1,889 Matching Annotations
  1. Jan 2022
  2. Dec 2021
    1. RRID:ZFIN_ZDB-GENO-071217-3

      DOI: 10.7554/eLife.72345

      Resource: (ZFIN Cat# ZDB-GENO-071217-3,RRID:ZFIN_ZDB-GENO-071217-3)

      Curator: @scibot

      SciCrunch record: RRID:ZFIN_ZDB-GENO-071217-3

      What is this?

    1. Second Amendment freedom of expression?

      it should be regarding the first amendment not second!

  3. Nov 2021
    1. painted an American flag on his bare chest, but painted it upside down.

      incorrect- the act being charged is "Unfurled the glad, doused it with kerosene and set it on fire"

    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. a stoichiometry of approximately one complex molecule per actin monomer

      This seems like an unreasonably high stoichiometry of Arp2/3 complexes per actin subunit; we now know that each Arp2/3 complex covers a few subunits, so there shouldn't be enough room on the filament to fit 1:1 Arp2/3 complex/actin

  4. Oct 2021
    1. List only the first author’s name followed by “et al.” in every citation, even the first, unless doing so would create ambiguity between different sources.

      APA 7 3 or more authors

    1. There will be three billion gamers by next year, according to a Newzoo study. And as Loftus puts it: “People are going to need to wear something.”

      THIS is it - web 3 is making consumers mutiplicitous - opens marketts WITHIN games, subworlds that can be exploited / marketed to / fashion trends will sweep games, online subcultures (maybe) - people have markeable personas on and off the web, new context for targeted advertising / commerce.

      Will cannabalize physical economies?

      Accessorize for a zoom meeting - digital suits, etc digital costumes. Something to wear at digital concerts, in games; your Perona will not be birthed into the metaverse clothed, accessorized...

      Assets will be portable across platforms.

    1. the power to destroy may defeat and render useless the power to create

      Is this the foundational argument for the Supremacy Clause? For this example, does this mean if the states had the power to influence federal programs or policies, that the powers delegated to the federal government would be redundantly delegated?

    2. is not supreme.

      When do state laws take effect over federal laws? How do you know when which is supreme? or vice versa?

    3. But if the full application of this argument could be admitted, it might bring into question the right of Congress to tax the State banks, and could not prove the rights of the States to tax the Bank of the United States.

      Based on the relationship between State, banks, and General Government, is this suggesting a contradiction or non mutual relationship between the 3 in this line of argument?

    4. Its means are adequate to its ends, and on those means alone was it expected to rely for the accomplishment of its ends.

      This whole phrase is really confusing me? I'm not able to even figure out what any of the parts mean, could anybody help me out?

    5. but by people over whom they claim no control.

      Is this suggesting that if States were allowed to tax the Federal Government, would States hold power and authority over citizens outside of their state? Would State laws apply universally?

    6. This is, we think, the unavoidable consequence of that supremacy which the Constitution has declared.

      A lot of this argument seems centered around the idea that the people, via its representatives, declared the Constitution has supreme power over states. In this, a State (and by extension, representatives of the people) is arguing the apparent supremacy of the federal gov't over the state, doesn't this contradict the argument of the Supreme Court a bit, that the will of the people was for a federal gov't to hold supreme over state?

    7. The powers of the General Government, it has been said, are delegated by the States, who alone are truly sovereign, and must be exercised in subordination to the States, who alone possess supreme dominion.

      Does Maryland grapple with the idea that the sovereignty and power of the states comes from the people in its argument that the Constitution receives its power from the states, as opposed to the people?

    8. we shall find it capable of changing totally the character of that instrument

      I don't understand what this is saying? So on the basis of Marylands contends it changes the meaning of what the Bank means in the constitution?? I'm not sure if I even read it correctly the end of the sentence isn't making sense to me. Changes the character of what instrument? Constitution?

    9. Taxation, it is said, does not necessarily and unavoidably destroy.

      Taxation seems to be a big pinpoint of this argument. In history we have seen many problems with "taxation without representation" or misuse of taxing in this case, but we also see problems today with people not agreeing with where the tax money goes, or especially problems with getting tax returns. Do you think taxation is going to continue being a problem?

    10. Would the people of any one State trust those of another with a power to control the most insignificant operations of their State Government?

      This question is more a thought for questions. Do you think that this idea of one government have power over another government issues still stand in to days modern world? or do you think that states and governments the the US are more lacs about this issues then they where in 1819?

    11. which another Government may furnish or withhold

      Is 'another government' directly referring to the state vs. the national government?

    12. But is this a case of confidence?

      What constitutes a case of confidence? Is Marshall saying here that the national and state governments need to just have confidence in one another that there will be no abuse of power from either end? When does the Court decide a case is to be considered a case of confidence?

    13. But the two cases are not on the same reason.

      Kind of seperate, but do federal buildings pay taxes to the state they are in? Or to the government? Or do they pay at all? If they dont does that mean that the state tax payer does?

    14. unanimously of opinion

      In the Epstein text, it says that one of the seven Supreme Court Justices, Thomas Todd, did not participate in the decision. So it was unanimous, but only among 6 of the 7. I wonder why Todd didn't participate? Does anyone know from the text or other history?

    15. We shall find it capable of arresting all the measures of the Government, and of prostrating it at the foot of the States.

      I feel like this is a tad dramatic. How would allowing Maryland to tax a government institution arrest "all the measures of the Government"? I understand that taxing the bank would likely lead to the closing of that bank - but how would it interrupt the rest of our governence?

    1. NFTs are compatible with anything built using Ethereum. An NFT ticket for an event can be traded on every Ethereum marketplace, for an entirely different NFT. You could trade a piece of art for a ticket!

      There - opens up GIGANTIC barter economy possibilities - will shut out old middle men & create new ones - swap airline tickets / hotel reservations for concert tickets or memorabilia

    1. Truman's action can be upheld as an exercise of the president's inherent military power as commander-in-chief.

      incorrect: The Court held that the President's military power as Commander in Chief of the Armed Forces did not extend to labor disputes.

    1. We do not need images here. you can use some kind of icon if you wish. But this is largely text. Also FYI this section might need a headline. Such as summary overview - tbd.

    1. It is also not entirely unworthy of observation, that in declaring what shall be the supreme law of the land, the constitution itself is first mentioned; and not the laws of the United States generally, but those only which shall be made in pursuance of the constitution, have that rank.

      Now that Marshall observes the supreme law of the land to be the constitution, this makes me wonder how the Judiciary Act of 1789 was passed when it altered the original jurisdiction that was written in the constitution. Did the creators of the this act not consult or care about the constitution?

  5. Sep 2021
    1. Why does a judge swear to discharge his duties agreeably to the constitution of the United States, if that constitution forms no rule for his government? If it is closed upon him, and cannot be inspected by him?

      This concludes Marshall's habit of asking questions that he proceeds to answer. Are many modern day decisions written similarly? Or is this style more suited to an early justice seeking to define the role of the court, including the types of questions a Justice should be asking and answering?

    2. If congress remains at liberty to give this court appellate jurisdiction, where the constitution has declared their jurisdiction shall be original; and original jurisdiction where the constitution has declared it shall be appellate; the distribution of jurisdiction, made in the constitution, is form without substance.

      Okay help me clarify this. In this phrasing saying that if Congress gives appellate jurisdiction over the constitution witch already holds original jurisdiction than that new law or rule makes no sense?

    3. mandamus should be used for that purpose, that will must be obeyed. This is true, yet the jurisdiction must be appellate, not original.

      So a mandamus can only be applied to Appellate jurisdiction and not Original? Or can it be applied to both if met with the "specificity" of the original jurisdiction requirements?

    4. no bill of attainder or ex post facto law

      I realize this is just an example he's using to prove his point, that every word of the constitution is important and that the constitution should trump other laws. However, I've never heard of either of these - A "bill of attainder" or an "ex post facto" law. Does anyone know what these are?

    5. If, however, such a bill should be passed and a person should be prosecuted under it; must the court condemn to death those victims whom the constitution endeavors to preserve?

      I'm a little confused on how this analogy fits in. Is this meaning that the bill passed should be enforced by the courts? If this is true how exactly does this fit in the argument?

    6. If any other construction would render the clause inoperative, that is an additional reason for rejecting such other construction, and for adhering to their obvious meaning.

      I do not understand how this statement relates to what was said before. If I'm correct, the previous statements was suggesting that the supreme and inferior courts have certain jurisdiction. Are they saying that how jurisdiction is determined may change over time?

    7. Neither is it necessary in such a case as this, to enable the court to exercise its appellate jurisdiction.

      I thought the whole point of this case was to review laws or precedents within the constitution and deem whether or not they were truly viable. Doesn't appellate jurisdiction become necessary then? A higher court overturning a lower courts decision?

    8. It is then the opinion of the court,

      How can a court's opinion change? When there are different people on the court would that maybe produce a different outcome? How would you be able to be sure that things are standard?

    9. he power remains to the legislature, to assign original jurisdiction to that court in other cases than those specified in the article which has been recited; provided those cases belong to the judicial power of the United States.

      I am confused on how the legislature fits into the court's original jurisdiction. Did the legislature have to approve each of the cases that the supreme court took? Why only for original jurisdiction and not appellate jurisdiction as well? Does this still apply today?

    10. to issue writs of mandamus to public officers, appears not to be warranted by the constitution

      Where does the issue writs of mandamus come from then if not the constitution? I thought that the way judicial courts act were all centered around the constitutional law. But the way this is worded, it appears that mandamus is not in reference to the constitution.

    11. Here the language of the constitution is addressed especially to the courts. It prescribes, directly for them, a rule of evidence not to be departed from. If the legislature should change that rule, and declare one witness, or a confession out of court, sufficient for conviction, must the constitutional principle yield to the legislative act?

      I'm curious as to how the writers of the Constitution had not considered that something similar could occur? Why did the writers not introduce some sort of specific method where one of the branches could declare an action of another unconstitutional?

    1. Voluntary Cessation Doctrine,

      I looked up the Voluntary Cessation Doctrine, but I am still unsure what it means and how it pertains to this case? I wish they would have elaborated more on it. I could be mistaken, but I believe the Doctrine deals with exceptions to mootness, however, I thought Justice Breyer said this case was not moot.

    2. Locke v. Davey

      Does Lockey vs Davey involve the qustion of scholarship (of religious schools) provided from government money? Thats what I'm gathering but I'm not sure. Not familiar with the case but before I look it up I'm guessing it has to do with government funded school scholarships secular vs non secular?

    3. Well -- well, for -- if the political winds change, we have -- we have this policy by Facebook or press release. So it can easily be changed back if political --

      How often does social media play a part into political bias? How are judges or trial members able to keep their rulings separate from the bias they're hearing in the news or headlines?

