Interesting. This is a general problem with historical movies. There is almost no such thing as objectivity. By making a movie, you make choices, you select what makes the cut and what doesn't. and by doing so, you form a certain image of the prophet, in this case. The free personal impressions of Majid are in fact a way to represent a certain image of Muhammad.

From what I have read, Majid is blamed for putting forward a Shi'ite Muhammed forward in the movie. Perhaps his free personal impressions are expressed in this regard?

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

This is an interesting and potentially important paper, which however has some deficiencies.

Strengths:

A significant amount of potentially useful data.

Weaknesses:

One issue is a confusion of thermal stability with solubility. While thermal stability of a protein is a thermodynamic parameter that can be described by the Gibbs-Helmholtz equation, which relates the free energy difference between the folded and unfolded states as a function of temperature, as well as the entropy of unfolding. What is actually measured in PISA is a change in protein solubility, which is an empirical parameter affected by a great many variables, including the presence and concentration of other ambient proteins and other molecules. One might possibly argue that in TPP, where one measures the melting temperature change ∆Tm, thermal stability plays a decisive or at least an important role, but no such assertion can be made in PISA analysis that measures the solubility shift.

We completely agree with the insightful comment from the reviewer and we are very grateful that the point was raised. Our goal was to make this manuscript easily accessible to the entire scientific community, not just experts in the field. In an attempt to simplify the language, we likely also simplified the underlying physical principles that these assays exploit. In defense of our initial manuscript, we did state that PISA measures “a fold change in the abundance of soluble protein in a compound-treated sample vs. a vehicle-treated control after thermal denaturation and high-speed centrifugation.” Despite this attempt to accurately communicate the reviewer’s point, we seem to have not been sufficiently clear. Therefore, we tried to further elaborate on this point and made it clear that we are measuring differences in solubility and interpreting these differences as changes in thermal stability. 

In the revised version of the manuscript, we elaborated significantly on our original explanation. The following excerpt appears in the introduction (p. 3):

“So, while CETSA and TPP measure a change in melting temperature (∆TM), PISA measures a change in solubility (∆SM).  Critically, there is a strong correlation between ∆TM and ∆SM, which makes PISA a reliable, if still imperfect, surrogate for measuring direct changes in protein thermal stability (Gaetani et al., 2019; Li et al., 2020). Thus, in the context of PISA, a change in protein thermal stability (or a thermal shift) can be defined as a fold change in the abundance of soluble protein in a compoundtreated sample vs. a vehicle-treated control after thermal denaturation and high-speed centrifugation. Therefore, an increase in melting temperature, which one could determine using CETSA or TPP, will lead to an increase in the area under the curve and an increase in the soluble protein abundance relative to controls (positive log2 fold change). Conversely, a decrease in melting temperature will result in a decrease in the area under the curve and a decrease in the soluble protein abundance relative to controls (negative log2 fold change).”

And the following excerpt appears in the results section (p. 4): 

“In a PISA experiment, a change in melting temperature or a thermal shift is approximated as a

significant deviation in soluble protein abundance following thermal melting and high-speed centrifugation. Throughout this manuscript, we will interpret these observed alterations in solubility as changes in protein thermal stability. Most commonly this is manifested as a log2 fold change comparing the soluble protein abundance of a compound treated sample to a vehicle-treated control (Figure 1 – figure supplement 1A).”

We have now drawn a clear distinction between what we were actually measuring (changes in solubility) and how we were interpreting these changes (as thermal shifts). We trust that the Reviewer will agree with this point, as they rightly claim that many of the observations presented in our work, which measures thermal stability, indirectly, are consistent with previous studies that measured thermal stability, directly. Again, we thank the reviewer for raising the point and feel that these changes have significantly improved the manuscript. 

Another important issue is that the authors claim to have discovered for the first time a number of effects well described in prior literature, sometimes a decade ago. For instance, they marvel at the differences between the solubility changes observed in lysate versus intact cells, while this difference has been investigated in a number of prior studies. No reference to these studies is given during the relevant discussion.

We thank the reviewer for raising this point. Our aim with this paper was to test the proficiency of this assay in high-throughput screening-type applications. We considered these observations as validation of our workflow, but admit that our choice of wording was not always appropriate and that we should have included more references to previous work. It was certainly never our intention to take credit for these discoveries. Therefore, we were more than happy to include more references in the revised version. We think that this makes the paper considerably better and will help readers better understand the context of our study.  

The validity of statistical analysis raises concern. In fact, no calculation of statistical power is provided.

As only two replicates were used in most cases, the statistical power must have been pretty limited. Also, there seems to be an absence of the multiple-hypothesis correction.

We agree with the reviewer that a classical comparison using a t-test would be underpowered comparing all log2 normalized fold changes. We know from the data and our validation experiments that stability changes that generate log2 fold changes of 0.2 are indicative of compound engagement. When we use 0.2 to calculate power for a standard two-sample t-test with duplicates, we estimated this to have a power of 19.1%. Importantly, increasing this to n=3 resulted in a power estimate of only 39.9%, which would canonically still be considered to be underpowered. Thus, it is important to note that we instead use the distribution of all measurements for a single protein across all compound treatments to calculate standard deviations (nSD) as presented in this work. Thus, rather than a 2-by-2 comparison, we are comparing two duplicate compound treatments to 94 other compound treatments and 18 DMSO vehicle controls. Moreover, we are using this larger sample set to estimate the sampling distribution. Estimating this with a standard z-test would result in a p-value estimate <<< 0.0001 using the population standard deviation. Additionally, rather than estimate an FDR using say a BenjaminiHochberg correction, we estimated an empirical FDR for target calls based on applying the same cutoffs to our DMSO controls and measuring the proportion of hits called in control samples at each set of thresholds. Finally, we note that several other PISA-based methods have used fold-change thresholds similar to, or less than, those employed in this work (PMID: 35506705, 36377428, 34878405, 38293219).  

Also, the authors forgot that whatever results PISA produces, even at high statistical significance, represent just a prediction that needs to be validated by orthogonal means. In the absolute majority of cases such validation is missing.

We appreciate this point and we can assure the reviewer that this point was not lost on us. To this point, we state throughout the paper that the primary purpose of this paper was to execute a chemical screen. Furthermore, we do not claim to present a definitive list of protein targets for each compound. Instead, our intention is to provide a framework for performing PISA studies at scale. In total, we quantified thousands of changes and feel that it would be unreasonable to validate the majority of these cases. Instead, as has been done for CETSA (PMID: 34265272), PISA (PMID: 31545609), and TPP (PMID: 25278616) experiments before, we chose to highlight a few examples and provide a reasonable amount of validation for these specific observations. In Figure 2, we show that two screening compounds—palbociclib and NVP-TAE-226—have a similar impact on PLK1 solubility as the two know PLK1 inhibitors. We then assay each of these compounds, alongside BI 2536, and show that the same compounds that impact the solubility of PLK1, also inhibit its activity in cell-based assays. Finally, we model the structure of palbociclib (which is highly similar to BI 2536) in the PLK1 active site. In Figure 4, we show that AZD-5438 causes a change in solubility of RIPK1 in cell- and lysate-based assays to a similar extent as other compounds known to engage RIPK1. We then test these compounds in cellbased assays and show that they are capable of inhibiting RIPK1 activity in vivo. Finally, in Figure 5, we show that treatment with tyrosine kinase inhibitors and AZD-7762 result in a decrease in the solubility of CRKL. We showed that these compounds, specifically, prevented the phosphorylation of CRKL at Y207. Next, we show that AZD-7762, impacts the thermal stability of tyrosine kinases in lysate-based PISA. Finally, we performed phosphoproteomic profiling of cells treated with bafetinib and AZD-7762 and find that the abundance of many pY sites is decreased after treatment with each compound. It is also worth stating that an important goal of this study was to determine the proficiency of these methods in identifying the targets of each compound. We do not feel that comprehensive validation of the “absolute majority of cases” would significantly improve this manuscript. 

Finally, to be a community-useful resource the paper needs to provide the dataset with a user interface so that the users can data-mine on their own.

We agree and are working to develop an extensible resource for this. Owing to the size and complexities there, that work will need to be included in a follow-up manuscript. For now, we feel that the supplemental table we provide can be easily navigated the full dataset. Indeed, this has been the main resource that we have been emailed about since the preprint was first made public. We are glad that the Reviewer considers this dataset to be a highly valuable resource for the scientific community.  

Reviewer #2 (Public Review):

Summary:

Using K562 (Leukemia) cells as an experimental model, Van Vracken et. al. use Thermal Proteome Profiling (TPP) to investigate changes in protein stability after exposing either live cells or crude cell lysates to a library of anti-cancer drugs. This was a large-scale and highly ambitious study, involving thousands of hours of mass spectrometry instrument time. The authors used an innovative combination of TPP together with Proteome Integral Solubility Alternation (PISA) assays to reduce the amount of instrument time needed, without compromising on the amount of data obtained.

The paper is very well written, the relevance of this work is immediately apparent, and the results are well-explained and easy to follow even for a non-expert. The figures are well-presented. The methods appear to be explained in sufficient detail to allow others to reproduce the work.

We thank the reviewer. One of our major goals was to make these assays and the resulting data approachable, especially for non-experts. We are glad that this turned out to be the case. 

Strengths:

Using CDK4/6 inhibitors, the authors observe strong changes in protein stability upon exposure to the drug. This is expected and shows their methodology is robust. Further, it adds confidence when the authors report changes in protein stability for drugs whose targets are not well-known. Many of the drugs used in this study - even those whose protein targets are already known - display numerous offtarget effects. Although many of these are not rigorously followed up in this current study, the authors rightly highlight this point as a focus for future work.

Weaknesses:

While the off-target effects of several drugs could've been more rigorously investigated, it is clear the authors have already put a tremendous amount of time and effort into this study. The authors have made their entire dataset available to the scientific community - this will be a valuable resource to others working in the fields of cancer biology/drug discovery.

We agree with the reviewer that there are more leads here that could be followed and we look forward to both exploring these in future work and seeing what the community does with these data.

Reviewer #3 (Public Review):

Summary:

This work aims to demonstrate how recent advances in thermal stability assays can be utilised to screen chemical libraries and determine the compound mechanism of action. Focusing on 96 compounds with known mechanisms of action, they use the PISA assay to measure changes in protein stability upon treatment with a high dose (10uM) in live K562 cells and whole cell lysates from K562 or HCT116. They intend this work to showcase a robust workflow that can serve as a roadmap for future studies.

Strengths:

The major strength of this study is the combination of live and whole cell lysates experiments. This allows the authors to compare the results from these two approaches to identify novel ligand-induced changes in thermal stability with greater confidence. More usefully, this also enables the authors to separate the primary and secondary effects of the compounds within the live cell assay.

The study also benefits from the number of compounds tested within the same framework, which allows the authors to make direct comparisons between compounds.

These two strengths are combined when they compare CHEK1 inhibitors and suggest that AZD-7762 likely induces secondary destabilisation of CRKL through off-target engagement with tyrosine kinases.

Weaknesses:

One of the stated benefits of PISA compared to the TPP in the original publication (Gaetani et al 2019) was that the reduced number of samples required allows more replicate experiments to be performed. Despite this, the authors of this study performed only duplicate experiments. They acknowledge this precludes the use of frequentist statistical tests to identify significant changes in protein stability. Instead, they apply an 'empirically derived framework' in which they apply two thresholds to the fold change vs DMSO: absolute z-score (calculated from all compounds for a protein) > 3.5 and absolute log2 fold-change > 0.2. They state that the fold-change threshold was necessary to exclude nonspecific interactors. While the thresholds appear relatively stringent, this approach will likely reduce the robustness of their findings in comparison to an experimental design incorporating more replicates. Firstly, the magnitude of the effect size should not be taken as a proxy for the importance of the effect.

They acknowledge this and demonstrate it using their data for PIK3CB and p38α inhibitors (Figures 2BC). They have thus likely missed many small, but biologically relevant changes in thermal stability due to the fold-change threshold. Secondly, this approach relies upon the fold-changes between DMSO and compound for each protein being comparable, despite them being drawn from samples spread across 16 TMT multiplexes. Each multiplex necessitates a separate MS run and the quantification of a distinct set of peptides, from which the protein-level abundances are estimated. Thus, it is unlikely the fold changes for unaffected proteins are drawn from the same distribution, which is an unstated assumption of their thresholding approach. The authors could alleviate the second concern by demonstrating that there is very little or no batch effect across the TMT multiplexes. However, the first concern would remain. The limitations of their approach could have been avoided with more replicates and the use of an appropriate statistical test. It would be helpful if the authors could clarify if any of the missed targets passed the z-score threshold but fell below the fold-change threshold.

The authors use a single, high, concentration of 10uM for all compounds. Given that many of the compounds likely have low nM IC50s, this concentration will often be multiple orders of magnitude above the one at which they inhibit their target. This makes it difficult to assess the relevance of the offtarget effects identified to clinical applications of the compounds or biological experiments. The authors acknowledge this and use ranges of concentrations for follow-up studies (e.g. Figure 2E-F). Nonetheless, this weakness is present for the vast bulk of the data presented.

We agree that there is potential to drive off-target effects at such high-concentrations. However, we note that the concentration we employ is in the same range as previous PISA/CETSA/TPP studies. For example, 10 µM treatments were used in the initial descriptions of TPP (Savitski et al., 2014) and PISA (Gaetani et al., 2019). We also note that temperature may affect off-rates and binding interactions (PMID: 32946682) potentiating the need to use compound concentrations to overcome these effects.

Additionally, these compounds likely accumulate in human plasma/tissues at concentrations that far exceed the compound IC50 values. For example, in patients treated with a standard clinical dose of ribocicilb, the concentration of the compound in the plasma fluctuates between 1 µM and 10 µM. (Bao, X., Wu, J., Sanai, N., & Li, J. (2019). Determination of total and unbound ribociclib in human plasma and brain tumor tissues using liquid chromatography coupled with tandem mass spectrometry. Journal of pharmaceutical and biomedical analysis, 166, 197–204. https://doi.org/10.1016/j.jpba.2019.01.017)

The authors claim that combining cell-based and lysate-based assays increases coverage (Figure 3F) is not supported by their data. The '% targets' presented in Figure 3F have a different denominator for each bar. As it stands, all 49 targets quantified in both assays which have a significant change in thermal stability may be significant in the cell-based assay. If so, the apparent increase in % targets when combining reflects only the subsetting of the data. To alleviate this lack of clarity, the authors could update Figure 3F so that all three bars present the % targets figure for just the 60 compounds present in both assays.

We spent much time debating the best way to present this data, so we are grateful for the feedback. Consistent with the Reviewer’s suggestion, we have included a figure that only considers the 60 compounds for which a target was quantified in both cell-based and lysate-based PISA (now Figure 3E). In addition, we included a pie chart that further illustrates our point (now Figure 3 – figure supplement 2A). Of the 60 compounds, there were 37 compounds that had a known target pass as a hit using both approaches, 6 compounds that had a known target pass as a hit in only cell-based experiments, and 6 compounds that had a known target pass as a hit in only lysate-based experiments.

Within the Venn diagram, we also included a few examples of compounds that fit into each category. Furthermore, we highlighted two examples of compound-target pairs that pass as a hit with one approach, but not the other (Figure 3 – figure supplement 2B,C). We would also like to refer the reviewer to Figure 4D, which indicates that BRAF inhibitors cause a significant change in BRAF thermal stability in lysates but not cells. 

Aims achieved, impact and utility:

The authors have achieved their main aim of presenting a workflow that serves to demonstrate the potential value of this approach. However, by using a single high dose of each compound and failing to adequately replicate their experiments and instead applying heuristic thresholds, they have limited the impact of their findings. Their results will be a useful resource for researchers wishing to explore potential off-target interactions and/or mechanisms of action for these 96 compounds, but are expected to be superseded by more robust datasets in the near future. The most valuable aspect of the study is the demonstration that combining live cell and whole cell lysate PISA assays across multiple related compounds can help to elucidate the mechanisms of action.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

More specifically:

P 1 l 20, we quantified 1.498 million thermal stability measurements.

It's a staggering assertion, and it takes some reading to realize that the authors mean the total number of proteins identified and quantified in all experiments. But far from all of these proteins were quantified with enough precision to provide meaningful solubility shifts.

We can assure the reviewer that we were not trying to deceive the readers. We stated ‘1.498 million thermal stability measurements.’ We did not say 1.498 million compound-specific thermal stability shifts.’ We assume that most readers will appreciate that the overall quality of the measurements will be variable across the dataset, e.g., in any work that describes quantitation of thousands of proteins in a proteomics dataset. In accordance with the Reviewer’s suggestion, we have weakened this statement. The revised version of the manuscript now reads as follows (p. 1): 

“Taking advantage of this advance, we quantified more than one million thermal stability measurements in response to multiple classes of therapeutic and tool compounds (96 compounds in living cells and 70 compounds in lysates).”

P 7 l 28. We observed a large range of thermal stability measurements for known compound-target pairs, from a four-fold reduction in protein stability to a four-fold increase in protein stability upon compound engagement (Figure 2A).

PISA-derived solubility shift cannot be interpreted simply as a "four-fold reduction/increase in protein stability".

We thank the Reviewer for highlighting this specific passage and agree that it was worded poorly. As such, we have modified the manuscript to the following (p. 8): 

“We observed a large range of thermal stability measurements for known compound-target pairs, from a four-fold reduction in protein solubility after thermal denaturation to a four-fold increase in protein solubility upon compound engagement (Figure 2A).”

P 8, l 6. Instead, we posit that maximum ligand-induced change in thermal stability is target-specific.

Yes, that's right, but this has been shown in a number of prior studies.

We agree with the reviewer and accept that we made a mistake in how we worded this sentence, which we regret upon reflection. As such, we have modified this sentence to the following:

“Instead, our data appears to be consistent with the previous observation that the maximum ligandinduced change in thermal stability is target-specific (Savitski et al., 2014; Becher et al., 2016).”

P 11 l 7. Combining the two approaches allows for greater coverage of the cellular proteome and provides a better chance of observing the protein target for a compound of interest. In fact, the main difference is that in-cell PISA provides targets in cases when the compound is a pro-drug that needs to be metabolically processed before engaging the intended target. This has been shown in a number of prior studies, but not mentioned in this manuscript.

While our study was not focused on the issue of pro-drugs, this is an important point and we would be happy to re-iterate it in our manuscript. We thank the Reviewer for the suggestion and have modified the manuscript to reflect this point (p. 19): 

“Cell-based studies, on the other hand, have the added potential to identify the targets of pro-drugs that must be metabolized in the cell to become active and secondary changes that occur independent of direct engagement (Savitski et al., 2014; Franken et al., 2015; Almqvist et al., 2016; Becher et al., 2016; Liang et al., 2022).”

While we are happy to make this change, we also would like to point out that the reviewer’s assertions that, “the main difference is that in-cell PISA provides targets in cases when the compound is a prodrug that needs to be metabolically processed before engaging the intended target” also may not fully capture the nuances of protein engagement effectors in the cellular context. Thus, we believe it is important to highlight the ability of cell-based assays to identify secondary changes in thermal stability.  

P 11 l 28. These data suggest that the thermal destabilization observed in cell-based experiments might stem from a complex biophysical rearrangement. That's right because it is not about thermal stability, but about protein solubility which is much affected by the environment.

We agree that the readout of solubility is an important caveat for nearly every experiment in the family of assays associated with ‘thermal proteome profiling’. Inherently complex biophysical arrangements could affect the inherent stability and solubility of a protein or complex. Thus, we would be happy to make the following change consistent with the reviewer’s suggestion (p. 12): 

“These data suggest that the decrease in solubility observed in cell-based experiments might stem from a complex biophysical rearrangement.”

P 12 l 7 A). Thus, certain protein targets are more prone to thermal stability changes in one experimental setting compared to the other. Same thing - it's about solubility, not stability.

We thank the Reviewer for the recommendation and have modified the revised manuscript as follows (p. 13):

“Thus, certain protein targets were more prone to solubility (thermal stability) changes in one experimental setting compared to the other (Huber et al., 2015).”

P13 l 15. While the data suggests that cell- and lysate-based PISA are equally valuable in screening the proteome for evidence of target engagement... No, they are not equally valuable - cell-based PISA can provide targets of prodrugs, which lysate PISA cannot.

We have removed this sentence to avoid any confusion. We will not place any value judgments on the two approaches. 

P 18 l 10. In general, a compound-dependent thermal shift that occurs in a lysate-based experiment is almost certain to stem from direct target engagement. That's true and has been known for a decade. Reference needed.

We recognize this oversight and would be happy to include references. The revised manuscript reads as follows: 

“In general, a compound-dependent thermal shift that occurs in a lysate-based experiment is almost certain to stem from direct target engagement (Savitski et al., 2014; Becher et al., 2016). This is because cell signaling pathways and cellular structures are disrupted and diluted. Cell-based studies, on the other hand, have the added potential to identify the targets of pro-drugs that must be metabolized in the cell to become active and secondary changes that occur independent of direct engagement (Savitski et al., 2014; Franken et al., 2015; Almqvist et al., 2016; Becher et al., 2016; Liang et al., 2022).”

P 18 l 29. the data seemed to indicate that the maximal PISA fold change is protein-specific. Therefore, a log2 fold change of 2 for one compound-protein pair could be just as meaningful as a log2 fold change of 0.2 for another. This is also not new information.

We again appreciate the Reviewer for highlighting this oversight. The revised manuscript reads as follows: 

“Ultimately, the data seemed to be consistent with previous studies that indicate the maximal change in thermal stability in protein specific (Savitski et al., 2014; Becher et al., 2016; Sabatier et al., 2022). Therefore, a log2 fold change of 2 for one compound-protein pair could be just as meaningful as a log2 fold change of 0.2 for another.”

P 19 l 5. Specifically, the compounds that most strongly impacted the thermal stability of targets, also acted as the most potent inhibitors. I wish this was true, but this is not always so. For instance, in Nat Meth 2019, 16, 894-901 it was postulated that large ∆Tm correspond to biologically most important sites ("hot spots") - the idea that was later challenged and largely discredited in subsequent studies.

Indeed, we agree with the Reviewer that there may be no essential connection between these. Rather, we are simply drawing conclusions from observations within the presented dataset. 

Saying nothing about the work presented in the paper that the reviewer notes above, the referenced definition is also more nuanced “…we hypothesized that ‘hotspot’ modification sites identified in this screen (namely, those significantly shifted relative to the unmodified, bulk and even other phosphomodiforms of the same protein) may represent sites with disproportionate effects on protein structure and function under specific cellular conditions.” Indeed, in the response to that work, Potel et al. (https://doi.org/10.1038/s41592-021-01177-5) “agree with the premise of the Huang et al. study that phosphorylation sites that have a significant effect on protein thermal stability are more likely to be functionally relevant, for example, by modulating protein conformation, localization and protein interactions.” 

Anecdotally, we also speculate that if we observe proteome engagement for two compounds (let’s say two ATP-competitive kinase inhibitors) that bind in the same pocket (let’s say the ATP binding site) and one causes a greater change in solubility, then it is reasonable to assume that it is a stronger evidence and we see evidence supporting this claim in Figure 2, Figure 3, Figure 4, and Figure 5.

It is also important to point out that previous work has also made similar points. This is highlighted in a review article by Mateus et al. (10.1186/s12953-017-0122-4). The authors state, “To obtain affinity estimates with TPP, a compound concentration range TPP (TPP-CCR) can be performed. In TPPCCR, cells are incubated with a range of concentrations of compound and heated to a single temperature.” In support of this claim, the authors reference two papers—Savitski et al., 2014 and Becher et al., 2016. We have updated this section in the revised manuscript (p. 20): 

“While the primary screen was carried out at fixed dose, the increased throughput of PISA allowed for certain compounds to be assayed at multiple doses in a single experiment. In these instances, there was a clear dose-dependent change in thermal stability of primary targets, off-targets, and secondary targets. This not only helped corroborate observations from the primary screen, but also seemed to provide a qualitative assessment of relative compound potency in agreement with previous studies (Savitski et al., 2014; Becher et al., 2016; Mateus et al., 2017). Specifically, the compounds that most strongly impacted the thermal stability of targets, also acted as the most potent inhibitors. In order to be a candidate for this type of study, a target must have a large maximal thermal shift (magnitude of log2 fold change) because there must be a large enough dynamic range to clearly resolve different doses.”

Also, the compound efficacy is strongly dependent upon the residence time of the drug, which may or may not correlate with the PISA shift. Also important is the concentration at which target engagement occurs (Anal Chem 2022, 94, 15772-15780).

In our study, the time and concentration of treatment and was fixed for all compounds at 30 minutes and 10 µM, respectively. Therefore, we do not believe these parameters will affect our conclusions.  

P 19 l 19. For example, we found that the clinically-deployed CDK4/6 inhibitor palbociclib is capable of directly engaging and inhibiting PLK1. This is a PISA-based prediction that needs to be validated by orthogonal means.

As we demonstrate in this work, the PISA assays serve as powerful screening methods, thus we agree that validation is important for these types of studies. To this end, we show the following:  

• Proteomics: Palbociclib causes a decrease in solubility following thermal melting in cells.

• Chemical Informatic: Palbociclib is structurally similar to BI 2536.

• Protein informatics: Modeling of palbociclib in empirical structures of the PLK1 active site generates negligible steric clashes. 

• Biochemical: Palbociclib inhibits PLK1 activity in cells.

