10.1126/science.abf9302

    1. Reviewer #2 (Public Review):

      Bhat et al. study transport mechanism of three members of the SLC6 family, i.e. DAT, NET and SERT, using a combination of cellular electrophysiology, fluorescence measurements - taking advantage of a fluorescent substrate (APP+) that can be transported by each of these different transporters - and kinetic modelling. They find that DAT, NET and SERT differ in intracellular K+ binding. In DAT and NET, intracellular K+ binding is transient, resulting in voltage-dependent transport. In contrast, SERT transports K+, and the addition of a charged substrate to the transport cycle makes serotonin transport voltage-independent.

      This is an extremely nice and interesting manuscript, based on a series of beautifully designed and executed experiments that are convincingly analyzed via a kinetic model. I have only some suggestions:

      1) Fig. 4: I find the description of Fig. 4 extremely difficult to understand. In clear contrast to the introductory sentence "Previous studies showed that Kin+ was antiported by SERT, but not by NET or DAT (Rudnick & Nelson, 1978; Gu et al., 1996; Erreger et al.,2008), SERT appears to be able to transport APP+ without K+ in Fig. 4. I was trying to understand this obvious discrepancy for a long time, until I found the authors coming back to this point in the discussion "However steady-state assessment of transporter mediated substrate uptake is hindered by the fact that all three monoamine transporters can also transport substrate in the absence of Kin+". This is a little late, and the author should address this point more explicitly in the result section, close to the description of Fig. 4.

      2) Throughout the whole manuscript I am missing statistical details in comparisons.

      3) Since APP+ might also only bind to the transporter or even only bind to the cell membrane, the authors might want to look at how the time course of the cellular APP+ signal depends on the size of the cells or on the ratio of transport currents and capacitance. It is of course possible that the tested cells do not differ sufficiently in size to permit such comparison. The authors should at least comment on this possibiliy.

      4) Another set of results one might look at are the time courses of fluorescence decay after the end of the APP+ perfusion (Fig. 2 and 4). Substrate (APP+) outward transport should have a comparable voltage dependence as substrate uptake, moreover it should depend on the amount of substrate that entered to the cell before. Could the authors provide such result and use them to exclude specific/unspecific APP+ binding?

    1. Reviewer #2 (Public Review):

      In this study, the authors investigate the long-appreciated but little understood link between chronic infection with Epstein-Barr virus and rheumatoid arthritis (RA). Using a collagen-induced (CI)-model of arthritis and a natural murine analog of EBV (gammaherpesvirus 68, HV68), the authors demonstrate that latent infection with HV68 exacerbates clinical progression of CI-arthritis and is associated with changes in the immune cell and cytokine profile in the spleens and joints of HV68 infected mice. The most compelling finding is that an infection can indeed exacerbate the progression of secondary diseases, and the requirement of age-associated B-cells (ABCs) to the severe disease progression. While this study addresses a timely and important question-how chronic infections affect subsequent or secondary disease progression-additional work as well as a clarification of the experimental design is encouraged to understand some of the key conclusions.

    1. Reviewer #2 (Public Review):

      Here the authors explore the role of PKA signaling in signaling downstream of the Moody GPCR in the BBB. The discovery that PKA is involved is interesting but not entirely surprising, as it functions downstream of many GPCRs to execute function (the really interesting question is how the same signal, changes in cAMP, causes PKA to do different things). The authors make the claim of a monotonic relationship between septate junctions (SJs) and cell-cell contact zones. I do not think they have measured the necessary parameters in a way that allows them to claim a "monotonic relationship between PKA activity, membrane overlap and the amount of SJ components in the area of cell contact." There is a correlation, but that is probably overstating it. There is an interesting analysis of several markers. These cells are very small and it is not clear what do the cytoskeletal markers really tell us. The markers change, no doubt, and do so in a way that correlates with the proposed Moody/PKA antagonistic relationship. The markers do change at the edges of cells and in regions of overlap, but wouldn't that be expected based on the changes in morphology? Again, the claim for "monotonic" changes is probably overstating the relationship. Doesn't the fact the total SJ area covered remains at 30% whether there is more or less overlap also argue against this (i.e. 30% of more overlap is not the same of 30% of less overlap...so more or less SJs are being made)?a

      The study would need to be strengthened by more rigorous quantification. There is no quantification in figure 3. This is a primary point in the manuscript-that cytoskeletal markers change (in a claimed "monotonic" way) in subperineurial glia when PKA is altered. There is also no quantification or statistics in Figure 5, which is among the most interesting observations.