    4. David A Cortman

      He doesn't really answer the justices question of how he doesn't see the other way of questioning as discrimination IN FAVOR of the church against other non religious people which I think is sort of telling and again why I think justice sotomayor is one of the dissenting judges. One question I have is whether or not there actually is anything in the constitution that protects discrimination in favor of religion? I always hear about it the other way around and I remember in one instance not long ago about a christian baker who refused to bake a wedding cake for a gay couple based on the argument that it violates his ability to practice his religious beliefs. The court sided with him, which to me is discrimination in favor of "religious beliefs" that was ruled by the supreme courts as justified and constitutional.

    5. There's also entanglement.

      Having no prior knowledge of the Establishment Clause, I believe I've been able to glean a little of what it might say from the course of listening to this case. The application of it seems to be broken into two halves of conditionality, "endorsement" and "entanglement." Does anyone on the thread have a definition of what these two conditions mean?

    6. Locke, right? Locke drew a distinction between assistance for devotional, theological education and scholarship and others.

      This mention of Locke has been used numerous times throughout this conversation, and I have yet to understand where the meaning of this is being drawn from. Are they referring to something John Locke wrote? Is this the name of something or someone I may have missed?

    1. ZFIN: ZDB-ALT-110503-3

      DOI: 10.1016/j.cub.2021.08.049

      Resource: (ZFIN Cat# ZDB-ALT-110503-3,RRID:ZFIN_ZDB-ALT-110503-3)

      Curator: @Naa003

      SciCrunch record: RRID:ZFIN_ZDB-ALT-110503-3

      What is this?

    2. ZFIN: ZDB-ALT-120320-3

      DOI: 10.1016/j.cub.2021.08.049

      Resource: (ZFIN Cat# ZDB-ALT-120320-3,RRID:ZFIN_ZDB-ALT-120320-3)

      Curator: @Naa003

      SciCrunch record: RRID:ZFIN_ZDB-ALT-120320-3

      What is this?

    3. ZFIN: ZDB-ALT-071129-3

      DOI: 10.1016/j.cub.2021.08.049

      Resource: (ZFIN Cat# ZDB-ALT-071129-3,RRID:ZFIN_ZDB-ALT-071129-3)

      Curator: @Naa003

      SciCrunch record: RRID:ZFIN_ZDB-ALT-071129-3

      What is this?

    1. polyphonic

      polyphonic: style of musical composition employing two or more simultaneous but relatively independent melodic lines


    2. primordial

      primordial: existing in or persisting from the beginning (as of a solar system or universe)


    1. Dampness and mould growth were also a feature of some houses on the Whiteway and Twerton council

      dampness of homes in council estates in Bath. research for community advocacy. Collective action example.



    1. he teacher proceeded to belittle everyone from German Eva, who hated laziness, to Japanese Yukari, who lovedpaintbrushes and soap. Italian, Thai, Dutch, Korean, Chinese--we all left class foolishly believing that the worstwas over. We didn't know it then, but the coming months would teach us what it is like to spend time in thepresence of a wild animal. We soon learned to dodge chalk and to cover our heads and stomachs whenever sheapproached us with a question. She hadn't yet punched anyone, but it seemed wise to prepare ourselves againstthe inevitable.

      he is transitioning from what he is going through to what he went through through the class. teacher seems pretty mean towards the students and a little bit aggressive

    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. I remind myself that I am now a full-grown man. No one will ever again card me for a drink or demand that Iweave a oor mat out of newspapers. At my age, a reasonable person should have completed his sentence in theprison of the nervous and the insecure--isn't that the great promise of adulthood? I can't help but think that,somewhere along the way, I made a wrong turn. My fears have not vanished. Rather, they have seasoned andmultiplied with age. I am now twice as frightened as I was when, at the age of twenty, I allowed a failed nursingstudent to inject me with a horse tranquilizer, and eight times more anxious than I was the day my kindergartenteacher pried my ngers off my mother's ankle and led me screaming toward my desk. "You'll get used to it," thewoman had said.

      feeling very scared. seems like he is regretting it a little bit?

  6. Aug 2021
    1. RRID:ZFIN_ZDB-ALT-140521-3

      DOI: 10.7554/eLife.44431

      Resource: (ZFIN Cat# ZDB-ALT-140521-3,RRID:ZFIN_ZDB-ALT-140521-3)

      Curator: @scibot

      SciCrunch record: RRID:ZFIN_ZDB-ALT-140521-3

      What is this?

    1. RRID:_ZFIN_ZDB-GENO-090923-3

      DOI: 10.7554/eLife.38911

      Resource: (ZFIN Cat# ZDB-GENO-090923-3,RRID:ZFIN_ZDB-GENO-090923-3)

      Curator: @scibot

      SciCrunch record: RRID:ZFIN_ZDB-GENO-090923-3

      What is this?

    1. RRID:ZFIN_ZDB-ALT-071109-3

      DOI: 10.7554/eLife.68755

      Resource: (ZFIN Cat# ZDB-ALT-071109-3,RRID:ZFIN_ZDB-ALT-071109-3)

      Curator: @scibot

      SciCrunch record: RRID:ZFIN_ZDB-ALT-071109-3

      What is this?

  7. Jul 2021
    1. RRID:ZFIN_ZDB-GENO-170619-3

      DOI: 10.7554/eLife.35796

      Resource: (ZFIN Cat# ZDB-GENO-170619-3,RRID:ZFIN_ZDB-GENO-170619-3)

      Curator: @evieth

      SciCrunch record: RRID:ZFIN_ZDB-GENO-170619-3

      What is this?

    1. Those are some typical metadata elements: Title and description, Tags and categories, Who created and when, Who last modified and when, Who can access or update.

      This list makes sense based on the definition of metadata above in the article saying that it helps organize, find and understand data. I would expect these elements to be a part of metadata because they all give information as to what you are looking at and who created it.

    2. Metadata is simply data about data. It means it is a description and context of the data. It helps to organize, find and understand data.

      The word metadata sounded way more advanced than my skill level in computers. I didn't really know what the word metadata meant until reading this article. The fact that it is just data about data makes it way easier to understand. I am finding that a lot of terms that we have used so far in class seemed hard to understand but are actually simple to do once they're broken down.

    1. IaskquestionsaboutthestructureandresultsofwebsearchesfromthestandpointofaBlackwoman—astandpointthatdrivesmetoaskdifferentquestionsthanhavebeenpreviouslyposedabouthowGoogleSearchworks.

      I agree with this. I think we should be asking and questioning things because that is how we learn and develop our ideas and opinions. Once we start questioning all of these things we can take this new knowledge and start to make changes.

    2. AtthecoreofmyargumentisthewayinwhichGooglebiasessearchtoitsowneconomicinterests—foritsprofitabilityandtobolsteritsmarketdominanceatanyexpense.

      I don't believe that Google should be biasing search to its own economic interests. I find that when I am searching things that my ads are targeted to what I have looked at previously or something that Google thinks I will like. As well, some of the other ads are completely random and not related to anything I have searched. I think that Google should base its ads on equality as well as its searches so you gain accurate information and not something put up to serve their own economic interests.

    3. TheGoogleSearchautosuggestionsfeaturedarangeofsexistideassuchasthefollowing:•Womencannot:drive,bebishops,betrusted,speakinchurch•Womenshouldnot:haverights,vote,work,box•Womenshould:stayathome,beslaves,beinthekitchen,notspeakinchurch•Womenneedto:beputintheirplaces,knowtheirplace,becontrolled,bedisciplined

      I can't believe that people are still thinking this way. Suggestions such as women cannot drive or be trusted and they should stay at home and be put in their places are not ones that we should be hearing anymore. Men and women are supposed to be viewed as equal and it shocked me that these came up as autosuggestions on Google Search. That shows that there is still a ways to go to fully achieve gender equality.

  8. 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. nternal forces create the potential for a production function with increasing returns.

      This is a shortened version of the full definition to avoid vocab words we haven't defined yet

    2. Growth Comes From Within

      Starting off with Giacomo's slide content

    1. ZFIN: ZDB-ALT-130718-3

      DOI: 10.1016/j.celrep.2021.109255

      Resource: (ZFIN Cat# ZDB-ALT-130718-3,RRID:ZFIN_ZDB-ALT-130718-3)

      Curator: @Naa003

      SciCrunch record: RRID:ZFIN_ZDB-ALT-130718-3

      What is this?

    2. ZFIN: ZDB-ALT-140521-3

      DOI: 10.1016/j.celrep.2021.109255

      Resource: (ZFIN Cat# ZDB-ALT-140521-3,RRID:ZFIN_ZDB-ALT-140521-3)

      Curator: @Naa003

      SciCrunch record: RRID:ZFIN_ZDB-ALT-140521-3

      What is this?

    3. ZFIN: ZDB-ALT-120723-3

      DOI: 10.1016/j.celrep.2021.109255

      Resource: (ZFIN Cat# ZDB-ALT-120723-3,RRID:ZFIN_ZDB-ALT-120723-3)

      Curator: @Naa003

      SciCrunch record: RRID:ZFIN_ZDB-ALT-120723-3

      What is this?

    4. ZFIN: ZDB-ALT-100525-3

      DOI: 10.1016/j.celrep.2021.109255

      Resource: (ZFIN Cat# ZDB-ALT-100525-3,RRID:ZFIN_ZDB-ALT-100525-3)

      Curator: @Naa003

      SciCrunch record: RRID:ZFIN_ZDB-ALT-100525-3

      What is this?

  9. May 2021
    1. My advice is if you are looking for a quick and accurate answer ask to have the trouble ticket elevated immediately and to speak with an engineer that will recognize your knowledge and speak with you on your level.
    2. I typically request to speak with an engineer when I find myself detecting an inexperienced support person.
    3. In one of my internship, I got to befriend a level 2 tech support, so learned a couple thing of how it worked (in that company). Level 1 was out-sourced, and they had a script to go from, regularly updated. From statistics, this took care of 90% of issues. Level 2 was a double handful of tech people, they had basic troubleshooting tools and knowledge and would solve 90% of the remaining issues. Level 3 was the engineering department (where I was), and as a result of level 1 and 2 efficiency less than 1% of issues ever got escalated. The process worked!
    1. Reviewer #3 (Public Review):

      Barone, Paul et al. present a new computational method, named T-REX, to detect changes in immune cell populations from repeated cytometry measurements (before and after infection or treatment). The proposed method is designed to detect changes in rare and common cells with particular focus on the former. T-REX detects subpopulations of cells showing marked differences in abundance between the proportion of cells from different time points (before and after infection) from a single individual. The method relies of a dimensionality reduction step using UMAP followed by a K-nearest neighbor (KNN) search to identify cells that have a large fraction (>0.95) of neighbors from one time point, indicating expansion or shrinkage of certain cell populations. Areas in the UMAP with clustered expanding or shrinking neighborhoods are labeled as hotspots. Cells in these hotspots were further characterized and enriched markers were identified using MEM, a method published earlier by the same authors. T-REX was applied to a newly collected dataset of rhinovirus infection and three publicly available datasets of SARS-CoV-2 infections, melanoma immunotherapy and AML chemotherapy. The results are presented clearly and the authors discuss in details several examples in which the cells identified by T-REX have a phenotypic profile which align with previous knowledge, indicating the relevance of the results.


