See 9:02 for snapshots of the new animation style

WIT Studio is working on a new One Piece series that has better animation/pacing. They seem to also collaborate with Netflix here?

List of people working on it.

foster collaboration intersection

for - sub0symbolic.artificail.intellgence

symbolic0representation Knowledge.Graphs GKs

for - Sub-Symbolic.Methods

The sole purpose of the SC Pump (Steam generator Self Cleaning pump) is to empty the steam generator.

In Basic settings:

The length of time the pump is required to run to empty the steam generator can be adjusted. (Normally set at 45 seconds)

The amount of time since the last Self Clean Automatic is recorded.for reference purposes.

The Self cleaning maximum interval time can be set (Normally 7,200 seconds (2 hours)).

The pumps operational functionality (XS ovens only) can be set between Continuous running or Pulsing

The CDS (Calcium Diagnostic Sensor) S11 is a hall sensor device, sensing movement of water though any of the five solenoid valves in the 5 port valve block.

In Basic Settings the number of pulses per litre can be adjusted.

Must be set at 1350 pulses per litre for 6-1/1 to 20-2/1

Must be set at 1000 pulses per litre for 6-2/3 (XS model)

The CDS sensor is connected to the A10 I/O board at connection X15 and when water is flowing, measures the volume by sending electrical pulses via the A10 I/O board through the bus system (Data Highway) to the main processor (A11).

The CDS provides a preset number of pulses for each litre of water passing through the valve block.

The processor (A11) uses this information to confirm:

a) That any specific valve is in fact working by sending a signal to open the valve and then waiting for pulses to appear from the CDS sensor as in confirmation that the valve has in fact opened.

b) The volume of water stored in the steam tank each time the SC Automaitic system operates..

Certain Launcher screens have "Module test" facilities which aid as a diagnostic tool by running specific processes and providing either confirmation that the system is fully functioning or indicating via service error codes which components may be faulty.

Basic settings on SCCWE required a machine to be switched off at the on/off button to confirm any changes.

iCombi Pro basic setting changes are active as soon as they are changed. It is no longer necessary to switch thje machine off and back on again to confirm the changes.

The diagnostic tab enables you to see real time functionality such as activation of components like door switch, level probe, fan motor speeds etc.

Error code description:

Faulty data communication to automatic ignition controller.

Condition for error detection:

BUS signal from automatic ignition controller is missing or is not transmitted for at least 5 seconds at a time.

Error area:

Data transfer cable, automatic ignition controller.

Relevant causes/components:

• Electrical connection to components
• Automatic ignition controller

Error code description:

Faulty data communication to the I/O board.

Condition for error detection:

BUS signal from I/O board is missing or is not transmitted for at least 5 seconds at a time.

Error area:

Data transfer cable, I/O board

Relevant causes/components:

• Electrical connection to components
• I/O board

Error code description:

Faulty data communication to pump PCB.

Condition for error detection:

BUS signal from pump PCB is missing or is not transmitted for at least 5 seconds at a time.

Error area:

Data transfer cable, pump PCB

Relevant causes/components:

• Electrical connection to components
• Pump PCB

Error code description:

Faulty data communication to bottom fan motor.

Condition for error detection:

BUS signal from bottom fan motor is missing or is not transmitted for at least 5 seconds at a time.

Error area:

Data transfer cable, control electronics for bottom fan motor

Relevant causes/components:

• eSTL has initialised
• Electrical connection to components
• Control electronics for bottom fan motor

Communications error with STM32

STM32 is a family of 32-bit microcontroller integrated circuits

Error code description:

Faulty data communication to the middle fan motor on floor units and the bottom fan motor on 10 tray table top units.

Condition for error detection:

BUS signal from middle fan motor on floor units and the bottom fan motor on 10 tray table top units is missing or is not transmitted for at least 5 seconds at a time.

Error area:

Data transfer cable, control electronics for middle fan motor on floor units and the bottom fan motor on 10 tray table top units

Relevant causes/components:

• eSTL has initialised
• Electrical connection to components
• Control electronics for middle fan motor

Fan motor bottom on floor units

"...Христианские проповедники говорят, что любая добродетель обращается чудовищем, если в ней нет любви. Справедливость становится жестокостью, искренность – хамством, вежливость – лицемерием, вера – фанатизмом. Даже самоотдача без любви – лишь инструмент для манипуляций.

Но я говорю, что любая добродетель – и даже сама любовь – обращается чудовищем, если в ней нет уважения. Там же, где уважение царит и властвует, даже ненависть и зависть обретают человеческое лицо."

"as (not THAN) it was to the Biden presidency

for - The roundtable on the human future - Club of Rome - Roundtable

for - Call for world action on multiple threats - Roundtable on the Human Future

Ну и что

https://www.liberation.fr/environnement/climat/bilan-carbone-de-paris-2024-vraiment-plus-verts-ces-jeux-20240811_OX6AGZH7KVGXFIWVX7MVXMXCEA/

Feels like what I do with Drafts/JS— solve problems programmatically. Maybe that’s a gateway to development beyond that stack; there’s only so much I can do in a text editor, however superpowered it might be…

https://www.theguardian.com/world/article/2024/aug/10/thousands-of-serbians-protest-in-belgrade-against-lithium-mine

“平衡态”和“偏离平衡”是古人用直觉就把握的世界理论。人们从这一观点出发，逐渐建立一个differential theory（newton laws）。

Aristotelian (384-322 BC): Violent motion (Need persistent causing by an agent) / Natural Motion. Eg. object naturally falls down and stay at rest. Eg. Projectile motion sustained by some intermediate carrier of motion, continuously exercising force to it. Denies empty space. Potentiality and Actuality.

Hipparchus (2CE): force is "transferred" to the body and dissipates as the body moves.

Philoponus (6CE): "inclination" is gained from the agent and then dissipated. Upon exhaustion, the object is back to natural motion. Objects continue to move in empty space. Concept of energy: dynamis, energeia.

(Iran) Avicenna (11CE) The Book of Healing. Distinction between Force and Inclination (Mayl). Object gains Mayl when it is in opposition to its natural motion. (close to potential energy). Similar idea as Newton's concept of Inertia. Dissipation of Mayl requires external force.

(Arabic) Hilbat Allah Abu'l-Barakat al-Baghdaadi (12CE). Like Philoponus' idea, unlike Sina: Mayl self-extinguishes.

Acceleration of falling body: The falling body itself provides mayl, one after another.

Jean Buridan (14CE), impetus = weight x velocity. Similar to today's concept of momentum. When something moves an object by violence, it provides 1) force - that makes the object move, 2) impetus - that keeps exerting force to the object. Still distinguishes moving and at rest. Distinguishes linear and circular impetus.

Posits that God provides celestial bodies with impetuses, that are never damped or resisted. Could not well explain why these bodies are rotating at a constant speed, rather than infinitely fast.

Tunnel Experiment distinguishes between 1) Aristotelian theory 2) H-P Variant of Aristotelian theory, 3) Buridan impetus theory.

1) Predicts that the object becomes at rest when reaches the center of the Earth, since no force would be there to move it. 2) Cannot reach the surface again, since by the time the ball reaches center of the Earth, the initial upward force of impetus is either exhausted, or produce a force opposite to the direction of motion of the ball. 3) Predicts that the object would perform pendulum motion. During the natural motion downward, the object would accumulate enough impetus, greater than gravity, to pull it up toward the surface after reaching the center.

Pendulum motion is not explainable by 1) or 2), only explainable by 3).

This thought experiment is the origin of all oscillations in the history of dynamics. Analogy is quickly drawn between pendula and vibrating strings.

Video summary start-timeflag - end-timeflag: Generate a succinct and informative video summary in one paragraph of about 150 words according to the video transcript. Capture the primary ideas, key insights, and notable information presented in the video transcript Highlights: + [00:00:29][^1^][1] La méditation de pleine conscience à l'école * Présentation de Stéphanie Devané, professeure des écoles, et Frédéric Lenoir, spécialiste d'hypnose. * Contexte : préoccupation croissante pour la santé mentale des enfants à l'école. * Promesses de la méditation de pleine conscience : amélioration des apprentissages, bien-être, réduction du stress, renforcement des compétences psychosociales. + [00:19:18][^2^][2] Origine de la méditation de pleine conscience * Remonte au 19e siècle dans le bouddhisme. * Propagation en Birmanie suite à la colonisation anglaise. * Laïcité remise en question : la méditation de pleine conscience est-elle vraiment laïque?

Video summary start-timeflag - end-timeflag: Generate a succinct and informative video summary in one paragraph of about 150 words according to the video transcript. Capture the primary ideas, key insights, and notable information presented in the video transcript.

Highlights: + [00:23:29][^1^][1] Origines de la méditation de pleine conscience * La méditation de pleine conscience a évolué à partir des pratiques bouddhistes en Birmanie et a été popularisée par des Occidentaux intéressés par l'Orient. * Elle est devenue plus répandue et a finalement atteint les écoles, mais avec des influences New Age. + [00:30:07][^2^][2] Laïcité et méditation à l'école * La question de savoir si la méditation est appropriée à l'école est complexe. * L'agrément éducation nationale ne garantit pas l'absence de biais ou de dérives sectaires. * La vigilance est nécessaire pour éviter tout impact sur la liberté de conscience des élèves.

epistemic dynamics / belief dynamics.

c.f. Borel-Cantelli.

adjective

pronoun

verb

noun

noun

conjuctive adverb

noun

prepositions

verb

adverb

noun

verb

verb

conjuctive adverb/conjuction

noun

pronoun

noun

verb

noun

transition word (conjuctions)

verb

subject

pronoun

transition word(likely this one as it isnt in the beggiinig of a sentence, i would say is a coordinating cunjuction belonging to FANBOYS)/conditional adverb

noun

preposition

何をやってもダメだったのにこれで回復した。 自分は一般的にはPCの知識がある方だと思っていたけど、これは知らなかった。 本当に感謝。

I see Collaboration as an essential pivot for a social constructivist environment. It is such a great vehicle for allowing people to engage with a multiple of different perspectives which can challenge assumptions.

Se aborda una crítica a una vision sesgada de los conflictos y actos de violencia global. Se cuestiona la tendencia a "satanizar" al Estado Islámico, destacando la influencia de los medios de comunicación en nuestra percepción de estos eventos. Tambien una falta de atención hacia las víctimas de los bombardeos occidentales en Siria e Irak y se argumenta que la violencia y barbarie tienden a engendrar más violencia. Además menciona que la historia ha demostrado repetidamente cómo las subjetividades y sus producciones racionales, por ser inherentemente conflictivas y subjetivas, pueden conducir a la barbarie. Esta perspectiva invita a reflexionar sobre las causas subyacentes y la responsabilidad compartida en los conflictos globales

We, in the Grafoscopio community, had a similar need, but our approach was different: to keep the format simple and to use Pandoc combined with metasystem capabilities of Pharo (for example the fact that a document can be used to program itself) to address publishing formats and reproducibility.

More details as comments ahead.

Should I be concerned that these really don't look like they match up well at all?

This is a table, not a Figure. The are referenced and numbered independently.

