1,917 Matching Annotations
  1. May 2022
  2. Apr 2022
    1. # Input Input: 123, Output: Input: 121, Output: Input: 111, Output: Input: 123454321, Output: Input 123123, Output: # Instruction Output true if input is a palindrome # Output Input: 123, Output: false Input: 121, Output: true Input: 111, Output: true Input: 123454321, Output: true Input 123123, Output: false

      Example of using GPT-3 for programming

  3. Mar 2022
  4. learn-us-east-1-prod-fleet02-xythos.content.blackboardcdn.com learn-us-east-1-prod-fleet02-xythos.content.blackboardcdn.com
    1. fighting ability was one way that this prayer was answered for some

      fighting and honor and protection

    2. Most bondsmen would have to mask their honor when confronted withaffronts and wait until a potential window of opportunity to seek redress; oth-ers displayed their honor at times by resisting dishonor

      honor by resisting dishonor

    3. the code of honor among bondsmen assumed a “sacredness,”and transgressors would, among other retributions, have “their names cast outas evil from among their brethren, and being subjected to scorn, and perhapspersonal violence.”

      honor

    4. In Africa fighters often exhibited their power on behalf of the enslavedcommunity in the enforcement of honor.

      enforcement

    5. Others commanded respect fortheir demonstrated skill in physical contests such as climbing poles, wrestling,or knocking and kicking. Through these various forms of display, enslavedAfricans and their descendants exclaimed their individual worth and honor.The desire to seek such communal honor was clearly strong

      honor

    6. an enslaved woman could gain honor in the eyes of the communityfor her ability to sing, hypnotize the community with engaging stories, or dancewith grace

      honor for all regardless of gender

    7. black drivers who felt compelled by the honor code not to carry out orderedwhipping if the plantation owner was not immediately present.2

      how far honor goes

    8. loyalty while enslaved

      concept

    9. enslaved showed this respect toone another by hiding and feeding runaways and refusing to betray otherbondsmen

      how remained honorable

    10. honor was loyalty to fellowbondsmen.

      honor

    11. loyalty to peers, personal displays, vengeance, and resistance to dishonor

      .

    12. honor system of Africansand their descendants in North America was most clearly exhibited through

      .

    13. Europeans and their descendants in North America did not always recog-nize this bonded honor code, but southern elites lived by their own reflexivehonor system that led to violent gouging matches and later duels.2

      white ppl are dumb

    14. Enslaved Africans carried strong concepts of honor to North America. As wehave seen, the pastoral Angolans held a tradition of reflexive honor in whichaffronts to honor were settled in stick fights.

      honor

  5. ivanov-petrov.livejournal.com ivanov-petrov.livejournal.com
    1. Есть маргинальная теория, на стыке психологии и философии, активно эксплуатирующая принцип 3+1. Теория предлагает рассматривать каждый аспект человеческой жизни как адаптацию ...Когда удаётся сформулировать, что именно в природе человека не совпадает с природой окружающего мира, то почти на каждую формулировку приходится 1+3 стратегии адаптации (одна прямая и 3 косвенных)...Для человека довольно естественно полагать себя бессмертным (в том или ином смысле) или, как минимум, полагать что он должен быть связан с чем-то не подверженным разрушению. В то же время, наблюдаемый бренный мир постоянно и непрерывно движется к распаду. ...Можно кратко обозначить начальные условия адаптации: мы пытаемся ощутить свою бессмертную природу в умирающем бренном мире. Психологически, такого рода адаптация регулируется чувством ничтожности. ...Если чувство собственной ничтожности невыносимо, значит бренный мир хорошенько нам врезал, разрушив ту часть нас, которую мы считали незыблемой.По теории, должны быть 4 стратегии, как адаптировать знание/представление о своей бессмертной природе к наблюдаемому бренному миру. Прямая стратегия - ориентация на вечное. Попытаться связать свою жизнь с тем что выходит за пределы времени, найти в себе то что принадлежит вечности. Например, ощутить себя частью замысла творца, найти смысл жизни и т.п. Прямая стратегия - стратегия независимости, она почти всегда игнорирует природу наблюдаемого мира, т.е. в данном случае - время. Три оставшихся стратегии, по теории, должны быть вспомогательными и уж они ничего не игнорируют. Стратегия ориентации на прошлое борется с чувством ничтожности через поиск артефактов, прошедших проверку временем. Связь с семьёй, устойчивые черты характера, проверенные временем таланты, память о незабываемых событиях, написанные статьи. Пока всё это живо, жив и я и чуть менее ничтожен в мире, где время всё разрушает. Стратегия ориентации на настоящее борется с чувством ничтожности через поиск лучшего места в бренном мире. Тот кто прямо сейчас находится в лучшем для себя месте и в лучших условиях, ощущает себя менее ничтожным, чем все остальные. Стратегия ориентация на будущее борется с ничтожностью с помощью понимания логики времени, видения текущих процессов. Тот кто знает, где мир окажется завтра, более живуч и утонет последним.Получается красиво: 3+1 (прошлое/кто я?, настоящее/где я?, будущее/куда я иду?) и вечность/зачем я?
  6. Feb 2022
    1. But it isn’t. This is because over such a long period a message can easily be distorted or altered without this being in any way intended. (This distortion or alteration in the meaning or method of transmission of a message, whether intended or not, is called “noise.”) Languages, both written and spoken, always change. The meanings of symbols are often lost in the passage of time. In fact, most messages are bound so closely to a particular period and place that even a short time later they cannot be understood. Therefore, ensuring that a message created now can be decoded by future generations is highly problematic.

