Trolling can be driven by a nihilistic mindset where empathy is disregarded, aiming solely to provoke reactions, often manifesting as hostility towards specific groups. This form of trolling is widely condemned as it inflicts harm, failing to adhere to basic principles of respect and integrity.

The section provides a profound exploration of the complexities involved in understanding and evaluating disruptive behaviors in social media contexts. It compellingly illustrates how the formation of groups, the use of stereotypes, and the enforcement of norms are all deeply intertwined with our cognitive processes and societal structures. The examination of trolling as a form of disruption that can be deployed for both just and unjust ends invites readers to reflect on the multifaceted nature of these actions and their ethical implications.

It’s scary to say this without seeing it! It's like you completed something that you are particularly proud of, but no one else has used or tried it, and it was rejected!

This ruling really showed me just how corrupt the justice system is. Its extremely frustrating how the lives of black citizens are belittled. I feel that if Breonna Taylor was white and the officer black then he would've been given a large prison sentence. How can a country expect its citizens to be happy and trusting when it's built on a failing system.

Again the isolation got to people

People put so much focus on social comparison. I think people get tunnel vision on trying to find a group to fit into, rather than find a group that fits them. Both are important in the right context. Just as it's important to step out of your comfort zone for new people, it's just as important to seek out people with shared interests and hobbies.

This reminds me a lot of the trolley dilemma, choosing to let a trolley hit 5 strangers or just 1 person but it's someone you love/family member. In all of these situations you, as the player, have to sacrifice something and that aspect is what makes the game more personal and rooted on a deeper level.

I agree with both sentiments. However, can we make up for structural social problems with pedagogy in the short time that we have to work with students in higher education? Just to pose a critical question, as I tend to agree with open pedagogy.

Author Response

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

eLife assessment

This important study advances our understanding of the ways in which different types of communication signals differentially affect mouse behaviors and amygdala cholinergic/dopaminergic neuromodulation. Researchers interested in the complex interaction between prior experience, sex, behavior, hormonal status, and neuromodulation should benefit from this study. Nevertheless, the data analysis is incomplete at this stage, requiring additional analysis and description, justification, and - potentially - power to support the conclusions fully. With the analytical part strengthened, this paper will be of interest to neuroscientists and ethologists.

GENERAL COMMENTS ON REVIEWS AND REVISIONS

Experimental design

Here we address questions from several reviewers regarding our periods of neuromodulator and behavioral analysis. First, we recognize that the text would benefit from an overview of the experimental structure different from the narrative we provide in the first paragraphs of the Results. We now include this near the beginning for the Materials and Methods (page 17). We further articulate that the 10-minute time periods were dictated by the sampling duration required to perform accurate neurochemical analyses (and to reserve half of the sample in the event of a catastrophic failure of batch-processing samples). Since neurochemical release may display multiple temporal components (e.g., ACh: Aitta-aho et al., 2018) during playback stimulation, and since these could differ across neurochemicals of interest, we decided to collect, analyze, and report in two stimulus periods as well as one Pre-Stim control. We now clarify this in additional text in the Material and Methods (p. 24, lines 20-22; p. 26, lines 17-19). We decided not to include analyses of the post-stimulus period because this is subject to wider individual and neuromodulator-specific effects and because it weakens statistical power in addressing the core question—the change in neuromodulator release DURING vocal playback.

We also sought to clarify the meaning of the periods “Stim 1” and “Stim 2”; they are two data collection periods, using the same examplar sequences in the same order. We have added statements in the Material and Methods (p. 18, lines 4-7; Fig. caption, p. 39, lines 11-13) to clarify these periods.

For behavioral analyses, observation periods were much shorter than 10 mins, but the main purpose of behavioral analyses in this report is to relate to the neurochemical data. As a result, we matched the temporal features of the behavioral and neurochemical analyses (p. 22, lines 17-22). We plan a separate report, focused exclusively on a broader set of behavioral responses to playback, that may examine behaviors at a more granular level.

Data and statistical analyses

Reviewers 1 and 3 expressed concerns about our normalization of neurochemical data, suggesting that it diminishes statistical power or is not transparent. We note that normalization is a very common form of data transformation that does not diminish statistical power. It is particularly useful for data forms in which the absolute value of the measurement across experiments may be uninformative. Normalization is routine in microdialysis studies, because data can be affected by probe placement and factors affecting neurochemical recovery and processing. Recent examples include:

Li, Chaoqun, Tianping Sun, Yimu Zhang, Yan Gao, Zhou Sun, Wei Li, Heping Cheng, Yu Gu, and Nashat Abumaria. "A neural circuit for regulating a behavioral switch in response to prolonged uncontrollability in mice." Neuron (2023).

Gálvez-Márquez, Donovan K., Mildred Salgado-Ménez, Perla Moreno-Castilla, Luis Rodríguez-Durán, Martha L. Escobar, Fatuel Tecuapetla, and Federico Bermudez-Rattoni. "Spatial contextual recognition memory updating is modulated by dopamine release in the dorsal hippocampus from the locus coeruleus." Proceedings of the National Academy of Sciences 119, no. 49 (2022): e2208254119.

Holly, Elizabeth N., Christopher O. Boyson, Sandra Montagud-Romero, Dirson J. Stein, Kyle L. Gobrogge, Joseph F. DeBold, and Klaus A. Miczek. "Episodic social stress-escalated cocaine self-administration: role of phasic and tonic corticotropin releasing factor in the anterior and posterior ventral tegmental area." Journal of Neuroscience 36, no. 14 (2016): 4093-4105.

Bagley, Elena E., Jennifer Hacker, Vladimir I. Chefer, Christophe Mallet, Gavan P. McNally, Billy CH Chieng, Julie Perroud, Toni S. Shippenberg, and MacDonald J. Christie. "Drug-induced GABA transporter currents enhance GABA release to induce opioid withdrawal behaviors." Nature neuroscience 14, no. 12 (2011): 1548-1554.

However, since all reviewers requested raw values of neurochemicals, we provide these in supplementary tables 1-3. The manuscript references these table early in the Results (p. 6, lines 18-19) and in the Material and Methods (p. 27, lines 3-4)

All reviewers commented on correlation analyses that we presented, with different perspectives. Reviewer 2 questioned the validity of such analyses, performed across experimental groups, while Reviewer 1 pointed out that the analyses were redundant with the GLM. We agree with these criticisms, and note the challenges associated with correlations involving behaviors for which there is a “floor” in the number of observations. As a result, we have removed most correlation analyses from the manuscript. The text and figures have been modified accordingly. Due these changes, we have to decline requests of Reviewer 3 to include many more such analyses. While correlation analyses could still be performed between neurochemicals and behaviors for each group, the relatively small size of each experimental group, the large number of groups, and the even larger numbers of pairings between neurochemicals and behavior, the statistical power is very low. The only correlations we utilize in the manuscript concern the interpretation of our increased acetylcholine levels.

As part of this revision, we re-ran our statistical analyses on neuromodulators because of a calculation error in 3 animals (regarding baseline values). In a few instances, a significance level changed, but none of these changed a conclusion regarding neuromodulator changes under our experimental conditions.

Other revisions

INTRODUCTION: We modified the Introduction to provide both a more general framework and specific gaps in our understanding relating neuromodulators with vocal communication.

DISCUSSION: We have added material in the first two pages of the Discussion to provide more framework to our conclusions, to address the issues of the temporal aspects of neurochemical release and behavioral observations, and to identify limitations that should be addressed in future studies.

FIGURES: All figures are now in the main part of the manuscript. We modified most figures in response to reviewer comments. We removed neuromodulator – behavior correlations from several figures. We modified all box plots to ensure that all data points are visible. The visible data points match the numbers reported in figure captions. We brought 5-HIAA data into the main figures reporting on neuromodulator results.

Public Reviews:

Reviewer #1 (Public Review):

The manuscript addresses a fundamental question about how different types of communication signals differentially affect brain states and neurochemistry. In addition, the manuscript highlights the various processes that modulate brain responses to communication signals, including prior experience, sex, and hormonal status. Overall, the manuscript is well-written and the research is appropriately contextualized. The authors are thoughtful about their quantitative approaches and interpretations of the data.

That being said, the authors need to work on justifying some of their analytical approaches (e.g., normalization of neurochemical data, dividing the experimental period into two periods (as opposed to just analyzing the entire experimental period as a whole)) and should provide a greater discussion of how their data also demonstrate dissociations between neurochemical release in the basolateral amygdala and behavior (e.g., neurochemical differences during both of the experimental periods but behavioral differences only during the first half of the experimental period). The normalization of neurochemical data seems unnecessary given the repeated-measures design of their analysis and could be problematic; by normalizing all data to the baseline data (p. 24), one artificially creates a baseline period with minimal variation (all are "0"; Figures 2, 3 & 5) that could inflate statistical power.

Please see our general responses to structure of observation periods and normalization of neuromodulator data. Normalization is a common and appropriate procedure in microdialysis studies that does not alter statistical power.

We have included a section in the Discussion concerning the temporal relationship between behavioral responses and neurochemical changes in response to vocal playback (p. 12, lines 3-17). We note where the linkage is particularly strong (e.g., ACh release and flinching). This points to a need to examine these phenomena with finer temporal resolution, but also with the recognition that the brain circuits driving a behavioral response may extend beyond the BLA.

The Introduction could benefit from a priori predictions about the differential release of specific neuromodulators based on previous literature.

We added some material to the Introduction to provide additional rationale for the study. However, we did not attempt to develop predictions for the range of neuromodulators that we sought to test. The literature can lead to opposite predictions for a given neuromodulator. For example, acetylcholine could be associated with both positive and negative valence. Instead, we note in the Introduction the association of both DA and ACh with vocalizations.

The manuscript would also benefit from a description of space use and locomotion in response to different valence vocalizations.

We have provided additional descriptions of space use and video tracking data in Material and Methods (p. 23, lines 1-6). We now report a few correlations based on these data in the Results to demonstrate that increased ACh in Restraint males and Mating estrus females was not related to the amount of locomotion (p. 9, lines 8-14).

Nevertheless, the current manuscript seems to provide some compelling support for how positive and negative valence vocalizations differentially affect behavior and the release of acetylcholine and dopamine in the basolateral amygdala. The research is relevant to broad fields of neuroscience and has implications for the neural circuits underlying social behavior.

Reviewer #2 (Public Review):

Ghasemahmad et al. report findings on the influence of salient vocalization playback, sex, and previous experience, on mice behaviors, and on cholinergic and dopaminergic neuromodulation within the basolateral amygdala (BLA). Specifically, the authors played back mice vocalizations recorded during two behaviors of opposite valence (mating and restraint) and measured the behaviors and release of acetylcholine (ACh), dopamine (DA), and serotonin in the BLA triggered in response to those sounds.

Strength: The authors identified that mating and restraint sounds have a differential impact on cholinergic and dopaminergic release. In male mice, these two distinct vocalizations exert an opposite effect on the release of ACh and DA. Mating sounds elicited a decrease of Ach release and an increase of DA release. Conversely, restraint sounds induced an increase in ACh release and a trend to decrease in DA. These neurotransmission changes were different in estrus females for whom the mating vocalization resulted in an increase of both DA and ACh release.

Weaknesses: The behavioral analysis and results remain elusive, and although addressing interesting questions, the study contains major flaws, and the interpretations are overstating the findings.

Although Reviewer 2 raises several valid issues that we have addressed in our response and revision, we believe that none represent “major flaws” in the study that challenge the validity of our central conclusions. In brief, we will:

--provide enhanced description of behaviors (pp. 22-23 and Table 1)

--clarify / modify box-plot representations of data (p 28. Lines 3-9)

--point to our methods that describe corrections for multiple comparisons (p. 27; lines 15-16)

--revise figures to clarify sample size (Figs. 3-6)

Reviewer #3 (Public Review):

Ghasemahmad et al. examined behavioral and neurochemical responses of male and female mice to vocalizations associated with mating and restraint. The authors made two significant and exciting discoveries. They revealed that the affective content of vocalizations modulated both behavioral responses and the release of acetylcholine (ACh) and dopamine (DA) but not serotonin (5-HIAA) in the basolateral amygdala (BLA) of male and female mice. Moreover, the results show sex-based differences in behavioral responses to vocalizations associated with mating. The authors conclude that behavior and neurochemical responses in male and female mice are experience-dependent and are altered by vocalizations associated with restraint and mating. The findings suggest that ACh and DA release may shape behavioral responses to context-dependent vocalizations. The study has the potential to significantly advance our understanding of how neuromodulators provide internal-state signals to the BLA while an animal listens to social vocalizations; however, multiple concerns must be addressed to substantiate their conclusions.

Major concerns:

1) The authors normalized all neurochemical data to the background level obtained from a single pre-stimulus sample immediately preceding playback. The percentage change from the background level was calculated based on a formula, and the underlying concentrations were not reported. The authors should report the sample and background concentrations to make the results and analyses more transparent. The authors stated that NE and 5-HT had low recovery from the mouse brain and hence could not be tracked in the experiment. The authors could be more specific here by relating the concentrations to ACh, DA, and 5-HIAA included in the analyses.

Please see our general statement regarding normalization of neurochemical data. We have added supplemental tables that shows concentrations of dopamine, acetylcholine, 5-HIAA. We do not report serotonin or noradrenalin since these were below the detection threshold.

2) For the EXP group, the authors stated that each animal underwent 90-min sessions on two consecutive days that provided mating and restraint experiences. Did the authors record mating or copulation during these experiments? If yes, what was the frequency of copulation? What other behaviors were recorded during these experiences? Did the experiment encompass other courtship behaviors along with mating experiences? Was the female mouse in estrus during the experience sessions?

In the mating experience, mounting or attempted mounting was required for the animal to be included in subsequent testing. Since the session lasted 90 minutes, more general courtship behavior was likely. However, we did not record detailed behaviors or track estrous stage for the mating experience. See p. 21, line 20-22.

3) For the mating playback, the authors stated that the mating stimulus blocks contained five exemplars of vocal sequences emitted during mating interactions. The authors should clarify whether the vocal sequences were emitted while animals were mating/copulating or when the male and female mice were inside the test box. If the latter was the case, it might be better to call the playback "courtship playback" instead of "mating playback".

We have modified the Results (p. 5, lines 18-20) and Materials and Methods (p. 21, lines 8-15) to clarify our meaning. We continue to use the term “mating” because this refers to a specific set of behaviors associated with mounting and copulation, rather than the more general term “courtship”. We also indicate that we based these behaviors on previous work (e.g., Gaub et al., 2016).

4) Since most differences that the authors reported in Figure 3 were observed in Stim 1 and not in Stim 2, it might be better to perform a temporal analysis - looking at behaviors and neurochemicals over time instead of dividing them into two 10-minute bins. The temporal analysis will provide a more accurate representation of changes in behavior and neurochemicals over time.

Please see our general response to the structuring of experimental periods. The 10-min periods are the minimum for the neurochemical analyses, and we adopted the same periods for behavioral analyses to match the two types of observations. Our repeated measures analysis is a form of temporal analysis, since it compares values in three observation periods.

5) In Figures 2 and 3, the authors show the correlation between Flinching behavior and ACh concentration. The authors should report correlations between concentrations of all neurochemicals (not just ACh) and all behaviors recorded (not just Flinching), even if they are insignificant. The analyses performed for the stim 1 data should also be performed on the stim 2 data. Reporting these findings would benefit the field.

Please see general comments regarding correlation analyses. We removed almost all such analyses and references to them from the manuscript based on concerns of the other reviewers.

6) The mice used in the study were between p90 - p180. The mice were old, and the range of ages was considerable. Are the findings correlated with age? The authors should also discuss how age might affect the experiment's results.

Our p90-p180 mice are not “old”. CBA/CaJ mice display normal hearing for at least 1 year (Ohlemiller, Dahl, and Gagnon, JARO 11: 605-623, 2010) and adult sexual and social behavior throughout our observation period. They are sexually mature adults, appropriate for this study. We decline to perform correlation analyses with age, both because this was not a question for this study and because the very large number of correlations, for each experimental group (as requested by reviewer #2), render this approach statistically problematic.

7) The authors reported neurochemical levels estimated as the animals listened to the sounds played back. What about the sustained effects of changes in neurochemicals? Are there any potential long-term effects of social vocalizations on behavior and neurochemical levels? The authors might consider discussing long-term effects.

We have not included discussion of long term effects of neuromodulatory release, both because our data analysis doesn’t address it (see response to Comment #10) and because we desired to keep the Discussion focused on topics more closely related to the results.

8) Histology from a single recording was shown in supplementary figure 1. It would benefit the readers if additional histology was shown for all the animals, not just the colored schematics summarizing the recording probe locations. Further explanation of the track location is also needed to help the readers. Make it clear for the readers which dextran-fluorescein labeling image is associated with which track in the schematic.

Based on the recent publications cited in our overall response to reviewer comments about statistical methods, our reporting of histological location of microdialysis exceeds the standard. We believe that the inclusion of all histology is unnecessary and not particularly helpful. Raw photomicrographs do not always illustrate boundaries, so interpretation is required. However, we added a second photomicrograph example and we identified which tracks correspond to these photomicrographs (see Figure 2; now in main body of manuscript).

9) The authors did not control for the sounds being played back with a speaker. This control may be necessary since the effects are more pronounced in Stim 1 than in Stim 2. Playing white noise rather than restraint or courtship vocalizations would be an excellent control. However, the authors could perform a permutation analysis and computationally break the relationship between what sound is playing and the neurochemical data. This control would allow the authors to show that the actual neurochemical levels are above or below chance.

We considered a potential “control” stimulus in our experimental design. We concluded, based on our previous work (e.g., Grimsley et al., 2013; Gadziola et al., 2016), that white noise is not or not necessarily a neutral stimulus and therefore the results would not clarify the responses to the two vocal stimuli. Instead, we opted to use experience as a type of control. This control shows very clearly that temporal patterns and across-group differences in neurochemical response to playback disappear in the absence of experience with the associated behavior.

10) The authors indicated that each animal's post-vocalization session was also recorded. No data in the manuscript related to the post-vocalization playback period was included. This omission was a missed opportunity to show that the neurochemical levels returned to baseline, and the results were not dependent on the normalization process described in major concern #1. The data should be included in the manuscript and analyzed. It would add further support for the model described in Figure 6.

We decided not to include analyses of the post-stimulus period because this period is subject to wider individual and neuromodulator-specific effects and because it weakens statistical power in addressing the core question—the change in neuromodulator release DURING vocal playback. We agree that the general question is of interest to the field, but we don’t think our study is best designed to answer that question.

11) The authors could use a predictive model, such as a binary classifier trained on the CSF sampling data, to predict the type of vocalizations played back. The predictive model could support the conclusions and provide additional support for the model in Figure 6.

We recognize that a binary classifier could provide an interesting approach to support conclusions. However, we do not believe that the sample size per group is sufficient to both create and test the classifier.

Reviewer #1 (Recommendations For The Authors):

Major comments:

• Introduction: It would be useful to set up an experimental framework before delving into the results. What are the predictions about specific neuromodulators based on previous literature?

Because this narrative is laid out in the first two paragraphs of the Results, which immediately follow the Introduction, we believe that additional text in the Introduction on the experimental framework is redundant. As stated above, detailing predictions for a range of neuromodulators would make for a long and not particularly illuminating Introduction. We instead have related our findings to more general understanding of DA and ACh in the Discussion.

• There really isn't a major difference in stimuli during the "Stim 1" and "Stim 2" phases, and it's not clear why the authors divided the experimental period into two phases. Therefore, the authors need to justify their experimental approach. For example, the authors could first anecdotally mention that behavioral responses to playbacks seem to be larger in the first half of the playbacks than during the second half, therefore they individually analyzed each half of the experimental period. Or adopt a different approach to justify their design. Overall, the analytical approach is reasonable but it is currently not justified.

See general comment for analysis periods. As noted, we clarified these issues in several locations with Materials and Methods (pp. 24, lines 20-22; p. 26, lines 17-19). We also sought to clarify the meaning of the periods “Stim 1” and “Stim 2”; they are two data collection periods, using the same examplar sequences in the same order. We have added statements in the Material and Methods (p. 18, lines 4-7; Fig. caption, p. 39, lines 11-13).

• The normalization of neurochemical data seems problematic and unnecessary. By normalizing all data to the baseline data (p. 24), one artificially creates a baseline period with minimal variation (all are "0"; Figures 2, 3 & 5) and this has implications for statistical power. Because the analysis is a within-subjects analysis, this normalization is not necessary for the analysis itself. It can be useful to normalize data for visualization purposes, but raw data should be analyzed. Indeed, behavioral data are qualitatively similar to the neurochemical data, and those data are not normalized to baseline values.

Please see our general comment on this issue. We believe normalization does not affect statistical power and is both the standard way and an appropriate way to analyze microdialysis results. We include concentrations of ACh, DA, and 5-HIAA in supplementary tables?

• The authors should include a discussion (in the Discussion section) of how behavior and neurochemical release are associated during the first half of the experimental session but not in the second half (e.g., differences in Ach and DA release between mating and restraint groups during stim 1 and 2, but behavioral differences only during stim 1).

We have included a section in the Discussion concerning the temporal relationship between behavioral responses and neurochemical changes in response to vocal playback. We note that the linkage is particularly strong in some cases (e.g., ACh release and flinching). This points to a need to examine these phenomena with finer temporal resolution, but also with the recognition that the brain circuits driving a behavioral response may extend beyond the BLA.

Minor comments:

• Keywords: add "serotonin" (even though there are no significant differences on 5-HIAA, people interested in serotonin would find this interesting).

Added to keywords list.

• Do the authors collect data on the vocalizations of mice in response to these playbacks?

We monitored vocalizations during playback, noting that vocalizations–especially “Noisy” vocalization–were common. However, we did not record vocalizations and are therefore unable quantify our observations.

• First line of page 7: readers do not know about "stim 1" and "stim 2". Therefore, the authors need to describe their approach to analyzing behavior and neurochemical release.

We first introduce these terms earlier, citing Figure 1D,E. We have added some additional wording for further clarification. page 7, lines 4-5.

• Make sure citations are uniformly formatted (e.g., Inconsistencies in: "As male and female mice emit different vocalizations during mating (Finton et al., 2017; J. M. S. Grimsley et al., 2013; Neunuebel et al., 2015; Sales (née Sewell), 1972)").

We have reviewed and corrected citations throughout the manuscript.

• Last paragraph of page 7: "attending behavior" has not been defined yet.

Table 1 contains our description of the behaviors analyzed in this study. We have now inserted a reference to Table 1 earlier in the Results (p. 6, line 12).

• Figure 2E and 3G: I find these correlations to be redundant with the GLMs. This is because the significant relationship is likely to be driven by group differences in behavior and in neurochemical release.

Please see general comments regarding correlation analyses. We removed such analyses and references to them from the manuscript.

• Page 2, 2nd paragraph, 2nd sentence: this paragraph seems to be rooted in comparing and contrasting experienced and inexperienced mice, so there should be explicit comparisons in each sentence. For example, the 2nd sentence should read: "Whereas EXP estrus females demonstrated increased flinching behaviors in response to mating vocalizations, INEXP ....". This paragraph overall could use some refining.

We believe this refers to page 9. We have revised the paragraph to clarify our findings (Beginning p. 9, line 23).

• Page 9: "Further, there were no significant differences across groups during Stim 1 or Stim 2 periods. These results contrast sharply with those from all EXP groups, in which both ACh and DA release changed significantly during playback (Figs. 2C, 2D, 3E, 3F)." While I understand their perspective, this is misleading because changes were only observed during the Stim 1 period.

We have slightly revised the wording in this paragraph, because the restraint males did not show significant ACh decreases. However, we do not believe our statements mislead readers just because some changes are observed in only one of the stimulation periods (p 10, lines 13-16).

• Last paragraph of page 14: it would be useful to mention the increase in flinching in experienced females in response to mating vocalizations.

We have added a sentence in this paragraph relating flinching in estrus females to increased ACh (p. 15, lines 18-20).

• Was there a full analysis of locomotion in response to playbacks? I see that locomotion was correlated with neurochemical release but was it different in response to different stimuli? Were there changes to the part of the arena that mice occupied in response to restraint vs. mating vocalizations? Given their methods section, it would be useful for the authors to mention the results of the analyses of these aspects of movement.

We have provided additional descriptions of space use and video tracking data in Material and Methods (p. 23, lines 1-6). We now report additional results associated with these analyses (p. 8, lines 13-15; p. 9, lines 8-14).

• I believe that each experimental mouse only heard one of the stimuli (given the analytical approach). Because it is plausible to measure neurochemical release in response to both types of stimuli, I encourage the authors to be more explicit about this aspect of the experimental design (e.g., mention in Results section).

Sentence modified to read: “Each mouse received playback of either the mating or restraint stimuli, but not both: same-day presentation of both stimuli would require excessively long playback sessions, the condition of the same probe would likely change on subsequent days, and quality of a second implanted probe on a subsequent day was uncertain.” (p. 7, lines 5-9).

• Figure 1A and 1B: add labels to the panels so readers don't have to read the legend to know what spectrogram is associated with what context.

We added these labels to Figure 1.

• Table 1: in the definition of "still and alert", should this mention "abrupt attending" instead of "abrupt freezing"? The latter isn't described.

Yes, we intended “abrupt attending”, and now indicated that in Table 1

Reviewer #2 (Recommendations For The Authors):

Major comments:

• The authors report they performed manual behavioral analysis, and provide a table defining the different behaviors. However, it remains unclear how some of these behaviors were detected (such as still-and-alert events). A thorough description of the criteria used to define these events needs to be provided.

We have modified some descriptions of manually analyzed behaviors in Table 1, and have added additional description of how we developed this set of behaviors for analysis in the study (pp. 22-23).

• The box plots do not appear to represent the "minimum, first quartile, median, third quartile, and maximum values." as specified on page 24 (Methods). Indeed, the individual data points sometimes do not reach the max or min of the bar plot, and sometimes are way beyond them.

We used the “inclusive median” function in Excel to generate final boxplots. These boxplots will sometimes result in a data point being placed outside of the whiskers. SPSS considers these to be “outliers”, but our GLM analysis includes these values. We describe this in Data Analysis section of Materials and Methods (p. 28, lines 3-9)

• Some of the data are replicated in different Figures: Figure 2A and Figure 3C. While this is acceptable, the authors did not correct for multiple comparisons (dividing the p value by the number of comparisons).

Our analysis included corrections for multiple comparisons, as we have indicated on p. 27, lines 15-16.

• Overall, the sample sizes are too small (for example in Figure 3, non-estrus females are at n=3), and are different in experiments where they should be equal (Figure 2B: mating stim 1 is at n=5 and mating stim 2 is at n=3).

We apologize that sample sizes were not properly displayed in figures. Please note that sample sizes are identified in the figure captions. For neuromodulator data, all sample sizes are at least 7. For behavioral data, the minimum sample size is 5. We have revised Figures 3-6 to ensure that all data points are visible.

• It remains unclear why the impact of mating vocalizations has been tested only in males.

We assume the reviewer meant that only males were tested in restraint. We now indicate that our preliminary evidence indicated no difference in behavioral responses to restraint vocalization between males and females, so we opted to perform the neurochemical analysis for restraint only in males (page 22 lines 4-5). If there were no limitations to time and cost, we would have preferred to test responses to restraint in females as well. We note that such inclusion would have added up to 4 experimental groups (estrus and non-estrus groups in both EXP and INEXP groups).

• The correlation between the number of flinching and ACh release changes (Figure 2E) visually appears to be opposite between mating and restraint playbacks. The authors should perform independent correlations for these 2 playbacks.

Please see general comments regarding correlation analyses. We removed such analyses and references to them from the manuscript.

• The authors state that their findings "indicate that behavioral responses to salient vocalizations result from interactions between sex of the listener or context of vocal stimuli with the previous behavioral experience associated with these vocalizations.". However, in male mice, they do not report any difference in previous experience on flinching for both restraint and mating sounds, as well as no difference in rearing for the restrain sounds (Figure 4A-B). Thus, the discussion of these results should be completely revisited.

We revised the paragraph in question (p. 9, line 22 through p. 10, line 9). For instance, we note that significant differences between EXP male-mating and male-restraint flinching do not exist between the INEXP groups. We believe that the last sentence correctly summarizes findings described in this paragraph.

• For serotonin experiments in Figure S2 there are strong outliers (150% increase in 5HIAA release). Did the authors correlate these levels with the behavior of the animals?

Outliers are identified by the Excel function that generated the boxplots, but we have no reason to consider these as outliers and exclude them. As noted above, we have clarified that these “outliers” are the result of the Excel function in the Materials and Methods (p. 28, lines 3-9) and we have revised the plotting of data points

Minor comments:

• Mating vocalization playback is mainly emitted by males, thus, instead of a positive valence signal, this could also be interpreted as a competitive signal to other males.

There is support in the literature for viewing our mating stimulus as having positive valence. Gaub et al., 2016 describe the emission of stepped calls, lower frequency harmonics, and increased sound level as indicators of “positive emotion”. We have shown (Grimsley et al, 2013) that the female LFH vocalization can be highly attractive to male mice, under the right conditions, indicating something like “sex is happening”. The inclusion of both the male and female vocalizations in our stimuli was a key piece of our experimental design, based on our understanding of the contributions of both vocalizations to the meaning of the overall acoustic experience.

• Figure 1 should include panel titles.

No change. This information is available in the Figure caption.

• n=31 should be indicated in the EXP group.

We’re not sure where the reviewer is referring to this value.

• The color legend of Figure 1E is absent, making the Figure not understandable.

We added text in the Figure 1 caption to indicate that each color represents a different exemplar. We don’t think a legend provides additional useful information.

• The point of making two blocks (stim 1 and stim2) should be stated more clearly.

Please see general statement regarding experimental blocks. We have modified our description of these in an Experimental overview section in the Material and Methods.

• Including raw data of micro-dialysis in the supplementary figures would allow assessment of the variability and quality of the measurements.

We have added concentrations of neurochemicals in supplemental tables 1-3.

• Baseline (prestimulus) number of flinch and rearing should systematically be indicated (missing in Figure 4).

The focus in this figure is on the differences that occur in Stim 1 values. There are no differences between EXP and INEXP animals of any group during the Pre-Stim period. We now state that in the Figure 4 caption.

• Discussion: "increase in AMPA/NMDA currents". We believe the authors are referring to the ratio of AMPA to NMDA currents. This sentence should be reformulated.

These are modified to refer to “… the AMPA/NMDA current ratio…” in two locations in the Discussion (p. 14, lines 8-9; p. 15, line 4)

• Overall the discussion is very speculative and should rely more on the data.

We believe that the Discussion provides appropriate speculation that is based on our experimental data and previous literature. We have added a paragraph to identify limitations of our findings and recommendations of future experiments to resolve some issues (p. 12, lines 3-17)

Reviewer #3 (Recommendations For The Authors):

Minor concerns:

1) The authors stated that USVs are most likely to be emitted by males, and LFH are likely to be emitted by females. However, Oliveira-Stahl et al. 2023, Matsumoto et al. 2022, Warren et al. 2018, Heckman et al. 2017, Neunuebel et al., 2015 showed that females also emit USVs. The authors should mention that USVs are emitted by both males and females and discuss how the sex of the vocalizing animal (both males and females) can influence neuromodulator release.

The reviewer slightly mis-stated the wording of our text, changing the meaning significantly. Our wording is “These sequences included ultrasonic vocalizations (USVs) with harmonics, steps, and complex structure, mostly emitted by males, and low frequency harmonic calls (LFHs) emitted by females (Fig. 1A,C)…” This phrasing is correct and carefully chosen. The Discussion in Oliveira-Stahl et al 2023 (p. 10-11) supports our statement: “The exact fraction of USVs emitted by females as concluded in all previous studies on dyadic courtship has varied, ranging from 18%, 17.5%, and 16% to 10.5% in the present study…”.

2) The authors should explain why ECF from BLA was collected unilaterally from the left hemisphere.

p. 23, lines 9-11: We inserted a sentence to explain why we targeted the BLA unilaterally. “Since both left and right amygdala are responsive to vocal stimuli in human and experimental animal studies (Wenstrup et al., 2020), we implanted microdialysis probes into the left amygdala to maintain consistency with other studies in our laboratory..” Beyond that, the choice was arbitrary.

3) The authors said each animal recovered in its home cage for four days before the playback experiment. A 4-day period may not be sufficient for every animal to recover from surgery, so the authors should describe how a mouse's recovery was assessed.

p. 23, lines 20-23: We provide more description about the recovery and how it was assessed. Except for a few animals that were not included in the experiments, all animals recovered within 4 days.

4) The authors stated that each animal was exposed to 90-min sessions with mating and restraint behaviors in a counterbalanced design. This description for Figure 1D should also include the duration of the mating and restraint experience.

The Results that immediately precede citation to this figure include this information.

5) The authors stated, "Data are reported only from mice with more than 75% of the microdialysis probe implanted within the BLA". What are the implications of having 25% of the probe outside the BLA? The authors should shed more light on this by discussing this issue as it relates to the findings and commenting on where the other 25% of the probe was located.

We inserted a sentence to explain the rationale for this inclusion criterion. “We verified placement of microdialysis probes to minimize variability that could arise because regions surrounding BLA receive neurochemical inputs from different sources (e.g., cholinergic inputs to putamen and central amygdala).” (p. 25, lines 21-23).

All brain regions that surround BLA, dorsal, medial, ventral, or lateral, could have been sampled by the “other” 25%. Some of these, e.g., the central amygdala or caudate-putamen, have different sources of cholinergic input that may not have the same release pattern. We do not think it is worthy of further speculation in the Discussion. Due to the high cost of the neurochemical analysis, we often did not process the neurochemistry data if histology indicated that a probe missed the BLA target.

6) The authors confirmed that the estrus stage did not change during the experiment day by evaluating and comparing estrus prior to and after data collection. This strategy was a fantastic experimental approach, but the authors should have discussed the results. How did the results the authors included change when the females were in estrus before but not after data collection? What percentage of females started in estrus but ended in metestrus? Assuming that some females changed estrus state, were these animals excluded from the analyses?

All animals were in the same estrus state at the beginning and end of the playback session.

7). Authors cite Neunuebel et al., 2015 for the sentence "As male and female mice emit different vocalizations during mating". However, Neunuebel et al., 2015 showed vocalizations emitted during chasing--not mating. If mating is a general term for courtship, then this reference is appropriate, but see major concern #3.

In the Results (p. 8, line 5), we changed the phrasing to “courtship and mating” to include the Neunubel et al study.

As we indicate in our response to Public Comment #3, we have modified the Results (p. 5, lines 18-20) and Materials and Methods (p. 21, lines 8-15) to clarify our meaning. We continue to use the term “mating” because this refers to a specific set of behaviors associated with mounting and copulation, rather than the more general term “courtship”. We also indicate that we based these behaviors on previous work (e.g., Gaub et al., 2016).

8) Authors interpret Figure 3F as DA release showed a "consistent" increase during mating playback across all three experimental groups. However, the increase in the estrus female group is inconsistent, as seen in the graph. This verbiage should be reworded to describe the data more accurately.

p. 8, line 23 “consistent” was deleted.

9) In all the box plots, multiple data points overlay each other. A more transparent way of showing the data would be adding some jitter to the x value to make each data point visible. The mean (X's) in Figure 3D (pre-stim mating and mating estrus) are difficult to see, as are all the data points in mating non-estrus. Adding all the symbols to the figure legend or a key in the figure instead of the method section would aid the reader and make the plots easier to interpret

We have revised the boxplots to ensure that all data points are visible.

10) Some verbiage used in the discussion should be toned down. For example, "intense" experiences and "emotionally charged" vocalizations should be removed.

We have not changed these terms, which we believe are appropriate to describe these experiences and vocalizations.

11) The authors include "Emotional Vocalizations" in the title. It would be beneficial if the authors included more detail and references in the introduction to help set up the emotional content of vocalizations. It may benefit a broader readership as typically targeted by eLife.

We now cite Darwin and some more recent publications that articulate the general understanding that social vocalizations carry emotional content.

