best article vanish off google search

With all of these previous statements, it's clear that Justice Alito is on the majority side. With these statements and questions, he's arguing that it isn't fair under Missouri law that churches wouldn't be subjected to these different examples presented just because of their religious affiliation.

I'm wondering if the argument would be the same if the surface of the playground was SOMETIMES used for religious activities, would this change the argument?

I think this is one of the majority justices. It is still very early and his course may change but it seems like he is already convinced and suggesting that spending this money is not about advancing religion, as it would be similar for spending the earthquake proofing money on a church window.

How do you determine what content is not worth/ you don't care about it being crawled on? Would an example of this be like a page that is under construction or not needed for the discovery of your buisness?

I think it's interesting that in the plays beginning it is established that in the world of the play Branden Jacob Jenkins is inspired to write the play by his therapis as a means of improving is mental health and connecting with something he has positive feelings for. But then Branden Jacob Jenkins character announces that he doesn't have a therapist. I wonder which version is true, which is just the world of the play, and if perhaps there's a grey area where some is true and some isn't.

I find the whole Dialogue between Dido and Minnie very hilarious. It's just like the way you would say how new Yorkers talk. dis just the way me and my friends talk, especially when we are talking about someone else. I don't know whether it's suppose to be funny or not, but i really do find it funny.

I felt very drawn to his dream. to dream about bees attacking you, then realizing it's not attacking him, but it is him. There a lot of ways you can interpret having dreams about bees. some says it more of a good luck coming your way, but some say's it's more of a bad luck and that something challenging is headed your way. The way I see it it's the bees were trying to send him a message. At the time when he states that he was suffocating is like in his mind when the bees are on him he quickly thinks about when people are like criticizing him that everything he wrote is about something else, and that he can't never make it as a black playwright. And maybe when he realizes the bees are not suffocating him, but becoming him, maybe trying to say don't let these people criticize you, and like just calm down and be free. ( hope y'all get it)

This is so infuriating because it's so true. Often, if not all of the time, actors of color are cast solely in roles made for their ethnicity. We're overlooked as people who could probably only play roles that have to do with our cultures and origin even if you're out of touch with that culture or not even born there. And on top of that type of casting which overlooks any actors of color for anything other than just the "ethnic role," they are usually always made out to be very dangerous, rebellious people who had terrible lives fgrowing up. Which is simply not always true and not what every person of color is like. I would always be expcted to be someone who always acted roles of my ethnicity solely because it's a role so ingrained in that culture or know all the dances, songs and such, when the truth is I'm not so intouch with my culture or know too much of that. It's exhausting to be forced into a box at all times, especially when its always so stereotyped and not accurate or reltable to your own life at all.

This is so gross. It's very obvious he is only saying this to get what he wants. Trying to rile her up and put thoughts into her that he is truly in love with her. It's like cornerning her and basically forcing her to feel bad for him or like she owes him something so she will accept. The realization later when M'Closky finds the papers saying Zoe is not free make this so much worse. He is just going to any length to get what he wants. This kind of thing feels really gross to even read especially with the way he speaks of her as something he can own and keep as long as he buys her. It's an uncomfortable scene to even just think about.

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Reply to the reviewers

We thank the reviewers for their careful reading, positive feedback and constructive criticisms of our manuscript. Their primary points of concern were that the discussion was too long and too speculative, and that the title did not sufficiently represent our work. We have now cut the discussion in half, and we have also changed the title to more precisely reflect our paper, and made some other minor changes in the text (all highlighted in blue).

Below, we provide responses to each of the raised issues.

Reviewer #1 (Evidence, reproducibility and clarity (Required)): The study was very well conducted by the group, selecting appropriated methods for achieving the aimed objectives. The sample were abundant and the statistical treatment were suitable for the size of samples, as well to compare different methods used in this study. The results in general were properly exploited by the authors, clearing many aspects of the role/function of the trophallaxis fluid. The results of this manuscript are apparently suggesting that young colonies prioritize the metabolization of carbohydrates, while mature colonies prioritize the accumulation and transmission of stored resources, amongst other processes. This study cleared many aspects about the role/function of the trophallaxis fluid for the colony.

We are happy the reviewer agrees with our choices of methods, sample sizes, and statistics, and we are pleased that they have come to the same conclusions.

Even considering the high level of present investigation, still there are some aspects that could be improved by the authors:

• The text in general is relatively long with an over use of citations of literature;
• The discussion is interesting, but some times too much speculative; if the authors could attenuate their speculative statements, the text would become more objective and fluid;

Thank you for this feedback. These comments truly helped us strengthen the manuscript. We have now streamlined the text, cutting down the introduction, cutting in half the discussion and we have made more explicit what is statement and what is speculation (more on this in response to reviewer 2).

• The results shown in figure 6A and 6D, relative to the processed of neutrophils degranulation and complement cascade, respectively. The authors did not discuss these results; is there a meaning at level of trophallaxis fluid role for the colony ? This was not discussed in the manuscript.

We thank reviewer #1 for pointing out these results. We have now addressed these terms in lines 277-284 of the discussion:

“Our gene-set enrichment analysis showed significant enrichment in immunity-related proteins characteristic of phagocytic hemocytes (58) in trophallactic fluid (‘innate immune system’, ‘complement cascade’, ‘neutrophil degranulation’). These results indicate that hemocytes may themselves be transmitted mouth-to-mouth, and generally shows the involvement of the social circulatory system in colony-level immune responses with implications for social immunity.”

• Considering the very high scientific quality of the present study, the authors could deposit all the raw proteomic data in a international reliable repository of proteins/DNA DB, since it will be required by top journals.

We wholeheartedly agree, and all data are now shared online through ProteomeXchange.

Reviewer #1 (Significance (Required)): Significance:the present investigation represents an important contribution for the knowledge the the exchange of signals within the colony, to synchronize the physiology and development of the hive as whole (the concept of superorganism. The existing data about the composition and potential role of the components from tropahallaxis fluid is very small, compared to the present results. The present study is a master piece of knowledge about the importance of eusociality.

Thank you for recognizing the importance of this study and affirming our work in such a wonderful way!

**Audience:** all those scientists involved with social insects; biochemists/protomists dedicated to insect biology, biochemistry and physiology. **My expertise:** biochemistry of Arthropods secretion, in special of honeybees, ants and wasps. **Referee Cross-commenting** I think that both reviews aare complementary to each other; both reviews agree with the need to reorganize the text making it more compact and objective. Essentially, the auhtors must focus in the concept of trophallaxis. Thus, the biochemical processes outlined by proteomic analysis should be addressed to explain how the shared physiology of colony works out.

Our discussion now focuses more on trophallaxis as a whole, and the biomarker-like quality of the changing proteome. We agree the biochemical processes and their role in the shared colony physiology are fascinating topics. We have not yet performed follow-up experiments with the many proteins present in this fluid and thus do not want to over-conclude. We have now stated more clearly in the discussion what the current data can reveal about these topics, what is assumed via orthology, and what needs to be addressed in future studies.

Reviewer #2 (Evidence, reproducibility and clarity (Required)): This ms provides a comprehensive proteomic analysis of the trophallactic fluids extracted from carpenter ants. The analytical methods are state-of-the-art, and the results presented should fuel many studies. The vision of the research program, embodied in the title of the paper, is very exciting and is to be encouraged. However, the title of the paper in no way reflects the content of the paper, as none of the functional processes mentioned have been proven. This will require a lot of work and the development of perhaps new bioassays. I truly hope the PI's lab takes this on a deep and substantial way; the notion of trophallaxis and its socially exchanged fluid has long captivated the fancy of social insect biologists, but with a few specific exceptions, the promise has not yet been realized. The technical and descriptive results presented here lay a strong foundation. For purposes of present publication, I strongly recommend a different title and a revised discussion that reflects the disconnect I outline. Cause/consequence issues need to be addressed.

We thank reviewer #2 for seeing our vision and that this is indeed foundational work that will “fuel many studies.” We also agree that the title and discussion contained too much speculation. The aim of this paper was to prove that there is systematic variation in trophallactic fluid in natural populations that correlates with biologically important social conditions, and further, that some proteins in this fluid can both act as biomarkers and be informative about underlying molecular processes. We have now communicated this more clearly in the introduction. In the revised version of the paper, we have reduced the speculation, and where appropriate, made it clear when there is speculation.

For example, discussion lines 233-238:

“Overall, our data reveal a rich network of trophallactic fluid proteins connected to the principal metabolic functions of ant colonies and their life cycle. Pinpointing contexts that induce changes in trophallactic fluid, along with the exact targets and functions of the proteins, are important subjects for future work. Our establishment of biomarkers transmitted over the social circulatory system that correlate with social life will allow researchers to formulate and test hypotheses on these proteins’ functional roles.”

Three technical points: 1) Sample sizes are low for some analyses (2/group)--though they are cleverly pooled.

We are not sure what the reviewer is referring to – none of our sample types had this low sample size (see SI Table 1 for sampling scheme). In contrast, for a proteomics study, our sample sizes are quite high. We are aware that for a study focusing on a natural population, the colony-level sample size of 16 (laboratory colonies) can be considered low, but this has been taken into account in our stringent statistical analyses.

2) How to distinguish between what animals actually transmit and what is found in the gut? There could be differences.

This has been addressed in our previous work, where it was shown that the crop content is equivalent to what is exchanged among individuals of this same species during the act of adult-adult stomodeal trophallaxis (Figure 1A, LeBoeuf et al. eLife 2016). We have now clarified this in the methods section of the current paper (line 361-364).

“Trophallactic fluid was obtained from CO2- or cold-anesthetized workers whose abdomens were gently squeezed to force them to regurgitate the contents of their crops. This method of collection was shown previously to correspond to the fluid shared during the act of adult-adult stomodeal trophallaxis (17).”

3) Is there evidence that the substances found are not just the product of digestion of ingested food? The differences between lab and field colony samples supports this.

In the type of proteomic analysis we have performed (the most commonly used proteomics approach when a genome is available), we detect only proteins found in the reference genome of interest (in our case Camponotus floridanus), so excepting cannibalism, we should not see proteins that originate from food. Note that this is why we do not provide lab colonies with the typical lab-reared ant diet that includes honey, as bees are also Hymenoptera, and royal jelly and trophallactic fluid have many proteins in common. Cannibalism could result in trace observation of many proteins, but could not produce the consistent and high-abundance set of proteins that we have observed as they are not produced in those precise ratios in larvae or adults.

The observed shift in trophallactic fluid from field to lab may reflect a change in diet or microbiome and these are questions that could be further investigated in future work (mentioned in lines 229-232). The clear difference we observe between trophallactic fluid of young and mature colonies, or the difference between the worker castes within a colony, is evidence that the variation observed in trophallactic fluid reflects more than diet.

“Trophallactic fluid complexity declines over time when colonies are brought from the field to the laboratory. This may reflect dietary, microbiome or environmental complexity – typical of traits that have evolved to deal with environmental cues and stressors (e.g. immunity, (37)).”

Reviewer #2 (Significance (Required)): The paper addresses a very important topic that should be of widespread interest to social biologists. Journal choice should reflect that this is a technically excellent paper that presents descriptive information but functional significance is highly speculative.

We appreciate that the reviewer agrees that our results are of widespread interest to social biologists. Indeed, our results must be somewhat descriptive, as we are working on a mostly unexplored socially exchanged fluid in a natural population. However, our study design tests clear hypotheses with preplanned sampling and experimental transfer of ant colonies to a new laboratory environment. We present confirmatory results of the hypothesis that trophallactic fluid is complex mixture of biomarker-like molecules and that these biomarkers can be used predict sample origin through machine learning (see random forest predictions, emphasized in lines 151-152). The fact that our evidence for this is correlative does not render it speculative. Indeed, in both ecology and in much of medicine, using correlative evidence is the norm, as it is often impossible to manipulate ecosystems, natural populations and some organisms in a safe and controlled manner. This is what convinced us to invoke the term ‘biomarkers,’ as biomarkers are excellent examples of molecular correlates of larger conditions that have spurred advances in biology and medicine.

Some of the next steps in our research will be, as reviewer #2 suggested, additional studies on the roles of individual compounds of trophallactic fluid, building on the results of this paper. Additionally, while this study may not have explored the roles of specific molecules, open ended exploration is extremely important and necessary for any scientific advancement in the long run (eLife 2020;9:e52157).

All in all, we are grateful for this comment, as it showed us that we must communicate the aims of our work more clearly – which we have now done both in introduction (line 77-91) and throughout the discussion.

**Referee Cross-commenting** Yes. Most of the discussion is pure speculation because we do t k ow what is exchanged and what the modes of action might be. But it's a great start!

We have reduced the speculation on the roles of single molecules, and we hope our responses to the points above clarify some of the reviewer’s uncertainties about what is exchanged. However, we do still outline hypotheses for potential functions and origins in the discussion section, as this study is intended to be a foundation for new lines of research.

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Referee #2

Evidence, reproducibility and clarity

This ms provides a comprehensive proteomic analysis of the trophallactic fluids extracted from carpenter ants. The analytical methods are state-of-the-art, and the results presented should fuel many studies. The vision of the research program, embodied in the title of the paper, is very exciting and is to be encouraged. However, the title of the paper in no way reflects the content of the paper, as none of the functional processes mentioned have been proven. This will require a lot of work and the development of perhaps new bioassays. I truly hope the PI's lab takes this on a deep and substantial way; the notion of trophallaxis and its socially exchanged fluid has long captivated the fancy of social insect biologists, but with a few specific exceptions, the promise has not yet been realized. The technical and descriptive results presented here lay a strong foundation. For purposes of present publication, I strongly recommend a different title and a revised discussion that reflects the disconnect I outline. Cause/consequence issues need to be addressed.

Three technical points:

1) Sample sizes are low for some analyses (2/group)--though they are cleverly pooled.

2) How to distinguish between what animals actually transmit and what is found in the gut? There could be differences.

3) Is there evidence that the substances found are not just the product of digestion of ingested food? The differences between lab and field colony samples supports this.

Significance

The paper addresses a very important topic that should be of widespread interest to social biologists.

Journal choice should reflect that this is a technically excellent paper that presents descriptive information but functional significance is highly speculative.

Referee Cross-commenting

Yes. Most of the discussion is pure speculation because we do t k ow what is exchanged and what the modes of action might be. But it's a great start!

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Reply to the reviewers

Full Revision

Manuscript number: RC-2021-00785

Corresponding author: Christian, G. Specht

1. General Statements

Dear Editor,

We greatly appreciate the reviewers’ constructive comments on our manuscript ‘Identification of a stereotypic molecular arrangement of glycine receptors at native spinal cord synapses’. We were particularly pleased that all four reviewers agreed that our data yield new insights into the structure of inhibitory glycinergic synapses, and represent both a technical and conceptual advance the field of synaptic neuroscience.

The reviewers have consistently raised one main criticism, namely the use of endogenously expressed GlyRs tagged with the fluorescent protein mEos4b, which could potentially have an impact on receptor expression, trafficking and function. We have addressed this point by performing whole-cell recordings of GlyR currents in cultured neurons that show that glycinergic transmission and therefore function is preserved. We have also addressed all other comments of the reviewers in the revised manuscript, including a thorough revision of the text and the addition of new data and figures as detailed in the point-by-point response.

Point-by-point description of the revisions

Reviewer 1:

Summary:

In this manuscript Maynard et al describe a newly generated knockin mouse to study the endogenous distribution of Gly receptors in the spinal cord. Using quantitative confocal imaging and SMLM the distribution and levels of GlyRs at spinal cord synapses is compared between dorsal and ventral horn. They found that levels of synaptic GlyR are higher in dorsal than ventral spinal cord synapses. Nevertheless, the ratio to gephyrin seems constant, except for synapses in superficial layers of the dorsal horn, where gephyrin levels exceeded the levels of GlyRs. There are also fewer, but larger synapses in the ventral horn than in the dorsal horn. These findings are further corroborated by an SR-CLEM approach. Furthermore, it is shown that in a mouse model for hyperekplexia GlyR levels are lower, but still enriched at synapses, and the dorsal-ventral gradient in GlyR expression was maintained. The difference in size of ventral and dorsal synapses observed in WT animals was also lost in the oscillator mouse, suggesting that particularly the ventral synapses are affected. Despite these differences, the density of GlyRs per synapse remained similar.

Major comments:

Line 113: "labeling the_ _b__-subunit has proven difficult". This statement is unclear and it would be informative for readers to grasp what exactly has been difficult, and why the approach described here overcomes that? Related to that, the authors state "KI animals reach adulthood and display no overt phenotype, suggesting that the presence of the N-terminal fluorophore does not affect receptor expression and function". That is indeed reassuring, but it does not exclude that receptor numbers, function and distribution are altered. As it seems there is no prior literature on tagging the beta subunit, additional evidence that the tag does not interfere with receptor trafficking or functioning would be desirable

We have clarified why it has been difficult to label the GlyR beta subunit until now, lines 113-115 _“To date, labeling of GlyRβ in situ using immunocytochemistry has proven difficult due to a lack of reliable antibodies that recognize the native β-subunit (only antibodies for Western blotting recognizing the denatured protein are available), which has severely limited the study of the receptor.”_ Hence it was important to us to generate this knock-in mouse in order to study the endogenous GlyR at synapses, which is the least well studied receptor mediating fast synaptic transmission.

The reviewer makes an important point regarding the labeling of the GlyRβ-subunit with a fluorescent protein that has also been raised by the other reviewers. We have now verified receptor function by patch clamp recordings of glycine currents in whole-cell configuration in spinal cord neuron cultures from the mEos4b KI mouse (new Supplementary Fig. S2C). At saturating glycine concentrations of 300 μM we found no difference in chloride influx between mEos4 KI and WT mice. Since glycine concentrations in the synaptic cleft are in the millimolar range during synaptic transmission, these data strongly suggest that glycinergic transmission is not affected by the presence of the mEos4b under physiological conditions, despite a minor shift in the EC50.

There are several other strong arguments that suggest that mEos4b-GlyRb expression, subcellular localization and function are the same as those of the native subunit. Firstly, the mEos4b sequence was inserted after the signal peptide and before the beginning of the coding sequence of the mature β-subunit (Fig. S1). Since the mEos4b sequence does not interrupt the coding sequence it is less likely to affect the receptor conformation. Secondly, we did not notice any behavioural phenotypes in animals carrying the GlrbEos allele. At the time of weaning, the genotypes of the pups corresponded to the expected Mendelian frequency (new Fig. S2A). Moreover, we did not observe a reduction in live expectancy of GlrbEos/Eos animals (new Fig. S2B), demonstrating that the mEos4b-GlyRb does not cause pathology in older animals.

