Showing a critical or disrespectful attitude.

Encourage or promote the development of (something, typically something regarded as good).

A snack

Python-object (streepje)

kan misschien tussen haakjes dat picklen, serialiseren betekent

protocolversie (aaneen)

Voeg 1 voorbeeldzin toe: “Gebruik dump() als je direct naar een bestand wilt schrijven, dumps() als je bytes in een variabele wilt.” iets in die trend

geeft terug/returnt

geïnteresseerdeN

worden

Misschien praktisch maken? Zegt een gemiddelde student misschein weinig.

"Pickle wordt gebruikt om python-objecten op te slaan en later weer terug te laden" iets in deze trend er bijzetten. Maakt het praktisch voor studenten

Het is ideaal voor onder andere studenten van klinische technologie. Dit voorbeeld kan wat minderen beladen zijn, er wordt nu bijna gesuggereerd dat dit het meest voornamelijke gebruik voor is. Beter misschein algemeen maken zoals "handig voor opslaan van python resultaten". daarna kan een specifiek voorbeeld wel genoemd worden

grootte

python-objecten

taalfout python-module hoort het te zijn

distinction essentielle : le numérique n'est pas en soi du virtuel psychique

numérique n'est pas égal à virtuel

Le numérique augmente le risque de figer la relation dans le virtuel

Rvo correspond à un refus de l'ajustement au réel

Rov est une composante normale de toute relation

avatare médiatise et déforme la relation à autrui

numérique amplifie les mécanismes anticipatoires

numérique accélère radicalement l'actualisation du virtuel

immersion renforce la plausibilité perceptive des représentations

numérique transforme pronfondement le rapport aux représentations

numérique n'est pas égal à virtuel ou irréel

usages numériques compensent un retrait déjà présent

numérique n'est pas la cause du Hikikomori

sujet évite toute situation évaluative

refus des attentes sociales structure le retrait relationnel

retrait implique une neutralisation affective défensive

désarticulation touche la capacité d'auto-représentation

retrait = agit comme défense contre éffondrement psychopathologique

actes persistent mais désinvestis de finalité symbolique

estime de soi pas indexée à reconnaissance sociale

retrait évite la responsabilité de l'action et de la transformation

dynamique actualisation / visualisation bloquée

retrait psychique précède et entraine le retrait social

Retrait durable socialement et temporellement défini

Hikikomori correspond rupture entre virtuel et perceptif

retrait = défense sontre tension psychique

ecart entre attente et expérience est structurel et potentiellement conflictuel

virtuel psychique appartient à la réalité du sujet

substitut défensif face à une réalité insupportable

saturation perceptive

compense impuissance réelle

continuité identitaire malgré les ruptures

angoisse d'abandon

Avatar = Moi idéal

dépendance affective

L’avatar comme support identitaire → difficulté de séparation.

Distinction essentielle : dépendance = mode de fonctionnement psychique

Thèse centrale : ce n’est pas le numérique qui rend addict, mais la problématique interne

consequences usages excessifs

conséquences usage excessifs

effet pervers des jeux par nature

Dispositifs incitatifs ≠ cause suffisante de l’addiction.

refus du changement interne

fuite face à la puberté

L’avatar soutient narcissiquement le sujet → fragilités identitaires.

Usage non pathologique

L’objet numérique comme prothèse psychique face à l’angoisse de séparation.

L’adolescence amplifie les usages : période de vulnérabilité narcissique.

L’addiction n’est pas liée au support mais au mode de relation adhésif à l’objet.

Critère du pathologique : disparition du jeu → collage à l’objet

Usage sain du virtuel = espace transitionnel (jeu, créativité, symbolisation).

Ambivalence du virtuel : peut nourrir la créativité ou fragiliser le rapport à la réalité.

Le virtuel mobilise surtout le registre perceptif → illusion sensorielle ≠ confusion avec le réel

Déconstruction du fantasme de nouveauté : le virtuel prolonge une histoire ancienne des images.

Idée clé : le numérique peut être créatif OU destructif → pas de déterminisme technologique.

Problématique centrale du devoir : le numérique modifie le rapport à soi, à l’autre et au réel → enjeu psychique, pas seulement technique.

références du texte

口譯待遇低賤無底限：

Nativecall360 這家低端公司提供一小時 USD 3 的醫療口譯（西語），顯然在招募美國外貧窮國家的線上口譯

台灣也有一票服務美國市場的線上英中醫療口譯，待遇大約 USD9-11，而且是大夜班。

text editor -> teksteditor

zet dit als hoofdregel: altijd eerst openen in teksteditor (bijv. VS Code/Notepad++)

Misschien ook zo'n warning box voor excel?

Waarom onstaan die errors?

Kan nog bij dat dit wordt veroorzaakt door conversiefouten (bijv. string ->float) als numpy numerieke waarden verwacht.

"doet ontstaan" kan beter neergezet worden als veroorzaakt.

Verkeerde woordkeuze. Gescheiden hoort het te zijn.

De header begint met #. Dat is handig als commentaar, maar dan wordt de header vaak niet als kolomnamen ingelezen.

Wil je de kolomnamen echt gebruiken in je code? Zet de header dan niet als commentaar, of lees hem apart in (anders gooit numpy hem weg).

Wordt --> worden. Data is meervoud.

De inhoud wordt inderdaad met python automatisch ingezet in strings, alleen de data in het initiele csv bestand zijn geen strings. Tekst \( \neq \) string

I Totally missed this. It’s an obligation now?

oof

On retrouve pour la première fois ici le terme « dits-documents ». Il serait préférable d'opter pour un autre terme (simplement « documents » ?).

Peut-être serait-il bon de rappeler de quel département il est question ici.

Plutôt : « Galerie d'Orléans » ?

La répétition est-elle volontaire ? On s'attendrait à lire seulement « Car l'utilité sociale du voyage se mesure à... »

Il faudrait indiquer le numéro de page de cette citation, si l'information est disponible.

La citation est ici donnée sans numéro de page.

update as follows Fall 2026 and Fall 2027(i.e. Fall 2026, Spring 2027, Summer 2027, Fall 2027).

update--

Projects implemented in Fall 2026 will be disbursed after the kickoff meeting in Summer 2026.<br /> Payment for projects implemented in later semesters will be disbursed Spring 2027.

update-- Spring 2026

delete this when updates after updates

https://www.reddit.com/r/Scorigami/comments/1nvrua1/new_4part_scorigami_youtube_series/

这是谁的网页

How Rosetta Stone unravelled the history of ancient Egypt?