We have changed this text to the following to clarify these points:

“For example, we found that the clinically-deployed CDK4/6 inhibitor palbociclib has a dramatic impact on PLK1 thermal stability in live cells, is capable of inhibiting PLK1 activity in cell-based assays, and can be modelled into the PLK1 active site.”

Reviewer #2 (Recommendations For The Authors):

I am wondering why the authors chose to use K562 (leukaemia) cells in this work as opposed to a different cancer cell line (HeLa? Panc1?). It would be helpful if the authors could present some rationale for this decision.

This is a great question. Two reasons really. First, they are commonly used in various fields of research, especially previous studies using proteome-wide thermal shift assays (PMID: 25278616, 32060372) and large scale chemical perturbations screens (PMID: 31806696). Second, they are a suspension line that makes executing the experiments easier because they do not need to be detached from a plate prior to thermal melting. We think this is a valuable point to make in the manuscript, such that non-experts understand this concept. We tried to communicate this succinctly in the revised manuscript, but would be happy to elaborate further if the Reviewer would like us to. 

“To enable large-scale chemical perturbation screening, we first sought to establish a robust workflow for assessing protein thermal stability changes in living cells. We chose K562 cells, which grow in suspension, because they have been frequently used in similar studies and can easily be transferred from a culture flask to PCR tubes for thermal melting (Savitski et al., 2014; Jarzab et al., 2020).”

I note that integral membrane proteins are over-represented among targets for anti-cancer therapeutics. To what extent is the membrane proteome (plasma membrane in particular) identified in this work? After examining the methods, I would expect at least some integral membrane proteins to be identified. Do the authors observe any differences in the behaviour of water-soluble proteins versus integral membrane proteins in their assays? It would be helpful if the authors could comment on this in a potential revision.

We agree this is an important point when considering the usage of PISA and thermal stability assays in general for specific classes of therapeutics. To address this, we explored what effect the analysis of thermal stability/solubility had on the proportion of membrane proteins in our data (Author response image 1). Annotations were extracted from Uniprot based on each protein being assigned to the “plasma membrane” (07/2024). We quantified 1,448 (16.5% of total proteins) and 1,558 (17.3% of total proteins) membrane proteins in our cell and lysate PISA datasets, respectively. We also compared the proportion of annotated proteins in these datasets to a recent TMTpro dataset (Lin et al.; PMID: 38853901) and found that the PISA datasets recovered a slightly lower proportion of membrane proteins (~17% in PISA versus 18.9% in total proteome analysis). Yet, we note that we expect more membrane proteins in urea/SDS based lysis methods compared to 0.5% NP-40 extractions.

Author response image 1.

We were not able to find an appropriate place to insert this data into the manuscript, so we have left is here in the response. If the Reviewer feels strongly that this data should be included in the manuscript, we would be happy to include these data.  

A final note: I commend the authors for making their full dataset publicly available upon submission to this journal. This data promises to be a very useful resource for those working in the field.

We thank the Reviewer for this and note that we are excited for this data to be of use to the community.

Reviewer #3 (Recommendations For The Authors):

There is no dataset PDX048009 in ProteomeXchange Consortium. I assume this is because it's under an embargo which needs to be released.

We can confirm that data was uploaded to ProteomeXchange.

MS data added to the manuscript during revisions was submitted to ProteomeXchange with the identifier – PDX053138.

Page 9 line 5 refers to 59 compounds quantified in both cell-based and lysate-based, but Figure 3E shows 60 compounds quantified in both. I believe these numbers should match.

We thank the Reviewer for catching this. In response to critiques from this Reviewer in the Public Review, we re-worked this section considerably. Please see the above critique/response for more details. 

Page 10, lines 26-28: It would help the reader if some of the potential 'artefactual effects of lysatebased analyses' were described briefly.

We thank the Reviewer for raising this point. The truth is, that we are not exactly sure what is happening here, but we know that, at least, for vorinostat, this excess of changes in lysate-based PISA is consistent across experiments. We also do not see pervasive issues within the plexes containing these compounds. Therefore, we do not think this is due to a mistake or other experimental error. We hypothesize that the effect might result from a change in pH or other similar property that occurs upon addition of the molecule, though we note that we have previously seen that vorinostat can induce large numbers of solubility changes in a related solvent shift assays (doi: 10.7554/eLife.70784). We have modified the text to indicate that we do not fully understand the reason for the observation (p. 11):

“It is highly unlikely that these three molecules actively engage so many proteins and, therefore, the 2,176 hits in the lysate-based screen were likely affected in part by consistent, but artefactual effects of lysate-based analyses that we do not fully understand (Van Vranken et al., 2021).”

Page 24, lines 29-30 appear to contain a typo. I believe the '>' should be '<' or the 'exclude' should be 'retain'.

The Reviewer is completely correct. We appreciate the attention to detail. This mistake has been corrected in the revised manuscript.  

Page 25, lines 5-7: The methods need to explain how the trimmed standard deviation is calculated.

We apologize for this oversight. To calculate the trimmed standard deviation, we used proteins that were measured in at least 30 conditions. For these, we then removed the top 5% of absolute log2 foldchanges (compared to DMSO controls) and calculated the standard deviation of the resulting set of log2 fold-changes. This is similar in concept to the utilization of “trimmed means” in proteomics data (https://doi.org/10.15252/msb.20145625), which helps to overcome issues due to extreme outliers in datasets. We have added the following statement to the methods to clarify this point (p. 27):

“Second, for each protein across all cells or lysate assays, the number of standard deviations away from the mean thermal stability measurement (z-score) for a given protein was quantified based on a trimmed standard deviation. Briefly, the trimmed standard deviation was calculated for proteins that were measured in at least 30 conditions. For these, we removed the top 5% of absolute log2 foldchanges (compared to DMSO controls) and calculated the standard deviation of the resulting set of log2 fold-changes.”

Page 25, lines 9-11 needs editing for clarity.

We tested empirical hit rates for estimation of mean and trimmed standard deviation (trimmedSD) thresholds to apply, to maximize sensitivity and minimizing the ‘False Hit Rate’, or the number of proteins in the DMSO control samples called as hits divided by the total number of proteins called as hits with a given threshold applied. 

Author response image 2.

Hit calling threshold setting based on maximizing the total hits called and minimizing the False Hit Rate in cells (number of DMSO hits divided by the total number of hits).

Author response image 3.

Hit calling threshold setting based on maximizing the total hits called and minimizing the False Hit Rate in lysates (number of DMSO hits divided by the total number of hits).

Figure 1 supplementary 2a legend states: '32 DMSO controls'. Should that be 64?

We thank the Reviewer for catching our mistake. This has been corrected in the revised manuscript. 

I suggest removing Figure 1 supplementary 3c which is superfluous as only the number it presents is already stated in the text (page 5, line 9).

We thank the Reviewer for the suggestion and agree that this panel is superfluous. It has been removed from the revised manuscript.

New data and tables added during revisions:  

(1) Table 3 – All log2 fold change values for the cell-based screen. Using this table, proteincentric solubility profiles can be plotted (as in Figures 2D and others). 

(2) Table 4 – All log2 fold change values for the lysate-based screen. Using this table, proteincentric solubility profiles can be plotted (as in Figures 2D and others). 

(3) Figure 1 – Figure supplement 3H – Table highlighting proteins that pass log2 fold change cutoffs, but not nSD cutoffs and vice versa. 

(4) Figure 2 – Panels H and I were updated with a new color scheme. 

(5) Figure 3 – Updated main figure and supplement at the request of Reviewer 3. 

• Figure 3E – Compares on-target hits for the cell- and lysate-based screens for all compounds for which a target was quantified in both screens. 

• Figure 3 – Figure supplement 2 – Highlights on-target hits in both screens, exclusively in cells, and exclusively in lysates. 

(6) Figure 5 – PISA data for K562 lysates treated with AZD-7762 at multiple concentrations.

• Figure 5F

• Figure 5 – Figure supplement 3A-C

• Figure 5 – Source data 2

(7) Figure 5 – Phosphoproteomic profiling of K562 cells treated with AZD7762 or Bafetinib. 

• Figure 5G

• Figure 5 – Figure supplement 4A-F

• Figure 5 – Source data 3 (phosphoproteome)

• Figure 5 – Source data 4 (associated proteome data)

Reviewer #1 (Public review):

This paper describes proteome solubility analysis (PISA) of 96 compounds in living cells and 70 compounds in cell lysates. A wealth of information related to on- and off-target engagement is uncovered. This work fits well the eLife profile, will be of interest to a large community of proteomics researchers, and thus is likely to be reasonably highly cited.

Reviewer #3 (Public review):

Summary:

This work aims to demonstrate how recent advances in thermal stability assays can be utilised to screen chemical libraries and determine compound mechanism of action. Focusing on 96 compounds with known mechanisms of action, they use the PISA assay to measure changes in protein stability upon treatment with a high dose (10uM) in live K562 cells and whole cell lysates from K562 or HCT116. They intend this work to showcase a robust workflow which can serve as a roadmap for future studies.

Strengths:

The major strength of this study is the combination of live and whole cell lysates experiments. This allows the authors to compare the results from these two approaches to identify novel ligand-induced changes in thermal stability with greater confidence. More usefully, this also enables the authors to separate primary and secondary effects of the compounds within the live cell assay.

The study also benefits from the number of compounds tested within the same framework, which allows the authors to make direct comparisons between compounds.

These two strengths are combined when they compare between CHEK1 inhibitors and suggest that AZD-7762 likely induces secondary destabilisation of CRKL through off-target engagement with tyrosine kinases.

Weaknesses:

One of the stated benefits of PISA compared to the TPP in the original publication (Gaetani et al 2019) was that the reduced number of samples required allows more replicate experiments to be performed. Despite this, the authors of this study performed only duplicate experiments. They acknowledge this precludes use of frequentist statistical tests to identify significant changes in protein stability. Instead, they apply an 'empirically derived framework' in which they apply two thresholds to the fold change vs DMSO: absolute z-score (calculated from all compounds for a protein) > 3.5 and absolute log2 fold-change > 0.2. They state that the fold-change threshold was necessary to exclude non-specific interactors. While the thresholds appear relatively stringent, this approach will likely reduce the robustness of their findings in comparison to an experimental design incorporating more replicates. Firstly, the magnitude of the effect size should not be taken as a proxy for the importance of the effect. They acknowledge this and demonstrate it using their own data for PIK3CB and p38α inhibitors (Figure 2B-C). They have thus likely missed many small, but biological relevant changes in thermal stability due to the fold-change threshold. Secondly, this approach relies upon the fold-changes between DMSO and compound for each protein being comparable, despite them being drawn from samples spread across 16 TMT multiplexes. Each multiplex necessitates a separate MS run and the quantification of a distinct set of peptides, from which the protein-level abundances are estimated. Thus, it is unlikely the fold-changes for unaffected proteins are drawn from the same distribution, which is an unstated assumption of their thresholding approach. The authors could alleviate the second concern by demonstrating that there is very little or no batch effect across the TMT multiplexes. However, the first concern would remain. The limitations of their approach could have been avoided with more replicates and use of an appropriate statistical test. It would be helpful if the authors could clarify if any of the missed targets passed the z-score threshold but fell below the fold-change threshold.

The authors use a single, high, concentration of 10uM for all compounds. Given that many of the compounds may have low nM IC50s, this concentration could be orders of magnitude above the one at which they inhibit their target. This makes it difficult to assess the relevance of the off-target effects identified to clinical applications of the compounds or biological experiments. The authors acknowledge this and use ranges of concentrations for follow-up studies (e.g. Figure 2E-F). Nonetheless, this weakness is present for the vast bulk of the data presented.

Aims achieved, impact and utility:

The authors have achieved their main aim of presenting a workflow which serves to demonstrate the potential value of this approach. However, by using a single high dose of each compound and failing to adequately replicate their experiments and instead applying heuristic thresholds, they have limited the impact of their findings. Their results will be a useful resource for researchers wishing to explore potential off-target interactions and/or mechanisms of action for these 96 compounds but are expected to be superseded by more robust datasets in the near future. The most valuable aspect of the study is the demonstration that combining live cell and whole cell lysate PISA assays across multiple related compounds can help to elucidate the mechanisms of action.

exchanging things for mutual benefit

to: https://hyp.is/xf-nwoSMEe-Obw9pt77C3A/brave.com/blog/ipfs-support/

search - ipfs support brave

These birds are mentioned as ebabil in Surah al-Fil.

eLife Assessment

Research on push-pull systems often focuses on controlled environments, limiting our understanding of their effectiveness under real-world conditions. This important study has validated how push-pull systems work in natural settings. However, the manuscript remains incomplete, since the findings have only been partially supported, as acknowledged by the authors.

Reviewer #1 (Public review):

Summary:

The manuscript of Odermatt et al. investigates the volatiles released by two species of Desmodium plants and the response of herbivores to maize plants alone or in combination with these species. The results show that Desmodium releases volatiles in both the laboratory and the field. Maize grown in the laboratory also released volatiles, in a similar range. While female moths preferred to oviposit on maize, the authors found no evidence that Desmodium volatiles played a role in lowering attraction to or oviposition on maize.

Strengths:

The manuscript is a response to recently published papers that presented conflicting results with respect to whether Desmodium releases volatiles constitutively or in response to biotic stress, the level at which such volatiles are released, and the behavioral effect it has on the fall armyworm. These questions are relevant as Desmodium is used in a textbook example of pest-suppressive sustainable intercropping technology called push-pull, which has supported tens of thousands of smallholder farmers in suppressing moth pests in maize. A large number of research papers over more than two decades have implied that Desmodium suppresses herbivores in push-pull intercropping through the release of large amounts of volatiles that repel herbivores. This premise has been questioned in recent papers. Odermatt et al. thus contribute to this discussion by testing the role of odors in oviposition choice. The paper confirms that ovipositing FAW preferred maize, and also confirmed that odors released from Desmodium appeared not important in their bioassays.

The paper is a welcome addition to the literature and adds quality headspace analyses of Desmodium from the laboratory and the field. Furthermore, the authors, some of whom have since long contributed to developing push-pull, also find that Desmodium odors are not significant in their choice between maize plants. This advances our knowledge of the mechanisms through which push-pull suppresses herbivores, which is critically important to evolving the technique to fit different farming systems and translating this mechanism to fit with other crops and in other geographical areas.

Weaknesses:

Below I outline the major concerns:

(1) Clear induction of the experimental plants, and lack of reflective discussion around this: from literature data and previous studies of maize and Desmodium, it is clear that the plants used in this study, particularly the Desmodium, were induced. Maize appeared to be primarily manually damaged, possibly due to sampling (release of GLV, but little to no terpenoids, which is indicative of mostly physical stress and damage, for example, one of the coauthor's own paper Tamiru et al. 2011), whereas Desmodium releases a blend of many compounds (many terpenoids indicative of herbivore induction). Erdei et al. also clearly show that under controlled conditions maize, silver leaf and green leaf Desmodium release volatiles in very low amounts. While the condition of the plants in Odermatt et al. may be reflective of situations in push-pull fields, the authors should elaborate on the above in the discussion (see comments) such that the readers understand that the plant's condition during the experiments. This is particularly important because it has been assumed that Desmodium releases typical herbivore-induced volatiles constitutively, which is not the case (see Erdei et al. 2024). This reflection is currently lacking in the manuscript.

(2) Lack of controls that would have provided context to the data: The experiments lack important controls that would have helped in the interpretation:

(2a) The authors did not control the conditions of the plants. To understand the release of volatiles and their importance in the field, the authors should have included controlled herbivory in both maize and Desmodium. This would have placed the current volatile profiles in a herbivory context. Now the volatile measurements hang in midair, leading to discussions that are not well anchored (and should be rephrased thoroughly, see eg lines 183-188). It is well known that maize releases only very low levels of volatiles without abiotic and biotic stressors. However, this changes upon stress (GLVs by direct, physical damage and eg terpenoids upon herbivory, see above). Erdei et al. confirm this pattern in Desmodium. Not having these controls, means that the authors need to put the data in the context of what has been published (see above).

(2b) It would also have been better if the authors had sampled maize from the field while sampling Desmodium. Together with the above point (inclusion of herbivore-induced maize and Desmodium), the levels of volatile release by Desmodium would have been placed into context.

(2c) To put the volatiles release in the context of push-pull, it would have been important to sample other plants which are frequently used as intercrop by smallholder farmers, but which are not considered effective as push crops, particularly edible legumes. Sampling the headspace of these plants, both 'clean' and herbivore-induced, would have provided a context to the volatiles that Desmodium (induced) releases in the field - one would expect unsuccessful push crops to not release any of these 'bioactive' volatiles (although 'bioactive' should be avoided) if these odors are responsible for the pest suppressive effect of Desmodium. Many edible intercrops have been tested to increase the adoption of push-pull technology but with little success.

Because of the lack of the above, the conclusions the authors can draw from their data are weakened. The data are still valuable in the current discussion around push-pull, provided that a proper context is given in the discussion along the points above.

(3) 'Tendency' of the authors to accept the odor hypothesis (i.e. that Desmodium odors are responsible for repelling FAW and thereby reduce infestation in maize under push-pull management) in spite of their own data: The authors tested the effects of odor in oviposition choice, both in a cage assay and in a 'wind tunnel'. From the cage experiments, it is clear that FAW preferred maize over Desmodium, confirming other reports (including Erdei et al. 2024). However, when choosing between two maize plants, one of which was placed next to Desmodium to which FAW has no tactile (taste, structure, etc), FAW chose equally. Similarly in their wind tunnel setup (this term should not be used to describe the assay, see below), no preference was found either between maize odor in the presence or absence of Desmodium. This too confirms results obtained by Erdei et al. (but add an important element to it by using Desmodium plants that had been induced and released volatiles, contrary to Erdei et al. 2024). Even though no support was found for repellency by Desmodium odors, the authors in many instances in the manuscript (lines 30-33, 164-169, 202, 279, 284, 304-307, 311-312, 320) appear to elevate non-significant tendencies as being important. This is misleading readers into thinking that these interactions were significant and in fact confirming this in the discussion. The authors should stay true to their own data obtained when testing the hypothesis of whether odors play a role in the pest-suppressive effect of push-pull.

(4) Oviposition bioassay: with so many assays in close proximity, it is hard to certify that the experiments are independent. Please discuss this in the appropriate place in the discussion.

(5) The wind tunnel has a number of issues (besides being poorly detailed):

(5a) The setup which the authors refer to as a 'wind tunnel' does not qualify as a wind tunnel. First, there is no directional flow: there are two flows entering the setup at opposite sides. Second, the flow is way too low for moths to orient in (in a wind tunnel wind should be presented as a directional cue. Only around 1.5 l/min enters the wind tunnel in a volume of 90 l approximately, which does not create any directional flow. Solution: change 'wind tunnel' throughout the text to a dual choice setup /assay.

(5b) There is no control over the flows in the flight section of the setup. It is very well possible that moths at the release point may only sense one of the 'options'. Please discuss this.

(5c) Too low a flow (1,5 l per minute) implies a largely stagnant air, which means cross-contamination between experiments. An experiment takes 5 minutes, but it takes minimally 1.5 hours at these flows to replace the flight chamber air (but in reality much longer as the fresh air does not replace the old air, but mixes with it). The setup does not seem to be equipped with e.g. fans to quickly vent the air out of the setup. See comments in the text. Please discuss the limitations of the experimental setup at the appropriate place in the discussion.

(5d) The stimulus air enters through a tube (what type of tube, diameter, length, etc) containing pressurized air (how was the air obtained into bags (type of bag, how is it sealed?), and the efflux directly into the flight chamber (how, nozzle?). However, it seems that there is no control of the efflux. How was leakage prevented, particularly how the bags were airtight sealed around the plants?

(5e) The plants were bagged in very narrowly fitting bags. The maize plants look bent and damaged, which probably explains the GLVs found in the samples. The Desmodium in the picture (Figure 5 supplement), which we should assume is at least a representative picture?) appears to be rather crammed into the bag with maize and looks in rather poor condition to start with (perhaps also indicating why they release these volatiles?). It would be good to describe the sampling of the plants in detail and explain that the way they were handled may have caused the release of GLVs.

(6) Figure 1 seems redundant as a main figure in the text. Much of the information is not pertinent to the paper. It can be used in a review on the topic. Or perhaps if the authors strongly wish to keep it, it could be placed in the supplemental material.

Reviewer #2 (Public review):

Based on the controversy of whether the Desmodium intercrop emits bioactive volatiles that repel the fall armyworm, the authors conducted this study to assess the effects of the volatiles from Desmodium plants in the push-pull system on behavior of FAW oviposition. This topic is interesting and the results are valuable for understanding the push-pull system for the management of FAW, the serious pest. The methodology used in this study is valid, leading to reliable results and conclusions. I just have a few concerns and suggestions for improvement of this paper:

(1) The volatiles emitted from D. incanum were analyzed and their effects on the oviposition behavior of FAW moth were confirmed. However, it would be better and useful to identify the specific compounds that are crucial for the success of the push-pull system.

(2) That would be good to add "symbols" of significance in Figure 4 (D).

(3) Figure A is difficult for readers to understand.

(4) It will be good to deeply discuss the functions of important volatile compounds identified here with comparison with results in previous studies in the discussion better.

Author response:

We thank both reviewers for their thorough and insightful feedback, which will contribute to improving our manuscript. In summary, the key concerns raised include the potential induction of GLV volatiles due to plant handling, limitations in the design of the "wind tunnel" bioassay, and the need for a deeper analysis of specific volatile compounds that contribute to the success of push-pull systems. We are happy to revise the entire manuscript according to all comments of the reviewers. This includes clarification of our methodology and providing a more reflective discussion on how physical stress might have influenced volatile emissions. Additionally, we will conduct new experiments with a modified bioassay setup to address concerns about directional cues and airflow control, minimizing cross-contamination. While the identification of individual compounds was beyond the scope of this study, we acknowledge its importance and propose it as a direction for future research.

Reviewer #1 (Public review):

Summary:

The manuscript of Odermatt et al. investigates the volatiles released by two species of Desmodium plants and the response of herbivores to maize plants alone or in combination with these species. The results show that Desmodium releases volatiles in both the laboratory and the field. Maize grown in the laboratory also released volatiles, in a similar range. While female moths preferred to oviposit on maize, the authors found no evidence that Desmodium volatiles played a role in lowering attraction to or oviposition on maize.

Strengths:

The manuscript is a response to recently published papers that presented conflicting results with respect to whether Desmodium releases volatiles constitutively or in response to biotic stress, the level at which such volatiles are released, and the behavioral effect it has on the fall armyworm. These questions are relevant as Desmodium is used in a textbook example of pest-suppressive sustainable intercropping technology called push-pull, which has supported tens of thousands of smallholder farmers in suppressing moth pests in maize. A large number of research papers over more than two decades have implied that Desmodium suppresses herbivores in push-pull intercropping through the release of large amounts of volatiles that repel herbivores. This premise has been questioned in recent papers. Odermatt et al. thus contribute to this discussion by testing the role of odors in oviposition choice. The paper confirms that ovipositing FAW preferred maize, and also confirmed that odors released from Desmodium appeared not important in their bioassays.

The paper is a welcome addition to the literature and adds quality headspace analyses of Desmodium from the laboratory and the field. Furthermore, the authors, some of whom have since long contributed to developing push-pull, also find that Desmodium odors are not significant in their choice between maize plants. This advances our knowledge of the mechanisms through which push-pull suppresses herbivores, which is critically important to evolving the technique to fit different farming systems and translating this mechanism to fit with other crops and in other geographical areas.

Thank you for your careful assessment of our manuscript.

Weaknesses:

Below I outline the major concerns:

(1) Clear induction of the experimental plants, and lack of reflective discussion around this: from literature data and previous studies of maize and Desmodium, it is clear that the plants used in this study, particularly the Desmodium, were induced. Maize appeared to be primarily manually damaged, possibly due to sampling (release of GLV, but little to no terpenoids, which is indicative of mostly physical stress and damage, for example, one of the coauthor's own paper Tamiru et al. 2011), whereas Desmodium releases a blend of many compounds (many terpenoids indicative of herbivore induction). Erdei et al. also clearly show that under controlled conditions maize, silver leaf and green leaf Desmodium release volatiles in very low amounts. While the condition of the plants in Odermatt et al. may be reflective of situations in push-pull fields, the authors should elaborate on the above in the discussion (see comments) such that the readers understand that the plant's condition during the experiments. This is particularly important because it has been assumed that Desmodium releases typical herbivore-induced volatiles constitutively, which is not the case (see Erdei et al. 2024). This reflection is currently lacking in the manuscript.

We acknowledge the need for a more reflective discussion on the possible causes of GLV (green leaf volatiles) emission, particularly regarding physical damage. Although the field plants were carefully handled, it is possible that some physical stress may have contributed to the release of GLVs. We will ensure the revised manuscript reflects this nuanced interpretation. However, we will also explain more clearly that our aim was to capture the volatile emission of plants used by farmers under realistic conditions and moth responses to these plants, not to be able to attribute the volatile emission to a specific cause. We think that this is also clear in the manuscript. However, we plan to revise relevant passages throughout the manuscript to ensure that we do not make any claims about the reason for volatile emissions, and that our claims regarding these plants and their headspace being representative of the system as practiced by farmers are supported. In the revised manuscript we will explain better that the volatile profiles comprise a majority of non-GLV compounds. As shown in figure 1, the majority of the substances that were found in the headspace of the sampled plants of Desmodium intortum or Desmodium incanum are non-GLV monoterpenes, sesquiterpenes, or aromatic compounds. We will also note that the experimental plants used in the study were grown in insect proof screenhouses and were checked for any insect damage before volatile collection and bioassay.

