How come the Prepositional Phrase isn't its own thing here? Normally the preposition would be the head of its own prepositional phrase that's an adjct to the Verb Phrase and containing the Noun Phrase as a comp, no?

3:1 이상

definition of the color contrast https://www.w3.org/TR/UNDERSTANDING-WCAG20/visual-audio-contrast-contrast.html#visual-audio-contrast-contrast-resources-head

At this point in the chapter, my interpretation is that you're making a comparison between the equalization of the rate of profit (from labour-intensive to capital intensive industries) to the current division between manual and mental labour.

I feel like the topic sentence introducing this paragraph needs to more deliberately attend to this argument.

So diligent is the head word? if so is it because its describing the workers to be diligent?

I think the significances of these lines are that depending on the way you look, dress and the state of your body can sometimes express the type of person you are or your family.

In these lines the speaker is trying to express that men do not only express themselves with face emotions. Men can express or show emotion with any part of their body. For example, when a man is sad they have their head down, hump on their back, slow walking etc.

Reviewer #1 (Public Review):

This study by Bhandare et al. applies endoscopic fluorescence imaging with head-mounted miniscopes and genetically encoded calcium sensors in awake behaving mice to document patterns of multicellular (including neurons and glial cells) calcium activity within circumscribed regions of the medulla oblongata (retrotrapezoid nucleus-RTN, Raphe magnus and pallidus nuclei, lateral parafacial region- pFL). These regions are proposed to have important chemosensory functions for the regulation of respiratory responses to elevated systemic CO2 (hypercapnia) and are critical for homeostatic regulation of breathing in mammals. Analyzing chemosensory properties of these medullary regions has been the focus of numerous studies, but the problem of analyzing regional multi-cellular chemosensory responses in the awake freely behaving rodent has not been previously addressed, so this paper represents an advance for the field. The authors importantly demonstrate chemosensory responses of neurons and astrocytes in RTN and Raphe, and activity profiles of neurons in pFL, and they describe substantial regional heterogeneity of cellular responses to hypercapnia in the RTN and Raphe regions. From this heterogeneity and distinct regional differences in chemosensory responses, the authors propose functional roles of these regions for detecting different aspects of the hypercapnic stimulus. In general all of the results presented suggest as justified by the data presented that cellular activity profiles in these various regions are more complex and encode different features for respiratory regulation than would be predicted from neuronal activity recordings in anesthetized animals. 

Strengths: (1) The authors demonstrate the successful technical application of the deep endoscopic fluorescence imaging approach for multicellular calcium activity imaging from key medullary structures involved in respiratory control in freely behaving mice. (2) Their novel results indicate functional diversity of neuronal responses and complexity in the encoding of chemosensory signals in key regions that have not been previously described from in vivo studies in anesthetized animals. 

Weaknesses: While the experiments are technically well executed for the most part in terms of implementing this imaging approach in freely behaving mice, important clarifications about experimental design and data analyses are required, and the authors do not fully discuss important technical limitations that may significantly affect interpretation of the results. A major concern with this imaging approach is whether the targeted regional neuronal and glial populations have been adequately sampled throughout the regions studied to reasonably understand the spatial and functional heterogeneity of the neuronal or glial hypercapnic responses. There is also the problem that cell identify has not been adequately established in some regions (e.g. RTN, pFL), which the authors need to specifically address to fully justify their conclusions about significance of their results. This is a particular problem for RTN neurons in which the chemosensory neurons that encode graded elevations of CO2, which were rarely found in this study, have an established molecular phenotype and this phenotype has not be sufficiently verified to know if the various response patterns identified in the freely behaving mouse are actually associated with this functional cell type. 

So while these new results indicate diversity of regional neuronal responses, many of these responses differ from the existing literature on how these cells are proposed to respond to CO2 from experiments with anesthetized rodents. Most notably, neurons in the RTN region were found to have an adapting excitatory response or were inhibited, rather than the expected preponderance of graded neuronal responses encoding the level of CO2; pFL neurons failed to exhibit a sustained expiratory-related oscillation predicted from studies suggesting that this area contains a conditional expiratory oscillator; and astrocytes failed to respond in terms of calcium signaling with elevated CO2. This underscores the need for additional technical clarifications to ensure reliability of the results, and for the authors to amplify discussion of the important caveats with their experimental design and functional interpretations.

Author Response:

Reviewer #1:

By presenting the detrimental effect of accumulative heterozygous mutations on the sperm head morphology, this report by Martinez and colleagues brings new attention to a wildly accepted paradigm in male germ cells that genetically haploid spermatids are phenotypically diploid, suggesting that multiple heterozygous mutations can lead to unexplained male infertility. The merit of this manuscript is the conceptual advance - oligogenic mutations as the possible cause of male infertility - the manuscript proposes, the strong rationale and reasoning of the motivation of the study, and development of a new tool to visually and quantitatively assess sperm head morphology which will benefit the field in general. The weakness that offsets these strengths is that the sperm phenotypes of the multiple heterozygous mice - while significant - are quite subtle in morphological changes and lack physiological phenotype. The study also does not provide data to support molecular mechanisms such as changes in the protein levels or localizations in their animal models. Due to these limitations, at currently presented the study remains rather descriptive and speculative. It would also be better to avoid excessive novelty claims.

Thank you for highlighting the conceptual advance of our MS. Regarding the weakness you mentioned, we would like to nuance your statement on the lack of physiological phenotype. Indeed, we clearly show that accumulation of heterozygote mutations led to a significant decrease of sperm motility, mutated sperm being 3 times slower.

Reviewer #2:

Digenic and oligogenic inheritance are extensions of monogenic disease models, in which effects of variation at two loci (digenic) or a few loci (oligogenic) contribute to the overall phenotype of an individual. The existence of oligogenic inheritance has been appreciated in human genetics for decades, and has been especially well documented for rare disorders with extensive locus heterogeneity, such as retinal degeneration, a condition for which more than 250 loci have been identified (Kousi and Katsanis 2015). Male infertility, itself a collection of diverse and often severe disorders affecting sperm count and sperm morphology, is likely to be driven by as many or even more loci as retinal degeneration, and is thus likely to feature oligogenic inheritance in some familial cases. Indeed, hypogonadic hypogonadism is one of the earliest and best examples of a human disease displaying digenic inheritance. Nonetheless, numerous challenges abound in the identification of digenic or oligogenic causes of male infertility, and validated examples in humans and model organisms are badly needed. In this study, Martinez et al. demonstrate oligogenic inheritance of sperm abnormalities by breeding a series of KO strains known to feature multiple morphological abnormalities of the flagella (MMAF). This is a significant paper for both the sperm abnormality field and for the broader male infertility community. the experiments and analyses are straightforward and the manuscript is well written.

My primary concerns are simply about the description of the experiments and analyses themselves.

1. There are numerous references to the "% of abnormal cells", "% of head abnormalities", "% flagellum abnormalities" (Figures 1B, 2B, 3B, 4B, 5, 6 and elsewhere). There are no clear definitions of how a cell is classified as "abnormal" or a head is classified as "abnormal" or a flagellum is classified as "abnormal". Are these all defined from manual classification of images? This seems essential to know if someone would like to reproduce this experiment.

We thank the reviewer for his remark and agree that this part was not sufficiently detailed. To allow an easy replication of the experiments, the material and methods now specify:

“Morphology was visually assessed on a Nikon Eclipse 80i microscope equipped with a Nikon DS-Ri1 camera with NIS-ElementsD (version 3.1.) software by trained experimenters. At least 200 spermatozoa were counted per slide at a magnification of ×1000. Cells are classified as abnormal when they bear at least one morphological defect, either on the head or the flagellum. Normal head morphology is defined by a typical murine overall shape with a pointy hook tip, a well-defined flagellum insertion notch in continuation of a smooth central region, a prominent caudal bulge and a dorsal region without notches. Normal flagellum must be continuous, of regular size and caliber, without angulation or excessive curling. Examples of normal and abnormal morphologies are provided in Appendix 1-Figure 7.”

We also added a new supplementary figure (Appendix 1-Figure 7) with light microscopy pictures of Harris-Schorr stained spermatozoa with typical and abnormal morphology. Legend section is modified accordingly.

1. In order to be of most value to the community, it would be helpful to provide the individual-level data behind Figures 5,6,7, indexed by genotype. Currently the supplementary tables just contain the summary statistics for each group. Further, for the individual level data, it would be good to decode the labels from "two genes" and "three genes" to the actual genotypes, since there are multiple genotypes in those groups. These data could be used for fitting genetic models to each of the traits (e.g. to estimate additive effects, epistatic effects, etc).

As requested by the reviewer, raw data behind figures 5 to 7 and data by genotype for the “two genes” and “three genes” groups have been added as source data files.

꡵

Perfect way of iteration is through repetition. The bad stuff will start out as minute "ehhh's" that soon turn into "fuck this part".

You don't need to be perfect the first time through, but by the 1000th time through you'll have perfection

TODO find pictures of these

The word "covenant" is often brought up when making a deal or negotiation as it portrays the religious views of the time which was Christianity. Even the Knight's code of chivalry is an example of this as it is a list of rules that ultimately relate to biblical morality. There are two distinct covenants in the Bible, the Old Testament and the New Testament. Upholding the laws is a crucial aspect of the OT while the NT focuses more on living by the spirit. The Green Knight is adamant on having Gawain keep his promise, and Gawain is determined to go through with the agreement, even if it means death, to uphold his duty as a knight. A shift happens at the end of the story that resembles more of a New Testament theme though, when Gawain breaks his covenant with the Green Knight, but Instead of cutting Gawain’s head off, "Bertilak calls it his right to spare Gawain and only nicks his neck," which is an act of mercy. In the Bible, one of the many characteristics of God is that he is merciful. In Titus 3:4-7 (NT) it states, "He saved us, not because of works done by us in righteousness, but according to his own mercy, by the washing of regeneration and renewal of the Holy Spirit, whom he poured out on us richly through Jesus Christ our Savior," and, therefore, the knights of this time considering themselves followers of Christ would be living by the spirit given to them by God and that is portrayed through the Green Knights mercy towards Gawain by not killing him. By demonstrating both of these themes relating to covenants in the story, insight into the religious beliefs held by the knights are shown in alignment with the overall theme and culture of the time.

https://www.sparknotes.com/lit/gawain/themes/ https://www.christianity.com/wiki/christian-life/what-is-the-spiritual-gift-of-mercy.html

Reviewer #2 (Public Review):

A major aspect of the authors' manuscript is their "sCRilecs-seq" method. This method sorts a CRISPRi library according to cell size and identifies mutants with significant cell size defects. Despite the promise of this novel, fundamentally sound method, much optimization of the experimental and/or analytical methods is required before this method will be of broad utility to the microbiological community. The major issue is regarding the reproducibility of the data, which then causes major issues with hit identification and limits the power of the method. To the extent that the authors wish to emphasize the method as part of the manuscripts' significance, these issued must be addressed head on and resolved.