Where was Rosetta Stone discovered?

The Rosetta Stone, a pivotal artifact discovered in 1799, unlocked ancient Egyptian hieroglyphics through its trilingual inscription, sparking a race among scholars like Young and Champollion to decipher its secrets, ultimately revealing a vanished world. <center>

Highlights

• Rosetta Stone was created during the reign of King Ptolemy V in ancient Egypt in 196 BCE and was eventually discovered by French engineers in 1799. This discovery played a crucial role in deciphering Egyptian hieroglyphics.
• An artisan inscribed the "Memphis Decree on the stone, which grants tax exemptions to the priestly class, aiming to stabilize Ptolemy V's rule.
• Fast forward to 1799, French military engineer Pierre Francois Bouchard discovers the stone while repairing a fort, unaware of its historical importance.

How the scholars deciphered the script on Rosetta Stone

• After Napoleon abandoned the expedition, the scholars were left with the stone and a pressing need to disseminate its information despite military challenges.
• The team devised a new method to capture the stone's inscriptions by using ink and paper, which proved successful.
• Following the surrender of the French forces, the Rosetta Stone was claimed as a spoil of war by the British and eventually donated to the British Museum.
-
• Despite initial expectations, matching the Greek text with hieroglyphics did not lead to immediate decipherment of ancient Egyptian.
• The quest to decode the Rosetta Stone saw numerous attempts throughout history, culminating in significant breakthroughs by Thomas Young and Jean-François Champollion, who recognized the phonetic nature of hieroglyphics.- The misunderstanding of hieroglyphics persisted until the 1800s, despite efforts by medieval Muslim researchers.
• Thomas Young made initial progress in translating the Rosetta Stone by focusing on the Demotic section and recognizing the phonetic writing of Greek names.</l>
• Jean-François Champollion, a talented linguist who understood Coptic, began his own translation efforts and ultimately surpassed Young's work. He utilized various sources, including artifacts and inscriptions from Egypt, to further his understanding of hieroglyphics.

Champollion's groundbreaking work

Jean-François Champollion's groundbreaking work on deciphering Egyptian hieroglyphics using the Rosetta Stone highlights his struggles, rivalries, and eventual success in unlocking the secrets of ancient Egypt. He utilizes his knowledge of Coptic and previous research to reconstruct Egyptian royal names in cartouches, aiming to decode hieroglyphics. Despite facing rivalries and political challenges, Champollion perseveres in his studies, leading to a significant breakthrough in understanding hieroglyphics. In 1822, Champollion successfully read the name Thutmose from an inscription, confirming his theories and dramatically celebrating his discovery. Champollion's journey to Egypt allowed him to read inscriptions and uncover the history of ancient kings and common people, further solidifying his achievements.

引数無しで

引数無しで

これデフォルト設定だからなー。 クォートをシングルにできるけど、設定なしの運用でいいんじゃない?みたいな記述がいいかなーって思ってます。

カンマを付けたい派

これはストーリーとしては、この前に ruff check --fix を実行していないといけないので、そのように手前で実行している必要がある

使われていないモジュールだとF401が出ちゃうのでisortの意味がない(fixできない)

無理やり使うモジュールを入れましょう

datetimeとかなにかをいれてメッセージに追加する感じで

文章（文章）。

が正しい書き方(のはず)

トル

この手前でfixされたあとのexample.pyを表示して、import osが削除されていることを確認したい

す。

系なくてもいい派です

下の設定例だとpyproject.tomlなので

前述の設定ファイルを用いて、にしてコードのキャプションに「説明 - pyproject.toml」とか書くのはどうでしょうか

checkコマンド

とか

長いので、トルでも通じるかなと

表のタイトルを入れてください。

https://myst-parser.readthedocs.io/en/latest/syntax/tables.html#table-with-captions

と、

ここで、を打たないと「一部のルール」がFにもかかっているように読める。

もしくはE系の一部とF系のルールが

とか

Flake8 (以下同様

Flake8と最初は大文字かなと

https://flake8.pycqa.org/en/latest/

同様にデフォルト設定っぽいものは除外して必要最低限にしてほしい

デフォルト設定を設定例に書いているのが気になります。

デフォルト設定はここにあることを示して、設定ファイルとしては必要最低限の記述例でいいかなと思います https://docs.astral.sh/ruff/configuration/

整形

他の箇所と表現を合わせる

を記述します。

前の文は主体が自分だったので

他のライブラリとかでuvを使ったインストールには触れていないので、ここはなくてもいいかなぁ

Suggestion:

Given a URL, check whether a page (specified by another URL or by file upload) links to that URL, and if so where/how.

need to link the log in and trial buttons in the nav

need to hide this section for now until we have actual social proof.

good section, just need to add the real symbols

the orange check over the blue background is a little disorienting, but otherwise I think this is a fine section.

we can probably remove compare page, just leaving the pricing page. Customer stories can be removed as well, since we probably won't have many at launch.

remove API page

remove press and careers for now

remove social links for now

need to remove the amount of professionals

remove hipaa page

This section is good, we'll just want to make sure the buttons go to the appropriate pages.

We'll have to figure out if this is the free trial model we want to go with.

We can probably expand on this by adding more questions and lengthening the answers

I'm questioning if we need this section at all

We can just remove the number of clinicians

Remove this

We can change these to info about the app. How many possible relationships? How many different symbols? How many modalities?

This will link to app.genogramplus.com

We'll want this to open up the video in a lightbox

Like Guyana, maybe?

https://nflscorigami.com/

via https://www.sportingnews.com/us/cfl/news/cfl-rouge-explained-one-point-score-canadian-football-league/cbd1hmplzoqhhwccffvapzx1

http://sports-reference.com/stathead/<br /> Stathead

La conclusion souligne les implications organisationnelles de l’apprentissage informel numérique, en invitant les entreprises à s’adapter aux pratiques réelles des professionnels.

Cette typologie permet de structurer et de mieux comprendre la diversité des pratiques d’apprentissage informel numérique.

L’auteur met en évidence le rôle du numérique comme catalyseur des pratiques d’apprentissage informel.