      • T-REX is based on a simple pipeline including UMAP and KNN. This is an advantage especially given the large number of cells collected. Further, the proposed approach has a key advantage since it allows the analysis of one sample at a time, which is practical if one wants to analyze a new sample. There is no need to rerun the analysis on an aggregate of a large number of samples.

      • The new rhinovirus dataset is of great value to the community.


      • The paper lacks a comparison to other methods for differential abundance testing. In particular, it is not clear how T-REX differs from the Differential abundance test proposed by Lun et al. (https://doi.org/10.1038/nmeth.4295). Similarly, there are no experiments or results to support the authors' initial claim that T-REX outperforms current clustering-based methods (SPADE, FLOWSOM, Phenograph,...etc.) in capturing changes in rare (<1%) cell populations.

      • T-REX relies on arbitrary cutoffs (0.95 and 0.5 %) to define expansion or shrinkage in the neighborhood of each cell (0.95 and 0.5 %) rather than a formal statistical test. These cut-offs were defined based on the ability to detect tetramer positive cells in one subject only. This greatly limits the generalizability of the method.

      • The authors do not motivate the use of UMAP prior to the KNN graph reconstruction. While UMAP is a clearly powerful method to visualize single cell data, the resulting embedding can potentially show distinct groups of points when the high dimensional manifold is more continuous. For this reason, KNN graphs are usually built using the high-dimensional data (or principal components).

      • Given that T-REX is mainly developed to detect changes in rare cell populations, the paper lacks an assessment of the method's sensitivity. For instance, cells were subsampled equally from each time point. An assessment of the effects of this subsampling step is necessary. In general, a guide to the users indicating the limitations of T-REX will be greatly helpful.

      • Given that the main aim of T-REX is to detect differences in rare cells, the rational to perform a separate analysis for CD4 positive cells is not clear. One would expect these differences to be identified also in the analysis performed using all cells.

      • The paper lacks a discussion on the effects of batch effects between the different time points on the performance of T-REX.

    1. Reviewer #3 (Public Review):

      This is an important manuscript on COVID-19 convalescent plasma (CCP) that challenges the findings of the larger Mayo Clinic CCP study demonstrating a lack of efficacy. Their main findings are that there is a strong inverse correlation between CCP use and mortality for admitted patients in the USA. Overall this is a well written manuscript without any overt weaknesses.

    1. Reviewer #3 (Public Review):

      In this study, Alhussein and Smith provide two strong tests of competing hypotheses about motor planning under uncertainty: Averaging of multiple alternative plans (MA) versus optimization of motor performance (PO). In this first study, they used a force field adaptation paradigm to test this question, asking if observed intermediate movements between competing reach goals reflected the average of adapted plans to each goal, or a deliberate plan toward the middle direction. In the second experiment, they tested an obstacle avoidance task, asking if obstacle avoidance behaviors were averaged with respect to movements to non-obstructed targets, or modulated to afford optimal intermediate movements based on a commuted "safety margin." In both experiments the authors observed data consistent with the PO hypothesis, and contradictory of the MA hypothesis. The authors thus conclude that MA is not a feasible hypothesis concerning motor planning under uncertainty; rather, people appear to generate a single plan that is optimized for the task at hand.

      I am of two minds about this (very nice) study. On the one hand, I think it is probably the most elegant examination of the MA idea to date, and presents perhaps the strongest behavioral evidence (within a single study) against it. The methods are sound, the analysis is rigorous, and it is clearly written/presented. Moreover, it seems to stress-test the PO idea more than previous work. On the other hand, it is hard for me to see a high degree of novelty here, given recent studies on the same topic (e.g. Haith et al., 2015; Wong & Haith, 2017; Dekleva et al., 2018). That is, I think these would be more novel findings if the motor-averaging concept had not been very recently "wounded" multiple times.

      The authors dutifully cite these papers, and offer the following reasons that one of those particular studies fell short (I acknowledge that there may be other reasons that are not as explicitly stated): On line 628, it is argued that Wong & Haith (2017) allowed for across-condition (i.e., timing/spacing constraints) strategic adjustments, such as guessing the cued target location at the start of the trial. It is then stated that, "While this would indeed improve performance and could therefore be considered a type of performance-optimization, such strategic decision making does not provide information about the implicit neural processing involved in programming the motor output for the intermediate movements that are normally planned under uncertain conditions." I'm not quite sure the current paper does this either? For example, in Exp 1, if people deliberately strategize to simply plan towards the middle on 2-target trials and feedback-correct after the cue is revealed (there is no clear evidence against them doing this), what do the results necessarily say about "implicit neural processing?" If I deliberately plan to the intermediate direction, is it surprising that my responses would inherit the implicit FF adaption responses from the associated intermediate learning trials, especially in light of evidence for movement- and/or plan-based representations in motor adaptation (Castro et al., 2011; Hirashima & Nozacki, 2012; Day et al., 2016; Sheahan et a., 2016)?

      In that same vein, the Gallivan et al 2017 study is cited as evidence that intermediate movements are by nature implicit. First, it seems that this consideration would be necessarily task/design-dependent. Second, that original assumption rests on the idea that a 30˚ gradual visuomotor rotation would never reach explicit awareness or alter deliberate planning, an assumption which I'm not convinced is solid.

      The Haith et al., 2015 study does not receive the same attention as the 2017 study, though I imagine the critique would be similar. However, that study uses unpredictable target jumps and short preparation times which, in theory, should limit explicit planning while also getting at uncertainty. I think the authors could describe further reasons that that paper does not convince them about a PO mechanism.

      If the participants in Exp 2 were asked both "did you switch which side of the obstacle you went around" and "why did you do that [if yes to question 1]", what do the authors suppose they would say? It's possible that they would typically be aware of their decision to alter their plan (i.e., swoop around the other way) to optimize success. This is of course an empirical question. If true, it wouldn't hurt the authors' analysis in any way. However, I think it might de-tooth the complaint that e.g. the Wong & Haith study is too "explicit."

    1. Reviewer #3 (Public Review):

      This is a well-presented study on the development of the CNS in the octopus O. vulgaris. The aim of the study is to identify the origin of the neural progenitors of the brain. The authors provide an excellent gene expression study of conserved neural genes to identify the location of these progenitors. Furthermore, by cell lineage tracing, they confirm the results of a previous study by Koenig et al. showing that the progeny of neural progenitors generated in the so-called lateral lips, a region adjacent to the eyes, migrate to different brain areas. The neural precursor location in the brain can be correlated with their spatial origin from the neural progenitors in the lateral lips. The authors suggest that the spatial map of the lateral lips is conserved in cephalopods. Furthermore, they analyse the mitotic activity in the developing brain by Ov-pcna in situ hybridisation and anti-PH3 immunohistochemistry. The authors conclude that "grossly, the embryonic octopus brain does not contain dividing progenitor cells." Based on the cell lineage studies, the strong expression of neural genes in the lateral lip and the observed mitotic activity, the authors overall conclude that the lateral lips represent the neurogenic zone in the developing brain of the octopus, i.e. that the neural progenitors of the brain derive from this area. I agree with the authors that the lateral lips are neurogenic regions, however, it is also possible that neural progenitors do arise from other regions of the developing brain. Overall this is a valuable contribution to our knowledge of neurogenesis in deuterostomian invertebrates and in a wider context the evolution of neural developmental processes.

    1. Reviewer #3 (Public Review):

      The manuscript of Oggenfuss et al presents a comprehensive analysis of TE insertion polymorphisms detected in the genome of ~300 isolates of the wheat fungus Zymoseptoria tritici. The article shows that numerous TE families generated thousands of polymorphic insertions and the authors propose that some of these insertions might potentially be linked to adaptation. They identified a recent burst of transposition in a rapidly expanding population, providing empirical evidence that drastic demographic process shape TE dynamics in nature. Last, they show that intra-specific variation in genome size can be accounted by variation in the number of polymorphic TE insertions, which recapitulate the well stablished association between TE content and genome size variation observed across the diversity of life forms.

      The article is well written, present novel as well as relevant results, and provide insights to our understanding of the role of TEs in microevolutionary processes. In addition, it provides an important amount of population genomic data that will serve as a resource. Thus, this manuscript is of potential interest to a broad audience on evolutionary and population genomics.

      My major concern is the lack of strong evidence supporting positive selection and/or functional relevance of the TE insertions detected. In particular, the selective sweep scans performed ignored other types of variants (such as SNPs and INDELs), preventing the identification of the actual targets of natural selection.

    1. Reviewer #3 (Public Review):

      In "Assembly of higher-order SMN oligomers is essential for animal viability, requiring a motif exposed in TG zipper dimers," Gupta et al. present an impressive amount of data regarding the solution behavior of constructs of the protein SMN1 (or just SMN) from Homo sapiens, Drosophila melanogaster, and Schizosaccharomyces pombe. Defects in the Hs protein are known to cause the neuromuscular disease "Spinal Muscular Atrophy" (SMA). They also present experiments in genetically modified organisms (fission yeast and fruit flies) to test their hypotheses. Bioinformatics are used to generate and refine hypotheses. The potential power of these complementary methods is substantial, if employed well.

      The main finding of these researchers is that the oligomerization potential of SMN and its disease-causing variants (usually in complex with the protein Gemin 2 or "G2") mostly correlates with phenotype severity. In humans, this is correlated with the Type of SMA (I/0 for severe disease, ranging to IV for a milder form), and in fruit flies and yeast, it is correlated with viability and, in some cases, animal behavior. The results are extended through the creation of a model that purports to show how higher-order SMN oligomers can form.


      The experiments appear to have been carried out competently. There is a virtual mountain of data presented in this paper, and, for the most part, they are summarized in a digestible fashion. The effort to correlate the biophysical solution data with observable phenotypes in human patients or genetically modified organisms is laudable, and it is done in a thoughtful fashion. The authors' structural intuition and savvy enables the generation of testable models that are explored in the paper. A plausible model for higher-order oligomers is presented.