No mention of what the dashed vs solid lines mean in the figure or the caption.

why should a reader care about this?

This was not defined earlier

explain before figure

Why did you do that here but not for the earlier situation? What is the motivation behind this?

Does this also mean that the drama and docu-series curves are individually significantly different?

what should a reader take away from this? Also, why the shading? That wasn't apparent in the previous plot.

explain before figure.

This is across all the platforms correct. The log rank test works both across groups or across pairs?

Explain before the image.

And so what should a reader be concluding from this image?

It would probably be worth lighboxing these images so that they don't take you to a different page when clicked, but instead get larger on this page.

Why this over the ratings, for instance?

You can write definitions in markdown with markdown word : definition which might be better here than subheadings.

I appreciate this, as I haven't taken all these classes!

I saw no statistics in this section. Only EDA visualization. So don't conflate the two.

You need a legend explaining the blue and green on these still.

Move the explanation before the figures.

I would stack these into the same figure if what you are wanting to do is compare across them.

To get a consistent x-axis I'd change it to days before or after release. Then the two should be comparable.

This is better than Fig 4, but it is still difficult to see some of the smaller variations b/c of the dominance of Netflix's all-at-once. Would a vertical log scale help this at all? That would seem preferential to making a cut on the vertical scale.

No sense of footnoting when it shows up on the next line. Just include your explanation as part of the caption.

I think I'd just skip straight to Figure 5, as Figure 4 is mostly not giving anything important because, as mentioned, of the Netflix dominance.

Above figure.

Also, this figure I have a little bit of a harder time understanding exactly how it is going to factor into your research question. So all the more reason to perhaps explain that.

It is important to relate your EDA back to your research question. What are the implications of this massive lead Netflix has on your research question? (Because I suspect there very much are implications) Keep a reader thinking about the data in the context that you want to evaluate it.

Are you referencing these in the Quarto method? Usually it will link them if you are doing that.

Your caption should also explain what the main thing you wanted a reader to get out of the image was. Here presumably that is the huge lead Netflix has in this area.

Again, I'd move the discussion about the image to before the image actually appears.

I think I'd move much of this up ABOVE your Figure. It is always better to explain the figure fully in the text before showing it. If someone wants to look ahead at the figure as they read then, that is fine. But it keeps someone from looking at a figure, being confused and taking a bunch of time to figure it out, only to continue reading and have you explain it all.

You should basically always introduce a section with some text before an image, other section heading, table or etc appears.

Also, I think I'd change this name to Analysis, or Data Analysis.

Good mention, but then you need to explain what is happening in the figure in the text.

Yeah, this is always tricky and difficult to automate.

write this out at least the first time you mention it

I would try to center a bit more of this around how this data interacts around your research question and possible ethical concerns in that direction. This isn't initially a topic that screams out ethics, so I think you need to think a bit harder about this. Maybe think about what the results of your study might be and if they could affect the streaming landscape in a way that might be unethical? If it turns out that all-at-once is substantially better for generating buzz, could that have unequal ramifications in some way to customers? Is the act of binge-watching entire seasons the healthiest way to consume entertainment content? I'm not 100% sure here, I'm just thinking about possible side-effects that might fall under ethical concerns. But I think you can expand this some, even if just to mention that you have considered some of these things and found there to be no ethical problems.

This feels like a many-to-many relationship type of dynamic, where you'd frequently use a "middle table" to store the connections, where it would just have two columns: show id and genre id, and if a show had multiple it would just show up on multiple rows (the connecting table often has no primary key)

I'm assuming this means as the only available language? Because many you can get dubbed or with sub-titles? Or does this just mean the original language?

Could one show be in multiple of these bins? Like a dramatic comedy?

EDIT: Oh ok, you address this later.

thetvdb gives this one Action, Drama, Science Fiction. So maybe those would have been more generalizable at least. Oh, and sub-genre of superhero

Are single quotes just rendering in this odd way? These actually feel like back-tics to me, except that they are going the wrong direction.

Also, on thetvdb, though I don't know how they compare

Need to name that source!

thetvdb has an API and includes release dates of every single episode.

Are these ratings of the streaming platform itself? Or like an average rating of shows shown on the platform? What is being rated here I guess is my question.

I don't love subheadings for blocks of text that only end up being a paragraph long. You might reconsider how you've structured this with the subheadings or see if there is more you could like to add about this and FlixPatrol.

show name?

might be worth pointing out that Google Trends does not offer an API, so this was the best (and only) approach

From your earlier descriptions, it seemed like most of the hybrid release systems did some sort of bulk release at the start before switching to weekly or similar. So it seems especially surprising that they do so much worse so quickly, since the initial episodes seem like they are essentially all-at-once released.

what are the oldest shows that you gathered data on? You haven't made that clear yet.

Given the timelines you mentioned above, having a timeframe for this would help me place it within that context

you can just leave this off and call this the Introduction. That is totally fine

Though release schedule and platform are highly correlated in this dataset correct?

what should a reader take away from this?

explain first.

good, since this kinda felt like the whole objective based on your research question.

explain first

why should a reader care?

Yeah, this felt very surprising to me, since docuseries in general was among the worst performing earlier. And yet here all of its variants seem to perform better. What would cause that?

Explain first.

Are the shaded regions indicating confidence or error? Or just arbitrary? I can't figure it out.

Also, I wonder if this graph would be more readable with panels, similar to the below figure.

How much of some of these curves are influenced by small numbers of shows fitting in that bin? Would it be helpful to show counts of the number of shows analyzed in each figure?

What should a reader take away from this graphic?

This is of all shows that have multiple seasons? Or were some show's scheduled changed mid-season? You'd only mentioned the season changes so far I think.

Exactly what difference are you talking about here. Presumably between shows that have changed and those that have done, but what is the variable you are comparing?

how to interpret this?

Oh, so these categories have been encoded as different features now?

It is interesting that you chose to not include the release schedule in this, given the general target of your research question. Why was that?

Two sentences isn't much of a paragraph.

We are still looking at original content right? So you are saying that Peacock had the least amount of original content and Netflix the most.

Wait, maybe I am confused about how these yearly top 10 ratings are working. They are broken down by platform? So you are asking if the show shows up on that particular platform's top 10? Isn't the platform then always going to play a strong role, given the relative amount of non-sharing of content that goes on?

I'd adjust the label on your plot to make this more clear.

Also, these values come to 173, which is a far cry from the numbers mentioned earlier. Why is this so much smaller?

How balanced is your data though?

Clarify this: you happen to have a small number in your testing dataset? Even over runs with different seeds? Or compared to the total number of shows relatively few make top 10 status? (Which I'm still not sure if it is by platform or not)

Ironically you do not meant release schedule here, even after saying that was your research question. So maybe you wanted something like: "In the end, we also ended up looking at extra potentially contributing factors, including genre and streaming platform."

continuing popularity. Not necessarily for its popularity at any one time, because you didn't really measure that correct?

at least by some metric.

I think you can flesh your conclusions out a bit more. What could be done differently in the future in make this sort of analysis easier, more reliable, faster, etc. What new areas would you like to add. Is critical reception one of those areas? Or is that something that is tough to quantify and thus you want to stay away from it? What should the takeaway be for streaming platforms?

Oooh, where is the real evidence of this? I don't think this was called out to me at any point during the analysis. Make sure you aren't making conclusions that you have not adequately shown or supported.

The speaker doesn't grade because he believes it allows students to create something to their own standard. He believes that when a student does an assignment it should be done with creativity and personality rather than uniformity.

These sentences contain many of the central ideas of the article in a nutshell. Laymon goes on to explore ideas of friendship (or its lack), the possibilities of forging friendships through reading, and the relationships he wishes to forge with his readers and larger audience.

Do you ever need to be?

Looking back I have done this before with a lot of my assignments. I don't necessarily ignore the comments and feedback, but I do give the number or letter grade more value. I think giving feedback without attaching a grade to it could be a reasonable solution to this issue. It would help students grow instead of basing their success on a letter in the grade book. I'm just not sure if/how this would work in schools.

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

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Reply to the reviewers

Point-to-point answer to reviewers comments

Reviewer #1

Evidence, reproducibility and clarity

*Summary: *

*The study by Cottignies-Calamarte et al. describes that AMP-activated protein kinase (AMPK) regulates cell energy balance by suppressing energy-consuming pathways like lipid and protein synthesis and promoting nutrient availability through autophagy. These pathways contribute to SARS-CoV-2 infection by hijacking autophagy and accumulating lipid droplets for viral replication. The antiviral activity of MK-8722, a direct pan-AMPK allosteric activator, was evaluated in vitro. MK-8722 effectively inhibited Alpha and Omicron SARS-CoV-2 variants in Vero76 and human bronchial epithelial Calu-3 cells at micromolar concentrations. This inhibition restored autophagic flux, degrading newly synthesized viral proteins, and reduced lipid metabolism, affecting viral factories. Additionally, MK-8722 treatment increased the type I interferon (IFN-I) response. Post-infection treatment with MK-8722 efficiently suppressed viral replication and restored the IFN-I response without altering the SARS-CoV-2-specific CD8+ T cell response elicited by Spike vaccination. The authors concluded that, MK-8722 acts as an effective antiviral against SARS-CoV-2 infection, even when applied post-exposure, suggesting potential for preclinical tests to inhibit viral replication and alleviate patient symptoms. *

__Major comments: __

• • Are the key conclusions convincing?** Partially. See comments below! *

- Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

From my perspective, the title "Direct pharmacological AMPK activation inhibits mucosal SARS-CoV-2 infection by reducing lipid metabolism, restoring autophagy flux and the type I IFN response" is a clear overstatement. In no way can the authors make statements about the autophagic flux, as it simply was not measured. The study would greatly benefit from conducting an autophagy/autophagic flux assay. See specific comments below!

Answer: As suggested by the author, we have now investigated the autophagic flux by staining the cells for LC3b expression and colocalization with the lysosomal marker LAMP1. Results are shown in the new Fig4.D and E and detailed in the results section to read lines 468-475 page 23-24:

“To assess directly the impact of MK-8722 on the autophagic flux, Calu3 cells were infected by SARS-CoV-2 without and with MK-8722 (5uM), double labelled with LC3b and LAMP1, and co-localisation of the two marker quantified (Fig.4D). MK-8722 treatment, compared with no treatment, increased LC3b colocalization in the LAMP1 compartment as shown by the increase in MOCs in treated versus non treated infected cells (LAMP1 signal in LC3b signal 0.078±0.014 vs 0.01±0.006, Mann-Whitney pand lines 480-481 page 24:

“This result indicates that MK-8722 restores the autophagic flux to address viral components to the lysosome, where they are degraded.”

- Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

See below specific comments regarding cell line consistency and autophagy measurements.

- Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

This question depends on various factors such as access to relevant biosafety labs, availability of required reagents, etc. In my estimation, experiments involving WT viruses and autophagy measurements could be conducted within 3-4 months. The proposed experiments with the delta-N-SARS-CoV-2 mutants, of course, depend on access to such viruses. Overall, I believe all experiments could be completed within 6 months. The costs of those assays are not very high.

