The author is building up her argument between every paragraph that she introduces to the audience. I enjoy this slow build up upon such a huge conflict that the author is writing about. It helps ease the audience in and increases attention.

Initially I believed that this article was only about the mistreatment that women receive when it comes to medical attention but with this sentence, I can tell that the author wants to talk about the discrimination that exists in the medical world overall.

Because the author is including such a personal story to introduce us into her article, her credibility towards the audience is built upon this story. The audience also feels a sense of empathy that would keep them engaged to read further on in the article.

I like that the author had included this paragraph that essentially teaches the audience about what endometriosis is about for the audience members that do not have prior knowledge on endometriosis. It is very helpful and would enable the audience to feel more engaged rather than exiting the article due to feeling not knowledgeable enough to read on.

I immediately was drawn into this article as the author had written in a style as if I was reading a novel. Very story-like and novel-like where it feels like I was reading a fiction book rather than a real article.

penguin - pen (head) guin (white)

bard - bardd (poet)

Avon - afon (river)

corgi - cor (dwarf) + ci (dog)

flannel - gwlanen (fabric from Wales)

coombe/combe - cwm (valley)

balderdash - baldorddu (noisy talk or chatter)

adder - neidr (snake)

crockery - crochan (cauldron)

iron - haearn (both steam instrument and the element)

crumpet - crempog (crepe/pancake)

London - Llundain (maybe from llyn + dain or pool of the river)

yes but a hard time understanding Bile's flaws?...ignores the rumors??

EZH2 is ubiquitinated.

Lu Bu slaughters his own father- from the situation, it seems like Lu Bu killed Ding Yuan because he wanted more power, and Li Su convinced Lu Bu that Dong Zhuo will give him that power based on their conversation

I find it shocking that women had so few rights and respect in society that men here are being compared to Christ. The author goes on to say the importance of men and the obedience of women- obviously very demeaning, but fitting to the time. I'm happy with how far away we've moved from these thoughts in today's time.

Why is Ogechi so motivated to have a successful baby? Even after horrible experiences, she still continues to persevere in having a baby. What is her motivation? The jealousy she gets when seeing other babies get praise and glory? Is the story's purpose to explain how parents feel about their children, and they are desperate to do anything for the child, even if irrational?

The old woman shaking her head is keep repeating which makes me wonder if it's going to be an important element throughout the story.

It is sad to see how human remembers the worst parts in his life until he dies. Like the past of this old woman, probably a traumatic moment from an war, it keeps repeating in her head and actions.

This is interesting, as it cannot really be based on anything other than a complete guess

Was trying to figure out where the canonical repo even is. Hard to figure out. Could be made clearer (like a prominent notice on one saying this is an unofficial clone with a link to the canonical source).

Ended up here via link from https://unix.stackexchange.com/questions/86879/suppress-rsync-warning-some-files-vanished-before-they-could-be-transferred to https://git.samba.org/?p=rsync.git;a=blob_plain;f=support/rsync-no-vanished;hb=HEAD

But then found https://github.com/WayneD/rsync, which I now believe to be canonical based on:

• last change here is Mon, 15 Mar 2021 09:35:39 -0700 (09:35 -0700) but on https://github.com/WayneD/rsync it was April 3.
• https://rsync.samba.org/bug-tracking.html links to: create an issue on GitHub

I never noticed this before, but now that I realize it, it is very true. Even other than chess this applies to sports, tabletop games and even work in some cases

bis236

In order to play video games, there is a lot of experimentation to figure out the best possible solution to a game. For example in chess, you are looking at the board and testing each possible move and its outcome in your head, in order to play the best possible move.

Again, I am myself supporting the Varieties of Capitalism approach as a valuable approach for revealing national socio-economic and political divergences in the organisation of production. However, in particular for a study on the global economic system and the multinational enterprise, I would like to see a stronger justification on the almost exclusive focus on Europe and North America as the important Variants for handling global economic constitutions. I would invite further research that includes the Varieties of Capitalism in Asia, Africa and South America. Relatedly, I would also find it useful to expand the focus from the relevance of a capitalist variant that applies to the "head" of a multinational group to the importance of national capitalist institutions of those countries that are affected by the operations of global corporations. There are a number of interesting empirical studies that highlight how national and local specifics affect the way in which global private regulation by multinationals is perceived, practiced and contested in local communities in which corporate subsidiaries or supplyy chain operate (cf for instance Tim Bartley, Rules without Rights, Oxford University Press 2018).

Again, I am myself supporting the Varieties of Capitalism approach as a valuable approach for revealing national socio-economic and political divergences in the organisation of production. However, in particular for a study on the global economic system and the multinational enterprise, I would like to see a stronger justification on the almost exclusive focus on Europe and North America as the important Variants for handling global economic constitutions. I would invite further research that includes the Varieties of Capitalism in Asia, Africa and South America. Relatedly, I would also find it useful to expand the focus from the relevance of a capitalist variant that applies to the "head" of a multinational group to the importance of national capitalist institutions of those countries that are affected by the operations of global corporations. There are a number of interesting empirical studies that highlight how national and local specifics affect the way in which global private regulation by multinationals is perceived, practiced and contested in local communities in which corporate subsidiaries or supplyy chain operate (cf for instance Tim Bartley, Rules without Rights, Oxford University Press 2018).

I often wonder why he chose the word "deferred" but the phrase a "dream deferred" and the alliteration of it also creates a musical element that sticks in your head easier. His jazz background shines through in this poem, as his word choices are intentional and critical in providing movement to the poem. A "dream deferred" rolls off the tongue easier and can be committed to memory better and I also think the words appear casual and harmless, when, just like a dream that is deferred, it's impact lies beneath the surface.

This sentence is nicely worded. The deep description gives the reader the opportunity to picture this sentence in their head.

Here the author asks a series of rhetorical questions to the reader. This is a very effective method because in my head while reading I find myself answering the questions.

I can really visualize this scene in my head because of the descriptive language used by the author.

The old man is very aware of his surroundings.

I think O brien is overhinking because he is so scared of being found out as a draft dodger

This old man is very sharp and notices everything and his keen senses to what he does makes him uncomfortable.

This imagery is depicting a pessimistic experience of O'Brien being drafted.

Characterization of Elroy, he's someone that pays attention and can catch onto things quickly.

Elroy may be a former veteran or someone who is wise enough to know that the Vietnam War is not only a dumb war, but also that war is not something one should be forced into.

He was scared and is very doubtful to the point he was uncomfortable due to the old man being sharp. But still he tried to doubt himself because the old man has helped him so far, but still he would've ran away if he was cornered.

It is unfair to be forced into fighting for a cause that you know little to known about, It is unfair to be forced to help finish a fight that you didn't start let alone it possibly taking your life.

Using imagery to show afraid he was when he was drafted for the war as he thought this war wasn't for him.

He feels like he doesn’t deserve to fight in the war.He thinks he’s destined for something more in life.

O'Brien tells how he felt when he got drafted and it was a bad feelings this is imagery

that fear and panic that rushes to us with new comes like

Often wonder if subconcious work is ok if it doesn't cause any stress

It's very interesting and somewhat surprising to me that one of the first smart fridges was introduced in 2000, over 20 years ago. In my head, I always saw this technology as somewhat new and modern. My thought was that touch screen fridges were an invention from within the past 6-8 years or so, but they have been around much longer than that!

Skin head: aggressively masculine youth subculture present during the 1960s dead head: Grateful Dead groupies, representative of hippies in the 1960s

The diction here, focusing on the word "black" repeatedly, aids in the idea of the sentence overall that regardless of the position these people are still being stripped down to the color of their skin as if that's the main deciding factor.

Reviewer #4 (Public Review):

Higashi et al. provide a new "Brownian ratchet" model for DNA loop extrusion mechanism by cohesin, a member of SMC protein family complexes. Based on previous works on crystal structures, cryo-EM structures, and DNA-protein crosslinking experiments, they shed light on two HEAT-repeat DNA binding modules on cohesin - Scc2-head and Scc3-hinge - and their relationships. They hypothesized that the association between Scc2-head and Scc3-hinge modules were dissociated and Scc2-head released DNA upon ATP hydrolysis, driving DNA slipping. By performing FRET experiments, they found that Scc2 and hinge modules indeed come close only in ATP-bound "Gripping" state, while hinge and Scc3 are always close to each other. Therefore, they suggest that, for DNA loop extrusion model, 1) upon ATP binding to the head domains, both Scc2-head and Scc3-hinge modules grip DNA, 2) when ATPs are hydrolyzed, Scc2-head module releases DNA so that DNA-associating Scc3-hinge module pulls DNA depending on stochastic Brownian motion of Scc3-hinge module, then 3) both Scc2-head and Scc3-hinge modules release DNA and go back to the state 1). This "Brownian ratchet" model also provides an explanation of how cohesin entraps DNA by opening the gate between Smc3 and Scc1, which also nicely explains the known facts regarding Scc1 cleavage-dependent DNA release and in vitro behaviors of single cohesin molecules that topologically bound to DNA. In addition, by performing computational modeling, they showed that the Brownian ratchet model well fits all previously reported in vitro loop extrusion assays by cohesin and condensin, making their model rigid and reliable.

Their model is mostly well supported by data, but several detailed points need to be explained or clarified.

1) In Figure 2C FRET experiments, proximity of Scc3-C and Scc2-N does not seem to be drastically increased in Gripping state compared to the case of hinge and Scc2-N. This could be because the FRET pairs (Scc3-C and Scc2-N) are still far. If the authors could label internal part in Scc3, this could solve the problem. In addition, if Scc3-C and Scc2-N are always close to each other irrespective of Gripping state, the authors should consider this fact in their modeling.

2) Major differences between topological loading and loop extrusion is kleisin-gate opening and head gate passage. Even if kleisin-gate wouldn't be opened, DNA should be released after head opening like in the topological loading. In case it happens, DNA and Scc1 would be tangled and it seems to be difficult to come back to next gripping state again. It would be helpful to add the explanation of why such tangling DNAs do not have to be considered in the model.

3) In the manuscript line 338, the authors mention "After DNA dissociation from the Scc3-hinge module, there is a time without tight contact between the cohesin ring and the DNA loop." However, both in Figure 3B and 4F, it seems that head-Scc2 always associates with DNA. This could be discrepancy. The authors should clarify the point if certain free time without any contact to DNA is assumed in the modeling.

4) Generally, initial DNA bending is the most challenging part in loop extrusion models. Especially in Figure 3B-a, such a bent DNA seems to be impossible if we consider the persistence length of DNA is 50 nm. The authors should discuss how DNA loop extrusion could be initiated.

Reviewer #2 (Public Review):

How the genome chromatin fiber is folded into loops and topologically associating domains (TADs) remains unclear. A recent attractive model is that these genomic structures are formed by a loop extrusion process mediated by cohesin. While the Uhlmann group has proposed an alternative mechanism, the diffusion capture model, to make loops (Cheng et al., 2015; Gerguri et al., 2021), in this paper, Higashi et al. proposed a structure-based model providing mechanistic insight into the reported loop extrusion activity of cohesin. For its topological DNA binding, cohesin inserts DNA into the cohesin ring by sequential passage through a kleisin N-gate and an ATPase head gate. Hisgashi et al. suggested that the gripping state in which DNA has not passed the kleisin N-gate might facilitate the loop extrusion activity reported. This paper is very intriguing, and informative to the chromatin/chromosome field. My specific comments are the following:

1) Since this paper is primarily based on the detailed structural information on cohesin loading onto DNA, which the Uhlmann group published in Mol Cell (2020), it might be hard for general readers to follow the whole story in this paper. For better understanding, the authors should provide readers with Supplemental Fig. corresponding to the Graphical abstract and Figs. 6E/7G in the Mol Cell paper, and adequately explain it first. Structural models such as Fig. 1 are accurate but might be difficult to capture cohesin's dynamic behavior with DNA.

2) Although this paper is very intriguing, it looks like a review paper, and the authors' message is not so clear. Given that the Uhlmann group has proposed an alternative mechanism to make loops, I wonder whether the main message might be that the loop extrusion, like reported in vitro, is unlikely to occur in vivo. If so, the authors should clearly state the point and shorten the Discussion part to enhance the paper's impact.

3) Page 24. The critical issue of the loop extrusion mechanism proposed is "not opening" of kleisin N-gate. The authors discussed that the low salt condition in vitro could be a reason: " For instance, electrostatic interactions contribute to keeping the kleisin N-gate closed and these are augmented in a low salt buffer." However, I assume that the condition also helps the topological loading, and this explanation is not so convincing.

4) While I agree with the authors' loop extrusion mechanism, there are other models to explain cohesin loading onto DNA (e.g., Shi et al., 2020; Collier et al.). They might want to discuss its compatibility with them.

Reviewer #1 (Public Review):

Higashi et al. present a molecular mechanism of how the cohesin complex, a supramolecular assembly of several proteins, can topologically embrace DNA and actively extrude DNA into loops. The loop-extruding activity of cohesin and of related condensin complexes have been proposed to represent a cornerstone of genome organization. While recent in vitro studies demonstrate that cohesin and condensin complexes are capable of extruding loops, the molecular mechanisms driving loop extrusion, i.e. how ATP energy is utilized, and what underlies the processivity of the loop extrusion, remains enigmatic. The cohesin complex consist of two long flexible protein "arms" connected at the 'hinge' ends. The other, 'head' ends are linked by the kleisin protein and also can dimerize to form an ATP-binding chamber. Defining how transitions in the cohesin complex structure and its ATPase activity underlies known cohesin functions has been the object of numerous studies for over two decades. Here, the authors build upon these studies.

The authors start by analyzing available structural data for cohesin domains and associated loading factors. First, by combining the structure of the cohesin-head-domain complex engaged with DNA in ATP-bound state and the corresponding free crystal structures, they show that the 'head module' in the ATP-bound state can tightly wrap around DNA, and upon ATP hydrolysis the DNA-embracing cavity will dilate. In other words, the complex transitions from a 'DNA-gripping state' into a 'DNA-slipping' state after ATP is hydrolyzed. Next, they show that the other DNA-binding module, the 'hinge module', does not change its interaction with the DNA after ATP hydrolysis. The authors also conclude that ATP hydrolysis weakens the interaction between the head and hinge modules, suggesting that the cohesin ring alternates between folded (with head and hinge closed) and unfolded ('free' hinge) states. The authors next carried out FRET experiments to provide experimental evidence for the predicted change in spatial arrangement between the head and hinge modules. Based on this structural analysis, they propose that whether DNA is passed (or not) through the 'kleisin gate' before binding to the head module (into the gripping state) determines if the DNA will be released inside the cohesin ring (i.e. 'topological entry') or if the DNA will remain loosely associated with the head module (i.e. 'loop extrusion') upon ATP hydrolysis. In the latter case, repetitive simultaneous binding of DNA to the head and hinge modules in a folded state followed by relaxation of the cohesin ring while DNA remains bound to the hinge module, may result in a overall 'inward' directed motion of the DNA thread relative to the head domain, i.e. loop extrusion. Stochastic simulations of a coarse-grain model further support that such a model can give rise to loop extrusion.

The real strength of the paper is in its combination of several pieces of structural and biophysical data that results in a compelling mechanism for cohesin function. The outcome is a united model for cohesin's two characteristic activities - topological engagement of the DNA and DNA loop extrusion. Importantly, the authors explore the role of ATP hydrolysis in driving conformational changes, and, thus, the translocation of DNA, as well as the role of the DNA binding kinetics. The authors go on to relate these findings to the consequences for cohesin function inside cells, where it must content with chromatized substrates. For example, they suggest that while a single nucleosome probably can be bypassed by the cohesin complex, an array of the nucleosome may present a significant hindrance.

Given its interdisciplinary nature and important conclusions, I believe that this paper will be of broad interest to scientists across disciplines and will influence and stimulate future consideration of how cohesin contributions to the spatiotemporal organization of chromatin.

This is an example of an conclusion. The fact that Buffalo is extremely far from saltwater is a fact. This is inferring that it is difficult to find good shellfish in Buffalo. The conclusion is stating that even though it is so far from salt water, the shell fish is worth a trip to this restaurant.

Indeed. When you have the space to figure things out, the writing can happen quickly.

Maybe it helps to not think of these things as separate and isolated activities. When they're all part of a continuum that represents knowledge work then the reading/thinking/note-taking/writing aspects all merge into each other.

foreshadowing 2

I think of this often when I drive through Franklin. There is a stone there, carves by the native people who lived there maybe 300 years ago. One of the most prominent images was a shad, a fish that spends part of it's life in the sea, returning to spawn, similar to a salmon. I had never head of shad in land before I found out about this stone. Apparently, the fish were incredibly plentiful, and was considered a staple of the native diet. Damming and over exploitation have stopped this natural cycle.

When I walk in the woods today, I find so much dead wood on the ground, that I sometimes wish the town would allow people to take some of it. IT is piled so high now in my local woods, that I think if there were to be a fire, it would be basically an inferno. I feel that maybe it is time for people to consider managing with more natural means.

I'm puzzled by how you'd conduct this survey. Would you just take sample size N and ask them if they know about the hornbill and its fragility, and then come back after the campaign has run for a while and ask the same N size the same question? If so, then how can you attribute any increase in knowledge of hornbills to the efforts of the flagship species marketing done by the Pride campaigns? I'm sure there is a straight forward answer to this but for now it's just going right over my head.

For ancient humans, some kinds of social situations can both be dealt with by choice-talk and source-talk, such as head injuries, seizures (aka "demonic possessions"), etc. Generally, these are inherently low-dimensional social situations, allowing source-talk to handle social situations that normally requires choice-talk.

In situations like that, since source-talk is more adaptive than choice-talk due to being more accurate, evolution selected for an extra trait: "When both choice-talk and source-talk applies, use source-talk."

This process of creating notes not only helps create a long-term archive of information but includes encoding and consolidation of information in memory.

this reminded me of the hawaiian term piko which can also be looked as your belly button, the giddys on your head, and your reproductive organs. The piko on your head symbolized your connection to your ancestors. The piko as your belly button symbolized the connection to you parents. The piko as your reproductive organs symbolized the future. Thereʻs also another piko that symbolized your deep and personal connection to a place or particular land. Like for example oneʻs piko could be Mauna a Wākea. I thought it was very beautiful to see another key cultural component shared with another ethnic group throughout Oceania.

Note this does not remove changes in the files, it just rolls back the 'commit' that was made

quick switches between branches without losing content

The entire reason Jesus taught with stories is because they have the ability to transcend the head and mind to the soul and heart, effecting everyone, regardless of age, education or status of life. The story allows us to embody ourselves within it, blasting through our compartments and our desire to control our presentation.

John le Carré's last novel, Agent Running in the Field, features the main character, Nat, meeting a enigmatic man at a suburban badminton club.

main point

It's a little funny that Sloppy happened to meet Jenny on accident but they almost immediately seem to get along really well. It's possible that at the end of the story Jenny gets to find the romance she's been "looking" for.

Each of these ingredients adds a distinct metaphysical component to the spell; look up the correspondences?