(2) Lack of controls that would have provided context to the data: The experiments lack important controls that would have helped in the interpretation:

(2a) The authors did not control the conditions of the plants. To understand the release of volatiles and their importance in the field, the authors should have included controlled herbivory in both maize and Desmodium. This would have placed the current volatile profiles in a herbivory context. Now the volatile measurements hang in midair, leading to discussions that are not well anchored (and should be rephrased thoroughly, see eg lines 183-188). It is well known that maize releases only very low levels of volatiles without abiotic and biotic stressors. However, this changes upon stress (GLVs by direct, physical damage and eg terpenoids upon herbivory, see above). Erdei et al. confirm this pattern in Desmodium. Not having these controls, means that the authors need to put the data in the context of what has been published (see above).

We appreciate this concern. Our study aimed to capture the real-world conditions of push-pull fields, where Desmodium and maize grow in natural environments without the direct induction of herbivory for experimental purposes. We will update the discussion to provide better context based on existing literature regarding the volatile release under stress conditions. We agree that in further studies it would be important to carry out experiments under different environmental conditions, including herbivore damage. However, this was not within the scope of the present study.

(2b) It would also have been better if the authors had sampled maize from the field while sampling Desmodium. Together with the above point (inclusion of herbivore-induced maize and Desmodium), the levels of volatile release by Desmodium would have been placed into context.

We acknowledge that sampling maize and other intercrop plants, such as edible legumes, alongside Desmodium in the push-pull field would have allowed us to make direct comparisons of the volatile profiles of different plants in the push-pull system under shared field conditions. Again, this should be done in future experiments but was beyond the scope of the present study. Due to the amount of samples, we could handle given cost and workload, we chose to focus on Desmodium because there is much less literature on the volatile profiles of field-grown Desmodium than maize plants in the field: we are aware of one study attempting to measure field volatile profiles from Desmodium intortum (Erdei et al. 2024) and no study attempting this for Desmodium incanum. We will point out this justification for our focus on Desmodium in the manuscript. Additionally, we will suggest in the discussion that future studies should measure volatile profiles from maize and intercrop legumes alongside Desmodium and border grass in push-pull fields.

(2c) To put the volatiles release in the context of push-pull, it would have been important to sample other plants which are frequently used as intercrop by smallholder farmers, but which are not considered effective as push crops, particularly edible legumes. Sampling the headspace of these plants, both 'clean' and herbivore-induced, would have provided a context to the volatiles that Desmodium (induced) releases in the field - one would expect unsuccessful push crops to not release any of these 'bioactive' volatiles (although 'bioactive' should be avoided) if these odors are responsible for the pest suppressive effect of Desmodium. Many edible intercrops have been tested to increase the adoption of push-pull technology but with little success.

Again, we very much agree that such measurements are important for the longer-term research program in this field. But again, for the current study this would have exploded the size of the required experiment. Regarding bioactivity, we have been careful to use the phrase "potentially bioactive", or to cite other studies showing bioactivity, where we have not demonstrated bioactivity ourselves.

Because of the lack of the above, the conclusions the authors can draw from their data are weakened. The data are still valuable in the current discussion around push-pull, provided that a proper context is given in the discussion along the points above.

We agree that our study is limited to its specific aims. Therefore, we think the revisions will make these more explicit and help to avoid misleading claims.

(3) 'Tendency' of the authors to accept the odor hypothesis (i.e. that Desmodium odors are responsible for repelling FAW and thereby reduce infestation in maize under push-pull management) in spite of their own data: The authors tested the effects of odor in oviposition choice, both in a cage assay and in a 'wind tunnel'. From the cage experiments, it is clear that FAW preferred maize over Desmodium, confirming other reports (including Erdei et al. 2024). However, when choosing between two maize plants, one of which was placed next to Desmodium to which FAW has no tactile (taste, structure, etc), FAW chose equally. Similarly in their wind tunnel setup (this term should not be used to describe the assay, see below), no preference was found either between maize odor in the presence or absence of Desmodium. This too confirms results obtained by Erdei et al. (but add an important element to it by using Desmodium plants that had been induced and released volatiles, contrary to Erdei et al. 2024). Even though no support was found for repellency by Desmodium odors, the authors in many instances in the manuscript (lines 30-33, 164-169, 202, 279, 284, 304-307, 311-312, 320) appear to elevate non-significant tendencies as being important. This is misleading readers into thinking that these interactions were significant and in fact confirming this in the discussion. The authors should stay true to their own data obtained when testing the hypothesis of whether odors play a role in the pest-suppressive effect of push-pull.

We appreciate this feedback and agree that we may have overstated claims that could not be supported by strict significance tests. However, we believe that non-significant tendencies can still provide valuable insights. In the revised version of the manuscript, we will ensure a clear distinction between statistically significant findings and non-significant trends and remove any language that may imply stronger support for the odor hypothesis that what the data show.

(4) Oviposition bioassay: with so many assays in close proximity, it is hard to certify that the experiments are independent. Please discuss this in the appropriate place in the discussion.

We have pointed this out in the submitted manuscript in the lines 275 – 279. Furthermore, we include detailed captions to figure 4 - supporting figure 3 & figure 4 - supporting figure 4. We are aware that in all such experiments there is a danger of between-treatment interference, which we will point out for our specific case. We will also mention that this common caveat does not invalidate experimental designs when practicing replication and randomization and assume insect’s ability to select suitable oviposition site in the background of such confounding factors under realistic conditions. We will also mention explicitly that with our experimental setup we tried to minimize interference between treatments by spacing and temporal staggering.

(5) The wind tunnel has a number of issues (besides being poorly detailed):

(5a) The setup which the authors refer to as a 'wind tunnel' does not qualify as a wind tunnel. First, there is no directional flow: there are two flows entering the setup at opposite sides. Second, the flow is way too low for moths to orient in (in a wind tunnel wind should be presented as a directional cue. Only around 1.5 l/min enters the wind tunnel in a volume of 90 l approximately, which does not create any directional flow. Solution: change 'wind tunnel' throughout the text to a dual choice setup /assay.)

We agree with these criticisms and will change the terminology accordingly. We also plan to conduct an additional experiment with a no-choice arena that provides conditions closer to a true wind tunnel. The setup of the added experiment features an odor entry point at only one side of the chamber to create a more directional airflow. Each treatment (maize alone, maize + D. intortum, maize + D. incanum, and a control with no plants) will be tested separately, with only one treatment conducted per evening to avoid cross-contamination.

(5b) There is no control over the flows in the flight section of the setup. It is very well possible that moths at the release point may only sense one of the 'options'. Please discuss this.

We will add this to the discussion. The newly planned assays also address this concern by using a setup with laminar flow.

(5c) Too low a flow (1,5 l per minute) implies a largely stagnant air, which means cross-contamination between experiments. An experiment takes 5 minutes, but it takes minimally 1.5 hours at these flows to replace the flight chamber air (but in reality much longer as the fresh air does not replace the old air, but mixes with it). The setup does not seem to be equipped with e.g. fans to quickly vent the air out of the setup. See comments in the text. Please discuss the limitations of the experimental setup at the appropriate place in the discussion.

We will add these limitations to the discussion and will address these concerns with new experiments (see answer 5a).

(5d) The stimulus air enters through a tube (what type of tube, diameter, length, etc) containing pressurized air (how was the air obtained into bags (type of bag, how is it sealed?), and the efflux directly into the flight chamber (how, nozzle?). However, it seems that there is no control of the efflux. How was leakage prevented, particularly how the bags were airtight sealed around the plants? 

We will add the missing information to the methods and provide details about types of bags, manufacturers, and pre-treatments. In short, Teflon tubes connected bagged plants to the bioassay setup and air was pumped in at an overpressure, so leakage was not eliminated but contamination from ambient air was avoided.

(5e) The plants were bagged in very narrowly fitting bags. The maize plants look bent and damaged, which probably explains the GLVs found in the samples. The Desmodium in the picture (Figure 5 supplement), which we should assume is at least a representative picture?) appears to be rather crammed into the bag with maize and looks in rather poor condition to start with (perhaps also indicating why they release these volatiles?). It would be good to describe the sampling of the plants in detail and explain that the way they were handled may have caused the release of GLVs.

We will include a more detailed description of the plant handling and bagging processes to the methods to clarify how the plants were treated during all assays reported in the submitted manuscript and the newly planned assays. This will address concerns about the possible influence of plant stress, such as GLV emission due to bagging, on the results. We politely disagree that the maize plants were damaged and the Desmodium plants not representative of those encountered in the field. The Desmodium plant pictured was D. incanum, which has sparser foliage and smaller leaves than D. intortum.

(6) Figure 1 seems redundant as a main figure in the text. Much of the information is not pertinent to the paper. It can be used in a review on the topic. Or perhaps if the authors strongly wish to keep it, it could be placed in the supplemental material.

We think that Figure 1 provides essential information about the push-pull system and the FAW. To our knowledge, this partly contradictory evidence so far has not been synthesized in the literature. We realize that such a figure would more commonly be provided in a review article, but we do not think that the small number of studies on this topic so far justify a stand-alone review. Instead, the introduction to our manuscript includes a brief review of these few studies, complemented by the visual summary provided in Figure 1 and a detailed supplementary table. We will revise the figure and associated text in the introduction to highlight its relevance for the current study and to reduce redundant information.

Reviewer #2 (Public review):

Based on the controversy of whether the Desmodium intercrop emits bioactive volatiles that repel the fall armyworm, the authors conducted this study to assess the effects of the volatiles from Desmodium plants in the push-pull system on behavior of FAW oviposition. This topic is interesting and the results are valuable for understanding the push-pull system for the management of FAW, the serious pest. The methodology used in this study is valid, leading to reliable results and conclusions. I just have a few concerns and suggestions for improvement of this paper:

(1) The volatiles emitted from D. incanum were analyzed and their effects on the oviposition behavior of FAW moth were confirmed. However, it would be better and useful to identify the specific compounds that are crucial for the success of the push-pull system.

We fully agree that identifying specific volatile compounds responsible for the push-pull effect would provide valuable insights into the underlying mechanisms of the system. However, the primary focus of this study was to address the still unresolved question whether Desmodium emits volatiles at all under field conditions, and the secondary aim was to test whether we could demonstrate a behavioral effect of Desmodium headspace on FAW moths. Before conducting our experiments, we carefully considered the option of using single volatile compounds and synthetic blends in bioassays. We decided against this because we judged that the contradictory evidence in the literature was not a sufficient basis for composing representative blends. Furthermore, we think it is an important first step to test for behavioral responses to the headspaces of real plants. We consider bioassays with pure compounds to be important for confirmation and more detailed investigation in future studies. There was also contradictory evidence in the literature regarding moth responses to plants. We thus opted to focus on experiments with whole plants to maintain ecological relevance.

(2) That would be good to add "symbols" of significance in Figure 4 (D).

We report the statistical significance of the parameters in Figure 4 (D) in Table 3. While testing significance between groups is a standard approach, we used a more robust model-based analysis to assess the effects of multiple factors simultaneously. We will clarify this in the figure legend and provide a cross-reference to Table 3 for readers to easily find the statistical details.

(3) Figure A is difficult for readers to understand.

Unfortunately, it is not entirely clear which specific figure is being referred to as "Figure A" in this comment. We kindly request further clarification on which figure needs improvement, and we will make adjustments accordingly to ensure that all figures are easily comprehensible for readers.

(4) It will be good to deeply discuss the functions of important volatile compounds identified here with comparison with results in previous studies in the discussion better.

Our study does not provide strong evidence that specific volatiles from Desmodium plants are important determinants of FAW oviposition or choice in the push-pull system. Therefore, we prefer to refrain from detailed discussions of the potential importance of individual compounds. However, in the revised version, we will indicate specifically which of the volatiles we identified overlap with those previously reported from Desmodium, as only the total numbers are summarized in the discussion of the submitted paper.

...

ερωτήματα για το τεστ:

1. τι συμβαίνει όταν κάνουμε ταυτόχρονα ένα διαφορετικό αρχείο στη λίστα;
2. πότε πρέπει να κάνουμε ριφρες;
3. στο εργαστήριο θα είμαστε όλα ως wordmord θα δουλεύουμε όλα στο ίδιο αρχείο ή σε διαφορετικά; μάλλον κάθε ομάδα θα δημιουργήσει ένα αρχείο να το τεστάρουμε

it works!

eLife Assessment

This study attempts to understand the functional roles of the human DCP1 paralogs in regulating RNA decay by DCP2. Using a combination of cellular-based assays and in vitro assays, the authors conclude that DCP1a/b plays a role in regulating DCP2 activity. While this revised version presents some new and interesting observations on human DCP1, the underlying data to support its claims remain incomplete. Overall, these results will be useful to the RNA community.

Reviewer #1 (Public review):

Summary & Assessment:

The catalytic core of the eukaryotic decapping complex consists of the decapping enzyme DCP2 and its key activator DCP1. In humans, there are two paralogs of DCP1, DCP1a and DCP1b, that are known to interact with DCP2 and recruit additional cofactors or coactivators to the decapping complex; however, the mechanisms by which DCP1 activates decapping and the specific roles of DCP1a versus DCP1b, remain poorly defined. In this manuscript, the authors used CRISPR/Cas9-generated DCP1a/b knockout cells to begin to unravel some of the differential roles for human DCP1a and DCP1b in mRNA decapping, gene regulation, and cellular metabolism. While this manuscript presents some new and interesting observations on human DCP1 (e.g. human DCP1a/b KO cells are viable and can be used to investigate DCP1 function; only the EVH1 domain, and not its disordered C-terminal region which recruits many decapping cofactors, is apparently required for efficient decapping in cells; DCP1a and b target different subsets of mRNAs for decay and may regulate different aspects of metabolism), there is one key claim about the role of DCP1 in regulating DCP2-mediated decapping that is still incompletely or inconsistently supported by the presented data in this revised version of the manuscript.

Strengths & well-supported claims:

• Through in vivo tethering assays in CRISPR/Cas9-generated DCP1a/b knockout cells, the authors show that DCP1 depletion leads to significant defects in decapping and the accumulation of capped, deadenylated mRNA decay intermediates.<br /> • DCP1 truncation experiments reveal that only the EVH1 domain of DCP1 is necessary to rescue decapping defects in DCP1a/b KO cells.<br /> • RNA and protein immunoprecipitation experiments suggest that DCP1 acts as a scaffold to help recruit multiple decapping cofactors to the decapping complex (e.g. EDC3, DDX6, PATL1 PNRC1, and PNRC2), but that none of these cofactors are essential for DCP2-mediated decapping in cells.<br /> • The authors investigated the differential roles of DCP1a and DCP1b in gene regulation through transcriptomic and metabolomic analysis and found that these DCP1 paralogs target different mRNA transcripts for decapping and have different roles in cellular metabolism and their apparent links to human cancers. (Although I will note that I can't comment on the experimental details and/or rigor of the transcriptomic and metabolomic analyses, as these are outside my expertise.)

Weaknesses & incompletely supported claims:

(1) One of the key mechanistic claims of the paper is that "DCP1a can regulate DCP2's cellular decapping activity by enhancing DCP2's affinity to RNA, in addition to bridging the interactions of DCP2 with other decapping factors. This represents a pivotal molecular mechanism by which DCP1a exerts its regulatory control over the mRNA decapping process." Similar versions of this claim are repeated in the abstract and discussion sections. However, this claim appears to be at odds with the observations that: (a) in vitro decapping assays with immunoprecipitated DCP2 show that DCP1 knockout does not significantly affect the enzymatic activity of DCP2 (Fig 2C&D; I note that there may be a very small change in DCP2 activity shown in panel D, but this may be due to slightly different amounts of immunoprecipitated DCP2 used in the assay); and (b) the authors show only weak changes in relative RNA levels immunoprecipitated by DCP2 with versus without DCP1 (~2-3 fold change in Fig 3H, where expression of the EVH1 domain, previously shown in this manuscript to fully rescue the DCP1 KO decapping defects in cells, looks to be almost within error of the control in terms of increasing RNA binding). If DCP1 pivotally regulates decapping activity by enhancing RNA binding to DCP2, why is no difference in in vitro decapping activity observed in the absence of DCP1, and very little change observed in the amounts of RNA immunoprecipitated by DCP2 with the addition of the DCP1 EVH1 domain?

In the revised manuscript and in their response to initial reviews, the authors rightly point out that in vivo effects may not always be fully reflected by or recapitulated in in vitro experiments due to the lack of cellular cofactors and simpler environment for the in vitro experiment, as compared to the complex environment in the cell. I fully agree with this of course! And further completely agree with the authors that this highlights the critical importance of in cell experiments to investigate biological functions and mechanisms! However, because the in vitro kinetic and IP/binding data both suggest that the DCP1 EVH1 domain has minimal to no effects on RNA decapping or binding affinity, while the in cell data suggest the EVH1 domain alone is sufficient to rescue large decapping defects in DCP1a/b KO cells (and that all the decapping cofactors tested were dispensable for this), I would argue there is insufficient evidence here to make a claim that (maybe weakly) enhanced RNA binding induced by DCP1 is what is regulating the cellular decapping activity. Maybe there are as-yet-untested cellular cofactors that bind to the EVH1 domain of DCP1 that change either RNA recruitment or the kinetics of RNA decapping in cells; we can't really tell from the presented data so far. Furthermore, even if it is the case that the EVH1 domain modestly enhances RNA binding to DCP2, the authors haven't shown that this effect is what actually regulates the large change in DCP2 activity upon DCP1 KO observed in the cell.

Overall, while I absolutely appreciate that there are many possible reasons for the differences observed in the in vitro versus in cell RNA decapping and binding assays, because this discrepancy between those data exists, it seems difficult to draw any clear conclusions about the actual mechanisms by which DCP1 helps regulate RNA decapping by DCP2. For example, in the cell it could be that DCP1 enhances RNA binding, or recruits unidentified cofactors that themselves enhance RNA binding, or that DCP1 allosterically enhances DCP2-mediated decapping kinetics, or a combination of these, etc; my point is that without in vitro data that clearly support one of those mechanisms and links this mechanism back to cellular DCP2 decapping activity (for example, in cell data that show EVH1 mutants that impair RNA binding fail to rescue DCP1 KO decapping defects), it's difficult to attribute the observed in cell effects of DCP1a/b KO and rescue by the EVH1 domain directly to enhancement of RNA binding (precisely because, as the authors describe, the decapping process and regulation may be very complex in the cell!).

This contradiction between the in vitro and in-cell decapping data undercuts one of the main mechanistic takeaways from the first half of the paper; I still think this conclusion is overstated in the revised manuscript.

Additional minor comment:

• Related to point (1) above, the kinetic analysis presented in Fig 2C shows that the large majority of transcript is mostly decapped at the first 5 minute timepoint; it may be that DCP2-mediated decapping activity is actually different in vitro with or without DCP1, but that this is being missed because the reaction is basically done in less than 5 minutes under the conditions being assayed (i.e. these are basically endpoint assays under these conditions). It may be that if kinetics were done under conditions to slow down the reaction somewhat (e.g. lower Dcp2 concentration, lower temperatures), so that more of the kinetic behavior is captured, the apparent discrepancy between in vitro and in-cell data would be much less. Indeed, previous studies have shown that in yeast, Dcp1 strongly activates the catalytic step (kcat) of decapping by ~10-fold, and reduces the KM by only ~2 fold (Floor et al, NSMB 2010). It might be beneficial to use purified proteins here, if possible, to better control reaction conditions.

In their response to initial reviews, the authors comment that they tried to purify human DCP2 from E coli, but were unable to obtain active enzyme in this way. Fair enough! I will only comment that just varying the relative concentration of immunoprecipitated DCP2 would likely be enough to slow down the reaction and see if activity differences are seen in different kinetic regimes, without the need to obtain fully purified / recombinant Dcp2.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public Review):

Weaknesses & incompletely supported claims:

(1) A central mechanistic claim of the paper is that "DCP1a can regulate DCP2's cellular decapping activity by enhancing DCP2's affinity to RNA, in addition to bridging the interactions of DCP2 with other decapping factors. This represents a pivotal molecular mechanism by which DCP1a exerts its regulatory control over the mRNA decapping process." Similar versions of this claim are repeated in the abstract and discussion sections. However, this appears to be entirely at odds with the observation from in vitro decapping assays with immunoprecipitated DCP2 that showed DCP1 knockout does not significantly affect the enzymatic activity of DCP2 (Figures 2B-D; I note that there may be a very small change in DCP2 activity shown in panel C, but this may be due to slightly different amounts of immunoprecipitated DCP2 used in the assay, as suggested by panel D). If DCP1 pivotally regulates decapping activity by enhancing RNA binding to DCP2, why is no difference in decapping activity observed in the absence of DCP1?

Furthermore, the authors show only weak changes in relative RNA levels immunoprecipitated by DCP2 with versus without DCP1 (~2-3 fold change; consistent with the Valkov 2016 NSMB paper, which shows what looks like only modest changes in RNA binding affinity for yeast Dcp2 +/- Dcp1). Is the argument that only a 2-3 fold change in RNA binding affinity is responsible for the sizable decapping defects and significant accumulation of deadenylated intermediates observed in cells upon Dcp1 depletion? (and if so, why is this the case for in-cell data, but not the immunoprecipitated in vitro data?)

We appreciate the reviewer's thoughtful comments on our paper. The reviewer points out an apparent contradiction between the claim that DCP1a regulates DCP2's cellular decapping activity and the observation that knocking out DCP1a does not significantly affect DCP2's enzymatic activity in vitro. However, it is important to underscore the challenge of reconciling differences between in vitro and in vivo experiments in scientific research. Although in vitro systems provide a controlled environment, they have inherent limitations that often fail to capture the complexities of cellular processes. Our in vitro experiments used immunoprecipitated proteins to ensure the presence of relevant factors, but these experiments cannot fully replicate the precise stoichiometry and dynamic interactions present in a cellular environment. Furthermore, the limited volume in vitro can actually facilitate reactions that may not occur as readily in the complex and heterogeneous environment of a cell. Therefore, the lack of a significant difference in decapping activity observed in vitro does not necessarily negate the regulatory role of DCP1 in the cellular context. Rather, it underscores our previous oversight of DCP1's importance in the decapping process under in vitro conditions. The conclusions regarding DCP1's regulatory mechanisms remain valid and supported by the presented evidence, especially when considering the inherent differences between in vitro and in vivo experimental conditions. It is precisely because of these differences that we recognized our previous underestimation of DCP1's significance. Therefore, our subsequent experiments focused on elucidating DCP1's regulatory mechanisms in the decapping process

The authors acknowledge this apparent discrepancy between the in vitro DCP2 decapping assays and in-cell decapping data, writing: "this observation could be attributed to the inherent constraints of in vitro assays, which often fall short of faithfully replicating the complexity of the cellular environment where multiple factors and cofactors are at play. To determine the underlying cause, we postulated that the observed cellular decapping defect in DCP1a/b knockout cells might be attributed to DCP1 functioning as a scaffold." This is fair. They next show that DCP1 acts as a scaffold to recruit multiple factors to DCP2 in cells (EDC3, DDX6, PatL1, and PNRC1 and 2). However, while DCP1 is shown to recruit multiple cofactors to DCP2 (consistent with other studies in the decapping field, and primarily through motifs in the Dcp1 C-terminal tail), the authors ultimately show that *none* of these cofactors are actually essential for DCP2-mediated decapping in cells (Figures 3A-F). More specifically, the authors showed that the EVH1 domain was sufficient to rescue decapping defects in DCP1a/b knockout cells, that PNRC1 and PNRC2 were the only cofactors that interact with the EVH1 domain, and finally that shRNA-mediated PNRC1 or PNCR2 knockdown has no effect on in-cell decapping (Figures 3E and F). Therefore, based on the presented data, while DCP1 certainly does act as a scaffold, it doesn't seem to be the case that the major cellular decapping defect observed in DCP1a/b knockout is due to DCP1's ability to recruit specific cofactors to DCP2.

The findings that none of the decapping cofactors recruited by DCP1 to DCP2 are essential for decapping in cells further underscore the complexity of the decapping process in vivo. This observation suggests that while DCP1's scaffolding function is crucial for recruiting cofactors, the decapping process likely involves additional layers of regulation that are not fully captured by our current understanding of DCP1. Furthermore, the reviewer mentions that the observed changes in RNA binding affinity (approximately 2-3 fold) in our in vitro experiments seem relatively modest. While these changes may appear insignificant in vitro, their cumulative impact in the dynamic cellular environment could be substantial. Even minor perturbations in RNA binding affinity can trigger cascading effects, leading to significant changes in decapping activity and the accumulation of deadenylated intermediates upon Dcp1 depletion. Cellular processes involve complex networks of interrelated events, and small molecular changes can result in amplified biological outcomes. The subtle molecular variations observed in vitro may translate into significant phenotypic outcomes within the complex cellular environment, underscoring the importance of DCP1a's regulatory role in the cellular decapping process.