- Are the data and the methods presented in such a way that they can be reproduced?

In the present study, a total of 3 cell lines and human PBMCs were utilized for various experiments. Please indicate why each cellular model was chosen and highlight the differences between these models considering what is known for SARS-CoV-2 infection and autophagy! Furthermore, the study would greatly benefit if the key findings were consistently demonstrated in a single cell line.

Answer: We agree with the reviewer that three cell lines and PBMC from patients were used but it was necessary for the experimental design of our experiments as detailed below.

In Figures 1 and 2, experiments use both Vero76 and Calu3 cells for the following reasons. We first used the simian Vero76 cells in order to validate the inhibitory effect of MK-8722 in widely a used cell line in virology, but which lacks the interferon response. This lack of the IFN response renders Vero cells a poor model for the pathophysiology of SARS-CoV-2. We therefore then used the IFN-competent human lung Calu-3 cell line as a more relevant cell model.

In Figures 3 both cells were again and western blots show a similar pattern of activation downstream AMPK after activation by MK-8722 and similar antiviral activity of MK-8722 in Vero76 and Calu3 cells. In Figure.4, we then mostly focused on deciphering the antiviral mechanism of MK-8722 and therefore focused on Calu3, which is more relevant from a pathophysiological point of view.

In Figure 5, we then investigated the T-cell response and therefore used primary human, namely PBMC/purified CD8 T-cells from healthy donors. As the reviewer knows, T-cell activation is restricted by HLA-TCR interaction, or in other words, only matched HLA cells can activate CD8 T-cells. As HLA-A2 is the main HLA expressed in human and only a HLA-A2 antibody is available on the market, for these experiments we used only CD8 T cells from HLA-A2 patients and had to find an additional HLA-A2-expressing epithelial cell susceptible to SARS-CoV-2 infection. It is the case of Caco2 cells, which are HLA-A2+ susceptible to infection and competent for IFN responses. We could not use simian Vero cells that are IFN-deficient and do not express human MHC, nor Calu3 cells being HLA-A2 negative. Altogether, in experiments in Figure 5 addressing HLA-A2 restricted antigen presentation, the use of Caco-2 cells was appropriate in contrast to that of Vero or Calu3.

Furthermore, for sake of clarification, we have now added the following p. 19 lines 390-392:

“The trends in modification of all markers by MK-8722 treatment by being conserved between cell lines indicates a common antiviral mechanism. We therefore focused our study on Calu-3 cells since they are more relevant to SARS-CoV-2 infection.”

The authors conclude that selective activation of AMPK has a pro-autophagic effect which in turn leads to a reduced SARS-CoV-2 replication. I would generally agree with this statement, but throughout the entire manuscript, no real autophagy assays are shown. This should definitely be rectified. It is important to demonstrate that (1) MK-8722 is capable of increasing autophagy and particularly autophagic flux in the cell models used, (2) that in the cell models employed, SARS-CoV-2 infection leads to modulation of autophagy, and (3) that SARS-CoV-2 infected cells, when co-treated with MK-8722, lead to a re-established autophagy. The autophagy assays should be performed according to the expert-curated guidelines by Klionsky et al. This is extremely important so that the results can be compared with the now vast number of existing autophagy-SARS-CoV-2 studies.

We fully agree with the reviewer that it was important for our study to formally address the impact of MK-8722 on the autophagic flux. Following reviewer recommendation and reading of the guidelines for autophagy study, we therefore have evaluated whether MK-8722 affected localization of LC3b, which is essential for autophagosome biogenesis/maturation and also functions as an adaptor protein for selective autophagy, in lysosomal compartment (labelled, by LAMP1). Therefore we labelled cells infected in the presence or absence of MK-8722 for LC3b expression and colocalization with the lysosomal marker LAMP1. Results are shown in the new Fig4.D, E. Please see our above answer (p1) for results description. These results indicate that MK-8722 restored the autophagic flux that had been interrupted by SARS-CoV-2 infection in Calu3 cells.

- Are the experiments adequately replicated and statistical analysis adequate?

Minor comments:

- Specific experimental issues that are easily addressable.

Typo: Line 288: It should be "MK-8722" instead of "MK-7288".

In general, a space between value and unit is not consistently used.

Please indicate always the phosphosite of substrate proteins when phosphorylation is described. E.g. line 288 and throughout the manuscript: regarding ACC phosphorylation.

Answer: We apologize for these typos, which have now been corrected in the revised MS.

• Are prior studies referenced appropriately?

See comment below. Fundmental work that describes the virus-autophagy relationship, such as the work by the Beth Levine lab would be important to add. Also the work bei Konstantin Sparrer and colleagues is important and leads to the current work presented here.

Answer: Konstantin Sparrer’s work is already cited as by ref 17. However, as suggested by the reviewer, the work by the Beth Livine lab has now been added in the revised MS in p29-30 lines 610-613, to read:

.“Convergence of Beclin-Atg14 and P62 activation could stimulated the selective clearance of viral components, in a process called virophagy64–66. We thus propose that AMPK pharmacological activation induce virophagy and is responsible, at least partially, for its antiviral effect.”

- Are the text and figures clear and accurate?

Introduction: In general, for some of the statements claimed in the introduction, which is in sum nicely written, informative and well structured, citations are required. For example - line 59 "...autophagy is sequentially activated and inhibited."

Answer: Citation has been added, namely: Koepke 2021 Autophagy ref 17

Lines 59-65. In the introduction, the interaction between autophagy and SARS-CoV-2 is primarily described in a one-sided manner. There are now several studies demonstrating that both inhibition of autophagy and also induction of autophagy in context of coronaviral infection. Both aspects should be illuminated and introduced here.

Answer: The reviewer points towards a crucial point of autophagy during ß-coronaviruses infection. Indeed, we fully agree that the autophagy is both inhibited and activated, at different levels, by different proteins as exemplified by, Sparrer’s group (ref 17). As we have mentioned in the initial version of our MS: “Throughout the SARS-CoV-2 viral cycle, autophagy is sequentially activated and inhibited 17–20”. As it may have not been clear enough, we have reformulated this paragraph as follows to read line 68-75 p5:

“Throughout the SARS-CoV-2 viral cycle, autophagy is sequentially activated and inhibited 17–20. Indeed, autophagy is initiated by the early expressed nsp6, resulting in the formation of autophagosomes that are essential for the establishment of viral factories 17,21 and subsequent viral proteins expression 17,21. In turn, viral proteins OFR3a and ORF7a expressed at a latter time post-infection, prevent the fusion between autophagosomes and lysosomes, thereby blocking completion of autophagy, as evidenced by increased LC3-B expression, activation of the ULK1 kinase and increase in the autophagy cargo receptor sequestosome-1/p62. Overall, this disruption of autophagy protects newly formed virus from degradation in the LAMP1+ lysosome 19,22–25”

Discussion: The selectivity of the compound should be discussed.

Answer: Manuscript have been revised as follows to discuss both specificity in regards of AMPK and tissue accessibility:

• lines 557-560, p27: “We show here that the blockade of AMPK activation upon infection can be reversed by MK-8722, the pharmacological allosteric pan-activator of AMPK, which blocks infection at a µM concentration, in agreement with the predicted role of AMPK activity on SARS-CoV-2 infection48 and with MK-8722 action on infection by other viruses49”

• Line 576-581, p28: “In contrast, MK-8722, as systemic drug, may reach these tissues with minimal side effects, as a daily treatment in diabetic Non-human primates (NHPs) with MK-8722 (10 mg/kg) for a month induced only a limited and reversible cardiac hypertrophy 36”

• Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

The presentation of the data is understandable. For reader with a less mechanistic background it would be helpful to present a schematic figure like a graphical abstract.

Answer: As suggested by the reviewer, we have now introduced a graphical abstract in the revised MS.

SECTION B – Significance

======================== - Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.

• Even though there is now a plethora of studies on autophagy and SARS-CoV-2, this study is important and of great interest to a broad readership. Not only virologists, immunologists, and autophagy researchers will eagerly anticipate this study, but especially researchers focusing on pharmacology around AMPK and autophagy will recognize the importance of the data presented here.*

Answer: We thank the reviewer for this positive appreciation of our work.

- Place the work in the context of the existing literature (provide references, where appropriate).

The work builds upon a variety of studies on the interplay between coronaviruses and the mechanism of autophagy. Foundational contributions to this research stem from groundbreaking preliminary work conducted in the laboratories of Gassen and Müller (Gassen et al. 2019 and Gassen et al. 2021), as well as general virus-autophagy studies from the laboratory of Beth Levine. Additionally, recent work by Konstantin Sparrer and colleagues would be important to cite, as it underscores the insights gained in this manuscript.

Answer: As suggested and previously mentioned, these references have now been added to the discussion p27 lines 557-560: “We show here that the blockade of AMPK activation upon infection can be reversed by MK-8722, the pharmacological allosteric pan-activator of AMPK, which blocks infection at a µM concentration, in agreement with the predicted role of AMPK activity on SARS-CoV-2 infection48 and with MK-8722 action on infection by other viruses49”.

In addition, Gassen et al. 2019 is already cited as ref 18

- State what audience might be interested in and influenced by the reported findings. See comment above!__ __

Reviewer #2

Evidence, reproducibility and clarity

In the current manuscript, Cottignes-Calamarte et al. have shown tha pharmacological activation of AMPK can be a strategy for overcoming SARS-CoV-2 infection induced reprogramming of host degradation pathways and innate immune response, without hindering the efficacy of spike expression from vaccine agents. Though the suggestion of selective activity of MK-8722 in degrading viral proteins in infection but not the ectopic spike peptides expression is interesting, the evaluation of the mechanism and providing therapeutic index for the drug will overall improve the study.

• Majorly, I have these suggestions;*

• • The manuscript did not clarify the mechanism clearly but correlated the reduction in viral proteins and their association to LAMP1 as the mechanisim of activity of MK-8722. In this way, the authors did not separate whether the reduction in SARS-CoV-2 infection could lead to the potentiation of these effects. There is mentioning of potential mechanism of the drug by inhibiting the activity of SARS-CoV-2 proteins that reduce the autophagic flux without directly showing this. SARS-CoV-2 Orf3a is a known inhibitor of autophagosome maturation and hence the authors should directly probe the activity of MK-8722 in overcoming the suppression of autophagic flux in cells by ectopically expressing Orf3a.* Answer: We fully agree with the reviewer that our work correlated the reduction in viral proteins and their association to LAMP1 to explain the mechanism of MK-8722 activity and did not focus on particular viral protein(s) that could induced autophagic flux.