Ce passage inscrit l’apprentissage informel numérique dans une logique plus large d’apprentissage continu tout au long de la vie.

L’auteur montre ici que l’apprentissage informel répond à un besoin d’adaptation continue face à l’évolution rapide des compétences professionnelles.

Ce résultat remet en question la place centrale accordée aux formations formelles, en soulignant le poids majoritaire de l’apprentissage informel.

La référence à Bandura ancre l’apprentissage informel dans des théories reconnues de l’apprentissage social.

Ce passage permet de relativiser la nouveauté du phénomène en montrant que le numérique amplifie des pratiques d’apprentissage déjà existantes.

L’auteur s’appuie ici sur des données empiriques pour objectiver l’importance de l’apprentissage informel numérique dans le monde professionnel.

Ce passage introduit l’idée centrale de l’article en montrant que l’apprentissage informel numérique occupe une place croissante dans le développement des compétences professionnelles.

No NFL Game Has Ever Ended in a Score of 36–23 - The Atlantic by [[Josh Levin]] accessed on 2025-12-17T23:05:52

Описываемые примеры скорее описалбы "невозможно" вернее шансы настолько малы что их НЕ стоит даже пытаться учитывать.

Наверное в этом и был в свое время фурор концепции нуля, что "ничто" может быть чемто", и до сихпор стандартная настройка что "невозможно" ощущается как ничто по шансам, хотя математически практичиски ничто и практически никогда не бывает 0% и 100%, максимум может к ним стремиться.

Можно попробовть учитывать порядки нулей после запятой, и тип связей в комбинаторике, чтобы или учесть в принципе весомой пещинки или отбросить вообще приняв для простоты максиму "невозможно"

Факт это гипотезы сомнения в которых находятся за гранью разумных сомнений.

La conclusion réaffirme la thèse défendue dans l’article : seule une formation explicite et structurée permet de développer durablement les compétences d’évaluation de l’information

Cette phrase nuance le débat en rappelant que l’éducation à l’évaluation de l’information repose sur des dispositifs progressifs et structurés.

L’auteur adopte ici une position critique en montrant que certaines méthodes largement utilisées à l’école manquent d’efficacité pédagogique.

Ce passage montre que les difficultés des élèves ne relèvent pas uniquement de leurs capacités, mais aussi du cadre pédagogique proposé.

Ce résultat de recherche souligne un problème majeur : les élèves évaluent le contenu sans questionner l’origine ni les intérêts des sources.

L’auteur met en évidence ici les limites des compétences informationnelles des élèves, même après plusieurs années de scolarité.

Cet exemple montre que les élèves s’appuient souvent sur des indices superficiels pour évaluer l’information, ce qui peut conduire à des erreurs d’interprétation.

Ce passage met en évidence la controverse centrale : face à la complexité de l’évaluation de l’information, l’école est-elle en mesure de former efficacement les élèves ?

L’auteur souligne ici que la désinformation ne concerne pas seulement certains publics, mais l’ensemble des citoyens exposés aux contenus numériques.

Ce passage introduit clairement l’enjeu central de l’article en montrant que l’évaluation de l’information est devenue une compétence essentielle dans un contexte de désinformation numérique.

Muy buena la conclusión. Solamente dos comentarios: 1.- me hace un poco de ruido definir como neutralización de clase el hecho de que estas condiciones objetivas no logren predecir pension mjp. Siguiendo la idea, me referiría más bien a un patrón generalizado de creencias entre clases 2.- En el párrafo de trabajos futuros mencionaría integrar nuevas mediciones de meritocracia (Castillo et al., 2023) en los estudios, pues se podrían develar otras configuraciones de creencias entre mjp y merit que la escala actual no permite observar

charge (lead)

I would appreciate if could add one example question, so students will be more encouraged to share their thoughts.

is it possible to add a fixed timeframe like first two weeks or first month to understand the structure?

This is great way to let the students share their different ideas and thoughts. I appreciate this.

Good!

Good!

Good!

can we add - team engagement as well.

can we add - "explain your growth with examples"

上の文章と似た内容だけど微妙に情報が増えて箇条書きになっている。この箇条書きでなにを伝えたいかを直前に文章で書いて欲しい。

以下は〜〜です。 以下に〜〜を示します。

みたいな

例なのでしょうがないけど、自分でファイル名を指定しつつ、拡張子も指定するというコマンドは、オプションの用途としてピンとこないなと思いました。

何か数え方のルールとかを変えられるといいんだけど。文字数でカウントするかバイト数でカウントするか、とか

これも = をトルでいいと思う

キーハード引数を示すときに = って付けるのあんまり見たことないので、トルでいいと思います

nits: 表の文章は最後の。はトルでいいかなぁ

typo?

19.2.3と内容が被ってますね？

「インタプリタごとに独立してロードされる」 ということですかね？であれば上記のように記載すると明確になるかなと思いました。

他とそろえて「Python 3.14」 とした方が良さそうですかね。

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

Learn more at Review Commons

Reply to the reviewers

1. General Statements

We thank the reviewers for their overall support, thorough review, and thoughtful comments. The points raised were all warranted and we feel that addressing them has improved the quality of our manuscript. Below we respond to each of the points raised.

2. Point-by-point description of the revisions

Reviewer #1

Minor comments:

Are the lgl-1; pac-1 M-Z- double mutants dead? Only the phenotype of pac-1(M-Z-); lgl-1 (M+Z-) is shown. In figures and text throughout, it should be clear whether mutants are referring to zygotic loss or both maternal and zygotic loss, as this distinction could have major implications on the interpretation of experiments.

Almost all experiments we performed used a combination of RNAi of lgl-1 in a homozygous pac-1 null mutant background, or the other way around. RNAi should eliminate maternal product, but we hesitate to use the terminology M/Z since it has previously been used for protein degradation strategies.

We have updated the text and figure 1 to address the potential of maternal product masking earlier phenotypes, and performed additional RNAi experiments to demonstrate that the phenotypes obtained by RNAi for either pac-1 or lgl-1 in a homozygous mutant background for the other are the same as for the genetic double mutant. The results are shown as additional images and quantifications in figure 1B,C. We also updated the legend to figure 1 to make it clear that double genetic mutants are obtained from heterozygous lgl-1/+ parents.

Regarding the phenotype of lgl-1; pac-1 M-Z- double mutants: assuming the reviewer refers to M-Z- double genetic mutants, we cannot make such embryos as the pac-1(M-Z-); lgl-1(M+Z-) animals are already lethal.