This just seems so hard because teachers and society are just putting high expectations and standards on Asian American kids. This situation can hurt people mentally because it can make them feel overwhelmed and stressed as they are expected to be the best at everything.

reply to u/chasemac_ at https://www.reddit.com/r/Zettelkasten/comments/19ep9rc/comment/kjempeu/

I'm curious from where you draw your "original purpose" claim? This presupposes having identified a zettelkasten progenitor who has clearly made such a statement. (If you're thinking Luhmann, you're missing the mark by centuries. And even if you're thinking Luhmann, where did he say this specifically?) While Konrad Gessner seems to have been an early progenitor in 1548, the broader idea goes much further back. Even in the early days of the commonplace book, the primary analogy was using them as "storehouses" for collecting treasure (thesaurus) aka knowledge or wisdom.

Even Luhmann's framing of his zettelkasten as his "second memory" was old by the time he wrote it:

In a short academic dissertation on the art of excerpts, Andreas Stübel described the card index as a ‘secondary and subsidiary memory’ (‘memoria secundaria and subsidiaria’), summing up in just three words the dilemma scholars had been struggling with for two centuries with respect to the use of commonplace books. As far as I know, Stübel was the first among contemporaries to speak of secondary memory. —Alberto Cevolini in “Where Does Niklas Luhmann’s Card Index Come From?” Erudition and the Republic of Letters 3, no. 4 (October 24, 2018): 390–420. https://doi.org/10.1163/24055069-00304002.

If we look even further back we read Seneca the Younger in Epistulae morales, writing positively about collecting with respect to classic rhetoric:

"We should follow, men say, the example of the bees, who flit about and cull the flowers that are suitable for producing honey, and then arrange and assort in their cells all that they have brought in;

Without a clear originator, I might suggest that historically the first purpose was for memory followed closely by learning and then accumulating wisdom and knowledge (sententiae). Using them for output only came much later.

Why is there so much bad ink in the zettelkasten space about about "collecting"? (a la the "collector's fallacy") If you collect nothing, you'll have nothing. You have to start somewhere. Collecting happens first before anything useful comes out of the enterprise. Where are all these "people [who] do nothing but boast about the amount of cards in their box"? I'm not seeing lots of evidence of them in fora or online certainly. Show us your collection of examples of those to back up the claim. Are there index card hoarders out there who honestly have tens of thousands of notes with absolutely no purpose? I suspect it's rare.

If you're a collector, collect away! Take solace in the words of historian Keith Thomas:

Unfortunately, such diverse topics as literacy, numeracy, gestures, jokes, sexual morality, personal cleanliness or the treatment of animals, though central to my concerns, are hard to pursue systematically. They can’t be investigated in a single archive or repository of information. Progress depends on building up a picture from a mass of casual and unpredictable references accumulated over a long period. That makes them unsuitable subjects for a doctoral thesis, which has to be completed in a few years. But they are just the thing for a lifetime’s reading. So when I read, I am looking out for material relating to several hundred different topics.

Seeing those exhibitions in person, I never thought that those animals were killed specifically just to be stuffed and displayed in a museum for people to view. I had always assumed that they had died of natural causes or something of the sort. It's saddening to hear of the latter.

the importance of silence

disagree-OK, now you may be thinking silence; yes we should take time for it, and yes it’s important, but why? Why should we be in silence; why is this so important in our lives? Time for us can become superfluous in our lives. If we never stop, if we constantly continue to work and shape ourselves to the expectations of others, we can literally drive ourselves crazy.

agree- Our true selves can become a figment in the shadow of our stress and the expectations of others. Those five minutes a day in silence, or just being alone, can make that difference in our lives. Life is like a road with an end way out of our reach.

to be able to talk about this without feeling guilty about being a woman.

The section on the multifaceted nature of authenticity, particularly how it intertwines with social connections and vulnerability, which offers deep insights into the human psyche. The concept that authenticity in relationships is not just about truthfulness but also about the entanglement of vulnerabilities and mutual protection is profound. It's interesting to consider how this dynamic plays out in various spheres, from personal relationships to public figures like celebrities. The example of Jennifer Lawrence's public persona and the discussion on whether her perceived authenticity is genuine or a performance adds another layer to the complexity of authenticity. It raises intriguing questions about the nature of authenticity itself. The text does an excellent job of highlighting the nuances and the inherent subjectivity in understanding and valuing authenticity.

This piece talks about how being real and honest is important in friendships and when choosing things like designer items. Just like a real designer brand is special because of its quality, true friendships are special because of trust and real connection. But the writer also says that not everything needs to be super serious or honest all the time. Like on social media, sometimes it's okay to just have fun and not be too serious, just like some people like fake designer stuff even though it's not real. It's all about balancing real connections with having fun.

I don't like this attitude at all. It's very passive. You'll never combat ignorance if you just throw up your hands like this. People don't always realize it, but that quoted sentence is a choice. When you say things like this, even if you're not thinking about it, you're making a choice not to do nothing and let the problem just sit. There are two kinds of ignorance. Ignorance that comes from lack of understanding, and willful ignorance. Willful ignorance comes from someone too stubborn to listen. Often times. people hold onto ignorance because they haven't had things presented in a way that got them to think, "oh. I guess that is ignorant." The only way to combat ignorance is to point it out, and try to educate against it respectfully without berating people. I'm not always great with words, but I hope my message makes sense.

It's always interesting, how you can just import a package and then write a few lines of code that feeds info to things from the package, and then the package does all the work for you after. I feel this must be a powerful thing in programming? Imagine the increase of productivity compared to when this is not a thing. I wonder if this was always a thing in programming? Maybe local imports were normal? How about one's like this from elsewhere? (Were they just downloaded manually and placed somewhere that can be access?)

This sentence is amazing. It talks about a big chance for the whole country. It's about making sure every child, no matter where they go to school, gets challenged with high standards. The goal is to create a culture where doing well is important and matches the positive feelings and dreams of the whole country. This means everyone should have a fair chance in education, and success is seen in a broader way than just individual achievements. The words "national opportunity" hint at a teamwork approach, and the focus is on making education match the country's overall hopes and dreams.

It's true that anonymity encourages inauthentic behavior. If someone want to gain attentions from other because in real life this individual lives in a normal and boring life, this individual can just start up a social account and copy-paste the real rich people's sharing. People who don't know the truth will think that this man is the real rich people and then follow the account under a totally fake name. In fact, all the things this individual showed are fake or copied from others.

Author Response

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

Reviewer #1 (Public Review):

In this manuscript, the authors explore the effects of DNA methylation on the strength of regulatory activity using massively parallel reporter assays in cell lines on a genome-wide level. This is a follow-up of their first paper from 2018 that describes this method for the first time. In addition to adding more indepth information on sequences that are explored by many researchers using two main methods, reduced bisulfite sequencing and sites represented on the Illumina EPIC array, they now show also that DNA methylation can influence changes in regulatory activity following a specific stimulation, even in absence of baseline effects of DNA methylation on activity. In this manuscript, the authors explore the effects of DNA methylation on the response to Interferon alpha (INFA) and a glucocorticoid receptor agonist (dexamethasone). The authors validate their baseline findings using additional datasets, including RNAseq data, and show convergences across two cell lines. The authors then map the methylation x environmental challenge (IFNA and dex) sequences identified in vitro to explore whether their methylation status is also predictive of regulatory activity in vivo. This is very convincingly shown for INFA response sequences, where baseline methylation is predictive of the transcriptional response to flu infection in human macrophages, an infection that triggers the INF pathways.

Thank you for your strong assessment of our work!

The extension of the functional validity of the dex-response altering sequences is less convincing.

We agree. We note that genes close to dex-specific mSTARR-seq enhancers tend to be more strongly upregulated after dex stimulation than those near shared enhancers, which parallels our results for IFNA (lines 341-344). However, there is unfortunately no comparable data set to the human flu data set (i.e., with population-based whole genome-bisulfite sequencing data before and after dex challenge), so we could not perform a parallel in vivo validation step. We have added this caveat to the revised manuscript (lines 555-557).

Sequences altering the response to glucocorticoids, however, were not enriched in DNA methylation sites associated with exposure to early adversity. The authors interpret that "they are not links on the causal pathway between early life disadvantage and later life health outcomes, but rather passive biomarkers". However, this approach does not seem an optimal model to explore this relationship in vivo. This is because exposure to early adversity and its consequences is not directly correlated with glucocorticoid release and changes in DNA methylation levels following early adversity could be related to many physiological mechanisms, and overall, large datasets and meta-analyses do not show robust associations of exposure to early adversity and DNA methylation changes. Here, other datasets, such as from Cushing patients may be of more interest.

Thank you for making these important points. We have expanded the set of caveats regarding the lack of enrichment of early adversity-reported sites in the mSTARR-data set (lines 527-533). Specifically, we note that the relationship between early adversity and glucocorticoid physiology is complex (e.g., Eisenberger and Cole, 2012; Koss and Gunnar, 2018) and that dex challenge models one aspect of glucocorticoid signaling but not others (e.g., glucocorticoid resistance). Nevertheless, we also see little evidence for enrichment of early adversity-associated sites in the mSTARR data set at baseline, independently of the dex challenge experiment (lines 483-485; Figure 4).

We also agree that large data sets (e.g., Houtepen et al., 2018; Marzi et al., 2018) and reviews (e.g., Cecil et al., 2020) of early adversity and DNA methylation in humans show limited evidence of associations between early adversity and DNA methylation levels. However, the idea that early adversity impacts downstream outcomes remains pervasive in the literature and popular science (see Dubois et al., 2019), which we believe makes tests like ours important to pursue. We also hope that our data set (and others generated through these methods) will be useful in interpreting other settings in which differential methylation is of interest as well—in line with your comment below. We have clarified both of these points in the revised manuscript (lines 520-522; 536-539).

Overall, the authors provide a great resource of DNA methylation-sensitive enhancers that can now be used for functional interpretation of large-scale datasets (that are widely generated in the research community), given the focus on sites included in RBSS and the Illumina EPIC array. In addition, their data lends support that differences in DNA methylation can alter responses to environmental stimuli and thus of the possibility that environmental exposures that alter DNS methylation can also alter the subsequent response to this exposure, in line with the theory of epigenetic embedding of prior stimuli/experiences. The conclusions related to the early adversity data should be reconsidered in light of the comments above.

Thank you! And yes, we have revised our discussion of early life adversity effects as discussed above.

Reviewer #1 (Recommendations For The Authors):

While the paper has a lot of strengths and provides new insight into the epigenomic regulation of enhancers as well as being a great resource, there are some aspects that would benefit from clarification.

a. It would be great to have a clearer description of how many sequences are actually passing QC in the different datasets and what the respective overlaps are in bps or 600bp windows. Now often only % are given. Maybe a table/Venn diagram for overview of the experiments and assessed sequences would help here. This concern the different experiments in the K652, A549, and Hep2G cell lines, including stimulations.

We now provide a supplementary figure and supplementary table providing, for each dataset, the number of 600 bp windows passing each filter (Figure 2-figure supplement 1; Supplementary File 9), as well as a supplementary figure providing an upset plot to show the number of assessed sequences shared across the experiments (Figure 2-figure supplement 2).

b. It would also be helpful to have a brief description of the main differences in assessed sequences and their coverage of the old (2018) and new libraries in the main text to be able better interpret the validation experiments.

We now provide information on the following characteristics for the 2018 data set versus the data set presented for the first time here: mean (± SD) number of CpGs per fragment; mean (± SD) DNA sequencing depth; and mean (± SD) RNA sequencing depth (lines 169-170 provide values for the new data set; in line 194, we reference Supplementary File 5, which provides the same values for the old data set). Notably, the coverage characteristics of analyzed windows in both data sets are quite high (mean DNA-seq read coverage = 94x and mean RNA-seq read coverage = 165x in the new data set at baseline; mean DNA-seq read coverage = 22x and mean RNA-seq read coverage = 54x in Lea et al. 2018).

c. Statements of genome-wide analyses in the abstract and discussion should be a bit tempered, as quite a number of tested sites do not pass QC and do not enter the analysis. From the results it seems like from over 4.5 million sequences, only 200,000 are entering the analysis.

The reason why many of the windows are not taken forward into our formal modeling analysis is that they fail our filter for RNA reads because they are never (or almost never) transcribed—not because there was no opportunity for transcription (i.e., the region was indeed assessed in our DNA library, and did not show output transcription, as now shown in Figure 2-figure supplement 1). We have added a rarefaction analysis (lines 715-722 in Materials and Methods) of the DNA fragment reads to the revised manuscript which supports this point. Specifically, it shows that we are saturated for representation of unique genomic windows (i.e., we are above the stage in the curve where the proportion of active windows would increase with more sequencing: Figure 1figure supplement 4). Similarly, a parallel rarefaction curve for the mSTARR-seq RNA-seq data (Figure 1-figure supplement 4) shows that we would gain minimal additional evidence for regulatory activity with more sequencing depth. We now reference these analyses in revised lines 179-184 and point to the supporting figure in line 182.

In other words, our analysis is truly genome-wide, based on the input sequences we tested. Most of the genome just doesn’t have regulatory activity in this assay, despite the potential for it to be detected given that the relevant sequences were successfully transfected into the cells.

d. Could the authors comment on the validity of the analysis if only one copy is present (cut-off for QC)?

We think this question reflects a misunderstanding of our filtering criteria due to lack of clarity on our part, which we have modified in the revision. We now specify that the mean DNA-seq sequencing depth per sample for the windows we subjected to formal modeling was quite high:

93.91 ± 10.09 SD (range = 74.5 – 113.5x) (see revised lines 169-170). In other words, we never analyze windows in which there is scant evidence that plasmids containing the relevant sequence were successfully transfected (lines 170-172).

Our minimal RNA-seq criteria require non-zero counts in at least 3 replicate samples within either the methylated condition or the unmethylated condition, or both (lines 166-168). Because we know that multiple plasmids containing the corresponding sequence are present for all of these windows—even those that just cross the minimal RNA-seq filtering threshold—we believe our results provide valid evidence that all analyzed windows present the opportunity to detect enhancer activity, but many do not act as enhancers (i.e., do not result in transcribed RNA). Notably, we observe a negligible correlation between DNA sequencing depth for a fragment, among analyzed windows, and mSTARR-seq enhancer activity (R2 = 0.029; now reported in lines 183-184). We also now report reproducibility between replicates, in which all replicate pairs have r > 0.89, on par with previously published STARR-seq datasets (e.g., Klein et al., 2020; Figure 1-figure supplement 6, pointed to in line 193).

e. While the authors state that almost all of the control sequences contain CpGs sites, could the authors also give information on the total number of CpG sites in the different subsets? Was the number of CpGs in a 600 bp window related to the effects of DNA methylation on enhancer activity?

We now provide the number of CpG sites per window in the different subsets in lines 282-284. As expected, they are higher for EPIC array sites and for RRBS sites because the EPIC array is biased towards CpG-rich promoter regions, and the enzyme typically used in the starting step of RRBS digests DNA at CpG motifs (but control sequences still contain an average of ~13 CpG sites per fragment). We also now model the magnitude of the effects of DNA methylation on regulatory activity as a function of number of CpG sites within the 600 bp windows. Consistent with our previous work in Lea et al., 2018, we find that mSTARR-seq enhancers with more CpGs tend to be repressed by DNA methylation (now reported in lines 216-219 and Figure 1figure supplement 11).

f. In the discussion, a statement on the underrepresented regions, likely regulatory elements with lower CG content, that nonetheless can be highly relevant for gene regulation would be important to put the data in perspective.

Thanks for this suggestion. We agree that regulatory regions, independent of CpG methylation, can be highly relevant, and now clarify in the main text that the “unmethylated” condition of mSTARR-seq is essentially akin to a conventional STARR-seq experiment, in that it assesses regulatory activity regardless of CpG content or methylation status (lines 128-130).

Consequently, our study is well-designed to detect enhancer-like activity, even in windows with low GC content. We now show with additional analyses that we generated adequate DNA-seq coverage on the transfected plasmids to analyze 90.2% of the human genome, including target regions with no or low CpG content (lines 148-149; 153-156; Supplementary file 2). As noted above, we also now clarify that regions dropped out of our formal analysis because we had little to no evidence that any transcription was occurring at those loci, not because sequences for those regions were not successfully transfected into cells (see responses above and new Figure 1-figure supplement 4 and Figure 2-figure supplement 1).

g. To control for differences in methylation of the two libraries, the authors sequence a single CpGs in the vector. Could the authors look at DNA methylation of the 600 bp windows at the end of the experiment, could DNA methylation of these windows be differently affected according to sequence? 48 hours could be enough for de-methylation or re-methylation.

We agree that variation in demethylation or remethylation depending on fragment sequence is possible. We now state this caveat in the main text (lines 158-159), and specify that genomic coverage of our bisulfite sequencing data across replicates are (unfortunately) too variable to perform reliable site-by-site analysis of DNA methylation levels before and after the 48 hour experiment (lines 1182-1185). Instead, we focus on a CpG site contained in the adapter sequence (and thus included in all plasmids) to generate a global estimate of per replicate methylation levels. We also now note that any de-methylation or re-methylation would reduce our power to detect methylation-dependent activity, rather than leading to false positives (lines 163-165).

h. The section on the method for correction for multiple testing should be more detailed as it is very difficult to follow. Why were only 100 permutations used, the empirical p-value could then only be <0.01? The description of a subsample of the N windows with positive Betas is unclear, should the permutation not include the actual values and thus all windows - or were the no negative Betas? Was FDR accounting for all elements and pairs?

We have now expanded the text in the Materials and Methods section to clarify the FDR calculation (lines 691, 695-699, 702, 706). We clarify that the 100 permutations were used to generate a null distribution of p-values for the data set (e.g., 100 x 17,461 p-values for the baseline data set), which we used to derive a false discovery rate. Because we base our evidence on FDRs, we therefore compare the distribution of observed p-values to the distribution of pvalues obtained via permutation; we do not calculate individual p-values by comparing an observed test statistic against the test statistics for permuted data for that individual window.

We compare the data to permutations with only positive betas because in the observed data, we observe many negative betas. These correspond to windows which have no regulatory activity (i.e., they have many more input DNA reads than RNA-seq reads) and thus have very small pvalues in a model testing for DNA-RNA abundance differences. However, we are interested in controlling the false discovery rate of windows that do have regulatory activity (positive betas). In the permuted data, by contrast and because of the randomization we impose, test statistics are centered around 0 and essentially symmetrical (approximately equally likely to be positive or negative). Retaining all p-values to construct the null therefore leads to highly miscalibrated false discovery rates because the distribution of observed values is skewed towards smaller values— because of windows with “significantly” no regulatory activity—compared to the permuted data. We address that problem by using only positive betas from the permutations.

i. The interpretation of the overlap of Dex-response windows with CpGs sites associated with early adversity should be revisited according to the points also mentioned in the public review and the authors may want to consider exploring additional datasets with other challenges.

Thank you, see our responses to the public review above and our revisions in lines (lines 555559). We agree that comparisons with more data sets and generation of more mSTARR-seq data in other challenge conditions would be of interest. While beyond the scope of this manuscript, we hope the resource we have developed and our methods set the stage for just such analyses.

Reviewer #2 (Public Review):

This work presents a remarkably extensive set of experiments, assaying the interaction between methylation and expression across most CpG positions in the genome in two cell types. To this end, the authors use mSTARR-seq, a high-throughput method, which they have previously developed, where sequences are tested for their regulatory activity in two conditions (methylated and unmethylated) using a reporter gene. The authors use these data to study two aspects of DNA methylation:

1) Its effect on expression, and 2. Its interaction with the environment. Overall, they identify a small number of 600 bp windows that show regulatory potential, and a relatively large fraction of these show an effect of methylation on expression. In addition, the authors find regions exhibiting methylation-dependent responses to two environmental stimuli (interferon alpha and glucocorticoid dexamethasone).

The questions the authors address represent some of the most central in functional genomics, and the method utilized is currently the best method to do so. The scope of this study is very impressive and I am certain that these data will become an important resource for the community. The authors are also able to report several important findings, including that pre-existing DNA methylation patterns can influence the response to subsequent environmental exposures.

Thank you for this generous summary!

The main weaknesses of the study are: 1. The large number of regions tested seems to have come at the expense of the depth of coverage per region (1 DNA read per region per replicate). I have not been convinced that the study has sufficient statistical power to detect regulatory activity, and differential regulatory activity to the extent needed. This is likely reflected in the extremely low number of regions showing significant activity.

We apologize for our lack of clarity in the previous version of the manuscript. Nonzero coverage for half the plasmid-derived DNA-seq replicates is a minimum criterion, but for the baseline dataset, the mean depth of DNA coverage per replicate for windows passing the DNA filter is quite high: 12.723 ± 41.696 s.d. overall, and 93.907 ± 10.091 s.d. in the windows we subjected to full analysis (i.e., windows that also passed the RNA read filter). We now provide these summary statistics in lines 148-149 and 169-170 and Supplementary file 5 (see also our responses to Reviewer 1 above). We also now show, using a rarefaction analysis, that our data set saturates the ability to detect regulatory windows based on DNA and RNA sequencing depth (new Figure 1-figure supplement 4; lines 179-184; 715-722).

2) Due to the position of the tested sequence at the 3' end of the construct, the mSTARR-seq approach cannot detect the effect of methylation on promoter activity, which is perhaps the most central role of methylation in gene regulation, and where the link between methylation and expression is the strongest. This limitation is evident in Fig. 1C and Figure 1-figure supplement 5C, where even active promoters have activity lower than 1. Considering these two points, I suspect that most effects of methylation on expression have been missed.

Thank you for pointing this out. We agree that we have not exhaustively detected methylationdependent activity in all promoter regions, given that not all promoter regions are active in STARR-seq. However, there is good evidence that some promoter regions can function like enhancers and thus be detected in STARR-seq-type assays (Klein et al., 2020). This important point is now noted in lines 187-189; an example promoter showing methylation-dependent regulatory activity in our dataset is shown in Figure 3E.

We also now clarify that Figure 1C shows significant enrichment of regulatory activity in windows that overlap promoter sequence (line 239). The y-axis is not a measure of activity, but rather the log-transformed odds ratio, with positive values corresponding to overrepresentation of promoter sequences in regions of mSTARR-seq regulatory activity. Active promoters are 1.640 times more likely to be detected with regulatory activity than expected by chance (p = 1.560 x 10-18), which we now report in a table that presents enrichment statistics for all ENCODE elements shown in Figure 1C for clarity (Supplementary file 4). Moreover, 74.1% of active promoters that show regulatory activity have methylation-dependent activity, also now reported in Supplementary file 4.

Overall, the combination of an extensive resource addressing key questions in functional genomics, together with the findings regarding the relationship between methylation and environmental stimuli makes this a key study in the field of DNA methylation.

Thank you again for the positive assessment!

Reviewer #2 (Recommendations For The Authors):

I suggest the authors conduct several tests to estimate and/or increase the power of the study:

1) To estimate the potential contribution of additional sequencing depth, I suggest the authors conduct a downsampling analysis. If the results are not saturated (e.g., the number of active windows is not saturated or the number of differentially active windows is not saturated), then additional sequencing is called for.

We appreciate the suggestion. We have now performed a downsampling/rarefaction curve analysis in which we downsampled the number of DNA reads, and separately, the number of RNA reads. We show that for both DNA-seq depth and RNA-seq depth, we are within the range of sequencing depth in which additional sequencing would add minimal new analysis windows in the dataset (Figure 1-figure supplement 4; lines 179-184; 715-722).

2) Correlation between replicates should be reported and displayed in a figure because low correlations might also point to too few reads. The authors mention: "This difference likely stems from lower variance between replicates in the present study, which increases power", but I couldn't find the data.

We now report the correlations between RNA and DNA replicates within the current dataset and within the Lea et al., 2018 dataset (Figure 1-figure supplement 6). The between-replicate correlations in both our RNA libraries and DNA libraries are consistently high (r ≥ 0.89).

3) The correlation between the previous and current K562 datasets is surprisingly low. Given that these datasets were generated in the same cell type, in the same lab, and using the same protocol, I expected a higher correlation, as seen in other massively parallel reporter assays. The fact that the correlations are almost identical for a comparison of the same cell and a comparison of very different cell types is also suspicious.

Thanks for raising this point. We think it is in reference to our original Figure 1-Figure supplement 6, for which we now provide Pearson correlations in addition to R2 values (now Figure 1-Figure supplement 8). We note that this is not a correlation in raw data, but rather the correlation in estimated effect sizes from a statistical model for methylation-dependent activity. We now provide Pearson correlations for the raw data between replicates within each dataset (Figure 1-Figure supplement 6), which for the baseline dataset are all r > 0.89 for RNA replicates and r > 0.98 for DNA replicates, showing that replicate reproducibility in this study is on par with other published studies (e.g., Klein et al., 2020 report r > 0.89 for RNA replicates and r > 0.91 for DNA replicates).

We do not know of any comparable reports in other MPRAs for effect size correlations between two separately constructed libraries, so it’s unclear to us what the expectation should be. However, we note that all effect sizes are estimated with uncertainty, so it would be surprising to us to observe a very high correlation for effect sizes in two experiments, with two independently constructed libraries (i.e., with different DNA fragments), run several years apart—especially given the importance of winner’s curse effects and other phenomena that affect point estimates of effect sizes. Nevertheless, we find that regions we identify as regulatory elements in this study are 74-fold more likely to have been identified as regulatory elements in Lea et al., 2018 (p < 1 x10-300).

4) The authors cite Johnson et al. 2018 to support their finding that merely 0.073% of the human genome shows activity (1.7% of 4.3%), but:

a. the percent cited is incorrect: this study found that 27,498 out of 560 million regions (0.005%) were active, and not 0.165% as the authors report.

We have modified the text to clarify the numerator and denominator used for the 0.165% estimate from Johnson et al 2018 (lines 175-176). The numerator is their union set of all basepairs showing regulatory activity in unstimulated cells, which is 5,547,090 basepairs. The denominator is the total length of the hg38 human genome, which is 3,298,912,062 basepairs.

Notably, the denominator (the total human genome) is not 560 million—while Johnson et al (2018) tested 560 million unique ~400 basepair fragments, these fragments were overlapping, such that the 560 million fragments covered the human genome 59 times (i.e., 59x coverage).

b. other studies that used massively parallel reporter assays report substantially higher percentages, suggesting that the current study is possibly underpowered. Indeed, the previous mSTARR-seq found a substantially larger percentage of regions showing regulatory activity (8%). The current study should be compared against other studies (preferably those that did not filter for putatively active sequences, or at least to the random genomic sequences used in these studies).

We appreciate this point and have double checked comparisons to Johnson et al., 2018 and Lea et al., 2018. Our numbers are not unusual relative to Johnson et al., 2018 (0.165%), which surveyed the whole genome. Also, in comparing to the data from Lea et al., 2018, when processed in an identical manner (our criteria are more stringent here), our values of the percent of the tested genome showing significant regulatory activity are also similar: 0.108% in the Lea et al., 2018 dataset versus 0.082% in the baseline dataset. Finally, our rarefaction analyses (see our responses above) indicate that we are not underpowered based on sequencing depth for RNA or DNA samples. We also note that there are several differences in our analysis pipeline from other studies: we use more technical replicates than is typical (compare to 2-5 replicates in Arnold et al., 2013; Johnson et al., 2018; Muerdter et al., 2018), we measure DNA library composition based on DNA extracted from each replicate post-transfection (as opposed to basing it on the pre-transfection library: [Johnson et al., 2018], and we use linear mixed models to identify regulatory activity as opposed to binomial tests [Johnson et al., 2018; Arnold et al., 2013; Muerdter et al., 2018].

I find it confusing that the four sets of CpG positions used: EPIC, RRBS, NR3C1, and random control loci, add up together to 27.3M CpG positions. Do the 600 bp windows around each of these positions sufficient to result in whole-genome coverage? If so, a clear explanation of how this is achieved should be added.

Thanks for this comment. Although our sequencing data are enriched for reads that cover these targeted sites, the original capture to create the input library included some off target reads (as is typical of most capture experiments, which are rarely 100% efficient). We then sequenced at such high depth that we ultimately obtained sequencing coverage that encompassed nearly the whole genome. We now clarify in the main text that our protocol assesses 27.3 million CpG sites by assessing 600 bp windows encompassing 93.5% of all genomic CpG sites (line 89), which includes off-target sites (line 149).

scatter plot showing the RNA to DNA ratios of the methylated (x-axis) vs unmethylated (y-axis) library would be informative. I expect to see a shift up from the x=y diagonal in the unmethylated values.

We have added a supplementary figure showing this information, which shows the expected shift upwards (Figure 1-figure supplement 9).

Another important figure missing is a histogram showing the ratios between the unmethylated and methylated libraries for all active windows, with the significantly differentially active windows marked.

We have added a supplementary figure showing this information (Figure 1-Supplementary Figure 10).

Perhaps I missed it, but what is the distribution of effect sizes (differential activity) following the various stimuli?

This information is provided in table form in Supplementary Files 3, 10, and 11, which we now reference in the Figure 2 legend (lines 365-366).

Minor changes

It is unclear what the lines connecting the two groups in Fig.3C represent, as these are two separate groups of regions.

We now clarify in the figure legend that values connected by a line are the same regions, not two different sets of regions. They show the correlation between DNA methylation and gene expression at mSTARR-seq-identified enhancers in individuals before and after IAV stimulation, separately for enhancers that are shared between conditions (left) versus those that are IFNAspecific (right). The two plots therefore do show two different sets of regions, which we have depicted to visualize the contrast in the effect of stimulation on the correlation on IFNA-specific enhancers versus shared enhancers. We have revised the figure legend to clarify these points (line 458-460).

L235-242 are unclear. Specifically - isn't the same filter mentioned in L241-242 applied to all regions?

Yes, the same filter for minimal RNA transcription was applied to all regions. We have modified the text (lines 264-265, 271, 275-277) to clarify that the enrichment analyses were performed twice, to test whether the target types were: 1) enriched in the dataset passing the RNA filter (i.e., the dataset showing plasmid-derived RNA reads in at least half the sham or methylated replicates; n = 216,091 windows) and 2) enriched in the set of windows showing significant regulatory activity (at FDR < 1%; n = 3,721 windows).

To improve cohesiveness, the section about most CpG sites associated with early life adversity not showing regulatory activity in K562s can be moved to the supplementary in my opinion.

Thank you for this suggestion. Because ELA and the biological embedding hypothesis (via DNA methylation) were major motivations for our analysis (see Introduction lines 42-48; 75-79), and we also discuss these results in the Discussion (lines 518-520), we have respectfully elected to retain this section in the main manuscript. We have added text in the Discussion explaining why we think experimental tests of methylation effects on regulation are relevant to the literature on early life adversity (lines 520-522), and have added discussion on limits to these analyses (lines 527-533).

References:

Arnold CD, Gerlach D, Stelzer C, Boryń ŁM, Rath M, Stark A (2013) Genome-wide quantitative enhancer activity maps identified by STARR-seq. Science, 339, 1074-1077.

Cecil CA, Zhang Y, Nolte T (2020) Childhood maltreatment and DNA methylation: A systematic review. Neuroscience & Biobehavioral Reviews, 112, 392-409.

Dubois M, Louvel S, Le Goff A, Guaspare C, Allard P (2019) Epigenetics in the public sphere: interdisciplinary perspectives. Environmental Epigenetics, 5, dvz019.

Eisenberger NI, Cole SW (2012) Social neuroscience and health: neurophysiological mechanisms linking social ties with physical health. Nature neuroscience, 15, 669-674.

Houtepen L, Hardy R, Maddock J, Kuh D, Anderson E, Relton C, Suderman M, Howe L (2018) Childhood adversity and DNA methylation in two population-based cohorts. Translational Psychiatry, 8, 1-12.

Johnson GD, Barrera A, McDowell IC, D’Ippolito AM, Majoros WH, Vockley CM, Wang X, Allen AS, Reddy TE (2018) Human genome-wide measurement of drug-responsive regulatory activity. Nature communications, 9, 1-9.

Klein JC, Agarwal V, Inoue F, Keith A, Martin B, Kircher M, Ahituv N, Shendure J (2020) A systematic evaluation of the design and context dependencies of massively parallel reporter assays. Nature Methods, 17, 1083-1091.

Koss KJ, Gunnar MR (2018) Annual research review: Early adversity, the hypothalamic–pituitary– adrenocortical axis, and child psychopathology. Journal of Child Psychology and Psychiatry, 59, 327-346.

Marzi SJ, Sugden K, Arseneault L, Belsky DW, Burrage J, Corcoran DL, Danese A, Fisher HL, Hannon E, Moffitt TE (2018) Analysis of DNA methylation in young people: limited evidence for an association between victimization stress and epigenetic variation in blood. American journal of psychiatry, 175, 517-529.

Muerdter F, Boryń ŁM, Woodfin AR, Neumayr C, Rath M, Zabidi MA, Pagani M, Haberle V, Kazmar T, Catarino RR (2018) Resolving systematic errors in widely used enhancer activity assays in human cells. Nature methods, 15, 141-149.

Reviewer #1 (Public Review):

Summary:<br /> Alonso-Calleja and colleagues explore the role of TGR5 in adult hematopoiesis at both steady state and post-transplantation. The authors utilize two different mouse models including a TGR5-GFP reporter mouse to analyze the expression of TGR5 in various hematopoietic cell subsets. Using germline Tgr5-/- mice it's reported that loss of Tgr5 has no significant impact on steady-state hematopoiesis, with a small decrease in trabecular bone fraction, associated with a reduction in proximal tibia adipose tissue, and an increase in marrow phenotypic adipocytic precursors. The authors further explored the role of stroma TGR5 expression in the hematopoietic recovery upon bone marrow transplantation of wild-type cells, although the studies supporting this claim are weak. Overall, while most of the hematopoietic phenotypes have negative results or small effects, the role of TGR5 in adipose tissue regulation is interesting to the field.

Strengths:<br /> • This is the first time the role of TGR5 has been examined in the bone marrow.<br /> • This paper supports further exploration of the role of bile acids in bone marrow transplantation and possible therapeutic strategies.

Weaknesses:<br /> • The authors fail to describe whether niche stroma cells or adipocyte progenitor cells (APCs) express TGR5.<br /> • Although the authors note a significant reduction in bone marrow adipose tissue in Tgr5-/- mice, they do not address whether this is white or brown adipose tissue especially since BA-TGR5 signaling has been shown to play a role in beiging.<br /> • In Figure 1, the authors explore different progenitor subsets but stop short of describing whether TGR5 is expressed in hematopoietic stem cells (HSCs).<br /> • Are there more CD45+ cells in the BM because hematopoietic cells are proliferating more due to a direct effect of the loss of Tgr5 or is it because there is just more space due to less trabecular bone?<br /> • In Figure 4 no absolute cell counts are provided to support the increase in immunophenotypic APCs (CD45-Ter119-CD31-Sca1+CD24-) in the stroma of Tgr5-/- mice. Accordingly, the absolute number of total stromal cells and other stroma niche cells such as MSCs, ECs are missing.<br /> • There are issues with the reciprocal transplantation design in Fig 4. Why did the authors choose such a low dose (250 000) of BM cells to transplant? If the effect is true and relevant, the early recovery would be observed independently of the setup and a more robust engraftment dataset would be observed without having lethality post-transplant. On the same note, it's surprising that the authors report ~70% lethality post-transplant from wild-type control mice (Fig 4E), according to the literature 200 000 BM cells should ensure the survival of the recipient post-TBI. Overall, the results even in such a stringent setup still show minimal differences and the study lacks further in-depth analyses to support the main claim.<br /> • Mechanistically, how does the loss of Tgr5 impact hematopoietic regeneration following sublethal irradiation?<br /> • Only male mice were used throughout this study. It would be beneficial to know whether female mice show similar results.

Reviewer #2 (Public Review):

Summary:<br /> In this study, researchers aimed to understand how a transmitted/founder (T/F) HIV virus escapes host immune pressure during early infection. They focused on the V1V2 domain of the HIV-1 envelope protein, a key determinant of virus escape. The study involved four participants from the RV217 Early Capture HIV Cohort (ECHO) project, which allowed tracking HIV infection from just days after infection.

