very user-write oriented—people still have to do roughly the same amount of work

possible MM use case: ask your teammate who left a question

not enough

Hmm. I can't find anything about this! It would be nice if we had a scan.

(Since philsp.com links are unstable, this is cited as "Best Stories of All Time [v1 #6, November 1925]")

Not social media, but Google is egregious at using dystopian tactics to determine your profile. There is a famous study (that has sense been replicated in a livestream) where typing the number of letters required to autofill for "dog toys" in google is highly dependent on how frequently your device's microphone has picked up dog related words. Anecdotally, I know it's not just microphone data, but keystroke data as well.

This shows how social media data can reveal personal traits, such as political views or susceptibility to scams, and societal trends, like misinformation. While unconventional data can provide insights, such as linking COVID-19 to bad candle reviews, concerns about privacy, bias, and the use of flawed methods remain significant in the world of social media and content

This part is interesting about raising socil media, as it potentially let's us know abput the rough definitiion and meaning of data mining which is using data to present it in a way

I find it fascinating that data mining specifically regarding social media data can be used to determine public opinion by obtaining emotions, ideas and motives behind posts. In which case this application of data mining can be applied to the social media aspect of brands when they want to gauge their audience's interests.

This is really creative in leveraging unusual data to find a trend. Who would have imagined that COVID-19 cases are correlated with negative reviews of Yankee Candle-a proxy for not being able to smell, a known symptom-from product reviews? Indeed, an interesting case of how sometimes unrelated streams of data can point to a pattern or predict a trend in public health. It also underlines that any data correlations have to be subject to critical examination in order not to jump to misleading conclusions, as in the case of negative reviews, the reasons could be altogether different. This is a good example of how strong and/or limited data interpretation can be in a real-life setting.

It’s alarming that even interests or vulnerabilities, such as a susceptibility to financial scams or addiction, can be guessed from online behavior. What’s even more disturbing is the use of AI to repackage debunked, racist theories about facial features to make judgments about people. Social media also plays a role in tracking broader trends, like how misinformation spreads, which affects public opinion on a massive scale. It's a reminder of how our online data is used in ways we might not even realize, sometimes with harmful consequences.

z

See I may not have the full story of Congo, the virus, and the people, but they told me pretty much everything I want to know in that one paragraph.

Lots of signs here, we're being offered a very sad image here, which sets the scene for dealing with the problem we're talking about.

I picked this because it has a non-politicized topic about something that still very strongly affects the lives of a lot of people. The values being offered are timeliness and human interest.

shifted topic from the headline.

Doesn't dive much deeper into this. Probably because it isn't related to Trump working at Mcdonald's but still an interesting addition.

Factual comment. Although this statement might sound like it has some sort of bias,I don't believe it does. Trump has disputed Kamala's claims of being a former Mcdonald's employee and hasn't offered evidence for that claim. In regard to the uncertainty of whether she has or hasn't worked for Mcdonald's before, there is no evidence that she has or hasn't.

I find it interesting that the Mcdonald's is making it clear to the public that they have no political affiliation in the race. Again, I do think it is necessary, but it leads me to ask further questions such as were the crew working with Trump happy with his attendance? Were employees given the option to work with him, or was it randomly mandatory regardless of the employee political opinions.

Clearly, the Mcdonald's that hosted Trump made it clear that they are not biased or in favor of one party over the other. They may have had to make this clear for the sake of their business. I wouldn't find it hard to believe that some people may boycott or even damage, or threaten the restaurant just for having Trump.

This clearly defines to the reader the circumstances of Trump's "shift". We now know that the people in the drive through weren't just random but "pre-selected". We can now infer that he really was handing food out to real people, but they weren't just anybody.

The first thing I noticed about this article was obviously the title. I like how it doesn't feel like there is an underlying agenda or bias. The title is very straightforward and factual. Clearly and simply states that Trump worked a drive-thru before town hall.

bootstrap peer list

I believe that API's are very important as they like Reddit’s PRAW library, enable structured access to data. While the PRAW documentation offers deeper insights into its capabilities, it may be challenging to navigate, requiring patience and exploration to master its use to ensure that its full potential and purpose is being utilized.

I think data poisoning can have severe consequences which are often overlooked including the idea that misleading information and drastically reduced accuracy can lead to big companies or brands especially on social media making big decisions leading to losses and failures.

This part raises an important point; If the purpose of data mining is usually to use data and present it in a way that gies insights and understandings, what would the point of having online surveys that use fake data and poisoning be as it will generate an inaccurate data representation?

I wonder how people working with data sets that may be poisoned go about fixing them or determining how detrimental the poison really is in the data set. It makes sense how one person with a specfic demographic fan base would be able to mess up the data in a survey but its kind of obvious thinking about it now. I wonder why i never heard of this story or ones similar.

This poisoning of the dataset is going to connect with the Deepfake issue in Korea, as data manipulation and biased inputs have severe consequences in terms of outcomes. Applications in deepfakes rely on enormous datasets of images and videos that feed into algorithms that generate realistic fake content. If these datasets are contaminated with biased or skewed data-intentional or unintentional-such as overrepresentation within certain demographics, then the outcomes will be very problematic. On one hand, it is similar that viral survey participation undermines data reliability, while deepfake datasets poisoned with biased material lead to applications considered very unethical or harmful, such as creating non-consensual videos or misinformation. In fact, these two examples drive the message home regarding the risk involved with unregulated data inputs in critical systems.

It made me realize how unpredictable, even unexpected, the influence of social media can be. A seemingly innocuous video can have such a profound impact on scientific research that the data can no longer be used. It also made me think about the importance of data quality and diversity, and how to prevent this kind of “data pollution” from happening again.

Unintentional and intentional data poisoning can severely compromise the usefulness of datasets. Whether caused by viral social media trends or deliberate sabotage, such as spamming job applications, these incidents highlight the vulnerability of data collection processes and the potential for disruption, often undermining research or organizational operations.

I did not use the google account that used UW email too much. However, surprisingly, google knows a lot of my private information. And a lot of them are pretty accurate. This also make me to think whether I need to improve my awareness on data security.

I did not use the google account that used UW email too much. However, surprisingly, google knows a lot of my private information. And a lot of them are pretty accurate. This also make me to think whether I need to improve my awareness on data security.