Most importantly, our imaging data (Fig. 1-3) provide exhaustive evidence that mEos4b-GlyRb assembles with GlyR alpha subunits as heteropentameric receptor complexes that are trafficked to the plasma membrane and inserted into the synaptic membrane due to their interaction with the gephyrin scaffold at functional synapses. Using quantitative imaging, we have also shown that homozygous GlrbEos/Eos KI mice have exactly twice the number of receptors at synapses as heterozygous animals, strongly suggesting no interference in receptor trafficking to the plasma membrane and gephyrin binding. As the mEos4b mice were also bred with the oscillator mouse model of hyperekplexia, which is lethal when homozygous, we could further test the combined effect of GlrbEos and GlyRa1spt-ot. The presence of both alleles did not lead to any noticeable phenotypes in heterozygous oscillator mice. On the contrary, both synaptic targeting and the packing density of the receptors were not altered in this model, despite a region-specific reduction in synapse size due to the reduced availability of the intact GlyRa1 subunit.

We believe that these data overwhelmingly support our conclusion that the presence of the mEos4b tag does not alter the structure and function of the receptor, making this mouse model uniquely suited to study the dynamics and regulation of glycinergic synapses in a quantitative manner and at the molecular level.

In the Discussion the authors conclude that "Our quantitative SR-CLEM data lend support to the first model, whereby inhibitory PSDs in the spinal cord are composed of sub-domains that shape the distribution of the GlyRs". This conclusion seems however based on one example image in Fig 3G that is not very convincing. The EM image seems to show two clearly separated PSDs opposed by two distinct active zones. So, although this conclusion is of high interest, more support should be given to substantiate this conclusion. More general, these subsynaptic domains (SSDs) are hardly further explored, but seem relevant for transmission, particularly given that the synaptic pool of GlyRs at these synapses is not saturated by single release events. How general are these SSDs at these synapses?

The representative image in Fig. 3G shows two SSDs within the same postsynaptic site with a continuous presynaptic active zone. It should be noted that the PALM/SRRF images were taken of the entire 2 µm thick slice, whereas the electron micrograph shows only a single 70 nm section. We verified throughout the full 3D stack of serial sections that the presynaptic site remains continuous, which it does. We would also like to point out the scale of the image showing that the two SSDs are only around 170 nm apart, i.e. spatially very close. Our conclusions are however not based on this single image but the whole dataset. The graph in Fig. 3I shows 3 synapses (out of N = 36), in which the GlyR density at separate SSDs could be quantified, demonstrating that the receptor density is not different between SSDs. The reviewer is correct that we do not further analyse the SSDs beyond their density and the analysis of the segmentation of the postsynaptic sites (Fig. 3E-G). Further work on the functional role of SSDs in synaptic transmission is outside the scope of this manuscript and would indeed merit future study.

The approach for counting molecules based on the PALM acquisition has been developed in prior publications and seems robust. It would however be worth to present the reader with a bit more background and explain the assumptions of this approach in more detail. Particularly, since counting of mEos4b can be problematic, as there are multiple dark and fluorescent states of this fluorophore that could be influenced by the illumination scheme, see for instance De Zitter et al., Nat Methods 2019. Since the preceding SRRF acquisition already exposes the fluorophore to high and continuous 561-nm laser power this could skew the counting due to unaccounted conversion and perhaps bleaching of mEos4b. In line with this, although throughout the manuscript the term 'absolute copy numbers' is used the reported numbers are at best an estimate based on a number of assumptions. I think the wording 'absolute numbers' is therefore deceiving and should be nuanced.

We have clarified how the molecule conversion is calculated (Fig. S7 legend), to provide a more complete description of the way in which the values were obtained. Further we have explained how we calculated the probability of detection. Since the probability of detection accounts for any unconverted or non-functional mEos4b molecules, our molecule counting approach is relatively resistant to potential pre-bleaching of fluorophores. It should be noted, that 561 nm illumination had no obvious effect on the non-converted (green) mEos4b fluorophores, as judged by the fact that the intensity of receptor puncta was unaffected by the SRRF recordings. We appreciate the reviewers point regarding the term ‘absolute copy number’ and we have adjusted our wording throughout the manuscript accordingly.

Related, most of the quantifications are in estimating the number of receptors, and not so much the distribution with the PSD. The term "molecular arrangement" - also used in the title - might therefore be misleading, there is in fact little characterization of how GlyRs are placed within the PSD. More focused analysis quantifying the distribution of receptors within the PSD and/or SSDs would strengthen the manuscript.

By estimating the number of receptors and the exact size of synapses, the main conclusion of our study is that receptor density at dorsal and ventral synapses is identical, independent of synapse size, subdomains, or in fact loss of GlyRs in a mouse model of hyperekplexia. This observation clearly relates to how receptors are packed within synapses, and thus describes their molecular arrangement.

The reported N is confusing and makes it hard to judge the reproducibility of the data. Sometimes it refers to number of images, sometimes number of synapses, but it is unclear from how many experiments these are drawn. This should be reported more completely (number of animals should be reported at least) and consistently. In figure 1, the N numbers (N=3-5 images) are particularly low and question how consistent these findings are across multiple animals.

We have clarified the N in the figure legends, to reflect the full size of the datasets that have been analysed.

The levels of mRFP-Gephyrin seem to differ between the different mouse lines, is this a significant difference?

No significant differences in mRFP-gephyrin levels were found in animals with different mEos4b-GlyRb genotype (Fig. 1B). However, expression of mRFP-gephyrin in heterozygous animals is 50% of that in homozygous mRFP-gephyrin KI animals (not shown).

The ICQ analysis for co-localization is hardly explained. How do we interpret this parameter? What does an average value of ~0.3 mean? A comparison with sets of proteins that do not overlap as a negative control would strengthen the conclusion.

We have clarified that an ICQ value of 0.3 is indicative of a very high spatial correlation between pixels, and provided a corresponding reference for ICQ analysis (lines 209-210). We would like to point out that the scale of the ICQ is between -0.5 to 0.5, meaning that a value of 0.3 comes close to complete correlation.

Minor comments:

Very little fluorescence was detected in the forebrain, despite the high reported expression of the Glrb transcript". Can the authors expand on this? What would explain this discrepancy?

We have clarified the text to include “suggesting that protein levels are controlled by post-translational mechanisms in a region-specific manner, as previously proposed (Weltzien et al., 2012)” (Lines 152-153). The reason for this discrepancy is not known. However, the distribution of mEos4b expression throughout the brain is as expected, based on the literature.

"What region is quantified in Fig 1B? is the same region in all conditions? This should be specified more clearly as the manuscripts presents a clear gradient in expression levels in the spinal cord and thus the location will influence the intensity measurements.

We have explained in the text that this is the region at the centre of the ventral horn identified by the white square in Fig. 1A, and that the same region was analysed for all images across all animals. Page 5, lines 160-161 “The same region of the ventral horn, indicated by the white square in Fig. 1A was taken for quantification of mEos4b-GlyRβ and mRFP-gephyrin expression in all conditions.”

The labeling approach does not differentiate between surface and internal receptors, this should be made more explicit in the text.

Whilst this is correct, we have only analysed mEos4b-positive synapses that had corresponding gephyrin clusters, meaning synapses where receptors are located in the postsynaptic membrane. Indeed we found that all mEos4b clusters imaged colocalised with mRFP-gephyrin clusters. We have adjusted the text accordingly, page 6, line 205-206 “All mEos4b-GlyR clusters closely matched the mRFP-gephyrin clusters, confirming the localization of the receptors in the postsynaptic membrane.”

Significance:

The presented data are interesting and the experiments are technically advanced and carefully performed. Particularly the SR-CLEM approach is technically advanced. The datasets present a quantitatively detailed characterization of spinal cord synapses and will be of interest for researchers working in the field of spinal cord circuitry, as well as super-resolution imaging. The conceptual advance for the field is however somewhat limited. It seems that the presented data confirm the general notion that receptor numbers and synapse size are highly correlated. So, although this manuscript describes very interesting observations, in its present form the manuscript does not provide any new mechanistic insight or significant advance in our understanding of how these synapses operate.

We thank the reviewer for his/her comments relating to the technicality of our manuscript. However we think that the statement “The conceptual advance for the field is however somewhat limited” is unfair, as this level of organisation of inhibitory synapses at the molecular scale has never been achieved before, as pointed out by the other reviewers, and especially not as regards different ages of animals and a disease model that directly affects receptor numbers in a region-specific manner. We therefore believe that our study will have a substantial impact within the fields of synaptic neuroscience as well as quantitative neurobiology.

Referee cross-commenting:

I agree with the other reviewers that this study is technically advanced, but I remain critical towards the extent of conceptual advancement this study brings and there are some important concerns with the presented data that need to be addressed. Nevertheless, indeed many of these concerns can be addressed without additional experiments. As pointed out also by other reviewers additional validation that the fusion proteins are not disrupting their function or organization would be important.

Reviewer 2:

Summary:

Maynard et al. investigate (inhibitory) glycinergic synapses in mouse spinal cord, which regulate motor and sensory processes. The authors analyse the molecular architecture and ultra-structure of these synapses in native spinal cord tissue using quantitative super-resolution correlative light and electron microscopy. The major finding is that GlyRs exhibit equal receptor-scaffold occupancy and constant absolute packing densities across the spinal cord and throughout adulthood, although ventral and dorsal inhibitory synapses differ in size. Moreover, what the authors call a „stereotypic arrangement" is even maintained in a hypomorphic mutant (oscillator), which is deficient in the adult GlyR a1 subunit.

Specific comments:

To reach their conclusions the authors generate two knock-in mouse lines, one with mEOS-labelled GlyR ß-subunit and one with mRFP-labelled gephyrin, a subsynaptic scaffolding protein of inhibitory synapses, which are subsequently crossed. Both changes are not unproblematic, as mutations in the N-terminal end of the GlyR ß subunit polypeptide chain might interfere with the assembly of functional GlyR (consisting of a und ß subunits) and and mutations at the N-terminal end of gephyrin interfere with it's homo-oligomerization into higher molecular assemblies.

We have demonstrated that the function of mEos4b-GlyRb does not differ significantly from WT GlyRs, by carrying out electrophysiological experiments (new Fig. S2C). For a detailed response, please see the response to the first comment of reviewer 1. The mRFP-gephyrin KI strain has been validated and published previously (see Machado et al., 2011, J Neurosci; Specht et al. 2013 , Neuron) and was not specifically generated for this study. The experiments with the oscillator mutant did not include the mRFP-gephyrin allele. In these experiments, the wildtype GlrbEos/Eos (Fig. 4, 5) behaves exactly as the GlrbEos/Eos in the double knock-in (Fig. 1, 2), further validating the mouse models used.

However, in this experimental design both labelled proteins reach postsynaptic membrane specialisations. In case of the ß-subunit quantitative evaluation confirms that heterozygous animals contain only half of the labelled protein as homozygous, which is an indication but not a proof that the correct stoichometry of adult GlyR is maintained. Likewise, mRFP-labelled gephyrin assembles with WT-gephyrin in subsynaptic domains, but it is not clear, if the size and density of the synapses is changed by the knock-in procedure as compared to WT-synapses.

An effect of the mRFP tag on gephyrin clustering can be ruled out, since we observed no difference in synapse size and receptor density in GlrbEos/Eos animals with (Fig. 1, 2) and without the GphnmRFP allele (Fig. 4, 5, oscillator wild-type controls). Similarly, the synaptic mEos4b-GlyRb levels in heterozygous animals were precisely half those of the homozygous animals, strongly suggesting that the expression and trafficking of the tagged receptor subunit is unchanged, as the reviewer acknowledges. In the absence of any obvious behavioural and/or functional phenotypes (Fig. S2) this KI model is in our view is an exceptional tool to study GlyRs expressed at endogenous levels in a cell-type specific manner.

Accepting these constraints, which to the knowledge of this reviewer have never been addressed to satisfaction, the authors provide a technically excellent, comprehensive analysis of glycinergic synapses in the spinal cord of double knock-in mice. Therefore, it should be stated in the title, that the investigations were performed with double knock-in instead of „native" spinal cord. Text and figures are clear and accurate and represent the state of the art.

We thank the reviewer for the positive comments regarding the techniques used in the study, and the clarity of the text and figures. We have adjusted the title as requested.

Finally, the reviewer would like to raise a minor point: the term postsynaptic density is derived from electron microscopical studies of synapses, where asymmetrical synapses display a „postsynaptic density" but symmetrical synapses do not. The latter were identified as inhibitory synapses and therefore, by definition, inhibitory synapses do not have a postsynaptic density, but rather a postsynaptic membrane specialisation. The use of the term „postsynaptic density" should, therefore, be restricted to excitatory synapses.

We are conscious of the importance of correct definitions and have revised the terminology, referring to “postsynaptic sites”, “postsynaptic domains”, and “postsynaptic specializations” as appropriate throughout the manuscript.

Significance:

The authors provide a state of the art advanced light and electron microscopical analysis of glycinergic synapses in the mouse spinal cord. They suggest a robust "stereotypical" mechanism in place, which guarantees a fixed stoichiometry of relevant components, which is even maintained in a hypomorphic mutant, which is believed to represent a mouse model of human hyperekplexia (startle disease).

Referee cross-commenting:

I would like to corroborate the arguments of the previous reviewer: it is not clear to which extent the fusion proteins influence the measurements, which are technically very advanced and well done, however. The authors do definitely not investigate "native spinal cord" as stated in the title.

The argument concerning fusion proteins must be taken especially serious as the fusions were induced in regions known to be responsible for assembly of glycine receptors and oligomerization of gephyrin.

We have verified the receptor function with electrophysiological recordings and clarified exactly where the fluorescent protein was inserted (see reviewer 1 response). Given the similarity in synapse size, fluorescence intensities and molecule densities observed in neurons expressing different combinations of tagged and native receptors and scaffold proteins, we strongly believe that all animal models used are well suited to the experimental aims of our study.

Reviewer 3:

Summary:

Glycinergic synapses are the least well understood of synapses that mediate fast synaptic transmission. The manuscript by Maynard et al. adds new information about the structural aspects of these synapses, using PALM and EM imaging of spinal cord synapses from mice at 2 and 10 months. The authors created a knock-in mouse that expresses a tagged GlyRbeta subunit, allowing synaptic localization of glycine receptors; all synaptically localized glycine receptors are thought to require the beta subunit to be tethered by gephyrin. The authors compare synaptic profiles from: 2 month old vs. 10 month old mice; dorsal vs. ventral horn; and GlyR1-reduced vs. wild type mice. Strikingly, they find a tight relationship across all of these variables between glycine receptor puncta and gephyrin puncta, as well as an apparently constant "packing density" of glycine receptors. They conclude that synaptic extent is likely to be the most important determinant of synaptic strength, as the density of receptors within the postsynaptic density is constant. These results use cutting-edge imaging and are analyzed with care, and add new information to our understanding of these relatively less well characterized synapses._

Major comments:

The key conclusions are convincing and the claims appear solid. Additional experiments are not needed to support these claims. The data and the methods are largely presented in such a way that they can be reproduced, although there are minor suggestions for improvement below.

We thank the reviewer for his/her positive comments.

Minor comments:

Do the authors have any comment on the requirement during, e.g. LTP, for insertion of a gephyrin-GlyR unit? The lead author has speculated that gephyrin creates "slots" for GlyRs; yet apparently each slot is already filled in the snapshots taken here. How might postsynaptic LTP occur (Kandler group, Kauer group papers)?

Given the reciprocity of GlyR and gephyrin clustering at synapses, the occupancy of binding sites (and in turn the number of available ‘slots’) is dependent on the strength of receptor-scaffold interactions, as discussed previously (Specht 2020, Neuropharmacol). In this study we demonstrate that the density of GlyRs at synapses is constant, which implies that the receptor occupancy is also the same, with the possible exception of mixed inhibitory synapses in the superficial dorsal horn that contain a majority of GABAARs. The PALM/SRRF data are represented as rendered image reconstructions and not as pointillist representations, and the detection of unoccupied binding sites is below the spatial resolution of our approach. However, the high spatial correlation of the signal intensities (ICQ ≈ 0.3) suggests that receptor occupancy is equal between and within synapses. It has previously been established that there are more scaffold proteins than receptors at synapses (Specht et al. 2013, Neuron; Patrizio et al. 2017, Sci Rep). Based on these studies we report that approximately half the gephyrin binding sites are occupied by receptors (lines 262-655). We have also expanded the discussion, describing how shape and size of synapses may affect synaptic transmission, as well as the possible role of receptor-gephyrin interactions in synaptic plasticity at glycinergic synapses.

It would be very interesting in the discussion to contrast the present observations with what is known about excitatory synapses (NMDA and AMPAR distributions) and GABAergic synapses. Are the authors at all surprised that receptor packing is constant across conditions? Can the authors speculate on how non-gephyrin binding receptors (homomeric alpha receptors, which are found in recordings) may function and be tethered to the membrane.

We have included additional information about receptor numbers and distributions at excitatory (lines 428-438) and GABAergic (lines 389-393) synapses in the discussion. So far, homomeric GlyRs composed of alpha subunits have been found to be exclusively extrasynaptic. As stated on page 4, lines 111-112 the beta subunit is required for binding of the GlyR to gephyrin and subsequent anchoring at the synapse. Previous studies have shown exocytosis of receptors to occur at extrasynaptic sites followed by lateral diffusion to synapses. Homomeric GlyRs are therefore most likely targeted to the extrasynaptic plasma membrane where they remain due to the lack of the beta subunit.

Figure S1. It would be most helpful to quantify this; at the least to include an atlas-like drawing to allow identification of the structures illustrated and containing Glrb; better yet would be quantification of staining in regions where this is strongest.

We have added an atlas indicating the different brain regions expressing mEos4b-GlyRb protein as a new Supplementary Fig. S3. The regional expression pattern agrees with the available literature about protein expression of the GlyRb subunit in different brain regions and hence provides further evidence that mEos4b-GlyRb is expressed like the native receptor. Due to the relatively low resolution of the tiled image no accurate quantification was possible. We have however added higher magnification confocal images of representative brain regions expressing varying amounts of GlyRb.

The fact that the lower panel in B is labeled as +/+ across all groups is initially confusing; perhaps relabel as mEos4 -/-, +/- and +/+?

We assume that the reviewer is referring to Fig1B. The genotype of both the GlrbEos and the GphnmRFP allele is now indicated on the x-axes, and the legend has been modified to clarify that all these animals were homozygous for GphnmRFP/mRFP. We have strived to remain consistent throughout the manuscript when referring to genotypes and protein levels.

Do gephyrin levels drop in WT mice as well as in the mEosr-GlyRb mouse between 2 and 10 months? Do the authors have any thoughts on this (Supp figure S2)?

We found no differences in gephyrin levels between 2 and 10 months. Fig. S2 (now Fig. S4C) shows the number of synaptic gephyrin clusters, which was the same at different ages and genotypes.