The authors use their method to identify 17 operons with significantly larger cell size. Both operons encoding components of the mevalonate pathway are in this set, which suggests that inhibiting mevalonate synthesis may synergize with amoxicillin, an aminopenicillin antibiotic. In a series of thoughtful and elegant experiments, the authors show that indeed, inhibiting UppS (which synthesizes Und-PP from Farnesyl-PP for which mevalonate is an upstream precursor) using clomiphene sensitizes cells to amoxicillin. Although this finding may be of some clinical relevance, its significance is lessened by the fact that clomiphene has been shown to sensitize both Staphylococcus aureus MRSA and B. subtilis to beta-lactams, including amino-penicillins since at least 2015. Given the conservation of this synergy between MRSA and B. subtills, it is not surprising that it holds for Streptococcus. Moreover, since this synergy has been previously observed, it is not clear that the authors' sCRilecs-seq method was necessary to discover it.

Considered as a whole, the authors' method and follow-up experiments have the potential to uncover novel aspects of Streptococcus biology, but at the current level of analysis, it falls short of this goal. In my opinion, this paper could be substantially improved if the sCRilecs-seq method and/or analysis were improved, and if the authors' findings were better contextualized to highlight differences and similarities to MRSA and B. subtilis.

there is no closing parenthesis in the first argument of the toUppercase function:

haskell toUppercase :: String -> String -> String toUppercase processedString remainingString = if (remainingString == []) then processedString else toUppercase (processedString ++ [toUpper (head remainingString)]*)* (tail remainingString)

got stuck on one ideas but unclear what

traditional learning platform vs AI driven learning.

SciScore for 10.1101/2022.03.04.22271830: (What is this?)

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

Table 1: Rigor

Table 2: Resources

No key resources detected.

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

Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

There are several limitations of this study. The evaluation of a 100 µg booster was open- label, and the study was not designed to evaluate booster doses at varying intervals or compare different booster dose levels head-to-head. Additionally, neutralization results from different groups were not generated in the lab at the same time, the historical control group is not the same group of participants enrolled in the present study, and the study was not designed to evaluate vaccine effectiveness. However, neutralizing antibody responses have been correlated to reduction of risk for breakthrough COVID-19.12, 21, 22 The strengths of the study include that over 300 participants received a booster dose, allowing for a robust evaluation of possible adverse reactions, and the antibody response against SARS-CoV-2 variants was evaluated. The same laboratory was used to perform all neutralizing antibody assessments in a formally validated assay. The results from this study provide evidence that a 100 µg mRNA-1273 booster dose administered at least 6 months after the primary series induces a robust immune response against the ancestral SARS-CoV-2 and against variants of concern. The results also suggest that the 100 µg booster dose enhances the breadth of the neutralizing antibody response and elicits even numerically higher neutralizing antibody titers against antigenically divergent variants compared to a 50 µg booster dose. Although observed to have a clinically-acceptable safety pro...

Results from TrialIdentifier: We found the following clinical trial numbers in your paper:<br><table><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Identifier</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Status</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Title</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT04927065</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Recruiting</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">A Study to Evaluate the Immunogenicity and Safety of mRNA Va…</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT04405076</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Completed</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Dose-Confirmation Study to Evaluate the Safety, Reactogenici…</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT04470427</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Active, not recruiting</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">A Study to Evaluate Efficacy, Safety, and Immunogenicity of …</td></tr></table>

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

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

Results from rtransparent:

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

Results from scite Reference Check: We found no unreliable references.

About SciScore

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

what does it really mean when an idea becomes the political belief or political thought of the majority? Tocqueville does not really go into this as he is more concerned with the consequence of the popularity of the idea-the danger or experience of the majority.

test

Author Response:

Reviewer #2 (Public Review):

Zhang et al. describe a new method for inducing traumatic optic neuropathy in larger mammalian models that offers the additional advantage of allowing rapid administration of local therapies to the site of optic nerve injury. Furthermore, the authors build on their prior work which has demonstrated a neuroprotective effect of hypothermia provided that protease inhibitors are employed to protect against cold-induced microtubule damage. In the present manuscript, they show that an endonasal approach to accessing the optic nerve within the optic canal can be performed safely in goat without inducing optic nerve damage. They then demonstrate that experimental crush of the optic nerve within the optic canal results in progressive degeneration of retinal ganglion cell (RGC) neurons and of their axons which form the optic nerve; this occurs over a period of several months, a similar time course to traumatic optic neuropathy in humans. Transcriptional profiling of mRNA obtained from the optic nerve at its site of injury identified changes in gene expression related to molecular pathways involved in inflammation, ischemia, and cellular metabolism. The authors then proceed to apply local hypothermia or protease inhibitor administration (individually or in combination) to the site of optic nerve crush for two minutes and observed a decrease in axonal degeneration in the subsequent months, although without an improvement of conduction of visual information by the affected optic nerve. Finally, the authors describe a computer program which uses computed tomography scans to evaluate thousands of potential endonasal approaches to access the prechiasmal optic nerve in large and medium sized mammals, and they proceed to successfully perform optic nerve exposure and experimental crush in a macaque model.

The authors' interpretation of the data is generally accurate; however, their conclusions about the impact of the work are somewhat overstated.

We appreciate the reviewer’s insightful and precise comments, which definitely help improve our manuscript. We have scaled back the conclusions about the impact of our work, and revised our manuscript point by point according to the reviewer’s comments.

Strengths:

The prevailing use of rodent models of traumatic optic neuropathy in the field is problematic, and the authors' efforts to use larger mammalian models may be helpful in understanding the pathophysiology of and developing treatments for traumatic optic neuropathy in humans. The computer program that evaluates and recommends detailed surgical approaches and instrumentation is novel and would be quite useful to other investigators attempting to perform similar endonasal procedures. The authors make use of multimodal assessments of the goats and macaques [e.g. quantitative retina and optic nerve histology; optical coherence tomography measurements of retinal layers; pupillary light reflex assessment; and electrophysiology studies including visual evoked potential (VEP) and pattern electroretinography (PERG)] to convincingly demonstrate that the endonasal procedure itself can be performed without inducing progressive optic nerve damage and that experimental optic nerve crush using this procedure induces the expected profound decrease in optic nerve function, associated with progressive degeneration of RGC cell bodies and axons. The histological assessment of goat optic nerves following the local hypothermia/protease inhibitor treatment demonstrates a convincing reduction in the absolute number of RGC axons that are lost at 1 month after optic nerve crush.

Thank you very much.

Weaknesses:

The premise that optic nerve crush within the optic canal is much more physiologically relevant than other existing animal models is overstated: while the optic canal is believed to be the most common site of injury to the optic nerve, most human cases of traumatic optic neuropathy occur as a result of indirect mechanisms rather than compression/crush-namely, stretching and shearing forces are applied to the optic nerve where it is tethered to the periosteum of the optic canal by its dura. The authors' example of a bony fragment compressing the optic nerve within the optic canal (Figure 1B) is relatively rare and would actually represent one of the few cases where a surgical intervention (to relieve acute optic nerve compression) might be considered clinically justifiable.

We agree with the reviewer that “most human cases of traumatic optic neuropathy occur as a result of indirect mechanisms rather than compression/crush”. To address the reviewer’s concern, we updated our discussion as: “Recently, rodent models have been developed using indirect mechanisms (apply periorbital ultrasound or skull weight drop) to induce distal ON injury. Compared with direct optic nerve compression or crush, these models are likely more clinically relevant since most clinical TON cases are indirect and due to force transmission (39-41). However, due to force scattering, unwanted and uncontrolled collateral damage to the eyeball, contralateral optic nerve, orbit or skull often occur in these models (39,40). Additionally, the success rate of these modeling methods is not as high as direct optic nerve crush; for example, 10% mice died immediately after head weight drop (39). Moreover, extension of these modelling methods to large animal species has not been reported. Therefore, clinically translatable, local treatment of injured ONs via trans-nasal endoscopy cannot be performed in these small animal models.”

To further address the reviewer’s concern, we have added the following statement to the “Limitations of this study” section in the revised manuscript: “Our TON model is clinically relevant in terms of injury site, subsequent spatiotemporal pattern of retrograde axonal degeneration, and availability of trans-nasal local treatment. However, the mechanism of optic nerve injury in our model differs from that in most clinical TON cases, in which the intra-canalicular optic nerve is injured by an indirect mechanism (stretching and shearing forces), rather than by direct compressing forces.”.

We respectfully disagree that “a bony fragment compressing the optic nerve within the optic canal (Figure 1B) is relatively rare”, at least in China. According to our previous clinical study of 1275 patients with indirect traumatic optic neuropathy (PMID: 27267448), bony fracture of the optic canal is not rare: 50% of patients had a visible optic canal fracture on high-resolution CT scans, and an additional 20% had a visible optic canal fracture under trans-nasal endoscopy (because endoscopy provides excellent illumination and a magnified view of the optic canal).