Yes, for example health data (especially mental health ) is very risky for users. Anxiety, depression, or suicidal ideation language on net can be identified using data mining. Social media exploit this to push targeted content or manipulate users during vulnerable times.

Whoa, are the 1920s issues really not that widely available?

Continuing to scroll down, "Our Core Team" [Minor edit: Capitalization]....

What is the "Resume Review" tab [at the top of your page] does not, however, appear sequentially, as everything else does, on this page.

Again, from the perspective of "job seeker" who might be unclear as to the breadth of services that you provide, not seeing the "Resume Review" as I scroll down the page - despite everything else appearing there - might leave me a bit confused.

On this "Home" tab....

I'd suggest inverting the order of the "Work: What We Do" and "Success: How We Help You Get the Right Team" sections.

As I read [through the eyes of "Job Seeker"], I was initially thrown off given that I don't need help "find[ing] the right people for the job", rather, to BE that "right person" who is found for a job!!

I feel like the information that is given to these social media sights is very private. They should make sure to ask for the proper permission when sharing sensitive data like search history or medical history.

I feel like i knew a lot about how different companies collect data on users but im always shocked to learn different ones that i didnt think about. I never would of thought that knowing when users are logged on and off is something that these companies tracked. Im curious to what other methods they use that people might not know about,

Personally, I do not like such targeted advertisements, because it is a violation of privacy to let advertisers know too much about users' privacy. Advertisers are using social media to collect our information and make profits for themselves. I think the advertising push on social media can only refer to the age and gender information in each user's profile.

This is very true, and I believe almost all people who use social media can experience it. A social media will detect users' reactions when watching different types of videos, such as whether a video has been played completely, or if they swiped away at the moment they saw it; or whether they frequently liked certain types of works; even the frequency of leaving comments in others' posts. Social media will push the content that different users are most interested in based on the collected information. To be more specific, if I like food, then social media may often push some videos about food reviews and cooking tutorials to me. This mechanism has two sides. On the one hand, people can see the content they like more frequently, and on the other hand, this mechanism also limits the information that everyone sees.

I find it interesting that the goal of mining data is to collect information about users to make them stay longer on social media. Some may consider this collection of information an invasion of privacy.

Social media platforms use data to increase user engagement and profits through targeted advertising. While this can be useful for businesses and consumers, it raises ethical concerns when ads are directed at vulnerable groups, like children or addicts, exploiting their weaknesses for financial gain.

Reviewer #2 (Public review):

Summary:

Juvenile hormone (JH) is a pleiotropic terpenoid hormone in insects that mainly regulates their development and reproduction. In particular, its developmental functions are described as the "status quo" action, as its presence in the hemolymph (the insect blood) prevents metamorphosis-initiating effects of ecdysone, another important hormone in insect development, and maintains the juvenile status of insects.

While such canonical functions of JH are known to be mediated by its intracellular receptor complex composed of Met and Tai, there have been multiple reports suggesting the presence of cell membrane receptor(s) for JH, which mediate non-genomic effects of this terpenoid hormone. In particular, the presence of receptor tyrosine kinase(s) that phosphorylate Met/Tai in response to JH and thus indirectly affect the canonical JH signaling pathway has been strongly suggested. Given the importance of JH in insect physiology and the fact that the JH signaling pathway is a major target of insect growth regulators, elucidating the identify and functions of putative JH membrane receptors is of great significance from both basic and applied perspectives.

In the present study, the authors identified candidate receptors for such cell membrane JH receptors, CAD96CA and FGFR1, in the cotton bollworm Helicoverpa armigera.

Strengths:

Their in vitro analyses are conducted thoroughly using multiple methods, which overall supports their claim that these receptors can bind to JH and mediate their non-genomic effects.

Weaknesses:

Results of their in vivo experiments, particularly those of their loss-of-function analyses using CRISPR mutants are still preliminary, and the results rather indicate that these membrane receptors do not have any physiologically significant roles in vivo. More specifically, previous studies in lepidopteran species have clearly and repeatedly shown that precocious metamorphosis is the hallmark phenotype for all JH signaling-deficient larvae. In contrast, the present study showed that Cad96ca and Fgfr1 G0 mutants only showed slight acceleration in their pupation timing, which is not a typical phenotype one would expect from JH signaling deficiency. This is inconsistent with their working model provided in Figure 6, which indicates that these cell membrane JH receptors promote the canonical JH signaling by phosphorylating Met/Tai.

If the authors argue that this slight acceleration of pupation is indeed a major JH signaling-deficient phenotype in Helicoverpa, they need to provide more data to support their claim by analyzing CRISPR mutants of other genes involved in JH signaling, such as Jhamt and Met. An alternative explanation is that there is functional redundancy between CAD96CA and FGFR1 in mediating phosphorylation of Met/Tai. This possibility can be tested by analyzing double knockouts of these two receptors.

Currently, the validity of their calcium imaging analysis in Figure 5 is also questionable. When performing calcium imaging in cultured cells, it is critically important to treat all the cells at the end of each experiment with a hormone or other chemical reagents that universally induce calcium increase in each particular cell line. Without such positive control, the validity of calcium imaging data remains unknown, and readers cannot properly evaluate their results.

Reviewer #3 (Public review):

Summary:

In this study, Li et al. identified CAD96CA and FGF1 among 20 receptor tyrosine kinase receptors as mediators of JH signaling. By performing a screen in HaEpi cells with overactivated JH signaling, the authors pinpointed two main RTKs that contribute to the transduction of JH. Using the CRISPR/Cas9 system to generate mutants, the authors confirmed that these RTKs are required for normal JH activation, as precocious pupariation was observed in their absence. Additionally, the authors demonstrated that both CAD96CA and FGF1 exhibit a high affinity for JH, and their activation is necessary for the proper phosphorylation of Tai and Met, transcription factors that promote the transcriptional response. Finally, the authors provided evidence suggesting that the function of CAD96CA and FGF1 as JH receptors is conserved across insects.

Strengths:

The data provided by the authors are convincing and support the main conclusions of the study, providing ample evidence to demonstrate that phosphorylation of the transducers Met and Tai mainly depends on the activity of two RTKs. Additionally, the binding assays conducted by the authors support the function of CAD96CA and FGF1 as membrane receptors of JH. The study's results validate, at least in H. amigera, the predicted existence of membrane receptors for JH.