She sneaks out of the palace, unnoticed, with this likely being preplanned, using forethought. Another note with the end of this paragraph describes her doing things in patterns of three. In Wicca, a religion that goes along with many that use witchcraft, the energy put out into the world will be returned threefold. In this way, she's eliciting this pattern of three to gain favor with the Triple Goddess, Hecate, in her plans for the future.

SciScore for 10.1101/2021.03.31.437955: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

While our predictions using 2D folding programs and chemical reactivity data do not identify other conformations besides the two pseudoknots and the 3-way junction, other possibilities cannot be excluded due to limitations in both the folding programs and reactivity experiments. Results for other works suggest other possible conformations, including two 3_8 pseudoknots, a different 3_3 pseudoknot (with Stem 2 formed by Stem 3 loop and 3′ end), a two-stem 2_1, and a two-stem-with-internal-loop 2_2 (Table S1 and Fig. S13). The length-dependent and context-specific conformations for the FSE emphasized in our work could be exploited biologically by the insidious virus in mechanisms of interactions with the ribosome. The bulky pseudo-knot may block and encourage ribosome pausing as the ribosome rolls over the FSE region, and any structural transitions may similarly interfere with this template-directed process. In the 2.3–7 Å-resolution Cryo-EM study by Bhatt et al., 21 the researchers observe the 3_6 pseudoknot wedged between the head and body of the small ribosomal subunit. Specifically, the residues before Stem 1 are found to be located at the mRNA entry tunnel so that, as translation proceeds, the GC-rich Stem 1 residues are unwound upon entering the mRNA tunnel. Stem 2 of the pseudoknot is found to be shorter by two base pairs than that predicted by computational 2D structure methods. To confirm these inferred structural interactions, the researchers mutated single residues i...

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

Results from JetFighter: We did not find any issues relating to colormaps.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

About SciScore

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Referee #4

Evidence, reproducibility and clarity

Summary:

The freshwater polyp Hydra possess the remarkable ability to regenerate a fully functional head within a few days after amputation, however when e.g., Notch signaling is inhibited the animals fail to regenerate the original head pattern. In the manuscript by Moneer et al. the authors aim to identify Notch responsive genes by RNA sequencing. 48 hours after Notch signaling inhibition with DAPT, 624 genes were up- and 207 genes downregulated. To identify putative direct Notch target genes the authors generated RNA-seq datasets at 3 and 6 hours after DAPT removal and propose that the expression of direct target genes is rapidly recovered within 3 hours as shown by the re-expression of HyHES. Furthermore, by performing motif enrichment analyses the authors propose that e.g., HyAlx and HySp5 could be direct Notch target genes.

Major comments:

1) It is not clear why the authors chose 48 hours as a time point for RNA sequencing. Why not 12 or 24 hours after DAPT exposure? Is the expression of HyHES or CnASH not downregulated at earlier time points? Furthermore, why did the authors use whole animals and not just the head tissue for RNA-seq to enrich the transcripts?

2) Why did the authors not perform RNA sequencing on head regenerating DAPT-treated animals? This would help to better understand the relationship between Notch and Wnt signaling especially as the authors showed in 2013 (Mündner et al) that the expression of Wnt3 is strongly affected in head regenerating DAPT-treated animals.

3) It is currently very difficult to fully evaluate the data. One single excel file with all up- and downregulated candidates should be provided (Trinity ID, fold change, False Discovery Rate, annotation etc.). I would have assumed that genes such as Wnt8 that are expressed at the base of the tentacles (Philipp et al., 2009) could be affected by DAPT. Is Wnt3 not affected at all in intact animals?

4) The silencing of Sp5 induces the formation of ectopic heads in intact and regenerating conditions and it has clearly been shown that Sp5 inhibits Wnt/β-catenin signaling. To call Sp5 a tentacle patterning gene just based on the identification of RBPJ-motifs in the Sp5 regulatory region is misleading, as it is currently not supported by experimental data. The fact that a regulatory motif is present in a promoter region does not mean that this regulatory motif is active.

5) This manuscript would be much more interesting and of greater importance if the authors would have added functional data for one or two candidate genes.

Minor comments:

1) Figure S1: Individual data points for the qPCR analysis should be shown and arrow bars added.

2) Figure 6: Scale bars are missing.

Significance

The manuscript is well written, and the presented results could be of interest for the Hydra field but they will not have a broad impact in the present state. I find it unfortunate that the authors did not use the datasets produced to better understand the complex regulatory network that is active during the patterning of the Hydra head.

Kai Kupferschmidt. ‘“Our Position Has Not Changed”, Says @EMA_News Head Emer Cooke about AZ Vaccine. “According to the Current Scientific Knowledge, There Is No Evidence That Would Support Restricting the Use of This Vaccine in Any Population."’. Tweet. @kakape (blog), 31 March 2021. https://twitter.com/kakape/status/1377268296739913728.

similar to the father? little hair?

Edwards syndrome, also known as trisomy 18, is a genetic disorder caused by the presence of a third copy of all or part of chromosome 18.[3] Many parts of the body are affected.[3] Babies are often born small and have heart defects.[3] Other features include a small head, small jaw, clenched fists with overlapping fingers, and severe intellectual disability.[3]

Do these go anywhere? Let's see.

Basically, the idea is to ask a closed (yes or no) question, which will spotlight the difference between how someone is behaving with how they would like to be. This creates cognitive dissonance, which creates discomfort, which they are then motivated to resolve.

Critically, all this is happening in the head of the person who needs to change.

When reading these words today, calling a group of people more "advanced" reads like a pseudoscientific description meant to put a lab coat and reading glasses on a racist statement; this isn't what the line means in this context, of course, but I had to re-read this sentence to get that image out of my head. Perhaps "advanced" meant something more class-related in 1926?

Iago is the master at planting ideas and images into Othello's head. You don't necessarily need to say a fact or the truth out right to convince someone of something. If you plant the seed and let the person imagination run wild is just as detrimental.

Just stating this shows how Othello is paying so much attention to what Cassio does and acts and over analyzes every small gesture and this is all thanks to Iago. He played with Othello's head and now has him noticing every little thing

This whole scene is very interesting. When I think deeply about the scene and Cassio speaking to this clown it seems very metaphorical. Especially this line. It almost feels like the "clown" is an inner voice fighting with Cassio in his head, asking him if he has made the right choice to let Iago control his destiny with Desdemona. The clown is clearly used by Shakespeare to reveal the stupidity and level of desperate that Cassio has reached in his attempt to reach Desdemona. Once again, Shakespeare develops his ongoing theme of dramatic irony.

Author Response:

Reviewer #1 (Public Review):

In this project, the authors set out to create an easy to use piece of software with the following properties: The software should be capable of creating immersive (closed loop) virtual environments across display hardware and display geometries. The software should permit easy distribution of formal experiment descriptions with minimal changes required to adapt a particular experimental workflow to the hardware present in any given lab while maintaining world-coordinates and physical properties (e.g. luminance levels and refresh rates) of visual stimuli. The software should provide equal or superior performance for generating complex visual cues and/or immersive visual environments in comparison with existing options. The software should be automatically integrated with many other potential data streams produced by 2-photon imaging, electrophysiology, behavioral measurements, markerless pose estimation processing, behavioral sensors, etc.

To accomplish these goals, the authors created two major software libraries. The first is a package for the Bonsai visual programming language called "Bonsai.Shaders" that brings traditionally low-level, imperative OpenGL programming into Bonsai's reactive framework. This library allows shader programs running on the GPU to seamlessly interact, using drag and drop visual programming, with the multitude of other processing and IO elements already present in numerous Bonsai packages. The creation of this library alone is quite a feat given the complexities of mapping the procedural, imperative, and stateful design of OpenGL libraries to Bonsai's event driven, reactive architecture. However, this library is not mentioned in the manuscript despite its power for tasks far beyond the creation of visual stimuli (e.g. GPU-based coprocessing) and, unlike BonVision itself, is largely undocumented. I don't think that this library should take center stage in this manuscript, but I do think its use in the creation of BonVision as well as some documentation on its operators would be very useful for understanding BonVision itself.

We have added a reference to the Shaders package at multiple points in the manuscript including lines 58-59 and in Supplementary Details. We will be adding documentation of key Shaders nodes that are important for the creation of BonVision stimuli to the documentation on the BonVision website.

Following the creation of Bonsai.Shaders, the authors used it to create BonVision which is an abstraction on top of the Shaders library that allows plug and play creation of visual stimuli and immersive visual environments that react to input from the outside world. Impressively, this library was implemented almost entirely using the Bonsai visual programming language itself, showcasing its power as a domain-specific language. However, this fact was not mentioned in the manuscript and I feel it is a worthwhile point to make.

Thank you - we have now added clarification on this in Supplementary details (section Customised nodes and new stimuli)

The design of BonVision, combined with the functional nature of Bonsai, enforces hard boundaries between the experimental design of visual stimuli and (1) the behavioral input hardware used to drive them, (2) the dimensionality of the stimuli (i.e. 2D textures via 3D objects), (3) the specific geometry of 3D displays (e.g. dual monitors, versus spherical projection, versus head mounted stereo vision hardware), and (4) automated hardware calibration routines. Because of these boundaries, experiments designed using BonVision become easy to share across labs even if they have very different experimental setups. Since Bonsai has integrated and standardized mechanisms for sharing entire workflows (via copy paste of XML descriptions or upload of workflows to publicly accessible Nuget package servers), this feature is immediately usable by labs in the real world.

After creating these pieces of software, the authors benchmarked them against other widely used alternatives. IonVisoin met or exceeded frame rate and rendering latency performance measures when compared to other single purpose libraries. BonVision is able to do this while maintaining its generality by taking advantage of advanced JIT compilation features provided by the .NET runtime and using bindings to low-level graphics libraries that were written with performance in mind. The authors go on to show the real-world utility of BonVision's performance by mapping the visual receptive fields of LFP in mouse superior colliculus and spiking in V1. The fact that they were able to obtain receptive fields indicates that visual stimuli had sufficient temporal precision. However, I do not follow the logic as to why this is because the receptive fields seem to have been created using post-hoc aligned stimulus-ephys data, that was created by measuring the physical onset times of each frame using a photodiode (line 389). Wouldn't this preclude any need for accurate stimulus timing presentation?

We thank the reviewer for this suggestion. We now include receptive field maps calculated using the BonVision timing log in Figure5 – figure supplement 1. Using the BonVision timing alone was also effective in identifying receptive fields.

Finally the authors use BonVision to perform one human psychophysical and several animal VR experiments to prove the functionality of the package in real-world scenarios. This includes an object size discrimination task with humans that relies on non-local cues to determine the efficacy of the cube map projection approach to 3D spaces (Fig 5D). Although the results seem reasonable to me (a non-expert in this domain), I feel it would be useful for the authors to compare this psychophysical discrimination curve to other comparable results. The animal experiments prove the utility of BonVision for common rodent VR tasks.

The psychometric test we performed on human subjects was primarily to test the ability of BonVision to present VR stimuli on a head-mounted display. We have edited the text to reflect this. The efficacy of the cube map approach for 3D spaces is well-established in computer graphics and gaming and is currently the industry standard, which was the reason for our choice.

In summary, the professionalism of the code base, the functional nature of Bonsai workflows, the removal of overhead via advanced JIT compilation techniques, the abstraction of shader programming to high-level drag and drop workflows, integration with a multitude of input and output hardware, integrated and standardized calibration routines, and integrated package management and workflow sharing capabilities make Bonsai/BonVision serious competitors to widely-used, closed-source visual programming tools for experiment control such as LabView and Simulink. BonVision showcases the power of the Bonsai language and package management ecosystem while providing superior design to alternatives in terms of ease of integration with data sources and facilitation of sharing standardized experiments. The authors exceeded the apparent aims of the project and I believe BonVision will become a widely used tool that has major benefits for improving experiment reproducibility across laboratories.

Reviewer #2 (Public Review):

BonVision is a package to create virtual visual environments, as well as classic visual stimuli. Running on top of Bonsai-RX it tries and succeeds in removing the complexity of the above mentioned task and creating a framework that allows non-programmers the opportunity to create complex, closed loop experiments. Including enough speed to capture receptive fields while recording different brain areas.

At the time of the review, the paper benchmarks the system using 60Hz stimuli, which is more than sufficient for the species tested, but leaves an open question on whether it could be used for other animal models that have faster visual systems, such as flies, bees etc.

Thank you for prompting us to do this - we have now added new benchmarks for a faster refresh rate (144 Hz; new Figure 4 - figure supplement 1).

The authors do show in a nice way how the system works and give examples for interested readers to start their first workflows with it. Moreover, they compare it to other existing software, making sure that readers know exactly what "they are buying" so they can make an informed decision when starting with the package.

Being written to run on top of Bonsai-RX, BonVision directly benefits from the great community effort that exists in expanding Bonsai, such as its integration with DeepLabCut and Auto-pi-lot. Showing that developing open source tools and fostering a community is a great way to bring research forward in an additive and less competitive way.

Reviewer #3 (Public Review):

Major comments:

While much of the classic literature on visual systems studies have utilized egocentrically defined ("2D") stimuli, it seems logical to project that present and future research will extend to not only 3D objects but also 3D environments where subjects can control their virtual locations and viewing perspectives. A single software package that easily supports both modalities can therefore be of particular interest to neuroscientists who wish to study brain function in 3D viewing conditions while also referencing findings to canonical 2D stimulus responses. Although other software packages exist that are specialized for each of the individual functionalities of BonVision, I think that the unifying nature of the package is appealing for reasons of reducing user training and experimental setup time costs, especially with the semi-automated calibration tools provided as part of the package. The provisions of documentation, demo experiments, and performance benchmarks are all highly welcome and one would hope that with community interest and contributions, this could make BonVision very friendly to entry by new users.

Given that one function of this manuscript is to describe the software in enough detail for users to judge whether it would be suited to their purposes, I feel that the writing should be fleshed out to be more precise and detailed about what the algorithms and functionalities are. This includes not shying away from stating limitations -- which as I see it, is just the reality of no tool being universal, but because of that is one of the most important information to be transmitted to potential users. My following comments point out various directions in which I think the manuscript can be improved.

We thank the reviewer for this suggestion. We have added a major new section, “Supplementary Details”, where we have highlighted known limitations and available workarounds. We also added new rows in the Supplementary Table that make these limitations transparent (eg. web-based deployment).

The biggest point of confusion for me was whether the 3D environment functionality of BonVision is the same as that provided by virtual spatial environment packages such as ViRMEn and gaming engines such as Unity. In the latter software, the virtual environment is specified by geometrically laying out the shape of the traversable world and locations of objects in it. The subject then essentially controls an avatar in this virtual world that can move and turn, and the software engine computes the effects of this movement (i.e. without any additional user code) then renders what the avatar should see onto a display device. I cannot figure out if this is how BonVision also works. My confusion can probably be cured by some additional description of what exactly the user has to do to specify the placement of 3D objects. From the text on cube mapping (lines 43 and onwards), I guessed that perhaps objects should be specified by their vectorial displacement from the subject, but I have very little confidence in my guess and also cannot locate this information either in the Methods or the software website. For Figure 5F it is mentioned that BonVision can be used to implement running down a virtual corridor for a mouse, so if some description can be provided of what the user has to do to implement this and what is done by the software package, that may address my confusion. If BonVision is indeed not a full 3D spatial engine, it would be important to mention these design/intent differences in the introduction as well as Supplementary Table 1.

Thank you for prompting us to do this. BonVision does indeed essentially render the view of an avatar in a virtual world (or multiple views, of multiple avatars), without any additional coding required by the user. We have now included in the new “Supplementary Details” specific pathways to the construction and rendering of 3D scenes. We have avoided the use of the terminology ‘game-engine’ as it has a particular definition that most softwares do not satisfy.

More generally, it would be useful to provide an overview of what the closed-loop rendering procedure is, perhaps including a Figure (different from Supplementary Figure 2, which seems to be regarding workflow but not the software platform structure). For example, I imagine that after the user-specified texture/object resources have been loaded, then some engine runs a continual loop where it somehow decides the current scene. As a user, I would want to know what this loop is and how I can control it. For example, can I induce changes in the presented stimuli as a function of time, whether this time-dependence has to be prespecified before runtime, or can I add some code that triggers events based on the specific history of what the subject has done in the experiment, and so forth. The ability to log experiment events, including any viewpoint changes in 3D scenes, is also critical, and most experimenters who intend to use it for neurophysiological recordings would want to know how the visual display information can be synchronized with their neurophysiological recording instrumental clocks. In sum, I would like to see a section added to the text to provide a high-level summary of how the package runs an experiment loop, explaining customizable vs. non-customizable (without directly editing the open source code) parts, and guide the user through the available experiment control and data logging options.

We have now added a brief paragraph regarding the basic structure of a BonVision program, and how to ‘close the loop’ in the new “Supplementary Details”.

Having some experience myself with the tedium (and human-dependent quality) of having to adjust either the experimental hardware or write custom software to calibrate display devices, I found the semi-automated calibration capabilities of BonVision to be a strong selling point. However I did not manage to really understand what these procedures are from the text and Figure 2C-F. In particular, I'm not sure what I have to do as a user to provide the information required by the calibration software (surely it is not the pieces of paper in Fig. 2C and 2E..?). If for example, the subject is a mouse head-fixed on a ball as in Figure 1E, do I have to somehow take a photo from the vantage of the mouse's head to provide to the system? What about the augmented reality rig where the subject is free to move? How can the calibration tool work with a single 2D snapshot of the rig when e.g. projection surfaces can be arbitrarily curved (e.g. toroidal and not spherical, or conical, or even more distorted for whatever reasons)? Do head-mounted displays require calibration, and if so how is this done? If the authors feel all this to be too technical to include in the main text, then the information can be provided in the Methods. I would however vote for this as being a major and important aspect of the software that should be given air time.

We have a dedicated webpage going through the step-by-step protocol for the automated screen calibration. We now explicitly point to this page in the new Supplementary Details section.

As the hardware-limited speed of BonVision is also an important feature, I wonder if the same ~2 frame latency holds also for the augmented reality rendering where the software has to run both pose tracking (DeepLabCut) as well as compute whole-scene changes before the next render. It would be beneficial to provide more information about which directions BonVision can be stressed before frame-dropping, which may perhaps be different for the different types of display options (2D vs. 3D, and the various display device types). Does the software maintain as strictly as possible the user-specified timing of events by dropping frames, or can it run into a situation where lags can accumulate? This type of technical information would seem critical to some experiments where timings of stimuli have to be carefully controlled, and regardless one would usually want to have the actual display times logged as previously mentioned. Some discussion of how a user might keep track of actual lags in their own setups would be appreciated.

We now provide this as part of the Supplementary Details, specifically animation and timing lags.

On the augmented reality mode, I am a little puzzled by the layout of Figure 3 and the attendant video, and I wonder if this is the best way to showcase this functionality. In particular, I'm not entirely sure what the main scene display is although it looks like some kind of software rendering — perhaps of what things might look like inside an actual rig looking in from the top? One way to make this Figure and Movie easier to grasp is to have the scene display be the different panels that would actually be rendered on each physical panel of the experiment box. The inset image of the rig should then have the projection turned on, so that the reader can judge what an actual experiment looks like. Right now it seems for some reason that the walls of the rig in the inset of the movie remain blank except for some lighting shadows. I don't know if this is intentional.