The transcriptomic profiling at three locations along the visual pathway in post-trauma goats only showed differences in expression at the location of optic nerve injury, and not within the retina or proximal optic nerve on the affected side. The authors assert that the high rate of expression changes in pathways relevant to ischemia, inflammation and metabolism indicates "that targeting these pathways with local treatment could alleviate secondary damage." This is overstated. While such profiling may be useful for hypothesis generation, it was not followed up by any experiments to determine whether these expression changes are actually detrimental to the optic nerve. Some of them may very well be compensatory, such that inhibiting them may only exacerbate damage. Furthermore, given that these transcriptional changes are not seen in the retina (where RGC nuclei reside) or in the proximal optic nerve, this would suggest that the observed transcriptional changes at the site of injury are actually occurring in non-neuronal cells (e.g. astrocytes, oligodendrocytes, microglia). The authors should convey that the changes they observe are unlikely intrinsic to the optic nerve axons.

We appreciate the reviewer’s insightful explanation and agree that our original claim is premature. We deleted the statement " targeting these pathways with local treatment could alleviate secondary damage" in the Results section. Additionally, we have replaced the previous sentence in the Discussion, which stated: " Instead, we targeted hypothermia to the injured pre-chiasmatic ON to prevent early changes in ischemia, inflammation, and metabolism transcripts (as revealed by RNA-sequencing at 1 dpi)." with “Instead, we targeted hypothermia to the injured pre-chiasmatic ON according to early transcriptomic changes in ischemia, inflammation, and metabolism pathways.”.

We also agree with the reviewer’s comment that these transcriptomic changes at the injured optic nerve mainly occurred in the micro-environment of non-neuronal cells. The major advantage of our large animal model is the ability to modulate the micro-environment around the axons of the distal optic nerve, including glial cells, vasculature, connective tissues and the extracellular matrix. To incorporate the reviewer’s comment, we have added the following statement in the Results section: “These transcriptomic changes at the injury site were unlikely intrinsic to the distal optic nerve, and more likely occurred mostly in non-neuronal cells in the micro-environment.”

The lack of any rescue of the pupillary light reflex or of visual evoked potential responses after local hypothermia/protease inhibitor treatment suggests that the physiological significance of any anatomical rescue by this treatment is minimal. If a number of axons survive but cannot conduct sufficient visual signal to stimulate the pupil response or stimulate the visual cortex, then the local treatment (even when applied immediately after trauma in this model, unlike in human patients in which a delay of a number of hours would be required at the very least) cannot be considered a substantial success. The authors also characterize the goats at 1 month post-injury, so one cannot say whether the statistically significant improvement in axon loss with the combined treatment would be durable at the later 3-month time point.

We agree with the reviewer that the local treatment with hypothermia/protease inhibitor did not achieve functional recovery of the visual pathway, and thus it could not be considered a substantial success. Our work provides a safe and clinically translatable approach to modulate the inhibitory extrinsic environment at the injury site. Although in our current study, application of local treatment with hypothermia/protease inhibitor does not achieve eye-to-brain functional recovery, and its long-term therapeutic effect is unclear, these results are encouraging because they show some benefit of local treatment. Another important feature of our large animal model is that local treatment of the extrinsic environment at the distal optic nerve can be combined with currently available intravitreal treatments, such as gene therapy to boost intrinsic axon regrowth capacity, to target both extrinsic and intrinsic factors. To address the reviewer’s comment, we have added the following to the discussion in the “Limitations of this study” section: “Additionally, the current local treatment did not achieve functional recovery of the eye-to-brain pathway, and its long-term therapeutic effect is unclear.”

As the authors acknowledge, the use of larger mammals prevented them from conducting studies with a large n. As a result, their analyses are underpowered.

We agree with the reviewer that, in an ideal scenario, increasing the sample size would improve the power of the analysis. In this study, however, ethical issues and limitations of housing space and other resources, constrain the sample size of large mammals. We include this point in our “Limitations of this study” section.

Because of the dissimilarities between their model and the most common mechanism of human traumatic optic neuropathy and because of the lack of a clinically significant rescue of the optic neuropathy when the local hypothermia/protease inhibitor treatment was applied, the authors' assertion that their model may "trigger a paradigm shift " for traumatic optic neuropathy research should be scaled back.

We agree with the reviewer that it is too early to state that our model may “trigger a paradigm shift”. We need more effective local treatments to prove the value of this model and the novel therapeutic approach. Therefore, we have deleted “trigger a paradigm shift” in the Introduction and Discussion.

Also here a bit of a sudden transition, maybe prolongue it w/o generating expectation of what's tc come.

This is a case of the past rearing its head to the present.It's a hard pill to swallow.

Reviewer #1 (Public Review):

This report includes the identification and subsequent molecular dynamics analysis of posttranslational modifications of specific residues in beta-MHC in normal hearts and hearts in cardiac failure. The study is prompted by the possible regulatory roles of beta MHC in the generation of mechanical activity and possible reversion to fetal isoforms in cardiac failure. The identifications are followed by molecular dynamics simulations bearing on the stability of thick filament tail interactions and myosin head positioning.

I need to research it.

mutual dependence, linked fates

• syntax - catastrophic events revealed in close proximity to interior thoughts about mundane topics
• death - violently interruptive (selection of detail - looking for earring juxtaposed with "taking with it a quarter of Saeed's mother's head)

Genius: The Life And Science of Richard Feynman,” James Gleick, Pantheon Books, 1992 (see pg. 409).

Should this be "A", "B", rather than "1", "2".

He was reconditioned back not to fear rats but was it a long term conditioning? Probably not. Did the reconditioning leave life long effects on little Albert?

This shows the brutal reality of how black people were treated. They never asked for harm and whenever they got it a little altercation life blew up for them. Black people constantly lived in fear.

love

so would each part of speech be the head? why are there multiple heads in a sentence?

I don't think we should present that growth as neutral or inevitable.

If you started seeing a python slithering around in your backyard, and your neighbor poked his head over the fence and was like "whoops that's my python, my bad," you would probably tell him to come get the python.

But if you saw twenty pythons in your backyard,

and you asked your neighbor and he told you that he has twenty thousand pythons in his house,

and that he has really good processes in place for keeping them inside that have a great success rate,

and that some of them are going to get through but that's just how it goes when you have twenty thousand pythons,

you would probably tell him that the point at which the pythons started escaping was the point at which he should have stopped getting pythons.

Does this mean that a phrase has no predicate? Is a phrase an incomplete thought with certain gramatical components, but incomplete

I remember being taught this in high school too! From the perspective of that old mindset, I would think that "from California" doesn't belong as a part of the subject because it could be easily removed from the sentence without changing it's meaning, therefore not being an important part of the sentence or subject.

Classification

Description

Definition

where: History

role of the wars in the transformation to feudalism

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Referee #3

Evidence, reproducibility and clarity

The article by Skokan and coworkers studies the regulation of macropinocytosis in the Hydra. They design a clever assay to image the formation of macropinosomes in the ectodermal cells of the Hydra body, by amputating the head and the foot of the animal and then helving it onto a thin glass rod, allowing them to study the dynamics of actin rings formation, associated with uptake of external fluid phase. They also observe the cyclic formation of macropinosomes during the oscillatory contractions of spheroids formed from amputated animals during regeneration. By using agonist and antagonist drugs targeting mechano-sensitive calcium channels, they show that the formation of macropinosomes correlates with the reduction of cell tension. Overall, the article is succint, but clear and convincing. However, in my opinion, two major points should be clarified, if not solved before considering publication.

Major points:

1. the function of macropinocytosis in the Hydra is not known. The author postulates that it could be linked to a regulation of membrane area during animal contractions. However, one may wonder if the membrane cell surface really changes during contractions. I wonder if another explanation is possible: most of the organisms leaving in fresh water require an efficient mechanism to remove excess water that comes in the cells through osmosis. The hydra regular contractile movement are part of this, and I am wondering the macropinocytosis could be linked to this mechanism. Would the author be able to apply osmotic shocks, in particular hypertonic shocks, and see how it changes the formation rate and the dynamics of macropinosomes? On the reverse, in paralyzed animal, I am wondering if macropinosomes are still formed? Results from these experiments may give a clue about the function of macropinocytosis in the Hydra.
2. Because of the role of Piezo and other mechano-sensitive calcium channels, the author conclude that the factor that limits macropinocytosis is membrane tension. However, unless I am mistaken, actin cytoskeleton has also been involved in mechano-sensing channels, it could be that cortical tension, rather than membrane tension is playing a regulatory role. A direct proof of membrane tension (by measuring it) changes would be required to conclude as the authors do. The role of membrane tension versus macropinocytosis could be directly assessed using membrane tension probes such as FliptR or flipper probes. Otherwise, a less clearly defined term, that combines both cortical tension and membrane tension, such as cell surface tension or cell tension would be preferable.
3. Number of macropinocytic cups(actin rings) per cell is used as a readout for rate of macropinocytosis. Yet in addition to the number of cups parameters the diameter increases in certain conditions such as GdCi3. It would ideally be interesting to show the changes in diameter of cups and how this varies per in different conditions. For example, in videos of Jedi1 treated body columns the cups seem bigger in size. Supporting experiments of monitoring macropinosomes via dextran uptake assays needs to be performed for quantifications a rate of change in macropinocytosis is proposed. Alternatively, dextran beads of different molecular sizes with different fluorophores could also be used to assess the differences in rate and volume of uptake via macropinocytosis in various conditions of this study.
4. If membrane tension is altered upon dissecting Hydra fragments, would it make sense to study potential changes in macropinocytosis within the regenerating body column? Such as differences in actin ring formation in cells close to wound edges versus equatorial regions of regenerating body columns and spheroids?
5. Reasoning for selection of Piezo as molecular target over other stretch activated channels has not been provided. Piezo activators have been used, on the contrary depletion of Piezo via RNAi could be performed in intact animals to assess increased macropinocytosis. Furthermore, rate of macropinocytosis could be assessed in body columns generated from Piezo depleted animals. This would further support the direct role of Piezo in the process.