      The most serious weakness of the paper is that the data cannot support the conclusion stated in the title, i.e. that multimerization of SMN is necessary for organismic viability. Instead, the data support an already-stated, decades-old conclusion (see their reference 21): that multimerization correlates with disease (viability). Even if the reader takes into account the new information about a multimerization interface that is separate from the dimerization one, the advance seems incremental.

      The large amount of data leads to numerous difficulties for the reader in the text:

      1) Complex biophysical measurements, due to space, are usually summarized by one or two words in tabular format.

      2) When these measurements are shown, there is no visual context for the reader to assess the pre-digested conclusions that are included in the figures. For example, all SEC-MALS data show a conclusion ("Tetramer-Octamer"), but there is no visual cue for the reader to know what the theoretical masses for these species are (so that the reader may draw an independent conclusion).

      In some cases, the conclusions reached in the paper are not clearly supported by the data or are self-contradictory. An example is the discussion of the residue H273 (human numbering). In Fig. 4B, the mutation H273R is said to have a wild-type "Oligomer Status". But in Fig. 5B, it is "Dimer-Tetramer+". The text says that H273R is "only partially impaired" in forming oligomers; the authors apparently mean the data presented in Fig. 5B but refer to the contradictory result in Fig. 4B. Another example centers on the discussion of the putative "dominant-negative" effect of some human missense mutations. But they do not point to any human data that support this contention (SMA-associated missense mutations are usually discovered in mixed heterozygotes have a deletion in the other copy of the Smn gene), but they cite data that suggest a more nuanced position regarding negative dominance would be appropriate.

      Finally, the paper suffers throughout from a lack of precision of language that undercuts its conclusions at numerous points. They continually rely on qualitative statements rather than hard, statistically rigorous facts, e.g. "more intimate," "a bit of a sequence outlier," "very modest."

    1. Reviewer #3 (Public Review):

      This study investigates the temporal orientation abilities of cerebellar degeneration and control subjects during an orientation discrimination task of visual stimuli with showed a contrast near threshold. Participants were queried to express their discrimination decision with a response only after a random delay following target offset, which decreases the motor preparation component of the task in the interval-based condition. CD subjects showed similar visual discrimination performance to controls when cued by a rhythmic set of stimuli but showed no benefit when the target interval was presented aperiodically. The authors interpret these findings as evidence supporting the notion that the cerebellum plays a role in interval based attentional orienting to proactively modulate perception. This is an elegantly simple experiment providing a novel observation in the field.

    1. Reviewer #3 (Public Review):

      The authors use a synthetic light-controlled transcription factor (GAVPO) to test a model of bistable gene expression that is hypothesized to originate from positive feedback via local histone modifications by trans-activator recruitment of CBP/p300 to facilitate open chromatin, which facilitates GAVPO binding, etc... Their proposed model for the origin of bistability is important because it should apply to any trans-activator that recruits CBP/p300 to modify chromatin and active gene expression. The authors show that periodic modulation of light reduces the bimodal distribution at intermediate light-intensity levels to a unimodal distribution. This is an elegant demonstration of how GAVPO and different temporal patterns of light can reduce cell-to-cell variability in gene expression, if needed.


      The authors generate an impressive amount of single-cell data of gene expression and chromatin state (flow cytometry, single-cell sequencing, live-cell MS2-tagging) at different intensity levels. The periodic modulation of GAVPO activity by light is a practical demonstration of how to sculpt the gene expression output in useful ways. This may be a very useful tool for future biologists.


      The proposed model for bistability is not convincingly tested or supported by the existing data. Each reporter should exhibit a bistable response because the positive feedback is localized to the promoter via cis-effects on gene expression by local chromatin state/GAVPO binding. The authors show a bimodal distribution of gene expression in a population of cells, which is consistent with a bistable response in a single reporter gene. However, their strain has 9 independent reporters integrated into the genome. Thus, I would expect to see up to 10 peaks, not 2 peaks. Moreover, the mathematical model used to validate their observations does not model the total expression from 9 independent promoters, which is a critical omission given the cis-nature of the positive feedback loop. The fact that these 9 promoters generate 2 peaks at intermediate light intensity suggests that the GAVPO bistability likely originates from a trans-effect, i.e., either all 9 promoters are OFF or all 9 promoters are ON, not a cis-effect.

    1. Reviewer #3 (Public Review):

      In this report the authors characterize a mechanism that plays a role in inducing the rhythmic depolarizations that are observed in identified neurons that are part of the feeding CPG in Aplysia. The neurons studied (B63 neurons) are of interest because previous work has established that they play an important role in triggering cycles of motor activity. Further, previous work from this group has demonstrated that activity in the B63 neurons can be modified by operant conditioning.

      The authors present this study as though previous work had established that plateau potentials generated in the B63 neurons play an important role in driving network activity. For example, in line 102 they state "This essential role played by B63 is partly mediated by a bistable membrane property, which allows the sudden switching of the neuron's resting membrane potential to a depolarized plateau..." To support this statement, they reference Susswein et al. 2002, which does not support this statement. In the Susswein et al. study it is the B31/32 neurons that are modeled as having plateau properties.

      If previous work has not established the role of the B63 plateau potentials, the only data that speak to this issue are presumably in the current report. In this study the authors do provide data that indicate that the B63 neurons generate low amplitude oscillations that are not likely to depend on input from the electrically coupled neurons studied (notably B31). The authors also show that in some instances, these depolarizations do trigger plateau potentials in B63. It is, however, not clear that the B63 generated plateau potentials are then responsible for triggering network activity (e.g., as opposed to a situation where depolarizing input from B63 triggers plateau potentials in B31/32 and the depolarization in B31/32 drives the rest of the feeding circuit). For example, in Figs. 6A and Supplemental Fig. 4A it does not appear that the plateau depolarization in B63 is being transmitted to other electrically coupled neurons to any large extent.

      A clarification of this issue is important because it potentially impacts thinking concerning how 'decision making' is occurring. If decision making means induction of a motor program and this does not occur unless the depolarization in B63 is transmitted to B31/32, the process is more complicated than what the manuscript currently suggests.

      The title is misleading since there are no studies of behavior in this report.

      In part, interest in the mechanisms that drive spontaneous oscillatory activity in the B63 neurons stems from the overall context of this work. Namely the authors have previously established that oscillatory activity can be modified through associative learning. In the Sieling et al. 2014 study they demonstrate that two aspects of plasticity are accounted for by changes in synaptic properties and an effect on a leak current. For readers trying to understand this body of work as a whole, the Discussion should more clearly indicated how the results of the present study integrate with these previous findings.

    1. Reviewer #3 (Public Review):

      Some Gram-negative bacteria synthesize acyl-homoserine lactone molecules, which are secreted into the environment and then transported into nearby bacteria, where they are detected by receptors. Different species make acyl-homoserine lactones that differ in chain length and oxidation state at the C-3 position. The manuscript by Wellington et al. reports an elegant and compelling investigation of the specificity determinants involved in quorum sensing, using a combination of bioinformatics and experimental approaches.

      Over the course of evolution, if an amino acid change occurs in one protein, then a compensating change can occur in a partner protein to restore/retain a functional interaction between the two. Analyses of evolutionarily covarying positions between two interacting proteins, or within a single protein, have long been used to identify positions that directly interact. Wellington et al. applied the same approach to two protein families (the synthases and receptors for acyl-homoserine lactones) to identify positions that are connected not by direct physical interaction between the two proteins but rather by interaction with the same acyl-homoserine lactone. The covariation analysis was made possible by the fortuitous case (and reasonable assumption) that genes encoding partner synthases and receptors are located close to one another within bacterial genomes.

      The covarying residues turn out to be in the active site of the synthase and the binding site of the receptor, in positions that directly interact with the acyl-homoserine lactone. The authors made a variety of single amino acid substitutions at positions with high covariation scores in the Pseudomonas aeruginosa LasI synthase and LasR receptor proteins. The mutant proteins exhibited altered synthetic and detection specificities for acyl-homoserine lactones. Altering three residues simultaneously resulted in substantial changes in specificity.

      This paper constitutes a proof of principle for an approach that could be used to investigate other families of proteins connected by interactions with small molecules (e.g. metabolic pathways). Furthermore, it suggests a path toward rational engineering of quorum sensing systems for synthetic biology, as well as specificity prediction for uncharacterized quorum sensing pathways based simply on the primary amino acid sequences of the synthase and receptor proteins.

    1. Reviewer #3 (Public Review):

      The manuscript titled "The Shu complex prevents mutagenesis and cytotoxicity of single-strand specific alkylation lesions" investigates the biological function of the Shu complex in S. cerevisiae. The Shu complex, containing a DNA binding module comprised of the Csm2-Psy3 heterodimer, is conserved from budding yeast to man, and contributes to the defense against DNA damage caused by DNA alkylation. DNA alkylation occurs due to spontaneous reactions with metabolites and can be greatly increased by exogenous exposure to DNA alkylating agents. Therefore, it is an important question for how the Shu complex acts to detect and direct repair of alkylation damage. It has been well established that loss of the Shu complex sensitizes cells to alkylation damage, but the mechanism by which this complex locates sites of DNA damage and directs repair is not fully understood. This paper measures the methylation-induced mutation spectrum and uses genetic interactions to argue that the Shu complex may be involved in detecting and directing error-free repair of 3-methyl cytosine. This is a plausible hypothesis based on the body of previous work, however the evidence that Csm2-Psy3 directly detects 3-methyl cytosine sites is indirect. It would be highly significant if this complex recognizes many different structures, but future structural information is needed to understand how this could be possible.

      The strengths of the paper are in the use of whole genome sequencing to map mutation type and location in different genetic backgrounds and in the systematic testing for genetic interactions between csm2 and other DNA repair factors. It appears that the mutation spectra are very similar in the presence and absence of csm2, which suggests a broad role of the Shu complex in the cellular response to MMS.

      The impact of the work is that it could help to explain the cellular program for protection against DNA alkylating agents in budding yeast which has been a very valuable model eukaryotic organism, and raise new questions about how DNA alkylation repair pathways might function in humans that differ from yeast in important features such as in the presence of a direct repair pathway performed by ALKBH2 and ALKBH3.

    1. Reviewer #3 (Public Review):

      The authors tackle an interesting question - whether the dentate gyrus is a locus of pathology in Scn1a+/- mice and uncover a strong phenotype - the granule cells of the dentate gyrus are over-activated and the EC to dentate pathway is prone to seizure genesis. In the discussion, they suggest that their results support the idea that the DG may be a common locus to several different types of epilepsy... an attractive hypothesis! There are several strengths of the paper. The team has done a nice job of presenting 'ground-truth' data that their measurements of dF/F across a large population of granule cells correlates with action potentials in these cells. As the authors point out, this is especially important when working in disease models in which the dF/F-action potential relationship may be altered. Throughout, the authors were also careful about considering the limitations of their various techniques and analyze the data in several ways to account for possible artifacts (e.g. ensuring that differences in activation are not arising because of slicing and consideration of kindling in later in vivo seizure threshold experiments). The experiments were well designed and appropriately interpreted.