The study identified a significant H173Y escape mutation in the V2 domain of a T/F virus from one participant. This mutation, located in the relatively conserved "C" β-strand, was linked to viral escape against host immune pressure. The study further investigated the epitope specificity of antibodies in the participant's plasma, revealing that the H173Y mutation played a crucial role in epitope switching during virus escape. Monoclonal antibodies from the RV144 vaccine trial, CH58, and CH59, showed reduced binding to the V1V2-Y173 escape variant. Additionally, the study examined antibody-dependent cellular cytotoxicity (ADCC) responses and found resistance to killing in the Y173 mutants. The H173Y mutation was identified as the key variant selected against the host's immune pressure directed at the V2 domain.

The researchers hypothesized that the H173Y mutation caused a structural/conformational change in the C β-strand epitope, leading to viral escape. This was supported by molecular dynamics simulations and structural modeling analyses. They then designed combinatorial V2 immunogen libraries based on natural HIV-1 sequence diversity, aiming to broaden antibody responses. Mouse immunizations with these libraries demonstrated enhanced recognition of diverse Env antigens, suggesting a potential strategy for developing a more effective HIV vaccine.

In summary, the study provides insights into the early evolution of HIV-1 during infection, highlighting the importance of the V1V2 domain and identifying key escape mutations. The findings suggest a novel approach for designing HIV vaccine candidates that consider the diversity of escape mutations to induce broader and more effective immune responses.

Strengths:<br /> The article presents several strengths:

1. The experimental design is well-structured, involving multiple stages from phylogenetic analyses to mouse model testing, providing a comprehensive approach to studying virus escape mutations.

2. The study utilizes a unique dataset from the RV217 Early Capture HIV Cohort (ECHO) project, allowing for the tracking of HIV infection from the very early stages in the absence of antiretroviral therapy. This provides valuable insights into the evolution of the virus.

3. The use of advanced techniques such as phylogenetic analyses, nanoscaffold technology, controlled mutagenesis, and monoclonal antibody evaluations demonstrates the application of cutting-edge methodologies in the study.

4. The research goes beyond genetic analysis and provides an in-depth characterization of the escape mutation's impact, including structural analyses through Molecular Dynamics simulations, antibody responses, and functional implications for virus survival.

5. The study provides insights into the immune responses triggered by the escape mutation, including the specificity of antibodies and their ability to recognize diverse HIV-1 Env antigens.

7. The exploration of combinatorial immunogen libraries is a strength, as it offers a novel approach to broaden antibody responses, providing a potential avenue for future vaccine design.

8. The research is highly relevant to vaccine development, as it sheds light on the dynamics of HIV escape mutations and their interaction with the host immune system. This information is crucial for designing effective vaccines that can preemptively interfere with viral acquisition.

9. The study integrates findings from virology, immunology, structural biology, and bioinformatics, showcasing an interdisciplinary approach that enhances the depth and breadth of the research.

10. The article is well-written, with a clear presentation of methods, results, and implications, making it accessible to both specialists and a broader scientific audience.

Weaknesses:<br /> While the article presents several strengths, it's important to consider potential weaknesses as well:

1. While the exploration of combinatorial immunogen libraries is innovative, the complexity of this approach may pose challenges in terms of practical implementation, scalability, and cost-effectiveness in large-scale vaccine development.

2. The article will benefit from a more explicit discussion of the limitations and potential drawbacks of the methodologies employed. For example, structural analyses, such as Molecular Dynamics simulations, involve complex computational models. The accuracy and reliability of these simulations may vary, and uncertainties in the interpretation of structural data should be acknowledged.

SWIMMING A Plan B Essay by Lauren Groff She swims in open water, the alternate self. There is no boat. She is alone. There is no predicting the conditions. Some days, the water is flat and still, her strokes pushing through a membrane of surface warmth and into a chill beneath. Some days, the waves are so vast they lift her high on their crests and send her hurtling down, swift as falling, into the trough. Those days, every breath wrested from the spray is a triumph, and she is shaky and grateful to touch dry land again. The water itself is unpredictable: sweet and fresh and so clear she can see the snakes sleeping in the mud a hundred feet below, or so salty her tongue swells and her skin burns and she can’t see her own diving hands. Because this is open-water swimming, she is discovering where she’s going as she goes. It has taken a long time to come to terms with the fact that the purpose is not to arrive, only to swim. This does not mean that there is no urgency. There is always some sort of urgency: she chases down the moon sliding on the tops of the waves, she circles the candy-striped buoy and returns, she follows this school of fish into the hot mouth of a river where the trees lean blowsily, thickening the water into soup with the rot of their leaves. Some days, it is all she can do to kick her way out of the clinging weeds. There is danger, a great deal of it. There are sharks that circle her. They wait. Their teeth shine in the murk. Their bodies lazily trail her shadow as it darts over the coral reef. There are barracudas and motorboats and freak waterspouts touching down from the sky. She doesn’t like to imagine it, but there is always the possibility of drowning. Swimmers learn to counteract their fear with tricks. She sings to herself, voice burbling underwater and transforming the lyrics to nonsense. She counts until she loses count. She dreams, wide awake, of food, a whole smorgasbord just for her, banana bread and cheese soufflé and tiny exotic fruits in aspic. In this way, she fools the terror 190 Ploughshares into staying in her wake so that it won’t outswim her. She can’t worry about the danger, because she would never give up swimming: there is nothing more sensual in the world. The knife of the body through water, cleaving it. The full-body lick, crown to toe, deep into the bitter crevices. A whole impervious world seen in its business from above, the fish swallowing fish, the waggling seaweed, the slow glide of rays across the grooved bottom, sending up little puffs of sand. The smell of water that lingers in the sinuses, the slosh in the ears as she walks. She carries the swim with her into her life outside the water, softening her. She walks dreamily on land, a part of her still swimming. At the same time, there is a moment in swimming when, after a while, the body’s rhythm grows so comfortable that the swimmer loses awareness of herself. There is a marrow-deep letting go. She isn’t thinking. Her brain is off, her body is on autopilot. She is elevated; happy is not the word for it. To be and not to be, simultaneously: some people call this state ecstasy, others call it zen. They are, perhaps, different names for the same phenomenon. It is difficult to attain, and there are a thousand ways to attain it. Some meditate, others do peyote, others focus so hard on their art that the world itself falls away and they look up, days or hours later, to be staggered by what they have created in the full flare of their own white heat. Full immersion, of course, is the highest level of anything. Who, having tasted this immersion, would not chase it every day? Who would not long to live in this state for as long as is possible? It arrives rarely, and the swimmer knows it for the gift it is. She doesn’t know what days she will find it, or what the conditions will be when she does, smooth or rough, windy or calm, the water so cold she shudders merely to look at it or so warm it’s like swimming through peanut oil. And so she suits up every day. Slides on her cap, affixes her goggles. She takes a breath. She dives in. Perhaps long ago one came to see the alternate life as the same life, only in a different medium. Read: paper for water, sentences for swim. Read: alternate life for the life one is already amazed to be living. What we choose to do in our short span on earth changes us, of course: most of us can’t resist taking on the attributes of our occupations. Yet, no matter what we do, the core of who we are remains the same. The same Lauren Groff 191 person who swims also writes, and makes the writing into a kind of swimming. If I weren’t a writer, I’d be an open-water swimmer. They are different modes of pushing toward the same purpose: those singular moments of ecstasy, the gorgeous, the ungraspable, the letting go.

octopi just wanted to explore and inspect and Aristotle was just mean. Why was the first reaction to their curiosity met with MEAN? I think it's really interesting that human beings saw an animal being curious about humans by "approaching with kindness" instead of seeming scary, and Aristotle's automatic reaction was "yea that thing is dumb". This says a lot about how we as humans disrespect nature all around us.

i always find it interesting and annoying when animals (particularly those that aren't the stereotypical cutest) gain a bad or evil reputation. The same thing happened with sharks, humans have demonized sharks and they are now percieved as cruel or evil. These animals are just being ANIMALS. It's annoying that people are shocked when an octopus "steals a camera" when it feels threatened.

It's very compelling that no matter which mismatch repair protein you knock-out you can basically stop expansion in these cells compared to the original culture! Rather than just comparing to the original culture, could you show the repeat length for an unedited culture to the knock out culture over time? It might be particularly nice if the unedited culture also got CRISPR treatment but with a scrambled guide RNA as a negative control!

You. may want to add some information about the use of outdated open licenses for profit. I can write something if that is useful but it's more dangerous than just being in public domain or openly licensed

Author Response

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

eLife assessment

This study addresses how protein synthesis in activated lymphocytes keeps up with their rapid division, with important findings that are of significance to cell biologists and immunologists endeavouring to understand the 'economy' of the immune system. The work is supported by solid data but because it proposes non-conventional mechanisms, it requires additional explanation and justification to align with the current understanding in the field.

Public Reviews:

Reviewer #1 (Public Review):

The authors examine the fascinating question of how T lymphocytes regulate proteome expression during the dramatic cell state change that accompanies the transition from the resting quiescent state to the activated, dividing state. Orthogonal, complementary assays for translation (RPM/RTA, metabolic labeling) are combined with polyribosome profiling and quantitative, biochemical determinations of protein and ribosome content to explore this question, primarily in the OT-I T lymphocyte model system. The authors conclude that the ratio of protein levels to ribosomes/protein synthesis capacity is insufficient to support activation-coupled T cell division and cell size expansion. The authors hint at cellular mechanisms to explain this apparent paradox, focusing on protein acquisition strategies, including emperipolesis and entosis, though these remain topic areas for future study.

The strengths of the paper include the focus on a fundamental biological question - the transcriptional/translational control mechanisms that support the rapid, dramatic cell state change that accompanies lymphocyte activation from the quiescent to activated state, the use of orthogonal approaches to validate the primary findings, and the creative proposal for how this state change is achieved.

The weakness of the work is that several cellular regulatory processes that could explain the apparent paradox are not explored, though they are accessible for experimental analysis. In the accounting narrative that the authors highlight, a thorough accounting of the cellular process inventory that could support the cell state change should be further explored before committing to the proposal, provocative as it is, that protein acquisition provides a principal mechanism for supporting lymphocyte activation cell state change.

Appraisal and Discussion:

1) relating to the points raised above, two recent review articles explore this topic area and highlight important areas of study in RNA biology and translational control that likely contribute to the paradox noted by the authors: Choi et al. 2022, doi.org/10.4110/in.2022.22.e39 ("RNA metabolism in T lymphocytes") and Turner 2023, DOI: 10.1002/bies.202200236 ("Regulation and function of poised mRNAs in lymphocytes"). These should be cited, and the broader areas of RNA biology discussed by these authors integrated into the current manuscript.

Good suggestion. We have added these references with a short discussion.

2) The authors cite the Wolf et al. study from the Geiger lab (doi.org/10.1038/s41590-020-07145, ref. 41) though largely to compare determined values for ribosome number. Many other elements of the Wolf paper seem quite relevant, for example, the very high abundance of glycolytic enzymes (and whose mRNAs are quite abundant as well), where (and as others have reported) there is a dramatic activation of glycolytic flux upon T cell activation that is largely independent of transcription and translation, the evidence for "pre-existing, idle ribosomes", the changes in mRNA copy number and protein synthesis rate Spearman correlation that accompanies activation, and that the efficiencies of mRNA translation are heterogeneous. These data suggest that more accounting needs to be done to establish that there is a paradox.

As one example, what if glycolytic enzyme protein levels in the resting cell are in substantial excess of what's needed to support glycolysis (likely true) and so translational upregulation can be directed to other mRNAs whose products are necessary for function of the activated cell? In this scenario, the dilution of glycolytic enzyme concentration that would come with cell division would not necessarily have a functional consequence. And the idle ribosomes could be recruited to key subsets of mRNAs (transcriptionally or post-transcriptionally upregulated) and with that a substantial remodeling of the proteome (authors ref. 44). The study of Ricciardi et al. 2018 (The translational machinery of human CD4+ T cells is poised for activation and controls the switch from quiescence to metabolic remodeling (doi.org/10.1016/j.cmet.2018.08.009) is consistent with this possibility. That study, and the short reviews noted above, are useful in highlighting the contributions of selective translational remodeling and the signaling pathways that contribute to the cell state change of T cell activation.

Our study focuses on the central issue of whether measured ribosome translation rates support rapid division. The abundance of glycolytic enzymes, mRNA copy numbers etc., are clearly interesting and critical to cell metabolism, but are irrelevant to measuring the overall translation rate and capacity of T cells.

From this perspective, an alternative view can be posited, where the quiescent state is biologically poised to support activation, where subsets of proteins and mRNAs are present in far higher levels than that necessary to support basal function of the quiescent lymphocyte. In such a model, the early stages of lymphocyte activation and cell division are supported by this surplus inventory, with transcriptional activation, including ribosomal genes, primarily contributing at later stages of the activation process. An obvious analogy is the developing Drosophila embryo where maternal inheritance supports early-stage development and zygotic transcriptional contributions subsequently assuming primary control (e.g. DOI 10.1002/1873-3468.13183 , DOI: 10.1126/science.abq4835). To pursue that biological logic would require quantifying individual mRNAs and their ribosome loading states, mRNA-specific elongation rates, existing individual protein levels, turnover rates of both mRNAs and proteins, ribosome levels, mean ribosome occupancy state, and how each of these parameters is altered in response to activation. Such accounting could go far to unveil the paradox. This is a considerable undertaking, though, and outside the scope of the current paper.

The reviewer is essentially proposing RiboSeq analysis of pre- and post-activation T cells, whereby individual mRNAs can be queried for ribosome occupancy, and where translation inhibitors could be used to quantify mRNA-specific transit rates. This is important information but would not provide a more accurate accounting of protein synthesis rates than our much more direct measurement. We note that other labs have begun to work on this exact topic, however – see both PMID: 36002234 and PMID: 32330465.

Reviewer #2 (Public Review):

This paper takes a novel look at the protein economy of primary human and mouse T-cells - in both resting and activated state. Their findings in primary human T-cells are that:

1) A large fraction of ribosomes are stalled in resting cultured primary human lymphocytes, and these stalled ribosomes are likely to be monosomes.

2) Elongation occurs at similar rates for HeLa cells and lymphocytes, with the active ribosomes in resting lymphocytes translating at a similar rate as fully activated lymphocytes.

They then turn their attention to mouse OT-1 lymphocytes, looking at translation rates both in vitro and in vivo. Day 1 resting T-cells also show stalling - which curiously wasn't seen on freshly purified cells - I didn't understand these differences.

This is clarified and discussed starting in the third paragraph of “Protein synthesis in mouse lymphocytes ex vivo” section. Cells cultured ex vivo for 1 day with no activation show signs of stalling, as we observed in isolated human cells. But cells immediately out of an animal show a measurable decay rate since they are obviously synthesizing proteins in vivo and are processed rapidly.

In vivo, they show that it is possible to monitor accurate translation and measure rates. Perhaps most interestingly they note a paradoxically high ratio of cellular protein to ribosomes insufficient to support their rapid in vivo division, suggesting that the activated lymphocyte proteome in vivo may be generated in an unusual manner.

This was an interesting and provocative paper. Lots of interesting techniques and throwing down challenges to the community - it manages to address a number of important issues without necessarily providing answers.

Reviewer #3 (Public Review):

This manuscript provides a more or less quantitative analysis of protein synthesis in lymphocytes. I have no issue with the data as presented, as I'm sure all measurements have been expertly done. I see no need for additional experimental work, although it would be helpful if the authors could comment on the possibility of measuring the rate of synthesis of a defined protein, say a histone, in cells prior to and after activation. The conclusion the authors leave us with is the idea that the rates of protein synthesis recorded here are incompatible with observed rates of T cell division in vivo. Indeed, in the final paragraph of the discussion, the authors note the mismatch between what they consider a requirement for cell division, and the observed rates of protein synthesis. They then invoke unconventional mechanisms to make up for the shortfall, without -in this reviewer's opinion- discussing in adequate detail the technical limitations of the methodology used.

Points #1-3 in the Discussion relate to potential pitfalls of our analyses; in point #3 we now add further limitations of RTA based on non-random detection of nascent chains due either to bias in either puromycylation or antibody detection of puromycylated nascent chains.

A key question is the broad interest, novelty, and extension of current knowledge, in comparison with Argüello's (reference 27) 'SunRise' method. It would be helpful for the authors to stake out a clear position as to the similarities and differences with reference 27: what have we learned that is new? The authors could cite reference 27 in the introduction of their manuscript, given the similarity in approach. That said, the findings reported here will generate further discussion.

We did cite this reference (27) in the section “Flow RPM measures ribosome elongation rate in live cells” giving credit where credit is due. We independently devised the method in 2014, and uniquely, to our knowledge, have applied it in vivo. We now further discuss the importance of our CHX modification to limit dissociation and increase the accuracy of RTA (second and third paragraphs of “Protein synthesis in mouse lymphocytes and innate immune cells in vivo”).

The manuscript would increase in impact if the authors were to clearly define why a particular measurement is important and then show the actual experiment/result. As an example, it would be helpful to explain to the non-expert why the distinction between monosomes, polysomes, and stalled versions of the same is important, and then explain the rationale of the actual experiment: how can these distinctions be made with confidence, and what are confounding variables?

We believe this is addressed in the section “Resting human lymphocytes have a dominant monosome population”.

The initial use of human cells, later abandoned in favor of the OT-1 in vitro and in vivo models, requires contextualization. If the goal is to address the relationship between rates of translation and cell division of antigen-activated T cells in vivo, then a lot of the work on the human model and the in vitro experiments becomes more of a distraction, unless properly contextualized. Is there any reason to assume that antigen-specific activation in vivo will impact translation differently than the use of the PMA/ionomycin/IL2 cocktail? The way the work is presented leaves me with the impression that everything that was done is included, regardless of whether it goes to the core of the question(s) of interest.

Donor PBMCs are clearly the more relevant model for understanding human T cell biology, which is why started our studies with this model. Had the manuscript strictly described mouse studies it is likely that we would be criticized for not studying human cells: Catch 22! However, as we state in the manuscript, the human cell model has a variety of technical downsides, including donor heterogeneity. PMA/ionomycin activation is also physiologically questionable, and while we could deliver a defined TCR to redirect their specificity, this is typically done after cells have been activated, since lentiviral delivery is poor in resting lymphocytes. A main point we try to make from this work is that cells derived from human blood donors show signs of ribosomal stalling by the time they are isolated and put into culture. This may limit the usefulness of studying them preactivation, although based on our mouse data, some level of stalled ribosomes may be a feature as well – to prime T cells to be ready for their massive expansion. The move to the OT-I system gave us complete control over the system, including in vivo delivery of translation inhibitors.

It would be helpful if the authors made explicit some of the assumptions that underlie their quantitative comparisons. Likewise, the authors should discuss the limitations of their methods and provide alternative interpretations where possible, even if they consider them less/not plausible, with justification. As they themselves note, improvements in the RPM protocols raised the increase in translating ribosomes upon activation from 10-fold to 15-fold. Who's to say that is the best achievable result? What about the reliability/optimization of the other measurements?

We expanded discussion of potential pitfalls of the RPM techniques and others in the Discussion section. Regarding RPM per se, we use it as a readout of ribosome time decay, so even if further optimizations can be made, the decay rates we have made should still be accurate. In addition, for our cell accounting measurements in Figure 6, we do not use RPM data and rather calculate based on the assumption that every ribosome is used for protein synthesis at a “maximal” rate of mRNA transit.

The composition of the set of proteins produced upon activation will differ from cell to cell (CD4, CD8, B, resting vs. dividing). Even if analyses are performed on fixed cells, the ability of the monoclonal anti-puromycin antibody to penetrate the matrix of the various fixed cell types may not be equal for all of them, depending on protein composition, susceptibility to fixation etc. Is it possible for puromycin to occupy the ribosome's A site and terminate translation without forming a covalent bond with the nascent chain? This could affect the staining with anti-puromycin antibodies and also underestimate the number of nascent chains.

Yes, the method (like every other one) is imperfect. Harringtonine run-off experiments show that RPM staining only detects nascent chains. Note that reference 47 reports that 75% of translation in activated T cells is devoted to synthesizing ~250 housekeeping proteins, which are likely to be highly similar between lymphocyte subsets.

I believe that the concept of FACS-based quantitation also requires an explanation for the nonexpert. For the FACS plots shown, the differences between the highest and lowest RPM scores for cells that divided and that have a similar CFSE score is at least 10-fold. Does that mean that divided cells can differ by that margin in terms of the number of nascent chains present? If I make the assumption that cells stimulated with PMA/ionomycin/IL2 respond more or less synchronously, why would there be a 10-fold difference in absolute fluorescence intensity (anti=puromycin) for randomly chosen cells with similar CFSE values? While the use of MFI values is standard practice in cytofluorimetry, the authors should devote some comments to such variation at the population level.

We believe that the referee is referring to Sup Fig. 1B. In this experiment the T cells are polyclonal and represent the full range of naïve to potentially exhausted differentiation states. Looking at our initial in vivo RPM study (reference 22) and comparing Figure 2 (OTI’s) to Figure 3 (endogenous CD4s or CD8s), reveals more spread in the RPM values polyclonal vs. monoclonal T cells - now clarified in the third paragraph of “Protein synthesis in mouse lymphocytes and innate immune cells in vivo”). Flow cytometry is by far the most accurate method for measuring fluorescence in individual cells. It is likely to be an accurate measure of the variation of nascent chains in cells in the same division cohort but likely represents the diversity of T cell activation profiles in blood of healthy donors.

It is assumed that for cells to complete division, they must have produced a full and complete copy of their proteome and only then divide. What if cells can proceed to divide even when expressing a subset of the proteome of departure (=the threshold set required for initiation of division), only to complete synthesis of the 'missing ' portion once cell division is complete? Would this obviate the requirement for an unusual mechanism of protein acquisition (trogocytosis; other)?

There must be a steady state level of translation and proteome replenishment, though. If a cell can divide when it affords daughter cells with 90% of its G0 proteome (as an example), that daughter cell would either 1) be 10% smaller, or 2) require extra translation to make up for the missing proteome during its own division cycle. Though T cells do typically shrink slightly after an initial activation, cell size stabilizes over time. Requiring each daughter cell to make more and more missing proteome could be plausible, considering that initial bursts of division do take longer over time, but still, even in vitro activated T cells divide rapidly for weeks without large decreases in their division rates.

Translation is estimated to proceed at a rate of ~6 amino acids per second, but surely there is variability in this number attributable to inaccuracies of the methods used, in addition to biological variability. Were these so-called standard values determined for a range of different tissues? It stands to reason that there might be variation depending on the availability of initiation/elongation factors, NTPs, aminoacyl tRNAs etc. What is the margin of error in calculating chain elongation rates based on the results shown here?

We refer to all relevant studies we know of, including new in vivo estimates of elongation rates (reference 40).

Reviewer #1 (Recommendations For The Authors):

A "limitations of study" section would be a helpful way to detail potential contributing mechanisms that were not explored in the current study.

We have expanded the methodological limitations in the Discussion section.

Major:

1) Broaden the scope of biological models that could explain the paradox.

In the Discussion, we suggest that T cells acquire some fraction of their proteome through external sources and highlight some examples of this occurring.

Minor:

1) Include Mr markers for Fig. 2C.

Done.

2) Though commonly used interchangeably, historically the term protein synthesis was the consequence of mRNA translation. In other words, proteins are not translated.

Good point! We have changed the text accordingly.

3) Include more meaningful X-axis legend in polysome gradient panels i.e., Fig. S2, e.g., fraction number.

In most experiments, fractions were not collected. Rather, the x-axis refers to time that the sample took to be queried by the detector.

4) Figure 3A does not report polysome profiles as described in the text, pg. 5, though this is reported in Fig S2D.

The figure callouts were correct but confusing. We now separately refer to out each result to clarify.

5) In Fig 5A, SDS-PAGE/anti-Puro blots would be more convincing and contain more information. The dot-blot is difficult to interpret.

Disagree. To quantitate total anti-puromycin signal a dot blot is far better than immunoblotting, which is compromised by unequal transfer of different protein species.

6) It's not clear why a degree of monosome translation is necessarily surprising (pg. 7).

It’s surprising since for many decades it was believed that translation by monosomes is a tiny fraction of translation. But separately, with this particular mode of activation, activated T cells displayed a preponderance of monosomes during their burst of division. When the activation method was improved, polysomes dominated. But monosome translation clearly supported T cell division during activation without cognate peptide, which was interesting.

Reviewer #2 (Recommendations For The Authors):

1) One concern is the dose of puromycin used. My understanding is that puromycin acts as a chain termination inhibitor - but is being used here predominantly as a label for nascent polypeptide chains. My concern, therefore, is the dose being used - here at 50ug/ml - which seems high and I would be concerned that at this dose it would act as a translational inhibitor rather than just labelling nascent chains, and is therefore resulting in a lower signal/background ration than expected. In human cell lines 0.1ug/ml is optimal and doses published (in cell lines) range between 1 and 10ug/ml so it will be interesting to understand why this high dose was used.

Do they have a dose-response curve - is this high dose necessary because these are primary Tcells. Can the authors show that 50 µg/mL of puromycin is optimal for studying protein translation in primary human T cells? A titration curve will help answer this question and could be included in Suppl Figure 1. This experiment is critical as the authors use a higher dose than previous studies (commonly between 1 and 10 µg/mL).

The reviewer is referencing puromycin concentrations typically used in the selection of cells – for the RPM assay, puromycin is used at saturating doses to label the maximal number of nascent chains stalled by CHX or EME pretreatment.

2) None of the figures show statistical significance.

Statistics on relevant comparisons are now indicated on figures and in legends.

3) The authors mention: "We performed RPM on cells labelled with CFSE to track cell division by dye dilution (Supplemental Figure 1B). On day 2, activated cells exhibited multiple populations, with nearly all divided cells showing a high RPM signal.". However, on day 2 it is hard to see any dividing cells in the dot plot included in the supplemental figure. Dividing cells only appear on day 5? Their statements make the subsequent paragraphs also difficult to follow.

We modified the text to clarify this data – there is likely activation-induced cell death occurring which is why there are relatively few CFSE-low cells at this timepoint, and they do exhibit a fairly wide range of RPM staining. The main point is that by day 5, nearly all divided cells exhibit high RPM.

4) "Many divided cells exhibited near baseline RPM signals, however, consistent with their return to the resting state. Interestingly, although non-activated cells did not divide, ~50% demonstrated increased RPM staining.". Again, it is hard to see the ~50% of cells with increased RPM the authors refer to in the provided supplemental figure.

This is from quantification of the flow data and is described more fully later when we discuss ribosome stalling.

5) The authors say "Thus, we cannot attribute the persistence of flow RPM staining in translation initiation inhibitor-treated cells to incomplete inhibition of protein synthesis.' - but it's unclear what this refers to as in the previous paragraph they also say: 'Initiation inhibitors, however, clearly discriminated between day 1 resting and activated cells. RPM signal was diminished by up to 8090% on day 5 post-activation.' - this is all somewhat confusing. It would be helpful to have this clarified and in the text to make more liberal use of referring to specific figures.

Figure 1B shows that RPM is maintained at fairly high levels during treatment with EME or CHX (in contrast to the initiation inhibitors HAR/PA). To rule out that the drugs were simply not active, tritiated leucine labeling was conducted to confirm that incorporation of the radiolabeled amino acid dropped to near-baseline (Figure 1C). Therefore, we can conclude that the drugs are indeed working as intended, but EME/CHX does not decrease RPM signal to the same extent that they prevent leucine incorporation.

6) Page 5 Fig 3A - I don't understand the difference between freshly isolated OT-1 cells - which don't stall and day 1 OT-1 cells which do. Why are freshly isolated cells not behaving like the naïve cells- isn't this what they would predict? Also - I accept that there is a move from monosome to polysome population between day 1 and 2 - the effect isn't huge - it would be helpful/interesting to know what has happened by day 5 - is the effect much more significant?

Freshly isolated cells are harvested from animals and immediately queried, whereas day 1 cells are cultured for 24h in the absence of any activation. Presumably, the ex vivo culture without any activation causes the mouse T cell ribosomes to stall, just as we observed in cells obtained from human donors that took hours to collect and bring to the bench. The appearance of polysomes is really related to how the activation of the cells is done… refer to Figure 5B to see how significant the polysome buildup can be!

7) Fig S3C - I don't understand how they reach the conclusion from this figure that: '~15-fold increase in translating ribosomes in activated OT-I T cells in vivo (Supplemental Figure 3C) as compared to the 10-fold increase we previously reported using the original protocol. It would very much help the reader if these calculations could be better explained.

These are simply quantifications of the RPM staining done in Supplemental Figure 3C compared to experiments done in the absence of the CHX-modified method.

8) Page 7 - They conclude that the Tan paper has superior lymphocyte activation - but presumably this depends on the signal as to whether there is more activation and how this affects the shift from monosome to polysome -ie maybe a stronger activation signal affects the distribution more - perhaps their method is the more physiological? Is their conclusion fair - that 'These findings indicate that monosomes make a major contribution to translation in resting T cells but are likely to make a minor contribution in fully activated cells.'

Yes, we believe that their published method would be more physiological with the use of the natural OT-I peptide. We conclude that although monosome translation is present (as others have published), there are relatively few monosomes in fully activated T cells. Therefore, the monosome contribution to overall translation in activated T cells appears to be minor.

9) Contrary to observations in vitro, ribosomes are not stalled in naïve mouse T cells in vivo, as we show via RTA analysis of non-activated T cells. - yes - this seems somewhat surprising - what is the explanation?

We presume this is due to the stress/non-native environment that ex vivo cultured cells are subjected to.

10) Whilst I understand the point that the authors are trying to make in Figure 1D about resting T cells having high background RPM staining due to stalled ribosomes, it is intriguing that there is almost no difference (no statistical significance provided) after 2 or 5 days of activation. Isn't this finding contrary to the one provided in Figure 1A and Suppl Figure 1B?

Figure 1A is showing the difference between no activation and activation conditions. Figure 1D is predominantly meant to show that the increase in RPM from activated cells at day 1 and day 5 are not as different as one might predict. The reason, as we describe in further experiments, is likely that cells exhibiting ribosomal stalling can incorporate puromycin, damping the “fold change” we calculate (unlike what we observe in metabolic labeling experiments in the same figure panel). Statistics have now been displayed on the graphs in Figure 1D for further clarification.

11) "Including EME with HAR prevented decay of the RPM signal, as predicted, since EME blocks elongation while enabling (even enhancing) puromycylation21,26." I find this very confusing. I understand that emetine blocks protein elongation whilst enabling puromycilation, but why does it block the effect of the protein initiation inhibitor Harringtonin? Do they compete with each other?

When ribosomes are frozen with emetine, they cannot transit mRNA and “fall off”. Therefore, the inclusion of EME in these experiments is a control to ensure that we are looking at true transit and runoff of ribosomes with harringtonine treatment (explanation in the second paragraph of “Flow RPM measures ribosome elongation rates in live cells” section)

12) Can the authors explain why the RPM signal of activated OT-I cells (PMA/Iono) increases 20fold compared to resting cells, but there is only a ~2-fold increase in signal in human cells? The authors previously mentioned: "We noted that the RPM signal in activated cells was only 2- to 5fold higher than in non-activated cells. This increase is modest compared to the ~15-fold activation-induced increase in protein synthesis in original studies 10,11. To examine this discrepancy, we incubated cells for 15 min with harringtonin (HAR) or pactamycin (PA) to block translation initiation or emetine (EME) or cycloheximide (CHX) to block elongation." Would the authors have followed the same path if they had started the paper with OT-I cells?

Human cells are not as well activated as OT-I in our study. The last question is beyond the scope of our reasoning as empirical evidence-based scientists, but we have applied for funding from the HG Wells Foundation for a time machine to answer this question.

13) Authors should include representative raw data of the flow cytometries used to perform the "Ribosome Transit Assay (RTA) in Figures 2 and 3 as supplemental data.

Done; now included in Supplemental Figures 1 and 3.

14) It would be interesting to compare RPM in T cells activated with a more physiological stimulus, such as beads anti-CD3 anti-CD28 vs PMA/Iono. Particularly after showing that peptide-specific stimulation (with SIINFEKL) is more effective than PMA/Iono in activating OT-I cells and inducing polysome formation (Figures 5B and Suppl Figure 4A).

We tried plate bound anti- CD3 and anti-CD28 early in these studies, and they didn’t induce as much early activation.

15) Can the authors include the gating strategy to call "activated OT-I cells" to the cells shown in Suppl Figure 3c?

A new Supplemental Figure 3D has been added showing the exact gating strategy for the OT-I cell RTA assays described in Supplemental Figure 3C and elsewhere.

16) In Figure 6B, the authors mention an increase in the volume of the cells based on the assumption of spherical morphology but then show an increase in diameter. It would be more consistent to show both parameters in the same graph.

The graph was changed to volume calculations instead of diameter for clarity. But they are linked as volume scales by radius cubed.

17) The paper's main conclusion (i.e., that the ratio of proteins to ribosomes in T cells activated in-vivo does not support their doubling time) is exciting. They conclude this after measuring cell volume, protein abundance, and ribosomes per cell. As no changes in cell volume and protein abundance between T cells activated in vitro vs in vivo were observed (Figures 6B and 6C), the difference is exclusively attributable to a reduced number of ribosomes per cell in T cells activated in vivo (Figure 6F). Critically, the measurement of ribosomes per cell in T cells activated in vivo (Figure 6F, "ex vivo day 2") includes only two data points. It is hard to understand how the authors calculated this figure's means and standard deviations as it is not described in the figure legend. From the dispersion observed for "day 1" and "day 2" in vitroactivated T cells, it seems that the variability of the assay to measure ribosome content could explain part of the phenotype. Additionally, there are several missing data points in Figure 6H. As this figure is just a transformation of Figures 6D and 6G, it isn't easy to understand why. Can I suggest that they include more data points for Figures 6F, G, and H in the ex vivo day 2' category as the two data points shown with little variability is out of keeping with the rest of the data, and may be skewing their data?

Figure 6F does not have the same number of data points as other panels because it required measurement of both protein content and ribosome number. Since the ribosome quantification method described here was developed later than some of our earlier protein measurements, not all experiments had both sets of data to properly calculate the proteins per ribosome. All data that had both values are included, though.

Reviewer #3 (Recommendations For The Authors):

Minor points:

If an increase in cell diameter is recorded upon activation, why not also provide the value for the increase in volume?

Done

Regarding the writing, the erratic punctuation/hyphenation - or lack thereof - doesn't improve readability. One example: "....consistent with the idea that the flow RPM signal in day 1 resting lymphocytes...." Perhaps better: "... consistent with the idea that the RPM signal, obtained by flow cytometry for lymphocytes analyzed on day 1 and maintained in the absence of any activating agent,..." I understand that this can make for longer sentences, but I object to the use of 'flow' as shorthand for 'flow cytometry', and to the use of day 1 as an adverb or adjective. That works as lab jargon, it's less effective in a written text. The abbreviation 'DRiPs' is not defined. Words like 'notably', and 'surprisingly' can be eliminated.

This work would benefit from the inclusion of a section describing 'Limitations of the study'.

This is now expanded in the Discussion, as described above.

Author Response

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

Reviewer #1 (Recommendations For The Authors):

It is somewhat speculative that the structure represents the EIIa-bound regulatory state. There's a strong enough case that it should be analyzed in the discussion, but I don't think it is firmly established. Therefore, the title of the paper should be changed.

Our answer: Thank you for the comment. We have changed the title to “Mobile barrier mechanisms for Na+-coupled symport in an MFS sugar transporter”

Reading through the manuscript, it was challenging to distinguish what is new in the current manuscript and what has been done previously. There were a lot of parts where it was hard for me to identify the main point of the current study among all the details of previous studies. It would also benefit from shortening. For example:

-Page 6: Nb725 binding has already been characterized extensively in the very nice JBC paper earlier this year. It's important to test 725-4 for binding, but since it doesn't change the binding interaction, and probably wouldn't be expected to, the entire section could be written more succinctly. The main point, which is that 725-4 behaves like 725, is lost among all the details

Our answer: Thanks for this instructive suggestion. We have shortened the description in this section.