Minor points:

1. The authors report differences in macropinocytosis based on different parts of the animal (Fig.S1), upon treating intact animals with GdCI3 (Fig.2C) how does this vary? Do the differences still persist in spite of increased macropinocytosis?
2. Hydra are animals with an elongated body column. Dissecting body columns of different lengths could give rise to spheroids of different volume, these could then be inflated to establish a comparative volume study with different volumes and macropinocytosis.
3. For all graphical representation it would also be ideal to state the p-values for each significant comparison to better appreciate differences instead of stars.
4. For better understanding of figures, highlight in graph legends and figure panels which tissue sample has been used i.e intact animal, body column or spheroid.
5. A graphical representation could be given for the comparison of macropinocytic cups between intact hydra versus body column samples with statistical analysis. To appreciate the claims made by the authors regarding the trend of more cups being observed in body columns versus intact animals, as only the mean values are stated in the text.
6. In Fig.1F, there exist streaks of dextran distinctly outlining apical membranes of cell sets in the hydra epithelia, what are these suggestive of?
7. Fig.S1 No p-values

Significance

Overall, the work is of interest for several research communities. The significance could be increase by providing a few more experiments about the physiological role of macropinocytosis in the Hydra.

The son is now too busy to spend time with his father. THE TABLES HAVE TURNED. I feel sad and pitiful for the singer as he is probably full of regret and wishes to turn back time so he could have spent more time with his son. If he had built a better relationship with his son, he would not have wanted to turn back time. Thus we can now see that "I'm gonna be like you, Dad You know I'm gonna be like you" has a negative connotation as the son is now neglecting his father

The words "he's spinning round in my head" further highlights how getting over the relationship is tough. However this time, when the memories come flashing back again, it comes "burning red" showing the persona's hatred and anger with "him" breaking up and ending the relationship with her. This shows the progression of the persona's breakup since the start of it till now, and also highlights the persona's change of feelings. This makes me feel proud for the persona as she is enduring all of this pain and slowly making it out without harming anyone nor herself.

The use of the brackets shows the voices in her head that are constantly ranting the words " red" and interrupting her song. This emphasises her true feelings and the sheer amount of pain she was actually facing, but just masked it all with melodies and music. This makes me reflect upon the theme of "expectations vs reality" and how not everything may seem fine until you experience it yourself.

The hyperbole of the speakers head ‘spinning’ suggests his extreme confusion and the imagery of an unfocused picture similar to his relationship with his partner. This emphasised how difficult it is to make her his lover but also his determination to love her no matter what

He is becoming akin to his father. When offered to spend time with his father. He refuses as he is more involved with his own life.

Turning the ongoing trend of their relationship on its heads. The Son has grown up and like he said "I'm gonna be like him". When the Son wanted to spend time with his father, he refused. Now when the father wants to spend time with his son, like his father, he refused. Not because of any intention to neglect but because they were caught up with their own lives.

Revealing the irony in the song where the son would sadly grow up to be like his father.

'head on the pillow' suggests someone sleeping, further connoting that the person is unaware about the things around it. 'sneaking' shows movement in a stealthy way, rather quiet and furtive, creeping in. From these, they suggest how the man entering the writer's life came without warning and quietly entering her life, without the writer realizing it since there was no indication of him entering her life. This further suggests how the writer's love interest came into her life in a way that she didn't expect, sneaking into her life in a way which she was unaware of how it happened.

This quote shows that the speaker is frustrated. The Literary Device here of structure of a poem, is the words ‘gotta and outta’ which shows that the speaker wants distractions to get out of his mind and stop distracting him.

I thought that this quote makes a really good point about plagiarizing. When we were younger, we were merely told that plagiarizing was bad because it's stealing someone's work and I think this quote really hits the nail on the head by explaining it's importance. Not only is it a big deal, but it's not giving credit where it's due to further help the original poster's work

Anubis is the Greek name of the god of death, mummification, embalming, the afterlife, cemeteries, tombs, and the Underworld, in ancient Egyptian religion, usually depicted as a man with a jackal head.

the demon Humbaba (Huwawa), who was a monster featured and slaughtered in the Epic of Gilgamesh. This example of his features was used in the interpretation of omens. The convolutions of the mask represent the intestines of a sheep examined for divination.

Humbaba/Huwawa - ProQuest

Writing on a terracotta plaque in the British Museum (cat. no. 127), Sidney Smith interpreted its inscription as an explanatory caption that identifies the grotesque face on its other side as that of Humbaba. The text reads:

If the coils of the colon resemble the head of Huwawa, [this is] an omen of Sargon who ruled the land. If [the omen is] for a poor man, the house of the interested party will expand. [Written by] the hand of Warad-Marduk, diviner (bārû), son of Kubburum, diviner.

The sinuous lines that make up the face on the front of this plaque clearly recall the convolutions of the small intestine. When taken together with the text inscribed on the back, it is irresistible to link this distinctive appearance with the persona of Humbaba

Pazuzu is a fictional character who is the main antagonist in The Exorcist horror novels and film series, created by William Peter Blatty. Blatty derived the character from Assyrian and Babylonian mythology, where the historic Pazuzu was considered the king of the demons of the wind, and the son of the god Hanbi.

In The Exorcist, Pazuzu appears as a demon who possesses Regan MacNeil. Pazuzu is often depicted as a combination of animal and human parts with his right hand pointing upwards and his left hand downwards. He has the body of a man, the head of a lion or dog, eagle-like taloned feet, two pairs of wings, a scorpion's tail, and a serpentine penis.

The beginning of their love story seems so true and carefree as if they just happened to find each other and Armand's demeanor comes off as sweet, but we read pieces like this that brings a twisted view on his character. It makes me realize how terrible the racism in this time was and how much ones personal life affected their slaves and how they would treat them. The fact that Desiree is so happy and shocked that he is not being as brutal as normal is a scary thought.

the gruesome imagery of “head explodes” depicts the human likeness of corrupt leaders being rapidly and violently destroyed, causing them to lose their emotions and morals

Simile. I can see from this phrase that the speaker was blinded by love. He was head over heels for his love that he had lost awareness and his sense of surroundings and danger.

individuals excluded often for drug abuse

이렇게 무료로 쓸 수 있게 해도 돈을 벌 수 있을까?

This was interesting to know, it makes me wonder what made him think this way to go as far as treating his own sons that way. Honestly, it makes no sense to me because treating one's own child like that is just cruel and can lead to things like depression when they grow up.

"Like inhumane creatures" is ambiguous because you could read it as both a recognition of their humanity and subsequent scolding of their character for not behaving like humans. Also interesting that their humanity is measured by their reaction to white mother and child being harmed

What is up with this recurring euphemism for being killed? The author is already recounting the perils of war and its bloody consequences yet feels the need to sugarcoat death itself.

This activity was routine

Like this, but don't explain why he has a minute of hazy confusion. Maybe "stared blankly and blurred" -> more impactful and implies unsettled attitude both physically AND mentally

SciScore for 10.1101/2022.02.17.22271142: (What is this?)

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

Table 1: Rigor

Table 2: Resources

No key resources detected.

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

Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

The major limitation of our study is the sample size, which limits drawing statistically significant conclusions regarding small differences in test performance. A larger study is warranted to further investigate the differences seen between our RADTs, as even small differences can have a large impact when scaled to the population level. Another limitation is our use of frozen samples in universal transport media rather than direct testing from a patient, but we would not expect there to be a major impact from one to two freeze-thaws on assay performance and the volume of analyte used in each assay was optimized in an earlier study for mimicking real-world performance12. A major strength of this study was the ability to compare three different RADTs using identical clinical samples, which allows for a robust comparison of performance. In summary, the analytic sensitivity versus Omicron remains stable in our head-to-head comparison of three of the most common RADTs in use in the United States. However, there were differences in inter-assay performance that warrant further study. Our findings will provide a degree of assurance that at-home testing should perform as expected compared to prior waves and also sets a baseline for comparison with future SARS-CoV-2 variants.

Results from TrialIdentifier: No clinical trial numbers were referenced.

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

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

Results from rtransparent:

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

Results from scite Reference Check: We found no unreliable references.

About SciScore

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

This could be an even more terrifying sensory experience

“Our every moment, I started to replace, ‘Cause now that they’re gone All I hear are the words I needed to say” trying to remember the memories of their time spent together, but can’t remember as what is filling his mind is questions, words that he feels should he should have spoken but didn’t and now he needs to get it all out, emphasising his regret. This makes me reflect on the theme of regret, how the speaker is regretting not helping his friend, and now is trying to make peace with the fact that their friend is gone and has no choice but to replace their memories, and thus I feel pity for the speaker.

“Our every moment, I started to replay.” The use of visual imagery tells me that the speaker is recalling all the memories that he has of his friend, and replaying them in his head, as he is no longer able to make new memories with his friend. Also, the use of the phrase, “our every” tells me that the speaker is also recalling the times when he was given the chance to help his friend but didn’t, and hence feels guilty. This makes me reflect on the theme of guilt, how some people feel guilty for their actions and keep recalling them, and hence I feel pity for the speaker as he is unable to bring himself to forgive himself, and thus I feel pity for the speaker.

I am glad the reading pointed out this useful tool for individuals who cannot use mouses. This can make learning easier and accessible, therefore encouraging students to stay motivated and make their learning experience much enjoyable. This is a tool that I will keep in mind and further research.

This phrase is a refrain throughout the whole song which is the lit device of repetition. This shows that this phrase was probably always playing or repeating in Brandon's head to encourage himself and that it was a very important phrase to him and that it helped him overcome his challenges while trying to reach his goals. This phrase also has alliteration of the "H" sound is in a relaxing tone which may have helped him calm down or remind himself of the goals that he wants to reach and is to also encourage the listener of this song. This makes me reflect on the theme of hope and how powerful of a force it can be.