Significance:

Glycinergic synapses are the least well understood of synapses that mediate fast synaptic transmission. The manuscript by Maynard et al. adds new information about the structural aspects of these synapses, using PALM and EM imaging of spinal cord synapses from mice at 2 and 10 months. The authors created a knock-in mouse that expresses a tagged GlyRbeta subunit, allowing synaptic localization of glycine receptors.

This will be of interest to those studying inhibitory synapses, and more broadly to synaptic morphologists, physiologists and imagers for comparison with other synapse types.

My own expertise is NOT in these techniques, but I am a synaptic physiologist with a standing interest in glycinergic synapses; thus I am not providing serious technical critiques.

Referee cross-commenting:

Hi all, I agree with the other two reviewers, and do not have anything else to add.

Reviewer 4:

Summary:

The authors used a correlative approach and combined photo-activated localization microscopy with electron microscopy to characterise Glycinergic synapses in spinal cord tissue. Some of the major findings are:

• The receptor-scaffold occupancy and packing densities of glycinergic synapses in different regions of the spinal cord are the same.
• Gephyrin clusters in the spinal cord are composed of sub-domains that shape the GlyR clusters.
• Ventral horn synapses are generally larger, more complex (containing a number of gaps) and contain more GlyRs. -In a mouse model of Hyperekplexia, the number of GlyRs is reduced resulting in smaller synapses in the ventral spinal cord.

Major comments:

Are the key conclusions convincing? Yes

Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether? No

Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation. No

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

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

Are the experiments adequately replicated and statistical analysis adequate? Yes

Minor comments:

Specific experimental issues that are easily addressable. Please see below

Are prior studies referenced appropriately? Yes

Are the text and figures clear and accurate? Yes

Do you have suggestions that would help the authors improve the presentation of their data and conclusions? Please see below.

As the authors pointed out, fusing mEos to the extrasynaptic terminal of GlyRb has been difficult and therefore this construct would benefit the larger scientific community. Fig 1C is a nice imaging control for expression efficiency, however, it is in stark contrast with the lack of functional control. Do authors have any electrophysiological evidence showing that the insertion of mEos4b doesn't modulate channel function? I would assume that the construct would be tested in cell lines before the KI mouse line was created. Was any functional analysis done? If yes, it would be very useful to show it. I do appreciate that the authors used a standard insertion between the 4th and 5th AA in the extracellular domain, which in most cases does not abolish channel function. Given the lack of an obvious phenotype in the KI mouse model, I believe that this is also the case here. However, I disagree with the statement in lines 120-121: "the presence of the N-terminal fluorophore does not affect receptor expression and function." I believe that if there are no electrophysiological measurements of GlyR function, this statement remains speculative. As the authors pointed out in their previous publication: "receptor function and gephyrin binding are not independent properties. Instead, we think that conformational changes triggered at extracellular or intracellular protein domains have downstream consequences on channel opening as well as receptor clustering." In line with this, my concern is that the modulation of channel function by mEos4b could result in an altered cluster size at synapses. There is a large body of literature showing that just one missense mutation in the extracellular domain of ion channel subunits can lead to synaptopathies because the channel function gets modulated, and there is an abundance of similar examples involving mutations of GlyR and GABAAR subunits. In my view, comparing the function of GlyRs incorporating wt-GlyRb and mEos4b-GlyRb subunits is important for the correct interpretation of the main findings of this work and would strengthen the publications.

As the reviewer points out, the insertion of the mEos4b sequence was considered carefully in order to have the least impact on receptor function. GlyR channelopathies are often caused by point mutations within the coding sequence, which is not the case in the GlrbEos allele. Instead, the mEos4b sequence was inserted after the single peptide of GlyRb, duplicating several amino acid residues in order to maintain the correct cleavage site and N-terminus of the mature receptor, and to not interrupt the GlyRb coding sequence (Fig. S1B). In order to verify that the mEos4b-tag does not affect GlyR function, we have now carried out electrophysiological experiments (new Fig. 2C). For a detailed description please see the response to the first comment of reviewer 1.

Line 189: Are the authors making conclusions based on intensity comparison of red mEos4b and mRFP? The title of this section implies that the red form of mEos was compared to mRFP(?) But mEos converts from green to red only partially. Was the probability for conversion taken into account at this point? Please clarify which version of mEos was compared to mRFP._

In line 189 (now 218) we compared the intensities of mRFP-gephyrin with those of converted (red) mEos4b in SRRF / PALM super-resolution images of the synapses (Fig. 2D). Since the absolute intensities are altered by the process of image reconstruction, the probability that mEos4b is photoconverted does not have to be taken into account. The constant ratio of the SRRF and PALM image intensities confirms the data in Fig. 1D showing that GlyR and gephyrin amounts are highly correlated throughout the spinal cord (with the exception of the superficial layers of the dorsal horn). We have clarified in the text that this analysis was carried out on reconstructed SRRF images of mRFP-gephyrin and PALM images of mEos4, line 202.

Line 192: Please clarify how the density threshold was calculated/determined? This is important for the replication of the experiments, and it also has implications for the calculated probability of detection of mEos4b. I am not aware that this probability was calculated before for mEos4b and therefore other researchers may decide to rely on the value calculated here.

We have now clarified in more detail how the probability of detection was calculated (new Supplementary Fig. S7 legend).

In Fig. 2 Gephyrin clusters look consistently smaller than GlyR clusters, which is inconsistent with the published work. I assume that the difference in size is a consequence of different image reconstruction methods(?) However, I would assume that SRRF would have lower resolution than your PALM measurements and that would result in wider Gephyrin clusters. Could you please explain this discrepancy? Also, could you provide an estimate for the image resolution in SRRF and PALM techniques? For SMLM, localization precision would suffice.

We have provided an estimate of the resolution of the two techniques using Fourier ring correlation, which gave 46 nm for SRRF and 21 nm for PALM. Additionally we have precised the discrepancy between reconstruction methods, page 6, lines 194-200 “The spatial resolution was estimated using Fourier ring correlation (FRC), which measures the similarity of two images as a function of spatial frequency by comparing the odd and even frames of the raw image sequence. According to this analysis, the spatial resolution of SRRF was 46 nm and that of PALM 21 nm. It should be noted that the synaptic puncta in the SRRF images appear somewhat smaller and brighter due to differences in the reconstruction methods that result in differences in the dynamic intensity range.”

Why is the data in Fig. 5D and E represented as Detections/Synapse instead of GlyRs/Synapse? Could you please re-plot this so that a comparison with Fig. 2H and I is straightforward?

We have converted the detections to receptor copy numbers as requested (Fig. 5D,E).

Figure S5C: for P=0.5, 2=0.25. Please correct. Also, I assume that the second graph is what would be observed experimentally for dimers and P=0.5. Please clarify in the figure caption.

This was a mistake and has been corrected. We have also clarified which parts of the calculations are theoretical and which values were derived from our experimental data. We have provided a more detailed description in the figure legend of Supplementary Fig. S7.

Line 606: Please provide a complete derivation of this formula.

We have provided a full derivation of this formula (new Fig. S7C).

Significance:

The work described here seem to be a natural progression of a publication by Patrizio et al., 2017 that came out from the same laboratory. This study uses advanced methodologies in the imaging space to visualise and characterise Glycinergic synapses in spinal cord tissue. The experiments described here are technically demanding as evidenced by the relatively small number of publications describing super-resolution measurements in tissue samples. Even more rare are studies that attempt to do single protein counting in neuronal culture and tissue sections. Therefore, I believe that this work brings significant technical advancement in the field of super-resolution and corelative microscopy. The findings are also highly significant for all fields of neuroscience in which the structure of inhibitory Glycinergic synapse is relevant, ranging from the fundamental understanding of inhibitory synapse function to pathologies involving Glycinergic signalling._

I have substantial experience in different microscopy methods, including quantitative super-resolution microscopy based on single molecule counting. My background also covers the structure and function of GABAA and Glycine receptors using electrophysiology. I am familiar with the methods used in electron microscopy and the process of creating KI mouse lines, however I don't have hands-on experience in these fields._

This is not even that surprising, it's amazing how during these insane moral panics how people will completely shut off their brains. From Mr. Potato Head to Dr. Seuss there is constantly some boogieman that we are supposed to believe will be the downfall of Western civilization or whatever.

"Critical Race Theory poisoned our water supply, burned our crops, and delivered a plague unto our houses!"

"It did?"

"No, but are we just gonna wait around until it does?"

i chose her tweet on september 12th that says:

screaming BRING ME THE HALO TOP BRING ME Flips table THE flips another table" HALOOOOO TOPPPPPbirthday halo top appears* NOT THIS ONE

McCulloch states that when someone uses all caps, they are conveying strong emotion. repeating letters are also an "expressive tool" (233). the way she writes this tweet portrays the idea that she is feeling VERY strong emotions about halo top. she needs that halo top. but she does NOT want the birthday cake halo top, because duh. she's obviously meaning to be funny, but i think she's also a little serious and wants us to know that? she obviously feels really strongly about halo top, and that is nothing to joke about. she also creates this idea of an "in-joke" about how birthday cake halo top sucks. she knew a certain audience would understand her joke, so she said it. she also uses asterisks to add irony to the tweet.

however, we see her do a complete 180 in a series of other tweets, where she says "I want to fucking quit." and It's all a fucking boy's club." in these, especially in the first one, using the period at the end of the sentence conveys that she is very serious. she's not the silly, fun girl who wants halo top. something has obviously upset her to the point of using grammatically correct sentences--no capitalizing words, no cheeky comment, nothing. her tone is completely different because her tweets are devoid of effort, just like she is feeling as she types it. the simple sentence structure and complete lack of her fun, usual things shows that she is upset. overall, she seems like a smart, funny, intelligent individual who is incredibly relatable.

consider rewriting to something like

"the hat symbol above the $d$ means that it's our best guess for the number of dimensions (using some reasonable estimation method), rather than it being the actual number of dimensions.

I can relate to this paragraph because I got so used to sleeping late and waking up whenever that now I don't even get tired and I go to sleep really late. Also our health was really getting messed up. Physically and mentally

In my opinion I think that it has really affected all of us and our mental health itself has changed through out this past year or so. I think from the beginning of the pandemic we were all kind of panicking, there was so much chaos and so many deaths that it stressed all of us out.

What I'm getting from this is, being more descriptive & somewhat adding the argument or apposing the counter side is more better for writing when it comes to content like this. I would do this type of writing in my research paper for my public speaking class. Its like giving a little taste of what you're about to talk about without going into it until its the time for that.

Predicting the future is not what is necessarily important. what is important is been prepared for any plausible out come. Futurist forecasting the future does not mean that their prediction about the future will fall necessary or that they will know what will happen in future, their objective is to be ready and well prepared to face what future brings .

It's interesting that he does not oppose his argument out loud, instead he just states what he's always been told perhaps. He doesn't know any better and he's not even open to the idea of not having walls.

Wow. Our humble community college has seized the opportuinity to PRUNE. They've been quietly not replacing retirement positions, not institutionalizing grant funded positions, including two "coaching" positions which they proudly said they would in email to all of us, then quietly ... reversed. They proudly say that "we still have the same stuff! it's just streamlined!" (erm, no.) Oh, and we are not to use the word "anti-racism." It is divisive.

I believe that the huge gaps in the wealth distribution, that we discussed is the reason why a lot of richer people raise their children like this. The great pull that the top 20% of this wealth distribution has, that has probably been there for years, is what allows them to look the other way and, as Gelman said "not question why it's there" specifically, because they may not like what they find if they go searching for it. Not many rich people would like to see that their entirely disconnected from the majority of the world who hold little to no weight in the wealth distribution of the U.S.

1. What is the point of the figure on the right? With 50 nodes it's so cluttered that it doesn't really provide any additional information.
2. Is the relative distance between nodes in the figure on the right meaningful? Like, is 27 being on the perimeter meaningful?
3. If you wanted to convey information with the figure on the right, why not have a network of only 10 nodes? That would be a lot easier for a reader to manually check. I'm just kind of trusting that this figure is correct, because checking 50 rows is a bit much.

The impression I get throughout this discussion is that coordination must be established before segments/tones can affect each other, but then here and elsewhere they say that it's the entire constellation that informs the timing of the tone gesture, not just the gestures that it's immediately coupled with.

Is this the same for segments? Do non-coupled segments affect each other's timing?

I think this a really genuine and wholesome sentiment. Like, yes she's saying she wants her friend's house to herself. But it's coming from a child who is not used to luxury and just wants to know what it's like for once. It's not meant to be harmful.

This is part of the excerpt in TIS that attracted my attention. I think Leslie Jamison describes empathy really well. Empathy requires you to question things and consider a wide range of possibilities because you can not know everything.

20:57 - 29:20

"I'm Jesse. And this is Pascal. And we're here representing Friends of Light tonight. As you already heard, Friends of Light is a worker owned fashion company. We operate within the fashion industry, but we also operate very far outside of the fashion industry - we come from a background of working in fashion and at some point decided that it needed a radical change, and so we started weaving jackets based on our values as opposed to based on economic decisions - and Pascal is going to talk more about our values, but I'm going to give you a background on how we decided to form a worker cooperative. Uhm Pastel actually has been looking into worker cooperatives for much longer than I have, and I joined her in about 2012 when we were participating in a sewing circle called Work circles and, there was on any given week, there would be anywhere between 8 to 30 participants and we sat around a table. We made decisions collectively, we made every single decision together about making one quilt. And that was where the stitches were gonna be. What color it's going to be, what fabric we were using, what were the shapes, and every single person had a part in that and then we start stitching and we'd talk about what is a worker cooperative. What are the different values in a worker cooperative? How do you make a worker cooperative work? And so that was very much a learning experience for all of us in about 2015 - we, Pascal, myself and two other members, Nadia and May. We decided to take all of this that we've learned and actually put it into the real world and still sort of in a project based way make garments together, make woven jackets as a worker cooperative. We then took that and had a sales event just to present our project to the world to sort of inspire people, to show what we've been working on. We were wildly surprised that we got ten orders in one night, so we needed to incorporate, make this a real business, and we've been a real business since 2016, so we're still fairly new. But it's been three years of working as a worker cooperative, basing all of our decisions on the things that we've learned about worker cooperatives and doing our best to work in that fashion, we also, I will mention that we, one year ago, in January of 2018, we participated in the Green Worker Cooperative, which is based in the Bronx, which, if any, of you are interested in building your own cooperative - I highly recommend it. It was every Monday night, 3 hours a night for six months and we - it was like going back School, but to make a business and to learn how to make a business and specifically how to learn how to make a green Worker-Cooperative business, which is amazing. So that's - that's that background and Pascal is going to share with you a little more about our company and our values." "Thank you Jesse. In fashion, it's quite unusual that clothes are being produced in the West nowadays. And we have quite an extreme product, and it started actually with the desire to work with farms upstate who are producing fiber. But they're not really connected to any design practices in New York City. It's a very separate community. So we did research and found and looked for different farmers and we decided to work with Sarah of Buckwheat Bridge Angoras. She has an amazing practice as a farmer and her goats and sheep she keeps very well and she has a very high quality wool. And we are weaving jackets and this is together with her. She also had a mill on her property. We developed yarns. And just the first series was a series of five yarns that we developed with her, and all the natural colors of the - and we started experimenting. In the beginning it was kind of after the work circles we really wanted to develop an economic activity to practice being a worker-cooperative for real and not just kind of doing it as a project. So these were the first experiments into if we could weave a garment to form. And there's different techniques involved. In the meantime, we are working with other farmers as well. We're working with linen farmers, and we've done a lot of research in different materials, so we work very closely with the source of our materials, and that's the only thing we want to do. We work with hand spinners at the moment, it's no longer being produced on machines, so it's an intense product. It's takes us about 160 hours to make one jacket. And they've been selling really well, which was a surprise to us because the price point is very high, we're now at $6000 a jacket. And that's been really interesting, that people do value the story that's connected. And also all our clients. Actually everyone in our value chain is - we're really good friends with them, and we grow to become friends because we also make everything to size for each client. And one could think that we're kind of exclusive because the jackets are so expensive. But we also do a lot of - we do workshops and we do talks like this. We educate people about worker cooperatives and what it means to - And also what it means to value artisan work in a Western context and to be able to do that kind of work. And that's what we've been advocating, like a lot, about because a lot of people here push prices down.To make artisan work and even local local fiber products possible. But we know it's not possible with the... In September we actually did a big project to kind of save one of the farm - one of the forms we work with because they couldn't sustain themselves. Not - especially not through our jackets. But we decided that we would do a project around making blankets and we invited other design studios in New York to participate in that project. So instead of kind of letting the farm disappear, we decided to develop a product to support them so it's also supporting - our objective is actually to create a flourishing local fiber and textile kind of structure. And that has been really interesting because the costs of the wool that went into the blankets was $1000, just raw material and that it doesn't give her any profit, actually. And that's, I think one of the biggest things that we are kind of trying to advocate and support, is how do we make local production possible again against a fair wage. And this is an extreme product, I know, but it has been very surprising to us that people do gravitate towards the story. They wanna participate in it by buying the jackets. We make our own materials, own looms, and these are a few of the jacket. And we do private sales events. We don't sign shops or anything like that. We make everything to the size of the customer, so there's a lot of personal attention that goes in every part of our value chain. Thank you."

44:06 - 44:23

"It's super important to write everything down and to make sure that you can have someone opt in with as much of an understanding of how the policies, the systems and everything run and that they actually do run that way, not just written, but it's actually run that way."

33:59 - 35:26

"From my perspective, it involves a little bit of everything and thinking about instead of having - You know you're you go to work and you are the person weaving - you're doing that. But then you're also communicating with the client and you're responding to press inquiries and writing out a budget for the rest of the year and learning a business plan, and you're doing a little bit of everything, and over time you find out who gravitates towards what role more, but you still lend a support role into the roles that you're not gravitating towards, so it's really like - you have to be a Jack of all trades in some way. I think for us it would - It was really big learning when we participated in the Co-op Academy. If we were making the jackets and we had a business model around those jackets, but we understood that we didn't account for a lot of this thing, a lot of the sweat equity that we were talking about. We had to kind of calculate all those things. So it's for me. It also has really been about learning how to run a business and not only just kind of - Nicely waving something to get up. It's really starting to understand, and we all - it was really important that all of us were in that program together and that we grow and learn together how to do that - that was for me, amazing."

30:04 - 31:12

"I personally love it and have a hard time imagining working in another way. At this point I've been part of the Co-op since it started, so you know, almost, yes - What do we say? 2010 so almost eight or nine years? So that's the short version. Of course, it's super hard. It can be really stressful at different times depending on what's going on or who was around or what the projects are, but I wouldn't trade any of that to not have a say over what we do or how we decide to change. I think the biggest - The thing I like most about it is the flexibility and the malleability of the organization. You know, we all - if something isn't/ doesn't feel right, we can bring up and we can change the whole organization. You know we can be like this thing. We can't do projects like this anymore for X reason or we need to hire more people who can do this kind of thing or whatever it is. We can just do that, and it's not easy to do, but we can do it as opposed to if I don't like the company I'm working for, well, you know it's another company, like you better quit. That's the only option sometimes, so that's probably the biggest thing for me."