      One of most intriguing results of the work is that PV interneurons in the DG of Scn1a+/- show only very minor impairments in young adult animals (they show more spike accommodation than in control animals). Rather, it seems that the GCs receive enhanced excitation from the entorhinal cortex. They perform a set of pharmacological experiments to prove that PV interneurons (and more generally inhibition) do not account for the difference in granule cell activation - however, here it would be useful to see the data summarized more consistently. It is difficult to interpret the pharmacological results (both of which are presented as changes in dF/F0) with respect to the initial findings of the manuscript (presented as estimated activation across the entire population). A beautiful aspect of this work is that it goes from cells to circuits to intact brain (in vivo). They nicely show that the heightened excitation from the EC to the DG is sufficient to drive seizures in the Scn1a+/- mice, and finally that since PVs are intact, they can be harnessed to balance out the over activation of GC via optogenetic stimulation of PVs.

    1. Reviewer #3 (Public Review):

      The manuscript endeavors to explain the mechanism of action of a Gram-negative bacterial outer membrane (OM) TonB-dependent transporter (TBDT), that acquires metabolites (in this case vitamin B12) from the external environment. The authors use electron paramagnetic resonance spectroscopy to monitor the proximity of different parts of OM protein to one another during the binding of B12. Their data show that different conformations of the target protein occur during the binding of B12.

    1. Reviewer #3 (Public Review):

      Inamdar et al. used biochemical and microscopy assays to investigate the role of I-BAR domain host proteins on HIV-1 assembly and release from HEK 293T and Jurkat cells. They show that siRNA knockdown of IRSp53, but not a similar I-BAR domain protein IRTKS, inhibits HIV-1 particle release from 293T cells after transfection of the HIV-1 provirus or HIV-1 Gag in cells. The authors then show that HIV-1 Gag associates with IRSp53 in the host cell membrane and cytoplasm, using biochemical assays and super resolution microscopy. In addition, IRSp53 is incorporated into HIV-1 particles along with other previously identified host proteins. Then using in vitro-derived membrane vesicles ("giant unilamellar vesicles" or GUVs), the authors indicate that HIV-1 Gag can associate with IRSp53, particularly on highly curved structures.

      The conclusions are largely supported data, with the virology and biochemical results being particularly strong, but the mechanistic studies in GUVs appear somewhat preliminary and are not entirely clear. The GUV experiments would benefit from better quantification of measurements and manipulation to simulate actual cellular scenarios. In addition, while it is appreciated that the HEK 293T cell line is convenient for biochemical and imaging studies, they are not biologically relevant HIV-1 target cells. While the authors present examples of reproducibility of their results in a CD4+ T cell line, these data are buried in the supplemental figures, whilst it would have been better to highlight them and perhaps include primary CD4+ T cells.

      1) Immortalized cell lines do not always recapitulate primary cells. It is unclear what the role of IRSp53 is in the membrane curvature of CD4+ T cells and whether expression levels and localization are consistent with Jurkat T cells.

      2) Description of some of the microscopy measurements could be improved. In lines 204-206 of the text and Figure S5, it is unclear how the localization of precision was determined to be approximately 16 nm for PALM-STORM. In Figure 4b, it is understood from the text (lines 252-256) that the red bars denote the Mander's coefficient for colocalization of the GFP-tagged proteins with Gag-mCherry (presumably the average of multiple experiments with standard deviations or errors of the mean, although this is not stated in the figure legend), it is unclear what the green bars are showing. Also, the histograms for IRSp53 and IRTKS colocalized with Gag look similar in Figure S10, suggesting that they are not different in Jurkat cells, but this is not addressed.

      3) GUVs are first referenced on page 7 after description of Figure 2, the significance of which is confusing to the reader. However, the actual experimental data are described on pages 12-13 and Figures 5 and S11. A better description of these structures would be warranted for an audience that is unfamiliar with them. In addition, the biologic concentrations of I-BAR proteins at cell membranes are not provided and it is unclear what conditions used in Figures 5 and S11 represent a "normal CD4+ T cell" situation. It appears that the advantage of this in vitro system is that different factors can be provided or removed to simulate different cellular scenarios. For example, relatively low IRSp53 concentrations may simulate siRNA knockdown experiments in Figure 1, which could recapitulate those results that less viral particles are released from the membrane. In addition, the authors state that HIV-1 Gag preferentially colocalizes with IRSp53 as the tips of the GUV tubular structures (Figure 5b,c), but this is not actually shown or quantified. Similar quantification as shown in Figure 1e could be performed to strengthen this argument.

    1. Reviewer #3 (Public Review):

      Gentile, A. et al. generated snai1b mutant zebrafish embryos and showed that loss of Snai1b led to two mutant phenotypes in the heart: i) hearts with clear looping defects, ii) hearts without looping defects that displayed abnormal cardiomyocyte (CM) extrusion. The authors focused on the second class of mutants and found that loss of Snai1b led to reduction of N-cadherin at cell junctions and basal accumulation of phosphorylated myosin light chain and the α-18 epitope of α-catenin, indicative of mechanical activation. Bulk RNA-sequencing of isolated hearts revealed an upregulation of intermediate filament (IF) genes in Snai1b mutants, and of particular interest, the authors identified upregulation of the muscle-specific IF gene desmin b. Immunofluorescent imaging revealed that Desmin was not only upregulated in Snai1b mutants, but mis-localized away from cell junctions and accumulated at the basal side of extruding cells along with actomyosin machinery. Accordingly, CM-specific overexpression of Desmin was sufficient to promote cell extrusion.

      The presented work is particularly interesting because it identifies a new role for the Snai1b transcription factor in maintaining proper tissue structure, independent of its typical function in regulating epithelial to mesenchymal transition (EMT). Overall, the experiments were well designed and controlled, and the data is clearly and logically presented. However, some of the findings could be explained by alternative hypotheses and other interesting aspects of the data were left unexplored.

      One hypothesis that was not sufficiently discussed is that loss of Snai1b may prevent cardiomyocytes from undergoing the EMT that is necessary for normal delamination and trabeculation, and thus cells are instead extruded away from the lumen to prevent overcrowding in the developing myocardium. In fact, the authors present evidence that EMT is blocked and acknowledge that extrusion is a known mechanism for preventing overcrowding. It would be interesting to see whether extrusion away from the lumen also occurs if EMT is blocked through other means.

      The authors show that extruding cells do not seem to be dead or dying, and that a small number of CMs do extrude in wild type embryos. This raises the intriguing possibility that some amount of CM extrusion is necessary for normal development and that these cells may give rise to epicardial or other cell types. Live-imaging and lineage-tracing studies would inform whether the extrusion observed in mutant embryos is an enhancement of a normal morphogenetic process or an additional abnormal response to loss of Snai1 function.

      One particularly interesting observation that was left unexplored was the identification of a second class of Snai1b mutants with defective heart looping. It isn't clear whether these embryos also display enhanced CM extrusion, or if there are other clearly aberrant cell behaviors. Furthermore, it would be very interesting to know whether there is any evidence that the defective looping is due to the same changes in cytoskeletal gene expression and protein organization observed in the class of Snai1b mutants that were detailed throughout the manuscript.

      The authors suggest that Snai1b regulates Desmin in two ways: 1) overall expression levels, and 2) post-translationally to control its localization at cell junctions. Although the first claim is sufficiently supported, the second claim lacks experimental evidence. An alternative explanation is that overexpression of Desmin in response to loss of Snai1b leads to mislocalization independent of an interaction with Snai1b. This point could be clarified by examining Desmin localization in the desmb overexpression system. In addition, assaying for co-IP of Snai1b and Desmin could demonstrate a direct interaction between the two and better support a role for Snai1 in regulating post-translational localization of Desmin.

      Although the authors convincingly show that Desmin accumulates with other contractile machinery at the basal side of extruding CMs in Snai1b muntants, additional evidence is needed to support a causal link between basal Desmin accumulation and extrusion. For instance, if knockdown or inhibition of Desmin prevents extrusion in the Snai1b mutants, the causal relationship would be much clearer.

    1. Reviewer #3 (Public Review):

      This manuscript investigates the structure, electrophysiological and biological functions of a novel mechanosensitive channel from the parasitic protist Trypanosoma cruzi. The channel was identified bioinformatically as being significantly related in sequence to known mechanosensitive channels of the McsS superfamily. Studies on channel proteins in pathogenic protists such as trypanosomes are limited, and this investigation focuses on a previously unstudied mechanosensitive channel which is of potential interest to parasite biology, because trypanosomes are subjected to various mechanical stress forces during their life cycles.

      Analysis of the sequence of the TcMscS protein by bioinformatics and structural prediction concludes that it is relatively divergent from previously studied members of the family from other microorganisms. Of particular interest, the divergent C-terminal domain contains a proposed novel cytoplasmic gate that could filter solutes on the cytosolic side. These observations are predictive rather than data driven. The recombinant protein was expressed in E. coli giant spheroplasts and studied by cell-detached patch clamp electrophysiology. The authors have clearly demonstrated that the channel is activated by pressure steps and fluxes K+, Cl-, Ca2+, and they speculate that it may be able to transport osmolytes such as amino acids. Other well supported conclusions are that the channel is located primarily in the contractive vacuole complex (CVC) in insect vector stage epimastigotes, an organelle that expels water from the parasite, but it occupies a wider range of subcellular sites in infective metacyclic and bloodstream trypomastigotes, and it localizes primarily to the plasma membrane of amastigotes. Thus, the channel changes its location during the life cycle. A MscS knockout line was generated and demonstrably shown to have impaired cell volume regulatory responses when subjected to changes in extracellular osmolarity, decreased motility, reduced ability to transform into infective stages such as metacyclic trypomastigotes, amastigotes, and bloodstream trypomastigotes, and altered Ca2+ homeostasis. Hence, the biological impacts of this channel are broad and significant.

      A strength of the manuscript is that it is well-executed study and examines many aspects of channel function and biology. Precisely how the channel mediates the biological functions is less clear and will require future investigations. For instance, whether the channel has functions related to shear stresses encountered by the parasites when they enter host cells or extravasate through vasculature is currently rather speculative, albeit of considerable potential interest. How the channel mediates volume changes or affects motility is also unclear. In addition, the manuscript would benefit from some editing to make several points or interpretations clearer to the readers.