Weaknesses:

The authors have provided evidences that the Cad96Ca and FGF1 RTK receptors contribute to JH signaling through CRISPR/Cas9, inducing precocious metamorphosis, although not to the same extent as absence of JH. Therefore, it still remains unclear whether these RTKs are completely required for pathway activation or only necessary for high activation levels during the last larval stage.

While the authors have included some additional data, the mechanism by which different RTKs function in transducing JH signaling in a tissue specific manner is still unclear. As the authors note in the discussion, it is possible that other RTKs may also play a role in facilitating the transduction of JH signaling.

Lastly, the study does not yet explain how RTKs with known ligands could also bind JH and contribute to JH signaling activation. Although receptor promiscuity has been suggested as a possible mechanism, future studies could explore whether activation of RTK pathways by their known ligands induces certain levels of JH transducer phosphorylation, which, in the presence of JH, could contribute to full pathway activation without the need for direct JH-RTK binding.

Author response:

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

Reviewer #1 (Public Review):

Summary:

Juvenile Hormone (JH) plays a key role in insect development and physiology. Although the intracellular receptor for JH was identified long ago, a number of studies have shown that part of JH functions should be fulfilled through binding to an unknown membrane receptor, which was proposed to belong to the RTK family. In this study, the authors screened all RTKs from the H. armigera genome for their ability to mediate responses to JH III treatment both in cultured cells and in developing animals. They also present convincing evidence that CAD96CA and FGFR1 directly bind JH III, and that their role might be conserved in other insect species.

Strengths:

Altogether, the experimental approach is very complete and elegant, providing evidence for the role of CAD96CA and FGFR1 in JH signalling using different techniques and in different contexts. I believe that this work will open new perspectives to study the role of JH and better understand what is the contribution of signalling through membrane receptors for JH-dependent developmental processes.

Weaknesses:

I don't see major weaknesses in this study. However, I think that the manuscript would benefit from further information or discussion regarding the relationship between the two newly identified receptors. Experiments (especially in HEK-293T cells) suggest that CAD96CA and FGFR1 are sufficient on their own to transduce JH signalling. However, they are also necessary since loss-of-function conditions for each of them are sufficient to trigger strong effects (while the other is supposed to be still present).

Thank you for the suggestion. We have added the discussion in the text: "CAD96CA and FGFR1 have similar functions in JH signaling, including transmitting JH signal for Kr-h1 expression, larval status maintaining, rapid intracellular calcium increase, phosphorylation of transcription factors MET1 and TAI, and high affinity to JH III. CAD96CA and FGFR1 are essential in the JH signal pathway, and loss-of-function for each is sufficient to trigger strong effects on pupation. The difference is that CAD96CA expression has no tissue specificity, and the Fgfr1 gene is highly expressed in the midgut; possibly, it plays a significant role in the midgut. Other possibility is that they play roles by forming heterodimer with each other or other RTKs, which needs to be addressed in future study. CAD96CA and FGFR1 transmit JH III signals in three different insect cell lines, suggesting their conserved roles in other insects.".

In addition, despite showing different expression patterns, the two receptors seem to display similar developmental functions according to loss-of-function phenotypes. It is therefore unclear how to draw a model for membrane receptor-mediated JH signalling that includes both CAD96CA and FGFR1.

Thank you for your question. We have modified the figure and the legends to make the conception clear.

Reviewer #2 (Public Review):

Summary:

Juvenile hormone (JH) is a pleiotropic terpenoid hormone in insects that mainly regulates their development and reproduction. In particular, its developmental functions are described as the "status quo" action, as its presence in the hemolymph (the insect blood) prevents metamorphosis-initiating effects of ecdysone, another important hormone in insect development, and maintains the juvenile status of insects. While such canonical functions of JH are known to be mediated by its intracellular receptor complex composed of Met and Tai, there have been multiple reports suggesting the presence of cell membrane receptor(s) for JH, which mediate non-genomic effects of this terpenoid hormone. In particular, the presence of receptor tyrosine kinase(s) that phosphorylate Met/Tai in response to JH and thus indirectly affect the canonical JH signaling pathway has been strongly suggested. Given the importance of JH in insect physiology and the fact that the JH signaling pathway is a major target of insect growth regulators, elucidating the identification and functions of putative JH membrane receptors is of great significance from both basic and applied perspectives. In the present study, the authors identified candidate receptors for such cell membrane JH receptors, CAD96CA and FGFR1, in the cotton bollworm Helicoverpa armigera.

Strengths:

Their in vitro analyses are conducted thoroughly using multiple methods, which overall supports their claim that these receptors can bind to JH and mediate their non-genomic effects.

Weaknesses:

Results of their in vivo experiments, particularly those of their loss-of-function analyses using CRISPR mutants are still preliminary, and the results rather indicate that these membrane receptors do not have any physiologically significant roles in vivo. More specifically, previous studies in lepidopteran species have clearly and repeatedly shown that precocious metamorphosis is the hallmark phenotype for all JH signaling-deficient larvae. In contrast, the present study showed that Cad96ca and Fgfr1 G0 mutants only showed a slight acceleration in their pupation timing, which is not a typical phenotype one would expect from JH signaling deficiency. This is inconsistent with their working model provided in Figure 6, which indicates that these cell membrane JH receptors promote the canonical JH signaling by phosphorylating Met/Tai.

If the authors argue that this slight acceleration of pupation is indeed a major JH signaling-deficient phenotype in Helicoverpa, they need to provide more data to support their claim by analyzing CRISPR mutants of other genes involved in JH signaling, such as Jhamt and Met. An alternative explanation is that there is functional redundancy between CAD96CA and FGFR1 in mediating phosphorylation of Met/Tai. This possibility can be tested by analyzing double knockouts of these two receptors.

Thank you for your question and suggestion. The cadherin 96ca (CAD96CA) and fibroblast growth factor receptor 1 (FGFR1) were finally determined as JH cell membrane receptors by their roles in JH regulated-gene expression, maintaining larval status, JH induced-rapid increase of intracellular calcium levels, JH induced-phosphorylation of MET and TAI, and their JH-binding affinity. Their roles as JH cell membrane receptors were further determined by knockdown and knockout of them in vivo and in cell lines, and overexpression of them in mammal HEK-293T heterogeneously. Figure 6 is drafted by these solidate evidences.