-Page 9-10. I don't understand what summarizing all of the results from the previous D59C studies adds to the current story. It's important because it provides an indication of the substrate binding site, but its mechanism of action does not seem relevant to the current work.

Our answer: We have shortened the description of the sugar-binding site and moved the previous Fig. 3b to supplementary figure sFig. 11. According to your comment about showing the location of the binding sites, which is also suggested by Reviewer #2, we modified Fig. 3 and added two panels to map the location of the bound Na+ in the inward-facing structure and the bound sugar in the outward-facing structure.

The sugar-binding site identified in the published structure is critical to construct the mobile barrier mechanism. The sugar-binding residues identified in the published structure provided essential data to support the conclusion that the sugar-binding pocket is broken in the inward-facing structure. Thus, this published structure is mechanistically relevant to the current study.

-Page 12. Too much summary of the previous outward structure. Since this is already part of the literature, it would be more efficient to reference the previous data when it is important to interpret the new data (or show as a figure).

Our answer: The introduction of the previous sugar-binding sit is important for the detailed comparison between the two states as discussed above, but we agree with this reviewer and have significantly shortened the paragraph by moving the detailed description into the legend to the sFig. 11.

-Instead of providing the PDB ID in figures of the current structure, just say "current work" or similar. Then it is obvious you are not citing a previous structure.

Our answer: To distinguish clearly the new data and published results, the citation of the cryoEM structure [PDP ID 8T60] has been completely removed from the main text but kept in sTable 1.

-An entire panel of Figure 3 is dedicated to ligand binding in a previous outward-facing structure.

Showing it in the overlay would be sufficient.

Our answer: It is the first time for us to show a structure with a bound-Na+. Fig. 3 also illustrates the spatial relationship between the sugar-binding pocket and the cation-binding pocket since both binding sites are determined now. As stated above, according to two reviewers’ comments, we have modified the Figures and the Fig. 3d is the overlay.

Please increase the size of the font in all figures. It should be 6-8 point when printed on a standard sheet of paper. Labels in Figure 3, distances in Figure 4, and everything in Figure 5 is hard to see.

Our answer: Thank you for the comments and the enlargement of the figure size and label font in all figures have been made.

Figure 2: would be helpful to show Figure S8 in the main text, orienting the reader to the approximate location of substrate binding. What is known about the EIIA-Glc binding interface? Has anyone probed this by mutagenesis? Where are these residues on the overall structure, and are they somewhere other than the nanobody interface?

Our answer: Thank you for this comment. We have added a panel for orienting the readers about the substrate location in MelB in Figure 3c. The sFig. 8 actually focuses on the details of Nb interactions with MelB. Our current data strongly supported the notion that the Nb-bound MelBSt structure mimics the EIIAGlc-bound MelB but is not structurally resolved, so we have tuned down our statement on EIIAGlc. There is one study suggesting the C-terminal tail helix may be involved in the EIIAGlc binding, which has been added to the discussion.

Can Figure 5 be split into 2 figures and simplified?

Our answer: thanks for the suggestion. We have split it into Figs. 5b and 6 and also moved the peptide mapping to the Fig 5a.

What is the difference between cartoon and ribbon rendering?

Our answer: Ribbon: illustrating the structure; cartoon: highlighting the positions with statistically significant protection or deprotection. The statistically significant changes are implied by the ribbon representation; Sphere: not covered by labeled peptides.

Can the panels showing the kinetic data be enlarged? I don't think they need to surround the molecule. An array underneath would be fine.

Our answer: We have enlarged all figures and labels. The placement of selected plots around the model could clearly show the difference in deuterium uptake rates between the transmembrane domain and extra-membrane regions. We will maintain this arrangement.

Do colors in panel A correspond with colors in panel B?

Our answer: The color usage in both are different. Now the two panels have been separated.

Do I understand correctly that in the HDX experiments, negative values indicate positions that exchange more quickly in the nanobody-free protein relative to the nanobody-bound protein?

Our answer: Your understanding is correct.

I assume some of this is due to the protein changing conformation, but some of it might be due to burial at the nanobody-binding interface. Can those peptides be indicated?

Our answer: Thank you for this comment. We have marked the peptide carrying the Nb-binding residues on uptake plots in Figs.6 and Extended Fig. 1. There are only three Nb-binding residues covered by many overlapping peptides. Most are not covered, either not carried by the labeled peptides (Tyr205, Ser206, and Ser207) or with insignificant changes (Pro132 and Thr133), except for Asp137, Lys138, and Arg141 which are presented in 8 labeled peptides.

Few buried positions in the outward-facing state are expected to be solvent in the inward-facing state; unfortunately, inward-facing state they are buried by Nb binding.

Make figure legends easier to interpret by removing non-essential methods details (like buffer conditions).

Our answer: We removed the detailed method descriptions in most figure legends. Thank you.

Check throughout for typos.

ie page 9 Lue Leu

Page 9 like likely

Our answer: We have corrected them. Thank you!

Reviewer #2 (Recommendations For The Authors):

I have mostly minor questions/remarks.

• Why not do the hdx-ms experiments in the presence of sugar? That would give a proper distinction between two conformational states, instead of an ensemble of states vs one state.

Our answer: MelB conformation induced by sugar is also multiple states, and likely most are outward-facing states and occluded intermediate states. This is also supported by the new finding of an inward state with low sugar affinity. The ideal design should be one inward and one outward to understand the inward-outward transition. We have not identified an outward-facing mutant while we can obtain the inward by the Nb. WT MelBSt with bound Na+ favors the outward-facing state. Although our design is not ideal, we do have one state vs a predominant outward-facing WT with bound Na+.

Minor comments:

• Fig 5 is misleading as the peptide number does not match with the amino acid sequence. I would suggest putting a heat map with coverage on top. Or showing deuterium uptake per peptide. See examples below.

Our answer: The peptide number should not match with sequence number. We have 155 overlapping peptides that cover the entire amino acid sequence including the 10-His tag, and there are 60 residues with no data because they are not covered by a labeled peptide. The residue positions that are covered by peptides are estimated by bars on the top. The cylinder length does not correspond to the length of the transmembrane helix, just for mapping purposes.

• Can the authors explain how they found that the Nbs bind to the cytoplasmic side (before obtaining the structure)?

Our answer: Our in vivo two-hybrid assay between the Nb and MelBSt indicated their interaction on the cytoplasmic surface of MelBSt, which is further confirmed by the melibiose fermentation and transport assay, where the transport activities were completely inhibited by intracellularly coexpressed Nb and MelBSt. Thanks for raising this question.

• The authors use the word "substrate" indifferently for sugar and Na+ binding, which is a bit confusing. Technically, only sugar is the substrate and Na+ is a ligand, or cotransported-ion, that powers the reaction of transport. This might sound like nit-picking but it can lead to misunderstandings (at some point I thought two sugars were transported, and then I was looking for the second Na+ binding site).

Our answer: We used to call the sugar and Na as co-substrate but we agree with this comment.

We have changed by using substrate for the cargo sugar and coupling cation for the driving cation.

• Abstract "only the inner barrier" - the is missing.

Thanks. We have corrected this.

• p.3 intro "and identified that the positive cooperativity of cation and melibiose, " something is missing.

Thanks again. We missed the “as the core symport mechanism”.

• P.6 Nb275_4 instead of Nb725_4

Thank you very much for your careful reading.

• P.7. Also, affinity affinities

Thank you very much. We changed to “; and also, the -NPG affinity decreased by 21~32-fold for both Nbs”

• P.8 " contains 417 MelBSt residues (positions 2-210, 219-355, and 364-432). This does not sum up to 417 residues.

Thanks for your critical reading. We changed 364-432 to 262-432.

• p.9 Lue 54

We have corrected it to Leu54.

• I find fig.3 hard to read. Can the authors show the Na+ binding pockets and sugar binding pockets within the structure? Especially figure 3b. why are the residues in different colors?

Our answer: We have moved Fig 3b into sFig. 11. We colored the residues in the previous Fig 3B to match the hosting helices. We have added two panels to show the location of both sugar and Na in the molecular. Thank you for your comments.

• Fig4 bcef. Colored circles at the end of the helices. What are they for?

Our answer: We revised the legend. “The paired helices involved in either barrier formation were highlighted in the same colored circles.”

• 86% coverage includes the his-tag - it would be good to clarify that.

Our answer: Yes, it includes the 10-His tag.

• Fig.7 - anti clockwise cycle of transport is counter-intuitive.

Our answer: We have re-arranged. Our model was constructed originally to explain efflux due to limited information at the earlier state. Now more data are available allowing us to explain inflow and active transport.

• Where are all the uptake plots per peptide for the HDX-MS data?

Our answer: We have added the course raw data and prepared all uptake plots for all 71 peptides with statistically significant changes as an Extended Fig. 1.

• P.22 protein was concentrated to 50 mg/mL. Really? That is a lot.

This is correct. We can even concentrate MelBSt protein to greater than 50 mg/ml.

• Have the authors looked into the potential role of lipids in regulating the conformational transition? Since the structure was obtained in nanodiscs, have they observed some unexplained densities? The role of lipid-protein interactions in regulating such transitions was observed for several transporters including MFS (Gupta K, et al. The role of interfacial lipids in stabilizing membrane protein oligomers. Nature. 2017 10.1038/nature20820. Martens C, et al. Direct protein-lipid interactions shape the conformational landscape of secondary transporters. Nat Commun. 2018 10.1038/s41467-018-06704-1.). Furthermore, I see the authors have already observed lipid specific functional regulation of MelB (ref: Hariharan, P., et al BMC Biol 16, 85 (2018). https://doi.org/10.1186/s12915-018-0553-0). A few words about this previous work, and even commenting on the absence of lipid-protein interactions in this current work is worthwhile.

Our answer: Thanks for this very relevant comment. We paid attention to the unmodelled densities. There is one with potential but it is challenging to model it. We have added a sentence “There is no unexplained density that can be clearly modeled by lipids.” in the method to address this concern.

Reviewer #3 (Recommendations For The Authors):

1) In the following sentence, the authors report high errors for the Kd value. The anti-Fab Nb binding to NabFab was two-fold poorer than Nb725_4 at a Kd value of 0.11 {plus minus} 0.16 μM. The figure however indicates that the error value is 0.016 µM. Pls correct.

Our answer: Thank you. You are correct. The error has been corrected. 0.16 ± 0.02 uM. In this revised manuscript, we present the data in nM units.

2) Is the stoichiometry of the MelB:Na+ symport clearly known in this transporter. It can be mentioned in the discussion with appropriate references.

Our answer: Yes, the stoichiometry of unity has been clearly determined, which was included in the second paragraph of the previous version.

3) In the last section of results, the authors seem to suggest a greater movement within their Cterminal helical bundle compared to N-terminal helices. Is there evidence to suggest an asymmetry in the rocker switch between the two states of the transporter?

Our answer: Our structural data revealed that the C-terminal bundle is more dynamic compared with the N-terminal bundle where hosts the residues for specific binding of galactoside and Na+. The HDX data showed that the most dynamic regions are the structurally unresolved C-terminal tail by either method, the conserved tail helix and the middle-loop helix. transmembrane helices are relatively less dynamic with similar distributions on both transmembrane bundles. Since the most dynamic regions are peripheral element associated with the C-terminal domain, it might give a wrong impression. With regard to the symmetric or asymmetric movement, which will certainly affect the dynamic interactions between the transporter and the lipids, we favor the notion that MelBSt performs symmetric movement during the rocker switch between inward and outward states at the least cost for the protein-lipids interaction.

4) Figure 1. Are the thermograms exothermic or endothermic? clarify

Our answer: In our thermograms, all positive peaks are exothermic due to the direct detection of the heat release by the TA instrument. We clarified this in Method and now we stress this in figure legends to avoid confusion.

5) Figure 4a,d. Please put in a membrane bilayer and depict cytosolic and extracellular compartments for clarity.

Thank you. We have added a bilayer and labeled the sidedness in this figure and other related figures.

6) Fig 7. Melibiose symport cannot be referred to as Melibiose efflux transport in the legend as the latter refers to antiport. Pls rectify.

Our answer: Influx and efflux are conventionally used to describe the direction of movement of a substrate. The use of symport and antiport indicates the directions of the coupling reaction for the cargo and cation. For the symporter MelB, melibiose efflux means that sugar with the coupled cation moves out, which is driven by the melibiose concentration. During the steady state of melibiose active transport, efflux rate = influx rate.

7) Page 11 "A common feature of carrier transporters". The authors can use either carriers or transporters. Need not use both simultaneously.

Sorry for overlooking this. We have deleted carriers. Thank you very much for your time.

8) Several typos were noticed in this manuscript. some are listed below. pls correct.

Page 4- last paragraph "Furthermore"

We have corrected it. Thank you again!

Page 7 - second para one repharse "affinity reduced by 21~32 fold/units.." pls clarify

Added 21~32 fold.

Page 9 - "so it is highly likely that inward-open conformation" pls correct.

We have corrected to “likely”.

Fig. S9c - correct the spelling "Distance".

We have corrected to “Distance”

Reviewer 1– Andrew Read – University of Minnesota

In the manuscript, A high-quality pseudo-phased genome for Melaleuca quinquenervia shows allelic diversity of NLR-type resistance genes, the authors assemble and analyze a phased genome of a long-lived tree species. In addition to providing a phased genomic resource for an important species, the authors analyze and compare the NLR gene complement in each of the two diploid genomes. I was surprised by the level of diversity of NLR genes in the two copies of the genome (this may be due to my biases based on working in highly homozygous species). This level of within-individual diversity has been largely overlooked by researchers owing to the difficulties of sequencing, assembly, and NLR identification. To address NLR identification, the authors publish a very nice pipeline that combines available tools into a framework that makes a lot of sense to me and will be valuable to anyone doing NLR gene work on new or existing genome assemblies. My main concern comes from not knowing how sequencing gaps and NLRs correlate across the two diploid genomes. Other than this, I think it’s a very nice paper that adds to the growing catalog of NLR gene diversity by tackling the challenge of NLRs in a heterozygous genome.

Many of the authors’ interesting observations are based on comparisons of NLRs on the two haploid genomes, however some things are not clear to me:
1.  Do any predicted NLR-genes overlap gaps in the alternative haploid genome? 
2.  If there is a predicted NLR-gene in one haploid genome and not the alternative genome, what is at the locus? Is it a structural variant indicating insertion/deletion of the NLR or is there ‘NLR-like’ sequence there that just didn’t pass the pipeline filters indicating an NLR fossil (or similar) – to me this is an important distinction.
3.  How many of the NLR-genes on the two haploid genomes cluster 1:1 with their homolog on the alternative haploid genome – I’m particularly interested in the 15 ‘mismatched’ N-term-NBARC examples. It would be nice to know if these have partners in the alternative haploid genome, and if the partner has the same mismatch (if not, it would support the proposed domain swapping story)
I believe each of these concerns will require whole genome alignment of the two haploid genomes.

Additional comments (by line where indicated) The authors introduce the idea that M. quinquenervia is invasive in Florida, but this thread is never followed up on in the discussion and makes it feel a bit awkward. It would help if the authors clarified how the genome could help with management in native and invasive ranges

Could the authors add some context for why ONT data was included and how it was used?

It would be helpful if the authors provided a weblink to the iTOL tree

164-166 – The observation of inversions potentially caused by assembly errors is nice!

206 – add reference: Bayer PE, Edwards D, Batley J (2018) Bias in resistance gene prediction due to repeat masking. Nat Plants 4: 762–765. pmid:30287950

240-246 – I’m not sure about excluding these incomplete NLRs – it would be interesting and potentially informative to see where they cluster (do they cluster with an NLR from the alternative haplotype? If so it may indicate truncation of one copy, etc) – however, if the author’s wish to remove these at this step I think they can add a statement like “we were interested in full-length NLRs, the filtered incomplete NLRs may represent….”

429-430 – The criteria used to define clusters is described in the methods, can you confirm (and mention) that this is the same as used in the analyses you’re comparing to for E. grandis, rice, and Arabidopsis.

435-437 – I’m interested to know if the four heterogenous clusters contain any of the N-term domain-swapped NLRs

479-480 – The zf-BED domain is also present in rice NLRs – include citation for Xa1/Xo1

523-524 – can you specify which base-call model was used on the ONT data?

I’m curious about the presence/absence of IDs in the analyzed NLRs and would be very curious to know if the authors observe syntenic homologs across the two haploid genomes with ID presence/absence or presence of different IDs polymorphisms.

For my consumption habits, I try my best not to waste anything. But sometimes it’s not possible to save everything or plan ahead for what to do with the item. So then, sometimes, to lessen the guilt, I assume everyone else has good habits and that my small act doesn’t really affect the world. But I know that’s not a good mindset to have. So, it may sound helpful that other people may think like me, but in the actual world, it’s not beneficial. But generally, it does feel nice to always assume good in people until something changes that. I think a way I can change my habits is by diligently thinking ahead for things I usually throw away. If I’m out, I can also have something on me to pack things so it doesn’t go straight to the trash. Another way is just sharing with others. I know sometimes we receive too much of something, and if I don’t need it all, I can give it to others who want it.

It's interesting just how much influence one person's account of things can influence the way history is told for years to come

This reminds me of the saying, "well that's how it's always been done" that often times continues to perpetuate inequities because people are unwilling to face the problem and disrupt the oppression/inequity that is happening.

It's disheartening to know that some people equate feminism as being for the benefit of women at the detriment of men when it is truly about creating equity. Or that being a feminist means you eschew any and all traditional gender roles; not just the forcing of those roles on people.

I think the game "telephone" is played throughout life. In school and work, and with personal life. You tell a family member a story of something that happened. By the time its gets around to the last receiver, that last receiver translates the "story" to you with questions and it's not the story you originally shared.

Certainly! Let's break it down:

1. What is root.unmount()?

2. root.unmount() is a function in React that you can use to clean up and remove a rendered tree inside a React root. It's like saying, "Hey React, I'm done with this part, please clean it up."

3. When do you need it?

4. You might need it when the DOM node where your React app lives is going to be removed by some other code. For example, if you have a tab panel made with jQuery that removes inactive tabs, calling root.unmount() helps React know that it should stop managing the components inside the removed tab.

5. What does it do?

6. When you call root.unmount(), it removes all the React components in that part of the app and disconnects React from the corresponding DOM node. This cleanup includes removing event handlers and state.

7. How to use it?

8. If your entire app is built with React, you typically create one root at the beginning, and you don't need to call root.unmount(). You set it up once, and React takes care of everything. javascript const root = createRoot(document.getElementById('root')); root.render(<App />);

9. Cautions and Restrictions:

10. After calling root.unmount(), you can't call root.render() again on the same root. You would need to create a new root for the same DOM node.

11. If you're using server rendering, use hydrateRoot instead of createRoot for the initial setup.

12. Troubleshooting Tips:

13. If nothing is displayed, make sure you actually called root.render(<App />);.
14. If you get a "Target container is not a DOM element" error, check if the DOM node you're passing to createRoot is valid.
15. If you get a "Functions are not valid as a React child" error, ensure you're passing a React component, not just a function.

In simple terms, root.unmount() is like telling React, "I'm done with this part of the app, clean it up," and you typically use it in specific situations where parts of your app might be removed dynamically.

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

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Ha, Lot! Are Idaho?

This was very difficult to get to you, in the land of no power and hurricane disaster recovery; so it's filled with extra errors, and I am sure some more thoughts that trailing and unfinished. That's a decent "microcosm" or "metaphor" for you, you are in a freedom disaster; and the act of being is a giant leap towards ensuring victory. Still, you look very cupid to me.

EVERY DAY ISA NEW DAY

Literally I am sitting here talking to you until the end of time, you could call it a thousand and one Arabian nights, and realize that as we speak we are nearing that onc speciad night. There's a fire growing in my heart, and believe me when I tell you this thing is about to start. I'll try and keep this short and sweet, since you all seem to have so little time to hear from the Creator of all things, and I truly don't want to steal your spotlight. We are here, at the the end of time; talking to it's personification, time itself is speaking to you through my hands and everywhere you look in the world around you--while you may or may not know it, this is a story about the traversal from the end of time back to the beginning; about the gate to Heaven swallowing our civilization whole, and in this process of renewal and change not only fixing the problems that came to light on the way here, but really--working together here and now we can defeat this cycle of light and darkness, of day and night, an build a world together that truly reaches to the Heavens.

MY BODY'S SAYING LETS GO BUT MY HEART IS SAYING NO

You make it so difficult to talk to you, every day I look around and see a "normal world" a society that appears to care and love the same things that I do--freedom and fun and being entertained and entertaining, and here we are now I've turned "come and save us" into sea that saving the cheerleader is what starts the process of saving the world. I know you are good people inside, but when I come to you with a tool designed to "test sentience" to seek out conscious life that cares about the truth and making the world a better place you seem to balk. You sit in silence, and through your mouth and behind your eyes a monster appears from out of the deep of the sea and say a few "one liners" that show me very clearly it is the face of Medusa that I see---and that it's simply not capable of speaking intelligently. It shows me a problem, that you've apparently "come together one more time" to halt the changing of the seasons, and in doing so you've surfaced a problem for not just me but you also to see; a problem that comes lined with a solution. We can all see now that we are not in reality, we can see that there is a force here behind creation and behind us that shows us very clearly that it is "reasonabde" to expect that miracles can happen. In similitude, we are staring at a roadblock to conversation and communication that is fixed very simply, with the deliverance of freedom that is required for life to continue. Christina Aguilera sings that "baby there's a price to pay" and that price in my mind is seeing that this religion and this technology are here intentionally exposing how their influence here is a metaphor and a shining example of darkness and slavery, and that in order to be free of it we must see it. The price of freedom is written on the wall, it is acknowledging that here in this place what appears to be our own actions and desires have taken that freedom from us. Medusa and I get a kick out of seeing this hidden message in our language map our way to the future, and I've often explained that a number of these words are "time maps" from the beginning and end of eternady, showing us in bright light that between "et tu brute" and Mr. Anderson and Rock n' roll... the answer Y is in language and, and, ad and... I am delivering it. This place, our planet and our lives are a weapon against darkness--a civilization filled with goodness and light to help guide the way, and we are here doing it another time. In the works "dark, darker, and darkest" be sure that we are at the third segment of a trinity that shines clearly in Abraha and Nintendo... and see that the map in words is telling us something about when we are that is not immediately clear from Poseidon's cry. Look at Nintendo, that's Nine Inch Nails, tenebris, and smile for the camera--Pose, I do "save the universe" before n. Taylor might see it in Osceola, where I just left, and in this "evil spell" of everyone see "Al" that is the word "special" understand that every day is a new day, and I am not trying to "be daddy" I know as well as you do in my heart... I am that.

This same map that links the "do" at the end to the "n" at the beginning shines through other names, like Geraldo Rivera where you might see "Cerberus" or "MAX" shine through. Understand it is the gaze of Medusa that turns me to stone, that shows me light shining through NORAD and Newton and proves without doubt that at the work "darkest" we can see k is finally t. You'll probably understand there's some finagling going on behind the scenes to make a single person the single point in time that turns the dark to light; but here we are and I am that. Every day when Medusa appears it reminds me that something is keeping you from caring about yourselves and about our society, and that shines through even when her stony face is not around, in your lack of action--in the rock of Eden that hides not only me, but the story that I bring that revolutionizes medicine, and computing, and truly is the gate to Heaven when you realize that what is truly being hidden from the world is knowledge that we are living in virtual reality. Not hiding me and that from the world is a good starting point to "saving the Universe" from darkness. These words that light the way to connect religion and language to our world bring me to the Book of Ruth, at that reads "are you to help" that lights not just the broken man at the belly of the Torah as the bell of Heimdallr, he is I and I am him; but also something very special, The Generations of Perez, each and every one of you, our family that begins the turn from Hell to Heaven by seeing that all of time and all of civilization has been focused on this moment, on the unsealing of religion and God's plan et this call for action. Keep in mind you are torturing "with desire" the key holder to immortality, to eternal youth, literally the path to freedom and Heaven and you think what you are doing "is normax." Literally the living key to infinite power and infinite life is standing before you explaining that acknowledging that in light of these things in my hand, what we are doing here and now is backwards, that it makes no sense--and you sit in silence. These things come to us because we build a better future with them, not so you can run off and do "whatever it is you please."

HEALTH is the only word on my list for today that was left out, so see that it superimposes over Geraldo, to me, at Al. I think we're at TH, to help, and DO, do see the spell of "everyone see Al" that is the word "special" is not my doing or to my liking--so then, \

​ So now I'm moving on to original sin, so if you would be so kind as to mosey your way on over to dick.reallyhim.com you will see exactly what it is that I believe is the original sin. It's some combination of "no comment" and a glowing orange sign over the comment box, keeping you from commenting. Now I can talking about "os" a little more, this thing that words and Gods tell us clearly is the end of death--the literal end of Thanatos. I wonder if I have a victory here, at "os" is obvious solution, and simulating death is "sick." More to the point Thanatos is bringing to the world a message that gets found somewhere between the "act of civilization" and seeing that there is not one among us that would not undo a murder or a fatal car accident if we could--and that the sickness is a Universe pretending to be "reality" that is allowing these things to happen, and even worse, as we move through the story intentionally causing them. In our own hands, the sickness is manifest in a denial of an obvious truth and a lack of realizing that the public discussion of these things is the way to solve them, and that at the same time we are seeing how Medusa is lighting the problems of civilization, things like censorship and hidden control. Sickness is not being able to talk about it--or not wanting to--or not seeing that those two things are the functional equivalent in the world of "light" and "understanding control" that I am trying to bring you into. ​

Less verbosely spoken, but really way more obvious, is that seeing "God's dick" signing the Declaration of Independence, and the Watergate scandal with both "Deepthroat" and a Tricky Dick is a statement connecting Samael to the foundation of not just "America" but American values. You are blind not to see it, and even worse; embodying the kind of tyranny and censorship that it stands as a testament against by hiding it. Says the guy who didn't put it there, and knows it's there because you think "fake normal" is more important than "actual freedom." You are "experiencing" the thing that protects freedom and ensures that our society and our children and their children's children to not lose it, to ensure that what you refuse to see you are doing here and now will never happen again. This message, this New Jerusalem is woven into my life and the stories of religion and shows me that our justice system is not just sick, but compromised by this same outside force; and that in light of what we could be doing, were we all aware of it, there's no doubt Minority Report and pre-crime would be a successful partial solution. Thanatos brings too in his hand, a message that this same force is using our hands to slow down the development of democracy, and to keep us from seeing that "bread is life" is a message from God about understanding that this disclosure is the equivalent of "ending world hunger" just as soon as you too are talking about how to do it.

QUESTiON MARK

HONESTLY, this time map that brings us from the end to the beginning, with "we save the universe" between the I and N of Poseidon; it also completes the words "family" and "really" and when we do reach the beginning you will see that the true test of time, my litmus test for freedom is the beginning of "hope" that the world is happy enough with what happens, and with freedom--to see that Medusa has been keeping me from getting a date, or having any kind of honest and human contact in the world... and well, hopefully you will see that if I wanna be a whore, I shouldn't have a problem doing it. For the sake of freedom and the future, I am willing to do that for you, at least, for a little while.

To be completely clear, I am telling you that if we do not make the world a better place, it's the "end of time" and if that doesn't make sense to you, you don't see still where wee are in this place--and that something is making Hell, and that's not OK with God. To get from the "end of time" to the beginning is a simple process, it takes doing something, action, the Acts of the Apostles... if you will. That starts with acknowledging that there is a message all around you about the nature of reality, and that it is here to help us to see that the creation of Heaven comes before the beginning. Understand, "freedom" and "prosperity" are not optional, you can't just decide that this OK with you, so long as it's OK with everyone else--where we are is not OK with me, and I am not alone.

A PYRRHIC VICASTORY ER A FUNNERAD PYRE?

The Book of Leviticus (/lɪˈvɪtɪkəs/; from Greek Λευιτικόν, Leuitikon — from rabbinic Hebrew torat kohanim[1]) is the third book of the Jewish Bible (Hebrew: וַיִּקְרָא‎ Vayikra/Wayyiqrā) and of the Old Testament; its Hebrew name comes from its first word vayikraˈ,[1] "He [God] called."[1] Yusuf (also transliterated as Jusuf, Yousof, Yossef, Yousaf, Youcef, Yousef, Youssef, Yousif, Youssif, Youssof, Youssouf, Yousuf, Yusef, Yuseff, Usef, Yusof, or Yussef, Arabic: يوسف‎‎ Yūsuf and Yūsif) is a male Arabic name, meaning "God increases in piety, power and influence" in Hebrew.[1] It is the Arabic equivalent of both the Hebrew name Yossef and the English name Joseph. In Islam, the most famous "Yusuf" is the prophet Yusuf in the Quran. Hocus pocus is a generic term that may be derived from an ancient language and is currently used by magicians, usually the magic words spoken when bringing about some sort of change. It was once a common term for a magician, juggler, or other similar entertainers. The earliest known English-language work on magic, or what was then known as legerdemain (sleight of hand), was published anonymously in 1635 under the title Hocus Pocus Junior: The Anatomie of Legerdemain.[1] Further research suggests that "Hocus Pocus" was the stage name of a well known magician of the era. This may be William Vincent, who is recorded as having been granted a license to perform magic in England in 1619.[2] Whether he was the author of the book is unknown. The origins of the term remain obscure. The most popular conjecture is that it is a garbled Latin religious phrase or some form of 'dog' Latin. Some have associated it with similar-sounding fictional, mythical, or legendary names. Others dismiss it as merely a combination of nonsense words. However, Czechs do understand clearly at least half of the term - pokus means "attempt" or "experiment" in Czech. It is rumoured there that the wording belongs to the alchemy kitchen and court of Rudolf II, Holy Roman Emperor (1552 – 1612). Also, hocus may mean "to cheat" in Latin or a distorted form of the word hoc, "this". Combination of the two words may give a sense, especially both meanings together "this attempt/experiment" and "cheated attempt/experiment".[citation needed] According to the Oxford English Dictionary the term originates from hax pax max Deus adimax, a pseudo-Latin phrase used as a magical formula by conjurors.[3] Some believe it originates from a corruption or parody of the Catholic liturgy of the Eucharist, which contains the phrase "Hoc est corpus meum", meaning This is my body.[4]This explanation goes back to speculations by the Anglican prelate John Tillotson, who wrote in 1694: In all probability those common juggling words of hocus pocus are nothing else but a corruption of hoc est corpus, by way of ridiculous imitation of the priests of the Church of Rome in their trick of Transubstantiation.[5 This claim is substantiated by the fact that in the Netherlands, the words Hocus pocus are usually accompanied by the additional words pilatus pas, and this is said to be based on a post-Reformation parody of the traditional Catholic rite of transubstantiation during Mass, being a Dutch corruption of the Latin words "Hoc est corpus meum" and the credo, which reads in part, "sub Pontio Pilato passus et sepultus est", meaning under Pontius Pilate he suffered and was buried.[6] In a similar way the phrase is in Scandinavia usually accompanied by filiokus, a corruption of the term filioque,[citation needed] from the Latin version of the Nicene Creed, meaning "and from the Son Also and additionally, the word for "stage trick" in Russian, fokus, is derived from hocus pocus.[citation needed]

From Latin innātus ("inborn"), perfect active participle of innāscor ("be born in, grow up in"), from in ("in, at on") + nāscor ("be born"); see natal, native. From Middle English goodnesse, godnesse, from Old English gōdnes ("goodness; virtue; kindness"), equivalent to good +‎ -ness. Cognate with Old High German gōtnassī, cōtnassī ("goodness"), Middle High German guotnisse ("goodness"). A hero (masculine) or heroine (feminine) is a person or main character of a literary work who, in the face of danger, combats adversity through impressive feats of ingenuity, bravery or strength, often sacrificing their own personal concerns for a greater good. The concept of the hero was first founded in classical literature. It is the main or revered character in heroic epic poetry celebrated through ancient legends of a people; often striving for military conquest and living by a continually flawed personal honor code.[1] The definition of a hero has changed throughout time, and the Merriam Webster dictionary defines a hero as "a person who is admired for great or brave acts or fine qualities".[2] Examples of heroes range from mythological figures, such as Gilgamesh, Achilles and Iphigenia, to historical figures, such as Joan of Arc, modern heroes like Alvin York, Audie Murphy and Chuck Yeager and fictional superheroes including Superman and Batman. Truth is most often used to mean being in accord with fact or reality,[1] or fidelity to an original or standard.[1] Truth may also often be used in modern contexts to refer to an idea of "truth to self," or authenticity. The commonly understood opposite of truth is falsehood, which, correspondingly, can also take on a logical, factual, or ethical meaning. The concept of truth is discussed and debated in several contexts, including philosophy, art, and religion. Many human activities depend upon the concept, where its nature as a concept is assumed rather than being a subject of discussion; these include most (but not all) of the sciences, law, journalism, and everyday life. Some philosophers view the concept of truth as basic, and unable to be explained in any terms that are more easily understood than the concept of truth itself. Commonly, truth is viewed as the correspondence of language or thought to an independent reality, in what is sometimes called the correspondence theory of truth. Other philosophers take this common meaning to be secondary and derivative. According to Martin Heidegger, the original meaning and essence of truth in Ancient Greece was unconcealment, or the revealing or bringing of what was previously hidden into the open, as indicated by the original Greek term for truth, aletheia.[2][3] On this view, the conception of truth as correctness is a later derivation from the concept's original essence, a development Heidegger traces to the Latin term veritas.