So as far as I can tell, the discrepancy between the in vitro (DCP1 not required) and in-cell (DCP1 required) decapping data, remains entirely unresolved. Therefore, I don't think that the conclusions that DCP1 regulates decapping by (a) changing RNA binding affinity (authors show this doesn't matter in vitro, and that the change in RNA binding affinity is very small) or (b) by bridging interactions of cofactors with DCP2 (authors show all tested cofactors are dispensable for robust in-cell decapping activity), are supported by the evidence presented in the paper (or convincingly supported by previous structural and functional studies of the decapping complex).

We have addressed the reconciliation of differences between in vitro and in vivo experiments in the revised manuscript and emphasized the importance of considering cellular interactions when interpreting our findings.

(2) Related to the RNA binding claims mentioned above, are the differences shown in Figure 3H statistically significant? Why are there no error bars shown for the MBP control? (I understand this was normalized to 1, but presumably, there were 3 biological replicates here that have some spread of values?). The individual data points for each replicate should be displayed for each bar so that readers can better assess the spread of data and the significance of the observed differences. I've listed these points as major because of the key mechanistic claim that DCP1 enhances RNA binding to DCP2 hinges in large part on this data.

Thank you for your feedback. Regarding your comments on the statistical significance of the differences shown in Figure 3H and the absence of error bars for the MBP control, we will address these concerns in the revised manuscript. We’ll include individual data points for the three biological replicates and corresponding statistical analysis to more clearly demonstrate the data spread and significance of the observed differences.

(3) Also related to point (1) above, the kinetic analysis presented in Figure 2C shows that the large majority of transcript is mostly decapped at the first 5-minute timepoint; it may be that DCP2-mediated decapping activity is actually different in vitro with or without DCP1, but that this is being missed because the reaction is basically done in less than 5 minutes under the conditions being assayed (i.e. these are basically endpoint assays under these conditions). It may be that if kinetics were done under conditions to slow down the reaction somewhat (e.g. lower Dcp2 concentration, lower temperatures), so that more of the kinetic behavior is captured, the apparent discrepancy between in vitro and in-cell data would be much less. Indeed, previous studies have shown that in yeast, Dcp1 strongly activates the catalytic step (kcat) of decapping by ~10-fold, and reduces the KM by only ~2 fold (Floor et al, NSMB 2010). It might be beneficial to use purified proteins here (only a Western blot is used in Figure 2D to show the presence of DCP2 and/or DCP1, but do these complexes have other, and different, components immunoprecipitated along with them?), if possible, to better control reaction conditions.

This contradiction between the in vitro and in-cell decapping data undercuts one of the main mechanistic takeaways from the first half of the paper. This needs to be addressed/resolved with further experiments to better define the role of DCP1-mediated activation, or the mechanistic conclusions significantly changed or removed.

We genuinely appreciate the reviewer’s insightful comments on the kinetic analysis presented in Figure 2C. Your astute observation regarding the potential influence of reaction duration on the interpretation of in vitro decapping activity, especially in the absence of DCP1, is well-received. The time-sensitive nature of our experiments, as you rightly pointed out, might not fully capture the nuanced kinetic behaviors. In addition, the DCP2 complex purified from cells could not be precisely quantified. In response to your suggestion, we attempted to purify human DCP2 protein from E. coli; however, regrettably, the purified protein failed to exhibit any enzymatic activity. This disparity may be attributed to species differences.

Considering the reviewer’s valuable insights, our revised manuscript emphasized that purified DCP2 from cells exhibits activity regardless of the presence of DCP1. This adjustment aims to provide a clearer perspective on our findings and to better align with the nuances of our experimental design and the meticulous consideration of the results.

(4) The second half of the paper compares the transcriptomic and metabolic profiles of DCP1a versus DCP1b knockouts to reveal that these target a different subset of mRNAs for degradation and have different levels of cellular metabolites. This is a great application of the DCP1a/b KO cells developed in this paper and provides new information about DCP1a vs b function in metazoans, which to my knowledge has not really been explored at all. However, the analysis of DCP1 function/expression levels in human cancer seems superficial and inconclusive: for example, the authors conclude that "...these findings indicate that DCP1a and DCP1b likely have distinct and non-redundant roles in the development and progression of cancer", but what is the evidence for this? I see that DCP1a and b levels vary in different cancer cell types, but is there any evidence that these changes are actually linked to cancer development, progression, or tumorigenesis? If not, these broader conclusions should be removed.

Thank you to the reviewer for pointing out that such a description may be misleading. We have removed our previous broader conclusion and revised our sentences. To further explore the potential impact of DCP1a and DCP1b on cancer progression, we examined the association between the expression levels of DCP1a and DCP1b and progression-free interval (PFI). We have incorporated this information into our revised manuscript.

(5) The authors used CRISPR-Cas9 to introduce frameshift mutations that result in premature termination codons in DCP1a/b knockout cells (verified by Sanger sequencing). They then use Western blotting with DCP1a or DCP1b antibodies to confirm the absence of DCP1 in the knockout cell lines. However, the DCP1a antibody used in this study (Sigma D5444) is targeted to the C-terminal end of DCP1a. Can the authors conclusively rule out that the CRISPR/Cas-generated mutations do not result in the production of truncated DCP1a that is just unable to be detected by the C-terminally targeted antibody? While it is likely the introduced premature termination codon in the DCP1a gene results in nonsense-mediated decay of the resulting transcript, this outcome is indeed supported by the knockout results showing large defects in cellular decapping which can be rescued by the addition of the EVH1 domain, it would be better to carefully validate the success of the DCP1a knockout and conclusively show no truncated DCP1a is produced by using N-terminally targeted DCP1a antibodies (as was the case for DCP1b).

Thank you for your insightful comment regarding the validation of our DCP1a/b knockout cell line. We acknowledge your point about the DCP1a C-terminal targeting of the Sigma D5444 antibody used in our Western blot analysis. We agree that we cannot definitively rule out the possibility of truncated DCP1a protein production solely based on the lack of full-length protein detection. To address this limitation, we utilized a commercial information available N-terminally targeted DCP1a antibody (aviva ARP39353_T100) in a Western blot analysis. This will allow us to comprehensively detect any truncated protein fragments remaining after the CRISPR-Cas9-generated frameshift mutation.

Some additional minor comments:

• More information would be helpful on the choice of DCP1 truncation boundaries; why was 1-254 chosen as one of the truncations?

Thank you for the reviewer's comment and suggestion. Regarding the choice of DCP1 1-254 truncation boundaries based on the predicted structure from AlphaFoldDB (A0A087WT55). We will include this information in the revised manuscript.

• Figure S2D is a pretty important experiment because it suggests that the observed deadenylated intermediates are in fact still capped; can a positive control be added to these experiments to show that removal of cap results in rapid terminator-mediated degradation?

Unfortunately, due to our institution's current laboratory safety policies, we are unable to perform experiments involving the use of radioactive isotopes such as 32P. Therefore, while adding the suggested positive control experiment to demonstrate rapid RNA degradation upon decapping would further validate our interpretation, we regret that we cannot carry out this experiment at the moment. However, the observed deadenylated intermediates in Figure S2D match the predicted size of capped RNA fragments, and not the expected sizes of degradation products after decapping. Furthermore, previous literature has well-established that for these types of RNAs, decapping leads directly to rapid 5' to 3' exonuclease-mediated degradation, without producing stable deadenylated intermediates. Thus, we believe that the current data is sufficient to support our conclusion that the deadenylated intermediates retain the 5' cap structure.

Reviewer #2 (Public Review):

Weaknesses:

The direct targets of DCP1a and/or DCP1b were not determined as the analysis was restricted to RNA-seq to assess RNA abundance, which can be a result of direct or indirect regulation by DCP1a/b.

Thank you for raising this important point. In our study, we acknowledge that the use of RNA-seq to assess RNA abundance provides a broad overview of the regulatory impacts of DCP1a and DCP1b. This method captures changes in RNA levels that may arise from both direct and indirect regulatory actions of these proteins. While we did not directly determine the targets of DCP1a and DCP1b, the data obtained from our RNA-seq analysis serve as a foundational step for future targeted experiments, which could include techniques such as RIP-seq, to delineate the direct targets of DCP1a and DCP1b more precisely. We believe that our current findings contribute valuable information to the field and pave the way for these subsequent analyses.

P-bodies appear to be larger in human cells lacking DCP1a and DCP1b but a lack of image quantification prevents this conclusion from being drawn.

Thank you for the reviewer’s valuable feedback. We have addressed the reviewer’s concern regarding P-bodies' size in human cells lacking DCP1a and DCP1b. We have now performed image quantification and can confirm that P-bodies are indeed larger in these cells.

The lack of details in the methodology and figure legends limit reader understanding.

We acknowledge the reviewer's concerns regarding the level of detail provided in the methodology and figure legends. To address this, we are committed to enhancing both sections with additional details and clarifications in our revised manuscript. Thank you for bringing this to our attention.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

(1) To me, the second half of the paper comparing DCP1a and DCP1b is in many ways distinct from the first half and could stand on its own as an interesting paper if this comparative analysis is explored a little deeper (maybe by validating some of the differences in decay observed for individual mRNAs targeted by DCP1a versus DCP1b, by measuring and comparing the decay rates of some individual transcripts under differential control by DCP1a vs b?), and revising the conclusions about links to cancer as mentioned above. I think these later comparative results in the paper present the most new and interesting data concerning DCP1 function in humans (especially since I think the mechanistic conclusions from the first half aren't well supported yet or are at least inconsistent), but when I read these later sections of the paper I struggle to understand the key takeaways from the transcriptomic and metabolomic data.

Thank you for the reviewer's suggestions. Estimating the decay rates of individual transcripts within the transcriptomes of DCP1a_KO, DCP1b_KO, and wild type can provide insight into the direct targets of DCP1a or DCP1b. However, this requires either time-series RNA-seq or specialized sequencing technologies such as Precision Run-On sequencing (PRO-seq) or RNA Approach to Equilibrium Sequencing (RATE-Seq). Unfortunately, we lack the necessary dataset in our project to estimate the decay rates for the potential targets identified in our RNA-seq data. Despite this limitation, we acknowledge the potential of this approach in identifying the true targets of DCP1a and DCP1b and have included this idea in our discussion.

(2) I think it would be helpful to add a little more descriptive or narrative language to the figure legends (I know some of them are already quite long!) so that readers can follow the general idea of the experiment through the figure legend as well as the main text; as written, the figure legends are mostly exclusively technical details, so it can be hard to parse what experiment is being carried out in some cases.

Thank you for the reviewer’s suggestion, we will strive to improve the language of the figure legends to include technical details while clearly conveying the main idea of the experiment. We will ensure that the language of the figure legends is more readable and comprehensible so that readers can more easily parse what experiment is being carried out.

Reviewer #2 (Recommendations For The Authors):

Suggestions for improved or additional experiments, data, or analyses:

The use of RNA-seq to measure RNA abundance in DCP1a and/or b knockout cells can give some insight into both the indirect and direct effects of DCP1a/b on gene expression but cannot identify the direct targets of these genes. Rather, global analysis of RNA stability or capturing uncapped RNA decay intermediates would allow the authors to conclude they have identified direct targets of DCP1a and/or b. Without such analyses, the interpretation of these data should be scaled back to clearly state that RNA levels can be altered through indirect effects of DCP1a/b absence throughout the text.

We appreciate the reviewer's suggestion. We have modified our sentences to emphasize that the dysregulated genes could be caused by both direct and indirect effects.

A control/randomly generated gene list should be analyzed for GO terms to determine whether the enrichment of cancer-related pathways in the differentially expressed genes in the DCP1a/b knockout cells is meaningful.

Thank you for the reviewer's comment. We shuffled our gene list and reperformed the pathway enrichment analysis in Figure 4C and 4D 1,000 times. We focused on the following cancer-related pathways: E2F targets, MTORC1 signaling, G2M checkpoint, MYC target V1, EMT transition, KRAS signaling DN, P53 pathway, and NOTCH signaling pathways. We then calculated how many times the q-values obtained from the shuffled gene list were more significant than the q-value obtained from our real data. In four of the eight pathways (E2F targets, MTORC1 signaling, G2M checkpoint, and MYC target v1), none of the shuffled gene lists resulted in a q-value smaller than the real one. In the other four pathways (EMT transition, KRAS signaling DN, P53 pathway, and NOTCH signaling pathways), the q-values were smaller than the real q-value 2, 11, 4, and 4 times out of the 1000 shuffles. Based on the shuffled results, we conclude that the transcriptome of DCP1a/b knockout cells is statistically enriched in these cancer-related pathways.

Author response image 1.

Distribution of q-values resulting from the Gene Set Enrichment Analysis (GSEA) conducted on 1,000 shuffled gene lists for eight cancer-related pathways. The q-values derived from Figure 4C and 4D are indicated by red (DCP1a_KO) and blue (DCP1b_KO) dashed lines, respectively. Some q-values derived from Figure 4C are too small to be labeled on the plots, such as in E2F targets (q value: 5.87E-07), MTORC1 signaling (q values: 6.59E-07 and 1.58E-06 for DCP1a_KO and DCP1b_KO, respectively), MYC target V1 (q value: 0.004644174 for DCP1a_KO), etc. The numbers x/1000 indicate how often the shuffled q-values were smaller than the real q-value out of 1,000 permutations.

Comparisons of the DCP1a and/or b knockout RNA-seq results should be done to published datasets such as those published by Luo et al., Cell Chemical Biology (2021) to determine whether there are common targets with DCP2 and validate the reported findings.

Thank you for reviewer’s suggestion. We compared the upregulated genes from DCP1a_KO, DCP1b_KO, and DCP1a/b_KO cell lines with the 91 targets of DPC2 identified by Luo et al. in Cell Chemical Biology (2021). Only EPPK1 was found to be overlapped between the potential DCP1b_KO targets and the targets of DCP2. No genes were found to be overlapped between the potential DCP1a_KO targets and the targets of DCP2. However, three genes, TES, PAX6, and C18orf21, were found to be overlapped between the significantly upregulated DEGs of DCP1a/b_KO and the targets of DCP2. We have included this information in the discussion section.

The RNA tethering assays are not clear and are difficult to interpret without further controls to delineate the polyadenylated and deadenylated species.

Thank you for the reviewer’s feedback. We acknowledge that the reviewer might harbor some doubts regarding the outcomes of the RNA tethering assays. Nonetheless, this methodology is well-established and has also found extensive application across many studies. We are committed to enhancing the clarity of our experiment’s details and results within the figure legends and textual descriptions.

The representative images of p-bodies clearly show that DCP1a/b KO cells have larger p-bodies than the wild-type cells. The authors should quantify p-body size in each image set as the current interpretation of the data is that there is no difference in size or number of p-bodies, but the data suggest otherwise.

Thank you very much for the reviewer’s insightful comments and for drawing our attention to the need to quantify p-body sizes in DCP1a/b KO and wild-type cells. We agree with the reviewer’s assessment that the representative images suggest a difference in p-body size between DCP1a/b KO cells and wild-type cells, which we initially overlooked. We will revise our manuscript accordingly to include these findings, ensuring that our interpretation of the data aligns with the observed differences.

Statistical analysis of the Figure 2C results should be included because the difference between the wild-type and Dco1a/b KO cells with GFP-DCP2 looks significantly different but is interpreted in the text as not significant.

Thank you for pointing out the need for a statistical analysis of the results shown in Figure 2C. We acknowledge that the visual difference between the wild-type and Dco1a/b KO cells with GFP-DCP2 suggests a significant variation, which may not have been clearly communicated in our text. We will conduct the necessary statistical analysis to substantiate the observations made in Figure 2C. Furthermore, we would like to emphasize that our primary focus was to demonstrate that purified DCP2 within cells retains its activity even in the absence of DCP1. This critical point will be highlighted and clarified in the revised version of our manuscript to prevent any misunderstanding.

Recommendations for improving the writing and presentation:

Additional context including what is known about the role of dcp1 in decapping from the decades of work in yeast and other model organisms should be incorporated into the introduction and discussion sections.

Thank you for the reviewer’s suggestion. We will incorporate additional context about the function and significance of DCP1 in decapping processes within our revised manuscript's introduction and discussion sections.

Details should be provided within the figure legends and methods section on experimental approaches and the number of replicates and statistical analyses used throughout the manuscript. For example, it is not clear whether western blots or RNA-IP experiments were performed more than once as representative images are shown.

Thank you for the reviewer’s suggestion. In the figure legends and methods section, we will provide more details about the experimental methods, number of replicates, and statistical analyses. Regarding the Western blots and RNA-IP experiments the reviewer mentioned, we performed multiple experiments and presented representative images in the manuscript. We will clarify this in the revised manuscript to eliminate potential confusion.

The rationale for performing metabolic profiling is not clear.

We appreciate the reviewer's thoughtful feedback. The rationale behind conducting metabolic profiling in our study is rooted in its efficacy as a valuable tool for deciphering the consequences of specific gene mutations, particularly those closely associated with phenotypic changes or final metabolic pathways. Our objective is to utilize metabolic profiling to unravel the distinct biofunctions of DCP1a and DCP1b. By employing this approach, we aim to gain insights into the intricate metabolic alterations that result from the absence of these genes, thereby enhancing our understanding of their roles in cellular processes. We recognize the necessity of clearly presenting this rationale and promise to bolster the articulation of these points in the revised version of our manuscript to ensure the clarity and transparency of our research motivation.

Details in the methods section should be included for the CRISPR/Cas9-mediated gene editing validation. The Sangar sequencing results presented in Figure S1b should be explained. The entire western blot(s) should be shown in Figure S1A to give confidence the Dcp1a/b KO cells are not expressing truncated proteins and the epitopes of the antibodies used to detect Dcp1a/b should be described. The northern blot probes should be described and sequences included. The transcriptomics method should be detailed.

Thank you for your feedback, in the revised manuscript we will detail the CRISPR/Cas9 gene editing validation, explain the Sanger sequencing results in Figure S1b, show the full Western blot in Figure S1A to confirm that the Dcp1a/b knockout cells are not expressing truncated proteins, describe the Northern blot probes used, and detail the transcriptomics method, all to ensure clarity and comprehensiveness in our experimental procedures and results.

A diagram showing the RNA tethering assays with labels corresponding to all blots/gels should be provided.

Thank you for your suggestion. We will provide a diagram showing the RNA tethering assays with labels corresponding to all blots/gels in our revised manuscript. This will help readers better understand our experimental design and results.

The statement, "This suggests that the disruption of the decapping process in DCP1a/b-knockout cells results in the accumulation of unprocessed mRNA intermediates" regarding the results of the RNA-seq assay is not supported by the evidence as RNA-seq does not measure RNA decay intermediates or RNA decay rates.

Thank you for the reviewer’s comment. We agree with that RNA-seq experiments indeed do not directly measure RNA decay intermediates or RNA decay rates. Our statement could have caused confusion, and we have therefore removed this sentence from the manuscript.

Minor corrections to the text and figures:

Figure S6A is uninterpretable as presented.

Thank you for the reviewer’s valuable feedback. We have taken note and made improvements. We have simplified Figure S6A to enhance its interpretability, hoping that the current version will make it easier for the readers to understand.

eLife Assessment

In this manuscript, the authors present valuable findings on the apparent role of a salience-network anterior insula node in directing fronto-parietal and default-mode network activity within a tripartite network during control of memory, drawn from an impressive invasive human neurophysiological dataset. Overall, the authors have presented a convincing set of analyses. We also commend the use of a large intracranial EEG dataset to approach this question.

Reviewer #1 (Public review):

Summary

Das and Menon describe an analysis of a large open-source iEEG dataset (UPENN-RAM). From encoding and recall phases of memory tasks, they analyzed power and phase-transfer entropy as a measure of directed information flow in regions across a hypothesized tripartite network system. The anterior insula (AI) was found to have heightened high gamma power during encoding and retrieval, which corresponded to suppression of high gamma power in medial prefrontal cortex (mPFC) and posterior cingulate cortex (PCC) during encoding but not recall. In contrast, directed information flow from (but not to) AI to mPFC and PCC is high during both time periods when PTE is analyzed with broadband but not narrowband activity. They claim that these findings significantly advance an understanding of how network communication facilitates cognitive operations during memory tasks, and that the AI of the salience network (SN) is responsible for influencing both the frontoparietal network (FPN) and default-mode network (DMN) during memory encoding and retrieval.

I find this question interesting and important and agree with the authors that iEEG presents a unique opportunity to investigate the temporal dynamics within network nodes. Their findings convey intriguing information about the structure and order of communication between network regions during on-task cognition in general (though, perhaps not specific to memory - see Weaknesses), with the AI of the SN ostensibly playing an important role in possibly influencing the DMN and FPN.

Strengths

- The authors present results from an impressively-sized iEEG sample. For reader context, this type of invasive human data is difficult and time-consuming to collect and many similar studies in high-level journals include 5-20 participants, typically not all of whom have electrodes in all regions of interest. It is excellent that they have been able to leverage open-source data in this way.<br /> - Preprocessing of iEEG data also seems sensible and appropriate based on field standards.<br /> - The authors tackle the replication issues inherent in much of the literature by replicating findings across task contexts, demonstrating that the principles of network communication evidenced by their results generalize in multiple task memory contexts. Again, the number of iEEG patients who have multiple tasks' worth of data is impressive.<br /> - Though the revised manuscript presents a broader and more novel investigation of the tripartite network's role in memory encoding and retrieval (as opposed to cognitive control of memory) the authors now thoroughly review the literature motivating this investigation of open-source data.

Weaknesses

- As the authors discuss, it is currently unclear if the directed information flow from AI to DMN and FPN nodes truly arises from memory-associated processes as opposed to more general attentional and cognitive demands, especially given that information flow does not relate meaningfully to task performance (whether memory retrieval is successful or not). I also note this is a concern because - though the authors have now demonstrated that information flow is increased compared to an off-task baseline - influences of AI on DMN or FPN were not increased relative to baseline epochs during the task in the original preprint version, again suggesting these effects may not be specific to the memory component of the analyzed tasks. The authors have thoughtfully noted in the Discussion several ways that experimental design can be improved in future studies to address this limitation.

Author response:

The following is the authors’ response to the previous reviews.

Removing claims of causality: To avoid confusion, we have now removed claims of causality from our manuscript and also changed the title of the manuscript accordingly

"Electrophysiological dynamics of salience, default mode, and frontoparietal networks during episodic memory formation and recall: A multi-experiment iEEG replication".

Control analyses directly comparing AI and IFG: As per the reviewer’s suggestion, we have carried out additional control analyses by directly comparing the net inward/outward balance between the AI and the IFG. Our analysis revealed that the net outflow for the AI is significantly higher compared to the IFG during both encoding and recall phases, a pattern that was replicated across all four experiments. 

These findings further highlight the unique role of the AI as a key hub in coordinating network interactions during episodic memory formation and retrieval, distinguishing it from a key anatomically adjacent prefrontal region implicated in cognitive control.

We have incorporated these results into the manuscript (see new Figure S6 and updated Results section). 

Control analyses directly comparing task with resting state: As per the reviewer’s suggestion, we compared the AI's net outflow during task periods to resting state, finding significantly higher outflow during both encoding and recall across all experiments (ps < 0.05). These results provide further evidence for enhanced role of AI net directed information flow to the DMN and FPN during memory processing compared to the resting state. 

We have incorporated these results into the manuscript (see new Figure S9 and updated Results section). 

Control analysis using every region of the brain outside the considered networks: We appreciate the reviewer's suggestion to conduct additional control analyses. However, we have concerns about implementing this approach for several reasons:

(1) Hypothesis-driven research: Our study was designed based on a strong hypothesis derived from prior fMRI studies, which have consistently shown that the salience network (SN), anchored by the anterior insula (AI), plays a critical role in regulating the engagement and disengagement of the default mode network (DMN) and frontoparietal network (FPN) across diverse cognitive tasks.

(2) Risk of p-hacking: Running analyses on a large number of brain regions outside our networks of interest without a priori hypotheses could lead to p-hacking, a practice strongly criticized in the scientific community, including by eLife editors (Makin & Orban de Xivry, 2019). Such an approach could potentially yield spurious results and undermine the validity of our findings.

(3) Principled control region selection: Our choice of the inferior frontal gyrus (IFG) as a control region was hypothesis-driven, based on its: a) Anatomical adjacency to the AI b) Involvement in cognitive control functions, including response inhibition c) Frequent coactivation with the AI in fMRI studies. 

(4) Robustness of current findings: Our PTE analysis involving the IFG, along with the additional control analyses requested by the reviewer (comparing the task-related net balance of the AI with the IFG and with resting state, see response to reviewer comment 2.1), strongly support a key role for the AI in orchestrating large-scale network dynamics during memory processes.

(5) Specificity of findings: The contrast between AI and IFG results demonstrates that our observed patterns are not general to all task-active regions but are specific to the AI's role in network coordination. 

We believe that our current analyses, including the additional controls, provide a comprehensive and rigorous examination of the AI's role in memory-related network dynamics. Adding analyses of numerous additional regions without clear hypotheses could potentially dilute the focus and interpretability of our results. 

However, we acknowledge the importance of considering broader network interactions. In future studies, we could explore the role of other key regions in a hypothesis-driven manner, potentially expanding our understanding of the complex interactions between multiple brain networks during memory processes.

These revisions, combined with our rigorous methodologies and comprehensive analyses, provide compelling support for the central claims of our manuscript. We believe these changes significantly enhance the scientific contribution of our work.

Our point-by-point responses to the reviewers' comments are provided below.