In Figure 1C, it would be more appropriate to show a fully elongated WT embryo to contrast with arrested elongation in mutant embryos.

We agree with the reviewer and have replaced the 2-fold WT embryo with a 3-fold embryo.

Is the lateral spread of DLG-1 in double mutant embryos a result of failure to polarize DLG-1, or failure to maintain polarity? This should be straightforward to address in higher time resolution movies.

We have analyzed additional embryos at early stages of development. In lgl-1; pac-1 embryos we never see the appearance of complete junctions: defects are apparent already at dorsal intercalation. We interpret these results as a failure to properly polarize DLG-1. We have added additional images to Figure S2 and added this sentence to the text: Imaging of embryos from early stages of development on showed that normal continuous junctional DLG-1 bands are never established in pac-1(RNAi); lgl-1(mib201) embryos (Fig. S2B).

The lack of enhancement of hmp-1(fe4) by lgl-1(RNAi) is quite interesting, given that pac-1 does enhance hmp-1(fe4). To rule out the possibility that this result stems from incomplete lgl-1 RNAi, this experiment should be repeated using the lgl-1 null mutant.

We have done this experiment by recreating the fe4 S823F mutation in the lgl-1(null) mutant background as well as in the wild-type CGC1 background using CRISPR/Cas9. The phenotype of both was similar, but differs from that of the original PE97 strain. In the original strain, there is ~50% embryonic lethality but worms that complete embryogenesis grow up to be fertile adults. In our new "fe4" strains, nearly all animals are severely malformed with little to no elongation taking place. We are able to maintain both strains (with and without lgl-1) homozygous but with difficulty as only ~5% of animals grow up and give progeny. Apparently, there are genetic differences between PE97 and our CGC1 background that cause phenotypic differences despite having the same amino acid change in HMP-1.

Nevertheless, using our original embryonic viability criterium of 'hatching', loss of lgl-1 does not enhance the S823F mutation. We have included the following text in the manuscript:

To rule out that the lack of enhancement by lgl-1(RNAi) is due to incomplete inactivation of lgl-1, we also re-created the hmp-1(fe4) mutation (S823F) by CRISPR in lgl-1(mib201) mutant animals and wild-type controls. The phenotype of the S823F mutant we created is more severe than that of the original PE97 hmp-1(fe4) strain, with only ~5% of animals becoming fertile adults (Fig. S2F). This likely represents the presence of compensatory changes that have accumulated over time in PE97. Nevertheless, consistent with our RNAi results, the presence of lgl-1(mib201) did not further exacerbate the phenotype of HMP-1(S823F) (Fig. S2E, F). Taken together, the lack of enhancement of hmp-1(S823F) mutants by inactivation of loss of lgl-1 This observation argues against a primary role for lgl-1 in regulating cell junctions.

• Related to point 4, do pac-1 or lgl-1 null mutants enhance partial knockdown of junction protein DLG-1, or is this effect (of pac-1) specific to HMP-1/AJs?*

We have attempted to address this point using feeding RNAi against dlg-1. However, we were not able to obtain partial depletion of DLG-1. On RNAi feeding plates, control, pac-1, and lgl-1 animals did not show significant embryonic lethality. We checked RNAi effectiveness with a DLG-1::mCherry strain and found RNAi by feeding to be very ineffective. Since we could not deplete DLG-1 to a level that results in partial embryonic lethality, we were not able to address this question properly.

Does lgl-1 loss affect PAC-1 protein localization and vice versa?

It does not. We have added the following text and a figure panel: Loss-of-function mutants that strongly enhance a phenotype are often interpreted as acting in parallel pathways. We therefore examined whether loss of lgl-1 or pac-1 alters the localization of endogenously GFP-tagged LGL-1 or PAC-1. In neither null background did we detect changes in the subcellular localization of the other protein, consistent with LGL-1 and PAC-1 functioning in parallel pathways (Fig. S1D).

Reviewer #2

Very little of the imaging data are analyzed quantitatively, and in many cases it is not clear how many embryos were analyzed. While the images that are presented show clear defects, readers cannot determine how reproducible, strong or significant the phenotypes are.

We completely agree with the reviewer that interpretation of our data requires this information and apologize for the omission in the first manuscript version. The phenotypes are highly penetrant and consistent (timing of arrest, % lethality, junctional defects), and we have now added quantifications throughout the manuscript.

In particular, the data below should be quantified and, where possible, analyzed statistically:

• The frequency of the various junctional phenotypes shown in 2C

We have now quantified the junctional phenotypes. The junctional defects are highly penetrant: >90% of lgl-1; pac-1 embryos have junctional defects (new Fig. 2B). We used airy-scan confocal imaging to analyze the distribution of the different phenotypes (unaffected, spread laterally, and ring-like pattern). The results are shown in Fig. 2G.

• The expansion of DLG-1::mCherry in pac-1 lgl-1 embryos should be quantified (related to Figure 2B). For example, the percentage of membrane (marked by PH::GFP) occupied by DLG-1 could be quantified.

We have performed this quantification, shown in Fig. 2D.

- Similarly, the expansion of the aPKC domain should be quantified (Figure 3A).

An objective quantification of aPKC signal is difficult due to the relatively weak expression of aPKC::GFP and the lack of a clear demarcating boundary. This is part of the reason we measured tortuosity as a more quantifyable indicator of apical domain expansion. We have now added a qualitative observation table as Figure 3B. In addition, we have expanded the quantification of cell geometry by measuring lateral and basal surfaces. Lateral surfaces were decreased. We added the following text:

To better understand the reason for the change in geometry, we also measured the lengths of the lateral and basal surfaces (Fig. 3F). We found that the absolute lengths of the apical surfaces were not significantly different between pac-1(RNAi); lgl-1(mib201) and control animals. Instead, the lengths of the lateral domain were reduced (Fig. 3F). Hence, the more dome-shaped appearance of epidermal cells in pac-1; lgl-1 double mutant animals is due to a decrease in lateral domain size, which is consistent with the observed lateral spreading of aPKC.

• How many embryos were analyzed for each marker shown in Figure 2A, and what proportion showed the described phenotypes? This could be given in the text or in a panel.

We have added these numbers to panel 2B, and indicated the percentage in the text.

