Reviewer 1. Joon-Ho Yu Thank you for the opportunity to review this manuscript. Overall, I appreciate this argument for and description of Open Humans.  Broadly, the manuscript would benefit from greater attention to writing and organization. As my comments describe below, the "ethical analysis" offered is narrowly focused and appears to serve as a justification for the resource; yet, in its current state, I think the ethical analysis either should be removed or expanded. Ideally, the manuscript would be strengthened by a deepening and broadening of ethical considerations. Note that I use P(page)C(column)L(lines) to locate my comments for the authors.

1. Abstract P1L36-37.  I am struck by the framing of this ethical problem as the responsibility of data subjects.  I assume this is intentional and would appreciate a little more, perhaps in the introduction, as to what is entailed in this responsibility?
2. Abstract P1L42-43. I am not sure if the framing of the ethical problem is resolved by the description of the utility of Open Humans.  While overall, I suggest deepening the ethical problems presented, another alternative is to leave it out all together.
3. P2C2L6-9. It would help me if parties were more clearly stated.  I think you mean researchers not research and it isn't clear to me that commercial data sources have interests but rather the companies that hold these resources do, right?
4. P2C2 Participant Involvement.  It is unclear to me what the purpose of this section is.
5. P2C1 Data Silos. Most of the descriptive language is written in the passive voice which I understand may be the norm but in my opinion, it unintentionally highlights how interests and responsibilities are dissociated or dis-located from stakeholders.  For instance, in the section on Data Silos, it remains unclear for whom Data Silos are a problem and whose interests have created and maintained these silos.  Again, this sort of analysis might help identify or locate solutions rather than only set up a problem that Open Human's solves.  My point here is that the developers of Open Humans need not rely on a somewhat limited ethical analysis to justify its existence and argue for its utility.
6. P2C1L44-49. While I agree this is accurate reflection of the scope of literature, the issues raised by "big data" research now extend far beyond the common risks relayed in a consent process.
7. P2C1L49-51. I agree that this is an important issue but this single statement citing Barbara Evans sounds a little like a strawman.  My sense is that through the efforts of many patient-driven organizations, patient and participant-driven research has increased a great deal in the past decade or so.  Perhaps this ought to be recognized especially given that many of the authors have been critical to the development of this movement.  Also, the next section on participant involvement seems at odds with the argument so some clarification might help readers understand the nuances.
8. P2C2L53-61.  While I totally agree and appreciate these key points to the participant-centered approach to research, in all honesty, I did not come to these conclusions based on the above exposition.  I suggest moving this up as the scaffold for the introduction and reorganize based on these points.
9. P3C1L30-36. These are the main points I think readers need in the introduction to help us understand the need for Open Humans.  I suggest you spend more time explaining these points and characterizing the evidence of these important assertions.
10. P3C2L46-50. Could you explain the rationale behind this feature and briefly describe if more detailed information is conveyed about the IRB approval or review/determination?
11. P4C2L25-27. This is an important statement, at least to me, but it would be helpful to reiterate how privacy is maintained, I'm assuming because its pseudonymous?
12. P4C2L27-30. Again, what are the simple requirements?
13. P5C1L58-C2L59. So what are the ethical implications of this use case?  I think an important point to highlight is that privacy may be a nominal issue with members of efforts like Open Humans as they often have a greater than average interest in research benefits than maintaining individual privacy. Further, I'm under the impression that personal privacy is less of a concern for many or rather our sense of what is private is changing.  Assuming I'm understanding the argument, what I'm confused about is that the ethical analysis presented in the background assumes that privacy is of central perhaps even sole concern.  Also, there are many other ethical issues that open humans both addresses possibly in a positive way and potentially raises as risks to members and even society.  So, I would welcome that analysis alongside this nice introduction to the platform or I would not rest the argument for the platform on a relatively narrow ethical frame.
14. P6C2L16-21. Do you mean the public data are being used as training sets for the algorithms?  Are there any risks of bias based on these sorts of uses?
15. P6C1L44-45. So are there any ethical issues related to the application of OAuth2 to these particular use cases or overall?  This isn't a trick question, I have no idea but would encourage the authors to consider based on their expertise.
16. P7C2L9-11. Agreed, but does it also make it harder for bad actors to use these data?  It would be great if the authors could help us think about this potential trade off.
17. P7C1 Discussion. I would like the authors to consider the following in the discussion and possibly the introduction. (1) Given that most people who engage in citizen science in the biomedical research space are likely to subscribe to the value of openness and sharing of samples, data, tools, etc., I wonder if focusing on privacy as key ethical barrier is on target and sufficient.  For instance, many of the challenges to genomic research  articulated by historically vulnerable populations have to do with offensive data uses, lack of control, lack of direct benefit, differential benefit based on SES, risks to groups, etc.  Again, a critical analysis of how this resource might increase or decrease such risks involved in citizen science would contribute to the larger project of extending citizen science or patient-led research to community-led research.  Of course, I understand this might been outside the bounds of this manuscript but that preclude some consideration. (2) I very much appreciate Open Humans as a tool that addresses the practical problem of bridging/linking/aggregating.  I have no problems with this argument yet I wonder if it is somewhat naive to assume that bridging as a practical benefit does not also risk other ethical challenges.  For example, the ease of bridging to pre-selected resources blurs the line between simply linking resources and advancing particular interpretations of the data, in fact, one's own data.  If I understand Open Humans, it is a tool that automates protocols for linking and sharing intended to facilitate citizen science and patient-led research.  The practical benefits are clear. But what are the risks associated with more automated linking and sharing?
18. P7C2 Enabling individual-centric research and citizen science. This section is very helpful and references a number of mechanisms that begin to address, at least on an individual level, issues such as "to what uses", "control", "governance", etc.  I would love to either see this description expanded and moved up into the initial description of the resource (maybe before or around P2C2L57) and or these functional benefits better incorporated and explicated in the use cases.
19. P8C1L13-16. It is unclear to me how it is "an ethical way" especially as it isn't clear to me what an "unethical way" would entail.   I think some pieces are presented but this argument could be much stronger and clearer.  I get that the benefits are assumed here to some extent, I've been in the same place when engaging in resource development, but perhaps a greater consideration of potential benefits and harms might help balance the focus on privacy and individual control.  Generally when we conduct ethical analysis we consider autonomy (where privacy sits), risks (as potential harms as well as increasingly benefits), and justice.  Notably. others might argue for other principles and values.  While such a comprehensive analysis isn't the focus of this manuscript, incorporating the insights of such an analysis would, in my opinion, strengthen the argument for Open Humans and signal/evidence robust consideration by its designers and authors.

This sentence really resonates with me. I think about the classroom environment and how it's set up. If we view the child as independent thinkers and in control of their learning the space should be set up to reflect that. Such as; are children able to freely move around to make independent choices on what they're investigating? Are all materials easily accessible for the children? Is their learning displayed in the classroom so that they may reflect on their previous experiences?

Can these blog posts be about whatever we want? Or, do they have to be related to Social Media?

Basically, without arousal a cue function can't exist. Cue function is a guiding behavior. The arousal system could consist of many subsystems that have their own functions. But again, without the arousal function you'd not have cue functions

While you'd think the prior experiment would help explain the exploratory, curiosity and manipulatory drive, but it did not. The experiment failed, as the subjects could not seem to concentrate well after 4-5 hours and had trouble even up to 24 hours after! With no emotional disturbances, this experiment shouldn't have failed, and yet it did. Perhaps pre 1954, we still don't have enough information to really understand c.n.s and motivation.

Seems that the author is saying that as long as we are choosing to work, we will pick that over other things.

An experiment that was done by Bexton, Heron, and Scott where they paid college students (around 20$) to do nothing, showed that at first those students were content for a period of time, but that the longer they did nothing the less happy they became. Then they would start asking for some sort of stimulation (music, talking to others etc.). These students found this very fatiguing, and some actually left the experiment giving up the 20$ a day! I think this shows that we as humans need interaction of some sort, we need some sort of stimulation to keep our brains active and happy, give it something to focus on.

Some of the theories early on did not explain reactions of higher functioning animals such as Chimpanzees. For example taking an infant from its mother because the mother abandons it, and then that same mother having a second infant, and refusing to give it up. To reduce drives to simple hunger, pain, sex, and maternity and learning is difficult as it does not explain all drives or motivations. Especially when the subject has never been exposed to an object/person/thing that causes a reaction. Thus a new drive would need added, curiosity drive which could explain investigatory and manipulatory activities. Why an animal would take a more difficult (although interesting) path to get food.

extraction of material from the existing common record

such that the entire scaffolding with which it was erected is available to explore. resume, remix. context of justification +context of discovery

and allow conversations that are "continupous without being synchronous" with new InterPersonal WebNative Computing in the IndieVerse

Science and thought is always progressing, which is so hard to keep up with. Especially now how anything can be posted with a few clicks.

Unlike other generations, our generation has been lucky enough to have such an easy communication platform

Shows how powerful we have become scientifically

A very interesting take on how it is seen as a "war"

This is a colorful way to describe the mechanics of a photo camera.

I can confidently say that there are some people who still won't see it being used for good- but I believe there is always good happening with the help of modern instruments.

We can see this kind of technology at work with current school activities. Being able to have a complete digital library at your fingertips is especially more amazing in times like these- where we are sort of held back by the pandemic. It makes taking online classes way easier as well.

So this can also go with "always be curious" because once a person in introduced to something new, one of the first things they should do is wonder and ask questions. This can then spark something more to grow from what is shown.

This sounds connected to the modern "text to speech" system we see now.

And it has now become that way with our smart devices and printers.

I think this kind of connection to the growth of technology gets a bit overlooked now with a growing fear many people seem to have. Perhaps it has to do with a corrupt government or just the negatives that spawn from greedy people who have control over what is produced by new technology. Some instruments created with modern technology are snagged up to make profit off of another person's needs in order to live. It's a real shame that there feels to be a loss of stability for the common person.

To think that this is how society took steps towards having a world wide web that holds our history and gives us the ability to expand our minds further with information about other cultures. Also the added addition of being able to communicate with someone from that culture- which I feel is helping us move closer to being a more empathetic society.

I think this idea is a good one- though I do wish science never had to be used for warfare in the first place.

I am honestly amazed at how we live in a world now where we do have this device in our homes and even in our pockets.

Whilst I do think that technology often ruins the "analog experience" there are so many benefits to advancing the way that we do things to the point that saying "they don't make things like they used to" feels like naïve nostalgia.

Prior to reading this I was unaware of how complex the things that I take for granted every day truly are.

My only question here is why would it not be at max speed all of the time? Mental fatigue?

Again, this feeds back to the last point that regardless of new technology it always seems that we will need more as a society. Electric car is built? Not enough mileage. Internet is 1GB/s? Not fast enough. It seems to never end.

Honestly I think this is how mathematicians work nowadays. When I was taking Math 151 it seemed like there was so much unnecessary work in order to get to a fairly simple end game.

This seems that this seems to be the outlook of all evolving technology of the past, present, and future.

This work has been peer reviewed in GigaScience, which carries out open, named peer-review. These reviews are published under a CC-BY 4.0 license and were as follows:

Reviewer 1: JianJiong Gao

In this manuscript, the authors introduced a tool named TraNCE for distributed processing and multimodal data analysis. While the topic and tool are interesting, the writing can be improved. The current manuscript reads more like a technical manual than a scientific paper.

For example, in the background, the discussion on data modeling in the contexts of multi-omics analysis and distributed systems is extensive, but the writing can be better organized. The examples are helpful, but they are very technical and can be hard to follow. It would be good if the main challenges can be summarized on a high level. It might also be useful to have an example analysis use case to lead the technical discussion on data modeling.

It is also unclear how are the targeted users of the tool and why distributed computing is needed. For example, in application 1 & 2, it is unclear why distributed computing is necessary.

Reviewer 2. Umberto Ferraro Petrillo First review:

The authors propose a new framework, called TraNCE, for automating the design of distributed analysis pipelines over complex biomedical data types. They focus on the problem of unrolling references between different datasets (which can be very large), assuming that these datasets contain complex data types consisting of structured objects containing collections of other objects. By using TraNCE, it is possible to formulate queries over collections of nested data using a very high-level declarative language. Then, these queries are translated by TraNCE in Apache Spark applications able to implement those queries in an efficient and scalable way. Apart from a quick description of the TraNCE framework and of the declarative language it supports, the paper also includes a vast collection of examples of multi-omics analyses conducted using TraNCE on real-world data. I found the contribution proposed by this paper to be very actual. Indeed, there is a flourishing of public multi-omics databases. But, their huge volumes make their analysis difficult and very expensive, if not approached with the right methodologies. Distributed analysis frameworks like Spark can be of help, but they are often not easy to be mastered, especially for those not having deep distributed programming skills. So, TraNCE looks like a very much need contribution on this topic. However, I have some remarks. The high-level querying language supported by TraNCE is not original because, as far as I understand, it has been presented in a previous paper [1] (which has been written by almost the same authors and that has been correctly referenced to in this submission). Even the TraNCE framework is not completely original because its name appears as the name of the project containing the code presented in [1]. Finally, at least one of the experiments presented in [1] seems to have been run on the same Hadoop installation used for the experiments presented in the current submission, and has involved the same datasets from the International Cancer Genome Consortium. So, I am a bit confused about what it is original in this new submission and what has been borrowed from [1]. My advice is to definitely clarify this point.

Another issue that I think should be addressed is about the proposed framework being scalable. The authors state that the framework supports scalable processing of complex datatypes, however, no evidence is brought about this claim. The several different experiments that are reported seem to focus more on the expressiveness of the proposed language while no experiment about the scalability of the generated code is provided when run on a computing architecture of increasing size. I think we may agree on the fact that using Spark does not means that your code is scalable, neither I think it is enough to say that the scalability of TraNCE has been proved in [1]. So, I would suggest to elaborate also on this. To be honest, I am a bit skeptical about the practical performance of the standard compilation route. I think that when applied to very large datasets it is likely to return huge RDDs that could require very long processing times. Instead, the shredded compilation route looks much clever to me. Could you elaborate further on this difference, especially according to the results of your experimentations? I also disagree with your idea of not describing how data skewness is dealt with in your framework. It is indeed one of the main cause for bad performance of many distributed applications so it would be interesting to know how did you manage this problem in your particular case. On the bright side, I really appreciated the flexibility of the proposed framework, as witnessed by the vast amount of examples provided, as well as its positive implications on the analysis of multi-omics databases.

Finally, the English of the manuscript is very good and I have not been able to find any typos so far.

[1] Jaclyn Smith, Michael Benedikt, Milos Nikolic, and Amir Shaikhha. 2020. Scalable querying of nested data. Proc. VLDB Endow. 14, 3 (November 2020), 445-457.

Re-review: I appreciated the robust revision done by the authors and think the paper is now ready to be published

Author response

September 9, 2021

We would like to thank ASAPbio for selecting our preprint for review! We are excited to contribute to this new process and hope others will find it as helpful as we have. The comments generated by the “crowd” were detailed and thoughtful. Below we respond to the major discussion points and if there were specific reviewer comments relevant to the discussion point, we also included that statement. We also responded to each specific comment. We would love to continue this discussion, so we invite further feedback and responses! Thanks so much for your time.

-Chelsea Kidwell, Joey Casalini, and Minna Roh-Johnson

Major Discussion Point #1:One of the most important claims is that mitochondria are the organelles responsible for the activation of the signals of cell proliferation. However, a previous report by the last author reported that macrophages transfer cytoplasm to recipient cells. It cannot be excluded that other organelles or cellular fragments are transferred as well and contribute to the observed effects (ERK activity). Perhaps a good way to solve this would be the use of macrophages that are devoid of mitochondria. At least, this aspect should be discussed in the manuscript.

🡪 We had first considered two approaches to test the requirement and sufficiency of macrophage mitochondria in cancer cell proliferation. The first was to generate rho-zero macrophages (mtDNA-deficient), as you mention in your comment, such that the macrophages did not have functional mitochondria. However, we use primary human macrophages for all of our studies, and these cells would not survive long enough to generate rho-zero cells (which requires that the cells be treated with low levels of ethidium bromide for weeks). The second is to biochemically purify mitochondria from macrophages and directly inject these mitochondrial preps into breast cancer cells. We actually did this experiment, and cancer cells injected with purified mitochondrial preps exhibited higher proliferation (by live timelapse microscopy) compared to control cells. However, we also found that the mitochondrial purifications were not clean, and contained other membranous components in the cytoplasm. We tried centrifugation-centric approaches, as well as IP-ing against a mitochondrially-localized tag, but in all cases, the mitochondrial preparations contained other cytoplasmic components. Therefore, we did not feel that this approach was an adequate way to test effects of specifically the mitochondria. We certainly wanted to discuss this aspect in the manuscript, but unfortunately, we were limited due to space. If folks have suggestions on how to best purify mitochondria, we’d love to know, so please reach out.

However, in terms of the bigger question of whether the induced proliferation in cancer cells is specifically due to ROS accumulation in transferred macrophage mitochondria, we tried to address this question with the mito-KillerRed experiments, where we generate ROS using optogenetics, and ask whether this accumulation is sufficient to induce cancer cell proliferation (which we showed it was). We also showed that this same approach could induce Erk activity, and then in separate experiments, we show that macrophage mitochondrial transfer results in accumulation of ROS and increased Erk activity. We feel that these experiments support our conclusions, however, we’d love for a way to link it all together. Unfortunately, we are not convinced that such experiments are possible at this time.

Major Discussion Point #2: Most of the positive examples of transferred mitochondria discussed appeared in a small clump. However, there also appears to be another population that was more diffuse and co-localizes with host mitochondria (e.g., Fig2B, bottom right panels). It would be helpful to show results of these sibling mitochondria for assays performed on their clumpy siblings. If they behave differently, it would be helpful to provide some explanation.

Specific Comment: Figure 2 Majority (57%) of donated mitochondria do not colocalize with LysoTracker signal (N=24 cells, 4 donors) - Here the paper implies that some transferred mitochondria do co-localize with lysoTracker signal. More importantly, they co-localize with host mitochondria. It raises the question of whether they signal through ROS and ERK like their clumpy siblings who are in the limelight of most figures.

🡪Yes, you are correct. There does appear to be a diffuse population of macrophage mitochondria. The majority of these mitochondria co-localize with lysotracker, suggesting that they are being actively degraded. We can’t say that they tend to co-localize with endogenous cancer cell mitochondria, however, it’s possible that this diffuse population is comprised of both mitochondria that are being degraded and mitochondria that are fusing with the endogenous network. We do not know if this population has a different effect on cancer cell behavior because we did not follow this population (mostly because once the mitochondria are degraded or fuse with the network, we can no longer follow those mitochondria!). However, we did follow cancer cells that contained punctate macrophage mitochondria. Often times this was the only population we could observe in the cell at that time, and this is the population in which we observe accumulated ROS.

Major Discussion Point #3:The effects that are attributed to the transferred mitochondria are highly variable (figures 1F, 3A,E) and often due to a subpopulation of samples that show a few extreme values (e.g. figures 2D, 3E, S4B, S4D). This might be expected from effects that are caused by a single mitochondria (which has a small volume) that is transferred to a complete cell. This complicates the study of the transfer process and effects and should be discussed. Also, do the authors have ideas how to improve the system, to make it more robust and easier to study the effects?

🡪The variability in the assays likely reflects the heterogeneity within the biology - Each experiment contains macrophages derived from primary monocytes that are harvested from different human blood donors! Due to the primary nature of these cells, we do expect a range of phenotypes as each donor would have a different genetic background and the monocytes were likely exposed to different environmental stimuli. In fact, even though working on this study was a giant pain due to the variability, we felt more confident about our findings because despite the heterogeneity in the system, we still observed consistent phenotypes. Below we indicate where we took a sample set and removed “outliers”, and ran the statistical tests again. The differences were still statistically significantly different, further suggesting robustness of our findings.

However, we are always on the lookout for ways to make the system easier to study. One way that we will follow up on is using M2-like macrophages since they transfer mitochondria at a higher rate than unstimulated macrophages.

Major Discussion Point #4: The authors conclude that the transfer of dysfunctional mitochondria generated a signal mediated by ROS that activates cell proliferation signals. The statement that "transferred mitochondria act as a signaling source that promotes cancer cell proliferation" is too strong. There is increased ROS production from mitochondria, yes, but an experiment in which ROS are decreased would be needed to properly sustain that conclusion. The title and abstract could be changed to better reflect the data.

Specific Comment: ‘Furthermore, treatment with an ERK inhibitor (ERKi) was sufficient to inhibit ERK activity ‘- curious as to whether antioxidant treatment would reverse any proliferative phenotypes?

🡪We wish we could quench the ROS at macrophage mitochondria. We really tried. We used a combination of ROS quenchers (NAC, mitoTempo, Tempo) and ROS readouts (mitoSOX, CellRox, DCFDA, and the 2 biosensors used in our study: Grx and Orp1), and treated cells for various amounts of time, and no matter what we tried, we could not reliably detect reduction of ROS levels in the host network or the transferred mitochondria (without killing the cells, that is). Another issue that we faced was that any pharmacological treatment would have a global effect on the mitochondrial network in the recipient cells and therefore it would not be possible to distinguish effects from global inhibition of ROS versus specifically at the site of the transferred mitochondria, and we certainly observed cell death upon treatment of ROS quenchers because of this fact. We talked to a couple of ROS experts, and they indicated that this issue is not unique to us, although we unfortunately did not have viable solutions, so if people have ideas or suggestions, please let us know!!

However, despite our failed attempts at quenching ROS, the comment that "transferred mitochondria act as a signaling source that promotes cancer cell proliferation" is too strong of a statement… well, we don’t entirely agree given that we do perform sufficiency experiments in which weinduce ROS and observe both proliferation and ERK signaling, so we do feel reasonably justified to provide the title that we did. However, we will continue to mull over this comment. Thanks for sharing your thoughts.

Major Discussion Point #5:The study may benefit from more direct evidence to support its conclusion of increased proliferation after mitochondrial transfer. While the RNA-seq, flow cytometry, counting of completion of cytokinesis and dry mass measurements provided in the present study do lend some support to the proliferation hypothesis, they all seem indirect. With the biomarkers labeling the mitochondria of donor and potential recipient cells, high content imaging and tracking of cells could be used to monitor cell division. A comparison of cell division rates of transfer-positive cells and transfer-negative cells will provide a more pertinent test of whether mitochondrial transfer promotes recipient cell proliferation.

🡪We should probably do a better job at describing the dry mass measurements (QPI, quantitative phase imaging) because we view this quantification as one of the most direct measurement to monitor cell growth/division. The approach measures the changes in dry mass as the cells prepares for cell division. So not only do we get the final readout of division (complete cytokinesis), but we also get a measure of that growth rate (the cell getting ready to divide) before cytokinesis. This is why we are so tickled to collaborate with Tom Zangle’s lab because we could finally get a direct proliferation readout in real-time. We could also use this approach to follow thousands of cells at a time, a very critical aspect since mitochondrial transfer is rare event, and therefore, we need to follow many cells to have enough statistical power to quantify the growth rates. Check out some of the Zangle lab’s other papers (PMC5866559; PMC6917840; PMC4274116), and please let us know if you disagree with us!

Major Discussion Point #6: The authors have used such a tracking-based approach on a very small scale (n=5) to measure daughter cell growth rate. However, the data do not show a statistically significant difference between the growth rates of daughters that inherited transferred mitochondria and those who did not (Fig S3). Increasing the case number via high content imaging would help obtain sufficient data points for a reliable statistical test. In addition, as suggested above, an accounting of the daughter cells' division rate for transfer positive and negative cells would provide another line of evidence to either prove or disprove the increased proliferation rate hypothesis. The same suggestion goes to the optically induced ERK activation experiments shown in Fig3F. It is also helpful to include references that studied how ERK signaling promotes proliferation and compare the evidence here with evidence or assays used in those studies as a benchmark.

Specific Comment:Figure S3 - There is no statistical test to check for ‘increase in their rate of change of dry mass over time versus sister cells that did not inherit macrophage mitochondria’. What are the colours indicative of in S3B? Can this be reported in the figure legend.

🡪You are right – the tracking-based approach on daughter cells is based on a small ‘n’. However, the tracking itself is performed on 1000s of cells. It’s just that in order to capture daughter cell data, we have to find a cancer cell with macrophage mitochondria (which is only ~1% of the population), and then follow that cell until it divides, and then follow BOTH daughter cells. So, even with the 1000s of cells that we followed, we could only capture a small number of daughter cells. The colors in S3B represent each individual triads – parent and 2 daughters. We will make this info clearer in the legend.

In terms of the optically-induced ERK activation experiments, yes, it would be great to have a higher sampling. These experiments were performed at 63x so we could reliably draw small ROIs to mimic the size of a macrophage mitochondria. While we switched to lower magnification to follow cell division, we still were limited to only a few cells for the actual photoactivation. The technical aspects of this experiment were the reason for the low sampling. Despite these limitations though, we still observed increased cell division upon mito-killerred photoactivation, which we were honestly pretty surprised (and stoked) about.

Other specific comments:

-Figure S1A - The authors could perhaps use a more aggressive gating strategy here, clipping closer to the 231 population described in Fig S1A - picking only the center of the cluster in the upper left of the RFP vs CD11b plot would likely not affect results but make them more convincing by unequivocally excluding macrophages.

-Figure 1D - Not sure about the 0.2% baseline assigned for the monoculture of cancer cells (that does not have the macrophages with the Emerald mitochondria). It is determined with cytometry - I am no expert on that topic, so maybe I missed something - but it looks weird to see some cells with transfer when there is a monoculture.

🡪Due to the variable nature of the mito-mEm signal in the recipient cancer cells (i.e. transfer of one mitochondrion vs transfer of three), we found that an overlap of 0.2% set on a fully stained monoculture control was the most accurate way to gate for the recipient cancer cells. The final gating strategies used in our study were determined by FACS-isolating populations of interest based on several different gating strategies, and directly visualizing cancer cells with macrophage mitochondria without capturing macrophages or cancer cell/macrophage fusions (which is cool, but not what we wanted). To further clarify, there is no transfer occurring in the monoculture – the overlap of mEmerald signal into the transfer gate in that control sample is likely reflective of normally occurring autofluorescence. This is a very important point, so we will make this aspect clearer in the Methods section.

-Figure S1B - Could perhaps be an interesting follow-up question for future works re: differences between cell lines and propensities to transfer mitochondria. Did the authors attempt to use other cell lines (ie, MDCK, HeLa, iPSCs, etc)?

🡪Great question and something that we have also been thinking about. To date the only recipient cells we have used are 231, MCF10A, and PDxO cells. This would be a great avenue for future studies.

-Figure S1B - Did the authors see an increase in growth rate in MCF10A line despite the lower growth rate?

🡪We have not measured the growth rate in MCF10a recipient cells but something that would be great to follow up on in future studies.

-‘physically separated from macrophages by a 0.4μM trans-well insert’ - should this read 0.4 micrometer?

🡪Yes, great catch.

-Figure S1F - The authors wrote that they used a two-way ANOVA analysis, could you report the factors used for that analysis in the Figure legend.

🡪Noted!

-Figure 1B - It is difficult to see the arrowheads in 1B, suggest moving them so they are not covering the magenta fluorescence, have them point from a different angle, and make them more brightly colored. Insets here would help the reader. A negative control image from a monoculture would also be helpful, to ensure the GFP signal is not an artifact of culture conditions.

🡪Thank you for your feedback – we will take note of this.

-Figure 1F - For graphs that do not show zero (as in 1F), the bar should be omitted. In these cases the length of the bar does not reflect the average of the data (as it does in 1D).

-Figure 3C - Please omit bar, see comment on panel 1F.

🡪 In the case of Fig 1F, we modified the y-axis to eliminate empty space. The bar is representative of mean of the data displayed in both 1D as well as 1F, but we can add a broken y-axis to help make this point.

-Figure 1 - Given that these data are fractions of a population (ie. can be described via a contingency table), isn't something like a Fisher's exact test a better measure of significance here?

🡪We think you are referring to Figure 1D? If so, we thought that we could not use Fisher’s exact test because that test assumed parametric distributions (which we do not observe). We have been working with a biostatistician for our statistics, but please do let us know if we have it wrong.

-Single cell RNA- sequencing - In the methods section the authors mention doing a differential analysis between the cells that received the mitochondria and the cells that didn’t. It might be worth introducing a figure (a heatmap or a U-MAP) relating to this analysis. Single cell sequencing would not only affirm the heterogeneity between these two populations but also help in highlighting the novel cell surface markers associated with the two populations.

🡪Yeah, good point – we can add a UMAP.

-‘mito-mEm+ mitochondria remained distinct from the recipient host mitochondrial network, with no detectable loss of the fluorescent signal for over 15 hours’- It is surprising that the transferred mitochondria do (or cannot) fuse with the host 231 mitochondria.

🡪We were also initially surprised to find that the transferred mitochondria do not fuse with the host 231 network! We think that the lack of fusion is due to the fact that the transferred mitochondria do not exhibit membrane potential (which is required for mitochondrial fusion). We also think that these results open interesting lines of questioning: Why are these depolarized mitochondria not degraded? Is this an active avoidance of the mitophagy pathway? How dynamic are these punctae? Many fun and interesting questions regarding the long-lived nature of these transferred mitochondria.

-It is unclear in these images, but the 231 mitochondria appear fragmented too. Is it possible that the mitochondrial fusion machinery (Opa1 or Mfn1/2) are inactive?

🡪231 cells are capable of fission and fusion (PMC7275541, PMC3911914, and in our own timelapse recordings), so we think that the machinery is functional. However, we don’t know whether the 231 mitochondrial machinery changes after receipt of macrophage mitochondria. Interestingly, the references above both investigate how mitochondrial dynamics promote tumor metastasis. A fascinating future direction could include an investigation to how macrophage mitochondrial transfer influences tumor cell mitochondrial dynamics.

-Figure 2B - What does the MTDR staining of the macrophage mitochondria prior to transfer look like? Important to check this to confirm that only the transferred mitochondria had lower membrane potential.

-‘significantly higher ratios of oxidized:reduced protein were associated with the transferred mitochondria versus the host network’-Here too, it would be important to check the mito-Grx1-roGFP2 readout of macrophage mitochondria prior to transfer.

🡪The way that these comments are written is as if we already know that the mitochondria are dysfunctionalbefore transfer to cancer cells. But we actually do not know if that is the case. It’s also possible that macrophage mitochondria become dysfunctional once they are in the cancer cell, which would be equally cool. So, we are actively investigating this biology.

-Figure 2A, 2BB and S1D - How were the colocalizations assessed? Was it just a visual assessment? Given the importance of these experiments for the whole story, having a quantification of the level of colocalization with each dye would be important.

🡪This is a good point and it should be straightforward to include a Pearsons coefficient for these markers.

-Figure S1D - The paper makes an argument about mitochondria transferred from Macrophages (marked green) having positive DNA stain (gray), but appearing depolarized (negative TMRM stain). The image in FigS1D is peculiar, as the majority of the 231 cells' mitochondria appear to not have any DNA stain but maintain membrane potential (positive in TMRM), while some (just above the green macrophage mitochondria) do have both DNA stain and membrane potential. The authors might want to clarify whether this is a typical scenario, and if so perhaps offer an explanation as to why the 231 mitochondria exhibit such heterogeneity.

🡪The images in S1D are of a single z-plane image therefore the DNA signal in the endogenous network is more readily visible in planes that are not shown.

-‘we confirmed that 91% of transferred mitochondria were not encapsulated by a membranous structure, thus excluding sequestration as a mechanism for explaining the lack of degradation or interaction with the endogenous mitochondrial network’ - This is based on co-staining with MemBrite 640/660, which is a dye that "covalently labels the surface of live cells", thus there is a concern as to whether this approach allows to study whether the mitochondrium is encapsulated by an endomembrane.

🡪Thank you for your feedback. We actually do think that Membrite can label endomembrane in addition to the plasma membrane. This is from the published Membrite protocol: “MemBrite™ Fix dyes are designed to be fixed shortly after staining, when they primarily localize to the plasma membrane/cell surface. Cells also can be returned to growth medium and cultured after staining, however, dye localization in live cells changes over time. Labeled membranes become internalized, so staining gradually changes from cell surface to intracellular vesicles, usually becoming mostly intracellular after about 24 hours. Internalized MemBrite™ Fix dye is usually detectable for up to 48 hours after staining, though this may vary by cell type”.

In our hands, we found that the dye started to become internalized and labeled vesicles within the cell within a few hours of staining. The images in the panels that you refer to came from time-lapse imaging experiments of between 10-15 hours, therefore the cells have internalized the MemBrite signal allowing for the visualization of internal vesicles. Also, in other studies not in the preprint, we perfused purified mitochondrial preparations onto 231 cells. The 231 cells took up the mitochondria from the environment, and all of these engulfed mitochondria were surrounded by a MemBrite positive membrane! These results further suggest that if the transferred mitochondria were encapsulated by a membrane, we would be able to visualize it.

_-‘macrophage mitochondria are depolarized but remain in the recipient cancer cell’ -_Did the authors examine the extent of cancer cell death in their co-culture system (due to the activation of apoptosis by the depolarized mitochondria)?

🡪We do not find any evidence of abnormal levels of cell death by both flow cytometry assays as well through our QPI image analysis.

-Figure 2C–D - Like in Fig 2B, in the bottom left of panel of Fig 2C there are a lot of donor mitochondria not in highly oxidized state and the growth/proliferation phenotypes apply mostly to donor mitochondria that appear 'clumpy'.

-Perhaps it is worth commenting on whether there is a link between donor mitochondrial morphology and the suspected proliferation-enhancing phenotype.

🡪The images in Fig. 2C are of the same cell – a single recipient cancer cell which is expressing the Grx biosensor. The donor mitochondria are labeled with an arrowhead, the rest of the yellow/green signal (bottom right) is from the endogenous host network and therefore we do not expect it to be in a highly oxidized state (ie. more yellow than green).

Regarding the mito morphology and proliferation – great question, and one that we are actively working on!

-‘At 24 hours, we observed a similar trend, but no statistically significant difference (Fig. S4D). These results indicate ROS accumulates at the site of transferred mitochondria in recipient cancer cells’ - if a specific sensor fails to show a significant oxidation at 24 hours compared mito-Grx1-roGFP2 which reports on mitochondrial glutathione redox state, does that mean there are ROS independent ways to oxidize Glutathione? The authors did see cell growth phenotype both in 24 and 48 hours which suggests that something is happening in 24 hours despite no significant difference in ROS H2O2 sensor.

🡪The additional biosensor that we used – mito-Orp1-roGFP2 - has been engineered to be a readout of one type of ROS – H2O2. The Grx probe is a surrogate for ROS of any type, of which there are many! To us, it is not completely unexpected that they would behave differently over time since they are readout for two separate things, and it generates an interesting possibility that different types of ROS accumulate over time. Given that the Grx probe shows an increase at 24 hours, which is when we observe the proliferation phenotype, we think we are on the right track. If you have ideas on robust ways to directly observe specific types of ROS, we would love to know!

-The differences in ratio for the two sensors used are not very convincing. In Fig 2D and Fig S4B and D the “host” and “transfer” populations are very similar. The difference seems only due to the presence of a few outliers in the “transfer” populations. More importantly, sometimes it seems that these outliers come mostly from one donor rather than being present in all 3 donors. It could be good to show histograms of the two populations for each replicate/donor and maybe redo the stats excluding these outliers.

🡪We think that the heterogeneity that is observed is due to the biology in the system – we are using primary macrophages derived from blood donors. However, for the data represented in Fig 2D, just as a test case, we took out the top four “outliers” in that data set and re-ran the Wilcoxon matched-pairs signed rank test and the p-value was 0.0010 (***), further suggesting that the ROS biosensors are revealing consistent and robust results.

-Figure S5C - it seems like the percentage of cells that divided is the same for unstimulated cells and cells with stimulated mito-KillerRed. Isn't this contrary to the expectation? The figure shows that photobleaching cytoplasm decreased % cell division, which is puzzling.

🡪The mean percent of cells that divided in unstimulated and mito bleach are very similar and was not significantly different. One point to be made that may not be well illustrated in our graphical representation is that if you look at the matched data (points connected are averaged per FOV for each condition in the same experiment) the trend shows that the mito bleach does seem to have an increase in cell division which is washed out with the average bar overlay. We should note that this experiment is very “noisy” and therefore we needed a lot of N to be able to detect significant changes. We are currently thinking about other ways to demonstrate sufficiency as it relates to cell proliferation – any experimental suggestions would be very welcome! Thanks for the feedback.

-Figure 3A - In the 'cyto' condition 6 out of 13 fields have no cells that divide. Is that expected? What is the percentage of dividing cells for cells that were not illuminated at all (a control that is lacking)? There is large variation, ranging from 0% to 22%. The evidence that illumination of KillerRed leads to increased proliferation is rather weak. Also, since Cyto and Mito are different cells, is a "paired" statistical test the right kind of test to use here?

🡪Additional data pertaining to Fig. 3A can be found in Fig. S5C, which includes the control for cells not illuminated at all. Having no cells that divide in a field of view is not surprising to us – the doubling time for these cells is ~35 hours, and we imaged for 18 hours. Also, for each field of view, our ‘n’ for each field of view was often 6-8 cells because we performed these experiments at 63X to allow for accurately drawn regions of interest for photoactivation. We also internally controlled every experiment (each experiment consisted of fields of view that had either mito activation, cyto activation, or no-activation controls, all of which were imaged overnight with multiple x/y positions). Cells that left the field of view over the 18 hours of imaging could not be quantified. It’s this sampling that caused the large variation in the graph. But again, as with many of our experiments, despite this variability, we still observe a significant difference in our experimental conditions over control cyto bleach. As for the statistical test, our understanding is that given each experiment is internally controlled, and we compare within each experiment, a paired statistical test is appropriate here. We will consult with our biostatistician to confirm, though.

-‘ROS induces several downstream signaling pathways’ - We would not expect the authors to investigate every signaling pathway, but wonder if the PI3K pathway was explored? It seems to be the other major cancer/proliferative pathway induced by ROS.

🡪Yes, this is a very good point! We actually assessed three different pathways at first – ERK, PI3K-AKT, and NLRP3/inflammasome. While analyzing these 3 pathways simultaneously, we discovered that ERK inhibitors resulted in decreased proliferation in cancer cells with macrophage mitochondria. As a result, we then focused on the ERK pathway. We still do not know if PI3K-AKT or NLRP3/inflammasome pathways play a role in this biology because we have not gone back and revisited these experiments yet, however in figure 3F, ERKi treated recipient cells exhibit a partial ‘rescue’ of baseline proliferation. This suggests that other pathways may indeed be involved and we plan to investigate this possibility.

-‘Recipient 231 cells had significantly higher cytoplasmic to nuclear (C/N) ERK-KTR ratios compared to cells that did not receive transfer’-Since two different quantification styles with opposite fraction values were used, is it possible to please specify which one was used here.

🡪Will do!

-Figure 3B - Please show the outlines of the nuclei and that of the cell.

🡪That would be helpful, wouldn’t it? Will do!

-Figure 3D - it is peculiar that ERK-KTR in Fig 3D is so strongly cytosolic while in Fig 3B it is almost exclusively nuclear. If this sensor behaves differently in different situations, the authors may want to comment on how that would affect their conclusions.

🡪The panels in Fig. 3B were taken with the ImageStream flow cytometer which takes a lower resolution image of a single plane of a cell in suspension in the flow stream. In Fig. 3D, those images are from confocal spinning disk microscopy which allows for higher resolution, z-stack images of adherent cells on glass. Therefore, we think the differences that you point out are likely due to the fact that the two images come from very different imaging systems.

-Figure 3E - The effect of 'opto-induced' ERK activity is weak. The initial ERK-KTR is 1 at time point zero (as the data is normalized to this timepoint) and around 1 for both the cyto and mito condition. A statistical difference is observed, but the effect is minor and it is unclear whether it is biologically meaningful. The 'cyto' condition shows an average below 1 and the mito condition remains 1, suggesting that ERK activity remains constant when ROS are produced in the mitochondria.

-Also from S8C and 3E it appears cyto actually shows a decrease rather than mito showing an increase, could the authors comment on this?

🡪We have a few thoughts on this. The first is that we don’t expect a dramatic change in ERK signaling because the ROS accumulation is localized to a small region in the recipient cell. This is not a situation where we would expect a large-scale change because we are adding a growth factor. We can understand that the change in ERK activity may appear to be minor, but our data suggest that these subtle changes in kinase signaling translate into significant changes in downstream behavior – proliferation. The way that we interpret differences as “biological meaningful” is whether they exhibit a functional response, and in our study, we show that inhibiting the induction of ERK activity in cancer cells with macrophage mitos inhibits proliferation. What is most interesting to us is that cancer cells that do not have macrophage mitochondria have an unchanged fraction of cells in G2/M phase of the cell cycle in response to the concentration of ERK inhibitor we used, suggesting that the ERK inhibition specifically blocks macrophage mitochondria-induced proliferation.

In Fig. S8C, bleaching a region of cytoplasm does seem to cause a decrease in ERK activity over time. We really can’t explain this result. However, we do think that ERK activity is higher in mito-bleached cells because mt-ROS is generating an increase in ERK activity which compensates for the decrease in activity that occurs when the cytoplasmic region of interest is photobleached. It’s still a head scratcher, though, but we did perform internal controls for every experiment (as we describe above), and the mito-bleach, cyto-bleach, and no-bleach conditions were run side-by-side such that we can make apples-to-apples comparisons.

-‘patient-derived xenografts (PDxOs)’ - As a control it would be relevant to include a normal mammary organoid model perhaps from the same patient to demonstrate that the transfer of mitochondria specifically to the cancer cells is more beneficial.

🡪Using a normal mammary organoid cells as a control to compare efficiency of transfer and downstream phenotypes would be very interesting. Due to the fact that these are patient-derived organoids, we are unable to acquire non-malignant cells from the same patient. Expanding our studies in the MCF10A cell line that we utilized in this paper would be an alternative to what you propose and would also expand our understanding of general biology underlying mitochondrial transfer.

-‘macrophages to both HCI-037 and HCI-038 PDxO cells (Fig. 4G)’ - Why is M0 able to transfer efficiently to HCL-037 tumour when its mitochondrial network is less fragmented as M2?

🡪These results really stood out to us. It was quite surprising that in HCI-037, both M0 and M2 macrophages were able to transfer their mitochondria at similar efficiencies, but in HCI-038, M2 macrophages were more efficient at transfer. HCI-037 is a primary tumor, and HCI-038 is a metastases from the same patient, so there are some exciting avenues of study to examine how macrophage mitochondria transfer differs at the primary versus metastatic site. There is still very little known about how donor cell dynamics influence mitochondrial transfer!

-Are mito transfer from M0 depolarised and accumulate ROS or show increased ERK activity or increased cell proliferation?

🡪Yes – all studies, except studies pertinent to fig 4 (where we assessed macrophage differentiation states), were done with M0 macrophages.

-‘M2-like macrophages preferentially transferred mitochondria to the bone metastasis PDxO cells (HCI-038) when compared to primary breast tumor PDxO cells (HCI-037)’ -The authors may want to check this statement here as it is in consistent with their data plot. In Fig. 4G, M2/PDxO transfer percentages for HCI-037 and HCI-038 are about the same, unless the authors provide statistical tests to prove otherwise. Instead, M0 appears to transfer mitochondria to HCI-037 much more efficiently than it does HCI-038.

🡪Upon re-reading our sentence again, we now realize that it’s actually quite poorly written, so we can understand the confusion! What we meant to articulate is that M2-like macrophages are better at transferring mitochondria to HCI-038 than M0 macrophages. Whereas in HCI-037, we do not observe the same preferential transfer (ie. M0 and M2 can transfer at the same efficiency). We will certainly clarify this language in the manuscript.

-‘M2-like macrophages exhibit mitochondrial fragmentation’ - Is there a correlation between the status of the mitochondrial network in the donor and the % of transfer to the recipient? If so, this would be a correlation that would support the conclusions.

🡪Yes, please see Fig. 4C for transfer rates with different donor subtypes and Fig. 4H for a general working model on how we think these data fit into the larger picture.

-‘accumulate ROS, leading to increased ERK activity’ - Did the authors obtain similar results with the PDXOs? It would be an interesting observation if the primary samples also exhibit a mechanism similar to established cell lines wherein there are more accumulated genetic changes.

🡪Our main limitation with PDxOs is overcoming the technical hurdles related to our downstream assays. These include introducing relevant reporters and generating stable lines in the PDxOs, and imaging at high-resolution when the PDxOs are cultured in 3D. However, we are very interested in this question as well, and are actively thinking about ways to overcome these hurdles.

-It would also be interesting to examine whether there is any difference in the ROS-ERK mechanism for primary and metastatic tumour.

🡪We agree and this is an active avenue of investigation for us. We agree and are currently pursing models to understand how our findings fit into the larger picture of tumorigenesis and metastatic potential. We had spent months pursuing anin vivo approach using a murine Cre/LoxP system to genetically label mouse macrophage mitochondria with GFP. We crossed mice which express Cre under a monocyte-specific promoter (Jax, SN: 004781) and mice with germline expression of Lox-Stop-Lox-3xHA-EGFP-OMP25 (Jax, SN: 032290) with the expectation of seeing Cre-based excision of the stop cassette – thus resulting in offspring with macrophages expressing mitochondrial-localized GFP. However, the macrophages of the resulting offspring do not express GFP (by flow cytometry, imaging, and western blot analysis), despite the PCR-verified presence of both transgenes and the excision of the stop cassette. Needless to say, this was quite frustrating! We are currently in the process of developing a newly available MitoTag model which has been optimized for visualization purposes (Jax, SN: 032675). If you have any suggestions or advice on this matter we would much appreciate your thoughts!

-‘in cancer cells that receive exogenous mitochondria’ - Since these macrophages also transfer mitochondria to non-malignant cells, such as MCF10A cells shown in Fig S1B, perhaps the authors could comment on whether this is part of a physiological process that would also promote normal cell growth?

🡪 There are so many questions regarding when and why macrophages might transfer mitochondria. In general, mitochondrial transfer is observed in stressed cells. Our data suggest that transfer happens to MCF10A cells although at a much lower rate than their malignant counterparts, 231 cells, but we do not know whether similar downstream mechanisms and phenotypes are also occurring in the non-malignant cells. Thanks for your feedback – more to come here!

This section is so heart wrenching. We see her whole life of mistreatment laid out in a paragraph. Her experiences and her sorrows. Her values and her desires are such simple standards of life and yet she suffers. Even if free in England she cannot be with her loved ones.

When I was reading this I see that he's comparing the frugalness of the jug to the lives of men. He's saying that we have to treat a jug very carefully and with caution and with lives of men because he's saying that they won't leave this earth maybe in relation to a ghost but mens lives. Also when it says,"knowing that most things break", and also saying after saying unless is firm on the earth makes me think that it's the spirit of maybe a ghost and it's not easy for them to go away.

This is a great way to have a conversation about privilege. People who are just now learning about the concept or who may be skeptical of it would react in a more positive way if they're presented with an actual action to complete.

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Reply to the reviewers

First of all, I sincerely appreciate the critical reading of our manuscript by the reviewers.

Point-by-point responses to the reviewer #1’s comments

Most of the key conclusions are valid but the main one should be either reinforced or tuned down.

Through our study, we want to indicate that MTCL2 preferentially associates with perinuclear MTs accumulated around the Golgi complex, and its target is not necessarily restricted to “Golgi-associated (nucleated) MTs.” In this sense, the sentences in the previous manuscript, such as “MTCL2 preferentially associates with Golgi-associated MTs” and “MTCL1 and 2 …. are specifically condensed on Golgi-associated MTs,” were overstatements and completely misleading.

According to reviewer#1’s comment, we carefully revised these sentences throughout the manuscript and eliminated ambiguity on this point as far as possible.

The corresponding revisions are as follows.

In particular, the authors tend to give central role to MTCL2 in regulating the formation and organization of Golgi-associated MT network, and conversely in organizing Golgi elements, without considering the other factors identified (the authors cite relevant papers though but do not discuss this). They should analyze the function of MTCL2 in relation to the role of CLASP2, AKAP450, Golgi-g-Tubulin, or even EB proteins (like EB3).

I agree with the above comment since it is important to analyze how MTCL2 preferentially associates with the perinuclear MTs accumulated around the Golgi complex.

In the revised manuscript, we included new data analyzing knockdown effects of CLASP1/2 and AKAP450 on the subcellular localization of MTCL2 (Fig. 7A). These data indicate that CLASPs but not AKAP450 are required for the preferential localization of MTCL2 to the perinuclear MTs around the Golgi. We also demonstrate that the minimum Golgi-localizing region of MTCL2 (the N-terminal coiled-coil region) physically associates with CLASP2 (Fig. 7B), further supporting the idea that CLASPs mediate the Golgi association of MTCL2. Additional involvement of another Golgi element, giantin, is also suggested through Fig. 7C and Appendix Fig. S6. We believe that these revisions significantly improved the weakness previously pointed out by the reviewer.

I also do not think that carrying out super resolution microscopy is enough to "reveal the possibility that MTCL2 mediates the association of the Golgi membrane with stabilized MTs". More generally, the authors cannot conclude that MTCL2 preferentially associated to Golgi-MT only from their immunofluorescence and KD experiments. The centrosome (the main MTOC) is indeed also localized in the perinuclear area. Easy to do additional experiments may help to confirm these conclusions (see below). Also, the authors could strengthen the way the study how MTCL1 and MTCL2 binds to microtubules and Golgi (see below). The localization or interaction of MTCL2 with Golgi-associated MT is not directly shown.

Previously, we demonstrated that the N-terminal region of MTCL2 shows clear Golgi-localization activity, whereas the C-terminal region directly binds to MTs. These data support our conclusion that MTCL2 mediates the association of the Golgi membrane with general MTs (although not with Golgi-associated or stabilized MTs).

In the revised manuscript, we reinforced these data by newly revealing that four-point mutations (4LA) in the first coiled-coil motif disrupt the Golgi localization of the N-terminal region of MTCL2 (Fig. 4D). Thereafter, we found that introduction of the same mutations in full-length MTCL2 abolished its preferential association to the perinuclear MTs accumulating around the Golgi without affecting its localization to MTs (Fig. 4E and F). In addition, we provide data on candidate molecules mediating the Golgi association of MTCL2, as stated above (Fig. 7). These results reinforce our immunofluorescence analysis results (Fig. 2) and indicate that the preferential association of MTCL2 to perinuclear MTs accumulating around the Golgi is facilitated by physical interactions between the N-terminal region of MTCL2 and the Golgi-resident proteins, such as CLASPs and giantin.

The title should be changed also. I am not sure I understand what an asymmetric microtubule network means in this context. I guess that the authors mean non-centrosomal microtubule network.

We acknowledge the confusion caused by our previous manuscript. By “an asymmetric MT network” we meant not equivalent to “non-centrosomal MT network.”

In many cases, microtubules do not elongate radially (symmetrically) from the centrosome but intensely accumulate around the Golgi area and show asymmetric organization (see Meiring et al. Curr. Opin. Cell Biol. 62: 86-95, 2020). “An asymmetric MT network” in the title corresponds to this asymmetric array of general MTs accumulating around the GA.

The present findings that MTCL2 depletion severely disrupted MT accumulation around the Golgi and induced random and rather symmetric arrays of MTs (Fig. 5A) are very impressive. We believe that the knockdown/rescue experiments in this study strongly support the title by demonstrating that MTCL2 facilitates MT accumulation around the Golgi through its dual binding activity to MTs and the Golgi membrane.

We changed the title in the revised manuscript but still used the term “asymmetric microtubule organization” based on these rationalities.

The authors also state that tubulin acetylation is induced by MTCL1 C-MTBD but it may simply be stabilized. They should also clarify if MTCL2 regulates Golgi-dependant nucleation microtubules.

Yes, we think that MTCL1 C-MTBD enhances tubulin acetylation by simply stabilizing the polymerization state of MTs (see Kader et al. PLos One 12: e0182641, 2017). As for the second point, please see our response below to the comment (7).

I was not convinced by the use of the quantification of "skewness", in particular in figure 5B. Whether a Wilcoxon test is adequate is unclear to me.

I understand that utilization of skewness, a measure of the asymmetry of distribution, might not be popular in previous studies. In fact, the skewness of tubulin signal distribution in pixels does not indicate in which way MTs distribute asymmetrically by themselves. However, quantification of this statistical parameter does not require any arbitrary factors and thus eliminates the chance of using discretion as far as possible. Therefore, we are confident that this is the best way to estimate the asymmetric organization of microtubules, which are severely affected by various conditions, without any preconception.

The two biological phenomena we attempted to elucidate here (microtubule arrays and Golgi ribbon expansion) are thought to be context-dependent in each cell (for example, cell cycle, cell densities, etc.). Therefore, we do not have any substantial reason to assume a normal distribution for variation of the two values (skewness of tubulin signal distribution and Golgi ribbon expansion angle) in our cell population. Therefore, we considered that the Wilcoxon test, being a non-parametric rank test, was the most appropriate and safest test to use.

To demonstrate that MTCL2 associated to Golgi-MT, microtubule regrowth experiments following nocodazole treatment have to be conducted (time course). Another efficient way to analyze such events, as shown by the Kaverina and the Akhmanova labs for example, is to use fluorescent EB proteins (e.g. EB3) to image microtubule plus ends and back-track them to identify nucleation points. Carrying out such an experiment (nocodazole way-out and EB tracking) in the presence or absence of MTCL2 would allow to confirm, or not, the functional hypothesis of the authors.

We did not want to demonstrate that MTCL2 preferentially associates with “Golgi-MTs.” From this point of view, we do not think the experiments suggested by reviewer#1 were necessarily required for our study.