Indeed, our approach has been to describe the MK-8722 antiviral activity against full primary SARS-CoV-2 viruses (not ectopically expressed viral proteins or recombinant viruses) in a pathologically relevant cell model including primary CD8+ T cells to mimic at best the initial steps of SARS-CoV-2 infection. Although the ectopical expression of Orf3a is recognized as a potent tool to study the principle of autophagy in the guidelines of autophagy measurement methods, when applied to infection, this ectopical expression of Orf3a will modify the endogenous level of Orf3a (as compared to infection level) and probably affect by itself the autophagy kinetics. To address the question of autophagic flux as suggested by the reviewer, we therefore preferred to use another recommended technique from the guidelines of autophagy measurement methods directly applicable to infected cells. We now evaluated the colocalization of LC3b with LAMP1 during treatment and infection of Calu-3 cells by primary viruses as now described in figure 4.D and E and discussed lines 468-475 pages 23-24 :

“To assess directly the impact of MK-8722 on the autophagic flux, Calu3 cells were infected by SARS-CoV-2 without and with MK-8722 (5uM), double labelled with LC3b and LAMP1, and co-localisation of the two marker quantified (Fig.4D, E). MK-8722 treatment, compared with no treatment, increased LC3b colocalization in the LAMP1 compartment as shown by the increase in MOCs in treated versus non treated infected cells (LAMP1 signal in LC3b signal 0.078±0.014 vs 0.01±0.006, Mann-Whitney p * As the authors propose MK-8722 as a preclinical candidate, they should present therapeutic measures and indexes. All across the data presented, there was no mentioning or measurement of drug toxicity for extended uses up to 36h pi.*

Answer: Our study aimed to describe the antiviral activity of MK-8722 in a cellular model mimicking only the initial steps of infection up to 3 dpi. Complementary experiments were conducted as required and show that treatment with MK-8722 up to 10mM did not result in an increase in cell death at any time post treatment as shown in the new Fig S2I and corresponding description in line 328-332 p17, which read__: __

__“__Furthermore, we also investigated MK-8722 toxicity at 4h, 24h and 96h (Fig. S2I) and found that the drug was not toxic up to 10mM. The 50% toxicity dose was calculated to be 57mM at 96h in Calu3 cells. This allows us to determine a therapeutic index of 76 against the SARS-CoV-2 Alpha variant and 36 against the Omicron variant, thus, placing MK-8722 as an attractive antiviral candidate.”

• Also, it was not clarified if the drug reduced the replication or exclusively worked by restoration of autophagic flux. For the earlier, the authors can consider two assays, i.e., a direct assay of looking into the replicon of SARS-CoV-2 (Bigotti et al 2024; PMID 38387750), or looking at earlier time points of infection (up to 8h pi; Twu et al. 2021; PMID 34788596) with intracellular sgRNA specific RNA probes.In this regards, 24h or 32h (Fig 1F-G) is too long to only measure single-round of infection.*

Answer: We thank the reviewer for this interesting question. The drug likely acts on both steps. Concerning replication, as shown in figure 1 GH at 24 and 32hrs, there is a block in replication but not in virus entry into the cell, since there is no difference in viral N cellular content after 1hr chase. Concerning the autophagic flux, the new Fig4 D and E shows that MK-8722 restores the autophagic flux in infected cells, which had been interrupted by the infection in the absence of the drug.

The viral cycle of ß-coronaviruses is highly dependent on hijacking cellular autophagy and lipid biosynthesis, which are both necessary for the assembly of DMV, essential for viral replication. Since MK-8722 treatment reduces cellular lipid content and increases autophagic flux, the use of viral replicons deficient in structural proteins or early post-infection timepoints will most likely show a decrease in viral genome replication under the effect of MK-8722 due to the inability of the replicons to establish such favourable environments (indicated in the corresponding Fig3A, B and C). However, the effect of MK-8722 on the replicon system will not distinguish between whether MK-8722 affects the viral replication complex and whether it is unable to induce viral factory formation.

• Are the viral components degraded by restored lysosomal activity include replicase or replication organelle components? This needs to be shown with intracellular nsp3/nsp4 levels in infection or ectopic expression.*

Answer: The reviewer raises a question highly relevant to the biology of coronaviruses, which we have addressed in Figure 4. Indeed, the Replication-Transcription Complex (RTC) is a multifactorial complex, in which N protein bound to the viral RNA is believed to help recruiting RdRp (Cong 2020 J Virol 10.1128/jvi.01925-19, Scherer 2022 Sci Adv DOI: 10.1126/sciadv.abl4895). The increased colocalisation of in N staining in LAMP-1+ compartments observed after MK-8722 treatment indicates that MK-8722 addresses RTC to the lysosomes and therefore our results indicate that replication-transcription organelles could be degraded in lysosomes.

• Previous studies suggested the decrease in AMPK phospharhorylation in SARS-CoV-2 infection What is inconsistent to previous studies should be commented by the authors. Eg, why is AMPK suppression not see in SARS-CoV- 2 inefction as reported before (Parthasarathy et al 2023; PMID 36417940)*

Answer: We agree with the reviewer and had already mentioned in the discussion that SARS-CoV-2 infection can have different outcome on AMPK activation as reported in ref 45 and 46 in the original version. Indeed, Gassen and colleagues reports that activation of AMPK and resulting phosphorylation are decreased in cells infected by SARS-CoV-2, as shown in (Ref 18), whereas Parthasarathy reports an increase in AMPK phosphorylation which is clear only at 96 h p.i. (Ref 47). Interestingly in this later study at earlier time point (24hr p.i.), authors report a tendency of AMPK phosphorylation to decrease although not in a statistically significant manner but for only n=3. In our present study, no statistically significant differences were found in AMPK phosphorylation although a small increase tendency is observed. Furthermore, cells used in all these studies differ: Gassen et al. used Vero-FM cells whereas Parthasarathy et al. used intestinal Caco2 cells, and in our study Vero 76 and lung Calu3 cells. Thus the differences reported on the effect of SARS-CoV-2 infection on AMPK activation are likely due to a question of kinetics and/or cellular background. Of note, the level of AMPK phosphorylation observed after SARS-CoV-2 infection is not to compare with the AMPK phosphoryalton induced by drug activation such as MK-8722.

This heterogenity in AMPK activation during SARS-CoV-2 infection might contribute to the various effect of AMPK activator as antiviral reported in the literature. Indeed, as mentioned p27 lines 560-563 “metformin, a drug approved by FDA since 1994, and the adenosine analogue AICAR that activates AMPK indirectly can inhibit replication of SARS-CoV-2 as well as Flaviviruses, but at a concentrations of 10 and 1 mM, respectively 35,47 ”. The reported discrepancy on the antiviral effect of AMPK activation might rely then on an activation threshold, as 10mM metformin and 1mM AICAR blocks infection, while 25__m__M AICAR does not (ref 18, 47). This later concentration was not evaluated in Gassen et al. on AMPK activation. Furthermore; the time frame evaluated in each study is different and could also be a confusing factor. Finally, as shown by Myers and colleagues (Myers 2017, Science ref 36), MK-8722 activates AMPK more efficiently than AICAR, most probably due to its direct activation of AMPK. We believe that direct AMPK activation by MK-8722 and the higher activation level of AMPK is responsible for the antiviral effect reported in our study.

As suggested by the reviewer, we have now clarified this section in the revised MS to read p27-28, lines 557-568:

“We show here that the blockade of AMPK activation upon infection can be reversed by MK-8722, the pharmacological allosteric pan-activator of AMPK, which, at a µM concentration, blocks infection, in agreement with the predicted role of AMPK activity on SARS-CoV-2 infection48 and with MK-8722 action on infection by other viruses49” .In line, metformin, a drug approved by FDA since 1994, and the adenosine analogue AICAR that activates AMPK indirectly can inhibit replication of SARS-CoV-2 as well as Flaviviruses, but at concentrations of 10 and 1 mM, respectively 35,47. These AMPK-sensitive viruses replicate all in viral factories, disturbing lipid synthesis and escaping autophagy 50,51. AMPK activation by metformin at high concentration (10mM) inhibits SARS-CoV-2 replication in vitro 47. However, AMPK activation with 5-amino-imidazolecarboxamide riboside (AICAR), a non-metabolised analogue of AMP able to activate AMPK, used at 25mM is unable to inhibit SARS-CoV-2 infection 18 while at 1mM has been proven to reduce by 10-fold viral production 47, suggesting that AMPK needs to reach an activation threshold to inhibit viral replication. “

• *

• Although AMPK activation is shown to be antiviral for SARS-CoV-2 before, e.g., with Metformin (Parthasarathy et al 2023; PMID 36417940), the role of AMPK-related kinases are shown to be pro-viral (e.g., NUAK2; Prasad et al. 2023; PMID 37421942). The authors should discuss these points to provide the reader a context in this sub-field.*

Answer: As suggested by the reviewer, we have now included in the discussion the following p28 lines 568-571:

“Conversely, NUAK2, an AMPK-related kinase, was reported to stimulate viral replication in A549 and Calu3 cells50. Altogether our results, in agreement with the literature, indicate that AMPK-dependent antiviral activity is restricted to AMPK-members only, confirming their distance with AMPK-related kinases such as NUAK2 53”

Minor

1. • Was there an increase of lipid droplets in SARS-CoV-2 infected cells to non-infected condition? It is not mentioned here.* Answer: Yes indeed, lipid droplets content was increased in Infected non treated cells as indicated in Figure Suppl. 4B (p21 lines 409-411) and as stated in the Result section as follows:

” Infection increased the overall Nile Red staining by 40±10% compared to non-infected cells (Fig. S4B, ANOVA: p * There are several typos and grammatical errors.*

Answer: We have now carefully checked the text for typographical and grammatical errors, which have been corrected.

There is no Fig S2I but is mentioend in the legend.

Answer: We apologize for this mistake in labelling figure S2 panel and have corrected their labelling as follows:

“____G-H: __ACE2 expression and viability were evaluated in Vero76 cells after 24h or in Calu-3 cells after 4days of MK-8722 continuous treatment (1 µM or 5 µM respectively) by flow cytometry. ACE2 expression is expressed as MFI (__G) and viability as frequency of cells non-stained by the amine-reactive dye Viobility (H). n=3 independent experiments. “

• Fig 4A - degradation is not shown, only association.*

Answer: We agree with the reviewer and have modified the text accordingly to read p37 lines 873-874:

Figure 4: MK-8722 treatment increases the ____autophagic flux directing viral components ____to ____lysosomes and restores type I interferon response.”

---

• Line 333 - there is no 3d pi post exposure treatment with MK-8722 in Fig 1l.*

Answer: We meant that treatment initiated at 1dpi lasted until harvest at 3dpi. This is clarified now to read, p18 line 351

“Post-exposure treatment at 1dpi and until harvest at 3dpi, …”

Significance

General assessment*: *

Strengths and limitations: The study provides evidence supporting previous results that the activation AMPK can be harnessed as a strategy to combat SARS-CoV2- infection. Whereas the results suggesting that this is targeted in infection by restoration of autopahgic flux and hence is selective is interesting, but the authors did not directly investigated the SARS-CoV-2 protein that is implicated in this effect. The study also does not discuss the context of AMPK and related kinases in discussion.*

*

Advance -* The study makes a pre-clinical advance, and has potential to make it fundamentally or conceptually sound. *

Audience - Virologists and Clinicians.