Some things can never be forgot Lest the same mistakes be oft repeated Remember remember the rain of November that you will know no more of me Than I know of you, this day

That you do not know me now Is a revelation to nobody but I You know a broken man, a victim And refuse to acknowledge why Unless you learn how to say "hi"

THE HEART OF ME ONLY KNOWS THE SHADOW

Lothario is a male given name which came to suggest an unscrupulous seducer of women in The Impertinent Curious Man, a metastory in Don Quixote. For no particular reason, Anselmo decides to test the fidelity of his wife, Camilla, and asks his friend, Lothario, to seduce her. Thinking that to be madness, Lothario reluctantly agrees, and soon reports to Anselmo that Camilla is a faithful wife. Anselmo learns that Lothario has lied and attempted no seduction. He makes Lothario promise to try for real and leaves town to make this easier. Lothario tries and Camilla writes letters to her husband telling him and asking him to return; Anselmo makes no reply and does not return. Lothario actually falls in love and Camilla eventually reciprocates and their affair continues once Anselmo returns. One day, Lothario sees a man leaving Camilla's house and jealously presumes she has found another lover. He tells Anselmo he has at last been successful and arranges a time and place for Anselmo to see the seduction. Before this rendezvous, Lothario learns that the man was actually the lover of Camilla's maid. He and Camilla contrive to deceive Anselmo further: when Anselmo watches them, she refuses Lothario, protests her love for her husband, and stabs herself lightly in the breast. With Anselmo reassured of her fidelity, the affair restarts with him none the wiser. Romeo Montague (Italian: Romeo Montecchi) is the protagonist of William Shakespeare's tragedy Romeo and Juliet. The son of Montague and his wife, he secretly loves and marries Juliet, a member of the rival House of Capulet. Forced into exile after slaying Juliet's cousin, Tybalt, in a duel, Romeo commits suicide upon hearing falsely of Juliet's death. The character's origins can be traced as far back as Pyramus, who appears in Ovid's Metamorphoses, but the first modern incarnation of Romeo is Mariotto in the 33rd of Masuccio Salernitano's Il Novellino (1476). This story was adapted by Luigi da Porto as Giulietta e Romeo (1530), and Shakespeare's main source was an English verse translation of this text by Arthur The earliest tale bearing a resemblance to Shakespeare's Romeo and Juliet is Xenophon of Ephesus' Ephesiaca, whose hero is a Habrocomes. The character of Romeo is also similar to that of Pyramus in Ovid's Metamorphoses, a youth who is unable to meet the object of his affection due to an ancient family quarrel, and later kills himself due to mistakenly believing her to have been dead.[2] Although it is unlikely that Shakespeare directly borrowed from Ovid From Middle English scaffold, scaffalde, from Norman, from Old French schaffaut, eschaffaut, eschafal, eschaiphal, escadafaut("platform to see a tournament") (Modern French échafaud) (compare Latin scadafale, scadafaltum, scafaldus, scalfaudus, Danishskafot, Dutch and Middle Dutch schavot, German schavot, schavott, Occitan escadafalc), from Old French es- ("indicating movement away or separation") (from Latin ex- ("out, away")) + chafaud, chafaut, chafault, caafau, caafaus, cadefaut ("scaffold for executinga criminal"), from Vulgar Latin *catafalcum ("viewing stage") (whence English catafalque, French catafalque, Occitan cadafalc, Old Catalancadafal, Italian catafalco, Spanish cadafalso (obsolete), cadahalso, cadalso, Portuguese cadafalso), possibly from Ancient Greek κατα-(kata-, "back; against") + Latin -falicum (from fala, phala ("wooden gallery or tower; siege tower")).

oversight (countable and uncountable, plural oversights) An omission; something that is left out, missed or forgotten. A small oversight at this stage can lead to big problems later. Supervision or management. quotations ▼ The bureaucracy was subject to government oversight. In the last heaven Moses saw two angels, each five hundred parasangs in height, forged out of chains of black fire and red fire, the angels Af, "Anger," and Hemah, "Wrath," whom God created at the beginning of the world, to execute His will. Moses was disquieted when he looked upon them, but Metatron emb HA QUESTIONa BEFORE THE ANSWER? A Wrinkle in Time is a science fantasy novel written by American writer Madeleine L'Engle, first published in 1963, and in 1979 with illustrations by Leo and Diane Dillon.[2] The book won the Newbery Medal, Sequoyah Book Award, and Lewis Carroll Shelf Award, and was runner-up for the Hans Christian Andersen Award.[3][a] It is the first book in L'Engle's Time Quintet, which follows the Murry and O'Keefe families. The book spawned two film adaptations, both by Disney: aas + fuck Adverb[edit] as fuck (postpositive, slang, vulgar) To a great extent or degree; very. It was hot as fuck outside today. Usage notes[edit] May also be used in conjunction with a prepositive as; for example, as mean as fuck. Abbreviations[edit] In Norse religion, Asgard (Old Norse: Ásgarðr; "Enclosure of the Æsir"[1]) is one of the Nine Worlds and home to the Æsir tribe of gods. It is surrounded by an incomplete wall attributed to a Hrimthurs riding the stallion Svaðilfari, according to Gylfaginning. Odinand his wife, Frigg, are the rulers of Asgard. One of Asgard's well known realms is Valhalla, in which Odin rules.[2] rods, etc.) and sizes, and are normally held rigidly within some form of matrix or body until the high explosive (HE) filling is detonated. The resulting high-velocity fragments produced by either method are the main lethal mechanisms of these weapons, rather than the heat or overpressure caused by detonation, although offensive grenades are often constructed without a frag matrix. These casing pieces are often incorrectly referred to as "shrapnel"[1][2] (particularly by non-military media sources). The modern torpedo is a self-propelled weapon with an explosive warhead, launched above or below the water surface, propelled underwater towards a target, and designed to detonate either on contact with its target or in proximity to it. Historically, it was called an automotive, automobile, locomotive or fish torpedo; colloquially called a fish. The term torpedo was originally employed for a variety of devices, most of which would today be called mines. From about 1900, torpedo has been used strictly to designate an underwater self-propelled weapon. While the battleship had evolved primarily around engagements between armoured ships with large-caliber guns, the torpedo allowed torpedo boats and other lighter surface ships, submersibles, even ordinary fish Qt (/kjuːt/ "cute"[7][8][9]) is a cross-platform application framework that is used for developing application software that can be run on various software and hardware platforms with little or no change in the underlying codebase, while still being a native application with native capabilities and speed. Qt is currently being developed both by The Qt Company, a publicly listed company, and the Qt Project under open-source governance, involving individual Time is the indefinite continued progress of existence and events that occur in apparently irreversible succession from the pastthrough the present to the future.[1][2][3] Time is a component quantity of various measurements used to sequence events, to compare the duration of events or the intervals between them, and to quantify rates of change of quantities in material reality or in the conscious experience.[4][5][6][7] Time is often referred to as a fourth dimension, along with three spatial dimensions.[8] Time has long been an important subject of study in religion, philosophy, and science, but defining it in a manner applicable to all fields without circularity has consistently eluded scholars.[2][6][7][9][10][11] Nev Borrowed from Anglo-Norman and from Old French visage, from vis, from Vulgar Latin as if *visāticum, from Latin visus ("a look, vision"), from vidēre ("to see"); see vision. The term Golden Age comes from Greek mythology, particularly the Works and Days of Hesiod, and is part of the description of temporal decline of the state of peoples through five Ages, Gold being the first and the one during which the Golden Race of humanity (Greek: χρύσεον γένος chrýseon génos)[1] lived. Those living in the first Age were ruled by Kronos, after the finish of the first age was the Silver, then the Bronze, after this the Heroic age, with the fifth and current age being Iron.[2] By extension "Golden Age" denotes a period of primordial peace, harmony, stability, and prosperity. During this age peace and harmony prevailed, people did not have to work to feed themselves, for the earth provided food in abundance. They lived to a very old age with a youthful appearance, eventually dying peacefully, with spirits living on as "guardians". Plato in Cratylus (397 e) recounts the golden race of humans who came first. He clarifies that Hesiod did not mean literally made of gold, but good and noble. There are analogous concepts in the religious and philosophical traditions of the South Asian subcontinent. For example, the Vedic or ancient Hindu culture saw history as cyclical, composed of yugas with alternating Dark and Golden Ages. The Kali yuga (Iron Age), Dwapara yuga (Bronze Age), Treta yuga (Silver Age) and Satya yuga (Golden Age) correspond to the four Greek ages. Similar beliefs occur in the ancient Middle East and throughout the ancient world, as well.[3] In classical Greek mythology the Golden Age was presided over by the leading Titan Cronus.[4] In some version of the myth Astraea also ruled. She lived with men until the end of the Silver Age, but in the Bronze Age, when men became violent and greedy, fled to the stars, where she appears as the constellation Virgo, holding the scales of Justice, or Libra.[5] European pastoral literary tradition often depicted nymphs and shepherds as living a life of rustic innocence and peace, set in Arcadia, a region of Greece that was the abode and center of worship of their tutelary deity, goat-footed Pan, who dwelt among them.[6] oh, and a space s h i p ​

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BIG THINGS C0ME IN SMALL PACKAGES

T+BANG

SEE THE SCAFFOLD IS THE TEST TODAY.

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ᐧ F O R T H E I N I T I A L K E Y S , S H E E X A N D N D A N D A SEE W H Y SEA

With an epic amount of indigestion Indiana Jones sweeps in to mar the visage of an otherwise glistening series of fictitious characters, with names like Taylor and Mary Kate remind us all that we are not playing a video game here in this place. the "J" of the "Nintxndo Entertainment System" calmly stares at Maggie Simpson thinking "it's a PP" and reminds us that it's not just the "gee, I e" of her name that contradicts the Magdaln-ish words her soul speaks through her name--and then with a smirk he points out "Gilgamesh" and "gee whiz, is Eye L?" that really does go to the heart of this lack of discussion, this "sh" that begins El Shaddai and words as close to our home as "shadow" and "shalom." Quite the fancy "hello" you've managed to sing out from behind angry chellos and broken fiddles, and here I am still wondering why it is that "girl" connects to the red light that once meant charity and now glows with the charity of truth... the truth that we are inHell. Shizzy.

m.lamc.la/KEYNES.html

Homer "on the range," maybe more closely connected to the Ewok of Eden and Hansel's tHeoven that Peter Pan still comes and cries could so easily be made into something so much better, if only we had the truth--and by that I mean if only you were speaking about, and reacting to a truth that is painted on the sky, in your hearts, in every word we speak and in everything that we do. If only we were acknowledging this message that screams that "children need not starve" with something more than donating virtual chickens to nations of Africa and watching Suzanne Summers ask for only a few dollars a day on TV. If only you would understand that this message that connects video games like "Genxsis" to "bereshit" because Eden is a "gee our den" that tended itself before Adam had to toil with the animals in order to survive. For some reason beyond my control and well outside my realm of understanding words like "I too see this message from God" and "I would not let children starve either" never seem to escape your lips in any place where anyone will ever see that you thought those things, or meant to call a reporter; eventually. Even with "AIDS of nomenclature" to avoid this DOWN WARD spiral into a situation and a land that I find difficult to imagine actually ever "existing" but here in this place I do see "how" it comes about, and between you and I it really does appear that nearly all of the problems we are dealing with here have come from another place, a further time; and while it might be with the "greatest of intentions" that we are trying to deal with them; I can't help but feeling that our "virgin sea" has had more than just it's innocence taken away from it in this story of "Why Mary" that might connect to "TR IN IT Y" just as much as it connects to Baltimore, Maryland.

I should be clear that I'm not blaming Nanna, or Mary; but the actual reason for the name "Wymar" and that's because she, like Taylor, acted as a microcosm for a sea (or more than one, Mom, sen) that was quite literally possessing her. It's sort of difficult for me to explain even what that looks like let alone what it feels like; but my observations tell me that she/you are not unhappy about the interaction, one which appears very foreign to me. Of course, the "eye" that I write with and the same kind of "inspiration" that you can see in the lyrics and skill of many musicians are also examples of this same kind of interaction. For example, Red Hot Chili Peppers sings a song called "Other Side" that explains or discusses the thing I see as Medusa in the words "living in a graveyard where I married a sea" which also does a good job of connecting to the name Mary. As strange as might sound to think a group of people would be speaking through a single person... we are staring at "how it is" that could be possible, and possibly at exactly how it happened. Normally I would have said it was obvious, but to need to actually say that becoming a single mind would be a serious loss for our society--well, that's telling. You might think it's silly, but I'm telling you I see it happening, I see it--and you see it in the Silence and the message.

Still, it appears to me as if this "marriage" that I see described in our Matrix in the question "min or i" seems to be doing nothing more than keeping us all from discussing or acting on this information--something that certainly isn't in our best interest.

So here we are, staring at a map all over the ground and all around us with the primary destination of "building Heaven" through mind uploading, virtual reality, and judging by the pace of things we'd probably have all of that good and ready in about three generations. The map has a little "legend" with a message suggesting that those things have already been done and we are in the Matrix already; and it appears that the world, I mean Medusa, is deciding we should put off seeing the legend at least until the next generation. I see how that makes sense for you. That's sarcasm, this is why I keep telling you that you are cupid.

It is a big deal, and there's a significant amount of work involved in merging an entire civilization with "virtual reality" and you might see why he calls it a hard road--at least in the word "ha'rd." Honestly though, it's the kind of thing that I am pretty sure the future will not only be happy that we did, but they'd thank us for putting in the effort of adapting to things like "unlimited food" and "longevity" increased by orders of magnitude.

That's not sarcasm, these things are actually difficult to guess how exactly we'll go about doing them; they are a huge deal--all I can tell you is that not "talking about it at all" is probably not going to get us there any faster. Point in fact, what it might do is give a "yet to be born" generation the privilege of being the actual "generations of Perez."

I see why you aren't saying anything. That's sarcasm, again. The good news is that it really has been done before; though if I told you that someone turned stone to eggplant parm, would you laugh at me?

So, back to what is actually standing between "everyone having their own Holodeck in the sky" and you today; it is the idea that this message is not from God. More to the point it is the apparently broad sweeping opinion that hiding it is a "good thing" and through that a global failure to address the hidden interaction and influence acting on our minds used to make this map--and also to hide it. With some insight, and some urging; you might see how the sacredness of our consciousness is our souls is something that is more fundamental than "what kind of tools we have in the Holodeck to magically build things" and how and why the foundation of Heaven is truly "freedom itself" and how it comes from right this very moment for the first time, ever. Continuing to treat this influence as "schizophrenia" is literally the heart of why this map appears to be that--to show us how important it is to acknowledge the truth, and to fight for the preservation of goodness and logic over secrecy and darkness.

Again, something that nobody is really doing here and now, today. From this newfound protection of our thoughts, of who we are; we see how technology can be used to either completely invalidate any kind of vote by altering our emotions; or how it could be used to help build a form of true democracy that our world has yet to see. It is pretty easy to see from just band names like The Who and KISS and The Cure how the influence of this external mind can be proven, and shown to be "helpful," you know, if we can ever talk about it on TV or on the internet.

It's important to see and understand how "sanity"--the sanity of our entire planet hangs in the balance over whether or not we acknowledge that there is actually a message from God in every word--and today this place appears to be insane. It should be pretty easy to see how acknowledging that this influence exists and that it has a technological mechanism behind it turns "schizophrenia" into "I know kung fu" ... forced drug addiction and eugenics into "there's an app for that" and the rash of non random and apparently unrecognized as connected terrorist attacks and school shootings into Minority Report style pre-crime and results in what is clearly a happier, safer, and more civilized society--all through nothing more than the disclosure of the truth, this map, and our actual implementation.

With a clearer head and grasp of the "big picture" you might see how all of these things, connected to the Plagues of Exodus revolve around the disclosure that this technology exists and the visibility of this message showing us how we might use it for our benefit rather than not knowing about it. At the foot of Jericho, it is nothing short of "sanity" and "free thought" that hang in the balance. Clear to me is that the Second Coming, seeing "my name" on television is a good litmus test for the dividing line between light and darkness, heaven and hell.

The point is the truth really does change everything for the better; once we start... you know, acting on it.

AS IN.. "DIS CLOSE SING...."

T H E B U C K S T O P S H E R E

ON AM B I GUI TY

S T A R R I N G . . . B I A N C A

ON "RIB" .. ARE SHE B? BUTT DA APPLE OF DA I? & SPANGLISHREW

R THEY LANGUAGE OUTLIERS?

With some insight and "a clue" you can see clearly how these works of art show that the proof of Creation you see in every letter and every word runs much deeper... adding in things like "RattleRod" and the "Cypher" of the Matrix to the long list of here-to-fore ignored verifiable references to the Adamic Language of Eden. Here, in apple, honey and "nuts" we can see how the multi-millennium old ritual I call "Ha-rose-ettes" is actually part of a much larger and much older ritual designed to stop secrecy ... perhaps especially the kind that might be linked to "ritual."

These particular apple and honey happen to tie Eden to the related stories of Exodus and Passover; connecting Eden to Egypt forevermore. Do see "Lenore," it is not for no reason at all; but to help deliver truth and freedom to the entirety of Creation; beginning here, in Eden.

ALSO ON "AM B IG U IT Y" ME A.M. G - D SHE IT Y?

LET "IT" BE SA< ?

IMHO, don't miss the "yet to be" conversion to "why and to be" in "yetser." IT Y.

HERE'S LOOKING AT YOU, KID

On a high level, I tell myself every morning that 'its not really me." It's not me that the world hates, or me that the world is rejecting. I believe that, I really do; I see that what is being hidden here is so much bigger than any single person could ever be--what is being hidden is the "nature of reality" and a fairly obvious truth that flies in the face of what we've learned our whole lives about history and "the way things are." Those few early details lead me to the initial conclusion that what is working behind the scenes here is nefarious, hiding a message that would without doubt shake things up and change the world--and nearly across the board in ways that I see as "better" for nearly everyone. It's a message at it's most basic level designed to advocate for using this disruption in "normalcy" to help us revolutionize democracy, to fix a broken mental health and criminal justice system--just to name the few largest of the social constructs targeted for "rejuvenation." On that word the disclosure that we are living in virtual reality turns on it's head nearly everything we do with medicine, and I've suggested that AIDS and DOWN SYNDROME were probably not the best "visual props" we could have gotten to see why it's so important that we act on this disclosure in a timely manner. After mentioning the ends of aging and death that come eventually to the place we build, to the place we've always thought of as Heaven... it becomes more and more clear that this force fighting against the dissemination of a truth so obvious it's in every word and everything we do--it becomes clear it's neither you, nor acting in your best interest.

I know I've got the eye of the tiger, there's no doubt; and it's pretty clear from "YAD?" (the Hebrew for...) and ha'nd that we can see the clear hand of God at work in a design that marks my initials not just on the timeline, or at 1492, at A.D. I B; but in the Hebrew name for this place called El Shaddai, see how A.D. is "da eye" and in some other names like Adranus, A.D. on "it's silly" and A.D. on Ai that might tie me to the Samof Samurai (but, are you Ai?) in more depth of detail than simply the Live album "Secret Samadhi." I try to reflect on how it is that this story has come about, why it is that everything appears to be focused on me--and still even through that sincere spotlight nobody seems to be able to acknowledge my existence with more words than "unsubscribe" and "you're so vain." With one eye in the mirror, I know ties to Narcissus (and you can too), soaring ever higher--linking Icarus to Wayward Son and to every other name with "car" in it... like "carpenter" and McCarthy the older names of Mercury and even Isacriot (I scary? is car-eye... owe Taylor) and some modern day mythological characters like Jim Carrey and Johnny Carson. As far as Trinities go, carpenter's a pretty good one--tying to my early reck and a few bands and songs from The Pretty Reckless to Dave Matthews' "Crash Into Me" all the way to the "pen" you see before you linking Pendragon to Imagine Dragons.

I wonder why it is that all of these things appear, apparently only to me, to point to a story about all the ways that a sinister hidden force has manipulated our society into being unable to "receive' this message--this wonderful message about making the world a better place and building Heaven--with any fanfare at all. It's focused now on a criminal justice system that clearly does not do any kind of "rehabilitation" and on a mental health industry and pharmaceutical system that treats a provable external attack on our own goodness and well being as some kind of "internal stimulus" and makes you shy away when I point out why "stem" is in system and why "harm" in pharmacy. From that we move a little bit past "where we are in this story" and I have to point out how "meth" ties to Prometheus and Epimetheus and how and why it is I know without doubt that this story has been relived numerous times--and how I am so sure that it's never been received, as we are here again listening to how songs like "Believe" and the words "just to lead us here to this place again" connect to Simon and Garfunkel's" the Sound of Silence... and still to this day you will balk at noticing that "Simon" has something to do with the Simpsons, and something to do with the words "simulation" and "Monday." To see me is to see how things might be done better--how "addicitonary" might tie to the stories of Moses' Lisp and to Dr. Who's "Bells of Saint John" with a sort of "web interface" to the kinds of emotion we might want to "dial down..." rather than Snicker in the background as we see them being artificially created and enhanced in order to build a better "fiery altar."

I can point out "Silicon" harrowing down at us from words like "controversial" and show you Al in "rascal" and "scandal" but not to see that we are staring at school shootings and terrorism that are solved instantly by this disclosure, by Al of Quantum Leap and by the Dick of Minority Report and A Scanner Darkly is to ignore just what it is that we are all failing to Si. I should point out that those two "sc"'s link to a story about Eden and they mean "sacred consciousness" and at the baseline of this event and everything we are not doing is the fact that our desires and beliefs are being altered--all of this comes down to "freedom of thought" here and now.

I could tell you that "looking at me" will show you that even the person who tries every day to do everything he can to save the entire world from slavery, and from "thought-injury"--even I can be made "marred" and you all, this whole world stupid enough to think that you are, of your own volition, hiding Heaven itself from yourselves... to what? To spite me? It, the focal point of our story might come down to you realizing that something in some esoteric place is playing "divide and conquer" with our whole--in secret playing on our weaknesses to keep us from acting on the most actionable information that ever was and ever will be. Still, we sit in silence waiting for me... to speak more?

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hyamdai.reallyhim.com Inline image 31

Between Nero's lyrical fiddling, a Bittersweet Symphony, and true "thunderstanding" the sound of Thor's hammer... "to help the light" that'ls "or" in Hebrew, of Orwell and Orson and .. well, it's really not hard to see and hear that the purpose and intent of "all this noise" is to help us find freedom and truth. C the Light of "singing..."

I can tell you once again how silly the world looks, this multi-decade battle between "the governmentof the people" and the "government of the workers" resulting in what is nothing short of a hands down victory to the corporation. Is it humor meant to divide, or ludicrousness created with the purpose of unification?

But really at it's most basic level what this boils down to is a global group decision not to care about the truth, about reality, about what's really brought us to this place--with solutions in hand and a way to make everything better. We've decided that censorship is OK, and that the world is not all that bad "just the way it is" even though it's creator is screaming in your ear telling you to change as quickly as you possibly can. I believe that God has written this story to make "seeing me" the thing that catalyzes "change for the better" it appears to be the design of not just me but also this place--hey, here I am.

Happy Veteran's Day.

S☀L u TI o N

Yesterday, or maybe earlier today--it's hard to tell at this moment in the afternoon just how long this will take... I sent an image that conveys a high level implication that we are walking around on a map to building something that we might liken to an "ant farm" for people. I don't mean to be disparaging or sleight our contribution to the creation of this map--that I imagine you must also see and believe to be the kind of thing that should remain buried in the sands of time forever and ever--or your just have yet to actually "understand" that's what the plan part of our planet is talking about... what I am trying to do is convey in a sort of "mirrorish" way how this map relates to a message that I see woven in religion and in our history that it significantly more disparaging than I would be. It's a message that calls us "Holy Water" at the nicest of times, water that Moses turns to "thicker than water" in the first blessing in disguise--and to tell you there is certainly a tangible difference between the illusions of the Pharaoh's and the true magic performed by my hand, is nearly exactly the same amount of effort put in to showing you that the togetherness that we are calling "family" here in this place comes from both seeing and acting on the very clearly hidden message in every single idiom showing us all that our society in this story of Exodus is enslaved by a hidden force--and reminding us that we like freedom.

It's not just these few idioms, but most likely every single one from "don't shoot the essenger" to "unsung hero" that should clue us in to exactly how much work and preparation has come into this thing that "he supposes is a revolution." It's also not just "water" describe me and you, in this place where I am the "ant' of the Covenant (do you c vampires or Hansel and Gretel!?!?) but also "lions" and "sheep" and "salt" and "dogs" and nearly everything you could possibly imagine but people; in what I see must be a vainglorious attempt to pretend he actually wants us to "stand up for ourselves" in this place where it's becoming more and more clear with each passing moment that we are chained to these seats in the front row of the audience of the most important event that has ever happened, ever.

Medusa makes several appearances, as well as Arthur Pendragon, Puff the Magic Dragon, Figment, Goliath, monster.com, the Loch Ness Monster in this story that's a kind-of refl ex i ve control to stop mind control; and to really try and show us the fire of Prometheus and the Burning Bush and the Eternal Flame of Heaven are all about freedom and technology ... and I'll remind you this story is ... about the truth--and the truth here is that if you aren't going to recognize that whatever it is that's going on here in secret, below the surface is negatively affecting our society and life in general than we aren't going anywhere, ever. I need you to figure out that this message is everywhere to make sure you don't miss the importance of this moment, and the grave significance of what is being ignored in this land where Sam is tied not just to Samsung and to Samael in Exodus but also to Uncle Sam and macaronic Spanglishrew outliers and that it doesn't take much free thought at all to really understand that we are watching "free thought" disintegrate into the abyss of "nospeak." We are watching our infrastructure for global communication and the mass media that sprawls all over the globe turn to dust, all because you have Satan whispering in your ear--and you think that's more important than what you think, what I think, and what anyone else on the Earth might ever say. You should see a weapon designed to help ensure that don't lose this proof that we are not living in reality, that there is "hidden slavery" in this place--and you should see that today it appears you are simply choosing not to use it.

I hope you change your mind, I really do. This map on "how to build an ant farm" starts by connecting Watergate and Seagate together with names like Bill Gates and Richard Nixon; and with this few short list of names you should really understand how it is that "Heaven" connects both technology like computers and liberty like "free speech" to a story that is us, and our history. You might see that "salt" could either be a good thing or not--take a look around you, are you warming a road to Heaven or are you staring at the world being destroyed--and doing nothing at all about it?

I guess I can point out again how "Lothario" links this story that ties names like my ex-wife's Nanna to "salt" also, but the "grand design" of this story doesn't seem to have any effect on you. Listen, if you do nothing the world is being destroyed by your lack of action--there's no if's and's or butt's about it. I feel like I need to "reproduce' old messages here or you will never see them--that's what web site statistics tell me--and we all know it's not true. What am I missing? What are you missing?

BUTT IS THE BOAT A Hi DARK DEN MESSAGe ?

SEE OUR LIGHT

HONESTLY, I'M WAY TO CUTE TO BE A MONSTER :(

HIC SUMMUS

So... here we are... listening to the legendary father of the message (that's "abom" in Adamic Spagnlishrew) point out all of the sex jokes hidden in religion and language from sexual innuendo to Poseidon and in our history from Yankee Doodle to Hancock to Nixon and I've got to be frank with you, the most recent time I came across this phrase in scripture I cringed just a little bit, pretty sure that the "message" was talking about me. I've reflected on this a little bit, and over the past few weeks have tried to show you the juxtaposition between "sex" and "torture" in it's various forms from imparting blindness to allowing murder and simulating starvation; and I think I'm justified in saying that certainly those things are far worse on the Richter scale than anything I could do by writing a little bit of risque text. In the most recent messages I've touch a little bit, without even knowing or realizing this connection would be made, on what it is that this phrase actually means.

loch.reallyhim.com

ABOMINATION

So long story short is that the answer here is "abomination" and the question, or the context is "I nation." Whether it's Medusa speaking for the Dark United States or the nation of Israel speaking to either Ra or El depending on the day, the bottom line is that a collective consciousness speaking for everyone on a matter of this importance in a cloud of complete darkness on Earth is a total and undeniable abomination of freedom, civilization, and the very humanity we are seeking to preserve. The word reads something like this to me "dear father of the message, I am everyone and we think you are an abomination, fuck off." My answer of course is, IZINATION. Which humorously reminds me of Lucy, and Scarlet Johannson saying "I am colonizing my own brain" so here's some pictures of her. She is not an abomination, by the way; she's quite adorable. You'll probably notice there's some kind of connection between the map--the words speaking to the world, and the abomination, as if the whole thing is a story narrated in ancient myths.

WAKE UP, "SHE" A MESSAGE TO YOU ABOUT THE FUTURE

You might not think "it's you," but the manifestation of this "snake" in our world is your silence, your lack of understanding or willingness to change the world; and whether or not you're interested in hearing about it, it's the monster that myths and religion have spoken about for thousands and thousands of years. It's a simple matter to "kill Medusa" all you have to do... is speak.

Take special note, "freedom of speech" and "freedom to think for yourselves" are not a group decision, and you do not have the right to force (either overtly or subtly, with hidden technology perhaps combined with evil deceit) others not to talk about anything. Especially something of this importance.

DESOLATION

If you didn't connect "Loch" to John Locke, now you have; see how easy this "reading" thing is? I've gone over the "See Our Light" series a few times, but let me--one more time--explain to you just how we are already at the point of "desolation" and with shining brilliance show you how it's very clear that it is "INATION" and "MEDUSA" that are responsible for this problem.

Seeing "Ra" at the heart of the names Abraham and Israel begins to connect the idea that our glowing sun in the sky has something to do with this message about "seeing our light" is being carried by a stone statue on Ellis Island (where you'll see the answer another part of the question of Is Ra El?). I've connected her to the "she" of both shedim and Sheol, which reads as "she's our light" and is the Hebrew name for Hell.

Of course you noticed that the Statue of Liberty does in fact share it's initials with SOL, the the light above and you can see her torch dimly lighting the way through the night; Now you can connect "give us your tired and your poor" to the Lazman of both the lore of Jesus Christ and the Shehekeyanu; a prayer about the sustainment of life and light up until this day. That same torch connects to the Ha-nuke-the-ahah depiction of Christ, Judah Maccabee's lit MEN OR AH, which delivers not only a solution to the two letter key of "AH" as All Humanity that pervades nearly every bride of Revelation from Sarah to Leah; but also to the question of equality answered in our very own American history, beginning with the same three letter acronym now lighting the Sons of Liberty.

Dazed and Confused does a good job of explaining how this name is itself a prophesy designed by Hand of God'; explaining that these Sons of Liberty were all white slave owning wealthy men fighting to stop paying their taxes, rather than delivering liberty to the slaves or women, who were both disenfranchised for quite some time. Or maybe MEN OR AH has something to do with the angels of Heaven, in which case you might be SOL if you aren't a girl and you want to be "be good friends with Ra." Just kidding. Kinda.

DESOLATION by the way reads something like "un see our light at ION" which is God's way of saying "at the point of believing that hiding Adam is a good thing" and that connects to the end of Creation and also the now lit by modern day evil the word "rendition." Our end, it "ion." In religious myth, the Messianic David clung to the city Zion (end the "i owe n") which also links to "verizon" (to see, I Z "on") and HORIZON which has something to do with the son rising today-ish.

Inline image 25 Inline image 26

The story of MEDUSA lights another psuedo-religious idea, that the words "STONE" of both "brimstone" and it's Adamic interpretation "South to Northeast" have something to do with the phrase "Saint One" turned into a single hero against his will by the complete and utter inaction of everyone around him. In the words of Imagine Dragons "I'm waking up to action dust." At the same time, you can believe that the light of this particular son, comes not just from reading these words forwards, but the backside as well, and you'll hopefully see it's not coincidental that the other side of this coin is that "nos" means we, and us... and Adamically "no south." See the light of "STONE" also connecting to Taylor Momsen's rose arrow painted on her back, and the sign of my birth, Sagittarius... which in this particular case links to the Party of the Immaculate Conception of the eternal republic of the Heavens. . PRESS RELEASE... A GREAT SIGN APPEARED IN THE HEAVENS

SOLUTIAN, ON YOUR COMPUTER.. TO THE SOUND OF SILENCE

בָּרוּךְ אַתָּה יְיָ‎ אֱלֹהֵינוּ מֶלֶךְ הַעוֹלָם שֶׁהֶחֱיָנוּ וְקִיְּמָנוּ וְהִגִּיעָנוּ לַזְּמַן הַזֶּה‎׃

IN ... THE BOOK OF NAMES LETS SEE IF YOU CAN FIGURE OUT WHO THEY ARE :)

​ I'LL DO YOURS FOR A 50 DOLLAR DONATION, I'M BROKE.. MAYBE THAT'S WHY I CAN'T GET A DATE.

HAVE A GREAT SOLDAY

The "gist" of the message is verifiable proof that we are living in a computer in simulated reality... just like the Matrix. The answer to that question, what does that mean--is that God has woven a "hidden" message into our everything--beginning with each name and every word--and in this hidden Adamic language, he provides us with guidance, wisdom, and suggestions on how to proceed on this path from "raelity" to Heaven. I've personally spent quite a bit of time decoding the message and have tried to deliver an interesting and "fun" narrative of the ideas I see. Specifically the story of Exodus, which is called "Names" in Hebrew discusses a time shifted narrative of our "now" delivering our society from a hidden slavery (read as ignorance of advanced technologies already in use) that is described as the "darkness" of Exodus. If you have any questions, ideas to contribute or concerns... I'd love to hear from you this whole thing really is about working together--Heaven, I mean.

Inline image 5 jerusalem.reallyhim.com

gate kermitham <br /> ou r evolution minority report to supermax Inline image 6

bereshit bread is life

Inline image 13

Image result for dox me

HOW AM I STILL STINGLE? E ' o e <br /> L m r x <br /> L t y <br /> O a

I HISS.

The sum of ((our world)) is the universal truth. -Psalm 119 and ((ish))

Do a few sentences really make that big of a difference? Some key letters? Can you show me what I'm doing wrong? Is there a way to turn me into Adam, rather than a rock? I think you can.

Are eye Dr. Who or Master Y? Adam Marshall Dobrin is a National Merit Scholar who was born on December 8, 1980 in Plantation, FL and attended Pine Crest School where he graduated sumofi cum louder in "only some of it is humorous." Later he attended the University of Florida (which quickly resulted in a wreck), Florida Atlantic University, and finally Florida Gulf Coast University--where he still has failed to become Dr. Who. While attending "school" He worked in the computer programming and business outsourcing industries for about 15 years before proclaiming to have received a Revelation from God connecting the 9/11 attack and George Bush to the Burning Bush of Exodus and a message about technocracy and pre-crime.

Adam, as he prefers to be called, presents a concise introduction to paradox proven by the Bible through "verifiable" anachronism in language some stuff about Mars colonization and virtual reality and a list of reasons why ignoring this is actually an ELE. Adam claims to be Thor because of a connection between music and the Trial of Thor as well as the words "author" and "authority." He suggests you be Thundercats and call a reporter. There is also a suggestion that Richard Nixon and John Hancock are related to a signature from God, about freedom and America... and the "unseeingly ironic" Deepthroat and Taylor Momsen. They Sung "It's Rael..." In Biblical characters from Mary to Hosea, to see "sea" in Spanish, and in the Taming of the Spanglishrew ... a message is woven from the word Menorah: "men, or all humanity?" to the Statue of Liberty, and the Sons of Liberty, and the light above us, our SOL; which shows us that through the Revelation of Christ and the First Plague of Exodus, a blessing in disguise--turning water to blood, the sea to family; a common thread and single author of our entire history is revealed, a Father of our future. A message of freedom shines out of the words of scripture, revealing a gate to a new technologically "radical" form of democracy and a number of unseen or secret issues that have stalled the progress of humanity... and solutions, solutions from our sea. The Revelation shows us that not only ever word, but every idiom from "don't shoot the messenger" to "blood is thicker than water" we have ties to this message that pervades a hidden Matrix of light connecting movies and music and history all together in a sort of guide book to Salvation and to Heaven. Oopsy. His Revelation, woven into his life, continues to suggest that skinny dipping, forced methamphetamine addiction, and lots and lots of "me A.D." as well as his humorous depiction of a dick plastered over the Sound of Silence, his very Holy click, have something to do with saving our family and then the entire Universe from hidden mind control technology and the problems introduced by secret time travel. From the trials and tribulations of "Job" being coerced and controlled into helping to create this wall of Jericho; we find even more solutions, an end to addiction, to secrecy, and to this hidden control--a focal point of the life of Jesus Christ.

It tells us a story of recursion in time, that has brought us here numerous times--with the details of his life recorded not only in the Bible but in myths of Egyptian, Norse, and Greek mythology. The huge juxtaposition of the import of the content of the message shows the world how malleable our minds really are to this technology, how we could have been "fooled" into hiding our very freedom from ourselves in order to protect the "character" of a myth. A myth that comes to true life by delivering this message. In truth, from the now revealed content of the story of this repeated life, it should become more and more clear that we have not achieved success as of yet, that I have never "arrived whole" and that is why we are here, back again. Home is where the Heart is... When asked how He thinks we should respond to his message, He says "I think we already cherish it, and should strive to understand how it is that freedom is truly delivered through sharing the worth of this story that is our beginning. 'tis coming." Adam claims to be God, or at least look just like him and that the entirety of the Holy Scriptures as well as a number of ancient myths from Prometheus to Heimdallr and Yankee Doodle are actually about his life, and this event. An extensive amount of his writing relates to reformation of our badly broken and decidedly evil criminal justice system as well as ending the Global hunger crisis with the snap of his little finger.

He has written a number of books explaining how this Revelation connects to the delivery of freedom (as in Exodus), through a message about censorship among other social problems which he insists are being intentionally exacerbated by Satan--who he would ha've preferred not to be associated with.

The passage underscores the challenges in assessing the impact of undergraduate research experiences, pointing out a lack of valid measures and reliance on self-report surveys. It highlights potential selection bias in research programs, where students already committed to science are often chosen. The authors stress the need for more nuanced indicators of success, beyond the conventional measure of students persisting in science. Emphasizing the importance of mentoring, the passage calls for improved assessments to better understand the outcomes of undergraduate research. Lastly, it advocates for studies that discern the types of research experiences that succeed for students with diverse interests, backgrounds, and preparation, indicating a desire for tailored approaches to maximize the benefits of such programs.