Cad96ca and Fgfr1 G0 mutants caused slight acceleration of pupation is one of the types of evidence of JH signaling-deficient. Othe evidences include a set of gene expression and the block of JH induced-rapid intracellular calcium increase.

Kr-h1 is a typical indicator gene at the downstream of Jhamt and in JH signaling, so we used it as an indicator to examine JH signaling. Jhamt and Met or other genes might be affected in Cad96ca and Fgfr1 G0 mutants, which can be examined in future study.

We have discussed the question that Cad96ca and Fgfr1 G0 mutants only showed a slight acceleration in their pupation timing: "Homozygous Cad96ca null Drosophila die at late pupal stages (Wang et al., 2009). However, we found that 86% of the larvae of the Cad96ca mutant successfully pupated in G0 generation, although earlier than the control. Similarly, null mutation of Fgfr1 or Fgfr2 in mouse is embryonic lethal (Arman et al., 1998; Deng et al., 1994; Yamaguchi et al., 1994). In D. melanogaster, homozygous Htl (Fgfr) mutant embryos die during late embryogenesis, too (Beati et al., 2020; Beiman et al., 1996; Gisselbrecht et al., 1996). However, in H. armigera, 91% of larvae successfully pupated in G0 generation after Fgfr1 knockout. The low death rate after Cad96ca and Fgfr1 knockout might be because of following reasons, including the editing efficiency (67% and 61% for Cad96ca mutant and Fgfr1 mutant, respectively), the chimera of the gene knockout at the G0 generation, and the redundant RTKs that play similar roles in JH signaling, similar to the redundant roles of MET and Germ-cell expressed bHLH-PAS (GCE) in JH signaling (Liu et al., 2009), which needs to obtain alive G1 homozygote mutants and double knockout of these two receptors in future study. We indeed observed that the eggs did not hatch successfully after mixed-mating of G0 Cad96ca mutant or Fgfr1 mutant, respectively, but the reason was not addressed further due to the embryonic death. By the similar reasons, most of the Cad96ca and Fgfr1 mutants showed a slight acceleration of pupation (about one day) without the typical precocious metamorphosis (at least one instar earlier) phenotype caused by JH signaling defects (Daimon et al., 2012; Fukuda, 1944; Riddiford et al., 2010) and JH pathway gene deletions (Abdou et al., 2011; Liu et al., 2009). On other side, JH can regulate gene transcription by diffusing into cells and binding to the intracellular receptor MET to conduct JH signal, which might affect the results of gene knockdown and knockout.".

Currently, the validity of their calcium imaging analysis in Figure 5 is also questionable. When performing calcium imaging in cultured cells, it is critically important to treat all the cells at the end of each experiment with a hormone or other chemical reagents that universally induce calcium increase in each particular cell line. Without such positive control, the validity of calcium imaging data remains unknown, and readers cannot properly evaluate their results.

Thank you for your question. For Figure 5, our goal was to demonstrate that JH can induce calcium mobilization through CAD96CA and FGFR1. Controls have been established between different experimental groups within the same cell, as well as between different cells. Increasing the positive experimental group would make the results more complex.

Reviewer #3 (Public Review):

Summary:

In this study, Li et al. identified CAD96CA and FGF1 among 20 receptor tyrosine kinase receptors as mediators of JH signaling. By performing a screen in HaEpi cells with overactivated JH signaling, the authors pinpointed two main RTKs that contribute to the transduction of JH. Using the CRISPR/Cas9 system to generate mutants, the authors confirmed that these RTKs are required for normal JH activation, as precocious pupariation was observed in their absence. Additionally, the authors demonstrated that both CAD96CA and FGF1 exhibit a high affinity for JH, and their activation is necessary for the proper phosphorylation of Tai and Met, transcription factors that promote the transcriptional response. Finally, the authors provided evidence suggesting that the function of CAD96CA and FGF1 as JH receptors is conserved across insects.

Strengths:

The data provided by the authors are convincing and support the main conclusions of the study, providing ample evidence to demonstrate that phosphorylation of the transducers Met and Tai mainly depends on the activity of two RTKs. Additionally, the binding assays conducted by the authors support the function of CAD96CA and FGF1 as membrane receptors of JH. The study's results validate, at least in H. amigera, the predicted existence of membrane receptors for JH.

Weaknesses:

The study has several weaknesses that need to be addressed. Firstly, it is not clear what criteria were used by the authors to discard several other RTKs that were identified as repressors of JH signaling. For example, while NRK and Wsck may not fulfill all the requirements to become JH receptors, other evidence, such as depletion analysis and target gene expression, suggests they are involved in proper JH signaling activation.

Thank you for your question. We screened the RTKs sequentially, including examining the roles of 20 RTKs identified in the H. armigera genome in JH regulated-gene expression to obtain primary candidates, followed by screening of the candidates by their roles in maintaining larval status, JH induced-rapid increase of intracellular calcium levels, JH induced-phosphorylation of MET and TAI, and affinity to JH. WSCK was not involved in the phosphorylation of MET and TAI and was discarded during subsequent screening. NRK did not bind to JH III, did not meet the screening strategy, and was discarded.

We increased the information in the Introduction: "We screened the RTKs sequentially, including examining the roles of 20 RTKs identified in the H. armigera genome in JH regulated-gene expression to obtain primary candidates, followed by screening of the candidates by their roles in maintaining larval status, JH induced-rapid increase of intracellular calcium levels, JH induced-phosphorylation of MET and TAI, and affinity to JH. The cadherin 96ca (CAD96CA) and fibroblast growth factor receptor 1 (FGFR1) were finally determined as JH cell membrane receptors by their roles in JH regulated-gene expression, maintaining larval status, JH induced-rapid increase of intracellular calcium levels, JH induced-phosphorylation of MET and TAI, and their JH-binding affinity. Their roles as JH cell membrane receptors were further determined by knockdown and knockout of them in vivo and cell lines, and overexpression of them in mammal HEK-293T heterogeneously.".