Reviewer 1:

(1.1) Because phase-transfer entropy is referenced as a "causal" analysis in this investigation (PTE), I believe it is important to highlight for readers recent discussions surrounding the description of "causal mechanisms" in neuroscience (see "Confusion about causation" section from Ross and Bassett, 2024, Nature Neuroscience). A large proportion of neuroscientists (myself included) use "causal" only to refer to a mechanism whose modulation or removal (with direct manipulation, such as by lesion or stimulation) is known to change or control a given outcome (such as a successful behavior). As Ross and Bassett highlight, it is debatable whether such mechanistic causality is captured by Granger "causality" (a.k.a. Granger prediction) or the parametric PTE, and imprecise use of "causation" may be confusing. The authors have defined in the revised Introduction what their definition of "causality" is within the context of this investigation. 

We appreciate the reviewer's feedback in terms of the terminology used in our manuscript. To avoid confusion, we have now removed claims of causality from our manuscript and also changed the title of the manuscript accordingly. 

Reviewer 2:

(2.1) Clarifying the new control analyses. The authors have been responsive to our feedback and implemented several new analyses. The use of a pre-task baseline period and a control brain region (IFG) definitively help to contextualize their results, and the findings shown in the revision do suggest that (1) relative to a pre-task baseline, directed interactions from the AI are stronger and (2) relative to a nearby region, the IFG, the AI exhibits greater outward-directed influence. 

However, it is difficult to draw strong quantitative conclusions from the analyses as presented, because they do not directly statistically contrast the effect in question (directed interactions with the FPN and DMN) between two conditions (e.g. during baseline vs. during memory encoding/retrieval). As I understand it, in their main figures the authors ask, "Is there statistically greater influence from the AI to the DMN/FPN in one direction versus another?" And in the AI they show greater "outward" PTE than "inward" PTE from other networks during encoding/retrieval. The balance of directed information favors an outward influence from the AI to DMN/FPN. 

But in their new analyses, they simply show that the degree of "outward" PTE is greater during task relative to baseline in (almost) all tasks. I believe a more appropriately matched analysis would be to quantify the inward/outward balance during task states, quantify the inward/outward balance during rest states, and then directly statistically compare the two. It could be that the relative balance of directed information flow is nonsignificantly changed between task and rest states, which would be important to know. 

We thank the reviewer for this suggestion. We have now run additional analysis by directly comparing the inward/outward balance during the task versus the rest states. To calculate the net inward/outward balance, we calculated the net outflow as the difference between the total outgoing information and total incoming information (PTE(out)–PTE(in)). This analysis revealed that net outflow during task periods is significantly higher compared to rest, during both encoding and recall, and across the four experiments (ps < 0.05). These results provide further evidence for enhanced role of AI net directed information flow to the DMN and FPN during memory processing compared to the resting state. These new results have now been included in the revised manuscript (page 12). 

Likewise, a similar principle applies to their IFG analysis. They show that the IFG tends to have an "inward" balance of influence from the DMN/FPN (the opposite of the AIs effect), but this does not directly answer whether the AI occupies a statistically unique position in terms of the magnitude of its influence on other regions. More appropriate, as I suggest above, would be to quantify the relative balance inward/outward influence, both for the IFG and the AI, and then directly compare those two quantities. (Given the inversion of the direction of effect, this is likely to be a significant result, but I think it deserves a careful approach regardless.) 

We appreciate the reviewer's suggestion. As per the reviewer’s suggestion, we directly compared the net inward/outward balance between the AI and the IFG. Specifically, we compared the net outflow (PTE(out)–PTE(in)) for the AI with the IFG. This analysis revealed that the net outflow for the AI is significantly higher compared to the IFG during both encoding and recall, and across the four experiments. These findings further highlight a key role for the AI in orchestrating large-scale network dynamics during memory processes. The AI's pattern of directed information flow stands in contrast to that of the IFG, despite their anatomical proximity and shared involvement in cognitive control processes. This dissociation underscores the specificity of the AI's function in coordinating network interactions during memory formation and retrieval. These new results have now been included in our revised manuscript (page 11). 

(2.2) Consider additional control regions. The authors justify their choice of IFG as a control region very well. In my original comments, I perhaps should have been more clear that the most compelling control analyses here would be to subject every region of the brain outside these networks (with good coverage) to the same analysis, quantify the degree of inward/outward balance, and then see how the magnitude of the AI effect stacks up against all possible other options. If the assertion is that the AI plays a uniquely important role in these memory processes, showing how its influence stacks up against all possible "competitors" would be a very compelling demonstration of their argument. 

We thank the reviewer for this suggestion. However, please note that running a large number of random analysis by including a large number of brain regions (every region of the brain outside these networks) and comparing their dynamics to the AI without a hypothesis or solid principle amounts to p-hacking, which has been previously strongly criticized by the eLife editors (Makin & Orban de Xivry, 2019). Our study was strongly driven by a solid hypothesis based on prior fMRI studies that have shown that the SN, anchored by the anterior insula (AI), plays a critical role in regulating the engagement and disengagement of the DMN and FPN across diverse cognitive tasks (Bressler & Menon, 2010; Cai et al., 2016; Cai, Ryali, Pasumarthy, Talasila, & Menon, 2021; Chen, Cai, Ryali, Supekar, & Menon, 2016; Kronemer et al., 2022; Raichle et al., 2001; Seeley et al., 2007; Sridharan, Levitin, & Menon, 2008). Moreover, our selection of the IFG as a control region for comparison was also very strongly hypothesis driven, due to its anatomical adjacency to the AI, its involvement in a wide range of cognitive control functions including response inhibition (Cai, Ryali, Chen, Li, & Menon, 2014), and its frequent co-activation with the AI in fMRI studies. Furthermore, the IFG has been associated with controlled retrieval of memory (Badre, Poldrack, Paré-Blagoev, Insler, & Wagner, 2005; Badre & Wagner, 2007; Wagner, Paré-Blagoev, Clark, & Poldrack, 2001), making it a compelling region for comparison. Our findings related to the PTE analysis involving the IFG and also the additional control analyses requested by the reviewer (directly comparing the task-related net balance of the AI with the IFG and also to resting state, please see response to reviewer comment 2.1) strongly highlight a key role of the AI in orchestrating large-scale network dynamics during memory processes. 

We believe that our current analyses, including the additional controls, provide a comprehensive and rigorous examination of the AI's role in memory-related network dynamics. Adding analyses of numerous additional regions without clear hypotheses could potentially dilute the focus and interpretability of our results.

However, we acknowledge the importance of considering broader network interactions. In future studies, we could explore the role of other key regions in a hypothesis-driven manner, potentially expanding our understanding of the complex interactions between multiple brain networks during memory processes.

(2.3) Reporting of successful vs. unsuccessful memory results. I apologize if I was not clear in my original comment (2.7, pg. 13 of the response document) regarding successful vs. unsuccessful memory. The fact that no significant difference was found in PTE between successful/unsuccessful memory is a very important finding that adds valuable context to the rest of the manuscript. I believe it deserves a figure, at least in the Supplement, so that readers can visualize the extent of the effect in successful/unsuccessful trials. This is especially important now that the manuscript has been reframed to focus more directly on claims regarding episodic memory processing; if that is indeed the focus, and their central analysis does not show a significant effect conditionalized on the success of memory encoding/retrieval, it is important that readers can see these data directly.

As per the reviewer’s suggestion, we have now included a Figure related to the results for the successful versus unsuccessful comparison in the Supplementary materials of the revised manuscript (Figures S10, S11).   

(2.4) Claims regarding causal relationships in the brain. I understand that the authors have defined "causal" in a specific way in the context of their manuscript; I do believe that as a matter of clear and transparent scientific communication, the authors nonetheless bear a responsibility to appreciate how this word may be erroneously interpreted/overinterpreted and I would urge further review of the manuscript to tone down claims of causality. Reflective of this, I was very surprised that even as both reviewers remarked on the need to use the word "causal" with extreme caution, the authors added it to the title in their revised manuscript.

We thank the reviewer for this suggestion. To avoid confusion, we have now removed claims of causality from our manuscript and also changed the title of the manuscript accordingly. 

References 

Badre, D., Poldrack, R. A., Paré-Blagoev, E. J., Insler, R. Z., & Wagner, A. D. (2005). Dissociable controlled retrieval and generalized selection mechanisms in ventrolateral prefrontal cortex. Neuron, 47(6), 907-918. doi:10.1016/j.neuron.2005.07.023

Badre, D., & Wagner, A. D. (2007). Left ventrolateral prefrontal cortex and the cognitive control of memory. Neuropsychologia, 45(13), 2883-2901. doi:10.1016/j.neuropsychologia.2007.06.015

Bressler, S. L., & Menon, V. (2010). Large-scale brain networks in cognition: emerging methods and principles. Trends in Cognitive Sciences, 14(6), 277-290. doi:10.1016/j.tics.2010.04.004

Cai, W., Chen, T., Ryali, S., Kochalka, J., Li, C. S., & Menon, V. (2016). Causal Interactions Within a Frontal-Cingulate-Parietal Network During Cognitive Control: Convergent Evidence from a Multisite-Multitask Investigation. Cereb Cortex, 26(5), 2140-2153. doi:10.1093/cercor/bhv046

Cai, W., Ryali, S., Chen, T., Li, C. S., & Menon, V. (2014). Dissociable roles of right inferior frontal cortex and anterior insula in inhibitory control: evidence from intrinsic and taskrelated functional parcellation, connectivity, and response profile analyses across multiple datasets. J Neurosci, 34(44), 14652-14667. doi:10.1523/jneurosci.3048-14.2014

Cai, W., Ryali, S., Pasumarthy, R., Talasila, V., & Menon, V. (2021). Dynamic causal brain circuits during working memory and their functional controllability. Nat Commun, 12(1), 3314. doi:10.1038/s41467-021-23509-x

Chen, T., Cai, W., Ryali, S., Supekar, K., & Menon, V. (2016). Distinct Global Brain Dynamics and Spatiotemporal Organization of the Salience Network. PLOS Biology, 14(6), e1002469. doi:10.1371/journal.pbio.1002469

Kronemer, S. I., Aksen, M., Ding, J. Z., Ryu, J. H., Xin, Q., Ding, Z., . . . Blumenfeld, H. (2022). Human visual consciousness involves large scale cortical and subcortical networks independent of task report and eye movement activity. Nat Commun, 13(1), 7342. doi:10.1038/s41467-022-35117-4

Makin, T. R., & Orban de Xivry, J. J. (2019). Ten common statistical mistakes to watch out for when writing or reviewing a manuscript. Elife, 8. doi:10.7554/eLife.48175

Raichle, M. E., MacLeod, A. M., Snyder, A. Z., Powers, W. J., Gusnard, D. A., & Shulman, G. L. (2001). A default mode of brain function. Proc Natl Acad Sci U S A, 98(2), 676-682. doi:10.1073/pnas.98.2.676

Seeley, W. W., Menon, V., Schatzberg, A. F., Keller, J., Glover, G. H., Kenna, H., . . . Greicius, M. D. (2007). Dissociable Intrinsic Connectivity Networks for Salience Processing and Executive Control. Journal of Neuroscience, 27(9), 2349-2356. doi:10.1523/JNEUROSCI.5587-06.2007

Sridharan, D., Levitin, D. J., & Menon, V. (2008). A critical role for the right fronto-insular cortex in switching between central-executive and default-mode networks. Proceedings of the National Academy of Sciences, 105(34), 12569-12574. doi:10.1073/pnas.0800005105

Wagner, A. D., Paré-Blagoev, E. J., Clark, J., & Poldrack, R. A. (2001). Recovering meaning: left prefrontal cortex guides controlled semantic retrieval. Neuron, 31(2), 329-338. doi:10.1016/s0896-6273(01)00359-2

public

q2

Together Works

social science is a big topic, it involve many aspects of knowledge, so I think it can't easy to define, which cause some controversies.

Reviewer #1 (Public review):

The authors survey the ultrastructural organization of glutamatergic synapses by cryo-ET and image processing tools using two complementary experimental approaches. The first approach employs so-called "ultra-fresh" preparations of brain homogenates from a knock-in mouse expressing a GFP-tagged version of PSD-95, allowing Peukes and colleagues to specifically target excitatory glutamatergic synapses. In the second approach, direct in-tissue (using cortical and hippocampal regions) targeting of the glutamatergic synapses employing the same mouse model is presented. In order to ascertain whether the isolation procedure causes any significant changes in the ultrastructural organization (and possibly synaptic macromolecular organization) the authors compare their findings using both of these approaches. The quantitation of the synaptic cleft height reveals an unexpected variability, while the STA analysis of the ionotropic receptors provides insights into their distribution with respect to the synaptic cleft.

The main novelty of this study lies in the continuous claims by the authors that the sample preservation methods developed here are superior to any others previously used. This leads them as well to systematically downplay or directly ignore a substantial body of previous cryo-ET studies of synaptic structure. Without comparisons with the cryo-ET literature, it is very hard to judge the impact of this work in the field. Furthermore, the data does not show any better preservation in the so-called "ultra-fresh" preparation than in the literature, perhaps to the contrary as synapses with strangely elongated vesicles are often seen. Such synapses have been regularly discarded for further analysis in previous synaptosome studies (e.g. Martinez-Sanchez 2021). Whilst the targeting approach using a fluorescent PSD95 marker is novel and seems sufficiently precise, the authors use a somewhat outdated approach (cryo-sectioning) to generate in-tissue tomograms of poor quality. To what extent such tomograms can be interpreted in molecular terms is highly questionable. The authors also don't discuss the physiological influence of 20% dextran used for high-pressure freezing of these "very native" specimens.

Lastly, a large part of the paper is devoted to image analysis of the PSD which is not convincing (including a somewhat forced comparison with the fixed and heavy-metal staining room temperature approach). Despite being a technically challenging study, the results fall short of expectations.

Reviewer #2 (Public review):

Summary:

The authors set out to visualize the molecular architecture of the adult forebrain glutamatergic synapses in a near-native state. To this end, they use a rapid workflow to extract and plunge-freeze mouse synapses for cryo-electron tomography. In addition, the authors use knockin mice expression PSD95-GFP in order to perform correlated light and electron microscopy to clearly identify pre- and synaptic membranes. By thorough quantification of tomograms from plunge- and high-pressure frozen samples, the authors show that the previously reported 'post-synaptic density' does not occur at high frequency and therefore not a defining feature of a glutamatergic synapse.

Subsequently, the authors are able to reproduce the frequency of post-synaptic density when preparing conventional electron microscopy samples, thus indicating that density prevalence is an artifact of sample preparation. The authors go on to describe the arrangement of cytoskeletal components, membraneous compartments, and ionotropic receptor clusters across synapses.

Demonstrating that the frequency of the post-synaptic density in prior work is likely an artifact and not a defining feature of glutamatergic synapses is significant. The descriptions of distributions and morphologies of proteins and membranes in this work may serve as a basis for the future of investigation for readers interested in these features.

Strengths:

The authors perform a rigorous quantification of the molecular density profiles across synapses to determine the frequency of the post-synaptic density. They prepare samples using two cryogenic electron microscopy sample preparation methods, as well as one set of samples using conventional electron microscopy methods. The authors can reproduce previous reports of the frequency of the post-synaptic density by conventional sample preparation, but not by either of the cryogenic methods, thus strongly supporting their claim.

Reviewer #3 (Public review):

Summary:

The authors use cryo-electron tomography to thoroughly investigate the complexity of purified, excitatory synapses. They make several major interesting discoveries: polyhedral vesicles that have not been observed before in neurons; analysis of the intermembrane distance, and a link to potentiation, essentially updating distances reported from plastic-embedded specimen; and find that the postsynaptic density does not appear as a dense accumulation of proteins in all vitrified samples (less than half), a feature which served as a hallmark feature to identify excitatory plastic-embedded synapses.

Strengths:

(1) The presented work is thorough: the authors compare purified, endogenously labeled synapses to wild-type synapses to exclude artifacts that could arise through the homogenation step, and, in addition, analyse plastic embedded, stained synapses prepared using the same quick workflow, to ensure their findings have not been caused by way of purification of the synapses. Interestingly, the 'thick lines of PSD' are evident in most of their stained synapses.

(2) I commend the authors on the exceptional technical achievement of preparing frozen specimens from a mouse within two minutes.

(3) The approaches highlighted here can be used in other fields studying cell-cell junctions.

(4) The tomograms will be deposited upon publication which will enable neurobiologists and researchers from other fields to carry on data evaluation in their field of expertise since tomography is still a specialized skill and they collected and reconstructed over 100 excellent tomograms of synapses, which generates a wealth of information to be also used in future studies.

(5) The authors have identified ionotropic receptor positions and that they are linked to actin filaments, and appear to be associated with membrane and other cytosolic scaffolds, which is highly exciting.

(6) The authors achieved their aims to study neuronal excitatory synapses in great detail, were thorough in their experiments, and made multiple fascinating discoveries. They challenge dogmas that have been in place for decades and highlight the benefit of implementing and developing new methods to carefully understand the underlying molecular machines of synapses.

Weaknesses:

The authors show informative segmentations in their figures but none have been overlayed with any of the tomograms in the submitted videos. It would be helpful for data evaluation to a broad audience to be able to view these together as videos to study these tomograms and extract more information. Deposition of segmentations associated with the tomgrams would be tremendously helpful to Neurobiologists, cryo-ET method developers, and others to push the boundaries.

Impact on community:

The findings presented by Peukes et al. pertaining to synapse biology change dogmas about the fundamental understanding of synaptic ultrastructure. The work presented by the authors, particularly the associated change of intermembrane distance with potentiation and the distinct appearance of the PSD as an irregular amorphous 'cloud' will provide food for thought and an incentive for more analysis and additional studies, as will the discovery of large membranous and cytosolic protein complexes linked to ionotropic receptors within and outside of the synaptic cleft, which are ripe for investigation. The findings and tomograms available will carry far in the synapse fields and the approach and methods will move other fields outside of neurobiology forward. The method and impactful results of preparing cryogenic, unlabeled, unstained, near-native synapses may enable the study of how synapses function at high resolution in the future.

Author response:

Reviewer #1 (Public review): 

The authors survey the ultrastructural organization of glutamatergic synapses by cryo-ET and image processing tools using two complementary experimental approaches. The first approach employs so-called "ultra-fresh" preparations of brain homogenates from a knock-in mouse expressing a GFP-tagged version of PSD-95, allowing Peukes and colleagues to specifically target excitatory glutamatergic synapses. In the second approach, direct in-tissue (using cortical and hippocampal regions) targeting of the glutamatergic synapses employing the same mouse model is presented. In order to ascertain whether the isolation procedure causes any significant changes in the ultrastructural organization (and possibly synaptic macromolecular organization) the authors compare their findings using both of these approaches. The quantitation of the synaptic cleft height reveals an unexpected variability, while the STA analysis of the ionotropic receptors provides insights into their distribution with respect to the synaptic cleft.

The main novelty of this study lies in the continuous claims by the authors that the sample preservation methods developed here are superior to any others previously used. This leads them as well to systematically downplay or directly ignore a substantial body of previous cryo-ET studies of synaptic structure. Without comparisons with the cryo-ET literature, it is very hard to judge the impact of this work in the field. Furthermore, the data does not show any better preservation in the so-called "ultra-fresh" preparation than in the literature, perhaps to the contrary as synapses with strangely elongated vesicles are often seen. Such synapses have been regularly discarded for further analysis in previous synaptosome studies (e.g. Martinez-Sanchez 2021). Whilst the targeting approach using a fluorescent PSD95 marker is novel and seems sufficiently precise, the authors use a somewhat outdated approach (cryo-sectioning) to generate in-tissue tomograms of poor quality. To what extent such tomograms can be interpreted in molecular terms is highly questionable. The authors also don't discuss the physiological influence of 20% dextran used for high-pressure freezing of these "very native" specimens.

Lastly, a large part of the paper is devoted to image analysis of the PSD which is not convincing (including a somewhat forced comparison with the fixed and heavy-metal staining room temperature approach). Despite being a technically challenging study, the results fall short of expectations. 

Our manuscript contains a discussion of both conventional EM and cryoET of synapses. We apologise if we have omitted referencing or discussing any earlier cryoET work. This was certainly not our intention, and we include a more complete discussion of published cryoET work on synapses in our revised manuscript.

The reviewer is concerned that the synaptic vesicles in some synapse tomograms are “stretched” and that this may reflect poor preservation.  We would like to point out that such non-spherical synaptic vesicles have also been previously reported in cryoET of primary neurons grown on EM grids (Tao et al., J. Neuro, 2018). Indeed, there is no reason per se to suppose synaptic vesicles are always spherical and there are many diverse families of proteins expressed at the synapse that shape membrane curvature (BAR domain proteins, synaptotagmin, epsins, endophilins and others). We will add further discussion of this issue in the revised manuscript.

The reviewer regards ‘cryo-sectioning’ as outdated and cryoET data from these preparations as “poor quality”. We respectfully disagree. Preparing brain tissues for cryoET is generally considered to be challenging. The first successful demonstration of preparing such samples was before the advent of the cryoEM resolution revolution (with electron counting detectors) by Zuber et al (Proc. Natl. Acad. Sci.,2005) preparing cryo-sections/CEMOVIS of in vitro brain cultures. We followed this technique to prepare tissue cryo-sections for cryoET in our manuscript. Recently, cryoFIB-SEM liftout has been developed as an alternative method to prepare tissue samples for cryoET (Mahamid et al., J. Struct. Biol., 2015) and only more recently this method became available to more laboratories. Both techniques introduce damage as has been described (Han et al., J. Microsc., 2008; Lucas et al., Proc. Natl. Acad. Sci., 2023). Importantly no like-for-like, quantitative comparison of these two methodologies has yet been performed. We have recently demonstrated that the molecular structure of amyloid fibrils within human brain is preserved down to the protein fold level in samples prepared by cryo-sectioning (Gilbert et al., Nature, 2024). We will add further detail on the process by which we excluded poor quality tomograms from our analysis, which we described in detail in our methods section.

The reviewer asks what the physiological effect is of adding 20% w/v ~40,000 Da dextran? This is a reasonable concern since this could in principle exert osmotic pressure on the tissue sample. While we did not investigate this ourselves, earlier studies have (Zuber et al, 2005) showing cell membranes were not damaged by and did not have any detectable effect on cell structure in the presence of this concentration of dextran.

The reviewer is not convinced by our analysis of the apparent molecular density of macromolecules in the postsynaptic compartment that in conventional EM is called the postsynaptic density. However, the reviewer provides no reasoning for this assessment nor alternative approaches that could be attempted. We would like to add that we have tested multiple different approaches to objectively measure molecular crowding in cryoET data, that give comparable results. We believe that our conclusion – that we do not observe an increased molecular density conserved at the postsynaptic membrane, and that the PSD that we and others observed by conventional EM does not correspond to a region of increased molecular density - is well supported by our data.  We and the other reviewers consider this an important and novel observation.

Reviewer #2 (Public review): 

Summary: 

The authors set out to visualize the molecular architecture of the adult forebrain glutamatergic synapses in a near-native state. To this end, they use a rapid workflow to extract and plunge-freeze mouse synapses for cryo-electron tomography. In addition, the authors use knockin mice expression PSD95-GFP in order to perform correlated light and electron microscopy to clearly identify pre- and synaptic membranes. By thorough quantification of tomograms from plunge- and high-pressure frozen samples, the authors show that the previously reported 'post-synaptic density' does not occur at high frequency and therefore not a defining feature of a glutamatergic synapse.

Subsequently, the authors are able to reproduce the frequency of post-synaptic density when preparing conventional electron microscopy samples, thus indicating that density prevalence is an artifact of sample preparation. The authors go on to describe the arrangement of cytoskeletal components, membraneous compartments, and ionotropic receptor clusters across synapses.

Demonstrating that the frequency of the post-synaptic density in prior work is likely an artifact and not a defining feature of glutamatergic synapses is significant. The descriptions of distributions and morphologies of proteins and membranes in this work may serve as a basis for the future of investigation for readers interested in these features.

Strengths: 

The authors perform a rigorous quantification of the molecular density profiles across synapses to determine the frequency of the post-synaptic density. They prepare samples using two cryogenic electron microscopy sample preparation methods, as well as one set of samples using conventional electron microscopy methods. The authors can reproduce previous reports of the frequency of the post-synaptic density by conventional sample preparation, but not by either of the cryogenic methods, thus strongly supporting their claim. 

We thank the reviewer for their generous assessment of our manuscript.

Reviewer #3 (Public review): 

Summary: 

The authors use cryo-electron tomography to thoroughly investigate the complexity of purified, excitatory synapses. They make several major interesting discoveries: polyhedral vesicles that have not been observed before in neurons; analysis of the intermembrane distance, and a link to potentiation, essentially updating distances reported from plastic-embedded specimen; and find that the postsynaptic density does not appear as a dense accumulation of proteins in all vitrified samples (less than half), a feature which served as a hallmark feature to identify excitatory plastic-embedded synapses. 

Strengths: 

(1) The presented work is thorough: the authors compare purified, endogenously labeled synapses to wild-type synapses to exclude artifacts that could arise through the homogenation step, and, in addition, analyse plastic embedded, stained synapses prepared using the same quick workflow, to ensure their findings have not been caused by way of purification of the synapses. Interestingly, the 'thick lines of PSD' are evident in most of their stained synapses.

(2) I commend the authors on the exceptional technical achievement of preparing frozen specimens from a mouse within two minutes.