Expertise - SARS-CoV-2 infection biology, Cell biology an Molecular virology__ __

__Reviewer #3 __

Evidence, reproducibility and clarity (Required):

• Summary.** The authors address a topic of great importance to the development of antivirals, particularly in light of the possibility of future pandemics due to hitherto understudied viruses: the evaluation of interfering with host pathways (host-directed antivirals), potentially leading to compounds that can be used against a broad spectrum of viruses which require the same host cell functions for their life cycle. The authors studied the effects of activating AMPK with a small molecule (MK-8722) on various aspects of infectivity of SARS-CoV-2. They find that the compound indeed markedly reduced viral infectivity in two cell lines (Vero and Calu3), which correlated with the ability of the compound to activate signaling downstream of AMPK and was associated with increased lysosomal genesis/function and reduced density of "lipid viral factories". *

• *

General assessment. The study provides important proof of concept in reductionist cellular models, which may lead to pharmacologically more conclusive in vivo studies later on. It is limited by the use of cell lines, instead of primary human cells, for viral infectivity. The caption "MK-8722 restores the IFN I pathway" is an overstatement, as the observed increase in ISG expression is quite modest.

Answer: We agree with the reviewer that we did not use primary cells in our study, which was designed to evaluate the antiviral activity of MK-8722 in a simple epithelial cell model still relevant for the pathophysiology. However, we believe that MK-8722 antiviral effect we observed in the present study will be conserved using primary cells given the breadth of cell lines we used such as Vero, pulmonary Calu-3 and intestinal Caco2 cells. Furthermore, concerning the magnitude of the IFN I response we report, we remind the reviewer that Calu-3 cells are infected with an MOI of 0.05, which does not result in 100% cells infected after 24h. Consequently, the IFN-I response as limited to the sole infected cells in a limited amount in this setup and thus, bulk RT-qPCR of Type I IFN and targeted ISG mRNA is expected to remain modest.

In future studies, as suggested by the reviewer, we plan to include few ALI culture treated primary cells. We have clarified this limitation of our study in the Discussion p. 31 line 636-638 to read:

“The antiviral effect of AMPK activation in primary lung reconstruction and preclinical models such as hamster remains to be tested.”

__ Significance (Required): __

Taken as an early proof-of-concept study, the findings are quite important to investigators aiming to develop host-directed antivirals. However, the overall interest and impact of the manuscript would greatly benefit from verifying key findings in a primary cell model. Also, checking at least one other viral species (the authors mention flaviviruses) would be helpful to test how broadly applicable the current compound (or others to be developed in the future) would be as host-directed antivirals.

Answer: We thank the reviewer for the positive evaluation of our work. We fully agree with the reviewer in that preparedness is the key to fighting future pandemics. However to our knowledge, the literature about Flaviviruses is already stating that AMPK activation by metformin is an antiviral strategy with regards to its lipid metabolism normalization and autophagy activation (Farfan-Morales 2021 Sci Rep doi: 10.1038/s41598-021-87707-9. Ref 50). Furthermore in a previous review, we discussed the role of AMPK activation on viral infection that could be either pro- or antiviral depending on the viral family and even within a viral family such as HSV-1 which first inhibits AMPK in early infection while activating AMPK in the latter stage (Moreira, D. et al. Curr Drug Targets 17, 942–953 (2016), ref 34). Hence, adding more viruses to our study will not add novelty to the study, even though the molecule is much stronger compared to metformin.

• *

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Referee #3

Evidence, reproducibility and clarity

Summary. The authors address a topic of great importance to the development of antivirals, particularly in light of the possibility of future pandemics due to hitherto understudied viruses: the evaluation of interfering with host pathways (host-directed antivirals), potentially leading to compounds that can be used against a broad spectrum of viruses which require the same host cell functions for their life cycle. The authors studied the effects of activating AMPK with a small molecule (MK-8722) on various aspects of infectivity of SARS-CoV-2. They find that the compound indeed markedly reduced viral infectivity in two cell lines (Vero and Calu3), which correlated with the ability of the compound to activate signaling downstream of AMPK and was associated with increased lysosomal genesis/function and reduced density of "lipid viral factories".

General assessment. The study provides important proof of concept in reductionist cellular models, which may lead to pharmacologically more conclusive in vivo studies later on. It is limited by the use of cell lines, instead of primary human cells, for viral infectivity. The caption "MK-8722 restores the IFN I pathway" is an overstatement, as the observed increase in ISG expression is quite modest.

Significance

Taken as an early proof-of-concept study, the findings are quite important to investigators aiming to develop host-directed antivirals. However, the overall interest and impact of the manuscript would greatly benefit from verifying key findings in a primary cell model. Also, checking at least one other viral species (the authors mention flaviviruses) would be helpful to test how broadly applicable the current compound (or others to be developed in the future) would be as host-directed antivirals.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #2

Evidence, reproducibility and clarity

In the current manuscript, Cottignes-Calamarte et al. have shown tha pharmacological activation of AMPK can be a strategy for overcoming SARS-CoV-2 infection induced reprogramming of host degradation pathways and innate immune response, without hindering the efficacy of spike expression from vaccine agents. Though the suggestion of selective activity of MK-8722 in degrading viral proteins in infection but not the ectopic spike peptides expression is interesting, the evaluation of the mechanism and providing therapeutic index for the drug will overall improve the study.

Majorly, I have these suggestions;

1. The manuscript did not clarify the mechanism clearly but correlated the reduction in viral proteins and their association to LAMP1 as the mechanisim of activity of MK-8722. In this way, the authors did not separate whether the reduction in SARS-CoV-2 infection could lead to the potentiation of these effects. There is mentioning of potential mechanism of the drug by inhibiting the activity of SARS-CoV-2 proteins that reduce the autophagic flux without directly showing this. SARS-CoV-2 Orf3a is a known inhibitor of autophagosome maturation and hence the authors should directly probe the activity of MK-8722 in overcoming the suppression of autophagic flux in cells by ectopically expressing Orf3a.
2. As the authors propose MK-8722 as a preclinical candidate, they should present therapeutic measures and indexes. All across the data presented, there was no mentioning or measurement of drug toxicity for extended uses up to 36h pi.
3. Also, it was not clarified if the drug reduced the replication or exclusively worked by restoration of autophagic flux. For the earlier, the authors can consider two assays, i.e., a direct assay of looking into the replicon of SARS-CoV-2 (Bigotti et al 2024; PMID 38387750), or looking at earlier time points of infection (up to 8h pi; Twu et al. 2021; PMID 34788596) with intracellular sgRNA specific RNA probes.In this regards, 24h or 32h (Fig 1F-G) is too long to only measure single-round of infection.
4. Are the viral components degraded by restored lysosomal activity include replicase or replication organelle components? This needs to be shown with intracellular nsp3/nsp4 levels in infection or ectopic expression.
5. Previous studies suggested the decrease in AMPK phospharhorylation in SARS-CoV-2 infection What is inconsistent to previous studies should be commented by the authors. Eg, why is AMPK suppression not see in SARS-CoV- 2 inefction as reported before (Parthasarathy et al 2023; PMID 36417940)
6. Although AMPK activation is shown to be antiviral for SARS-CoV-2 before, e.g., with Metformin (Parthasarathy et al 2023; PMID 36417940), the role of AMPK-related kinases are shown to be pro-viral (e.g., NUAK2; Prasad et al. 2023; PMID 37421942). The authors should discuss these points to provide the reader a context in this sub-field.

Minor

1. Was there an increase of lipid droplets in SARS-CoV-2 infected cells to non-infected condition? It is not mentioned here.
2. There are several typos and grammatical errors.
3. There is no Fig S2I but is mentioend in the legend.
4. Fig 4A - degradation is not shown, only association.
5. Line 333 - there is no 3d pi post exposure treatment with MK-8722 in Fig 1l.

Significance

General assessment

Strengths and limitations: The study provides evidence supporting previous results that the activation AMPK can be harnessed as a strategy to combat SARS-CoV2- infection. Whereas the results suggesting that this is targeted in infection by restoration of autopahgic flux and hence is selective is interesting, but the authors did not directly investigated the SARS-CoV-2 protein that is implicated in this effect. The study also does not discuss the context of AMPK and related kinases in discussion.

Advance - The study makes a pre-clinical advance, and has potential to make it fundamentally or conceptually sound.

Audience - Virologists and Clinicians.

Expertise - SARS-CoV-2 infection biology, Cell biology an Molecular virology

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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Referee #1

Evidence, reproducibility and clarity

Summary:

Provide a short summary of the findings and key conclusions (including methodology and model system(s) where appropriate).

The study by Cottignies-Calamarte et al. describes that AMP-activated protein kinase (AMPK) regulates cell energy balance by suppressing energy-consuming pathways like lipid and protein synthesis and promoting nutrient availability through autophagy. These pathways contribute to SARS-CoV-2 infection by hijacking autophagy and accumulating lipid droplets for viral replication. The antiviral activity of MK-8722, a direct pan-AMPK allosteric activator, was evaluated in vitro. MK-8722 effectively inhibited Alpha and Omicron SARS-CoV-2 variants in Vero76 and human bronchial epithelial Calu-3 cells at micromolar concentrations. This inhibition restored autophagic flux, degrading newly synthesized viral proteins, and reduced lipid metabolism, affecting viral factories. Additionally, MK-8722 treatment increased the type I interferon (IFN-I) response. Post-infection treatment with MK-8722 efficiently suppressed viral replication and restored the IFN-I response without altering the SARS-CoV-2-specific CD8+ T cell response elicited by Spike vaccination. The authors concluded that, MK-8722 acts as an effective antiviral against SARS-CoV-2 infection, even when applied post-exposure, suggesting potential for preclinical tests to inhibit viral replication and alleviate patient symptoms.

Major comments:

• Are the key conclusions convincing?

Partially. See comments below! - Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

From my perspective, the title "Direct pharmacological AMPK activation inhibits mucosal SARS-CoV-2 infection by reducing lipid metabolism, restoring autophagy flux and the type I IFN response" is a clear overstatement. In no way can the authors make statements about the autophagic flux, as it simply was not measured. The study would greatly benefit from conducting an autophagy/autophagic flux assay. See specific comments below! - Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

See below specific comments regarding cell line consistency and autophagy measurements. - Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

This question depends on various factors such as access to relevant biosafety labs, availability of required reagents, etc. In my estimation, experiments involving WT viruses and autophagy measurements could be conducted within 3-4 months. The proposed experiments with the delta-N-SARS-CoV-2 mutants, of course, depend on access to such viruses. Overall, I believe all experiments could be completed within 6 months. The costs of those assays are not very high. - Are the data and the methods presented in such a way that they can be reproduced?

In the present study, a total of 3 cell lines and human PBMCs were utilized for various experiments. Please indicate why each cellular model was chosen and highlight the differences between these models considering what is known for SARS-CoV-2 infection and autophagy! Furthermore, the study would greatly benefit if the key findings were consistently demonstrated in a single cell line.

The authors conclude that selective activation of AMPK has a pro-autophagic effect which in turn leads to a reduced SARS-CoV-2 replication. I would generally agree with this statement, but throughout the entire manuscript, no real autophagy assays are shown. This should definitely be rectified. It is important to demonstrate that (1) MK-8722 is capable of increasing autophagy and particularly autophagic flux in the cell models used, (2) that in the cell models employed, SARS-CoV-2 infection leads to modulation of autophagy, and (3) that SARS-CoV-2 infected cells, when co-treated with MK-8722, lead to a re-established autophagy. The autophagy assays should be performed according to the expert-curated guidelines by Klionsky et al. This is extremely important so that the results can be compared with the now vast number of existing autophagy-SARS-CoV-2 studies. - Are the experiments adequately replicated and statistical analysis adequate?