We increased discussion: "This study found six RTKs that respond to JH induction by participating in JH induced-gene expression and intracellular calcium increase, however; they exert different functions in JH signaling, and finally CAD96CA and FGFR1 are determined as JH cell membrane receptors by their roles in JH induced-phosphorylation of MET and TAI and binding to JH III. We screen the RTKs transmitting JH signal primarily by examining some of JH induced-gene expression. By examining other genes or by other strategies to screen the RTKs might find new RTKs functioning as JH cell membrane receptors; however, the key evaluation indicators, such as the binding affinity of the RTKs to JH and the function in transmitting JH signal to maintain larval status are essential.".

Secondly, the expression of the six RTKs, which, when knocked down, were able to revert JH signaling activation, was mainly detected in the last larval stage of H. amigera. However, since JH signaling is active throughout larval development, it is unclear whether these RTKs are completely required for pathway activation or only needed for high activation levels at the last larval stage.

Thank you for the question. We knocked down the genes at last larval stage to observe pupation, which is a relatively simple and easily to be observed target to examine the role of the gene in JH-maintained larval status. The results from CRISPR/Cas9 experiments showed: "Most wild-type larvae showed a phenotype of pupation on time. However, in the Cad96ca mutant, 86% of the larvae (an editing efficiency of 67% by TA clone analysis) had a shortened feeding stage in the sixth instar and entered the metamorphic molting stage earlier, showing early pupation, with the pupation time being 24 h earlier. In the Fgfr1 mutant, 91% of the larvae (an editing efficiency of 61%) had a shortened feeding stage in the sixth instar and entered the metamorphic molting stage earlier, showing early pupation, with the pupation time being 23 h earlier (Figure 4D and E). The data suggested that CAD96CA and FGFR1 support larval growth and prevent pupation in vivo.".

Additionally, the mechanism by which different RTKs exert their functions in a specific manner is not clear. According to the expression profile of the different RTKs, one might expect some redundant role of those receptors. In fact the no reversion of phosphorilation of tai and met upon depletion of Wsck in cells with overactivated JH signalling seems to support this idea.

Nevertheless, and despite the overlapping expression of the different receptors, all RTKs seem to be required for proper pathway activation, even in the case of FGF1 which seems to be only expressed in the midgut. This is an intriguing point unresolved in the study.

Thank you for your comments. Yes, from our study, different RTKs exert their functions in a specific manner. We have increased discussion: "This study found six RTKs that respond to JH induction by participating in JH induced-gene expression and intracellular calcium increase, however; they exert different functions in JH signaling, and finally CAD96CA and FGFR1 are determined as JH cell membrane receptors by their roles in JH induced-phosphorylation of MET and TAI and binding to JH III. We screen the RTKs transmitting JH signal primarily by examining some of JH induced-gene expression. By examining other genes or by other strategies to screen the RTKs might find new RTKs functioning as JH cell membrane receptors; however, the key evaluation indicators, such as the binding affinity of the RTKs to JH and the function in transmitting JH signal to maintain larval status are essential.".

Finally, the study does not explain how RTKs with known ligands could also bind JH and contribute to JH signaling activation. in Drosophila, FGF1 is activated by pyramus and thisbe for mesoderm development, while CAD96CA is activated by collagen during wound healing. Now the authors claim that in addition to these ligands, the receptors also bind to JH. However, it is unclear whether these RTKs are activated by JH independently of their known ligands, suggesting a specific binding site for JH, or if they are only induced by JH activation when those ligands are present in a synergistic manner. Alternatively, another explanation could be that the RTK pathways by their known ligands activation may induce certain levels of JH transducer phosphorylation, which, in the presence of JH, contributes to the full pathway activation without JH-RTK binding being necessary.

Thank you for your professional questions. It is an exciting and challenging to explore the molecular mechanism by which multiple ligands transmit signals through the same receptor. It requires a long-term research plan and in-depth studies. We added discussion in the text: "CAD96CA (also known as Stitcher, Ret-like receptor tyrosine kinase) activates upon epidermal wounding in Drosophila embryos (Tsarouhas et al., 2014) and promotes growth and suppresses autophagy in the Drosophila epithelial imaginal wing discs (O'Farrell et al., 2013). There is a CAD96CA in the genome of the H. armigera, which is without function study. Here, we reported that CAD96CA prevents pupation by transmitting JH signal as a JH cell membrane receptor. We also showed that CAD96CA of other insects has a universal function of transmitting JH signal to trigger Ca2+ mobilization, as demonstrated by the study in Sf9 cell lines of S. frugiperda and S2 cell lines of D. melanogaster.

FGFRs control cell migration and differentiation in the developing embryo of D. melanogaster (Muha and Muller, 2013). The ligand of FGFR is FGF in D. melanogaste_r (Du et al., 2018_). FGF binds FGFR and triggers cell proliferation, differentiation, migration, and survival (Beenken and Mohammadi, 2009; Lemmon and Schlessinger, 2010). Three FGF ligands and two FGF receptors (FGFRs) are identified in Drosophila (Huang and Stern, 2005). The Drosophila FGF-FGFR interaction is specific. Different ligands have different functions. The activation of FGFRs by specific ligands can affect specific biological processes (Kadam et al., 2009). The FGFR in the membrane of Sf9 cells can bind to Vip3Aa (Jiang et al., 2018). One FGF and one FGFR are in the H. armigera genome, which has yet to be studied functionally. The study found that FGFR prevents insect pupation by transmitting JH signal as a JH cell membrane receptor. Exploring the molecular mechanism and output by which multiple ligands transmit signals through the same receptor is exciting and challenging.".

Reviewer #1 (Recommendations For The Authors):

As an experimental suggestion, I will only propose that authors test the double knock-down/knock-out or overexpression of CAD96CA and FGFR1 to give some hints into how redundant/independent the two receptors are.

Thank you very much for your professional advice. We agree with your point of view that double knockout of CAD96CA and FGFR1 is very important to resolve the redundant/independent of the two receptors, which can make our research more complete. Unfortunately, due to experimental difficulty and time constraints, we did not provide supplementary experiments. In this study, we aim to screen the cell membrane receptors of JH. Therefore, we focused on which RTKs can function as receptors. This article is a preliminary study to identify the cell membrane receptors of JH. To further understand the relationship between the two membrane receptors, we will conduct in-depth research in future work.