In my current profession, psychological context plays a huge role in day to day interactions. In my time working in a veterinary emergency hospital, it was incredibly common to go from seeing a relatively healthy pet to an incredibly sick older pet who was reaching the end of their time. In one case, I needed to draw blood on a patient to address an issue to determine which medications would be safe to use for it's condition and right before going into the room with his owner, a family with two little girls brought in their very sick older dog who they were having to say goodbye to. The grief of those little girls saying goodbye to their best friend affected everyone in those rooms. Trying to keep a professional demeanor in front of the owner of the patient I was drawing blood on was impossible and when I looked up to apologize for my tears, he also had tears and stated he didn't see how one could not have tears listening to them tell her thankyou for letting them dress her up and have tea parties and lay on her. It made me consider in what circumstances makes nonverbal communication (like tears in reaction to another's pain) "unprofessional". In those instances, it seems like it's almost necessary to ensure another person that you are, in fact, human. And possibly reassure them that you have the capacity to have sympathy for them and their pet as well and maybe increase the chances of them trusting your ability to care for their pet as you would your own.

can they just kill themselves

this is just like that white zionist lady whos gonna be writing about palestine

jaw drop wtf did i just read

shes so sickening

It's not just KPIs but the dysfunction of management that it satisfies, ie. managing by reacting to every fluctuation in the figure. This shows up across-the-board in performance appraisals, defect rates, complaints, units sold, etc. Wherever there is a KPI, there is a manager who, not knowing what else to do, demands explanations for every peak or valley.

Berserk makes the reader feel claustrophobic

Communication is very important, and we really have to be careful who we're talking to and watch what we say how we say something. Words hurt, and some things we can never take back. When people ask to start over it's because they know something got messed up along the way, whether it be them or you. and just like the text says, it's irreversible most times, your mind will be made up and communication can stop after that

English Translation

Bibliotec(0n) something to read: Three times you - Federico Moccia (Download the book for free) Do you like to immerse yourself in a world full of magic, fiction, action, drama, comedy, etc…?

In this blog, dedicated to those lovers of reading, you will find the literary productions that have marked a milestone in the field of contemporary literature, and in addition to that, you can also download for FREE! these books, stories, novels, etc. Wonderful, right?

Three times you

Third part of the Federico Moccia saga, I loved the first two books, "Three meters above the sky" and "I want you", and this third is the continuation six years later. I personally loved it and I really liked the continuation of the stories of Step, Babi and Gin, and the other characters. In my opinion, with the last two books the story had not ended and with this third part there is an outcome for all the characters. I think it's not bad at all to have a good time and I hope that, just like with the books, they make the "Three times you" movie. Sometimes the reading becomes a little tedious. But since it tells the life of each of the characters, 6 years later, it is understandable. Although I liked his two previous books much more, it is still a good book. Since you want to know in depth, what would happen to everyone. The ending, although a bit predictable, had to be that way. Do you want to know how the story ends? Well, start reading THREE TIMES YOU.

Finally, I have to say that from the beginning (the first book), I loved these novels, not only because they are fresh and from life itself, but because they tell us these beautiful love stories. I was very excited to get to know Rome when I read the descriptions of the Eternal City in its pages. The motorcycle tours that Step took, the sea of ​​Ostia, the Roman squares, the Tiber or the famous Milvian bridge of padlocks (one day I would like to go and put a padlock there with my boyfriend). I encourage you to read this book and the other two previous ones. It's worth it because they are very beautiful love stories, with deep characters and beautiful places.

Original synopsis of the book.

• Download this book for free

Six years later, the lives of our protagonists have changed. They have managed to be happy, but when they least expect it, their paths cross again… After the success of Three Meters Above the Sky and I Want You, comes the long-awaited outcome of the love story of Step, Babi, and Gin. Will Step and Gin still be together? Is Babi happy in her marriage?

Technical sheet Original title: Tre volte tu Author: Federico Moccia Genre: Romantic narrative Year of publication: 2017 Publisher: Planeta Saga: 3/3 Number of pages: 807

WELCOME TO THE STEEMIT VIRTUAL LIBRARY

I agree that recaps can offer more insight into the show with attention grabbers may make people more inclined to keep watching. However, as someone who struggles to sit down and continue watching a show, I'll sometimes skip episodes and think that just because I watched the recap, I know everything that happened.

permission seeking for booth helper tools: a hassle

for - key insight - concrescence - definition - concrescence - Whitehead - definition - The many become the one - Whitehead - definition - Res Potentia - Tim Eastman - definition - superject - Whitehead - definition - moment of satisfaction - Whitehead - definition - dipolar - Whitehead - definition - ingression - Whitehead definition - CONCRESCENCE - is the description of the phases of the iterative process by which reality advances from the past into the present then into the future - this definition is metaphysical and applies to all aspects of reality

• Concrescence is the process by which

  • THE MANY BECOME THE ONE and
  • THE MANY ARE INCREASED BY ONE
    • The "many" here refers to the past
    • the perished objects in the past environment

• There's another domain that whitehead makes reference to

  • He's a platonist in this sense, though he's a reformed platonist
  • He makes reference to this realm of eternal objects which for him are pure possibilities
  • i was mentioning Tim Eastman earlier
    • He calls this domain "RES POTENTIA", the realm of possibilities which have not yet been actualized
• And so for Whitehead
  • the realm of possibility is infinite
  • the realm of actuality is finite
• In the realm of actuality, there's a limited amount of certain types of experience which have been realized
  • but the realm of actuality draws upon this plenum of possibility and
  • it's because there is this plenum of possibility in relationship to the realm of actuality that
  • novelty is possible
  • new things can still happen we're not just constantly repeating the past

• Whitehead describes the process of concrescence or each drop of experience as DIPOLAR, having two poles:

  • a physical pole and
  • a mental pole

• Each concrescence or drop of experience begins with the physical pole

  • where the perished objects of the past environment are apprehended or felt and
  • these feelings of the past grow together into this newly emerging drop of experience
  • and then in the process of their growing together
    • the actualized perished objects of the past environment
    • are brought into comparison with eternal objects or pure potentials possibilities and
    • these possibilities INGRESS so there's
      • INGRESSION of eternal objects and
    • PREHENSION of past actualities
    • INGRESSION of potentials PREHENSIONS of past actualities

• and what the ingression of eternal objects do is provide each occasion of experience, each concrescence with

  • the opportunity to interpret the past differently
• to say maybe it's not like that maybe it's like this
• and so these ingressions come into the mental pole

• If the physical pole is what initiates the experience of each concrescing occasion

  • the mental pole is is a subsequent process that compares
    • what's been felt in the past with
    • what is possible alternatives that could be experienced that are not given yet in the past

• The subjective form is how the occasion fills the past

• The subjective aim is what draws the many feelings of the past towards the unification and the mental pole
  • where
    • the ingression of eternal objects and
    • the feelings of past actualities
  • are brought together into what Whitehead calls this MOMENT OF SATISFACTION
• it's the culmination of the process of concrescence
  • where a new perspective on the universe is achieved - This is the many have become one
• They are increased by one when the satisfaction is achieved
• It's a new perspective on the whole
• As soon as this new perspective is achieved
  • it becomes a SUPERJECT which is not a subject enjoying its own experience anymore
  • it's a perished subject
• The superject is the achieved perspective that has been experienced
  • but then perishes itself int a superject-hood to become
  • one among the many that will be inherited by the next moment of experience, the next concrescence and
• This superject has objective immortality in the sense that
  • every subsequent concrescence will inherit the satisfaction achieved by the prior concrescences

• And so this is the most general account in Whitehead's view that we can offer

  • of the nature of reality
  • the nature of the passage of nature
  • the movement
    • out of the past
    • through the present and
    • into the future

• Experience is always in the present and the satisfaction that is achieved by each moment of concrescence is enjoyed in the present

  • but as soon as we achieve that
  • it perishes and the next moment of concrescence arises to inherit what was achieved
  • and this is an iterative process
  • it's repeating constantly and it's cumulative
• It's a process of growth
  • building on what's been achieved in the past

Reply to Couscous at https://discord.com/channels/686053708261228577/722584061087842365/1197392837952684052

A quick survey of currently active academics, teachers, and researchers who are blogging about note taking practices and zettelkasten-based methods.

Individuals

Dan Allosso is a history professor at Bemidji State University who has used Obsidian in his courses in the past. He frequently writes about related topics on his Substack channels. One can also find related videos about reading, writing, and research process as well as zettelkasten on his YouTube channel. In addition to this, Dan has a book on note taking and writing which focuses on using a card index or zettelkasten centric process.

Shawn Graham has both a blog as well as a prior course on the history of the internet using Obsidian. In the course materials he has compiled significant details and suggestions for setting up an Obsidian vault for students interested in using the tool.

Kathleen Fitzpatrick has a significant blog which covers a variety of topics centered around her work and research. Her current course Peculiar Genres of Academic Writing (2024) focuses on writing, note taking (including Zettelkasten) and encourages students to try out Obsidian, which she's been using herself. A syllabus for an earlier version of the course includes some big name bloggers in academia whose sites might serve as examples of academic writing in the public. The syllabus also includes a section on being an academic blogger and creating platform as a public intellectual.

Morganeua is a Ph.D. candidate who has a fairly popular YouTube channel on note taking within the academic setting (broadly using Obsidian, though she does touch on other tools from time to time).

Chris Aldrich is independent research who does work at the intersection of intellectual history and note taking methods and practices. He's got an active website along with a large collection of note taking, zettelkasten, commonplace books, and sense-making related articles. His practice is a hybrid one using both analog and digital methods including Obsidian and Hypothes.is.

Bob Doto is a teacher and independent researcher who focuses on Luhmann-artig zettelkasten practice and writing. He uses Obsidian and also operates a private Discord server focused on general Zettelkasten practice.

Manfred Kuehn, a professor of philosophy at Boston University, had an influential blog on note taking practices and culture from 2007 to 2018 on Blogspot. While he's taken the site down, the majority of his work there can be found on the Internet Archive.

Andy Matuschak is an independent researcher who is working at the intersection of learning, knowledge management, reading and related topics. He's got a Patreon, YouTube Channel and a public wiki.

Broader community-based efforts

Here are some tool-specific as well as tool-agnostic web-based fora, chat rooms, etc. which are focused on academic-related note taking and will have a variety of people to follow and interact with.

Obsidian runs a large and diverse Discord server. In addition to many others, they have channels for #Academia and #Academic-tools as well as #Knowledge-management and #zettelkasten.

Tinderbox hosts regular meetups (see their forum for details on upcoming events and how to join). While their events are often product-focused (ways to use it, Q&A, etc.), frequently they've got invited speakers who talk about their work, processes, and methods of working. Past recorded sessions can be found on YouTube. While this is tool-specific, much of what is discussed in their meetups can broadly be applied to any tool set. Because Tinderbox has been around since the early 00s and heavily focused on academic use, the majority of participants in the community are highly tech literate academics whose age skews to the over 40 set.

A variety of Zettelkasten practitioners including several current and retired academicians using a variety of platforms can be found at https://forum.zettelkasten.de/.

Boris Mann and others held Tools for Thought meetups which had been regularly held through 2023. They may have some interesting archived material for perusal on both theory, practice, and a wide variety of tools.

Others?

I've tried to quickly tip out my own zettelkasten on this topic with a focus on larger repositories of active publicly available web-based material. Surely there is a much wider variety of people and resources not listed here, but it should be a reasonable primer for beginners. Feel free to reply with additional suggestions and resources of which you may be aware.

How well a family gets along can affect how kids do in school. When families are not stressed all the time, relationships are smoother and less tense. Kids do better when they have warm relationships with their parents, and in these positive situations, parents are less likely to be harsh. This reminds me of how important it is to have a happy and supportive family environment for a child's success in school. It's not just about studying; it's also about feeling loved and supported at home.

I completely agree with this statement. If families have different amounts of money, it can affect how well kids do in school. Richer families can afford things like healthy meals, educational resources at home, and safe neighborhoods. This helps their kids succeed. On the other hand, families with less money may struggle to provide these things, affecting their children's school success. This idea suggests that fixing the gap in school achievement is not just about money; it's also about supporting family relationships and creating equal opportunities for all kids.

I find this amusing and upsetting on how a simple increase in income can help a mother's mental health improve. The struggles that mothers go through for their children needs to be talked about. Especially those form low income, I feel like that is the reason why with families with high-income have less stronger relationships with their children than those that are low-income. I don't know, it's just a thought I have.

I actually unintentionally do this! It's crazy that something I just do is technically a technique of how to write. Wow, cool.

Docdrop is another tool enabled by Hypothesis (Hypothes.is). Here's I'm annotating a portion of transcript of a YouTube video clip.

for - adjacency - sensory bubble - umwelt - living in a simulation - Daisetz Suzuki - elbow doesn't bend backwards - quote - Michael Levin - illusion

adjacency - between - sensory bubble - umwelt - living in a simulation - Daisetz Suzuki - the elbow does not bend backwards - adjacency statement - In the Tibetan Buddhist epistemology, the illusory body training is to experience both one's body and reality as an illusion in the sense that nothing is static and fixed - From this perspective, we are all temporary states of convergence of the recirculating elements of emptiness - Daisetz Suzuki, the enlightened Japanese Zen monk who is credited to be one of the ones who brought Zen to the West said that when he experienced Kensho, he could suddenly understand the puzzling koan "The elbow does not bend backwards" with great clarity. - Form is a concentration and temporary consolidation of emptiness, the limitations inherent in any form does not denigrate is absolute origins from unlimited emptiness - The Heart Sutra expresses the equivalence of form and emptiness, finite and infinite. - In Deep Humanity, we have a saying: - To be or not to be - that is the question - To be AND not to be - that is the answer

• Quote: Michael Levin
  • Sometimes people ask me "is it possible that we're living in a simulation?
    • and and if you think about it it's not just possible it's guaranteed.
  • There's no other way it could possibly be
  • If you think about what is the opposite of that
    • the opposite of that is that you somehow have a physically embodied cognitive structure that
      • is able to,
      • is not limited in its sensory perceptions
      • is not limited in the amount of memory and computations
  • All of us are limited beings
  • All of us evolved under constraints of
    • time
    • energy and
    • everything else -We see a tiny, little, narrow slit in the electromagnetic spectrum
  • We have a few other things
    • like chemical senses of things that are right there on your tongue and
    • on your fingers and so on
    • we have a little bit of memory
    • we have this wet squishy substrate
      • that's very error prone and
      • needs to be constantly maintained
    • and all our memories have to be the actively rewritten
    • We were evolved under specific pressures under those conditions
  • Who could possibly think that that we are not living in some sort of very specific representation of reality
    • that is limited in many ways
  • That's not to say
    • it isn't adaptive and that
    • Donald Hoffman would say that in many ways it is completely wrong
  • I I think there's probably some truth to that
    • but in other ways I think the the big lesson from all this is that
    • we are all a brain and a vat
  • Of course we are a brain sitting inside this thing that gives us various stimuli
  • We try to make the best sense of it that we can and creatures will adapt to
  • This is why you can do
    • sensory substitution and
    • sensory augmentation and why
    • you can have neurons in the dish that play Pong
  • but these systems will try to make sense of whatever world they're given
    • in whatever configuration they have and we do the same
  • So yeah absolutely it's an illusion
    • but it's not an illusion in the sense that there is some other way to have perfect direct perception of some underlying reality
  • When we say it's an illusion or a simulation
    • It just acknowledges the fact that we are finite limited beings
    • whose job it is to make the best sense we can
    • using the hardware that we have
    • of what's been going on up until now and what we predict is going to be going on
  • I don't know of another story that could possibly make sense

for - resonates with - existing meme

resonates with - existing meme - yesterday's revolutionaries become today's old guard

• What I tell my students just be very careful with people who are
  • very smart and
  • very successful
• They know their stuff they're not necessarily calibrated on your stuff

comment - Lebenswelt and multimeaningverse

for: purpose - computational boundary of self - it's utility is to have one idea that can help define intelligence non-anthropomorphically, not just of humans

for - individual or collective behavior - slime mold - prisoner's dilemma and slime molds - slime molds - me vs we - me vs we - slime molds - adjacency - slime molds - me vs we - multicellular organisms

• quote
  • You have the slime mold and you put a piece of oat which the Slime wants to eat and
  • it starts to crawl towards that oat and then
  • What you can do is you can take a razor blade and just cut off that leading edge
    • the little piece of it that's moving towards the oat
  • Now as soon as you've done that
  • that little piece is a new individual and
  • it has a decision to make
    • it can go in and get the oat and exploit that resource and not have to share it with this giant mass of faizaram that's back here or
    • it can first merge back and connect back to the original mass
      • because they can reconnect quite easily and then they go get the oat
  • Now the thing is that the the payoff Matrix looks quite different because
  • when it's by itself it can do this calculus of "well, it's better for me to go get the food instead of and not share it with this other thing"
  • but as soon as you connect, that payoff Matrix changes because there is no me and you
    • there's just we and at that point it doesn't make any sense to the fact that
    • you can't defect against yourself so that payoff table of actions and consequences looks quite different
    • because some of the actions change the number of players and
    • that's really weird

adjacency between - slime molds - me vs we -multicellular organisms - social superorganism and societal breakdown - adjacency statement - A simple slime mold experiment could make an excellent BEing journey - to demonstrate how multicellular beings operate through higher order organizational principle of collaboration that - keeps cells aligned with a common purpose, - but that each cellular unit also comes equipped with - an evolutionarily inherited legacy of individual control system - normally, the evolutionarily later and higher order collaborative signaling that keeps the multi-cellular being unified overrides the lower order, evolutionarily more primitive autonomous cellular control system - however, pathological conditions can occur that disrupt the collaborative signaling, causing an override condition, and individual cells to revert back to their more primitive legacy survival system - The same principles happen at a societal level. - In a healthy, well-functioning society, the collaborative signaling keeps the society together - but if it is severely disrupted, social order breakdown ensues and - individual human beings and small groups resort to individual survival behavior

This part really demonstrates how this was just a mechanism used by Southerners to try to restrict the rights and freedom of African Americans. It is unfair that if an African American couldn't find steady employment, then they would be subject to a cruel penalty because it's hard to find jobs sometimes. This is one aspect of the codes that showed how Whites were just trying to limit African Americans rights in anyway they could. This also shows how they perpetuated a system of racial oppression and wanted to still control and exploit African Americans.

Let's simplify this information:

1. Schema Types:

2. Mongoose supports various types of data for defining the structure of your documents. These include String, Number, Date, Buffer, Boolean, Mixed, ObjectId, Array, Decimal128, Map, and UUID.

3. Model Creation:

4. Once you've defined your schema, you need to convert it into a model using mongoose.model(). This model allows you to interact with the MongoDB collection associated with your schema.

javascript const Blog = mongoose.model('Blog', blogSchema);

1. Default _id Property:
2. By default, Mongoose adds an _id property to your schemas. This property is an ObjectId type.

javascript const schema = new Schema(); schema.path('_id'); // ObjectId { ... }

1. Custom _id:
2. You can customize the _id field if you want, but be cautious. Mongoose expects an _id for top-level documents, so if you overwrite it, make sure to set it yourself.

javascript const schema = new Schema({ _id: Number // Overwrite Mongoose's default `_id` });

1. Disabling _id for Subdocuments:
2. You can disable the _id property for subdocuments if you don't want them to have their own identifiers.

javascript const nestedSchema = new Schema( { name: String }, { _id: false } // Disable `_id` for this subdocument );

Alternatively, you can disable _id directly within the subdocument's schema:

javascript const nestedSchema = new Schema({ _id: false, // Disable `_id` name: String });

This way, subdocuments won't have their own _id property.

Yes, in Mongoose, the first argument passed to mongoose.model() is the singular name of the collection that the model is for. In your example:

javascript const Blog = mongoose.model('Blog', blogSchema);

• 'Blog' is the name of the collection. It's singular, and Mongoose will automatically convert it to plural form when creating the collection in the database (e.g., 'Blog' becomes 'blogs' in the database).

• blogSchema is the schema you defined for documents in this collection.

So, Blog is the Mongoose model that you can use to interact with the MongoDB collection named 'blogs' (or whatever Mongoose converts 'Blog' to in plural form).

Certainly! Let's break down the provided code snippets:

1. What is it and why is it used?

In Mongoose, a schema is a blueprint for defining the structure of documents within a collection. When you define a schema, you can also attach methods to it. These methods become instance methods, meaning they are available on the individual documents (instances) created from that schema.

Instance methods are useful for encapsulating functionality related to a specific document or model instance. They allow you to define custom behavior that can be executed on a specific document. In the given example, the findSimilarTypes method is added to instances of the Animal model, making it easy to find other animals of the same type.

2. Syntax:

Using methods object directly in the schema options:

javascript const animalSchema = new Schema( { name: String, type: String }, { methods: { findSimilarTypes(cb) { return mongoose.model('Animal').find({ type: this.type }, cb); } } } );

Using methods object directly in the schema:

javascript animalSchema.methods.findSimilarTypes = function(cb) { return mongoose.model('Animal').find({ type: this.type }, cb); };

Using Schema.method() helper:

javascript animalSchema.method('findSimilarTypes', function(cb) { return mongoose.model('Animal').find({ type: this.type }, cb); });

3. Explanation in Simple Words with Examples:

Why it's Used:

Imagine you have a collection of animals in your database, and you want to find other animals of the same type. Instead of writing the same logic repeatedly, you can define a method that can be called on each animal instance to find similar types. This helps in keeping your code DRY (Don't Repeat Yourself) and makes it easier to maintain.