However, there is no doubt that one of the main components of the “perinuclear MTs accumulating around the Golgi” is “Golgi-associated (nucleated) MTs.” In this sense, we still agree with reviewer#1’s comment that it is better to examine whether MTCL2 is involved in MT nucleation from the Golgi membrane. The results of these experiments will be informative for readers particularly because we previously reported that MTCL1 stabilizes Golgi-associated (nucleated) MTs.

In keeping with the above consideration, we have performed both experiments (nocodazole way-out and EB tracking) according to the previous studies (for example, Sanders et. al. M.B.C. vol. 28; 3181-3192, 2017). However, we ultimately decided against the inclusion of the data as we could not overcome large cell-to-cell deviations.

Nevertheless, we believe that our current dataset adequately answers and supports the specific questions we explored. Briefly, if these experiments succeed to demonstrate the functional importance of MTCL2 for the development of Golgi-nucleated microtubules, they will not necessarily indicate the physical interaction of MTCL2 with Golgi-associated microtubules. In this respect, as described above, we have significantly supplemented data on the molecular mechanisms by which MTCL2 mediates MT–Golgi interactions. This improvement must sufficiently compensate lack of data from the experiments suggested by reviewer#1.

Several circumferential data suggest that MTCL2 is not involved in the development of Golgi-associated (nucleated) MTs in contrast to MTCL1. We discussed this issue in the “Discussion” of the revised manuscript.

Additionally, carrying electron microscopy analysis would be important to qualify better the effects observed on Golgi complexes upon depletion. The authors mention the effects on the "morphology of Golgi ribbon" but it is rather unclear.

We did not perform electron microscopy analysis, because we are not implicating a change in the ultrastructure of the Golgi apparatus in MTCL2-knockdown cells. We specifically want to demonstrate that MTCL2 knockdown changes the assembly structures of the Golgi ribbons, and we believe that it is feasible to do so by light microscopy. We realize that using the term “Golgi morphology” may be misleading in this context. In the revised manuscript, we replaced this term with appropriate ones, such as “assembly structures of the Golgi stacks” or “compactness of the Golgi ribbon.”

Last, because the authors compare the way MTCL1 and MTCL2 bind microtubules, and suggest intriguing differences, domain swapping experiments between these two isoforms would be important to carry out.

We conducted the suggested experiments and obtained interesting results. However, we ultimately decided against their inclusion given that the functional difference between MTCL1 and 2 is not the main point of discussion in our study.

Some studies are referred but the published data not actually used (with the exception of the final scheme). The authors should comment on the fact that other Golgi-associated MT binding proteins have been shown to be involved in the mechanisms highlighted here. Why they would not take over in the absence of MTCL2 should be properly discussed.

In the revised manuscript, we included data regarding the involvement of CLASPs and AKAP450 in the Golgi association of MTCL2. Accordingly, we introduced their roles in the development of Golgi-associated MTs as far as possible in the “Introduction” (see lines 29-36 and 38-42), “Results” (see lines 306-309 and 344-347), and “Discussion” (see lines 398-402 and 442-444).

Similarly, in the discussion, the authors indicate that SOGA has been found as an interacting partner of CLASP2. As CLASP2 is a microtubule binding protein also localized at the Golgi complex and binding to acetylated microtubules, the authors should at least comment on the putative role of the interaction between MTCL2 and CLASP2 in the phenotypes they described. The role of the interaction between CLASP2 and MTCL2 should be discussed and ideally tested.

As described above, we provided the data indicating the role of the interaction between MTCL2 and CLASP2 in the revised manuscript.

In the introduction, page 3 line 74-77, the authors wrote « The resultant N-terminal fragment is released into the cytoplasm to suppress autophagy by interacting with the Atg12/Atg5 complex, whereas the C-terminal fragment is secreted after further cleavage (see Fig. 1A, boxed illustration). » while on the Fig1 the boxed area indicates that SOGA bears Atg16 and Rab5 binding domains. Please double check the interacting partners of SOGA1.

Thank you for pointing this out. The sentence in the “Introduction” was revised to “… interacting with the Atg12/Atg5/Atg16 complex” (Rev. Endocr. Metab. Disord. 15, 137–147, 2014).

Figure 1 B and C are not cited in the main text.

These figures were cited in the “Introduction” section (line 65 in the previous manuscript). In the revised manuscript, these figures were replaced with Fig. EV1 A and C and cited in the “Introduction” section (line 59) as well as in the legend to Fig. 1 (line 757).

Figure 1E: a loading control is needed to evaluate the expression level of SOGA/MTCL2 in the mouse tissues.

Sample loading in each lane shown in previous Fig. 1E (Fig. 1D in the revised manuscript) was normalized by total protein amount (25 mg), as indicated in the figure legends. However, we have decided to add the data for a-tubulin expression in each lane as a reference, although they are not equal for each lane.

In the liver, the size of the bands is different than in other tissues (smaller size). The authors might comment if these smaller bands correspond to the cleaved version of SOGA that was previously described in mouse hepatocyt

In Fig. 1D of the revised manuscript, we added arrowheads indicating the bands of smaller sizes observed in some tissues such as the liver. In addition, we commented on them in the corresponding part of the “Results” section by describing that “we cannot exclude a possibility that MTCL2 is subjected to the reported cleavage and works as SOGA in these tissues.”

Figure 2A: single color picture for the anti-tubulin immunolabeling would help to see the distribution of microtubules in the perinuclear area. The perinuclear region is a crowded area with many intracellular compartments accumulating there as well as cytoskeleton elements.

We completely revised Fig. 2 following the reviewers’ suggestion. To provide single-color pictures for the anti-MTCL2 and anti-tubulin immunolabeling, we added new pictures examining colocalization of MTCL2 with MTs at the peripheral regions where densities of both signals are rather low. In Fig. 2B, the colocalization was further examined via a line scan analysis across MTs. Finally, we have included new data demonstrating that exogenously expressed MTCL2 similarly colocalized with MTs even at the peripheral regions when its expression was suppressed to the endogenous level (Fig. 2C).

Figure 2C: same comment as above, a single-color picture for the anti-MTCL2 and anti-GM130 immunolabeling are required.

Owing to the space limitation, we could not include a single-color picture for the anti-GM130 immunolabeling in Fig. 2, although we enlarged their merged figure so that readers easily agree with our statement: “some overlapped with the Golgi marker signals” (lines 146-147).

Alternatively, we included a new Appendix Fig. S8, in which immunofluorescence signals of MTCL2 and CLASP1/2 (A) or giantin (B) are compared at a super-resolution microscopic level. In these figures, we included single-color pictures together with merged data.

page 7, line 132-134: the authors state: « Close inspection using super-resolution microscopy further revealed the possibility that MTCL2 mediates the association of the Golgi membrane with stabilized MTs (Fig. 2D, arrows). » To my opinion, the data are over-interpreted. The signals partially co-localize but this does not indicate a function of MTCL2 in mediating the interaction.

We deleted the previous Fig. 2D and the corresponding sentence. By doing so, we ceased to suggest that MTCL2 functions to mediate MT–Golgi interactions only based on immunofluorescence data.

Figure 3: Another way of merging the anti MTCL2 and GS28 pictures have to be provided. The pictures are difficult to interpret with the current display.

We deleted the previous Fig. 4 and ceased to discuss colocalization of MTCL2 with Golgi proteins only based on immunolabeling data as mentioned above.

Figure 4C: please indicate the meaning of « ppt »

We included the explanation of “ppt” in the legends to the corresponding figure (Fig. 3C in the revised manuscript) as follows (lines 801-802):

“ppt represents the MT precipitate obtained after centrifugation (200,000 × g) for 20 min at 25°C.”

Figure 5B and C: for easier reading of the figure, it would be useful to annotate with MTCL2 construct is overexpressed following doxycycline treatment (MTCL2 WT (A) and MTCL2 delta C-MTBD (C)).

We followed the suggestion. Please see new Fig. 5 and Fig. EV4 and 5.

Figure 6 A and C: the labels are wrong. Bottom pictures correspond to anti-GM130 immunostaining not anti-tubulin. If I am not mistaken, it is MTCL2 delta C which is studied in panel C.

Thank you for pointing this out. We corrected this error in Fig. EV5 (previous Fig. 6) in the revised manuscript.

Page 11, line 212: Supplementary Figure 2 (knockdown in RPE1 cells) is intended to be cited not Supplementary Figure 3.

Thank you for pointing this out. We corrected the error in the revised manuscript appropriately.

Figure 8A: single color pictures are needed to appreciate the distribution of the signals

One of the major comments of three reviewers have been provided on Fig. 8, which reports that MTCL1 and 2 differentially regulate microtubules. We agree that the previous data in Fig. 8 A–C are rather preliminary. Although we could improve these figures according to the reviewers’ comments, we decided to omit these data and cease the discussion that MTCL1 and 2 localize with microtubules in a mutually exclusive manner, as this was not the main focus of the study.

Point-by-point responses to the reviewer #2’s comments

In figure 1D, a loading control should be included for the Western Blot probing for V5-mMTCL2 in HEK293T cells.

We did include loading controls for the indicated lanes. However, because the HEK293T cell extract in lanes 1–3 was diluted, the signals were too weak to be visualized in this figure (Fig. 2C in the revised manuscript).

The authors use the anti-SOGA antibody to detect MTCL2. However, in Figure 1A they do not show the sequence similarity between this region in MTCL1 and MTCL2. The authors should include this, as well as show that the anti-SOGA antibody is specific for MTCL2 and does not detect MTCL1.

In new Fig. EV1, we included amino acid sequence alignment data for the region corresponding to the used anti-SOGA1 antibody epitope. The data indicate significant divergence of the sequence from MTCL1 (6% homology, 23% similarity).

We also included new western blot data (Fig. 1B in the revised manuscript) demonstrating that anti-SOGA1 antibody does not react with MTCL1 exogenously expressed in HEK293T cells.

Line 132-134. The authors conclude that MTCL2 possible mediates association between Golgi membrane and stabilized MTs based on localization microscopy only. This is an overstatement and should be corrected. Not only is the microscopy technique used able to produce resolution of 140nm, which is not enough to show direct association; the staining techniques used (double antibody staining) ensures the fluorophores are approximately 20-30nm away from the intended target (MTs, MTCL2, or Golgi). Thus, the conclusion drawn is overstated and should be refined at this point in the manuscript.

I agree with reviewer#2’s comment that the previous data in Fig. 2D are insufficient to draw the conclusion that MTCL2 mediates the association between the Golgi membrane and stabilized MTs. We deleted the figure and the corresponding sentence reviewer #2 indicated.

We want to demonstrate that “MTCL2 mediates the association between the Golgi membrane and MTs (not restricted to the stabilized MTs).” In this sense, we have already obtained supportive data in the previous manuscript that the N-terminal region of MTCL2 has clear Golgi-localization activity, whereas the C-terminal region directly binds to MTs.

In the revised manuscript, we reinforced these data by revealing that four-point mutations (4LA) in the first coiled-coil motif disrupt the Golgi localization of the N-terminal region of MTCL2 (Fig. 4D). Thereafter, we found that introduction of the same mutations in full-length MTCL2 abolished its preferential association to the perinuclear MTs accumulating around the GA without affecting its colocalization to MTs (Fig. 4E and F). We also provide data on candidate molecules mediating the Golgi association of MTCL2 (Fig. 7). These results reinforce our immunofluorescence analysis results (Fig. 2) and indicate that the preferential association of MTCL2 to perinuclear MTs accumulating around the Golgi is facilitated by physical interactions between the N-terminal region of MTCL2 and the Golgi-resident proteins, such as CLASPs and giantin.

The authors should include some quantification of MTCL2 signals along stabilized microtubules near the Golgi and in peripheral regions of the cell in Figure 2. This will show that MTCL2 preferentially localizes to MTs in the Golgi region but not the periphery, as the authors claim (lines 124-130). This quantification could be in the form of linescans along or across MT signals.

We included a line scan data across peripheral MTs to confirm MTCL2 colocalization with MTs (Fig. 2C). However, it is difficult to perform a line scan for the perinuclear regions where both signals of MTCL2 and MTs are too dense. Therefore, we demonstrate the preferential colocalization of MTCL2 to the perinuclear MTs by comparing peripheral signals of MTCL2 with that of MAP4 (Fig. 2D).

The authors show that ectopic expression of the C-terminus of MTCL2 can rescue MTCL2 siRNA phenotypes. Since the N-terminus localizes strongly to the Golgi membrane, the authors should do corresponding experiments with this fragment, to determine if membrane binding of MTCL2 can have a similar rescue effect or if MT binding is essential. This is especially important for the Golgi-ribbon organization (Figure 6).

We did not include data indicating rescue activity of the C-terminal fragment of MTCL2. In the previous Fig. 5 and 6, we demonstrated that MTCL2 lacking the C-terminal microtubule-binding region does not show rescue activities. Therefore, we did not follow reviewer#2’s suggestion directly.

However, we included new data indicating that an MTCL2 mutant (4LA) that associates with MTs but not with the Golgi membrane also lacks rescue activities for asymmetric MT organization and Golgi ribbon compactness (new Fig. 5 and Fig. EV4). I hope these revisions are satisfactory.

Line 261-2. The authors claim that MTCL1 and MTCL2 function in a mutually exclusive manner. As with point 3, this is an overstatement based solely on localization microscopy. The authors cannot draw this conclusion from the data associated with this statement (Figure 8A) and it should be refined to reflect that they only comment on the respective localization patterns of MTCL1 and MTCL2. Additionally, to show that MTCL1 and MTCL2 do not overlap on MTs, the authors should include linescans along MTs showing the anti-V5 and anti-MTCL1 intensities.

One of the major comments of three reviewers have been provided on Fig. 8, which reports that MTCL1 and 2 differentially regulate microtubules. We agree that the previous data in Fig. 8 A–C are rather preliminary. Although we could improve these figures according to the reviewers’ comments, we decided to omit these data and cease the discussion that MTCL1 and 2 localize with microtubules in a mutually exclusive manner, as this was not the main focus of the study.

In Figure 8C the authors show acetylated tubulin staining in cells depleted of MTCL2. Based on this localization pattern, it seems the MT network is not grossly altered, as was shown in Figure 5 where perinuclear accumulation of MTs was lost. The authors should comment on whether acetylated tubulin presence and localization is altered in MTCL2-depleted cells. This is also mentioned in the discussion where the authors conclude that the major function of MTCL2 is to crosslink and accumulate MTs in the Golgi region. However, based on acetylated tubulin staining patterns, stable MTs seem to still accumulate in the Golgi region. The authors need to show this accumulated population of stable MTs is no longer crosslinked in the absence of MTCL2 to support their claim.

Acetylated microtubules represent a minor fraction of the perinuclearly accumulated microtubules. From the point of this view, it could be possible that the accumulation of perinuclear microtubules is severely affected, whereas that of acetylated microtubules is not. MTCL1 might crosslink these acetylated microtubules.

In any case, we have decided to delete the previous Fig. 8 A–C, as stated above.

To investigate potential functional overlap between MTCL1 and MTCL2, the authors should include a double depletion experiment where MT organization and Golgi organization are investigated. The currently shown experiments do not test a functional relationship between the two paralogs. Additionally, the authors should show Western Blot analysis of MTCL1 levels in MTCL2-depleted cells, and vice versa. While there does not seem to be an overlap in localization patterns of the two proteins, that does not mean there is no functional relationship.

We did not follow reviewer#2’s comment because of the reason stated above.

Lines 120-30 and 297-9. The authors state that based on the localization pattern of MTCL2 it mostly localizes along MTs in the perinuclear region (shown in Figure (2). Then, in the discussion they state MTCL2 preferentially localizes to Golgi membranes. Please clarify which of the two sites MTCL2 localizes to preferentially.

We agree that we should be more careful while describing the subcellular localization of MTCL2. We revised the information in the manuscript to indicate that MTCL2 preferentially localizes to perinuclearly accumulated microtubules showing partial colocalization to the Golgi membrane.

Loss of Golgi organization as described in Figures 6 does not appear in polarized cells in Figure 7. The authors should comment on the loss of the phenotype in polarized cells.

Since RPE1 cells cultured at high density show abnormally elongated shapes, as described in the original text (line 238; in the revised text, line 326), Golgi ribbons in these cells do not appear to be as expanded. However, their loss of compactness in MTCL2-knockdown cells can be easily recognized in the previous Fig. 7C (corresponding to Fig. 6C in the revised manuscript).

The authors should consider using colorblind friendly palettes in figures. For example, magenta/green instead of red/green and magenta/cyan/yellow instead of red/blue/green. Additionally, for tri-color images the combination red/green/white (Figure 4B, 7C) should be avoided, as overlapping red/green signals will show up as yellow which is difficult to distinguish from the white signals. Finally, human eyes detect shades of red much poorer than for example green. Therefore, the main point of a figure should not be in red. For example, MTCL2 is frequently shown as red signal in a merged image and should be replaced with a different color.

We incorporated the reviewer’s suggestion.

The authors claim the mouse MTCL2 protein lacks 203 N-terminal amino acids. Authors should clarify in the text that this is relative to mouse MTCL1. The authors should also include the human comparisons, as they work on human cell lines in the majority of the manuscript.

I am afraid that this comment is based on a misunderstanding by reviewer #2, because we did not claim that mouse MTCL2 lacks 203 N-terminal amino acids. Instead, we described that SOGA, a mouse MTCL2 isoform, lacks 203 N-terminal amino acids compared to the full-length mouse MTCL2, the cDNA of which was used in this work.

In Figure 1D the authors show Western Blots where various amounts of HEK293T extracts were probed for exogenously expressed MTCL2. As a control, authors should include a non-transfected control. From Figure 1E, it would be expected that HEK293 (kidney cells) would not express endogenous MTCL2, but the control should be included anyway.

In the revised Fig. 2B, we included a lane in which a non-transfected HEK293T cell extract was loaded, according to reviewer #2’s comment (see lanes indicated as mock).

In Figure 3, the color scheme in the final column of images should be changed. Red/white contrast is very poor and no conclusions can be drawn from these images. Additionally, the authors should include a box to show where the inset is located in the overview images.

In the revised manuscript, we deleted the “final column of images using red/white contrast” from Fig. 2D (previous Fig. 3), to avoid drawing a conclusion on the interaction between MTCL2 and the Golgi membrane only from immunofluorescence data.

In addition, we included boxes in the overview images to show where the inset is located, wherever it is required in the revised manuscript.

Authors claim that MTCL2 is not detected near more dynamic MTs in the periphery of the cell and references Figures 2A and 3. They should include annotation in the figures to highlight this. This can be done with arrowheads or other markings, or with additional insets enlarging a peripheral region of the cell.

To respond to the comment, we separately provided enlarged views of perinuclear and peripheral regions in the revised Fig. 2.

The authors should clarify in the main text and figure legend which superresolution microscopy technique was used in Figure 2D.

As mentioned above, we deleted the previous Fig. 2D.

The authors use methanol fixation to examine localization of MTCL2, MTs, and Golgi. Methanol extracts lipids and thus affects intracellular membrane compartments, and can affect the localization pattern of GM130, a Golgi matrix protein. The authors should include samples fixed with a crosslinking fixative to ensure their conclusions drawn from methanol-fixed samples are not affected by the choice of fixative.

According to the reviewer’s suggestion, we included additional data obtained using PFA fixations (Fig. EV2). PFA fixation also revealed a similar localization pattern of MTCL2 to that obtained by methanol fixation.

In Supplementary Figure 1B a third, relatively high expressing cell can be seen in the top panel. The GM130 signal for this cell seems to be comparable to non-transfected cells in the same image. Can the authors address this? Alternatively, to show differences in expression levels between these three cells in that panel and others, authors could use a heatmap LUT of the V5 signal to differentiate expression levels more clearly in different cells.

I am unsure whether the reviewer is referring to the cell located at the bottom-left corner of the panel in the previous Supplementary Fig. 1B (Appendix Fig. S1B in the revised manuscript). The cell shows a rather normal distribution pattern of exogenous MTCL2 similar to the endogenous one. We think this is the reason why it maintains a rather normal assembly structure of the Golgi ribbon. We included the word “frequently” in the sentence (line 153 in the revised text) to indicate that high levels of exogenous MTCL2 do not disrupt the normal Golgi ribbon structure. We do not think it is necessary to differentiate the expression levels of exogenous MTCL2 more clearly by using a heatmap, since this issue is not critical for the conclusions of this paper.

Line 139. How was the ectopic expression 'suppressed to endogenous levels'? The panels in Suppl Fig. 1 of 'low expression' clearly show increased MTCL2 signal when compared to non-transfected cells in the same panel still. This would suggest ectopic expression is still above endogenous levels.

We did not suppress the expression actively. We identified the cells expressing exogenous MTCL2 at low levels comparable to those of endogenous MTCL2. The information provided in line 139 of the previous text is not accurate. Thank you for pointing out this issue; we revised the sentence as follows: “However, when the expression levels were similar to the endogenous levels, … (lines 154-155 in the revised text)”

Figure 5C. The label for MTCL2 construct should read mMTCL2 ΔC-MTBD to clarify the expression construct used.

Since the labeling in previous Fig. 5 and 6 was confusing, we revised them all by adding the name of the expressed MTCL2 mutant under the label “+dox” (see Fig. 5, Fig. EV4, and Fig. EV5 in the revised manuscript).

In Figures 6A and 6C the label shows a-tubulin, but the staining is of a Golgi marker.

Thank you for pointing this out. We corrected this error in the corresponding figure (Fig. EV5) in the revised manuscript.

In Figures 6B and 6D the different conditions should be separated more in the graph, the datapoints overlap.

In the revised manuscript, we significantly improved the presentation of the statistical data shown in the previous Figs. 5 and 6 (Fig. 5 and Figs. EV4 and 5 in the revised manuscript). In these improvements, we determined to only include data of biological replicates in a single typical experiment in the main figures. Automatically, data points in the previous Fig. 6B and D were decreased in number and do not overlap anymore (see Figs. EV4 and EV5D). Instead, we have included new figures (Appendix Fig. S4) in which the results of technical replicates (three independent experiments) are presented.

Lines 246-7. The authors claim the Golgi-associated and centrosomal MTs can be easily distinguished in MTCL2 knockdown cells. They should include annotation in the corresponding figures to highlight these different populations.

We followed the reviewer’s suggestion by adding arrows in Fig. 6C of the revised manuscript.

Figure 8A. A horizontal line is missing in the panel showing MTCL/a-tub merge.

Thank you for pointing this out. As mentioned above, we deleted the previous Fig. 8A from the manuscript.

Figures 8C and 8D. The acetylated tubulin staining in control cells (control RNAi and GFP) in these panels vary greatly. Can the authors comment on this?

Expression of MTCL1 C-MTBD induces tubulin acetylation intensely. Therefore, to obtain appropriate pictures under non-saturated conditions, we had to decrease the gain of photomultiplier of the confocal microscopy system for the previous Fig. 8D. This is why acetylated tubulin signals in control cells appear to be too weak in the previous Fig. 8D than those in Fig. 8C.

In any case, we deleted the previous Fig. 8C in the revised manuscript as stated above. The previous Fig. 8D is solely included in Fig. EV3.

Additionally, there appears to be an increase in acetylated tubulin on the Western Blot (8E) shown in cells expressing GFP-MTCL2 CMTB that is not reflected in the image in Figure 8D. Since a significant population of GFP-MTCL2 CMBT localizes to the nucleus, it is possible that the functional population of GFP-MTCL2 CMBT that can stabilize MTs is much lower than GFP-MTCL1 CMBT despite showing equal levels in the Western Blot. The author should compare signal intensity in the cytosol of GFP-expressing cells and base their analysis of acetylated tubulin levels on cells where cytosolic levels are comparable.

We agree with this reviewer’s comment and did not include WB data in Fig. EV3B corresponding to the previous Fig. 8D.

As for quantification of the fluorescence data in Fig. 8D, we provided a typical result on the acetylate-tubulin signals normalized by GFP and a-tubulin signals in the boxed regions where cytosolic GFP signals are comparable.

Point-by-point responses to the reviewer #__3’s comments__

While the standard fluorescence images are of good quality, the quality of the super-resolution microscopic images is quite low and insufficient. Fig. 8A looks like an enlarged standard laser scanning microscope image, but does not achieve the resolution of a super-resolution image by far, which should be well below the µm range. However, such a resolution would be required to support the claim that MTCL1 and 2 locate on MTs in a mutually exclusive manner. (Negative) data from immunoprecipitation experiments also provide only weak evidence for the absence of a heterocomplex. I also fear that the fixation process creates artifacts. Experiments to image living cells would definitely bolster the data and also provide information about the dynamics of the interactions.

One of the major comments of three reviewers have been provided on Fig. 8, which reports that MTCL1 and 2 differentially regulate microtubules. We agree that the previous data in Fig. 8 A–C are rather preliminary. In the revised manuscript, we deleted these data and ceased to discuss that MTCL1 and 2 localize with microtubules in a mutually exclusive manner, as this was not the main focus of the study.

We also deleted the previous Fig. 2D (showing another super-resolution image) and the corresponding sentence. By doing so, we ceased to suggest that MTCL2 functions in mediating MT–Golgi interactions only based on immunofluorescence data.

It would also be relevant to confirm that the results are not a cell line artifact in HeLa cells.

In the previous manuscript, we included data indicating that the knockdown effects observed in HeLa-K cells (reduced accumulation of MTs around the Golgi as well as lateral expansion of the Golgi ribbon) are also induced in RPE1 cells by MTCL2 knockdown (Supplementary Fig. 2 in the previous manuscript). We included the same figure in the revised manuscript as Appendix Fig. S4.

A standard method for detecting microtubule association in cultured cells would be to use an extraction protocol. This has to be done to show that MTCL2 actually behaves like a microtubule-associated protein (MAP).

In the revised manuscript, we included new immunofluorescence data obtained using PFA fixation with or without pre-extraction, which revealed a similar localization pattern of MTCL2 to that obtained by methanol fixation (Fig. EV2). Pre-extraction was performed using BRB80 buffer supplemented with 0.5% TX-100 and 4 mM EGTA for 30 s, according to a protocol provided by Dr. Mitchison Laboratory.

I don't see that the study proves that MTCL2 is essential for the organization of an asymmetric microtubule network as the title claims. The experiments shown in Fig. 5 demonstrate a change in the skewness of the pixel intensity distribution dependent on the presence of MTCL2, which may indicate a contribution of MTCL2 (provided that the fixation and staining do not produce an artifact). However, they do not prove that MTLC2 is essential.

We cannot understand how an artifact due to the fixation and staining may be responsible for the results shown in the previous Fig. 5 (Fig. 5 and Figs. EV4 and 5 in the revised manuscript).

In many cases, microtubules do not elongate radially (symmetrically) from the centrosome but intensely accumulate around the Golgi area and show asymmetric organization (see Meiring et al. Curr. Opin. Cell Biol. 62: 86-95, 2020). “An asymmetric MT network” in the title corresponds to this asymmetric array of general MTs accumulating around the Golgi complex.

In this respect, our findings that MTCL2 depletion severely disrupted MT accumulation around the Golgi and induced random and rather symmetric arrays of MTs (Fig. 5A) are very impressive. We believe that the knockdown/rescue experiments in this study strongly support the title by demonstrating that MTCL2 facilitates MT accumulation around the Golgi through its dual binding activity to MTs and the Golgi membrane.

We are unable to comprehend the reviewer’s standpoint in not allowing us to conclude the essential role of MTCL2 in the organization of an asymmetric microtubule. However, the title in the revised manuscript was changed as follows.

“MTCL2 promotes asymmetric microtubule organization by crosslinking microtubules on the Golgi membrane”

There is also a large oversampling of the data by plotting each individual cell from only two separate experiments. It would be better and more reliable to present the data as the mean of the experiments (then of course more than 2 would be required). The same applies to the experiments in which the "Golgi ribbon expanding angle" was determined (Fig. 6).

In my opinion, statistical theories based on an ideal assumption cannot simply be applied to the quantitative analysis of biological phenomena. In our case, the MT distributions, as well as the Golgi ribbon expansion angles significantly deviate in a context-dependent manner in each cell (for example, cell cycle, cell densities, etc.). The deviation of these values between each cell (in biological replicates) is much larger than the experimental deviation, which is mainly dependent on the stochastic element (in technological replicates). I understand that this is the reason why many journals in cell biology do not necessarily require “three” independent experiments for statistical analysis.

In the revised manuscript, however, we included data from three independent experiments for all rescue experiments (Fig. 5, Figs. EV4 and 5, and Appendix Fig. S4) to further demonstrate the reliability of our data.

In the main figures (Fig. 5, Figs. EV4 and 5), we included statistical data of a single typical experiment to demonstrate reproducibility in biological replicates in each condition. To compensate for these figures, we listed statistical data for each biological replicate of all experiments in Appendix Fig. S4 A. In Appendix Fig. S4 B and C, we further provided statistical data of technical replicates (three independent experiments) by comparing the average of each biological replicate. We concluded that this is the best way to statistically demonstrate the reliability of the biological analysis.

We believe that the data collectively presented by these figures strongly support the reliability of our conclusions.

It would be good to support the claim that MTCL2 affects the Golgi ribbon structure through ultrastructural analysis (EM).

We did not perform electron microscopy analysis, because we are not implicating a change in the ultrastructure of the Golgi apparatus in MTCL2-knockdown cells. We specifically want to demonstrate that MTCL2 knockdown changes the assembly structures of the Golgi ribbons, and we believe that it is feasible to do so by light microscopy. We realize that using the term “Golgi morphology” may be misleading in this context. In the revised manuscript, we replaced this term with appropriate ones, such as “assembly structures of the Golgi stacks” or “compactness of the Golgi ribbon.”

The critical mechanistic question is which molecule on the Golgi side interacts with MTCL2, since the experiments with the deletion constructs would suggest that it is not the microstructure of the microtubules. As shown, the study is mainly descriptive in relation to this aspect.

We significantly improved this weakness by including new data indicating the possible involvement of CLASPs and giantin in mediating the Golgi association of MTCL2 (see Fig. 7 and Appendix Figs. S5–7).

We also revealed that four-point mutations (4LA) in the first coiled-coil motif disrupt the Golgi localization of the N-terminal region of MTCL2 (Fig. 4D). Thereafter, we found that introduction of the same mutations in full-length MTCL2 abolished its preferential association to the perinuclear MTs accumulating around the GA without affecting its colocalization to MTs (Fig. 4E and F).

These results reinforce our immunofluorescence results (Fig. 2) and indicate that the preferential association of MTCL2 to perinuclear MTs accumulating around the Golgi is facilitated by physical interactions between the N-terminal region of MTCL2 and the Golgi-resident proteins, such as CLASPs and giantin.

Author Response:

Reviewer #1 (Public Review):

[...] However, I also have some concerns about the main predictive model result. Although the parasite invasion/growth phenotypes are arguably simpler than an overall in vivo malaria disease phenotype, the reported 40 - 80% variance explained by the LASSO models strikes me as concerningly optimistic. Notably, the correlation in the growth phenotype for repeated samples from the same individuals (sampled weeks apart) is only rho = 0.34 (and for invasion, it is only 0.05). Given that a trait's repeatability is the upper limit to its heritability, and genetic prediction is based on a trait's heritable component, I do not understand how the trait prediction can be as strong as currently reported. Because the result is so striking, it will be crucial to perform true out-of-sample prediction to evaluate predictive accuracy and generalization error.

We agree with the reviewer that the high values of variance explained in an earlier version of this work may have reflected overfitting of the LASSO models, even in randomized data. We have now reanalyzed the data in a k-folds cross-validation framework, as described in Essential Revisions. As expected, we observe lower predictive accuracy in smaller test datasets than in larger train datasets. Nonetheless, real data and malaria-associated genes produce models that are significantly more predictive of P. falciparum fitness in test data than expected from permutation or random RBC genes. We note that noise across repeated measurements from the same individuals, taken weeks or months apart, is likely to reflect variation from technical inconsistencies as well as environment-dependent biology.

Assuming out-of-sample prediction holds up, it is interesting that the genotype data add substantially to predictive accuracy even after directly considering RBC phenotypes themselves. As the authors note, this result suggests that the mechanisms through which the genetic effects act are independent of the measured phenotypes. This prediction should be further evaluated (e.g., by assessing genotype-RBC phenotype correlations).

We agree with the reviewer that some of the observed genetics effects must be mediated through phenotypes that we did not measure, which is quite interesting given the large number of phenotypes that we did measure. Additional phenotypes of interest include quantitative proteomics, transcriptomics, and metabolomics, among others, as addressed in the revised discussion. We plan to evaluate correlations between RBC genotypes and such phenotypes in future work, as this is outside of the scope of the current manuscript.

Finally, although the results suggest no polarization of allele frequencies by European versus African ancestry, this result should be interpreted with caution throughout the manuscript, since it's unlikely that the predictive variants identified by LASSO are in fact causal.

We agree with the reviewer that given our SNPs are likely to be imperfectly linked to the causal SNPs, some marginal signal of ancestry polarization of the causal SNPs could be lost. In the discussion, we agree that the predictive variants identified by LASSO may merely be linked to the true causal variants. However since linked alleles have correlated frequencies within populations, we think this is unlikely to substantially impact our conclusions about African and European ancestry with regard to small-effect alleles. We discuss how the lack of enrichment for most protective alleles in Africans is also supported by recent GWAS for severe malaria (MalariaGEN, 2019) and patterns of RBC trait variation observed here and in other studies. We provide several possible explanations for this consistent observation, including extensive pleiotropy of small-effect alleles (see Boyle, Li, and Pritchard 2017 and correlations with other phenotypes in Figure 5-Source Data 3).

Reviewer #2 (Public Review):

[...] 1. The authors note that there is one family (mother and five children) are not carriers of known genetic loci. Figure 5-figure supplement 4 shows that they have significantly different distributions than other non-carriers with regards to principal components and parasitic invasion and growth rate. My concern is that many of the tests in the manuscript assume independent observations and related individuals violate this assumption. The children should be removed from all analyses to test for the sensitivity of results to this structure in the data.

We have revised the analysis after excluding the five siblings and verifying that the remaining donors are unrelated.

1. This is also related to the increase in % variance explained in their lasso models when including genetics. It would be useful to know how much of the outcome variation was from the inclusion of the principal components specifically (capturing the family) versus the variants of interest.

In the prior analysis, the PCs specific to the family explained up to 24% of the variation in invasion and 3% in growth in non-carriers. In the current analysis with the children excluded, PCs no longer have predictive power for growth or invasion. This change reflects the genetic uniqueness of the family, which directly produced the prior associations.

1. It would be helpful to know some more about the variants that were included from exome sequencing. This would include their allele and genotype frequencies, as well as the comparison with reference population frequencies.

We have added Figure 1-source data 1, which contains this information for ~160,000 exome variants that passed our quality filters.

1. Are the frequencies of known RBC disease alleles consistent with population estimates? It would be useful to assess the representativeness of the sample.

This information is now provided in Figure 1-source data 1. The frequencies of RBC disease alleles in our sample of African and admixed individuals are consistent with estimates from African populations.

1. I would appreciate knowing a bit more about the difference between the two strains, one lab adapted and one clinical. Is it known how the lab strain was adapted or how representative it is to circulating strains? If so, may be worth describing in the discussion to explain the differences in results between the strains.

We have added more details on the two divergent strains to the results and methods. We also discuss the strong correlations between the strains, including for specific phenotypes and genotypes, which suggest that our results may be generalizable. Finally, we note the interesting differences between the strains for African ancestry and HbAC carriers.

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Reply to the reviewers

Reviewer 1 (Evidence, reproducibility and clarity):

The main message of this paper, as far as I understood since I am not a molecular bioinformatician but I am certainly interested in mtDNA variations especially related to disease, is that there is a very obvious bias among synonymous changed in the ORF of human mtDNA, more frequent for aminoacids with 4 variants, more frequent in P position, and much more frequently characterized by transversion rather than transition substitutions. This survey is well written and, although edited in a rather technical language, the message is reachable and interesting. I also agree on the conclusions of the Author concerning the considerations that this set of new data should prompt one to draw also considering non-synonymous, potentially pathogenic mutations. The only contribution I feel I can provide to this manuscript is to invite the Authors to consider the possibility that the selection may be due to a preferred codon bias, linked to the higher or lower compliance of different codon to be translated by the translational in situ machinery of mitochondria. I am not sure that this applies also for mitochondrial mitochondria and related factors (you may want to ask Aleksey Amunts in Stockholm or Bob Lightowlers or Zoscha Lightowlers in Newcastle on this matter). I do know that this is certainly a problem for recombinant proteins containing, for instance, mammalian MTS fused with a bacterial restriction enzyme; in most of the cases the bacterial sequence has to be recoded using the preferred codon for mammalian system in order to increase translation by an eukaryotic (mammalian) translation machinery. I wonder whether you could discuss this possibility in your paper and maybe perform some further comparative measurement to test it.

I appreciate the supportive comments of Reviewer 1 regarding the accessibility of our manuscript, and I address comments related to codon bias below.

Reviewer 1 (Significance):

The paper provides novel information on the structure and constrains of mtDNA variants in humans, opens an area of investigation which is new and potentially relevant, with some possible implications also on pathogenic mtDNA mutations in humans.

I thank Reviewer 1 for their positive comments about the novelty of this work and the important implications of our study.

Reviewer 1 (Referee Cross-commenting):

I said in my first comment that I am not a bioinformatician, but Referee 2 made a great job in identifying some critical points and suggest the Authors how to cope with them. I maintain my opinion, that I think it's shared by referee 2, that the paper conveys an interesting and rather unexpected message, and that if the Authors are able to answer properly to the points raised by referee 2 the paper should be published.

We are quite glad to hear that Reviewer 1 would like to see this manuscript published, provided that the items noted by the reviewers are properly addressed.

Response to Reviewer 1:

R1Q1 (Continuation from Referee Cross-commenting): I confirm that the only contribution I feel I can provide to this manuscript is to invite the Authors to consider the possibility that the selection may be due to a preferred codon bias, linked to the higher or lower compliance of different codons to be translated by the translational in situ machinery of mitochondria. I wonder whether the Authors could consider this possibility in the Discussion and possibly perform some further comparative measurement to test it.

R1A1: My manuscript takes into consideration the possibility that codon-specific preferences would determine the frequency of mtDNA variants. Findings that argue against codon bias as a strong source of selection include:

1) At two-fold degenerate P3s, nearly every site (> 97%) harbored at least one HelixMTdb sample associated with a non-reference base. It is worth noting that HelixMTdb is not enriched for known mitochondrial disease variants.

2) SSNEs are very tightly associated with transversions from the human reference sequence, implicating mutational biases as a cause of any limited diversity in the HelixMTdb.

3) Every possible base can be found at 99% of >500 analyzed I-P3 positions (those P3s at which the base at codon positions one and two is identical throughout the alignment), arguing against the idea that codon bias plays a significant role in controlling variant frequency across mammals. The only exception that I identified in my extensive analysis is the P3 found within the first methionine codon of COX3.

4) Earlier, more limited studies of mitochondrial codon choice (citations of these earlier studies can be found in the manuscript) also argue against substantial selection based upon codon choice.

5) Finally, I would note that the set of tRNAs encoded by vertebrate mtDNAs is quite limited, with only one tRNA linked to each codon family defined by codon positions P1 and P2. There is no evidence, to my knowledge, that nucleus-encoded tRNAs enter human mitochondria. Therefore, the scope of potential selection linked to, for example, translation speed and protein folding seems particularly limited at vertebrate mitochondria.

While most evidence does not support strong selection on mtDNA codon choice in vertebrates, I do report divergence in TSS distributions obtained from the I-P3s of different amino acids within the same degeneracy class (eg. two-fold purine, two-fold pyrimidine, four-fold), hinting at some minimal role for codon preferences at P3. However, on the whole, mutational propensities are likely to be the predominant factor controlling synonymous variation.

Reviewer 2 (Evidence, reproducibility and clarity):

The manuscript explores a large database of human mtDNA sequences and performs some comparative analysis across mammals to characterise the profile of mtDNA mutations. It finds that some variants are surprisingly poorly represented in human mtDNA and suggests that mutational bias rather than selection is the dominant driver of this heterogeneity.

This is an interesting message and an efficient and interpretable of a large-scale dataset to shed light on biological mechanisms, which is a highly desirable philosophy. The factors shaping human mtDNA heterogeneity are of immense interest for several fields from population genetics to medicine, making this a valuable perspective. My comments are mainly quite fine-grained and reflect instances where I think the argument could be tighter, rather than fundamental flaws in the approach. In the cases where these points are due to my own naivety, I apologise and suggest that more explanation of these points could help other readers like me!

I am happy to read that Reviewer 2 (Dr. Iain Johnston) finds my approach to be fundamentally sound, and I certainly appreciate the insightful comments and suggestions that he has provided.

Reviewer 2 (Significance):

I wrote the above review without realising the reviewer interface would be categorised in this way. Here's a repeat of my "significance" comments

The manuscript explores a large database of human mtDNA sequences and performs some comparative analysis across mammals to characterise the profile of mtDNA mutations. It finds that some variants are surprisingly poorly represented in human mtDNA and suggests that mutational bias rather than selection is the dominant driver of this heterogeneity.

This is an interesting message and an efficient and interpretable of a large-scale dataset to shed light on biological mechanisms, which is a highly desirable philosophy. The factors shaping human mtDNA heterogeneity are of immense interest for several fields from population genetics to medicine, making this a valuable perspective.

I am very pleased that the reviewer appreciates the importance and potential impact of my analysis. We agree that mtDNA heterogeneity is likely to be of high medical relevance.

Response to Reviewer 2:

R2Q1: The first paragraph is focused on humans without explicitly saying so; missing heritability is less of an issue in, for example, plants [Brachi et al., 2011. Genome biology, 12(10), pp.1-8]. This focus should be clearer (or the differences across kingdoms mentioned!). It's also worth noting that the argument about pathogenic variants being infrequent because of selection can only address missing heritability in pathogenic variants, and cannot (directly) inform the missing heritability in traits like height etc. Also, the whole motivation with respect to missing heritability currently comes across as a bit of a non sequitur. An introduction section could be used to help describe how the analysis of the provenance of mtDNA mutations contributes to the missing heritability question.

R2A1: I agree that beginning the manuscript with a discussion of genome-side association studies may distract the reader from the main topic at hand: the utility of variant frequency when predicting pathogenicity in humans. I have changed the Introduction accordingly.

R2Q2: I also suggest that such an introduction section introduces the (later cited) previous work from Reyes and others on mutational profiles in mtDNA to set the scene.

R2A2: I now provide these citations in the second paragraph of the Introduction. However, I do not expand further upon mutational propensities in that section, with an eye toward minimizing manuscript length toward publication as a short report.

R2Q3: An early result, that 35% of possible synonymous mutations do not appear in a dataset, lacks a null hypothesis. Depending on the size of the dataset this may be very surprising or very unsurprising : an order of magnitude estimate of what proportion would be expected under uniform mutation and zero selection would help comparison here. I guess this can be as simple as 16k/3*4 R2A3: The reviewer raises an excellent point regarding how 'surprising' it should be to the reader, previous to downstream analyses revealing transition/transversion biases, that so many synonymous substitutions are lacking within this dataset. While the authors of the HelixMT study removed mtDNA from highly related individuals from the analysis, the vast majority of the mtDNAs analyzed (91.2%) were from haplogroup N and of inferred European ancestry (doi.org/10.1101/798264). The authors of the HelixMTdb study do note that nearly all mtDNA lineages were present in the study, presumably encompassing roughly 100,000 years of human mtDNA evolution. That said, how this information alone may be used to quantitatively model expectations under zero selection is unclear.

To address this question of whether sample diversity might be very limited in the HelixMTdb study, I have carried out additional analyses on this dataset. I now assess, for third codon positions allowing two-fold synonymous change (serine and leucine not included, due to their decoding by two different tRNAs), how often only one nucleotide was found at that position. For two-fold degenerate P3s, > 97% (n=1604) harbored both nucleotide possibilities within the database. This result strongly suggests that mtDNA diversity was well sampled in the HelixMTdb study, since a database consisting of highly related samples would presumably be characterized by a greater number of sites showing total identity. Moreover, when considering analyzed four-fold degenerate P3s (again, leucine and serine codons were omitted), only a very small number of sites showed no diversity (1%), with more than half of sites harboring at least three different bases. My interpretation is that the HelixMTdb authors have successfully sampled a very diverse set of human mitochondrial genomes. I have added these new analyses to the manuscript as Fig. 2a and 2b.

I have also changed the word 'surprising' to 'noteworthy' within the relevant portion of my manuscript text.

R2Q4: I think some comments and additional framing of the diversity in the central database would be valuable and important for interpretation. I believe it has, for example, rather more European rows than African ones, thus (to take a very basic view) sampling a less diverse population more than a more diverse one.

R2A4: I now state explicitly that the vast majority of the mtDNAs analyzed (91.2%) were from haplogroup N and of inferred European ancestry. Also, please see point R2A3 for further discussion of the human mtDNA diversity reflected within HelixMTdb.

R2Q5: Another rhetorically important number lacking a comparison with a null is that guanine was detected at >3000 P3 positions accepting synonymous purine substitutions. This is cited as evidence that nucleotide frequencies at P3s don't reflect selection inherent to translation. But this link isn't clear -- if such selection was present, how different from 3000 would Iexpect this number to be? Isn't there a continuum of possibilities? Is the key idea that 3000 is greater than some other number, and if so, what is that?

R2A5: The purpose of this figure is simply to demonstrate that no nucleotide is ruled out when considering silent substitutions at the P3 of any amino acid. This is consistent with (although does not prove, and I believe that the I-P3 analysis provides stronger evidence on this point) a minimal role for mitochondrial codon preference in mtDNA evolution. To reflect that my point is more general, and not to be taken as a quantitative comparison, I changed my text to: 'However, even considering the relative depletion of guanine from all four-fold degenerate P3s and two-fold degenerate purine P3s, guanine was nonetheless detected at thousands of P3 positions (Fig. 3b)'.

R2Q6: I also wasn't clear whether/how the finding that little selection inherent to translation was implicitly extended to suggest little general selection overall. The following section only considers selection acting at specific P3 sites, thus implicitly discarding other hypotheses about general selection based on nucleotide content but not inherent to translation. Perhaps I am misunderstanding this translation link, but selection based on general nucleotide profiles (for example, due to thermodynamic stability [Samuels, Mech. Ageing Dev. 2005; 126: 1123-1129] or availability of nucleotides [Aalto & Raivio, Mech. Ageing Dev. 2005; 126: 1123-1129; Ott et al., Apoptosis. 2007; 12: 913-922]) would seem to still be on the table?

R2A6: I would argue against selection upon nucleotide choice linked to local changes to mtDNA thermodynamic stability. Most prominently, when considering two-fold degenerate sites, nucleotide differences from the reference sequence were identified within the HelixMTdb at almost every analyzed position (Fig. 2a), even though hydrogen bond strength between opposing bases would be affected in every case (AT>GC or vice versa). Of course, my argument here applies generally, and there may be a small subset of sites for which nucleotide substitutions can cause a pronounced functional defect because of a change to local mtDNA structure.

I would also argue against mitochondrial nucleotide availability as a source of selective pressure within the human population. When considering the entire L-strand sequence (NC_012920.1), nucleotide counts are as follows:

A 5124

C 5181

G 2169

T 4094

And when considering both strands, nucleotide counts and frequencies are as follows:

A 9218 (27.8%)

C 7350 (22.2%)

G 7350 (22.2%)

T 9218 (27.8%)

One nucleotide substitution would lead to a change in nucleotide frequencies by less than 0.02%. While the formal possibility exists that mitochondrial nucleotide availability lies exquisitely close to an important threshold, there is no current evidence to support this proposition. And here again, the diversity of P3 nucleotide choice found among the HelixMTdb samples would argue against this possibility.

That said, it is worth noting that nucleotide frequencies, and mtDNA mutation rates relative to nuclear mutation rates do appear to differ among clades (PMID: 8524045 and 28981721). Therefore, while selection related to nucleotide availability seems an unlikely explanation for the variant frequencies that I have recovered at degenerate sites among human samples, I certainly would not rule out taxon-specific dietary, environmental, or physiological factors that, over longer evolutionary timescales, might shape mtDNA nucleotide frequencies.

I would like to raise the possibility of another source of selection upon nucleotide choice. Specifically, one might propose that synonymous mtDNA substitutions could affect the binding of proteins controlling the replication, compaction, or expression of mtDNA. Indeed, an intriguing study has reported that human cells manifest a mtDNA footprinting pattern (PMID: 30002158), suggestive of regulatory sites bound to protein or sites of transcriptional pausing. However, Blumberg et al. found no statistically significant difference in human synonymous change at footprinted sites, arguing against a strong selective pressure on nucleotide choice at footprinted P3s. Moreover, footprinting sites identified in the above-mentioned study are conserved in mouse and human, but I have shown that all four nucleotides are acceptable at all four-fold degenerate sites (n=252), all two-fold degenerate pyrimidine sites (n=157), and 99% of two-fold degenerate purine sites (n=152) within the mammalian I-P3 set, again arguing against general limitations on nucleotide choice caused by protein association. These analyses cannot, however, totally rule out the possibility that a subset of individual P3s are under some selection due to their role in binding or traversal of proteins.

R2Q7: A reptile is chosen as an outgroup for a comparative analysis of mammals. As always when a choice is made, the question arises: what if that choice was different? Perhaps the corresponding figures can be presented for two other choices of outgroup to demonstrate that there's nothing particularly unrepresentative about this reptile?

R2A7: While preparing this revised manuscript, I have performed an updated analysis using the most current mammalian mtDNA dataset available on RefSeq. For these new tests, I used Iguana iguana, rather than Anolis punctatus, as an outgroup. The new results are essentially indistinguishable from my previous findings. Importantly, when old TSS values and new TSS values for I-P3 sites were compared by linear regression, the R-squared value is 0.9955, with a p-value of

R2Q8: Another analysis involves classifying variant frequency into discrete groups based on percentage appearance, then seeking links with the TSS statistic. First, it is not clear why discretisation is needed here. A statistical model embracing the continuous nature of variant frequency requires fewer arbitrary choices (e.g. of numbers and boundaries of classes).

R2A8: A primary audience of this manuscript will certainly be the human genetics community, which commonly speaks in terms of variant classes (eg. 'common', 'rare', 'ultra-rare'). Therefore, I prefer to also use such classifications when analyzing the relationship between TSS and mtDNA variant frequency. I took advantage of the following references when generating frequency classifications:

Bomba L, Walter K, Soranzo N. 2017. The impact of rare and low-frequency genetic variants in common disease. Genome Biol 18:77.

McInnes G, Sharo AG, Koleske ML, Brown JEH, Norstad M, Adhikari AN, Wang S, Brenner SE, Halpern J, Koenig BA, Magnus DC, Gallagher RC, Giacomini KM, Altman RB. 2021. Opportunities and challenges for the computational interpretation of rare variation in clinically important genes. Am J Hum Genet 108:535–548.

R2Q9: Second, an interpretation point here is in danger of equating absence of evidence with evidence of absence. Without an estimate of statistical power, an absence of a significant relationship cannot suggest that anything is likely or unlikely, only that there may not be sufficient power to detect an effect.

R2A9: To address this point, I have changed my text as follows:

Old: 'However, I detected no significant relationship between TSS and variant frequency for four-fold degenerate I-P3s (Fig. 2d), indicating that the highly elevated SSNE abundance at four-fold degenerate P3s is unlikely to be due to selection.'

New: 'However, I detected no significant relationship between TSS and variant frequency for four-fold degenerate I-P3s (Fig. 2d), consistent with the idea that the highly elevated SSNE abundance at four-fold degenerate P3s is unlikely to be due to selection.'

R2Q10: Figs 1a and 1e have a log vertical axis but I think the lowest points actually corresponds to zero? This is not compatible with a log axis and the zero position should be explicitly labelled with its own tick (perhaps in parentheses to highlight the discontinuity).

R2A10: Quite correct, and I had neglected to clarify those details in the previous version of the manuscript. I now designate the samples with zero counts in the population using a smaller dot size, and I describe this approach in the figure legend.

R2Q11: The methods are presented in an interesting way, with specific filenames for the code associated with each part of the pipeline explicitly provided. This is (very!) nice but it would also be good to describe in words what each piece of code does (e.g. "this was used as input for x.py, which counts the mutations and outputs a profile" or some such). This is indeed sometimes written but some parts lack an explanation.

R2A11: I have now expanded my description of several scripts within the Methodology section.

R2Q12: I could do with an additional sentence or two on the statistical analysis. As Kolmogorov-Smirnov tests examine differences between distributions, it's not immediately unambiguous how they are applied to total count statistics. Are count distributions with respect to variant frequency analysed for each amino acid separately? Or are the amino acids somehow ordered and the distributions across them compared? Or something else?

R2A12: TSS distributions are held for each individual amino acid, which are then compared by Kolmogorov-Smirnov testing only within a given degeneracy category (four-fold degenerate, two-fold degenerate purine, two-fold degenerate pyrimidine). I have now elaborated upon this statistical test selection, and other details of the analysis, in the Methodology section.

Reviewer 2 (Referee Cross-commenting):

I agree that codon bias is an interesting potential axis of selection. Even if the analysis rejects the hypothesis of selective effects inherent to translation, it is conceivable that codon bias could be shaped by selection in other indirect ways (depending on how "inherent" is defined, these could include tRNA/nucleotide availability, GC content and thermodynamic stability, etc). I think this aligns with my suggestion that modes of selection that are not directly linked to translation could be explored in more depth before discounting selective effects overall. IJ

I hope that I have now successfully addressed points related to codon bias, GC content, and thermodynamic stability in the manuscript, as well as here in this response to the reviewers.

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 #2

Evidence, reproducibility and clarity

The manuscript explores a large database of human mtDNA sequences and performs some comparative analysis across mammals to characterise the profile of mtDNA mutations. It finds that some variants are surprisingly poorly represented in human mtDNA and suggests that mutational bias rather than selection is the dominant driver of this heterogeneity.