Because we have had limited experimental capacity in this period, we only simulated a real-time augmented reality environment off-line, using pre-existing videos of animal behaviour. We think that the comment above reflects a misunderstanding of what the Figure and associated Supplementary Movie represents, and we realise that their legends were not clear enough. We have now made sure that these legends make clear that these are based on simulations (new legends for Figure 3 and Figure 3 - video supplement 1).

Reviewer #3 (Public Review):

Summary:

This is a tools paper that describes an open source software package, BonVision, which aims to provide a non-programmer-friendly interface for configuring and presenting 2D as well as 3D visual stimuli to experimental subjects. A major design emphasis of the software is to allow users to define visual stimuli at a high level independent of the actual rendering physical devices, which can range from monitors to curved projection surfaces, binocular displays, and also augmented reality setups where the position of the subject relative to the display surfaces can vary and needs to be adjusted for. The package provides a number of semi-automated software calibration tools to significantly simplify the experimental job of setting up different rigs to faithfully present the intended stimuli, and is capable of running at hardware-limited speeds comparable to and in some conditions better than existing packages such as Psychtoolbox and PsychoPy.

Major comments:

While much of the classic literature on visual systems studies have utilized egocentrically defined ("2D") stimuli, it seems logical to project that present and future research will extend to not only 3D objects but also 3D environments where subjects can control their virtual locations and viewing perspectives. A single software package that easily supports both modalities can therefore be of particular interest to neuroscientists who wish to study brain function in 3D viewing conditions while also referencing findings to canonical 2D stimulus responses. Although other software packages exist that are specialized for each of the individual functionalities of BonVision, I think that the unifying nature of the package is appealing for reasons of reducing user training and experimental setup time costs, especially with the semi-automated calibration tools provided as part of the package. The provisions of documentation, demo experiments, and performance benchmarks are all highly welcome and one would hope that with community interest and contributions, this could make BonVision very friendly to entry by new users.

Given that one function of this manuscript is to describe the software in enough detail for users to judge whether it would be suited to their purposes, I feel that the writing should be fleshed out to be more precise and detailed about what the algorithms and functionalities are. This includes not shying away from stating limitations -- which as I see it, is just the reality of no tool being universal, but because of that is one of the most important information to be transmitted to potential users. My following comments point out various directions in which I think the manuscript can be improved.

The biggest point of confusion for me was whether the 3D environment functionality of BonVision is the same as that provided by virtual spatial environment packages such as ViRMEn and gaming engines such as Unity. In the latter software, the virtual environment is specified by geometrically laying out the shape of the traversable world and locations of objects in it. The subject then essentially controls an avatar in this virtual world that can move and turn, and the software engine computes the effects of this movement (i.e. without any additional user code) then renders what the avatar should see onto a display device. I cannot figure out if this is how BonVision also works. My confusion can probably be cured by some additional description of what exactly the user has to do to specify the placement of 3D objects. From the text on cube mapping (lines 43 and onwards), I guessed that perhaps objects should be specified by their vectorial displacement from the subject, but I have very little confidence in my guess and also cannot locate this information either in the Methods or the software website. For Figure 5F it is mentioned that BonVision can be used to implement running down a virtual corridor for a mouse, so if some description can be provided of what the user has to do to implement this and what is done by the software package, that may address my confusion. If BonVision is indeed not a full 3D spatial engine, it would be important to mention these design/intent differences in the introduction as well as Supplementary Table 1.

More generally, it would be useful to provide an overview of what the closed-loop rendering procedure is, perhaps including a Figure (different from Supplementary Figure 2, which seems to be regarding workflow but not the software platform structure). For example, I imagine that after the user-specified texture/object resources have been loaded, then some engine runs a continual loop where it somehow decides the current scene. As a user, I would want to know what this loop is and how I can control it. For example, can I induce changes in the presented stimuli as a function of time, whether this time-dependence has to be prespecified before runtime, or can I add some code that triggers events based on the specific history of what the subject has done in the experiment, and so forth. The ability to log experiment events, including any viewpoint changes in 3D scenes, is also critical, and most experimenters who intend to use it for neurophysiological recordings would want to know how the visual display information can be synchronized with their neurophysiological recording instrumental clocks. In sum, I would like to see a section added to the text to provide a high-level summary of how the package runs an experiment loop, explaining customizable vs. non-customizable (without directly editing the open source code) parts, and guide the user through the available experiment control and data logging options.

Having some experience myself with the tedium (and human-dependent quality) of having to adjust either the experimental hardware or write custom software to calibrate display devices, I found the semi-automated calibration capabilities of BonVision to be a strong selling point. However I did not manage to really understand what these procedures are from the text and Figure 2C-F. In particular, I'm not sure what I have to do as a user to provide the information required by the calibration software (surely it is not the pieces of paper in Fig. 2C and 2E..?). If for example, the subject is a mouse head-fixed on a ball as in Figure 1E, do I have to somehow take a photo from the vantage of the mouse's head to provide to the system? What about the augmented reality rig where the subject is free to move? How can the calibration tool work with a single 2D snapshot of the rig when e.g. projection surfaces can be arbitrarily curved (e.g. toroidal and not spherical, or conical, or even more distorted for whatever reasons)? Do head-mounted displays require calibration, and if so how is this done? If the authors feel all this to be too technical to include in the main text, then the information can be provided in the Methods. I would however vote for this as being a major and important aspect of the software that should be given air time.

As the hardware-limited speed of BonVision is also an important feature, I wonder if the same ~2 frame latency holds also for the augmented reality rendering where the software has to run both pose tracking (DeepLabCut) as well as compute whole-scene changes before the next render. It would be beneficial to provide more information about which directions BonVision can be stressed before frame-dropping, which may perhaps be different for the different types of display options (2D vs. 3D, and the various display device types). Does the software maintain as strictly as possible the user-specified timing of events by dropping frames, or can it run into a situation where lags can accumulate? This type of technical information would seem critical to some experiments where timings of stimuli have to be carefully controlled, and regardless one would usually want to have the actual display times logged as previously mentioned. Some discussion of how a user might keep track of actual lags in their own setups would be appreciated.

On the augmented reality mode, I am a little puzzled by the layout of Figure 3 and the attendant video, and I wonder if this is the best way to showcase this functionality. In particular, I'm not entirely sure what the main scene display is although it looks like some kind of software rendering — perhaps of what things might look like inside an actual rig looking in from the top? One way to make this Figure and Movie easier to grasp is to have the scene display be the different panels that would actually be rendered on each physical panel of the experiment box. The inset image of the rig should then have the projection turned on, so that the reader can judge what an actual experiment looks like. Right now it seems for some reason that the walls of the rig in the inset of the movie remain blank except for some lighting shadows. I don't know if this is intentional.

Reviewer #1 (Public Review):

In this project, the authors set out to create an easy to use piece of software with the following properties: The software should be capable of creating immersive (closed loop) virtual environments across display hardware and display geometries. The software should permit easy distribution of formal experiment descriptions with minimal changes required to adapt a particular experimental workflow to the hardware present in any given lab while maintaining world-coordinates and physical properties (e.g. luminance levels and refresh rates) of visual stimuli. The software should provide equal or superior performance for generating complex visual cues and/or immersive visual environments in comparison with existing options. The software should be automatically integrated with many other potential data streams produced by 2-photon imaging, electrophysiology, behavioral measurements, markerless pose estimation processing, behavioral sensors, etc.

To accomplish these goals, the authors created two major software libraries. The first is a package for the Bonsai visual programming language called "Bonsai.Shaders" that brings traditionally low-level, imperative OpenGL programming into Bonsai's reactive framework. This library allows shader programs running on the GPU to seamlessly interact, using drag and drop visual programming, with the multitude of other processing and IO elements already present in numerous Bonsai packages. The creation of this library alone is quite a feat given the complexities of mapping the procedural, imperative, and stateful design of OpenGL libraries to Bonsai's event driven, reactive architecture. However, this library is not mentioned in the manuscript despite its power for tasks far beyond the creation of visual stimuli (e.g. GPU-based coprocessing) and, unlike BonVision itself, is largely undocumented. I don't think that this library should take center stage in this manuscript, but I do think its use in the creation of BonVision as well as some documentation on its operators would be very useful for understanding BonVision itself.

Following the creation of Bonsai.Shaders, the authors used it to create BonVision which is an abstraction on top of the Shaders library that allows plug and play creation of visual stimuli and immersive visual environments that react to input from the outside world. Impressively, this library was implemented almost entirely using the Bonsai visual programming language itself, showcasing its power as a domain-specific language. However, this fact was not mentioned in the manuscript and I feel it is a worthwhile point to make. The design of BonVision, combined with the functional nature of Bonsai, enforces hard boundaries between the experimental design of visual stimuli and (1) the behavioral input hardware used to drive them, (2) the dimensionality of the stimuli (i.e. 2D textures via 3D objects), (3) the specific geometry of 3D displays (e.g. dual monitors, versus spherical projection, versus head mounted stereo vision hardware), and (4) automated hardware calibration routines. Because of these boundaries, experiments designed using BonVision become easy to share across labs even if they have very different experimental setups. Since Bonsai has integrated and standardized mechanisms for sharing entire workflows (via copy paste of XML descriptions or upload of workflows to publicly accessible Nuget package servers), this feature is immediately usable by labs in the real world.

After creating these pieces of software, the authors benchmarked them against other widely used alternatives. IonVisoin met or exceeded frame rate and rendering latency performance measures when compared to other single purpose libraries. BonVision is able to do this while maintaining its generality by taking advantage of advanced JIT compilation features provided by the .NET runtime and using bindings to low-level graphics libraries that were written with performance in mind. The authors go on to show the real-world utility of BonVision's performance by mapping the visual receptive fields of LFP in mouse superior colliculus and spiking in V1. The fact that they were able to obtain receptive fields indicates that visual stimuli had sufficient temporal precision. However, I do not follow the logic as to why this is because the receptive fields seem to have been created using post-hoc aligned stimulus-ephys data, that was created by measuring the physical onset times of each frame using a photodiode (line 389). Wouldn't this preclude any need for accurate stimulus timing presentation?

Finally the authors use BonVision to perform one human psychophysical and several animal VR experiments to prove the functionality of the package in real-world scenarios. This includes an object size discrimination task with humans that relies on non-local cues to determine the efficacy of the cube map projection approach to 3D spaces (Fig 5D). Although the results seem reasonable to me (a non-expert in this domain), I feel it would be useful for the authors to compare this psychophysical discrimination curve to other comparable results. The animal experiments prove the utility of BonVision for common rodent VR tasks.

In summary, the professionalism of the code base, the functional nature of Bonsai workflows, the removal of overhead via advanced JIT compilation techniques, the abstraction of shader programming to high-level drag and drop workflows, integration with a multitude of input and output hardware, integrated and standardized calibration routines, and integrated package management and workflow sharing capabilities make Bonsai/BonVision serious competitors to widely-used, closed-source visual programming tools for experiment control such as LabView and Simulink. BonVision showcases the power of the Bonsai language and package management ecosystem while providing superior design to alternatives in terms of ease of integration with data sources and facilitation of sharing standardized experiments. The authors exceeded the apparent aims of the project and I believe BonVision will become a widely used tool that has major benefits for improving experiment reproducibility across laboratories.

stratigies

Small individual communities all making and promoting things can be a powerful thing.

Where have we gone wrong?

I wonder just how successful the production of natural medicines can/will be. There is such a large pharmacological industry, and I can only imagine what would happen if those drugs were to be replaced rapidly by natural medicines. In turn, I think it would be amazing to get more natural medicines that work, in my head I have this idea that there would be fewer side effects, and if that was the case then that would be reason enough to work towards a more natural medicine path.

Here, the author makes reference to how Hurston uses narration to slip in and out of the past whilst telling of the story of Janie's life. Usually, I don't like when the author chooses to do this. However, in this book I almost felt like it added to the story by showing how far Janie had come with being comfortable with her true self. Just wondering what other people think here. -Ollie

I find this moment rather beautiful but rather sad and somber still. It is very obvious Lizzie loves Eugene even though she believes they could not be together due to status. Eugene believes this yet wanting one last time almost as an act of closure, he asks her that perhaps in a different life could they be together. Although the answer Lizzie gives is vague, I think Eugene believes it to be true. He follows on his promise and leaves her with a bittersweet feeling having grown so much after meeting with Lizzie.

I read this part in my head like a movie scene. It sounds like a mystery at first, but then you hear the whole situation which makes more sense. It is also kind of creepy at the same time.

I forgot that Bella doesn't know John's real identity yet. I wonder whether this will cause problems later on when she presumably does find out, or whether Bella will understand why he kept it from her.

This is a touching moment when Mr. Rokesmith shows up and I quote "takes Bella in his arms."

Charles Dickens is incredible for his eye for romance, he explains in the writing that Rokesmith realizes that Bella has given up her life of fortune for him, and the two of them decide they'll be together forever. This is a big scene and shoulf be noted as such. Obviously with Mr, Wilfer standing right there witnessing everything, the two of them turn to him first and ask his consent, which he gives and after reading I was filled with joy.

I think it's really interesting how we get to see two men being rather despicable with each other in different but similar forms. Jenny's father wishes to sate his desire for more alcohol and is willing to go long and somewhat evil ways to do so, providing information about Lizzie that Eugene shouldn't have for beer money. Eugene on the other hand wishes to sate his desire to find and speak to Lizzie and similarly is welcome to oblige in the somewhat evil ways of fueling a drunkard's addiction and paying for information on the girl he wishes to speak to.

In this passage, I felt the tension between these two men rising at a very quick rate. Dickens does a good job at creating a bickering atmosphere between Wegg and Venus. It is almost as if a clock struck "blackmail time" during this long conversation. Their final conclusion that they come to seems the most fair for the two of them, given the utmost complications.

Betty's death is both a relief and a tragedy at the same time. She dies completely free and without being a burden to anyone. Her character is very adamant about remaining independent and standing on her own. However, her desire for independence also became her insistence for isolation. Betty very nearly dies alone, and the only reason she doesn't is because Lizzie just happens to come across her.

OK WHOA! The guy is obviously taking a lonely stroll by himself and he is taking about the things in his head. He goes over different things that have happened to him and then he talks to himself in third person and IT'S JOHN HARMON! He is the heir of the Harmon fortune and we all thought that he was dead. This is a huge moment and my jaw dropped.

This stranger has created an enormous amount of suspense for the reader with these remarks he leaves. The tension between him and Riderhood in this entire excerpt is at an all time high in these last few statements of their conversation. What could he possibly mean by "sharing" the reward with Riderhood? It seems to me from his evil tones that this cannot be good news for Riderhood.

Here Sloppy combines good etiquette and just being a child. He makes sure to be respectful and show he's grateful then immediately loses his composure for a second to celebrate in a very silly and child-like manner. It's quite a funny scene in a novel that deals with a lot of intense topics.

Imagery O’Brien is describing the day he got drafted by the government to go to war and describe how he felt being drafted there which was bad feeling to him

Reviewer #1 (Public Review):

It is difficult to overestimate the importance of this paper. The full connectome of the Drosophila central complex is both the beginning and the end of an era. It provides the first comprehensive dataset of arguably the most enigmatic brain region in the insect brain. This endeavor has generated ground truth data for years of functional work on the neural circuits the connectome outlines, and constitutes an unparalleled foundation for exploring the structure function relations in nervous systems in general. This will be of great importance far beyond work on the Drosophila brain, and will have far reaching implications for comparative research on insect brains and likely also smoothen the path toward understanding navigation circuits in vertebrate nervous systems. Based on presented data, the paper develops overarching ideas (at exquisite detail) of how sensory information is transformed into head direction signals, how these signals are used to enable goal direction behavior, how goals are represented, and how internal state can modulate these processes. The connectome enables the authors to base these ideas and their detailed models on actual biological data, where earlier work was forced to indirectly infer or speculate. While significantly going beyond models of central-complex function that existed previously, the authors have to be much credited for incorporating huge amounts of existing knowledge and data into their interpretations, not only work from Drosophila, but also from many other insects. This makes this paper not only an invaluable resource on the connectome of the Drosophila central complex, but also a most comprehensive review on the current state of the art in central-complex research. This unifying approach of the paper clearly marks a reset of central-complex research, essentially providing a starting point of hundreds of new lines of enquiry, probably for decades to come.

Given the type and amount of data presented, the paper is clearly overwhelming. That said, it also clearly needs to be presented in the way it was done, mostly because no single aspect of the function of this neuropil makes as much sense in isolation as it makes sense when viewed in conjunction of all its other functions. The complexity of the neural circuits discussed is clearly reflected in the enormous scope of the paper. Nevertheless, the authors have done a fantastic job in breaking the circuits and their function down into digestible bits. The manuscript is very systematic in its approach and starts with sensory pathways leading to the CX, covering the clearly delineated head direction circuits and then moving on to the more complex and less understood parts, always maintaining a clear link between structure and function. As function is necessarily based on previous work, including that from other species, the results part is interwoven with interpretation, but this is clearly necessary to keep the text readable. The authors have made considerable efforts to provide additional introductions and summaries whenever needed, almost creating nested papers embedded within the overall paper.

The figures are equally overwhelming as the text at first sight, but when taking the time to digest each one in detail, they present the data in a rich and clear manner. The figures are often encyclopedic and will serve as reference about the central complex for years. The summary graphs that are presented in regular intervals are welcome resting places for the reader, helping to digest all the detailed information that has preceded or that will follow.

The analysis performed in the paper is excellent, comprehensive and should set the standard for any future work on this topic. Also, the text is very honest about the limits of the conclusions that can be reached based on this kind of data, which is important in generating realistic and feasible hypotheses for future experiments.

Hox genes are responsible for putting the genes that are needed for the basic structure of an organism together.

Reviewer #4 (Public Review):

Using a transgenic line of Platynereis, in which GFP is expressed under the control of cis-regulatory elements for r-opsin, the study isolates r-opsin expressing cells from the head (eye photoreceptors) and trunk region (a population of segmentally repeated r-opsin expressing cells associated with the parapodia) by FACS. Subsequent RNA-Seq establishes that both populations of cells express genes for all components of the rhabdomeric phototransduction cascade, while the population of trunk sensory cells additionally expresses genes encoding proteins involved in mechanosensation. Using heterologous expression in a mammalian cell line, it is shown that the Platynereis r-opsin responds to blue light via coupling to Gαq suggesting that it mediates photoresponses via a canonical rhabdomeric phototransduction cascade. Transcriptomic analysis of an r-opsin mutant created by TALEN mediated gene editing then reveals that expression levels of the mechanosensory Atp2b channel are modulated by protracted exposure to blue light, a response abolished in the mutant. Behavioral analysis further suggests that undulatory movements of the worms are equally altered under these illumination conditions. Taken together this suggests that the r-opsin expressing trunk sensory cells act as both photo- and mechanoreceptors and that their mechanosensory properties are modulated in response to light. In combining the transcriptomic analysis of cell types with experimental studies of gene function and behavioral analyses, this study provides exciting new insights into the evolution of sensory cells. Several prior studies have found co-expression of photosensory and mechanosensory proteins in sensory cells of various bilaterians, and comparative studies suggested that photo- and mechanosensory cells may share a common evolutionary origin. However, the current study goes far beyond these findings in establishing a direct functional link between photo-and mechanosensation in a population of sensory cells suggesting that these sensory cells function as multimodal cells and that their mechanosensory properties are altered in response to light. Furthermore, the behavioral data (based on a novel machine-learning based tool of analysing the animals' movement) suggest that these cells have a behaviorally relevant function. Because r-opsin was found to be expressed in mechanoreceptors not only in lophotrochozoans (including Platynereis) but also in ecdysozoans and vertebrates (although functional studies are lacking here) and r-opsins belong to a large family of opsins, almost all of which are responsive to light, the present study suggests that r-opsins may have an ancestral bilaterian role in modulating mechanosensory function in response to light (in addition to their purely photosensory role in the photoreceptors of the eyes). Light-independent functions of r-opsin as recently revealed in Drosophila may, thus, be secondarily derived.