      The complementary localization of Moody and the PKA catalytic (activated) subunit is very nice. It shows a very interesting cellular polarity. However, it is unclear whether this is altered in Moody mutants (the authors only did knockdown) and whether catalytic (activated) PKA now goes everywhere.

      Throughout, the authors use some very nice genetic studies, using loss-of-function, gain-of-function, and enhancer/suppressor approaches, and their findings are consistent with polarized localization of Moody being important.

    1. Reviewer #2 (Public Review):

      A key genomic study on emerging, nutritious, alternative grain crop.

      Deep genomic data on hundreds of land races/accessions.

      Population structure analysis, could be enhanced.

      Agronomic growth and yield traits are correlated and environmentally sensitive.

      Genomic dissection via GWAS to multigenic loci with candidate genes add genomic prediction and selection.

      Inference on domestication.

    1. Reviewer #2 (Public Review):

      Methods to characterize cell types in intact tissue using large scale analysis of molecular expression profiles are now readily available, with the best example being in situ RNA sequencing (spatial transcriptomics). However, these methods depend on separate immunohistochemical investigations to define the precise cellular and subcellular distribution of the protein products. Cole et al use iterative indirect immunofluorescence imaging (4i, Gut et al Science 2018) to compare the immunoreactivity of an impressive 18 different molecules within the same brain sections containing the dentate gyrus from young and old mice. First, they demonstrate that the method can be applied to not only adult mouse brain tissue, but also to human embryonic stem cell derived organoids and mouse embryonic tissue, which is an advance on the original report (Gut et al 2018). This demonstration is particularly important as it shows the potential for applying 4i to different biological disciplines. The rest of the manuscript focuses on the mouse dentate gyrus (DG) at 2, 6 and 12 months of age in order to map the complex changes and associations in the tissue across age. Various combinations of the 18 molecules are used to define different cell types and it incredibly informative to be able to view so many molecules in exactly the same area and will advance the field. This is the greatest strength of the manuscript. They find that neurogenic, radial glia-like stem cells (R cells) and proliferating cells are reduced in aged animals, as are immature (DCX+) cells, but claim that fluorescence intensity increases for the remaining R cells in 12 month old mice. They report that the density of vasculature also decreased with age, as did the associated pericytes, but astrocytes associated with the blood vessels increased. The last part of the manuscript defines 'microniches' (random or targeted regions of interest within the DG) and attempts to show how cell types, especially Nestin+ R cells, change in their associations with vasculature within these sub-regions at 2, 6 and 12 months of age. It is a commendable approach and the authors use a variety of statistical tests to compare the different cell types. However, there are several parts of the methods, along with insufficient details of the results that prevent full interpretation of the data, meaning that it is difficult to determine whether all conclusions are supported.

      1) There are many factors that can affect the measurements of immunoreactive structures (Fritschy, Eur J Neurosci, 2008 vol 28, p. 2365-70). The main limitation is not providing sufficient detail for the immunolabelling design and imaging parameters but providing some unclear details for the imaging analysis (below).

      a. In terms of immunohistochemistry, with the impressive number of tested antibodies, there is potential for variation due to antibody antibody penetration, unreported combinations of secondary antibodies, tissue quality (variations in fixation), etc. It is difficult to have confidence in the conclusions based on a total of 3 mice per age group for a single 40 um section per mouse. Ideally, to increase confidence in individual section variability, it is recommended that measurements should be taken from at least 3 sections per mouse then averaged, before averaging for the age group.

      b. Assuming there were 3 primary antibodies with 3 secondary antibodies per cycle before elution, were the combinations used consistent for all brain sections and mice? Was the testing and elution order the same (i.e. systematic)? There is a risk of cross-excitation and mis-interpretation of true immunoreactivity if spectrally close fluorophores for the secondary antibodies were selected for primary antibodies that recognize spatially overlapping structures. Can the authors show the cycle number and fluorophore for the examples in figures 1 and 2 to determine which markers were imaged together in the same cycle? This would give confidence to the methods for colocalisation and cell type descriptions. For example, can cross-excitation be ruled out for some of the signals in the images used in Fig 2 (duplicated in Fig 4) such as intensely immunopositive Laminin-B1 cells in the MT3 and Sox2 channels (2A) and Ki167, SOX2 and phospho-histone 3 channels (2C)?

      c. For image acquisition, details are required on the resolution (numerical aperture of the lenses) in order to interpret colocalisation measurements in the later figures. Which beamsplitters/filters were used, and was the same laser power used for the same markers over different specimens (important for interpreting figure 4 data)?

      d. For the analysis of ROIs (figures 3-6), were the 20x or 40x images used?

      e. Details of the antibody specificity controls should be provided.