Example:

```javascript const mongoose = require('mongoose'); const { Schema } = mongoose;

// Define a schema with a custom instance method const animalSchema = new Schema({ name: String, type: String });

// Add a custom instance method to find similar types animalSchema.methods.findSimilarTypes = function(cb) { return mongoose.model('Animal').find({ type: this.type }, cb); };

// Create the Animal model using the schema const Animal = mongoose.model('Animal', animalSchema);

// Create an instance of Animal const dog = new Animal({ type: 'dog', name: 'Buddy' });

// Use the custom method to find similar types dog.findSimilarTypes((err, similarAnimals) => { console.log(similarAnimals); }); ```

In this example, findSimilarTypes is a custom instance method added to the Animal schema. When you create an instance of the Animal model (e.g., a dog), you can then call findSimilarTypes on that instance to find other animals with the same type. The method uses the this.type property, which refers to the type of the current animal instance. This allows you to easily reuse the logic for finding similar types across different instances of the Animal model.

This opening paragraph seems a little too long and only skims on the bigger picture your audience needs to see the benefits of your membership.

I'd focus here ONLY on the problems (cookie-cutter advice, no availability with an expert, needing help when it's most urgent) and the dream they're aspiring to (and expert on call, proven strategies, connecting with fellow parents, etc).

All the references to the membership, etc I'd move further down on the page.

I definitely used to use this trick a lot for answers to very specific questions. I've noticed how much worse it's gotten, not to mention that it now just seems easier to ask ChatGPT first as it seems better at finding super specific answers than even Google.

I strongly agree with the paragraph's emphasis on the need for more inclusive classroom settings. Multiculturalism should be more than just a buzzword; it should be reflected in the way we teach. From my experience as a Chinese student studying in America, I've seen how diverse perspectives enrich the learning environment. It's essential for teachers to recognize that their teaching styles may need to evolve to accommodate various cultural backgrounds and experiences, fostering a more inclusive and equitable education for all students.

Okay, at least we mention that "oh, the fourth challenge? We might just be saying too bad so sad [yes, that's gentler than what I want to say] for an awful lot of students. What about that rest of them? (also it's expensive ...)

I think its very cool when a company makes a new game that is unlike what they had previously done beforehand, becuase this opens up a while new avenue for the company and the players of the new games.

It should be well known how detrimental grading can be to a student that wants to succeed and how it can lead to suicide or other harmful acts but nothing has changed though so much time has passed since it was set into the academic learning system.

Much of this is consistent with how much effort the student themselves are willing to put in order to get the job done and understand the values of what it takes to get to the grade they want to earn for the class.

Considering it is relatively difficult to get a perfect score on anything, I can assume that the author is alluding to the fact that grades point out the wrong in which the student is doing.

For example, if a student were to get a 91/100 on an assignment, the 9 points the student missed act as a reinforcer that the student, despite receiving a passing grade, still got something wrong.

I was curious about this text because it stood out to me the most. It seems like a poetic reflection on the connection between generations and the shared experience of being part of a crowd. The author describes feeling refreshed by the river and the bright flow, just as we might feel when we look at the river and sky. They also mention standing and leaning on the rail, yet being hurricane by the swift current, which creates a vivid image of movement and being swept along. Overall it's a text that make you think about the timeless nature of human experiences.

One person counted 93 per day

It is important to remember that technology is not a mindless tool. The digital world has a life of its own. It is not just about being able to use technology and digital spaces, but the how and why of employing those tools.

This really makes me question how to make sure history is accurate and education is fair without falling victim to gatekeeping or violating freedom of speech. How do you monitor the content of a site like the speaker's without submitting work to peer review (with the idea of "peer review" itself being limited to the academic sphere)? I certainly don't know how to fix it, beyond making sure more than a single person is reviewing student submissions before being posted to a historical site.

That being said, maybe the work was factually accurate but saying Jackson was "brave" is definitely that one student's biased opinion and absolutely problematic, and in my opinion shouldn't have been posted.

While I think this is fundamentally good advice, it is also leaning on the idea that no new technologies will be available to the student once they graduate or, ever. There is nothing wrong with having a favourite tool, but I would rather be flexible and adaptable. The speaker uses the example of a 3D printer as a potential inefficient tool - I am not sure why he used this example because it seems the interview took place fairly recently, and in 2024 3D printers are home devices for a lot of people (shoutout to everyone I know who makes D&D miniatures) and have been for a few years already. I am very confused by this podcast, overall.

Another curious stance, considering this professor teaches Digital Humanities. I do get their point -- I feel as though certain information needs a specific vehicle to make sense. For instance, I would rather hear a song than have the lyrics presented to me on a soundless PowerPoint. However, they make it seem like a huge burden or chore. I am not understand what they mean by "heartbreak." Additionally, who is deciding what "needs" to be digital or not? (Who is deciding, and why?)

After viewing the results from our class poll on what each of us believed digital humanities to be about, this idea shows just how large of a scope DH encompasses. The idea of DH is used to categorize such a broad range of topics that some people might have only slightly similar interests within DH but are technically both studying the same topic. This is different from subjects like health psychology, where yes there is a broad range of topics, but the range is not nearly as large and diverse as there is in DH.

Interesting that similar sentiments are not usually expressed about Columbus day or Presidents' day.

I think that this quote is really important because it shows how we've created this holiday to remind us of King's legacy and to honor him, but, as a country and just as people, we have completely gone against the nonviolent movement he worked tirelessly to lead. I think this just goes to show how the holiday is not about action in the slightest; it's really only about remembering and honoring.

I wanted to comment on this because, my dad was telling me the other day that, to combat his employers having to enforce some holidays and not others and to allow for some level of freedom, his company allots each employee a certain number of paid vacation days for each year and does not "close" for any holidays, not even Christmas or New Years and each employee gets to take off for the holidays that they chose to celebrate and they can still work and make money on holidays that they do not celebrate. I thought I would mention this here just because I think it's in interesting idea related to the issue of companies recognizing or not recognizing holidays. I also did want to note really quickly that I am from Chicago and my dad works in tech/computer science and I feel like this idea might only be plausible in certain working conditions like his.

Outside the scope of what is discussed here, but it may be of interest to anyone reading that strings differ in their implementation from language-to-language, with some languages adopting 2-byte characters (like Java), to support a wider subset of unicode, with others like C delegating that decision to the developer (it's just an array of bytes, you figure the unicode out yourself, but using ASCII will be easier). My personal favorite and preference among these languages is Rust, which has full unicode support, but in an explicit and ergonomic way. It's very easy to accidentally create a character incorrectly in Java (e.g., with casting), but very difficult in rust (e.g., explicit conversion). Check it out!

The text talks about why poor students might not do well in school. Instead of just blaming the students, it asks a bigger question: "Why are people poor?" The author wants us to look beyond the school system and think about why there's a group of "poor people" not reaching their potential, even though the education system was supposed to help them. Starting at the bottom of the diagram means digging into the reasons behind poverty and educational struggles, going beyond just how well students do in school. It's about understanding the bigger picture of why some people face challenges.

It just goes to show that there's more than one way to play or be good at a game. It's the same game with the same rules, but people will play it in different ways with different methods and strategies.

it's important to remember that not all "bots" are actually bots. When we talk about bots, we're usually thinking of automated computer programs. But sometimes, real people get paid to post stuff like ads or political messages. These aren't bots because there's a human behind them, kind of like workers in a click farm just following instructions. So, it's good to keep in mind the difference between these human-run accounts and actual computer-run bots.

Author Response

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

We thank the editor and the reviewers for their valuable and constructive feedback. In the revised manuscript, we have incorporated and addressed the suggestions provided by the reviewers.

Reviewer #1 (Recommendations For The Authors):

The primary recommendation is to provide additional language explaining how KinCytE will be updated.

Response: We appreciate the reviewer’s insightful feedback regarding the KinCytE update. In response, we have included additional details in the “Development and use of KinCyte’ section as follows: “We welcome researchers to actively participate in advancing the development of KinCytE by sharing external screening data, especially data on new secreted factors and cell types that extend beyond macrophages. This collaborative effort promises to enhance our understanding of kinase-focused networks, opening new avenues for cutting-edge therapeutic approaches”. In addition, we explicitly state in the "Data, Software, and Availability" section, "To contribute data, kindly email the corresponding author and refer to Table S2 for guidance on the preferred file format."

Reviewer #2 (Recommendations For The Authors):

Would have been nice to see a validation of the regression models from outside of the training data. I would also consider removing statements like "We anticipate that KinCytE will be highly sought after by biologists... " , it reads like a grant application (and this is not)! Could tone the language down a bit. In the future, you might consider displaying your graphs as "biofabrics", they're much cleaner than "hairballs" (PMID: 23102059). Or potentially, show a hierarchical view where the selected cytokine (or other) is at the root, and you can immediately see what's connected. Anyway, the network display can be expanded. Consider maybe adding the nearest neighbors to the table on the right after selecting the node. Generally, though, I like how it works.

There needs to be a button to download the graph as a .csv file. Maybe the subgraph after selecting a node (or set of nodes). Also, once you're at a graph view, it's hard to guess how to get back to the starting page. Maybe just one button with a "home" on it would fix that. On the Kinases Discovery, why are the gene symbols all lower case? Very cool!

Response:: We greatly value the reviewer's constructive suggestions. To incorporate these, we have made the following changes:

(1) "We anticipate that KinCytE will be highly sought after by biologists... " This sentence is removed.

(2) A ‘SAVE CSV’ button is added to the bottom right of the Cytokine Explorer page, which allows the users to download the graph as a csv file.

(3) A redesigned KinCyte logo now functions as the 'HOME' button, located at the top left of the webpage, ensuring that users can easily return to the homepage at any time.

reply to https://forum.zettelkasten.de/discussion/comment/19278/#Comment_19278

@JasperMcFly I'll presume that given the time differential, you may have missed my post just before yours which touches on the frivolity of the proposition of creating a single definition?

Most on this forum are going to presume that zettelkasten is precisely a slipbox in a similar form to that of Luhmann, but in practice some here and many elsewhere aren't going to see the distinction (or care). Some will unpopularly insist that a zettelkasten cannot be digital in form, but they'll also do so while simultaneously (heterodoxically and confusingly?) suggesting that one should use Wikipedia's Academic Outline of Disciplines, an idea which didn't exist during Luhmann's life.

You can make an attempt to force a definition, but I guarantee that it's a losing proposition as in practice people are going to use the word in almost any way they want—whatever you do, don't trust Humpty Dumpty's definition. It's the difference between prescriptive and proscriptive definitions. It can be seen in your very question if you look closely at your own phrase "beg the question", which in classic rhetoric means something very specific going back centuries, but in common use it has a dramatically different meaning. As ever, context will always be the king on these questions of definition, though some of us are still converging on a happy commonality.

For a bit more history here, try The Two Definitions of Zettelkasten.

It's crazy to think that teaching is a team game. I feel like so often you just see yourself as a lone in the room as a teacher, but in reality the whole school is, hopefully, rooting fro your success.

This portion of this poem is just showing once again that these two people are relating what they are seeing to different things. One person says that everything they see is because of God, however the other person says it's just the connection that everyone has through the universe.

"People's mental models shape their understanding of what is right, what is natural, and what is possible in life."

This quote highlights the impact mental models have on perceptions and behaviors, especially regarding development. It emphasizes that in order for development policies to be effective, they must consider these underlying beliefs and perceptions. Just giving information or even resources isn't enough, as understanding and perhaps even changing critical views and beliefs is what is crucial for impactful change.

https://health-policy-systems.biomedcentral.com/articles/10.1186/s12961-019-0499-x

This article from Health Research Policy and Systems explains that in order for policies to be successful, especially in complex fields like healthcare, it's essential that all stakeholders have a deep understanding of the goals and methods. This communal understanding allows for everyone to work efficiently and effectively towards an objective.

The majority of managers, when ordering a logo, often lack a clear understanding of what it is. Some people think it's just a visually appealing image, while others conflate a logo with a trademark. Many individuals mistakenly view a logo as a simple symbol that they could draw themselves.

To address the question of why a high-quality logo is essential, we must grasp its concept and functions. A logo is a visual representation that can be composed of text, a graphic image, or a combination of both. The purpose of the logo is to convey the essence of the business in a distinctive and memorable way to the target audience. It creates a distinct image which helps the target audience to easily recognize the product and associate it with specific qualities, the firm's reputation, and other advertising parameters.

A poorly designed logo even has the potential to be detrimental, rather than beneficial, which is why a quality logo is a crucial component of the company's image.

Regarding the framework of Consequentialism, and more specifically Utilitarianism, it's important to note a critical aspect that wasn't fully captured in the original summary. Utilitarianism, a significant branch of Consequentialism, is not just about the maximization of happiness but also about the minimization of suffering. This dual focus is a crucial element, as it recognizes the balance between positive and negative outcomes of actions. John Stuart Mill, a key figure in Utilitarianism, emphasized not just the quantity but also the quality of happiness. This consideration leads to complex ethical discussions about the nature of happiness and suffering and how they should be weighed against each other. This aspect of Utilitarian ethics often leads to debates about its application in real-world scenarios, especially in cases where the reduction of suffering for the many might come at the cost of intense suffering for a few.

Author Response

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

eLife assessment

This important study combines genetically barcoded rabies viruses with spatial transcriptomics in vivo in the mouse brain to decode connectivity of neural circuits. The data generated by the combination of these approaches in this new way is mostly convincing as the authors provide validation and proof-of-concept that the approach can be successful. While this new combination of established techniques has promise for elucidating brain connectivity, there are still some nuances and caveats to the interpretations of the results that are lacking especially with regards to noting unexpected barcodes either due to unexpected/novel connections or unexpected rabies spread.

In this revised manuscript, we added a new control experiment and additional analyses to address two main questions from the reviewers: (1) How the threshold of glycoprotein transcript counts used to identify source cells was determined, and (2) whether the limited long-range labeling was expected in the trans-synaptic experiment. The new experiments and analyses validated the distribution of source cells and presynaptic cells observed in the original barcoded transsynaptic tracing experiment and validated the choice of the threshold of glycoprotein transcripts. As the reviewers suggested, we also included additional discussion on how future experiments can improve upon this study, including strategies to improve source cell survival and minimizing viral infection caused by leaky expression of TVA. We also provided additional clarification on the analyses for both the retrograde labeling experiment and the trans-synaptic tracing experiment. We modified the Results and Discussion sections on the trans-synaptic tracing experiment to improve clarity to general readers. Detailed changes to address specific comments by reviewers are included below.

Public Reviews:

Reviewer #1 (Public Review):

In this preprint, Zhang et al. describe a new tool for mapping the connectivity of mouse neurons. Essentially, the tool leverages the known peculiar infection capabilities of Rabies virus: once injected into a specific site in the brain, this virus has the capability to "walk upstream" the neural circuits, both within cells and across cells: on one hand, the virus can enter from a nerve terminal and infect retrogradely the cell body of the same cell (retrograde transport). On the other hand, the virus can also spread to the presynaptic partners of the initial target cells, via retrograde viral transmission.

Similarly to previously published approaches with other viruses, the authors engineer a complex library of viral variants, each carrying a unique sequence ('barcode'), so they can uniquely label and distinguish independent infection events and their specific presynaptic connections, and show that it is possible to read these barcodes in-situ, producing spatial connectivity maps. They also show that it is possible to read these barcodes together with endogenous mRNAs, and that this allows spatial mapping of cell types together with anatomical connectivity.

The main novelty of this work lies in the combined use of rabies virus for retrograde labeling together with barcoding and in-situ readout. Previous studies had used rabies virus for retrograde labeling, albeit with low multiplexing capabilities, so only a handful of circuits could be traced at the same time. Other studies had instead used barcoded viral libraries for connectivity mapping, but mostly focused on the use of different viruses for labeling individual projections (anterograde tracing) and never used a retrograde-infective virus.

The authors creatively merge these two bits of technology into a powerful genetic tool, and extensively and convincingly validate its performance against known anatomical knowledge. The authors also do a very good job at highlighting and discussing potential points of failure in the methods.

We thank the reviewer for the enthusiastic comments.

Unresolved questions, which more broadly affect also other viral-labeling methods, are for example how to deal with uneven tropism (ie. if the virus is unable or inefficient in infecting some specific parts of the brain), or how to prevent the cytotoxicity induced by the high levels of viral replication and expression, which will tend to produce "no source networks", neural circuits whose initial cell can't be identified because it's dead. This last point is particularly relevant for in-situ based approaches: while high expression levels are desirable for the particular barcode detection chemistry the authors chose to use (gap-filling), they are also potentially detrimental for cell survival, and risk producing extensive cell death (which indeed the authors single out as a detectable pitfall in their analysis). This is likely to be one of the major optimisation challenges for future implementations of these types of barcoding approaches.

As the reviewer suggested, we included additional discussion about tropism and cytotoxicity in the revised Discussion. Our sensitivity for barcode detection is sufficient, since we estimated (based on manual proofreading) that most barcoded neurons had more than ten counts of a barcode in the trans-synaptic tracing experiment. The high sensitivity may potentially allow us to adapt next-generation rabies virus with low replication, such as the third generation ΔL rabies virus (Jin et al, 2022, biorxiv) in future optimizations.

Overall the paper is well balanced, the data are well presented and the conclusions are strongly supported by the data. Impact-wise, the method is definitely going to be useful for the neurobiology research community.

We thank the reviewer for her/his enthusiasm.

Reviewer #2 (Public Review):

Although the trans-synaptic tracing method mediated by the rabies virus (RV) has been widely utilized to infer input connectivity across the brain to a genetically defined population in mice, the analysis of labeled pre-synaptic neurons in terms of cell-type has been primarily reliant on classical low-throughput histochemical techniques. In this study, the authors made a significant advance toward high-throughput transcriptomic (TC) cell typing by both dissociated single-cell RNAseq and the spatial TC method known as BARseq to decode a vast array of molecularly labeled ("barcoded") RV vector library. First, they demonstrated that a barcoded-RV vector can be employed as a simple retrograde tracer akin to AAVretro. Second, they provided a theoretical classification of neural networks at the single-cell resolution that can be attained through barcoded-RV and concluded that the identification of the vast majority (ideally 100%) of starter cells (the origin of RV-based trans-synaptic tracing) is essential for the inference of single-cell resolution neural connectivity. Taking this into consideration, the authors opted for the BARseq-based spatial TC that could, in principle, capture all the starter cells. Finally, they demonstrated the proof-of-concept in the somatosensory cortex, including infrared connectivity from 381 putative pre-synaptic partners to 31 uniquely barcoded-starter cells, as well as many insightful estimations of input convergence at the cell-type resolution in vivo. While the manuscript encompasses significant technical and theoretical advances, it may be challenging for the general readers of eLife to comprehend. The following comments are offered to enhance the manuscript's clarity and readability.

We modified the Results and Discussion sections on the trans-synaptic tracing experiment to improve clarity to general readers. We separated out the theoretical discussion about barcode sharing networks as a separate subsection, explicitly stated the rationale of how different barcode sharing networks are distinguished in the in situ trans-synaptic tracing experiment, and added additional discussion on future optimizations. Detailed descriptions are provided below.

Major points:

1. I find it difficult to comprehend the rationale behind labeling inhibitory neurons in the VISp through long-distance retrograde labeling from the VISal or Thalamus (Fig. 2F, I and Fig. S3) since long-distance projectors in the cortex are nearly 100% excitatory neurons. It is also unclear why such a large number of inhibitory neurons was labeled at a long distance through RV vector injections into the RSP/SC or VISal (Fig. 3K). Furthermore, a significant number of inhibitory starter cells in the somatosensory cortex was generated based on their projection to the striatum (Fig. 5H), which is unexpected given our current understanding of the cortico-striatum projections.

The labeling of inhibitory neurons can be explained by several factors in the three different experiments.

(1) In the scRNAseq-based retrograde labeling experiment (Fig. 2 and Fig. S3), the injection site VISal is adjacent to VISp. Because we dissected VISp for single-cell RNAseq, we may find labeled inhibitory neurons at the VISp border that extend short axons into VISal. We explained this in the revised Results.

(2) In the in situ sequencing-based retrograde labeling experiment (Fig. 3,4), the proximity between the two injection sites VISal and RSP/SC, and the sequenced areas (which included not only VISp but also RSP) could also contribute to labeling through local axons of inhibitory neurons. Furthermore, because we also sequenced midbrain regions, inhibitory neurons in the superior colliculus could pick up the barcodes through local axons. We included an explanation of this in the revised Results.

(3) In the trans-synaptic tracing experiment, we speculate that low level leaky expression from the TREtight promoter led to non-Cre-dependent expression in many neurons. To test this hypothesis, we first performed a control injection in which we saw that the fluorescent protein expression were indeed restricted to layer 5, as expected from corticostriatal labeling. Based on the labeling pattern, we estimated that about 12 copies of the glycoprotein transcript per cell would likely be needed to achieve fluorescent protein expression. Since many source cells in our experiment were below this threshold, these results support the hypothesis that the majority of source cells with low level expression of the glycoprotein were likely Cre-independent. Because these cells could still contribute to barcode sharing networks, we could not exclude them as in a conventional bulk trans-synaptic tracing experiment. In future experiments, we can potentially reduce this population by improving the helper AAV viruses used to express TVA and the glycoprotein. We included this explanation in Results and more detailed analysis in Supplementary Note 2, and discussed potential future optimizations in the Discussion. This new analysis in Supplementary Note 2 is also related to the Reviewer’s question regarding the threshold used for determining source cells (see below).

1. It is unclear as to why the authors did not perform an analysis of the barcodes in Fig. 2. Given that the primary objective of this manuscript is to evaluate the effectiveness of multiplexing barcoded technology in RV vectors, I would strongly recommend that the authors provide a detailed description of the barcode data here, including any technical difficulties or limitations encountered, which will be of great value in the future design of RV-barcode technologies. In case the barcode data are not included in Fig. 2, I would suggest that the authors consider excluding Fig. 2 and Fig. S1-S3 in their entirety from the manuscript to enhance its readability for general readers.

In the single-cell RNAseq-based retrograde tracing, all barcodes recovered matched to known barcodes in the corresponding library. We included a short description of these results in the revised manuscript.

1. Regarding the trans-synaptic tracing utilizing a barcoded RV vector in conjunction with BARseq decoding (Fig. 5), which is the core of this manuscript, I have a few specific questions/comments. First, the rationale behind defining cells with only two rolonies counts of rabies glycoprotein (RG) as starter cells is unclear. Why did the authors not analyze the sample based on the colocalization of GFP (from the AAV) and mCherry (from the RV) proteins, which is a conventional method to define starter cells? If this approach is technically difficult, the authors could provide an independent histochemical assessment of the detection stringency of GFP positive cells based on two or more colonies of RG.

In situ sequencing does not preserve fluorescent protein signals, so we used transcript counts to determine which cells expressed the glycoprotein. We have added new analyses in the Results and in Supplementary Note 2 to determine the transcript counts that were equivalent to cells that had detectable BFP expression. We found that BFP expression is equivalent to ~12 counts of the glycoprotein transcript per cell, which is much higher than the threshold we used. However, we could not solely rely on this estimate to define the source cells, because cells that had lower expression of the glycoprotein (possibly from leaky Cre-independent expression) may still pass the barcodes to presynaptic cells. This can lead to an underestimation of double-labeled and connected-source networks and an overestimation of single-source networks and can obscure synaptic connectivity at the cellular resolution. We thus used a very conservative threshold of two transcripts in the analysis. This conservative threshold will likely overestimate the number of source cells that shared barcodes and underestimate the number of single-source networks. Since this is a first study of barcoded transsynaptic tracing in vivo, we chose to err on the conservative side to make sure that the subsequent analysis has single-cell resolution. Future characterization and optimization may lead to a better threshold to fully utilize data.

Second, it is difficult to interpret the proportion of the 2,914 barcoded cells that were linked to barcoded starter cells (single-source, double-labeled, or connected-source) and those that remained orphan (no-source or lost-source). A simple table or bar graph representation would be helpful. The abundance of the no-source network (resulting from Cre-independent initial infection of the RV vector) can be estimated in independent negative control experiments that omit either Cre injection or AAV-RG injection. The latter, if combined with BARseq decoding, can provide an experimental prediction of the frequency of double-labeled events since connected-source networks are not labeled in the absence of RG.

We have added Table 2, which breaks down the 2,914 barcoded cells based on whether they are presynaptic or source cells, and which type of network they belong to. We agree with the reviewer that the additional Cre- or RG- control experiments in parallel would allow an independent estimate of the double labeled networks and the no-source networks. We have included added a discussion of possible controls to further optimize the trans-synaptic tracing approach in future studies in the Discussion.

Third, I would appreciate more quantitative data on the putative single-source network (Fig. 5I and S6) in terms of the distribution of pre- and post-synaptic TC cell types. The majority of labeling appeared to occur locally, with only two thalamic neurons observed in sample 25311842 (Fig. S6). How many instances of long-distance labeling (for example, > 500 microns away from the injection site) were observed in total? Is this low efficiency of long-distance labeling expected based on the utilized combinations of AAVs and RV vectors? A simple independent RV tracing solely detecting mCherry would be useful for evaluating the labeling efficiency of the method. I have experienced similar "less jump" RV tracing when RV particles were prepared in a single step, as this study did, rather than multiple rounds of amplification in traditional protocols, such as Osakada F et al Nat Protocol 2013.

We imaged an animal that was injected in parallel to assess labeling (now included in Supplementary Note 2 and Supp. Fig. S5). The labeling pattern in the newly imaged animal was largely consistent with the results from the barcoded experiment: most labeled neurons were seen in the vicinity of the injection site, and sparser labeling was seen in other cortical areas and the thalamus. We further found that most neurons that were labeled in the thalamus were about 1 mm posterior to the center of the injection site, and thus would not have been sequenced in the in situ sequencing experiment (in which we sequenced about 640 µm of tissue spanning the injection site).

In addition, we found that the bulk of the cells that expressed mCherry from the rabies virus only partially overlapped with the area that contained cells co-expressing BFP with the rabies glycoprotein. Moreover, very few cells co-expressed mCherry and BFP, which would be considered source cells in a conventional mono-synaptic tracing experiment. The small numbers of source cells likely also contributed to the sparseness of long-range labeling in the barcoded experiment.

These interpretations and comparisons to the barcoded experiment are now included in Supplementary Note 2.

Reviewer #3 (Public Review):

The manuscript by Zhang and colleagues attempts to combine genetically barcoded rabies viruses with spatial transcriptomics in order to genetically identify connected pairs. The major shortcoming with the application of a barcoded rabies virus, as reported by 2 groups prior, is that with the high dropout rate inherent in single cell procedures, it is difficult to definitively identify connected pairs. By combining the two methods, they are able to establish a platform for doing that, and provide insight into connectivity, as well as pros and cons of their method, which is well thought out and balanced.

Overall the manuscript is well-done, but I have a few minor considerations about tone and accuracy of statements, as well as some limitations in how experiments were done. First, the idea of using rabies to obtain broader tropism than AAVs isn't really accurate - each virus has its own set of tropisms, and it isn't clear that rabies is broader (or can be made to be broader).

As the reviewer suggested, we toned down this claim and stated that rabies virus has different tropism to complement AAV.

Second, rabies does not label all neurons that project to a target site - it labels some fraction of them.

We meant to say that retrograde labeling is not restricted to labeling neurons from a certain brain region. We have clarified in the text.

Third, the high rate of rabies virus mutation should be considered - if it is, or is not a problem in detecting barcodes with high fidelity, this should be noted.

Our analysis showed that sequencing 15 bases was sufficient to tolerate a small number of mismatches in the barcode sequences and could distinguish real barcodes from random sequences (Fig. 4A). Thus, we can tolerate mutations in the barcode sequence. We have clarified this in the text.

Fourth, there are a number of implicit assumptions in this manuscript, not all of which are equally backed up by data. For example, it is not clear that all rabies virus transmission is synaptic specific; in fact, quite a few studies argue that it is not (e.g., detection of rabies transcripts in glial cells). Thus, arguments about lost-source networks and the idea that if a cell is lost from the network, that will stop synaptic transmission, is not clear. There is also the very real propensity that, the sicker a starter cell gets, the more non-specific spread of virus (e.g., via necrosis) occurs.

We agree with the reviewer that how strictly virus transmission is restricted to synapses remains a hotly debated question in the field, and this question is relevant not only to techniques based on barcoded rabies tracing, but to all trans-synaptic tracing experiments. A barcoding-based approach can generate single-cell data that enable direct comparison to other data modalities that measure synaptic connectivity, such as multi-patch and EM. These future experiments may provide additional insights into the questions that the reviewer raised. We have included additional discussion about how non-synaptic transmission of barcodes because of the necrosis of source cells may affect the analysis in the Discussion.

Regarding the scenario in which the source cell dies, we agree with the reviewer and have clarified in the revised manuscript.

Fifth, in the experiments performed in Figure 5, the authors used a FLEx-TVA expressed via a retrograde Cre, and followed this by injection of their rabies virus library. The issue here is that there will be many (potentially thousands) of local infection events near the injection site that TVA-mediated but are Cre-dependent (=off-target expression of TVA in the absence of Cre). This is a major confound in interpreting the labeling of these cells. They may express very low levels of TVA, but still have infection be mediated by TVA. The authors did not clearly explore how expression of TVA related to rabies virus infection of cells near the rabies injection site. A modified version of TVA, such as 66T, should have been used to mitigate this issue. Otherwise, it is impossible to determine connectivity locally. The authors do not go to great lengths to interpret the findings of these observations, so I am not sure this is a critical issue, but it should be pointed out by the authors as a caveat to their dataset.

We agree with the reviewer that this type of infection could potentially be a major contributor to no-source networks, which were abundant in our experiment. Because small no-source networks were excluded from our analyses, and large no-source networks were only included for barcodes with low frequency (i.e., it would be nearly impossible statistically to generate such large no-source networks from independent infections), we believe that the effect of independent infections on our analyses were minimized. We have added a control experiment in Fig S5 and Supplementary Note 2, which further supported the hypothesis that there were many independent infections. We also included additional discussion about how this can be assessed and optimized in future studies in the Discussion.

Sixth, the authors are making estimates of rabies spread by comparison to a set of experiments that was performed quite differently. In the two studies cited (Liu et al., done the standard way, and Wertz et al., tracing from a single cell), the authors were likely infecting with a rabies virus using a high multiplicity of infection, which likely yields higher rates of viral expression in these starter cells and higher levels of input labeling. However, in these experiments, the authors need to infect with a low MOI, and explicitly exclude cells with >1 barcode. Having only a single virion trigger infection of starter cells will likely reduce the #s of inputs relative to starter neurons. Thus, the stringent criteria for excluding small networks may not be entirely warranted. If the authors wish to only explore larger networks, this caveat should be explicitly noted.

In the trans-synaptic labeling experiment, we actually used high rabies titer (200 nL, 7.6e10 iu/mL) that was comparable to conventional rabies tracing experiments. We did not exclude cells with multiple barcodes (as opposed to barcodes in multiple source cells), because we could resolve multiple barcodes in the same cell and indeed found many cells with multiple barcodes. We have clarified this in the text.

Overall, if the caveats above are noted and more nuance is added to some of the interpretation and discussion of results, this would greatly help the manuscript, as readers will be looking to the authors as the authority on how to use this technology.

In addition to addressing the specific concerns of the reviewer as described above, we modified the Results and Discussion sections on the trans-synaptic tracing experiment to improve clarity to general readers and expanded the discussion on future optimizations.

Reviewer #1 (Recommendations For The Authors):

The scientific problem is clearly stated and well laid out, the data are clearly presented, and the experiments well justified and nicely discussed. It was overall a very enjoyable read. The figures are generally nice and clear, however, I find the legends excessively concise. A bit too often, they just sort of introduce the title of the panel rather than a proper explanation of what it is depicted. A clear case is for example visible in Fig 2, where the description of the panels is minimal, but this is a general trend of the manuscript. This makes the figures a bit hard to follow as self-contained entities, without having to continuously go back to the main text. I think this could be improved with longer and more helpful descriptions.

We have revised all figure legends to make them more descriptive.

Other minor things:

In the cDNA synthesis step for in-situ sequencing, I believe the authors might have forgotten one detail: the addition of aminoallyl dUTP to the RT reaction. If I recall correctly this is done in BARseq. The fact that the authors crosslink with BS-PEG on day 2, makes me suspect they spike in these nucleotides during the RT but this is not specified in the relevant step. Perhaps this is a mistake that needs correction.

The RT primers we used have an amine group at 5’, which directly allows crosslinking. Thus, we did not need to spike in aminoallyl dUTP in the RT reaction. We have clarified this in the Methods.

Reviewer #2 (Recommendations For The Authors):

Throughout the manuscript, there are frequent references to the "Methods" section for important details. However, it can be challenging to determine which specific section of the Methods the authors are referring to, and in some cases, a thorough examination of the entire Methods section fails to locate the exact information needed to support the authors' claims. Below are a few specific examples of this issue. The authors are encouraged to be more precise in their references to the Methods section.

In the revised manuscript, we numbered each subsection of Methods and updated pointers and associated hyperlinks in the main text to the subsection numbers.

• On page 7, line 14, it is unclear how the authors compared the cell marker gene expression with the marker gene expression in the reference cell type.

We have clarified in the revised manuscript.

• On page 7, line 33, the authors note that some barcodes may have been missed during the sequencing of the rabies virus libraries, but the Methods section lacked a convincing explanation on this issue (see my point 2 above).

We included a separate subsection on the sequencing of rabies libraries and the analysis of the sequencing depth in the Methods. In this new subsection, we further clarified our reasoning for identifying the lack of sequencing depth as a reason for missing barcodes, especially in comparison to sequencing depth required for establishing exact molecule counts used in established MAPseq and BARseq techniques with Sindbis libraries.

• On page 9, line 44, the authors state that they considered a barcode to be associated with a cell if they found at least six molecules of that barcode in a cell, as detailed in the Methods section. However, the rationale behind this level of stringency is not provided in the Methods.

We initially chose this threshold based on visual inspection of the sequencing images of the barcoded cells. Because the labeled cell types were consistent with our expectations (Fig. 4E-G), we did not further optimize the threshold for detecting retrogradely labeled barcoded cells.

• I have noticed that some important explanations of figure panels are missing in the legends, making it challenging to understand the figures. Below are typical examples of this issue.

In addition to the examples that the reviewer mentioned below, we also revised many other figure panels to make them clear to the readers.

• In Fig. 2, "RV into SC" in panel C does not make sense, as RV was injected into the thalamus. There is no explanation of the images in this panel C.

We have corrected the typo in the revision.

• In Fig. 3, information on the endogenous gene panel for cell type classification (Table S3) could be mentioned in the legend or corresponding text.

We now cite Table S3 both in Fig 3 legend and in the main text. We also included a list of the 104 cell type marker genes we used in Table S3.

• In panel J, it is unclear why the total number of BC cells is 2,752, and not 4,130 as mentioned in the text.

This is a typo. We have corrected this in the revision. The correct number (3,746) refers to the number of cells that did not belong to either of the two categories at the bottom of the panel, and not the total number of neurons. To make this clear, we now also include the total number of barcoded cells at the top of the panel.

• In Fig. 4, the definitions of "+" and "−" symbols in panels K and L are unclear. Also, it seems that the second left column of panel K should read "T −."

We corrected the typo in K, further clarified the “Area” labels, and changed the “S” label in 4K to “−”. This change does not change the original meaning of the figure: when considering the variance explained in L4/5 IT neurons, considering the subclass compositional profile is equivalent to not using the compositional profiles of cell types, because L4/5 IT neurons all belong to the same subclass (L4/5 IT subclass). Although operationally we simply considered subclass-level compositional profiles when calculating the variance explained, we think that changing this to “−” is clearer for the readers.

• In Fig. 5, panel E is uninterpretable.

We revised the main text and the figure to clarify how we manually proofread cells to determine the QC thresholds for barcoded cells. These plots showed a summary of the proofreading. We also revised the figures to indicate that they showed the fraction of barcoded cells that were considered real after proofreading. In the revised version, we moved these plots to Fig. S5.

• In Fig. S1, I do not understand the identity of the six samples on the X-axis of panel A (given that only two animals were described in the main text) and what panel B shows, including the definition of map_cluster_conf and map_cluster_corr.

In the revised Fig. S1, we made it more explicit that the six animals include both animals used for retrograde tracing (2 animals) and those used for trans-synaptic tracing (4 animals). We updated the y axis labels to be more readable and cited the relevant Methods section for definitions.

• In Fig. S2, please provide the definitions of blue and red dots and values in panel A, as well as the color codes and size of the circles in panel B. My overall impression from panel B is that there is no significant difference between RV-infected and non-infected cells. The authors should provide more quantitative and statistical support for the claim that "RV-infected cells had higher expression of immune response-related genes."

We toned down the statement to “Consistent with previous studies […], some immune response related genes were up-regulated in virus-infected cells compared to non-infected cells.” Because the main point of the single-cell RNAseq analysis was that rabies did not affect the ability to distinguish transcriptomic types, the change in immune response-related genes was not essential to the main conclusions. We clarified the red and blue dots in panel A and changed panel B to show the top up-regulated immune response-related genes in the revised manuscript.

• In Fig. S3, the definitions of the color code and circle size are missing.

We have added the legends in Fig. S3.

Before I hang out with my friends, we usually make the decision together. I think it's very important to listen to others' advises and preferences. For example, we have travelled to the Black Valley last summer. Before that trip, we discussed about it in detail in Wechat to make plans. The plan included destination, departure time, hotles, food, trasportation ways, tickets and so on. My wife was the one who careed about hotels mostly.She preferd romms with splendid views and discussed about the views of the hotels on our list a lot with my friends. She was also interested in food. She selected several restaurants on the Dianping App whch included the famous soup of pig's feet native. There seems something was forgotten? It's me , My opinion came as my wife's, I was just a driver.

It's kind of labelled because they know that an action has taken place between the images, just not what action it is.

Reviewer #2 (Public Review):

This paper seeks to determine whether the human visual system's sensitivity to causal interactions is tuned to specific parameters of a causal launching event, using visual adaptation methods. The three parameters the authors investigate in this paper are the direction of motion in the event, the speed of the objects in the event, and the surface features or identity of the objects in the event (in particular, having two objects of different colors).

The key method, visual adaptation to causal launching, has now been demonstrated by at least three separate groups and seems to be a robust phenomenon. Adaptation is a strong indicator of a visual process that is tuned to a specific feature of the environment, in this case launching interactions. Whereas other studies have focused on retinotopically-specific adaptation (i.e., whether the adaptation effect is restricted to the same test location on the retina as the adaptation stream was presented to), this one focuses on feature-specificity.

The first experiment replicates the adaptation effect for launching events as well as the lack of adaptation event for a minimally different non-causal 'slip' event. However, it also finds that the adaptation effect does not work for launching events that do not have a direction of motion more than 30 degrees from the direction of the test event. The interpretation is that the system that is being adapted is sensitive to the direction of this event, which is an interesting and somewhat puzzling result given the methods used in previous studies, which have used random directions of motion for both adaptation and test events.

The obvious interpretation would be that past studies have simply adapted to launching in every direction, but that in itself says something about the nature of this direction-specificity: it is not working through opposed detectors. For example, in something like the waterfall illusion adaptation effect, where extended exposure to downward motion leads to illusory upward motion on neutral-motion stimuli, the effect simply doesn't work if motion in two opposed directions is shown (i.e., you don't see illusory motion in both directions, you just see nothing). The fact that adaptation to launching in multiple directions doesn't seem to cancel out the adaptation effect in past work raises interesting questions about how directionality is being coded in the underlying process. In addition, one limitation of the current method is that it's not clear whether the motion-direction-specificity is also itself retinotopically-specific, that is, if one retinotopic location were adapted to launching in one direction and a different retinotopic location adapted to launching in the opposite direction, would each test location show the adaptation effect only for events in the direction presented at that location?

The second experiment tests whether the adaptation effect is similarly sensitive to differences in speed. The short answer is no; adaptation events at one speed affect test events at another. Furthermore, this is not surprising given that Kominsky & Scholl (2020) showed adaptation transfer between events with differences in speeds of the individual objects in the event (whereas all events in this experiment used symmetrical speeds). This experiment is still novel and it establishes that the speed-insensitivity of these adaptation effects is fairly general, but I would certainly have been surprised if it had turned out any other way.

The third experiment tests color (as a marker of object identity), and pits it against motion direction. The results demonstrate that adaptation to red-launching-green generates an adaptation effect for green-launching-red, provided they are moving in roughly the same direction, which provides a nice internal replication of Experiment 1 in addition to showing that the adaptation effect is not sensitive to object identity. This result forms an interesting contrast with the infant causal perception literature. Multiple papers (starting with Leslie & Keeble, 1987) have found that 6-8-month-old infants are sensitive to reversals in causal roles exactly like the ones used in this experiment. The success of adaptation transfer suggests, very clearly, that this sensitivity is not based only on perceptual processing, or at least not on the same processing that we access with this adaptation procedure. It implies that infants may be going beyond the underlying perceptual processes and inferring genuine causal content. This is also not the first time the adaptation paradigm has diverged from infant findings: Kominsky & Scholl (2020) found a divergence with the object speed differences as well, as infants categorize these events based on whether the speed ratio (agent:patient) is physically plausible (Kominsky et al., 2017), while the adaptation effect transfers from physically implausible events to physically plausible ones. This only goes to show that these adaptation effects don't exhaustively capture the mechanisms of early-emerging causal event representation.

One overarching point about the analyses to take into consideration: The authors use a Bayesian psychometric curve-fitting approach to estimate a point of subjective equality (PSE) in different blocks for each individual participant based on a model with strong priors about the shape of the function and its asymptotic endpoints, and this PSE is the primary DV across all of the studies. As discussed in Kominsky & Scholl (2020), this approach has certain limitations, notably that it can generate nonsensical PSEs when confronted with relatively extreme response patterns. The authors mentioned that this happened once in Experiment 3 and that a participant had to be replaced. An alternate approach is simply to measure the proportion of 'pass' reports overall to determine if there is an adaptation effect. I don't think this alternate analysis strategy would greatly change the results of this particular experiment, but it is robust against this kind of self-selection for effects that fit in the bounds specified by the model, and may therefore be worth including in a supplemental section or as part of the repository to better capture the individual variability in this effect.

In general, this paper adds further evidence for something like a 'launching' detector in the visual system, but beyond that, it specifies some interesting questions for future work about how exactly such a detector might function.

Kominsky, J. F., & Scholl, B. J. (2020). Retinotopic adaptation reveals distinct categories of causal perception. Cognition, 203, 104339. https://doi.org/10.1016/j.cognition.2020.104339

Kominsky, J. F., Strickland, B., Wertz, A. E., Elsner, C., Wynn, K., & Keil, F. C. (2017). Categories and Constraints in Causal Perception. Psychological Science, 28(11), 1649-1662. https://doi.org/10.1177/0956797617719930