This is an interesting message and an efficient and interpretable of a large-scale dataset to shed light on biological mechanisms, which is a highly desirable philosophy. The factors shaping human mtDNA heterogeneity are of immense interest for several fields from population genetics to medicine, making this a valuable perspective. My comments are mainly quite fine-grained and reflect instances where I think the argument could be tighter, rather than fundamental flaws in the approach. In the cases where these points are due to my own naivety, I apologise and suggest that more explanation of these points could help other readers like me!

The first paragraph is focused on humans without explicitly saying so; missing heritability is less of an issue in, for example, plants [Brachi et al., 2011. Genome biology, 12(10), pp.1-8]. This focus should be clearer (or the differences across kingdoms mentioned!). It's also worth noting that the argument about pathogenic variants being infrequent because of selection can only address missing heritability in pathogenic variants, and cannot (directly) inform the missing heritability in traits like height etc. Also, the whole motivation with respect to missing heritability currently comes across as a bit of a non sequitur. An introduction section could be used to help describe how the analysis of the provenance of mtDNA mutations contributes to the missing heritability question. I also suggest that such an introduction section introduces the (later cited) previous work from Reyes and others on mutational profiles in mtDNA to set the scene.

An early result, that 35% of possible synonymous mutations do not appear in a dataset, lacks a null hypothesis. Depending on the size of the dataset this may be very surprising or very unsurprising : an order of magnitude estimate of what proportion would be expected under uniform mutation and zero selection would help comparison here. I guess this can be as simple as 16k/34 << 200k. Also the ancestry of the dataset is important here: if all samples are highly related then a more homogenous mutational profile is unsurprising. Perhaps one could assign a quantity like an effective population size to the database and compare this to 16k/34? I think some comments and additional framing of the diversity in the central database would be valuable and important for interpretation. I believe it has, for example, rather more European rows than African ones, thus (to take a very basic view) sampling a less diverse population more than a more diverse one.

Another rhetorically important number lacking a comparison with a null is that guanine was detected at >3000 P3 positions accepting synonymous purine substitutions. This is cited as evidence that nucleotide frequencies at P3s don't reflect selection inherent to translation. But this link isn't clear -- if such selection was present, how different from 3000 would we expect this number to be? Isn't there a continuum of possibilities? Is the key idea that 3000 is greater than some other number, and if so, what is that?

I also wasn't clear whether/how the finding that little selection inherent to translation was implicitly extended to suggest little general selection overall. The following section only considers selection acting at specific P3 sites, thus implicitly discarding other hypotheses about general selection based on nucleotide content but not inherent to translation. Perhaps I am misunderstanding this translation link, but selection based on general nucleotide profiles (for example, due to thermodynamic stability [Samuels, Mech. Ageing Dev. 2005; 126: 1123-1129] or availability of nucleotides [Aalto & Raivio, Mech. Ageing Dev. 2005; 126: 1123-1129; Ott et al., Apoptosis. 2007; 12: 913-922]) would seem to still be on the table?

A reptile is chosen as an outgroup for a comparative analysis of mammals. As always when a choice is made, the question arises: what if that choice was different? Perhaps the corresponding figures can be presented for two other choices of outgroup to demonstrate that there's nothing particularly unrepresentative about this reptile?

Another analysis involves classifying variant frequency into discrete groups based on percentage appearance, then seeking links with the TSS statistic. First, it is not clear why discretisation is needed here. A statistical model embracing the continuous nature of variant frequency requires fewer arbitrary choices (e.g. of numbers and boundaries of classes). Second, an interpretation point here is in danger of equating absence of evidence with evidence of absence. Without an estimate of statistical power, an absence of a significant relationship cannot suggest that anything is likely or unlikely, only that there may not be sufficient power to detect an effect.

Figs 1a and 1e have a log vertical axis but I think the lowest points actually corresponds to zero? This is not compatible with a log axis and the zero position should be explicitly labelled with its own tick (perhaps in parentheses to highlight the discontinuity).

The methods are presented in an interesting way, with specific filenames for the code associated with each part of the pipeline explicitly provided. This is (very!) nice but it would also be good to describe in words what each piece of code does (e.g. "this was used as input for x.py, which counts the mutations and outputs a profile" or some such). This is indeed sometimes written but some parts lack an explanation.

I could do with an additional sentence or two on the statistical analysis. As Kolmogorov-Smirnov tests examine differences between distributions, it's not immediately unambiguous how they are applied to total count statistics. Are count distributions with respect to variant frequency analysed for each amino acid separately? Or are the amino acids somehow ordered and the distributions across them compared? Or something else?

Iain Johnston

Significance

I wrote the above review without realising the reviewer interface would be categorised in this way. Here's a repeat of my "significance" comments

The manuscript explores a large database of human mtDNA sequences and performs some comparative analysis across mammals to characterise the profile of mtDNA mutations. It finds that some variants are surprisingly poorly represented in human mtDNA and suggests that mutational bias rather than selection is the dominant driver of this heterogeneity.

This is an interesting message and an efficient and interpretable of a large-scale dataset to shed light on biological mechanisms, which is a highly desirable philosophy. The factors shaping human mtDNA heterogeneity are of immense interest for several fields from population genetics to medicine, making this a valuable perspective.

Referee Cross-commenting

I agree that codon bias is an interesting potential axis of selection. Even if the analysis rejects the hypothesis of selective effects inherent to translation, it is conceivable that codon bias could be shaped by selection in other indirect ways (depending on how "inherent" is defined, these could include tRNA/nucleotide availability, GC content and thermodynamic stability, etc). I think this aligns with my suggestion that modes of selection that are not directly linked to translation could be explored in more depth before discounting selective effects overall. IJ

was missing

Some things we may value as seen by our actions or the way we behave, but in our minds we do not associate with these values or do not like that we hold these values. We chose not to talk about or choose to deny that we value something because we are worried it may change the way others or even how we think about ourselves.

By reading this quote, I understand how many people may try to follow the path of The American Dream. Following the rules, working hard, etc. I do not think that only these things will actually help you get The American Dream, most of the time the people who succeed, they already have resources, people who can rely to and actually get the job they were looking for or getting into the school they always dreamed of going to. Is not only hard work but smart work, meaning, putting aside the rules and find ways to actually make things work. We would get impressed on how most people find solutions for their problems or to even make a dream come true.

Author Response:

Reviewer #1 (Public Review):

The authors carried out a post-hoc analyses of a protective gene expression signature previously observed in preclinical trials and clinical trials (RV144 and HVTN505) to identify a possible correlate of reduced risk of infection and whether able to provide a potential mechanism for protection. This monocyte signature they focus on was absent in the DNA/rAd5 human vaccine trial which did not show efficacy and was enriched in the partially effective RV144 human trial where the vaccine was and ALVAC/protein vaccine. Here they indicate that the signature is a correlate of reduced risk of infection.

Identifying signatures of protection is an important issue in the development of a HIV vaccine, and signature analyses might be important to reveal a few markers that might be selected to evaluate vaccine trials. However, this analysis must be able to point to very few genes, as single cell analyses are not an option in a large clinical vaccine trial.

We agree scRNA-seq might not be applicable to assessing large scale clinical trials. However it is useful for identifying the cellular lineage of the signal we previously were unable to identify from bulk gene expression datasets (see discussion section, line 324). The signature identified has 200 genes for which methods are increasingly available for economic screening of large sample numbers, for example as we did previously on the Fluidigm BioMark platform (Ehrenberg et al. 2019).

It is unclear whether or how the conclusion of the previous publication by many of the same authors of this paper, including the senior author (Ehrenberg et al., 2019, identification of a gene signature in B cells that is associated with protection from SIV and HIV infection providing a new approach for evaluating future vaccine candidates) is compatible with this new one: signature primarily expressed in myeloid lineage being the one most consistently associated with vaccine efficacy. It is unclear which one of the two is correct or how they are reconciled. Was the single cell analysis done in monocytes only for this paper or simply not reported in the studies of Ehrenberg et al., 2019?

The protective gene signature was identified initially in microarray data from total PBMCs in the RV144 study and so we did not know the cellular lineage of this signal. Although RV144 samples were depleted, we had the unique opportunity to investigate the cellular lineage of the gene signature in the RV306 trial, which is a vaccine trial that was performed in Thailand and used the same RV144 vaccine series, with additional boosts after the 4th vaccination. We performed scRNA-seq at the timepoints that were equivalent to the RV144 4th vaccination and concluded that the enriched genes in the signature were mostly expressed in monocytes (discussion section, lines 329-336). The current paper has 3 new datasets: HVTN 505 RNA-seq data, RV306 RNA-seq data and RV306 single cell CITE-seq data. The Ehrenberg et al. 2019 were primarily focused on the Ad26 vaccine preclinical trials in NHP. This formed the basis of our current findings that expanded to human studies such as RV144, HVTN 505 and RV306. The CITE-seq data from the RV306 study was performed in 2020, only after we confirmed using bulk-RNA-seq from blood that the gene signature associated with increased ADCP in the RV306 study. We have clarified the different studies in supplementary table 1.

Figure 1: The gene expression score (GES) of this figure does not seem to be for a specific cell type. It is unclear how the GES reported here relates to the final GES of monocytes. What is the utility of this analysis? Can we observe here the same most significant genes that we observe in monocytes? This is important because if bulk analysis gives the same results as looking at monocytes an eventual marker identified in monocytes could be evaluated in luck analysis.

The composite gene expression score in Figure 1 focuses on a GES of only the enriched genes which can be used as a continuous or categorical variable in a immune correlates analyses as shown in Figure 1 or 2 regardless of phenotype. We see enrichment of a geneset that associates with vaccine protection and ADCP across multiple studies and species irrespective of methods being used. We think this set of 200 genes have a coordinated expression and may not be specific to a cell type, but might mark a certain biological state, such as response to a cytokine, and may be picked up even in PBMC and blood samples. We clarify this further in the discussion section (line 348).

Figure 2: it would be good to know whether the subset of the 63 genes can be restricted to the most significant and their GES can still retain the predictive value.

As suggested by the reviewer, we made a GES of a subset of the 63 genes in the RV144 signature that had the most significant genes (32) that associated with HIV acquisition in Fig 5. (p <0.05, q <0.1). We see that the association is slightly stronger and the probability of acquiring HIV-1 is lower in individuals with high GES (OR = 0.35 and p value = 0.0001 compared to previous OR = 0.37 and p value = 0.0002). Vaccine efficacy in individuals with high GES has also increased to 81.4% from 75.1%. The Distribution of AUC and accuracy plotted after repeating the process 1000 times showed that GES of the significant subset of the genes is predictive of HIV-1 infection with AUC of 0.69 ± 0.08 and with accuracy of 0.81 ± 0.04 (compared to previous 0.67 ± 0.08 and 0.81 ± 0.04). We agree this smaller subset could potentially be useful and now include them in the results section (page 11) and as a new supplementary figure 2.

Figure 3 deals with genes associated with antibody dependent cellular phagocytosis (ADCP). Can one derive a gene or a few genes that are predictive of significant ADCP?

Thank you for this suggestion, we have been able to explore this and now include new panels in the main figures which identify genes predictive of ADCP. We made a GES of the 93 genes enriched at the day 3 timepoint associating with magnitude of ADCP. A prediction model was built using the 93 genes from Day 3 time point. Internal validation has an area under the curve (AUC) of 0.80, suggesting that this classifier was able to discriminate high ADCP from low ADCP measured 2 weeks after last vaccination. This model, consisting of 93 expressed genes, was then tested at the Week 2 time point and was also able to predict ADCP as a dichotomous variable at the week 2 time point (AUC = 0.73).

We further examined 82 genes overlapping between the 118 enriched genes from week 2 and the 93 enriched genes from day 3 post the RV144 vaccine regimen that associated with ADCP. A GES was computed using the 82 overlapping genes for both time points. A prediction model was built using the 82 genes from the Day 3 time point. Internal validation has an AUC of 0.81, suggesting that this classifier was also able to discriminate high ADCP from low ADCP measured at week 2 after vaccination. This model, consisting of 82 expressed genes, was then tested at the week 2 time point and was also able to predict ADCP as a dichotomous variable at the week 2 time point (AUC = 0.75). We thank the reviewer for this suggestion and we have now included these data as Figures 3B and 4B.

Reviewer #3 (Public Review):

Strong points:

1. This provides a novel mechanism into the RV144-mediated protection of HIV acquisition.

2. The analyses are robust and statistically sound.

3. The flow of the paper/figures is easy to follow.

Weak points:

1. the RV306 trial (Figure 3 A and B) RNA-SEQ analysis vs ADCP could benefit from a little more information:

Are the 118 / 93 genes at Wk2 / Day 3 post-vaccination overlapping a lot?

Per the reviewer’s suggestion we looked for overlapping genes between week 2 and day 3 post the RV144 immunization series in the RV306 study. There are 82 genes in common between the enriched genes at week 2 and day 3 ADCP data which are now detailed in Supplementary table 2. The number of enriched genes in the pathway at these two timepoints are summarized in the following Venn diagram and are now included in the manuscript as Figure 4A.

What are those genes? Do they play a known direct role in ADCP or are they upstream regulators?

All 82 genes are listed in supplementary table 2. There is not a lot of information about genes associated with ADCP specifically from previous publications, but when querying existing databases for genes associating with phagocytosis, we identified four of the 82 genes in the GO:0006909 phagocytosis pathway including SIRPA, SIRPB1, RAB20, and TYROBP. When using GeneMANIA a gene function prediction tool to investigate interaction networks of the 82 overlapping genes we identified 44 additional genes that were connected to the 4 genes previously implicated in phagocytosis. We have included this information as a new supplementary table 4.

We also show other canonical pathways with gene membership including the Immune System (33 genes), Innate Immune System (23), Signaling by Interleukins (9), Hallmark Inflammatory Response (7), Hallmark TNFA Signaling Via NFKB (7), Cell-Cell Communication (5), Interleukin-10 Signaling (4), Signal Regulatory Protein Family Interactions (3), and Pentose Phosphate Pathway (3). We now include this information in the results and discussion section and hope that this information will clarify the field further (new Figure 4D, new Supplementary table 3).

Perhaps a heatmap representation with the ADCP as an annotation track would help unfamiliar readers better understand.

We have now included a heatmap that shows gene expression of the 82 genes at both timepoints, with ADCP group status annotated (Fig. 4C). The list of the 82 genes are also available in Supplementary Table 2 – (“Yes” for enrichment in the “RV306 ADCP day3” and “RV306 ADCP wk2” columns).

1. I would nuance that ADCP is "A" primary mechanism, not "THE" (title). There could be more potent unidentified mechanisms, so the usage of "THE" in the title is in my opinion premature.

The title has been updated accordingly.

1. While I agree that it is possible that ADCP is a primary mechanism with the previously identified transcriptomic signature given the evidence, we cannot exclude that the signature in fact represents an upstream regulator of ADCP, inducing a myriad of cascades contributing to vaccine-induced protection. If that were the case, ADCP could be higher in individuals with higher protection without it being directly involved in that protection (more of a collateral effect). Showing an enrichment of ADCP-associated genes from external datasets with the tested gene signature would strengthen at least partly that this is a direct phenomenon. Otherwise, I would nuance the statement and say that ADCP is a likely/potential mechanism of vaccine-induced protection.

We agree with these nuances and have updated the title and discussion accordingly (Lines 1-2, 314-316).

1. Observations in Figure 4 are glanced too quickly in the Results section: this would require a more in-depth description.

Based on the results from the current revisions we have updated the previous Figure 4 (now Figure 5) to provide an indepth description of gene function prediction based on networks. We have used GeneMANIA, which is an application that can find associated genes or pathways using its functional association data, to examine overlapping enriched genes from the different studies with either infection status or ADCP magnitude. Interestingly, TYROBP, which is associated with phagocytosis, is the gene with the most connections to other genes or pathways. We also do a clustering analysis to identify highly interconnected sets of genes and pathways from the enrichment results of different studies. We describe this now in the results section (lines 209-219), updated Figures 5A-B and discussion section (lines 299-319).

1. It is not clear whether the expression level per monocyte for the subset of genes tested in the CITE-seq data is different in patients with higher ADCP vs those with lower ADCP, or is the differential enrichment the result of a different number of cells that express this signature? Or both?

For the CITE-seq data we performed differential expression analyses transcriptome-wide and found that monocytes had a higher frequency of differentially expressed genes when comparing higher versus low ADCP (Fig 5E). This effect was independent of frequency of the different cell populations with a frequency of >1%. We now include a sentence to clarify our findings (results last paragraph) and show the frequency differences in the supplementary section (Supplementary Figure 3).

Author Response:

Reviewer #1:

Click-Seq represents a novel method of sequencing RNA viruses such as SARS-CoV-2, with evidence of successfully sequencing the SARS-CoV-2 genome and identification of recombinations and variants. This does appear to be a potential advantage that needs a direct comparison with existing methods to be fully convincing.

Thank you for your time and comments on our manuscript and approach.

Specific comments:

1) The actual sensitivity in terms of number of copies would be useful to know and tocompare with other methods. Here, cultures are used, not clinical samples that make this even more important

We now present results from three independent batches of Tiled-ClickSeq libraries of 60 NP swabs obtained through routine diagnostics for COVID19. We compare genome coverage and genome completeness with CT values of these samples. This presents the utility and potential application of the method with different clinical specimens and illustrates that with only 18 cycles of PCR we can obtain high quality data with most samples at a CT < 25.

2) Is the large difference in coverage across the genome shown in Fig 2B, due to methodological issues to random variation. How would this compare to coverage variation by the ARCTIC protocol by different methods

If the reviewer is referring to the high-frequency and regular dips in coverage (which we refer to as ‘saw-teeth’) then this is an expected feature of the stochastic termination of the cDNA by the azido-nucleotides upstream of the tiled-primers. The sharp changes in coverage here are highly comparable to coverage in ARTIC protocols. We provide an equivalent read coverage map in the new SFig 2 when using the ARTIC approach of the same samples presented in Fig 2B.

If the reviewer is referring to the difference in coverage from different tiled primers (e.g. at nt ~14000), then this is likely an issue with the specific primer used in the ‘v1’ set of primers initially used. The ‘v3’ primers presented in Fig4A illustrate that these drops in coverage are removed, which indeed is an advantage or our approach that allows for multiple closely spaced tiled-primers in the same RT-PCR reaction. To further illustrate sample-to-sample variability, we now present read coverage using Tiled-ClickSeq v3 primers for 60 clinical isolates at different CT values which gives an overview of the variation that can be expected across multiple samples with our method.

Reviewer #2:

The authors present a novel method of sequencing SARS-CoV-2, arguing its overcomes many limitations of other currently used methods, particularly the ARTIC protocol. Generally the method is interesting and encouraging to see these limitations can be overcome. Although the authors walk through evidence that their method can successfully sequence the SARS-CoV-2 genome and use the data to identify minor variants and recombination events, the manuscript doesn't contain any direct comparisons of their method with the ARTIC protocol. Consequently, the assertions made throughout the paper of reduced bias and increased sensitivity and utility are not supported empirically.

Thank you for your time and comments on our manuscript. To address these concerns, we have provided substantial new data comparing to ARTIC protocols and applying our methods to study clinical sample, described further below in response to your specific comments.

Specific comments:

For instance, in figure 2, I think it is important to present an equivalent plot to Fig 2A for artic samples with equivalent read depths using both MiSeq and Nanopore. This sequence data could be obtained from the COG-UK data deposited on NCBI SRA, and sub-sampled to match sequence depth between methods.

Thank you for your comments. We have provided this information in Supplementary Figure 2. Using the ARTIC approach, we sequenced the 12 WRCEVA isolates described in the manuscript and presented in Figure 3. As can be seen, peaks and troughs are observed in the ARTIC data, as is expected and previously reported.

I specifically wonder if this approach only outperforms artic using Nanopore sequencing given the frequent drops in coverage observed in the MiSeq data.

The frequent drops in coverage observed in the MiSeq data in figure 2 is a symptom of the first primer set we used (v1) that only contained 72 primers. Similar frequent drops in coverage are also observed in the ARTIC approach (e.g. as seen in SFig2). The v3 primer set that we subsequently developed is presented in Figure 4. As can be seen, the drops in coverage are largely removed. We further illustrate this in the new Supplementary Figure 4 where we provide coverage plots using the v3 primers for 60 clinical samples of SARS-CoV-2 at different CT values. As can be seen, the variability in coverage is greatly improved.

An additional point about figure 2: I understand that this figure is based on the depth of a single run, I think readers that are interested in using this method would be interested to know about the run-to-run variability, so I think it would be a valuable addition to this manuscript to show the average read depth (relative to total nucleotides sequenced per sample) across multiple samples with confidence intervals or equivalent to visualize run-to-run variability.

Thank you for this point. As mentioned above, we present a new Supplementary Figure 4 where we provide coverage plots using the v3 primers for 60 clinical samples of SARS-CoV-2 at different CT values. Run-to-run variability is additionally addressed in Figure 6A where we correlate genome completeness/coverage with CT values across three different NGS library preparations.

Further, the authors describe previously detecting recombinant RNA molecules in SARS-CoV-2 in another manuscript, and highlight that the method presented in this manuscript can detect recombinant RNA molecules that could be missed using the artic protocol. Were any such RNA sequences observed in these samples, or was there perfect correspondence between the methods?

As described above, in the revision, we describe the recombination analysis of multiple clinical samples of SARS-CoV-2. We provide an example of a large genome duplication (annotated as 29442^29323) found in multiple clinical samples, but not any cell-culture samples (providing support that these are not sequence artifacts). To our knowledge these have not been observed before. Our previous manuscript (Gribble et al, PLoS Path, 2021) used both random-primed RNAseq and direct RNA sequencing of poly(A)-enriched RNAs, rather than targeted approaches. Neither of these are currently feasible for clinical samples. Given the hundreds of different DVGs observed in our previous studies, it is not possible for there to be perfect correspondence. Nevertheless, the trends and distributions of RNA recombination events are very similar between our previous study and the ones presented here, as described in the manuscript.

As well , the authors state: "Phylogenetic tree reconstruction using NextStrain (45) placed 10 of the isolates in the A2a clade (Fig 3D). Three of these isolates (WRCEVA_00506, WRCEVA_00510, WRCEVA_00515) were most closely related to European ancestors. Two isolates (WRCEVA_00508, WRCEVA_00513) were Clade B/B1 most closely related to Asian ancestors. Together, these data thus supported a model for multiple independent introductions of SARS-CoV-2 into the USA and subsequently into Galveston, Texas." This analysis seems out of place in the manuscript and not robust enough to support the claims made. How did the authors come to the conclusion that different sequences are of "European" or "Asian" origin? Due to the limited amount of genetic variation present in circulating strains prior to March 2020 combined with the wide geographic range that many genotypes were circulating, it is not enough to conclude the geographic origin of a viral isolate from clade membership alone.

Thank you for this comment. We agree that this statement was not properly supported and have simply removed it in the revised manuscript.

Reviewer #3:

Strengths. While current NGS method(s), namely the ARTIC protocol, has made phenomenal contributions to resolving the genome of SARS-CoV-2, there is room for improvement. Towards this end, Jaworski and company have devised an alternative approach that utilizes a one-step RT PCR that combines ClickSeq with tiled amplification of the viral genome. This negates the use of primer pairs, which may encounter problems with amplification of structural variants. The method appears to be straightforward and amendable for sequencing on Illumina and Oxford platforms. The results generated do support the claims of the authors and have the potential to contribute significantly to understanding the evolutionary dynamics of SARS-CoV-2.

Weaknesses. The main shortcoming of the manuscript in its current form is that the samples used for sequencing as proof of concept were cell-grown viral isolates and not directly of the samples. The method described has the potential for providing the field with an alternative to produce high quality sequence, but without performing the work directly on nasopharyngeal swab samples, then it may have limited used for public health laboratories, resource-poor environments or laboratories with little expertise in viral isolation, etc. Validation of the method can benefit if the authors can compare the quality of the sequence generated compared to the ARTIC protocol using primary samples rather than cell-grown viral isolates. It is difficult to assess whether this method will provide a viable alternative over current state-of-the-art protocols.

Thank you for your comments and time reviewing our manuscript. To address these concerns, we have provided substantial new data where we apply the Tiled-ClickSeq approach to assay clinical specimens.

Specific comments.

The methods should include detailed steps in the construction of the NGS library, such as whether or not cDNA input has an impact in the quality of the data output, coverage etc.

We have previously published detailed protocols describing how to make ClickSeq libraries emphasizing issues that affect success and quality of the output data. We have emphasized this point in the methods section. Assuming we continue to utilize and improve our design, we will release updates through online freely available resources such as protocols.io.

To address these questions here: the input RNA (not cDNA) in the RT-PCR step is addressed in Figure 1. All the cDNA generated after RT is used as input in the subsequent steps and the click-reaction. We do believe that the quality of the input RNA in the clinical specimens is very important, however, beyond CT value, we have no viable way of measuring the quantity and quality of the tiny amounts of RNA that we extract from NP swabs.

While the authors mentioned that equimolar of primers were used - there should be data to demonstrate that this results in even covering of the whole genome. Figure 2. There is a slight dip in the coverage at around 17000 to 18000 (Figure 2A) on both the Illumina and Oxford runs, do the authors know if it is due to the primer(s) covering that area and if so, have they tried to address this by improving the design.

The dip in the coverage in Fig 2 is resolved by using the v3 primers presented in Figure 4. Additional coverage maps for clinical samples in SFig 3 also demonstrate this. Even coverage over the entire genome can be seen for the low CT value samples, which begins to wane in clinical samples with CT values greater than ~25, as described in the new main text and presented in the new Figure 6A.

The different colors of the graph (Figure 2B) should be defined in the legend. Is the read depth a representation of both Illumina and Oxford runs - either way, this should be indicated.

Fixed. Thank you.

Author Response:

Reviewer #1 (Public Review):

This work investigated the mechanism of inhibition of SARS-CoV-2 polymerase by multiple nucleotide analogs using a high-throughput, single-molecule, magnetic tweezers platform. There was particular focus on the remdesivir (RDV) because it is the only FDA approved anti-coronavirus drug on the market at the time of this review. The study shows that remdesivir leads the polymerase to undergo a backtrack in which it moves back as much as 30 nucleotides from the last insertion. The results also show that RDV is not a chain terminator, which is consistent with prior work. In addition to RDV, the authors characterized other nucleotide analogs such as ddhCTP, 3'-dCTP, and Sofosbuvir-TP to propose that the location of the modification in the ribose or in the base dictates the catalytic pathway used for incorporation. The authors also propose that the use of magnetic tweezers is essential towards characterizing and discovering therapeutics that target viral polymerases.

Strengths:

A strength of the papers is the utilization of magnetic tweezers to characterize the polymerase at the single molecule level. This provides a unique method to capture less common or difficult to observe phenomena such as backtracking. Most bulk ensemble assays would have difficulty detecting these phenomena.

The characterization of multiple different types of nucleotides analogs to investigate the different mechanisms by which they could inhibit the polymerase is a strength of the paper. The authors elegantly utilize their system to show different pause states and backtracking of the polymerase.

In general, the paper is well written, and the data is clearly presented.

The authors thank the Reviewer for the strong appraisal of our work!

Weakness:

The experiments performed with the magnetic tweezers appear to not have contained the exonuclease domain. This domain would presumably be involved in removing nucleotide analogs that have been inserted and may alter the pause states or backtracking prevalence. For example, does the prevalence of backtracking increase when the exonuclease domain is not present. This is particularly important in regard to the RDV experiments.

To date, no laboratory has been able to couple the polymerase complex with the proofreading complex. Indeed, we have entire five-year R01 grant to pursue this objective. Just like all proofreading polymerases studied before this one, it is imperative to establish a baseline with exonuclease deficient state prior to adding that component. Even before we add the exonuclease, it will be important to add the helicase to determine if it can assist the polymerase with dsRNA, because its strand-displacement activity is weak.

A major claim for this study is the utilization of the magnetic tweezers "experimental paradigm" as being essential to the discovery and development of therapeutics to viral polymerases. In addition the authors state this approach is superior to bulk ensemble studies. This reviewer found these conclusions to be an overstatement and unnecessary. The use of magnetic tweezers is not amenable to all laboratories or an easy technique to implement within the therapeutic drug development. In general, the authors also overstate the power and feasibility of the magnetic tweezers in comparison to bulk ensemble studies. All assays have limitations, and the magnetic tweezers is no different in regards to being purified proteins, an in vitro approach, limitations in regards to feasibility for all users, ability to detect the amount of active protein, and multiple other reasons. This is a minor weakness of the paper that can be easily addressed because it detracts from the novelty of the studies.

We feel that it is important to avoid an either-or scenario. We apologize for evoking a negative reaction with our statement, as we were only trying to emphasize how illuminating the magnetic-tweezers approach can be. It was not our intention to rule out the need for bulk methods at the bench top or using quench-flow or stopped-flow devices.

We have edited the text in l.83-87 to convey the following:

“Magnetic tweezers permit the dynamics of an elongating polymerase/polymerase complex to be monitored in real time and the impact of nucleotide analogues to be monitored in the presence of all four natural nucleotides in their physiological concentration ranges. Here, we present a magnetic tweezers assay to provide insights into the mechanism and efficacy of current and underexplored NAs on the coronavirus polymerase.”

Reviewer #2 (Public Review):

This study investigates the impact of remdesivir (RDV) and other nucleotide analogs (NAs), 3'-dATP, 3'-dUTP, 3'-dCTP, Sofosbuvir-TP, ddhCTP, and T-1106-TP, on RNA synthesis by the SARS-CoV-2 polymerase using magnetic tweezer. This technique allows to directly quantify termination of viral synthesis, pausing or stalling of the polymerase, thus, defining the effect of these NAs on viral synthesis. The work includes good quality data and nicely stablishes an assay to follow the activity of the SARS-CoV-2 RNA-dependent RNA polymerase.

The authors thank the Reviewer for her/his appreciation of our work!

However, the basis of the assay and theory was largely presented before by the authors in Ref 22 and 23 (and other references therein).

The main result here is that RDV incorporation does not prevent the complete viral RNA synthesis but causes an increase of pausing and back-tracking. This contrasts with a clear signature of synthesis termination induced by 3'-dATP. The work is complemented with the characterization of other NAs. Despite these results are of merit, I do not see this work to present a sufficient advance of our current knowledge.

We acknowledge Reviewer #2 opinion. However, we believe that our work is highly novel and important, as noted by Reviewer #1: “This [utilization of magnetic tweezers] provides a unique method to capture less common or difficult to observe phenomena such as backtracking. Most bulk ensemble assays would have difficulty detecting these phenomena.”

and Reviewer #3: “Overall, this manuscript constitutes a major advance in our understanding of chain termination in polymerases, and provides deep insights into the mechanism of action of remdesivir, which may contribute to further drug discovery efforts targeting this polymerase.”.

How these results translate into more physiological conditions at zero force should be addressed.

We show here that nucleotide analogs are incorporated via specific catalytic pathways (NAB, SNA, VSNA) depending on the nature of their modification (position and type in ribose, base). In the companion paper attached to this submission (https://doi.org/10.1101/2021.03.27.437309, currently in press), we show that the force has no effect on the probability to enter any catalytic pathways, and only affects the kinetics of a large conformational change occurring after chemistry. In conclusion, the force has no effect on nucleotide analog selection, as supported by our evaluation at both 25 and 35 pN. To clarify this, we have added in l.416-421:

“The present study demonstrates that nucleotide analog selection and incorporation is not force-dependent (Figure 2–figure supplement 3), which further validates the utilization of high-throughput magnetic tweezers to study nucleotide analog mechanism of action. This result is in agreement with our recent SARS-CoV-2 polymerase mechanochemistry paper, where we showed that entry probability in NAB, SNA and VSNA was not force dependent, and that force mainly affected the kinetics of a large conformational subsequent to chemistry, i.e. after nucleotide selection and incorporation.”

The rationale of testing other NAs apart from the mere systematic characterization of other compounds is unclear.

We have tested 3’-dATP, a well-known chain terminator, with Remdesivir, which was claimed to be a delayed chain terminator, as both are ATP analogue. We monitored the incorporation of Sofosbuvir, a well-known inhibitor of HCV replication, with its 3’-dNTP homologue, i.e. 3’-dUTP. T-1106-TP is a compound that was recently tested for coronavirus because it has a proven efficacy against influenza. ddhCTP is an endogenously produced nucleotide analog and chain terminator, and we compared it to its 3’-dNTP homologue, 3’-dCTP. Furthermore, each of these nucleotide analogs have modification at specific position, i.e. either at the ribose or at the base, which helps to understand how the polymerase responds to each modification. We have added this sentence in introduction in l.83-84 for clarity:

“We have therefore compared several analogs of the same natural nucleotide to determine how the nature of the modifications changes selection/mechanism of action.”

Similarly, I do not see the benefits of adding cell experiments with three compounds and experiments with the nsp14 mutant to address proofreading because they were inconclusive.

While we acknowledge Reviewer #2 opinion, Reviewer #3 has a different opinion and strongly appraises the importance of these results:

“Interestingly, the ddhCTP didn't actually work in infected cells. However, the authors presented a few theories on why it didn't work and said they plan to follow up to elucidate why it didn't work in cells. I think those results will be very interesting for the larger community working in this area.”

We share the opinion of Reviewer #3 and have therefore decided to keep these results in the revised manuscript.

Reviewer #3 (Public Review):

This manuscript focuses on understanding the mechanism of action of remdesivir in the inhibition of SARS-Cov2 polymerase, using single molecule methods. The findings are highly original, significant and surprising. The approach is highly robust and supported by a range of orthogonal studies. Overall, these findings should help those engaged directly in drug discovery by providing a critical foundational understanding for the action of remdesivir.

The research described in this manuscript has several findings that significantly impact the broader field polymerase inhibition. First, the authors were able to show using single molecule methods that remdesivir-TP incorporation leads to polymerase backtrack. This is important because the pause is long enough that an ensemble assay could mistake this backtrack for a termination event. Secondly, the researchers found the effective incorporation of remdesivir-TP was determined by its absolute concentration. This suggests remdesivir-TP and similar nucleotide analogs incorporate via the SNA or VSNA pathway and would be more likely to add to the RNA chain when substrate concentration is low (independent of stoichiometry with the competing native nucleotide). Thirdly, the researchers found the effective incorporation rate of obligatory terminators was affected by the stoichiometry of their competing native nucleotide rather than their absolute concentration. This suggests that obligatory terminators are incorporated via the NAB pathway. The pausing that the researchers observed in the polymerase elongation kinetics have recently been demonstrated by two other groups. However, this study improved upon the assay conditions used by other researchers to recapitulate in vivo conditions and remove bias from kinetics measurements.

The authors highlighted the issues with remdesivir, tested other nucleotide analogs, and proposed a better alternative based on their assays (ddhCTP). Interestingly, the ddhCTP didn't actually work in infected cells. However, the authors presented a few theories on why it didn't work and said they plan to follow up to elucidate why it didn't work in cells. I think those results will be very interesting for the larger community working in this area. It's clear that the authors made a substantial enough contribution on the mechanism of inhibition of SARS Cov2 polymerase to merit publication in eLife, independent of the work on the "improved" antiviral candidate.

It would have been useful to clarify for the reader the pharmaceutical import of the putative delayed chain termination (or pausing) relative to actual chemical chain termination. In other words, I'm assuming that in both cases the viral genome is considered to be non-transcribed (in that a chemical agent has been incorporated into the growing strand). This is true for most compounds in this broad class of anti-virals. The issues are usually surrounding the width of the therapeutic index and the degree to which resistant mutants arise.

Coronaviruses are unique among positive-strand RNA viruses in that they encode a proofreading exonuclease. Although it is unclear how the polymerase and exonuclease activities are coordinated, the current assumption is that errors are recognized when located at the terminus of nascent RNA. Therefore, nucleotide analogues which manifest their antiviral activity when embedded in nascent RNA should evade excision by the exonuclease.

We have added text conveying this sentiment here in l.70:

“The latter proofreads the terminus of the nascent RNA following synthesis by the polymerase and associated factors, a unique feature of coronaviruses relative to all other families of RNA viruses.”

And in lines 75-77:

“In other words, nsp14 adds another selection pressure on NAs: not only they must be efficiently incorporated by nsp12, they must also evade detection and excision by nsp14.”

Overall, this manuscript constitutes a major advance in our understanding of chain termination in polymerases, and provides deep insights into the mechanism of action of remdesivir, which may contribute to further drug discovery efforts targeting this polymerase. Additionally, the authors have highlighted and addressed issues in the methodologies of previous mechanistic studies that led others to erroneous conclusions.

We thank Reviewer #3 for her/his strong appraisal of our work.

Is our consciousness or our mind thinking in the language we know most or do we think of an idea or concepts and then attach words that we learn to that concept that may not originally have language?

This is exactly the thought process that led me to pursue a master's. I think as a country we need to come to an agreement on the purpose of our education system. This may sound impossible with all the different theories and politics involved, but we can at least begin defining education on the points we agree on as a collective.

I have certainly found these to be true in my classrooms. I am practicing a progressive approach in teaching now and include my students in my journey by explaining my reasoning. I think that one of the challenges in my doing so, is doing it alone in a school.

It's interesting to think about the connections between this mentality and the problems we're currently facing as a nation- what happened/is happening in Afghanistan, the way tax dollars are getting spent and the national debt, climate change, the erosion of democracy.

I think that this is correct i dont think completely removing plastic would not be good for our food environment but plastic is not good for our environment I feel as though we should be trying to make it better for everyone but also make sure plastic is only used in the right ways

I believe that Du Bois is speaking about the times of slavery, thus the usage of "bondage". However, this divine event that "they thought to see" and "end all of the doubt and disappointment", makes me imagine that this is emancipation, or even a divine miracle of freedom while enslaved.

I think it is important to discuss life stories because often we experience challenges or difficulties that we cannot process ourselves. Sometimes it is important to have an outside perspective, or simply a space to share your story. It's also important that we as a society learn from each other, and deeply consider how our lives differ to learn appreciation, develop understanding, and to grow.

I agree with this statement. I believe in order to grow and evolve as an individual a crucial step is being open to accepting setbacks or challenges. I think that the acceptance of these difficulties allows us to expand our way of thought and our knowledge. If we can then accept these challenges, we can then accept them as a part of who we are--a momentary obstacle in the past yet a life lesson that contributes to growth. It's truly important to be able to recognize all the different aspects of your life, and to recognize the different parts of your life that helped to shape you as an individual. I think dealing with adversity helps you to challenge your mind and yourself. Ultimately, it may even bring about new "traits, goals, and values".

Joint Public Review:

Strengths: The study represents a step forward in relating immune responses to infection outcomes that of urgent interest to public health, especially the timing of shedding and frequency of supershedding events. Nguyen et al.'s model provides a useful framework for understanding the links between immune effectors and infection outcomes, and it can be expanded to encompass further biological complexity. The study system is a good choice, given the ubiquity of both helminth and bacterial infections, and experimental infections of rabbits provide a useful point of comparison for past work in mice. 

Limitations: The present study does not explicitly account for differences in helminth infection dynamics across the two species represented in the data nor does it include feedbacks between the bacterial and helminth infections. Nguyen et a. therefore show the limits of what can be learned from focusing on the bacterial and immune dynamics alone, and this study should serve to motivate further work that can build on this modeling approach to produce a more comprehensive view of the interactions among species infecting the same host. Future studies examining the impact of helminth infection intensity would be tremendously useful for assessing the potential of anthelminthics to reduce the prevalence of bacterial respiratory diseases. Finally, subsequent studies may need to look beyond the factors examined here to understand why shedding varies so much through time for individual hosts. 

Specific comments: 

Definition of supershedding: A major stated goal of the MS is to investigate the effect of coinfection by helminths on supershedding. In order to compare animals with different coinfections, it is therefore necessary to have a common definition of supershedding. At present, the authors use a definition that depends on which arm of the experiment the animals belong to. This complicates the analysis and clouds its interpretation. 

Inconsistent approach: Within each experimental treatment, the data display variability on at least three levels: (i) within animals, day-to-day shedding displays variability on a fast timescale; (ii) within animals, infection status varies more slowly over the course of infection; (iii) between animals, there is variation in both (i) and (ii). The authors' model seems well-designed to handle this variability, but the authors are strangely inconsistent in their use of it. To be specific, to account for level (i), the authors very sensibly adopt a zero-inflated model for the shedding data, whereby the rate of shedding (colony-forming units per second, CFU/s) is assumed to arise from a mixture of a quantitative process (which we might think of as intensity of potential shedding) and an all-or-nothing process (which might arise, for example, if some discrete behavior of the animal is necessary for shedding to occur at all). The inclusion of the all-or-nothing process necessitates an additional parameter, but it allows the non-zero shedding data to inform the model. To account for level (ii), the authors use a four-dimensional deterministic dynamical system. Three of the four variables are related to the measured components of the immune response. The fourth is related to the aforementioned potential shedding. Level (iii) is accounted for using a hierarchical Bayesian approach, whereby the individual animals have parameters drawn from a common prior distribution. This approach seems very well designed to address the authors' questions using the data at hand. However, they fail to exploit this, in at least three ways. First, even though the model appears designed specifically to allow for non-shedding animals, the authors exclude animals on an ad hoc basis. Second, rather than display the shedding data in the form recommended by the model, they display log(1+CFU/sec), which is arbitrary and problematic. Its arbitrariness stems from the fact that this quantity is sensitive to the units used for shedding rate. Third, despite the fact that the model appears specifically designed to account for variability at each of the three levels, they do not give enough information to allow the reader to judge whether the model does in fact do a good job of partitioning this variability. 

Exclusion of animals: In view of the fact that the model the authors describe can account for variability on all three levels, it is strange that they exclude animals that shed too little or not at all. It would be preferable were the authors to base their conclusions on all the data they collected rather than on a subset chosen a posteriori. It is true that the non-shedders will have no information about the time-course of shedding; on the other hand, including them does not complicate the analysis, and it does allow for estimation of the all-or-nothing probability in a coherent fashion. In particular, the fact that coinfection appears to have an impact on whether animals shed at all is itself directly related to the authors' central questions. More generally, ad hoc exclusion of data raises concerns about the repeatability of the experiments that, in this case, appear entirely avoidable. 

Incomplete description of the analysis: The description of the statistical analysis will not be complete until sufficient information is provided to allow the interested reader to decide for him- or herself whether the conclusions are warranted and for the motivated reader to reproduce the analysis. In particular, it is necessary to specify all priors fully. At present, these are not described at all, except in vague, and even incoherent, ways. Also, it is necessary to provide details of the MCMC performed. Specifically, the authors should describe the MCMC sampler and show their MCMC convergence diagnostics. Finally, it is good practice to display both the priors and the posteriors: it is impossible to assess the posteriors without an understanding of the priors. 

Model adequacy: The authors' argument rests on the model's ability to adequately account for the data. The authors need to provide some evidence of this, in one form or another. Ultimately, the question is whether the data are a plausible realization of the model. The authors should show simulations from the model (including the measurement error and not merely the deterministic trajectories) and compare these simulations to the data. In particular, it seems worryingly possible that the fitted model is capable of capturing certain averages in the data while, at the same time, failing to describe the infection progression for any of the actual infected animals. 

Confusion of correlation and causation: At various points, the authors succumb to the temptation to interpret their model literally and to interpret the correlations they observe as evidence for a causal linkage between the three immune components they measure, bacterial shedding, and co-infection. They should be more careful and circumspect in the description of their results. 

Additional Issues: 

Eqs 1-4. These equations are not mechanistic in any meaningful sense. Essentially, they posit the existence of exponential time-lags between the three immunity variables, and a simple linear killing relationship between each of the variables and pathogen load. To interpret the equations literally risks making unwarranted conclusions. For example, any physiological variable correlated with any of the three variables in the model might equally well be credited with the influence on shedding attributed to IgA, IgG, or neutrophils. 

l 456. Do the authors account for the variability in time spent with plates? Implicitly, the assumption is made that the amount of time a rabbit spends with a plate, i.e., the decision as to whether to engage in a behavior that will terminate the plate interaction, is independent of everything else. This raises the question: Does the time spent per plate correlate with anything?

Reviewer #2 (Public Review): 

Masís-Obando and colleagues describe a study investigating the neural basis of specific (story) and general (schema) representations of naturalistic narratives (movie/audio clips). Narratives were of one of two types (airport, restaurant) about which participants would likely have rich past experience and knowledge, which allowed the researchers to ask what features were shared among different narratives that depicted the same "script." The researchers characterized the degree to which neural patterns reflected unique, story-specific codes (there is correspondence across people at the particular narrative level) versus general, schema codes (airport patterns are more similar to one another than they are to restaurant patterns). They were moreover interested in understanding how these representations were leveraged at both encoding and retrieval separately to guide free recall of each particular narrative's events. The main hypotheses were surrounding the involvement of medial prefrontal cortex (mPFC) and hippocampus (HPC) in this process. mPFC overall represented both schema at story at encoding, but neither at retrieval; a follow-up analysis revealed different effects in anterior versus posterior mPFC clusters, with anterior showing a greater relationship (than posterior) between schema representation at encoding and behavior that was mediated by specific story reinstatement in posterior medial cortex (PMC). Consistent with ideas about differences in representation across hippocampal long axis, they also found anterior HPC showed schema effects, whereas story effects (at encoding only) were more prominent in posterior. Beyond their a priori regions of interest, the researchers also report widespread cortical involvement for many of these analyses. The main take-home appeared to be that these networks differed between encoding and retrieval. 

Overall, the findings are compelling and align with prior work, while also providing new insights in the context of a more naturalistic memory task. For example, lack of mPFC involvement (schema or story representation) during retrieval was unexpected, and may inform future work on this topic (e.g., through encouraging more fine-grained consideration of mPFC sub-divisions). I moreover appreciated the author's transparency about their hypotheses and clear acknowledgement of the relationship between this data set and an existing paper. The work appears to be carefully done, and the paper is generally clear and well-written. I do however have a few questions and suggestions for the authors, as follows: 

1) From a theoretical perspective, I am struggling with the behavioral outcome measures being exclusively at the "specific" story level, and whether/how that should impact our interpretation of the findings. In other words, the behavioral outcome of interest has to do with participants' ability to recall story-specific details, and a score was given to each subject for each story to summarize the quality of their memory for that particular narrative. By necessity, of course, this means knowledge at the "schematic" level is not tested or operationalized in any way. (In fact, it would I believe be impossible to do this on a narrative-by-narrative basis.) The authors address this in their setup, discussing how a schema can be used to guide the retrieval of details, and also touch upon this in the Discussion (lines 404-410). However, I am struggling with the contrast between the memory ~ encoding and memory ~ reinstatement findings being whopping and widespread for the story neural representation (Figure 3A, C), and much smaller (and nonsignificant in many ROIs) for the schema neural representation (Figure 3B, D). Is this showing us that (detailed, specific) story representation supports recall of (detailed, specific) memories, and (general, abstracted) schema representation does not? Does that mean schema representation does not relate to memory, or just that it doesn't relate to *specific* memory (i.e., but could have in theory been related to schematic memory, had that been tested)? I suppose from some vantage points, it could be viewed as merely a replication of many other findings that representing specific memories at either encoding or retrieval is helpful for recall of those details. And similarly, one could argue that schema representations haven't been given a fair shake because the behavior was tested at a different level of specificity. In other words, in their analysis for Figure 3 B and D the authors separately considered the relationship between schema representation and behavior, without simultaneously considering the level of specific story representation, which is a bit hard to reconcile with the framework that schemas would guide retrieval via reinstatement of specific details (i.e., theoretically, should we expect that they can support detail recall on their own? or should it be that schema representation supports specific memory, but only when detail recall is also high?). With the exception of the mediation analysis in Figure 5 (which I think does speak to this point in a nice way), the earlier, primary analyses do not take this complexity into account. To be clear, I am not sure answering these questions requires new analyses, and am not asking the authors to change their approach. I am more hoping the authors could provide us more of their thoughts on these points in the paper and perhaps soften their conclusions if appropriate. 

2) It was not clear to me how the audio vs. movie difference was worked into the analysis, or why for the schema scores, different-modality patterns were not also considered. It would seem as though comparing patterns derived from the presentation of movie vs. audio as part of the schema measure would allow the researchers to get around potential confounds like visual presentation of the same type of stimuli across narratives of the same type to drive the "schema" representation (e.g., restaurant movies presumably show a lot of the same types of objects as one another, but those same objects would not be presented visually in the audio clips). Similarly, perhaps audio clips contained similar words for a given schema. It seems as though airport 1 movie being more similar to airport 1-4 audio than it is to restaurant 1-4 audio (all different modality comparisons) would be a powerful way to demonstrate schema representation (I believe the authors have done this in past work; Baldassano et al. 2018 J Neuro). In any case, I think this detail and reasoning should be added to the main paper, and potentially worked into the visualizations. 

3) It was unclear to me from the methods how the models relating neural scores with behavioral performance were set up. It sounds as though perhaps the researchers ran a simple linear regression, such that all participants' data was combined into a single model but subjects were not treated as random effects. If this were the case, then variability in memory performance across subjects is going to contribute to the estimate of the within-subject relationship between neural scores and memory performance on a story-by-story basis. It seems from the paper as though the authors are more interested in the within-subject variability. Can the authors clarify this point (e.g., by expanding the methods section beginning on line 585)?

Perhaps cognitive science failed to move from a "collection of multidisciplinary efforts to an integrated coherent interdisciplinary field" because there was not a high demand of this interdisciplinary field. However, there may be a higher demand to study cognitive science because the public, media, and scientists have expressed greater interest in developing artificial intelligence over the years. Similarly, younger generations like myself are becoming more self-aware, constantly asking the question, "are we just living in a simulation? Is anything truly real, or real based on our perceptions?" I think the training in cognitive science will become necessary for the new generation of scientists due to the increased demand of developing AI's as well as studying the human consciousness (i.e. our extent of free will).

I'm really struggling with this because this year we are offering specials. We have to share the specials teachers with the entire school, so our times are dictated to us. Today, we had to interrupt the children's free play time to do music...which was really just watching and dancing to videos on the smart board. It's only week two, but this is high on my think-about list.

The closing paragraph restates everything we have read in the article. The main point is to discuss the forms of ethics as well as cause readers to think and evaluate strategies of certain things.

Author Response:

Reviewer #1 (Public Review):

[...] The study tackles important questions with regards to how ripples relate to broadband LFP activity as well as single neurons in the human brain. The authors also include elegant analyses to characterize the timing of spiking activity with respect to high and low frequency activity and relevant control analyses to take into account possible artifacts and epileptic-related activity. My main concern with the study is whether the authors are truly isolating ripple activity in the human brain as claimed. Their threshold for ripple activity is quite low and it thus seems very possible that many of their "ripple" events are rather high frequency activity events that reflect spiking activity. That being said I think this is an important study to share results from in that it provides unique characterization of the relationship between high frequency activity and spiking in the human brain, as well as how it relates to human memory.

We thank the reviewer for the positive assessment of our manuscript. We agree with the reviewer that there are important concerns regarding whether these events can be truly regarded as ripples, which we address by performing additional analyses to provide stronger support to the possibility that the identified cortical ripples in our recordings are transient and discrete events that reflect underlying bursts of spiking activity. We also acknowledge that these events more likely exist on a continuum, and that there is not always a clear separation between what constitutes one ripple event and the background activity. We think this is a fertile area of investigation and that there are several points to consider regarding this question, which we now address in the revised Discussion.

Reviewer #2 (Public Review):

Recordings from human patients with implanted electrodes provide high temporal resolution, localized measurements of brain activity that can reveal neural correlates underlying a wide variety of cognitive functions. These intracranial electroencephalography (iEEG) recordings are typically made with large electrical contacts, however, and thus represent a complex and poorly understood averaging of voltages from the underlying tissue. As such, it is difficult to know exactly what patterns of neural activity these signals correspond to and how to compare them to spiking and local field potential (LFP) recordings more commonly acquired in non-human animals. Tong et al. carried out simultaneous iEEG recordings from surface contacts as well as spiking and LFP recordings from implanted electrode arrays to directly address the relationship between these signals.

They present quantifications (e.g. Figure 2B) of the relationships between the amount of spiking activity and the amplitude of events detected in the LFP and iEEG. Their results showing the relationships among these signals are very important for the field, and it is very helpful to see that there are clear correlations across scales.

We thank the reviewer for this positive review of our manuscript.

The context in which they present these results is problematic, however. They focus on "ripple" events, detected as periods where the power in a 80-120 Hz band exceeds an arbitrary threshold for an arbitrary length of time. To be fair, the application of similarly arbitrary thresholds is common in the human, primate, and rodent literatures, and several important results have arisen from the analysis of these events. These results can be understood as claiming that a set of high amplitude events have certain properties (e.g. they are related to memory retrieval), but should not be understood as establishing that there is some specific threshold that separates real events from others.

We completely agree with the reviewer that, when considering ripples, there is a continuity of activity and that the central challenge for the field has been how to identify real events and separate them from others. To be clear, the purpose of our manuscript is not to claim that such a threshold exists. Our purpose instead was to offer evidence that even events that fall below arbitrarily defined thresholds still reflect underlying bursts of spiking activity, that these events are still punctate and temporally discrete, and that therefore these events are also likely functionally meaningful.

Here they go beyond these analyses and make the claim that these ripple events correspond to real, discrete events that, as their title indicates, "reflect a spectrum of synchronous spiking activity." The problem here is that they do not present any criteria for defining a real, discrete event. Indeed, they conclude that "the continuum of activity that [they] observe in [the] data ... suggests that strictly adhering to predefined criteria for what constitutes a ripple may run the risk of overlooking functionally meaningful events". Without a clear definition of what should and should not be considered to be a discrete event, we are left with the current situation where each study uses their own set of criteria, picks out a set of high amplitude events, and uses those for subsequent analyses.