The study is very carefully conducted and well presented. The only minor flaw is that in its present form, the discussion of the evolutionary implications of the finding lacks in clarity and specificity. The authors here often refer ambiguously to an "ancient" or "ancestral" role of r-opsins without specifying the lineage referred to (ancestral for lophotrochozoans? bilaterians? eumetazoans? metazoans?). The discussion should, therefore be revised with an explicit phylogenetic framework in mind.

Reviewer #3 (Public Review):

Opsin proteins are ancient light-sensitive molecules found in photoreceptor cells throughout the animal kingdom. Recent discoveries including those made in the current paper have revealed that besides r-opsins, some classes of photoreceptor cell also express genes that are found in mechanosensory cells, and that r-opsins have both light-dependent and light-independent effects on mechanical force transduction or motion. A question remains as to whether or not: 1) a protosensory cell of animals existed which contained both photoreceptor and mechanoreceptor-like features and, 2) whether the original function of opsin included light-dependent mechanosensory features? The authors consider three competing hypotheses for the cellular evolution of photoreceptor and mechanosensory function. Two of the hypotheses envision either photo- or mechanosensory function for opsins evolving first, the third imagines them evolving simultaneously. The authors note that the majority of what we know about rhabdomeric opsins comes from studying the eye photoreceptors of the fruit fly, Drosophila melanogaster. But might this kind of photoreceptor have functions that are derived compared to the ancestral photoreceptor cell? To investigate this question, the authors turn to the non-model system, Platynereis dumerilii, which has both head and non-head photoreceptors. Here the authors use 1) a fluorescent cell sorting method to perform RNA profiling of eye and trunk photoreceptor cells of a mutant marine worm and find evidence of co-expression of photo- and mechanosensory genes in photoreceptor cells. They also compare the genes that are expressed in Platyneris photoreceptors with genes expressed in Drosophila JO (hearing organ in flies), Zebrafish lateral lines and mouse IEH (inner ear hair) cells, and again they find some commonly-expressed genes. 2) The authors use cell culture to express the opsin, demonstrate that it interacts with G-alphaq, and that it's peak sensitivity is in the blue range. 3) They use in situ hybridization to validate the RNA-seq and detect select enriched transcripts in the photoreceptor tissues. 4) They use a new method, which should be widely useful to other researchers, to detect undulation behavior of the opsin mutant vs. wildtype worms and show that the mutant worm behavior is perturbed in altered light cycles. Taken together, the authors suggest that an ancient light-dependent function of opsin was linked to mechanosensation and that light-independent mechanosensory functions of opsins evolved secondarily. The interpretation is somewhat reasonable given the available data but does not yet entirely rule out other possibilities (see below).

This paper is a tour-de-force and a really impressive collection of experiments which examines the function of r-opsin in Platyneris. There's lots of innovation here from the use of fluorescent cell sorting and cell-specific RNA-Seq on a non-model system to the deep-learning based approach to examining behavior. Overall, the authors' interpretation of their data seems reasonable however I do believe a even stronger case could be made that what we are talking about is shared ancestry vs. recent recruitment if the authors made phylogenetic trees of the numerous TRE genes that are enriched between Drosophila JO and mouse IEH cells. If a significant number of these genes were true orthologs vs. paralogs across all three species then this would provide stronger evidence of an ancient light-dependent mechanosensory function for r-opsin. GO enrichment terms, while intriguing and suggestive, don't go far enough into the weeds. Also, I think the estimate of there being only 12 genes involved in making a photoreceptor cell able to detect light is probably an underestimate, as this ignores, for example, the understudied molecular machinery required for chromophore metabolism and transport. At the very least, the work should help inspire vigorous debate between vision and auditory neuroscience communities (which do not usually converse with one another) to more carefully consider the ways in which their systems overlap and why.

Reviewer #2 (Public Review):

Rhabdomeric Opsins (r-Opsins) are well known for their role in photon detection by photosensory cells which are commonly found in eyes. However, r-Opsin expression has also been detected in non-photosensory cells (e.g., mechanosensors), but their function(s) in these other sensory cells is less well understood. To explore the function of r-Opsins outside the context of an eye/head (non-cephalic function) as well as to investigate the potential evolutionary path by which sensory systems that rely on r-Opsins have evolved, Revilla-i-Domingo et al. have investigated gene expression in two distinct subsets of r-Opsin expressing cells in the marine bristle worm Platynereis dumerilii : EP (eye photoreceptor) and TRE (trunk r-opsin1 expressing) cells. The authors also generate two Pdu-r-Opsin1 mutant strains in order to investigate how the loss of r-Opsin function affects gene expression and behavior.

The question of what role r-Opsins play outside of photoreceptors is an interesting one that remains poorly understood. In this manuscript, the authors demonstrate a powerful protocol for FACS sorting and sequencing different cell populations from an important evolutionary model organism.

The transcriptomic analysis presented here demonstrates that both the cephalic EP cells and the non-cephalic TRE cells express components of the photosensory transduction pathway. This observation, together with heterologous cell expression data presented demonstrating sensitivity of Pdu-r-Opsin1 to blue light, suggests that both EP and TRE cells are likely to be light sensitive. The authors also suggest that they observe "mechanosensory signatures" in the transcriptomes, which, together with the analysis of undulatory movements in headless animals, lead them to suggst that r-Opsin in TRE cells functions as an evolutionarily conserved light-dependent modulator of mechanosensation, a conclusion that is not well-supported by the data presented.

Overall, many of the conclusions drawn from the transcriptome data are inferential and based on weak evidence. Key limitations are listed below:

1) The apparent overlap between the phototransduction and mechanosensory systems has already been shown (in Drosophila for instance) and the current work adds limited information to this story, and what is added is weakened by the absence of functional and physiological analyses. This is particularly true for supporting the claims of mechanosensory signatures in these cells. For example, genes whose expression is suggested in the text as being indicative of a mechanosensory function (glass and waterwitch) are, in fact, expressed in multiple sensory cell types. Glass (gl) is a transcription factor best known for regulating the expression of phototransduction proteins in photoreceptors. The function of waterwitch (wtrw) is not fully understood, but it is broadly expressed in sensory cells in Drosophila. It would be more compelling if mechanotransduction channels like Piezo and NompC were expressed in the TREs, but there is no mention of this.

2) The suggestion that the TRE cells share similarity with the mechanosensitive mammalian inner ear is provocative, but lacks strong support. For instance, physiological characterization of the response properties of these sensory cells or identification of anatomical similarities analogous to the stereocilia upon which hair cell mechanosensitivity is based would greatly increase plausibility of this claim. Particularly for a species that diverged from mice and flies many hundreds of millions of years ago, speculation based largely on transcriptome analysis is risky. Careful validation is required as identified genes might not share a conserved function with their assigned orthologs in mice and Drosophila.

3) The current analysis lacks sufficient power to make compelling claims with regard to potential ancestral protosensory cells. The investigators are examining a single species of marine worm and doing so without detailed anatomical and functional studies of the r-Opsin-expressing cells in the worm.

4) The behavioral experiments require more functional data to interpret unambiguously. The data indicate that r-opsin1 is required for light to surpress the undulation of decapitated worms. Does this mean that the TREs are photosensors whose activity inhibits locomotion or that the TREs are light-sensitive mechanosensors ?

5) It is assumed that the TREs constitute a homogenous cell population, but this is not demonstrated. This means that the TREs could be a mixed population (for example, distinct sets of photosensors and mechanosensors) and some of the TRE-expressed genes identified could be expressed in different specific subset of TREs.

Add elem to Head is O(1) while add to tail is O(n), n is the elem in the list.

EZH2 is ubiquitinated.

The output does not seem to be correct here.

You can simplify the task of learning 30 words by simply going over them in your head over and over again until you get them right, and of course, putting each word into practice as it will retain in your memory better.

I am glad that this detail was included in the article. Being someone who has worked with a disabled family member my entire life, I have seen how frustrating it can be when they are not provided the necessary assistive technology.

SciScore for 10.1101/2020.06.24.20139006: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

This study has several limitations. Although this study was based on well characterized controls, we cannot definitively extrapolate findings to other populations [28]. For example, the sensitivity of these assays was assessed in samples from RT-PCR confirmed hospitalized patients with samples collected a mean of 16 days (SD 5) after symptom onset. Given most SARS-CoV-2 infections are mild or asymptomatic and do not require hospitalization, and given that little is known about humoral kinetics >2 months post infection, the sensitivity of these assays may be lower in individuals > 2 months post-infection and/or individuals with mild or asymptomatic infections that likely mount less durable immune responses [26,28]. We did not include negative control samples from individuals with confirmed common coronaviruses, which may increase cross reactivity and false positives. Our study findings align with manufacturer data for the Roche Diagnostics’ Elecsys Anti-SARS-CoV-2 IgG immunoassay and confirm that false positives are rare, specificity is high, and sensitivity is excellent > 3 weeks after symptom onset, but lower early in the disease course and among immunocompromised individuals. The Epitope Diagnostics IgG immunoassay has lower specificity and sensitivity than data released by the manufacturer, but overall performed well with few false positives and high sensitivity. The Epitope IgM had more false positives and lower sensitivity and given IgM does not appear to rise substantia...

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

SciScore for 10.1101/2020.07.22.20160432: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

No key resources detected.

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Limitations of this review are that conclusions are mostly from indirectness evidence, with direct evidence available from only four head-to-head trials, and that it was not possible to estimate the dose of hand hygiene for some trials. A recent Cochrane review of the effect of rinse-free handwashing, compared to traditional hand hygiene, on absenteeism for ARI in preschool and school children reported a significant reduction in absenteeism of 9 days per 1000 available days for children in the rinse-free group, with the results coming from six randomised trials [4]. The effectiveness of handwashing with materials other than sanitiser or soap and water, such as ash, which may be used in low-income countries has mostly been examined in observational studies with uncertain effects [25].

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

SciScore for 10.1101/2020.08.12.20173856: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

To overcome the limitation of an imperfect positive reference standard and to improve inferences, the relative performance of these tests was evaluated by head-to-head comparison. RBD ELISA and DiaSorin CLIA were able to identify more positive IgG sera/plasma than Zydus Kavach. However, RBD ELISA is an in-house ELISA developed using similar sample collections. While the sample panel used for evaluation was independent of that used in the development of the RBD ELISA, its true test would be when it is evaluated externally. Highly sensitive serological assays can assess immune response to SARS-CoV-2 and are needed to determine the extent of spread of the virus, which in turn is critical for assessing case fatality rates and herd immunity. Serological assays also help in assessing development of herd immunity to devise community management strategies that are of crucial importance at this time, and will continue to be relevant in the coming years. Other uses of serological assays can be to assess exposure in high-risk populations such as healthcare workers and assess vaccination strategy at state or national level. Cross-reactivity with SARS-CoV and seasonal coronaviruses in different population and timing of IgM and IgG responses need to continue to be considered. Till date, studies on comparative performance serological assays for SARS-CoV-2 show a range of sensitivity of 84-98% and specificity of 96-99% [12-29]. Two of the three IgG assays in this study used the Spike protein...

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

SciScore for 10.1101/2020.08.13.248351: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Nevertheless, the inclusion of immunoadhesin potentials of the antiviral may have caveats. While it may certainly boost immune clearance of the virus, such as through FcγR’s selective binding of clustered Fc, it may also elevate complement and cytokine responses to further aggravate the inflammation. Although these adverse side effects can be mitigated through modifications of the Fc domain, the ultimate therapeutic effects in the context of individual patients’ conditions can only be determined through rigorous clinical studies. With regard to drug toxicity, our mouse study of repeated doses of ACE2-Fc in mice had showed the biologic to be well tolerated for up to two months[13]. However, we cannot extrapolate that its will also be safe COVID-19 patients. As we consider it is not a simple neutralizing agent of the virus, its bifurcated ACE2 head groups can possibly trigger agglutination of the virus that can potentially aggravate the hypercoagulable state, making the drug less tolerable in these conditions. From the perspective of recombinant manufacturing, there are challenges ahead for the simple fact that ACE2-Fc is a large protein (∼130 kDa as a monomer and ∼260 kDa as a dimer). Furthermore, cocrystal structures of ACE2 with an inhibitor showed large movements of the two lobes as compared to the Apo structures[40], suggesting an intrinsic instability of ACE2 protein. Also of note is an earlier study by Lei et al using double mutations of His374 and His378 of the zinc-bin...

Results from TrialIdentifier: We found the following clinical trial numbers in your paper:<br><table><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Identifier</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Status</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Title</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT04335136</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Completed</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Recombinant Human Angiotensin-converting Enzyme 2 (rhACE2) a…</td></tr></table>

Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 19, 20 and 21. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• No funding statement was detected.
• No protocol registration statement was detected.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

SciScore for 10.1101/2020.10.19.20213140: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

There are some limitations in this study. The technique evaluated considered swab position but did not evaluate duration of swab and number of swab twists performed. Participants were not asked if they had a swab performed on themselves, which may have improved their knowledge of accurate technique. Although an angle of 70 degrees was chosen to reflect clinical practice, alteration of head position in clinical settings may allow improved access to the nasopharynx. Only technique in nasopharyngeal swab was assessed but testing of specimens from multiple sites may improve overall sensitivity.20

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

SciScore for 10.1101/2020.11.02.364497: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

SciScore for 10.1101/2020.11.12.20230748: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

While we assess aggregate data from different studies to gain insight into these variables, a limitation of this meta-analysis is that true comparison is precluded in the absence of head-to-head studies. Furthermore, while there are trends we observe in our subgroup analyses, these findings may be population-related and should be interpreted with caution. Timing of sampling from symptom onset was also quite variable (collection occurred within days to weeks in some studies), and was inconsistently reported, which likely had a major impact on diagnostic performance given decreasing viral load over time. Head-to-head studies are urgently needed of flocked vs unflocked swabs (and specialized vs unspecialized swabs for MT collection), collected at different times in disease and with different sampling methods, and also in important subpopulations (e.g. children), to resolve the persistent uncertainty. We note that the reporting quality of studies was low, STARD guidelines (74) were not consistently followed, and study bias was considered moderate to high on QUADAS 2. Lastly, in this study we chose to report the % positive alternative-specimen, % positive comparator-specimen, and % dual positives instead of the positive percent agreement (PPA). This decision was motivated by our presumption regarding the low rate of false-positives using NAAT, and the potential for an alternative to yield more positive results than the comparator NP, which would otherwise not be considered. In sum...

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a protocol registration statement.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

SciScore for 10.1101/2020.12.07.20245696: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

A limitation of this work is that the timing of serum collection was not standardised, and that samples obtained were not tested equally across all assays due to limitations in sample volume and dead volume requirements of the automated EIAs. Nonetheless, the relatively large number of samples run remains a strength of this study, and allows for a head-to-head comparison of commercially available SARS-CoV-2 serological tests seen in few other studies. We and others have shown that commercially available serological assays for SARS-CoV-2 have varying performance that is dependent on both the platform and marker used. The assay chosen by end-users should be tailored to specific applications - for example, assays measuring antibodies to the nucelocapsid antigen might be best suited to disease surveillence as spike-protein based vaccines become available. During the COVID-19 pandemic, serological assays will be crucial in answering questions of immune protection against reinfection. If convalescent plasma or COVID-19 immunoglobulin is found to be a potentially effective therapeutic intervention, high throughput serological assays that closely correlate with neutralisation antibody levels are vital for scalability. As further testing platforms become available, validation studies such as this are needed to identify assays that inform on antibody titre and functionality.

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

SciScore for 10.1101/2020.12.08.20233056: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

No key resources detected.

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Increasing air flow rate from rates on one side led to more aerosols leaving the room per time interval, but on the other side induced more turbulent room air flow at the given geometry, favoring air mixing and thus potentially transporting aerosol-loaded air from non-breathing zones towards breathing zones; therefore, the benefit of just increasing ventilation thus has limitations that depend on the specific characteristics of a room and A/C system. Cough shields: Cough “shields” were partly effective in stopping forward-directed cough droplets but led to a redirection of the air stream carrying aerosols, including in the direction of adjacent beds and nurse workplaces at the side of the patient bed, an effect which is not desired. Also, air flow around obstacles is a well-known physical phenomenon36 that limits the effectiveness of such shields for aerosol plumes. Air filtering: Air purification by filtering with a high flow, HEPA filter equipped commercially available air filtering device, was examined. We found in modeling as well as in real-world experiments (E-cigarette smoke distribution) that such an air filtering device alone, even when positioned near the patient head, only has a limited capabililty for directly and quantitatively removing droplets/aerosols from the cough jet because the jet velocities away from the device were sufficiently high to overcome the modest pressure/velocity gradient produced by the device. Non patient centered air filtering has been show...

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

SciScore for 10.1101/2021.01.13.21249735: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

No key resources detected.

Results from OddPub: Thank you for sharing your code and data.

It may also be preferable to have a more sensitive cut-off for a primary test and confirm the positives with a highly-specific secondary test system in low prevalence settings.(17) Our study has several of limitations. Firstly, the samples are mostly derived from subjects with mild to no symptoms and therefore conclusions drawn here might best apply to an epidemiological rather than a diagnostic approach. Further evaluation using specimens from severely ill subjects or from high-prevalence settings will need to be conducted and compared to the enclosed results. Secondly, the dataset used to estimate optimised cut-offs and performance of the tests is not an unbiased random sample, as all were sampled in the city of Munich, Germany. It cannot be ruled out that blood donors are generally healthier than the general population. Furthermore, not all confirmatory tests were performed on all samples; only a subset, namely those with positive results in at least one primary test and a dedicated negative/positive cohort, were tested using these systems. Finally, we did not have information on underlying health conditions known to increase the quantity of polyclonal antibodies. In conclusion, our study provides a head-to-head cross-comparison of tests and can be used as a resource to enable the refinement of testing-strategies for individual and public-health use. We also investigated the correlations of the different test systems in-depth, considering confounders such as seasonality an...

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

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SciScore for 10.1101/2021.01.05.20248973: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

No key resources detected.