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    1. Reviewer #3 (Public Review):

      The sodium-coupled biogenic transporters DAT, NET and SERT, terminate the synaptic actions of dopamine, norepinephrine and serotonin, respectively. They belong to the family of Neurotransmitter:sodium:symporters. These transporters have very similar sequences and this is reflected at the structural level as judged by similarity of the crystal structures of the outward-facing conformations DAT and SERT. However, earlier functional studies indicated that transport by SERT is electroneutral because the charges sodium ions and substrate moving into the cell are compensated by the outward movement of potassium ions (or protons) to complete the transport cycle. On the other hand, DAT and NET are electrogenic. Moreover, potassium ions are not extruded by these transporters and the Authors set out to investigate if the electrogenicity is related to difference in potassium handling between SERT and the two other biogenic transporters. This was done by analyzing the role of intracellular cations and voltage on substrate transport by the three biogenic amine transporters. This was achieved by the simultaneous recording of uptake of the fluorescent substrate APP+ and the current induced by this process under voltage-clamp conditions by single HEK293 cells expressing the transporters. The Authors found that even though uptake by NET and DAT did not require internal potassium, these transporters could actually interact with internal potassium as judged by the voltage dependence of the so-called peak current. This voltage dependence was very steep in the absence of both sodium and potassium. However, in the presence of either cation this voltage dependence became less steep when either of these cations was present in the internal milieu, indicating that not only sodium but also potassium could bind from the inside. The same result was obtained with SERT. However, uptake by SERT was found to be much less dependent on the membrane voltage than that by DAT and NET and was stimulated by internal potassium, consistent with the proposed electroneutrality of the former. The observations indicate that the structural similarity of the three biogenic amine transporters is also reflected in their ability to bind potassium, even though this cation can translocate to the outside only in SERT.


      Development of a sophisticated technique to interrogate the mechanism of sodium coupled biogenic amine transport in single cells. Rigorous analysis of the data. Conclusions supported by the data. The methodology can be used to obtain novel insights into the mechanism of other transporters.


      The presentation could be made more "user friendly" by explaining in more detail what is happening as we go through the data. For instance, peak and steady state currents are shown already in Figure 1, but an (too brief) explanation is only provided when describing Figure 5. A schematic in the first part of the Results would be useful. Some information of on the structural background should be provided as well as a full description of the transport cycle, namely the number of sodium ions translocated per cycle and the argument why chloride remains bound to the transporter throughout the cycle. The control that in contrast to potassium, lithium is inert should be performed not only for DAT, but also for the two other transporters.

    1. Reviewer #3 (Public Review):

      The authors developed an in vivo model of EBV's contribution to RA that recapitulates aspects of human disease. They examined the role of age-associated B cells and find that they are critical mediators of the viral-enhancement of arthritis.

      The manuscript is written in a well-structured form that facilitates the reading and following the incremental experimental setups. The manuscript is appropriate for publication after revisions.

      Some of the statistical measures did not show significant values while the author based several statements as if there is a difference (they rather used phrases as increased/fold change). Whether this is strong enough to support their statements is not clear.

      Overall, this report provides important insights regarding the association between latency, age-associated B cells, and the enhancement of RA in a mouse model. If these insights are translatable to RA immunology in humans is to be further investigated.

    1. Reviewer #3 (Public Review):

      Proper sealing of the blood brain barrier (BBB) is essential for viability in many animals, including humans and Drosophila. In Li et al., the authors used Drosophila as a simple genetic system to define the signaling pathways that control BBB formation and maintenance. In Drosophila, the BBB is composed of a thin epithelial sheath of subperineural glia (SPG) that are connected by septate junctions. Previously, the authors found that the G protein-coupled receptor Moody is essential for BBB formation during embryogenesis, but the downstream signaling pathways that facilitate septate junction assembly were not known. Here, they performed a series of genetic screens and epistasis experiments to uncover that Moody and PKA antagonistic signaling drives BBB assembly and expansion throughout organismal development. In the present study, they show that loss of PKA signaling components results in a leaky BBB both during development, and during adulthood. They further show that these functions of PKA are dependent on downstream suppression of Rho and changes in cytoskeletal dynamics. Interestingly, overexpression of PKA also causes BBB permeability, indicating that PKA signaling levels must be tightly regulated for BBB integrity. The authors then use serial section TEM to visualize the intact SPG sheath for the first time at ultrastructural resolution, and show that overexpression of PKA results in an enlarged yet patchy septate junction, accounting for the leakiness. In sum, the authors show that the combined signaling of Moody (apically located) and PKA (basally located) shapes the cytoskeleton to drive efficient assembly and maintenance of septate junctions, and thus, the BBB.

      The conclusions of this paper are mostly well supported by the data, but the study would be improved by some expanded analyses and descriptions of statistical assessment.

    1. Reviewer #3 (Public Review):

      The authors have re-sequenced 310 quinoa accessions and carried out field phenotyping of the same set of accessions for two years in order to characterize genetic diversity and analyze the genetic basis of agronomically important traits.

      The main strength of the manuscript is that the authors have carefully characterized more than 300 quinoa accessions, achieving a sufficiently large population size for GWAS analysis with good statistical power. It is especially promising that the phenotypes all show high heritability. This indicates that the field phenotyping was of high quality and provides a good starting point for discovering relevant marker-trait associations. In addition, the authors provide convincing evidence for distinct population characteristics of highland and lowland quinoa, adding additional information compared to previous work (Maughan, 2012).

      The weak points are related to the genotype data and the conclusions drawn based on the GWAS analysis.

      1) An important issue is related to the relatively low depth of coverage (4-10x) that was used for re-sequencing. Across the accessions, there is a pronounced negative correlation between the mean sequencing depth and the heterozygosity level, indicating that heterozygotes are overcalled in individuals with low coverage. This also results in heterozygosity levels that are generally higher than expected for what is assumed to be mainly homozygous inbred lines.

      2) Another potential issue concerns SNPs called in repetitive regions. Among the significant GWAS SNPs identified, a very large proportion appears to be found in intergenic regions. While this does not rule out that some of them are genuinely important associations, it does suggest a potentially high level of noise in the GWAS results. In addition to the filtering already imposed, which includes a filter for mapping quality, the SNPs called in intergenic regions with unusually high coverage could be more closely examined to determine the extent of the issue. Masking repetitive genomic regions using RepeatMasker or similar programs could be useful.

      3) When the authors discuss their GWAS results, they frequently focus on cherry-picked candidate genes, although, in several cases, the top SNPs in the region in question are not found within these candidates. A more broad focus on all genes within the LD blocks, while still mentioning the candidate genes, would be more informative.

      4) The manuscript includes statements that a particular genotype "results in" some phenotypic outcome, although no causal relationship has been demonstrated. In general, there is a tendency to draw too strong conclusions based on the GWAS results.

      5) As this is primarily a resource paper, the authors should make the complete genotype and phenotype data as well as the layout of the field trials available. It would not be possible to reproduce the GWAS analysis based on the data included with the current version. They should also clarify how the quinoa accessions described will be made accessible to the community and provide all scripts used for data analysis through GitHub or a similar repository.

    1. Reviewer #3 (Public Review):

      Cole and co-authors report the development of a novel immunofluorescence technique, where targets of interest are analysed over iterative cycles of staining-imaging-elution(stripping). This method allows for the multiplexed analysis of protein targets, well beyond the usual constraints of such technique (limited by availability of filters and non-overlapping wavelengths of fluorophores). The authors also present several applications of such technique, highlighting how the advantage of being able to record additional parameters (such as cell morphology) can be an advantage over more high-throughput methods such as spatial-resolved transcriptomics.

      The technique has been carefully tested. Staining for the same markers after several rounds of stripping/reprobing shows high concordance, indicating that the iterative treatment and staining of the same tissue section is not altering the detection of protein markers.

      The authors tested staining with a total of 18 antibodies, and suggest that this number can be increased arbitrarily, as the number of iterations is not limited. Further, they suggest that this technique can be applied to virtually any tissue. It is quite possible that this technique can be readily applied to any other tissue, as the only constraint seem to be the robustness of antibodies. The authors may include the suggestion that previous success of immunofluorescence on a particular tissue type could be a good indication for the success of the iterative staining.

      The proposed 4i method is quite interesting, has great potential and is likely to be of very wide interest.

    1. Reviewer #3 (Public Review):

      This manuscript seeks to provide mechanistic insight into the role of GJA1-20k in mitochondrial changes that protect against ischemia-reperfusion damage. In previous studies, this group has shown that GJA1-20k protein increases in response to ischemic stress, localizes to mitochondria, promotes mitochondrial biogenesis, and mimics ischemic preconditioning protection in the heart. These changes did not coincide with changes in mitochondrial dynamics proteins, but the increase in GJA1-20k provides protection through an unknown mechanism. This makes it a potentially attractive therapeutic candidate for protection against ischemia.

      The evidence in this manuscript shows that mitochondrial size is affected by GJA1-20k, as over-expression of this fragment reduced mitochondrial area. The authors argue that this change in morphology is independent of Drp1 activity, and actin dynamics drive mitochondrial division. These ultrastructural changes coincide with cytoprotective effects during reperfusion following ischemic events by limiting ROS production.


      The data on ultrastructural changes is convincing, and GJA1-20k induced a decrease in mitochondrial size. The imaging looks good and quantification is helpful in the evaluating the impact of these changes.

      To complement the use of proposed Drp1 inhibitors, the authors use genetic knock-down (KD) of Drp1, and the KD looks robust. Still see some Drp1 colocalization on the mitochondria in the KD, but the levels are diminished.

      The decrease in ROS when HEK cells were treated with H2O2 is convincing. And this coincides with the decreased respiration capacity observed in the Seahorse analysis. This provides some mechanistic insight about a specific change in mitochondrial function that contributes to the protective effects observed.


      With the introduction of GJA1-20k, there is clearly a difference in mitochondrial size, and total mitochondrial content appears unaltered (i.e. Tom20 does not increase). Previously it was suggested that mitochondrial biogenesis was increased with increased levels of GJA1-20k. Is this a difference in the cellular model (HEK) and do the changes in cell culture accurately recapitulate the changes seen in animals? Having more mitochondrial mass despite decrease in the avg. size of these organelles may represent an important difference.

      Mdivi-1 is not a selective Drp1 inhibitor. It is a Complex I inhibitor, leading to unintended changes in mitochondrial dynamics in response to ETC stress. Rather than Mdivi-1, a dominant negative Drp1 mutant K38A could be overexpressed to see whether this prevents GJA1-20k-mediated fission. If it still goes through, then I agree that Drp1 is not involved at all.

      For the kinetics studies (see Fig 4), I think it is important to measure the timing of the actin recruitment and eventual fission when Drp1 is knocked down and/or when a DN mutant (K38A) is involved. Again, I do not trust the chemical inhibitor (Mdivi-1) data since this does not inhibit Drp1 activity.