      2) Numerous markers have been used to define different cells, but the proportions are not reported. For example, R cells are defined differently in figures 3 and 4. How many types of R cells (based on combinations of markers) were observed? High resolution examples of each defined cell type (neuronal and glial) would assist the reader in the confidence of the measurements (ideally as single channels side by side, with arrows indicating areas of detectable immunoreactivity that the authors would use to define each cell).

      3) The authors use HOPX and GFAP immunoreactivity and a lack of detectable S100beta immunoreactivity to distinguish R cells from triple immunopositive mature astrocytes. In Figure 3, the images are too low power to be able to confirm this. This part would benefit from some single cell examples showing the separate channels.

      a. Furthermore, the results (paragraph 2, page 7) report changes in cell number, but rather density is reported. Please either state the numbers or refer to density.

      b. Related to Fig 3, there are no details of the number of R cells counted in supplementary table 1. How were the density measurements obtained? How thick were the image stacks and how many R cells per section? Similarly, as stated in methods, for glial cells, 100 cells were randomly counted in each section (presumably the same count for each age), so how was it reported that specifically the numbers of astrocytes were reduced and no significant differences in other glial cell types? (bottom of p.7)

      4) An increase in fluorescence intensity for HOPX and MT3 (also marks R cells) was observed with age (Fig 4), with methods stating that the 5 ROIs used to calculate the background intensity were measured at each [optical?] slice for where the cells were measured, to account for unequal antibody penetrance. Several clarifications are required in order to interpret these results: For the example HOPX images in Fig 4A, for the 2 month old mouse, the background is low, whereas for 12 months, the background is far higher, meaning different background ROI values. Can this difference be explained by differences in laser power, contrast adjustments, optical slice thickness, or whether these are maximum intensity projections of different z thickness? These values must be reported, and for each image presented in the manuscript, details must be included as to what type of image (z-projection or single optical slice, z thickness). Was the optical section(s) of the 12 month mouse imaged closer to the surface of the section for this example in Fig 4A? Were cells sampled at all depths of the imaged volume? Did the antibody show better penetration in the 12 month old mice than the 2 month old mice? How many optical slices would a cell soma cover? In these cases, how was the fluorescence intensity measured? If a soma covered several optical slices, which one was selected for the ROI measurement?

      5) The described methods for studying cellular interactions are not clear, making it difficult to interpret the associations between vasculature, cell types, and age. How was colocalisation defined, and at what resolution? For example, it is expected that GFAP would be associated with but not directly colocalized with collagen IV (Fig 5). In these cases, the manuscript would benefit from high resolution examples of this colocalization/interaction. How many ROIs were taken, how exactly were the ROIs for cell types associated with collagen IV selected, was this in 2D or 3D?

      6) The methods for random microniches are difficult to follow, as are the methods for investigating the associations of other markers to radial processes of R cells. Please provide a definition of a 'spot'. Again, details of the micron per pixel resolution and optical slice thickness would help in the interpretation of results. Additionally, if possible, illustrated examples of the full procedure for niche mapping should be provided in order to follow how the measurements were collected.

    1. Reviewer #2 (Public Review):

      The paper uses computer modeling and simulations to show how a radially growing circular plant organ, such as a hypocotyl, can develop and maintain its organization into tissues including, in particular, cambium, xylem and phloem. The results are illustrated with useful movies representing the simulations. The paper is organized as a sequence of models, which has some rationale - it presumably depicts the path of refinements through which the authors arrived at the final model - but the intermediate steps are of limited interest. At the same time, mathematical details of the models are not presented to the full extent. Fortunately, the models can be downloaded over the Internet, and the supplementary materials include detailed instructions for executing them (using the VirtualLeaf framework). Consequently, the paper and its results can potentially serve as a stepping stone for further model-assisted studies of radial tissue organization and growth.