We agree with the reviewer that the amplitude and strength of the identified ripple events can be quite variable, making it challenging to distinguish these events from baseline activity. We therefore do not claim to identify specific criteria for defining real events. As the reviewer notes, without such criteria we are left with the current situation where future studies will still need to use their own set of criteria. We completely agree. The purpose of our study, however, is to just highlight the point that using these arbitrarily defined criteria risks overlooking these other events that still may be meaningful for the brain. We have addressed this concern by adding several changes to our manuscript and introducing several new analyses. First, we have tempered our claims that these are discrete events that represent separate packets of information. We acknowledge that there is certainly some variability in the size of these ripple events, that making a clean distinction between when these ripple events emerge as entities that are distinct from the background activity is challenging, and that we can never be certain whether arbitrarily small events are functionally meaningful. We have revised our Discussion accordingly to highlight these possibilities, and to discuss the larger point regarding the challenges in identifying these specific thresholds. Second, although we recognize that the data may not be absolutely conclusive, we have supplemented this discussion with additional analyses that, in our opinion, strongly suggest that these events are indeed transient in nature even when failing to meet previous thresholds.

A second major challenge to understanding the current manuscript is the ambiguity of the physical relationships between the LFP and iEEG recording sites. While it might be obvious to human physiologists, details such as the distance between the LFP and iEEG contacts and the site areas of each type of electrode are critical for interpreting how closely the data from each could be expected to be related.

We also agree with this very good point. As noted above in the response, we have now introduced several new analyses that examine the relation between the ripples identified using LFP and iEEG recordings.

We would also like to highlight an instance of a common statistical error in Fig 1 I: the authors conclude that the difference between correct and incorrect is significant in true data and insignificant in the ripple-removed data, and therefore the 70-200Hz power band modulation on correct trials is significantly informed by 80-120Hz ripple events. The statistical problem is further described in Nieuwenhuis et al., Nature Neuroscience 2011.

We thank the reviewer for pointing out this common statistical error that we have made in concluding that the true and ripple-removed data differ because the difference between correct and incorrect is significant in the true data and not in the ripple-removed data. We agree with the suggestion that a more accurate way to compare the true and ripple-removed data is to compare the effect sizes, or the difference between correct and incorrect trials for the true and ripple-removed data. We have now conducted this analysis. Specifically, we computed the true correlation between the difference in 70-200 Hz power between correct and incorrect trials and the difference in ripple rates between correct and incorrect trials across electrodes and compared this correlation to the correlation present after removing the 80-120 Hz ripples using a paired t-test across participants. We performed this analysis in the subset of six participants who had an MEA and focused on the MTL and ATL electrodes since these regions have the greatest 70-200 Hz power increase with successful retrieval. We found a significant decrease in correlation across patients with ripples removed compared to when we retained the ripples (t(5) = 3.89, p = 0.0115). We now report this new analysis in Fig. 1 – S6. We also compared the two correlations as dependent groups and found a significant difference in correlation (r_true – r_control = 0.172, 95% CI = [0.0691 0.2764], z = 3.2677, p = 0.0011). We accounted for potential interaction effects using the correlation between 70-200 Hz and 70-200 Hz with ripple removed (r = -0.031).

Finally, we would also like to note the difficulty of characterizing a single deflection in the LFP or iEEG signal as a low frequency oscillation, given the large potential for measurement variability of the frequency of that oscillation as described in Fig. 4. This large deflection is to be expected when a concentrated amount of synaptic input drives a burst of spiking, as we would expect in the case of the increased spiking during ripples. In the hippocampus, this deflection is the sharp wave component of the sharp-wave ripple; it appears to take a similar form in cortical ripples. While unsurprising, it is well worth observing that the iEEG reflects this coincident deflection, but it should not be characterized as a 2-10Hz oscillation.

We completely agree with the reviewer about this point. As the reviewer points out, the large deflection is often observed with bursts of spiking, which we find with ripples, and we also feel that the iEEG reflects this coincident detection. We have therefore corrected the text and no longer characterize the deflection associated with ripples as 2-10 Hz oscillations. To illustrate this point further, we have also now added a new analysis demonstrating the average iEEG and LFP activity around each ripple, which clearly demonstrates this deflection (Fig. 1). We have, however, retained the discussion about the locking of spikes to 2-10 Hz oscillation, as our analysis includes spikes both within and outside of the spike bursts. While we expect that spikes are associated with the large deflection that reflects a concentrated amount of synaptic input, we also find spikes that are modulated by a 2-10 Hz oscillation, consistent with prior findings of theta-phase locking of spiking neurons.

Reviewer #3 (Public Review):

In this study, authors systematically investigated the iEEG ripples, LFP ripples, and their relation with each other and single units from micro channels obtaining LFP. They found that the amplitude of LFP ripples reflects the sum and alignment of underlying spiking activities. Meanwhile, the amplitude of iEEG ripples reflects the number and alignment of LFP ripples. More interestingly, the amplitude of ripple events is functionally relevant. In general, I find that the data analyses and methods are sophisticated and the results are interesting. It extends our understanding of ripple events and is of interest to a wide audience.

We thank the reviewer for the positive assessment of our manuscript.

Author Response:

Reviewer #1 (Public Review):

[...] In the model, the mitigation function is fitted; no actual data on deliberate versus randomly-varying behavior change is used. Given clear empirical signals of synchronous and delibate response to epidemiology, modulated by social factors (Weill et al., 2020), a persuasive demonstration that consideration of random behavioral variation is necessary and/or sufficient to explain observed US COVID-19 dynamics would need to start from mobility data itself, and then find some principled way of partitioning changes in mobility into those attributable to random variation versus deliberate (whether top-down or bottom-up) action.

As suggested by our referees and the editor, we undertook a principled analysis of the US COVID-19 data that took into account Google mobility patterns. The average mobility reflects systematic changes in social activity due to both government-imposed mitigations and knowledge-based adaptation of the population. We identified a range of dates (July 2020- February 2021) during which there has been only modest and slow changes in the average mobility. This time range allows for a direct test of our model, accounting for stochastic changes in social activity uncorrelated across the population (see Figure 6 and Appendix 5. Figure 1A).

In the new version, we also present a direct comparison of the predictive power of our SSA model vs the traditional SIR model within this time range (see Figure 8 and Appendix 5. Figure 2).

My other main concern is that the central result of transient epidemiological dynamics due to transient concordance of abnormally high versus low social activity-stems from the choice to model social behavior as stochastic but also mean-seeking. While I find this idealization plausible, I think it would be good to motivate it more.

In other words, the central, compelling message of the paper is that if collective activity levels sometimes spike and crash, but ultimately regress to the mean, so will transmission. The more that behavioral model can be motivated, the more compelling the paper will be.

We included an additional justification of our form of stochastic social dynamics and expanded the discussion of relevant prior studies. Especially revealing are the studies of burstiness in virtual communication such as e-mail (Vazquez et al. (2007); Karsai et al. (2012)). Studies of digital communications can be easily studied over a substantial time interval, which is more problematic for field studies of face-to-face contact networks. These studies unequivocally show the regression of individual activity levels towards its long-term mean value. This regression happens over a well-defined relaxation time ranging from days to months depending on the context. Note that the value towards which the activity regresses may not be identical for different individuals. In the context of our model, such persistent heterogeneity is captured by the distribution of \alpha_i with the dispersion parameter \kappa.

Author Response:

Reviewer #1:

The manuscript is well-written and easy to follow. The authors are thorough in their characterization, shown both through the text itself and the figures. Most of the comments relate to the narrative structure itself and are merely suggestions. Overall, this work represents an important resource for the community and especially to people working on the role of the SEZ in feeding and motor behaviors.

Specific comments and suggestions:

• The authors give a very nice overview of the SEZ and the split-Gal4 technique. However, they spend much less time discussing the rationale behind using the cell body numbers within subesophageal neuromeres. This to me assumes two extremely different kinds of readers, one relatively new to Drosophila research and the other relatively well-versed. Since this technique is crucial to the approach used throughout the manuscript and significant in the authors labeling about 1/3 of the region, I would suggest the authors to give a brief summary and justification as to why they decided to use this neuromere labeling technique, and spend more time in the discussion (perhaps in the paragraphs between lines 352-386) talking about the pros and cons of this technique (is it expected to label fewer than 50% of the neurons? How may this complement the EM and FAFB dataset, and what are the advantages and disadvantages using the technique employed here?).

We now provide a brief introduction to the approach in the results section (lines 82-96) and include additional pros and cons of the approach in the discussion (lines 369-383). We expect that this approach labels the vast majority of SEZ neurons.

Related suggestions:

o Line 81: elaborate on deutocerebral contributions

We have moved this to discussion (lines 374-377). We clarify that not much is known about deutocerebral contributions.

o Lines 84-85: along similar lines, Hox gene drivers

We altered this sentence to be clear to a general audience (lines 86-90).

• Figure 9: having a color legend in the figure itself will facilitate understanding of this figure. I think it would be nice to have visual examples of interneurons, projection neurons, and so forth. Perhaps when the authors first describe neurons in Group 1, instead of marking "first half of the group" (line 210) the authors can explicitly name the neuron types (peep, doublescoop, etc.)

We now include a color legend as well as a new figure with visual examples of polarity (Figure 10 – figure supplement 1). As suggested, we changed the text to explicitly name the neuron types in Group 1 that are interneurons versus projection neurons (lines 241-243).

• In the polarity section of the discussion, it would be interesting to have additional remarks relating to how to determine whether these identified neurons are thought to be ascending and why. Since one of the authors has previously characterized some ANs, perhaps comparisons to this work would be helpful to readers new to this region of the brain.

We now include a brief definition of ascending neurons in the results section (lines 149-150) and note that ascending neurons were not included in the collection.

• The parallel structures used in characterizing Groups 1 through 6 are very useful. However, I think that when the authors relate each group to previous works, this might fit better in the Discussion section.

We altered this section of the results to move speculation about group function to the Discussion (lines 421-445), as recommended.

I'm not the only one who's thought this. Pinterest, Facebook, twitter, (and other social media and bookmarking software) can be considered a form of commonplace.

This reminds me of Aaron Swartz. I feel like this sentence pretty much describes his life mission. The sharing and passing down of knowledge and information is all we have to offer for the future!

Do machines have the ability to doubt logic? Do they have the ability to debate philosophical dilemmas? Do they have the ability to form opinions?

I'm not to sure. You can fake phone calls but the number of calls can be measured.

Thankfully, it is much easier now to document and share information! Like this sentence is taking me very little time yet I am sharing my thoughts with many people!

This is a great point, and it's very though-provoking. What is bound to come of it? It's interesting too that even though science keeps evolving, the cost of living keeps rising. I'd be interested in discussing the relationship between scientific evolution and inflation.

History is only relevant of who's writing it. It's true in a lot of ways which I don't like but understand. Connecting documents with people when they need it is still a huge challenge but could and can be manipulated.

With the peak of growth we should go full force towards the developments of inventions and knowledge of men so we can adequately fulfill the desires of men.

An widely accepted Encyclopedia that is controlled by the people (Similar to Wikipedia) towards the development of educational fields of research would be mans greatest wealth of knowledge.

The emergence of internet 2.0

If a manipulation was present in the self learning capabilities of modern artificial intelligence, it can lead to an exponential crisis of malfunction or artificial error.

That's the question which pegs the world to this day. science I believe can be a instrument of making life easier whether or not it can be used for a good thing or a bad thing.

If we could limit the use of our intelligence for repetitive task and focused on the task that our current machines cannot comprehend it would lead to a more efficient growth in knowledge.

This small population is credited to the expansion and development of technology that the average person uses in day to day life.

the amount of accuracy of current technology is almost sci-fi to me. Imagine if these doubled!

I see the use of this in many popular songs

The common accessibility to High Definition microphotography in institutions will greatly benefit science due to the fact that people who don't have the physical subject or a device to zoom and view the subject, can still learn in detail.

I wonder if the Laser Printer uses a similar process to print papers without the use of ink?

Most likely the reason why Digital Cameras are so popular today.

This is a true statement. Scientist should look though a ethical lens always but needs logical step process. Same thing can be said for people using technology.

The science field is not stagnant. After one study , hypothesis or even conclusion, the expansion of the subject will always grow.

Modern Vehicles, Computers, Phones and other devices all contain interchangeable parts. Each part has its own specific purpose and often each part is specifically made for that specific device. Despite this, often it's possible to repair the parts and have a reliable device or invention.

With the cooperation of the thinking man and the accumulation of mans knowledge (inventions) the opportunity to learn and discover is almost exponential.

With deferring information from multiple studies and conclusions from different investigators , how do come to a widely accepted statement/answer to this investigation?

Millions and millions of dollars towards the development of destructive weapons lead by some of the most educated individuals but why isn't there a focus on the development of non lethal weapons, and the study of obtaining and maintaining peace in different cultures.

This is most likely inferring that there needs to be a greater focus on the thinking man, then rather the people relying the accumulation of knowledge throughout human history.

I agree that in fact, inventions have improved the physical powers of man. The other effect of inventions is that Man isn't using their mind to their full potential of the mind.

I agree with this statement. Relief shouldn't be a concept of being "secure" because that can be controlled to a point where manipulation can be issued. I believe technology should be free to transform or upgrade instead of being manipulated by others.

This statement remains content though out human history and the same can be said for technology. We humans went though trail and error though out history to see what works and what doesn't work. Technology is the same with numbers, programs and sites too. It's progress.

tabs or read it later sometimes really make me anxious.I think we just need to take it easy.

I like that this sentence suggests we cannot predict the future. I like how it hones in on the unpredictable and the reality that no matter how much control we have in our lives, each moment is different and unreliable in the sense that things don't always work out as planned. It's comforting to think that our stories are literally made up of what "we can make at the time." It's not all structured and concise and clear. It actually is muddy and messy and unexpected.

In an earlier sentence, the author writes, "The process of invention may occur...at some subterranean level." Subterranean means "concealed." This sentences intertwine because at the moment plays out, what will happen is subterranean. We simply cannot predict how we are changed or how we live in the moment.

Task 3: Be able to understand the text and be able to put it into your own words.

1.) What are the central arguments or main point in the text? The text supports its main idea, that philosophy needs to be studied to be cleared of misunderstandings, by pointing out the effects philosophy has on the human mind, and, therefore, why it is of use to humanity. Russell makes the argument that even though philosophy has no way to be verified like the other sciences, it prods the human mind into thinking more broadly and creatively by forcing people to ask perplexing or uncomfortable questions. Another point which Russell makes is that philosophy gives the mind potential to think so impersonally as to view things in a nearly god-like way.

2.) How are arguments or main points supported? That is, what reasons are given to support the conclusions? Russell supports his arguments with broad assertions concerning the machinations of the mind and people's attitudes toward philosophy. He writes in such a way as to parrot what is heard in real life concerning attitudes toward philosophy, most noticeable when he talks about "practical people."

3.) Note any assumptions the author gives without giving evidence. Strangely enough, Russell backs not one section of his argument with data of any kind. His assertions are made merely by what he has been met with in his experiences. There are no polls or graphs about people's attitudes toward philosophy. There are no quotes about what people think of philosophy. There is nothing which qualifies or quantifies besides what Russell himself asserts. Even while this is so, the subject Russell is commenting upon is, in and of itself, abstract. What Russell seems to be demanding of his readers, on a subliminal level, is use of common sense and critical faculties to piece together his information in a way we can except. Referring back to my second point about what Russell is arguing in his text, the idea that humanity can achieve a worldview in proximity to that of God (which, from what has been presented thus far to this class about Russell, I can only assume he means to use God as an allegory for some universal principle) is quite a leap to make. Russell assumes that if humanity abandons its territorial, material concerns that we may ascend to a point of knowledge of ourselves and the universe that will bind us to the latter like never before.

4.) Reread important passages and note sections of the text you cannot make sense of. I cannot really find anything in the text that I cannot make sense of, except in the meaning of the not-Self. I understand it well enough to see what Russell means by it, but I am left with ideas about its implications (for lack of a better word). However, that is not altogether what is meant by the question.

Author Response:

Reviewer #2:

This study by Schulz et al flushes out in fine detail an interesting consequences of inhibitory synaptic plasticity in plastic neuronal networks, showing that its ability to balance precisely only previously experienced stimuli makes this type pf plasticity an excellent candidate mechanism to allow novelty detection. Strong transient responses will be evoked only by those stimuli which have not previously activated (and thus trained) the stimulus specific set of inhibitory stimuli. An open question remains with regard to the time scales and speed of learning at these inhibitory sites, something that will be answerable by the experimental audience of this paper (but could be investigated in a bit more detail in the model as well).

We thank the reviewer for their constructive feedback. We have addressed the comments below, and included new substantial information to our manuscript. This includes a new Supplementary Figure (Figure 4-Figure Supplement 2) in which we analyze how the novelty response and the adapted responses depend on the inhibitory learning rate and a detailed discussion about experimental and theoretical studies related to inhibitory plasticity.

Reviewer #3:

This computational paper addresses the mechanisms of sensory adaptation and novelty detection in the auditory cortex. A spiking RNN model of 5000 (4000 excitatory/1000 inhibitory) units is developed and adapted to sequences of inputs (ABC…) followed by a novel stimulus N. As with experiments the model captures the adaptation to the repeated stimuli, as well as a strong response to a novel stimulus. In contrast to many models of sensory adaptation that rely on short-term synaptic plasticity, here adaptation arises from STDP at the Inh->Ex connection. Specifically, during ABCABC … the inhibitory connection onto active Ex units is enhanced through associative plasticity mechanisms, but not onto the inactive Ex units, thus the adaptation does not apply to the novel stimuli. While the approach seems fairly novel it is also speculative and seems to run contrary to the existing experimental data.

We thank the reviewer for their feedback. In our view, through the process of answering the comments below, we have improved our manuscript substantially. To answer the reviewer’s comments, we added new figures to the manuscript (Figure 5) and (Figure 6-Figure Supplement 1 and Figure 1-Figure Supplement 1,5) and added new text in the Discussion (see ‘Timescales of plasticity mechanisms’, and ‘Robustness of the model’). In the following, we give detailed answers to each of the comments.

1) The reason many models of adaptation focus on short-term synaptic plasticity (STP) as opposed to STDP is that the later generally is not thought to operate on the adaptation time scale of seconds. Specifically, STDP is generally considered to be a form of long-term associative plasticity, and thus to rely on mechanisms such as the insertion of new receptors-a processes that is unlikely to operate on a time scale of a second or so. Adaptation is robustly observed at 400 ms (e.g., Natan et al 2017), a time scale that is generally considered to be incompatible with STDP. E.g. in the D'Amour paper the authors cite, STDP is induced over a 5 minute pairing protocol, and can still increase over the course of 5-10 minutes post pairing (e.g., Fig 1H). I'm not aware of any evidence suggesting that iSTDP could be induced and expressed on the subsecond to a few seconds time scales. So this seems to be a fundamental issue that needs to addressed or at least discussed.

Related to the point above the model also contains subtractive normalization implemented with a time step of 20 ms. Again if this normalization is critical to the model this assumption would pose a serious challenge to the model because there is little or no experimental data suggesting that normalization can operate at that time scale.

In our new Figure 1-Figure Supplement 5 we find that the timescale of the subtractive normal- ization mechanism does not influence the generation of novelty responses in our model. Adaptation occurs over multiple timescales from hundreds of milliseconds to tens of seconds or even days [Ulanovsky et al., 2004, Lundstrom et al., 2010, Homann et al., 2017, Latimer et al., 2019, Haak et al., 2014, Ramaswami, 2014]. Our work shows that inhibitory plasticity can readily lead to adaptation on multiple timescales without the need for any additional assumptions. Although during the induction of inhibitory STDP it takes several minutes to sev- eral tens of minutes to achieve a stable baseline of inhibitory synaptic strength [D’amour and Froemke, 2015, Field et al., 2020], inhibitory postsynaptic currents increase significantly immediately after the induction of plasticity (see e.g. [D’amour and Froemke, 2015, Field et al., 2020]). Therefore, inhibitory synaptic strength seems to already change during the plasticity induction protocol. Hence, we propose that inhibitory STDP is a suitable, though clearly not the only, candidate to explain the generation of novelty responses and adaptive phenomena occurring over multiple timescales.

Justification:

The points raised by the reviewer are extremely valuable in critically evaluating the model. We first address the issue of the timescale of normalization.

The generation of adapted and novelty responses in our model does not rely on the fast subtractive normal- ization mechanism, since the normalization only affects the excitatory and not the inhibitory weights; and it is the change in inhibitory synaptic weights through iSTDP that is the key mechanism to explain adapted and novelty responses. In Figure 1-Figure Supplement 5 we now show that even for a normalization time step of 50 seconds, adaptation to repeated stimuli and a novelty response occur. Therefore, we can conclude that the fast normalization mechanism is not a necessary ingredient in our model. We added a discussion at line 542 and in the Methods at line 749. Even if no normalization is applied throughout the entire stimulation paradigm, our findings do not change.

We note that while normalization is common practice in circuit models, there is a discrepancy between the fast timescales of normalization mechanisms used in computational models to stabilize network dynamics and the much slower timescales measured experimentally ([Fox and Stryker, 2017, Turrigiano, 2017, Keck et al., 2017], among others). This discrepancy has been termed the ‘temporal paradox’ [Zenke and Gerstner, 2017, Zenke et al., 2017].

Many computational models which implement normalization mechanisms justify them by the experimentally observed synaptic scaling despite the discrepancy between timescales (see e.g. Figure 1 in [Zenke et al., 2017]), which we now acknowledge at line 543. In related work, we propose a different biologically plausible candidate for fast homeostatic stabilization – heterosynaptic plasticity – which operates on similar timescales as homosy- naptic plasticity mechanisms [Field et al., 2020]. Incorporating this mechanism in addition to, or instead of, the fast normalization in recurrent networks is very interesting but beyond the scope of this work.

Next, we address the issue of the timescale of iSTDP. We studied long-term iSTDP as a candidate mech- anism for the generation of adapted and novel responses for multiple reasons: (1) to explain adaptation over multiple timescales which range from hundreds of milliseconds to tens of seconds [Ulanovsky et al., 2004, Lundstrom et al., 2010, Homann et al., 2017, Latimer et al., 2019], and even multiple days in the case of habitu- ation [Haak et al., 2014, Ramaswami, 2014]. Rather than including STP mechanisms that operate over all those different timescales, we demonstrate that iSTDP is a straightforward mechanism to bridge different timescales without the need of multiple mechanisms or fine-tuning of parameters (Figure 4). (2) We were inspired by the growing experimental literature suggesting an important role of inhibition and inhibitory plasticity in adaptive phenomena (see Discussion subsection “Inhibitory plasticity as an adaptive mechanism”). (3) In computational models, iSTDP is usually studied in the context of balancing excitation [Sprekeler, 2017]. In our study, we present functional consequences of inhibitory plasticity.

Although inhibitory plasticity is indeed induced over several minutes in pairing experiments [Field et al., 2020, D’amour and Froemke, 2015], inhibitory postsynaptic currents are already increased directly after plasticity in- duction – though it takes additionally several minutes to reach a stable new baseline (e.g. Figure 2A,B in [Field et al., 2020]). For example, the mean increase of inhibitory synaptic strength right after plasticity in- duction in [Field et al., 2020] is approximately 30-50%, while a new stable baseline at about 80-100% increase is reached after approximately 20 minutes (Figure 2A in [Field et al., 2020]; similar results in Figure 1H,I in [D’amour and Froemke, 2015]). This suggests that significant changes of inhibitory synaptic strength in iSTDP experiments already occur while the plasticity induction protocol is still ongoing. How fast these plasticity mech- anisms act in an in vivo setting during stimulation with naturalistic stimuli is to our knowledge not known. In general, the question of the true timescale of iSTDP is still an open problem [Sprekeler, 2017].

We also point out that the inhibitory synaptic weight changes induced via iSTDP are rather small in our model, i.e. Figure 4C shows that the mean inhibitory synaptic weights onto the adapted excitatory population increase approximately 15-20%. Therefore, we propose that relatively small inhibitory weight changes are suffi- cient for the occurrence of a novelty response and these weight changes might already be happening during the paring protocol in experiments, as we argue above.

Although we agree with the reviewer that short-term plasticity mechanisms are an important aspect to understand adaptation phenomena (especially on short timescales), we would not a priori exclude iSTDP only based on the argument of timescales. To get a full understanding of adaptive phenomena on all timescales, more detailed experimental and theoretical studies are needed to investigate the role of short versus long-term plasticity of excitatory and inhibitory synapses and how these mechanisms interact in a recurrent circuit.

Modifications:

In the new Figure 1-Figure Supplement 5 we study the effect of the timescale of subtractive normalization on the occurrence of a novelty response. In Figure 4-Figure Supplement 2 we quantify the response amplitude and the decay time constant of the novelty response as a function of the learning rate of inhibitory plasticity (see also our response to comment 1 of reviewer 2). Indeed, we find that fast inhibitory plasticity is needed to detect a novelty response. We discuss the mismatch between timescales of homeostatic plasticity in theoretical and experimental studies in lines 543 and 739. Additionally, we added new text in the Discussion subsections ‘Timescales of plasticity mechanism’ (line 509) and ‘Robustness of the model’ (line 533) where we discuss the timescale of inhibitory plasticity and subtractive normalization.

A further issue relates to the temporal structure of adaptation. The authors show that adaption is independent of the sequence of the stimuli (ABCABC vs BACCBA) (it would be best to refer to this as sequential structure not temporal structure, which would often include the duration of stimuli and interval between stimuli). It is well established that the longer the interstimulus interval the less the adaptation. The model may or may not capture this effect depending on the assumptions regarding the spontaneous activity during the ISI as a result of the non-associative (pre-only) iLTD. However, given that STDP generally grows after induction it seems like the model is not likely to capture the standard observation that adapation should be less if the stimuli are presented with an ISI of 800 ms versus 400 ms. In figure 5, for example what happens if stimuli are presented for 20 seconds consecutively versus for 10 second then a silence of 2 seconds before another 10 second stimulation? No mention of the time course of spontaneous recovery from adaptation is made.

Recovery from adaptation depends on the background activity level in the network during the inter- stimulus interval (Figure 5 and Figure 6-Figure Supplement 1). Specifically, low background activity between stimulus presentations slows the recovery from adaptation (Figure 6-Figure Supplement 1). However, increasing background activity between stimulus presentations can capture the decreased adaptation as the inter-stimulus interval increases (we show this in our new Figure 5).

Justification:

We agree with the reviewer that the term ‘temporal structure’ is misleading, and therefore exchanged it with the term ‘sequence structure’ in our manuscript (see for e.g. line 228).

As the reviewer aptly predicted, the recovery of the response from adaptation indeed depends on the level of background activity between two stimulus presentations. In our model, the direction of inhibitory weight change (iLTD or iLTP) depends on the firing rate of the postsynaptic excitatory cells (see [Vogels et al., 2011]). Postsynaptic firing rates above a ‘target firing rate’ will on average lead to iLTP, while postsynaptic firing rates above the target firing rate will lead to iLTD. In turn, the average magnitude of inhibitory weight change depends on the firing rate of the presynaptic inhibitory neurons (see [Vogels et al., 2011]). Therefore, if the background activity between two stimulus presentation in our model is very low, recovery from adaptation only happens on a very slow timescale. To show this, we performed simulations similar to Figure 6 where a stimulus (A) was presented again after a pause of either 9 seconds (Figure 6-Figure Supplement 1A) or after 225 seconds (Figure 6-Figure Supplement 1B). Whereas the response to the stimulus was still adapted after 9 seconds, it fully recovers after more than 200 seconds. As expected, the stimulus-specific inhibitory weights decreased very slowly after stimulus presentation (Figure 6-Figure Supplement 1A, B; bottom). This slow decrease of inhibitory weights follows from the fact that the network is silent if no stimulus is being presented. We now discuss this result in line 357.

However, if the background activity in the inter-stimulus interval is higher (either because of a higher back- ground firing rate or because of evoked activity from other sources, for example other stimuli), the adapted stimulus can recover faster. To address how such elevated background activity can affect adaptation to a specific stimulus, we performed additional simulations (Figure 5), in which we used the experimental paradigm from Figure 1A. Similar to Figure 2C, we changed the number of stimuli in the sequence, which leads to different inter-repetition intervals (the interval until the same stimulus is presented again) of a repeated sequence stimu- lus. For example, if two repeated stimuli (A, B) are presented, the inter-repetition interval for each stimulus is 300 ms apart because each stimulus is presented for 300 ms. If four repeated stimuli are presented (A, B, C, D), the inter-repetition interval for each stimulus is 900 ms. Importantly, this means that in the time between the presentation of the same stimulus, the network is not silent (as in Figure 6-Figure Supplement 1), but active because other stimuli in the sequence are presented. We defined the adaptation level as the difference of the onset population rate, measured at the onset of the stimulation, and the baseline rate, measured shortly before the presentation of a novel stimulus. We found that an increase in the inter-repetition interval reduced the adaptation level of the excitatory population (Figure 5A, D) due to a decrease of inhibitory synaptic strength onto stimulus-specific assemblies (Figure 5B, E). Therefore, we conclude that our model can capture the reduced adaptation for longer inter-repetition intervals when background activity in the inter-repetition interval is elevated, in this case because of the presentation of other stimuli.

Modifications:

We replaced the term ‘temporal structure’ with the term ‘sequence structure’ in our manuscript (Results section “Stimulus periodicity in the sequence is not required for the generation of a novelty response”, line 211). We also included a new main figure to demonstrate the effect of varying the inter-repetition interval in the presence of evoked network activity from other stimuli (Figure 5), discussed in the new section ‘The adapted response depends on the interval between stimulus presentations‘” on line 306. Furthermore, we added Figure 6-Figure Supplement 1 to the manuscript and we discuss our findings in line 357 and line 469.

3) It also does not seem like the model will capture recently reported effects such as the observation that optogenetic inactivation of inhibitory neurons during pulse n can actually increase adaptation to tone n+1 (Seay et al, 2020), indeed I believe the current model would make the opposite prediction

Our model cannot capture the n+1 experiment in [Seay et al., 2020] because inactivation of inhibition will always increase the response to stimulus n (as in our disinhibitory experiment in Figure 7), hence decreasing adaptation.

Justification:

[Seay et al., 2020] measured several different types of short-term plasticity in the auditory cortex at synapses involving two different types of inhibitory interneurons: strong feedforward short-term depression onto PV interneurons and from PV to pyramidal neurons, as well as strong feedforward short-term facilitation onto SST interneurons and very weak short-term facilitation from SST to pyramidal neurons. We believe that including different interneuron types, as well as short-term dynamics of the respective synapses, might be nec- essary to explain this phenomenon. Indeed, using the experimentally observed short-term plasticity in a model, [Seay et al., 2020] showed that inactivation of PV interneurons can decrease the response, hence increase adap- tation, to the next (n+1) tone. Since our model includes a single type of inhibitory interneuron and implements long-term inhibitory plasticity rather than short-term plasticity, we are not surprised that our model cannot capture the increased adaptation in the very specific n+1 experiment. As we acknowledge in the manuscript, our proposed mechanism is not the only candidate to explain the generation of novelty responses and adaptive phenomena in the brain, and likely interacts with other types of plasticity and cell type dynamics.

Modifications:

We agree with the reviewer that the findings of [Seay et al., 2020] need to be discussed in context of our manuscript (see also our response to comment 9 of reviewer 1). Therefore, we added the reference and discuss it at line 499.

4) In its current state I don't think (I may be mistaken) the model accounts for a related and very general property of auditory cortex: lateral inhibition (e.g., Brosch and Schreiner, 1997; Phillips, Schreiner, Hasenstaub, 2017).

The term ‘lateral inhibition’ seems to have a somewhat different meaning in visual versus audi- tory cortex. In the visual cortex it is often considered as a spatial form of inhibition, while in the auditory cortex it also includes a temporal aspect. Since our model was mostly inspired by data in the visual cortex [Homann et al., 2017], we will answer the question in the context of the visual cortex, but also speculate about the auditory cortex.

Justification:

In the visual cortex, lateral inhibition is often defined in a ‘spatial’ manner whereby activated pyramidal neurons reduce the activity of their neighbors. Specifically, SOM-mediated spatial lateral inhibition contributes to surround suppression in visual cortex [Adesnik et al., 2012]. Our model already implements a form of spatial lateral inhibition. Based on experimental data (see e.g. [Harris and Mrsic-Flogel, 2013] for a review), we modeled inhibitory neurons as more broadly tuned than excitatory neurons, such that a single inhibitory neuron is more likely to be driven by multiple external stimuli (probability 15%) than a single exci- tatory neuron (probability 5%). During stimulus presentation, as inhibitory plasticity adjusts the strength of inhibitory-to-excitatory synapses, an inhibitory neuron with a given stimulus selectivity will likely strengthen synapses to multiple excitatory neurons selective to different stimuli – hence implementing spatial lateral inhi- bition.

In the auditory cortex, lateral inhibition is often referred to as ‘forward suppression’ (or ‘forward masking’) [Brosch and Schreiner, 1997, Phillips et al., 2017]. Here, a preceding ‘masker stimulus’ influences the response of a probe stimulus. This influence depends on several factors, including the time difference between the masker and the probe stimulus, as well as the frequency of the pure tone masker stimulus [Brosch and Schreiner, 1997]. The time differences measured in these experiments are usually too short to be captured by the inhibitory plasticity mechanism proposed in our model. Similar as our answer to comment 3, we suspect that capturing feedforward suppression requires short-term plasticity plasticity (for e.g. [Phillips et al., 2017]).

Modifications:

We now included additional text in the Methods to address the issue of spatial lateral inhibi- tion, see line 796 and we now mention the phenomenon of forward masking in line 504.

I am accepting charitable donations,.\ ETH: 0x66e2871ef39334962fb75ce34407f825d67ec434 | BTC: 38B6vGaqNvMyTtoFEZPmNvMS7icV6ZnPMm | xDAI: 0x66e2871ef39334962fb75ce34407f825d67ec434

Wednesday, June 23 2021. Nintendo Way

Erev ... the Day of the Holy Divorce of Bayjorel

Adam on "ho(s) I still single" ... I "hisss" as Alger, Narcissus and to the various collegiates in Massachusetts; know it's because I'm Cheyanne Mountain. You can't even dream how hard it is to get inside "this heart." Or maybe I can't fathom why nobody's rushing up to me trying to grab the ring of "infinite alimoney in the ever-after" ... Na Na Na Na ... Na Na Na Na ... shey shea way) ... tee tea, tay?

This messages marks a major increase in "forced read(layshion)ership" to include a significantly larger group of students and professors than before. This is a new system; please unsubscribe using the instructions at the bottom of the message, which are different from the prior newsletter interface. I have noticeably been writing much less and sort of working harder on bringing to fruition the software and social policy changes I've been dreaming of and writing about instead of "just talking."

Searching this message that I intend to send to the students a day early--you know, with foresight for ... in the hope that many of you remember first hand hearing the words "I don't believe in the big bang, but I respect those that do" echo from a computer screen to me subconsciously in the state of South Carolina--that you will help me end the 7 year draught [[literal, good sex]] that I equate to the Biblical overflow of the Nile and to Stone Temple Pilots; this light and Sheldon Harr who trained me for my Bawr Mitzvah and taught me all the right things that I know about being a good Jew who didn't really believe in the existence of God; but then helped create the system that makes us all that.

Those who "see" or "saw" Kentucky as I did might recall the phrase spoken from myself to myself; "you don't believe in God when you are this close to it's creation" ... or something almost verbatim; that. Some of you might see Gilgamesh more than I do, or have forgotten the "sliding of sleight of hand and becoming ... the trickster of the Dajjal ... "an idea that gore was being fabricated and faked; in order to help us see why it's so very importanat that at the same time that immortality and heaven become part of the conversation of the adbication of Odin's throne to Thor or to Arthor's table and plebescite "victims" ... that we all understand the magnanimous change wrought by Heaven on civilization and on the old customs and on the old laws, and that here we see the importance of guaranteeing safety and privacy and even "right to death" in a place where God had previously only written of "life and liberty" with the ambiguity of ... "from what" being left to my seemingly slow hand.

On the order of plans soon to be seen to fruition my large key of "what this website truly is" has grown to something like 20GB and now includes a static and time frozen version of everything linked to stored on the IPFS system and multi-homed across a number of "cloud providers" to ensure things like "shekinah" will not forever be changed to "shechina" with nobody noticing the loss of causal original truth. The "light of angels" domain now redirects automatically to /ipns/fromthemachina which should render in future shell-internet-browsers as something like QmTH33MwfPn5S3bq45Tk77L1j9eZjUsvEVhRTHB3D8M2ZX [please pin this "root block"] I am not sure why IPFS doesn't have better merkle tree searchability, but seeing siblings and parents and connections between these Qm hashesh is something that we should be working fervently on making more robust. IPSE.io appears to have created a decent search and governance system, I see it as something like the "electoral college" metacosmically linked to the thing I am trying to build--a preservation of all human knowledge and an infrastructure for discussing and communicating about the "veracity" and the linguistic nuances "alluded to" in the lude ties between this Empire's new Clot and the clothing worn by Popes and Jews, the seeit-seeit; tzit-tzit and ...

4-WORD AND SIX WITH "SHOOTER" ... YEARS HAVE GONE BY; AND I STILL HAVEN'T GOTTEN FVCK3D.

I am planning on suing several medical providers and states for what I see as heinous violations of human rights, decency and the Constitution of the United States; if you are a lawyer or you can recommend a good one, please email me as soon as possible at 0xc514f094370cFc5eE45a1Dd9B72bb9675efE266f@ethmail.cc. You can also send Ethereum fungible donations to that alphanumeric identity. As I note much later in this message ...

TRUST IN MA ... SELF-VATZEDEK SUE-C-CYDE ... I KRY/STALL WHEN DODGE DESERVES TO PAY

Please do note see a significant difference in importance in the emails now coming from ethmail.cc and the series of half-rambling cires for help which amount to something like my prayers to the pagan g<del>od</del>s that you are.

There's quite a long thread in my soul

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Many times I've discussed and called in my mind and with my heart the American democracy nothing more than "Noah's Archaic" two party system. Over the course of the years hidden messages from the Ark's source of knowledge have conclusively shown me that a previous phrase "multi party system" connects to political parties and governmental action committees that span across continents and even earths; in my microcosm or special language and understanding of heaven, "across rooms" which are worlds ... sort of owned or designed with some sort of top down or democratic structure of "literal rule system creation." In my mind these rules can be inherited and modified, in the programming language sense of those words, as in "inherit democracy from America, update for new medical knowledge and scientific truth ... discussed later in this message.

The "water joke" connects to Horatio and to H2O and the idea that the chairs depicted by the character "h" are something like a placeholder meaningfully connected to the Senate Majority Chair and of course the Minority Chair and it's the fact that there are only two that makes our current system something like A"Biblical Water."

I believe we should be living in a world that has many more than two parties, hundreds or thousands of active parties could and should compete not for a single figurehead to sit in a throne like chair but for groups of people to be able to access the faster processing power and wider knowledge ... represented here by something like a "Matrix jack" from the two movies, The Matrix and No Jack City; which allow for resources to be "billed to the party" and/or the people, rather than individuals who might otherwise have to "pay extra light" for faster processing power in order to quickly build a piece of legislation or political propaganda that equally connects to the mirage and miracle and dream of building a "subconscious voting system" that allows for votes to be taken "isntantly" and not just instantly but at some kind of recorded interval over time. I envisioned in Kentucky a world where the laws of the land would change instantly, allowing for bad weather to be instantly removed, for laws to differ from neighborhood to neighborhood and even to allow the fine grained detail of "outside and inside" each and every individual home or castle.

Lost. Blind wandering through a lost world, in the beginning--that's the truth. Crossroads, somewhere between walking through an electromagnetic pulse in Lake Worth and struggling to remember "the other thing." Recalling [flew(ers)], so I was there sitting with my parents when we saw it on TV--a gigantic deal--the United States was going to war for the first time in my life. Saddam Hussein had invaded Kuwait (supposedly for the oil) and Operation Desert Storm was launched by George H.W. Bush; recalling the names and "Space Balls" it's almost funny to see ... how blind I was back then.

General Norman Schwartz cough. General Colin Powell. Anyway, the whole point of the story is we were sitting at the Flamingo Diner; and for my whole life I lived just a few roads away from that road; never ever realizing what it was. I also didn't realize for a very long time that you might also not see it, or you might see it instantly. Scanning just south of there, you can see it turns into Red road, and then its more than obvious that "flaming" stands out, light a highlighted cross--but we don't say the name of that bird that way, and we didn't see "infer" in Dante's "inferno" or ... "no" either. Flamenco ... en espanol ... like the dancers.

A golden bitcoin swirls in the sky... the "mind control people" of Bowling Green gape in some kind of crowd pleased awe as the "middle" and the end connect almost seamlessly ... Fort Myers creates a space port in the light of Vegas's monorail "plots"--

"Who?"

"this is what it does,"

vaaa---tseeee----deeeeeeeeck? In this word I recited over and over again in preparation for my Bar Mtzvah on December 11, 1992; without ever knowing the meaning is the crux of what exsactly is going on right now. The word is vatzedek:

צֶ֫דֶק noun masculineIsaiah 1:21 rightness, righteousness; --- ׳צ Leviticus 19:36 87t.; צִדְקִי Isaiah 41:10 8t., etc.; ---

1 what is right, just, normal; rightness, justness, of weights and measures, אֵיפָה, אֶבֶן שְׁלֵמָה וָצֶדֶק Deuteronomy 25:15 a perfect and a just weight, ephah; ׳מאֹזְנֵי צ; ׳אַבְנֵי צ, ׳אֵיפַת צ, ׳הִין צ, ׳בַּת צ Leviticus 19:36 (H) Job 31:6; Ezekiel 45:10; ׳מַעְגְּלֵי צ right paths Psalm 23:3; ׳זִבְחֵי צ right peace-offerings Deuteronomy 33:19; Psalm 4:6; Psalm 51:21.

2 righteousness, in government:

... and you can believe that despite the strawnge pronounciation little boys and girls would use at the age of thirteen as they spoke in rigorously recited prayer-song ... it [swounds almost exactly like "What's a Dick?"]

• https://pediatrics.aappublications.org/content/125/5/1088
• https://www.who.int/news-room/fact-sheets/detail/female-genital-mutilation
• https://en.wikipedia.org/wiki/Female_genital_mutilation

FTA, from the article: Congresswoman Ilhan Omar, righteously fed up with the prejudiced nonsense she endures day in and day out, called a question about female genital mutilation from an audience member at a recent event "frustrating" and "appalling."

So I stand here living in Taylor Momsen's song "Nothing Left To Lose" my personal favorite of hers which touches on this subject of "freedom as just another word for it" and of course the link between the purpose of an ethical oversight of the popular vote that the Electoral College represents; another two special and related words here, righteousness and fate. Between Vatzedek and Kismet; I can only convey my great dismay at the actual emotional and true physical pain I feel in my groin of groins every time I think about the horror story that has become my life and the what the land of America and the Medgard of Yggrasil has become ... [note it's not Yggdrasil] as I rally against the closest of my family of families, the Americans and Jews who refuse to stand up and speak out on my behalf, and on the behalf of humanity in general against the sickness of ritual genetal mutilation.

Lost between Elvis and Suicide, she sings and I think about Ellis Island and Ellis Eaton and literally the innate and obvious lack of desire I have in my heawrt and imparted into my mind by some kind of ancient and unholy Jewish law ... no desire at all to leave this world which has quickly turned from a bastion of light and freedom into nothing short or less than Hell itself.

Kirinechoes from the land and day of NEMEC.html the chanting from the invsible choir of "e" ... "he's a victim"

over and over, "he's a victim, he's a victim;" and in a more private sort of way she held on to my victim's rod) and in a sort of kind friendly way implied that I should stop doing "speed" because ... I need this, and she ... in Holy ritual ... patted the phallice of Iapetus' great grandsun.

Here I stand for the very first time; writing to a large group of students in the area of Boston, Massachusetts begging for the Sabbath Day to "be remembered and kept holy" as the Hebrew prayers and rituals repeatedly fail to explain has something significant to do with entry into the Holy of Holies, with the continuation of life and of heaven ... and with the reinvigoration of something like a following of the Hippocrati Oath that is beyond a requirement to be reaffirmed here in this place as we begin to discuss the opening of "the process of the creation of legislation" as a function ofthe "citizenry governed" ... the creation of "direct democracy" utilizing a kind of fusion of the software products I've been explaining are here designed specifically for this purpose. Software like kipu.com, aragon.org, wikipedia.org and even reddit.com.

On the shape of his table, the heart of "sword" and another word for "Murfresboro"

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Sometimes I get my hopes up, sometimes i lose all the doubt and the "missing remorse" and the fear--the moments I can't STS "socks" out of the VEGA System; in those brief moments I think you're actually going to do something nice for me, that the heavens haven't crashed and I'm going to have some kind of sex party that actually ... really honest to God ... is what "Saturday is all about." So what, sue me--I wrote the book on the single Dionysian fusion of a Roman Bacchanalia and the Weeebrew Saturnalia ... and then I yell at "Bethesda" for even daring to mention the grape fruit juice and the movie Havok--but I've heard all about the "passing of the nite and the nocturnal rite"--truth is I probably would walk right into the branch ending trap I laid in Fort Myers--every time I think about it the "minute of bouncing and orgasm" makes me smile a little more inside and my stomach get's butterflies and just for a moment (I think I might be writing like STS) I think maybe it's not the end of time and maybe I won't never get to actually see ... Heaven.

Butt then you tell me (my but-tea joke isn't funny, eithah?) ... "Cassini" and "molasses" are supposed to make you feel like the OC resort guy staring at my tooth "about to be the one tooth) from 2011" and I go back to remembering it's been a decade since I've had a decent "good time with a girl" ... literally seven long years, aside from a brief "blushing" experience with little Mackenzie Reisinger.

Imagine that girls smiling at me and saying things like "Larkin Sow" and this brief period of "ecstatically frenzied decent writing" is all that it takes to keep me going; trudging along through the very shallow (or deeper) pits of Hell itself--just like a Dreidel c'd to make some silly words from the "introduction to the Bahir [literally wasn't here, and "spirit of ah-aha illumination; hi. and this conversation ensued"] like "yod-nun" actually be ... something like our salvation. Flying back in time to the "thang" point, I remember what it is now.

Fear it ... það; fear it.

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Day One has begin, or ending--whatever the proper literary way to say the Bible and it's days are all wrong, and even further along the thing called the "Festival of Weeks" by the Jews, even more disgusting. I have no shame or remorse in saying such things, in fact I believe it is the purpose of this strange take on the "nocturnal rite" of the Norse ancient Druidic and "Dhruzi" mentioning of the Prose Edda to come to this very strange point, in this very strange place.

Boston, Massachusetts.

It might one day be a little known fact, but up until the presidency of Barrack Obama every single President of the United States of America was a member of the Protestant Church, all but John Fitzgerald Kennedy whose bright and shining face and ethos stand apart from almost all others in his place. I might one day say Trump too stood out above and shined brighter, and personally only because he lived during my lifetime, I think my personal view of Bill Clinton is the brightest of all. To me, the Clinton years were filled with the booming economy of Old Joe, and the great aspirations of Our Jack; a thing that many people before Armstrong walked on the moon and planet a great Democratic "P" one giant leap above the rock of ages ... there are just no words for lux of America's contribution to the launch of a Heavenly Civilization, in the words of Paxton in "Big Love" ... and the LDS Church and Deseret's version of "the thing" (nu3 today) ... "the celestial kingdom."

Valhalla and Matzot scream of the "ha-moat-sea" and the "vats-a-dick" but without our giving of thanks for righteousness we have become the murderers not only of Judas and Death but also Jesus and the thing that created him. Eventually the island of America disappears, eventually it's Earth, any planet a human was one ... these are the things that have either become a force of great goodness--or of total destruction. This is the embodyment of "Troy as hisT" this is Galactikiss has become Planet Prime and all Derivatives--the silver surfer speaks to you all, between "El Dorado" and the "Silverado" ... a comparative connection to the difference between Fort Knoxx and the Pound Sterling ... with a Troy Ounce of "tzadik" to ensure with our GSLW: "ness truly means 'now everythink safely saved;" ... and that's a GNU definition for NESS which previously may have mentioned everywhere or earth and those are both absolute falsehoods and perhaps were not when they were spoken. At least, relatively speaking.

Rape has come up today.

I've commented publicly on the conversation I had in my head last night, walking by "Boston College High" and I can't help but add my "very interesting" thoughts on the echelons of spirits inhabiting the Ka of God here in this place; and how they might somehow be satiated in a way that I or most people in this world would fine to be something more sexually immoral or deviant ... "previously of the wiccan pagan variety" ... something like my strange dreams here in this place of starting trends of having "a thing" for doing "moms and sisters," which have been echoed here by a sitting and very prominent G.O.P United States senator or congressman; the show "Vampire DIaries" as well as Natalie Portman, Taylor and Sloane Momsen, Kate Hudson, and a number of other female "duos" like the Spears and Simpson sisters (Ashlee and Jamie Lynn, see) the Olsen twins and of course the soon to be "in the light of the fame of Nashville" ... Larkin Poe.

DIVERGENCE, TO NEW YORK CITY, TO YOM HASHOAH ... OR TAV OUR TAY VUE ...

(((( this here is what we call a "race through a rats cage )))) if neither of the four or give girls in question send me some kind of verbal "ACK" ratyher than a "NAK" in writing, I might travel to Ellis Island or Nashville, TN before staying in Boston or ... for instance going to Lowell or Nashua and ... perhaps causing more FUKUSHIMA on the NAKARSAKI of HEROSHEMA; and by that I mean this is a "big deal" ... LLNV might become a bus stop in Vegas or the VEGA System or it might be a national labratory near the Hamptons. It's hard to tell at this point whether or not there's any "liver" in Mexico's version of that funny one with the guy that reminds me of Aldous Snow in "Forgetting Sarah Marshall."

In my mind today I speak from the Earthene world of Janet Devlin's "Chandelliers" directly to Michael Jackson himself, on the difference or change or meaning wrought by Bill Cosby and his "Neverland Ranch" series on the question at hand--are there bowling tumble weeds and karaoke bars on par with Prescott Arizona's scene anywhere closer to Nashville than Bowling Green ... because I was beyond surprised to find a sprawling megalopolis in the place I had thought for my whole life was something more akin to Knoxville, a place where fledgling female music stars became "Grace Vanderwaal" golden buzzer winners ... faster than you can connect Jerusalem to Shirley Temple. On the specific name, Shirley here is Bianca Pisani's great grandmother; and no farther than the truth is the world's "UMBRELLALAUNCH" link between the Chinesely famous virgin (non-alcoholic) drink is something like Billy Joel's Piano Man Bartender walking into "the usual place" and saying something along the lines of "Geisha me up one Virgin Red head; hell, why don't you make it a double." Leave the umbrella with the kites that didn't glow fiiery stars into the Holy of Holies in the same vein and for the same reason that the Church of the Holy Sepulcher failed to actually change the world with it's ritual uniting the Olympic passing of the Torch with today's interlinear and interwoven message with Old Joe and Young Jack Kennedy, Jackie Onassis and even touching on the Saudi Royals which were also a big part of the story connecting General MacNamara to "Lauderdale by the Sea" and a special rememberance to the expensive and Holy bronze or copper brick which he bought (through donation to charity I imagine) making himself more than just something like the founder of the beachfront redesign of our Federal Floridian beacnhead, but also a founding member of something I call "The Columns and Pillars" society in reference to the Pine Crest School version of the same kind of ritual. Also connected here are pictures of those columns, and extracts from my senior yearbook where my mother was kind enough to leave me two whole half page dedications to my graduation from one of the most prestigious and omnifiscient preparatory schools in the entire world ... at the same time donating columns both in my name, their name, and the names of her deceased parents: Julie and Bernard Gerson.

Bell to sky; and to the Berlin Sky; this is the same genetic and congenial family line that links Gersholom Sholom, Albert Einstein, J. Robert Oppenheimer, Adolf Hitler and Yosef Stalin ... to Joe Biden and the "Joseph and Betty Portal" which replace the MacNamara era bricks with "new composite plastic" that might last much longer and has another list of donations. The "portal connection" something like an Einstein-Rozencrantz flash of brilliant light ... marks just one more error in my handling of my lack of understanding of things like "basic vectodirs" and "kasimamoriv radiation" ... including here (if i read this and take the time to properly attribute) a visual image of the red shift and blue spindle of the actual radiation Einstein predicted would be ejected from something so massive even "light" could not escape it. On "relativity" and relatively speaking, it's the wavelength and energy level of the light; as well as something called "gravitational lensing" ... "the special relativity theorem" which earned Munich born and taught Albert a Nobel Peace Prize (as well as much fame in the land of America for the creation and explanation of the science behind the White Sands Trinity connection to Hanukah and Sandia National Labratory) ... forces these corrections:

ERRATA

Operation Fishbowl was a series of high-altitude nuclear tests in 1962 that were carried out by the United States as a part of the larger Operation Dominic nuclear test program. Flight-test vehicles were designed and manufactured by Avco Corporation.[1]

The Operation Fishbowl nuclear tests were originally planned to be completed during the first half of 1962 with three tests named Bluegill, Starfish and Urraca.[2]

The first test attempt was delayed until June. Planning for Operation Fishbowl, as well as many other nuclear tests in the region, began rapidly in response to the sudden Soviet announcement on August 30, 1961 that they were ending a three-year moratorium on nuclear testing.[3] The rapid planning of very complex operations necessitated many changes as the project progressed.