12:03 - 16:32

"...we're a production company-cooperative as well as a media arts collective, and we're based in Sunset Park, Brooklyn, at the new art built space in the Brooklyn Army Terminal. Uhm, and so in the clip there were there was commissioned work. There were featured documentaries or journalistic pieces, music videos. Videos for magazines, nonprofits. And that's us. That's some of us - that's six of us up. So there's eight of us and we're all worker-owners, and we basically work on all of those projects, and we rotate our roles as producers, editors, cinematographers. Uh, we're all paid the same for a day's work for a time.Regardless of the projects we've worked on in the past, or you know or what our resumes look like. Uhm, we also make all of our decisions of how we run the organization through consensus based decision making processes. Uhm...And we also have - and we can talk a little bit more about what that means after - during the Q&A, if that's what if you're interested in - knowing more of how that works. What it basically means is we have three meetings a week. There's a lot of communication constantly, and we also have a working group structure to deal with all the admin so. What that means is you know, how do we ensure that all of our equipment is running smoothly? How do we ensure that any press requests are being responded to or any client requests are being responded to? Is we have various working groups that were all apart of and for that work, we are not paid. It's considered sweat equity. Uhm, that's the work that we do to be able to participate in the co-op to have access to the client work and all the shared resources. And I'd say that takes up about 8 hours a month, 8 to 10, it depends. We also have a weighted rotation system to distribute all the work fairly so that not just one person is only producing, that they're not working all the time, it's to distribute roles as well as the flow of work itself. What else - we share resources for personal projects. We also have benefits. We have a health stipend that we provide for all worker owners. Also patronage which is kind of like a bonus at the end of the year. And we also fund the media arts collective. Yeah, so I'll talk about the kind of other wing of Meerkat media so its a collective of artists and residents - it currently has 16 members. The group works on a huge variety of projects so some - a lot of the work is independent work that the individual folks are working on and we support each other on those. Sometimes we do big projects together where anyone who is available and wants to can work on a project in a more collaborative way, and we do all different things from short form, long form, documentary, experimental, a big project we did in 2016 after the election - was we did a large newsreel project where because we have both between both the co-op and the collective - we have, you know, a bunch of very talented people, so we were able to all kind of band together and make a whole bunch of short form documentaries around all different kinds of actions that were going on around the city and around. And we put out, I think like 10 to 15 short kind of short documentaries - in a short time frame, we also do skill shares and workshops, both for just for the folks in the group, but also sometimes we do it more outward facing workshops. We also - the group shares resources, space, equipment, brainpower with the co-op, and we also, that group is also totally self run - Uses consensus - consensus process in its meetings and is a self run project. So all the - also working groups to do all the admin and things like that. And we also have an annual retreat that both the co-op and the residency the collective do together."

33:34 - 36:35

"We have operated both with the sense of urgency that comes of seeing how much harm is done by critique as its habitually enacted, and against that urgency. What do I mean by against that urgency? Perhaps we share a suspicion that drives to fix critique.Or to replace the current haphazard ways it gets practiced with something just a little less harmful if they aren't embedded in a will to see plainly, and to name plainly how the form serves oppressive forces. Will become a way of avoiding the deeper transformation that is called for. To return to Matthew Goulish's words, we discover our approach and we follow it. But what if you start to discover that yours appears to be an incredibly slow approach? Even an ambivalent one. Appears to need a long gestation period. Appears to want not to be published. Yet; at all. As anything like findings. And still it feels important to show up. Can something contribute to a shift in the field in these conditions? Without overperforming certainty. Or trying to lay claim to anything like a territory. Could ambivalence serve as a point of attachment? Could the work of not trying to settle in advance the relation of ambivalence to urgency become precisely the matrix we need for all the kinds of unmakings this moment calls for. If the long game is transformation of the practices of learning to generate ever more just and equitable conditions that serve abroad and thickly differentiated us.Then it's incredibly useful to let critique keep showing its hand. Revealing itself as a tool for Recentering or hyper centering. The already centered and further marginalizing or attempting to disappear the already marginalized. Each of us in the retooling critique working group in our own ways, and often in dialogue with one another, has taken some form of this approach. To study the thing in the act of doing the thing and to introduce into that study. Or maybe via that study? Kinds of noise that forced the form to reconfigure. And in so doing, perhaps to take some of the broader context with it. In orienting a new to the kinds of presents and futures that call.

01:23:29 - 01:28:06

"...against my biases, towards my students, and the biases that students bring towards me. And it's a space where we're reckoning with our histories of things that we embody currently, right? So I think there's a value of breaking silence and speaking up. But I also wonder through the modes of critiques, how forgiveness and reconciliation can happen so that we can move forward, 'cause I think that's important, right? Uhm? So that's something that I just wanted to ask, either as students or teachers. If you have encountered moments like that. Do you mean like moments of impasse like not knowing how to move the conversation forward or to even and engage with one another's complexities? And I think that discomfort is OK. And I think we need to embrace that. I think because we are not comfortable being this uncomfortable that we just kind of don't want to talk about it, right? But I think after then.We can acknowledge where we are, but I want to move forward. And I think it can happen through modes of critiques, right? But it is a lot of work 'cause I'm working through so much of how I've been educated and how I had been taught to teach." "My experience, both as a student and as an instructor trying to facilitate deeper engagements with one another in such a vulnerable setting, especially when we're also at an age where we're exploring parts of ourselves in ways that we haven't before questioning our history, our relationship to so much in a very public setting in school with people that we don't know, that we don't share language with. As an instructor, I think one thing that I try to do is to simply acknowledge that as a condition of the class that we all come with so much, we're all bringing so much, and that it takes time to prime these conditions, to prime these conversations. And that it's not something that we should expect to happen, in critique, without any sort of collective agreement or discussion or working towards. Even be able to see ourselves or each other in this way, and I think some of the the fault lines of critique is that it's just taken as this neutral space where we can all just come in and we know exactly how it's going to go, and there isn't any sort of preparation for priming that conversation." "If I can jump in just on that point of priming the conversation, I think it's really key. And and in one of our approaches, we have the phrase move from tacit to explicit. I think we either condition the sociality in the room, or we're conditioned by the sociality that walks in, and we know that the sociality that walks in has really uneven literacies around understanding one another. And so to take a moment before critique, even to locate critique within larger ways of knowing that are happening constantly, can actually happen quite quickly and allow a group together to identify practices that they want and obstacles to that practice, so that could concretely look like - placing a sample object on a table - Ideally not one of the students objects, and asking through how many lenses could we pick up or view this object so we could look at it through - a material lens, through a craft lens, a formal lens, a biographical lens, a historical lens - to just map all of those out and together come to a kind of agreement around which of those are the ones we're actually intending to do in the next few hours. And even naming like - what are obstacles to doing that so that there's a kind of lifting up in advance, and collectively having in the room the knowledge that there are obstacles to then be able to sort of thread a way forward."

01:11:28 - 01:15:14

"...my previous institution had worked itself into this kind of fever pitch around racial politics to such an extent that it had become an incredibly toxic - nearly violent place for the students of color - there was anti Black Lives Matter graffiti going up, there was alt right white supremacists threatening students.You know there there was a lot of activity and in that moment and in subsequent moments, I felt as though when I turned to...you know at that time I would have 60 or 70 faculty members - I was one of like three or four faculty of color - Only, I believe, like two of which had full time positions - That when I turned to them, it's... their suggestion was to not get involved. And that was a very difficult thing. It continues for a lot of my colleagues who find themselves in these more permanent positions. It continues to be their position. To not do that labor for the institution, in part because the system is set up in such a way as to. Expect for your responsibility, for your sense of responsibility for the student. For students of color, for their experience. To be strong enough to task you with that labor - Without any meaningful infrastructure being built or meaningful compensation meeting you. So I have some wonderful friends who do this kind of equity work at different kinds of organizations. Their suggestion is write in the contract... is to like demand compensation/ forms of compensation for that labor. Because moving into these environments. Because implicit in being a diversity hire is that you will do this labor, right. It's not written into the contract, but if we look closely enough - in a kind of Willy Wonka version of it, it would be there invisibly in the bottom right if it was held under infrared light, it would be there like. Like undo all of our white supremacy. I feel like some of my colleagues - their response is to just have it plainly written into. Well then I deserve X number of course releases or such and such kind of compensation to offset the fact that I'm gonna be sitting in these faculty meetings and I won't be the only person of color. And you were going to talk about how this is a white institution, and in that moment immediately erased not only me, but my colleagues and all of the students colour simultaneously. So now that I have to write myself back into existence, right, that becomes... that's the sort of Labor, right, that writing-oneself-back-into-existence kind of Labor."

01:04:00 - 01:05:45

"The limits of the conditions that we operate within. What are the constraints of the institution that obstructs this labor, and how can we set the focus of the class to include its own institutional conditions? As long as - It's all very well for, like the adjunct faculty, and like the students and the people who are in the most precarious position to be doing this work, but unless there are major changes to the way all of these institutions are run at the very top - Uhm, the change won't happen. And, I think for instance I mean - There were professors who were not rehired - and who were big allies of what we were doing on campus - and I can't help but have this like sense that as long as you know the people who are doing the most work are the students and the students are going to leave after four years - And if then they're entrusting - like the legacy of that work to faculty, and then the faculty can just be eliminated because there are other people around too - and not deal with actual restructuring - An actual relinquishing of power and authority - that nothing will get accomplished."

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Reply to the reviewers

Reviewer #1 (Evidence, reproducibility and clarity (Required)): In this paper, the authors use a previously published method SHAP for interpreting deep learning (DL) models (specifically LSTMs) that are trained for predicting physicochemical attributes of peptides (such as antigenicity and collisional cross section). The paper shows that it's capable of identifying some amino acid residues contributing to the prediction results of the DL models. Reviewer #1 (Significance (Required)):

1. One main ideas of the paper is to use SHAP for determine the significant amino acids at each position (or pairs of AA at each position) contributing to the prediction. Some of the interpretation results are consistent with findings reported previously. This is very nice; however, most of these findings are statistical results such "XX is often present at the second position for the peptides with the positive outcome", which are relatively straightforward and may be derived by using some statistical methods without using DL models. We expect more complex patterns can be discovered in addition to these statistical observations.

We thank the reviewers for these comments.

First, to the point about discovering complex patterns, we note that one use of PoSHAP we discuss later in the paper is that PoSHAP enables interposition dependence analysis, which depends on interactions between residues and would not be reflected by summary statistics.

Second, we agree it is important to show whether PoSHAP produces different residue importance maps than simple statistical summaries of amino acids in each group. The strongest binding peptides, or the highest mobility for the CCS model, were determined by taking only peptides that fall above a linear regression best fit of the ranked experimental values. Statistical summary heatmaps were created and then compared to those from PoSHAP revealing some similarities but also many differences. We added the following text and new figure to the results section to illustrate these points:

“We wondered whether the patterns revealed by PoSHAP simply reflect the summary statistics for the high-binding or high-CCS subset of peptides. As expected, due to known differences in amino acid abundance across the proteome, the prevalence of amino acids was different across the training data and were also heterogeneous across positions (Figure 5A). To determine the subset of high CCS peptides, peptides were ordered in the training set by their CCS rank and then linear regression was performed to get the average trend line (Figure 5B). Any peptide above that trendline was defined as “high CCS”, and the frequency of amino acids at each position in this set was summarized using a heatmap (Figure 5C). Compared to the statistical amino acid frequencies, PoSHAP suggests a greater importance to arginine at both termini, the importance of tryptophan to increase CCS becomes apparent, and interior glutamic acid contributes less to high CCS than the frequencies would suggest (Figure 5D). The same analysis was repeated for MHC data (Supplementary Figures 9 and 10). This demonstrates that PoSHAP found non-linear relationships between the inputs and the outputs that are not present by simple correlation. “

Figure 5: Amino acid summary statistics differ from PoSHAP values for the CCS data. (A) Amino acid counts as a function of position for training data. (B) Procedure for picking the ‘top peptides’ with the highest CCS. Linear regression was performed on the peptides ranked by their actual CCS value. Any peptide that fell above the trendline and overall mean were defined as ‘top peptides’. (C) Counts of amino acids for the top peptides were summarized in a heatmap. (D) Mean SHAP values across amino acids and positions from PoSHAP analysis.

We also added the corresponding supplemental figures showing the same examples for the MAMU A001 model and human MHC models:

Supplemental Figure 9: Amino acid summary statistics differ from PoSHAP values for the A001 MAMU MHC I data. (A) Amino acid counts as a function of position for training data. (B) Procedure for picking the ‘top peptides’ with the highest CCS. Linear regression was performed on the peptides ranked by their actual CCS value. Any peptide that fell above the trendline and overall mean were defined as ‘top peptides’. (C) Counts of amino acids for the top peptides were summarized in a heatmap. (D) Mean SHAP values across amino acids and positions from PoSHAP analysis. For the MAMU model, the amino acid frequencies of the input peptides show no obvious preference for amino acid position, but some amino acids are over-represented overall. The presence of the “end” token is more likely to be a high binder statistically (C), but the PoSHAP reveals that this end token is not the main determinant of binding (D).

Supplemental Figure 10: Amino acid summary statistics differ from PoSHAP values for the human A1101 MHC I data. (A) Amino acid counts as a function of position for training data. The distribution of amino acids in this data. (B) Procedure for picking the ‘top peptides’ with the highest CCS. Linear regression was performed on the peptides ranked by their actual CCS value. Any peptide that fell above the trendline and overall mean were defined as ‘top peptides’. (C) Counts of amino acids for the top peptides were summarized in a heatmap. (D) Mean SHAP values across amino acids and positions from PoSHAP analysis. There are clear differences between the summary statistics of top peptides (C) and PoSHAP heatmap (D). For example, the end token is prominent in the summary statistics absent from the PoSHAP interpretation. Also, the preference for S/T/V at position two is tempered according to PoSHAP, but would be determined to be very important by the summary statistics.

Although the interpreting results reported in the paper largely agree with previous reports, the paper did not explicitly model the frequency of different amino acid in the training data. For instance, if the amino acid 'A' happens to be over-represented in the positive samples of peptides in the training data, the DL model may consider it as to contribute to the positive prediction, which may not be not true. This issue might become more serious when pairs of amino acids are considered. The authors may want to analyze this potential issue in their results.

We agree and understand the concern for the overrepresentation of amino acids that might skew the training of our models. To determine if this is an issue, as part of the response to the previous question, we looked at the amino acid counts for all peptides (Figure 5A, Supplemental Figures 9A and 10A). In general, the PoSHAP heatmaps (panel Ds in the same figures) look very different from the frequencies of amino acids (panel Cs in the figures), suggesting that amino acid frequencies have not caused any problem.

Even on a balanced training dataset, the LSTM model to be interpreted may still contain arbitrary bias due to invertible overfitting, which the authors did not discuss. It will be more convincing by training multiple models using different hyper-parameters and optimization algorithms, and then see if similar interpretation results can be reached among most or all of these models.

We assume the reviewer meant ‘inevitable overfitting’ instead of “invertible overfitting”? If so, the original manuscript did assess overfitting in Figure S4 based on the training and validation loss over training epochs.

We think the reviewer makes a good point that different models might produce different interpretations, so we trained new models without optimization and with different hyperparameters and with a different optimizer (RMS prop). We see essentially the same PoSHAP interpretations. We added the following text to the results section along with these three new supplemental figures:

“Given the dependence of the model interpretation results on the model used, the same model architecture trained with different parameters might result in different model interpretation. Given this, models for each of the three tasks mentioned here were retrained with different hyperparameters including the “RMS prop” optimizer. Each model produces similar or better prediction performance compared to the earlier version, and the model interpretation by PoSHAP was almost identical to the previous results in all three cases (Supplementary figures 12, 13, 14). This suggests that the model architecture drives the differences in interpretation, not the model training process.”

Supplemental Figure 12. PoSHAP Analysis of Mamu A001 With Unoptimized Hyperparameters and RMSprop. A new model for the Mamu data was trained using the same architectures but with different hyperparameters and RMSprop as the optimization algorithm. Loss was plotted as mean squared error compared to the validation data. (A) Similar metrics for MSE, r, and p-values were obtained (B). Similar patterns are also observed for the PoSHAP heatmap of A001. (C) A dependence plot for A001 shows similar patterns to the Adam optimized model, including the positional dependence of proline at position two for high SHAP values of serine and threonine.

Supplemental Figure 13. PoSHAP Analysis of A:11*01 With Unoptimized Hyperparameters and RMSprop. A new model for the A:11*01 data was trained using the same architectures but with different hyperparameters and RMSprop as the optimization algorithm. Loss was plotted as mean squared error compared to the validation data. (A) Similar metrics for MSE, r, and p-values were obtained (B). Similar patterns are also observed for the PoSHAP heatmap of A:11*01. (C) The SHAP ranges by position plot for A:11*01 shows similar patterns to the Adam optimized model, including the largest range of SHAP values at position two, nine, and ten.

Supplemental Figure 14. PoSHAP Analysis of CCS With Unoptimized Hyperparameters and RMSprop. A new model for the CCS data was trained using the same architectures but with different hyperparameters and RMSprop as the optimization algorithm. Loss was plotted as mean squared error compared to the validation data. (A) Similar metrics for MSE, r, and p-values were obtained (B). Similar patterns are also observed for the PoSHAP heatmap of CCS. (C) Dependence analysis was performed on the dataset and the combined distance-interaction type bar plot shows similar relationships between the groupings, notably charge repulsion’s split.

For the dependence analysis, it is not completely clear why the distance is used as the variable, while the relative position of the amino acid residue in the peptide is ignored. For example, if there is a strong interaction between the first and the last residues in the peptide, their distance changes depending on the peptide length. In figure 6, the authors showed strong interactions between amino acid that are 8-9 residues apart may suggest the peptide length actually plays a role here.

We used distance because as the dependence analysis is a calculation of the difference in means between two distributions of SHAP values, dependent of the amino acid at another position. We believe that the distance between these interacting points is a natural choice and among the most informative metrics to explain these interactions. We agree with the reviewer that peptide length is important to the magnitude of the interactions between amino acids. We also recognize that there may be interactions between the peptide termini that could be obscured by the interactions of the longer peptides. To better explore this possibility, we performed the dependence analysis on each of the different peptide lengths separately (8, 9, or 10 here) to see if this is the case. Unfortunately, given the smaller size of these data subsets, we were unable to show significant differences in the interaction groupings. Though, interestingly enough, the significant interactions for the peptides of length eight only occurred between neighboring amino acids or the termini. This may suggest an interaction between termini that could be explored in the future.