(3) The approaches highlighted here can be used in other fields studying cell-cell junctions.

(4) The tomograms will be deposited upon publication which will enable neurobiologists and researchers from other fields to carry on data evaluation in their field of expertise since tomography is still a specialized skill and they collected and reconstructed over 100 excellent tomograms of synapses, which generates a wealth of information to be also used in future studies.

(5) The authors have identified ionotropic receptor positions and that they are linked to actin filaments, and appear to be associated with membrane and other cytosolic scaffolds, which is highly exciting.

(6) The authors achieved their aims to study neuronal excitatory synapses in great detail, were thorough in their experiments, and made multiple fascinating discoveries. They challenge dogmas that have been in place for decades and highlight the benefit of implementing and developing new methods to carefully understand the underlying molecular machines of synapses.

Weaknesses: 

The authors show informative segmentations in their figures but none have been overlayed with any of the tomograms in the submitted videos. It would be helpful for data evaluation to a broad audience to be able to view these together as videos to study these tomograms and extract more information. Deposition of segmentations associated with the tomgrams would be tremendously helpful to Neurobiologists, cryo-ET method developers, and others to push the boundaries.

Impact on community: 

The findings presented by Peukes et al. pertaining to synapse biology change dogmas about the fundamental understanding of synaptic ultrastructure. The work presented by the authors, particularly the associated change of intermembrane distance with potentiation and the distinct appearance of the PSD as an irregular amorphous 'cloud' will provide food for thought and an incentive for more analysis and additional studies, as will the discovery of large membranous and cytosolic protein complexes linked to ionotropic receptors within and outside of the synaptic cleft, which are ripe for investigation. The findings and tomograms available will carry far in the synapse fields and the approach and methods will move other fields outside of neurobiology forward. The method and impactful results of preparing cryogenic, unlabelled, unstained, near-native synapses may enable the study of how synapses function at high resolution in the future.

We thank the reviewer for their supportive assessment of our manuscript.  We thank the reviewer for suggesting overlaying segmentations with videos of the raw tomographic volumes. We will include this in our revised manuscript.

eLife Assessment

This important study uses the Jurkat T cell model to study the role of Formin-like 1 β phosphorylation at S1086 on actin dynamics and exosome release at the immunological synapse. The evidence supporting these findings is compelling within the framework of the Jurkat model. As the Jurkat model is known to have a bias toward formin-mediated actin filament formation at the expense of Arp2/3-mediated branched F-actin foci observed in primary T cells, it will be beneficial in the future to confirm major findings in primary T cells.

Joint Public Review:

Summary

Based on i) the documented role of FMNL1 proteins in IS formation; ii) their ability to regulate F-actin dynamics; iii) the implication of PKCdelta in MVB polarization to the IS and FMNL1beta phosphorylation; and iv) the homology of the C-terminal DAD domain of FMNL1beta with FMNL2, where a phosphorylatable serine residue regulating its auto-inhibitory function had been previously identified, the authors have addressed the role of S1086 in the FMNL1beta DAD domain in F-actin dynamics, MVB polarization and exosome secretion, and investigated the potential implication of PKCdelta, which they had previously shown to regulate these processes, in FMNL1beta S1086 phosphorylation. They demonstrate that FMNL1beta is indeed phosphorylated on S1086 in a PKCdelta-dependent manner and that S1086-phosphorylated FMNL1beta acts downstream of PKCdelta to regulate centrosome and MVB polarization to the IS and exosome release. They provide evidence that FMNL1beta accumulates at the IS where it promotes F-actin clearance from the IS center, thus allowing for MVB secretion.

Strengths

The work is based on a solid rationale, which includes previous findings by the authors establishing a link between PKCdelta, FMNL1beta phosphorylation, synaptic F-actin clearance and MVB polarization to the IS. The authors have thoroughly addressed the working hypotheses using robust tools. Among these, of particular value is an expression vector that allows for simultaneous RNAi-based knockdown of the endogenous protein of interest (here all FMNL1 isoforms) and expression of wild-type or mutated versions of the protein as YFP-tagged proteins to facilitate imaging studies. The imaging analyses, which are the core of the manuscript, have been complemented by immunoblot and immunoprecipitation studies, as well as by the measurement of exosome release (using a transfected MVB/exosome reporter to discriminate exosomes secreted by T cells).

Weaknesses

As stated in the title of the article, the main findings have been obtained in clones of Jurkat cells and have not been confirmed in primary T cells.

from: search - ipfs support brave

from: https://hypothes.is/a/0eW1dISKEe-OemfFVhhVsQ

skeuomorphisme - étudie ce concept et évoque-le

virtuel n'est pas immatériel ;)

mmm je ne ferais pas le lien entre les deux. pour bachi la raison computationnelle n'est pas pauvre. Et computationnelle est bcp plus large que pwp... numérique n'est pas synonyme de microsoft

considération générale: il me semble que tu devrais sectionner davantage en mettant des intertitres et soigner encore plus le fil rouge - en rajoutant des paragraphes de passage.

pas d'accord du tout. Pourquoi le support serait l'écran? Le support c'est la RAM et puis le disque... bien sûr que c'est une inscription directe sur un support... fin, directe... quand tu écris avec une plume c'est pas direct non plus. Veux-tu écrire avec ton sang sur une pierre? T'as passé trop de temps à l'armée!

dommage d'avoir commencé et puis de revenir à des questions plus générales

comprends pas ce que tu veux dire

mieux expliquer pourquoi

attention! rhétorique pub!

attention à ne pas avoir une rhétorique publicitaire. cette affirmation n'est pas appuyée par des faits

comprends pas

Here are the key points regarding Rama's new "instant PState migration" feature and the context around it:

Status Quo of Database Migrations

• SQL Databases: Support schema evolution through Data Definition Language (DDL) and Data Manipulation Language (DML).
• Support alterations in table structures but may require complex transactions and can incur downtime.

• Issues include re-implementation of logic in SQL, extended migration times, and potential locking of tables.

• NoSQL Databases: Limited built-in support for schema migrations; often require custom solutions or third-party tools.

• Many document databases are "schemaless,” making it cumbersome to change existing data structures.

• Common approaches include eating migration efforts during read time or using third-party tools like Mongock.

• NewSQL Databases: Aim to combine NoSQL's scalability with SQL's transactional integrity.

• Though effective, they still retain many limitations of traditional SQL migrations.

Features of Instant PState Migrations in Rama

• Expressive: Migrations can be performed with arbitrary transformations written in the user’s programming language, offering more flexibility than SQL.

• Instantaneous: The data migration process is quick; immediately after deployment, all PState reads reflect the migrated data regardless of data volume.

• Durable and Fault-Tolerant: Automatically handles persistent changes in a consistent manner without manual intervention. Migrated data is rewritten in the background during ongoing operations, maintaining overall application performance.

Migration Process

1. Schema Definition: Developers define the new schema for PStates and specify migration functions using existing application logic.
2. Deployment: Deployments use CLI commands that spin up new workers, allowing for seamless transitions.
3. Progress Monitoring: Migration status is available in the UI, allowing for real-time tracking of the migration process without interrupting service.

Advantages of Using Rama

• Simplifies schema evolution, reducing operational pain compared to traditional databases.
• Retains all historical data through an event sourcing architecture.
• Enables easy updates and adjustments in response to changing business needs without downtime.

Conclusion

Rama's instant PState migration significantly enhances the responsiveness and flexibility of application development, providing powerful tools for developers to manage schema changes efficiently.

!search - ipfs support brave

> for

for - Book - Society of the Spectacle - 1967 - Guy Debord - Advertising - critique

Summary - This is a youtube that presents the work of French Marxist theorist Guy Debord and his important book "The society of the spectacle" that critically examines the power of mass media to shape our reality and transform us - from an active participant to - a passive spectator (hence the "spectacle" and consumer - When mass media fabricates images that become the aspirations for large swaths or the population,<br /> - it can implant market ideology that channels their future consumerist behaviour to conform with elitist hidden agenda - The idea emerged from a group of leftist scholars and activists called the Situationist International that dissolved in 1972 but - the idea is quite relevant to describing global capitalism and information systems in modernity

to - Wikipedia - Situationist International - https://hyp.is/L4ObqISEEe-gJpNANP04Mw/en.wikipedia.org/wiki/Situationist_International

for - society of the spectacle - insight - quote - illusion and free will

society of the spectacle - insight - quote - illusion and free will - The false reality governed by images - facilitates the work of the capitalist system - The system gives you the illusion of having Free Will and choosing what you consume - but in reality everything has already been decided for you

for - to - Roman poet Juvenal - phrase - bread and circuses

to - Roman poet Juvenal - phrase - bread and circuses - https://hyp.is/Q9uOEISGEe-ZTp8-GdVfwQ/drlindaellis.net/a-brief-look-at-the-juvenals-bread-circuses/ - This phrase refers to how easily people are pacified and controlled through distractions

for - question - the society of the spectacle - is it just another critique of capitalism?

question - the society of the spectacle - is it just another critique of capitalism? - In short, no. It adds something new. - The new subtlety added by the creation of the spectacle by the market economy or by capitalism is that - the spectacle aims to produce the same passive and predictable individual everywhere - ** A SPECTATOR!" - This new being is - a passive consumer instead of - an active participant in society - The Spectator - sacrifices his authenticity to fit in society and - isn't a decision maker in his life anymore - The spectator is a passive human being who just awaits orders to execute (and consume)

for - the society of the spectacle - global brands

for - adjacency - the largest companies in the world have more capital than many countries - the society of the spectacle - lobby industry

adjacency - between - the largest multi-national companies in the world have more capital than many countries - the society of the spectacle - adjacency relationship - It is a well publicized fact that the world's largest multi-national companies have more capital than many countries - This fact is a prime example of the conclusions of the society of the spectacle, - Governments are coopted to serve the needs of the multi-nationals through corporate lobbyists - In fact, multi-national corporations are called "multi-national" precisely because they are so large that they exceed the boundaries of nation states, they are LARGER than nation states - Advertising, movies and products all flow trans-nationally across political boundaries - Military weapons developed by the military industrial complex and sold to nation states make modern warefare between them exponentially more harmful - In the end, the elites within such corporations benefit from the most from the consumption - The diversion is towards maximizing their profits at the expense of all else: - people - the environment - life on earth

for - adjacency - the society of the spectacle - internet society of modernity - Deep Humanity - BEing journey - to discover the society of the spectacle

Being journey - to discover the society of the spectacle - Modernity, so steeped in social media and the internet is INDEED a new mode of living and perceiving the world - To discover the extend to which we have socially normalized a social pathology, we can introduce BEing journeys that help us explore how a life that is freed from the social norm feels like

for - definition - the spectacle - context - the society of the spectacle - cacooning - the spectacle - social media - the spectacle

definition - the spectacle - context - the society of the spectacle - A society where images presented by mass media / mass entertainment not only dominate - but replaces real experiences with a superficial reality that is - focused on appearances designed primarily to distract people from reality - This ultimately disconnects them from - themselves and - those around them

comment - How much does our interaction with virtual reality of - written symbols - audio - video - two dimensional images - derived from our screens both large and small affect our direct experience of life? - When people are distracted by such manufactured entertainment, they have less time to devote to important issues and connecting with real people - We can sit for hours in social isolation, ignoring our bodies need for exercise and our emotional need for real social connection - We can ignore the real crisis going on in the world and instead numb ourselves out with contrived entertainment

for - insight - society of the spectacle - secrecy and diversion is inherent to it

insight - society of the spectacle - secrecy and diversion is inherent to it - it's a society where things happen normally like in any other society but - where we don't know who is pulling the strings - Its main objective is - to divert people's attention by - hiding the real and - promoting the Irrelevant

for - who is - Guy Debord - to - Wikipedia - Situationist International

who is - Guy Debord - Guy Debord was - French - born in 1931 in Paris - Marxist theorist, - philosopher - filmmaker - founding member of the Situationist International

to - Wikipedia - Situationist International - https://hyp.is/muSjgIR4Ee-IdBeTNYoyrg/en.wikipedia.org/wiki/Situationist_International

Good job!

for - from youtube - society of the spectacle - A Brief Look at the Juvenal’s “Bread & Circuses”

for - from - youtube - the society of the spectacle

from - youtube - the society of the spectacle - https://hyp.is/aJX4NoRsEe-7c5M0eZf09w/www.youtube.com/watch?v=93jXDJhi6_c

for - definition - the spectacle - Situationist International - adjacency - the spectacle - manufacturing consent

definition - the spectacle - Sittuationist International - A unified critique offered by the Situationist International of advanced capitalism - The critique was concerned with the insidious use of mass media and entertainment to subvert individual expression through lived experience by - substituting it with mass media images as proxies to directly lived experiences - which leads to mass consumption of commodities produced by advanced captalism - creates far-reaching passive second-hand alienation that harms both the individual and society

adjacency - between - the spectacle - manufacturing consent - adjacency relationship - The spectacle is closely related to Noam Chomsky's work on manufacturing consent

for - book - The Society of the Spectacle - definition - recuperation - from - youtube - The Society of the Spectacle - politics - Marxist group - Situationist International

definition - recuperation - A technique of the spectacle whereby - Official culture is considered a "rigged game" - Conservative powers forbid subversive ideas to have direct access to public discourse - Subversive ideas must first - get trivialized - get sterilized - before they are safely incorporated back within mainstream society - where they lose their agential power and - they can be exploited to add new flavors and bolster the status quo dominant ideas of the rigged game

from - youtube - The Society of the Spectacle - https://hyp.is/K2b2OIR5Ee-khSfaPJUKWg/www.youtube.com/watch?v=93jXDJhi6_c

eLife Assessment

This is an important piece of work that sheds light on our understanding of early lung development. There is solid evidence that there is a key new role for Svep1, which may be acting via FGF9. A more precise understanding of the interactions between Svep1 and FGF9, with a possibility of other ECM factors, would add value.

Reviewer #1 (Public review):

Summary:

This is an important contribution to the field of molecular embryology of the lung. The authors introduce a novel mesenchymally expressed molecule Svep1. Knocking it out in mice produces a profoundly hypoplastic phenotype which can be rescued in vitro. Svep1 interacts with the FGF signaling complex to control differentiation and expression of smooth muscle in lung mesenchyme, thereby affecting proximal-distal patterning of the airway branches by acting as a putative branch suppressor.

Strengths:

The study shows strong evidence in mouse knockouts, in vitro embryonic lung culture as well as gene expression and in vitro rescue studies that confirm a key role for Svep1. It is a beautiful piece of work and an important contribution to our understanding of early lung branching morphogenesis.

Weaknesses:

Claiming a possible therapeutic role for this gene is a bit far-fetched at the present state of the art.

Reviewer #2 (Public review):

Summary:

In an effort to elucidate the role of the ECM protein Svep1 in lung development, Foxworth and colleagues have generated a Svep1 mutant (lacking exon 8). Based on their developmental analyses of branching morphogenesis and expression of epithelial, mesenchymal, and mesothelial markers in these mutants, the authors conclude that Svep1 is essential for normal lung growth, morphogenesis, and patterning. They propose that the Svep1 protein regulates, in part, FGF9 signalling. Overall, the paper demonstrates that the ECM is important for lung development and tries to implicate the ECM in the regulation of epithelial-mesenchymal interactions during lung development.

Strengths:

The strengths of this paper are the careful spatiotemporal characterization of 1) lung growth 2) branching morphogenesis 3) epithelial marker expression. The differential perturbation of growth and branching morphogenesis along the D-V axis and the progressive perturbation of branching morphogenesis are both very interesting and noteworthy phenotypes.

Weaknesses:

The weakness of this paper is that it does not present a convincing explanation for how Svep1 regulates any of the phenotypes described. In this regard, a demonstration of a genetic interaction between Svep1 and FGF9 mutants or a careful characterization of a tissue-specific knockdown of Svep1, could be insightful. In addition, a comparison of the phenotype of Svep1 mutants and the phenotypes of other mutants affecting ECM components would be worthwhile.

A minor weakness is that the title of the paper is not fully supported by the data presented. While the defects in the morphogenesis of the distal lung in Svep1 mutants presage a defect in alveolar differentiation, this cannot be formally demonstrated since the animals die soon after birth.

Author response:

Response to Reviewer #1:

“Claiming a possible therapeutic role for this gene is a bit far-fetched at the present state of the art”.

We agree that while the therapeutic relevance of Svep1 is not clear at this point, this potential is always something we consider in interpreting our data.

Response to Reviewer #2:

a. “The weakness of this paper is that it does not present a convincing explanation for how Svep1 regulates any of the phenotypes described. In this regard, a demonstration of a genetic interaction between Svep1 and FGF9 mutants or a careful characterization of a tissue-specific knockdown of Svep1, could be insightful. In addition, a comparison of the phenotype of Svep1 mutants and the phenotypes of other mutants affecting ECM components would be worthwhile”. 

We agree that additional experiments are needed to determine how exactly Svep1 contributes to the phenotypes described. While our preliminary data point to an interaction of Svep1 and Fgf9, we agree that additional data are needed to prove that such interaction is a primary driver of the phenotypes observed.

b. “A minor weakness is that the title of the paper is not fully supported by the data presented. While the defects in the morphogenesis of the distal lung in Svep1 mutants presage a defect in alveolar differentiation, this cannot be formally demonstrated since the animals die soon after birth”

The reviewer is correct that we cannot formally demonstrate this in the current model. The profound defects observed in Svep1 mutants lead to early death, making it challenging to study the full process of alveolarization. However, it is important to note that lung morphogenesis is a continuous process in which earlier developmental phases lay the groundwork for subsequent stages. During the branching phase, the fate of alveolar cell types is established, while the saccular stage serves as a critical foundation for alveolar development, where alveolar cells begin to differentiate. We believe that the significant abnormalities in cellular differentiation observed prior to the bulk of alveolarization indicate likely defects in the later stages of alveolar differentiation. Therefore, while the model limits our ability to directly assess alveolarization, we anticipate that defects in cellular differentiation will continue to manifest beyond the saccular stage in Svep1 mice.

We're already there; GPT injected with enough domain-specific contexts beats out incompetent software engineers -- many of whom have real jobs at real companies. Asking AI to roll an implementation is far easier than asking an intern today.

Author is dodging the topic: AI will replace many agronomists. A language model competent enough to surface any data you need with a question can replace people; the productivity of one person is multiplied, eliminating the need for other people in that role. Language models disproportionately reward experts, and multiplying the productivity of an expert eliminates jobs.

What is manas Sarovar ?

What is Angsi

What is Barzakh ??

Who is Juno? How this name came from?

(Experimental question)

I don't believe this. You can't develop these models without web-scale data.

eLife Assessment

The study presents a very well-illustrated specimens of the artiopodan Cindarella eucalla from the Chengjiang Biota, using computer tomography (CT) scanning to illustrate multiple specimens with preserved appendages, a rarity in artiopodans. The description of these fossils is important for expanding our understanding of this taxon and its relatives. The imaging and morphological description are followed by a discussion of how this morphology relates to other Cambrian arthropods and its potential ecological function. The evidence provided in this section about resulting function and ecology is presently incomplete and the conclusions are put forward too strongly. This assessment could be improved if the work is revised with more careful wording and additional data.

Reviewer #1 (Public review):

Summary:

The manuscript by Zhang et al. analyzed 17 specimens of Cindarella eucalla with 3D technology and discussed the anatomical findings, the relationship to other artiopods, and the ecology of the animal. The results are excellent and the findings are very interesting. However, the discussion needs to be extended, as the point the authors are trying to make is not always clear. I also recommend some restructuring of the discussion. Overall this is an important manuscript, and I'm looking forward to reading the edited version.

Strengths:

The analyses, the 3D data is excellent and provides new information.

Weaknesses:

The discussion - the authors provide information for the findings, but do not discuss them in detail. More information is needed.

Reviewer #2 (Public review):

Summary:

Zhang et al. present very well-illustrated specimens of the artiopodan Cinderella eucalla from the Chengjiang Biota. Multiple specimens are shown with preserved appendages, which is rare for artiopodans and will greatly help our understanding of this taxon. The authors use CT scanning to reveal the ventral organization of this taxon. The description of the taxon needs some modification, specifically expansion of the gut and limb morphology. The conclusion that Cinderella was a fast-moving animal is very weak, comparisons with extant fast animals and possibly FEA analyses are necessary to support such a claim. Although the potential insights provided by such well-preserved fossils could be valuable, the claims made are tenuous and based on the available evidence presented herein.

Strengths:

The images produced through CT scanning specimens reveal the very fine detail of the appendages and are well illustrated. Specimens preserve guts and limbs, which are informative both for the phylogenetic position and ecology of this taxon. The limbs are very well preserved, with protopodite, exopodite, and endopodites visible. Addressing the weaknesses below will make the most of this compelling data that demonstrates the morphology of the limbs well.

Weaknesses:

Although this paper includes very well-illustrated fossils, including new information on the eyes, guts, and limbs of Cinderella, the data are not fully explained, and the conclusions are weakly supported.

The authors suggest the preservation of complex ramifying diverticular, but it should be better illustrated and the discussion of the gut diverticulae should be longer, especially as gut morphology can provide insights into the feeding strategy.

The conclusion that Cinderella eucalla was fast, sediment feeding in a muddy environment, is not well supported. These claims seem to be tenuously made without any evidence to support them. The authors should add a new section in the discussion focused on feeding ecology where they explicitly compare the morphology to suspension-feeding artiopodans to justify whether it fed that way or not. To further explore feeding, the protopodite morphology needs to be more carefully described and compared to other known taxa. The function of endites on the endopodite to stir up sediment for particle feeding in a muddy environment would also need to be more thoroughly discussed and compared with modern analogs. The impact of their findings is not highlighted in the discussion, which is currently more of a review of what has been previously said and should focus more on what insights are provided by the great fossils illustrated by the authors.

The authors argue that their data supports fast escaping capabilities, but it is not clear how they reached that conclusion based on the data available. Is there a way this can be further evaluated? The data is impressive, so including comparisons with extant taxa that display fast escaping strategies would help the authors make their case more compelling. The authors also claim that the limbs of Cinderella are strong, again this conclusion is unclear. Comparison with the limbs of other taxa to show their robustness would be useful. To actually test how these limbs deal with the force and strain applied to them by a sudden burst of movement, the authors could conduct Finite Element Analyses.

Reviewer #3 (Public review):

This paper provides an interesting description of the ventral parts of the Cambrian xandarellid Cindarella eucalla, derived from exceptionally preserved specimens of the Chengjiang Biota. These morphological data are useful for our broad understanding and future research on Xandarellida, and are generally well-represented in the description and accompanying figures. The strengths of this work rest in this morphological description of exceptional fossil material, and this is generally well supported. In addition, the authors put this description in the context of the morphology of other xandarellids and Cambrian arthropod groups, with most of these parallels being useful and reasonably supported, though in several places homology is assumed and this currently lacks evidence. The manuscript goes on to use these morphological data and comparisons to other groups (particularly trilobites) to make suggestions for the ecology of Cindarella eucalla and other xandarellids. The majority of my comments on this work relate to this latter aim - the ecological conclusions drawn are generally derived through morphological comparisons, where a specific morphology has been suggested as an adaption to a particular ecological function in another extinct arthropod group. However, the original suggestions for ecological function are untested, and so remain hypotheses. Despite this, they are frequently presented as truisms to enable ecological conclusions to be drawn for Cindarella eucalla. I have listed my comments and queries on the study below for the authors to address or respond to, and I hope they are useful to the authors.

Comments:

There are a number of ecological and functional morphology conclusions stated that seem put too strongly to be considered sufficiently supported by the evidence given. These relate to both the description of C. eucalla, and comparisons to other extinct arthropod taxa (notably trilobites). Many of these latter statements are assumptions of functional morphology, and should not be repeated as truisms, rather than they represent suggested functions and ecologies based on the known morphological descriptions. This aspect occurs throughout the article, and, for me, is the primary concern.

Author response:

Reviewer #1 (Public review):

Summary:

The manuscript by Zhang et al. analyzed 17 specimens of Cindarella eucalla with 3D technology and discussed the anatomical findings, the relationship to other artiopods, and the ecology of the animal. The results are excellent and the findings are very interesting. However, the discussion needs to be extended, as the point the authors are trying to make is not always clear. I also recommend some restructuring of the discussion. Overall this is an important manuscript, and I'm looking forward to reading the edited version.

Strengths:

The analyses, the 3D data is excellent and provides new information.

Weaknesses:

The discussion - the authors provide information for the findings, but do not discuss them in detail. More information is needed.

We are committed to enhancing the quality of our manuscript further and, in response to your comments, will implement the following improvements:

(1) Comparative Analysis of Eyes: We will expand our discussion to include a detailed comparative analysis of the eyes of Cindarella eucalla with those of other artiopods (e.g. Xandarellids, trilobites, living insects), focusing on morphology, size, and other relevant characteristics.

(2) Segmental Mismatch Discussion: We will provide an in-depth exploration of the specifics and significance of the segmental mismatch to offer a clearer understanding of its implications. We will also compare the characteristics of this mismatch in our focal species with those observed in extant arthropods, such as spiders and myriapods. This comparison will be further enriched by integrating our phylogenetic analysis, thereby providing a broader evolutionary context.

(3) Methodological Clarity: We will provide more detailed information on the parameters used for the analyses in the Methods section, especially the phylogenetic sections and the X-ray tomography section.