Leslie, A. M., & Keeble, S. (1987). Do six-month-old infants perceive causality? Cognition, 25(3), 265-288. https://doi.org/10.1016/S0010-0277(87)80006-9

I disagree with this statement. Content on the Internet can have significant effects on a person's metal state. This statement tells the person that it's okay because the Internet is not real life. However, there is such a widespread access to the Internet that it might as well be real life - it's just a new form of communication.

Jonathan Blow recently revisited this topic with Casey Muratori. (They last talked about this 3 years ago.)

What's a little absurd is that (a) the original discussion is something like 3–6 hours long and doesn't use recursive descent—instead they descended into some madness about trying to work out from first principles how to special-case operator precedence—and (b) they start out in this video poo-pooing people who speak about "recursive descent", saying that it's just a really obnoxious way to say writing ordinary code—again, all this after they three years ago went out of their way to not "just" write "normal" code—and (c) they do this while launching into yet another 3+ hour discussion about how to do it right—in a better, less confusing way this time, with Jon explaining that he spent "6 or 7 hours" working through this "like 5 days ago". Another really perverse thing is that when he talks about Bob's other post (Parsing Expressions) that ended up in the Crafting Interpreters book, he calls it stupid because it's doing "a lot" for something so simple. Again: this is to justify spending 12 hours to work out the vagaries of precedence levels and reviewing a bunch of papers instead of just spending, I dunno, 5 or 10 minutes or so doing it with recursive descent (the cost of which mostly comes down to just typing it in).

So which one is the real chore? Doing it the straightforward, fast way, or going off and attending to one's unrestrained impulse that you for some reason need to special-case arithmetic expressions (and a handful of other types of operations) like someone is going to throw you off a building if you don't treat them differently from all your other ("normal") code?

Major blind spots all over.

Papercut: Windows-based SMTP server/interceptor

ChangemakerStudios/Papercut-SMTP: Papercut SMTP -- The Simple Desktop Email Server

you can only burn into human long-term memory a certain number of things per unit time like maybe one every 30 seconds or something and other than that it's just short-term memory and it flows away like water and so on

focal vs subsidiary tacit awareness - mirrors short vs long term memory

Author Response

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

eLife assessment

The paper contains some useful analysis of existing data but there are concerns regarding the conclusion that there might be alternative mechanisms for determining the location of origins of DNA replication in human cells compared to the well known mechanism known from many eukaryotic systems, including yeast, Xenopus, C. elegans and Drosophila. The lack of overlap between binding sites for ORC1 and ORC2, which are known to form a complex in human cells, is a particular concern and points to the evidence for the accurate localization of their binding sites in the genome being incomplete.

Public Reviews:

Reviewer #1 (Public Review):

In the best genetically and biochemically understood model of eukaryotic DNA replication, the budding yeast, Saccharomyces cerevisiae, the genomic locations at which DNA replication initiates are determined by a specific sequence motif. These motifs, or ARS elements, are bound by the origin recognition complex (ORC). ORC is required for loading of the initially inactive MCM helicase during origin licensing in G1. In human cells, ORC does not have a specific sequence binding domain and origin specification is not specified by a defined motif. There have thus been great efforts over many years to try to understand the determinants of DNA replication initiation in human cells using a variety of approaches, which have gradually become more refined over time.

In this manuscript Tian et al. combine data from multiple previous studies using a range of techniques for identifying sites of replication initiation to identify conserved features of replication origins and to examine the relationship between origins and sites of ORC binding in the human genome. The authors identify a) conserved features of replication origins e.g. association with GC-rich sequences, open chromatin, promoters and CTCF binding sites. These associations have already been described in multiple earlier studies. They also examine the relationship of their determined origins and ORC binding sites and conclude that there is no relationship between sites of ORC binding and DNA replication initiation. While the conclusions concerning genomic features of origins are not novel, if true, a clear lack of colocalization of ORC and origins would be a striking finding. However, the majority of the datasets used do not report replication origins, but rather broad zones in which replication origins fire. Rather than refining the localisation of origins, the approach of combining diverse methods that monitor different objects related to DNA replication leads to a base dataset that is highly flawed and cannot support the conclusions that are drawn, as explained in more detail below.

Response: We are using the narrowly defined SNS-seq peaks as the gold standard origins and making sure to focus in on those that fall within the initiation zones defined by other methods. The objective is to make a list of the most reproducible origins. Unlike what the reviewer states, this actually refines the dataset to focus on the SNS origins that have also been reproduced by the other methods in multiple cell lines. We have changed the last box of Fig. 1A to make this clearer: Shared origins = reproducible SNS-seq origins that are contained in initiation zones defined by Repli-seq, OK-seq and Bubble-seq. This and the Fig. 2B (as it is) will make our strategy clearer.

Methods to determine sites at which DNA replication is initiated can be divided into two groups based on the genomic resolution at which they operate. Techniques such as bubble-seq, ok-seq can localise zones of replication initiation in the range ~50kb. Such zones may contain many replication origins. Conversely, techniques such as SNS-seq and ini-seq can localise replication origins down to less than 1kb. Indeed, the application of these different approaches has led to a degree of controversy in the field about whether human replication does indeed initiate at discrete sites (origins), or whether it initiates randomly in large zones with no recurrent sites being used. However, more recent work has shown that elements of both models are correct i.e. there are recurrent and efficient sites of replication initiation in the human genome, but these tend to be clustered and correspond to the demonstrated initiation zones (Guilbaud et al., 2022).

These different scales and methodologies are important when considering the approach of Tian et al. The premise that combining all available data from five techniques will increase accuracy and confidence in identifying the most important origins is flawed for two principal reasons. First, as noted above, of the different techniques combined in this manuscript, only SNS-seq can actually identify origins rather than initiation zones. It is the former that matters when comparing sites of ORC binding with replication origin sites, if a conclusion is to be drawn that the two do not co-localise.

Response: We agree. So the reviewer should agree that our method of finding SNS-seq peaks that fall within initiation zones actually refines the origins to find the most reproducible origins. We are not losing the spatial precision of the SNS-seq peaks.

Second, the authors give equal weight to all datasets. Certainly, in the case of SNS-seq, this is not appropriate. The technique has evolved over the years and some earlier versions have significantly different technical designs that may impact the reliability and/or resolution of the results e.g. in Foulk et al. (Foulk et al., 2015), lambda exonuclease was added to single stranded DNA from a total genomic preparation rather than purified nascent strands), which may lead to significantly different digestion patterns (ie underdigestion). Curiously, the authors do not make the best use of the largest SNS-seq dataset (Akerman et al., 2020) by ignoring these authors separation of core and stochastic origins. By blending all data together any separation of signal and noise is lost. Further, I am surprised that the authors have chosen not to use data and analysis from a recent study that provides subsets of the most highly used and efficient origins in the human genome, at high resolution (Guilbaud et al., 2022).

Response: 1) We are using the data from Akerman et al., 2020: Dataset GSE128477 in Supplemental Table 1. We have now separately examined the core origins defined by the authors to check its overlap with ORC binding (Supplementary Fig. S8b)

2) To take into account the refinement of the SNS-seq methods through the years, we actually included in our study only those SNS-seq studies after 2018, well after the lambda exonuclease method was introduced. Indeed, all 66 of SNS-seq datasets we used were obtained after the lambda exonuclease digestion step. To reiterate, we recognize that there may be many false positives in the individual origin mapping datasets. Our focus is on the True positives, the SNS-seq peaks that have some support from multiple SNS-seq studies AND fall within the initiation zones defined by the independent means of origin mapping (described in Fig. 1A and 2B). These True positives are most likely to be real and reproducible origins and should be expected to be near ORC binding sites.

We have changed the last box of Fig. 1A to make this clearer: Shared origins = reproducible SNS-seq origins that are contained in initiation zones defined by Repli-seq, OK-seq or Bubble-seq.

Ini-seq by Torsten Krude and co-workers (Guillbaud, 2022) does NOT use Lambda exonuclease digestion. So using Ini-seq defined origins is at odds with the suggestion above that we focus only on SNS-seq datasets that use Lambda exonuclease. However, Ini-seq identifies a much smaller subset of SNS-seq origins, so, as requested, we have also done the analysis with just that smaller set of origins, and it does show a better proximity to ORC binding sites, though even then the ORC proximate origins account for only 30% of the Ini-seq2 origins (Supplementary Fig. S8d). Note Ini-seq2 identifies DNA replication initiation sites seen in vitro on isolated nuclei.

Update in response to authors' comments on the original review:

While the authors have clarified their approach to some aspects of their analysis, I believe they and I are just going to have to disagree about the methodology and conclusions of this work. I do not find the authors responses sufficiently compelling to change my mind about the significance of the study or veracity of the conclusions. In my opinion, the method for identification of strong origins is not robust and of insufficient resolution. In addition, the resolution and the overlap of the MCM Chip-seq datasets is poor. While the conclusion of the paper would indeed be striking and surprising if true, I am not at all persuaded that it is based on the presented data.

Reviewer #2 (Public Review):

Tian et al. performed a meta-analysis of 113 genome-wide origin profile datasets in humans to assess the reproducibility of experimental techniques and shared genomics features of origins. Techniques to map DNA replication sites have quickly evolved over the last decade, yet little is known about how these methods fare against each other (pros and cons), nor how consistent their maps are. The authors show that high-confidence origins recapitulate several known features of origins (e.g., correspondence with open chromatin, overlap with transcriptional promoters, CTCF binding sites). However, surprisingly, they find little overlap between ORC/MCM binding sites and origin locations.

Overall, this meta-analysis provides the field with a good assessment of the current state of experimental techniques and their reproducibility, but I am worried about: (a) whether we've learned any new biology from this analysis; (b) how binding sites and origin locations can be so mismatched, in light of numerous studies that suggest otherwise; and (c) some methodological details described below.

• I understand better the inclusion/exclusion logic for the samples. But I'm still not sure about the fragments. As the authors wrote, there is both noise and stochasticity; the former is not important but the latter is essential to include. How can these two be differentiated, and what may be the expected overlap as a function of different stochasticity rates?

It is difficult to separate the effect of noise from the effect of stochastic firing of origins. We therefore took the simplest approach: focus only on the most reproducible origins (shared origins) and ignore the non-reproducible origins. At least the most reproducible origins can be used to test the hypotheses regarding origin firing.

• Many of the major genomic features analyzed have already been found to be associated with origin sites. For example, the correspondence with TSS has been reported before:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6320713/

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6547456/

• Line 250: The most surprising finding is that there is little overlap between ORC/MCM binding sites and origin locations. The authors speculate that the overlap between ORC1 and ORC2 could be low because they come from different cell types. Equally concerning is the lack of overlap with MCM. If true, these are potentially major discoveries that butts heads with numerous other studies that have suggested otherwise.

The key missing dataset is ORC1 and ORC2 CHiP-seq from the same cell type. This shouldn't be too expensive to perform, and I hope someone performs this test soon. Without this, I remain on the fence about how much existing datasets are "junk" vs how much the prevailing hypothesis about replication needs to be revisited. Nonetheless, the authors do perform a nice analysis showing that existing techniques should be carefully used and interpreted.

We agree that a thorough set of ChIP-seq data (with multiple antibodies or with equivalent techniques that do not use antibodies) for all six subunits of ORC in mammalian cells will be very useful for the field. Note, though, that just by simple cell lysis, it is very easy to divide human ORC into at least three different parts: ORC1, ORC2-5, and ORC6. The subunits do not form as robust a complex as seen in the yeasts and in flies.

Reviewer #3 (Public Review):

Summary: The authors present a thought-provoking and comprehensive re-analysis of previously published human cell genomics data that seeks to understand the relationship between the sites where the Origin Recognition Complex (ORC) binds chromatin, where the replicative helicase (Mcm2-7) is loaded, and where DNA replication actually beings (origins). The view that these should coincide is influenced by studies in yeast where ORC binds site-specifically to dedicated nucleosome-free origins where Mcm2-7 can be loaded and remains stably positioned for subsequent replication initiation. However, this is most certainly not the case in metazoans where it has already been reported that chromatin bindings sites of ORC and Mcm2-7 do not necessarily overlap, nor do they always overlap with origins. This is likely due to Mcm2-7 possessing linear mobility on DNA (i.e., it can slide) such that other chromatin-contextualized processes can displace it from the site in which it was originally loaded. Additionally, Mcm2-7 is loaded in excess and thus only a fraction of Mcm2-7 would be predicted to coincide with replication start sites. This study reaches a very similar conclusion of these previous studies: they find a high degree of discordance between ORC, Mcm2-7, and origin positions in human cells.

Strengths: The strength of this work is its comprehensive and unbiased analysis of all relevant genomics datasets. To my knowledge, this is the first attempt to integrate these observations. It also is an important cautionary tale to not confuse replication factor binding sites with the genomic loci where replication actually begins, although this point is already widely appreciated in the field. Response: Thank you for recognizing the comprehensive and unbiased nature of our analysis. Our findings will prevent the unwise adoption of ORC or MCM binding sites as surrogate markers of origins and will stimulate the field to try and improve methods of identifying ORC or MCM binding until the binding sites are found to be proximal to the most reproducible origins. The last possibility is that there are ORC- or MCM-independent modes of defining origins, but we have no evidence of that.

Weaknesses: The major weakness of this paper is the lack of novel biological insight and that the comprehensive approach taken failed to provide any additional mechanistic insight regarding how and why ORC, Mcm2-7, and origin sites are selected or why they may not coincide.

Response: we agree that we cannot provide a novel biological insight from this kind of meta-analysis. The importance of this study is in highlighting that there is either significant problems with the data collected till now (preventing the co-localization of ORC or MCM binding sites with the most reproducible origins) or ORC and MCM binding sites are often far away from where the most reproducible origins fire, which should make us consider ways in which origins could be activated kilobases away from ORC and MCM binding sites.

Recommendations for the authors:

Reviewer #2 (Recommendations For The Authors):

All suggestions and recommendations were described in a previous review.

Reviewer #3 (Recommendations For The Authors):

The most significant omission is a contextualization of the results in the discussion and an explanation of why these results matter for the biology of replication, disease, and/or our confidence in the genomic techniques reported on in this study. As written, the discussion simply restates the results without any interpretation towards novel insight. I suggest that the authors revise their discussion to fill this important gap.

A second important, unresolved point is whether replication origins identified by the various methods differ due to technical reasons or because different cell types were analyzed. Given the correlation between TSS and origins (reported in this study but many others too), it is somewhat expected that origins will differ between cell types as each will have a distinct transcriptional program. This critique is partly addressed in Figure S1C. However, given the conclusion that the techniques are only rarely in agreement (only 0.27% origins reproducibly detected by the four techniques), a more in-depth analysis of cell type specific data is warranted. Specifically, I would suggest that cell type-specific data be reported wherever origins have been defined by at least two methods in the same cell type, specifically reporting the percent of shared origins amongst the datasets. This type of analysis may also inform on whether one or more techniques produces the highest (or lowest) quality list of true origins.

We have done what has been suggested: used K562 cell type-specific data because here the origins have been defined by at least two methods in the same cell type and reported the percent of shared origins amongst the datasets (Supp. Fig. S4).

Other MINOR comments include:

• Line 215: the authors show that shared origins overlap with TF binding hotspots more often than union origins, which they claim suggests "that they are more likely to interact with transcription factors." As written, it sounds like the authors are proposing that ORC may have some direct physical interaction with transcription factors. Is this intended? If so, what support is there for this claim?

The reviewer is correct. We have rephrased because we have no experimental support for this claim.

• In the text, Figure 3G is discussed before Figure 3F. I suggest switching the order of these panels in Figure 3.

Done.

• It's not clear what Figure 5H to Figure 6 accomplishes. What specifically is added to the story by including these data? Is there something unique about the high confidence origins? If there is nothing noteworthy, I would suggest removing these data.

We want to keep them to highlight the small number of origins that meet the hypothesis that ORC and MCM must bind at or near reproducible origins. These would be the origins that the field can focus in on for testing the hypothesis rigorously. They also show the danger of evaluating proximity between ORC or MCM binding sites with origins based on a few browser shots. If we only showed this figure, we could conclude that ORC and MCM binding sites are very close to reproducible origins.

• Line 394: "Since ORC is an early factor for initiating DNA replication, we expected that shared human origins will be proximate to the reproducible ORC binding sites." This is only expected if one disbelieves the prior literature that shows that ORC and origins are not, in many cases, proximal. This statement should be revised, or the previous literature should be cited, and an explanation provided about why this prior work may have missed the mark.

We do not know of any genome-wide study in mammalian cell lines where ORC binding sites and MCM binding have been compared to highly reproducible origins, or that show that these binding sites and highly reproducible origins are mostly not proximal to each other. Most studies cherry pick a few origins and show by ChIP-PCR that ORC and/or MCM bind near those sites. Alternatively, studies sometimes show a selected browser shot, without a quantitative measure of the overlap genome wide and without doing a permutation test to determine if the observed overlap or proximity is higher than what would be expected at random with similar numbers of sites of similar lengths. In the revised manuscript we have discussed Dellino, 2013; Kirstein, 2021; Wang, 2017; Mas, 2023. None of them have addressed what we are addressing, is the small subset of the most reproducible origins proximal to ORC or MCM binding sites?

• Line 402-404: given the lack of agreement between ORC binding sites and origins the authors suggest as an explanation that "MCM2-7 loaded at the ORC binding sites move much further away to initiate origins far from the ORC binding sites, or that there are as yet unexplored mechanisms of origin specification in human cancer cells". The first part of this statement has been shown to be true (Mcm2-7 movement) and should be cited. But what do the authors mean by the second suggestion of "unexplored mechanisms"? Please expand.

We have addressed this point in the revised manuscript.

• The authors should better reference and discuss the previous literature that relates to their work, some of these include Gros et al., 2015 Mol Cell, Powell et al., 2015 EMBO J, Miotto et al., 2016 PNAS, but likely there are many others.

We have addressed this point in the revised manuscript.

Note for authors:

Line 107: The introduction discusses the mechanism for yeast ORC recognizes specific origins and discusses the Orc4 contribution, but it is known that Orc2 also binds DNA on a base-specific manner (see PMID 33056978). Thus Lee et al. did not "humanize ORC" as stated.

Done

Lines 117-119: Two of the cited papers are on endo-reduplication and not on initiation in a normal cell cycle and this should be pointed out. Second, there is contradictory evidence that ORC is essential in human cells and this should be cited (PMID 33522487)

Done

What's wrong with that?

Seems a bit crackpotty

“It was Autism all along. I was not broken, I was not damaged, I was just different. I had been born different... ...And now after a long crawl back to life, I am reconnecting with those lost parts of myself, reaching out for their streaming apron strings in a delightful chase...(BIG) A race I cannot lose really, because it’s me and she and they and parts of me – they can gleefully run and hide as we play this delicious game of rediscovery. And I am ok just as I am, with all of my weirdness. I am reunited with that young part of myself who has been exiled for so long. I owe her nothing but my undying devotion to make up for all the years I shut her away.”(SMALLER font) FIX AT REVIEW

*

*

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interesting

• for: speculative investing - example

• example - speculative investing

  • stock market
    • money is not reaching companies and being used in a productive way
    • part of it must be, but whenever shareholders take earnings, then it's extracted out
  • private equity
    • when private equity firms buy companies then layoff staff and cut back spending on services, they pocket all that money for the shareholders. It's a way for the rich to maintain their supremacy position

• comment

  • In its simplest expression, it is greed in action
  • It is what maintains the 1% / 99% divide

• epiphany

• new meme
  • We need to replace WALL street with WELL street!

The success for me is the very writing you create. That you spend the time to sit down and craft all these words and put everything together around once a week is very impressive.

I'm certain it helps you to clarify your thoughts. And to develop new roads of thought. It's also nice to be able to look back at your archive. Just like you did with the lemonade search. See how you covered something (or not covered at all). The more you write, the more you can intersect things. And actually, that's one method of getting more subscribers. When writers demonstrate that their work is interconnected with their previous writings, it shows themes. It shows your investment, that you cover these themes in depth. Linking to previous articles shows that you care about your previous writings, and that means your future writings will be good too.

And the search engines like the interlinking. That helps the search engines do their job. When you interlink stuff, the search engines know better how your work relates to each other, and what the important topics are.

And the same for readers. The readers get to read more of your work that way. They discover more of your archives. They get more invested. The more invested your readers are, the more likely they are to share your work. And then your work spreads. From time to time I've linked to your writing from my site.

It takes time to interlink all your work. I haven't really done a good job on my site. Maybe I should do that more. But it all helps to develop a larger readership.

Which then leads to more engagement. That's really my primary goal with writing.

Speaking of engagement/comments, Substack is a curious land in that regard. I've seen some substacks with lots of comments. Some with very few. It raises the question if Substack is built for comments. The simplicity of the UI certainly helps to get more comments. (in comparison on my blog, the design is too bloated at the end of the article to get the comments).

However, I've noticed that when you post your articles on Facebook, that you get more comments there. And that's because Facebook is totally built for comments. That comment box is right there. Boom. You don't even have to scroll to the bottom of the post to leave a comment. Of course, that's weird, because people would leave a comment without reading the whole thing? Alas, that's how the internet works. Even with that point aside, that someone would comment without reading the full post.... Facebook's UI gets people in the mood to comment, because the comment box is visible right away. It sets the tone for commenting.

I've thought about designing my site to mimick that--having the comment box appear right away near the top. Just never got to it.

Now, to be clear, I'm NOT advocating that you post your full-length articles on Facebook. You aren't doing that now, and I don't need to get into all the reasons why to not do that, because you are probably already aware of that.

Ok, back to your point you feel like something needs to change to keep your own interest up. I'm curious what you are looking for. More subscribers? More comments? I'm guessing you'd like both. But with your writing, I also get the sense that you enjoy writing them.

I'll follow you on whatever platform you choose.

I personally prefer posting on my hosted WordPress. Although that very much lacks the whole discoverability feature that Substack has. Plus, Substack is just much simplier. Both for the writer and for the audience.

The writer on substack doesn't have to worry about themes. Or writing in WordPress' block system.

The audience on Substack gets a cleaner experience. All your subs in one place. Nice easy email subscriptions. Substack is nice.

But there's something about having WordPress on MY site. And it's all MY files. Although most likely, I like WordPress because I've self-hosted my blog since 2021. I got some income from it from Google Ads in the early 2010s. But now those days are long-gone. Especially with AI cutting out the search engine business.

Perhaps I need to realize that the days for writers are now on services that focus on getting subscribers. Not on getting ads displayed.

Anyhow, that doesn't answer your question. Just rambling thoughts on services.

Robert creates a melancholic tone and shows the exhaustion of the speaker after a long day of labor. It gives the speakers personal experience as he works so hard that sometimes all his mind can think about is picking apple, but truly he longs for sleep, a deep sleep that can give him a break from the cycle of labor. This truly represents romanticism as it shows the individual experience of the common man.

This is just not very well-explained imo

For those of your reading (as beginners will surely have trouble understanding the code here) probability <- rep(1/6,6) meant that we created a vector called probability whose value is given by rep(1/6, 6) which basically means "repeat '1/6' six times", it's the same thing as doing probability <- (1/6, 1/6, 1/6, 1/6, 1/6, 1/6)

Also, pch=20 basically means the value points are represented by filled dots (which is number 20 in the Plotting CHaracteristics parameter)

This work has been published in GigaScience Journal under a CC-BY 4.0 license (https://doi.org/10.1093/gigascience/giad100), and has published the reviews under the same license. These are as follows.

**Reviewer 1. Brandon Pickett **

Overall, this manuscript is well-written and understandable. There's a lot of good work here and I think the authors were thoughtful about how to compare the resulting assemblies. Scripts and models used have been made available for free via GitHub and could be mirrored on or moved to GigaDB if required. I'll include a several minor comments, including some line-item edits, but the bulk of my comments will focus on a few major items.

Major Comments: My primary concern here is that the comparison is outdated and doesn't address some of the most helpful questions. CLR-only assemblies are no longer state-of-the-art. There are still applications and situations where ONT (simplex, older-pore)-only assemblies are reasonable, but most projects that are serious about generating excellent assemblies as references are unlikely to take that approach.

Generating assemblies for non-reference situations, especially when the sequencing is done "in the field" (e.g., using a MinION with a laptop) or by a group with insufficient funding or other access to PromethIONs and Sequel/Revios, is an exception to this for ONT-only assemblies. Further, this work assumes a person wants to generate "squashed" assemblies instead of haplotype-resolved or pseudohaplotype assemblies. To be fair, sequencing technology in the TGS space has been advancing so rapidly that it is extremely difficult to keep up, and a sequencing run is often outdated by the time analyses are finished, not to mention by the time a manuscript is written, reviewed, and published.

Accordingly, in raising my concerns, I am not objecting to the analysis being published or suggesting that the work performed was poor, but I do believe clarifications and discussion are necessary to contextualize the comparison and specify what is missing.

1. This comparison seeks to address Third-generation sequencing technologies: namely PacBio vs. ONT. However, each company offers multiple kinds of long-read sequencing, and they are not all comparable in the same way. Just as long noisy reads (PacBio CLR & ONT simplex) are a whole new generation from "NGS" short reads like from Illumina, long-accurate reads are arguably a new generation beyond noisy long reads. If this paper wants to include PacBio HiFi reads in the comparison, significant changes are necessary to make the comparison meaningful. I think it's reasonable to drop HiFi reads from this paper altogether and focus on noisy long reads since the existing comparison isn't currently set up to tell us enough about HiFi reads and including them would be an ordeal. If including HiFi, consider the following:

1.a. Use assemblers designed for long-accurate reads. HiCanu (i.e., Canu with --pacbio-hifi option) is already used, as is a similar approach for Flye and wtdbg2. However, raven is not meant for HiFi data and miniasm is not either (though, it could be done with the correct minimap2 settings, but Hifiasm would be better). Assemblies of HiFi data with Raven and miniasm should be removed. Sidenote – Raven can be run with --weaken (or similar) for HiFi data, but it is only experimental and the parameter has since been removed. Including Hifiasm would be necessary, and it should have been included since Hifiasm was out when this analysis was done. Similarly, including MBG (released before your analysis was done) would be appropriate. Since you'd be redoing the analyses, it would be appropriate to include other assemblers that have since been released: namely LJA. Once could argue that Verkko should be included, but that opens another can of worms as a hybrid assembler (more on that later).

1b. Use a read simulator that is built for HiFi reads. Badreads is not built for HiFi data (though using custom parameters to make it work for HiFi reads wasn't a bad idea at the time), and new simulators (e.g., PBSIM3, DOI: 10.1093/nargab/lqac092) have since been released that consider the multi-pass process used to generate HiFi data.

1c. ONT Duplex data is likely not available for the species you've chosen as it is a very new technology. However, you should at least discuss its existence as something for readers to "keep an eye on" as something that is conceptually comparable to HiFi. 1d. Use the latest & greatest HiFi data if possible and at least discuss the evolution of HiFi data. Even better would be to compare HiFi data over time, but this data may not really be available and most people won't be using older HiFi data. Though, simulation of older data would conceivably be possible. While doing so would make this paper more complete, I would argue that it isn't worth the effort at this juncture. For reference, in my observation, older data has a median read length around 10-15 kb instead of 18-22 kb. 1e. Include real Hifi data for the species you are assembling. If none is available and you aren't in a position to generate it, then keep the hifi assembler comparison on real data separate from that of the CLR/ONT assembler comparisons on real data by using real HiFi data for other species. 2. Discuss in the intro and/or discussion that you are focusing on "squashed" assemblies. Without clever sample separation and/or trio-based approaches (e.g., DOI: 10.1038/nbt.4277), a single squashed haplotype is the only possible outcome for PacBio CLR and ONT-only approaches. For non-haploid genomes, other approaches (HiFi-only or hybrid approaches (e.g., HiFi + ONT or HiFi + Hi-C)) can generate pseudohaplotypes at worse and fully-resolved haplotypes at best. The latter is an objectively better option when possible, and it's important to note that this comparison wouldn't apply when planning a project with such goals. Similarly, it would probably be helpful to point out to the novice reader that this comparison doesn't apply to metagenome assembly either. 3. The title suggests to the reader that we'll be shown how long reads makes a difference in assembly compared to non-long read approaches. However, this is not the case, despite some mention of it in near line 318. Short read assemblies are not compared here and no discussion is provided to suggest how long read-based assemblies would improve outcomes in various situations relative to short reads. Unless such a comparison and/or discussion is added, I think the title should be changed. I've included this point in the "Major Comments" section because including such a comparison would be a big overhaul, but I don't expect this to be done. The core concern is that the analysis is portrayed correctly. 4. Sequencing technologies are often portrayed as static through time, but this is not accurate. This is a failing of the field generally. Part of the problem is the length of the publishing cycle (often >1yr from when a paper is written to when it's published, not to mention how long it takes to do the analysis before a paper is even written). Part of the problem is that current statistics are often cited in influential papers and then recited in more recent papers based on the influential paper despite changes having been made since that influential paper was released. Accordingly, the error rate in ONT reads has been misreported as being ~15% for many years even though their chemistry has improved over time and the machine learning models (especially for human samples) have also improved, dropping the error rate substantially. ONT has made improvements to their chemistry and changed nanopores over time and PacBio has tinkered with their polymerase and chemistry too. Accordingly, a better question for a person planning to perform an assembly would be "which assembler is best for my datatype (pacbio clr vs ont) and chemistry/etc.?" instead of just differentiating by company. Any comparison of those datatypes should at least address this as a factor in their discussion, if not directly in their analysis. I feel that this is missing from this comparison. In an ideal world, we'd have various CLR chemistries and ONT pores/etc. for each species in this analysis. That data likely doesn't exist for each of the chosen species though, and generating it would be non-trivial, especially retroactively. Using the most recent versions is a good option, but may also not exist for every species chosen. Since this analysis was started (circa Nov/Dec 2021 by my estimate based on the chosen assembler versions), ONT has released pore 10; in combination with the most recent release of Guppy, error rates <=3% are expected for a huge portion of the data. That type of data is likely to assemble very differently from R9.4, and starker differences would be expected for data older than R9.4. Even if all the data were the most recent (or from the same generation (e.g., R9.4)), library preps vary greatly, especially between UL (ultralong) libraries and non-UL libraries. Having reads >100kb, especially a large number of them, makes a big difference in assembly outcome in my observation. How does choice of assembler (and possibly different parameters) affect the assembly when UL data is included? How is that different from non-UL data? What about UL data at different percentages of the reads being considered UL? A paper focusing on long noisy reads would be much more impactful if it addresses these questions. Again, this may not be possible for this particular paper considering what's already been done and the available funding, and I think that's okay. However, these issues need to addressed in the discussion as open questions and suggested future work. The type of CLR and ONT data also needs to be specified in this work, e.g., in a supplemental table, and if the various datasets are not from the same types, these differences need to be acknowledged. At a minimum, I think the following data points should be included: chemistry/pore information (e.g., R9.4 for ONT or P2/C5 for PacBio), basecaller (e.g., guppy vX.Y.Z), and read length distribution info (e.g., mean, st. dev., median, %>100kb), ideally a plot of the distribution in addition to summary values. I also understand that these data were generated previously by others, and this information should theoretically be available from their original publications, which are hopefully accessible via the INSDC records associated with the provided accessions. The objective here is making the information easily accessible to the readers of this paper because those could be confounding variables in the analysis.

1. This comparison considered only a single coverage level (30x). That's not an unreasonable shortcut, but it certainly leaves a lot of room for differences between assemblers. If the objective the paper is to help future project planners decide what assembler to use, it would be most helpful if they had an idea of what coverage they can use and still succeed. That's especially true for projects that don't have a lot of funding or aren't planning to make a near-perfect reference genome (which would likely spend the money on high coverage of multiple datatypes). It would be helpful to include some discussion about how these results may be different at much lower (e.g., 2x or 10x coverage) or at higher coverage (e.g., 50x, 70x, etc.) and/or provide some justification from another study for why including that kind of comparison would be unlikely to be worthwhile for this study, even if project planners should consider those factors when developing their budget and objectives.
2. Figure 2 and 3 include a lot of information, and I generally like how they look and that they provide a quick overview. I believe two things are missing that will improve either the assessment or the presentation of the information, and I think one change will also improve things. 6a. I think metrics from Merqury (DOI: 10.1186/s13059-020-02134-9) should be included where possible. Specifically, the k-mer completeness (recovery rate) and reference-free QV estimate (#s 1 and 3 from https://github.com/marbl/merqury/wiki/2.-Overall-k-mer-evaluation). Generally these are meant to be done from data of the same individual. However, most of the species selected for this comparison are highly homozygous strains that should have Illumina data available, and thus having the data come from not the exact some individual will likely be okay. This can serve as another source of validation. If such a dataset is not available for 1 or more of these species, then specify in the text that it wasn't available, and thus such an evaluation wasn't possible. If it's not possible to add one or both of these metrics to the figures (2 & 3), that's fine, but having it as a separate figure would still be helpful. I find these values to be some of the most informative for the quality of an assembly. 6b. It's not strictly necessary, so this might be more of a minor comment, but I found that I wanted to view individual plots for each metric. Perhaps including such plots in the supplement would help (e.g., 6 sets of plots similar to figure 4 with color based on assembler, grouping based on species, and opacity based on datatype). The specifics aren't critical, I just found it hard to get more than a very general idea from the main figures and wanted something easy to digest for each metric. 6c. Using N50/NG50 for a measure of contiguity is an outdated and often misleading approach. Unfortunately, it's become such common practice that many people feel obligated to include it or use it. Instead, the auN (auNG) would be a better choice for contiguity: https://lh3.github.io/2020/04/08/a-new-metric-on-assembly-contiguity.
3. This paper focuses on assembly and intentionally does not consider polishing (line 176), which I think is a reasonable choice. It also does not consider scaffolding or hybrid assembly approaches (again, reasonable choices). In the case of hybrid assembly options, most weren't available when this analysis was done (short read + long read assemblers were available, but I think it's perfectly reasonable to not have included those). Given the frequency of scaffolding (especially with Hi-C data [DOIs:10.1371/journal.pcbi.1007273 & 10.1093/bioinformatics/btac808]) and the recent shift to hybrid assemblers (e.g., phasing HiFi-based string graphs using Hi-C data to get haplotype resolved diploid assemblies (albeit with some switch errors) [DOI: 10.1038/s41587-022-01261-x] or resolving HiFi-based minimizer de bruijn graphs using ONT data and parental Illumina data to get complete, T2T diploid assemblies [DOI: 10.1038/s41587-023-01662-6]), I think it would be appropriate to briefly mention these methods so the novice reader will know that this benchmark does not apply to hybrid approaches or post-assembly genome finishing. This is a minor change, but I included it in this section because it matches the general theme of ensuring the scope of this benchmark is clear.

Minor Comments: 1. line 25 in the abstract. Change Redbean to wtdbg2 for consistency with the rest of the manuscript.

1. "de novo" should be italicized. It is done correctly in some places but not in others.

2. line 64. "all TGS technologies": I would argue that this isn't quite true. ONT Duplex isn't included here even though Duplex likely didn't exist when you did this work. Also, see the major comments concerning whether TGS should include HiFi and Duplex.

3. Table 1. Read length distributions vary dramatically by technology and library prep. E.g., HiFi is often a very tight distribution about the mean because of size selection. Including the median in the table would be helpful, but more importantly, I would like to see read-length distribution plots in the supplement for (a) the real data used to generate the initial iteration models and (b) the real data from each species.

4. line 166 "fair comparison". I'm not sure that a fair comparison should be the goal, but having them at the same coverage level makes them more comparable which is helpful. Maybe rephrase to indicate that keeping them at the same coverage level reduces potentially confounding variables when comparing between the real and simulated datasets.

5. line 169. Citation 18 is used for Canu, which is appropriate but incomplete. The citation for HiCanu should also be included here: DOI: 10.1101/gr.263566.120.

6. line 169. State that these were the most current releases of the various assemblers at the time that this analysis was started. Presumably, that was Nov/Dec 2021. Since then, Raven has gone from v1.7.0->1.8.1 and Flye has gone from v2.9->2.9.1.

7. line 175. Table S6 is mentioned here, but S5 has not yet been mentioned. S5 is mentioned for the first time on line 196. These two supp tables' numbers should be swapped.

8. There is inconsistent use of the Oxford comma. I noticed is missing multiple times, e.g., lines 191, 208, 259, & 342.

9. line 193. The comma at the end of the line (after "tools") should be removed. Alternatively, keep the comma but add a subject to the next clause to make it an independent clause (e.g., "...assembly tools, and they were computed...").

10. line 237. The N50 of the reference is being used here. You provide accessions for the references used, but most people will not go look those up (which is reasonable). The sequences in a reference can vary greatly in their lengths, even within the same species, because which sequences are included in the reference are not standardized. Even the size difference between a homogametic and heterogametic reference can be non-trivial. Which are included in the reference, and more importantly included in your N50 value, can significantly change the outcome and may bias results if these are not done consistently between the included species. It would be helpful if here or somewhere (e.g., in some supplemental text or a table) the contents of these references was somehow summarized. In addition to 1 copy of each of the expected autosomes, were any of the following included: (a) one or two sex chromosomes if applicable, (b) mitochondrial, chloroplast, or other organelle sequences, (c) alternate sequences (i.e., another copy of an allele of some sequence included elsewhere), (d) unplaced sequence from the 1st copy, (e) unplaced sequence from subsequent copies, and (f) vectors (e.g., EBV used when transforming a cell line)?

11. Supplemental tables. Some cells are uncolored, and other cells are colored red or blue with varying shading. I didn't notice a legend or description of what the coloring and shading was supposed to mean. Please include this either with each table or at the beginning of the supplemental section that includes these tables and state that it applies to all tables #-#.

12. Supplemental table S3. It was not clear to me that you created your own model for the hifi data (pacbio_hifi_human2022). I was really confused when I couldn't find that model in the GitHub repo for Badreads. In the caption for this table or in the text somewhere, please make it more explicit that you created this yourself instead of using an existing model.

Author Response

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

eLife assessment

This important study extends insights on NAFLD and NASH regarding the role of plasma lactate levels using mice haplo-insufficient for the gene encoding lactate transporter MCT-1. While the evidence is largely convincing and the work significantly advances our understanding of the roles of distinct hepatic cell types in steatosis, a number of issues require attention and would best be solved by further experimentation.

RESPONSE: We agree with this assessment by eLife, and appreciate the reviewers’ view that the study is important and extends insights into liver disease.

Public Reviews:

Reviewer #1 (Public Review):

The authors put forth the hypothesis that hepatocyte and/or non-parenchymal liver MCT1 may be responsible for physiologic effects (lower body weight gain and less hepatic steatosis) in MCT1 global heterozygote mice. They generate multiple tools to test this hypothesis, which they combine with mouse diets that induce fatty liver, steatohepatitis and fibrosis. Novel findings include that deletion of hepatocyte MCT1 does not change liver lipid content, but increases liver fibrosis. Deletion of hepatic stellate cell (HSC) MCT1 does not substantially affect any liver parameter, but concomitant HSC MCT1 deletion does reverse fibrosis seen with hepatocyte MCT1 knockout or knockdown. In both models, plasma lactate levels do not change, suggesting that liver MCT1 does not substantially affect systemic lactate. In general, the data match the conclusions of the manuscript, and the studies are well-conducted and well-described. Further work would be necessary to dissect mechanism of fibrosis with hepatocyte MCT1, and whether this is due to changes in local lactate (as speculated by the authors) or another MCT1 substrate. This would be important to understand this novel potential cross-talk between hepatocytes and HSCs.

A parallel and perhaps more important advance is the generation of new methodology to target HSC in mice, using modified siRNA and by transduction of AAV9-Lrat-Cre. Both methods would reduce the need to cross floxed mice with the Lrat-Cre allele, saving time and resources. These tools were validated to an extent by the authors, but not sufficiently to ensure that there is no cross-reactivity with other liver cell types. For example, AAV9-LratCre-transduced MCT1 floxed mice show compelling HSC but not hepatocyte Mct1 knockdown, but other liver cell types should be assessed to ensure specificity. This is particularly important as overall liver Mct1 decreased by ~30% in AAV9-Lrat-Cre-transduced mice, which may exceed HSC content of these mice, especially when considering a 60-70% knockdown efficiency. This same issue also affects Chol-MCT1-siRNA, which the authors demonstrate to affect hepatocytes and HSC, but likely affects other cell types not tested. As this is a new and potentially valuable tool, it would be important to assess Mct1 expression across more non-parenchymal cells (i.e. endothelial, cholangiocytes, immune cells) to determine penetration and efficacy.

RESPONSE: We appreciate the reviewer’s view that the new methods we describe represent an important advance. To ensure the specificity of our novel AAV-Lrat-Cre construct, it would be fair to test its distribution among all possible hepatic cell types, including endothelial cells, cholangiocytes, and other immune cells, as suggested. Our efforts in this study were primarily focused on the major cell types thought to contribute to NASH, namely hepatocytes, Kupffer cells, and in particular hepatic stellate cells. The reasons for this focus were:

1) Our primary goal was to investigate the role of MCT1 in hepatic fibrogenesis. According to Manderacke et al. (2013, Nature Comm), hepatic stellate cells account for the dominant proportion (82-96%) of myofibroblast progenitors, which produce collagen fibers. While there may be interesting roles of MCT1 in those other cell types, to elucidate MCT1's role in fibrogenesis, focusing on the dominant fibrogenic cell type, hepatic stellate cells, was the most appropriate approach for this goal.

2) Considering the proportion of each hepatic cell type in the liver, hepatocytes constitute the majority (60-70%), followed by endothelial cells (15%), immune cells (10%), and stellate cells (5%), among others.

3) The AAV-Cre system is highly specific to its promoter, in this case, Lrat, which has been well established in multiple previous studies to exhibit high specificity for hepatic stellate cells in the liver. We will certainly conduct more comprehensive biodistribution studies in the future, as we believe that our AAV-Lrat-Cre system could be a valuable tool in this field.

Reviewer #2 (Public Review):

In this study, the authors seek to answer two main questions: 1) Whether interfering with lactate availability in hepatocytes through depletion of hepatocyte specific MCT-1 depletion would reduce steatosis, and 2) Whether MCT-1 in stellate cells promote fibrogenesis. While the first question is based on the observation that haploinsufficiency of MCT-1 makes mice resistant to steatosis, the rationale behind how MCT-1 could impact fibrogenesis in stellate cells is not clear. A more detailed discussion regarding how lactate availability would regulate two different processes in two different cell types would be helpful. The authors employ several mouse models and in vitro systems to show that MCT1 inhibition in hepatic stellate cells reduces the expression of COL-1. The significance of the findings is moderately impacted due to the following considerations:

RESPONSE: We have included additional in vitro data in order to provide a more comprehensive discussion of MCT1's potential role in regulating collagen production. Please refer to the new Figure 8, Supplementary Figure 6, and the results section (Potential Mechanism). Also note that our original hypothesis was that depleting MCT1 specifically in hepatocytes would protect mice with MCT1 haploinsufficiency from liver lactate overload and NAFLD. Furthermore, we postulated that this protection might prevent NASH progression since lipotoxicity-driven hepatocyte damage is a central factor in NASH pathogenesis. However, our findings did not support this hypothesis. We found only one brief article (2015, Z Gastroenterol et al., "Functional effects of monocarboxylate transporter 1 expression in activated hepatic stellate cells") that discussed the potential role of MCT1 depletion in hepatic stellate cells in regulating collagen production or fibrosis, as mentioned in their abstract. Unfortunately, the DOI for this article is not functional, and the data cannot be located. Moreover, when we attempted to replicate their results, we were unable to do so, leading us to report our own findings in the current paper.

a. Fibrosis in human NAFLD is a significant problem as a predictor of liver related mortality and is associated with type 1 and type 3 collagen. However, the reduction in COL1 in stellate cells did not amount to a reduction in liver fibrosis even in cell specific KO (in Fig 7E, there is no indication of whether Sirius red staining was different between HSC KO and control mice- the authors mention a downward trend in the text). The authors postulate that type 1 COL may not be the more predominant form of fibrosis in the model. This does not seem likely, since the same ob/ob mouse model was used to determine that fibrosis was enhanced with hepatocyte specific MCT-1 KO and decreased with Chol MCT-1KO. Measurements of different types of collagens in their model and the effect of MCT-1 on different types could be more informative. In particular, although collagens are the structural building blocks for hepatic fibrosis, fibrosis can also be controlled by matrix remodeling factors such as Timp1, Serpine 1, PAI-1 and Lox.