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Some acts of corporal punishment reported in our study might not be acts of child abuse, and caveat is advised in the interpretation of these findings. The associations between stress and verbal abuse and corporal punishment were weakest among caregivers living in villages on quarantine or restriction of movement with quarantined or ill family members. In other words, there seem to be effect modification by lockdown experience, although we are not quite sure of the mechanism behind it. It is possible that some of the household’s children were quarantined or treated and placed in an area designated by the state with chaprones instead of staying at home, which reduced the amount of contact between the caregivers and the children and, subsequently, the opportunities where abuses could occur. It is also possible that lockdown experiences were associated with economic or material hardship, which was found to be associated with parenting stress (Xu et al., 2020). In that regard, we did not directly ask participants about the extent that they experienced material hardship, and future studies should consider making such measurements using tools recently developed for household surveys (Fallon et al., 2020). In addition, the study area has experienced by decades-long armed conflict with as many as 20,000 casualties(Deep South Watch, 2020). Casualty was particularly heavy during the first decade of the conflict (Abuza, 2015; Human Rights Watch, 2007). The security situation deterred in...

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

About SciScore

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SciScore for 10.1101/2020.06.29.179192: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

No key resources detected.

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

A limitation of this study was that the same samples were not tested by the three different assays, so a head-to-head comparison of the three assays was not performed. This was due to the limit of available kits for routine testing in patient care. However, we did a head-to-head comparison of the assay’s workflow. The NeuMoDx 96 molecular system is a sample-to-answer and random-access platform that automatically performs nucleic acid extraction, amplification, and signal detection and analysis requiring only very little human interaction for loading and scanning the samples (20). It has the shortest TAT and highest throughput of the three assays. DiaSorin Simplexa CoV-2 Direct assay involves a simple operation procedure that does not include an extraction step; it has, however, much lower throughput than NeuMoDx. The modified CDC SARS-CoV-2 assay is singleplex; each target must be run in different tubes, as opposed to the two commercial assays, which are multiplex. It requires separate extraction steps on the EasyMag which can extract up to 24 samples at a time, longer hands-on time, and has much lower throughput compared to NeuMoDx. It has comparable throughput to DiaSorin if nucleic acid extraction and PCR reaction tubes for the next sample batch are prepared before the PCR cycles of the previous batch ends. Therefore, although the three assays have the same accuracy, the overall workflow favors the commercial platforms. In conclusion, diagnostic laboratories around the wor...

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

• No conflict of interest statement was detected. If there are no conflicts, we encourage authors to explicit state so.
• No funding statement was detected.
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About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

Art projects can be so complex. As the author mentions before, there are so many moving parts to this.

• web - gotcha : fontboosting

He is asking her what she remembers, does she remember a house, or a certain person? when she looks back what is the image that is painted in her head?

Women were sometimes degraded to a level near or as low as children in Western society. It is expected then for this to be taken to a grave extreme to ensure an enslaved woman of a "lesser race" would abide by semi-arbitrary rules. One such rule showcased here was to not speak unless prompted. Yet another is the menial task assigned to her: to cook dinner. While she was busy performing her task, men were likely outside doing rougher work that her master would not suffer her to take on. Lest she do it to a lesser degree of quality than it was believed a man could.

Question 2: I would absolutely engage with him about his thoughts on God and religion. Being "rejected" by both is religious family and the deity they worship is a massive factor of who he is and how he moves through life. Using the strengths approach, I could use this to help bolster his individuality and his personal strength since he rejects the others. Also, refuting and distrusting God, doesn't necessarily mean that he is not spiritual so engaging him in this conversation might lead to a spiritual response / treatment that was previously unseen and lumped with "useless religion" in his head.

claim

this relates to what I wrote about

Referencing the folk tale of straightening curly hairs to deter a devil.

Straightening of curly head hair.

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

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Reply to the reviewers

We thank the reviewers for their insightful comments and suggestions. Addressing them will improve our work. Please find below our point-by-points answers to the issues raised. We also provide a partially revised version of the manuscript with changes indicated in blue.

Reviewer #1 (Evidence, reproducibility and clarity (Required

**Summary**

The authors propose a mechanism through which voltage dependent water pore formation is key to the internalization of Cell permeable peptides (CPPs). The claim is based on an in-silico study and on several experimental approaches. The authors compare 5 peptides (R9, TAT-48-57, Penetratin, MAP and Transportan and use 3 distinct cell lines (Raji, SKW6.4 and HeLa cells), plus neurons in primary cultures. The also present in vivo experiment (mouse skin and zebrafish embryo). All in all, it is an interesting study, but it raises several issues that need to be addressed. Moreover, the length and structure of the manuscript make it very difficult to read (see below under "Reviewer statement")

**Reviewer statement**

The instructions are to use the "Major comments" section to answer 6 precise questions. Unfortunately, this is not possible due to the structure of the document to review. The main manuscript (22 pages) comes with 4 primary figures and 19 supplemental ones. Most of these figures have an enormous number of panels and their legends occupy 17 pages. To this, are added 6 supplemental tables and 7 supplemental movies (with 2 pages of legends), 28 pages of Material and Methods, and 146 References (109 for the main manuscript and 37 for Supplemental information). To be frank, I was often tempted to send the manuscript back, asking for the authors to submit a document facilitating the task of the reviewers.

Because of this complexity, my "Major comments" will come after a page by page, paragraph ({section sign}s) by paragraph and figure by figure "Detailed analysis" of the manuscript.

**Detailed analysis**

Q1. Page 4 {section sign} 3

The test is based on the ability of TAT-RasGAP to kill the cells. Although controls exist, this is worrying since necrotic death might participate in the rupture of the membrane and artificially amplify internalization after a first physiological entry of the peptide. It is also a bit dangerous to add a FITC group to a short peptide without controlling that it has no effect on the interaction with the membrane (FITC-induced local hydrophobicity can provoke peptide tilting and membrane shearing). In the same vein, the very high peptide concentrations often used in the study (40µM for Raji and SKW6.4 cells and 80µM on HeLa cells) can be highly toxic.

A1. We took advantage of the fact that TAT-RasGAP317-326 can kill cells to design a CRISPR/Cas9 screen based on cell survival for the identification of genes encoding proteins involved in CPP uptake. For this purpose, it was important therefore that the peptide was able to kill wild-type cells. Even if we consider the possibility that “necrotic death might participate in the rupture of the membrane and artificially amplify internalization after a first physiological entry of the peptide”, it remains that the cells that survived the screen did so because they were carrying mutations in genes that encoded potassium channels required for CPP uptake. And since the cells that survived the screen, by definition, were not dying, the issue raised by the reviewer is void in this case. The reviewer mentions that we included controls to validate the observations made with FITC-TAT-RasGAP317-326. Indeed, these controls were performed to address the potential problem raised by the reviewer. These controls, listed below, demonstrate that the genes identified through the CRISPR/Cas9 screen were also involved in the uptake of CPPs devoid of killing properties as well as CPPs that were not labelled with fluorophores.

i) Three different cell lines, lacking specific potassium channels identified through the CRISPR/Cas9 screen, were unable to allow a non-labelled, non-toxic CPP (TAT-PNA) to enter cells (Supplementary Fig. 8a).

ii) The Cre recombinase hooked to TAT, a construct that is not labelled with a fluorochrome and that is not toxic, did not enter Raji cells lacking the KCNQ5 potassium channels, also identified through a CRISPR/Cas9 screen (Supplementary Fig. 8b).

iii) The internalization of a TAT-conjugated FITC-labelled cell-protective therapeutic compound was inhibited, sometimes fully, in three different cell lines, lacking specific potassium channels identified through the CRISPR/Cas9 screen (Supplementary Fig. 8c).

Additionally, we are now reporting that the entry of FITC-labelled TAT, R9, and penetratin, all non-toxic CPPs, is impaired in Raji cells lacking the KCNQ5 potassium channel identified in the CRISPR/Cas9 screen. These new results will be incorporated in the revised version of our manuscript.

As supportive evidence that a potential toxicity effect of TAT-RasGAP317-326 is not a confounding factor in experiments recording the initial uptake of the peptide is that internalization is measured after one hour of incubation with the cells (Figure 1), time at which the peptide only minimally impacts the survival of cells (PNAS December 15, 2020 117 31871-31881).

Finally, please note that depolarizing cells, which is what happens in cells lacking the potassium channels identified through the CRISPR/Cas9 screen, not only blocked the uptake of TAT-RasGAP317-326, but also the uptake of a series of non-toxic CPPs (using short-time incubation protocols; Figure 2).

Page 5 {section sign} 1

Q2. Supp. Fig.1a shows no differences between the 3 cell types, even though they differ in their modes of peptide internalization, some favoring vesicular staining and others cytoplasmic diffusion.

A2. The images shown in panel A of this figure depicts, for each cell line, examples of cells that do not take up the CPP, those that only display vesicular staining, and those that additionally take up the peptides in their cytosol. These images were picked to depict these uptake phenotypes and this is why they are similar in the three cells lines. Panel A does not provide any quantitative information on the prevalence of these different uptake modes in the three cell lines. This is shown in panel B of Supplementary Fig. 1. There is, therefore, no discrepancies between the two panels.

Q3. Multiplying cell and peptide types contributes to the complexity of the manuscript without increasing its interest. If there is a conceptual breakthrough, as might be the case, it is obscured by the accumulation of useless images and data. A step into simplifying the manuscript would be (i), to concentrate on Raji cells (leaving out SKW6.4 and HeLa cells) and (ii) to only discuss the R9, TAT (including TAT-RasGAP) and Penetratin peptides.

A3. We are sorry that the inclusion of several cell lines and several CPPs was seen as confusing by the reviewer. Our current vision is that our observations are strengthened if we show that the observed effects are seen in several cell lines and with a variety of CPPs. We would like therefore to not exclude supportive evidence presented in our work because if we do remove some of the data shown in the manuscript, we will definitely weaken some of our claims. We nevertheless remain open with this point that can be further discussed with the editors.

Q4. TAT and R9 are poly-R peptides, which is not the case for Penetratin that has only 3 Rs. These 3 Rs are important (cannot be replaced by 3 Ks), but the two Ws absent in R9 and TAT are equally important as they cannot be replaced by Fs. This must be considered by the authors when they tend to generalize their model.

A4. The point raised by the reviewer concerning the importance of W and R residues in CPPs is well taken. We have now developed this in the discussion with the addition of the paragraphs shown below.

“An additional potential explanation to the internalization differences observed between arginine- and lysine-rich peptides is that even though both arginine and lysine are basic amino acids, they differ in their ability to form hydrogen bonds, the guanidinium group of arginine being able to form two hydrogen bonds1** while the lysyl group of lysine can only form one. Compared to lysine, arginine would therefore form more stable electrostatic interactions with the plasma membrane.

Cationic residues are not the only determinant in CPP direct translocation. The presence of tryptophan residues also plays important roles in the ability of CPPs to cross cellular membranes. This can be inferred from the observation that Penetratin, despite only bearing 3 arginine residues penetrates cells with similar or even greater propensities compared to R9 or TAT that contain 9 and 8 arginine residues, respectively (Supplementary Fig. 9g). The aromatic characteristics of tryptophan is not sufficient to explain how it favors direct translocation as replacing tryptophan residue with the aromatic amino acid phenylalanine decreases the translocation potency of the RW9 (RRWWRRWRR) CPP2. Rather, differences in the direct translocation promoting activities of tryptophan and phenylalanine residues may come from the higher lipid bilayer insertion capability of tryptophan compared to phenylalanine3-5. There is a certain degree of interchangeability between arginine and tryptophan residues as demonstrated by the fact that replacing up to 4 arginine residues with tryptophan amino acids in the R9 CPP preserves its ability to enter cells6. It appears that loss of positive charges that contribute to water pore formation can be compensated by acquisition of strengthened lipid interactions when arginine residues are replaced with tryptophan residues. This can explain why a limited number of arginine/tryptophan substitutions does not compromise CPP translocation through membranes**.”

Q5. Supp. Fig1c-d is not necessary (very little information in it) and Supp. Fig 1e is misleading as it takes a lot of imagination to see a difference between homogenous (top) and focal (bottom) diffusion.

A5. Since we perform cytosolic quantitation to infer direct translocation, it appears important to us, for allowing others to potentially replicate our results, that we precisely report how methodologically we perform our experiments. For Supplementary Fig. 1e, we agree that the examples shown are not easily interpretable. We have now removed this panel, as well as the accompanying panel f, from the Supplementary Fig. 1.

Q6. Supp. Fig.1g: How many cells are we looking at? Given the high variance, the result cannot be interpreted easily. A distribution according to fluorescence bits would be a better way to present the data.

A6. Over 230 cells have been quantitated per condition, which includes all cells where CPP entry has occurred regardless of the intensity or the type of entry. We did not only focus on cells with strong cytosolic staining to avoid any bias with regards to detection limitations. High variance can also be explained by the fact that CPP cellular entry is not synchronized. We tested the way of showing the data as suggested by the reviewer but this did not improve the visualization of the results in our opinion. We will therefore keep the initial presentation. Note that regardless of the way the data are presented, the conclusion remains the same, namely that illumination in our hands is not the cause of CPP membrane translocation.

Q7. Supp. Fig2i. This panel confirms that Raji cells differ from the two other cell types by showing clear temperature dependency. The explanation will come later with the energy barrier for low Vm-induced pore formation. This contradicts earlier reports showing that Penetratin translocation is not temperature-dependent, possibly because it was done on neurons naturally hyperpolarized. Or else because mechanisms are, at least in part, different from the one proposed here for R9 and TAT. This requires some clarification and supports the suggestion that, instead of multiplying models and peptides, it would be more efficient to compare TAT, R9 and Penetratin internalization by Raji cells and primary neurons.

A7. Supplementary Fig. 1i (not Supplementary Fig. 2i as indicated by the reviewer) was reporting the overall CPP uptake, both through direct translocation and endocytosis as a function of temperature. As there is limited endocytosis in Raji cells, the data shown for this cell type mostly correspond to direct translocation. For Hela and SKW6.4, endocytosis is not marginal however and we will perform a new set of experiments to define the role of temperature (4, 20, 24, 28, 32°C) in CPP direct translocation (i.e. cytosolic acquisition) in HeLa cells and SKW6.4 (using the CPPs listed by the reviewer). We have partially performed this for HeLa cells already and this shows that direct translocation is indeed inhibited by low temperatures (more than 10-fold at 4°C compared to 37°C). Bear in mind that no endosomal escape occurs in our settings (see Supplementary Fig. 7c). This indicates that the decrease in cytoplasmic fluorescence induced by low temperature is not a consequence of diminished CPP endocytosis.

Q8. Supp. Fig. 2a-f. Last sentence of the legend "Concentrations above 40µM led to too extensive cell death preventing analysis of peptide internalization". This confirms the warning against the use of concentrations varying between 40 µM and 80 µM and partially jeopardizes the validity of some experiments.

A8. The reviewer has truncated this sentence that actually reads “Note: concentrations above 40 mM of TAT-RasGAP317-326 led to too extensive Raji and SKW6.4 cell death, preventing analysis of peptide internalization at these concentrations.” As different cell lines display various sensitivities to potential toxic effects induced by CPPs (Raji and SKW6.4 cells being more sensitive than HeLa cells for example), we have adapted the concentrations of CPPs used to monitor cellular uptake so that cell death was minimal or non-existent in order to prevent the potential confounding effects mentioned by the reviewer. Hence in contrast to what the reviewer is stating, we are taking care of the toxicity effect and perform our experiments in conditions were toxicity is minimal. The logic of the reviewer to state that we “jeopardize[d] the validity of some experiments” is therefore unclear to us as we did take care of not exposing our cells to toxic CPP concentrations.

Page 6 {section sign} 2

Q9. The authors advocate 2 modes of entry, opposing transport across the membrane and endocytosis. In contrast with R9, TAT and Penetratin, Transportan or MAP seem to be purely endocytosed but, if they reach the cytoplasm, they still have to cross a membrane (unless "a miracle happens"). For Penetratin and R9/TAT, the authors consider that water pore and inverted micelle formation are incompatible. This is a bit rapid as inverted micelles might induce water pores through W/lipids interactions requiring less R residues and, possibly, less energy. This provides the opportunity to signal that, in spite of their very high number, key references are missing or hidden in cited reviews, some of them written by colleagues who are not among the main contributors to the CPP field.

A9. Transportan in our hands indeed appear to enter cells via endocytosis mostly. As reported by the reviewer, how Transportan reaches the cytosol remains unresolved.

Our data support a model where CPPs enter cells via water pores that are not made by the CPPs themselves but that are created by the megapolarization state of the membrane. Our data therefore do not support toroidal or barrel-stave pore models because these pores would be built as a result of CPP assemblage.

Inverted micelles have been hypothesized to mediate CPP translocation across membranes7 but to our knowledge, there is no in silico or cellular experimental evidence for this in the literature. To us, the data on which the involvement of inverted micelle in CPP translocation is based are also fully consistent with the water pore model. CPP translocation through water pores has been seen by several authors during in silico experiments but, to the best of our knowledge, simulations have not reported the formation of inverted micelles during CPP translocation across membranes.

Finally, we would be grateful to this reviewer if the “key references” that are apparently missing from our manuscript are disclosed so that we could acknowledge them appropriately.

Page 7 {section sign} 1

Q10.Fig. 1b confirms that Raji cells provide a good model for loss and gain of function (lovely rescue experiment) and that the authors should drop the two other cell types that provide no decisive information.

A10. Raji and HeLa cells display a stronger direct CPP uptake impairment phenotype when lacking a given potassium channel (KCNQ5 and KCNN4, respectively). In these cell lines, it appears that one potassium channel predominantly controls the plasma membrane potential. In contrast, in SKW6.4 cells, several potassium channels (e.g. KCNN4 and KCNK5) appear to be equally or redundantly involved in the control of the membrane potential. This probably explains the intermediate impact on the Vm and on CPP direct translocation when knocking out a given potassium channel in this cell line. When pharmacologically inducing cellular depolarization, a clear impairment in CPP translocation is however observed in this cell line. Thus, even though the Vm in SKW6.4 cells, is controlled predominantly by several potassium channels, it remains that an appropriate membrane potential is crucially required for these cells to take up CPP across their membrane. We agree with the reviewer that the stronger phenotypic effect observed in Raji and HeLa cells allows easy interpretation. On the other hand, it seems important to us that we provide data reporting intermediate situations so that readers can appreciate the variability that can be observed in different cell lines. Nevertheless, we would like to propose along the reviewer’s suggestion to move the SKW6.4 data from figure 1 to the supplemental data. Feedback from the editors would also be appreciated in this particular instance.

Page 8 {section sign} 1

Q11. A) Supp. Fig. 6b (no serum conditions) allows for the use of "normal" CPP concentrations and suggests that a fraction of the peptides may bind to serum components. No arrows in Supp. Fig.6b (but in 6c), and the R/pyrene butyrate interaction is not in 6c but in 6a. Still for Supp. Fig. 6c, the death of cells at 20µM (or less) even in the absence of K+ channels, confirms that we are borderline in term of peptide toxicity.

B)There is a confusion between Supp. Fig. 6d and 6e and a legend problem (6e is not described). Cell death is assessed in % of PI-positive cells. Does this securely distinguish between death and holes allowing for PI entry without death?

C) The CPP is incubated in the presence of Pyrene butyrate, making the KO cells less resistant. How does that demonstrate that the potassium channels are not involved in the killing if the peptide is already in? Unless the KO is done after internalization (but the cells should be already dead or dying?). This lacks clarity.