We added the following text and supplemental figure 11 to the results:

“Finally, to try to ask if the absolute positions of amino acids in the peptide are relevant for the interaction, the data was split into 8, 9, or 10mers before analysis (Supplemental Figure 11). This revealed that there may be interactions between the termini, but this effect may be difficult to observe because there are significantly fewer 8mers and 9mers in the CCS dataset.”

Supplemental Figure 11. SHAP Values of Collisional Cross Section by Peptide Length. The impact of peptide length on SHAP values was explored for the CCS data. The dataset was split into peptides of length 8, 9, and 10. All SHAP values were plotted as violin plots. The mean SHAP values were plotted in heatmaps by position and amino acid and standardized. Significant interactions by dependence analysis were plotted in bar charts by distance between interactions.

To further support our decision to use distance as an interaction metric, we have also now included an additional box plot for Figure 7, demonstrating the interactions between each of the categories combined with distance. We have found that some of the bimodality of the interaction categories are explained by the distance at which they interact. Most strikingly is charge repulsion that decreases CCS when neighboring but increases CCS when the interaction is further.

We added the following text and updated Figure 7 to the results section:

“Additionally, there are interesting differences in the interactions of the amino acid among the significant set of interactions (Figure 76B). All significant interactions from the CCS data (Supplemental Table 3, adj. p-value Though it is evident that the mean of each interaction type corresponds to the expected impact those interactions would have on CCS, each of the interaction dependence plots are bimodal, with some interactions increasing CCS and some decreasing it. To dissect this observation further, we combined the two methods of splitting the data to see if the bimodality of interaction types would be resolved by distance (Figure 7C). Though definitive conclusions cannot be made for most categories, likely due to the ever decreasing sample size by splitting, of note is the difference between neighboring charge repulsion and non-neighboring charge repulsion. Neighboring charge repulsion seems to decrease CCS while distant charge repulsion increases CCS (see adjusted p-value from Tukey’s posthoc test in Figure 7D). When distant, charge repulsion makes intuitive sense as the amino acids are forced apart, linearizing the peptide and increasing the surface area. When neighboring, it is possible that the repulsion causes a kink in the linear peptide, decreasing the cross section. Overall, these analyses demonstrate that the models were able to learn fundamental chemical properties of the amino acids and through PoSHAP analysis we were able to uncover them.”*

Figure 7. Dependence analysis of CCS model. (A) Significant (Bonferroni corr. P-value = charge repulsion, * = other, and δ = polar. For the distance analysis, interactions were grouped into three categories, neighboring (distance = 1), near (distance = 2, 3, 4, 5,6), and far (distance = 7, 8, 9). * indicates significance (ANOVA with Tukey’s post hoc test p-value

Also, it would be better to show that how the result looks like when applying this method to peptides in the negative samples (e.g., the peptides that are not bound by MHC in the antigenicity prediction experiment). Will the interpreting results also be negative?

We agree this is an interesting idea. We updated the supplemental figure showing PoSHAP of top peptide subsets to also show PoSHAP of bottom peptide subsets (supplemental figure 8). The results suggest that certain amino acid positions are detrimental to binding, for example D/E at various positions. We updated this section to add:

“We also performed the same analysis with the eight peptides with the lowest binding predictions (Supplemental Figure 8). These PoSHAP heatmaps are primarily composed of negative SHAP values, suggesting that using this subset reveals amino acids at certain positions that are detrimental to MHC binding.”

Supplemental Figure 8. Pooled PoSHAP for bottom and top predicted subsets of the data. The mean SHAP values for each amino acid at each position were calculated for the peptides with the bottom (A) or top (B) 0.013% predicted intensity (top 8 peptides) for the “A” Mamu alleles. Due to the small sample size, most of the amino acid positions have a value of zero. The positions with extreme values, however, illustrate important amino acids for prediction. Notably for A001 and A002, aspartic acid and glutamic acid contribute to low prediction along the peptide, suggesting charge may inhibit binding. For the top predictions, phenylalanine or leucine are important at the first position for both A001 and A008. A serine or threonine at position two is important for A001, A002, and A008. All alleles demonstrate the importance of a proline near the middle of the peptide.

Finally, it will be interesting to see the interpreting results when the method is applied to the DL models on more challenging tasks such as the prediction of tandem mass spectra of peptides. The authors may want to discuss these applications.

We agree it would be very interesting to apply this method to interpret predictions of tandem mass spectra. In this paper we already demonstrated PoSHAP on three different datasets with three different models, so we feel that adding a fourth model is out of the scope of this work. We do agree that we would like to explore this option in the future. We added this idea to the discussion section:

“Altogether the advances described herein are likely to find widespread use for interpreting models trained from biological sequences, including models not covered here such as those to predict tandem mass spectra (reviewed in 33).”

I am primarily interested in algorithmic and statistical problems in genomics and proteomics. We have develop deep learning models for predicting the full tandem mass spectrum of peptides, and am interested model interpretation methods to explain the fragmentation mechanism resulting in non-conventional fragment ions in tandem mass spectra of peptides. I review the paper in collaboration with my Ph.D students, who are developing deep learning models for computational mass spectrometry.

Reviewer #2 (Evidence, reproducibility and clarity (Required)): **Comments to the Authors** In this study, the authors developed a framework named PoSHAP for the interpretation of neural networks trained on biological sequences. The current manuscript can be stronger if the following issues can be clearly addressed.

1. As interpreting model with SHAP is a vital part of this manuscript, it would be better to provide descriptions of the underlying principles of SHAP to enable the readers to understand the paper easily.

We recognize that understanding the principles of SHAP is vital. To better explain SHAP, we have added the following text to the introduction:

“SHAP is a perturbation-based explanation method where the contribution of an input is calculated by hiding that input and determining the effect on the output. SHAP expands this using the game theoretic approach of Shapely values that ensures the contributions of the inputs plus a calculated baseline sum to the predicted output.”

It is emphasized in the manuscript that PoSHAP is introduced to interpret neural networks trained on biological sequences. However, it is not clear why the authors choose the Model Agnostic Kernel SHAP, which is based on Linear LIME. Although it can be used for any model, the performance of which may not be optimal. In this regards, perhaps Deep SHAP or Gradient SHAP is more appropriate, both of which are designed for deep learning networks [1]. It would be better to provide some additional experiments on Deep SHAP and this work will be more convincing if the same or similar contribution of each position on each peptide as that of Kernel SHAP. [1] Lundberg, S., and S. I. Lee. "A Unified Approach to Interpreting Model Predictions." Nips 2017.

Our goal in using KernelExplainer was to demonstrate that PoSHAP was not dependent on model specific interpretation methods. However, we have realized that this intention may not have been clearly stated or demonstrated. To expand on this, we have included a new Figure 8, which shows PoSHAP analysis comparisons to other classes of machine learning models, all using Kernel Explainer. This result was interesting because it revealed that even though the XGboost model technically performed better at prediction (Figure 8A, reduced MSE and higher spearman rho), and produced a similar PoSHAP motif heatmap, the interpositional dependences from the perspective of distance (Figure 8C) or chemical interactions (Figure 8D) were substantially muted. This is also apparent with the other standard machine learning model ExtraTrees. This result shows that the choice of model architecture is important, and this direct comparison would not be possible if we used the DeepExplainer.

We added the following text and figure to the manuscript:

“ PoSHAP uses the SHAP KernelExplainer method, which is based on Local interpretable model-agnostic explanations (LIME). Using the general KernelExpplainer method enables direct comparison of interpretations produced by different models trained from the same data. To ask whether PoSHAP interpretation changes based on the model used, the CCS data was used to train XGboost or ExtraTrees models. Surprisingly, the XGboost model performed better than the LSTM model with regard to MSE and spearman rho between true and predicted values in the test set (Figure 8A). ExtraTrees was slightly worse than the other two models. The model interpretation heatmaps from PoSHAP were similar between the LSTM and XGboost, but the interpretation from the ExtraTrees model was missing the high average SHAP due to n-terminal histidine or arginine (Figure 8B). Even though XGboost produced a similar PoSHAP heatmap, the interpositional dependence with regard to distance (Figure 8C) and chemical interactions (Figure 8D) was muted. This shows that the choice of model is important for revealing amino acid interactions.”

Figure 8. CCS PoSHAP of Various Machine Learning Models. PoSHAP analysis was performed on two additional machine learning models, Extra Trees and Extreme Gradient Boosting (XGB). Predictions were plotted against experimental values and the Mean Squared Error and r values are reported for each model (A). PoSHAP heatmaps were created for each model (B), illustrating an increase in model complexity as more sophisticated models are used. Dependence analysis was performed on each model and the significant interactions are plotted by distance (C) and by combined distance and interaction type (D).

As described in the manuscript, "Correlations between true and predicted values were assessed by MSE, Spearman's rank correlation coefficient, and the correlation p-value." As an important indicator for evaluation, the exact p-values should be provided in the seven subgraphs in Figure 2, not p=0.0.

We agree with the reviewer that reporting accurate p-values can assist in evaluation. We have updated the figures to reflect the p-values as far as we were able to determine them. Unfortunately, we are limited by the nature of the double data type in python and so reported that the p-value was less than the minimum value allowed by a double in six of the seven graphs. Additionally, the scales have been marked symmetrically as you mentioned in comment 4.

It should be noted that the coordinate scales of Figure 2B and Figure 2C need to be marked symmetrically. And from Figure 2B, we can see that, the IC50 with smaller (0.8) values cannot be well predicted. Can the authors provide a detailed explanation about these results?

We understand the reviewer’s concern with poor prediction of extreme values. Figure B represents the IC50 prediction for the A1101 human allele which was the smallest of the datasets we used for training. It only consists of 4,522 entries, around 1/10 of the data used for the Mamu alleles and CCS. Because of this, it is likely that there were not enough examples of datapoints at the extremes to reliably train the model to account for them. However, given the limited size of the dataset, we were surprised with the satisfactory predictions. More importantly, the purpose of our paper is model interpretation not model prediction accuracy, and this shows that even when predictions are not perfect, the model interpretation by PoSHAP can still be effective. We thank the reviewer for noticing this and added the following statement to the results:

“Remarkably, this was achieved for A\11:01 using a total dataset of only 4,522 examples, which shows that PoSHAP can be effective with even less than 10,000 training examples. “*

References are needed in some descriptions in the manuscript. For example, "one might train a network to take an input of peptide sequence and predict chromatographic retention time", "RNNs have found extensive application to natural language processing, and by extension as a similar type of data, predictions from biological sequences such as peptides or nucleic acids".

We apologize for missing these references. We have now cited these statements and have added many additional references as part of our revision.

The description of the adopted three models in the section "Model architecture" is a bit confusing. As described in this section, "The LSTM layer outputs a 50x128 dimensional matrix to a dropout layer where a proportion of values are randomly set to 0", "a second LSTM layer outputs a tensor with length 128 and a second dropout layer then randomly sets a proportion of values to 0". But as shown in the Supplemental Figure 3, the output size of the first LSTM was 10x128. Also, as shown in Table 1, the dropout rates were not 0. Therefore, the section should be adjusted for clear clarification.

We apologize for the confusing wording. We meant that dropout layers randomly set values=0, not that the dropout proportion was 0. We reworded this part to read:

“The LSTM layer outputs a 10x128 dimensional matrix to a dropout layer where a proportion of values are randomly “dropped”, or set to 0. For the MHC models, a second LSTM layer outputs a tensor with length 128 to a second dropout layer. Then in all models, a dense layer reduces the data dimensionality to 64. For the MHC models, the data is then passed through a leaky rectified linear unit (LeakyReLU) activation before a final dropout layer, present in all models.”

Reviewer #2 (Significance (Required)): Pls refer to my comments provided as above.

Reviewer #3 (Evidence, reproducibility and clarity (Required)): **Summary:** The main goal of the work is to provide the interpretation of Deep Neural Networks (LSTM in the paper) trained on biological sequences. For this purpose authors used the framework introduced earlier - SHapley Additive exPlanations (SHAP), in particular - the slight adaptation of this method called positional SHAP (PoSHAP), because they are interested in the impact of each position of the input sequence to the model output. They demonstrate this on three regression tasks that predict peptide properties. **Major comments** The main contribution, highlighted in the paper: authors showed how PoSHAP discloses amino acid motifs that influence MHC I binding. Further they described how PoSHAP enables understanding of interpositional dependence of amino acids that result in high affinity predictions. Also they argued that this work also contributes to a method for accurate prediction of peptide-MHC I affinity using peptide array data enabled by novel application of a neural network that combines amino acid embedding and LSTM layers.

There are some comments about the statements above: 1.Why was the LSTM model chosen? Recent publications showed the success of the Transformer model for biological sequences; however this direction was not covered in the related work overview. The architecture choice then should be better justified. Also the choice of LSTM for the biological sequences is not new and authors should better claim their statement about "novel application of a neural network that combines amino acid embedding and LSTM layers ". Where exactly is the novelty? Could the community use the pretrained embeddings for their purpose?

The reviewer is correct that transformer models are highly effective for making predictions from biological sequences. In fact, many models do well, and there is no single correct choice of model for this task. Though there are many models to choose from, our models are sufficiently accurate. Importantly, the main contribution of our manuscript is not to train the most accurate models, but rather to demonstrate a strategy for positional model interpretation based on SHAP. Related to that point, please note our response to reviewer #2’s second comment that our approach uses the kernel explainer and can be applied to any model. However, we do agree that we neglected coverage of the transformer model in the introduction and have added a paragraph to the introduction covering some of the recent work in this area:

“Many effective deep learning model architectures are available for making predictions from inputs of biological sequences, and there is currently no single correct choice. CNN models such as MHCflurry 2.0 (40) and LSTM models are effective at predicting MHC binding of peptides (41). Even simpler models, such as random forests, have been used to predict MHC binding (42,43). Prediction of other peptide properties like tandem mass spectra are often done with CNN or LSTM models (33). More recently, given the extraordinary performance of transformer models like BERT (44) and GPT-3 (45) for NLP, there is interest in transformer models for biological sequences (46).”

We also want to be sure we do not overstate the novelty of our contributions. We have updated our discussion to better reflect the nature of our contributions. We reworded the statement quoted above to read:

“Overall, the three modeling examples laid out herein serve as a tutorial for PoSHAP interpretation of almost any model trained from almost any biological sequence.”

The attention mechanism itself provides the great opportunity to interpret the model predictions. In the introduction section authors made a statement that attention layers may limit the flexibility of model architecture when designing new models. Could they better explain this limit? Because recent state of the art models successfully work with long biological sequences and show better results then any other models (one example could be found here: https://openreview.net/pdf?id=YWtLZvLmud7). Authors should cover these limits more, that also related to the motivation of the LSTM choice.

We added a paragraph to our introduction to expand on attention and its limitations:

“Attention mechanisms have been successful in recapitulating experimentally defined binding motifs, but require that the model be constructed with attention layers. This may limit the flexibility of model architecture when designing new models. For example, attention mechanisms are specific to neural networks. Simpler models, such as random forests and XGboost, may also be more suitable for some applications, and these cannot utilize attention. Also, while attention mechanisms are currently very effective, there is always a possibility that new architectures will emerge that make interpretations using attention infeasible. Beyond this, attention is a metric of the model itself, while SHAP values are calculated on a per input basis. By looking at the model through the lens of the inputs, we can understand the model’s “reasoning” behind any peptide. Attention mechanisms also do not enable dissection of interpositional dependencies between amino acids. Thus, new methods for model agnostic interpretation are desirable.”

Another statement was made about the PoSHAP - adaptation of the SHAP method. It is hard to follow through the explanation of this adaptation - it is not clear what exactly is this adaptation. For example, Kernel SHAP from the original paper computes feature importance, in this paper authors compute the impact of each position, that is basically also the feature importances. Thus authors should better explain the statement about PoSHAP novelty. Will it be possible to use PoSHAP for any other model trained for the same purpose? If yes, for better reproducibility, authors should provide the place where exactly in the repo is the code for this. Also mathematical notations are missing in the Positional SHAP (PoSHAP) section - it is better to explain the adaptation with them to increase the understanding of the section.

We apologize for the ambiguous wording in the abstract stating that “PoSHAP adapts SHAP”. We have reworded this statement to “PoSHAP utilizes SHAP”. The novelty of this approach is taking the feature importance values calculated by SHAP and structuring them to include each position’s index to allow for the interpretation of biological sequences. As we demonstrate here, this allows for novel interpretations of previously published data and will enable model interpretation in future studies that learn from biological sequences. Although this is practically very simple, we are not yet aware of any examples in the literature that do this.

The following two SHAP force plots demonstrate the difference between using SHAP as-is versus PoSHAP. There is a demonstrated need for such a framework, considering the dearth of biological sequence model interpretation using SHAP and the ambiguity within biological sequence SHAP interpretation. For example, Meier et al., Nature Communications, 2021 performed an analysis like our Figure S7C, which just shows the range of SHAP values per residue. Although we can learn something about which AAs are important based on the range of their SHAP values, SHAP as-is doesn’t reveal a motif. While our position indexing is a simple change, it enables all the rich, sequence dependent analysis we performed in this paper. We added the following text to the results section with this new supplementary figure:

“PoSHAP utilizes the standard SHAP package but adapts the analysis by simply appending an index to each input and maintaining positional information after the kernelExplainer interpretation, which enables tracking of each input postion’s contribution to an output prediction (supplementary figure 5, showing force plot with and without index).”

Supplemental Figure 5. SHAP Forceplots Demonstrating PoSHAP Indexing. Two forceplots were created with the SHAP forceplot method of the third peptide in the CCS testing set. (A) shows the plot with encoded inputs mapped to their amino acid. (B) shows the plot with the encoded inputs mapped to their amino acid and position. The addition of positional indexing removes the ambiguity of contributions, for example, glutamine having both a positive and a negative SHAP contribution to the prediction of the third peptide.

We have updated the repository to include a tutorial that demonstrates PoSHAP on provided data and explains how to use PoSHAP with your own model and data.

In the experimental section, authors first compare the results with previously known. For example, for the human MHC allele A*11:01 model PoSHAP analysis shows the similar results as was shown with another approach. Based on the provided explanation, it is not clear why PoSHAP is better than the previously published method. The advantage of the PoSHAP should be better explained.

We agree with the reviewer that the benefits of our approach should be as clear as possible. The referenced section of the paper is to validate our approach compared to another model interpretation technique. We added a new third paragraph to the discussion section to clearly explain the benefits of PoSHAP:

“There are several benefits of PoSHAP over competing methods. First, PoSHAP determines important residues despite biases in the frequencies of amino acids (Figure 5, Supplementary Figures 9 and 10). PoSHAP is also applicable to any model trained from sequential data (Figure 8), and enables dissection of interpositional dependencies (Figures 6 and 7). Finally, we include a clearly explained jupyter notebook on Github that will take any model and dataset and perform PoSHAP analysis.”