Apart from that, here are some minor points about the manuscript:

Figure 2A: changing the scale on the y-axis would help to better see the different genotypes (similar to the way it is presented in Figure 5).

Thanks for your reminding, we have changed the scale in Figure 2A.

Figure 4J: image settings could be improved to better highlight the green fluorescence.

Thank you for your advice, we have improved the imaged in Figure 4J.

In general, the manuscript would benefit from some proofreading since a number of sentences are incorrect.

Thanks for your reminding, we have carefully revised the manuscript.

Reviewer #2 (Recommendations For The Authors):

(1) Although the authors note that there are 21 RTK genes in Drosophila (line 55), I can only see 16 Drosophila RTKs in Figure 1 - Figure Supplement 1. Some important Drosophila RTKs such as breathless are missing. The authors need to redraw the phylogenetic tree.

Thanks for your reminding, we have presented the new phylogenetic tree in Figure 1-figure supplement 1.

(2) The accelerated pupation phenotype in Cad96ca and Fgfr1 G0 mutants needs to be better described. In particular, it is critical to examine which developmental stage(s) are shortened in these mutant larvae. Refer to a similar study on a JH biosynthetic enzyme in Bombyx (PMID: 22412378) regarding how to describe the developmental timing phenotype.

Thank you for your advice. We have re-shown Figure 4E and added the explanation in the text: "In 61 survivors of Cas9 protein plus Cad96ca-gRNA injection, 30 mutants were sequenced, and a mutation efficiency was 49.2%. Similarly, in the 65 survivors of Cas9 protein plus Fgfr1-gRNA injection, 35 mutants were sequenced, and a mutation efficiency was 53.8% (Figure 4C). The DNA sequences, deduced amino acids and off–target were analyzed (Figure 4—figure supplement 1). Most wild-type larvae showed a phenotype of pupation on time. However, in the Cad96ca mutant, 86% of the larvae (an editing efficiency of 67% by TA clone analysis) had a shortened feeding stage in the sixth instar and entered the metamorphic molting stage earlier, showing early pupation, with the pupation time being 24 h earlier. In the Fgfr1 mutant, 91% of the larvae (an editing efficiency of 61%) had a shortened feeding stage in the sixth instar and entered the metamorphic molting stage earlier, showing early pupation, with the pupation time being 23 h earlier (Figure 4D and E). The data suggested that CAD96CA and FGFR1 support larval growth and prevent pupation in vivo.".

(3) The editing efficiency described in lines 211-213 is obscure. Does this indicate the percentage of animals with noisy sequencing spectra or the percentage of mutation rates analyzed by TA cloning?

Thanks for your reminder. We have revised the description in the text: "In 61 survivors of Cas9 protein plus Cad96ca-gRNA injection, 30 mutants were sequenced, and a mutation efficiency was 49.2%. Similarly, in the 65 survivors of Cas9 protein plus Fgfr1-gRNA injection, 35 mutants were sequenced, and a mutation efficiency was 53.8% (Figure 4C). The DNA sequences, deduced amino acids and off–target were analyzed (Figure 4—figure supplement 1). Most wild-type larvae showed a phenotype of pupation on time. However, in the Cad96ca mutant, 86% of the larvae (an editing efficiency of 67% by TA clone analysis) had a shortened feeding stage in the sixth instar and entered the metamorphic molting stage earlier, showing early pupation, with the pupation time being 24 h earlier. In the Fgfr1 mutant, 91% of the larvae (an editing efficiency of 61%) had a shortened feeding stage in the sixth instar and entered the metamorphic molting stage earlier, showing early pupation, with the pupation time being 23 h earlier (Figure 4D and E). The data suggested that CAD96CA and FGFR1 support larval growth and prevent pupation in vivo.".

(4) In Figures 4F and G, the authors examined expression levels of some JH/ecdysone responsive genes only at 0 hr-old 6th instar larvae. This single developmental stage is not enough for this analysis. In particular, the expression level of Fgfr1 only goes up in the mid-6th instar according to their own data (Figure 1-Figure Supplement 4), so it is critical to examine expression levels of these genes at least throughout the 6th larval instar.

Thank you for your advice. Indeed, it is essential to detect the expression levels of JH/ecdysone response genes in the whole sixth instar larvae. Because we observed that the mutation has a shorter feeding stage at the sixth instar, we examined the expression level of the JH/ecdysone response gene at the early sixth instar. Due to the number of mutants obtained in the experiment was small and non-destructive sampling could not be performed in sixth instar period, there were no enough samples to test. In the future, we will generate Cad96ca Fgfr1 double mutations to carry out studies and detect the expression level of JH/ecdysone response genes in the whole sixth instar.

(5) As mentioned above, some important Drosophila RTKs such as breathless are missing in their analyses. As breathless is a close paralog of heartless (Htl), I am sure that Drosophila breathless is also orthologous to Helicoverpa FGFR1. The authors therefore need to analyze breathless in Figure 5B in addition to Htl.

Thank you for your advice. We added experiments and the results are shown in Figure 5B and Figure 5—figure supplement 1.

(6) More discussion about the reason why dsNrk and dsWsck can provide resistance to JHIII in Figure 1 is required.

Thank you for your advice. We added explanation in the discussion: "It is generally believed that the primary role of JH is to antagonize 20E during larval molting (Riddiford, 2008). The knockdown of Cad96ca, Nrk, Fgfr1, and Wsck showed phenotypes resistant to JH III induction and the decrease of Kr-h1 and increase of Br-z7 expression, but knockdown of Vegfr and Drl only decrease Kr-h1, without increase of Br-z7. Br-z7 is involved in 20E-induced metamorphosis in H. armigera (Cai et al., 2014), whereas, Kr-h1 is a JH early response gene that mediates JH action (Minakuchi et al., 2009) and represses Br expression (Riddiford et al., 2010). The high expression of Br-z7 is possible due to the down-regulation of Kr-h1 in Cad96ca, Nrk, Fgfr1 and Wsck knockdown larvae. The different expression profiles of Br-z7 in Vegfr and Drl knockdown larvae suggest other roles of Vegfr and Drl in JH signaling, which need further study."