Minor comments: - Specific experimental issues that are easily addressable.

Typo: Line 288: It should be "MK-8722" instead of "MK-7288". In general, a space between value and unit is not consistently used. Please indicate always the phosphosite of substrate proteins when phosphorylation is described. E.g. line 288 and throughout the manuscript: regarding ACC phosphorylation. - Are prior studies referenced appropriately?

See comment below. Fundmental work that describes the virus-autophagy relationship, such as the work by the Beth Levine lab would be important to add. Also the work bei Konstantin Sparrer and colleagues is important and leads to the current work presented here. - Are the text and figures clear and accurate?

Introduction:

In general, for some of the statements claimed in the introduction, which is in sum nicely written, informative and well structured, citations are required. For example - line 59 "...autophagy is sequentially activated and inhibited." Lines 59-65. In the introduction, the interaction between autophagy and SARS-CoV-2 is primarily described in a one-sided manner. There are now several studies demonstrating that both inhibition of autophagy and also induction of autophagy in context of coronaviral infection. Both aspects should be illuminated and introduced here.

Discussion: The selectivity of the compound should be discussed. - Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

The presentation of the data is understandable. For reader with a less mechanistic background it would be helpful to present a schematic figure like a graphical abstract.

Significance

• Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.

Even though there is now a plethora of studies on autophagy and SARS-CoV-2, this study is important and of great interest to a broad readership. Not only virologists, immunologists, and autophagy researchers will eagerly anticipate this study, but especially researchers focusing on pharmacology around AMPK and autophagy will recognize the importance of the data presented here. - Place the work in the context of the existing literature (provide references, where appropriate).

The work builds upon a variety of studies on the interplay between coronaviruses and the mechanism of autophagy. Foundational contributions to this research stem from groundbreaking preliminary work conducted in the laboratories of Gassen and Müller (Gassen et al. 2019 and Gassen et al. 2021), as well as general virus-autophagy studies from the laboratory of Beth Levine. Additionally, recent work by Konstantin Sparrer and colleagues would be important to cite, as it underscores the insights gained in this manuscript. - State what audience might be interested in and influenced by the reported findings.

See comment above! - Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

My expertise is limited to the mechanistic aspects of autophagy, metabolism, and signaling cascades related to autophagy and endosomal-exosomal mechanisms. In some studies, I have been able to investigate the interplay between CoV and autophagy in collaborations with coronavirus experts. I can only provide a superficial assessment of the purely virological (methodological) aspects of the present study.

eLife assessment

This important study compared the brain development trajectories of humans and macaque monkeys to quantify different evolutionary effects of convergent and divergent neural pathways between the two species. The cross-species evidence is solid, based on brain age prediction models that were carefully developed by using public MRI datasets of both humans and macaque monkeys. The findings will be of interest to neuroscientists, developmental biologists, and evolutionary biologists.

Reviewer #1 (Public Review):

The authors conducted cross-species comparisons between the human brain and the macaque brain to disentangle the specific characteristics of structural development of the human brain. Although previous studies had revealed similarities and differences in brain anatomy between the two species by spatially aligning the brains, the authors made the comparison along the chronological axis by establishing models for predicting the chronological ages with the inputting brain structural features. The rationale is actually clear given that brain development occurs over time in both. More interestingly, the model trained on macaque data was better able to predict the age of humans than the human-trained model was at predicting macaque age. This revealed a brain cross-species age gap (BCAP) that quantified the discrepancy in brain development between the two species, and the authors even found this BCAP measure was associated with performance on behavioral tests in humans. Overall, this study provides important and novel insights into the unique characteristics of human brain development. The authors have employed a rigorous scientific approach, reflecting diligent efforts to scrutinize the patterns of brain age models across species. The clarity of the rationale, the interpretability of the methods, and the quality of the presentation all contribute to the strength of this work.

Reviewer #2 (Public Review):

In the current study, Li et al. developed a novel approach that aligns chronological age to a cross-species brain age prediction model to investigate the evolutionary effect. This method revealed some interesting findings, like the brain-age gap of the macaque model in predicting human age will increase as chronological age increases, suggesting an evolutionary alignment between the macaque brain and the human brain in the early stage of development. This study exhibits ample novelty and research significance. However, I still have some concerns regarding the reliability of the current findings.

(1) Although the authors named their new method a "cross-species" model, the current study only focused on the prediction between humans and macaques. It would be better to discuss whether their method can also generalize to cross-species examination of other species (e.g., C. elegans), which may provide more comprehensive evolutionary insights. Also, other future directions with their new method are worth discussing.

(2) Algorithm of prediction model. In the method section, the authors only described how they chose features, but did no description about the algorithm (e.g., supporting vector regression) they used. Please add relevant descriptions to the methods.

(3) Sex difference. The sex difference results are strange to me. For example, in the second row of Figure Supplement 3A, different models show different correlation patterns, but why their Pearson's r is all equal to 0.3939? If they are only typo errors, please correct them. The authors claimed that they found no sex difference. However, the results in Figure Supplement 3 show that, the female seems to have poorer performance in predicting macaque age from the human model. Moreover, accumulated studies have reported sex differences in developing brains (Hines, 2011; Kurth et al., 2021). I think it is also worth discussing why sex differences can't be found in the evolutionary effect.

Reference:<br /> Hines, M. (2011). Gender development and the human brain. Annual review of neuroscience, 34, 69-88.<br /> Kurth, F., Gaser, C., & Luders, E. (2021). Development of sex differences in the human brain. Cognitive Neuroscience, 12(3-4), 155-162.

Reviewer #3 (Public Review):

SUMMARY:

The authors identified a series of WM and GM features that correlated with age in human and macaque structural imaging data. The data was gathered from the HCP and WA studies, which was parcellated in order to yield a set of features. Features that correlated with age were used to train predictive intra and inter-species models of human and macaque age. Interestingly, while each model accurately predicted the corresponding species age, using the macaque model to predict human age was more accurate than the inverse (using the human model to predict macaque age). In addition, the prediction error of the macaque model in predicting human age increased with age, whereas the prediction error of the human model predicting macaque age decreased with age.

After elaboration of the predictive models, the authors classified the features for prediction into human-specific, macaque-specific and common to human and macaque, where they most notably found that macaque-only and common human-macaque areas were located mainly in gray matter, with only a few human-specific features found in gray matter. Furthermore, the authors found significant correlations between BCAP and picture vocabulary (positive correlation) test and visual sensitivity (negative correlation) test. Several white matter tracts (AF, OR, SLFII) were also identified showing a correlation with BCAP.

STRENGTHS AND WEAKNESSES

The paper brings an interesting perspective on the evolutionary trajectories of human and non-human primate brain structure, and its relation to behavior and cognition. Overall, the methods are robust and support the theoretical background of the paper. However, the overall clarity of the paper could be improved. There are many convoluted sentences and there seems to be both repetition across the different sections and unclear or missing information. For example, the Introduction does not clearly state the research questions, rather just briefly mentions research gaps existing in the literature and follows by describing the experimental method. It would be desirable to clearly state the theoretical background and research questions and leave out details on methodology.<br /> In addition, the results section repeats a lot of what is already stated in the methods. This could be further simplified and make the paper much easier to read.<br /> In the discussion, authors mention that "findings about cortex expansion are inconsistent and even contradictory", a more convincing argument could be made by elaborating on why the cortex expansion index is inadequate and how BCAP is more accurate.

STUDY AIMS AND STRENGTH OF CONCLUSIONS

Overall, the methods are robust and support the theoretical background of the paper, but it would be good to state the specific research questions -even if exploratory in nature- more specifically. Nevertheless, the results provide support for the research aims.

IMPACT OF THE WORK AND UTILITY OF METHODS AND DATA TO THE COMMUNITY

This study is a good first step in providing a new insight into the neurodevelopmental trajectories of humans and non-human primates besides the existing cortical expansion theories.

ADDITIONAL CONTEXT:

It should be clearly stated both in the abstract and methods that the data used for the experiment came from public databases.

eLife assessment

This important work offers an experimental structural characterization of the Tramtrack-like BTB/POZ domains in insects, revealing that these domains form stable hexameric assemblies. The structural evidence is convincing, and validated by fold prediction and evolutionary pathway analyses. This paper would be of interest to structural and evolutionary biologists.

Reviewer #1 (Public Review):

Using structural analysis, Bonchuk and colleagues demonstrate that the TTK-like BTB/POZs of insects form stable hexameric assemblies composed of trimers of POZ dimers, a configuration observed consistently across both homomultimers and heteromultimers, which are known to be formed by TTK-like BTB/POZ domains. The structural data is comprehensive, unambiguous, and further supported by theoretical fold prediction analyses. In particular the judicious complementation of experiments and fold prediction is commendable. This study now adds an important cog that might help generalize the general principles of the evolution of multimerization in members of this fold family.

I strongly feel that enhancing the inclusivity of the discussion would strengthen the paper. Below, I suggest some additional points for consideration for the same.

Major points.<br /> (1) It would be valuable to discuss alternative multimer assembly interfaces, considering the diverse ways POZs can multimerize. For instance, the Potassium channel POZ domains form tetramers. A comparison of their inter-subunit interface with that of TTK and non-TTK POZs could provide insightful contrasts.

(2) The so-called TTK motif, despite its unique sequence signature, essentially corresponds to the N-terminal extension observed in other "non-TTK" proteins such as Miz-1. Given Miz-1's structure, it becomes evident that the utilization of the N-terminal extension for dimerization is shared with the TTK family, suggesting a common evolutionary origin in metazoan transcription factors. Early phylogenetic trees (e.g. in PMID: 9917379) support the grouping of the TTK-like POZs with other animal Transcription factors containing POZ domains such as those with Kelch repeats further suggesting that the extension might be ancestral. Structural investigations by modeling prominent examples or comparing known structures of similar POZ domains, could support this inference. Control comparisons with POZ domains from fungi, plants and amoebozoans like Dictyostelium could offer additional insights.

(3) Exploring the ancestral presence of the aforementioned extension in metazoan transcription factors could serve as a foundation for understanding the evolutionary pathway of hexamerization. This analysis could shed light on exposed structural regions that had the potential to interact post-dimerization with the N-terminal extension and also might provide insights into the evolution of multimer interfaces, as observed in the Potassium channel.

(4) Considering the role of conserved residues in the multimer interface is crucial. Reference to conserved residues involved in multimer formation, such as discussed in PMID: 9917379, would enrich the discussion.

eLife assessment

This important study explores and delineates multivariate mappings between brain structure and functional measures with latent dimensions of psychopathology. This work provides solid evidence for the existence of such mappings and charts the relationship between different neurobiological measures and distinct dimensions of psychopathology. This work will be of broad interest within the neuroscience field.