These are scary passions however succumbing to them forces other men to face them head on.

"simultaneous exposure to multiple organic contaminants is the norm rather than the exception for developed-watershed head-water streams"

“But” shows how despite the previous line highlighting to us her hesitation and worry, the speaker is able to overcome her fears and anxieties and being able to muster up the courage in order to face her ex, and by association, her past mistakes and failures, therefore coming to terms with the mistakes she may have made in the past and taking accountability for them. This displays to the readers her growth and how she is able to start taking the initial small steps to improve her situation, in turn showing her willingness and hope to do so as well. “I'm” displays to the reader how she is able to make the brave decision to bear the responsibility of the situation on her own and is able to take accountability for what has happened. This shows the emotional maturity she takes the conscious effort to keep up and do what is right instead of what is easy, once more linking back to the title and showing how she is “trying” to improve the situation. “doorway” evokes the visual imagery of the speaker slightly stepping foot in the entrance of her ex’s home, ready to finally face him. This also works as a metaphor for how the speaker is ready to face and tackle her problems head on, even if she is just beginning to take the first step. This once again pushes to the readers the speaker’s desire and willingness to improve her situation. Admiration is evoked in me by the speaker’s bravery as she possesses the strength and courage to fight through the pain and sadness she is made to endure, to improve her situation and circumstance. This pushes me to reflect on the theme of courage and how much of it it takes to constantly overcome fears and pressures in order for us to flip the odds in our favour and improve any bad situation we may be in.

The absolute word “all” used conveys to the reader how others attempt to help her but they end up doing the opposite as they do not understand the situation well enough and up end over generalising the speaker’s struggles due to their ignorance yet still feel they have the right to tell her what to do, something that could be greatly detrimental to her situation. “my” shows us that despite the fact that the metaphorical “cages” belong to her, implying a deep connection only she has with them, she is still told by others through unsolicited advice what it is or isn’t, evoking great pity towards the speaker because her judgement of what is best for herself gets clouded by others who barely understand the situation and have nothing to do with it at all. The vivid tactile imagery evoked by “cages” creates a claustrophobic and uncomfortable sense of entrapment and a lack of freedom. This closely mirrors how trapped the speaker feels by her metaphorical “cages”, in this case referring to her “mental” setbacks caused by the situation. “mental” implies to us that others seem to be telling the speaker that all her problems are made up in her head and are therefore neglecting and belittling her true pain and suffering, therefore painting her out to be self-sabotaging and pushing the blame of her failures all onto her.

Here the author makes use of quotes in order to introduce the idea of words and images having profound impacts when used artistically and creatively. This gives the reader insight into how the reading as a whole will analyze the importance of certain artistic techniques, and how certain techniques can make art more profound.

Wrongfully accused of offending a white lady, he was then beaten and shot in the head, then dunked in the river.

this is so crazy to me. I love this because it shows the contrast between schools that are overpopulated and underpopulated. I think this adds nicely to the article.

I like this statistic a lot. it talks about the growth of students in Utah's public school enrollment, which is what my topic is. I think that it is fit in at a great spot in the article.

i like this because as humans we think about so many things and question about it. In theatre it's the same way.

I feel like this applies so much to Caitlin because of all the questions she's left with after Ingrid's death. Not only that, but the journal she finds, too; instead of finding answers in the journal she seems to be left with more questions.

If natural means 'as humans were in the Stone Age', then obviously not.

If natural means 'as a human would behave in a given environment', then probably.

A roof over your head, comfortable bed, mattress, duvets, pillows, and atmospheric regulation are not cheap - but it does seem human-like to value them as we do.

mayanila on sivan head

...even vishnu could not find it when siva sent vishnu and brahma to find who is greatest of the lords

https://www.linkedin.com/in/ACoAAABfDWoBXAvOBkBJp_nFSBDKvZZnHXwPIUM?lipi=urn%3Ali%3Apage%3Ad_flagship3_detail_base%3Bu%2BF35oelQQS613GcLeIUYg%3D%3D

https://www.abc.net.au/news/2022-01-28/index-history-invention-made-simultaneously-800-years-ago/100690782

The idea of the index was invented twice in roughly 1230.

Once by Hugh of Saint-Cher in Paris as a concordance of the Bible. The notes towards creating it still exist in a variety of hands. The project, executed by a group of friars at the Dominican Friary of Saint-Jacques, listed 10,000 words and 129,000 locations.

The second version was invented by Robert Grosseteste in Oxford who used marginal marks to create a "grand table".

The article doesn't mention florilegium, but the head words from them must have been a likely precursor. The article does mention lectures and sermons being key in their invention.

<small><cite class='h-cite via'>ᔥ <span class='p-author h-card'>Aaron Davis</span> in 📑 Monks, a polymath and an invention made by two people at the same time. It’s all in the history of the index | Read Write Collect (<time class='dt-published'>02/15/2022 21:22:10</time>)</cite></small>

the stanza's we're doing

This paragraph helps be decipher how taxes are taken out of someone's' paycheck and how you marital status changes how much you pay in taxes. I could use this information when i create a example and break down how much you get taken when taxes are taken from your paycheck. Having a real world example could help my peers understand what is really going on rather than just having to imagine.

May refer not only to his theory of relativity but also to imagination and creativity. As Einstein had quoted "Knowledge is limited. Imagination encircles the world"

This shows the two characters fighting about which one is better science or art? and which has a more profound impact.

despite Dr Amstrup knowing that we have to do something or it will mean the worse for us, the world isn't technically doing anything to help out

The forms of thinking, writing, and elaboration that go into creating permanent notes for a slip box are natural means of facilitating actual, long-term learning.

Proofreading while rewriting, structuring, or doing the thinking or creative parts of writing is a form of bikeshedding. It is easy to focus on the small and picayune fixes when writing, but this distracts from the more important parts of the work which really need one's attention to be successful.

Get your ideas down on paper and only afterwards work on proofreading at the end. Switching contexts from thinking and creativity to spelling, small bits of grammar, and typography can be taxing from the perspective of trying to multi-task.

Link: Draft #4 and using Webster's 1913 dictionary for choosing better words/verbiage as a discrete step within the rewrite.

Linked to above: Are there other dictionaries, thesauruses, books of quotations, or individual commonplace books, waste books that can serve as resources for finding better words, phrases, or phrasing when writing? Imagine searching through Thoreau's commonplace book for finding interesting turns of phrase. Naturally searching through one's own commonplace book is a great place to start, if you're saving those sorts of things, especially from fiction.

Link this to Robin Sloan's AI talk and using artificial intelligence and corpuses of literature to generate writing.

Whether it's for writing, to do lists, or other productivity spaces, a well designed system is something that one can put their absolute trust into. This allows one to free themselves from the burden of tracking and dealing with minutiae so they can get serious work done.

Additionally, on the topic of fully formedness, when I write, a possible outcome is that the result is an incomplete snapshot (a less fully formed version) of the full version in my head. Writing is almost always IO-bound.

cf Carl Sagan as Mr. X, and Ted Nelson on nonlinear structure.

automatically this idea that he know that he's ahead and the leader. but he knows that people will still see Jewel as the leader.

like i knoowwwww she's dying but... she's so unpleasant

1. president appoints the chairperson but jogorku kenesh gives concent
2. president Dismises memers of cabinet ministers but jk gives concent
3. Prsident is electing for 5 years term and he is a head of executive but jk (legislature) calls elections of the president
4. president dismisses chairman but jk ellect judges
5. President and parlant ellect judges but judiciary mwith deputies and prosecution may dismiss president

This might be referring to someone keeping their gun under the pillow? Guarding their head & sharing a pillow. to share a pillow is again, personification as a gun wouldnt normally have.

That's a lot of weight to be between your parent's issues especially at a young age. My heart would be crushed, especially feeling that betrayal from her father whom she looked after so much.

Diaz gives a description of her father who was more rebellious as opposed to her uncle. He is the problem child and eventually leads to him having to leave to the states.

Where I teach, our parent conferences are organized around a discussion of the child's "strengths" and "areas to develop." This shapes how I look at each child, always making a checklist in my head. Now I am noticing how counter this seems to the Reggio approach. What about "What knowledge is your child constructing?" How do some of you approach conferences at Reggio-inspired schools?

In this scene, the Magistrate states that if he had to listen to a woman who wears a veil, then to hang him, to which Lysistrata responds by putting her scarf on his head claiming "if that keeps pestering you, I've a veil here for your hair." Although this may seem like a small moment where she is embarrassing him in front of his guards, this moment is much more symbolic than one may think. The veil represents the lack of control and domestication that women face, as stated by Kenneth DelCastillo, "the Greek comedy Lysistrata by Aristophanes captures the woman’s veil under the motif conservatism, domesticity and chastity." To put it simply, when Lysistrata takes off her veil and gives it to the Magistrate, she is taking control over herself and fighting against the bias that women are not capable of being in a position of power, even over themselves.

DelCastillo, K., & Moy, O. L. (2017, May 1). The Power Behind The Veil. Lehman College eng 121: English composition ii. Retrieved February 12, 2022, https://lysistrata.commons.gc.cuny.edu/2017/05/01/the-power-behind-the-veil-symbolism-from-ancient-greece-to-muslim-societies-in-france-and-germany/

The most senior person is the one who gets interrupted the most. This seems true even among software engineers...

Work out mathematically at what rate the accumulation of notes would outstrip one's ability to memorize them solely using spaced repetition of a few generations.

this is just so fascinating.

Most of the times, we are stuck with boring daily lives, but sometimes, we are sensitive to supernatural horror, and no amount of rational mental defense can keep us from feeling it.

This sensitivity is probably biological and ancient, which is why there has always been a significant minority of humans who feel it from time to time.