• The frequency of the various junctional phenotypes shown in 4F.

To address this, we have changed figure 4F to show three types of phenotype (strong, mild, no phenotype) and added how frequently we observed each to the panels. In rescue experiments, 18/24 embryos showed no junctional defects, while 6/24 showed a mild defect (compared to 100% severe in non-rescued embryos). To make room for this and other quantifications in Figure 4, we moved the demonstration that PAC-1 is depleted by RNAi to supplemental figure S4.

Because the genetic perturbations used are global (either deletions or RNAi), it is not established whether PAC-1/LGL-1 act in epidermal epithelial cells per se (versus an earlier requirement that manifests in epidermal epithelial cells). While I agree that this is the most likely scenario, other mechanisms are possible.

Our experiments indeed use global depletion/deletion of lgl-1 and pac-1. We cannot exclude therefore that other tissues do not contribute to the epithelial phenotypes. We assume that other tissues would be affected as well, and in fact have observed abnormal looking pharynx tissue (see our response to reviewer 3 below for examples). As the epidermis is one of the first tissue to develop it is likely the first in which phenotypes become apparent.

In particular, the overall GFP::aPKC levels appear notably higher in pac-1 lgl-1 embryos in Figure 3A. aPKC levels should be quantified to determine if this is true of pac-1 lgl-1 embryos. If so, couldn't that explain (or at least contribute to) the observed phenotypes?

Overall higher levels could indeed contribute to the phenotype. However, we have now quantified total aPKC levels in control and pac-1; lgl-1 embryos found no difference between them. We have added the following text to the manuscript: To determine if increased expression of aPKC might explain the broadened apical localization, we measured total intensity levels of aPKC::GFP. However, we detected no differences in fluorescence levels between control and pac-1(RNAi); lgl-1(mib201) animals (Fig. S3B, C).

Minor

Figure 4: For completeness, please include the embryonic viability of pac-1 lgl-1 +/- embryos treated with EV and cdc-42(RNAi), as was done for pac-1 lgl-1 pkc-3(ts) in Figure 4E. Presumably the increased proportion of viable embryos with the lgl-1 deletion allele is reflected in an overall increase in embryonic viability.

The embryonic viability indeed increases, but not as much as one might think because 15% of embryos die from the cdc-42 RNAi itself. The most important rescue argument is that we can obtain adult pac-1; lgl-1 animals with cdc-42 RNAi.

We have now included the overall rescue and the following text: Overall, cdc-42 RNAi caused a mild increase in embryonic viability (Fig. 4A). However, total embryonic viability may underestimate rescue of pac-1; lgl-1 embryonic lethality, because it also includes the ~15% lethality caused by cdc-42 inactivation itself, even among animals wild type for lgl-1.

The orientation of the inset images in Figures 2C, 3A and 3D is confusing. An illustration showing how these images are oriented relative to each other would be helpful.

We have added a figure showing how the junctions are oriented in the figures (Fig. 2E). We have also added supplemental videos S3 and S4 that should illustrate the phenotype more clearly as well.

For completeness, it would be good to test whether lgl-1(delta) is also synthetically lethal with picc-1(RNAi) (Zilberman 2017).

We like this idea and had already looked into this. Lgl-1 and picc-1 are not synthetic lethal (see graph in word file submitted). However, PICC-1 is not the only junctional localization signal for PAC-1, as demonstrated by the Nance lab. We find the data interesting but feel that it deserves a more thorough structure/function investigation of PAC-1 than we can provide here. Therefore we would prefer not to include this data.

Reviewer #3

We thank the reviewer for their support of our manuscript.

A few small areas to improve this manuscript:

p. 6 like 139: "remain" should be "remaining"

We have fixed this typo.

Could the authors mention what is the phenotype of the 10% of pac-1 animals that die?

Yes. They die with pleotropic phenotypes not resembling those of our pac-1; lgl-1 double mutant embryos. We have added examples of these to Figure S1.

Based on the Supplemental figures, it made me curious to ask: Did the authors notice changes in dorsal epidermal fusions? Cadherin normally disappears in the dorsal hyp7 cells at this time. Did the timing of the fusions change at all?

We haven't analyzed this in detail but our time-lapse videos show that dorsal fusions still take place and do not seem to be particularly delayed (overall development is slightly delayed but the delay in fusion is consistent with overall delay).

Again, curiosity driven by the Supplemental figures: did the authors notice defects in apical regions of internal organs, like the pharynx or intestine? The CDC-42 biosensor is asymmetrical in the developing intestine. See: DOI: 10.1242/bio.056911

We did not pay much attention to the intestine as PAC-1 is barely detectable in this tissue. The pharynx is formed, which we can easily detect in arrested embryos as we use GFP or BFP expressed under the myo-2 promoter to mark the deletion of pac-1. While we did not look closely, we do observe defects in pharynx development.

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Referee #3

Evidence, reproducibility and clarity

This manuscript by Jarosinska and colleagues addresses a long-standing mystery in the apical/basal polarity field: why LGL-1 and PAC-1/RhoGAP19D, which are essential in Drosophila, and in some tissue culture contexts, are not essential in C. elegans embryos.

The authors take an open-ended approach by using genetics, in the form of a genome wide RNAi screen, to find other proteins that enhance the mild phenotypes of lgl-1 mutant embryos. They uncover strong synthetic lethality when they reduce pac-1, a well-documented CDC-42 GAP that supports apical/basal polarity during early embryogenesis, and yet is also only partially required during embryogenesis.

The phenotypic analysis to understand why the embryos die when missing both lgl-1 and pac-1 leads to a careful analysis of known junctional molecules in C. elegans. Using newly made endogenously tagged junctional proteins, including DLG-1 and AFD, so that they can examine all three C. elegans apical junction complexes, the authors find a penetrant defect in the epidermal junctions as the embryos undergo elongation, an actomyosin dependent contractile event that dramatically reshapes the embryos into long, skinny tubes. With disorganized junctions, the embryos die due to ruptures, or hernias, as shown in the Supplemental Movie 2. In addition, and quite excitingly, the apical domains of the embryos are expanded. These defects are then partially rescued by removing CDC-42 or aPKC using RNAi depletion.

Major comments:

The claims and conclusions are supported by the data.

The data is presented in such a way that it is easy to understand what was done, and how measurements were obtained and evaluated.