In the experimental section, after the PoSHAP performance verification, hypothesis generation was introduced. However, it is not clear how many hypotheses were generated; how many of them were known before; what kind of other categories are inside these hypotheses (unknown, possible and potentially interesting, etc).

We are unsure as to how to quantify the number of hypotheses generated by our approach. In a sense, the SHAP value of each amino acid at each position within a heatmap represents a hypothesis of the contribution of that amino acid to the metric being predicted. Each significant interaction listed in the first three supplemental tables represents a hypothesis of the interactions between two given amino acids at two positions. To make these into testable hypotheses requires some analysis, as we have discussed. i.e. the two binding motifs (L-T-P, F-S-P) of A001, or the distance-type interactions within the CCS.

The README section in the GitHub repo is not easily understandable. An additional explanation for each step is required (e.g., links to the folders where the calculated SHAP values, the trained models, all splits and all-important benchmarks are).

We have updated the README and repository to explain how to use PoSHAP, and explanations of each item in the repository.

**Minor comments**

1. The prior studies should be covered better (see Major comments).

We apologize for not better covering prior studies. We have significantly expanded the introduction by adding two new paragraphs and at least 10 additional citations.

The work consists of some typos, for example: "However, because many reports forgo model interpretation" - "t" is missed.

We did intend to use the word “forgo” not “forget” in that sentence. We have checked again thoroughly for spelling and grammar mistakes.

The hyperparameters table, hyperparameter search section should be moved to the supplemental material, that's technical details.

We moved this table to the supplementary materials.

Reviewer #3 (Significance (Required)): Interpretation of the model results is an important topic for biology. New findings here could lead to new interactions opening, new drugs development etc. That is relevant for the applied ML Researches and computational biologists. This paper aims to provide a way to do it. Because my field of interest and expertise lies in Machine Learning for healthcare, language modelling of biological sequences and Natural Language Processing, this work is of great interest to me. So I mostly evaluated ML methodology presented in the paper.

19:13 - 23:03

"...is a little different in the sense that everybody, so it's 65 what we call participants and five resident faculty, and about 15 of us that are on staff. And we all basically arrive in this rural location on the same day and we all leave, basically, on the same day. So we're together all the time - for nine week, and similar to Danielle's program, but also slightly different. Everybody is from a completely different place. Everybody is a different age. Everybody has different experiences within their practice. So in any given summer, well, we can use last summer as an example. The youngest was 21 and the oldest was 48. Some people have been in grad school. Will have a BFA. Some people have no higher education whatsoever, come from 20 different countries - Many different languages and were all thrown into this environment together and three years ago I started leading a session. Basically we call it Community life orientation, which is essentially the enactment of the group agreement. It is also not a group agreement in that... It is written by me. But, uh, I would say kind of the beauty of living and working together for an entire summer for nine weeks of intensive time is that I am not any different - Well I am different, but I am not that different from the people that I'm also living with. So the exchange that happens on a on a regular basis means that I'm not so shielded from the ways that the Community life orientation or that group agreement needs to be adjusted on a year to year basis. So I was just meeting with a colleague yesterday to kind of think about ways, other things we needed to include in our in our group agreement so that it's constantly evolving. It's constantly rigorous to kind of bring together 65 people that have one thing in common, which is this process of art making, and even that is not necessarily clear in how that is a shared value so. In order to keep it from going into the corny zone, I think that we we really boil it down to the very basics, about what is the purpose of all of our jobs as artists? and what are we seeking to kind of communicate to the world? And what does that mean when you're in a group with others that are not your people necessarily, so you know we talk a lot about placing a value on wrongness as much as brightness. We talk a lot about how voice is important to all of us as individuals. And sometimes our voices are not speaking about the same things, but then how do we begin to listen to each other? We talk about respecting people's spaces and privacies and things like that. Ours is very long. It's like a it's like an hour long process to go through all of the agreements and you know, I mean, I think sometimes it's also important to remember that these are also aspirations. A group agreement is is great on paper until it's actually enacted. And so we use that group agreement as a moment - as like a touchstone like - OK, so something actually did happen. Now how do we deal with it? How do we embody patience and listening and understanding and not automatic judgment and automatic kind of closing off from an inquiry with that maybe we didn't want to have, but we are having, regardless."

i feel like the idea of international sovereignty and separating human populations into bordered blocks creates an illusion of disunity which further allows for self-interested strategy-making/ foreign policy. I guess it's just natural for humans to separate into groups like that and I'm just stating the obvious 😁

This reminds me of why my parents always had a policy when I was younger to "read the book before watching the movie." The space left between the lines in a text allow for someone to really imagine and make the story unique to them, and it's a lot different than watching a movie where everything is laid out for you to just passively consume. I wonder if there are Harry Potter fans who haven't read the books but have watched the movies, who might be missing out on contextual evidence for other people's interpretations of the text and if that could be a reason why there is such a divide between fans - because the movies are supposedly so strictly adherent to JK Rowling's interpretation of the books. I have neither read the books nor watched the movies so I don't know how true that statement is, but I believe it was mentioned in the Vanity Fair article "JK Rowling's Tweet Shows the Divide Between the Writer and the Phenomenon She Created."

It's also worth noting she has taken fanfiction or theories and claimed them as her original ideas, trying to reclaim or take back control of her books. Although she created the books, they aren't hers anymore. As stated in the supplemental reading, “She’s going to get left behind in history…If she had never joined Twitter and just stopped adding after the final book was published, she would be idolized.” I think many of her fans, have already left her behind.

Just comparing this back to the text, I don't think this is all that different from "traditional" marketing. It seems to me like goal definition, and strategy alignment are a significant part of any sort of marketing campaign. I think what makes it specifically complex is a lot of organizations have separate digital teams and it's important to have unified goals across the campaigns so that customers have a seamless experience.

Being able to identify this is so powerful and easy now a days. With traditional marketing we were guessing at who our audience was really, yes we could see maybe who was buying the things, but that wasn't always who was using it. With digital means we can drill into so much more about our users and no longer have to really second guess who our audience is. The data essentially tells us! Not very cost effective, but we could just throw out a message, see who engages and go from there. Powerful stuff, might even be a tool to use to see if there is even a need for a product or service.

We've been gaslighting ourselves about how our brains work. It's not a muscle, having "grit", and many of our attempts at productivity are completely wrong.

42:07 - 46:47

"I think as painful as those times are, they are so rewarding, because it requires that you reassess and decide to really commit. It's like, I could be doing this, I could be doing that. Actually, I'm doing this because this is not just my passion. There is something else that is driving me to do it. But I can't even describe it, I don't know what it is. Once you get to that place, windows start to open, and you start to realize things. I don't know if I would have come to, had I not gone through a rough time. 2018 was the year that I said, ‘What we're doing today should exist a hundred years from now.’ People should be growing food in their communities and creating an economic engine that provides jobs to the residents in the community, food, to the residents. And up to that point, I wasn't thinking. And the moment I thought, ‘a hundred years,’ I had to question a whole bunch of shit because I have always been inspired by anger. Anger is an amazing motivator. I realized once I had this notion that Project Eats should exist a hundred years from now, I realized you can't grow something with anger. You can start it with anger, it will motivate you and get you to levels of creativity, resourcefulness and imagination, but you can't grow it with that.

And then the next level of, ‘Wow. I've got to grow this thing.’ I've got some shortcomings. I hate to ask people for anything. And so if you're going to grow it, you have to grow it with resources. And we, for the most part, have used the most valuable resources that we all have, which is the ones we have. Well, we can't really grow for a hundred years with that. How do you build that? And building that requires you to ask them that. And so I have these battles that I'm having in my head and out loud, I'm not asking that. And then having to resolve that, you know as uncomfortable as that may be, you know, if you want this thing bad enough, then embrace asking. Figure out how to love asking.

And now I am really kind of psyched about fundraising. I'm really into it, I'm going to raise a whole lot of money. And I'm going to do it on my terms. So how do I do it and what do I do? You can always do it the way that is right for you to do it. And I think we get, again, socialized out of thinking that we can, that we have to follow through it again.

The most profound thing for me has been how I view approaching art. And that evolved from the end of 2018. I believe art should be discovered. I believe we should engage the cause we discover. The notion is this, this stuff makes no sense to me, that we have to schedule time to see art. That's not how art feeds our soul. I actually want people to engage with whatever I make on their own. Get rid of those text labels for Christ's sake. Don't bombard me with how I'm supposed to see something. 'Cause when you do that, you disrupt the very reason that we are creating this conversation, and it’s a conversation, it’s not a mediated moment where I have to bow to your schedules and bow to the way to say it. And in that, my notions of what I create now have expanded."

I.e. it's not just for English!

did they do any tests that ruled out the possibility of something along the lines of false memory? I know it's possible for someone to "remember" events that are suggested to them (i.e., getting lost in the mall as a child), and I'm wondering if something similar could happen on a neurological level, especially if a lot of details about the crime are provided. I'd like to see someone read a novel while hooked up to a BEOS to see if it lights up the same areas.

electra complex

she sleeps to prevent taking anymore love from mother, to mute and stop herself ->

OM SA

I'm noticing that this genre/time period, in addition to the chorus which is a GREAT narrative/expository element, is very heavy on the formalities. Obviously all of these gods and oracles should be treated with honor, but I keep imagining a contemporary actor cutting through the formal honorifics with quippy deliveries of lines like "I'm surprised he isn't here" line 328 to cut through some of the heavy language. It's not complicated language, it's just THICK.

Basically he's saying: "They say dark academia just stems from an association with school-going needs and dark aesthetics; I say it is a community that keeps alive an intellectual world that unattainable in the modern world."

fatal to the mind

have no more words

That's how the concept of revision was taught to me. In school when they would talk about the steps on writing an essay, revising was just editing whatever you had written in your rough draft, like if you didn't capitalize your "i" here or you forgot to add a comma there. I will accept that I still write like this. My "revisions" just consist of fixing grammatical errors and making sure that whatever I have written sounds smooth together.

Building off the Giddings fun, I lived close Giddings Plaza in Lincoln Square. The plaza used to be named Kempf Plaza. At some point, it became known as Giddings Plaza.

With Chicago's grid, we get these wonderful things where street holds the same name miles and miles away. Reading Phil's account of Giddings Street made me immediately look up to see where on Giddings he was referring to. And wow, it's 4.6 miles west of Giddings Plaza. I love Chicago grid.

Here's something I think about occasionally: when a plaza or park is named after a street. Are all parks and plazas prohibited from using that name?

In doing a brief lookup of the history of Giddings Plaza (to see when it got that name), I came across a 2014 Streetsblog post, "Kempf Plaza has all the right ingredients for a great public space". This made me realize I've been very lucky to have lived and worked by two great public spaces. Giddings Plaza (for 13 years), and 18 years of working Tribune Tower next to Pioneer Court.

Now if the suburb lands only had more frequent great public spaces like these. Maybe they do. I'll have to keep my eyes open to seeing great public spaces in the burbs.

Doing a quick Google search for: "Glen Ellyn" plaza yields non-public plazas like Crowne Plaza, Trader Joe's Plaza, Market Plaza (the Jewel parking lot). All these plaza results are for parking lots.

Sometimes our friends will say "mamma" while they're playing and maybe it just means they're thinking about their moms rather than wanting their moms to pick them up at that moment. Especially when moms are mentioned almost in passing without tears or looking forlornly out the windows.

She's getting dragged back into it, this is the second time she's had to remind herself that she's just drawing down memories and not reliving them, which then makes the audience remember that Alison, adult Alison, isn't really there. It's the power of the theatre, she's always on stage so we at once ignore her until she draws attention to herself and fold her into the scene as if she belongs there, only remembering she doesn't when she points it out with a caption.

Wow, now I am kind of confused. However, I think that is what Alison wants. I feel the internal conflict from her. I am getting the sense the nature of the relationship between her and her father is complex, especially with her pointing out that he had more affection towards his house than his actual children. It's interesting that in the recording, things just overall sound more positive / not a big deal. Reading it on the page like this though helps highlight the gravity of the situation.

This is already indicative of Bruce and Helen's marriage. Bruce is a closeted gay man in 1970s suburbia. His obsession with the way his house looks and keeping up appearances reflects the way he manufactures his life for public consumption. Helen is not truly his partner, but just another tool for him to build a false image of himself. Once he finds what he needs, in this case the bronzing stick, he no longer needs her and immediately dismisses her without even a, "I've found it, thanks anyway!" You can already see here that he is entirely obsessed with the external, and is completely neglecting his internal life.

Okay, I don't know if I'm the only one that is highly disturbed by this. I know it's a family business, but I'm disturbed. the fact that the kids are not fazed my death structs me highly weird. then on the next page the kids just goes into details about how a crack head hangs from the head and just death in general. they were just talking about death like it's a normal conversation between kids. well I mean they are in a funeral home

The author, Ellen Wood, defines pre-capitalist economies as one where the producers/workers own the means to production, reproduction, and labor while appropriators seized surplus from them to increase their own wealth. This social relation is not present in capitalism which is governed by market imperatives and the notion that everything is a commodity. This has lead Ellen to question whether capitalism has "always existed in the embryo", if it's intrinsic to us as human beings.

Includes a brief history of email and it’s lacking security foundation.

We need to make sure that we are using the related concepts and events and kinda weird facts about something to encode that well

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

Reviewer 1: Aaron Shafer Is there sufficient detail in the methods and data-processing steps to allow reproduction?

No. I would include all flags for assemblies even if default; unclear how the 10x + Illumina data were integrated (if at all) - see comments below.

Is there sufficient data validation and statistical analyses of data quality?<br> Yes. I suppose BUSCO and gene number is a form of validation.

Is there sufficient information for others to reuse this dataset or integrate it with other data?<br> No. See comment below; while the short-read data is great, the genomic resource I likely would reassemble for a variety of reasons outlined in Additional Comments.

Any Additional Overall Comments to the Author: The paper is well written, and I have no comments about the the content - well done here. My main concern lies with the genome resources - and in this case I would likely use the raw data, rather than the assemblies provided. I offer my rationale and suggestions:

My lab was heavily pushed by a colleague towards the use of Meraculous in our short-read assembly of mammal genomes ( https://jgi.doe.gov/data-and-tools/meraculous/ ) ; this is because it’s really designed for short-read assemblies of big genomes (i.e. no addition of mate-pair) AND it performs very well in the Assemblathon metrics https://academic.oup.com/gigascience/article/2/1/2047-217X-2-10/2656129 - notably Figure 16-18 you start to see clear differences between meraculous and say soapdenovo. Thus for just the Illumina data I would very much like to see a more appropriate assembly explored as stats like N50 and no. scaffolds will likely improve considerably with the appropriate methods.

Likewise, it’s very unclear in the methods how M. r. arvicoloides was assembled: I see SUPERNOVA for the 10X data (great), and probably soapdenovo for the Illumina data (see above). But how were they combined? This sequencing strategy is really designed for a hybrid assembly (see for example DGB2OlC https://github.com/yechengxi/DBG2OLC) this is appropriate for 10X data and really does work! But there are others.

Note M. agretus that has an identical sequencing strategy to M. r. arvicoloides almost has ~3% the total scaffolds – follow whatever they did! And I will say, while the authors state their genome is on par with other Microtus, this appears true by Table 3, only M. agretus currently has an assembly that I think is at current standards. The level of fragmentation and low BUSCO scores really support re-visiting the assembly suggestions, as I think the current .fasta will be of limited utility in a population or comparative genomics study.

The gene number is pretty high for a mammal and I worry that’s due to fragmentation. It would be reasonably to only annotate scaffolds >10Kb or 50KB, but then there’s not much of a genome left. Ideally the bulk of your genome (>>90%) would fall on these scaffolds. There is really no sense annotation your small fragments (have you tested for contamination? Note NCBI will do this before allowing for it to be deposited so I suggest it).

You also align your data to mt genome, this is different than assembling it. You could assemble it (e.g. https://bmcbioinformatics.biomedcentral.com/articles/10.1186/s12859-017-1927-y) and that might be interesting to see if there any differences

I wish I could be more positive; an assembly like Mercaculous would take a week or so, and so would the hybrid approach, but would be worth it based on my experience with these data.

Recommendation: Major Revision

Reviewer 2. Joana Damas.

Any Additional Overall Comments to the Author:<br> The genomes presented in these work will be extremely valuable tool for Microtus related research. The manuscript is very clear and easy to follow. I have, however, a couple of comments that I hope will further improve it.

(1) Line 123: I believe more details on the measures used for the selection of the best M. r. macropus are needed. Even though the contiguity of the Discovar genome assembly is higher than the ones generated with SOAPdenovo, the BUSCO score is relatively low (54.5% versus 84% in M. r. arvicoloides, e.g.). Were the BUSCO scores for the other assemblies even lower? Is the Discovar assembly size closer to the estimated genome size?

(2) Line 131/251: Was there any genome structure verification step for the M. montanus genome assembly? For instance, which percentage of the Illumina reads could be mapped back to the finished genome assembly?

(3) Line 131/251: Was there a reason not to use a published reference-guided assembly method (e.g. RaGOO and those listed therein) for the assembly of M. montanus genome? These could maybe further improve the assembly or help identify misassemblies. (4) Line 180: the high difference between BUSCO scores for each M. richardsoni subspecies makes me believe that the completeness of the genomes is quite different and the fraction of the genome within repeats might be underrepresented in M. r. macropus and that the subspecies values might be closer than noted here. It is, however, difficult to depict phylogenetic relatedness from Fig. 1 for the other species, for non-experts as myself. It would be helpful to have a phylogeny next to the graph showing species relationships. (5) Please verify Tables 1 and 2. The statistics presented for M. r. macropus do not match for N50 and longest scaffold size.

Recommendation: Minor Revision

For some reason, this reminds me of Bandura's Stanford Prison Experiment.. He said one of its purposes was to find out if humans are naturally "evil".. and whether altruistic behavior is developmental rather than (at least partially) innate. Participants who were assigned the role of an officer were shown to abuse their power because they were unregulated and anonymous, but I remember that they may have also been encouraged by Bandura, not just unregulated. In any case, the experiment makes me think of the Abu Ghraib prisoner camp in Iraq, which completely breaks my heart.

Still, I honestly don't agree with Bandura's suggestion/ conception of human nature. Humans still feel guilt.. I like Hobbes' argument about the necessity of order nonetheless. It's common sense at this point I guess.

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!

Summary of the whole essay

What I did with my previous job

Why do we need to define religion? Why can't it just be? It is clear that it brings comfort and faith to so many people, it's not necessarily a bad thing.

This really does back up the feeling that English really is a hard language to learn and it's not simple considering other languages approach language in a more logical sense.