      The assessment of the impact of ischemic stress with the heterozygous animal (M213L/WT) is hard to interpret. How reduced is the expression of GJA1-20k in these animals and how is mitochondrial function impacted based on Seahorse analysis? The mitochondrial morphology is not altered in these animals, so would mitochondrial function be largely unchanged as well? It is not clear how much GJA1-20k is needed to observe changes in mitochondrial shape and function, and comparisons with the homozygous mutant (M213L/M213L) are not the same, making it difficult to resolve the interpretation of these data.

      It is still unclear to me how GJA1-20k is affecting mitochondrial size and function. Based on previous papers, this peptide localizes to the surface of mitochondria, but it is not clear how, or whether, it directly facilitates actin recruitment. The interplay with the endoplasmic reticulum (ER), which can nucleate actin at sites of mitochondrial fission, was not examined. If actin is driving membrane remodeling, is it mediated by ER crossover at these sites?

    1. Reviewer #3 (Public Review):

      Nguyen Lam Vuong et al performed a nested case control study of a multisite, multicountry prospective dengue study (IDAMS) to identify early biomarkers at day 1-3 of illness onset that predicts for severe dengue of ten biomarkers. Ten biomarkers from the inflammatory, immune or vascular pathways (VCAM-1,SDC-1, Ang-2, IL-8, IP-10, IL-1RA, sCD163, sTREM-1, ferritin, CRP) were chosen based on prior literature and understanding of dengue pathogenesis in severe disease. The biomarkers were measured at two time points: at enrollment (illness day 1-3) and after recovery(day 10-31 ). They find moderate-to strong positive correlations for some markers, particular IP-10 and IL-1RA, and IP-10 and VCAM-1, ( Spearman's rank correlation coefficients above 0.6). Interestingly, in their single modal analysis, they also find differences in biomarkers levels in children compared to adults, Associations between SDC-1 and IL-8 and the S/MD endpoint were stronger in adults than children, while the effects of IL-1RA and ferritin were stronger in children than adults. When global analysis was performed, only SDC-1 and IL-1RA were the most stable relative to the single models for both children and adults. And the the differences of the associations between children and adults were more marked, particularly for Ang-2, IL-8 and ferritin. When the biomarkers were combined, for children, the best subset that showed the clearest association with S/MD was the combination of the six markers IL-1RA, Ang-2, IL-8, ferritin, IP-10, and SDC-1 with an AIC of 465.9. For adults, the best subset included the seven markers SDC-1, IL-8, ferritin, sTREM-1, IL-1RA, IP-10, and sCD163 This manuscript certainly provides useful insight into the biomarkers that are involved in the early phase of dengue before onset of vascular leakage or severe dengue which is valuable as most previous publications mainly focused on measurement of these markers after onset of severe disease which was often too late for meaningful interpretation of the disease biology or of limited clinical utility. The conclusions of this paper are mostly well supported by data, but some aspects of study and data analysis need to be clarified in order to improve understanding of the statistical methodology and readability.

      Major Strengths:

      • More than 7000 participants ( children and adults) in eight countries across Asia and Latin America were enrolled in the IDAMS study
      • Prospective and systematic blood sampling starting from day 1 of illness onset
      • Cases were laboratory confirmed via PCR or NS1 testing
      • Cases and control were fairly well- matched
      • Strong rationale for selection of host biomarkers


      • Three quarter of cases from one country
      • Serotype-1 biased

      Specific comments to address:

      1) For general ease of readership, it would greatly help if the authors can explain the choice of the statistical method used in the data analysis and perhaps briefly explain the model and how AIC should be interpreted in the main rather than the supplementary text).

      2) While this reviewer understands that the authors want to focus on host immune and inflammatory biomarkers but it would be helpful if NS1 and viremia data are also shown ( at least in supplementary data) if these have been found not to correlate with disease severity.

      3) It is Interesting to note that some biomarkers ( particularly the vascular markers) in severe group do not return to the same baseline as mild cases at convalescence even after >20 days. Whether such individuals already are at higher inflammatory state at baseline (pre-infection) as a result of underlying co-morbidities such as obesity or diabetes? Table 1 did not provide such information but would be interesting to show if there is any difference in health state in the 2 groups especially for obesity.

      4) It is rather confusing that the 2nd paragraph of discussion stated "Balancing model fit, robustness, and parsimony, we suggest the combination of five biomarkers IL-1RA, Ang-2, IL-8, ferritin, and IP-10 for children, and the combination of three biomarkers SDC-1, IL-8, and ferritin for adults to be used in practice."

      But the concluding paragraph went on to state "The best biomarker combination for children includes IL-1RA, Ang-2, IL-8, ferritin, IP-10, and SDC-1; for adults, SDC-1, IL-8, ferritin, sTREM-1, IL-1RA, IP-10, and sCD163 were selected." This should be clarified further.

    1. Reviewer #3 (Public Review):

      In this paper McPherson and Bandres investigated temporal and regional features of spontaneous neural activity in the spinal cord of anesthetized unconscious rats from multi-unit electrophysiological recordings of neuronal activity in the lumbar spinal cord. Spontaneous temporally correlated neural activity in the mammalian central nervous system during unconsciousness is a feature of supraspinal circuits, and recent studies from resting-state fMRI in the spinal cord of non-human primate and in the human spinal cord indicate that spontaneous activity in the absence of sensory stimulus-evoked activity and spontaneous motor output is also a basic property of spinal cord circuits. Here the authors sought to provide more direct evidence of robust and temporally correlated spontaneous neuronal activity in the in vivo mammalian spinal cord by applying correlation-based analyses of activity with single neuron resolution from multi-unit electrophysiological recordings simultaneously in several dorsal and ventral regions of a lumbar spinal cord segment. They successfully demonstrated robust spontaneous activity in these regions, and their correlation analyses of temporal features of this activity, to infer functional connectivity between spontaneously co-active neuronal units, suggests functional connectivity between sensory- and motor-dominant regions of the spinal cord, in addition to intraregional connectivity. This includes finding evidence for mono- and di-synaptic neuronal interactions as well as excitatory and some inhibitory interactions. Evidence is also presented that the spatiotemporal patterns of this spontaneous activity could not be explained theoretically by randomly spiking interconnected neurons, leading the authors to speculate that the spontaneous activity is intrinsic to the spinal cord and may reflect some type of replay of more structured experience-dependent patterns occurring during conscious behavior. The origins and functional significance of this temporally correlated spontaneous activity, however, remain to be determined.

      Strengths of the paper include: (1) Clearly presented descriptions of the authors' procedures for recordings of multi-unit electrophysiological activity with a dual-shank 32 channel microelectrode array positioned at lateral and medial regions of the lumbar hemi-cord. (2) Novel reconstructions of functional connectivity maps, from correlation-based analyses, which enabled some topological features of the activity correlations to be represented from microelectrode array geometry and location within the rat spinal cord. (3) Novel results at the single neuron level potentially indicating spontaneous functional connectivity between sensory and motor regions in the unconscious animal. (4) Appropriate discussion of important caveats associated with technical aspects of their correlation analyses including problems for inferring functional connectivity in the presence of polysynaptic connection pathways and shared synaptic inputs as well as limitations of detecting inhibitory connections via correlation-based approaches. (5) Insightful discussion of possible functions of persistent spontaneous connectivity during unconsciousness in the spinal cord including latent activity in spinal central pattern generators or ongoing activity of circuits involved in maintenance/regulation of physiological processes under anesthesia.

      Weaknesses of the experimental approach and for potential functional interpretations include (1) the need for more elaboration of technical details about how the temporal correlations of neuronal activity was performed, and (2) electrophysiological measurements were confined to a single lumbar spinal segment, so that origins of the spontaneous activity including interactions between spinal and supraspinal regions, interactions between various spinal segments, and contributions of sensory afferent feedback despite anesthesia, could not be established. While attributing the patterns of spontaneous activity found to reflect intrinsic spinal circuit activity, the authors did not fully explore possible contributions of sensory afferent feedback, for example, by employing local deafferentation.

      The results presented in general suggest spontaneous temporally correlated neural activity in the spinal cord during unconsciousness, consistent with the concept that such activity may be a general property of central nervous system circuits.

    1. Reviewer #3 (Public Review):

      Wodeyar et al. suggest a new method for estimating the phase of oscillatory signals in real-time, based on a state-space objective. They test their approach in simulations and data and present evidence for higher accuracy compared to standard methods based on band-pass filtering. While I especially find the possibility of credible intervals highly interesting in this context, the relationship of credible intervals to an amplitude criterion threshold criterion, customary employed by standard approaches should be elucidated more, it's not clear to whether this practically results in very similar outcomes. In addition, it would be good to see clarifications on the underlying data generating process and physiological motivation for the provided simulations. It would increase accessibility of the manuscript, if the text would be more self-contained & more methodological details were included.

    1. Reviewer #3 (Public Review):

      The authors effectively utilized Beta5T-iCre to specifically manipulate beta-catenin expression in TECs and definitively showed that careful control of beta-catenin within TECs is needed for the proper development of TEC microenvironments critical for T cell development.


      1) The methods used allowed the authors to effectively targeted TECs while avoiding extrathymic side effects of manipulating beta-catenin in ways that impacted skin or other tissues leading to abortive development or improper separation of the thymus and parathyroid.

      2) The results showed that beta-catenin GOF specifically in TECs results in thymic dysplasia and loss of thymic T cell development.

      3) The results from the analysis of beta-catenin LOF indicate that beta-catenin in TECs is not essential for the generation of functional TECs that support T cell development but the loss of beta-catenin in TECs results in the reduction in the number of cTECs, which leads to the reduction in the number of thymocytes during the postnatal period.

      4) The results demonstrated that GOF of beta-catenin in TECs results in trans-differentiation of TECs into terminally differentiated keratinocytes.


      The fact that beta5T expression is restricted primarily to cTECs suggests that the models used may not accurately capture the impacts of gain of function and loss of function of beta-catenin to mTECs and the maintenance of the medulla in postnatal mice. Given that beta5T-expressing cells have been shown to give rise to both cTECs and mTECs during fetal development the models may more closely demonstrate the importance of fine-tuning beta-catenin expression during fetal development while missing impacts on postnatal mTECs.

      The authors achieved their aims and the results strongly support their conclusions.

      The work clearly demonstrates the importance of proper regulation of Wnt/beta-catenin signaling in the development and maintenance of TEC microenvironments and should lead to more interest in defining the specific Wnts and Frizzleds that are important in the development and postnatal maintenance of specific TEC subsets. This work will be important in identifying clinical strategies to counteract thymic involution and the subsequent loss of T cell function.