A11. We apologize for the lack of clarity in the legend of Supplementary Fig. 6. This will be corrected in the revised version of the manuscript.

A) Supp. Fig. 6b (no serum conditions) allows for the use of "normal" CPP concentrations and suggests that a fraction of the peptides may bind to serum components.

A) The reviewer is correct that CPPs interact with serum components. This is indeed what is reported in this figure. The presence or absence of serum has therefore an important impact in experiments performed with CPPs and should be reported to allow proper interpretation of our data.

No arrows in Supp. Fig.6b (but in 6c), and the R/pyrene butyrate interaction is not in 6c but in 6a.

Thank you for noting this. This is now corrected.

Still for Supp. Fig. 6c, the death of cells at 20µM (or less) even in the absence of K+ channels, confirms that we are borderline in term of peptide toxicity.

It has to be understood that in Supplementary Fig. 6c, we use the TAT‑RasGAP317‑326 peptide that is inducing cell death when translocating into cells8. This cell death response is not provided by the CPP portion of TAT‑RasGAP317‑326 (i.e. TAT) but by its bioactive cargo (i.e. RasGAP317‑326). The read-out in this particular experiment is therefore cell death and this should not be confused with general CPP toxicity.

B) There is a confusion between Supp. Fig. 6d and 6e and a legend problem (6e is not described).

B) This has now been fixed.

Cell death is assessed in % of PI-positive cells. Does this securely distinguish between death and holes allowing for PI entry without death?

The answer to this question is yes. In this manuscript we used PI in two very different experimental set-ups.

i) the conventional cell death detection assay where cells are incubated with 8 mg/ml PI prior to flow cytometry. In this set-up, dead cells with compromised membrane integrity have their nucleus brightly stained with PI.

ii) the detection of small pores in the plasma membrane (water pore) where cells are incubated with ~30 mg/ml PI and the fluorescence of PI measured in the cytosol by confocal microscopy. In this set-up, PI enters into the cytosol through small plasma membrane pores but PI does not stain the DNA in the nucleus. This protocol has been previously described9 and we have further validated it in the present work (Figure 3 and Supplementary Fig. 12).

PI does not fluoresce well unless it binds to DNA. In solution without cells, PI cannot be detected below 128 mg/ml (Supplementary Fig. 12e). At low PI concentrations (8 mg/ml), living cells (even when treated with compounds such as CPPs that create transitory pores) do not display cytosolic PI fluorescence. At high PI concentrations (32 mg/ml), the cytosol of CPP-treated cells becomes PI fluorescent. PI is positively charged and is attracted by the negative membrane potential of the cells. Its movement across the cell membrane is therefore unidirectional. This enables the PI molecules to accumulate/concentrate within the cytosol to values (> 64 mg/ml) allowing its detection (Supplementary Fig. 12a-c). PI and CPPs do no interact (Supplementary Figure 12d); hence they move independently from one another. If PI enters through the water pores induced by CPPs, the entry kinetics of PI and CPPs should be identical. Indeed, this is what we show now in a new figure (refer to our answer #31).

C) The CPP is incubated in the presence of Pyrene butyrate, making the KO cells less resistant. How does that demonstrate that the potassium channels are not involved in the killing if the peptide is already in? Unless the KO is done after internalization (but the cells should be already dead or dying?). This lacks clarity.

C) For the pyrene butyrate experiments the rationale was the following. The CRISPR/Cas9-identified potassium channels could either be involved in CPP internalization or they could be required for the killing activity of TAT-RasGAP317-326 when the peptide is already in the cytosol. To experimentally introduce TAT-RasGAP317-326 in the cytosol and to bypass any potential entry depending on potassium channels, we used pyrene butyrate that efficiently creates an artificial entry route for CPPs into cells. Our data show that when TAT-RasGAP317-326 is introduced in the cytosol through the use of pyrene butyrate, cells died whether they lack specific potassium channels or not. This led to our interpretation that potassium channels are not modulating the cell death activity of TAT-RasGAP317-326 once in the cytosol but that they are required for the entry of the CPP in the cytosol.

Page 9 {section sign} 1

Q12.The conclusion that the diffuse staining does not come from endosomal escape is based on the certainty that LLOME disrupts both endosomes and lysosomes. First, it should be verified with specific markers (rab5, rab7) that the fluorescent vesicles are endosomes. Second, the literature strongly suggests that LLOME primarily disrupts lysosomes and not endosomes. Finally, even if some endosomes are disrupted, the endosomal population is heterogenous and some CPPs may be in a subpopulation insensitive to LLOME. In addition, the importance of this issue is not well explained. In practice, access to the cytoplasm and nucleus requires crossing the plasma and/or the endosomal membrane and the latter, at least in early endosomes (thus the need of identifying the CPP-enriched vesicles), might not be very different from the plasma membrane.

A12. The conclusion that diffuse staining does not come from endosomal escape is based on experiments where HeLa cells were incubated in the presence of CPP for 30 minutes to allow CPP entry into cells, then the cells were washed to prevent further uptake (Supplementary Fig. 7c). We only monitored the cells that initially took up the CPP by endocytosis and not through direct translocation (for the HeLa cell line, there is always a substantial fraction of such cells; see Supplementary Fig. 1b). We measured the cytosolic CPP fluorescence intensity in these cells by time-lapse confocal microscopy for 4 ½ hours. The procedure to do this is now explained in new Supplementary Fig. 7c. We then assessed the CPP fluorescence intensity within the cytosol. No increase in cytosolic fluorescence was detected in this condition, speaking against the possibility that cytosolic acquisition of CPPs by the cells resulted from vesicular escape (the identity of the vesicles being unimportant in this context). Our set-up has the potential to detect CPPs in the cytosol if these CPPs leak out from vesicles because we could measure increased CPP fluorescence in the cytosol in cells treated with LLOME. It did not matter in this positive control experiment what types of CPP-containing vesicles are disrupted by LLOME. What was important to show in this control condition was that the disruption of at least some CPP-containing vesicles permitted us to detect a cytosolic signal.

Page 9 {section sign} 2

Q13. Is Supp. Fig. 7e really necessary? First, as mentioned several times, if 20 µM is a borderline concentration in term of toxicity, raising the concentration up to 100 µM is problematic. Secondly, what matters is not "binding" in general, but binding to the proper membrane components. As mentioned by the authors themselves (Supp. Fig. 1e and movie), there are privileged sites of entry that may correspond to the recognition of specific molecular entities/structures.

A13. The goal of the experiments presented in Supplementary Figure 7e was to determine whether the CRISPR/Cas9-identified potassium channels modulate CPP/membrane interaction. If those channels were to be required for the initial binding of the CPPs to the plasma membrane, this would have not hampered cells to take up the CPPs. Our data showed (Figure 7e) that Raji cells lacking the KCNQ5 potassium channel had a slightly decreased ability to bind TAT-RasGAP317-326 but importantly, these cells, at similar or even higher initial surface binding compared to wild-type cells (this was achieved by adequately varying the CPP concentrations), were still drastically impaired in taking up the peptide. Note that after one hour of incubation with TAT-RasGAP317-326 in the presence of serum there is only marginal amount of cell death (317-326, we have now performed an additional experiment with TAT that is not toxic to cells that confirms our data obtained with TAT-RasGAP317-326.

Page 9 {section sign} 3 and Page 10 {section sign} 1

Q14.The authors should have used a construct that does not kill the cells much earlier, just after the screening experiments based on resistance to necrosis induced by TAT-rasGAP. For Supp. Fig 8a and b: I am fully convinced by Raji cells and HeLa cells but not by the SKW6.4 cells.

A14. As mentioned in our answer to point 10, we agree that SKW6.4 cells present intermediate phenotypes probably because, unlike Raji and HeLa cells, a combination of ion channels seems to regulate the plasma membrane potential. As indicated above, we can move the SKW6.4 data to the supplementary information to clarify the message presented in the main text. Again, feedback from the editors is welcome here.

Page 10 {section sign} 2

Q15. A) Supp. Fig 9 is quite convincing but adds the information that 2 µM are sufficient in neurons. This again makes the 20 to 80 µM concentrations used on transformed cells unsatisfactory.

B) If one needs a cell line (more user friendly than primary cultures), there are several neural ones that can be differentiated (SHY, LHUMES, etc.) that may have an appropriate membrane potential (below -90mV). Indeed, it would then be important to verify if pore formation is still induced by TAT, R9 and Penetratin (separately) on "naturally" hyperpolarized cells.

C) Figure 2a confirms that changes in Vm are not solid for HeLa and SKW6.4 cells. This casts a doubt on the validity of the results obtained with the latter 2 cell lines.

A15. A) The experiments performed in Supplementary Fig. 9d with cortical rat neurons and HeLa cells were performed in the absence of serum accounting for the low concentrations used. We apologize for not emphasizing enough when experiments were performed in the presence or absence of serum, explaining the use of high CPP concentrations (40-80 mM) and low CPP concentrations (2-10 mM), respectively. We would like to emphasize however that we have adjusted the concentrations of CPPs in our study so as to get similar levels of CPP activity or CPP uptake between the different cell lines used. The concentrations used should not be compared as mere numbers, it is the CPP activity or uptake that should be considered.

B) We thank the reviewer for his/her suggestion. To address this point, we will perform a new experiment to determine if in neurons TAT, R9, and Penetratin induce pores (using the PI uptake approach).

C) Please see our answer to point 10.

Page 11 {section sign} 2

Q16. Why valinomycin was only tried on Raji cells?

A16. In this study, valinomycin was used on Raji and HeLa cells (Figure 2 and 3). We did not use valinomycin on SKW6.4 cells, as the drug-induced hyperpolarization levels were insufficient in this cell line. As we got a nice hyperpolarization in HeLa wild-type and KCNN4 KO cells through ectopic expression of the KCNJ2 potassium channels (which restored the ability of the KO cells to take up the CPPs), we did not perform the CPP uptake experiment with valinomycin in HeLa cells (although we had tested that valinomycin is able to hyperpolarize HeLa cells).

Page 12 {section sign} 2

Q17.A)Looking at Fig. 2c, it seems that low Vm increases the uptake of all CPPs, except Transportan. Is there any reason why this Figure does not provide the number of vesicles per cell in the hyperpolarized conditions?

B) In fact, if one goes to Supp. Fig. 9c, it appears that, among all peptides, only Penetratin is almost entirely cytoplasmic after 90' of incubation, whereas MAP and Transportan remain essentially vesicular. TAT and R9 are at mid-distance between these two extremes. This leads to send again the warning that all CPPs cannot be placed in a single category. The table that describes the sequences strongly suggests that, TAT and R9 uptake is due to the numerous Rs that cannot be replaced by Ks. In the case of Penetratin, that only has 3 Rs, the situation is thus different with the presence of 2 Ws previously shown to be mandatory for internalization, although absent in TAT ad R9.

C) In Supp. Fig9, panel g is useless.

D) A difference between peptides is also visible in Figure 2d where depolarization with KCl does not show the same efficiency on all peptides. The issue is whether these differences are significant and, if so, why? This discussion could be restricted to TAT, R9 and Penetratin.

E) Supp. Fig. 10a also suggests that all peptides do not respond similarly to depolarization and that the effects differ between cell types and concentrations used. However, given the high concentrations used and the high variance between replicates, this figure might not be a priority in the reorganization of the manuscript.

A17. A) As mentioned in the figure legend “Quantitation of vesicles was not performed in hyperpolarizing conditions due to masking from strong cytosolic signal.” This would create a bias towards underestimation of vesicles numbers in cells displaying strong cytosolic signal.

B) We agree with the reviewer that Transportan enters cells primarily through endocytosis. This is mentioned in the text as well as other differences that were observed with regards to the prevalence of endocytosis or direct translocation. These mentions are reported below.

Page 12: “With the notable exception of Transportan, depolarization led to decreased cytosolic fluorescence of all CPPs, while hyperpolarization favored CPP translocation in the cytosol (Fig. 2c, Supplementary Fig. 9h and 10a). Transportan, unlike the other tested CPPs, enters cells predominantly through endocytosis (Supplementary Fig. 9e), which could explain the difference in response to Vm modulation.”

Page 14: “Even though this extrapolation is likely to lack accuracy because of the well-known limitation of the MARTINI forcefield in describing the absolute kinetics of the molecular events, the values obtained are consistent with the kinetics of CPP direct translocation observed in living cells (Figure 1c and Supplementary Fig. 1b and 9e). With the exception of Transportan, the estimated CPP translocation occurred within minutes. This is consistent with our observation that Transportan enters cells predominantly through endocytosis and its internalization is therefore not affected by changes in Vm (Fig 2c-d and Supplemental Fig. 9e)”.

Page 20: “On the other hand, when endocytosis is the predominant type of entry, CPP cytosolic uptake will be less affected by both hyperpolarization and depolarization, which is what is observed for Transportan internalization in HeLa cells (Fig. 2c and Supplementary Fig. 10a).”

Concerning the roles of arginine and tryptophan residues, please refer to our answer #4.

C) We do not think this panel (now panel h) is useless as it shows representative examples of the quantitation shown in Figure 2c. We can however remove it if requested by the editors.

D) The reviewer is correct with the observation that KCl-induced depolarization does not lead to similar inhibition in uptake of the tested CPPs. As mentioned in the text, these differences can be explained by the prevalence of direct translocation in the cells. For example, transportan enters cells primarily through endocytosis, which as we show is not regulated/affected by the membrane potential (Figure 2c, lower graphs). Consequently, it is expected that KCl treatment will not impact on transportan cellular uptake.

E) The reviewer is correct in mentioning that there is quantitative heterogeneity between the different CPP tested. We mentioned these differences in the manuscript. These mentions are those that are reported under B, plus those listed below.

Page 19: “It is known for example that peptides made of 9 lysines (K9) poorly reaches the cytosol (Fig. 3f and Supplementary Fig. 9e) and that replacing arginine by lysine in Penetratin significantly diminishes its internalization10,11. According to our model, K9 should induce megapolarization and formation of water pores that should then allow their translocation into cells. However, it has been determined that, once embedded into membranes, lysine residues tend to lose protons12,13. This will thus dissipate the strong membrane potential required for the formation of water pores and leave the lysine-containing CPPs stuck within the phospholipids of the membrane. In contrast, arginine residues are not deprotonated in membranes and water pores can therefore be maintained allowing the arginine-rich CPPs to be taken up by cells.”

Page 21: “Therefore, the uptake kinetics of lysine-rich peptide, such as MAP, appears artefactually similar as the uptake kinetics of arginine-rich peptides such as R9 (Supplementary Fig. 11b).”

Page 21: “The differences between CPPs in terms of how efficiently direct translocation is modulated by the Vm (Fig. 2c-d and Supplementary Fig. 10a) could be explained by their relative dependence on direct translocation or endocytosis to penetrate cells. The more positively charged a CPP is, the more it will enter cells through direct translocation and consequently the more sensitive it will be to cell depolarization (Fig. 2c). On the other hand, when endocytosis is the predominant type of entry, CPP cytosolic uptake will be less affected by both hyperpolarization and depolarization, which is what is observed for Transportan internalization in HeLa cells (Fig. 2c and Supplementary Fig. 10a).”

However, what remains is that depolarization always affects CPP uptake, at most concentrations tested. The heterogeneity reported in Supplementary Fig. 10a for a given experimental condition in a given cell type is in itself of interest as it suggests that there are varying factors within a cell population (e.g. cell cycle, metabolism, etc.) that may impact on the ability of cells to take up CPPs. As per reviewer’s suggestion we may remove this panel from the figure if instructed to do so by the editors.

Page 12 {section sign} 3 and Page 13 {section sign} 1

Q18. The pH story is either too long or too short.

A18. One mechanism put forward to explain direct translocation relies on pH variation between the extracellular milieu and the cytosol14. It was therefore of interest in the context of the model we putting forward to see if pH is affecting the uptake of CPPs in our experimental model. Our data show that pH variations do not affect CPP direct translocation. This information should in our opinion be disclosed.

Page 14 {section sign} 2

Q19. At low Vm values, there is a decrease in free energy barrier. Does this modify temperature-dependency for internalization? Do cells really require energy when the Vm is very low, like is often the case for neurons?

A19. We thank the reviewer for this interesting comment. We will now address this by visualizing under a confocal microscope CPP direct translocation in rat cortical neurons incubated at various temperature (4°C, 24°C, 37°C).

Page 15 {section sign} 2

Q20. Figure 2e is not explained, not even in the legend while the statement that CPPs induce a local hyperpolarization is central to the study.

A20. As there is no Figure 2e, we believe that the reviewer is talking about Figure 3e, the legend of which was present in the initial version of the manuscript.

Page 16 {section sign} 1

Q21. It is confusing that the same agent, here PI, is used to measure internalization (2 nm pore formation in response to hyperpolarization,) and cell death. I have seen the explanation below, but I do not find it fully satisfactory.

A21. We have tried to explain this better under our answer to point 11B.

Page 16 {section sign} 2

Q22. Entry is not necessarily a size issue. Structure is an important parameter, including possible structure changes, for example in response to Vm modifications. Therefore, the statement that molecule with larger diameters are mostly prevented from internalization is not only vague ("mostly") but incorrect.

A22. We agree with the reviewer’s comment in the sense that the secondary structure of a molecule will also play an important role in its internalization. For that reason, we have used a series of molecules of identical structure (dextrans) but that have different molecular weights. In these experiments we saw that dextran of higher molecular weight enter less efficiently than that of lower molecular weight (Figure 3). We will rephrase some of our sentences so to precise that the size and the shape (structure) of molecules will determine their ability to enter cells through water pores that are characterized by a certain diameter.

Page 2: “Using dyes of varying sizes and shapes, we assessed the diameter of the water pores**.”

Page 4: “translocation and we characterize the diameter of the water pores used by CPPs**.”

Page 15: “cells were co-incubated with molecules of different sizes and structure and FITC-labelled CPPs at a peptide/lipid ratio of 0.012-0.018 (Supplementary Fig. 11c-d).”

Page 16: “3 kDa, 10 kDa, and 40 kDa dextrans, 2.3 ±0.38 nm, 4.5 nm and 8.6 nm (diameter estimation provided by Thermofisher), respectively, were used to estimate the diameter of the water pores formed in the presence of CPP.”

Page 16: “These results are in line with the in silico prediction of the water pore diameter obtained by analyzing the structure of the pore at the transition state.”

Page 16: “The marginal cytosolic co-internalization of dextrans was inversely correlated with their diameter.”

Page 35: “200 µg/ml dextran of different molecular weight in the presence or in the absence of the indicated CPPs in normal […]”.

Page 17 {section sign} 4 and Page 18 {section sign} 1

Q23. In Supp. Fig. 13b and c, since the GAP domain is mutated, death is not due to RasGAP activity. So what causes zebrafish death (hyperpolarization?) The results seem contradictory with those of Supp. Fig 13f where survival is 100% at 48 h.