Reviewer #1 (Public Review):

This report describes work aiming to delineate multi-modal MRI correlates of psychopathology from a large cohort of children of 9-11 years from the ABCD cohort. While uni-modal characterisations have been made, the authors rightly argue that multi-modal approaches in imaging are vital to comprehensively and robustly capture modes of large-scale brain variation that may be associated with pathology. The primary analysis integrates structural and resting-state functional data, while post-hoc analyses on subsamples incorporate task and diffusion data. Five latent components (LCs) are identified, with the first three, corresponding to p-factor, internal/externalising, and neurodevelopmental Michelini Factors, described in detail. In addition, associations of these components with primary and secondary RSFC functional gradients were identified, and LCs were validated in a replication sample via assessment of correlations of loadings.

This work is clearly novel and a comprehensive study of associations within this dataset. Multi-modal analyses are challenging to perform, but this work is methodologically rigorous, with careful implementation of discovery and replication assessments, and primary and exploratory analyses. The ABCD dataset is large, and behavioural and MRI protocols seem appropriate and extensive enough for this study. The study lays out comprehensive associations between MRI brain measures and behaviour that appear to recapitulate the established hierarchical structure of psychopathology.

The work does have weaknesses, some of them acknowledged. There is limited focus on the strength of observed associations. While the latent component loadings seem reliably reproducible in the behavourial domain, this is considerably less the case in the imaging modalities. A considerable proportion of statistical results focuses on spatial associations in loadings between modalities - it seems likely that these reflect intrinsic correlations between modalities, rather than associations specific to any latent component. Assessment of associations with functional gradients is similarly a little hard to interpret. Thus, it is hard to judge the implications for our understanding of the neurophysiological basis of psychopathology and the ability of MRI to provide clinical tools for, say, stratification. The observation of a recapitulation of psychopathology hierarchy may be somewhat undermined by the relatively modest strength of the components in the imaging domain. The task fMRI was assessed with a fairly basic functional connectivity approach, not using task timings to more specifically extract network responses.

Overall, the authors achieve their aim to provide a detailed multimodal characterisation of MRI correlations of psychopathology. Code and data are available and well organised and should provide a valuable resource for researchers wanting to understand MRI-based neural correlates of psycho-pathology-related behavioural traits in this important age group. It is largely a descriptive study, with comparisons to previous uni-modal work, but without particularly strong testing of neuroscience hypotheses.

Reviewer #2 (Public Review):

In "Multi-modal Neural Correlates of Childhood Psychopathology" Krebets et al. integrate multi-modal neuroimaging data using machine learning to delineate dissociable links to diverse dimensions of psychopathology in the ABCD sample. This paper had numerous strengths including a superb use of a large resource dataset, appropriate analyses, beautiful visualizations, clear writing, and highly interpretable results from a data-driven analysis. Overall, I think it would certainly be of interest to a general readership.

That being said, I do have several comments for the authors to consider.

- Out-of-sample testing: while the permutation testing procedure for the PLS is entirely appropriate, without out-of-sample testing the reported effect sizes are likely inflated.

- Site/family structure: it was unclear how site/family structure were handled as covariates.

- Anatomical features: I was a bit surprised to see volume, surface area, and thickness all evaluated - and that there were several comments on the correspondence between the SA and volume in the results section. Given that cortical volume is simply a product of SA and CT (and mainly driven by SA), this result may be pre-required.

- Ethnicity: the rationale for regressing ethnicity from the data was unclear and may conflict with current best practices.

- Data quality: the authors did an admirable job in controlling for data quality in the analyses of functional connectivity data. However, it is unclear if a comparable measure of data quality was used for the T1/dMRI analyses. This likely will result in inflated effect sizes in some cases; it has the potential to reduce sensitivity to real effects.

Reviewer #3 (Public Review):

In this study, the authors utilized the Adolescent Brain Cognitive Development dataset to investigate the relationship between structural and functional brain network patterns and dimensions of psychopathology. They identified multiple components, including a general psychopathology (p) factor that exhibited a strong association with multimodal imaging features. The connectivity signatures associated with the p factor and neurodevelopmental dimensions aligned with the sensory-to-transmodal axis of cortical organization, which is linked to complex cognition and psychopathology risk. The findings were consistent across two separate subsamples and remained robust when accounting for variations in analytical parameters, thus contributing to a better understanding of the biological mechanisms underlying psychopathology dimensions and offering potential brain-based vulnerability markers.

Strengths:<br /> - An intriguing aspect of this study is the integration of multiple neuroimaging modalities, combining structural and functional measures, to comprehensively assess the covariance with various symptom combinations. This approach provides a multidimensional understanding of the risk patterns associated with mental illness development.

- The paper delves deeper into established behavioral latent variables such as the p factor, internalizing, externalizing, and neurodevelopmental dimensions, revealing their distinct associations with morphological and intrinsic functional connectivity signatures. This sheds light on the neurobiological underpinnings of these dimensions.

- The robustness of the findings is a notable strength, as they were validated in a separate replication sample and remained consistent even when accounting for different parameter variations in the analysis methodology. This reinforces the generalizability and reliability of the results.

Weaknesses:

- Based on their findings, the authors suggest that the observed variations in resting-state functional connectivity may indicate shared neurobiological substrates specific to certain symptoms. However, it should be noted that differences in resting-state connectivity between groups can stem from various factors, as highlighted in the existing literature. For instance, discrepancies in the interpretation of instructions during the resting state scan can influence the results. Hence, while their findings may indicate biological distinctions, they could also reflect differences in behavior.

- The authors conducted several analyses to investigate the relationship between imaging loadings associated with latent components and the principal functional gradient. They found several associations between principal gradient scores and both within- and between-network resting-state functional connectivity (RSFC) loadings. Assessing the analysis presented here proves challenging due to the nature of relating loadings, which are partly based on the RSFC, to gradients derived from RSFC. Consequently, a certain level of correlation between these two variables would be expected, making it difficult to determine the significance of the authors' findings. It would be more intriguing if a direct correlation between the composite scores reflecting behavior and the gradients were to yield statistically significant results.

- Lastly, regarding the interpretation of the first identified latent component, I have some reservations. Upon examining the loadings, it appears that LC1 primarily reflects impulse control issues rather than representing a comprehensive p-factor. Furthermore, it is worth noting that within the field, there is an ongoing debate concerning the interpretation and utilization of the p-factor. An insightful publication on this topic is "The p factor is the sum of its parts, for now" (Fried et al, 2021), which explains that the p-factor emerges as a result of a positive manifold, but it does not necessarily provide insights into the underlying mechanisms that generated the data.

eLife assessment

This potentially important work presents a tool for performing phylogenetic taxonomic classification of DNA sequences. In terms of methodology, the work is compelling. The authors perform a benchmark experiment against current state-of-the-art tools using real and simulated datasets to demonstrate where the novel tool stands in the context of existing methods. However, the experimentation is still incomplete. It would benefit from a more thorough exploration of existing methods as well as data sets that better represent real-world use cases.

Reviewer #1 (Public Review):

In this manuscript, the authors present Tronko, a novel tool for performing phylogenetic assignment of DNA sequences using an approximate likelihood approach. Through a benchmark experiment utilizing several real datasets from mock communities with pre-known composition as well as simulated datasets, the authors show that Tronko is able to achieve higher accuracy than several existing best-practice methods with runtime comparable to the fastest existing method, albeit with significantly higher peak memory usage than existing methods. The benchmark experiment was thorough, and the results clearly support the authors' conclusions. However, the paper could be improved by exploring how certain design choices (e.g. tool selection and parameter choices) may impact Tronko's performance/accuracy, and some relevant existing phylogenetic placement tools are missing and should be included.

Reviewer #2 (Public Review):

This is, to my knowledge, the most scalable method for phylogenetic placement that uses likelihoods. The tool has an interesting and innovative means of using gaps, which I haven't seen before. In the validation the authors demonstrate superior performance to existing tools for taxonomic annotation (though there are questions about the setup of the validation as described below).

The program is written in C with no library dependencies. This is great. However, I wasn't able to try out the software because the linking failed on Debian 11, and the binary artifact made by the GitHub Actions pipeline was too recent for my GLIBC/kernel. It'd be nice to provide a binary for people stuck on older kernels (our cluster is still on Ubuntu 18.04). Also, would it be hard to publish your .zipped binaries as packages?

Thank you for publishing your source files for the validation on zenodo. Please provide a script that would enable the user to rerun the analysis using those files, either on zenodo or on GitHub somewhere.

The validations need further attention as follows.

First, the authors have not chosen data sets that are not well-aligned with real-world use cases for this software, and as a result, its applicability is difficult to determine. First, the leave-one-species-out experiment made use of COI gene sequences representing 253 species from the order Charadriiformes, which includes bird species such as gulls and terns. What is the reasoning for selecting this data set given the objective of demonstrating the utility of Tronko for large scale community profiling experiments which by their nature tend to include microorganisms as subjects? If the authors are interested in evaluating COI (or another gene target) as a marker for characterizing the composition of eukaryotic populations, is the heterogeneity and species distribution of bird species within order Charadriiformes comparable to what one would expect in populations of organisms that might actually be the target of a metagenomic analysis?

Second, It appears that experiments evaluating performance for 16S were limited to reclassification of sequencing data from mock communities described in two publications, Schirmer (2015, 49 bacteria and 10 archaea, all environmental), and Gohl (2016; 20 bacteria - this is the widely used commercial mock community from BEI, all well-known human pathogens or commensals). The authors performed a comparison with kraken2, metaphlan2, and MEGAN using both the default database for each as well as the same database used for Tronko (kudos for including the latter). This pair of experiments provide a reasonable high-level indication of Tronko's performance relative to other tools, but the total number of organisms is very limited, and particularly limited with respect to the human microbiome. It is also important to point out that these mock communities are composed primarily of type strains and provide limited species-level heterogeneity. The performance of these classification tools on type strains may not be representative of what one would find in natural samples. Thus, the leave-one-individual-out and leave-one-species-out experiments would have been more useful and informative had they been applied to extended 16S data sets representing more ecologically realistic populations.

Finally, the authors should describe the composition of the databases used for classification as well as the strategy (and toolchain) used to select reference sequences. What databases were the reference sequences drawn from and by what criteria? Were the reference databases designed to reflect the composition of the mock communities (and if so, are they limited to species in those communities, or are additional related species included), or have the authors constructed general purpose reference databases? How many representatives of each species were included (on average), and were there efforts to represent a diversity of strains for each species? The methods should include a section detailing the construction of the data sets: as illustrated in this very study, the choice of reference database influences the quality of classification results, and the authors should explain the process and design considerations for database construction.

Reviewer #3 (Public Review):

Pipes and Nielsen propose a valuable new computational method for assigning individual Next Generation Sequencing (NGS) reads to their taxonomic group of origin, based on comparison with a dataset of reference metabarcode sequences (i.e. using an existing known marker sequence such as COI or 16S). The underlying problem is an important one, with broad applications such as identifying species of origin of smuggled goods, identifying the composition of metagenomics/ microbiomics samples, or detecting the presence of pathogen variants of concern from wastewater surveillance samples. Pipes and Nielsen propose (and make available with open source software) new computational methods, apply those methods to a series of exemplar data analyses mirroring plausible real-life scenarios, and compare the new method's performance to that of various field-leading alternative methods.