    1. Reviewer #3 (Public Review):

      The manuscript by Xiang and Bartel explores the molecular coupling of poly(A) tail length and translational efficiency (TE) in frog oocytes and various mammalian cell lines. From their experiments they draw several broad conclusions. Firstly, it is that limiting amounts of PABPC in frog oocytes is the basis for coupling between poly(A) tail length and TE. Secondly, in mammalian somatic cell lines PABPC contributes little to TE and transcript with TUT4 and TUT7-mediated uridylation promoting degradation of transcript with short poly(A) tails. Overall, the experimental design is excellent. The conclusions drawn from the frog oocytes are strongly supported by the data provided whereas the cell line studies are more open to interpretation due to the drastic consequences of PABPC depletion.

    1. Reviewer #3 (Public Review):

      Ma et al investigate the effect of racial and ethnic differences in SARS-CoV-2 infection risk on the herd immunity threshold of each group. Using New York City and Long Island as model settings, they construct a race/ethnicity-structured SEIR model. Differential risk between racial and ethnic groups was parameterized by fitting each model to local seroprevalence data stratified demographically. The authors find that when herd immunity is reached, cumulative incidence varies by more than two fold between ethnic groups, at approximately 75% of Hispanics or Latinos and only 30% of non-Hispanic Whites.

      This result was robust to changing assumptions about the source of racial and ethnic disparities. The authors considered differences in disease susceptibility, exposure levels, as well as a census-driven model of assortative mixing. These results show the fundamentally inequitable outcome of achieving herd immunity in an unmitigated epidemic.

      The authors have only considered an unmitigated epidemic, without any social distancing, quarantine, masking, or vaccination. If herd immunity is achieved via one of these methods, particularly vaccination, the disparities may be mitigated somewhat but still exist. This will be an important question for epidemiologists and public health officials to consider throughout the vaccine rollout.

    1. Reviewer #3 (Public Review):

      In this work, Chattaraj and colleagues utilize simulation models to study collective behaviors of molecules with multiple binding sites (multivalency). When the concentrations are low, the molecules do not bind to each other frequently, and they are called free. On the other hand, if the concentrations increase, they start to bind and eventually form a wide network of molecules connected by molecular binding. This transition can be considered as a model for liquid-liquid phase separation. Their major claim is that the solubility product, a simple product of the concentrations of the free molecules, can be used as a proxy to the phase separation threshold (known as the saturation concentration). They observed in various simulation conditions that as the total concentration of molecules increases, the solubility product first increases but eventually converges to a certain value, and the value is consistent over different simulation conditions. The value is the upper limit of the solubility product, after which the molecules start to form a molecular network.

      After establishing the model, they tested systems with different valences. Higher valency leads to reduction of the threshold (and phase separation occurs at lower concentrations). The theory was also valid for systems with non-equal valences (e.g. pentavalent A + trivalent B). They applied their models to a three-component system, and found that the results qualitatively explain the published experimental patterns. Lastly, using off-lattice coarse-grained simulations, they show that the linker flexibility and the spacing of binding sites are important determinants of the threshold, which confirms the findings from other computational and experimental works.

      The authors successfully defend their claim by using different types of simulations, and their methods to crosscheck the physical validity of their models may be useful for other simulation works. For example, the authors checked if increasing the number of molecules and reducing the system size give the same results for equal concentrations. Also, they employed two different methods (so-called FTC and CMC in the manuscript) to determine the threshold concentrations. However, the conclusions are not easily transferable to real biopolymer systems, since it is hard to determine the valences (and binding affinities) of biopolymers such as intrinsically disordered proteins.

    1. Reviewer #3 (Public Review):

      This study sought to identify essential features of ESCRT-III subunits, with a focus on the yeast proteins Vps2 and Vps24, in order to reveal the required features of both subunits. The combined genetic and biochemical studies solidified the model that essential functions of ESCRT-III polymers - spiral formation, lateral association, and binding of Vps4 - are mostly distributed between different subunits (with some redundancy) and can be engineered into a single polypeptide. This study also sheds light on the long-standing and initially surprising finding that ESCRT-dependent budding of HIV does not require CHMP3 (Vps24), presumably because the distribution of distinct functions between different ESCRT-III subunits is not absolute.

      Inspired by earlier studies, the ability of overexpression of one ESCRT-III subunit to compensate for deletion of another subunit was explored using sorting assays. The demonstration of partial rescue inspired a mutagenesis approach that identified three residues that cluster on one face of a helix that enhanced rescue, and therefore confer functionality that in wt is primarily provided in the deleted subunits, which in this case is binding to Snf7. Extension of this analysis by protein engineering further demonstrated that the essential role of recruiting the Vps4 ATPase is normally performed by Vps2 but can be transferred to Vps24 by substitution of residues near the ESCRT-III subunit C-terminus. Similarly, it is shown that sequences that alter the propensity for bending of a helix at a point where open and closed ESCRT-III subunits differ in conformation contributed to the ability of Vps24 to substitute for deletion of Vps2, presumably by conferring the ability to adopt the open, activated conformation as well as the closed conformation.

      I don't have concerns about design or technical aspects of the experimental approach.

    1. Reviewer #3 (Public Review):

      Maltese et al performed 2p imaging of both dSPNs and iSPNs at the same time, while focusing on correlates of forward locomotion. The modulation of dSPN ensembles in response to DA agonism or antagonism was mostly consistent with classic models, although they also observed an 'inverted U-shape' response to D1R agonsists. In addition, they found distinct modulation of dSPN and iSPN ensembles in DA-intact and Parkinsonian mice.

    1. Reviewer #3 (Public Review):

      Mark and colleagues set out to examine the relationship between neuronal targeting and connectivity and the developmental history of neurons. Specifically, the authors examine if, how and to what extent hemilineage identity combined with temporal birth order can explain neuronal connectivity. To this end they use the fly larval nerve cord with its EM-level resolution of connectivity and prior knowledge about hemilineage identity and birth order as a model.

      General comments:

      The manuscript represents a comprehensive, thorough and deep analysis of the system. The descriptive elements are outstanding, and the analysis of how Notch activity correlates with hemilineage targeting is of great interest. While understanding the relationship between connectivity diagrams and developmental history, including the role of Notch signaling, is not new, the scale of the analysis presented here is key because it allows - in principle - the drawing of general conclusions. The main "weakness" of the manuscript is not in the work itself but rather some of the key conclusions drawn from the data which often somewhat beyond what the data alone would support, especially in terms of the developmental mechanisms involved in establishing connectivity. With one partial exception (Notch gain of function experiments), the work essentially represents (very important) correlation analysis between the various parameters. While the authors are of course free to interpret their data as per their own views and biases, they do need to either tone down their, often categorical, statements and soften their conclusions, or perform further analysis to examine whether some of the stronger conclusions they draw are justified.

      Specific comments:

      1) Figure 1; page 3: The authors refer to the "striking" similarity between EM reconstructions and GFP filled clones and yet there are clear differences in some of the clones in the extent and localization of arborization. This may be in part technical but almost certainly also reflects inter individual differences in single neuron morphology. Since EM reconstructions presumably come for, one animal, the use of GFP clones allows the authors to map the degree of variation between clones and it would be interesting for them to show this.

      2) Figures 2 and 4; pages 3-5: Along the same lines as above, the authors make categorical statements about the mapping of arbors to dorsal and ventral regions of the nerve cord and correlate that to hemilineage identity. Again, there is clear mixing in almost all neuroblast lineages, that seems to range from 15-30% as a rough estimate, and perhaps a bit more dorsally than ventrally, which the authors do not comment on (except to say it's "mostly non-overlapping"). This is a pity because they obviously have the tools to do so quantitatively and the information is already there in their data.

      3) The analysis of Notch activity in hemilineages is excellent and very interesting, as is the new tool they develop. However, the analysis lacks loss of Notch function data and where and when Notch signaling is required to segregate the connectivity space (i.e. in neurons or in precursors such as Nbs and GMCs). Is this a binary fate specification mechanism or lateral inhibition among competing neurons? What about Notch activity manipulation in single neurons? If the authors wish to draw strong conclusions about the role of Notch in segregating target space and its relation to hemilineage identity, these experiments are essential. Alternatively, drawing subtler conclusions and acknowledging these caveats would be very welcome.

      4) Figure 7; Page 7: The authors state that 75% of hemilineage neurons correlated by temporal identity are separated by 2 synapses or less, suggesting greater connectivity than expected. How are these data normalized? What is the expected connectivity between neurons that are less related along these two developmental axes?

      5) Figure 8; page 7 and discussion: The authors conclude that the combination between temporal identity and hemilineage identity predicts connectivity beyond what would be predicted by spatial proximity alone. This conclusion is problematic at least two levels. First, practically what really matters for proximity is proximity during the time in development when synapses are forming between neuronal pairs, not proximity at the end in the final pattern. Second, conceptually, opposing spatio-temporal mechanisms with proximity-based bias for connectivity makes no sense because that's exactly what spatio-temporal mechanisms achieve: getting neurons to the same space at the same time so connectivity can happen. At any rate, drawing strong conclusions about where and when neurons meet to form (or not form) synapses requires live imaging and absent that authors should refrain from making such a string statement about what their excellent correlative dataset means.

    1. Reviewer #3 (Public Review):

      In this manuscript Lituma and colleagues investigate a potential role for presynaptic NMDARs at hippocampal mossy fiber (MF) synapses in regulating synaptic transmission. The combined use of electron microscopy, electrophysiology, optogenetics, calcium imaging, and genetic manipulations expertly employed by the authors yields high quality compelling evidence that presynaptic NMDARs can participate in activity dependent short term facilitation of release onto postsynaptic CA3 pyramid and mossy cell targets but not onto inhibitory interneurons. Moreover, presynaptic NMDAR activation is demonstrated to be particularly effective in promoting BDNF release from MF boutons. The investigation is well designed with a clear hypothesis, appropriate methodological considerations, and logical flow yielding results that fully support he authors conclusions. The manuscript fills an important gap in our understanding of MF regulation by unambiguously confirming a functional role for presynaptic NMDARs that were first described anatomically at MF terminals nearly 30 years ago. Combined with a handful of other studies describing presynaptic NMDARs at various central synapses this study expands the role of NMDARs as critical players in synaptic plasticity on both sides of the cleft.

    1. Wh

      After examining one condition which renders an action involuntary (forced actions), Ar. moves on to examine the other condition: ignorance. His main claims in this section are thus: most actions performed in ignorance are involuntary, but can be counter-voluntary to the extent that the agent regrets being the cause of those wrongdoing; an action performed in ignorance is either involuntary or counter-voluntary, whereas an action performed because of general ignorance on the part of the agent is voluntary.

      1110b17-1111a21 What comes about by force...and involve regret.