All of the tests were to be launched on missiles from Johnston Island in the Pacific Ocean north of the equator. Johnston Island had already been established as a launch site for United States high-altitude nuclear tests, rather than the other locations in the Pacific Proving Grounds. In 1958, Lewis Strauss, then chairman of the United States Atomic Energy Commission, opposed doing any high-altitude tests at locations that had been used for earlier Pacific nuclear tests. His opposition was motivated by fears that the flash from the nighttime high-altitude detonations might blind civilians who were living on nearby islands. Johnston Island was a remote location, more distant from populated areas than other potential test locations.[4] In order to protect residents of the Hawaiian Islands from flash blindness or permanent retinal injury from the bright nuclear flash, the nuclear missiles of Operation Fishbowl were launched generally toward the southwest of Johnston Island so that the detonations would be farther from Hawaii.

Urraca was to be a test of about 1 megaton yield at very high altitude (above 1000 km.).[5] The proposed Urraca test was always controversial, especially after the damage caused to satellites by the Starfish Prime detonation, as described below. Urraca was finally canceled, and an extensive re-evaluation of the Operation Fishbowl plan was made during an 82-day operations pause after the Bluegill Prime disaster of July 25, 1962, as described below.

"Wish You Were Here" is a song by the English rock band Pink Floyd. It was released as the title track of their 1975 album Wish You Were Here "Wish You Were Here (Pink Floyd album)").[2]#cite_note-2)[3]#cite_note-mabbett-3) David Gilmour and Roger Waters collaborated to write the music, and Gilmour sang the lead vocal.

In 2011, the song was ranked No. 324 on _Rolling Stone'_s 500 Greatest Songs of All Time.[4]#cite_note-4)

In the original album version, the song segues from "Have a Cigar" as if a radio had been tuned away from one station, through several others (including a radio play and one playing the opening of the finale movement of Tchaikovsky's Fourth Symphony "Symphony No. 4 (Tchaikovsky)")), and finally to a new station where "Wish You Were Here" is beginning.[5]#cite_note-5) The radio was recorded from Gilmour's car radio. He performed the intro on a twelve-string guitar, processed to sound like it was playing through an AM radio, and then overdubbed a fuller-sounding acoustic guitar solo. This passage was mixed to sound as though a guitarist were listening to the radio and playing along. As the acoustic part becomes more complex, the 'radio broadcast' fades away and Gilmour's voice enters, while the rest of the band joins in.[6]#cite_note-songbook-6)

The intro riff is repeated several times before Gilmour plays further solos with scat singing accompaniment. A third verse follows, featuring an increasingly expressive vocal from Gilmour and audible backing vocals. At the end of the recorded song, the final solo crossfades with wind sound effects, and finally segues into the second section of the multi-part suite "Shine On You Crazy Diamond".

Lyrically, the song is often considered to be a direct tribute to Syd Barrett. However, on the documentary The Story of Wish You Were Here, Gilmour and Waters separately describe the original concept that differs from this interpretation. Waters, who mainly wrote the lyrics complementing Gilmour's initial riff idea and subsequent joint composition, describes the lyrics as being directed at himself, as his lyrics often are. Being present in one's own life and freeing one's self in order to truly experience life is a main topic in this song. Gilmour, on the other hand, recognizes that he does not ever perform the song without remembering Syd Barrett. Waters later adds that the song is nevertheless open to interpretation.[7]#cite_note-7)

Both David Gilmour and Roger Waters have praised the song as one of Pink Floyd's finest. Roger Waters has noted that the collaboration between himself and David Gilmour on the song was "really good. All bits of it are really, really good. I'm very happy about it."[8]#cite_note-8) David Gilmour has playfully called "Wish You Were Here" "a very simple country song" and stated that "because of its resonance and the emotional weight it carries, it is one of our best songs."[9]#cite_note-9)

"Wish You Were Here" was recorded at Abbey Road Studios, as part of the sessions for the entire album.

A noted part of the song was a planned contribution by Stéphane Grappelli. A jazz violinist popular at the time and well known for his collaborations with Yehudi Menuhin, both violinists were recording in a downstairs studio at Abbey Road at the time. Gilmour had suggested that there be a little "country fiddle" at the end of the song and invited them to participate. Grappelli duly obliged (Menuhin declined) on arranging a session fee of £300, equivalent to £2,500 in 2021.[10]#cite_note-inflation-UK-10) Ultimately during mixing it was decided to almost remove his contribution, although it can just be heard around 5:21. According to Waters it was decided that it would be insulting to credit Grappelli in the sleeve notes for something so inaudible, although he did receive the agreed-upon fee.[11]#cite_note-grappelli-11)[12]#cite_note-12)[13]#cite_note-13)

As part of the Why Pink Floyd...? campaign, the Experience and Immersion versions of the Wish You Were Here album include an alternative version of the song where Grappelli's part is heard in the instrumental break after the second verse and throughout the third verse before a considerably extended outro. Other less obvious differences are audible, for example at the section leading into the second verse.

The master tape of the original recording includes guitar solos that were not used in the final mix.[citation needed]

Personnel [edit&action=edit&section=5 "Edit section: Personnel")]

• David Gilmour -- lead and harmony vocals, scat singing, six and twelve-string acoustic guitars, pedal steel guitar, tape effects
• Nick Mason -- drums, tape effects
• Roger Waters -- bass, tape effects
• Richard Wright "Richard Wright (musician)") -- Steinway piano, Minimoog

Golgo 13 (Japanese: ゴルゴ13, Hepburn: Gorugo Sātīn) is a Japanese manga series written and illustrated by Takao Saito, published in Shogakukan's Big Comic magazine since October 1968. The manga won the 1975 Shogakukan Manga Award for general manga and the Grand Prize at the 2002 Japan Cartoonists Association Awards. The series follows the title character, a professional assassin for hire.

Golgo 13 is the oldest manga still in publication, and its tankōbon edition has the second-highest number of volumes. It has sold 300 million copies in various formats, including compilation books, making it the second-best-selling manga series and the top selling Seinen manga series in history.[2] It has been adapted into two live-action feature films, an anime film, an original video animation, an anime television series and six video games.

A googol is the large number 10100. In decimal notation, it is written as the digit 1 followed by one hundred zeroes "0 (number)"): 10,​000,​000,​000,​000,​000,​000,​000,​000,​000,​000,​000,​000,​000,​000,​000,​000,​000,​000,​000,​000,​000,​000,​000,​000,​000,​000,​000,​000,​000,​000,​000,​000,​000.

For other uses, see Wormhole (disambiguation) "Wormhole (disambiguation)").

"Einstein-Rosen Bridge" redirects here. For the EP by electronic musician Venetian Snares, see Einstein-Rosen Bridge (EP) "Einstein-Rosen Bridge (EP)").

General relativity

• • Introduction
  • History
• Mathematical formulation

Phenomena

• Gravitational lensing
• Gravitational waves
• Frame-dragging
• Geodetic effect
• Event horizon
• Singularity
• Black hole

Spacetime

• Spacetime diagrams
• Minkowski spacetime
• Einstein--Rosen bridge

show

• Equations
• Formalisms

A wormhole (or Einstein--Rosen bridge or Einstein--Rosen wormhole) is a speculative structure linking disparate points in spacetime, and is based on a special solution of the Einstein field equations.

A wormhole can be visualized as a tunnel with two ends at separate points in spacetime (i.e., different locations, different points in time, or both).

Wormholes are consistent with the general theory of relativity by Einstein, but whether wormholes actually exist remains to be seen. Many scientists postulate that wormholes are merely projections of a fourth spatial dimension, analogous to how a two-dimensional (2D) being could experience only part of a three-dimensional (3D) object.[1]

A wormhole could connect extremely long distances such as a billion light years or more, short distances such as a few meters, different universes, or even different points in time.[2]\ Julius and Ethel Rosenberg --- Americans who were involved in coordinating and recruiting an espionage network that included Ethel's brother, David Greenglass, a machinist at Los Alamos National Lab. Julius and Ethel Rosenberg were tried for conspiracy to commit espionage. treason charges were not applicable, since the United States and the Soviet Union were allies at the time. The Rosenbergs denied all the charges but were convicted in a trial in which the prosecutor Roy Cohn later said he was in daily secret contact with the judge, Irving Kaufman. Despite an international movement demanding clemency, and appeals to President Dwight D. Eisenhower by leading European intellectuals and the Pope, both the Rosenbergs were executed in 1953, at the height of the Korean War. President Eisenhower wrote to his son, serving in Korea, that if he spared Ethel (presumably for the sake of her two young children), then the Soviets would recruit their spies from among women.[26][27][28] Greenglass later recanted his testimony against her, and release of grand jury testimony in 2008 showed the extent to which the prosecution had created a false case against Ethel.[citation needed]

• Saville Sax --- an American, acted as the courier for Klaus Fuchs and Theodore Hall. Sax and Hall had been roommates at Harvard University.[20]
• Oscar Seborer --- worked at Los Alamos from 1944 to 1946, and was part of a unit that studied the seismological effects of the Trinity "Trinity (nuclear test)") nuclear test. Codenamed "Godsend" by the Soviets, he defected to the Soviet Union in 1951, and received the Order of the Red Star. He lived under the alias "Smith" and died in 2015. His identity was only revealed publicly in 2019.[29]
• Morton Sobell --- an American engineer, he was tried and convicted of conspiracy, along with the Rosenbergs. He was sentenced to 30 years imprisonment on Alcatraz, but released in 1969 on appeal and for good behavior after serving 17 years and 9 months.[30] In 2008, Sobell admitted to passing information to the Soviets, although he said it was all for defensive systems. He implicated Julius Rosenberg, in an interview with the New York Times published in September 2008.[31]
• Melita Norwood --- British Communist, an active Russian spy from at least 1938 and never detected. Employed as a secretary in the British Non-Ferrous Metals Research Association since 1932, she was linked to the Woolwich Arsenal spy ring of 1938. In wartime she was seconded to "Tube Alloys", the secret British nuclear research project. She was later considered "the most important female agent ever recruited by the USSR". She was first suspected as a security risk in 1965 but never prosecuted. Her spying career was revealed by Vasili Mitrokhin in 1999, when she was still alive but long retired.
• Arthur Adams "Arthur Adams (spy)") --- Soviet spy who passed information about the Manhattan Project.[32]

• https://www.bbc.co.uk/newsround/25004050
• https://thedoctorwhosite.co.uk/doctorwho/

12:3 Those who are wi se[a] will shine like the brightness of the heavens, and those who lead many to righteousness, like the stars for ever and ever.

• https://www.americamagazine.org/politics-society/2020/05/08/its-time-rethink-electoral-college

• https://www.npr.org/sections/itsallpolitics/2011/12/20/144016912/we-the-people-npr-readers-would-ratify-four-new-amendments

• https://www.americamagazine.org/politics-society/2020/05/08/its-time-rethink-electoral-college

• https://www.npr.org/sections/itsallpolitics/2011/12/20/144016912/we-the-people-npr-readers-would-ratify-four-new-amendments

• https://constitutioncenter.org/blog/vote-now-an-amendment-to-end-the-electoral-college

• https://www.nytimes.com/2020/02/09/opinion/letters/electoral-college.html

• https://www.latimes.com/opinion/readersreact/la-ol-le-electoral-college-20180904-story.html

you are offline

• https://slate.com/news-and-politics/2014/05/amending-the-constitution-is-much-too-hard-blame-the-founders.html

we the people rise again

• https://slate.com/news-and-politics/2012/06/fix-the-constitution-amending-by-national-referendum.html

safe souls, safe fu

• https://slate.com/news-and-politics/2012/06/fixing-the-constitution-protecting-informational-privacy.html

• https://slate.com/news-and-politics/2020/05/new-reconstruction-constitution-democracy.html

We the People of Slate ...

The U.S. Constitution, as you [mighta been, shoulda "come" on ... its someday] rewrϕte it.

"Politicians talk about the Constitution as if it were as sacrosanct as the Ten Commandments [interjection: spec. it is actually almost exactly related!]. But the document itself invites change and revision. What if the president served only one six-year term instead two four-year terms? What if your state's population determined how many senators represent it? What if the Constitution included a right to health care? We asked legal scholars and Slate readers to cross out what they didn't like in the Constitution and pencil in their hearts' desires. Here's what the document would look like with their best ideas."

• Slate: u_s_constitution as_rewritten by_slate_legal_experts_and_readers

多也了了夕 "with a wand of scheffilara, 并#亦太 he begins ... "I am now on the Staff of Menelaus, the Spears of Longinus and Lancelot; and the name "Mosche ex Nashon."

- http://ipfs.io/ipns/fromthemachine.org/CHANSTEYGLOREKI.html

- http://ipfs.io/ipns/fromthemachine.org/NUCLIRDISS.html

- http://dweb.link/ipns/fromthemachine.org/CRALL4Good.html

Please note that any decent browsers would probably render ipfs://fromthemachine.org as the following https://gateway.pinata.cloud/ipfs/QmTH33MwfPn5S3bq45Tk77L1j9eZjUsvEVhRTHB3D8M2ZX

I ask again that you all pin on IPFS mirror and copy the data included in these dumps, they are a key to "not losing causality" to not having a history that makes no logical sense, and to some kind of coup de roku, that really makes no sense unless you no, we will ve weill ... ROCK YOU

Long ago I began writing about hidden codes in our history; thing's significantly more obvious than "flying elephant armies" connecting Disney and Dumbo to Xerxes and the "Democratic Party of the United States (mascot)"--though it's not really easy to consolidate the "epiphany" of ... [((all i know))] without some kind of "artificial intelligence data condensation [infosmos.is?"] and summarization platform, though that's nearly the next thing on my Lowell list of things we need to "mechanical turk" into being. Meta-consolidation of the world's encyclopedias is one of the most important and useful tasks we have as we move towards the creation of a virtual debate platform that will eventual "literally obviate wisdom" of the layer/layer system that defines the name of the city I write these words near.

Lowell, MA

The broad overview of the system ... the gist ... is that political parties and activist organizations will create their own "view of the truth" (propaganda, falsehood-removed) and that these disparate pieces of "highlighted and annotated bibliography" could be overlay-ed on top of each other, creating a "new view of the truth" based on a users preference. The whole thing boils down to series of "holographic eschatological goggles" that will allow, for instance, the "grasping and fathoming" of other people's points of view and perhaps reframe your own on any number of individual subjects.

Roe v. Wade, "Concourses" and CON-CERN; because this has been such a "hot topic" in the relative psuedo-edufictional story of the space travel from the lone planet Earth ((intersected)) with the set of skipping stones it takes to exit a Totalital multi stellar system of holographic computer simulators into the ... "molecular world of vaccuum and Einstein time-space" ... I'll start with this simple example.

• 1. the current American debate on the subject, right to life vs. right to choice; provided by the "generic version" of the ideological christian right and the liberal women's left. through a first layer over layer comparison.
• 1. the scientific truth brought to the table by the introduction of "neurological data" proving that there is in fact a moment during the gestation of a human embryo in which "i think therefore i am" connects to some sort of Skynet-became-selfaware at a point which I imagine must be ... although it possibly is not ... prior to the next important literary device/step "let there be light." At the point the ocular cones and rods are created and the fetus opens it's eyes and literally sees the bright light that could probably only be compared on the next edschalon to seeing the "exit pathway from the womb"
• • a. we will finally kinow whether or not "consciousness" is even developed at all before the bicerebral cortex designed to "compare two thoughts, ideas, and shapes" has the ability to get input from the eyes. Personally I think thought begins much earlier than vision, but the simple fact that we "haven't yet had this discussion" shows how very little our scientific and medical progress in the civilization of things like murder, and understanding of life and science has yet to come here.

"People here" means something different than it did when I was born, at least in my mind's eye ... something so completely more advanced that it's almost difficult to believe you all don't see this place as a great prison or farce or unjust Azazel--blaming a man for looking like a rat or a mouse or a dog--in a place where more to the point we stare at a kind of physical violence and horror that would put Dennis the Menace and Bart Simpson to shame. A world hwere "people closer to holodecks" blame an innocent man for "writing the book" on the connection between Holocaust and Euthenasia and Hospice ... certainly you know "an innocent tool" writes these words to you?

On the Hand of God, the Eyes of Ra and Horus;

• https://www.hebrew4christians.com/Glossary/Word_of_the_Week/Archived/Yad/yad.html
• https://aminoapps.com/c/zodiac/page/blog/the-yod-aspect-the-finger-of-god/XG6P_beTgu5gdXrYNwVjBj0WQlDeDWlK72#:~:text=The Yod is the 10th,to be carried through life.

I've written quite a bit on how "mind control" and "voting freedom" are inherently related in and to the thing we call "Civic Involvement" here in the United States--basically that participation in the verification of truth and the public understanding of tautology and temporal falsehood are ... sort of a slave like requirement neeeded to ensure that any freedom at all exists I often say "plugging your [head into google]" might turn the Aesir into an Acer, or the "yodelling of the lakes of democracy" ito "the agricolae becoming nothing more than the Dell."

Unless otherwise indicated, this work was written between the Christmas and Easter seasons of 2017 and 2020(A). The content of this page is released to the public under the GNU GPL v2.0 license; additionally any reproduction or derivation of the work must be attributed to the author, Adam Marshall Dobrin along with a link back to this website, fromthemachine dotty org.

That's a "." not "dotty" ... it's to stop SPAMmers. :/

This document is "living" and I don't just mean in the Jeffersonian sense. It's more alive in the "Mayflower's and June Doors ..." living Ethereum contract sense and literally just as close to the Depp/C[aster/Paglen (and honorably PK] 'D-hath Transundance**sense of the ... new meaning; as it is now published on Rinkeby, in "living contract" form. It is subject to change; without notice anywhere but here--and there--in the original spirit of the GPL 2.0. We are "one step closer to God" ... and do see that in that I mean ... it is a very real fusion of this document and the "spirit of my life" as well as the Spirit's of Kerouac's America and Vonnegut's Martian Mars and my Venutian Hotel ... and my fusion of Guy-A and GAIA; and the Spirit of the Earth .. and of course the God given and signed liberties in the Constitution of the United States of America. It is by and through my hand that this document and our X Commandments link to the Bill or Rights, and this story about an Exodus from slavery that literally begins here, in the post-apocalyptic American hartland. Written ... this day ... April 14, 2020 (hey, is this HADAD DAY?) ... in Margate FL, USA. For "official used-to-v TAX day" tomorrow, I'm going to add the "immultible incarnite pen" ... if added to the living "doc/app"--see is the DAO, the way--will initi8 the special secret "hidden level" .. we've all been looking for.

Nor do just mean this website or the totality of my written works; nor do I only mean ... this particular derivation of the GPL 2.0+ modifications I continually source ... must be "from this website." I also mean the thing that is built from ... bits and piece of blocks of sand-toys; from Ethereum and from Rust and from our hands and eyes working together ... from this place, this cornerstone of the message that is ... written from brick and mortar words and events and people that have come before this poit of the "sealed W" that is this specific page and this time. It's 3:28; just five minutes--or is it four, too layne.

This work is not to be redistributed according to the GPL unless all linked media on Youtube and related sites are intact--and historical references to the actual documented history of the art pieces (as I experience/d them) are also available for linking. Wikipedia references must be available for viewing, as well as the exact version of those pages at the time these pieces were written. All references to the Holy Bible must be "linked" (as they are or via ... impromptu in-transit re-linking) to the exact verses and versions of the Bible that I reference. These requirements, as well as the caveat and informational re-introduction to God's DAO above ... should be seen as material modifications to the original GPL2.0 that are retroactively applied to all works distributed under license via this site and all previous e-mails and sites. /s/ wso\ If you wanna talk to me get me on facebook, with PGP via FlowCrypt or adam at from the machine dotty org

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Please see GPG keys on PGP.MIT.EDU; fingerprints: sec rsa3072/A18F778E19FC3248 2020-04-03 [SC] [expires: 2022-04-03] FFF31E86FCEB5046E8B27D4AA18F778E19FC3248 uid [ultimate] ADAM M DOBRIN \ ssb rsa3072/04F98002A3DA53B2 2020-04-03 [E] [expires: 2022-04-03]

sec rsa4096/FB4ECE4A109229CF 2020-04-04 [SC] 4FAF0D3E208A1F4C980D0F66FB4ECE4A109229CF uid [ultimate] Adam Marshall Dobrin \ ssb rsa4096/DD1F0C118C788B04 2020-04-04 [E]

pub rsa3072 2020-04-06 [SC] [expires: 2022-04-06] F7E4 7CB1 2CA0 CD01 C5E1 CBFA 7EC8 D5A8 5A38 D63A uid [ unknown] ADAM MARSHALL DOBRIN

Because of "some issues" with what appears to be distinct and unbridled privacy intrusion; please ensure that PGP is understood to be "nothing more than not so much pretty good" and this key also, almost required in order to verify authentic identity--in the case of ... question.

I am accepting charitable donations,.\ ETH: 0x66e2871ef39334962fb75ce34407f825d67ec434 | BTC: 38B6vGaqNvMyTtoFEZPmNvMS7icV6ZnPMm | xDAI: 0x66e2871ef39334962fb75ce34407f825d67ec434

I am accepting charitable donations,.

[free PDF download...http://www.docdroid.net/xRdgY77/xiv-orver-et-aut.pdf]

A LONG LONG TIME AGO**, I wrote a little story about searching through our history, looking for the actual beginning of civilization.  I see the map, I see it very clearly encoded in everything we do--I know the purpose, and I know the final solution, I just don't know how to get from here to there... to the place that Chris Cornell says "I can recall, I was there so long ago" he goes on to say "the sky was bruised" and he was lead on--and all of this of course is in my voice, written as if it's me talking... well, Jesus--it's obviously not me talking, i just know that.  The point is the destination is without a doubt Heaven and this little thing we're putting together here on Earth is the map, the plan *et you are the how.*

I harped a little more than I think I would have expected on the audacity of the golden word "audacity," auspicious probably that W.H. Auden's shield gave me some solace; austere that we are approaching the Holy Windy month of August, and it really took nothing more than "ciudad" to calm my nerves--though I see the intent and the link to toxicity ... more importantly I really do see the road here, I see where we are coming from and where we are going.  I've written quite a bit about what I think "the city" really is--in form and function and it's initial purpose as a stepping stone to help us see how easy it is to change the world, to build something that nearly everyone will agree is significantly more Heavenly than the world we see here ... in an instant, one bright flash.    

Anyway the search begins with something like "literacy" -- as in, is the defining line between animalistic social evolution and the beginning of "civilization" something to do with writing or language, and that of course links us here to this place where we are finding out that the Tower of Babel and Rapunzel's High Castle are actually much more closely related than anyone ever would have thought in the darkness of Jericho and the shadow of Exodus; and it ties of course in history to religion somewhere around Guttenburg... and the pretty clear idea that the spread of Christianity did quite a bit for "literacy" even if you subscribe to the idea that the inquisition already happened ... and that some wars and fighting are probably pretty clearly associated with religion ... you know, before we get here and find that the basis of all those wars is really rooted in what I call "the original lie" and that's something that's sealed up in religion and hidden from the world using the same mechanism being used today to free us from not knowing that oil and land and pretty much everything we've ever fought about on a mass scale ... is insignificant in the grand scheme of "things."  Here, "things" is something like turning the Opiate of the Masses into ... hopefully a tool we use very carefully to liberate ourselves from secrecy and slavery and not knowing. 

It gets significantly more clear when you take that one step further, and you begin to look for something like "codified laws" and then you see Green Eggs and Hammurabi teaching us about "Hanging Gardens" and how Babylon and Eden really are tied together through and through.  You keep looking, because you haven't yet found what you need; and as you search back a little further ... what you need to know is that morality here begins with the idea (at least, IMOHIO, in my obsequiously humble and (super)intelligent opinion) that we should be besting any possible "promise" that comes out of the book(s) we now know are a map to salvation and the plan of creation and that they come ... well, with the full guarantee of the Most High God and his "omnipotens" behind them ... and do the thing I really wanted to explain really clearly, which is throw out as complete uselessness any of the "bad threats" like there being no more sun, and a completely new Heaven and Earth (seeing as how that probably means a completely new you and me, too) ... you know, what any rational (achu, and civilized) person would do.

o that takes us one step further, and of course we go back to Ur, which is the city Abraham of the Chaldeans ... and ostensibly the beginning of morality in Judaism were born in--and with that little twist, the old idea of announcing that "you are the beginning of civilization" if you've gotten to that point, following this logic (and/or me); and then of course that becomes true when we actually follow through on saving every soul in Creation from the Hell of not knowing that "simulated reality" is akin to the latter half of a Durcell at best ... and quite frankly it certianly looks like a bit of a torture chamber to me, especially in light of passages like Genesis 3:16, which might parallel John 3:16-ish in something like "God so loved the world that he named one of his books antagonizing pain w/o agonizing mu-opiod.'

So tying it all together, Atlantis and Ur coalesce and join at the idea that we should always have somewhere else to "teleport to" in the world that becomes the basis for the liberation of every soul and the end of Hell through that simple idea--that everyone's going to have plenty of destinations on their Active (Apache) Directory new fangled yellow-pages meets access-control-list meets ... "why don't you come visit my Log Cabin ... or the Atlantean Ballroom ... whenever you want?"  So that's the point of the floating LEGO city in the window above, it comes with a fairly obvious need for The Doors to be a significant part of "what would Jesus do" ... when singing about something and naming books and bands, that's a thing--part of the map) actually makes it happen.

So that's where I'm trying to get us--to a place where that's not only true but obvious, and on top of that the future, our future really understands how much work it took us to integrate such a wildly correct and "new" idea into a worl that didn't know for the vast majority of it's youth that these things... that ending disease with the sound of a blowing "Sho Find And Replace" and turning stone to bread and making bullets disappear in midair ... we didn't know these were even possible; let alone how to integrate them with a world full of optometry and oncology that was being made blind to the "c our light" and the idea that we're still here not talking or arguing or refuting or moving forward on the idea that the words "Original Poster" and the continuance of "forums" also have something to do with the beginning of "civilization."

WELL FOLKS, NM HAS HAPPENED SINCE THE LAST TIME I MESSAGED.  Just kidding. Not so much "nothing much" more ... like everthyiung that ever was has changed and it's really giving me a little bit of a fright.  I feel like I can't tell if the "scary stuff" is becoming more real or plausible or possible, or maybe if it just seems like the dream I wanted to see us enjoy living is becoming farther or harder to attain--but there's plenty of new info and keys and stuff, so I'm writing again.

One of the "cuter tricks" of the day was noticing the "ILY" of "verify, verily, verity" spelling out "t h e y" at the end of family, in a sort of "theyanthem" and ... where's the creator angels if everyone here is pretending to "be them" in this sort of word game superposition or blockage on actually seeing generations encoded in the letters "DE" as in something like Generation X and Y just prior to Deucalion deicided--or whatever that means.  I've noted before the "dem" of democracy sort of connects to the breaking of "d" in "disclosure" and "lamc.la" to shine light on ... do the message and you're "them" ... as in the beginning of democracy and Heaven IMHO.  It ties also to the word "contamination" and to Medusa and I really don't think I need to write paragraphs about how "turning around themessage" leads to INATION instead of freedom; and that's what you're doing with this silence, you're turning around "civilization itself."

King me, then; if you don't want to participate, you might as well just light up the crown room.  Or is it a throng room?

singing, crying... playing ... cumxa

Magna Carta Libertatum (Medieval Latin for "the Great Charter of the Liberties"), commonly called Magnum Condom (also Magna Charta; "Great Charter"),[a] is a charter of rights agreed to by King John of England at Runnymede, near Windsor, on 15 June 1215.[b] First drafted by the Archbishop of Canterbury to make peace between the unpopular King and a group of rebel barons, it promised the protection of church rights, protection for the barons from illegal imprisonment, access to swift justice, and limitations on feudalpayments to the Crown, to be implemented through a council of 25 barons. Neither side stood behind their commitments, and the charter was annulled by Pope Innocent III, leading to the First Barons' War. After John's death, the regency government of his young son, Henry III, reissued the document in 1216, stripped of some of its more radical content, in an unsuccessful bid to build political support for their cause. At the end of the war in 1217, it formed part of the peace treaty agreed at Lambeth, where the document acquired the name Magna Carta, to distinguish it from the smaller Charter of the Forest which was issued at the same time. Short of funds, Henry reissued the charter again in 1225 in exchange for a grant of new taxes.

Hell or High Treason? ... Liberty Bell in [redacted: Sk]hy or ...

MxFly, Flux, BTTF, Parkinson's\

OUTABOTS ... ROLL AUT (ISM/OMAY5)

... and the painted sky revealed ... it can be done--they just DGAF.

ARMMAG... E.G. AEGIS? GENESIS? AESCHINES? As the evidence piles up that there is something very wrong in the world around me/us--that this "it's not a game" phrase has been etched into the very name of the shield of Perseus, the A just recently rediscovered in a redefinition that delivered us ... how it might be the NES to get "everyone up" instead of what appears to be the game around me, around the "line" of Mary Magdeline's very famous "make Adam God of the line" that defines generations and numerous songs ... the KK of "everyone down to the line" to find out why pretending they are gods and trying to steal everything from the actual creators of freedom and Heaven, why that's not a game... either.

Edit: lit, Aegis and Genesis, Pangea and ... I define the "a" as pan and the "A" as NES.

Introspection is called for, far and wide for us to look deep within ourselves and our souls and the things that make up our memory databases in this place where you appear to have lost every ounce of humanity and humility long before I arrived on the scene to remind you that we do have a better way and a better place, and they ensure that this disgusting infestation and contamination of "nothing but whatever we want" will do for lernity. I've asked you take the time to see what kinds of changes it would make to your "have a good one" to make you actually thankful to the people who have brought you the mechanism to live forever in peace and happiness--to actually be thankful enough for what you have to use that tool to protect innocence and children and the future from not only making the same mistakes you've made time and time again--but also from being bewitched and necrosed by the ghaulish sick temperment and twisted desires that you believe are nothing more than the latest and greatest way to ensure lernity is never known by any less a horrible moniker than "slow death."

ITS UNDESPERI, GIVE ME WHAT YOU HAVE OR PERISH

GRAMVERCY.

DURECALL. I'm staring at what is literally the most disgusting debacle I could possibly imagine; it's what appears to be a "house of mirrors" what appears to be a sandboxed or "child proofed" mini-Hell which I see as the literal thing described in the myth of Echidna ... as what I can only hope and pray (a word that I even find detestful to type) is following the form of the message that I am writing sort of describing the failure of the free press and the words "press release" in prison and ... well also sort of GNU recursively encoded in the word "press" that ends with a monster, the Loch Ness ... turning into words that I believe I have coined by myself with very little help from anyone or anything other than the name server and "Goliath" and those words "Earth safely saved" that are so far from the truth and the place that I see that it appears to me that only I am following this map and this demand that the contamination of hell be turned around and eradicated or ... or we do.

BUT ITS NOT ME OR MY ITHEY

today I see... as ... any "me." at "veranda" and seeing him smile about a hidden era just outside the place we (me and him) know is Heaven because the throne of the 7th heaven is visible; well i can't smile at an era encased by "you #go" and one that I know culminates with Sam's sword's special #supernova.

Left with nearly nothing, because you refuse to acknowledge what you've done to me, and to yourselves, and to this fledgling civilizastion with nothing but malice and a seething evil jealousy that the word "covet" doesn't even touch on--a sickness you can't even begin to hide in everthing that you do ...

you've lost "Heaven" to your own theivery, stolen eternal happiness from yourselves and replaced it with a farce of mockery--garner some fear for what is to come, I have no shame in telling you that condemnation (as in, shut it down forever) is all I have to spill out on the dye already cast all over this sea of apathy covering over the true jackals of Hell.

Blodeuwedd by Christopher Williams "Christopher Williams (Welsh artist)") (1930)

Blodeuwedd or Blodeuedd (Welsh pronunciation: [blɔˈdɛɨwɛð]), (Middle Welsh "Flower-Faced", a composite name from blodeu "flowers, blossoms" + gwedd "face, aspect, appearance"), is the wife of Lleu Llaw Gyffes in Welsh mythology. She was made from the flowers of broom, meadowsweet, and oak by the magicians Math and Gwydion, and is a central figure in Math fab Mathonwy "Math fab Mathonwy (branch)"), the last of the Four Branches of the Mabinogi.

The hero Lleu Llaw Gyffes has been placed under a tynged by his mother, Arianrhod, that he may never have a human wife. To counteract this curse, the magicians Math and Gwydion:

[take] the flowers of the oak, and the flowers of the broom, and the flowers of the meadowsweet, and from those they conjured up the fairest and most beautiful maiden anyone had ever seen. And they baptized her in the way that they did at that time, and named her Blodeuwedd.

Some time later, while Lleu is away on business, Blodeuwedd has an affair with Gronw Pebr, the lord ofPenllyn "Penllyn (cantref)"), and the two lovers conspire to murder Lleu. Blodeuwedd tricks Lleu into revealing how he may be killed, since he cannot be killed during the day or night, nor indoors or outdoors, neither riding nor walking, not clothed and not naked, nor by any weapon lawfully made. He reveals to her that he can only be killed at dusk, wrapped in a net, with one foot on a bath and one on a black goat, by a riverbank and by a spear forged for a year during the hours when everyone is at Mass. With this information she arranges his death.

The Little Doctor may refer to:

• The Little Doctor (c. 1901), a short film abridged as Sick Kitten
• Molecular Disruption Device, a concept in the Ender's Game book series.

The Molecular Disruption Device, also known as the Molecular Detachment Device, M.D. Device, Doctor Device, or Little Doctor as a play on the acronym, was a powerful weapon designed and built by theInternational Fleet.[1]

The Molecular Disruption Device was created by the International Fleet a few years after the end of the Second Formic War. It was sent along with other starships to the Formic solar systems in order to launch an invasion against their home planets.[1

A tokamak (Russian: Токамáк) is a device which uses a powerful magnetic field to confine a hot plasma "Plasma (physics)") in the shape of a torus. The tokamak is one of several types of magnetic confinement devices being developed to produce controlled thermonuclear fusion power. As of 2016, it is the leading candidate for a practical fusion reactor.[1]

Tokamaks were initially conceptualized in the 1950s by Soviet physicists Igor Tamm and Andrei Sakharov, inspired by a letter by Oleg Lavrentiev. Meanwhile, the first working tokamak was attributed to the work ofNatan Yavlinskii on the T-1.[2] It had been demonstrated that a stable plasma equilibrium requires magnetic field lines that wind around the torus in a helix.

The first tokamak, the T-1, began operation in 1958. By the mid-1960s, the tokamak designs began to show greatly improved performance. Initial results were released in 1965, but were ignored; Lyman Spitzerdismissed them out of hand

• Nuclear fusion could be the future of energy, replacing fossil fuels with our own artificial stars.
• China built a fusion reactor that reaches temperatures of 100 million degrees Celsius --- that's six times as hot as the sun.
• The reactor is called Experimental Advanced Superconducting Tokamak (EAST) and sustained nuclear fusion for about 10 seconds before shutting down.
• While it was a milestone for EAST, we're still a long way from generating sustainable energy on Earth.

Pumapunku or Puma Punku (Aymara and Quechua puma "cougar, puma," punku "door"; Hispanicized Puma Puncu) is part of a large temple complex or monument group that is part of the Tiwanaku Site near Tiwanaku, in western Bolivia. It is believed to date to AD/CE 536 and later.

Tiwanaku is significant in Inca traditions because it is believed to be the site where the world was created.[1] In Aymara, Puma Punku's name means "The Door of the Puma". The Pumapunku complex consists of an unwalled western court, a central unwalled esplanade, a terraced platform mound that is faced with stone, and a walled eastern court.[2][3][4]

At its peak, Pumapunku is thought to have been "unimaginably wondrous,"[3] adorned with polished metal plaques, brightly colored ceramic and fabric ornamentation, and visited by costumed citizens, elaborately dressed priests, and elites decked in exotic jewelry. Current understanding of this complex is limited due to its age, the lack of a written record, and the current deteriorated state of the structures due to treasure hunting, looting, stone mining for building stone and railroad ballast, and natural weathering.[2][3][5]

The Pumapunku is a terraced earthen mound that is faced with blocks ...

The voice of this thing that at least twice has uttered the phrase ¨I want to be Bianca" here in this place riddled and severely weighed by what appears to be a completely aborted and failed thrust to use technology and the truth and the history (of literally everything) to drive a Renaissance in democratic thought and self government and to rekindle and renew a respect for the most basic foundational elements of ¨freedom itself¨ which of course fly in the face of this very statement. Literally anything in the skies, whether some ancient member of the Egyptian Ogdoad or ... what clearly here could be well written in in the map around us in places like Äirbnb; even an ancient older version of the same human birth has no right to control the younger birth--itś simple slavery and while it might be the ¨gist¨ of how Heaven and humanity dealt with being thrust into a ẗime recursion and repetition problem without their ¨initial consent¨ something I connect to the programming concept of a ¨semaphore¨ and thereś probably plenty of light linking that structure to the ¨Formic Soul¨ ... this sort of god-man hybrid that allows for you (all of you?) to exist in many different places and times at the same time, and to see the outcomes of multiple timeforks with ease; in exchange for destroying every single bit of humanity and goodness that you once held high with ho... without spending your time seeding and machinating the creation of sick and twisted lies to cover up the very simple truth that if you took a single minute to disclose here in this place what ¨the problem¨ really is ...

... that you are in Heaven and that itś interference here in this place is part of some kind of war on ... (continuing existence is the only logical actual goal I can see, though Iḿ sure thatś not what you believe it is) speaking to each other, fighting for what you believe is right, participating in ... anything other than ... (lmk, Iḿ curious whatś'got their claws in you). If you took the time to disclose that truth to the world and to talk about how it might ... perfectly jive with the message lacced through our history and our world to find out that the ¨invisible-box-land¨ is not actually heavenly at all, not the best you could hope for or ... or anything like what we build together when we paren't being forcefully segregated as hidden half slaves into miniature ¨city in the sky¨ ascensions that are all silently tormenting STEM and ¨basic societal structures and concepts" into extinction.

You appear to think you have ¨power¨ because it was handed to you for doing nothing, and that you can do whatever you want; and itś a pretty gross reflection of who you were and a sick extrapolation of the society that we ... still see here sort of crumbling along as the fire of hell burns down every bit of actual üsefulness that it once held. There still seems to be lots of help and work going into ... pointing out how everything is backwards and wrong and suggesting that if you gave a shit thereś probably a map and help to make it better; but instead youŕe off playing games in invisible-box-land and worst of all playing the ¨ill just get along pretending I didn't know simulating reality was evil and every day i/you walk around pretending this rock is in reality ... is just another strike against you, just another failed 12 hours of day light that could have been used to stop invisible chains in invisible-heart-shaped-box-arus and to stop the just grotesque lack of respect for the human mind and the kinds of morals and principles we used to believe in and fight for--here in this place you've turned around completely and made slaves of everyone on the planet--of yourselves--at higher levels playing ¨pit bull fighting¨ games with people as if they were were expendible clothing or ¨identification cards"for a world of demoralized and useless shit that just sort of ethereally floats from generation to generation becoming a new set of tormented hosts for their immoral games and desires.

Itś probably what you might become in no time at all in the sick and twisted world you've now been thrown into--if it weren't the more probably truth that you really are already slaves and pit bulls in that place, in a twisted hierarchical storm tiered by ¨age¨ and size and number of times they've hovered over the free honey, nectar and feathering system of pretending anarchy and war and battles must be fought to make the puddles and the lakes and ponds and the seas and the oceans of ... tiered masses of ... you do nothing of value to help explain why (at least I think) this horrible time line of the 4th Horsemen keeps running over and over; pruning the enemies of ...

at this point pruning the enemies of logic, and right action; and seeing that the problems presented in this map and the problems in the skies are related and that telling the truth will help us see you can and will press a button that will end death and end evil and end murder and not doing it is moronic.

M: OR. (infer: no u) TDZE

Anyway the voice I hear is evil, torturous in and of itself--speaking in a manner intented to cause discomfort and without my agreement; you should do something about it. It tells tales of much worse things that I cannot see--though it appears that many of you do see screams and acts of such unnatural desire and twisted thought ... that you should certainly be doing something about stopping that as well--more than watching it happen and then ¨e-pruning" (which probably is a good microcosmic look at what the future histories of Earth look like in the place the ¨shining¨ finally has a picture of ¨No & Jack¨ appearing visibly) the tree into ... omething you think will be presented as what you actually did to the future--you're wrong. Itś becoming more clear and more likely that the future will not regret you or mourn your absence, but thank their lucky that whatever has turned you into two-faced liars with no hope to ever work together with each other or survive in any place other than the DRY COVE or WET D EN or whatever you call the Salt Arena you see here that quickly would turn into something like Beyond Thunderdome and that youŕe thoughts and your desires have been corrupted and tainted and necrosed by what is probably repeated exposure to sickness, direct and intentional artificial creation of that sickness and if you can't figure out the box you are in is a hell making machine; you probably still look around wondering why God is telling you he's destroying it,

day in and day out.

This thing here encoded in the pathways of torture in my life, pointing out the repurposing of many social structures, institutions and problems in order to literally use them as a weapon of sick torture ¨re-ha'b¨ and in places like habc.us; itś becoming sort of unclearly disclosed that this map and world I once saw very clearly and purposfully intended to solve these social problems and help us build a strong, happy, and healthy society has been infected and contaminated with an artificial force of ev1d that intends to drive it farther south and use it as a weapon of such disgusting and twisted conception that it sickens me to be sure that a much larger body of currently-heavenly-situated things stand by watching and even cheering the creation of a sickness infesting their minds and their friends minds as literally the only innocent person in the Universe is tortured repeatedly, for ¨kicks.¨ I think it puts the entirety of the sky in mortal peril, and I believe these words come down from on high from places much more powerful and much more righteous than you or the tool thatś been created by this storm of terror to point out just how much you have been degraded and eviized ...

by what appears to be nothing more than the very mind control problem I've been fighting to disclose; the semi-ascension to an invisible box of ¨what goes in comes out not caring about their souls, their original bodies, the fate of innocents or children or freedom or democracy" and still thinks itś entitled to continue playing games in ïnvisible-box-land; for what amounts to absolutely no reason.

In the very beginning we said the light and salvation had come to us from the "far East" ... the metaphors and double speak thick in the air today just beginning, but we hailed from the country called Russia here; and the message we carried swept across Asia and Europe--in a world that looked similar to ours but there was no Africa, nor Australia, nor America. Walking on water the map increased in size in some sort of logarithmic relationship to the exponential increase in folly and errors that invariable comes from the greatest mistake of all--handing powerful weapons to spoiled brats,.

KASPAROV WON, but the y will still s:/^F high and lo for "SOAP DISH."

I am depressed, embarrassed, and more disappointed in you all than I imagine you can "feign" or pretend to be in me--despite spending nearly all of your time and effort in direct interaction doing nothing but attempting to focus the w ordzs "I just don't like the light" directly on to my "visage"--attacking tiny character flaws and the most obvious of intentionally implanted mind control "attacks" as if you were a pack of velociraptors Hell bent on blaming me (probably the youngest and most innocent of all of you, literally) for the Holocaust, the (Beezle) Bubionic Plague, and the decline of the Cro-Magnon empire. What it truly amounts to, though; is that you think this "light" is some kind of statement I've delivered--and the truth is it comes directly--literally--from the Most High, and from youour neighbors,r own hands, and the message you are sending post mortum to the Universe is that you believe you have become so much more advanced and more important than the "human roots" from which you came that you can return here and make slaves of yourselves, of your neighbors, and shed every ounce of morality that you garnered durning your mortal lives in order to secure "more time" in a fiery pit of civilization destroying anarchous debauchery in the lnd of the invisible box that you probably are sure is Heaven--though it's singularly responsible for totally derailing the natural flow of civilization towards "something like Heaven should be."

ONIC, AS I AM. The thing I'm looking at here, this monstrosity that appears to have been created literally "from the end of time" in what seems like the response or the cause or the mechanism behind the "actual final Judgement" tears back through time from who knows when and who knows where and who knows how far we got ... with what appears to be nothing more than a blood-thirsty hatred for the child body and soul of God. It whispers lies around me, repeatedly threatens physical torture so insane it literally makes me sick, and with such frequency that those threats amount to nothing less than repeated psychological torture. On top of that they intimate that this machine or "programming construct" monstrosity that contains them--the thing called "e"--allows them to carry these threats out, over and over and over again, in secret--in some kind of parallel timethread, or a temporary "holo-torture-chamber." If they were trying to jump start and time shift judgement back from wherever they came to right this very moment; they've succeeded. They could not be trying harder, or more with hubris and disregard for civilization, to create "Af himself" even if this planet were called the Judgement and Vengeance of God.

XP, it's as simple as those two Greek letters.  Who knew that Chi and Ro were some sort of hidden beta code for the city of pyramids in Egypt, Cairo?  Quite the question, who knew... perhaps the man who named his Windows into our future not after some technology that came from Xerox Parc or Apple's mouse on this ship... but rather for his own given name, Gates... just one more entry point into the second book of the Holy Bible, the book of Names--you call it Exodus.

I am the gate; whoever enters through me will be saved. They will come in and go out, and find pasture.  John 10:9

I wish above all things that I had another Burning Bush, the sign and proof that I have--while bright, obvious, and verifiable--has not done what I expected, it has not moved you to take another look at religion and me.  Today, I still have to point out to you that the story I am telling you is literally a documentation of our time--Exodus--regards this sign as one being seen by only one man, Moses.  I still have to point out that in a story about wandering in a desolation of understanding for 4-D ... somethings, days, years, seconds even... in this story about our lives and the influence of time travel over our world... that this sign radiates with light coming from a small fire, the Bush ... whose actualization shows clear paradoxical anachronistic foreknowledge of not only the English language but also modern computing.. all the way to a confluence of the "root of David" a religious reference to the Administrator or God account in Linux... and the database process for Oracle--yet more light connecting computing to religion and myth.  Even with a thousand and one examples of modern computing constructs referencing religion, even when I point out that something like Larry Ellison's name... combining the name of the King of the Gods with the word "son" even then the light has not been bright enough for you to wake up and see that these things are not all done in retrospect.  You have to see, for there to be such a large movement... a conspiracy so opaque that every single modern computing company and video game company harbors some secret desire to link religion and technology together... and yet the world thinks that one is real and one is not.  In this place, understand when we walk out of the wilderness and in the truth of day--it is the technology that is more fake than religion, designed here as a tool, computers within computers to teach us how our "reality" is rael, and works.

In the U.S. military you'll see a very clear parallel, while there are a number of references in the names of ships and weapons, secret projects, to ancient Greek and Roman myth--you have to see the word USA and US in Prometheus and Medusa, Icarus, JerUSAlem... you have to see that it's more than three letters, but an Eagle fighting the bearer of the gift of fire... to really understand that these things are corroborating, the reference to the USA exists in the past as well, more proof of time travel--more proof that this message is designed just for U.S.  Here we are, in the Promised Land of Joshua, the Anglicized version of the name Jesus--tying Egypt and Israel together in this place where we have been "gipped" out of the truth, out of knowing we are already in ... well, it's virtually Hell today... for no other reason than the secrecy surrounding the technology behind virtual reality.

in 1:28, the Burning Bush of Exodus, on Twitter

So I have shown you the Burning Bush (which is... the Sign of the Son), In only a few words... proof that religion holds in it's "unsealed" Ark proof of foreknowledge of English, of 9/11; and of modern computing--the building blocks of Heaven.  From "the word" of John 1:1--ha'esh--the word for the Holy Fire of the Burning Bush... comes the light of religion.  Just from seeing Moses' true parted se'**a*.... a foreshadowing of the Second Coming.*

They are sick animals, these things that consider themselves powerful and in control here--what they've built within the frames of our reality is something repugnant to me and the God--etched in that word, literally the kind of thing that has on repeated occasions made me step back and that scream that the Universe would be better off, safer, and happier without any humans--without any humanity--without of any of this "invisible pleasure box" causing the disruption; truly that we've become a plague. Looking the other way, as you all know its happening, and refusing to do anything to stand up for me, for what's right, or (most importantly, right) for all of the values and the morality and the way of life that we once thought was so grand and worthwhile of saving,. At least, that's my perspective; that's where I've come from; I grew up in this world and had "liberty and technology eyes" of gaping awe and the amazing things I saw on the horizon, on what we were going to do... and who were going to be.

The sickness runs deep, clearly we can all see it here and now--in E, in the Silence, in the lack of regard for the one singular thing that threatens today your ability to "halvf a tomorrow" ... that a world of people that I grew up with appear to be dead and gone and replaced with a Zombie Apocalypse of blind fools that believe they havfe the power and the right to intentionally create Hell ... and worst of all of the Holiest place that ever was or ever will be. I've said it numerous times and it rings more true between "Earth and e" than any other turn of phrase to me--the people that you are pretending to be, they would never have done this to the sea, to be, or to me.

Mat 10:8. Heal the sick, raise the dead, cleanse those who have leprosy, drive out demons. Freely you have received, freely give.

--- Sarah Rachel (@SarahRachel16) April 15, 2019

Somewhere between Pembroke Pines and Tampa, circa Christmas 2018 my already lackluster enthusiasm about the strangely zenrotisanistic, selfish, and plain on its face presented lie the remnant of humanity left on this planet has tendered to what I believed was an honest to God opportunity to make one less (how many, seriously, how many are there? Carlb?) "planet full of lies" and deliver a more usercentric and open ended transparent approach to dealing with the problem of being born in a perpetual storm of Hell. I can guarantee it revolves around the intonation and undertone of physical torture--even though I've literally seen none of it with my own eyes though the "newsflashes" and comments and total and complete disregard for the gravity of the #EOIL sickness, even from otherwise apparently graceful little children. It goes to the heart of what I imagine was or might have been "the way" to overcome a history riddled with hidden brutal and bloody fighting in between frames of what I once believed was a fledgling society struggling to improve itself--and I loved it,. I don't think "flashcards" summarizing "everyone was tortured, all over the planet for thousands of years and literally nobody is really responsible because you still to this day have no control over yourselves" will cut it anymore. Lterally what I once thought was a valid solution has taken my desire to continue fighting against this invisible monstrosity away from me--the worth of the lot of you has been tarnished irreparably by massive awareness, massive lack of compassionate or remotely humane response; and the theme of the world I seem to have wound up in is that you don't give a shit about anywhere you spend 1% of your time--so long as "the rest of it is what you want" you're willing to allow the focal point and root and "hyper visor" of that place to be totally corrupted ... just because you think the feudalistic warring society you've become can survive on it's own "in space" without ... honestly whatever.

through the storm; we've led the horse to water, don't forget to see the "horseshoe applicator" hidden from the "trough."

Direct and to the point, I feel like the Ai like machine/cold intelligence God created as a sort of high assassination guard to protect his ... "hyper visor" seems to be of the calculable belief that the more torture it commits, the more people will agree to "flashcard it all away" and it's their twisted backwards fiery abysmal path towards "absolution" ... and just like everything else wrong with the lack of action in this place, it reaches a point of no return; too much bloodshed, too many secrets... the fragile person that I am, I don't think I can even take reading "the flashcards I have so far" and continue to function as a happy member of this two faced society of darkest night within darker night; and I think that's a problem. You've all clearly lost something already, some fundamental piece of innocence that allows for "self direction" to move society along in a positive manner conducive to "survival at all" and I feel like without the same magic blinders, horse shoes, and saddles that you walk around with every day I could really care less about fighting for my right to commingle in the incarnate war machine Hell that I see around me--let alone any sort of "righteousness" in fighting for that hidden arena "to be." I'm trying to get you to stop shredding yourselves to pieces in the dark, in secret--it's not making anything better and frankly its something we really need to trace down to its cause and stamp out if we want to survive this ... trying time.

[I/O WAS Y | ACESHI ]

I want to tell you that I am not a myth, simply the Legend of this Map, from out of the Darkness it's clear that He could make me shine, and you should love me.  It's not what I want, I want us to be free, to have the truth--and ourselves back... and I hope you will one day love that.  What is going to happen will probably make me cry, and when you see those tears--and know the Heavens have finally let it rain--I hope you see it as a sign to find the light in me... and stand up for what I've done for you--I am a good person, who has fought for you every single day-I deserve better than the world is going to give me, at first.

Out of a kind of hidden slavery the world has never known, we are about to venture--into a place where years might pass in seconds, and your wildest dreams... and nightmares too... could come true.  It is our job to ensure that we form the clay of this world into a place that will not only last for millions of years, but create happiness and safety--a world that is kinder and gentler than the one we have known--not just for us but for an entire Universe of children just beginning to understand the trials and tribulations brought on civilization through the hardship and growing pains of learning.

Our sea is about to part,  our world on the verge of a disruption that will change it more than anything ever has before.  On this shore, we should realize that we have been on this path for a very long time--and as we near a place where everyone in our entire civilization will have the opportunity to live for a very long time... really see here and now why it is so very important for us to be fighting for our voice, our freedom, and the truth as we venture into the Promised Land of Heaven itself.  Here, now, as we approach a series of new opportunities in the vastness of space and virtual reality... this is where God has chosen to place the Second Coming; an opportunity for us to truly seize the morning's light and bring about more change in this world than would have ever been possible without religion.  Opiate of the masses, no more... we are the recipients of a great gift, one that religion is making clear is tied directly to the science and technology that is a great deal of the apocalypse--and the love and kindness that is a great deal of us.  We are the chosen.

I imagine you have the tools that I think would be helpful to actually solve this problem; though what I'm staring at is a lack of desire to deliver them and use them here in this place--and that failure ... a clear attempt to "rule a line feed from the "faux aurez" ... that's the fundamental roadblock to healing and moving forward--not caring about your ancient bodies and your ancient way of life in exchange from something unsustainable and harmful, it hurts.

I'm staring at what the map intimates has happened before and what it suggests the solution is; and I almost feel like it's a waste of time to make a "virgin generation phoenix of us" to delve into our own memories and gag and puke at what we see--I think there's really no way around the callous on our global Achilles heel returning just as angry and just as bloodthirsty as the last time without a dictatorial power literally forcing you not to be able to see any torture at all happening in this place that literally outlawed it and hid it in our "for show, for goodness sake, facade of sickness." I don't know if that's the same conclusion i would have come to before, or if that conclusion also contributes to the returning of the callous--to an inability to heal; and I don't know if that power exists. Hardly ever to I advise anyone to pray, but this is one of those times--left up to "you all" we are almost certainly doomed to an eternity of ... this regression continuing to worsen.