In terms of methodology, the manuscript presents a novel computational analyses inspired by standard existing probabilistic phylogenetic models for the evolution of genome sequences. These form the basis for comparisons of each NGS read with a reference database of known examples spanning the taxonomic range of interest. The evolutionary aspects of the models are used (a) to statistically represent knowledge about the reference organisms (and uncertainty about their common ancestors) and their evolutionary relationships; and (b) to derive inferences about the relationship of the sample NGS reads that may be derived from reference organisms or from related organisms not represented in the reference dataset. This general approach has been considered previously and, while expected to be powerful in principle, the reliance of those methods on likelihood computations over a phylogenetic tree structure means they are slow to the point of useless on modern-sized problems that may have many thousands of reference sequences and many millions of NGS reads. Alternative methods that have been devised to be computationally feasible have had to sacrifice the phylogenetic approach, with a consequent loss of statistical power.

Pipes and Nielsen's methodology contribution in this manuscript is to make a series of approximations to the 'ideal' phylogenetic likelihood analysis, aimed at saving computational time and keeping computer memory requirements acceptable whilst retaining as much as possible of the expected power of phylogenetic methods. Their description of their novel methods is solid; as they are largely approximations to other existing methods, their value ultimately will rest with the success of the method in application.

Regarding the application of the new methods, to compare the accuracy of their method with a selection of existing methods the authors use 1) simulated datasets and 2) previously published mock community datasets to query sequencing reads against appropriate reference trees. The authors show that Tronko has a higher success at assigning query reads (at the species/genus/family level) than the existing tools with both datasets. In terms of computational performance, the authors show Tronko outperforms another phylogenetic tool, and is still within reasonable limits when compared with other 'lightweight' tools.

As a demonstration of the power of phylogeny-based methods for taxonomic assignment, this ms. could gain added importance by refocusing the community towards explicitly phylogenetic methods. We agree with the authors that this would be likely to give rise to the most powerful possible methods.

Strengths of this ms. are 1) the focus on phylogenetic approaches and 2) the reduction of a consequently difficult computational problem to a practical method (with freely available software); 3) the reminder that these approaches work well and are worthy of continued interest and development; and ultimately most-importantly 4) the creation of a powerful tool for taxonomic assignment that seems to be at least as good as any other and generally better.

Weaknesses of the manuscript at present are 1) lack of consideration of some other existing methods and approaches, as it would be interesting to know if other ideas had been tried and rejected, or were not compatible with the methods created; 2) some over-simplifications in the description of new methods, with some aspects difficult or impossible to reproduce and some claims unsubstantiated. Further, 3) we are not convinced enough weight has been given to the complexity of 'pre-processing' the reference dataset for each metabarcode (e.g. gene) of interest, which may give the impression that the method is easier to apply to new reference datasets than we think would be the case. Lastly, 4) we encountered some difficulties getting the software installed and running on our computers. It was not possible to resolve every issue in the time available to us to perform our review, and some processing options remain untested.

Overall, the methods that Pipes and Nielsen propose represent an important contribution that both creates a computational resource that is immediately valuable to the community, and emphasises the benefits of phylogenetic methods and provides encouragement for others to continue to work in this area to create still-better methods.

eLife assessment

In this study, Perez-Lopez and colleagues examine an important function of the chemokine CCL28 in mucosal host defenses against the gut bacterial pathogen Salmonella Typhimurium and lung pathogen Acinetobacter baumanii. They find that CCL28-CCR3 axis regulates neutrophil recruitment and function, and promotes bacterial clearance in one infectious context but exacerbates disease against the other pathogen. Therefore, CCL28 plays a critical role in mucosal immunity and neutrophil biology that differentially affects host defenses against pathogens.

Reviewer #1 (Public Review):

In this manuscript, Perez-Lopez et al. examine the function of the chemokine CCL28, which is expressed highly in mucosal tissues during infection, but its role during infection is poorly understood. They find that CCL28 promotes neutrophil accumulation in the intestines of mice infected with Salmonella and in the lungs of mice infected with Acinetobacter. They find that Ccl28-/- mice are highly susceptible to Salmonella infection, and highly resistant and protected from lethality following Acinetobacter infection. They find that neutrophils express the CCL28 receptors CCR3 and CCR10. CCR3 was pre-formed and intracellular and translocated to the cell surface following phagocytosis or inflammatory stimuli. They also find that CCL28 stimulation of CCR3 promoted neutrophil antimicrobial activity, ROS production, and NET formation, using a combination of primary mouse and human neutrophils for their studies. Overall, the authors' findings provide new and fundamental insight into the role of the CCL28:CCR3 chemokine:chemokine receptor pair in regulating neutrophil recruitment and effector function during infection with the intestinal pathogen Salmonella Typhimurium and the lung pathogen Acinetobacter baumanii.

Reviewer #2 (Public Review):

In this manuscript by Perez-Lopez et al., the authors investigate the role of the chemokine CCL28 during bacterial infections in mucosal tissues. This is a well-written study with exciting results. They show a role for CCL28 in promoting neutrophil accumulation to the guts of Salmonella-infected mice and to the lung of mice infected with Acinetobacter. Interestingly, the functional consequences of CCL28 deficiency differ between infections with the two different pathogens, with CCL28-deficiency increasing susceptibility to Salmonella, but increasing resistance to Acinetobacter. The underlying mechanistic reasons for this suggest roles for CCL28 in enhanced neutrophil antimicrobial activity, production of reactive oxygen species, and formation of extracellular traps. However, additional experiments are required to shore up these mechanisms, including addressing the role of other CCL28-dependent cell types and further characterization of neutrophils from CCL28-deficient mice.

Reviewer #3 (Public Review):

The manuscript by Perez-Lopez and colleagues uses a combination of in vivo studies using knockout mice and elegant in vitro studies to explore the role of the chemokine CCL28 during bacterial infection on mucosal surfaces. Using the streptomycin model of Salmonella Typhimurium (S. Tm) infection, the authors demonstrate that CCL28 is required for neutrophil influx in the intestinal mucosa to control pathogen burden both locally and systemically. Interestingly, CCL28 plays the opposite role in a model lung infection by Acinetobacter baumanii, as Ccl28-/- mice are protected from Acinetobacter infection. Authors suggest that the mechanism by which CCL28 plays a role during bacterial infection is due to its role in modulating neutrophil recruitment and function.

The major strengths of the manuscript are:

The novelty of the findings that are described in the manuscript. The role of the chemokine CCL28 in modulating neutrophil function and recruitment in mucosal surfaces is intriguing and novel.

Authors use Ccl28-/- mice in their studies, a mouse strain that has only recently been available. To assess the impact of CCL28 on mucosal surfaces during pathogen-induced inflammation, the authors choose not one but two models of bacterial infection (S. Tm and A. baumanii). This approach increases the rigor and impact of the data presented.

Authors combine the elegant in vivo studies using Ccl28 -/- with in vitro experiments that explore the mechanisms by which CCL28 affects neutrophil function.

The major weaknesses of the manuscript in its present form are:

Authors use different time points in the S. Tm model to characterize the influx of immune cells and pathology. They do not provide a clear justification as to why distinct time points were chosen for their analysis.

Authors provide puzzling data that Ccl28-/- mice have the same numbers of CCR3 and CCR10-expressing neutrophils in the mucosa during infection. It is unclear why the lack of CCL28 expression would not affect the recruitment of neutrophils that express the ligands (CCR3 and CCR10) for this chemokine. Thus, these results need to be better explained.

The in vitro studies focus primarily on characterizing how CCL28 affects the function of neutrophils in response to S. Tm infection. There is a lack of data to demonstrate whether Acinetobacter affects CCR3 and CCR10 expression and recruitment to the cell surface and whether CCL28 plays any role in this process.

Is it possible, knowing the input parameters, to model and artificially manipulate the equation by driving down certain parameters and driving up others to arrive to a satisfactory aggregate score?

democratic preconditions are not co-related to the democratic outcomes? Better analysis of preconditions?

good!

what are other forms of democracy and how connected they are

how is the power defined here?

https://newsela.com/ - a source for current events that offer the same stories at different levels. One argument is that a teacher with a wide variety of students could assign the same story so all could read via Mark Grabe @ DABC

His library contains more than 30k books for sure.

He said: "When I arrived 25 years ago, they were 30,000... I have no more time to count them."

Seems logical that his library contained more than 30k books for this reason.

( ~9:25)

Lol Umberto Eco was evicted from one of his houses because the engineers thought the floors would collapse because of his books.

( ~9:00)

Apparently, Eco was interested in occultism, or at least in understanding it.

( ~7:10)

Umberto Eco was apparently so famous and conceptualized as a master, and great person that hundreds (if not thousands) of people came to his funeral in a castle.

( ~4:16)

At the beginning of this video, Umberto Eco is seen walking in his library (0:48)

While the waterfall model can be used for large, complex projects, it's more commonly and effectively applied to smaller, simpler projects. This is because it's a rigid model that doesn't accommodate changes well. In larger, more complex projects, requirements are likely to change, making the waterfall model less suitable.  

So, the statement "the waterfall model is a software development model used in the context of large, complex projects" is inaccurate.

You do have to register if you want to annotate :|

This reviewer misinteprets "can you read signs?" as "can you read psalms?"

If this theory is true, what did the Aryans do after the conquered the land? Why was it abandoned after all? Also, why do so many people believe this if there is no actual evidence?

This is... very uncivilized 🙁

How did these civilizations find each other? Has this civilization been mentioned in other civilizations (e.g. within written records or art)?

How did they plan the city out? Did they plan by drawing/writing? Who planned and built it?

How did they trade with them? What did they trade? What caused the decrease in trade?

Did the large population somehow influence India's large population today?

Will deciphering it ever be possible? Are there other ways of understanding a language other than translating from another known language?

What are "Vedic" sources?

overall its pretty ironic that the environments and organisms that are being destroyed by human activity are still helping us by giving inspiration for advanced materials and innovations.

imagine getting beat by a shrimp, jokes aside its really amazing that something so small can contain so much strength and its leading to the development of more advanced materials.

Concrete is such an integrated part of our lives that it can be hard to remember the impacts. Its really good that the coral reefs are the inspiration for a more sustainable form of concrete.

for - climate change impacts - marine life - citizen-science - potential project - climate departure - ocean heating impacts - marine life - marine migration - migrating species face collapse - migration to escape warming oceans - population collapse

main research findings - Study involved 146 species of temperate or subpolar fish and 2,572 time series - Extremely fast moving species (17km/year) showed large declines in population while - fish that did not shift showed negligible decline - Those on the northernmost edge experienced the largest declines - There is speculation that the fastest moving ones are the also the one's with the least evolutionary adaptations for new environments

for - thermal battery - off-the-shelf

for - Cities 1.5 podcast - guests - Xuemei Bai - earth system boundaries downscaled to cities - cross-scale translation of earth system boundaries