A23. Indeed, it appears that valinomycin in water leads to zebrafish embryo death, as can be seen in Supplementary Fig. 13c. However, the main difference between Supplementary Fig. 13c and S13f is that in Supplementary Fig. 13f zebrafish were not incubated in valinomycin-containing water, but were locally injected with a CPP in the presence or in the absence of valinomycin. This has now been clarified in the text. We saw that local injections with the hyperpolarizing agent are much less toxic and are well tolerated by the zebrafish embryos.

Page 18 {section sign} 2

Q24. The formation of inverted micelles is not incompatible with that of pores. CPP-induced hyperpolarization (Vm) is not measured directly, but deduced from experiments involving artificial membranes and in silico modeling. It would be useful to distinguish between what takes place on live cells (in vitro and in vivo) and what is speculated (based on modeling and artificial systems).

A24.

The formation of inverted micelles is not incompatible with that of pores.

As mentioned above (point 9), we do also think that what has been presented as inverted micelles could have been in fact water pores.

CPP-induced hyperpolarization (Vm) is not measured directly, but deduced from experiments involving artificial membranes and in silico modeling. It would be useful to distinguish between what takes place on live cells (in vitro and in vivo) and what is speculated (based on modeling and artificial systems).

If we understand this point correctly, the reviewer is talking about the -150 mV hyperpolarization. This value is not a speculation but has been estimated from in silico experiments and also from experiments using live cells (not artificial membranes). In living cells, the hyperpolarization (megapolarization) has been estimated based on accumulation of intracellular PI over time in the presence or in the absence of CPP.

Page 19 {section sign} 3

Q25A. The model posits that the number of Rs influences the ability of the CPPs to hyperpolarize the membrane and, consequently, to induce pore formation. Since pore formation is key to the addressing to the cytoplasm, how can one explain that Penetratin which has only 3 Rs is transported to the cytoplasm more readily that TAT or R9? The authors should take this contradiction in consideration and should not leave aside, in the literature, what does not fit with their model.

A25A. We fully agree that this should be discussed and not left aside. Please refer to point 4 for detailed discussion about the role of arginine and tryptophan in the ability of CPPs to translocate across membranes.

Q25B. The fact that that Rs cannot be replaced by Ks, both in R9 and Penetratin is explained by differences in deprotonization. This is interesting but speculative. It might be that the interaction between Rs versus Ks with lipids and sugars are different and not only based on charge. After all their atomic structures, beyond charges, are different.

A25B. We do not claim that protonation differences between R and K is the definitive answer for their ability to promote CPP translocation. It is one possible explanation that we find sound. As suggested by the reviewer, the ability of K and R to bind lipids and sugars can also play a role. We can mention in this context that the guanidinium group of arginine residues can form two hydrogen bonds1, which allow for more stable electrostatic interactions while the lysyl group of lysine residues can only form one hydrogen bond. We have included these additional possibilities in the revised version of our manuscript as indicated under point 4.

Page 20 {section sign} 1 Q26. We still need to understand endosomal escape.

A26. We agree with the reviewer that endosomal escape is still poorly understood. This is an interesting research topic that deserves its own separate study.

**Major comments**

• The key conclusions are convincing for a subset of CPPs and cell types
• Yes, some claims should be qualified as speculative, but not preliminary
• Many experiments should be removed. Neuronal primary cultures should be introduced to verify the main conclusions, at least for the 3 mains CPPs (TAT, R9, Penetratin). Answers must be given to the concentration issue. Vesicles should be characterized as well as the localization of the peptides in or around the vesicles. See above for less decisive but still important experiments that would benefit to the study.
• Yes, the requested experiments correspond to a reasonable costs and amount of time (10 to 20,000 € and 3 to 5 months of work)
• Yes, the methods are presented with great details. -Yes, the experiments are adequately replicated and statistical analysis is adequate

**Minor comments (not so minor for some of them)**

• See "Detailed analysis"
• No, prior studies are not referenced appropriately (see above)
• No, the text and figures are not clear and not accurate (see above)
• (i) use Raji cells and primary neuronal cultures, plus in vivo model and forget the other cell types; (ii) forget MAP and Transportan and compare TAT/R9 and Penetratin; (iii) drastically reduce the number of figures, tables and movies (6 primary figures, 6 supplemental figures and 4 tables are reasonable numbers; movies are not absolutely necessary); (iv) limit to 6 (max) the number of panels per figure; (v) limit the number of references to less than 50 and cite the primary reports rather than reviews); (vi) reduce the size of the Material and Methods and the length of figure legends.

Reviewer #1 (Significance (Required)):

• The mode of CPP internalization is an unanswered question and the report, if revised, will represent a conceptual and technical advance.
• Bits and pieces of the conclusions can be found in previous reports. But the Vm-dependent pore formation as well as the CPP-induced "megapolarization" (even if only shown for a subset of CPPs) would be an important contribution. The authors must resist the tentation to generalize to all CPPs what might only be true for a few of them.
• I do not have the expertise for the in-silico work, but my field of expertise allows me to understand all other aspects of the manuscript.

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

In this manuscript, the authors investigated the effect of membrane potential on the internalization of CPPs into the cytosol of some cancer cell lines. Using a CRISPR/Cas9-based screening, they found that some potassium channels play an important role in the internalization of CPPs. The depolarization decreases the rate of internalization of CPPs and the hyperpolarization using valinomycin increases the rate. Using the coarse-grained MD simulations, the authors investigated the interaction of CPPs with a lipid bilayer in the presence of membrane potential. In the interaction of CPPs with the cells, propidium iodide (PI) enters the cytosol significantly. Based on this result, the authors concluded that pores with 2 nm diameter are formed in the plasma membrane.

This reviewer raises one main issue concerning CPP endocytosis. The reviewer challenges our method to investigate CPP direct translocation and specifically how do we make sure that what we consider direct translocation is not a combination of CPP endocytosis (followed or not by endosomal escape) and CPP plasma membrane translocation. As explained below in details our methodology is able to accurately distinguish CPP uptake by direct translocation from CPP endocytosis and we further demonstrate that endosomal escape does not occur in our experimental settings.

Q27. One of the defects in this manuscript is the method to determine the fraction of internalization of CPPs via direct translocation across plasma membrane. The authors estimated the fraction of the direct translocation of CPPs by the fluorescence intensity of the cytosolic region (devoid of endosomes) and the fraction of the internalization via endocytosis by the fluorescence intensity of vesicles. However, the CPPs can enter the cytoplasm via endocytosis, and thus the increase in the fluorescence intensity of the cytoplasm is due to two processes (via endocytosis and direct translocation). The authors should use inhibitors of clathrin-mediated endocytosis and macropinocytosis to determine the fraction of internalization of CPPs via direct translocation accurately. Low temperature (4 C) has been also used as the inhibitor of endocytosis (e.g., J. Biophysics, 414729, 2011; J. Biol. Chem., 284, 33957, 2009). Supplementary Figure 1i (the temperature dependence of internalization of TAT-RasGAP317-326) clearly shows that at 4 C the fraction of the internalization was very low, indicating that this peptide enters the cytosol mainly via endocytosis. The determination of the fraction of the internalization via endocytosis by the fluorescence intensity of vesicles in this manuscript is not accurate because it is difficult to examine all endosomes in cells and it is not easy to discriminate the fluorescence intensity due to the endosomes from that due to the cytosol.

It is important to follow a time course of the fluorescence intensity of single cells from the beginning of the interaction of CPPs with the cells (at least from 5 min) in the presence and absence of inhibitors of the endocytosis (J. Biol. Chem., 278, 585, 2003) to elucidate the process of the internalization of CPPs in the cytosol.

A27. The reviewer raises the possibility that the signal of fluorescent CPPs in endosomes somehow perturbs the acquisition of the signal in cytosol. This could occur in two ways: CPP endosomal escape and diffusion of the signal located in endosomes into adjacent cytosolic regions (halo effect). The second possibility can be readily dismissed because in situations where cells only take up fluorescent CPPs by endocytosis, the cytosol emits background fluorescence (autofluorescence). This can be seen in Supplementary Fig. 1a (“vesicular” condition) or in Supplementary Fig. 9h in the depolarized cells that cannot take up CPP by direct translocation. Also note that when we record the cytosolic signal we take great care of using regions of interest (ROI) that are distant from endosomes. In contrast to what the reviewer is saying (“it is not easy to discriminate the fluorescence intensity due to the endosomes from that due to the cytosol”), it is actually not difficult discriminating the cytosolic fluorescence from the endosome fluorescence. To illustrate this, we now provide examples of high magnification images of cells incubated with fluorescent CPPs (new Supplementary Fig. 1c, right[1]) to better explain/illustrate our methodology and to show that it is quite straightforward to find cytosolic areas devoid of endosomes. Such high magnification images are those that are used for our blinded quantitation. The other possibility is endosomal escape. We demonstrate in Supplementary Fig. 7c that in our experimental conditions, no endosomal escape is detected[2]. We may not have explained our methodology well enough in the earlier version. We will try and improve the description of our quantitation procedures better in the revised version. To this end, we have now added a scheme illustrating the experimental setup (now part of Supplementary Fig. 7c) that is used to assess endosomal escape.

The reviewer also questions the way we quantitate the CPP signals in endosomes. In the present paper, our goal is to characterize the direct translocation process of CPPs in to cells. We do not wish here to investigate in details the endocytic pathway taken by CPPs. This has been done in a separate study that we are currently submitting for publication. In a nutshell, this work shows that the endocytic pathway taken by CPPs is different from the classical Rab5- and Rab7-dependent pathway and that the CPP endocytic pathway is not inhibited by compounds that affect the classical pathway. Thus, even if we had wanted to use the inhibitors mentioned by the reviewer, they would not have blocked CPP endocytosis.

To sum up the issues raised under this point, we believe we have presented the reasons why there are no grounds to support the concerns raised by the reviewer.

[1] Supplementary Fig. 1c (right) is mentioned in the “Cell death and CPP internalization measurements” section of the methods.

[2] In this experiment, cells were incubated with CPPs for 30 minutes to allow CPP entry into cells. Then the cells were either washed (to prevent further uptake including uptake through direct translocation) or incubated in the continued presence of CPPs. In both conditions, cells where only endocytosis took place were followed by time-lapse confocal microscopy for 4 hours (i.e. these cells do not display any cytosolic CPP signal at the beginning of the recording). We then assessed the CPP fluorescence intensity within the cytosol (i.e. away from endosomes). From these experiments we saw that cytosolic fluorescence increased only in conditions where CPP was present in the media throughout the experiment. No increase of cytosolic fluorescence was detected in the condition where CPPs were washed out. In conclusion these results demonstrate that the cytosolic signal that we observed in our experiments is due to direct translocation and not endosomal escape. In these experiments we have used the LLOME lysosomotropic agent as a control to make sure that if endosomal escape had occurred (even if only from a subset of endosomes/lysosomes), we would have been able to detect it. Indeed, upon addition of LLOME we were able to record CPP release from endosomes to the cytosol. There is therefore no endosomal escape occurring in our experimental conditions. In conclusion, the observed cytosolic signal in our confocal experiments do not originate, even partly, from endosomal escape.

Supplementary Figure 1i (the temperature dependence of internalization of TAT-RasGAP317-326) clearly shows that at 4 C the fraction of the internalization was very low, indicating that this peptide enters the cytosol mainly via endocytosis.

The experiment shown in Supplementary Fig. 1i was analyzed by flow cytometry that cannot discriminate the cytosolic signal from the endosomal signal. We will therefore perform this experiment again but this time using confocal imaging to record the impact of temperature on CPP cytosolic acquisition. We have performed this for HeLa cells already and this shows that direct translocation is indeed inhibited by low temperatures (full blockage at 4°C). Bear in mind that no endosomal escape occurs in our settings (see Supplementary Fig. 7c). This indicates that the decrease in cytoplasmic fluorescence induced by low temperature is not a consequence of diminished CPP endocytosis.

Q28. Recently, it has been well recognized that membrane potential greatly affects the structure, dynamics and function of plasma membranes (e.g., Science, 349, 873, 2015; PNAS, 107, 12281, 2010). The results of the effect of membrane potential on the internalization of CPPs (depolarization decreases the rate of internalization and hyperpolarization increases the rate), which is main results of this manuscript, can be interpreted by various ways. For example, the rate of endocytosis may be greatly controlled by membrane potential, which can explain the authors' results.

A28. This reviewer may have missed the experiment presented in Figure 2c that clearly shows that CPP endocytosis is unaffected by depolarization or hyperpolarization of cells. We have also determined that transferrin uptake through endocytosis is not affected by potassium channel knockout (which also leads to depolarization). The possibility raised by the reviewer is therefore refuted by our experimental evidence.

Q29. A) The authors used the similar concentrations of various CPPs for their experiments (10 to 40 microM), and did not examine the peptide concentration dependence of the internalization. It has been recognized that the CPP concentration affects the mode of internalization of CPPs (e.g., J. Biol. Chem., 284, 33957, 2009). The authors should examine the peptide concentration dependence of the mode of internalization (less than 10 micorM, e. g., 1 microM).

B) In the case of depolarization, can higher concentrations of CPPs (e.g., 100 micorM) induce their internalization?

A29. A) We agree that CPPs/cell ratio might prompt one mode of entry over the other. It has been reported by imaging that at lower CPP concentrations endocytosis is favored since only vesicles were observed15-19. Our data confirm this (new Supplementary Fig. 9f).

B) In Supp. Fig. 7e we have incubated KCNQ5 KO Raji cells that are slightly more depolarized than WT cells in the presence of increasing CPP concentrations up to 100 m From the obtained results, we can see that at 100 mM, the uptake in depolarized cells is increased but does not reach the level of uptake seen in wild-type cells. Therefore, lack of hyperpolarization can be compensated to a mild extent by increased CPP availability.

Q30. A) The effects of membrane potential on plasma membranes and lipid bilayers have been extensively investigated experimentally and thus are well understood, although currently the coarse-grained MD simulations cannot provide quantitative results which can be compared with experimental results. In this manuscript, using the coarse-grained MD simulations, the authors applied 2.2 V to a lipid bilayer to examine the translocation of CPPs. However, it is well known the experimental results that application of such large voltage to a lipid bilayer induces pore formation in the membrane or its rupture (Bioelectrochem. Bioenerg., 41, 135, 1996; Sci. Rep., 7, 12509, 2017), but at low membrane potential (B) What is the probability of the existence of R9 in the surface of the membrane? R9 cannot bind to the electrically neutral lipid bilayers (such as PC) under a physiological ion concentration (Biochemistry, 55, 4154, 2016). Even if in the case of R9 the membrane potential reaches at -150 mV, the other CPPs have lower surface charge density than that of R9, and hence, the decrement of membrane potential is lower. The authors should provide the data of other CPPs.

C) It has been reported that the negative membrane potential increases the rate of entry of two kinds of CPPs into the lumen of giant unilamellar vesicles (GUVs) without leakage of water-soluble fluorescent probe (Stokes-Einstein radius; ~0.9 nm diameter), i.e., no pore formation in the GUV membrane (Biophys., 118, 57, 2020, J. Bacteriology, 2021, DOI: 10.1128/JB.00021-21). The authors should discuss the similarity and the difference between the results in these papers and the above results in this manuscript.

A30. A) As correctly stated by this Reviewer, we reported simulations with high transmembrane potential values, which is a common procedure in in silico simulations used to accelerate the kinetics of the studied process. In this manuscript we have additionally developed and carefully validated a novel protocol to estimate the free energy landscape of water pore formation and CPP translocation under physiological transmembrane potential (further details about the methodological procedure, the convergence and the validation of the free energy estimation are reported in Supplementary Fig. 15-19 of the manuscript). This protocol allowed us to demonstrate the impact of megapolarization (‑150 mV) on the free energy barrier corresponding to the CPP translocation process. The results exemplify how the megapolarization process modifies the uptake probability of the R9 peptide, reducing locally the free energy barrier of the membrane translocation (Fig. 3c-d). Moreover, we have also demonstrated how a single CPP produces a local transmembrane potential of about -150 mV, in agreement with our hypothesis (Fig. 3e).

Finally, the quantitative accuracy of the molecular simulations was found to be satisfactory because the water pore formation free energy in a symmetric DOPC membrane that we calculated is in excellent agreement with previous atomistic estimation (Table S5).

B) It has been demonstrated that CPP/membrane interactions are mostly electrostatic between positively charged amino acids carried by the CPPs and various negatively charged cell membrane components, such as glycosaminoglycans20-31 and phosphate groups32. It is in line with our model that the more positively charged CPPs are the better they should translocate into cells. Therefore, we agree with the reviewer that the level of megapolarization may vary according to the charges carried by the CPPs. However, our data clearly indicate that a certain membrane potential hyperpolarization threshold must be achieved to induce water pore formation. As suggested by the reviewer we will now conduct additional modeling experiments with other CPPs.

C) We have carefully read these papers and do not necessarily reach the same conclusions as the authors. In both papers, the translocation of CPPs in polarized GUVs is monitored through CPP acquisition on vesicles found within the GUVs (intraluminal vesicles; either smaller GUVs or LUVs). There is actually no evidence of the presence of luminal CPPs outside of the intraluminal vesicle membranes. We would therefore argue that these studies elegantly demonstrate that membrane potential increases CPP binding and insertion into the membrane of the mother GUVs but that the CPPs then move, by diffusion, from the lipidic boundary of the mother GUVs to the lipidic membranes of its intraluminal vesicles. This CPP diffusion would presumable occur when the intraluminal vesicles touch the outer membrane bilayer of the mother GUV. There is a marked lag between binding of the CPPs to the membrane of the mother GUV and appearance of CPPs on the intraluminal vesicles (Figure 3c of the Biophysical Journal paper). This lag is, according to us, more compatible with the explanation we are giving than with a translocation mechanism. If there were direct translocation of the CPP through the membrane of the mother GUV, such a large lag would not be expected to be seen (see next point). If there is no translocation of the CPPs across the GUV membrane, it could explain why the water soluble dye within the mother GUVs does not leak out.

Q31. The authors consider that the translocation of CPPs induces depolarization, and as a result, the pore closes immediately. This kind of transient pore cannot explain the authors' result of the significant entry of PI into the cytosol during the interaction of CPPs with the cells. The authors should explain this point.

A31. Our interpretation is that PI takes advantage of the water pore triggered by hyperpolarization to penetrate cells. PI is positively charged and is attracted by the negative membrane potential of the cells. Its movement across the cell membrane is therefore unidirectional. This enables the PI molecules to accumulate/concentrate within the cytosol (Supplementary Fig. 12). When PI is in the presence of a CPP, both molecules enter with similar kinetics (Supplementary Fig. 12a and the new quantitation provided in the partially revised version of the manuscript; Supplementary Fig. 12b). PI and CPPs do no interact (Supplementary Figure 12d); hence they move independently from one another.

Q32. In this manuscript, the authors used only cancer cell lines (Raji cell, SKW6.4 cell, and HeLa cell). The lipid compositions and the stability of the plasma membranes of these cells may be different from normal cells (e.g., 33; Cancer Res., 51, 3062, 1991). Is there a possibility that negatively charged lipids such as PS and PIP2 locate in the outer leaflet locally in these cells? At least, some discussions on this point is essential.