Rigorous documentation of how the strains were built and how the genome wide RNAi screen was conducted is included in the Supplemental files.

Beautiful use of CRISPR to do the genetics:

since when they made the deletion of lgl-1 they replaced the coding sequence with GFP, they could use GFP to count the animals carrying the deletion in their double mutant analysis with pac-1 deletion mutants.

Figures are very nicely done.

The writing is clear.

Minor comments:

A few small areas to improve this manuscript:

p. 6 like 139: "remain" should be "remaining"

Could the authors mention what is the phenotype of the 10% of pac-1 animals that die?

Based on the Supplemental figures, it made me curious to ask: Did the authors notice changes in dorsal epidermal fusions? Cadherin normally disappears in the dorsal hyp7 cells at this time. Did the timing of the fusions change at all?

Again, curiosity driven by the Supplemental figures: did the authors notice defects in apical regions of internal organs, like the pharynx or intestine? The CDC-42 biosensor is asymmetrical in the developing intestine. See: DOI: 10.1242/bio.056911

Significance

This study raises interesting and important questions for the general polarity field. Early embryos have hugely redundant methods to maintain apical/basal polarity, which in C. elegans masked the roles for lgl-1 and pac-1 at earlier events, like compaction, when apical/basal polarity is first established. However, during elongation, when healthy strong junctions are a requirement, the double mutant loss of LGL-1 and PAC-1 results in expanded apical domain, that is lethal.

The study will be of interest to the broader polarity community, and to developmental biologist interested in how the apical junctions are assembled and strengthened during morphogenesis. The Discussion does a good job of showing what aspects of this study are novel, and which support prior findings that suggested, for example, that PAC-1 may have roles independent of CDC-42. I appreciate the comment that our field needs more and more sensitive biosensors to fully address the changes of key polarity regulators.

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Referee #2

Evidence, reproducibility and clarity

Summary: This study focuses on the polarization of epidermal epithelial cells in C. elegans. Whereas the basolateral polarity protein is LGL-1 is required for epithelial polarity in flies, LGL-1 is dispensable for polarization and viability in C. elegans. Through a whole-genome RNAi screen, Jarosinska et al discover that the depletion of the RhoGAP PAC-1 is synthetically lethal with an lgl-1 deletion mutant. pac-1 lgl-1 double mutants have significant polarity defects in the epidermal epithelial, including mislocalization of junctional markers and expansion of the apical aPKC domain. As a result pac-1 lgl-1 double mutants fail to maintain surface epithelial and arrest development. Genetic interaction data suggest that increased CDC42 and aPKC activity in pac-1 lgl-1 contributes, as least in part, to the polarity defects and resulting embryonic lethality.

Major comments:

Very little of the imaging data are analyzed quantitatively, and in many cases it is not clear how many embryos were analyzed. While the images that are presented show clear defects, readers cannot determine how reproducible, strong or significant the phenotypes are. In particular, the data below should be quantified and, where possible, analyzed statistically:

• The frequency of the various junctional phenotypes shown in 2C
• The expansion of DLG-1::mCherry in pac-1 lgl-1 embryos should be quantified(related to Figure 2B). For example, the percentage of membrane (marked by PH::GFP) occupied by DLG-1 could be quantified.
• Similarly, the expansion of the aPKC domain should be quantified (Figure 3A).
• How many embryos were analyzed for each marker shown in Figure 2A, and what proportion showed the described phenotypes? This could be given in the text or in a panel.
• The frequency of the various junctional phenotypes shown in 4F.

Because the genetic perturbations used are global (either deletions or RNAi), it is not established whether PAC-1/LGL-1 act in epidermal epithelial cells per se (versus an earlier requirement that manifests in epidermal epithelial cells). While I agree that this is the most likely scenario, other mechanisms are possible. In particular, the overall GFP::aPKC levels appear notably higher in pac-1 lgl-1 embryos in Figure 3A. aPKC levels should be quantified to determine if this is true of pac-1 lgl-1 embryos. If so, couldn't that explain (or at least contribute to) the observed phenotypes?

Minor

Figure 4: For completeness, please include the embryonic viability of pac-1 lgl-1 +/- embryos treated with EV and cdc-42(RNAi), as was done for pac-1 lgl-1 pkc-3(ts) in Figure 4E. Presumably the increased proportion of viable embryos with the lgl-1 deletion allele is reflected in an overall increase in embryonic viability.

The orientation of the inset images in Figures 2C, 3A and 3D is confusing. An illustration showing how these images are oriented relative to each other would be helpful.

For completeness, it would be good to test whether lgl-1(delta) is also synthetically lethal with picc-1(RNAi) (Zilberman 2017).

Significance

LGL-1 is a conserved polarity protein that is essential for viability in Drosophila. In contrast, lgl-1 mutants are viable and have weak polarity phenotypes in C. elegans. A previous study showed that LGL-1 acts redundantly with the posterior polarity proteins PAR-2 during establishment of anterior/posterior polarity in the one-cell worm embryo. Here, Jarosinska et al show that LGL-1 acts redundantly with another protein, the RhoGAP protein PAC-1, in the polarization of the embryonic epidermal epithelial. The strength of this study is the identification of redundant roles for PAC-1 and LGL-1, the apparent strength of the polarity defects in the double mutant and the broader implication that LGL-1 may act in a range of redundant, cell/tissue specific pathways to regulate polarity. The primary weakness of this study is the lack of quantification. Additionally, the aPKC and CDC42 genetic interaction data hint at potential pathways, but fall short of establishing LGL-1's or PAC-1's mechanism of action.

Advance: This works identifies a redundant genetic interaction between LGL-1 and PAC-1. While the data require additional quantification, the phenotypes presented appear clear and strong. Although the molecular mechanism by which LGL-1 and PAC-1 act is not well established in the current work, the core observation is significant and should provide a foundation for future studies dissecting the molecular mechanisms.

Audience: This work will be of interest to a broad audience. LGL-1 is conserved and its role in cell polarization and epithelial polarity is very actively studied, including in mammalian systems.

Field of expertise. C elegans embryonic development; cell polarity.