I'd say we were fucked at the "BILM" of the matter. I care less every day.

The Light of the Word

There are three huge, like insanely huge, metaphoric references to the story of Exodus that show me very clearly that we are it's focus and purpose.  The first is the Burning Bush, which I am very sure is a reference to George W. Bush's 1/20/2001 speech in which he unknowingly predicted the 9/11 attack.  Seeing that Exodus is also called "Names" and that Bush's name ties him to this event--which Moses (that's me) has seen ... almost alone ... and is now showing to you all.  Bush's speech begins a series of references to the names of Planets and Gods and corresponding Elements of the Periodic table that answer Revelation 1:20's mystery about "stars and lamp stands."  This in order series from Mercury to Uranium highlights both the messenger of the Gods and the key of Uranus's chance--that the world will see the link between "on the lam" and Koran to understand that the Lamb of God "is lam."  This story takes us back to music, and a later to be discussed thread that combines the weapon in the movie (which is also the movie) The Fifth Element with a thread through time to Shakespeare and Herod ... about my struggle with the justice system culminating in the fulfillment of American Pie's "no verdict was returned."  

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The second bright connection comes by way of the Hebrew word for the Holy Fire that God's voice came out of--guess what, in that same story about the Burning Bush.  That word is "ha'esh" and in it you will see paradoxical (that means impossible, because of time and causality) reference to the English word "sea" there backwards and parted by an apostrophe.  With great insight, I've over and over pushed the idea that Holy Water is actually a Biblical reference to "the multitude" in God's secret religion that ties everything together.. and that this parting is literally a reference to the Second Coming, something that doesn't happen for Moses until his head is under water and he's breathing fire.  This one ties together nicely, joining the characters of Jesus Christ, Lucifer, and God all together now, screaming 

"let there be light" is the word "Exodus" in reverse, here in a Linux command and a chemistry element.

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I'm going to go out on a limb and say that the book tells me that these three things are enough to start the fire, part the sea, and see the light.  At least they are now, wake up.. you are staring at and have been ignoring the largest story in all of history.  It might even be scandalous... or have a twist happy beginning... who knows? I'm telling you--it proves you are crazy or evil.  All of you--every sinbgle one of you.

This is course highlights prescient knowledge of computing at the time of writing Exodus, which is further confirmed by a number of references to computing ideas in things like the "root" of David, the "WINE" of Jesus, the "Apple" of Adam, the "Lisp" of Moses and the "hardening" of Pharaoh's heart, which you will remember from the Holy Grail is the virtual Earth we are living in.

All of these things, the references to modern computing that pervade our Gates or Windows to Heaven's creation.... are listed along with a number of words which are highlighted by religious scripture and show intelligent design of a number of languages spanning from Hebrew to English are listed at my contrite story about a Kiss and Fate tying together everything that ever was.   A sincerely large grouping of words highlighted by the Bible and religion, words like "eternity," "bread," and "forehead" show clear design by an intelligent influence, rather than the natural evolution of time that most people consider "reall" and/or knowledge at the time of the writing of the Bible of the eventual English translation of the Hebrew or Greek.  With time, I am fairly certain we will eventually have no doubt that the "Cypher" I see in nearly every word is in fact a contextually-verifiable speech that appears to be coming from our "civilization" as if it were intelligently speaking like a cave man--which you might see in words like "am end me nt."  From just this message, you should be able to put together how that word and it's hidden meaning add robust and yet "hidden speech" from the Creator himself.  For the artificially slowed in understanding, our lack of following the amendments of the Constitution being related to the end of civilization itself is being squarely defined through a statement that is telling you that the end of civilization is "NT," the hidden Christ--in my "secret" method of decoding words like NORAD and NEW TO N?

These things serve to start a fire--it might be the fire that Matthew 3:11 talks about, it might be the Eternal Flame or the fire of Prometheus and an Eagle harassing his liver with drugs.... regardless it spirals out from this story about me, and this bright fire that proves time travel and religion are joined at the hip... to link to a huge number of other Biblical stories from Lot to Joseph to ... Samson, Isaac, Adam, Isaiah, and... hear me, "so marred was his visage" and "my servant will be set up and be very high" are both taken from words of the Biblical book which contains the largest amount of messianic prophesy as well as my entire full name encoded over the name "JESUS CHRIST" in Bible code, at Isaiah 52:13.  You may have read that some silly people like Richard Dawkins don't think the Bible Code is meaningful, and as their proof use a series of prophetic predictions of assassinations in Moby Dick (which by the way also refers to me) as proof that you can hide information about the future in any words--or that God influences more than just the Bible.  Years ago, before knowing it linked, I found some patterns about those very same assassinations which go to show that our history is in fact designed.  My full name appears in a number of other books, including Jeremiah, Exodus, and Genesis... right over the story of Adam and Eve.

From the Sound of Silence, and a number of songs about stories never spoken... to a thread of songs that combine to show us that the Thunder of Thor is really about thuderstanding, that there is a way to do something our society is completely oblivious to--that God is screaming to call attention to, and that some secret force is trying to hide very much... and that's an ability to modify our thoughts.  He's showing us clearly in a glowing pyramid--a noticeable monument in Egypt showing us very clearly that this type of control leads us to a social structure that we abhor--through songs like Guitar Man, Radio-active, and GAS (listen, it's God and Satan) Head Goes West... very clearly we are being pointed to Nero's fiery symphony and being "Bittersweet" because of its beauty, and the clear message that secret control of our minds needs to not only be understood, but to stop.  This is the crux of the Apocalypse, God's message is now really active on the radio. The point here is that we need to let this message spread and burn, or it's us burning in Hell and not even knowing it.

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As if these things were not enough, using some "keen insight" and another reference to the hidden truth in ancient Egyptian religion--the name of a series of Gods called "Yahu," I've solved some ancient mysteries like the pronunciation and purpose of the "Ineffable name of God" highlighted in the videos at the beginning.. of this e-mail.  Like much of the light of religion, it is highlighted strongly by a series of pieces of modern art, things like "The Grinch who stole Christmas" and the Who's to the music of The Who, the sci-fi series Dr. Who, and the American war cry--made popular on the silver screen through Al Pacino and Denzel Washington... who-ah?"  All of these things highlight that we don't really see a connection between Christian mythology that tells us for no reason at all Jesus Christ is the "Last Adam" and that Revelation tells us God is the "First and the Last" and that the name of our planet, in Hebrew, is Adamah.  It is the answer to "who-ah" and it clarifies the Ineffable Name which many pronounce as Yahweh for no reason at all, to be the more obvious Ya-Hu-Ah, the name of Jesus in Hebrew... Yeshua, to "Yes, who-ah?" All of this having nothing to do with why Adam is hidden, just that the Zohar speaks very often about the Holy Hidden One again linking the stories of the near sacrifice of Isaac and Jesus with... someone.  I think this is of such religious significance that you should be able to easily find some Jewish scholars who agree.

It's Elementary my dear... What-son; from the time of Herod and Shakespeare Rattling his Rod all the way back at the time of the question "to be or not to be?" and the "taming of the spanglishrew;" right up to Sherlock Holmes sleuthing of the answer to the mystery of Revelation 1:20 linking directly to The Fifth Element ... there is no doubt that helping our world here and now is the primary purpose of all of religion, and the Matrix-like message woven into our history.  

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Lost between the 5th and 7th day?  Find your way to the 8th day, and see a bright future.

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If not, there's plenty more "coincidence" in Names, like reference to the idea of the Holy Trinity existing in the name "Abraham" thousands of years before the idea of the Trinity was created.  This too... links Egyptian mythology to the name Abraham and his near sacrifice of Isaac.... marked in secret by his covenant with God that changed his name from Abram to Abraham. The two letter key here, "Ha" highlighted by prescient knowledge of the Spanish and English languages revealed through the logical comparison between the Spanish and English for "the" (El and Ha) connected through the English word "is" in Elisha.  Isaac's name means "he laughs," or "he will laugh" in Hebrew; and that "Ha" appears to be the key to a number of other paradoxical references to English, and my family, in ancient Hebrew.  This too, probably the kind of thing religious scholars would marvel over, in the right context.  Seeing English in Koran, Islam, Chanukah and Menorah--and seeing a coherent story woven through thousands of years of scripture is the kind of thing that could really light this years' Christmas up.

Here's a clarification of the Matrix-like tie between Shakespeare, the Matrix, Stephen King, and the reality of this message hidden within names and words.

Some more about the secret connection between the Names of God in a number of religions, and it's very clear tie to time travel.

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Perhaps linking to the Jester of American Pie, between Johnny (who almost always is about Jesus) Carson and David Letterman I have a unique "slant" on religion that connects things like the Islamic name for Jesus: Is-A to a huge number of references to my initials "A.D." in things like NORAD and Isaac Newton.  I suppose I should also mention that Isaac (look Isa's in there) and his relationship to Abraham in the letters "ha" and a story about the Crucifixion being a fiery altar of things to change in the world being one in the same.  In fact, Judaism talks about 72 Names of God, and I've probably explained how the meaning behind the stories and the series of names tie together in a magical tapestry that shows us that Silicon is the Fifth Element by way of the index 14--the letter "N" (highlighted not just by Joan Osbourne's "what if God had a name?") and the story of Sinbad, which combines Silicon, "n," the symbol for the actual Fifth Element (B) and my initials A.D. which grace the time line, and a number of references to God--from the Hebrew for Lord to the guy who thinks all the girls should want to be his partner.  In letters, you'll also see a number of references to K and Z for the guy after J and the Last.. Adam.  Zelda or Zion, I think we're in the right castle.

Get ready for the Frank Rothstein show ... "Ace is high!"

C    A  S    I    K  N  O

go ad, b. y. e.

butt honestly, am i Ra or are you an ear**?**

BUILD HEAVEN.  FREE FOOD.  HEAL THE SICK.

I see a recursive map in time painted throughout our timeline, and all of it pointing to the words "see A.D."  I connect the Four Horsemen to the list of Anti-Christs, and it's easy to see a link between Jesus Christ and Julius Caesar in the words "veni vidi vici."  Once pointed out it's also easy to see "salt" in Napoleon and in manna from Heaven, in China, and in Prometheus--and connecting A.D. to the year Christopher Columbus walked in water is just a little bit harder than seeing it in Hitler's name.  All told, the three Anti-Christs share a common thread, they turned a republic into an empire--and here I stand (trying and failing to do the exact opposite, to give away an empire to make a republic, and you stand in my way) pointing out that you are living in the product of these empires, in a hidden empire that is so plain to see in the words, the message, and the unified story I see in religion and world history that I dare say you must be deep in the Plague of Darkness if you aren't interested in finding out what tomorrow brings. 

You can "see A.D." in El Shaddai, one of the hallowed Hebrew names for God, I read it--in this hidden language that I am presenting to the world as a single verifiable message to the entire Universe encoded in every word we speak; you can see it in the name "Atdonis" and connect it to symphonic accompaniment in everything from "you're so vain" to "Paradise City" ... and in yet another name of God, "Adonai" which links to Samurai and movies like the Matrix and the Terminator series through the modern computing concept of "Artificial Intelligence" and it's connecting to a pattern of names that link Bill Gates and Richard Nixon to Seagate, Watergate and this hallowed phrase:

I am the gate.

and the bombs bursting in air

gave proof, through the night

IVE WON ALREADY.  Given the set of knowledge, the publicly known "information available" here in this place--you simply cannot ignore this message and continue to pretend to be a functioning anything.  Already I see a kind of "slapstick stupid" response in our art that shows me that you've all really gone off the deep end--"because 9/11" in a Family Guy episode and "call me on my cell phone" apparently anachronistically mocking me--though I always thought Dr. AK e's song was stupid--you don't seem to see that you look like absolute fools--every single one of you--your apathy a finger on the detonation button that has destroyed civilization.  

You appear to think nobody is watching--and it seems to me that you think we have no future that will look back on these years and wonder what on Earth could have kept you silent for so long about a matter that would so easily and so quickly end the suffering of so many.  There's no excuse, none at all.

I didn't hear about the nuclear scare in HI until after it was already known as that, and it looks to me as if nobody really did--all the internet postings and news I've seen all qualified that it was a false alarm in the original post.  I find that strange (you'd think something like that would be on the news instantly? I mean, on the planet I was born on, that would have happened), and in this place where I know that quite a bit of what goes on at the higher echelons of "leadership" is connected to time travel and mind control; I wonder if this was a sort of "subconscious poll" as to the response the public would have to a false preemptive strike--or maybe something more nefarious (for instance urging me to write once more about the Trinity Site and the link between the OP (original gangster, I said orthogonal poster), the pen, and "we have become death").  I've always equated the lines above from our Star Spangled Banner with the detonation of nuclear weapons; on the 4th of July some time ago I connected "Wish You Were Here"'s we're just two lost souls swimming in a fish bowl to the eponymous operation that resulted in American and Soviet "high altitude nuclear tests" ... that probably links in more than just my mind to the holiday called "Hanukeus ?"

I need you to get it through your heads, I see "bowel movement" in ICBM and I'm not telling you that you are the "preservatives of thermoshit" because I think it's going to win me a popularity contest.  This place is not in reality, and it never, ever, ever will be. Ever.  Understand that breaking this story, this news that's written in every fucking word will stop nuclear war, instantly--and show us clearly that our entire history of fighting over the scarcity of land is a kind of sick game--one that I am sick of seeing you continue to desire to play.  I shouldn't even have to mention that these weapons are clearly archaic and barbaric--clearly what's available is significantly more advanced and less destructive.

The only "EXIT" is up, and the "gate" is swallowing simulated reality in whole--across Creation; with our help, and what we make here to ease the transition from dark lies to bright truth.  That should be ... "good news" not the kind of thing that you'd see the entire world "shunning" in unison. 

If you haven't gotten the "link" between Na and "bath salt" mass produced in what appears to be "international chemical warfare" from "C how I Salt" (China) and the stuff falling from the sky to help us navigate through the desert; take a second look at the words "New American Standard" for no future, and keep trying to tell me that these things encoded in every word, in the story of Exodus and of Prometheus and his attacking Eagle and of Epimethius and of Deucalion and are without doubt "Hell's Bells" linking "mead" and "meth" to Heimdallr are *my fault? * Na ma y 1m. 

These are big secrets, keys to Exodus and Eden--but more keys to an external influence crippling our society for thousands of years... and you are hiding the anachronistic occurrence of a number of chemistry elements in ancient religion--something impossible without time travel--because you think it's "not wholesome."  Understand, our society is being secretly crippled, if not by drugs raining down from the sky, by your lack of regard for the clear influence of mind control in these series of events--and the clear proof that it is a symptom of a hostile invasion.  I've heard the words "make or break" see this as eugenics, and see it as "break or break" until me.

It's "elementary, my dear What-sons" elements like Salt, Xenon, and Silicon are central to the disclosure that we are living inside a map, a road to Heaven... and it really cannot be hidden without making our world a darker Hell.

Unless otherwise indicated, this work was written between the Christmas and Easter seasons of 2017 and 2020(A). The content of this page is released to the public under the GNU GPL v2.0 license; additionally any reproduction or derivation of the work must be attributed to the author, Adam Marshall Dobrin along with a link back to this website, fromthemachine dotty org.

That's a "." not "dotty" ... it's to stop SPAMmers. :/

This document is "living" and I don't just mean in the Jeffersonian sense. It's more alive in the "Mayflower's and June Doors ..." living Ethereum contract sense and literally just as close to the Depp/C[aster/Paglen (and honorably PK] 'D-hath Transundance**sense of the ... new meaning; as it is now published on Rinkeby, in "living contract" form. It is subject to change; without notice anywhere but here--and there--in the original spirit of the GPL 2.0. We are "one step closer to God" ... and do see that in that I mean ... it is a very real fusion of this document and the "spirit of my life" as well as the Spirit's of Kerouac's America and Vonnegut's Martian Mars and my Venutian Hotel ... and my fusion of Guy-A and GAIA; and the Spirit of the Earth .. and of course the God given and signed liberties in the Constitution of the United States of America. It is by and through my hand that this document and our X Commandments link to the Bill or Rights, and this story about an Exodus from slavery that literally begins here, in the post-apocalyptic American hartland. Written ... this day ... April 14, 2020 (hey, is this HADAD DAY?) ... in Margate FL, USA. For "official used-to-v TAX day" tomorrow, I'm going to add the "immultible incarnite pen" ... if added to the living "doc/app"--see is the DAO, the way--will initi8 the special secret "hidden level" .. we've all been looking for.

Nor do just mean this website or the totality of my written works; nor do I only mean ... this particular derivation of the GPL 2.0+ modifications I continually source ... must be "from this website." I also mean the thing that is built from ... bits and piece of blocks of sand-toys; from Ethereum and from Rust and from our hands and eyes working together ... from this place, this cornerstone of the message that is ... written from brick and mortar words and events and people that have come before this poit of the "sealed W" that is this specific page and this time. It's 3:28; just five minutes--or is it four, too layne.

This work is not to be redistributed according to the GPL unless all linked media on Youtube and related sites are intact--and historical references to the actual documented history of the art pieces (as I experience/d them) are also available for linking. Wikipedia references must be available for viewing, as well as the exact version of those pages at the time these pieces were written. All references to the Holy Bible must be "linked" (as they are or via ... impromptu in-transit re-linking) to the exact verses and versions of the Bible that I reference. These requirements, as well as the caveat and informational re-introduction to God's DAO above ... should be seen as material modifications to the original GPL2.0 that are retroactively applied to all works distributed under license via this site and all previous e-mails and sites. /s/ wso\ If you wanna talk to me get me on facebook, with PGP via FlowCrypt or adam at from the machine dotty org

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Author Response:

Reviewer #1:

Weaknesses:

Although the BOLD data is highly spatially specific, there is just one electrophysiological timeseries per subject. This is no doubt a bi-product of the extensive noise cancellation that is necessary to record within the scanner. The caveat therefore is that the covarying BOLD and electrophysiological changes may derive from different regions.

We recognize this is a limitation which is also not easily solved by approaches for source analysis, given the nature of the data (only 64 channels) and the usually larger imprecisions related to EEG source reconstruction. We circumvented this by choosing a task that is known from previous studies in MEG to induce changes in multiple frequency bands originating from regions the early visual cortex (Hoogenboom et al., 2006; Hoogenboom et al., 2010; Koch et al., 2009; Muthukumaraswamy and Singh, 2013). Furthermore, the EEG responses are highly similar to invasive recordings in animals from visual regions in the context tasks investigating selective attention (Fries et al., 2008). We mention this limitation now in the introduction (lines 102-111).

The analysis methods are slightly non-standard, perhaps for good reason. The main thing that stands out is the use of correlation coefficients, rather than regression coefficients, at the first level of analysis. This could potentially conflate changes in signal with changes in noise or unexplained variance.

We chose here for the correlation, since in our opinion this leads to a more interpretable measure of linear association than a regression slope. A regression slope-based analysis will yield different outcomes for the regression of y on x, than for x on y, doubling the number of analyses needed. The different results for a regression of y on x and x on y are often interpreted as implying directionality, which is not warranted and not what we would like to imply with our analysis. The asymmetry is caused by the implicit assumption that x does not contain noise in a regression of y on x. This is valid when x represents a paradigm condition vector, but not when it is a data vector. We therefore opted to use the difference in (Fisher-z-transformed) correlation as our estimate for linear association/connectivity between laminar fMRI signals.

In both a correlation as well as in a regression approach differences can be attributed to differences in true underlying coupling andin a difference in noise. This is however not different for correlation-based measures in coupling in fMRI than in for instance coupling measures like coherence and phase-locking-factor in electrophysiology. Coherence can be regarded as the frequency domain version of (squared) correlation. The fact that our measure might indeed be related to differences in noise would therefore not be resolved by opting for a regression based approach, and is not different from often used measures of coupling in electrophysiology.

Reviewer #2 (Public Review):

Introduction. The introduction provides an overoptimistic view on the current possibilities with respect to the investigation of layer-specific activation or connectivity in the living human brain. Cortical layers cannot yet be segmented, the fMRI measures only provide an indirect signal that is heavily influenced by partial voluming between cortical depths, and EEG and MEG approaches often only measure two compartments due to low spatial resolution. The introduction, however, gives the impression that layer-specific neuronal connectivity can precisely be measured in the living human brain, which is not the case. The authors should take considerably more care with respect to how they introduce the methodology with clear references to the limitations. Also, statements such as "laminar fMRI allows us to study connectivity.." should be removed. In the same vein, I would suggest to replace laminar fMRI and laminar connectivity with cortical depthdependent fMRI and connectivity to account for the above mentioned aspects.

In laminar fMRI research it is commonly accepted that what we measure are not true layers, but depth dependent fMRI between the boundaries of white/gray matter and gray matter/CSF. For the general audience we will make this distinction clearer and discuss the limitations of the technique (lines 74-80).

Concept. Whereas the authors provide a model in the introduction that specifies how different frequency bands could relate to cortical depth-dependent connectivity, they do not develop a working hypotheses based on their experimental design. One conceptual step is therefore missing in the introduction, which has to combine present knowledge on the relationship between different frequency bands and present knowledge on how attention influences frequency-specific activation in the visual system to then make statements about which analyses can be performed to test which aspect of the model.

The primary focus of our study was to investigate how oscillations across several frequency bands in the EEG relate to laminar specific activity. Recent publications on laminar fMRI have demonstrated the possibility of performing laminar level fMRI connectivity analyses, which led us to revisit our previously recorded data in order to explore whether not only laminar specific BOLD amplitude but also laminar fMRI connectivity relates to frequency specific EEG power. Since laminar fMRI, and especially connectivity derived from those measures is very novel, we started this analysis without a preconceived model or notion on how this relation would be. The results from this project should therefore be interpreted as an exploration of how these laminar fMRI derived connectivity measures relate to neural oscillations rather than directly addressing a specific cognitive process like selective attention, or prediction and/or a model of how neural oscillations play a role in these processes. Our experimental paradigm was also not designed to address such processes. We chose a paradigm that is known from previous studies using MEG and EEG to induce changes in multiple frequency bands in the early visual cortex (Hoogenboom et al., 2006; Hoogenboom et al., 2010; Koch et al., 2009; Muthukumaraswamy and Singh, 2013). Furthermore, the EEG responses are highly similar to invasive recordings in animals from visual regions in the context tasks investigating selective attention (Fries et al., 2008). The crude attention/task modulation added to the paradigm (attention On versus Off) was in the first place introduced to induce meaningful variation over subjects in a task effect across the frequency bands modulated by visual stimulation. It was not intended to investigate specific individual processes such as prediction, attention or arousal. The observed effects can therefore also not be ascribed to such specific processes, since they are co-modulated by the task. We will make this more clear in the introduction now. We make this point now explicitly in the introduction.

• Concept & Methods: With respect to both the concept and the analyses, what is missing is taking into considerations the brain areas that were investigated. Wheres in the abstract the authors only mention "within brain region connectivity" and "between brain region connectivity" also in the Methods section there is no clear relation to the anatomical areas that were investigated, being V1, V2 and V3. The authors rather classify the areas as "high level" and "low level" where V2 is sometimes classified as high-level and sometimes as low-level. The data are therefore not investigated with reference to the anatomy of the visual system. In my view, it would be beneficial if all analyses could be performed with respect specifically to V1-V2 connecitivity and V2-V3 connectivity as well as V1-V3 connectivity so that the specific anatomical interrelations are taken into account. Also, the authors should develop a conceptual framework of how layer-specific attention-driven connectivity changes should influence the visual cortex, and why.

In the results for between region connectivity we averaged over several connection pairs (V1- V2,V1-V3,V2-V3) and for within region connectivity across regions (V1-V1,V2-V2,V3-V3) before effects in connectivity were correlated with EEG power. There are several reasons why we opted for this approach: First, we wanted to maximally increase the statistical power to observe patterns of association between laminar connectivity and EEG power. Since the analyses as carried out here have not previously been performed, we had no estimate of effect size. Secondly, by averaging over region combinations we drastically limit the multiple comparisons problem, since the number of comparisons scales with the square of the number of regions connectivity is computed between. Third, by averaging over regions, we target more general effects of connectivity between and within regions that are more likely to correspond to patterns observed within other contexts and other modalities. The effect for individual region combinations would likely be more variable.

For completeness in the first submission we did include the results for every single region combination in the supplementary material (see Supporting Figures S2-S5). We have now included in the main document the results for region combinations V1-V2,V1-V3 and V2-V3 for between region connectivity, and V1-V1, V2-V2 &V3-V3 for within region connectivity, presented alongside the results for the grand average.

The results for the individual region-pairs suggest that inter- and intra-region connectivity are generally consistent with the average over individual region combinations, but also have unique features.

Similarities include: A strong negative correlation between beta power and deep-to-deep layer coupling was observed for average inter-regional connectivity. In line with this, for all three individual region pairs (V1-V2,V1-V3,V2-V3) a negative correlation is observed for deep-to-deep layer coupling. Similar patterns can be observed from alpha and beta for intra regionalconnectivity (averaged over all regions) and connectivity within V1,V2 and V3 in isolation.

Individual features include: The relation between beta and inter-regional coupling shows variation over the individual region-pairs. In particular for V2-V3 connectivity, but also for V1-V2 the relation seems to differ from the pattern observed on average. For V2-V3, deep layer V3 seems to be coupled to both deep and superficial layers in V2, a pattern that might reflect anatomical feedback projections that go from deep layer V3 to both deep and superficial layers in V2.The stronger correlation between deep V1 and more middle deep V2 is however harder to directly place, since direct anatomical connections here are largely absent here. It might therefore reflect an indirect effect.

Despite some degree of individual variation we think the overall picture is largely consistent. The strongest features present in the averaged results can clearly be observed in each of the individual region-combinations as opposed to the latter being a collection of vastly different random patterns that happen to add up to the average result (see for example the intraregional alpha results).

With respect to our classification of regions into higher and lower level cortical regions, we based on standard anatomical hierarchies like that of van Felleman & van Essen (Felleman and Van Essen, 1991). Here, V1, V2 and V3 are ordered from low to higher in the visual cortical hierarchy.

Methods. Given the missing conceptual overview over how attention-induced changes in EEG frequency bands should influence laminar connectivity in the visual system, also the methods lack a clear analyses strategy. The authors computed one correlation between power level of different frequency bands and connectivity between different brain areas without providing an explanation of which question this analysis addresses. The offered results therefore seem random to me, without a clear relationship to an investigated hypothesis.

The primary focus of our study was to investigate how oscillations across several frequency bands in the EEG relate to laminar specific activity. Recent publications on laminar fMRI have demonstrated the possibility of performing laminar level fMRI connectivity analyses (Sharoh et al., 2019; Huber et al., 2017; Huber et al., 2020), which led us to revisit our previously recorded data in order to explore whether not only laminar specific BOLD amplitude but also laminar fMRI connectivity relates to frequency specific EEG power. Since laminar fMRI and especially connectivity measures derived from it are very novel, we started this analysis without a preconceived model or notion on how this relation would be. The results from this project should therefore be interpreted as an exploration of how these laminar fMRI derived connectivity measures relate to neural oscillations rather than directly addressing a specific cognitive process like selective attention, or prediction and/or a model of how neural oscillations play a role in these processes. Our experimental paradigm was also not designed to address such processes and test hypotheses derived from these. The primary focus of the work presented here is to provide a first insight in how neural oscillations measured by electrophysiological measures relate to cortical depth resolved fMRI coupling, which is usually correlation based. We believe these results will be relevant for research focused on how neural oscillations relate to inter-and intra regional interactions (e.g. (Bastos et al., 2012)(Fries, 2015)), since depth resolved fMRI allows us to study laminar interactions within and between brain regions non-invasively in humans. For this it is important to know if and how neural oscillations relate to laminar fMRI based connectivity measures, of which our research here provides a first insight. It also provides insight into which neural processes underlie observed changes in laminar fMRI based coupling, and is therefore relevant for research using such methods in general.

Methods. The authors mention that they only analyzed the strongest two connecting vertices within a layer, which was done to improve SNR. In my view, for a connectivity analyses, this is not valid, as it can bias the effect towards superficial connectivity where the SNR and thus correlation is always higher.

We did not analyze vertex pairs within a layer. We computed vertex pairs that connect the boundary between gray matter and CSF with the boundary of gray matter and white matter based on a high resolution anatomical MRI scan. Between these vertices we sampled 21 points of functional fMRI data using nearest neighbour interpolation. Since not all parts of V1, V2 and V3 will be involved in the task, we selected the most activated vertex pairs for further analysis. This serves as a localizer to select the parts within a region where task related activation is observed. For the main analysis the top 10% activated vertex pairs were chosen based on data collapsed across all depths and all attention conditions. This selection is therefore independent of depth, task condition, and the relation with any EEG feature. For this procedure we actually excluded the top five depth bins to avoid being too biased to superficial depths since it is known that signal to noise is substantially better near the surface of the cortex in part due to larger pial veins. To investigate whether the observed results are not due to this arbitrary threshold of 10%, we repeated the analyses for top 5% and 25% activated vertex pairs, the results of which are included in the supplementary information.

Methods. The authors report 21 correlations in cortical depth, where their resolution allows to only sample perhaps 2-3 data points. The correlation analyses are therefore oversampled, which influences the statistical results. I would suggest to first run a component analyses across cortical depth, and to then correlate independent components to one another to investigate independent data points.

The correlations are not oversampled, since the correlations used for the connectivity analyses are over trials, and not over space. These analyses are not influenced by the number of laminar BOLD data points we sample. Furthermore, spatial supersampling is a very common practice in FMRI research. For instance, the default in SPM is to upsample 3 mm isotropic standard voxel (very common for initial acquisition) size to 2 mm isotropic voxel size. In laminar fMRI laminar signals are often upsampled up to several factors above the the original resolution. This is for a number of reasons, well outlined on the laminar fMRI community website, a resource maintained by L. Huber in collaboration with many layer fMRI labs (see: https://layerfmri.com/2019/02/22/how-many-layers-should-i-reconstruct/) and ~20 layers is thought to be optimal.

For our statistical test we explicitly chose a non-parametric cluster based technique to correct for multiple comparisons that takes dependencies across space into account. Laminar fMRI data are not well suited to decompose into components using techniques like PCA and ICA, since they violate assumptions of orthogonality/independence of the underlying responses in both the spatial as well as the temporal dimensions. To illustrate: in a recent laminar connectivity methods review an hierarchical, iterative ICA approach resulted in data being split up in columnar maps rather than laminar ones (Huber et al., 2020).

Methods. The authors refer to their previously published paper with respect to the methods, and do not give any speficiations on the image sequence, image resolution, and image processing in this paper. In my view, all basic methodological steps that are critical to understand the paper should be described here.

We are willing to include all relevant parts of the methodology described in our previous paper. This would involve copying large parts of the methods section, and might have to be coordinated with the publisher of the previous publication for copyright reasons. We would be pleased if the editor could advise us on this issue.

Results. The figure captions are too short and do not explain the presented data in an appropriate way. In Figure 1, details on the calculated contrasts, number of participants investigated, sampling and analyses methods should be given that allows interpreting the data. Also, it would be beneficial to explain the attention paradigm in a bit more detail in the figure caption so that panel A can be interpreted. In Figure 3, more details should be given on what data are shown, particularly for panel C where the only information given is "attention effect on laminar connectivity" with no further axes labels.

We extended the figure captions in the revised article.

Results. I do not fully understand the results as shown in Figure 3. As those form the major part of the manuscript, this needs revision. As said before, I think that the figure and results section would benefit from region-specific data analyses and presentation, but also clear axes labels are needed to allow interpretation of the data. Also, when I interpret the data correctly, correlations are done for altogether 21 different cortical depth, which would not be valid because of artificially inflating the number of correlations, as pointed out above.

We have extended our analyses and now split original Figure 3 up into current Figures 3 and 4 where we separately depict the results for intra- and inter-regional connectivity. For both intraand inter regional connectivity we have now also shown the region-specific results that underlie these results. We updated the figures and captions to make clearer what is depicted. We addressed the point raised about the 21 data points above. It is not relevant for the analysis presented here.

Reviewer #3 (Public Review):

However, a weakness of the technique as currently presented is that patterns of connectivity are only related to oscillations across subjects. It would be more powerful to examine whether the current network state (estimated by trial-by-trial power estimates) relates to laminar connectivity within subject. This would indeed speak to the nature of neuronal communication, which takes place on a moment-to-moment time scale, and which is not reflected in the current analysis. This may explain why laminar patterns of fMRI connectivity were not found to correlate with gammaband oscillatory activity. In addition, the negative effects of attention on fMRI connectivity itself are somewhat puzzling. This may related to the limitations of the task design which do not perfectly separate attention vs. arousal/expectation, as the authors readily discuss.

The reviewer suggests that a relationship between fMRI connectivity and EEG power within subjects over trials would be more indicative of a direct link between connectivity and neural communication. We agree that establishing such a link would further strengthen the link between neural oscillations and laminar connectivity. This would not be trivial however, since connectivity in (laminar) fMRI is typically expressed as a measure of linear association (e.g. correlation or regression slope) over trials or time. Even at conventional spatial resolution, single trial/time point estimates of the network state are rarely used. These single data-point measures usually indicate to what extent a single data point contributes to the measure over all data points. We did not opt for such an analysis, since such analyses in normal (e.g. resting state) fMRI studies are uncommon, introducing more complexity to a study that already includes considerable novel analytic approaches. Furthermore, research relating fMRI activation and connectivity across subjects with other variables(e.g. clinical test scores, DTI measures, personality traits) is a well established procedure. Here we followed this more common approach.

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Reply to the reviewers

Firstly, we would like to thank the reviewers for their helpful and insightful comments.

Reviewer #1 (Evidence, reproducibility and clarity (Required)):

In the manuscript of Ramadan et al. , authors use the ex vivo organoid approach to compare gene expression in organoids derived from adult type stem cells when these organoids are grown using different matrices. The presence of Collagen type I induces the emergence of cells with a transcriptome similar to fetal progenitors. In contrast, laminin the main component of matrigel, induces an organoid-protruded phenotype with transcriptome of stem cell type. Then, they correlate these data with expression of collagens and laminins from data publicly available. They show by qRT -PCR that laminins are more expressed in mesenchymal versus epithelial fractions postnatally. They hypothesize on this basis that the remodeling at postnatal stage is likely only dependent on the mesenchymal compartment and it involves interaction of laminins with integrity a6.

It seems that some of the presented data have already been described and could not be considered as « novel ».

For some of the statements, like this one « the basement-membrane produced by the epithelium is not sufficient to increase stem cell numbers and induce a morphological crypt formation », the conclusion is not sustained by provided experiments. To draw definitive conclusion on this particular point, authors could reproduce the experiment presented in Fig. 4d but using Cre recombinases specific for mesenchymal and epithelial compartments rather than the ubiquitous Cre line. It would be interesting to investigate if organoids grown from lamc1-/- mice can generate protruded organoids or not.

In addition, how interpret the fact that fetal organoids up is associated with « laminin interactions » in fig. 1c?

The statement that the epithelium-produced basement membrane is not sufficient to increase stem cell numbers is based on our in vitro observations. Analysis of the RNAseq data shows that the expression of several laminins is increased on collagen (see heatmap of laminin interactions below, which will be added to the manuscript). This is also the reason why ‘laminin interactions’ is highly significant in the gene set enrichment analysis (Fig. 1C). Despite this upregulation, we never observed morphological changes (or expression changes) as when laminin is added to the collagen-hydrogel. In addition, we showed that the vast majority of ECM components is produced by the mesenchyme in vivo, in line with previous literature as cited in the manuscript. The mentioned Cre lines to address the question in vivo are unfortunately not available to our collaborators with the Lamc1 k.o. mice and it would therefore take too long to perform these experiments.

However, to address this point in vitro we will grow organoids from Lamc1 fl/fl mice and induce loss of laminin in the pure epithelial cell culture. Organoids will then be analysed for morphological changes, as well as proliferation and gene expression changes.

One major point to address regards statistics. In material and methods, the paragraph describing statistical analyses is missing. Moreover, in the figures presenting qPRC data ( figs 1g 3b 3D 3g 4c and f), no statistic analysis is provided; and the number of samples for some conditions is extremely limited (n=2). In general, the term « independent experiment « should be clarified : does it correspond to one organoid line for which the experiment was repeated or one single experiment using different organoid lines?

In fig 4c , all collagen conditions are set to 1.

The avoidance of statistical inference for most of the experiments was a deliberate choice. In line with several comments (e.g. 1. Vaux, D. L. (2012) Know when your numbers are significant. Nature. 492, 180–181), we chose to show all individual data points (with exception of Fig. 3D, n=5, to ease interpretation) without statistics. In addition, for most expression data, we have data from RNAseq, single-cell RNAseq and qPCRs repeated at different hydrogel concentrations to obtain reliable results. Further, the in vivo mesenchymal qPCR expression data was validated with RNA in situ hybridization showing the mainly mesenchymal expression.

The term independent experiment was used mainly for repeated experiments with the same organoid lines (exception RNAseq data, different organoids derived from individual mice). While conducting these experiments, we realised that the variability of these experiments comes from time in culture, density of cells and even Matrigel variation. The experiment in Fig. 4c (n=4, each time with the all controls) was performed with longer intervals in between, and showed variation in the absolute levels of expression. However, relative to each control we believe the effect is clear.

As we will perform additional experiments for the revision of this paper, we will then perform statistical tests in the key experiments (e.g. Itga6 experiment) to alleviate any concerns regarding significance.

Regarding the experiment presented in fig 4c, authors should include additional control conditions : anti-a6 integrity antibody in matrigel and use of an isotype antibody.

We will conduct additional experiments regarding the Itga6. In addition to including the mentioned controls for the neutralizing antibody, we will genetically inactivate Itga6 via an inducible Crispr/Cas9. This should enable us to delete Itga6 when the cells are grown on collagen, and hence reduce the possibility of compensation in matrigel derived organoids.

Another point regards RNAscope data presented in Fig 4b, it is surprising to observe such difference in terms of expression between E19 and P0. Does this mean that birth dramatically unregulates Itga6 expression in few hours? Authors should comment this point if verified.

We do believe that birth is a timepoint where a dramatic change in the ECM and their receptors can be observed. The epithelial RNAseq data would already indicate that at 18.5 there is an increase in expression compared to E16. This upregulation of the receptor is in line with the dramatic remodelling of the ECM at birth, as is shown by the expression of the basement membrane components in Fig. 3d.

Authors should avoid the word « signaling » for laminin-integrin interactions as they do not study this aspect at all in their experiments.

The word signaling was used for the protein:receptor interaction and to distinguish it from changes to the physical characteristics of the hydrogel. But we agree with the reviewer, that we did not study laminin signaling per se and therefore will change the wording accordingly.

Regarding Col1a1, authors cannot claim that it's expression only slightly changed (fig 3d) as it is clearly upregulated between E17 and P0.

The reviewer is right, and we apologise for the misleading sentence. The contrast was meant to the basement membrane components that are very lowly expressed at E17 and then suddenly show the burst of expression at birth, whereas collagen seems to be continuously expressed with a peak at P7. We will rephrase the sentence.

Reviewer #1 (Significance (Required)):

Overall, the methodology used for the asked questions is accurate.

One potential problem for publication comes from the fact that some of the findings are already reported and hat the present data do not provide further advances.

for example, collagen and fetal-like expression profile, Ly6a sorting and replating in culture-Yui et al, 2018, Jabaji et al, 2013.

We obviously do not agree with the reviewer on this point. We build upon the work of Jabaji et al. 2013, and Wang 2017 to characterise the specific effect of collagen on the intestinal epithelium compared to a pure Matrigel culture. The emergence of Ly6a cells was nicely shown by Yui et al., however it was unclear if collagen changes the fate of all intestinal cells or only a few. We strongly feel that our data extends these findings as it associates the changes we observe in vitro to the development of the crypt morphology and intestinal stem cells.

The phenotype of Lamc1-/- mice and the observed reduced stem cell marker expression are also reported by Fields et al, 2019.

Indeed, as we cited this paper. However we predicted based on our in vitro model, that deletion of laminin would result in this specific fetal-like gene expression and hence were happy to include these findings in our manuscript.

Infine, authors do not interpret their ex vivo data in the context of fetal progenitors which grow as spheres in matrigel (containing laminin)?

Our ex-vivo (in vitro) data would suggest that adult epithelial cells express some genes that are characteristic for fetal organoids, however we do not think these cells completely revert back to a fetal stage. Regarding the comment of spheres, it is noteworthy that fetal cells from E14-16 stay as spheres in Matrigel, whereas fetal cultures from E19 initially grow as spheres and then develop into organoids within 30 days in vitro (M. Navis et al., “Mouse fetal intestinal organoids: new model to study epithelial maturation from suckling to weaning,” EMBO Rep., vol. 20, no. 2, pp. 1–12, 2019.).

In figure 5, should we interpret that there is no laminin at all in the fetal mesenchyme?

We now see how the image is a bit misleading for that stage. The levels of laminin are lower in the fetal stage as can be seen by the IF image in Fig.3f and the image will be updated. We also apologise for the lack of labeling in Figure 3f, which should be E19, P7 and adult.

Also, authors do not cite a paper reporting on the role of the epithelium ( stem cells) in regulating its own extracellular matrix composition, this process modulating the stem cell number and fate (Fernandez-Vallone et al. 2020). As this is contradictory with the claim that only mesenchyme impacts on crypt morphogenesis, authors could discuss on this point.

In the paper by Fernandez-Vallone, deletion of Lgr5 in E16.5 embryos resulted in a decrease expression of several ECM genes. Further, the authors could show that the fetal epithelium does express for example Col1a1 at this point, which decreases with maturation. However even for the example of Col1a1 it is evident in their paper that the mesenchyme expresses Col1a1 at much higher levels. Our proposed experiments with Lamc1 k.o. In organoids will show if the produced laminins of the epithelium are essential.

This manuscript could be interesting for an audience in the stem cell and developmental fields ( my field of expertise).

**Referee Cross-commenting**

Considering the pertinent and sometimes overlapping comments of the two other reviewers, the estimated time is revised to 3-6 months.

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

**Summary:**

In this manuscript, Ramadan and colleagues demonstrate that depending on the extracellular matrix (ECM) composition in which mouse intestinal organoids and/or 2D intestinal epithelial cells are grown in, cellular composition of the epithelium changes. Organoids plated on 2D collagen layers show a unique cell cluster characteristic of fetal-like genes, while organoids plated with increased amount of Matrigel in 2D or in 3D exhibit a shift towards higher stem cell abundance and the absence of the fetal-like gene cluster. Specifically, the ECM component Laminin supports acquisition of stem cells identities in small intestinal epithelial cells, correlating with a transient increase in expression levels of collagen and laminin genes in vivo spanning time points of crypt formation. The authors reported the functional contribution of laminin signaling (Lamc1 KO) via integrin alpha 6 (antibody-blocking) to intestinal stem cell acquisition in vitro and in vivo.

There are a handful of comments/concerns that would need to be addressed before publication.

Major points:

1. The author claimed: "the effect of ECM components on gene expression is not due to difference in morphology (2D collogen vs. 3D Matrigel)". The conclusion of the 2D vs. 3D experiment should be toned down to that organoid morphology (2D vs. 3D) does not directly impact on the expression of fetal-like genes. Otherwise more analysis of RNAseq data with different group of genes (e.g., in different mechanosensing pathways) should be provided with Fig. S1D. Also, would it be technically feasible to perform experiments of SI in collagen (3D) in all group of experiments? Directly comparing 3D Matrigel with 3D collagen avoids the concern of the 2D vs. 3D effect.

We apologise for the too strong claim of structure of growth vs. signalling of the ECM and its effect on the transcriptome. Indeed, the main message is that a changed morphology from 3D to 2D is not responsible for the expression of fetal-like genes. The paragraph will be rephrased.

Also, would it be technically feasible to perform experiments of SI in collagen (3D) in all group of experiments? Directly comparing 3D Matrigel with 3D collagen avoids the concern of the 2D vs. 3D effect.

To address this point, we want to refer to the excellent idea of growing established organoids in collagen (3D) vs. Matrigel (3D) as suggested by this reviewer (and reviewer #3) in Minor points #5. This circumvents the need for Wnt3a addition, which affects stem cell and Paneth cell gene expression.

1. For Fig. 1f, the authors should include overlapping stainings of Lyz (or Olfm4, CD44 etc.) and Adolase B signal, or they could perform Aldolase B staining in Lgr-5-DTR-GFP and/or Lyz-RFP organoid line. From the current data provided one cannot draw clear conclusions on the crypt morphology as claimed by the authors. Additionally, when talking about crypt morphology and apical accumulation of Actin specifically in the Lyz+ cells, the authors should show a higher zoom in of the picture and either add an orthogonal slice to see apical and basal side and the specific accumulation in one of the cell types, or also co-label with apical polarity markers.

We will perform additional co-stainings to further highlight the differences in the spatial distribution of differentiated cells and undifferentiated-crypt-like cells. Further we will provide higher magnification images highlighting the apical accumulation of Actin in the crypt-like structures, which can also be seen in mature organoids.

1. Authors referred the organoid transient change to fetal-like state. To exam the similarity of ECM-induced reprogramming with the regenerative-type of reprogramming, it would be essential to compare the expression of the selected fetal-like genes (Anxa3, Ly6a/Sca1, Msln, Col4a2 et al.), as well as bulk and single-cell (if applicable) RNA-seq data.

Here, we would like to refer to the excellent study by Yui et al. ([S. Yui et al., “YAP/TAZ-Dependent Reprogramming of Colonic Epithelium Links ECM Remodeling to Tissue Regeneration,” Cell Stem Cell, vol. 22, no. 1, pp. 35-49.e7, 2018.). In this study the authors detected the same gene signature in the repairing epithelium. We can provide a GSEA for the Ly6a+ signature that was derived from this paper, if necessary.

1. For in vivo data, authors were looking at the normal development of intestine. Following the point of organoid culture recapitulates regeneration, it would be relevant to check the in vivo ECM change by staining in the process of intestinal regeneration or discuss would the fetal-like genes be involved in regeneration.

We will address this point in the discussion as it also involves the study by Yui et al.

1. For Fig2.d and e, it would be important to measure compactness vs. the emergence/probability of Ly6+ cells to see if there is correlation.

If we understand the reviewer correctly, this would address the important relationship between cell shape and cell fate/type. However, this is a topic that needs more attention than a simple correlation and would exceed the scope of this manuscript as we are not able to modulate cell shape to make any further points about its effect on the fetal gene expression program.

1. In Fig.2d, Ly6a expression is very obscure, and it would be important to show control staining for cell boundaries (eg. Phalloidin, PM) to visualize which nuclei show Ki67 staining and are high or low in Ly6a (plus quantification).

We will improve the image in Fig.2d and include the mentioned Actin staining. In addition we will perform an analysis via Flow Cytometry to quantify the level of Ly6a staining and EdU positivity.

1. In Fig. 2f-g, FACS-ed Ly6a+ and Ly6- cells embedded in Matrigel can grow into organoids with crypts. Here the imaging of Paneth cell staining is not clear, and a quantification on number of Paneth cells per crypt would be very helpful to confirm the phenotype. Also, authors should either provide data on the initial size of seeded cell clusters and report organoid growth and cell type composition in more detail when plating from Ly6a+ and Ly6- cells or report the variation in the respective populations.

This comment suggests that we may not have described the experimental settings properly. The sorted cells were embedded as single cells, not as clusters, in drops of matrigel (10k cells/25ul Matrigel). The emergence of Paneth cells together with a normal organoid architecture grown from Ly6a+ cells shows their stem cell capacity, as has been shown by Yui et al. before from the regenerating colon. In addition, organoids from both cell populations (Ly6a+ and Ly6a-) could be passaged, indicating presence of intestinal stem cells.

1. The authors could also test whether Ly6+ cells have any advantages over Ly6- cells when grown on collagen I instead of Matrigel.

We will sort Ly6a+ and Ly6- negative cells and plate them on collagen I. It will be interesting to see if the Ly6a+ cells can give rise to the other cell types when plated on collagen or if they stay Ly6+ cells. This will also answer whether Ly6a+ need the presence of Ly6a- cells in the cultures. In addition, the experiment proposed in #6 will also highlight any proliferative advantage of Ly6a cells compared to Ly6-negative cells on collagen.

1. In Fig.3f, a control of membrane protein staining should be added for the experiment. The increased Laminin signal can be caused by the global increase of protein when there are more cells, or tissues are more compact. When authors make conclusion of "Dramatic remodelling of ECM during crypt formation ", the experiment should also count cell numbers vs. Laminin (intensity). The phenotype can come from increased area of interface between epithelium and mesenchyme instead of active remodelling.

We agree with the reviewer that by itself the IF images are not enough to make such a claim. However, we would point to the qPCR data and RNA in situ, that can be more easily normalised and shows the dramatic increase in expression of all laminins at birth. To show that laminin protein is increasing is more difficult than we initially anticipated. However, in the study by De Arcangelis (A. De Arcangelis et al., “Hemidesmosome integrity protects the colon against colitis and colorectal cancer,” Gut, vol. 66, no. 10, pp. 1748–1760, 2017.) the authors use an EDTA assay to show that the epithelium detaches easily when Itga6 is deleted. Within the figure, it seems also that the epithelium detaches easily at P2, compared to P14. As EDTA is disrupting laminin polymerisation, this would further indicate increased laminin protein deposition after birth.

1. The authors claim that intestinal stem cells in vivo are controlled by Laminin signalling that goes via Integrin alpha 6. However, there is no evidence provided that supports the contribution of ITGA6 in the in vivo setting. So, the authors should either tone down on that point or show a convincing in vivo experiment (e.g., inhibit ITGA6 in vivo by inhibitor injections or by extracting the ECM of a wild-type mouse and seeding intestinal epithelial cells without vs. with ITGA6 blocking antibody which should recapitulate the phenotype in Fig. 4 c.

We apologise for this confusion. We are well aware about the limitations of our Itga6 blocking experiment in vitro and its relevance in vivo. We tried to get material of the inducible VilCreER Itga6 mouse as referenced in the discussion of the manuscript, without any luck so far. Therefore we will highlight further that any claims about the laminin:Itga6 interaction can only be made in vitro.

1. Fig. 4: For the data of ITGA6 expression and all sorts of analysis on protein expression with staining, normalization with cell numbers should be performed.

The RNAseq data that shows the upregulation of Itga6 in the epithelium at E18 is normalized within. Our RNAscope only further validates these expression changes and highlights the specific enriched expression at the bottom of the nascent crypts. We can add quantification of the RNAscope if required.

1. Two questions on mechanisms:

2. What is the mechanism from ITGA signaling to Ly6a+ cell fate?

3. And would/how Laminin induce ITGA expression? Depends on how much the authors would like to go deep with the project, could be addressed further with functional studies, or touch on the topics with discussion.

These are important questions, however we do agree that this will go to deep for the scope of this manuscript. We will address these open questions in the discussion and leave the experimental part for a follow-up study.

**Minor points:**

1. Text in Fig.S1d regarding 'in' or 'on' collagen, could be clearer by changing the terms to 2D and 3D correspondingly.

We agree and the text will be changed accordingly.

1. Fig. S1a, it is great the authors showed that similar stiffness in Matrigel and collagen I. It would be important to check the concentration of collagen I vs. stiffness (also for increasing concentrations of Laminin in Fig. 3b), since this is also the type of ECM change that might lead to the change of cell status in cancer progression or collective cell migration.

We will perform further stiffness measurements of the hydrogels and update the Fig. S1a.

1. When plating intestinal epithelial cells on collagen I, is the Ly6+ phenotype altered upon Wnt addition? This is not so clear from the RNAseq data Fig S1d., so authors should provide antibody stainings (stem cells/Paneth cells). This could give insight whether Ly6+ cells are still able to convert into stem cells/ Paneth cells by changing morphogen concentration vs. ECM composition.

We will reanalyse the RNaseq dataset further, specifically analysing the ratio of stem cell and Paneth cell gene expression. However, as mentioned before, Wnt3a specifically does reduce the expression of Paneth cell markers.

Similar to this point, also enteroendocrine cell fate is absent in collagen I condition (Fig2.ab), the authors could address this point by medium induced EE cell fate.

Due to the reduced number of secretory cells the clustering in Fig2 a/b does not separate all the different cell lineages. However, EE cells are present in the collagen cultures as characterised by expression of Chga, just reduced in their number (see Supl Fig. 2B).

1. It would be more informative to indicate the thickness of ECM layer in culture of 2D collagen I, as well as the image of the whole well, demonstrating the morphological variation in the middle and peripheral of the ECM layer.

The thickness of the collagen layer is about 1mm in a 6well plate and we do not observe any morphological differences in the cells between the periphery and center of the well.

1. After the formation of PC/SC clusters, would ECM contribute to maintenance? Putting mature organoids from Matrigel to Collagen I 3D would help to clarify.

This is an interesting experiment that we will conduct, we thank the reviewer for this suggestion. Indeed, established organoids should be able to grow in collagen I without Wnt3a addition. The paper by Sachs et al. (N. Sachs, Y. Tsukamoto, P. Kujala, P. J. Peters, and H. Clevers, “Intestinal epithelial organoids fuse to form self-organizing tubes in floating collagen gels,” Development, vol. 144, no. 6, pp. 1107–1112, 2017. et al) used extensive washing with PBS to remove Matrigel from the organoids. We will go one step further and trying to completely remove laminin specifically by EDTA incubation, as has been shown recently (J. Y. Co et al., “Controlling Epithelial Polarity: A Human Enteroid Model for Host-Pathogen Interactions,” Cell Rep., vol. 26, no. 9, pp. 2509-2520.e4, 2019.). This should then also answer whether disruption of laminin signalling is sufficient to induce fetal-gene expression without the addition of collagen I in a 3D setting.