A32. We agree with the reviewer that plasma membrane composition may vary between cancerous and not cancerous cells and that this may impact on the ability of CPPs to cross cellular membranes. We now mention this in the discussion: “While the nature of the CPPs likely dictate their uptake efficiency as discussed in the precedent paragraph, the composition of the plasma membrane could also modulate how CPPs translocate into cells. In the present work, we have recorded CPP direct translocation in transformed or cancerous cell lines as well as in primary cells. These cells display various abilities to take up CPPs by direct translocation and the present work indicates that this is modulated by their Vm. But as cancer cells display abnormal plasma membrane composition33, it will be of interest in the future to determine how important this is on their capacity to take up CPPs”.

Q33. The authors found that PI enters the cytosol significantly when CPPs interact with these cells. Based on this result, the authors concluded that pores with 2 nm diameter are formed in the plasma membrane. However, they did not show the time courses of entry of PI and that of CPPs, and thus we cannot judge whether the pore formation in the plasma membrane is the cause of the entry of CPPs or the result of the entry of CPPs. We can reasonably consider that CPPs enters the cytosol via endocytosis and bind to the inner leaflet of the plasma membrane, inducing pore formation in the plasma membrane.

A33. The kinetics we are now showing in point A31 indicate co-entry of CPPs and PI, an observation that is in line with our model. Also note that we have demonstrated that CPPs do not escape endosomes (please see our answers to questions 12 and 28). These data are therefore not compatible with the reviewer’s interpretation.

Q34. It has been reported that the negative membrane potential increases the rate constant of antimicrobial peptide (AMP)-induced pore formation or local damage in the GUV membrane (J. Biol. Chem., 294, 10449, 2019; BBA-Biomembranes, 1862, 183381, 2020). These results are related to those in the present manuscript, because here the authors consider that CPPs induce pores in the plasma membrane in the presence of negative membrane potential.

A34. We thank the reviewer for mentioning these interesting articles. As we understand them, they demonstrate that antimicrobial peptides (AMPs) bind membranes better as a function of increasing negative membrane potential and that this favors their ability to form pores in the membrane, compromising membrane integrity and inducing the release of cytosolic or luminal content. These AMPs do not behave exactly like CPPs because the latter do not compromise the integrity of the membranes.

In conclusion, the results of the membrane potential dependence of the rate of the internalization of CPPs may be solid results, which is an important contribution. However, the other analyses and the interpretations are not conclusive at the current stage.

We thank the reviewer for the positive assessment of our results concerning the membrane potential dependence on CPP uptake. Hopefully we have clarified the remaining points with our answers developed above and with the new data we are presenting.

Reviewer #2 (Significance (Required)):

(1) Using a CRISPR/Cas9-based screening, the authors found that some potassium channels play an important role in the internalization of CPP TAT-RasGAP317-326. This result advances the field of CPPs.

(2) Several researches have suggested that the depolarization decreases the rate of internalization of CPPs into cell cytosol and the hyperpolarization increases the rate. It has been also reported that negative membrane potential increases the rate of entry of two kinds of CPPs into the lumen of GUVs of lipid bilayers. The authors provide a new genetic evidence that membrane potential plays an important role in the internalization of CPPs in the cytosol. However, modulation of membrane potential affects the structure, dynamics and function of plasma membranes greatly. At the current stage, it is difficult to judge which process of the internalization of CPPs is affected by the membrane potential.

(3) The researchers of CPPs and AMPs are interested in their results after they improve the contents of the manuscript.

(4) My field of expertise is membrane biophysics, especially the interaction of AMPs and CPPs with GUVs and cells.

References

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BETWEEN me and the other world there is ever an unasked question: unasked by some through feelings of delicacy; by others through the difficulty of rightly framing it. All, nevertheless, flutter round it. They approach me in a half-hesitant sort of way, eye me curiously or compassionately, and then, instead of saying directly, How does it feel to be a problem? they say, I know an excellent colored man in my town; or, I fought at Mechanicsville; or, Do not these Southern outrages make your blood boil? At these I smile, or am interested, or reduce the boiling to a simmer, as the occasion may require. To the real question, How does it feel to be a problem? I answer seldom a word. 1 And yet, being a problem is a strange experience,—peculiar even for one who has never been anything else, save perhaps in babyhood and in Europe. It is in the early days of rollicking boyhood that the revelation first bursts upon one, all in a day, as it were. I remember well when the shadow swept across me. I was a little thing, away up in the hills of New England, where the dark Housatonic winds between Hoosac and Taghkanic to the sea. In a wee wooden schoolhouse, something put it into the boys’ and girls’ heads to buy gorgeous visiting-cards—ten cents a package—and exchange. The exchange was merry, till one girl, a tall newcomer, refused my card,—refused it peremptorily, with a glance. Then it dawned upon me with a certain suddenness that I was different from the others; or like, mayhap, in heart and life and longing, but shut out from their world by a vast veil. I had thereafter no desire to tear down that veil, to creep through; I held all beyond it in common contempt, and lived above it in a region of blue sky and great wandering shadows. That sky was bluest when I could beat my mates at examination-time, or beat them at a foot-race, or even beat their stringy heads. Alas, with the years all this fine contempt began to fade; for the worlds I longed for, and all their dazzling opportunities, were theirs, not mine. But they should not keep these prizes, I said; some, all, I would wrest from them. Just how I would do it I could never decide: by reading law, by healing the sick, by telling the wonderful tales that swam in my head,—some way. With other black boys the strife was not so fiercelysunny: their youth shrunk into tasteless sycophancy, or into silent hatred of the pale world about them and mocking distrust of everything white; or wasted itself in a bitter cry, Why did God make me an outcast and a stranger in mine own house? The shades of the prison?house closed round about us all: walls strait and stubborn to the whitest, but relentlessly narrow, tall, and unscalable to sons of night who must plod darkly on in resignation, or beat unavailing palms against the stone, or steadily, half hopelessly, watch the streak of blue above. 2 After the Egyptian and Indian, the Greek and Roman, the Teuton and Mongolian, the Negro is a sort of seventh son, born with a veil, and gifted with second-sight in this American world,—a world which yields him no true self-consciousness, but only lets him see himself through the revelation of the other world. It is a peculiar sensation, this double?consciousness, this sense of always looking at one’s self through the eyes of others, of measuring one’s soul by the tape of a world that looks on in amused contempt and pity. One ever feels his two-ness,—an American, a Negro; two souls, two thoughts, two unreconciled strivings; two warring ideals in one dark body, whose dogged strength alone keeps it from being torn asunder. 3 The history of the American Negro is the history of this strife,—this longing to attain self?conscious manhood, to merge his double self into a better and truer self. In this merging he wishes neither of the older selves to be lost. He would not Africanize America, for America has too much to teach the world and Africa. He would not bleach his Negro soul in a flood of white Americanism, for he knows that Negro blood has a message for the world. He simply wishes to make it possible for a man to be both a Negro and an American, without being cursed and spit upon by his fellows, without having the doors of Opportunity closed roughly in his face. 4 This, then, is the end of his striving: to be a co-worker in the kingdom of culture, to escape both death and isolation, to husband and use his best powers and his latent genius. These powers of body and mind have in the past been strangely wasted, dispersed, or forgotten. The shadow of a mighty Negro past flits through the tale of Ethiopia the Shadowy and of Egypt the Sphinx. Throughout history, the powers of single black men flash here and there like falling stars, and die sometimes before the world has rightly gauged their brightness. Here in America, in the few days since Emancipation, the black man’s turning hither and thither in hesitant and doubtful striving has often made his very strength to lose effectiveness, to seem like absence of power, like weakness. And yet it is not weakness,—it is the contradiction of double aims. The double-aimed struggle of the black artisan—on the one hand to escape white contempt for a nation of mere hewers of wood and drawers of water, and on the other hand to plough and nail and dig for a poverty-stricken horde—could only result in making him a poor craftsman, for he had but half a heart in either cause. By the poverty and ignorance of his people, the Negro minister or doctor was tempted toward quackery and demagogy; and by the criticism of the other world, toward ideals that made him ashamed of his lowly tasks. The would-be black savant was confronted by the paradox that the knowledge his people needed was a twice-told tale to his white neighbors, while the knowledge which would teach the white world was Greek to his own flesh and blood. The innate love of harmony and beauty that set the ruder souls of his people a-dancing and a- singing raised but confusion and doubt in the soul of the black artist; for the beauty revealed to him was the soul-beauty of a race which his larger audience despised, and he could not articulate the message of another people. This waste of double aims, this seeking to satisfy two unreconciled ideals, has wrought sad havoc with the courage and faith and deeds of ten thousand thousand people,—has sent them often wooing false gods and invoking falsemeans of salvation, and at times has even seemed about to make them ashamed of themselves. 5 Away back in the days of bondage they thought to see in one divine event the end of all doubt and disappointment; few men ever worshipped Freedom with half such unquestioning faith as did the American Negro for two centuries. To him, so far as he thought and dreamed, slavery was indeed the sum of all villainies, the cause of all sorrow, the root of all prejudice; Emancipation was the key to a promised land of sweeter beauty than ever stretched before the eyes of wearied Israelites. In song and exhortation swelled one refrain—Liberty; in his tears and curses the God he implored had Freedom in his right hand. At last it came,—suddenly, fearfully, like a dream. With one wild carnival of blood and passion came the message in his own plaintive cadences:— “Shout, O children! Shout, you’re free! For God has bought your liberty!” 6 Years have passed away since then,—ten, twenty, forty; forty years of national life, forty years of renewal and development, and yet the swarthy spectre sits in its accustomed seat at the Nation’s feast. In vain do we cry to this our vastest social problem:— “Take any shape but that, and my firm nerves Shall never tremble!” 7 The Nation has not yet found peace from its sins; the freedman has not yet found in freedom his promised land. Whatever of good may have come in these years of change, the shadow of a deep disappointment rests upon the Negro people,—a disappointment all the more bitter because the unattained ideal was unbounded save by the simple ignorance of a lowly people. 8 The first decade was merely a prolongation of the vain search for freedom, the boon that seemed ever barely to elude their grasp,—like a tantalizing will-o’-the-wisp, maddening and misleading the headless host. The holocaust of war, the terrors of the Ku-Klux Klan, the lies of carpet-baggers, the disorganization of industry, and the contradictory advice of friends and foes, left the bewildered serf with no new watchword beyond the old cry for freedom. As the time flew, however, he began to grasp a new idea. The ideal of liberty demanded for its attainment powerful means, and these the Fifteenth Amendment gave him. The ballot, which before he had looked upon as a visible sign of freedom, he now regarded as the chief means of gaining and perfecting the liberty with which war had partially endowed him. And why not? Had not votes made war and emancipated millions? Had not votes enfranchised the freedmen? Was anything impossible to a power that had done all this? A million black men started with renewed zeal to vote themselves into the kingdom. So the decade flew away, the revolution of 1876 came, and left the half-free serf weary, wondering, but still inspired. Slowly but steadily, in the following years, a new vision began gradually to replace the dream of political power,—a powerful movement, the rise of another ideal to guide the unguided, another pillar of fire by night after a clouded day. It was the ideal of “book?learning”; the curiosity, born of compulsory ignorance, to know and test the power of the cabalistic letters of the white man, the longing to know. Here at last seemed to have been discovered the mountain path to Canaan; longer than the highway of Emancipation and law, steep and rugged, but straight, leading to heights high enough to overlook life. 9Up the new path the advance guard toiled, slowly, heavily, doggedly; only those who have watched and guided the faltering feet, the misty minds, the dull understandings, of the dark pupils of these schools know how faithfully, how piteously, this people strove to learn. It was weary work. The cold statistician wrote down the inches of progress here and there, noted also where here and there a foot had slipped or some one had fallen. To the tired climbers, the horizon was ever dark, the mists were often cold, the Canaan was always dim and far away. If, however, the vistas disclosed as yet no goal, no resting-place, little but flattery and criticism, the journey at least gave leisure for reflection and self-examination; it changed the child of Emancipation to the youth with dawning self-consciousness, self?realization, self-respect. In those sombre forests of his striving his own soul rose before him, and he saw himself,—darkly as through a veil; and yet he saw in himself some faint revelation of his power, of his mission. He began to have a dim feeling that, to attain his place in the world, he must be himself, and not another. For the first time he sought to analyze the burden he bore upon his back, that dead-weight of social degradation partially masked behind a half-named Negro problem. He felt his poverty; without a cent, without a home, without land, tools, or savings, he had entered into competition with rich, landed, skilled neighbors. To be a poor man is hard, but to be a poor race in a land of dollars is the very bottom of hardships. He felt the weight of his ignorance,—not simply of letters, but of life, of business, of the humanities; the accumulated sloth and shirking and awkwardness of decades and centuries shackled his hands and feet. Nor was his burden all poverty and ignorance. The red stain of bastardy, which two centuries of systematic legal defilement of Negro women had stamped upon his race, meant not only the loss of ancient African chastity, but also the hereditary weight of a mass of corruption from white adulterers, threatening almost the obliteration of the Negro home. 10 A people thus handicapped ought not to be asked to race with the world, but rather allowed to give all its time and thought to its own social problems. But alas! while sociologists gleefully count his bastards and his prostitutes, the very soul of the toiling, sweating black man is darkened by the shadow of a vast despair. Men call the shadow prejudice, and learnedly explain it as the natural defence of culture against barbarism, learning against ignorance, purity against crime, the “higher” against the “lower” races. To which the Negro cries Amen! and swears that to so much of this strange prejudice as is founded on just homage to civilization, culture, righteousness, and progress, he humbly bows and meekly does obeisance. But before that nameless prejudice that leaps beyond all this he stands helpless, dismayed, and well-nigh speechless; before that personal disrespect and mockery, the ridicule and systematic humiliation, the distortion of fact and wanton license of fancy, the cynical ignoring of the better and the boisterous welcoming of the worse, the all?pervading desire to inculcate disdain for everything black, from Toussaint to the devil,— before this there rises a sickening despair that would disarm and discourage any nation save that black host to whom “discouragement” is an unwritten word. 11 But the facing of so vast a prejudice could not but bring the inevitable self-questioning, self-disparagement, and lowering of ideals which ever accompany repression and breed in an atmosphere of contempt and hate. Whisperings and portents came borne upon the four winds: Lo! we are diseased and dying, cried the dark hosts; we cannot write, our voting is vain; what need of education, since we must always cook and serve? And the Nation echoed and enforced this self-criticism, saying: Be content to be servants, and nothing more; what need of higher culture for half-men? Away with the black man’s ballot, by force or fraud,— and behold the suicide of a race! Nevertheless, out of the evil came something of good,—the more careful adjustment of education to real life, the clearer perception of the Negroes’ social responsibilities, and the sobering realization of the meaning of progress. 12So dawned the time of Sturm und Drang: storm and stress to-day rocks our little boat on the mad waters of the world-sea; there is within and without the sound of conflict, the burning of body and rending of soul; inspiration strives with doubt, and faith with vain questionings. The bright ideals of the past,—physical freedom, political power, the training of brains and the training of hands,—all these in turn have waxed and waned, until even the last grows dim and overcast. Are they all wrong,—all false? No, not that, but each alone was over-simple and incomplete,—the dreams of a credulous race-childhood, or the fond imaginings of the other world which does not know and does not want to know our power. To be really true, all these ideals must be melted and welded into one. The training of the schools we need to-day more than ever,—the training of deft hands, quick eyes and ears, and above all the broader, deeper, higher culture of gifted minds and pure hearts. The power of the ballot we need in sheer self-defence,—else what shall save us from a second slavery? Freedom, too, the long-sought, we still seek,—the freedom of life and limb, the freedom to work and think, the freedom to love and aspire. Work, culture, liberty,—all these we need, not singly but together, not successively but together, each growing and aiding each, and all striving toward that vaster ideal that swims before the Negro people, the ideal of human brotherhood, gained through the unifying ideal of Race; the ideal of fostering and developing the traits and talents of the Negro, not in opposition to or contempt for other races, but rather in large conformity to the greater ideals of the American Republic, in order that some day on American soil two world-races may give each to each those characteristics both so sadly lack. We the darker ones come even now not altogether empty-handed: there are to-day no truer exponents of the pure human spirit of the Declaration of Independence than the American Negroes; there is no true American music but the wild sweet melodies of the Negro slave; the American fairy tales and folk-lore are Indian and African; and, all in all, we black men seem the sole oasis of simple faith and reverence in a dusty desert of dollars and smartness. Will America be poorer if she replace her brutal dyspeptic blundering with light-hearted but determined Negro humility? or her coarse and cruel wit with loving jovial good-humor? or her vulgar music with the soul of the Sorrow Songs? 13 Merely a concrete test of the underlying principles of the great republic is the Negro Problem, and the spiritual striving of the freedmen’s sons is the travail of souls whose burden is almost beyond the measure of their strength, but who bear it in the name of an historic race, in the name of this the land of their fathers’ fathers, and in the name of human opportunity.

14 And now what I have briefly sketched in large outline let me on coming pages tell again in many ways, with loving emphasis and deeper detail, that men may listen to the striving in th

有點特別的發現， 對於頭頸癌個案，即使是在診斷之前，可能就有比較高的憂鬱分數。 有可能是他們已經開始感到跟診斷有關的不適，因此已經在受苦

自殺風險最高的癌別。補充： larynx/pharynx: 喉 / 咽

頭頸 肺癌 胰腺癌

Once again we see the Sabres adding quotes that will cause less backlash to them in the future. Including this quote about how they are unsure of when they will hire a new head coach gives them more breathing room. If this article was written by an outside source, there would most likely be less bias, and more negative sentences/ words about the GM and the organization.

Many people were unhappy with how long it took the GM to fire the head coach since the Sabres have lost 12 games straight, so including this in the article is another way to try and inform the readers why it took so long, even if it may be a lie.

This is a great insight into the police officers eyes. This is so interesting to see and observe. I think it was very smart how the author of the article showed this insight. I also think that it puts a new light on the police force in general since a lot of recent events have caused a negative thought to be shown.

Ive never head of "Grindr" before, but I find it kind of interesting the there's a separate dating app for gay people but also wonder why there's a need for a separate service when on any other dating app you can just set what your preference is and only that would show up.

He has two contradicting voices going through his head. The good angel does not want him to make the agreement with Mephistophilis and the evil angel does.

I think there are very probably counterexamples to this. Not sure how much that would be technical nit-picking or important to keep in the back of one's head. But ok, perhaps under some mild assumptions or exclusion of special cases, I find it intuitively likely that such equilibria do exist.

Daunting question. How do you define who you are? Is it what you do and what you like? Is it how you interact with the world around you? Or is it the thoughts in your head?

💯

Examples of pain points when marketing is absent.

Examples of value added by marketing.

If this refers to the standard error, shouldnt it be without the "head"?! Otherwise this is not consistent with the previous naming scheme

Companies that have a larger crawl breadth than IA are one way to measure. e.g. Google.

pick your battles lol - but also the whole pupil and teacher power structure

many such cases

It seems as though he is feeling regret here, and rightfully so. He should have more confidence, bravery, and courage. You only live once. No?

Jesus died on the cross for us. He went through so much pain and torture to free us from all sin, that way we could have a better life than he did. They nailed him to the cross that they made him carry himself. Pressed a thorn cross onto his head to the point of it making him bleed, and was whipped across the back multiple times bleeding to death.