Author Response

Reviewer #2 (Public Review):

In this interesting and beautifully illustrated study, the authors are addressing the question of the emergence of craniofacial tissues by dissecting the interplay between skeletal muscle progenitors and associated connective tissue cells. By combining sophisticated lineage-tracing single cell RNA-seq experiments with potent computational analysis tools followed by in situ validations, the authors have identified a population of Myf5+ bipotent progenitors that give rise to both muscle and connective tissue. However, some conclusions are solely based on the RNA-Seq data that would require further experimental validations.

We thank the Reviewer for evaluating our work and their encouraging comments. We agreed with the assessment and have now added more in-situ validations and quantifications to support the in-silico analyses.

Reviewer #3 (Public Review):

In this manuscript, Grimaldi et al. present evidence for the existence of Myf5+ bipotent progenitors for myogenic and connective lineages in the dorsal regions of the mouse head, which is not populated by neural crest cell-derived connective tissue. The study relies heavily on scRNA-seq dataset obtained from cell populations sorted at defined time points, and refined computational analysis, including trajectory and gene network inference using the established tools RNA velocity and SCENIC, respectively. The proposed model is partially validated by in situ staining experiments, including genetic labeling, which identified Pdgfra+ non-myogenic cells within the Myf5+ lineages, notably in association with extraocular muscles (EOM). The authors propose a myogenic origin for the connective tissue, in regions devoid of neural crest cells, and show that loss of Myf5 function causes an increase in the proportion of Sox9+ cells among Myf5+ lineage cells, which is consistent with a binary fate choice from Myf5+ progenitors. The authors tentatively identify signaling molecules and transcription regulators underlying both fate decisions and cell-cell communications between myogenic and non-myogenic cell populations.

The general message of the study offers a potentially new paradigm to study neural crest cell-independent mesodermal fate decision in the vertebrate head, and is thus poised to augment our understanding of craniofacial development, and potential diseases.

Unfortunately, there are shortcomings that strongly reduce enthusiasm for this manuscript. Strictly speaking, there is no clear demonstration for the existence of bipotent progenitors in the absence of clonal analysis. The study relies excessively on computational analysis of descriptive scRNA-seq datasets, with a general paucity of secondary experimental validation. The manuscript would benefit from a refined focus on the key point, and addition of validation for the initial conclusions, at the expense of somewhat convoluted analyses (e.g. Figs. 6 and 7)

We thank the Reviewer for their constructive comments. We have now provided additional in-situ validations on embryos and quantifications to support the in-silico analyses.

Reviewer #3 (Public Review):

In this manuscript, Grimaldi et al. present evidence for the existence of Myf5+ bipotent progenitors for myogenic and connective lineages in the dorsal regions of the mouse head, which is not populated by neural crest cell-derived connective tissue. The study relies heavily on scRNA-seq dataset obtained from cell populations sorted at defined time points, and refined computational analysis, including trajectory and gene network inference using the established tools RNA velocity and SCENIC, respectively. The proposed model is partially validated by in situ staining experiments, including genetic labeling, which identified Pdgfra+ non-myogenic cells within the Myf5+ lineages, notably in association with extraocular muscles (EOM). The authors propose a myogenic origin for the connective tissue, in regions devoid of neural crest cells, and show that loss of Myf5 function causes an increase in the proportion of Sox9+ cells among Myf5+ lineage cells, which is consistent with a binary fate choice from Myf5+ progenitors. The authors tentatively identify signaling molecules and transcription regulators underlying both fate decisions and cell-cell communications between myogenic and non-myogenic cell populations.

The general message of the study offers a potentially new paradigm to study neural crest cell-independent mesodermal fate decision in the vertebrate head, and is thus poised to augment our understanding of craniofacial development, and potential diseases.

Unfortunately, there are shortcomings that strongly reduce enthusiasm for this manuscript. Strictly speaking, there is no clear demonstration for the existence of bipotent progenitors in the absence of clonal analysis. The study relies excessively on computational analysis of descriptive scRNA-seq datasets, with a general paucity of secondary experimental validation. The manuscript would benefit from a refined focus on the key point, and addition of validation for the initial conclusions, at the expense of somewhat convoluted analyses (e.g. Figs. 6 and 7)

I found this part of the reading very interesting as it not only shows the intricacies of Don Quixote's grandfather's armor, but it also shows Quixote's unwavering personality and stubbornness. He would rather sit, unable to eat, with his helmet still securely fastened to his head than remove it forcefully, metaphorically stripping him of any sense of knighthood that he is pursuing. By not removing his helmet, Don Quixote still clings onto the hope that he can become a honorable knight against all logic or reasoning.

The direct address to the reader during the prologue was most interesting to me, ex. "...for you are neither his kin nor his friend, and you have a soul in your body and a will...". I found myself arguing against Quixote's stepfather in my head, proposing that while I may not know not know him personally he certainly doesn't know me at all either. How would his stepfather know that I have a soul and will as free as anyone's, that I'm in my house, "where [I] am lord"? Maybe it's indicative of the assumptions Cervantes has to make because of the socioeconomic barriers to literacy that plagued Europe -- but he also later includes the theme of books as a tool for liberation. It's ironic, and I wonder how we can reconcile this paradox.

The tactile imagery of this phrase shows that the speaker wants his partner to know just how much he loves and appreciates her. The diction of the word “beating heart” also help to show that the speaker lives for his partner as she makes his heart beat faster around her.

Author Response:

Reviewer #2 (Public Review):

The authors have developed a new method that allows for two-color STED imaging. They have applied this method to measure spine head size and PSD95 changes following exposure to an enriched environment.

Strengths

-The new method is well-described and seems to have considerably less crosstalk than previous attempts at in vivo two-color STED imaging. The analyses and controls of the method are compelling. I think that this method could be valuable for examining how different components of the synapse are changing in response to sensory or environmental changes.

-The method is appropriate for measuring the size of PSD95 and spine head size in the enriched environment paradigm they use here. They find that in the short-term spine head size and PSD95 size are not always correlated.

-They also find that there is less variability in the spine head size in animals in an enriched environment.

Weaknesses<br /> -The authors use an enriched environment plasticity paradigm to showcase the method and measure spine head and PSD95 size and how they change over short periods of time. This particular biological study is not well-motivated and there is not a stated reason for studying the short-term (30-120 minutes) dynamics of PSD95 and spine head size, and their correlations. They also show that the variability in spine head size is decreased with the enriched environment, but do not show what the implications of that change would be from a biological point of view for synaptic dynamics or synaptic function.

-The authors show that there are differences in the morphology of PSD95 between mice reared in enriched environments and those in control environments. While this quantification is done blindly by three different analysts, it is not done in a quantitative way. Also the authors do not show or explain the biological relevance of differences in the morphologies of PSD95, thus it is not clear what this measure means for synaptic plasticity or function.

-The authors use a cranial window preparation, which is commonly used in the literature. However, it is not clear how long they wait to image the mice after the cranial window. Previous work from Xu et al. (PMID: 17417634) suggests that there is in an increase in glial activation for a period of up to a month after surgery. The authors have not shown the degree of glial activation that follows after their surgeries and if they have not waited a month, there may be upregulation of microglia, which may alter synaptic stability (also demonstrated in the same paper). The authors have not discussed this point or the implications for their findings.

We thank the reviewer for his/her valuable input.

The time-scale we study is similar to what is known from structural changes after LTP and thus we wanted to study the same time scale in vivo. We revised the motivation and explained better the biological relevance of the observed changes. We absolutely agree with the reviewer on his/her concern for chronic imaging. However, we performed acute experiments and imaged directly after implanting the window in the same session. After imaging the mice were sacrificed.

Reviewer #3 (Public Review):

Wegner et al. use two-color STED to follow spines and their PSDs in layer1 of mouse visual cortex over 2 hours under anesthesia. They compare mice that were kept in an enriched environment (EE) to control mice housed in standard laboratory cages. Spines in EE mice are larger and show larger fluctuations in size. PSDs in EE mice shrink during anesthesia and tend to change their nanostructure. Very importantly, changes in spine size were not driven by PSD size changes, or vice versa. Technologically, this is a landmark study, as tracking two different labeled structures in individual synapses at the nanoscale can obviously be applied to a large number of synaptic proteins and organelles, two at a time. Single-color superresolution microscopy is much less useful, as 'puncta in space', without cellular context, are difficult to interpret. This pioneering work is the first proof-of-concept of two-color in-vivo STED and of major importance for the community. Although stochastic processes seem to drive much of the synaptic dynamics under anesthesia, the environment shapes the spine size distribution and affects synaptic dynamics in a lasting fashion.

One major comment:

l.259: "These results suggest that Ctr housed mice undergo stronger morphological changes." This I find a bit misleading. What about: These results suggest that anesthesia induces stronger morphological changes in Ctr housed mice? Altogether, a discussion of the potential effects of anesthesia on spine/PSD dynamics is missing (see e.g. Yang et al., DOI: 10.1371/journal.pbio.3001146). The fact that there was weak correlation between spine head and PSD fluctuation could have something to do with the state of suppressed activity the system was in during imaging. Under conditions of intense processing of visual information, changes might have been more rapid and more tightly correlated. This could be mentioned as a perspective for the future - to visually stimulate the anesthetized animal.

We agree with the reviewer that it should be mentioned here that the morphological change was observed under anesthesia. However, the sentence suggested by the reviewer is also a bit misleading since it suggests that the anesthesia has triggered the change. We think that anesthesia might affect the amplitude and dynamic of the observed changes but does not induce the change. Thus we rephrased as follows: These results suggest that Ctr housed mice undergo stronger morphological changes under anesthesia.

We absolutely agree about the potential influence of the anesthesia on the spine and PSD95 nanoplasticity and added the following comment. Of course, we would like to perform the measurement in the future also in awake mice and after visual stimulation.

Added to discussion: However, it was shown that MMF anesthesia reduces spiking activity and mildly increases spine turnover in the hippocampus (Yang et al., 2021). Thus, the plasticity of spine heads and PSD95 assemblies might be different in the awake state and under intense processing of visual information.