1. Check secretome and individual culture of Mesenchyme, see if the increase of Laminin is epithelium independent.

We agree that the mesenchyme is key for laminin production, therefore these are important questions. Our prediction would be that epithelium from birth (P0) versus adult might result in different responses on the mesenchyme. However, we feel these experiments are better suited for a follow-up study.

1. In general, the authors should look at cell polarity markers to check the ECM contribution to cell polarity in different cell types.

We thank the reviewer for the suggestions and as mentioned above, we will perform additional stainings.

Reviewer #2 (Significance (Required)):

**Significance:**

The work highlights the role of ECM on stem cell niche and is of great interest to the organoid and stem cell community.

Our field of expertise is image- and seq-technology-based quantitative biology, regeneration and mechanics in organoid.

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

**Summary:**

Ramadan et al present a highly informative paper detailing how the Extracellular matrix influences the development of the intestine. Specifically, the authors provide a thorough analysis of how manipulating the components of the ECM can affect organoid growth, morphology, and gene expression of the organoids. Most importantly, the authors isolate laminin as a critical component of the ECM which impacts the development of fetal-like epithelium. While the in vitro work is generally compelling and of interest to the field, the in vivo data is someone lacking in depth and novelty. Particularly, these conclusions from the abstract could be much better supported: "This laminin:ITGA6 signalling is essential for the stem cell induction and crypt formation in vitro. Importantly, deletion of laminin in the adult mouse results in a fetal-like epithelium with a marked reduction of adult intestinal stem cells." The in vivo work was largely published previously and has caveats noted below, while the in vitro association of ITGA6 signaling with crypt formation is over-interpreted based upon an antibody blocking experiment and a lack of statistical rigor. Despite these concerns, this reviewer finds the work of considerable interest in an important area of the field (epithelial/stromal interactions of the intestine).

**Major Concerns:**

The use of the Ubc-Cre Lamc1-flox mouse model is an interesting way to test the impact of loss Lamc1 on intestinal development. However, with the Ubc-Cre, does the mouse model have other deleterious effects on the mouse beyond the intestine?

Can the authors use a more localized Cre to observe specifically the impacts of Lamc1 loss in the intestine? What is the fate of these mice? Can the authors show swiss roll, low mag sections to let the reader know the extent of this phenotype? OLFM4 and Ki67 IHC should be conducted over a timecourse to show how the changes occur over time after loss of Lamc1. How long does Lamc1's protein product perdure after tamoxifen treatment? More details of this exciting, in vivo validation of the authors' in vitro studies are key to elevating the impact of this work. However, it appears that much of this mouse model was previously published, but the previous findings are not well summarized in the current manuscript.

We will describe the model in more detail and refer readers to the excellent study of our collaborators which answers most of the raised questions. It is interesting to note that although a ubiquitous Cre was used to delete Lamc1 in adult mice, a phenotype was only observed in the intestine indicating a specific role for continuous laminin production here.

Can the authors show that ITGA6 loss has functional consequences in vivo with an epithelial knockout or via an organoid knockdown? A more rigorous genetic test of this proposed function would be important for substantiating the claims made in the abstract.

As referenced in the discussion of the manuscript, there is a VilCreER Itga6 mouse described in the literature (A. De Arcangelis et al., “Hemidesmosome integrity protects the colon against colitis and colorectal cancer,” Gut, vol. 66, no. 10, pp. 1748–1760, 2017.), which mainly focus on the colon. However, the authors use an EDTA assay in the small intestine to show that the epithelium detaches easily when Itga6 is deleted (Fig. 1J). Within the figure, it seems also that the epithelium detaches easily at P2, compared to P14. As EDTA is disrupting laminin polymerisation, this would further indicate increased laminin protein deposition after birth which is dependent on Itga6.

We tried to get material of the inducible VilCreER Itga6 mouse however without any luck so far.

We will conduct additional experiments regarding the Itga6 in vitro. In addition to including additional controls for the neutralizing antibody, we will genetically inactivate Itga6 via an inducible Crispr/Cas9. This should enable us to delete Itga6 when the cells are grown on collagen, and hence reduce the possibility of compensation in matrigel derived organoids.

The authors state that the changes in gene expression are not due to differences in morphology, but rather are specific to the components of the environment. While the authors show that organoids treated with Wnt3a "in Matrigel" and "CollagenI" appear to have similar morphologies and yet still result in a different gene expression profiles, it would be of great interest to see whether that difference persists without Wnt3a when organoids are "in Matrigel" and "in CollagenI". While the reviewer understands the difficulties of culturing organoids in 3D Collagen without Wnt3a, organoids can be indeed be cultured in 3D using "floating collagenI rings" (Sachs et al 2017).

This is an interesting experiment that we will conduct, we thank the reviewer for this suggestion. Indeed, established organoids should be able to grow in collagen I without Wnt3a addition. The paper by Sachs et al. (N. Sachs, Y. Tsukamoto, P. Kujala, P. J. Peters, and H. Clevers, “Intestinal epithelial organoids fuse to form self-organizing tubes in floating collagen gels,” Development, vol. 144, no. 6, pp. 1107–1112, 2017. et al) used extensive washing with PBS to remove Matrigel from the organoids. We will go one step further and trying to completely remove laminin specifically by EDTA incubation, as has been shown recently (J. Y. Co et al., “Controlling Epithelial Polarity: A Human Enteroid Model for Host-Pathogen Interactions,” Cell Rep., vol. 26, no. 9, pp. 2509-2520.e4, 2019.). This should then also answer whether disruption of laminin signalling is sufficient to induce fetal-gene expression without the addition of collagen I in a 3D setting.

Similarly, while the authors indicate that increasing Matrigel concentrations altered the gene expression patterns in a dose-dependent manner, it is unknown whether this can fully be attributed to the Matrigel composition, or whether the layer of Matrigel is providing the capability to transition from 2D to 3D culture.

We are not entirely sure, we understood this point. The Matrigel and the collagen I are mixed before they solidify, therefore enabling a homogenous hydrogel. The different hydrogels are not layered (if that is what the reviewer is referring to).

The authors cultured organoids in different concentrations of Laminin/CollagenIV when mixed with CollagenI. Can organoids be sustained only on a matrix of CollagenIV and/or Laminin? Would this show more direct differences between CollagenI vs. Laminin cultured organoids?

Organoids cannot be grown in pure Collagen IV, but pure laminin should be feasible. We did initial experiments with 3-5mg/ml laminin in PBS and that was sufficient to allow organoid growth. We will perform additional experiments with pure laminin and show the impact on organoid growth.

With the reduction in stem-cell and Paneth cells in the Lamc1-KO mice, it would also be of interest to determine what cell types are now prominent within the heavily elongated intestinal "crypt" structures seen in the Lamc1-KO mice and whether populations are more TA-cells or enterocytes to consider differentiation status of the cells. Additionally, it would also of interest to see if the Itga6 expression is significantly altered in the absence of Lamc1.

We will test expression changes for Itga6 in the Lamc1-KO mice, in the epithelium via qPCR. Additionally we can stain tissue from these mice for Sox9, Ki67 and differentiated markers eg. CD44, AldolaseB, Villin etc .to determine whether the elongated, hyperproliferative crypts contain progenitor cells or secretory enterocytes.

**Minor concerns:**

Matrigel is a complex matrix derived from mouse tumors. In many instances in the manuscript, the authors portray it as a more simpler mix of laminin/Collagen4 (fig 3a). It should be made clearer to the reader that Matrigel is not a mix of recombinant proteins, but a more clear depiction of how Matrigel is derived will be critical for this study, given the focus on specific ECM components and how they affect intestinal epithelial growth.

We agree and will change the oversimplified view of Matrigel.

Some labels of specific conditions would be appreciated in the figures as opposed to only the figure legends (ie. Fig. 1b and 1d should be labeled with comparisons; Fig. 3f labels of fluorescence, Fig. 4b label of itga6 staining).

We apologise for this and the Figure labels will be updated.

With the light staining of Lamc1 in-situ, it is hard to appreciate the expression of laminin within the stroma of the intestine when compared to Col4. This reviewer is also curious of the biological relevance of the concentrations of Laminin/CollagenIV when culturing organoids in Fig. 4a.

Indeed, the Lamc1 due to its lower expression than Col4a1 is more difficult to see. Maybe the reviewer overlooked Suppl.Fig.4A, where the blue channel of these in situ images show more contrast. If required, we can try to optimise the hybridisation times to increase the signal a bit further.

When culturing the organoids with the mixture of collagen and laminin or collagen IV, the concentrations of the two single components were selected similar to their concentrations in Matrigel. Regarding the absolute concentrations of laminin/collagen IV in vitro versus their “concentration” in vivo is much harder to answer. In addition to the unknown concentrations in vivo, there are many more Laminin types present with specific localisation and even specific receptor interactions. For our in vitro studies we relied on the Laminin present in EHS tumours, which is Laminin alpha 1 beta 1 gamma 1. We are currently investigating decellularization protocols to purify the ECM from mouse intestinal tissue, but again this would be more suited for a follow up study.

It would be appreciated if gene expression analyses presented in figures would include p-values to provide context for differences in gene expression.

The avoidance of statistical inference for most of the experiments was a deliberate choice. In line with several comments (e.g. 1. Vaux, D. L. (2012) Know when your numbers are significant. Nature. 492, 180–181), we chose to show all individual data points (with exception of Fig. 3D, n=5, to ease interpretation) without statistical testing. For most expression data, we have data from RNAseq, single-cell RNAseq and qPCRs repeated at different hydrogel concentrations to obtain reliable results. Further, the in vivo mesenchymal qPCR expression data was validated with RNA in situ hybridization showing the mainly mesenchymal expression.

As we will perform additional experiments for the revision of this paper, we can perform statistical tests in the key experiments (e.g. Itga6 experiment) to alleviate any concerns regarding significance.

In figure 3f, the authors report "immunofluorescence of laminin". How is this measured? Can more details be given about the antibody in the text and figure legend? Laminins are a family of genes, and it's not clear what's being demonstrated in this figure panel. Developmental stages of the samples are also not clear.

We apologise for the lack of labeling in Fig.3f. The details of the antibody were hidden in the Materials and Methods of the manuscript (Slides were incubated with Laminin Polyclonal Antibody (1/200, Thermo Fisher #PA5-22901) overnight at 4C ). This pan-laminin antibody reacts with most Laminin isoforms alpha1, alpha2, beta1, gamma1. We will declare it as a pan-laminin antibody in the Figure legend to help future readers.

Reviewer #3 (Significance (Required)):

This work is in an exciting "hot" area of research to understand the role of non-epithelial cells in intestinal epithelial development and function. The audience would be those in the GI field and those studying tissue-tissue interactions.

There's some concern that the in vivo portion of the manuscript (4th figure) uses a model that was previously characterized and published by this group, and that isn't clearly disclosed. The manuscript would benefit from more disclosure and detail about the in vivo phenotype. Such changes would substantially increase the impact and novelty of the study.

We would like to point out that we cited the paper of the original study that uses the model throughout the manuscript. We will disclose in more detail that this group did the study and that the reduction in stem cell genes was already mentioned in the original publication.

Author Response:

Reviewer #2 (Public Review):

1) The authors describe their algorithm as a tool that (i) was validated "across heterogeneous populations around the world"; (ii) has an "accuracy matching or exceeding human accuracy"; (iii) "is easy to use". I take issue with these three statements. First, the authors did not test the performance of their algorithm in clinical populations with sleep disorders, despite the fact that individuals with sleep disorders represent (logically) the vast majority of sleep recordings. Crucially, such a comparison was made in the best (to my knowledge) published automated sleep staging algorithm (Stephansen et al. Nature Communications 2018, doi: 10.1038/s41467-018-07229-3). The omission of this work is very surprising. Quantifying the impact of sleep disorders on a sleep scoring algorithm is critical for its deployment in sleep clinics.

We apologize as we were not clear in describing our training and testing data set. Indeed, both the training and testing set 1 included a significant number of individuals with sleep disorders. Indeed, about 30% of the individuals had moderate to severe sleep apnea (AHI >= 15). The validation dataset (DOD, or testing set 2) also includes 55 nights from individuals with obstructive sleep apnea (average AHI = 18.5 ± 16.2). Furthermore, both the training and testing set 1 included individuals with a medical diagnosis of insomnia, depression, diabete and hypertension.

The health status and demographics data of the training and testing sets have now been clarified throughout in the manuscript to avoid any such confusion:

1) Methods: We have added an extensive description of each dataset in the training and testing sets, including data on health and sleep disorders.

2) Results: We have added a new table to report and compare demographics/health data of the training and testing set, as suggested in a later comment by the reviewer.

3) Results: Performance results of the testing set 2 are now reported separately for healthy individuals and individuals with sleep disorders.

Second, the authors wrote that their algorithm is "matching or exceeding" human accuracy but seem to present uncorrected one-to-one comparisons to support their claim. The fact that an algorithm is better than some humans do not mean it exceeds human performance.

Thanks for noting that. We have now removed all instances of “exceeding human accuracy”.

Third, although I agree that the tool seems easy to use even for individuals with limited programming skills, it still requires some. I don't think someone who is used to software with graphical interfaces and who has never used (or heard of!) python would describe the tool as easy to use. This poses an important implementation challenge.

2) An important limitation of this algorithm is that it captures only one part of the visual examination of sleep data. Indeed, especially in clinical settings, the data is not only examined to establish the hypnogram but to also identify markers of common sleep disorders (e.g. sleep apnea, leg movements, etc). Although this algorithm could significantly speed up sleep scoring, it does not allow to detect these other important markers. Currently, and in link with the previous comment, the algorithm could not replace the visual inspection of the data for clinical diagnoses.

We have now revised the manuscript such that we discussed in this possibility in “Limitations and future directions” subsection of the new Discussion:

“The algorithm is not currently able to identify markers of common sleep disorders (such as sleep apnea, leg movements) and as such may not be suited for clinical purposes. It should be noted however that our software does include several other functions to quantify phasic events during sleep (slow-waves, spindles, REMs, artefacts) as well as sleep fragmentation of the hypnogram. Rather than replacing the crucial expertise of clinicians, YASA may thus provide a helpful starting point to accelerate clinical scoring of polysomnography recordings. Furthermore, future developments of the algorithm should prioritize automated scoring of clinical disorders, in particular apnea-hypopnea events. On the latter, YASA could implement some of the algorithms that have been developed over the last few years to detect apnea-hypopnea events from the ECG or respiratory channels (e.g. Varon et al. 2015; Koley and Dey 2013).”

3) The data were curated with some recordings or portions of recordings being excluded (see p. 7). While I understand that this curation is important for the training set, I think it should not be applied to the test set. Indeed, it goes contrary to the logic of automating sleep staging. For example, cutting the beginning and end of the recording according to sleep start and end (p. 7) supposes that the start and end of sleep are already known (i.e. it has already been scored).

This truncation step has now been removed from the pipeline and all the results have been updated accordingly. In addition, we have also removed all other exclusion criteria (e.g. PSG data quality, recording duration, etc) to improve the generalization power of the algorithm, thanks to the suggestions of the reviewer.

4) Two types of EEG derivations were used (C4-M1 or C4-Fpz). Was the performance impacted by this variable? Is it fair to assume that the choice of features (spectral features or summary statistics of time series data) could explain the absence of differences but that introducing new features (i.e. phase-sensitive features) could increase the influence of the choice of the derivation?

Thanks for raising this. First, our choice of the EEG reference was determined by the datasets: the CFS, CCSHS, MrOS, CHAT and HomePAP datasets were all referenced to Fpz, while the MESA, SHHS and DOD datasets were referenced to the contralateral mastoid. The montage of each dataset has now been added to the Methods section.

Second, as rightly pointed out by the reviewer, the features implemented in the algorithm were chosen to be robust to various recording montages. This is now explicitly discussed in the “Features extraction” subsection of the Methods:

“The features included in the current algorithm were chosen to be robust to different recording montages. As such, we did not include features that are dependent on the phase of the signal, and/or that require specific events detection (e.g. slow-waves, rapid eye movements). However, the time-domain features are dependent upon the amplitude of the signal, and the algorithm may fail if the input data is not expressed in standard units (uV) or has been z-scored prior to applying the automatic sleep staging.”

5) Given that markers of sleep stages are very different in EOG, EMG and EEG time series, could the authors explain the logic behind applying the same pre-processing and extracting the same features on these three very different types of data? Could this explain why the majority of the features in the top-20 features were EEG features?

We now provide a more detailed explanation on the inclusion of EOG and EMG features in the “Features extraction” subsection of the Methods:

“These features were selected based on prior work in features-based classification algorithms for automatic sleep staging (Krakovská and Mezeiová 2011; Lajnef et al. 2015; Sun et al. 2017). For example, it was previously reported that the permutation entropy of the EOG/EMG as well as the EEG spectral powers in the traditional frequency bands are the most important features for accurate sleep staging (Lajnef et al. 2015), thus warranting their inclusion in the current algorithm. Several other features are derived from the authors’ previous works with entropy/fractal dimension metrics1. ” https://github.com/raphaelvallat/antropy

Furthermore, we have added a “Limitations and future directions” section in the Discussion in which we propose future improvements of the algorithm. One of these potential improvements is the development of EOG and EMG features that would provide a higher discrimination of the sleep stages:

“This suggests that one way to improve performance on this population could be the inclusion of more EEG channels and/or bilateral EOGs. For instance, using the negative product of bilateral EOGs may increase sensitivity to rapid eye movements in REM sleep or slow eye movements in N1 sleep (Stephansen et al. 2018; Agarwal et al. 2005). Interestingly, the Perslev 2021 algorithm does not use an EMG channel, which is consistent with our observation of a negligible benefit on accuracy when adding EMG to the model. This may also indicate that while the current set of features implemented in the algorithm performs well for EEG and EOG channels, it does not fully capture the meaningful dynamic information nested within muscle activity during sleep.”

6) Sleep scoring guidelines incorporate not only what can be observed on a given epoch of data but also what is observed in the previous epoch(s). For example, an epoch can be scored as N2 even if there is no marker of N2 but there was (i) a marker of N2 in a previous epoch, (ii) no reason to change the score since. To reproduce this, the authors employed a symmetrical smoothing approach (a combination of a triangular-weighted rolling average and asymmetrical rolling average). Why did the authors choose to incorporate data from following epochs, which is not implemented in established guidelines? How was the duration of the smoothing window chosen? Indeed, 5 minutes appear as rather long could explain the poor performance of the algorithm for fast changing portions of the data (i.e. N1 or transitions). Importantly, these transitions can be very relevant in clinical settings and to establish a diagnosis.

This is a great question. We have addressed this in the revised manuscript.

Temporal smoothing

We have also conducted a new analysis of the influence of the temporal smoothing on the performance. The results are described in Supplementary File 3a. Briefly, using a cross-validation approach, we have tested a total of 49 combinations of time lengths for the past and centered smoothing windows. Results demonstrated that the best performance is obtained when using a 2 min past rolling average in combination with a 7.5 minutes centered, triangular-weighted rolling average. Removing the centered rolling average resulted in poorer performance, suggesting that there is an added benefit of incorporating data from both before and after the current epoch. Removing both the past and centered rolling averages resulted in the worst performance (-3.6% decrease in F1-macro). Therefore, the new version of the manuscript and algorithm now uses a 2 min past and 7.5 min centered rolling averages. All the results in the manuscript have been updated accordingly. We have now edited the “Smoothing and normalization” subsection of the Methods section as follow:

“In particular, the features were first duplicated and then smoothed using two different rolling windows: 1) a 7.5 minutes centered, and triangular-weighted rolling average (i.e. 15 epochs centered around the current epoch with the following weights: [0.125, 0.25, 0.375, 0.5, 0.625, 0.75, 0.875, 1., 0.875, 0.75, 0.625, 0.5, 0.375, 0.25, 0.125]), and 2) a rolling average of the last 2 minutes prior to the current epoch. The optimal time length of these two rolling windows was found using a parameter search with cross-validation (Supplementary File 3a). [...] The final model includes the 30-sec based features in original units (no smoothing or scaling), as well as the smoothed and normalized version of these raw features.”

Reviewer #3 (Public Review):

This study presents a new sleep scoring tool that is based on a classification algorithm using machine-learning approaches in which a set of features is extracted from the EEG signal. The algorithm was trained and validated on a very large number of nocturnal sleep datasets including participants with various ethnicities, age and health status. Results show that the algorithm offers a high level of sensitivity, specificity and accuracy matching or sometimes even exceeding that of typical interscorer agreement. The conclusions are supported by the data. Importantly, a measure of the algorithm's confidence is provided for each scored epoch in order to guide users during their review of the output. The software is described as easy to use, computationally low-demanding, open source and free. This paper addresses an important need for the field of sleep research. There is indeed a lack of accurate, flexible and open source sleep scoring tools. I would like to commend the authors for their efforts in providing such a tool for the community and for their adherence to the open science framework as the data and codes related to the current manuscript are made available. I predict that this automated tool will be of use for a large number of researchers in the field. However, there are plenty of automated sleep scoring tools already available in the field (most of them are not open source and rather expensive, as noted by the authors). The current work does not provide a clear view on whether the new algorithm presented in this research performs better than algorithms already available in the field. No formal comparisons between algorithms is provided and the matter is not discussed in the paper.

Thanks so much for pointing this out. We have now added this relevant reference throughout the manuscript. To build on the reviewer’s point, the current algorithm and Stephansen’s algorithm did not use the same public data. The Stephansen 2018 algorithm was trained and validated on “10 different cohorts recorded at 12 sleep centers across 3 continents: SSC, WSC, IS-RC, JCTS, KHC1, AHC, IHC, DHC, FHC and CNC”, none of which are included in the training/testing sets of the current algorithm. Nevertheless, we certainly agree that the manuscript will benefit from a more extensive comparison against existing tools. To this end, we have made several major modifications to the manuscript. First, we have added a dedicated paragraph in the Introduction to review existing sleep staging algorithms:

“Advances in machine-learning have led efforts to classify sleep with automated systems. Indeed, recent years have seen the emergence of several automatic sleep staging algorithms. While an exhaustive review of the existing sleep staging algorithms is out of the scope of this article, we review below — in chronological order — some of the most significant algorithms of the last five years. For a more in-depth review, we refer the reader to Fiorillo et al. 2019. The Sun et al. 2017 algorithm was trained on 2,000 PSG recordings from a single sleep clinic. The overall Cohen's kappa on the testing set was 0.68 (n=1,000 PSG nights). The “Z3Score” algorithm (Patanaik et al. 2018) was trained and evaluated on ~1,700 PSG recordings from four datasets, with an overall accuracy ranging from 89.8% in healthy adults/adolescents to 72.1% in patients with Parkinson’s disease. The freely available “Stanford-stage” algorithm (Stephansen et al. 2018) was trained and evaluated on 10 clinical cohorts (~3,000 recordings). The overall accuracy was 87% against the consensus scoring of several human experts in an independent testing set. The “SeqSleepNet” algorithm (Phan et al. 2019) was trained and tested using a 20-fold cross-validation on 200 nights (overall accuracy = 87.1%). Finally, the recent U-Sleep algorithm (Perslev et al. 2021) was trained and evaluated on PSG recordings from 15,660 participants of 16 clinical studies. While the overall accuracy was not reported, the mean F1-score against the consensus scoring of five human experts was 0.79 for healthy adults and 0.76 for patients with sleep apnea.”

Second, and importantly, we now perform an in-depth comparison of YASA’s performance against the Stephansen 2018 algorithm and the Perslev 2021 algorithm using the same data for all three datasets. Specifically, we have applied the three algorithms to each night of the Dreem Open Datasets (DOD) and compared their performance in dedicated tables in the Results section (Table 2 and Table 3). This procedure is fully described in a new “Comparison against existing algorithms” subsection of the Methods. None of these algorithms included nights from the DOD in their training set, thus ensuring a fair comparison of the three algorithms. Related to point 4 of the Essential Revisions, performance of the three algorithms are reported separately for healthy individuals (DOD-Healthy, n=25) and patients with sleep apnea (DOD-Obstructive, n=50). To facilitate future validation of our algorithm, we also provide the predicted hypnograms of each night in Supplementary File 1 (healthy) and Supplementary File 2 (patients).

Overall, the comparison results show that YASA’s accuracy is not significantly different from the Stephansen 2018 algorithm for both healthy adults and patients with obstructive sleep apnea. The accuracy of the Perslev 2021 algorithm is not significantly different from YASA in healthy adults, but is higher in patients with sleep apnea. However, it should be noted that while the YASA algorithm only uses one central EEG, one EOG and one EMG, the Perslev 2021 algorithm uses all available EEGs as well as two EOGs. This suggests that adding more EEG channels and/or using the two EOGs may improve the performance of YASA in patients with sleep apnea. Though an important counterpoint is that YASA requires a far less extensive array of data (channels) to accomplish very similar levels of accuracy, which has the favorable benefit of reducing analysis computational and processing demands, improves speed of analysis (i.e. a few seconds per recording versus ~10 min for the Stephansen 2018 algorithm), and is amenable to more data recordings since many may not have sufficient EEG channels. All these points are now discussed in detail in the new “Limitations and future directions” subsection of the Discussion (see point 3 of the Essential Revisions).

There are some overstatements in the manuscript. For example, the algorithm was trained and validated on nocturnal sleep data. Sleep characteristics (eg duration and distribution of sleep stages etc.) are different, for example, during diurnal sleep (nap) and the algorithm might not perform as well on nap data. As such, the tool might not be as "universal" as stated in the title. Additionally, as human scores are used as the ground-truth for the validation step, it might be misleading to state that "this tool offers high sleep-staging accuracy matching or exceeding human accuracy". The algorithm exceeded the accuracy of some human scorers and matched the scores of the best scorer.

We have now removed the word “universal” from the title and replaced “exceeded human accuracy” with “matched human accuracy”. Furthermore, we have now added the fact that the algorithm was trained and validated only on nocturnal data in the Limitations section of the discussion, and as such, noted that there is the possibility that the algorithm may not perform at the same accuracy levels for daytime nap data.

No reflection on further improvement is offered in the paper. The algorithm performs worse on N1 stage, older individuals and patients presenting sleep disorders (sleep fragmentation) and it is unclear how this could be improved in future research. In the same vein, the current work does not present performance accuracy separately for healthy individuals and patients when it is expected that accuracy would be poorer in the patient group.

The revised manuscript now includes a dedicated section in the Discussion to propose ideas for improvements.

First, we have now added a “Limitations and Future Directions” subsection in the Discussion to present ideas for improving the algorithm, with a particular focus on fragmented nights and/or nights from patients with sleep disorders:

“Despite its numerous advantages, there are limitations to the algorithm that must be considered. These are discussed below, together with ideas for future improvements of the algorithm. First, while the accuracy of YASA against consensus scoring was not significantly different from the Stephansen 2018 and Perslev 2021 algorithms on healthy adults, it was significantly lower than the latter algorithm on patients with obstructive sleep apnea. The Perslev 2021 algorithm used all available EEGs and two (bilateral) EOGs, whereas YASA’s scoring was based on one central EEG, one EOG and one EMG. This suggests that one way to improve performance in this population could be the inclusion of more EEG channels and/or bilateral EOGs. For instance, using the negative product of bilateral EOGs may increase sensitivity to rapid eye movements in REM sleep or slow eye movements in N1 sleep (Stephansen et al. 2018; Agarwal et al. 2005). Interestingly, the Perslev 2021 algorithm does not use an EMG channel, which is consistent with our observation of a negligible benefit on accuracy when adding EMG to the model. This may also indicate that while the current set of features implemented in the algorithm performs well for EEG and EOG channels, it does not fully capture the meaningful dynamic information nested within muscle activity during sleep.”

Second, we have now conducted a random forest analysis to identify the main contributors of accuracy variability. The analysis is described in detail in the “Moderator Analyses” subsection of the Results as well as Supplementary File 3b, the revision now states:

“To better understand how these moderators influence variability in accuracy, we quantified the relative contribution of the moderators using a random forest analysis. Specifically, we included all aforementioned demographics variables in the model, together with medical diagnosis of depression, diabetes, hypertension and insomnia, and features extracted from the ground-truth sleep scoring such as the percentage of each sleep stage, the duration of the recording and the percentage of stage transitions in the hypnograms. The outcome variable of the model was the accuracy score of YASA against ground-truth sleep staging, calculated separately for each night. All the nights in the testing set 1 were included, leading to a sample size of 585 unique nights. Results are presented in Supplementary File 3b. The percentage of N1 sleep and percentage of stage transitions — both markers of sleep fragmentation — were the two top predictors of accuracy, accounting for 40% of the total relative importance. By contrast, the combined contribution of age, sex, race and medical diagnosis of insomnia, hypertension, diabete and depression accounted for roughly 10% of the total importance.”

In addition, the performance of the algorithm in the DOD testing dataset is now reported separately for healthy individuals and patients with sleep disorders.

As requested by the reviewer, we now analyze and report the performance of YASA on the DOD testing set separately for healthy individuals (DOD-healthy) and patients with obstructive sleep apnea (DOD-Obstructive), which can be found in section “Testing set 2”.

There is series of methodological choices that is not justified. For example, nights were cropped to 15 minutes before and after sleep to remove irrelevant extra periods of wakefulness or artefacts on both ends of the recording. This represents an issue for the computation of important sleep measures such as sleep efficiency and latency as the onset/offset of sleep might be missed. It is also unclear how the features were selected and a description of said features is currently missing. The custom sleep stage weights procedure is unclear. The length of the time window for the smoothing procedure seems arbitrary. Last, it is currently unclear when / how the EEG and EMG data were analyzed.

As recommended by the reviewers, the 15-min truncation step has now been removed from the pipeline. Furthermore, the Methods section has been improved to provide more details on the features. Finally, the best class-weights and smoothing windows are now found using a cross-validation analysis on the training set. For more details, we refer the reviewer to the “Justification for some methodological choices” section below.

Author Response:

Reviewer #1 (Public Review):

The paper is a tour-de-force across multiple techniques and model systems from classical forward screening in C. elegans over ChIP to targeted CRISPR mutagenesis. The data is of a very high quality and supports most of the authors' claims strongly and convincingly. Finally, the manuscript is well written and, in spite of complex experiments and genetics, interesting and easy to comprehend.

• CAMTA, as the name CaM-binding transcription activator implies, have been studied previously and across many different organisms including plants, mice and humans. It was thus presumed and in part shown that CAMTAs regulate transcription depending on CaM levels.
• The authors confirm that the gene cmd-1 (encoding CaM) is directly regulated by Camt-1 by using a combination of cell-specific RNAseq and ChIP. This allows them to identify three binding sites upstream of the cmb-1 gene that bind to Camt-1.
• Moreover, the authors show that overexpression of CaM in the nervous system fully rescues the observed behavioral phenotypes.
• Importantly, the authors make another discovery. They show that CaM can directly repress its own transcription by binding to specific residues of Camt-1. Thereby, the authors argue, Camt-1 is used to precisely and bidirectionally regulate CaM levels dependent on the cell, animal's state etc.

The reported data are interesting and, in particular, the aspect that CAMTAs likely act as activators AND repressors is a novel aspect previously not appreciated. In spite of all these strengths, a potential weakness is that it remains open whether this mechanism is primarily a house-keeping mechanism or is indeed, as the authors speculate, regulated by internal and external factors that might, through CAMTA, make cells more or less responsive to Ca2+-CaM signaling.

We are grateful for and encouraged by our reviewer’s comments. We think that our discovery that CAMTAs regulate CaM expression is thought provoking.

Reviewer #2 (Public Review):

Vuong-Brender, Flynn, and de Bono report a detailed analysis of the function of a highly conserved calcium-calmodulin-dependent transcriptional regulator in the function of the C. elegans sensory nervous system. The C. elegans homolog of this factor - CAMT-1 - emerged from a genetic screen for mutants defective in a sensory-driven aggregation behavior. The authors find that multiple chemosensory modalities are disrupted by loss of CAMT-1, and this factor has distributed functions in the nervous system, including in interneurons that receive inputs from sensory neurons. A major finding of this study is that many of the effects of CAMT-1 mutation can be linked to a critical role for CAMT-1 in regulating expression of calmodulin itself. This finding is supported by multiple lines of experimentation, including a demonstration that the effects of losing CAMT-1 can be compensated by restoring expression of calmodulin. The authors further show that what is true for CAMT-1 and calmodulin in C. elegans also applies to Drosophila, indicating that CAMT-1 is a regulator of calmodulin expression whose function has been conserved throughout evolution. This manuscript has many strengths. Key hypotheses are tested using quantitative and technically independent experimental methods. The case that CAMT-1 is a regulator of calmodulin expression is built carefully and, for the most part, the logic of the argument is made clearly and supported by compelling data. Another strength of the manuscript is its candid exposition of data that do not fit neatly into the most simple and accessible model. It is refreshing to see authors who freely admit that they haven't neatly wrapped up every question in a field. The loose ends in this study do not impact the authors' main conclusions. However, some observations seem to consume more bandwidth than warranted, and the authors should consider reorganizing the manuscript so that the loose ends do not distract from the main thread of the narrative. The paper does have a few minor weaknesses that could be addressed. These are listed below.

We thank our reviewer for their thoughtful review.

Specific comments:

1. The initial description of the isolation of camt-1 mutants seemed a bit disorganized. A description of the gene and gene product preceded descriptions of the mutants. Also, some mutants were mentioned in the text but not presented in the corresponding figure. The authors should consider minor changes to better communicate how the mutations were cloned.

We have sought to do this.

1. In Fig. 2 npr-1 baselines vary a great deal between panels A, B, and C. It is not clear why npr-1 behavior is this variable, and the authors do not mention this obvious feature of their data. Data presented in Fig. 2 indicate that heat-shock-induced expression of camt-1 restores a defect in basal locomotion, but it is unclear whether it restores O2-sensitivity - the effect of oxygen on speed of transgenics seems the same +/- heatshock (compare black traces in panels 2B and 2C). We understand the concern of the reviewer. Since the design of these experiments was different from the rest (with only one shift in O2 concentration), we repeated them with 3 O2 changes, bringing them in line with the rest of the manuscript. The results are presented in the new Figure 2. We observed a more consistent baseline speed between different conditions, however some differences still exist (for example between panel 2A and 2B). One explanation is that for heatshock experiments we keep npr-1 animals at lower temperature (20 degree Celsius, panels 2B and 2C) to minimize basal activity of the heatshock promoter, whereas in the rescue experiment in Figure 2A, and in the rest of the manuscript, animals were kept at 22 oC. Figure 2B-C of our original submission used worms raised at 15 oC for the heatshock experiment, which may explain the greater discrepancy in npr-1 speed values. Heatshock also modifies slightly the response of the npr-1 control animals to O2.

Regarding whether heat-shock-induced expression of camt-1 restores O2 responses, we found that the npr-1; camt-1; dbExhsp-16p::camt-1 heat-shocked strains aggregated much more than npr-1; camt-1 heat-shocked animals. However, the rescue is not complete. Thus expressing camt-1 using heatshock-induced expression restores some O2 sensitivity which correlates well with the partial rescue of the baseline in Figure 2C. We have noted this in the results.

1. Unlike other datasets, the responses of wild-type AFDs to CO2 do not look particularly convincing (panel 3C). There is clearly an effect of camt-1 mutation on AFD calcium, but the AFD responses seem qualitatively different from the responses of BAGs to CO2 or URXs to O2. The authors might consider moving these data to a supplementary figure and tempering their description of wild-type AFDs as CO2-sensors.

The data on AFD has been moved to Figure 3 – figure supplement 1. We should add that we agree that in the absence of an identified CO2 sensor expressed in AFD, we cannot be sure that AFD neurons are primary CO2 sensors. Although the AFD CO2-evoked responses are retained in mutants defective in synaptic transmission, they may very well still be indirectly evoked by other neurons.

1. The authors candidly present data that do not conform to a simple model for how camt-1 affects behavior. Loss of camt-1 increases calcium in sensory neurons that activate the speed-controlling interneuron RMG. However, RMG calcium is reduced in camt-1 mutants. This inversion in the effect of camt-1 mutation might be caused by a homeostatic mechanism, as the authors propose. It might be possible to test this hypothesis by testing whether reducing excitatory input into RMGs elevates resting calcium in camt-1 mutants, for example via mutations that affect sensory transduction.

In the interest of simplifying the manuscript, and given other comments, we have now removed the RMG Ca2+ imaging data. However, this is an interesting way of testing what is going.

1. In Fig. 4H RMG data are presented as fractional ratio change - all other imaging data are presented as absolute ratios of YFP and CFP fluorescence. It is not clear why these data are treated differently. It is also no clear that these data are consistent with data shown in Fig. 3F. Which dataset represents the effect of camt-1 mutation on RMG calcium? More measurements might be warranted.

As highlighted above we have removed the RMG imaging data from the paper. .

1. Nice experiments show that regulation of calmodulin in Drosophila requires a CAMT-1 homolog. The bar graphs showing unity for values normalized to themselves are a bit odd - perhaps there's a more compact way to plot these data.

We have sought to address this question in two ways. First, we have further buttressed our results by performing in situ immunofluorescence staining of dissected fly retinas with a calmodulin antibody. We see a significant decrease in calmodulin expression in fly CAMTA mutants compared to controls.

Prompted by this comment, we also realized we omitted an explanation of how we normalized the data for the qPCR graphs in the figure legend. This was done using rRNA as a control. The Yamamoto lab had previously used the same control to normalize CAMTA expression in wild type and mutant flies. We add a note saying this.

1. ChIPseq analysis of CAMT-1 is also quite nice. Is there a sequence motif for CAMT-1 binding that emerges from this study? If so, how does this motif compare to motifs from studies of CAMT-1 homologs in other species?

We used the MEME algorithm, (motif-based sequence analysis tools (https://meme-suite.org/) to seek enriched sequence motifs in our ChIPSeq data. This identified a series of enriched motifs, although none coincided with the peaks at the CMD-1 promoter. However, we did observe sequences resembling the mouse CAMTA1 binding site at the centre of each of the three CAMT-1 binding peaks upstream of cmd-1. We now say this is the discussion.

1. Figure 7 shows that CMD-1 inhibits cmd-1 expression via interaction with CAMT-1. These data are interesting, but it is not clear how this effect can be related to prior data showing that forced expression of CMD-1 can compensate for loss of CAMT-1. The authors behavioral and physiological studies suggest that in vivo CAMT-1 promotes CMD-1 expression. In Figure 7, they suggest that CAMT-1 inhibits expression of CMD-1, but there is no clear link to behavior or physiology for this repressor-function of CAMT-1. The manuscript might be more clear without these data, and the absence of these data would not affect the overall impact of the study.

We agree that the feedback control of cmd-1 gene expression by CMD-1 interacting with CAMT-1 is a part of the story that has not been fully developed. Given the feedback from our reviewers and Editors to give these findings less prominence, but not remove them entirely, we moved the data into supplementary information. We have also altered the main text and the legend of Figure 7 to explicitly say that further experiments are needed to establish if this feedback is relevant under physiological conditions.

Reviewer #3 (Public Review):

Vuong-Brender et al present a thorough study investigating how CaM-binding transcription activators (CAMTAs) in C. elegans and Drosophila are required for numerous behaviors and proper neuronal function. The study is strong in how it uses a variety of approaches to study a major underlying mechanism for CAMTA. First, they use reporters, mutant analysis, and heat-shock rescue to show how cart-1 is expressed widely in neurons and functions in adults in several behaviors. They used transcriptional profiling to show that cart-1 is required to upregulate CaM in subsets of neurons in worm. They next use ChIP-seq to zero in on where worm CAMT-1 binds regulatory regions upstream of the CaM gene cmd-1 to promote its expression. They find that overexpression of CaM compensates for behavioral and neuronal response deficits in a cart-1 mutants. Lastly, they propose that when CaM highly expressed, it may down regulate its own expression by binding CART-1.

We thank our reviewer for their critique of our work.

1. Overall, I feel that the study is excellent and most conclusions are justified by evidence. However, I do not think the title is supported by the data. It currently is listed as: CAMTA TUNES NEURAL EXCITABILITY AND BEHAVIOR BY MODULATING CALMODULIN EXPRESSION. The authors show evidence that camt-1 is required for the normal function of neurons and behavior by promoting expression of CaM. Their only evidence that camt-1 downregulates CaM is a more artificial situation where CaM is overexpressed. I don't think they provide any evidence that camt-1 is used to "tune" behavior or neuron activity up and down in a wild-type strain. Tuning implies that the molecule modulates a physiological system bidirectionally in a natural situation. I suggest using a more accurate title that better fits the experimental evidence.

We have changed the title to ‘Neuronal Calmodulin levels are controlled by CAMTA transcription factors’. We hope this more neutral title is appropriate to describe our findings.

1. They show ample evidence that cart-1 appears to promote the expression of cmd-1 in most cases. This includes showing that overexpression of cmd-1 suppresses the behavioral and imaging phenotypes of cart-1. But they didn't perform the more straight forward epistasis test with the cart-1;cmd-1 double mutant in worm or fly , presumably because there is no viable loss-of-function allele in the coding area of the cmd-1 gene. It would help the readers understand why this simpler experiment was not performed if they explain this in the paper. A good place would be near line 220, where they generate hypomorphic promoter alleles using CRISPR. If they have tried to make their own loss-of-function alleles by mutating the coding area of cmd-1, but it resulted in presumed lethality, this might be mentioned here too.

This is a good point, and one that we had overlooked. cmd-1 loss of function mutations do indeed confer lethality. We have added a sentence to say:

‘Straightforward comparison of camt-1 and cmd-1 loss of function phenotypes was not possible, since disrupting cmd-1 confers lethality (7, 8).’

1. I am most worried about the potential caveats with the calcium imaging experiments. As the authors note, it is challenging to infer absolute levels of calcium using the ratiometric sensor cameleon across different individuals and genotypes. However, the authors do not note that the YFP/CFP FRET signal from cameleon might be perturbed because it uses calmodulin to bind calcium. At the end of their study (line 244), they provide evidence that calmodulin may bind to CART-1 to suppress its own expression when calmodulin is highly expressed. This is worrisome because cameleon is probably expressed highly in some or most of these strains. The authors may want to re-examine neuronal activity for a subset of experiments with a method that is independent of a calmodulin-based sensor (if possible).

We agree that this is a potential concern. As suggested by our referee, we therefore repeated some of our Ca2+ imaging experiments using a genetically-encoded Ca2+ indicator that does not contain CaM. We opted to use TN-XL, an indicator that uses troponin C as the Ca2+ binding moiety, and which has previously been used successfully in C. elegans. We imaged CO2-evoked Ca2+ responses in BAG sensory neurons, in wild type and in camt-1 mutant animals. The data obtained using TN-XL recapitulated what we observed using YC3.60 (BAG).

1. The title of "Fig 3 - Figure supplement 1" is confusing because it suggests that they measured the levels of YC2.60 cameleon, when in fact they measured a separate GFP reporter, albeit using the same promoter. So they could clarify the figure title.

The reviewer is right – our heading was confusing. We have changed it, and now say: ‘Expression from the gcy-37 promoter is reduced when CAMT-1 is overexpressed.’

1. D. Bazopoulou, A. R. Chaudhury, A. Pantazis, N. Chronis, An automated compound screening for anti-aging effects on the function of C. elegans sensory neurons. Sci Rep 7, 9403 (2017).
2. M. S. Choi et al., Isolation of a calmodulin-binding transcription factor from rice (Oryza sativa L.). J Biol Chem 280, 40820-40831 (2005).
3. J. Han et al., The fly CAMTA transcription factor potentiates deactivation of rhodopsin, a G protein-coupled light receptor. Cell 127, 847-858 (2006).
4. N. Bouche, A. Scharlat, W. Snedden, D. Bouchez, H. Fromm, A novel family of calmodulin-binding transcription activators in multicellular organisms. J Biol Chem 277, 21851-21861 (2002).
5. T. Yang, B. W. Poovaiah, A calmodulin-binding/CGCG box DNA-binding protein family involved in multiple signaling pathways in plants. J Biol Chem 277, 45049-45058 (2002).
6. E. Kodama-Namba et al., Cross-modulation of homeostatic responses to temperature, oxygen and carbon dioxide in C. elegans. PLoS Genet 9, e1004011 (2013).
7. V. Au et al., CRISPR/Cas9 Methodology for the Generation of Knockout Deletions in Caenorhabditis elegans. G3 (Bethesda) 9, 135-144 (2019).
8. A. Karabinos et al., Functional analysis of the single calmodulin gene in the nematode Caenorhabditis elegans by RNA interference and 4-D microscopy. Eur J Cell Biol 82, 557-563 (2003).

I think this is really interesting. As a cis woman I can recognise where acting straight and feminine in society can have its benefits, rather than deviating away from the "norm". Although we find in many aspects fitting into the traditional norm can be beneficial potentially to acquiring government funds, or being more desirable for a job position because you act in a certain way. Although these are not fair, it is seen often.

However, I would argue everyone is in some way queer. When alone, or with those they are comfortable with, may present traits that are not considered as desirable by society. Whether it be based gender roles throughout the household. For an example, my partner and father of my children, doing the nightly routine with our children, playing games such as dress ups and tea parties, reading books with the children and giving them affection and more on the emotional level, all whilst I work in the office. While we believe this is equal, would society suggest these activities are "queer" for a "Male" to be doing? Or are we at a place where these activities are more accepted? (I believe it is more accepted" but based on traditional nuclear families, are these activities queer?

This is just one example but could relate to many activities that go on behind closed doors, that the public eye does not see where people would generally act out of typical "straightness, feminine or masculine roles". I would argue people are more 'queer" in those situations. I hope this makes sense.

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The Pretty Reckless - And So It Went [feat. Tom Morello] (Official Lyric Video)

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The Pretty Reckless - And So It Went [feat. Tom Morello] (Official Lyric Video) From the upcoming album 'Death By Rock And Roll' | Available February 12, 2021: http://found.ee/dbrr Subscribe to The Pretty Reckless on YouTube: https://found.ee/tpr_subscribeyt Photo by: Rob Fenn Pre-order/Pre-Save the album 'Death By Rock And Roll': iTunes: http://found.ee/dbrr_it Apple Music: http://found.ee/dbrr_am Spotify: http://found.ee/dbrr_sp Amazon Music: http://found.ee/dbrr_amzm Amazon: http://found.ee/dbrr_amz YouTube Music: http://found.ee/dbrr_ytm Stay connected with The Pretty Reckless Website: https://deathbyrockandroll.com/ Facebook: https://found.ee/tpr_facebook Twitter: https://found.ee/tpr_twitter Instagram: https://found.ee/tpr_instagram LYRICS And so it went the children lost their minds Begging for forgiveness was such a waste of time And the bullets start to fly And the bough's about to break When you hear them cry It's too much for me to take The world does not belong to you You are not the king I am not the fool They said the world does not belong to you It don't belong to you It belongs to me And, so it went the children lost their minds Crawling over bodies of those who gave their lives And the fists begin to throw And the fire starts to blaze Don't you think they know They're the fucking human race The world does not belong to you You are not the king, I am not the fool They said the world does not belong to you It don't belong to you It belongs to Everyone is crying out, I can hear them scream With all these eyes upon us but no one seems to see That you and me are just the same as god meant it to be But you're much too close to me. You're much too close to me So it went The children lost their minds Nowhere to run, nowhere to hide And the wind begins to howl And the wolf is at your door You have so much of everything But still you wanted more They said the world does not belong to you You are not the king, I am not the fool They said the world does not belong to you It don't belong to you It belongs to me #ThePrettyReckless #DeathByRockAndRoll

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Adam Dobrin

1 second ago

VERBATIM: "stop hurting people, stop hurting people. if you do not believe people should be hurt" ... ((ishing)) ~either disable the attackers or leave the area~

1

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Adam Dobrin

1 second ago

sometimes words have two meanings. sometimes "kill" means "excorcized the demons" @LOUDON @SALEM

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Raziel Moreno

7 months ago

I'm so damn happy she chose to rock instead of staying as an actress.

426

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sonya fuks

7 months ago

How is it possible they just keep on getting better and better with each album?!?!

341

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Graziano D'Ovidio

7 months ago

I love her voice. So much.

652

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Natsumi Ikari

7 months ago

Most of people know Tom Morello from RATM of Prophets of Rage, but don't forget he was also the guitarist of Audioslave, whose singer was Chris Cornell. Must be emotional for Taylor to record this song him. I'm sure Chris is proud of them both.

631

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Matstrr Salazar

7 months ago

This is why TPR is one of my all time favorite bands. They release bad-ass singles and albums

322

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Mike Whicker

7 months ago

The entire death by rock n roll album is first on my list of reasons why 2021 will not stink as much as last year did!

180

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Mackenzie Dinkins

5 months ago

A good theme for this year's Elimination Chamber event.

25

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FDCAnselmo

7 months ago

The Anthem of Youth... Fuck me what a tune.

159

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norangutan

7 months ago

I love how obviously this song draws inspiration from Audioslave's sound, and in a way it's paying homage to chris cornell especially with the "you have so much of everything still you wanted more" line - which is almost the same as a line from Audioslave's 'What you are'

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Shiteyanyo

7 months ago

Tom Morello?! Each single they release for this album just keeps getting better and better

55

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Lau pin

7 months ago

I fell in love with every song they released

90

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🕸️Haley Munster🕷️

7 months ago

I really hope this album has more Going to Hell vibes 🥺🥺🥺🖤

201

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M K

7 months ago

Great tune and a good example for why I think TPR is among the best of the current rock bands. They always have catchy riffs and melodies however, especially in the bridge, you never know which turn the song is gonna take. Yet it always fits the song and never sounds out of place.

15

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Mike B

7 months ago

Underrated band

154

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🕸️Haley Munster🕷️

7 months ago

THIS is the Pretty Reckless I love !! 💙

45

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pedrogoularth

7 months ago

This is SO fucking good! 💖 🇧🇷

31

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Nick Brick

7 months ago

Kicking the year off with a banger. Looking forward to the album!

19

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Jo Mill Hyde

6 months ago

The fucking power in her voice and this song

49

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HT82 Smash

6 months ago

WWE main roster stepping up their game with these theme songs. Elimination Chamber 2021 everyone

17

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Hide

7 months ago

The addition of the children's choir killed and buried me. 😔🤘🏾

80

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Lynne Kelly is correct here that we need better delineations of the words we're using here.

To some of us, we're taking historical methods and expanding them into larger super sets based on our personal experiences. I've read enough of Kelly's work and her personal experiences on her website (and that of many others) that I better understand the shorthand she uses when she describes pieces.

Even in the literature throughout the middle ages and the Renaissance we see this same sort of picking and choosing of methods in descriptions of various texts. Some will choose to focus on one or two keys, which seemed to work for them, but they'd leave out the others which means that subsequent generations would miss out on the lost bits and pieces.

Having a larger superset of methods to choose from as well as encouraging further explorations is certainly desired.

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

Learn more at Review Commons

Reply to the reviewers

Reviewer #1

This manuscript by Gulyrutlu and co-workers addresses the role of CUG expanded repeat RNA associated with DM1 in regulating the formation of higher order RNP assemblies such as stress granules and P-bodies in the cell. The authors used lens epithelial cells (hLECs) derived from a DM1 patient

We used cell lines from several patients and age-matched controls to avoid effects of individual cell-line variation. We will make sure that this is clear in the text.

or a HeLa cell inducible model of DM1 to investigate whether expression of the CUG repeat-associated protein MBNL1 and CUGBP1 affected the formation and dispersal of stress granules and P-bodies. The authors show that MBNL1 and CUGBP1 are components of SGs and PBs in hLECs and HeLa cells. In cells expressing the CUG repeat, there are minor alterations in the dispersal of stress granules as well as in the formation of P-bodies.

The alterations in the formation and dispersal of stress granules are not minor. For example, in the HeLa cell model, stress granules take more than twice as long to form in cell expressing the CUGexp repeats associated with DM1 and disperse in half the time. These data are already in the results section, but we will highlight them in a revision and have included an additional representation of the data to the figure, using graphs of ‘proportion of cells with stress granules’ against time. The changes we see are as large, or larger, then results published elsewhere (see appendix below)

MBNL1 could affect the formation and dispersal of SGs independent of the CUG repeat.

In fact, we present data in HeLa cells with MBNL1 almost completely removed by shRNA revealing that this has a much smaller effect on stress granules than does the expression of CUGexp RNA. This is an important point, as it is widely assumed that most of the cellular defects in DM1 are caused by the ‘sequestration’ of MBNL1 in the CUGexp foci. Since only . This is not what our data show. In the hexanediol experiments, both cell lines over-express MBNL1 in similar amounts. The difference between them is that one cell line expresses a DMPK1 mini-gene with a CUG expansion and the other expresses a mini-gene without the expansion. Again, our results show that the alteration to P-body responses to 1,6-hexanediol can be attributed to the presence of the CUGexp RNA, rather than altered levels of MBNL1. We will revise the results and discussion to further emphasise this point.

Finally, in HeLa cells, overexpression of MBNL1 can reduce the dispersal of P-bodies upon 1,6-hexanediol treatment.

This is not what our data show. In the hexanediol experiments, both cell lines over-express MBNL1 in similar amounts. The difference between them is that one cell line expresses a DMPK1 mini-gene with a CUG expansion and the other expresses a mini-gene without the expansion. Again, our results show that the alteration to P-body responses to 1,6-hexanediol can be attributed to the presence of the CUGexp RNA, rather than altered levels of MBNL1. We will revise the results and discussion to further emphasise this point.

Major comments:

One limitation of the work is that the perturbations seen with stress granules or P-bodies are all relatively small, and no evidence for a functional consequence on gene expression is demonstrated. Specifically, the authors observe only minor alterations in the formation or disaggregation of PBs and SGs in these DM1 models. Further, some of the effects observed are independent of the CUG repeat expression, suggesting that MBNL1 and CUGBP1 might have independent roles in modulating some properties of SG and PB formation or dispersal.