      Can symbol that represents one thing change over a long period of time to mean something different?

    2. ea. It is only because there is already a well-established connection in our minds between the appearance of an apple and the idea of temptation that this fruit is used in the picture. It is this connection that makes the picture successful in terms of communicatio

      Why was the apple chosen as the representation of temptation?

    3. semiotician,

      A Theory of signs and symbols that deals especially with there function in both artificially constructed and natural languages.

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  7. Jan 2022
    1. Des initiatives pour protéger ces espaces :

      Arg3: Malgré les risques environnementaux qui pèsent sur les EM, il existe des initiatives pour protéger ces espaces

      • Réduction des émissions de CO2 GAS + niveau CO2 atm + température ++ océans = phytoplanctons moins utiles à l'homme Absorber 30% du CO2 généré par l'homme Produire 50% oxygène = indispensables pour la vie humaine sur terre.
    2. La lutte contre les trafics illégaux

      Arg3: Les Etats tentent également de lutter contre les trafics illégaux

      • Lutte contre la pêche illicite Selon FAO = 15 et 20 % des prises => 23 Milliards $ /an Moins de préservation du stock renouvelable = surpêche Concurrence déloyale = pecheurs + respect quotas

      • Lutte contre le trafic de cocaïne 2017 - 2019: Cocaïne saisie à bord d'embarcations commerciales x3 = 73 tonnes Moyens ? Sous marins artisanaux, Ou ? Antilles = plaque tournante Quelles saisies ? 3 tonnes sous marins par police espagnole en 2019

      • Lutte contre le trafic des déchets Cout traitement + législation contraignante = Etats exportent leurs déchets / filières illégales ==> Pays d'Asie et d'Afrique: ++ déchets électroniques Malaisie => FR: 43 conteneurs de plastique illégaux

    3. Des espaces qui suscitent des litiges et des contentieux

      Arg3: Les EM sont au coeur de tensions géopolitiques entre les Etats qui se disputent l'accès aux ressources halieutiques, hauturières et aux routes maritimes

      • Mer de Chine méridionale Chine // Philippines, Vietnam, Brunei, Indonésie, Malaison "langue de boeuf" => Archipel des Spratleys + Îles Paracels Zone de trafic CMM Territorialisation , militarisation des EM Mars 2020: percussion bateau de pêche vietnamien

      • Mer de Chine Orientale Chine // Japon Îles Senkaku et Diaoyu COVID19: Liaoning traverse ZEE jap

      • Mer méditerranée Turquie // Grèce // Chypre // Israël // Liban EM triangulaire de 850km² = réserves de gaz ?

    4. Les ressources énergétiques

      Arg3: les ressources énergétiques sont également exploitées dans les Mers et les Océans

      • les hydrocarbures 30% hydrocarbures proviennent de gisements offshore Mer du Nord, Mer Caspienne, Golfe de Guinée, Golfe Arabo-persique, Mer de Chine méridionale

      espaces inexploités: conditions extrêmes (Arctique), fonds marins profonds = ++ pression (Brésil qui se lance dans l'exploitation d'hydrocarbures au dela du plateau continental)

      • les énergies renouvelables off shore Usines marémotrices: UM de la Rance (Côtes d'Armor, FR) Parcs éoliens off shore: projets aux îles de Lérons, Europe = 1er prod mondial électricité = 5000 éoliennes off shore (Mer du Nord ++)

      • les avancées scientifiques possibles Bathymétrie est mal connue = couts ++ 90% des espèces marines resteraient à découvrir

    5. Argument 3 : Les routes maritimes, les points de passage stratégiques et les goulets d’étranglement : des espaces maritimes vitaux pour le commerce maritime

      Arg3: les enjeux vitaux des chokes points pour l'économie mondialisée et le CM Connecter espaces de prod°/conso° Espaces maritimes très réduits concentrent trafic maritime:

      • Canaux; Suez, Panama
      • Détroit, Ormuz, Gibraltar, Bab el Manbed, Bering, Malacca
      • Caps: Bonne espérance, Horn

      Détroit de Malacca selon Institut Supérieur d'économie marine (2017) 14M barils / jours Port Klang (Malaisie) = 12e port à conteneurs mondial "Dilemme de Malacca" selon Hu Jintao 20 - 25% du trafic mondial / an

      Dépendance = ouverture permanente des passages stratégiques Mauvaise situations: insécurité, piraterie, conflits, tensions géopolitiques sinon = perturber approvisionnement marchandises + matières premières = - économie mondiale EX: 1967 -> 1974 = pas Canal de Suez (G6jours) alternative = Cap de Bonne Espérance (+ temps, +argent)

  8. 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!

  9. 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

  10. 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?

  11. 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

      merriam

    2. primordial

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

      merriam

    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.

    Tags

    Annotators

    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?

  12. 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?

  13. 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.

  14. 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?

  15. 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.

      Strengths:

      • 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.

      Weaknesses:

      • 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.

      Strengths:

      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.

      Weaknesses:

      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.

      Strengths:

      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.

      Weakness:

      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.