Reviewer #3 (Recommendations For The Authors):

(1) The authors should consider optimizing their experimental approach by depleting the six candidate RTKs in an early larval stage rather than using a sensitized background with JH application in the last larval stage.

Thank you for your precious suggestion. We knocked down the genes at last larval stage to observe pupation, which is a relatively simple and easily to be observed target to examine the role of the gene in JH-maintained larval status. The results from CRISPR/Cas9 experiments showed: "Most wild-type larvae showed a phenotype of pupation on time. However, in the Cad96ca mutant, 86% of the larvae (an editing efficiency of 67% by TA clone analysis) had a shortened feeding stage in the sixth instar and entered the metamorphic molting stage earlier, showing early pupation, with the pupation time being 24 h earlier. In the Fgfr1 mutant, 91% of the larvae (an editing efficiency of 61%) had a shortened feeding stage in the sixth instar and entered the metamorphic molting stage earlier, showing early pupation, with the pupation time being 23 h earlier (Figure 4D and E). The data suggested that CAD96CA and FGFR1 support larval growth and prevent pupation in vivo.". To know the roles of other RTKs in the whole larval development needs future work since a lot of experiments are needed.

(2) Including a positive control for JH signaling, such as met or tai, would strengthen the assays and provide a benchmark for evaluating the downregulation of target genes and phenotype reversion upon JH application. This addition, especially in Figure 1, would enhance the interpretability of the results.

Thank you for your suggestion. We agree with your point of view that adding the detection of Met or Tai as a positive control. Our laboratory has reported in previous studies that knockdown of Met leads to decreased expression of genes in the JH signaling pathway and precocious pupation (PMID: 24872508), so we did not repeat this related experiment in this study. In the future, when performg Cad96ca and Fgfr1 double mutant experiments, Met mutant can be generated as a control to provide more references for the interpretation of the results.

(3) I recommend revising the manuscript to improve readability, particularly in the Results section, where descriptions of the binding part are particularly dense.

Thank you for your advice. We have carefully revised the manuscript.

(4) In line 122, please add the reference Wang et al., 2016.

Thank you for your reminding, we have added the reference in line 125 of the new manuscript.

(5) The authors should clarify why they chose to test the possible binding to JH of only Cad96CA, FGFR1, and NRK after conducting various assays while including OTK in the study as a negative control. This explanation should be included in the text.

Thank you for the suggestion. We added the explanation, as described in the text: "We screened the RTKs sequentially, including examining the roles of 20 RTKs identified in the H. armigera genome in JH regulated-gene expression to obtain primary candidates, followed by screening of the candidates by their roles in maintaining larval status, JH induced-rapid increase of intracellular calcium levels, JH induced-phosphorylation of MET and TAI, and affinity to JH. The cadherin 96ca (CAD96CA) and fibroblast growth factor receptor 1 (FGFR1) were finally determined as JH cell membrane receptors by their roles in JH regulated-gene expression, maintaining larval status, JH induced-rapid increase of intracellular calcium levels, JH induced-phosphorylation of MET and TAI, and their JH-binding affinity. Their roles as JH cell membrane receptors were further determined by knockdown and knockout of them in vivo and cell lines, and overexpression of them in mammal HEK-293T heterogeneously.".

"Since Cad96CA, FGFR1, and NRK were not only involved in JH-regulated Kr-h1 expression, JH III-induced delayed pupation, and calcium levels increase, but also involved in MET and TAI phosphorylation, we further analyzed their binding affinity to JH III. OTK did not respond to JH III, so we used it as a control protein on the cell membrane to exclude the possibility of nonspecific binding.".

(6) The observed embryonic lethality of cad96ca and FGF1 mutants in Drosophila contrasts with the ability of the respective mutants in H. armigera to reach the pupal stage. The authors should discuss this significant difference.

Thank you for the suggestion. We added the explanation in the discussion, as described in the text: "Homozygous Cad96ca null Drosophila die at late pupal stages (Wang et al., 2009). However, we found that 86% of the larvae of the Cad96ca mutant successfully pupated in G0 generation, although earlier than the control. Similarly, null mutation of Fgfr1 or Fgfr2 in mouse is embryonic lethal (Arman et al., 1998; Deng et al., 1994; Yamaguchi et al., 1994). In D. melanogaster, homozygous Htl (Fgfr) mutant embryos die during late embryogenesis, too (Beati et al., 2020; Beiman et al., 1996; Gisselbrecht et al., 1996). However, in H. armigera, 91% of larvae successfully pupated in G0 generation after Fgfr1 knockout. The low death rate after Cad96ca and Fgfr1 knockout might be because of following reasons, including the editing efficiency (67% and 61% for Cad96ca mutant and Fgfr1 mutant, respectively), the chimera of the gene knockout at the G0 generation, and the redundant RTKs that play similar roles in JH signaling, similar to the redundant roles of MET and Germ-cell expressed bHLH-PAS (GCE) in JH signaling (Liu et al., 2009), which needs to obtain alive G1 homozygote mutants and double knockout of these two receptors in future study. We indeed observed that the eggs did not hatch successfully after mixed-mating of G0 Cad96ca mutant or Fgfr1 mutant, respectively, but the reason was not addressed further due to the embryonic death. By the similar reasons, most of the Cad96ca and Fgfr1 mutants showed a slight acceleration of pupation (about one day) without the typical precocious metamorphosis (at least one instar earlier) phenotype caused by JH signaling defects (Daimon et al., 2012; Fukuda, 1944; Riddiford et al., 2010) and JH pathway gene deletions (Abdou et al., 2011; Liu et al., 2009). On other side, JH can regulate gene transcription by diffusing into cells and binding to the intracellular receptor MET to conduct JH signal, which might affect the results of gene knockdown and knockout.".

(7) Building upon the previous point, it is noteworthy that the cad96ca and FGF1 mutants exhibit only a 24-hour early pupation phenotype, contrasting with the 48-hour early pupation induced by Kr-h1 depletion. This discrepancy suggests that while the function of these RTKs is necessary, it may not be sufficient to fully activate JH signaling. The expression profile of these receptors, primarily observed in the last larval stage, supports this hypothesis.