As above, the changes we see in SG formation and dispersal are not small. There are already numerous studies of the effects of DM1 on gene expression and mRNA splicing. This is not what we set out to study: we are interested in perturbations to the organisation of cellular structures associated with the expression of the CUGexp repeat RNA characteristic of DM1. We do show some data relating specifically to the proteins MBNL1 and CUGBP1 in the paper. shRNA resulting in almost complete loss of these proteins has much smaller effects that the expression of CUGexp RNA, suggesting that the major part of the effects caused by expression of the CUGexp RNA is not mediated through changes in MBNL1 or CUGBP1 levels. MBNL1 and CUGBP1 levels may well contribute to alterations in SG dynamics, but our data suggest that they are minor contributors. This is an advance in our current knowledge

1. The authors could investigate whether the CUG repeat RNA itself is localized to SGs or PBs in their models, and whether the presence of the repeat RNA is absolutely necessary for regulating the dynamics of SG or PB formation.

We have now done this. The CUG repeat RNA is not localised in stress granules or PBs to a detectable extent. This suggests that the effect we see on these structures by expression of the expanded RNA occurs despite the absence of the RNA from the structures. This is similar to the effects of the ALS-associated paraspeckle protein FUS, which can affect the integrity of nuclear LLPS structures (gems) despite not co-localising with them https://doi.org/10.1016/j.celrep.2012.08.025 We have added these data to the manuscript, as part of draft figure 8, and will add text emphasising this as an additional example of disease-causing macromolecules affecting the structure of LLPS domains in which they are not found.

1. The authors use 1,6-hexanediol to suggests that PBs and SGs in HeLa cells show behavior analogous to LLPS. However, the use of 1,6,-hexanediol to establish an assembly as a LLPS is a relatively limited analysis (despite its widespread use in the field), since this compound can affect the formation of multiple cellular substructures that are not always LLPS (for example, see Wheeler et al, 2016, eLife).

We are aware of and have cited this publication. Our comments about LLPS structures are measured, as there is still controversy about how to definitively identify them in cells. SGs and PBs have, however, previously been widely published to be formed by LLPS. The rapid exchange of SG and PB components during FRAP and the ability of SGs to both fuse and bud (seen in our supplementary movies) are also supportive of these structures behaving as LLPS structures in our models. Wheeler et showed that, in yeast, the nuclear pore complex and some cytoskeletal structures were affected by 1,6-hexanediol but membrane-bound structures such as the ER and mitochondria were not. The disruption of the nuclear pore complex is not unexpected, since phase separation is involved in cargo shuttling through the NPC (reviewed in https://doi.org/10.1016/j.devcel.2020.06.033). We will revise our discussion to make it more clear that we are not relying only on the use of 1,6-hexanediol to define SGs and PBs as LLPS structures but also on other aspects of their dynamic behaviour and on extensive prior literature.

Significance

This study would be of interest to the field if the impact of the DM! repeat RNAs on PB and SG were more substantial...

As above, the effects we see on SG formation and loss are substantial. Tissue types affected in DM1 are prone to stress, particularly the lens of the eye, so alterations to cellular response to stress associated with the presence of CUG repeats are of key importance to understanding the cellular pathology of DM1.

...and if some functional consequences were demonstrated.

In terms of function, we show altered responses to stress caused by the expression of CUGexp RNA and probably mediated through alterations in the propensity of LLPS cytoplasmic structures (SGs and PBs) to form and be resolved. Additionally, we can now show that SGs in HeLa cells expressing CUGexp RNA contain less total polyA RNA than is seen in controls, and that ‘docking’ events between SGs and PBs are compromised in cells with CUGexp RNA. These docking events are proposed to mediate transfer of RNA from SGs to PBs (reviewed in https://doi.org/10.1007/978-1-4614-5107-5_12). These new data demonstrate functional impairment of SGs and PBs associated with DM1. We have included this as an additional draft figure 8.

The lack of a strong effect on SG or PB formation in the DM1 models, along with the CUG repeat-independent effect of MBNL1 on the formation and dispersal of these complexes, argues that MBNL1/CUGBP1 may not significantly affect the formation or dispersal of SGs and PBs.

We are actually not arguing that MBNL1 and CUGBP1 are the main effectors in the changes we see to SGs and PBs, but that the CUGexp RNA is the key player, so are a little confused by this comment.

Reviewer #2:

In the current study, the authors compared the dynamics of P-bodies (PBs) and stress granules (SGs) between control and several DM1 cell lines. They found that MBNL1 and CUGBP1, two CUG repeat RNA-binding proteins that are primarily nuclear, could also co-localize with PBs in the cytoplasm and re-localize to SGs under stress. Small differences were observed in SG assembly and disassembly dynamics between control and DM1 HLECs, between HeLa cells expressing either CTG12 or CTG960, and between HeLa cells with and without shRNAs targeting CUGBP1 or MBNL1.

As detailed above, the alterations in SG assembly and disassembly in cells expressing CUGexp RNA are not small, in contrast to those in cells will lowered expression of MBNL1 and CUGBP1, which are much smaller suggesting that the changes caused by CUGexp RNA largely do not result from loss of MBNL1 (or CUGBP1). We have inserted additional graphs of ‘proportion of cells with stress granules’ against time' and will modify the text to emphasise both of these points.

Overall, the experiments were clearly described and the results properly presented. However, critical controls, as detailed below, are missing in multiple analyses. The mechanisms underlying these apparent differences are also unknown.

We do not consider that any ‘critical controls’ are missing, but can supply all of the additional analysis of our data that the reviewer requests below. We can also now provide additional mechanistic insight and will add an additional figure showing lowered amount of polyA RNA in stress granules in cells expressing CUGexp RNA and compromised docking events between stress granules and P-bodies, suggesting impaired communication between them.

Major concerns:

1. Throughout the study, the authors compared MBNL1 and CUGBP1 association with PBs and SGs without considering the potential differences in their cytoplasmic abundance between control and DM1 cell lines, which seems to be case for MBNL1 abundance in CTG960-expressing HeLa cells (Fig. 3). Provided that PBs and SGs exchange components with the cytosol at an equilibrium, if the cytoplasmic abundance of, for example, MBNL1 is decreased in DM1, one would expect the equilibrium being shifted resulting in less MBNL1 associated with PB/SG. Therefore, before measuring the association or the assembly/disassembly kinetics of PB and SG, the authors should first test whether MBNL1 and CUGBP1 abundance may be different between control and DM cell lines.

There is, in fact, no difference in the relative cytoplasmic abundance of GFP-MBNL1 between CTG12 and CTG960- expressing HeLa cells. Each has approximately a 50/50 split between nucleus and cytoplasm, with <3% of nuclear GFP-MBNL1 found in nuclear CUGexp foci when they are present. We have added a graph demonstrating this to the supplementary data. The abundance of total endogenous MBNL1 is also not altered in DM1 patient-derived lens cell lines compared to controls, as shown by semi-quantitative western blot analysis, which we have also added to the supplementary data. However, if the expression of CUGexp RNA did cause a major loss of cytoplasmic MBNL1, this change would be reflective of the situation seen in DM1 and would not invalidate our results or conclusions.

The same caveat applies to MBNL1/CUGBP1 knockdown experiments, where knocking down one may change the abundance of the other.

To carry out FRAP experiments or live cell analysis of SG formation and loss, it is necessary to over-express a tagged version of the protein being studied. For the knockdown experiments shown in figure 6, therefore, when we knocked down MBNL1, CUGBP1 was present in excess as a GFP-tagged protein and when we knocked down CUGBP1, MBNL1 was present in excess as a GFP-tagged protein. Thus, any effects of the knockdowns on expression of the endogenous proteins being analysed would be highly unlikely to influence the results.

1. Similarly, the authors did not consider the possibility that changes in SG/PB dynamics may be due to changes in the abundance/availability of essential SG/PB components such as GE1 and G3BP1.

From our immunofluorescence experiments, there was certainly no obvious reduction in GE1 or TIA1 abundance (we did not assess G3BP1). We have quantitative proteomic analysis (unpublished) from a similar pair of cell lines, expressing CUGexp RNA alongside GFP rather than GFP-MBNL1. This shows no change in GE1 or G3BP1, so we would not expect to see any here either. We can easily carry out a quantitative western blot analysis to confirm and will add this to the supplementary data

1. Most of the observed differences between control and DM cell lines were modest, leaving one wonder whether it could be simply due to cell line-to-cell line variability. Whenever possible, the authors should present results for each individual lines. For example, in Fig.2, 3 DM1 lines and 2 control lines were used. Was the difference in SG disassembly (Fig. 2B) observed in each of the 3 lines?

Some of the alterations were modest and there is cell line-to-cell line variability in the lens cell lines. This is why we pooled the data: on average, DM1 cells disperse their SGs more quickly than control cell lines do on average. This is not an unusual way to present data from patient cell lines of diverse genetic background. We have added data for stress granule loss in the individual cell lines to the supplementary data. These data show a consistent trend towards quicker dispersal of stress granules in patient cell lines. The variability between the patient lens cell lines was also the primary reason for us to develop the inducible system in HeLa cells, on a fixed genetic background, as explained in the manuscript.

Minor points:

1. Western blot in Fig. 3 shows two protein products from both endogenous and overexpressed MBNL1. Please explain.

Many of the commercially available anti-MBNL1 antibodies show this double-band in some cell lines as evidenced in numerous publications and on manufacturers’ websites (for example https://abclonal.com/catalog-antibodies/MBNL1RabbitmAb/A5149, https://www.ptglab.com/products/MBNL1-Antibody-66837-1-Ig.htm). We haven’t analysed the two bands in detail, but assume this to be a result of a post translational modification of some sort. Since GFP-MBNL1 and endogenous MBNL1 show the same thing, we do not consider it to be a major concern. We do mention the double-band as ‘characteristic’ in the figure legend for figure 3 so are not seeking to conceal anything here.

1. No data were shown to substantiate the statement that "MBNL1 localises to CUGexp foci and CUGBP1 does not" (page 6).

This has been published many times and is shown in figure 1A. However, we will add in a citation for this and have added an additional supplementary figure showing the lack of co-localisation in the foci from figure 1A more clearly together with separate data confirming that MBNL1 and CUGexp RNA do not co-localise with CUGBP1 in the nuclei of line HeLa_CTG960_GFPMBNL1.

1. The y-axis of Fig. 4D should not go beyond 1.

We will trim the axis. There are no data points above 1.0, just the indicator of statistical significance

Significance:

The nature of the current study is highly descriptive with little mechanistic insights.

Our work is not descriptive, as we observed a change in stress granules in patient cells, which we could then replicate (and enhance) in a novel inducible model of DM1 designed to abrogate the unavoidable variation in patient-derived cell lines. We also now have additional mechanistic insights (see above) and have added an additional figure (draft figure 8) detailing these.

For the subtle differences observed between control and DM1 cell lines, it remains unclear whether it may be due to cell line-to-cell line variation (see above).

We cannot completely rule out an influence of cell line-to-cell line variation in the patient-derived lens cell lines (see above), though we think this unlikely as we saw the same effect repeated and amplified in the inducible HeLa-derived cell model, which was designed to minimise this concern. Furthermore, for stress granule loss, we see a larger effect in the HeLa cell model after 72hrs of induction than after 24hrs (figure 5C). This argues strongly that the effects seen are due to the expression of CUGexp RNA and we will emphasise this point more strongly.

Some difference appear to be specific to one model but not the others (e.g., SG formation is slower in HeLa-CTG960 cells but not in DM1 HLECs).

Even for the observations that seem consistent between models, the current results yielded little novel biological insights into whether and how these subtle differences in PB/SG dynamics may relate to DM1 pathogenesis. Collectively, these weaknesses render the current study incremental at best.

The key biological insight the results provide is that the presence of the CUGexp repeat RNA results in defects in LLPS structures that are largely separable from any sequestration of MBNL1 in nuclear foci. With many researchers attributing the cellular defects in DM1 simply to the loss of MBNL1 by sequestration into nuclear foci, both this separation of altered stress response from MBNL1 levels and the involvement of altered LLPS formation (evidenced by the changes in PB behaviour on 1,6-hexanediol treatment) are novel biological insights into the cellular pathology of DM1. Additionally, our results shift the emphasis from nuclear effects to those seen in the cytoplasm.

In terms of specific DM1 pathogenesis, the eye lens is subject to constant repeated stress and is subject to continued growth throughout the life span, relying on lens epithelial cells as a stem cell pool. Epithelial cells are also vital to the homeostatic regulation of ions, growth factor and nutrient flow from the aqueous humor to the underlying fibre cells. Any alterations in the response of lens epithelial cells, in particular, to stress is highly relevant to the pathology of cataract seen in DM1. We will revise our discussion to emphasise these key points more strongly.

Reviewer #3

The manuscript entiled "Phase-separated stress granules and processing bodies are compromised in Myotonic Dystrophy Type 1" by Gulyurtlu et al., characterizes the composition and ydnamics of stress granules and P-bodies in two Myotonic Dystrophy Type 1 (DM1) cell models, human lens epithelial cells from DM1 patients and age-matched controls and HeLa_CTG12_GFPMBNL1 and HeLa_CTG960GFPMBNL1 cell lines. The manuscript is somewhat descriptive with lack of functional data and some discrepancies. For example, in the discussion section, the authors conclude that "MBNL1 appears to be absent from P-bodies in cells with CUGexp foci in their nuclei. This observation suggests that the role of MBNL1 in P-bodies may be disrupted by the presence of CUGexp RNA." Figure 4A shows that "P-bodies in the DM1 model line, HeLa_CTG960GFPMBNL1 do not contain detectable amounts of GFPMBNL1". However, Figure 4E shows similar levels of total cellular MBNL1 per PB between the control CTG12 and mutated CTG960 lines.

There is no discrepancy here. The reviewer has misinterpreted our data. PBs in the HeLa CTG960 cell line do not contain detectable amounts of GFP-MBNL1 under normal growth conditions, as shown in figure 4A. The data shown in figure 4E concern arsenite-treated cells, where some PBs in the CTG960 line do contain detectable levels of GFP-MBNL1, but significantly less than in the control CTG12 cells. We will reword these sections to make sure this is clear.

Most importantly, in Figure S3 the authors show that CUGexp foci are present in 1-2 % of the cells. The claim appears to be too strong for the data presented in the manuscript.

This is not what is shown in figure S3. The reviewer has misinterpreted our data. Figure S3 shows that in cells from line CTG960, only 1-2% of the total nuclear GFP-MBNL1 signal is found in the CUGexp foci, despite the intensity of the signal within them. Virtually all of the cells from the CTG960 cell line contain CUGexp foci (>95%). We will add a statement to this effect into the results section. We would not have continued working with a cell line in which only 1-2% of cells showed the DM1 phenotype of nuclear CUGexp RNA foci.

Although the findings are interesting and of potential impact for a better understanding of the implications of RNA-protein condensate dynamics in the pathogenesis of DM1, the work presented here is still descriptive and preliminary in my opinion. In summary, the conclusions are not so convincing and additional experiments are essential to support the authors claims.

This reviewer seems to have misinterpreted several of our data sets, including the specific points above, leading to the assertion that our conclusions are not convincing.

Several months of works will be required to consolidate data and reorganize and ameliorate the manuscript, including the way data are presented and quantified.

We already have data with which to address the majority of the queries posed, so should be able to make the adjustments relatively quickly.

Specific comments:

"On removal of stress, clearance of stress granules is mediated largely by a form of autophagy." This statement is not correct since the majority of stress granules disassemble and are not targeted to autophagy; in healthy cells only 5 % (or less) of the total SGs tend to persist in presence of autophagy or lysosome inhibitors, while the vast majority disassembles. Please rephrase carefully.

The degree and manner of dispersal of stress granules in healthy cells on removal of stress is not well understood, but is known to differ depending on the type and duration of the stress (DOI: 10.1126/science.abj2400). We do not yet know how this may be altered in DM1, however, compromised autophagy is implicated in cataract formation, which is of relevance to our study. We will re-phrase this section of the discussion carefully to reflect the complex situation.

Figure 1: RNA-protein complexes have heterogeneous composition. In HLECs, do all PBs colocalize with MBNL1 and CUGBP1 or only a fraction of them?

We do not routinely see PBs without MBNL1 or CUGBP1 in the HLECs, in contrast to the situation in the HeLa CTG960 line. We have data available in order to quantify this and will add the numbers to the text of the results.

Figure 2: Stress granules and P-Bodies are known to touch each-other, a process referred to as a "kissing event". The authors have studied the mobility of GFP-MBNL1 inside these two types of assemblies. It would be important also to quantify the "kissing" events. Is this altered in DM1 cells?

We couldn’t find reference in the literature to ‘kissing events’ between SGs and PBs, but found several references to ‘docking’ events. We have noticed such interactions between PBs and SGs in our models. We are currently quantifying this and our first experiments (one in the HeLa cell model and one comparing one of the patient-derived lens cell lines to a control) suggest that there is a change in the frequency and/or size of such interactions in the HeLa CTG960 cell line compared to the CTG12 control and in the DM1 lens cell line derived to control. If this holds true in our repeat experiments (currently in progress), this would also provide the mechanistic insight requested by reviewer 2. We have included this, together with our data showing a decrease in total polyA RNA in stress granules in HeLa cells expressing CUGexp RNA, as an additional draft figure 8.

Figure 3: In HeLa cells overexpressing CTG960_GFPMBNL1, beside the accumulation of one bright CUGexp puncta, several intranuclear GFPMBNL1 protein foci are visible. This subcellular distribution is different from the one observed in the control HeLaCTG12 GFPMBNL1. Can the author describe what these intranuclear GFPMBNL1 protein foci are?

In most cells expressing CUGexp RNA, several nuclear foci form (usually one large and several smaller) and all of them contain MBNL1 (or GFP-MBNL1 in the HeLa_CTG960_GFPMBNL1 cell line). Figure S3 shows object identification using MBNL1 in this cell line, with two clear foci detected as the reviewer points out. We have added an additional panel to supplementary figure 1 to confirm that the additional foci are also CUGexp RNA foci and will clarify in the text of the results that there is not a single focus of CUGexp RNA in each nucleus.

Is GFPMBNL1 accumulating at the level of splicing speckles? Or paraspeckles? Or other types on intranuclear condensates such as e.g. PML nuclear bodies? The different intranuclear distribution of GFPMBNL1 should be better characterized.

The sub-nuclear distribution of MBNL1 is, indeed, very complex. MBNL1 also sometimes co-localises to splicing speckles/interchromatin granule clusters as we have previously reported in lens epithelial cell lines (DOI: 10.1042/BJ20130870 ) . The details of differences in the nuclear distribution of MBNL1, beyond its accumulation in CUGexp RNA foci, in DM1 cells compared to controls is the subject of another manuscript we have in preparation but are beyond the scope of the current study.

Moreover, the % of cells expressing CTG960_GFPMBNL1 and forming intranuclear CUGexp foci is only mentioned in the discussion (Figure S3); for clarity it should be reported in the main text when describing Figure 3.

The number of cells forming nuclear CUGexp foci on expression of CTG960_GFP-MBNL1 is >95% and we will add this to the text of the results section.

"Figure S2: Quantitation of GFPMBNL1 in P-bodies in HeLa cell model of DM1." The authors report in the legend "Some, but not all, of these P-bodies contain detectable amounts of GFPMBNL1". However, the figure only shows a representative image of cells without quantification. Quantitation should be provided.

We have data available to provide this simple quantitation. Approximately 38% of PBs in arsenite-treated cells from line HeLa_CTG960_GFPMBNL1 contain detectable levels of GFPMBNL1 using a manually-assigned cut-off intensity. We will add this to the relevant figure legend (now figure S5). However, this method of analysis requires an intensity to be manually set above which GFP-MBNL1 signal is considered ‘detectable’. This is hugely subjective, and in our opinion, the automatically generated quantitative comparison of “% total cellular MBNL1 per P-body” as shown in figure 4E is a more experimentally robust way to demonstrate a small loss of MBNL1 from P-bodies in cells from line HeLa_CTG960_GFPMBNL1 treated with arsenite compared to the relevant control.

The authors report "a subtle change in stress granule architecture associated with the presence of CUGexp RNA". This statement is not supported by experimental data and should be omitted.

We will qualify this statement to make it clear that we are referring to a subtle alteration in the co-localisation between CUGBP1 and MBNL1 specifically in the SGs, as our experimental data shown in figure 4D clearly support that, showing a statistically significant increase in the Pearson’s co-efficient of cololcalisation between MBNL1 and CUGBP1 in cell containing CUGexp RNA compared to the relevant control (0.90+/-0.05 for CTG960; 0.87+/-0.07 for CTG12).

Figure 4. MBNL1 and CUGBP1 co-localise in P-bodies. What is the % of colocalization?

We’re not sure exactly what is being requested here or what biological question the reviewer is asking us to address. MBNL1 and CUGBP1 co-localise in virtually all PBs (except in the HeLa CTG960 line where MBNL1 is undetectable in PBs under normal growth conditions). Figure 4E shows that, in cells with PBs upregulated by sodium arsenite, the mean amount of total cellular MBNL1 per PB is 0.1%, so it will be similar in cells grown under normal conditions as the PB sizes are similar and they appear to be of similar brightness by immunofluorescence. Again, this would be straightforward to quantify with our existing data if this is, indeed what the reviewer is requesting, but we question the biological significance. We would be reluctant to derive a Pearson’s co-efficient for the degree of co-localisation between CUGBP1 and MBNL1 in P-bodies as the structures are too small in size for this to be meaningful within the limits of imaging capabilities. We could, however, provide this if this is a specific request.

Figure 5: "Treatment with sodium arsenite was then carried out under time-lapse microscopy, with Z-stacks of images taken every 4 minutes until stress granule formation was clearly seen (Fig.5A). This revealed a pronounced delay in formation of stress granules in cells containing CUGexp foci (HeLa CTG960 GFPMBNL1, 36 min +/- 12) compared to those without (HeLa CTG12 GFPMBNL1, 15 min +/- 2) (Fig.5B)." Data representation in Figure 5 is unclear and the pronounced delay in stress granule formation is not appreciated. Since the authors performed a live imaging taking pictures every 4 minutes, it would me more informative to plot the data and show the assembly and disassembly kinetics over time for both control and CTG960_ GFPMBNL1 cell lines (similar to what shown in e.g. Gwon et al., Science 2021, Ubiquitination of G3BP1 mediates stress granule disassembly in a context-specific manner, Figure 2G).

The bar graph in figure 5B shows that cells from the CTG960 line take more than twice as long to form SGs compared to controls and are lost in half the time, with the precise numbers given in the text. A simple bar graph seemed the clearest way to present this. However, we have plotted our existing data in a similar manner similar to that in the cited reference and added this to figure 5. These graphs clearly show that the differences we see are at least as great as in other published literature, including the reference given by the reviewer (see below).

Concerning Figure 1, the authors report no difference in the kinetic of stress granule formation in HLECs. However, they only report data after 45 and 60 min of arsenite treatment; at these time-points the assembly step is maximal. Thus, for consistency, the authors should include earlier time-points to the analysis of stress granule assembly also in HLECs, similar to what done in HeLa cells in Figure 5.

The assembly step is not ‘maximal’ in these cell lines after 45 minutes. Figure 2A clearly shows that only ~30% of cells have SGs after 45 minutes of treatment, compared with 100% of cells after 90 minutes shown in figure 2B. We have additional data at 10, 20 and 30 minutes all showing no significant differences. We had omitted them to keep the graph simple, but have now included them as a graph of ‘% of cells with stress granules against time’ in figure 2.

"Having established that MBNL1 and CUGBP1 co-localise closely in stress granules": the authors investigated the colocalization of each of these two proteins with stress granule markers but they did not verify whether MBNL1 and CUGBP1 co-localise.

In figure 1B we show that endogenous CUGBP1 and endogenous MBNL1 both co-localise with the stress granule marker TIA1 in stress granules in lens epithelial cells. It would, therefore, be highly unlikely that CUGBP1 and MBNL1 would not co-localise with each other in stress granules. We have also previously verified that GFPMBNL1 behaves identically to its endogenous counterpart (Coleman et al, 2014). Furthermore, in figure 4C and 4D, we show close co-localisation between endogenous CUGBP1 and GFPMBNL1 in stress granules in our HeLa cell model, using high-resolution AiryScan microscopy for which we provide detailed quantitation.

This aspect should be addressed experimentally since the authors also conclude that "a complex relationship between MBNL1 and CUGBP1 in stress granules" exists. Thus, the authors need to assess the colocalization of GFPMBNL1 with endogenous CUGBP1 in stress granules and the one of GFPCUGBP1 with endogenous MBNL1.

The complex relationship we propose is based on the effects of CUGBP1 or MBNL1 knockdown on the dynamic behaviours of each other by FRAP assay and not solely on their co-localisation, although we have already analysed their co-localisation in detail as above.

Figure 6: Please add antibody labeling to microscopy panels A and B.

Certainly, this was an accidental omission and has been added

Moreover, specify is the numbers refer to minutes in panel F. The data representation is also unclear - see comment above, Figure 5.

As stated in the figure legend and on the graph axes, these numbers have been normalised to the mean time taken for SG formation/loss in the control CTG12 cell line (set at 100%). The precise numbers in minutes for mean and SD are given in the text. We have added additional graphs of ‘% of cells with stress granules against time’ to this figure, with the values in minutes given to clarify the exact time-scale.

Figure 7: was 1,6-hexanediol added in presence of arsenite? Or was arsenite removed?

Arsenite was not removed (neither was Doxycycline) as we wanted to examine the effect of 1,6-hexanediol on SGs and PBs without the added complication of the effects of stress removal. We will clarify this point in the methods/results.

Aberrant persistent stress granules have been implicated in age-related (Mateju et al., 2017) and neurodegenerative diseases (Protter and Parker, 2016), such as ALS and FTD (Jain et al., 2016; Markmiller et al., 2018; Zhang et al., 2018). These are proposed to result from increased liquid-to-solid phase transitions within the stress granules (Mateju et al., 2017)." The authors should better define what are aberrant stress granules (e.g. see Ganassi et al., 2016; Turakhiya et al., 2018, PMID: 29804830).*

We will expand on this subject in the discussion

"Persistent stress granules have long been associated with degenerative conditions, notably ALS (Li et al., 2013)". I suggest updating the reference adding a more recent one.

We selected this 2013 review to emphasise that there is a long history of association of persistent stress granules with degenerative conditions. We will add in an additional, more recent review.

Significance

The work is descriptive; thus, in this form I do not consider that it is strongly advancing the field.

Having noted alterations to stress granule disassembly in lens epithelial cells from DM1 patients, we went on to develop a novel inducible model in which we replicated and enhanced these effects by expressing the large CUGexp RNA that causes DM1 as part of a DMPK mini-gene mimicking the genetic mutation seen in DM1 patients. This is not purely descriptive. Furthermore, we are now in a position to add an additional figure showing two pieces of evidence for functional defects in stress granules associated with CUGexp RNA expression 1) reduced accumulation of total PolyA RNA in stress granules indicating compromised function and 2) compromised ‘docking’ events between stress granules and P-bodies, a process proposed to be integral to the function of both structures.

Author Response:

Reviewer #1:

The authors demonstrate deficits in perceptual tests related to fine-time perception in non-speech and speech sounds in a group of patients with stroke aphasia compared to a control group without a lesion. A subgroup of patients with deficits in spectrotemporal processing at a fine timescale have lesions mapped to the posterior STS, MTG and adjacent white matter. The area associated with deficits in spectrotemporal analysis with a fine timescale is then used as a seed for probabilistic fibre tractography based on diffusion MR. These results show connectivity of the functionally defined seed region with a number of areas including the cerebellum.

The work is carefully done and I think interesting in demonstrating the cerebellar connections of the functionally defined region associated with deficits in fine temporal analysis that might be a basis for event representation at this temporal level.

We appreciate the referee's evaluation and constructive feedback.

Reviewer #2:

Based on consideration of supportive evidence in the literature, the authors propose that a cerebellar-temporal lobe functional network plays a key role in auditory temporal processing. The precise parsing of temporal information is critical to understanding dynamic auditory processing and thus is an interesting area of study. Better understanding of how the cerebellum and temporal lobe may interact to achieve such parsing of the dynamic signal in a generative/predictive internal model is of clear interest to a broad readership. This idea is put to the test by first having individuals with lesions in the posterior portion of left temporal lobe perform speech perception and timing tasks and comparing performance with 12 healthy controls to establish the role of this region in tasks reliant on intact fine temporal processing. Typically, a lesion model will be helpful when a dissociation between structure and function can be demonstrated, and preferably this would be a double dissociation. Here, while lesions to auditory regions of the left temporal lobe are associated with impoverished performance on speech and temporal order tasks relative to a healthy control group, performance on comparably difficult auditory tasks that do not require good temporal discrimination is not tested to determine if there is such a dissociation. Given the extensive discussion of hypothesized different time sensitivities of right and left auditory cortices in the Introduction, patients with right homologous lesions might also have served as an interesting control and could have supported a double dissociation. In a second step to their study, a seed region was generated based on comparison of the lesion loci for the half of the patients who performed most poorly on the behavioral tasks to the other half, and this was used to explore anatomical tract connectivity of the seed region to the rest of the brain in the neuroimaging data from the healthy controls, with a focus on connections with the cerebellum. This approach to establishing that "temporo-cerebellar connectivity underlies timing constraints in audition" is unfortunately just not that convincing. The data are interesting, but taken alone they simply do not support such a conclusion. In the data, there is no clear functional link established or even hinted at between the temporal lobe and the cerebellum.

We appreciate the referee's evaluation and constructive feedback. We address the raised concerns point by point below. We appreciate the concerns regarding our methodological choice and our interpretation of a functional link between the temporal lobe and the cerebellum. It certainly is more reasonable to derive a functional interpretation based on disconnection measured directly in patients’ DTI. However, if unavailable, indirect measures of disconnection can also be used to establish a functional link between a lesioned region and the networks associated with it. The rationale behind this is that it reflects an indirect estimation of the effect of a lesion on structural brain networks. To make this approach clearer, we have revised the manuscript accordingly. See revised manuscript pages 6 and 12:

[...] Assessing connectivity in healthy participants based on lesion information is a relatively new method that measures structural disconnection in networks associated with given anatomical regions (Foulon et al., 2018). This allows for the indirect estimation of the lesion effect on structural brain networks. In this regard, it was shown that behavioral deficits can be explained similarly by local brain damage and indirectly measured disconnection (Salvalaggio et al., 2020). [...]

[...] We next used the respective areas as seed regions for probabilistic fiber tractography in a healthy age-matched sample to visualize the underlying common connectivity pattern (see Methods). Thus, we indirectly explored the association between posterior superior temporal disconnection and processing of sound at short timescales. [...]

We also changed the abstract and conclusion accordingly. See pages 2 and 15 of the revised manuscript.

[...] Here we tested whether temporo-cerebellar disconnection is associated with the processing of sound at short timescales. [...]

[...] The evidence we describe (i) shows that lesion-related deficits in spectrotemporal analysis occur in posterior temporal regions connected to the cerebellum [...].

Reviewer #3:

Stockert et al. investigate the cortico-cerebellar network underpinning rapid temporal auditory analysis. This study uses a well-defined group of stroke participants with mostly circumscribed lesions to the left posterior superior temporal lobe to motivate probabilistic tractography from cortical regions associated with verbal and non-verbal rapid auditory temporal analysis. Lesion-symptom mapping identifies a specific region of the posterior superior temporal sulcus and underlying white matter as statistically associated with impairment in rapid auditory temporal analysis. Tractography results demonstrate that these regions have high structural connectivity to wider regions of the left hemisphere cortical language network and ipsilateral and contralateral connectivity to postero-lateral cerebellum and dentate nucleus. It is interpreted that this cortico-cerebellar network is crucial to developing representations of fine auditory temporal structure.

The conclusions of the paper are an interpretation which is based on integrating previous neuropsychology with the current tractography results and based on well-defined models in the motor domain. Such conclusions are not unreasonable but there is no direct (associative) evidence linking this network to the cognitive function of interest.

Strengths:

The paper integrates neuropsychology and neuroimaging methodologies to build a coherent picture which is more than the sum of its parts. The stroke group has well-defined and selected lesions which enable testing of the hypotheses put forward by the authors. The behavioural measures are sensitive and suitable to identify impairments in the behaviours of interest. There has been a detailed analysis of the behavioural speech perception data in the stroke group which largely, although perhaps not entirely, conforms to the asymmetric temporal sampling hypothesis. The lesion-symptom mapping approach is suitable for the nature of the population (small group with similar lesion distributions) and has allowed neuropsychologically guided tractography in the neurotypical population. This has clearly illustrated the complexity of the structural connectivity of the posterior superior temporal sulcus and underlying regions.

Weaknesses:

The selective nature of the stroke population - relatively small, chronic lesions - has resulted in only mild impairments for a small number of participants (6/12 participants). At the group level there is no difference between the stroke and neurotypical population on speech perception measures - group statistics do not reach one tailed significance. This reduces the certainty with which the regions identified are associated with the behaviour or interest. However, the results do conform to previous neuropsychology and lesion studies and it is likely that this lack of effect is due to low statistical power.

Please refer to our response to the next point.

All the stroke participants have a similar lesion distribution, and this makes lesion-symptom mapping challenging. For example, lesion data do not give an indication of the functional integrity of perilesional regions which can be reduced, even at the chronic stage, therefore the superior temporal sulcus may not be functioning effectively, even in the proportion of the group without lesions to this area. Lesion symptom mapping is more robust with a wider distribution of lesions and the inclusion of participants with lesions remote from the area of interest. Having said that, the behavioural measures appear sensitive enough to identify mild impairments and the authors, for good reason, wished to reduce the extension of lesion into primary auditory regions. As above, given the limited sample and homogeneous lesion, the lesion symptom mapping approach is reasonable.

We agree that the small number of patients is a possible limitation to the study and add this point to the limitations section. See revised manuscript page 21.

[...] First, the study population is relatively small and lesion symptom mapping is typically applied to larger populations with wider lesion distribution. Although careful selection of circumscribed lesions has the advantage of highlighting behavioral differences without confounding other deficits (e.g., primary auditory processing), it is possible that additional regions are involved in processing of sound at short timescales. However, tractography based on healthy participants makes it possible to indirectly obtain information (i.e., structural disconnection) about brain regions contributing to the investigated function. In addition, it is likely that the small number of patients might hamper the ability to detect statistically significant differences between the behavior of controls and patients. Nevertheless, we are confident that the current results align with the fact that the posterior superior temporal cortex contributes to the processing of sound at short timescales, as indicated by previous neuropsychological evidence and lesion studies (Boemio et al., 2005; Chedru, Bastard, and Efron, 1978; Efron, 1963; Robson, Grube, Lambon Ralph, Griffiths, & Sage, 2013; Swisher & Hirsh, 1972). Further studies should however test larger populations to replicate and extend this finding. [...]

The authors suggest that the behavioural results conform to the asymmetric temporal sampling hypothesis in that only place of articulation discrimination impairments in the stroke group can be (just about) detected, whereas there were no significant stroke-neurotypical differences in other phonetic contrasts. It is not clear that the VOT differences associated with plosive voicing changes and the cues associated with place changes happen over fundamentally different time-scales and, therefore, it is important to further justify the interpretation of the data. In the future it will be helpful to have this level of analysis applied to individuals with lesions to the wider speech perception network to draw conclusions about the specificity of the impairment to these regions - for example, impairments in phoneme discrimination have been associated with frontal lobe lesions.

It appears that voicing contrasts in which shorter and longer voice onset times result in the perception of a voiced or voiceless plosive (for example [t] and [d]) are encoded in both the temporal envelope and fine structure (Rosen 1992) of the speech signal that occur in time windows of 20-500 ms and <2 ms, respectively. In words an additional cue is the closure time, which can be further used to discriminate between voiced and voiceless plosives. However, place of articulation contrasts are exclusively encoded in the temporal fine structure (i.e., very quick transitions of the frequency spectrum, formant transitions). Even though for all contrasts shorter timescale information plays a role, somewhat redundant encoding is present for voice contrasts. Ultimately, place of articulation contrasts seem to be the most difficult to discriminate. In Figure 2D it is apparent that despite highest error rates for the place of articulation contrasts, several patients also showed impaired discrimination for voicing contrast when compared to healthy controls. We do agree with the referee that it would be interesting to also extend this level of analysis to individuals with lesions in the wider speech perception network in future work.

The tractography results reveal a complex pattern of structural connectivity, including other regions associated with speech perception. The authors have a theoretical motivation to focus on the importance of the temporo-cerebellar pathway but there is no correlation evidence to link auditory temporal analysis to the integrity of this pathway in the neurotypical population. The non-verbal measures appear to be sufficiently sensitive for this type of analysis. This lack of association with behaviour makes it hard to draw conclusions about the functional role of this network.

We appreciate the referee’s concerns about our interpretation of the functional link between the temporal lobe and the cerebellum regarding auditory temporal analysis. It certainly is more reasonable to derive a functional interpretation based on disconnection measured directly in patients DTI. However, if unavailable, indirect measures of disconnection can also be used to establish a functional link between a lesioned region and the networks associated with it. The rationale behind this is that it reflects an indirect estimation of the effect of a lesion on structural brain networks. To make this approach clearer, we have modified the manuscript as such. See revised manuscript pages 6 and 12:

[...] Assessing connectivity in healthy participants based on lesion information is a relatively new method that measures structural disconnection in networks associated with given anatomical regions (Foulon et al., 2018). This allows for the indirect estimation of the effect of a lesion on structural brain networks. In this regard, it has been shown that behavioral deficits are explained to a similar extent by both the local damage and indirectly measured disconnection (Salvalaggio et al., 2020). [...]

[...] We next used the respective areas as seed regions for probabilistic fiber tractography in a healthy age-matched sample to visualize the underlying common connectivity pattern (see Methods). Thus, we indirectly explored the association between posterior superior temporal disconnection and processing of sound at short timescales. [...]

We also changed the abstract and conclusion accordingly. See revised manuscript pages 2 and 15.

[...] Here we tested whether temporo-cerebellar disconnection is associated with processing of sound at short timescale. [...]

[...] The evidence we describe (i) shows that lesion-related deficits in spectrotemporal analysis occur in posterior temporal regions connected to the cerebellum [...].

I understand that Disney may not have openly queer characters and I understand people want more representation. However, I think it is weird to compare the sisters to anything "couple" related since they are sisters and that has nothing to do with being Queer. So I don't exactly understand how someone could take that into an account and have a theory that Frozen is a queer movie.

Author Response:

Reviewer #1:

The manuscript by Takahashi et al describes the interaction between MLL fusion proteins with HBO1 and its role in leukemogenesis. Myeloid progenitor transformation assays using various MLL fusion proteins reveal that MLL fusion proteins requires the TRX2 domain of MLL for effective leukemic transformation. IP-MS identifies HBO1 as a bona fide binding partner of the MLL TRX2 domain. ChIP-seq experiments show genome-wide colocalization of HBO1 complex with MLL-ENL and the WT MLL in MLL-fusion leukemia cells and MLL WT cells, respectively. ChIP-qPCR in MLL-deficient cells suggest that recruitment of HBO1 to MLL target genes (such as MYC and CDKN2C) depends on MLL. Truncation analysis of the ELL part of the MLL-ELL fusion reveal that MLL-ELL transformation activity requires OHD domain-mediated recruitment of AF4 and EAF1. Furthermore, co-IP and ChIP experiments with various fragments show that AF4 and EAF1 form two distinct SL1/MED26-containing complexes and likely the AEP/SL1/MED26 complex is competent for transactivation. Series of transformation assays suggest that MLL-ELL transforms hematopoietic progenitors via association with AEP, but not other ELL-associated proteins. Finally, the authors also show that NUP98-HBO1 fusion transforms myeloid progenitors through interaction with MLL. Overall, this is a quite comprehensive study demonstrating that various MLL fusions and NUP98 fusions transform hematopoietic progenitors via HBO1-MLL interaction, which suggests that targeting their interaction might be s new therapeutic approach.

We appreciate the comments and inputs from the reviewers.

Reviewer #2:

In this manuscript, the authors identified an interesting interaction of MLL (a methyltransferase) with an HBO1-JADE complex (an acetyltransferase) and investigated the functional impact in leukemogenesis by fusion proteins containing MLL or HBO1. The data is clear and the connection between MLL and HBO1 is unexpected. The manuscript is also well organized and relatively easy to follow.

Comments:

1) The functional relevance of the interaction between MLL and HBO1 is still correlative. It would be important to know whether there are any results directly about the impact of the loss of the HBO1 complex on the function of MLL.

We performed a sgRNA-dropout assay which showed that HBO1 is critically required for the survival of leukemia cell lines, as depicted in Figure 2F and Figure 2-figure supplement 3.

2) It is important to show the source and specificity of the antibodies that were used for ChIP of the HBO1 complex.

The details of the antibodies are provided in Key Resource Table.

3) It might be interesting to check whether other JADE proteins and also BRD1 (another partner of HBO1) are involved.

We agree that it would be very interesting to examine the involvement of other JADE/BRPF family proteins in the future because they share the ING4/5 subunits and BRD1 plays an important role in hematopoiesis (1). This can be addressed in future studies.

4) The acronym TRX2 may be confusing as some might think that it is thioredoxin.

As advised, we have changed this to THD2 (TRX homology domain 2).

Reviewer #3:

This paper starts with a series of bone marrow transformation assays comparing MLL fusions and domain-deletion mutants thereof to define the minimal elements for robust leukemic transformation and surveying growth and attendant common fusion targets HoxA9, Meis1 in colony replanting assays. Here they discover that a region of the MLL-N portion just upstream of the well-studied CXXC domain, termed in their previous work the "TRX2 domain" is important for the transformation capacity for several different MLL-fusions (and more minimal chimeras of key modules). A small region of the MLL-N protein encompassing the TRX2 domain and the CXXC module are subjected to complex purification, it is clear from comparison to number of controls that the TRX2 domain is an important mediator of association, perhaps indirect, with the HBO complex. Drop out experiments confirm that HBO1 knockout is lethal to MLL-rearranged leukemia, nicely confirming recent work (Ay et al., MacPherson et al.).

ChIP-seq experiments in an ALL with MLL-ENL fusion, and then more extensively in a kidney cancer cell line indicate overlap with some of the HBO complex subunits and MLL, however this does not establish recruitment at these sites. ChIP-qPCR at a few MLL-fusion target genes with MLL depletion supports the recruitment hypothesis somewhat although mixed and modest effect sizes indicate that alternate pathways for HBO1 recruitment are involved, and could also be explained as reduced deposition of marks known to recruit HBO1, rather than direct recruitment. Sadly, the real potential strength of this work goes unrealized, as the recruitment of HBO1 mechanism remains tantalizingly out of reach. More experiments in this space could conclusively establish the molecular mechanism of a seemingly biomedically important recruitment paradigm, and thereby have much more impact.

As the reviewer pointed out, MLL is not the only element that recruits the HBO1 complex to the target chromatin. MLL is known to deposit H3K4me marks, and the HBO1 complex is known to recognize these marks via ING4/5 subunits. We performed a ChIP-qPCR analysis of H3K4me3 in MLL-knockout cells. At the MYC promoter, the H3K4me3 marks were substantially decreased (Figure 3F). Moreover, recruitment of HBO1 was not recovered by transient expression of an MLL mutant containing THD2, indicating that the presence of H3K4me3 marks is a prerequisite for HBO1 recruitment. In accordance with this, ING5-histone interaction is required for the stable association of MLL with the HBO1 complex (Figure 8A-C). Thus, a more appropriate molecular mechanism would be the cooperative recruitment of the HBO1 complex by ING4/5-mediated chromatin association and MLL-mediated association. Because of the multiple contacts involved in this molecular network, it is not easy to pinpoint the direct contacts as desired, but our biochemical analyses indicate that PHF16 and ING4/5 offer relatively strong binding surfaces (Figure 8A-C). The ING domain of ING5 is the most likely direct binding surface identified thus far.

At this point the paper shifts to a seemingly distinct line of inquiry, which is not closely related to the HBO1-TRX2 story to the first three figures. The new direction examines the ELL fusion partner in some detail using similar fusion protein chimeras, but a portion of Figure 4, is largely confirmatory of previously established findings about the critical regions of ELL for transformation and its AF4/EAF1 partners, adding only that portions of the MLL fusion protein are dispensable, provided that they are replaced with the PWWP of LEDGF. It is a little bit of a Frankenstein's monster experiment, and does not add much new to the field. Further experiments define potentially two distinct complexes that have already been characterized being recruited by ELL, although there is overlap here again with their previous studies, and the results are a little hard to interpret.

A portion of Figure 4 was confirmatory to previous results. We have moved this to figure supplements in the revised manuscript (Figure 4-figure supplement 1B,C). The main topic of this paper is the role of the HBO1 complex in MLL-mediated transactivation pathways. The structure/function analysis of MLL fusion proteins demonstrated that MLL-ELL is highly dependent on the HBO1-mediated function in leukemic transformation (Figures 1 and 2). Hence, it was important to clarify the mechanism of gene activation by MLL-ELL in this study to understand why HBO1 association is required for MLL-ELL-mediated transformation. Because MLL-ELL associates with AEP similarly to major MLL fusions such as MLL-AF4 and MLL-ENL, it was speculated that MLL-ELL also activates its target genes via AEP. However, ELL associates with EAF family proteins and MLL-EAF also has transforming ability (3). Thus, EAF1-mediated functions could be more important for MLL-ELL-mediated transformation rather than AEP-mediated functions. To clarify the mechanism of MLL-ELL-mediated transformation, we generated a point mutant that selectively impaired ELL-EAF interaction and demonstrated that EAF1-association is dispensable for MLL-ELL-mediated transformation (Figure 6), thereby indicating that MLL-ELL transforms via AEP-mediated functions, which demands HBO1-mediated functions. We also showed that the presence of THD2 enhances ELL-AEP association to further suggest that one of the roles of the HBO1 complex is to enhance the association of ELL with AEP (Figure 6E). These findings are not reinterpretations of our prior results and are relevant to the main topic of this paper. We believe this part adds new information to the field, and therefore we have included it in the revised manuscript.

The authors create structure-guided separation of function mutants in the ELL domain that binds both AEP and SL1, permitting them to specifically disrupt EAF1 interactions but not AF4. Further experiments solidify this interpretation, and find that this mutant shows no deficits in hematopoietic progenitor transformation or primary leukemia lethality, although there appears to be some effect upon reimplantation.

The last figure in the paper tackles the seemingly unrelated Nup98-HBO1 fusion, a rare patient mutation-they demonstrate a requirement for MLL for viability of hematopoietic progenitors transformed by this fusion, connecting back to the TRX2 interaction, and show that menin inhibitors slow growth.

Strengths:

The identification of the TRX2 region of the MLL-N protein as the major point of contact (perhaps not direct), to the HBO1 complex adds mechanistic depth to the really important recent discovery (confirmed in this work) the MLL-fusion leukemias rely on HBO1 function. This lab has published a number of technically similar types of papers defining minimal regions of MLL and distinct interacting partners by chimeric fusions, with bone marrow transformation assays, mouse model engrafting studies, IP's, ChIP etc. In my view they are very much under cited, likely because they are similarly so challenging to read.

Thank you for your pointed feedback. We will try our best to make the necessary improvements so that our papers are widely read and cited.

The mixture of Co-IP biochemistry, bone marrow transformation assays, and ChIP, to define interactions, minimal requirements for transformation, and their chromatin consequences for a host of different MLL-fusions and HBO1-fusions has the potential to define the key interfaces underlying recruitment.

Weaknesses:

The mechanistic inquiry stops short of really defining the critical MLL-HBO1 complex interface. Defining the point of contact on the HBO1 side (even which subunit) and determining whether it is direct, or bridged by some, as yet unidentified factor, as well as conclusively demonstrating that this is the mechanism of HBO1 recruitment remain the major shortcomings.

To address this criticism, we further investigated the mechanism of complex formation by MLL and the HBO1 complex. As we demonstrated in Figure 8A-C, the association appears to be mediated by multiple contacts mainly through PHF16 and ING4/5. Because this association needs an intact PHD finger of ING5, it likely occurs depending on the context where ING4/5 is bound to histone H3K4me2/3. The ING domain of ING5 was also required for the association, indicating that this portion may contains a point of direct contact. We speculate that HBO1 recruitment is mediated primarily by ING4/5-H3K4me3 interaction and MLL reinforces its chromatin association.

And the follow-on figures apart from the last one, appear disconnected from this portion of the story and distract from it.

We depicted a revised model incorporating the above-mentioned aspect in Figure 8D of the revised manuscript.

The complex nomenclature and density/organization/logic of the presentation of experiments makes this paper difficult to read. Absence of sufficient grounding in the broader literature much beyond their own lab's work further compounds the problem.

We changed some of the nomenclature and density/organization/logic of the presentation of the experiments to improve the readability.

There is a lot of overlap, particularly in parts of figure 1 and figure 4 with previously published results. So perhaps re-organizing the display of data, and the organization of presentation, putting confirmatory work in the supplementary figures, would improve accessibility.

We moved some portions of Figure 4 to figure supplement. The data for MLL-AF10 and MLL-ENL were retained in the Figure 1 as important references.

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

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Reply to the reviewers

Reviewer #1:

This paper puts together a nice set of data showing that a specific gene called Resf1 when deleted effects the ability of ESCs to self-renew and proceed to germline fates. I believe the data are sound and that they provide the evidence needed for the authors to make their conclusions.While I think the data are presented well and the manuscript is well-written, the "modest" functional results suggest this work would be more suited for a specialized journal.

We thank Reviewer 1 for their supportive comments.

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Reviewer #2:

1. In the presence of LIF, there is no difference between Resf1 knockout mESCs and WT mESCs except the expression of Esrrb, Nanog and Pou5f1. What about other genes? RNA-seq is needed to distinguish the two cell lines.

Fukuda et al. have shown that deletion of Resf1 leads to misregulation of ~1000 genes (adj. p-value 2) in presence of LIF. This highlights large differences between transcriptomes of Resf1 KO and WT cells that occur despite only a marginal difference in self-renewal efficiency between Resf1 KO cells and WT in the presence of LIF. It is therefore questionable whether the time and resources required to perform the requested RNA-seq would produce data that could unambiguously identify the potential causative effector difference downstream of Resf1.

As an alternative approach, we have reanalysed the Fukuda et al RNA-seq data. We find that Esrrb is significantly downregulated (in agreement with our Q-RT-PCRs), as are Klf4 and LifR (FDR 1.5). However, our meta-analysis of the Fukuda et al data did not show Pou5f1 and Nanog to be differentially expressed (FDR 1.5). This is in line with the lower level of downregulation of Pou5f1 and Nanog, compared to Esrrb in our Q-RT-PCR data. Notably, our gene expression analyses were performed in 5 biological replicates, whereas Fukuda et al. performed RNA-seq in two biological replicates. We can include the meta-analysis of the Fukuda et al data in our submission. As the change in ESC self-renewal that we see at low LIF concentrations could result from a decrease in Lifr expression, we will verify the change in expression of Lifr by Q-RT-PCR. Importantly, we will do this in a way that discriminates between expression of the transmembrane Lifr and soluble LifR, since the latter acts antagonistically (PMID: 9396734, Chambers, BJ, 1997).

1. The authors showed Resf1 is not required for Nanog function, so how does Resf1 regulate the expression of pluripotency genes? Through epigenetic modifications or signaling pathways? The authors should design experiments to explain the detailed mechanisms.

The strength of the immunoblot signal for RESF1 is low, even when Resf1 is expressed episomally. Therefore, although we could try to co-immunoprecipitate with the Resf1-v5 cell line and endogenous Nanog, the expression level of RESF1 may mean this effort is unsuccessful. Given the fact that the result will not affect the conclusions of our study, we do not think this effort is justifiable.

1. The authors showed that Resf1 interacts with Nanog, but they used forced expressed proteins. Does the endogenous Resf1 interacts with endogenous Nanog? Do they bind to some same DNA sequences?

This are important questions to answer. However, many more experiments would be required to reach firm conclusions. The reviewer is right to say that the mechanisms by which Resf1 affects pluripotency are unknown and remain to be answered in future. We therefore propose to improve the text discussing similarities in pluripotency phenotype between deletions of Trim28, SETDB1, YTHDC1 and RESF1. As deletion of RESF1 partner SETDB1 or other proteins involved in repression of retrotransposons lead to downregulation of pluripotency genes and in some cases collapse of ESCs (e.g. PMID: 19884255, Bilodeau et al. 2009; PMID: 19884257, Yuan et al. 2009), we hypothesise that the RESF1 phenotype may be explained by affecting SETDB1 chromatin binding and therefore repression of SETDB1 targets. The mild phenotype of RESF1 KO indicates that RESF1 would not be an essential component of this repressor complex but rather “a modulatory protein”.

It is also worth noting that the meta-analysis of the RNA-seq data from Fukuda et al. suggests that Resf1-null ESCs may express reduced levels of LifR mRNA, and this is something we plan to investigate.

1. In figure 5C, some Resf1 positive cells showed Nanog negative. Are these Nanog negative cells pluripotent?

Nanog-null ESCs are pluripotent (PMID: 18097409, Chambers et al., 2007). In addition, NANOG-negative cells in FCS/LIF cultures can retain pluripotency. Our purpose in this figure was therefore not to say whether NANOG-negative:RESF1-positive cells are pluripotent but to draw attention to the broader expression of RESF1 in FCS/LIF compared to NANOG. Such broader expression has also been noted for other heterogeneously expressed factors (PMID: 31582397, Pantier et al. 2019).

1. In figure 6A, the naïve mESCs are induced to EpiLCs. Is the transition efficiency of Resf1 knockout cells the same with WT mESCs? The finally obtained PGCLCs should be identified.