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Referee #1

Evidence, reproducibility and clarity

In this manuscript, Jarosinska and colleagues address the roles of two polarity regulators, pac-1 and lgl-1, in C. elegans epidermal polarity. Loss of function mutations in either of these gene individually does not block polarization, but through a genome-wide RNAi screen, the authors find that pac-1 and lgl-1 enhance each other to cause apical-basal polarity defects and arrest during epidermal morphogenesis. The remainder of the paper focuses on testing genetic interactions between both proteins and AJ proteins (HMP-1) as well as apical proteins (CDC-42, PKC-3). These experiments reveal some interesting differences in how lgl-1 and pac-1 interface with junctional proteins (pac-1 enhances hmp-1 but lgl-1 does not) and apical proteins (lgl-1 suppresses pkc-3 or cdc-42 partial loss but pac-1 does not).

Minor comments:

1. Are the lgl-1; pac-1 M-Z- double mutants dead? Only the phenotype of pac-1(M-Z-); lgl-1 (M+Z-) is shown. In figures and text throughout, it should be clear whether mutants are referring to zygotic loss or both maternal and zygotic loss, as this distinction could have major implications on the interpretation of experiments.
2. In Figure 1C, it would be more appropriate to show a fully elongated WT embryo to contrast with arrested elongation in mutant embryos.
3. Is the lateral spread of DLG-1 in double mutant embryos a result of failure to polarize DLG-1, or failure to maintain polarity? This should be straightforward to address in higher time resolution movies.
4. The lack of enhancement of hmp-1(fe4) by lgl-1(RNAi) is quite interesting, given that pac-1 does enhance hmp-1(fe4). To rule out the possibility that this result stems from incomplete lgl-1 RNAi, this experiment should be repeated using the lgl-1 null mutant.
5. Related to point 4, do pac-1 or lgl-1 null mutants enhance partial knockdown of junction protein DLG-1, or is this effect (of pac-1) specific to HMP-1/AJs?
6. Does lgl-1 loss affect PAC-1 protein localization and vice versa?

Significance

Overall, the manuscript provides additional insights into apical-basal polarization in C. elegans and demonstrates that lgl-1 is likely working in a similar way as in Drosophila, despite the lack of a phenotype in single lgl-1 mutants. I found the experiments to be done rigorously and interpretations of the data appropriate. All of my suggestions on improving the manuscript are minor; suggested experiments should be viewed as optional ways to strengthen the conclusions/impact of the study.

Thomas Kuhn’s view of scientific change is that science does not progress smoothly or simply by accumulating facts. Instead, it advances through periodic revolutions that transform how scientists understand the world.

https://practical-scheme.net/

Deze beschrijving begrijp ik niet. Nieuw te plaatsen objecten zijn dus ontworpen, maar nog niet gerealiseerd. In dat geval behoren ze toch niet tot de revisietekening?

Vraag: geldt dat ook als de object-id's van de bestaande objecten overgenomen wordt in de CAD tekeningen en de workflow tot en met de revisietekening?

IN 6.1.3.3 worden de statussen in de NLCS revisietekening benoemd. Daarmee kunnen deze objecten toch herkend worden?

je zou verwachten dat een geo-coordinaat hierbij kan helpen. Locatieaanduiding van putten staan ook in de BGT o.b.v. een coordinaat.

eLife Assessment

This important study demonstrates the significance of incorporating biological constraints in training neural networks to develop models that make accurate predictions under novel conditions. By comparing standard sigmoid recurrent neural networks (RNNs) with biologically constrained RNNs, the manuscript offers compelling evidence that biologically grounded inductive biases enhance generalization to perturbed conditions. This manuscript will appeal to a wide audience in systems and computational neuroscience.

Reviewer #1 (Public review):

This manuscript introduces a biologically informed RNN (bioRNN) that predicts the effects of optogenetic perturbations in both synthetic and in vivo datasets. By comparing standard sigmoid RNNs (σRNNs) and bioRNNs, the authors make a compelling case that biologically grounded inductive biases improve generalization to perturbed conditions. This work is innovative, technically strong, and grounded in relevant neuroscience, particularly the pressing need for data-constrained models that generalize causally.

Comments on revisions:

The authors have addressed all my concerns.

Reviewer #2 (Public review):

Sourmpis et al. present a study in which the importance of including certain inductive biases in the fitting of recurrent networks is evaluated with respect to the generalization ability of the networks when exposed to untrained perturbations.

The work proceeds in three stages:

(i) a simple illustration of the problem is made. Two reference (ground-truth) networks with qualitatively different connectivity, but similar observable network dynamics, are constructed, and recurrent networks with varying aspects of design similarity to the reference networks are trained to reproduce the reference dynamics. The activity of these trained networks during untrained perturbations is then compared to the activity of the perturbed reference networks. It is shown that, of the design characteristics that were varied, the enforced sign (Dale's law) and locality (spatial extent) of efference were especially important.

(ii) The intuition from the constructed example is then extended to networks that have been trained to reproduce certain aspects of multi-region neural activity recorded from mice during a detection task with a working-memory component. A similar pattern is demonstrated, in which enforcing the sign and locality of efference in the fitted networks has an influence on the ability of the trained networks to predict aspects of neural activity during unseen (untrained) perturbations.

(iii) The authors then illustrate the relationship between the gradient of the motor readout of trained networks with respect to the net inputs to the network units, and the sensitivity of the motor readout to small perturbations of the input currents to the units, which (in vivo) could be controlled optogenetically. The paper is concluded with a proposed use for trained networks, in which the models could be analyzed to determine the most sensitive directions of the network and, during online monitoring, inform a targeted optogenetic perturbation to bias behavior.

The authors do not overstate their claims, and in general, I find that I agree with their conclusions.

Author response:

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

Public Reviews:

Reviewer #1 (Public review)

Major:

(1) In line 76, the authors make a very powerful statement: 'σRNN simulation achieves higher similarity with unseen recorded trials before perturbation, but lower than the bioRNN on perturbed trials.' I couldn't find a figure showing this. This might be buried somewhere and, in my opinion, deserves some spotlight - maybe a figure or even inclusion in the abstract.

We agree with the reviewer that these results are important. The failure of σRNN on perturbed data could be inferred from the former Figures 1E, 2C-E, and 3D. Following the reviewers' comments, we have tried to make this the most prominent message of Figure 1, in particular with the addition of the new panel E. We also moved Table 1 from the  Supplementary to the main text to highlight this quantitatively. 