(4) Phylogenetic Analysis: We will engage in a more in-depth discussion of certain characters (e.g. anterior sclerite, hypostome, endopodite, segmental mismatch, etc.) within our phylogenetic analyses to clarify their relevance and contribution to our findings.

Reviewer #2 (Public review):

Summary:

Zhang et al. present very well-illustrated specimens of the artiopodan Cinderella eucalla from the Chengjiang Biota. Multiple specimens are shown with preserved appendages, which is rare for artiopodans and will greatly help our understanding of this taxon. The authors use CT scanning to reveal the ventral organization of this taxon. The description of the taxon needs some modification, specifically expansion of the gut and limb morphology. The conclusion that Cinderella was a fast-moving animal is very weak, comparisons with extant fast animals and possibly FEA analyses are necessary to support such a claim. Although the potential insights provided by such well-preserved fossils could be valuable, the claims made are tenuous and based on the available evidence presented herein.

Strengths:

The images produced through CT scanning specimens reveal the very fine detail of the appendages and are well illustrated. Specimens preserve guts and limbs, which are informative both for the phylogenetic position and ecology of this taxon. The limbs are very well preserved, with protopodite, exopodite, and endopodites visible. Addressing the weaknesses below will make the most of this compelling data that demonstrates the morphology of the limbs well.

Weaknesses:

Although this paper includes very well-illustrated fossils, including new information on the eyes, guts, and limbs of Cinderella, the data are not fully explained, and the conclusions are weakly supported.

The authors suggest the preservation of complex ramifying diverticular, but it should be better illustrated and the discussion of the gut diverticulae should be longer, especially as gut morphology can provide insights into the feeding strategy.

The conclusion that Cinderella eucalla was fast, sediment feeding in a muddy environment, is not well supported. These claims seem to be tenuously made without any evidence to support them. The authors should add a new section in the discussion focused on feeding ecology where they explicitly compare the morphology to suspension-feeding artiopodans to justify whether it fed that way or not. To further explore feeding, the protopodite morphology needs to be more carefully described and compared to other known taxa. The function of endites on the endopodite to stir up sediment for particle feeding in a muddy environment would also need to be more thoroughly discussed and compared with modern analogs. The impact of their findings is not highlighted in the discussion, which is currently more of a review of what has been previously said and should focus more on what insights are provided by the great fossils illustrated by the authors.

The authors argue that their data supports fast escaping capabilities, but it is not clear how they reached that conclusion based on the data available. Is there a way this can be further evaluated? The data is impressive, so including comparisons with extant taxa that display fast escaping strategies would help the authors make their case more compelling. The authors also claim that the limbs of Cinderella are strong, again this conclusion is unclear. Comparison with the limbs of other taxa to show their robustness would be useful. To actually test how these limbs deal with the force and strain applied to them by a sudden burst of movement, the authors could conduct Finite Element Analyses.

Here are the key points we plan to address:

(1) Gut and Limb Morphology: We will expand our description of the gut and limb morphology of C. eucalla, providing a more detailed comparison and analysis. This will include a revised discussion on the function and ecological implications of these features.

(2) Fast-Moving Animal Claim: We acknowledge your concern about the conclusion that C. eucalla was a fast-moving animal. We will conduct a more detailed comparison among C. eucalla and other Cambrian artiopods and living arthropods, focusing on morphological and functional aspects. We will also reconsider our claim and will be more cautious in our conclusions. If the comparison proves insufficient, we will remove this assertion from the manuscript. But we may no longer conduct Finite Element Analysis, as a comprehensive and cautious analysis would require a massive project to complete.

(3) Sediment Feeding in a Muddy Environment: We will revise the section discussing the feeding ecology of C. eucalla. We will enhance this section by comparing the morphology of C. eucalla to that of suspension-feeding artiopods, which will help to substantiate our claims. Additionally, we will expand the discussion to include a more detailed examination of endites, gnathobases, and other relevant anatomical structures.

(4) Impact of Findings: We will endeavor to highlight the impact of our findings in the discussion, focusing on the insights provided by the well-preserved fossils illustrated in our study.

Reviewer #3 (Public review):

This paper provides an interesting description of the ventral parts of the Cambrian xandarellid Cindarella eucalla, derived from exceptionally preserved specimens of the Chengjiang Biota. These morphological data are useful for our broad understanding and future research on Xandarellida, and are generally well-represented in the description and accompanying figures. The strengths of this work rest in this morphological description of exceptional fossil material, and this is generally well supported. In addition, the authors put this description in the context of the morphology of other xandarellids and Cambrian arthropod groups, with most of these parallels being useful and reasonably supported, though in several places homology is assumed and this currently lacks evidence. The manuscript goes on to use these morphological data and comparisons to other groups (particularly trilobites) to make suggestions for the ecology of Cindarella eucalla and other xandarellids. The majority of my comments on this work relate to this latter aim - the ecological conclusions drawn are generally derived through morphological comparisons, where a specific morphology has been suggested as an adaption to a particular ecological function in another extinct arthropod group. However, the original suggestions for ecological function are untested, and so remain hypotheses. Despite this, they are frequently presented as truisms to enable ecological conclusions to be drawn for Cindarella eucalla. I have listed my comments and queries on the study below for the authors to address or respond to, and I hope they are useful to the authors.

Comments:

There are a number of ecological and functional morphology conclusions stated that seem put too strongly to be considered sufficiently supported by the evidence given. These relate to both the description of C. eucalla, and comparisons to other extinct arthropod taxa (notably trilobites). Many of these latter statements are assumptions of functional morphology, and should not be repeated as truisms, rather than they represent suggested functions and ecologies based on the known morphological descriptions. This aspect occurs throughout the article, and, for me, is the primary concern.

We plan to address the following points in upon revision:

(1) Homology Assumptions: You pointed out that we have assumed homology in certain instances without sufficient evidence. We will revise the manuscript to include a more detailed analysis of the anterior sclerite and exite, considering phylogenetic relationships and morphological comparisons to provide a more robust discussion.

(2) Ecological and Functional Morphology: We acknowledge that our conclusions regarding the ecological function were presented with too much certainty. We will adopt a more cautious approach in our discussion, ensuring that our ideas are clearly labeled as such and are supported by a comparison of relevant studies on Cambrian artiopods and extant arthropods, including fluid dynamics, functional morphology, etc. We will re-evaluate the ecological function section, and if it does not adds value and clarity to the manuscript—our speculations do not contribute to the understanding of the specimen or may lead to misunderstandings—we will remove the relevant parts. We believe future changes reflect a more cautious and rigorous approach to the ecological and functional interpretations of C. eucalla.

Résumé de la vidéo [00:00:00][^1^][1] - [00:21:39][^2^][2]:

Cette vidéo traite de l'augmentation inquiétante des cancers du côlon chez les jeunes. Elle présente des témoignages de patients et des explications médicales sur les causes possibles et les efforts de recherche en cours.

Moments forts: + [00:00:45][^3^][3] Témoignage de Christina * Diagnostiquée à 31 ans avec un cancer du côlon stade 4 * Subit une opération majeure et une chimiothérapie * Rémission après 5 ans + [00:03:47][^4^][4] Augmentation des cas chez les jeunes * Augmentation des cancers du côlon chez les moins de 50 ans * 13% d'augmentation en 2022 en Europe * Nécessité d'une nouvelle approche médicale + [00:05:01][^5^][5] Symptômes et diagnostic * Symptômes courants : sang dans les selles, douleurs abdominales, perte de poids * Importance de consulter en cas de symptômes * Retard de diagnostic fréquent chez les jeunes + [00:09:01][^6^][6] Études et recherches * Études en cours pour comprendre les causes * Hypothèses sur l'inflammation et le microbiome * Différences immunitaires entre jeunes et âgés + [00:16:42][^7^][7] Facteurs de risque environnementaux * Pollution, pesticides, microplastiques * Études pour identifier les liens avec le cancer * Importance de la recherche pour des solutions préventives

Вот пример, и на него можно ответить ниже.

https://web.archive.org/web/20241007071434/https://www.dbreunig.com/2024/10/03/we-need-help-with-discovery-more-than-generation.html

Author says generation isn't a problem to solve for AI, there's enough 'content' as it is. Posits discovery as a bigger problem to solve. The issue there is, that's way more personal and less suited for VC funded efforts to create a generic tool that they can scale from the center. Discovery is not a thing, it's an individual act. It requires local stuff, tuned to my interests, networks etc. Curation is a personal thing, providing intent to discovery. Same why [[Algemene event discovery is moeilijk 20150926120836]], as [[Event discovery is sociale onderhandeling 20150926120120]] Still it's doable, but more agent like than central tool.

eLife Assessment

The paper presents a new pipeline for functional validation of genes known to underlie fragile bone disorders, using CRISPR-mediated knockouts and a number of phenotypic assessments in zebrafish. The solid data demonstrate the feasibility and validity of the approach, which presents a valuable tool for rapid functional validation of candidate gene(s) associated with heritable bone diseases identified from genetic studies.

Reviewer #1 (Public review):

Summary:

In this work, a screening platform is presented for rapid and cost-effective screening of candidate genes involved in Fragile Bone Disorders. The authors validate the approach of using crispants, generating FO mosaic mutants, to evaluate the function of specific target genes in this particular condition. The design of the guide RNAs is convincingly described, while the effectiveness of the method is evaluated to 60% to 92% of the respective target genes being presumably inactivated. Thus, injected F0 larvae can be directly used to investigate the consequences of this inactivation.

Skeletal formation is then evaluated at 7dpf and 14dpf, first using a transgenic reporter line revealing fluorescent osteoblasts, and second using alizarin-red staining of mineralized structures. In general, it appears that the osteoblast-positive areas are more often affected in the crispants compared to the mineralized areas, an observation that appears to correlate with the observed reduced expression of bglap, a marker for mature osteoblasts, and the increased expression of col1a1a in more immature osteoblasts.

Finally, the injected fish (except two lines that revealed high mortality) are also analyzed at 90dpf, using alizarin red staining and micro-CT analysis, revealing an increased incidence of skeletal deformities in the vertebral arches, fractures, as well as vertebral fusions and compressions for all crispants except those for daam2. Finally, the Tissue Mineral Density (TMD) as determined by micro-CT is proposed as an important marker for investigating genes involved in osteoporosis.

Taken together, this manuscript is well presented, the data are clear and well analyzed, and the methods are well described. It makes a compelling case for using the crispant technology to screen the function of candidate genes in a specific condition, as shown here for bone disorders.

Strengths:

Strengths are the clever combination of existing technologies from different fields to build a screening platform. All the required methods are comprehensively described.

Weaknesses:

One may have wished to bring one or two of the crispants to the stage of bona fide mutants, to confirm the results of the screening, however, this is done for some of the tested genes as laid out in the discussion.

Reviewer #2 (Public review):

Summary:

More and more genes and genetic loci are being linked to bone fragility disorders like osteoporosis and osteogenesis imperfecta through GWAS and clinical sequencing. In this study, the authors seek to develop a pipeline for validating these new candidate genes using crispant screening in zebrafish. Candidates were selected based on GWAS bone density evidence (4 genes) or linkage to OI cases plus some aspect of bone biology (6 genes). NGS was performed on embryos injected with different gRNAs/Cas9 to confirm high mutagenic efficacy and off-target cutting was verified to be low. Bone growth, mineralization, density, and gene expression levels were carefully measured and compared across crispants using a battery of assays at three different stages.

Strengths:

(1) The pipeline would be straightforward to replicate in other labs, and the study could thus make a real contribution towards resolving the major bottleneck of candidate gene validation.

(2) The study is clearly written and extensively quantified.

(3) The discussion attempts to place the phenotypes of different crispant lines into the context of what is already known about each gene's function.

(4) There is added value in seeing the results for the different crispant lines side by side for each assay.

Weaknesses:

(1) The study uses only well-established methods and is strategy-driven rather than question/hypothesis-driven.

(2) Some of the measurements are inadequately normalized and not as specific to bone as suggested:

(a) The measurements of surface area covered by osteoblasts or mineralized bone (Figure 1) should be normalized to body size. The authors note that such measures provide "insight into the formation of new skeletal tissue during early development" and reflect "the quantity of osteoblasts within a given structure and [is] a measure of the formation of bone matrix." I agree in principle, but these measures are also secondarily impacted by the overall growth and health of the larva. The surface area data are normalized to the control but not to the size/length of each fish - the esr1 line in particular appears quite developmentally advanced in some of the images shown, which could easily explain the larger bone areas. The fact that the images in Figure S5 were not all taken at the same magnification further complicates this interpretation.

(b) Some of the genes evaluated by RT-PCR in Figure 2 are expressed in other tissues in addition to bone (as are the candidate genes themselves); because whole-body samples were used for these assays, there is a nonzero possibility that observed changes may be rooted in other, non-skeletal cell types.

(3) Though the assays evaluate bone development and quality at several levels, it is still difficult to synthesize all the results for a given gene into a coherent model of its requirement.

(4) Several additional caveats to crispant analyses are worth noting:

(a) False negatives, i.e. individual fish may not carry many (or any!) mutant alleles. The crispant individuals used for most assays here were not directly genotyped, and no control appears to have been used to confirm successful injection. The authors therefore cannot rule out that some individuals were not, in fact, mutagenized at the loci of interest, potentially due to human error. While this doesn't invalidate the results, it is worth acknowledging the limitation.

(b) Many/most loci identified through GWAS are non-coding and not easily associated with a nearby gene. The authors should discuss whether their coding gene-focused pipeline could be applied in such cases and how that might work.

Reviewer #3 (Public review):

Summary:

The manuscript "Crispant analysis in zebrafish as a tool for rapid functional screening of disease-causing genes for bone fragility" describes the use of CRISPR gene editing coupled with phenotyping mosaic zebrafish larvae to characterize functions of genes implicated in heritable fragile bone disorders (FBDs). The authors targeted six high-confident candidate genes implicated in severe recessive forms of FBDs and four Osteoporosis GWAS-implicated genes and observed varied developmental phenotypes across all crispants, in addition to adult skeletal phenotypes.

A major strength of the paper is the streamlined method that produced significant phenotypes for all candidate genes tested.

A major weakness is a lack of new insights into underlying mechanisms that may contribute to disease phenotypes, nor any clear commonalities across gene sets. This was most evident in the qRT-PCR analysis of select skeletal developmental genes, which all showed varied changes in fold and direction, but with little insight into the implications of the results.

Ultimately, the authors were able to show their approach is capable of connecting candidate genes with perturbation of skeletal phenotypes. It was surprising that all four GWAS candidate genes (which presumably were lower confidence) also produced a result. These authors have previously demonstrated that crispants recapitulate skeletal phenotypes of stable mutant lines for a single gene, somewhat reducing the novelty of the study.

from: https://hypothes.is/a/M6gibIR6Ee-bBR9gG2hplA

!search - peergos ck editor spell checker

eLife Assessment

This important work advances our understanding of trained immunity, especially in the context of Bacillus Calmette-Guérin (BCG) administration and host-pathogen interactions. The evidence supporting the conclusions are convincing, based on a combination of state-of-the-art omics techniques such as bulk and single-cell RNA sequencing with the use of JAK/STAT signaling inhibitors. The work will be of broad interest to immunologists and infection biologists.

Reviewer #1 (Public review):

Summary:

In the submitted manuscript, Solomon et al carefully detail shifts in tissue-specific myeloid populations associated with trained immunity using intraperitoneal BCG injection as a model for induction. They define the kinetics of shifts in myeloid populations within the spleen and the transcriptional response associated with IP BCG exposure. In lineage tracing experiments, they demonstrate that tissue-resident macrophages, red-pulp macrophages (RPM) that are rapidly depleted after BCG exposure, are replenished from recruited monocytes and expansion of tissue-resident cells; they use transcriptional profiling to characterize those cells. In contrast to previous descriptions of BCG-driven immune training, they do not find BCG in the bone marrow in their model, suggesting that there is not direct training of myeloid precursor populations in the bone marrow. They then link the observed trained immunity phenotype (restriction of heterologous infection with ST) with early activation of STAT1 through IFN-γ.

Strengths:

The work includes careful detaining of shifts and origins of myeloid populations within tissue associated with trained immunity and is a meaningful advance for the field. Given that the temporality of exposure relative to trained immunity phenotypes is a major focus of the work, there are some additional experiments that would make the work stronger.

Weaknesses:

(1) The contribution of persistent BCG in spleen to the observed trained immunity phenotypes is not clear: The trained immunity phenotypes are interpreted as being driven by the early (within days) response to BCG exposure. While the fedratinib data generally support this interpretation, the authors show that BCG remains present in spleen albeit at low levels all the way out to 60 days post exposure. Given that the focus in the paper is on tissue-specific immune training, it would be helpful to know whether the ongoing presence of BCG at low levels in the profiled tissue contributes to the trained immunity phenotypes observed.

(2) Unclear temporality of STAT1/IFN-γ requirement for the trained immunity phenotype: The data demonstrate that STAT1/IFN-γ are required at the earliest time points post-BCG exposure for trained immunity to be initiated. Related to the point about BCG above, it would be helpful to understand whether this is a specifically time-limited requirement when trained immunity is first induced, or whether ongoing signaling through this axis is required for maintenance of the observed trained immunity phenotypes.

Reviewer #2 (Public review):

Summary:

In this study, Solomon and colleagues demonstrate that trained immunity induced by BCG can reprogram myeloid cells within localised tissue, which can sustain prolonged protective effects. The authors further demonstrate an activation of STAT1-dependent pathways.

Strengths:

The main strength of this paper is the in-depth investigation of cell populations affected by BCG training, and how their transcriptome changes at different time points post-training. Through use of flow cytometry and sequencing methods, the authors identify a new cell population derived from classical monocytes. They also show that long-term trained immunity protection in the spleen is dependent on resident cells. Through sequencing, drug and recombinant inhibition of IFNg pathways, the authors reveal STAT1-dependent responses are required for changes in the myeloid population upon training, and recruitment of trained monocytes.

Weaknesses:

A significant amount of work has already been performed for this study. No significant weaknesses found.

testing

Nice coffin

eLife Assessment

This useful study reports that the exogenous expression of the microRNA miR-195 can partially compensate in early B cell development for the loss of EBF1, one of the key transcription factors in B cells. While this finding will be of interest to those studying lymphocyte development, the evidence, particularly with regard to the molecular mechanisms that underpin the effect of miR-195, is currently incomplete.

Reviewer #1 (Public review):

Summary:

Here, the authors are proposing a role for miR-196, a microRNA that has been shown to bind and enhance the degradation of mRNA targets in the regulation of cell processes, and has a novel role in allowing the emergence of CD19+ cells in cells in which Ebf1, a critical B-cell transcription factor, has been genetically removed.

Strengths:

That over-expression of mR-195 can allow the emergence of CD19+ cells missing Ebf1 is somewhat novel.

Their data does perhaps support to a degree the emergence of a transcriptional network that may bypass the absence of Ebf1, including the FOXO1 transcription factor, but this data is not strong or definitive.

Weaknesses:

It is unclear whether this observation is in fact physiological. When the authors analyse a knockout model of miR-195, there is not much of a change in the B-cell phenotype. Their findings may therefore be an artefact of an overexpression system.

The authors have provided insufficient data to allow a thorough appraisal of the step-wise molecular changes that could account for their observed phenotype.

Reviewer #2 (Public review):

Summary:

The authors investigate miRNA miR-195 in the context of B-cell development. They demonstrate that ectopic expression of miR-195 in hematopoietic progenitor cells can, to a considerable extent, override the consequences of deletion of Ebf1, a central B-lineage defining transcription factor, in vitro and upon short-term transplantation into immunodeficient mice in vivo. In addition, the authors demonstrate that the reverse experiment, genetic deletion of miR-195, has virtually no effect on B-cell development. Mechanistically, the authors identify Foxo1 phosphorylation as one pathway partially contributing to the rescue effect of miR-195. An additional analysis of epigenetics by ATACseq adds potential additional factors that might also contribute to the effect of ectopic expression of miR-195.

Strengths:

The authors employ a robust assay system, Ebf1-KO HPC, to test for B-lineage promoting factors. The manuscript overall takes on an interesting perspective rarely employed for the analysis of miRNA by overexpressing the miRNA of interest. Ideally, this approach may reveal, if not the physiological function of this miRNA, the role of distinct pathways in developmental processes.

Weaknesses:

At the same time, this approach constitutes a major weakness: It does not reveal information on the physiological role of miR-195. In fact, the authors themselves demonstrate in their KO approach, that miR-195 has virtually no role in B-cell development, as has been demonstrated already in 2020 by Hutter and colleagues. While the authors cite this paper, unfortunately, they do so in a different context, hence omitting that their findings are not original.

Conceptually, the authors stress that a predominant function of miRNA (in contrast to transcription factors, as the authors suggest) lies in fine-tuning. However, there appears to be a misconception. Misregulation of fine-tuning of gene expression may result in substantial biological effects, especially in developmental processes. The authors want to highlight that miR-195 is somewhat of an exception in that regard, but this is clearly not the case. In addition to miR-150, as referenced by the authors, also the miR-17-92 or miR-221/222 families play a significant role in B-cell development, their absence resulting in stage-specific developmental blocks, and other miRNAs, such as miR-155, miR-142, miR-181, and miR-223 are critical regulators of leukocyte development and function. Thus, while in many instances a single miRNA moderately affects gene expression at the level of an individual target, quite frequently targets converge in common pathways, hence controlling critical biological processes.

The paper has some methodological weaknesses as well: For the most part, it lacks thorough statistical analysis, and only representative FACS plots are provided. Many bar graphs are based on heavy normalization making the T-tests employed inapplicable. No details are provided regarding the statistical analysis of microarrays. Generation of the miR-195-KO mice is insufficiently described and no validation of deletion is provided. Important controls are missing as well, the most important one being a direct rescue of Ebf1-KO cells by re-expression of Ebf1. This control is critical to quantify the extent of override of Ebf1-deficiency elicited by miR-195 and should essentially be included in all experiments. A quantitative comparison is essential to support the authors' main conclusion highlighted in the title of the manuscript. As the manuscript currently stands, only negative controls are provided, which, given the profound role of Ebf1, are insufficient, because many experiments, such as assessment of V(D)J recombination, IgM surface expression, or class-switch recombination, are completely negative in controls. In addition, the authors should also perform long-term reconstitution experiments. While it is somewhat surprising that the authors obtained splenic IgM+ B cells after just 10 days, these experiments would be certainly much more informative after longer periods of time. Using "classical" mixed bone marrow chimeras using a combination of B-cell defective (such as mb1/mb1) bone marrow and reconstituted Ebf1-KO progenitors would permit much more refined analyses.

With regard to mechanism, the authors show that the Foxo1 phosphorylation pathway accounts for the rescue of CD19 expression, but not for other factors, as mentioned in the discussion. The authors then resort to epigenetics analysis, but their rationale remains somewhat vague. It remains unclear how miR-195 is linked to epigenetic changes.

Reviewer #3 (Public review):

Summary:

In this study, Miyatake et al. present the interesting finding that ectopic expression of miR-195 in EBF1-deficient hematopoietic progenitor cells can partially rescue their developmental block and allow B cells to progress to a B220+ CD19+ cells stage. Notably, this is accompanied by an upregulation of B-cell-specific genes and, correspondingly, a downregulation of T, myeloid, and NK lineage-related genes, suggesting that miR-195 expression is at least in part equivalent to EBF1 activity in orchestrating the complex gene regulatory network underlying B cell development. Strengthening this point, ATAC sequencing of miR-195-expressing EBF1-deficient B220+CD19+ cells and a comparison of these data to public datasets of EBF1-deficient and -proficient cells suggest that miR-195 indirectly regulates gene expression and chromatin accessibility of some, but not all regions regulated by EBF1.

Mechanistically, the authors identify a subset of potential target genes of miR-195 involved in MAPK and PI3K signalling. Dampening of these pathways has previously been demonstrated to activate FOXO1, a key transcription factor for early B cells downstream of EBF1. Accordingly, the authors hypothesize that miR-195 exerts its function through FOXO1. Supporting this claim, also exogenous FOXO1 expression is able to promote the development of EBF1-deficient cells to the B220+CD19+ stage and thus recapitulates the miR-195 phenotype.

Strengths:

The strength of the presented study is the detailed assessment of the altered chromatin accessibility in response to ectopic miR-195 expression. This provides insight into how miR-195 impacts the gene regulatory network that governs B-cell development and allows the formation of mechanistic hypotheses.

Weaknesses:

The key weakness of this study is that its findings are based on the artificial and ectopic expression of a miRNA out of its normal context, which in my opinion strongly limits the biological relevance of the presented work.

While the authors performed qPCRs for miR-195 on different B cell populations and show that its relative expression peaks in early B cells, it remains unclear whether the absolute miR-195 expression is sufficiently high to have any meaningful biological activity. In fact, other miRNA expression data from immune cells (e.g. DOI 10.1182/blood-2010-10-316034 and DOI 10.1016/j.immuni.2010.05.009) suggest that miR-195 is only weakly, if at all, expressed in the hematopoietic system.

The authors support their finding by a CRISPR-derived miR-195 knockout mouse model which displays mild, but significant differences in the hematopoietic stem cell compartment and in B cell development. However, they fail to acknowledge and discuss a lymphocyte-specific miR-195 knockout mouse that does not show any B cell defects in the bone marrow or spleen and thus contradicts the authors' findings (DOI 10.1111/febs.15493). Of note, B-1 B cells in particular have been shown to be elevated upon loss of miR-15-16-1 and/or miR-15b-16-2, which contradicts the data presented here for loss of the family member miR-195.