Evaluation Summary:

Synapses convey information in the brain, including signals from the environment. The changes in the incoming signals can alter the efficacy of synaptic transmission, which in turn can be represented by the changes in synaptic structure that is particularly evident in the postsynaptic compartment called spines. This study uses a custom-built superresolution microscope to follow individual spine shape and the dynamics of the resident scaffolding protein PSD95 simultaneously, to study the effects of rearing mice in an enriched environment relative to a simple standard cage. The imaging data are of superb quality. The authors find that regardless of the rearing condition, dynamic changes in the sizes of spine head and PSD95 are detected that do not necessarily correlate with each other. Furthermore, mice reared in an enriched environment show less variable spine head size. While these findings may be of potential interest to neuroscientists studying synaptic network architecture, a clarification of the biological question being addressed, and validation of the method used to monitor PSD95, would considerably strengthen the study and enhance its overall impact.

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

Reviewer #1 (Public Review):

This study capitalizes on the crosstalk-free two-color STED developed by the authors (cf. Willg et al., Cell Rep 2021) to examine the dynamic changes in synapse structure in mouse visual cortex. Imaging the superficial dendrites of layer V pyramidal neurons, the authors report features of dendritic spine morphology and the dynamics of scaffolding protein contained within that are affected by rearing mice in an enriched environment (EE) compared to control housing. Curiously, EE mice show less variable spine head volumes and PSD95 areas compared to control mice, while the spine head volume is larger but not PSD95 area in EE group compared to the control group. Moreover, nano-organization of PSD95 displays more prominent changes in EE compared to controls. These findings may be of potential interest to neuroscientists studying synaptic network architecture.

Reviewer #2 (Public Review):

The authors have developed a new method that allows for two-color STED imaging. They have applied this method to measure spine head size and PSD95 changes following exposure to an enriched environment.

Strengths<br /> -The new method is well-described and seems to have considerably less crosstalk than previous attempts at in vivo two-color STED imaging. The analyses and controls of the method are compelling. I think that this method could be valuable for examining how different components of the synapse are changing in response to sensory or environmental changes.

-The method is appropriate for measuring the size of PSD95 and spine head size in the enriched environment paradigm they use here. They find that in the short-term spine head size and PSD95 size are not always correlated.

-They also find that there is less variability in the spine head size in animals in an enriched environment.

Weaknesses<br /> -The authors use an enriched environment plasticity paradigm to showcase the method and measure spine head and PSD95 size and how they change over short periods of time. This particular biological study is not well-motivated and there is not a stated reason for studying the short-term (30-120 minutes) dynamics of PSD95 and spine head size, and their correlations. They also show that the variability in spine head size is decreased with the enriched environment, but do not show what the implications of that change would be from a biological point of view for synaptic dynamics or synaptic function.

-The authors show that there are differences in the morphology of PSD95 between mice reared in enriched environments and those in control environments. While this quantification is done blindly by three different analysts, it is not done in a quantitative way. Also the authors do not show or explain the biological relevance of differences in the morphologies of PSD95, thus it is not clear what this measure means for synaptic plasticity or function.

-The authors use a cranial window preparation, which is commonly used in the literature. However, it is not clear how long they wait to image the mice after the cranial window. Previous work from Xu et al. (PMID: 17417634) suggests that there is in an increase in glial activation for a period of up to a month after surgery. The authors have not shown the degree of glial activation that follows after their surgeries and if they have not waited a month, there may be upregulation of microglia, which may alter synaptic stability (also demonstrated in the same paper). The authors have not discussed this point or the implications for their findings.

Reviewer #3 (Public Review):

Wegner et al. use two-color STED to follow spines and their PSDs in layer1 of mouse visual cortex over 2 hours under anesthesia. They compare mice that were kept in an enriched environment (EE) to control mice housed in standard laboratory cages. Spines in EE mice are larger and show larger fluctuations in size. PSDs in EE mice shrink during anesthesia and tend to change their nanostructure. Very importantly, changes in spine size were not driven by PSD size changes, or vice versa. Technologically, this is a landmark study, as tracking two different labeled structures in individual synapses at the nanoscale can obviously be applied to a large number of synaptic proteins and organelles, two at a time. Single-color superresolution microscopy is much less useful, as 'puncta in space', without cellular context, are difficult to interpret. This pioneering work is the first proof-of-concept of two-color in-vivo STED and of major importance for the community. Although stochastic processes seem to drive much of the synaptic dynamics under anesthesia, the environment shapes the spine size distribution and affects synaptic dynamics in a lasting fashion.<br /> One major comment:<br /> l.259: "These results suggest that Ctr housed mice undergo stronger morphological changes." This I find a bit misleading. What about: These results suggest that anesthesia induces stronger morphological changes in Ctr housed mice? Altogether, a discussion of the potential effects of anesthesia on spine/PSD dynamics is missing (see e.g. Yang et al., DOI: 10.1371/journal.pbio.3001146). The fact that there was weak correlation between spine head and PSD fluctuation could have something to do with the state of suppressed activity the system was in during imaging. Under conditions of intense processing of visual information, changes might have been more rapid and more tightly correlated. This could be mentioned as a perspective for the future - to visually stimulate the anesthetized animal.

Yes! This idea was hard for me to fully understand since all of my experience with computer music has been relatively compact whether "in the box" or with a mixture of outboard gear. The idea of having to travel from location to location for simply recording a piece sounds strenuous and if any errors were to occur the time to fix them would be much longer.

sea monster indicates water-nymph

When looking at these quotes from non-feminist historians, how is it that the argument of women having their own separate history doesn't concern men? Is this only in conversation with Western History, especially the US? I can think of multiple women throughout history that heavily impacted Western culture in Europe, the first one off the top of my head being Cleopatra of Egypt and her involvement with the Roman Empire.

Can't annotate the comments for some reason, so I am copying the below:

The Jerk talked about getting hit by a bus, etc. I said who cares about code if a guy’s in hospital or there’s a war, we’ve got better things to worry about than a few days of lost code.

I feel the pain. I do. I understand. I get scared. I want to check in early and often, but source control isn’t your personal unlimited undo. When I look at history I don’t want to see rubbish. I want to see snapshots of working code.

Don’t hold back, and don’t go dark, but use your f’ing head. It’s far better to locally integrate multiple times per day and merge back after a few days because guess what, no surprises.

He is definitely dedicated to getting rid of that eye

What a shallow attempt from Marie to look sorrowful for the things she has done, or at least seems to have done as far as Woyzeck knows. The part of the Bible that she's quoting is about a sinful woman who washes Jesus' feet. One of the Pharisees criticizes Jesus for this, and Jesus basically says that since she has a lot of sins to be forgiven, she loves much more. Maybe Marie is saying this in good faith but I have my doubts.

GLOSSARY

Red blood cell/Erythrocyte: contain a metalloprotein, hemoglobin, which serves to bind oxygen molecules to the erythrocyte.

Heme: the portion of hemoglobin that contains iron, and binds the oxygen.

Hemoglobin: serves to bind oxygen molecules to the erythrocyte.

Oxygen dissociation curve: a graph that describes the relationship of partial pressure to the binding of oxygen to heme and its subsequent dissociation from heme.

pO2: partial pressure of oxygen.

O2 saturation: when considering the blood as a whole, the percent of the available heme units that are bound to oxygen at a given time is called hemoglobin saturation.

Bohr effect: a phenomenon that arises from the relationship between pH and oxygen’s affinity for hemoglobin: A lower, more acidic pH promotes oxygen dissociation from hemoglobin.

Oxygen transport: Increasing oxygen transport allows cells to ramp up cellular respiration and thus ATP production, the energy needed to build new structures. (see Figure 3.1)

Carbon dioxide transport: Figure 3.6

Heart/Pumps/Chambers: The circulatory system is a network of vessels—the arteries, veins, and capillaries—and a pump, the heart. In humans, the heart is divided into four chambers: two atria and two ventricles.

Atrium: The right atrium receives deoxygenated blood from the systemic circulation through the major veins: the superior vena cava, which drains blood from the head and from the veins that come from the arms, as well as the inferior vena cava, which drains blood from the veins that come from the lower organs and the legs. The left atrium receives the oxygen-rich blood from the lungs.

Ventricle: The right ventricle contracts, pumping the blood to the lungs for reoxygenation. The left ventricle is where the blood is pumped into the aorta.

Open circulatory system: are found in invertebrate animals in which the circulatory fluid bathes the internal organs directly even though it may be moved about with a pumping heart.

Closed circulatory system: Blood circulates inside blood vessels and circulates unidirectionally from the heart around one of two circulatory routes, then returns to the heart again.

The pelisse, a popular garment most recently revived through the iconic yellow model worn by Ana Taylor-Joy in Autumn de Wilde’s Emma (2020), might be included as a footnote in the twin history of fashion and ecological degradation.

By donning a pelisse, Mrs. Allen and Mrs. Thorpe, whatever their rivalries, were both at the cusp of early nineteenth-century fashion. Austen herself owned at least two pelisses, as historian Hilary Davidson has demonstrated. The pelisse, an overdress, was developed partly in response to the new Empire-period silhouette and partly due the “muslin disease” or influenza that ailed young women wearing fashionable lightweight fabrics in freezing weather.

In the colder months, pelisses could be lined with fur, so Mrs. Allen’s observation that Mrs. Thorpe’s lace is not as handsome would indicate that this scene takes place in the warmer months. The pelisse’s popularity led it to replace the fur cloaks of the earlier eighteenth century. Soon, though, pelisses themselves would be replaced with fur coats, which gained popularity throughout the nineteenth century, reaching a high point in the 1850s. Their popularity was in large part due to new methods of processing fur, which made it more supple (Fashioned 86). The consumption of fur and sealskin jackets, as well as feathers and cotton, throughout the period would lead to the devastation (e.g., India’s cotton industry) of ecosystems (71).