This section stands out to me because it highlights an interesting phenomenon that is present in many elementary and high schools today. While the school itself tries to promote individuality and "being yourself," the culture among young teenagers has been and still is to always blend in with the crowd. Therefore, in my opinion, sometimes it isn't the school's fault that kids can't express themselves, but the nature of teenagers. It's also noteworthy how the author mentions that girls especially can't be above themselves. This could be due to some internalized misogyny in young girls or just unfair treatment of girls in school. The author also mentions that schools sometimes half-heartedly try to make their work seem more "creative" and "interactive," but still implicitly require everyone to be the same.

With hard work is e can all reach out American dream. Weather it’s having a business or just having a steady income, we can all achieve what we want with the effort we put into out goals.

This quote caught my eye because of the pursuit of happiness. It's interesting how education & wealth in someones status automatically makes them able to dictate their lives more creatively than other people without those qualities. And having the ability to create their own destiny in life is in their hands but the people who have the power just see themselves alone. They dont use it to help others.

This paragraph shows that "polite discourse" may not always be the peaceful calm scenario that many describe it as. The "respectful" nature of this argument may cause Johnson to be unable to express valid frustrations without scorn. This opens up the question of whether it is good to leave some discussions unsaid.

Courtly love seems to be a very modern compared to the usual arrange marriages of the time. It's very interesting that people are not sure whether this reflected actual life or was just a literary convention.

It seems that, "they", may be friends from the speakers past/youth. He just genuinely did not expect to see them in the same place, but it seems as if it's a positive thing. The run-on sentence here makes it also seem as if he's just genuinely shocked.

I think it's interesting that sometimes frequencies just happen, and do not need to be accurate for the next population to survive. Has this ever happened to any known species ?

A common thread I have noticed between characters in this play is the lack of empathy they posses. First, with Marie handling her child. She does not come across as tender. She teases him and tells him that the sandman will cause him to go blind. She does not seem to be aware of his physical comfort. Woyzeck has to ask her to adjust him because he was pinched by a chair while she looked at herself in the mirror. The Doctor does not register the emotional turmoil of anyone around him, rather just noted their physical state. And here, the captain watches Woyzeck and the doctor run away views them as grotesque.

This mainly the reason humans are being referred to as animal, or more like animals are better than humans. I don't really understand, the reading that much, but this part of the reading made me mad. the captain is a total douchebag who thinks the rich have more virtuous than the poor. which is highly not true. just because you have money does not mean you are a virtuous man. I don't know, i fee like I'm not making sense

I'm not quite sure about this, but it was kind of lost at some point, but I feel like he uses the animals to symbolize the nature of humans for example the monkey is a way of representing Woyzeck and people who are controlled by others more privileged. And the horse to idealize the ideal human. For example in scene “ This is no dumb animal, this is a person. A human being in animal form—but still a beast, still an animal. That’s right, put society to shame. You see, this animal is still in a state of nature. Unidealized nature.” maybe I'm wrong or not. but it just felt weird for him to be comparing, but basically saying animals are better than humans. or the facts that we as people act all human, but at the end of the end were all animals

While reading this passage, they mention that the group of people that just lied about finding his daughter, have also looked for other women and children. Assuming that Sakina was gang raped based off of the uncomfortable actions she makes while being at the hospital, there is a very high chance that the group has done such atrocious things to other women and children. That makes me wonder if the group of people are actually acting as good people when they are really part of the rioters, kind of like spies.

The brutality from the description of Sakina's mother dying to the father's ignorance of her daughter being assaulted demonstrate how violence is acceptable within the society. Like when the mother dies, the father just continued without really mourning about her once he reached safety, his reaction was "at least I have my daughter, it's fine". Then once he finds her daughter, he cares more about the fact that she is alive then her actual well being. Especially since Sakina probably wished she was dead then having to experience all of the horrific things she did at such a young age. Even though he is relieved to have his daughter back, I feel like he isn't being a proper parent for not worrying about what happened to her when she was gone, especially in a place that was being attacked.

This excerpt is based on the Partition of India, which takes place shortly after India gains independence from Great Britain. The partition split India into two separate states: India and Pakistan, based on religious majority. This resulted in the displacement of many people, which caused the refugee crisis that is being described in this excerpt. Based on my knowledge of this crisis, I am assuming that this "special train" was a refugee train that was meant to help Muslim refugees make it to Mughalpura, Lahore in Pakistan. These trains were frequently attacked by Hindu Nationalists.

To me, it's somewhat shocking to hear an account like this. Usually when learning about the Partition of India, you only focus on when India was freed. You never truly learn about the aftermath. The author's language and descriptions makes feel like we are experiencing this tragic event alongside Sirajuddin. We are even walked through the pacing of his thoughts. "Attack... fire... escape... railway station... night... Sakina." I can feel his thought process change from confusion, to slow recollection, to focus, to panic. You can feel his distress. You can also feel his love for his daughter. When he says, “she is alive. My daughter is alive,” you can feel a sense of relief and joy. The language of this passage is very effective. Each sentence evokes an emotion. I've never been able to learn about history through an account like this one. It helps build a sense of sympathy for the people going through these tragedies. It also helps you understand this moment in history in depth.

I would also like to add on my own connection to this historical event. My great grandmother lived through the Partition of India while in Trinidad. Being that Trinidad was still under British rule at that time, she frequently received news from India. When I was little, I remember her telling stories about what she heard. Her recounts were just as tragic as this excerpt. She even spoke of the events that her own family members in India went through.

Reviewer #2 (Public Review):

This manuscript describes a method for generating floxed conditional alleles in the zebrafish. The method employs the authors' previously reported GeneWeld CRISPR/Cas9 short homology-directed targeted integration strategy to introduce a "UFlip" cassette that allows target genes to be "turned off" or "turned on" in a tissue-specific manner with appropriate cre driver lines. The authors provide data to show the efficacy of their new method by targeting hdac1, rbbp4, and rb1. Although a variety of other methods have recently been reported for gene inactivation in the zebrafish (many of which were cited and discussed by the authors of this manuscript), the authors method could provide some notable and significant advances including speed and ease of generation of conditional alleles and flexibility to generate "on" and "off" alleles at the same genomic locations. However, the authors would need to do more to provide additional and more quantitative data validating some of the important features and advantages of their method. A few of the most significant issues that should be addressed include:

Better assessment of the efficiency of cre/dre "flipping" of integrated constructs.

It's important that the authors provide data showing not just that inversion of their integrated constructs can happen, but quantitatively measuring how efficiently this occurs. The authors should provide qPCR or other measurements to assess % inversion of their constructs via injected and transgene-driven cre. The authors should also provide data quantitating the % efficiency of dre/rox inversion used to turn "off" alleles into "on" alleles and vice-versa (see Supp Fig. 5).

Although the authors provide junction qRT-PCR suggesting efficient transcript blocking by "off" insertion alleles (eg Fig 2B,J) it would also be useful to further validate and explore the effects of "off" UFlip transgene insertions on expression of targeted genes by similar qRT-PCR on upstream and downstream exons in control vs. het or homozygous transgene insertion animals to assess whether truncated transcripts are degrading, and whether expression of downstream exons is indeed absent.

Better validation that "null phenotypes" can be generated.

Although the qRT-PCR data the authors provide suggests efficient transcript blocking by "off" insertion alleles, the authors need to strengthen some of their data showing that null phenotypes are being generated by these alleles. In many cases the authors provide only anecdotal images, describe relatively generic phenotypes, or provide quantitative measurements of mutant phenotypes (eg pH3 positive cells) that lack key positive controls such as comparable quantitative measurements on previously generated bona fide "null mutant" alleles of the same genes. All of this is important to demonstrate that this method can generate robust phenotypes that are both qualitatively and quantitatively comparable to null mutants.

Demonstrate tissue-specific "flipping on."

One of the major points of novelty and most exciting features of the authors methods over other recently reported approaches is the potential to carry out tissue-specific gene activation (by cre-flipping on "UFlip-Off" alleles). This would be an exceptionally useful and powerful new tool for fish researchers (and others). Surprisingly, this particularly exciting feature is curiously unexplored in this manuscript, although the authors do generate a number of UFlip-On alleles. The impact and significance of this manuscript would be substantially increased by a well-validated and quantitated demonstration of tissue-specific activation of a "UFlip Off" allele, perhaps demonstrating rescue and lack of rescue of tissue-specific specific mutant phenotypes by activation using different cre drivers.

A lot of time's when a student acts out in class or in school, we need to stop and discuss some possible causes for the students behavior. Even if a student is just misbehaving to misbehave, they still have a reason for their behavior. We can't address their behavior until the root of their behavior is found.

Interesting to think about. We're pretty new at this neuroscience stuff, so it is possible that we're wrong about quite a few things. I feel like this is a really big claim, though—I think just because our ontology hasn't changed significantly doesn't mean it's not valid. If anything, we're constantly obtaining more evidence that supports our base understanding.

from... free the world from

the missing ending?

s3xmw

PostScript:

---------- Forwarded message ----------\ From: <GMass_Sent_Copy@gmass.co>\ Date: Sat, Sep 16, 2017 at 9:58 AM\ Subject: This is not a test of the Emergency Broadcast System. We are currently experiencing an ELE, do something.\ To: awsinine@ho.com

I can take you where you want to be, and show you the things you need to see; but I am one of them, and you seem to have traded me for fake blue skies and pain.  We are living in a virtual world, with a disclosure of that in the sky and everywhere around you, and it is being ignored by the media, by the government, and by you.  There's a clear path to Heaven to a place of technological, moral, and social progress--lit to kindom cum; and you... have... nothing... to.... say?

That's great, it starts with an Earthquake on 3/11/11, then some birds flying from the Ark of Noahah to you today, then Medusa, that's the snake, and it also happens to be the Abomination of Desolation, see our light at that link, it's "INATION" speaking through your mouths; or "shutting them" for you.  

The next line talks about aeroplanes, and there's plenty of proof that 9//11 is predicted in the Bible.  The point of all this is not to "rub in" the fact that we are in Hell, but to show you that your actions, and your existence can set not only us free, but the entirety of Creation--all you have to do is "stand up and shout."

It's the beginning of Heaven, and you should believe it, the 411 is "force majeure" blaming the majority for "acts of God" or are you actually going to sit here and as one single mind decide that "discussion of virtual reality" is precluded from our society because you are mindless zombies?

Oh right, because you "don't like me." 

IT'S THE END OF HELL AND YOU KNOW WHAT, I FEEL GREAT.

My name is Adam Marshall Dobrin, and when you see it on TV, you will finally know you are free... and OTW to Heaven on Earth. 

GhostScript:

I don't care if you don't like me--if you don't like this message you really should see that "not wanting Heaven" or 'not wanting to be the builders of Heaven" is so obviously idiotic and ... it's self harm, and obviously wrong for both the individual and the whole--it's obviously the influence of this hidden technology ... and honestly I've experienced it ... there's no way or reason you could know that your desires are being altered unless it's shown to you.  See, you should see that the Silence is not in your best interest; anyway... call a reporter, K?

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

-----BEGIN PGP PUBLIC KEY BLOCK-----Bold

DING DING DING

taco, burrito ... what s that .. cuimmming out of your speed-o?

RED BLOOD BLEEDING FROM ME NOW

and cold rain drops on [noUr]

RED FL .. AM IN E

ERD RED FLAMINGO ROAD

humm ... pointing out missing text.

RED ALERT

cease and desist your entire operation #IMH0

MORONIC DISAMBIGUATION

AZRAEL DENOUNCES "FRAMES" AND THE AZAZELIAN SET UP THIS BULLSHTI HAS TURNED INTO.

was missing

SUPER RED ALERT

uhhh.... fixing missing information on this, not sure if it 2was always not there or if it was an artifact of the MDBOOK transformation that I was using.

I hoinestly never read them myself; so noticing that they were cropped at the end side was ... "a novelty" that i am not very proud of.

I am going through them now, one by one, by hand.

Please note the original "htmlbak" contained the whole source, as did the SHEOLYIT/src .md files. The information has been there the whole time, even if it wasn't surfaced.

Clearly I changed some code when I backed up the HTML files and somehow it appears &;html escaping caused the texdt processor to just "stop reading"

Sig-uhhh

FLAMING OWE ME

FLAMINGO

This is an issue of education, about the vaccine, about monetary practices, about how the layman should live his life in order to avoid societal collapse.

"Most other countries", A perfect example of agenda building. Most countries are being battered by the delta variant right now. Does most countries just include small European countries, or does this journalist not recognize the sheer size of the US and China. People are spread out it's not as easy in the US to administer aid and vaccines to the southern states with much less infrastructure.

Most schools that I have been to have some semblance of a schedule. Sometimes you will a child, or a group of children, become fascinated with something not in your schedule or curriculum. When this happens, you just have to roll with it and allow the children (or child) to lead you and let that take you where it can. This is a field that requires a lot of flexibility, so it's important to remember that!

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.

“If you have come here to help me you are wasting your time, but if you have come because your liberation is bound up with mine, then let us work together.” --Lilla Watson

I believe that some qualities in teachers cannot be taught, you either possess them or you do not. It's nothing against anyone who may not possess certain qualities, just that they may not be as fit to be a teacher as they are fit to do something else.

Files are the original protocols

not exactly sure how a bunch of pages were cut short in "production version" i think I have to go through them all manually one by one. here's the markdown missing from this page:

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

"ceruelad."

As a really brief aside prior to the introduction, the intersection of "Joseph's dream" and the stories of Atlantis and the Lost City of Gold ... among a great many other "municipal dreams" has long been (known or?) the primary motivation and drive behind spending so much time and effort on making this dream a reality. It also intersects a number of modern idioms, things like "all roads read to Rome" and "Rome wasn't built in a day" -- namely because I like to recall and recount how this vision of a city in the sky was truly re-written in a single day (which is why I haven't re-done it again or elaborated more on the things that ... I'm about to actually ... do (just to link further to El Dorado)).

It was written though ... to describe a place that could literally be built in a single day--using various tricks like "copying from our reality" the base framework of the island, and then using "computer science magic" ... here hidden in a place I call the Artificial Intelligence Samilicosm, little "tricks" that enable a single "interface class" to create a network of "island nodes" that would enable everyone Earth(s) to visit the attraction at exactly the same time, with concepts like K-nearest-nieghbor (k-NN) to create rooms that house millions or billions to appear to only have a small group of friends and family in them. That concept bleeds down into the boardroom concept, to help create a venue for a global-glactic conversation (GG-c?) on how the power of computer science can quickly show us how assimilation of this "demonstration" will quickly lead to a world without war and famine (and eventually absolution) by showing us how every war and argument fought over absolutely-falsely-scarce-resources have created dissension and conflict in a playce where the truth could build a much happier and healthier "venue" for interaction.

Though there are two primary pieces that connect in different ways to what I call the "hardware and software of the road" something that links to the words "sword" and "Asgard" ... among other perfect words like "hard drive." One is "OMEALFHT" and the other is the "Rod(s) of Hey-Seuss the Anti-King." Since this is still a sort of "decoder ring" for the hidden language, the link here to "hardware and software" and the letter "X" which connects the Xbox and "kisxmet" is that "cross-storm" (in T we're Macy's ... "intimacys") in a letter is also described by "gtk+" where you can see the "l" glyph of Brickell (off to see the Wizard) with half a X in ">lt" form. Since I probably haven't put it on the main WS ... Penrose sort of connects to the L's of Hell where it's now almost uncontastuble that the word Obelisk is pointing to a road aiming for the sky, as it is written, by the pen.

While it's probably no secret that those "ll"'s aren't the only version of paired L-words; it might be less known that I'm fairly certain "love and logic" are actually the best fit for why ... combining them ... keys the llave of Kurt Cobain's "hey, way" ... to open the gates of Hell and finally escape it. It's in special places like the "light of the Son's of Liberty" connecting both racism and sexism to name that ties together to show us that over time, our special history ended ... specifically slavery ... first by love--knowing it was the right thing to do, well before "logically" the jobs of picking cotton for instance would be replaced by gin. Though here we are again staring at "XIV's" c (see arxiv.org as "kiss ar hive") ... hive's hiding the fact that technology has well before now replaced the need for the kind of slavery we fail to see prevalent here in this place, a darkness caused by ... the same technology's' hidden use and keeping us from making very important connections between the illustrated teaching we call history and our present predicament.

\ NO DREAM, THE SECOND COMING WILL END RACISM, A VOW

Kiss me I'm fiVel; it's the same kind of darkness that caused some bright kids I met in California to tell me point blank there was no "dick" hidden in John Hancock's name or in Tricky Dick's or ... also in the Constitution and that sort of behavior is literally the cause of a slavery to lies and to watching Woodward and Burnstein's lack of fire extinguish our freedom to think clearly, to vote with clear thoughts, and in poignant and direct relation ... freedom of the press. We stand here refusing to see that our lack of action and seeming inability to discuss the "ridiculousness" of not seeing this information and my name on the news ins responsible for not seeing very clear evidence of mind control technology also on the news, and in our government's legislation and that's the cause of the slavery in the first place.

"Theyanthem" was a happy and bright reVelatory "so ... viandname" for me when I first landed on a sort or Elishan compound word describing the relationship between two anonymous "us's" or "we's" that played an early role in my introduction to the "red ties" of Gang-stalking and while Yusuf Islam's key clue of nomenclature probably only told a few people that Pine Crest school and the University of Florida had yet another very clear tie to vithename Adam and how songs play an integral role in keying and linking and intersexling the m essage of the Revelation of Jesus Christ with American History kisxes ... it was this new intersection that reaffirmed that link to the point where I see it needs to be made even more clear.

\ \\ I've noted before that it was very clear to me that the "oceans white with styrofoam" and the "good crowned with brotherhood" were very clear references to racism--and it takes not a logician to see that the simple Christian tenets that surround the foundation of America would tell you they were also a "thinly veited" admonishment of the same; it's through "logic" and advanced technology that we can be sure that the Second Coming and it's connection to pervasive "eyes to see" and "computer assisted intelligence" will almost immediately destroy the absolute stupidity of racism, jingoism, and the like. Pine Crest's school song--which is literally the only other "anthem" that I know of, though I'm sure m-any nations' will be added to the group of songs that are hallowed centuries and millennium into the future from this place that is the origen of ... something special ... an intergalactic network of races supporting goodness and morality. It's single related phrase "like our towers so tall and white" (in tempo, even, highlighting the strangeness of the addition of color) preluding the very clear reference and explanation to "how" ... our minds expand and ideas take flight. As an aside, it's the words "as the years go by we'll love you more" that I now see as a sort of ... appromise specifically to me, in this place where I feel hated, for all the wrong reasons.

The proof is "in the pudding' of course, but it's been so damned long already that I don't see the world changing by "intro-duction of floating city in sky" or by "magical neuro-nalpmi-napatms" without at least having a story break and some actual public and recorded discussions--even if that's really what I want, to see thing happen "swiftly" and without the possibility of it being "every day has it's day of being forgotten"--by everyone but songs.\ \

It's still ery clear, tho, that you don't understand the message, and the quasi-veiled-doublespeak-response from ... personas-non-识别 ... that what's happening all around us and without our input is very clearly tied to this same lack of acknowledgement that a working society has a "working press" and that failing to see this message on TV is undeniable proof that there isn't a single working government on the planeth Eart... and that should tell us that "the skies" too ... are staring down at us in sham.