(2) It's mentioned in the introduction (line 84) and elsewhere (e.g., line 259) that spiking has some advantage, but I don't see any figure supporting this claim. In fact, spiking seems not to matter (Figure 2C, E). Please clarify how spiking improves performance, and if it does not, acknowledge that. Relatedly, in line 246, the authors state that 'spiking is a better metric but not significant' when discussing simulations. Either remove this statement and assume spiking is not relevant, or increase the number of simulations.

We could not find the exact quote from the reviewer, and we believe that he intended to quote “spiking is better on all metrics, but without significant margins”. Indeed, spiking did not improve the fit significantly on perturbed trials, this is particularly true in comparison with the benefits of Dale’s law and local inhibition. As suggested by the reviewer, we rephrased the sentence from this quote and more generally the corresponding paragraphs in the intro (lines 83-87) and in the results (lines 245-271). Our corrections in the results sections are also intended to address the minor point (4) raised by the same reviewer.

(3) The authors prefer the metric of predicting hits over MSE, especially when looking at real data (Figure 3). I would bring the supplementary results into the main figures, as both metrics are very nicely complementary. Relatedly, why not add Pearson correlation or R2, and not just focus on MSE Loss?

In Figure 3 for the in-vivo data, we do not have simultaneous electrophysiological recordings and optogenetic stimulation in this dataset.  The two are performed on different recording sessions. Therefore, we can only compare the effect of optogenetics on the behavior, and we cannot compute Pearson correlation or R2 of the perturbed network activity. To avoid ambiguity, we wrote “For the sessions of the in vivo dataset with optogenetic perturbation that we considered, only the behavior of an animal is recorded” on line 294. 

(4) I really like the 'forward-looking' experiment in closed loop! But I felt that the relevance of micro perturbations is very unclear in the intro and results. This could be better motivated: why should an experimentalist care about this forward-looking experiment? Why exactly do we care about micro perturbation (e.g., in contrast to non-micro perturbation)? Relatedly, I would try to explain this in the intro without resorting to technical jargon like 'gradients'.

As suggested, we updated the last paragraph of the introduction (lines 88 - 95) to give better motivation for why algorithmically targeted acute spatio-temporal perturbations can be important to dissect the function of neural circuits. We also added citations to recent studies with targeted in vivo optogenetic stimulation. As far as we know the existing previous work targeted network stimulation mostly using linear models, while we used non-linear RNNs and their gradients.

Minor:

(1) In the intro, the authors refer to 'the field' twice. Personally, I find this term odd. I would opt for something like 'in neuroscience'.

We implemented the suggested change: l.27 and l.30

(2) Line 45: When referring to previous work using data-constrained RNN models, Valente et al. is missing (though it is well cited later when discussing regularization through low-rank constraints)

We added the citation: l.45

(3) Line 11: Method should be methods (missing an 's').

We fixed the typo.

(4) In line 250, starting with 'So far', is a strange choice of presentation order. After interpreting the results for other biological ingredients, the authors introduce a new one. I would first introduce all ingredients and then interpret. It's telling that the authors jump back to 2B after discussing 2C.

We restructured the last two paragraphs of section 2.1, and we hope that the presentation order is now more logical.

(5) The black dots in Figure 3E are not explained, or at least I couldn't find an explanation.

We added an explanation in the caption of Figure 3E.

Reviewer #2 (Public review):

(1) Some aspects of the methods are unclear. For comparisons between recurrent networks trained from randomly initialized weights, I would expect that many initializations were made for each model variant to be compared, and that the performance characteristics are constructed by aggregating over networks trained from multiple random initializations. I could not tell from the methods whether this was done or how many models were aggregated.

The expectation of the reviewer is correct, we trained multiple models with different random seeds (affecting both the weight initialization and the noise of our model) for each variant and aggregated the results. We have now clarified this in Methods 4.6. lines 658-662.

(2) It is possible that including perturbation trials in the training sets would improve model performance across conditions, including held-out (untrained) perturbations (for instance, to units that had not been perturbed during training). It could be noted that if perturbations are available, their use may alleviate some of the design decisions that are evaluated here.

In general, we agree with the reviewer that including perturbation trials in the training set would likely improve model performance across conditions. One practical limitation explaining partially why we did not do it with our dataset is the small quantity of perturbed trials for each targeted cortical area: the number of trials with light perturbations is too scarce to robustly train and test our models.

More profoundly, to test hard generalizations to perturbations (aka perturbation testing), it will always be necessary that the perturbations are not trivially represented in the training data. Including perturbation trials during training would compromise our main finding: some biological model constraints improve the generalization to perturbation. To test this claim, it was necessary to keep the perturbations out of the training data.

We agree that including all available data of perturbed and non-perturbed recordings would be useful to build the best generalist predictive system. It could help, for instance, for closed-loop circuit control as we studied in Figure 5. Yet, there too, it will be important for the scientific validation process to always keep some causal perturbations of interest out of the training set. This is necessary to fairly measure the real generalization capability of any model. Importantly, this is why we think out-of-distribution “perturbation testing” is likely to have a recurring impact in the years to come, even beyond the case of optogenetic inactivation studied in detail in our paper.

Recommendation for the authors:

Reviewer #1 (Recommendation for the authors):

The code is not very easy to follow. I know this is a lot to ask, but maybe make clear where the code is to train the different models, which I think is a great contribution of this work? I predict that many readers will want to use the code and so this will improve the impact of this work.

We updated the code to make it easier to train a model from scratch.

Reviewer #2 (Recommendation for the authors):

The figures are really tough to read. Some of that small font should be sized up, and it's tough to tell in the posted paper what's happening in Figure 2B.

We updated Figures 1 and 2 significantly, in part to increase their readability. We also implemented the "Superficialities" suggestions.

explicar doble dimensión: - Prácticas Corporales Tecno-Mediadas: Artísticas Explicar marco teórico de post-danza, post-fenomenología... - Prácticas Corporales Tecno-Mediadas: Fenómeno Social

Esta clasificación es útil como guía para posteriormente desarrollar un análisis de la corporalidad, considerando los tres aspectos.

Añadir que de esta clasificación, también consideramos lasa portaciones de Butler, y otras contemporáneas . Añadir un párrafo que explique la tesis busca comprender a cuerpo conectad en esas distintas dimensiones,

Añadir, que de estaclasificación, interesa acercarme a entender aspectos de usos intencionales del cuerpo, como los que ofrece Mauss y posteriormente Muñiz.