A second weakness is that some claims by the authors appear overstated or at least not fully backed up by the presented data. In particular, the findings that miR-195-expressing cells can undergo VDJ recombination, express the pre-BCR/BCR and class switch needs to be strengthened. It would be beneficial to include additional controls to these experiments, e.g. a RAG-deficient mouse as a reference/negative control for the ddPCR and the surface IgM staining, and cells deficient in class switching for the IgG1 flow cytometric staining.

Moreover, the manuscript would be strengthened by a more thorough investigation of the hypothesis that miR-195 promotes the stabilization and activity of FOXO1, e.g. by comparing the authors' ATACseq data to the FOXO1 signature.

eLife Assessment

This important study presents a new method for generating cell-type restricted knockouts in zebrafish and it reports several interesting applications of this method to study pigmentation and melanomagenesis. The evidence supporting the conclusions is convincing, with rigorous characterization of several knock out mutations that provide a proof of principle. The work will be of broad interest to cell, skin, and cancer biologists.

Reviewer #1 (Public review):

Summary:

Perlee et al. sought to generate a zebrafish line where CRISPR-based gene editing is exclusively limited to the melanocyte lineage, allowing assessment of cell-type restricted gene knockouts. To achieve this, they knocked in Cas9 to the endogenous mitfa locus, as mitfa is a master regulator of melanocyte development. The authors use multiple candidate genes - albino, sox10, tuba1a, ptena/ptenb, tp53 - to demonstrate their system induces lineage-restricted gene editing. This method allows researchers to bypass embryonic lethal and non-cell autonomous phenotypes emerging from whole body knockout (sox10, tuba1a), drive directed phenotypes, such as depigmentation (albino), and induce lineage-specific tumors, such as melanomas (ptena/ptenb, tp53, when accompanied with expression of BRAFV600E). While the genetic approaches are solid, the argued increase in efficiency of this model compared to current tools was untested, and therefore unable to be assessed. Furthermore, the mechanistic explanations proposed to underlie their phenotypes are mostly unfounded, as discussed further in the Weaknesses section. Despite these concerns, there is still a clear use for this genetic methodology and its implementation will be of value to many in vivo researchers.

Strengths:

The strongest component of this manuscript is the genetic control offered by the mitfa:Cas9 system and the ability to make stable, lineage-specific knockouts in zebrafish. This is exemplified by the studies of tuba1a, where the authors nicely show non-cell autonomous mechanisms have obfuscated the role of this gene in melanocyte development. In addition, the mitfa:Cas9 system is elegantly straightforward and can be easily implemented in many labs. Mostly, the figures are clean, controls are appropriate, and phenotypes are reproducible. The invented method is a welcomed addition to the arsenal of genetic tools used in zebrafish.

Weaknesses:

The major weaknesses of the manuscript include the overly bold descriptions of the value of the model and the superficial mechanistic explanations for each biological vignette.

The authors argue that a major advantage of this system is its high efficiency. However, no direct comparison is made with other tools that achieve the same genetic control, such as MAZERATI. This is a missed opportunity to provide researchers the ability to evaluate these two similar genetic approaches. In addition, Fig.1 shows that not all melanocytes express Cas9. This is a major caveat that goes unaddressed. It is of paramount importance to understand the percentage of mitfa+ cells that express Cas9. The histology shown is unclear and too zoomed out of a scale to make any insightful conclusions, especially in Fig.S1. It would also be beneficial to see data regarding Cas9 expression in adult melanocytes, which are distinct from embryonic melanocytes in zebrafish. Moreover, this system still requires the injection of a plasmid encoding gRNAs of interest, which will yield mosaicism. A prime example of this discrepancy is in Fig.6, where sox10 is clearly still present in "sox10 KO" tumors.

The authors argue that their model allows rapid manipulation of melanocyte gene expression. Enthusiasm for the speed of this model is diminished by minimal phenotypes in the F0, as exemplified in Fig.2. Although the authors say >90% of fish have loss of pigmentation, this is misleading as the phenotype is a very weak, partial loss. Only in the F1 generation do robust phenotypes emerge, which takes >6 months to generate. How this is more efficient than other tools that currently exist is unclear and should be discussed in more detail.

In Figure 3, the authors find that melanocyte-specific knockout of sox10 leads to only a 25% reduction in melanocytes in the F1 generation. This is in contradiction to prior literature cited describing sox10 as indispensable for melanocyte development. In addition, the authors argue that sox10 is required for melanocyte regeneration. This claim is not accurate, as >50% of melanocytes killed upon neocuproine treatment can regenerate. This data would indicate that sox10 is required for only a subset of melanocytes to develop (Fig.3C) and for only a subset to regenerate (Fig.3G). This is an interesting finding that is not discussed or interrogated further.

Tumor induction by this model is weak, as indicated by the tumor curves in Figs.5,6. This might be because these fish are mitfa heterozygous. Whereas the avoidance of mitfa overexpression driven by other models including MAZERATI is a benefit of this system, the effect of mitfa heterozygosity on tumor incidence was untested. This is an essential question unaddressed in the manuscript.

In Fig.6, the authors recapitulate previous findings with their model, showing sox10 KO inhibits tumor onset. The tumors that do develop are argued to be highly invasive, have mesenchymal morphology, and undergo phenotypic switching from sox10 to sox9 expression. The data presented do not sufficiently support these claims. The histology is not readily suggestive of invasive, mesenchymal melanomas. Sox10 is still present in many cells and sox9 expression is only found in a small subset (<20%). Whether sox10-null cells are the ones expressing sox9 is untested. If sox9-mediated phenotypic switching is the major driver of these tumors, the authors would need to knockout sox9 and sox10 simultaneously and test whether these "rare" types of tumors still emerge. Additional histological and genetic evaluation is required to make the conclusions presented in Fig.6. It feels like a missed opportunity that the authors did not attempt to study genes of unknown contribution to melanoma with their system.

Overall, this manuscript introduces a solid method to the arsenal of zebrafish genetic tools but falls short of justifying itself as a more efficient and robust approach than what currently exists. The mechanisms provided to explain observed phenotypes are tenuous. Nonetheless, the mitfa:Cas9 approach will certainly be of value to many in vivo biologists and lays the foundation to generate similar methods using other tissue-specific regulators and other Cas proteins.

Reviewer #2 (Public review):

Summary:

This manuscript describes a genetic tool utilizing mutant mitfa-Cas9 expressing zebrafish to knockout genes to analyze their function in melanocytes in a range of assays from developmental biology to tumorigenesis. Overall, the data are convincing and the authors cover potential caveats from their model that might impact its utility for future work.

Strengths:

The authors do an excellent job of characterizing several gene deletions that show the specificity and applicability of the genetic mitfa-Cas9 zebrafish to studying melanocytes.

Weaknesses:

Variability across animals not fully analyzed.

Reviewer #3 (Public review):

Summary:

Perlee et al. present a method for generating cell-type restricted knockouts in zebrafish, focusing on melanocytes. For this method, the authors knock-in a Cas9 encoding sequence into the mitfa locus. This mitfaCas9 line has restricted Cas9 expression, allowing the authors to generate melanocyte-specific knockouts rapidly by follow-up injection of sgRNA expressing transposon vectors.

The paper presents some interesting vignettes to illustrate the utility of their approach. These include 1) a derivation of albino mutant fish as a demonstration of the method's efficiency, 2) an interrogation and novel description of tuba1a as a potential non-autonomous contributor to melanocyte dispersion, and 3) the generation of sox10 deficient melanoma tumors that show "escape" of sox10 loss through upregulation of sox9. The latter two examples highlight the usefulness of cell-type targeted knockouts (Body-wide sox10 and tuba1a loss elicit developmental defects). Additionally, the tumor models involve highly multiplexed sgRNAs for tumor initiation which is nicely facilitated by the stable Cas9.

Strengths:

The approach is clever and could prove very useful for studying melanocytes and other cell types. As the authors hint at in their discussion, this approach would become even more powerful with the generation of other Cas9-restricted lineages so a single sgRNA construct can be screened across many lineages rapidly (or many sgRNA and fish lines screened combinatorially).

The biological findings used to demonstrate the power of the approach are interesting in their own right. If it proves true, tuba1a's non-autonomous effects on melanosome dispersion are striking, and this example demonstrates very nicely how one could use Perlee et al.'s approach to search for other non-autonomous mechanisms systematically. Similarly, the observation of the sox9 escape mechanism with sox10 loss is a beautiful demonstration of the relevance of SOX10/SOX9's reciprocal regulation in vivo. This system would be a very nice model for further interrogating mechanisms/interventions surrounding Sox10 in melanoma.

Finally, the figure presentation is very nice. This work involves complex genetic approaches including multiple fish generations and multiplexed construct injections. The vector diagrams and breeding schemes in the paper make everything very clear/"grok-able," and the paper was enjoyable to read.

Weaknesses:

The mitfa-driven GFP on their sgRNA-expressing cassette is elegant, but it makes one wonder why the endogenous knock-in is necessary. It would strengthen the motivation of the work if the authors could detail the potential advantages and disadvantages of their system compared to expressing Cas9 with a lineage-specific promoter from a transposon in their introduction or discussion.

Related to the above - is mitfa haplosufficient? If the mitfaCas9/+ fish have any notable phenotypes, it would be worth noting for others interested in using this approach to study melanoma and pigmentation.

A core weakness (and also potential strength) of the system is that introduced edits will always be non-clonal (Fig 2H/I). The activity of individual sgRNAs should always be validated in the absence of any noticeable phenotype to interpret a negative result. Additionally, caution should be taken when interpreting results from rare events involving positive outgrowth (like tumorogenesis) to account for the fact many cells in the population might not have biallelic null alleles (i.e., 100% of the gene product removed).

Along those lines: in my opinion, the tuba1a results are the most provocative finding in the paper, but they lack key validation. With respect to cutting activity, the Alt-R and transgenic sgRNA expression approaches are not directly comparable. Since there is no phenotype in the melanocyte specific tuba1a knockouts, the authors must confirm high knockout efficiency with this set of reagents before making the claim there is a non-autonomous phenotype. This can be achieved with GFP+ sorting and NGS like they performed with their albino melanocytes.

The whole-body tuba1a knockout phenotype is expected to be pleiotropic, and this expectation might mask off-target effects. Controls for knockout specificity should be included. For instance, confidence in the claims would greatly increase if the dispersed melanosome phenotype could be recovered with guide-resistant tuba1a re-expression and if melanocyte-restricted tuba1a re-expression failed to rescue. As a less definitive but adequate alternative, the authors could also test if another guide or a morpholino against tuba1a phenocopies the described Alt-R edited fish.

I have similar questions about the sox10 escapers, but these suggestions are less critical for supporting the authors claims (especially given the nice staining). Are the sox10 tumors relatively clonal with respect to sox10 mutations? And are the sox10 tumor mutations mostly biallelic frameshifts or potential missense mutations/single mutations that might not completely remove activity? I am particularly curious as SOX10 doesn't seem to be completely absent (and is still very high in some nuclei) in the immunohistochemistry.

Poverty is a big issue that affects opportunities but like stated in someone else anootation, it affects physical health. It takes a toll when it comes down to burning yourself out because there is so much financial stress that if you have a job, we can not afford missing work because that day will affect us. It makes us contemplate and think about so many things before actually putting ourselves first. It is sad to say that it is the hard reality even if we do not like it.

This is an example of literally reality today and i can put it in my perspective. I would be first generation graduating knowing that both of my parents did not complete school. It is harder because they had no other option but to work and find different ways to find income. I didn't have the opportunity to be fully raised by my parents because they. would always be working but i'm happy to say that i am able to see what they did for me. Seeing others have a different luxury does emphasize that ones circumstances shape pnes future on how they decide to manage situations.

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Statistics is always something that can be used to mislead or trick readers. In this example, it mentions only 5% of users being bots, but doesn't mention what counts as a user. There could be many inactive accounts lowering the overall number of bots, but not lowering the percentage of bots that users see.

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Amsterdam Typewriter began selling these around 2024-09-24: https://www.facebook.com/groups/typewritermaintenance/posts/3869850413252379/

One set consists of 45 normal keys (12.2mm) + 4 shift keys (16.2mm)

Résumé de la vidéo [00:00:00][^1^][1] - [00:16:30][^2^][2]:

Cette vidéo explore le dilemme moral de diffuser ou non une interview controversée avec un leader du Jihad islamique à Gaza. Elle aborde les thèmes de la responsabilité des créateurs de contenu, de la manipulation des écritures religieuses et de la quête de la confiance et du respect mutuel dans les discussions.

Temps forts: + [00:00:00][^3^][3] Introduction et dilemme * Présentation du dilemme moral * Rôle et responsabilité en tant que créateur de contenu * Impact potentiel de la diffusion de l'interview + [00:01:02][^4^][4] Développement personnel * Importance de la guérison sociale * Comparaison avec d'autres justifications de la violence * Importance de la confiance et du respect + [00:05:00][^5^][5] Expérience personnelle * Réflexion sur le passé de l'intervieweur * Impact des personnages publics sur la perception * Transition vers un rôle de guérisseur social + [00:10:00][^6^][6] Débat et discours * Différence entre débat et discours * Importance de l'écoute et du respect * Techniques pour des discussions constructives + [00:14:00][^7^][7] Conclusion et résolution * Décision de ne pas diffuser l'interview * Importance de créer un terrain d'entente * Réflexion sur les méthodes de communication efficaces

I've always felt weird seeing these kinds of posts on my feed. It feels weird morally to post about something like helping the less fortunate. The posts make the good deed feel disingenuous.

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• 4 single hole rubber stoppers and 1/4 20 bolts or studs. About 5$ per typewriter.

• multiple rubber washers (glued together)
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Dealing with stripped screws

I found this to be the main reason against grading. I understand that grading makes students study hard in order to get the grade but I often wonder if does effect the desire to learn in a lot of students.

This is probably actually true of a lot of criminal activity. None of it occurs in a vacuum due to "naturally bad people," and none of it is actually solved by our current punitive justice system.

This article was written in 2015. The narrative of gambling addition is much more common nowadays, especially in the sphere of online gaming.

This study has actually come under much controversy over the years for some flaws in methodology and the fact that its results have not been replicated (see "Environment Is Not the Most Important Variable in Determining Oral Morphine Consumption in Wistar Rats" by Bruce Petrie)

The real stories of addiction and treatment never make for as good TV as the damaging "radical" stories in shows like that. Obviously every story is different, but it often only perpetuates the cycle of harm further to be cold, cruel, and isolating to an addict, like Hari observes.

A lot of issues that hurt communities have their blame placed on individual failures, and ignore the need for societal change to benefit all. I appreciate this focus in the article.

I see where he's coming from with this; as much as the internet does to foster community, it is completely unmatched compared to the experience of actually going out in the real world and connecting with said communities in a tangible way.

Consistently, housing is the key for lowering recidivism and guaranteeing those in poverty are healthy and connected to their community.

Again, the author goes to great lengths to establish their credibility on the subject

It's interesting to see the author comparing soldiers to rats, especially considering they're specifically Vietnam War soldiers, probably most morally dubious war of its time.

Several appeals to logos made here, constructing the foundation for the upcoming argument on a solid base of evidence.

This passage and the previous one work to display the author's personal connection to the subject, establishing their credibility to discuss it.

I've heard lots of stories about people who start with a prescription and then turn to street drugs when their doctor stops prescribing. Did they stop using because the pain was cured or because the drugs were labeled medical? If the pain was cured through medical intervention, it makes sense they would be able to stop. But for people with chronic pain, would this be different?

preach

again this is a crazy leap to make without more details

good example of how use of language can greatly impact perspective

But it's not the 'same drug,' the author just said it's a more pure version that hasn't been tampered with.

And fighting what exactly? People who need help? I really wonder who/where this concept of "war" came from. Was it Reagan/federal government? The police? Wouldn't be surprised. The concept seems like it most likely did not come from any recovered addict.

The language that the author chose to use here is interesting. Their argument is that we can't criminalize addicts, but they go on to frame this phrase like this

This makes sense, and lines up with my previous knowledge about addiction. People with trauma or who live in difficult environments are much more likely to become addicted. There have been many studies about the relationship between ACE scores and addiction, physical/mental health issues, and early death.

this is so cool

Very interesting way to describe addiction, definitely agree and appreciate the new insight

The internet is not quite a travesty, but there is some truth in his point. Immersing myself in the internet as a kid did the opposite of motivating or cultivating friendships, so it wasn't able to be directly beneficial in that way. Still, it was a way of feeling that I was closer to understanding and having a chance to participate in a more reasonable and accepting social environment than could be found outside of it with as much abuse I got back then

I don't like the structure of this sentence/paragraph. When I read a paragraph where most sentences start with "From", I expect the last sentence to start with "To". I had to re-read in order to grasp the meaning, structure feels stilted

arguably either good for or damaging to the author's credibility

This sets up a tone for the author, and gives the reader an idea of what themes the author is going to discuss

Starting by introducing rat experiments then shifting to something similar with humans helped both pieces of evidence feel cohesive

The American dream is held accountable for each student as they go to school. I think seeing the difference back then in where it was more implemented to be college for certain and now they don't really push you to that extent. Like stated in the last paragraph it all comes down to individual success and how one decides to take initiative in education and goods.

BOOM.... THESE ARE COVID MICRO VESSICLES.. THEY COULD CONTAIN ANY COMPONENT OR COMPLETE VIRIONS OF COVID

13页--Q

while sports do move people to pursue things i dont think it can be compared to religion

i think it would be interesting to write an essay on that question

i dont think sports should be compared to religion

Sacrifice is not just a Christian thing, Aztecs and Greeks also had sacrifices to their gods.

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For those who need to hear it, even Duane Jensen of Phoenix Typewriter regularly asks questions about typewriter repair jobs which stump him.

Cor was not organized, caused enemy to go agaisnt them even stronger

commons wanted to keep some Athenian ships and send some of their people as proof of peace, when Athenians refused they took it as bad intention and armed themselves, Athenians tried to get peace again

Athenian attempt at peace

didn't think tear gas was needed at a sports game

There area many athletes that embody what it is like to love a sport such as babe ruth

sports are what keep us entertained and for some keep us going as it is their passion

An instance of completive rivalry going to far.

i find it interesting that the critics underestimated a determined man

teammates are all usually there to help defend and support one another

hockey games are usually full of violence but never so bad that the crowd can sense something bad

People will do crazy things for people they love, even if it is a fan celebrity relationship

If I read this 4 years ago I would wonder how could people do this, now how ever, I can easily see this happen.

Honestly that is not all that surprising since that can be true with a lot of things with fandoms, weather it be movies, video games, comics, and especially with sports judging how popular foot ball is.

crazy to think there was that many arrests

He didn't think that he did anything fully wrong and only took partial responsibly for his actions

This sentence shows that since 1980, Los Angeles has begun to increase a lot of immigrants.

This statement emphasizes the impact of the local economy on local schools. Although the percentage of funding each state provides to schools is similar, the amount of funding each state receives varies greatly because of the difference in revenue. This is very unfair to poor areas, whose schools end up being the same or worse anyway. This is one of the important reasons leading to the educational inequality in the country.

This statement shows the unequal treatment of different schools by the local legislature. Although they give money to poor school areas, but this only helps them maintain the status. In reality it doesn't work for them. It also reflects economic inequality that has led to schools being divided into "poor districts" and "rich districts." I can also see that poor and rich schools are divided into different districts, rather than randomly having poor and rich campuses in one place, which proves that the local economy also has a great impact on schools.

This is my favorite, I like how straightforward it is, I will probably use it on my own essay.

I agree with this point of view, it's important to be open minded and consider what we can gain, even from arguments we disagree with. This reminds me of a class discussion we had about critical thinking in my last English class, where we talked about how every argument, no matter how flawed, can teach us something.

I like how the text highlights the importance of considering different perspectives. It shows a good effort to include different views which makes a better and more balanced discussion.

Looking for strengths in a flawed argument helps you understand it better. Even if it's flawed, there could be good points worth considering. This balanced approach makes your critique more constructive, helps you find ways to improve the argument, and shows empathy for other perspectives.

follow

I think once you get to a point where you can communicate in a language, you can go out into a lot more places where that language takes center stage, and so you can iron out any quirks or imperfections. I feel like this type of learning primes you for immersion and so lets the rest of the learning be done on the fly, or in action.

This sounds like it would work great as an exercise that you would do in order to learn a language, I don't think that it would be the greatest method if it was your primary tool for learning a new language. I personally find the most trouble with grammar structures when learning a new language. If I were to try this I feel like I might have a lot of success on that front, but I don't think it should be someones only method.

I find that when I am learning a language, even when I am taught specific rules of structure and so on, I only truly grasp the concept when I repeatedly try and fail to apply it correctly. Ultimately I do think we have our own indiviual ways through which we understand the world, and by learning, but focusing on the remembering and the application of what we've learned we can come to better understandings.

I think this is great, I'm gonna start giving myself a reward whenever I learn a word in a separate language. Just jokes, but reward is a great motivator. When it comes to language learning I think it goes much farther than punishment, I mean, what would that look like? lol

I highly agree with this statement, as I have experienced it being used. some individuals rather win the argument by making a statement or question that baits the listener onto agreeing with the individual, it can be seen as a form of manipulation.

it guarantees the right of free speech but it doesn't free the person from the potential of consequences. an example of this could be yelling fire at a theater.

I highly agree with this statement, strong arguments need well researched facts. recently I saw a lot of arguments that are not backed up by facts are often misleading, I see a lot of arguments that obtain personal bias rather than facts.

Decline of classical knowledge in Europe due to the rise of Christianity.

Islamic scholars preserved and enhanced Greek knowledge.

I agree with Wynter that western philosophy is seen as the measure of humanity, and I would go as far as to say that the "Western Men" lifestyle is seen as the standard. Which is very disheartening when considering not everyone is from the west or shares the same experiences. Securing the western man as an ethnoclass has served a great disservice to the human species as a whole. How can we say we care about all humans as a whole and make western philosophy and ideology the standard? 

This is is something you see in Hispanic/latine discourse. The adherence that one's speaking of Spanish in a particular accent, dialect, or mode is more 'proper' or 'ideal' , alongside a host of other signifiers, serves to differentiate a people with a shared history of colonization. Someone from Mexico may not speak or look like someone from Puerto Rico or Haiti but that's not because there is some platonic form of each. The colonialist distinction of 'mestizos' and 'mulatto' is rooted into the ideas which these nations were founded on.

I think Chantal hit on an important point that often diasporic discourse ( for any peoples) can in attempting to reclaim some degree of identity ,stolen by colonization, can fit itself back into the same hegemonic orderings.

In ethnic studies and just in general discourse about 'diasporas', it is often the case that national and by necessity historically contingent differences between a peoples are treated as innate features to a particular group within the diaspora.

The invention of paper technology occurred through trade routes like the Silk Road.

The invention of paper technology occurred through trade routes like the Silk Road.

How long would this process take? It's a very interesting process and I'd love to know more.

Joseph Needham argued that science and technology evolved through cultural exchanges, which is something I can agree with.

There was significant collaboration between families and guilds, this highlights the community aspect of the production of goods.

Time was tracked by natural events like sunrise and sunset. Through the invention of clocks, time could be easily measured.

This resource delves deeper into the ways guilds balanced power between merchants and local authorities.

I think it is interesting how Gawain's belt (which is green) is reflective of nature, and also his defence against nature. There is a clear divide between Gawain's "safe" and comfortable experience in the castle, and the harshness and cruel nature that can often be associated with nature or the outside world. The natural world also goes against the idea of the idealized lifestyle that largely deals with courtly love, and everything that comes with that.

This is a very neat point. It is very interesting that the structures and natural elements that compliment this scene actually directly reflect the characterization of Gawain. His chivalry IS demonstrated through the grandness of the castle and the other buildings appearance. As we read on, this is contrasted quite beautifully with the delicateness of Guinevere's character that is reflected through the silks and more "soft" elements of her surroundings. I just think this is so cool and I have not put this much thought into setting's of stories previously, but it is wonderfully effective.

I think this is a very interesting way that Guinevere's "visibility" is perceived. Through her setting, she can be characterized and this is unlike any other character within the text. I find the little juxtapositions that Woods points out here to be fascinating as they're not blatant until you read deeper and understand what the setting around her represents for her. Prior to reading this, I was unaware just how much her setting amplifies her character as a whole.

I absolutley agree with this. Everyone has a different schedule at home and it is important to help them with their academic goals by adjusting to their schedule. The blatant school schedule that everyone abides to cannot fully help every single student equally. Their futures can be highly affected by just this simple fix. There needs to be a stronger relations with students and their teachers.

I feel like the hidden cirriculum is something that is just in general hard to relate to when the teachers are not inbursed in what some of these students go through. When it comes to solutions from this I believe that it is important to have people that the students to relate to. As the book states there could be a lot of people that can benefit from induvidual mentorship as this can help personalize education to help with maximizing results.

"boring" still, and as the "gravitrons" disappear from memory and the county fairs too; the days we had remembered things like "banks that used to have hyperloops in every teller drive through"

or the difference between what was "i can see the whole universe" and ... http://opensea.io/adobrin/created

The human mind is a powerful thing when it comes up against a problem it knows it needs to confront. This is why immersion can be a great way to learn. If you put up walls you can only scale with language you're bound to learn.