As we read these lines, then, we are reminded, of Austen’s critical eye for the consumer habits of her time. Although her critique here pertains to petty fashion rivalry, when reading about fashion items in her novels, we might find ourselves considering not only how little our fashion rivalries have changed but also how fashion and environmental degradation are historically linked.

For more on the pelisse, the spencer, and muslin, head over to Austenprose to read Hilary Davidson's post on Regency fashion in Emma (2020).

Works Cited

Seems like a very respectful way to address a person. Is she the head of the family? Is her husband dead?

Names at the head too

This paragraph explains that some animals display similar mental and physical reactions to humans, just within a different context. Allowing for psychology experiments to be used with studying animals, who by default, are more willing to participate and at larger scales.

I am having trouble understanding how to get my website/page crawled if it is new? How do I get my new page crawled in order to benefit?

According to the World History Encyclopedia, "Brahma is the Hindu creator god" (Cartwright). Brahma is one third of the great Hindu gods along side Shiva and Vishnu. Compared to the other two gods Brahma isn't worshipped nearly as much despite being responsible for creation. This is explained by one Hindu myth in which Brahma becomes so obsessed with a woman named Shatarupa that he grew five heads so he could always look at her. Shiva put an end to this by cutting off Brahma's fifth head and cursed Brahma to not be worshipped by people as repentance.

Upon reading this quote, I was curious as to exactly how The Bhagavad Gita imprinted and shaped Gandhi. Through the website American Vedantist, I was able to expand further upon this question. One of the first thing it states is that in regards to Gandhi, the Gita "was not only his constant companion, it was his “eternal mother” whom he esteemed even more than his earthly mother." The website goes on to say that after reading the Gita, Gandhi decided to study the Bible and even felt there was many parallels between the two. Specifically the sentiment of turning evil into good and giving oneself over to God. Proving that the Gita not only taught Gandhi many things, but also ignited a sense of wonderment in him about other religions and their teachings. One of the things Gandhi seemed to love most about the Gita was that it "appeals to both the head and the heart." Meaning that although it is "analytical", it also is deeply moving and empathetic. The American Vedantist also explains how Gandhi was so inspired by the Gita that he began implementing it in his daily life, stating, "Since he firmly believed in “being the change you want to see in the world,” he himself put into practice the Gita’s ideals of the yamas and niyamas such as truth, ahimsa (nonviolence, brahmacharya (celibacy), non-possession, and others." Through this source, it has become apparent exactly how moved and transformed Gandhi was by the Gita and how monumental he found it as a practice. This is specifically significant because it shows how intellectually and spiritually stimulating the Gita can be and how its contents can teach and shape minds.

Source: https://americanvedantist.org/2014/articles/mahatma-gandhi-and-the-bhagavad-gita/

这就是free的原因

Author Response:

Reviewer #1 (Public Review):

This manuscript is a follow-up of an earlier manuscript using the LRET technology, but extends the study by identifying a new "open" state and using experimental distance constraints to provide molecular models of the different states. All in all, the manuscript is well written, the experiments are described in sufficient details and experiments are done to high quality with the appropriate controls. The data corroborate the partially open state as published early, but extend the study to a second, open state. It is very good to see that the observed states are not only present in the catalytic head but the authors also use the full-length protein and find similar states. However, in the present manuscript, I find the conceptual advance with respect to the mechanism of MR somewhat limited. The authors curiously do not include any DNA in their structural studies, so the observed states are only relevant for the free MR complex, but not the complex "in action" bound to DNA where quite different conformations might occur. As one consequence, the structurally proposed states do not directly correlate with the functional nuclease states that are necessarily bound to DNA. Perhaps as a consequence, in the author's model, Rad50 is merely a gate-keeper for Mre11, but this is not the case as recent structural work shows that Rad50 forms a joint DNA binding surface with Mre11. Likewise, biochemical studies are done with physiologically unclear/less relevant 3' exonuclease activity only, but not with the physiological important 5' endonuclease activity. In my opinion, it is important for a publication in a journal with the scope of eLife and addressed to a broad audience to provide structural analysis in the presence of DNA and validate the structures using the endonuclease activity.

We thank the reviewer for these comments.

Specific recommendations:

1) Instead of using the physiological unclear exo activity, I suggest to use the more relevant endonuclease activity to validate the mutants.

We now include plate- and gel-based endonuclease activity assays, using a variety of DNA substrates, for all of the validation mutants. We have expanded Fig. 3 and included a new Supplemental Fig. S4 to show this data. We have expanded the Results section of the modified manuscript to present and discuss these findings.

2) Since the authors mutated one side of newly identified/proposed salt-bridges, I also suggest to test whether a charge reversal on both sides of the salt bridge rescues the phenoptype. I find this important because MR has quite many conformations, and mutating a single residue might not unambiguously validate the proposed conformation, a rescue by a charge reversed salt bridge is much stronger.

We thank the Reviewer for this suggested experiment, and we tried to do it. Although we were successful in generating each of the charge reversal mutations in full-length Rad50, all of the mutants unfortunately had issues with either expression or purification. For example, the 6x His-tag for several of the new Rad50 mutants was not accessible to the TEV protease for cleavage indicating that the mutated proteins were mis-folded (the His-tag of the WT full-length Rad50 is readily cleaved off by TEV). As such, we did not feel confident using these proteins in subsequent MR activity assays.

3) Since all LRET experiments are done without DNA, the authors do not capture relevant DNA processing states and comparison of structural (w/o DNA) and biochemical data (w/ DNA) is not really justified, in my opinion. Also, they might miss critical conformations. Is there a technical reason for not including DNA in the LRET studies?

We have collected LRET data on ATP-bound MRNBD in the presence of a hairpin DNA or a ssDNA as substrates. We still observe three states in the presence of both DNAs; however, the open conformation appears to be slightly more compact (i.e., closer distance between Rad50NBD protomers) in the presence of ssDNA. As described above, we have added to the Results section of the modified manuscript and included a new figure (Fig. 4) describing these data.

4) If the authors want to claim processive movement coupled to partially open/open state interchanges, they should provide experimental evidence. Where would the energy come from for such a movement, this is not clear from the model?

On the surface, ATP hydrolysis by Rad50 would seem to be the perfect source of energy for the conformational changes that drive the sequential and/or processive nuclease functions of the MR complex. However, the D313K mutant is not as good at ATP hydrolysis as the wild type enzyme (Fig. 3E), and the data in Fig. 3 and Supplemental Fig. S4 clearly demonstrate that D313K is by far the best nuclease. If the free energy for the movement does not come from ATP hydrolysis, where else could it come? Richardson and co-workers measured a release of -5.3 kcal mol-1 (-22.17 kJ mol-1) of free energy for the hydrolysis of a DNA phosphodiester bond (Dickson, K.S. et al. 2000 J. Biol. Chem. 275:15828–15831). Thus, the free energy released from the Mre11 nuclease activity could be the driving force for the conformational changes we propose. We have made this point in the Discussion of the revised manuscript.

5) The SAXS data for the "open" state do not validate the model, in my opinion. Experimental data and model are not inconsistent, but the curve looks to me as if the open state is perhaps much more flexible (i.e. an ensemble) or extended? Please comment.

We agree with the Reviewer on this point. We have updated Fig. 5A (original Fig. 4) to include the two-state fits to the experimental SAXS data. Although the multi-state fit to the apo MR SAXS data is better than any of the single model fits (2 = 1.05 vs. 1.26, respectively), the 2 is still larger than the multi-state fits to the ATP-bound MR SAXS data. Thus, an additional unobserved conformation (perhaps the so-called “extended”) might be present in solution for apo MRNBD. We have added a sentence to the revised manuscript with this point.

To explore the possibility that the previously described “extended” structure might be contributing to the SAXS data, we built a model of the extended conformation of Pf MRNBD based on the Tm MRNBD structure (PDB: 3QG5) and used Rosetta to connect the coiled-coils and add the linker to the Mre11 HLH. When this model was used in the FoXS calculations for the apo SAXS data, the 2 was 4.77 (versus 2 of 1.26 for the “open” model). The MultiFoXS two-state fit gave 90% open + 10% closed (2 of 1.04), whereas the three-state fit gave 65% open + 20% extended + 15% part open (2 of 0.84). Thus, there is some improvement when using the extended model, but since that model is not measurable in our LRET experiments and we are unsure of its validity as we have modeled it for Pf MR, we have chosen to omit it from the analysis.

6) Distance errors for the full complex are much smaller than those for the catalytic module only (Fig. 1d). Does that mean that the full complex is more rigid, please comment?

From looking at the data presented in Fig. 1D, it is logical to suggest that the full-length complex may be more rigid or better defined by the LRET data. However, we note that there are nearly as many distance errors which are similar between MRNBD and MR as there are MR errors less than MRNBD. And although many are not identical, most are of a similar magnitude. Because of this, we do not think the variations in LRET errors are systematic (i.e., related to a more rigid full-length complex).

Let me get this straight, they got drunk, had a little drunk sex, and now... what? All is doomed?<br /> Huh. Well. I guess I better flee myself then, because...but no! The shame! I must instead fall upon my sword! As soon as I chop the head off this bird.

Think about this narrator. Is he supposed to be in Alan's shoes? Is he an observer? Judging by the 'ain't', this is sort of supposed to be through Alan's eyes, so keep in the back of your head the details the details that Alan would focus on (as opposed to what a rational person would focus on).

Testing this out

the only vocabulary that is slightly "confusing" in this article is certain acronyms that I personally don't know off the top of my head

References back to the Haitian push for freedom by those who "tend the plough." I think with the use of "tend his plough" there's a sense of legacy.

Whether you are away and idle or you are dead

After git rebase -i HEAD~N or

git rebase -i [commit-hash] your editor of choice will pop up, showing the list of commits you want to merge and you'll mark a commit as squashable by changing the word pick into squash next to it (or s for brevity, as stated in the comments).

git rebase --interactive HEAD~N # or -i where N is the number of commits you want to join, starting from the most recent one.