    1. Reviewer #2 (Public Review):

      Shimura et al. have discovered that GJA1-20K may provide protection in ischemic hearts through polymerizing actin around mitochondria and inducing mitochondrial fission. The authors use a series of elegant genetic, chemical, biochemical and cell biology studies including the use of the Gja1 M213L mouse line, which is unable to generate the 20kD Gja1 isoform, in order to determine that the beneficial effects of GJA1-20K. Specifically, the authors discovered that this beneficial effect is due to decreased reactive oxygen species (ROS) generation from smaller mitochondria. The overall work is well done but additional discussion should be provided about the impact of the work, particularly how the work may help realize a goal of therapeutically achieving ischemic preconditioning that has not been achieved in more than 30 years since ischemic preconditioning was first recognized.

    1. Reviewer #2 (Public Review):

      Summary:

      In this study, Vuong and colleagues conducted a case control study nested within a larger longitudinal and multi-country observational study to evaluate 10 biomarkers. These biomarkers were selected based on the strength of evidence in the literature and the current understanding of dengue pathogenesis. Using a 1:2 ratio of severe/moderately severe dengue cases to uncomplicated dengue controls, the authors examined the trends of the expression of these biomarkers during acute illness (days 1-3 from illness onset) as well as early (10-20 days from illness onset) and late (>20 days from illness onset) convalescence. The identified several biomarkers that were expressed at higher levels during acute illness in cases than controls and showed that these could be used in combination to predict those at increased risk of severe/moderately severe dengue. Notably, the authors identified different sets of biomarkers for paediatric and adult dengue cases, suggesting that the underlying pathophysiology of severe disease may differ in these groups of dengue cases. The authors concluded that the biomarkers they identified would be a major public health benefit to allocate healthcare resources during dengue outbreaks, and suggested that these biomarkers could also be applied as biological endpoints in dengue clinical trials.

      Strengths:

      This is a fairly sizeable study involving 281 severe/moderately severe dengue cases and 556 uncomplicated dengue controls. The authors combined data clinical observation data with those derived from serum protein measurements and analysed them using sophisticated statistical approaches.

      The search for biomarkers predictive of the risk of severe dengue has spanned decades. This study distinguishes itself from others in its study design and the systematic selection of biomarkers for evaluation. Avoidance of untargeted screening reduces the likelihood of chance discovery and makes the findings more statistically robust.

      The findings have useful practical applications. Severe dengue manifests typically around the period of fever defervescence at around days 4-7 from fever onset. Application of biomarkers in the acute febrile phase of illness could thus provide a 2- to 3-day lead time to triage those at risk of severe dengue for closer monitoring and management.

      Weaknesses:

      The main weakness is the exclusion of virological markers, such as plasma/serum viral RNA levels or NS1 antigenaemia. Indeed, previous observations have found severe dengue patients to have higher viraemia in the acute phase of illness compared to those with uncomplicated dengue. More recently, several mechanistic studies have suggested that dengue virus NS1 protein could bind endothelial cells to disrupt its integrity, leading to vascular leakage. Indeed, the authors have pointed out these findings in lines 20-25 on page to lines 1-2 on page 6. Despite these reports, it is curious that the authors have not included either viraemia or NS1 antigenaemia as possible biomarkers for severe dengue.

      The manuscript in its present form may favour those with a strong statistical background to fully appreciate the nuances. Clearer explanations on the statistical findings would, I think, be helpful to those without such statistical background but who would nonetheless be in positions to translate these findings into clinical practice.

      Most of the cases included in this study had DENV-1 infection. The biomarkers identified in this study may thus be DENV-1 specific and may not be readily applied to triage dengue cases caused by other DENV infection.

      Overall impression:

      This study provides two interesting findings. Firstly, that there are biomarkers that can be further developed into clinical tests to triage dengue patients for management. Although this possibility will require further assay development - the Luminex platform used for multiplex measurements of these biomarkers is unlikely to be available in most clinical laboratories - this study does show proof-of-concept to justify the development of simpler and perhaps even point-of-care assays. Secondly, the finding that adult and paediatric dengue require different biomarkers to indicate risk of severe disease should also trigger more detailed clinical and basic science investigation into how age influence host response to infection.

    1. Reviewer #2 (Public Review):

      This manuscript reports a study that sought evidence of patterned inter-areal activity in the spinal cord of anesthetized rats. This could be a very significant finding, with potentially important scientific and therapeutic implications. However, the Methods lack necessary details, and the Results raise substantial issues that need to be resolved. Until these gaps and uncertainties are resolved, it is not possible to evaluate the results and their implications with confidence. Substantial revisions are essential.