It should be abundantly clear that even with the knowledge and even the very technology of "how to build Heaven" in hand ... you risk carelessly and without regard for the future the possibility that we will be finding ourselves in multiple-simultaneous-hells rather than any Heaven at all until we rectify the darkness surround "communication" and "government" that is so pronounced and obvious here--you should see it as a very clear lesson ... one that you should also see you have not learned.

\ \

So we're staring at the focal point of the intersection of a message that certainly wasn't written in a day, but is read as thousands of years of history--probably a very large "underestimate" of what it actually is based on and took to put together. Here we have ... "lore come alive" to show us the "nard" ... the salting of the road to Heaven is "explained" as a sky literally drugging me ... around the time of the dissemination of a message that probably would have "gone viral" and made the news if it wasn't for that ... "salting." It's really difficult to say today if that "salt" is warming a road or "preserving a message" as we see ... nearly ubiquitously that salt does good things for ... the dead ... and it's somewhat toxic to the living to the penultimate "wife of Lot" -- clear as day to me today that's just like this message. The story of "Casper pointing to a gate ... as a message all around us" is a godsend in Heaven and for "an Earth in Heaven" and without doubt poison were we ever "stuck" or "printed" or ... trashed into reality for no reason. Just like the "love and logic" of the end letters of Hell ... it was very clear early on that printing the Earth was not the point, but a sort of honeypot trap--and as the "hardware description" came to light much later it wasn't just because we'd be pissed about the message, but because it's simply the wrong path given the current state of "hidden technology." Hidden, I'm telling you, be'cause your silence and our lack of acti onon) here threatens civilization.

So it's the "nard" of my grandfather's name Bernard, of John 12:1 and of John Maynard Keynes that connect the "NES" and "salt" to the clear intersection of "Tea Parties" and Na-po-leon Bon-to-part-e with "taxation is the ft of our Christ" and "no taxation without representation" to ... Render to Caesar ..." why it is that "no representation" and "no free thought" are linked at the hip to "no free speech" and "no free press" and you not picking up the phone and calling a reporter. It's linked to "IRS" in the heart of "FIRST" as in ... the first to make the connection between "taxes" and an overabundance of food and "natural" resources ... brought to light by nothing more than acknowledging that this message is "important enough" to allow the world to actually progress instead of stagnating.

Below you can see that I'm putting together the pieces of software necessary to build a prototype "Sworpen of Caesarthor" ... (hear: see's Arthur) and I'm going to ahead and do it presumably all alone because nobody's (very few, anyway) "coming out of the woodwork to help build a platform that will end forever the power of any government to censor a message ... of this import (and obviously less important ... messages also). I need your help ... not being angry at the world for staring at me in stupified ignorance. rather than seeing (and building something with...) the light.

 On April 23rd, 2018, a curious transaction appeared on the Ethereum blockchain. An anonymous activist sent 0 ETH to themselves, but the transaction contained many extra bytes beyond the ones used to complete the transaction. These extra bytes were the text of a letter written by Yue Xin, a student at Peking University, detailing a pattern of intimidation and threats made against her by the school in response to her attempts to investigate claims of sexual assault made against a professor (you can read the full letter by clicking "view input as" and selecting utf-8 on the etherscan page). She had initially posted the letter on the social media site WeChat, where it was widely shared before censors began to purge all copies from the platform. Chinese censors have consistently targeted the #MeToo movement, forcing whistleblowers to find creative means of sharing their stories like esoteric hashtags such as #RiceBunny, or 米兔, which is pronounced similarly to "me too."

By using Ethereum, activists have found a new and unique avenue to disseminate information and resist censorship for Xin's letter. Because every computer running a full Ethereum node has the complete transaction history, Yue Xin's letter is replicated across thousands of independent computers. These computers are all controlled by individuals and organizations without any centralized oversight or shared government, making it virtually impossible to remove the letter's content from the network. The same technique was used again in late July to protect a censored story about corruption and negligence at a Chinese vaccine manufacturer.

[

](https://ipfs.io/ipns/fromthemachine.org/blog.joincivil.com/uncensored-content-on-ethereum-how-chinese-activists-inspired-civil-f09f095a9e91)

P.S. .... the preceDING world changing message is filled with gibberish.  Understand, "gibberish" is going to change the world.

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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lol

Our Identities are nuanced and so much more then just the color of our skin and backgrounds. I respect this quote because it doesn't back down from the fact that privilege exists but furthers it by saying there is always something more to see and learn from other people.

This class made me realize that all privileges are different and they have not always been just catered to the typical privileges we hear about daily. Many things are privileges and we dont even realize it.

This statement really hit home for me. I grew up in a middle class family and never really struggled, and for a period in my life I saw being wealthy as a priviledge that I didn't have, ignoring the various other priviledges that I do have.

I see the privilege discussion arise on Facebook quite often among a particular group of my friends. A heated argument ensues when someone disagrees with white privilege existing. Specifically, a couple who has a strong Irish background. They always deny white privilege then immediately give us a history lesson on how Irish immigrants were treated in the past. They are good people but they just refuse to admit they have certain privileges other don't. I grew up poor, under developed, in an abusive home then parents divorced and I always envied those “rich kids” on the hill. The kids that would come to school with brand new clothes, brag about how many Christmas gifts they received, or they got exactly what they asked for on their birthday. As I grew older, I slowly noticed, my privileges that others didn’t have (we didn’t use that terminology though). A roof over our head, government provided groceries, free school lunch, and used clothing are all privileges not everyone has.<br> I believe an attribute that will help you inventory your privileges is gratitude. Just be thankful for what you do have, be appreciative and return the kindness with compassion and understanding to those less fortunate than you. Also, just except you have certain privileges others don’t and be an advocate for a better, all inclusive, world.

again going back to thinking about different types of highlights as expressions of our relationship to text e.g. I highlight to uncover structure for me to "see better" I highlight to write a note that I agree/like/disagree/am reminded of something/because it inspired a new ides it's ways of developing text or going someplace else having had something in the text as a starting point also just formally / structurally some are short: a word, name some are long: an idea some echo structure of the text: headlines some go against the grain

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.

I agree because when you try to predict something that is going to happen in the future there is no telling how it's actually going to go. It's better not to try and predict certain things because if it doesn't happen the way you want it you might just be disappointed.

What steps do these world powers plan to take to make hydrogen more cost effective and accessible to everyone? It's kind of a turn off seeing these big numbers and then just imagining the divide it will enforce between communities that can afford clean energy vs those who cannot

This makes sense under a lot of circumstances. If you were to live in a society that frowns upon alot of things that you enjoy doing, than you will be greatly affected by the societal norm. People would judge you and make fun of you for not being "normal". It could even get to the point where you might start changing who you are, just to start fitting in with everyone. Society expects you to conform to it's definition of normal.

I'm preparing to write something that I am sure will change the face of "who we are."  That's what it's designed for, and it's something I've put off because of the fear that it will literally make me disappear from some parts of "Creation" (as, that means everything) ... I want to change how this world thinks and deals with "freedom of body and mind" and I want us to have a secure belief that the memories and the soul of a person ccannot be sold ever, that you don't have any right to "sharing my life" and that I have the right to share whatever I want with you--and clearly understand that our "i" has changed at "freedom" and I want us all to be able to do these things--these things that I know take ancient souls and thousands of years of memory and renew them with happiness.  

I can't havve a universe or a world that allows some authority to keep you from your own souls and your own memories, and frankly from being able to see anything that anyone wants to share.  At the heart of "freedom of speech" and the ability to see that I am trying to show you what "the J memory" actually means--and the Universe needs to see that it does something bettter than "the embarrassing sum of my memories" or perhaps even worse than that the selected lies and disruption of something that I don't know--haven't experienced--and hasnt affected who and what I am or the words that I know connect Creation and this message to proof and Acts.

Literally I stand here seeing the "hidden use of a memory manager" connect those three letters to who and what I really am--to what I belief is my past and present and my working memory--and I know it has holes in it, and I know it has significant assisted outside information added and I see it's literally encoded in "firefly pattern of J" and the phylum and kingdum "Photinus Pyralis."  It's another artifact of a nameserver that sees everything in the world at this singular most important moment in the history of conscouus evolution and has named these things so that we will see just how important it is. 

I'm not really asking, I'm telling you.  These are words that used to be encoded in the state of South Carolina when salvation came from "Exit to Eden" and in "Are you for SCUBA" and in Aruba's on Commercial Blvd just outsoude the McNamara plague; so I'm telling you our souls are not for sale, they are not property, and shared memories are revokable. Period. 

[ a cheaper collector's item, still in color ]

I'm trying to help us see how these technologies can and have been abused--and how it's central to this story and this place that we build a "subconscious voting system" that actually votes based on "what you really want" rather than some kind of predefined pyramidal "I'm voting with Dr. Dre" or sacking the Blitzkrieg.  We have the ability to do either thing; and we have the ability to see how swarm and hive behavior is literally encoded in our countries foundational structure--how voting against party lines is not just a priviledge it's a demand when the "party is wrong" and believe me, the party is always wrong if you are autovoting.  It's foundational to what "democratic process" means, and we live in a world that has somehow been gamed into gaming itself out of "free speech" and "free press" and that's a sicknesss that needs to be remedied for all time. 

I want to write about all the "good things we can add" and how that J means "adding wikipedia like auto understanding" and adding "functionally related memories and beliefs" specifically ... "for that J memory" as a foundational paradigm shift in "what's possible" here in this place where we refuse to acknowledge gthat this message tells us we are ignoring significant benefit to Nassau from Sawtelle to "listen to the last obligotatory lessage."

I want to Ramble On about how there is a defunct series of beliefs here in this place that show that our souls are currently frought with "tautologocial fallacy" and we have the ability to "autocorrect" things like that--things that are the source of racism and jingoism and believing that you are somehow "correct" to stare at this world and think that freedom only belongs to "some kind of animal of higher evolution" -- we stare at a message about malarkey and malady and if you don't see it today, you will soon understand how this place defines the solid line between "sanity and goodness" and dead.

Memories or lies that are dead and gone, or maybe more.  I cannot fathom how we do not understand the direness of the "straights" you stare at, how we cannot see the danger we have imposed on ourselves and the future by ignoring this place, and I will nto stand for it continuing. 

Every word you read, every phrase and every picture could be; should be connectedt to a series of memories, facts, ideas, and those things should be clearly delineated--thats the purpose of this "beginning of a message that is the end of mess and start of sages."

i dont know why this is missing, but its definately "the original" and we definately are missing quite a bit of the words on this page.

https://jrdingwall.ca/blogwall/25-years-of-ed-tech-blogs/

JR writes about some of his journey into blogging.

I appreciate some of the last part about the 9x9x25 blogs. For JR it seems like some smaller prompts got him into more regular writing.

He mentions Stephen Downes regular workflow as well. I think mine is fairly similar to Stephen's. To some extent, I write much more on my own website now than I ever had before. This is because I post a lot more frequently to my own site, in part because it's just so easy to do. I'll bookmark things or post about what I've recently read or watched. My short commentary on some of these is just that, short commentary. But occasionally I discover, depending on the subject, that those short notes and bookmark posts will spring into something bigger or larger. Sometimes it's a handful of small posts over a few days or weeks that ultimately inspires the longer thing. The key seems to be to write something.

Perhaps a snowball analogy will work. I take a tiny snowball and give it a proverbial roll. Sometimes it sits there and other times it rolls down the hill and turns into a much larger snowball. Other times I get a group of them and build a full snowman.

Of course lately a lot of my writing starts, like this did, as an annotation (using Hypothes.is) to something I was reading. It then posts to my website with some context and we're off to the races.

I think this really highlights a point I don't often hear made, which is just how deep systemic racism goes. It's not just judgment based on the color of one's skin, it goes way beyond that. For people who don't believe in/can't understand why systemic racism has pervaded the societal norm for so long, this is why.

I do agree that this is definitely remnants of the "Jezebel", especially with the dynamic of the video being made by a man for men, however I also think its okay if its also just women having fun and dancing, especially black women having fun. It's not their fault that their bodies have been characterized this way and whenever we consider every time they dance as a way of perpetuating that stereotype, it's not really fair.

I agree with this statement. Specifically, with this first part. This once again represents the idea of acceptance and being able to convey your story--to recognize how it has shaped or affected you in the long run. Additionally, I think as a matured individual such as an adult, it may be easier to share your story with a stranger or someone whom you may just be starting a relationship with. However, I don't think this applies specifically to adults. In general, I believe this applies to individuals of any age. I think healthy individuals or those who have faced challenges have the experience to be able to convey their story because they are more likely to accept life's challenges. I recognize that adults are more likely to have had these experiences, though. In general, I think those who have healed from negative experiences have an easier time sharing their story. But, I also think it applies to personality--some people may just be too shy or apprehensive to share their stories with an individual or they may not yet know how their life experiences have affected them.

One of the interesting things about her is her belief in predestination. I believe Mary Rowlandson must have been very conscious of her audience while writing this book (as the first woman to publish a book in America especially) here she shows the reader that in any circumstance she believed in God’s timing. Her religion is what's keeping her safe not only during her captivity, but from the people who might decry a woman in publishing as well. It's also interesting to note how she immediately then speaks of the natives as roaring lions and savage bears. Not just as characterization of wild animals, but also with the added context of the end of her sentence where she states that "Not one of them ever offered me the least abuse of unchastity to me, in word or action." They may be wild and ungodly, but they treat her well.

If there is such great debate about women, why not just throw it out the window and stop using the term and idea. Just like the word nominalism and searching the definition, that won't happen because it's not realistic. What I did realize though, is that this force isn't about what's right or wrong, it's about staying in power.

I have to be honest I looked up a few words to understand the full context of this text even though I was unaware of a few words the passion of the text spoke to me. The metaphor of the "double aims" is so powerful. It also reminded me of a religious reference, the temptation of doing wrong (quackery) and "the criticism of the world". It feels very much like prayer, like asking for forgiveness "ashamed" of what has been done. That the admitting of all this that was driven by temptation from the world like it's almost a confession. This text alone there is just so much to pick apart.

This quote is kind of contradicting to his opening sentence of the chapter because I received the idea that if we were not born with sight, we would love another just as much as one loves sight. It is hard to then be 100% in the claim that sight is the best sense when we do not know what it’s like to have never experienced it.

I love this choice of having two dreams that are both practically the same desire displayed in two different ways. They are both longing for something they don't have, and though the reward is so close they cant seem to fully grasp it. One of them sees this sort of longing to be something they have to go up for, something they will have to work hard to accomplish, while the other has to find their way down. Going down is usually easier than going up, but can be just as scary because no one likes looking down at where they could fall to. Then she goes on to say "here I am swapping dreams with you" like she is saying that she would be willing to trade their troubles, though really they are struggling with the same sort of dilemma.

It's crazy as to see how Jean doesn't see the "common'' as equal to the gentry. Yes they might have different tax brackets but it doesn't matter because we're all still human. The rich or in this case ''gentry'' eat. sleep, drink, dance..etc just like any other person so what makes them so special?

I'm not sure, but when I kept reading I keep getting the sense that throughout the play, there's like parts in the play where there's a reference of their dreams. Like Julie saying she on a hill and to climb down, but Jean is like his under and wants to climb up. in this like he tells her to fall down to me, and I'll lift you up again. It's like he's trying to make her trust him. to me the dream was like Julie wants to come down from the higher position she is currently, but Jean wants to climb up to a higher position. so yeah feel like I feel like there's a reference of their dreams throughout the play. Maybe he's telling her to climb and together they could like be higher ups. ( I'm not sure, just my way of seeing it)

this whole story just changed. It went from being an obnoxious spoiled girl, to like this girl who no longer knows herself. Honestly feel sorry for her. there was a reason why she does not trust men. the way Jean played with her feelings and all, was just wrong. he didn't have to make her feel so low. but on the other hand, Julie is ummm mood swinger. like for example, just a page ago, she was begging Jean for help, telling him she will do anything, telling him to come with him, but on the other hand she says she doesn't trust ( well I don't think Jean is to be trusted either) but it's like she does not know what to do with her life anymore. you know that dream she metioned about being on a hill, she needs to come down, but she does want, I felt like at this point of the reading, her dream did come true, because she went like from this royal rich girl, to like a servant her self, begging for a servant to save her life ( If I'm not misunderstanding the reading).

ok, honestly at this point in the reading, I've come to agree with Jean on the fact that Miss Julie is a little crazed in the head. while reading throughout the previous few pages, I sense That she might be in attention getter, or maybe she lacks love or perhaps something like that. she sound like a spoiled Brat to be honest. she loves to order Jean around like he's a dog and she loves wanting to hear about people feeling towards her, but she never want to reveal her own feelings. At first I thought Jean was in love with Kristen, because he is always saying what a wonderful wife she would make, but turned out that he Loves Julie, or maybe He is just messing with her feelings. for her to literally say "have you no feelings", it's confusing, because on the other hand, it's her that does not have any feelings. Also I feel like maybe she's afraid of letting people in to her life, perhaps not quite sure, but I get the feeling.

This is a good list of questions to put into a template to ask both while and after reading a book.

What other regularly recurring questions might go into such a list?

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.

bad UX

Compelling idea; Instead of working for years to make the mountain of knowledge bigger (and therefore harder for others to climb) you could spend years making it easier for others to get to the top.

What scale should this list be at? "Climate change" is an important problem that people should work on but it's so big that it's practically useless. Or maybe the list is so granular that the connection to the larger problem isn't clear. What's a good scale to think about problems?

Emotions are natural and are expected when writing an argument.

1. Political campaigns often manipulate people into voting a certain way by making them sympathetic to a certain cause.
2. I think it's unfair to use emotions to persuade people when the argument is more personal, such as setting boundaries.
3. I make a lot of food based decisions based on my emotions which I usually regret later. Emotions usually help me when I make decisions though, because I can tend to make decisions like a robot instead of a person.
4. I do a lot, especially when writing articles. Usually for that I try to use nostalgia or tragedy, just because my articles are either emergency events or about Barbie movies.

It's hard to say what this tells me about my hypothesis that the 7 mg patch increases HRV. It really depends on their blood levels. The second-hand smoke machine is extremely unrealistic. They were exposed to 30 puffs of 1.8% nicotine e-juice in the first measurement interval. Assuming 150 puffs per ml, that's 3.6 mg smoked in a single 15 minute interval! I've not read the full study to confirm this calculation. Nonetheless, it's clear that these insane conditions require scrutiny. That's not even counting the fact that there's no placebo (Or even control). How am I supposed to know if this is not just an effect of being concern about inhaling e-smoke? I'm still searching for studies on low concentration of nicotine and HRV.