Thank you for your suggestion. We added the explanation in the discussion, as described in the text: "Homozygous Cad96ca null Drosophila die at late pupal stages (Wang et al., 2009). However, we found that 86% of the larvae of the Cad96ca mutant successfully pupated in G0 generation, although earlier than the control. Similarly, null mutation of Fgfr1 or Fgfr2 in mouse is embryonic lethal (Arman et al., 1998; Deng et al., 1994; Yamaguchi et al., 1994). In D. melanogaster, homozygous Htl (Fgfr) mutant embryos die during late embryogenesis, too (Beati et al., 2020; Beiman et al., 1996; Gisselbrecht et al., 1996). However, in H. armigera, 91% of larvae successfully pupated in G0 generation after Fgfr1 knockout. The low death rate after Cad96ca and Fgfr1 knockout might be because of following reasons, including the editing efficiency (67% and 61% for Cad96ca mutant and Fgfr1 mutant, respectively), the chimera of the gene knockout at the G0 generation, and the redundant RTKs that play similar roles in JH signaling, similar to the redundant roles of MET and Germ-cell expressed bHLH-PAS (GCE) in JH signaling (Liu et al., 2009), which needs to obtain alive G1 homozygote mutants and double knockout of these two receptors in future study. We indeed observed that the eggs did not hatch successfully after mixed-mating of G0 Cad96ca mutant or Fgfr1 mutant, respectively, but the reason was not addressed further due to the embryonic death. By the similar reasons, most of the Cad96ca and Fgfr1 mutants showed a slight acceleration of pupation (about one day) without the typical precocious metamorphosis (at least one instar earlier) phenotype caused by JH signaling defects (Daimon et al., 2012; Fukuda, 1944; Riddiford et al., 2010) and JH pathway gene deletions (Abdou et al., 2011; Liu et al., 2009). On other side, JH can regulate gene transcription by diffusing into cells and binding to the intracellular receptor MET to conduct JH signal, which might affect the results of gene knockdown and knockout.".

(8) The expression profile of the RTK hits described in Supplementary Figure 4A appears to be limited to the last larval stage until pupation. The authors should clarify whether these receptors are expressed earlier, and the meaning of the letters in the plot should be described in the figure legend.

Thank you for the suggestion. We added the explanation in the Figure 1—figure supplement 4 legend, as described in the text: "The expression profiles of Vegfr1, Drl, Cad96ca, Nrk, Fgfr1, and Wsck during development. 5F: fifth instar feeding larvae; 5M: fifth instar molting larvae; 6th-6 h to 6th-120 h: sixth instar at 6 h to sixth instar 120 h larvae; P0 d to P8 d: pupal stage at 0-day to pupal stage at 8-day F: feeding stage; M: molting stage; MM: metamorphic molting stage; P: pupae.".

We are very sorry, but due to time limitations, we will investigate the expression profile of RTK throughout the larval stage in future work.

(9) In Figure 4, panels F and G, the levels of Kr-h1 are shown in cad96ca and FGF1 mutants in the last larval stage. The authors should indicate whether Kr-h1 levels are also low in earlier larval stages or only detected in the last larval stage, as this would imply that these RTKs are only required at this stage.

Thank you for your suggestion. In this study, the Cad96ca and Fgfr1 mutants' feeding stage was shortened in the sixth instar, and they entered the metamorphic molting stage earlier. So, we detected the expression of Kr-h1 in the sixth instar. It is an excellent idea to detect the expression of Kr-h1 at various larvae stages to analyze the stages in which CAD96CA and FGFR1 play a role and to study the relationship between CAD96CA and FGFR1 in future.

(10) While Figure 5 demonstrates JH-triggered calcium ion mobilization in Sf9 cells and S2 cells, the authors should also include data on JH signaling target genes, such as Kr-h1, for a more comprehensive analysis.

Thank you for your advice. We added experiments, as described in the text: "To demonstrate the universality of CAD96CA and FGFR1 in JH signaling in different insect cells, we investigated JH-triggered calcium ion mobilization and Kr-h1 expression in Sf9 cells developed from S. frugiperda and S2 cells developed from D. melanogaster. Knockdown of Cad96ca and Fgfr1 (named Htl or Btl in D. melanogaster), respectively, significantly decreased JH III-induced intracellular Ca2+ release and extracellular Ca2+ influx, and Kr-h1 expression (Figure 5A, B, Figure 5—figure supplement 1A and B). The efficacy of RNAi of Cad96ca and Fgfr1 was confirmed in the cells (Figure 5—figure supplement 1C and D), suggesting that CAD96CA and FGFR1 had a general function to transmit JH signal in S. frugiperda and D. melanogaster.".

(11) The authors should consider improving the quality of images and some plots, particularly enlarging panels showing larval and pupal phenotypes, such as Figure 1B and Supplementary Figure C. Additionally, adding a plot showing the statistical analysis of the phenotype in Supplementary Figure C would enhance clarity. Some plots are overly busy and difficult to read due to small size, such as Figure 1C, Figure 2A, and all the plots in Figure 3. Figure 4E also requires improvement for better readability.

Thank you for your suggestion. We have adjusted Figure 1B, Figure 1C, Figure 1—figure supplement 1C, Figure 2A and Figure 4E. However, for Figure 3, we have not found a better way to arrange and adapt them, considering the overall arrangement of the results and the page space, so we keep them in their original state.

I think this needs its own section for how to add students to a Cycle or how to add additional Forms.

GUARANTEES??

Are there any that you're ready to stand behind? Any that you'd be willing to offer your job seeker for choosing "MJL Projects LLC" over another résumé service?

Perhaps some type of "Post-Review Support Guarantee" that provides some type of ongoing support for a specified period (e.g., 30, 60, or 90 days) after the review is completed?

Again, with all the services and promises flung at desperate candidates, offering this type of limited/ongoing support might help to ease concerns about who to entrust with this #RésuméRevamp

"LINE BY LINE REVIEW":

But what, exactly, does that look like??

For job seekers that have sooooo many people pitching sooooo many different services, the one thing that I want to see is what this "line by line review" and the "detailed feedback on how...[to] improve" your résumé ACTUALLY looks like.

Is there a way that you can provide a couple of samples??

Just some thoughts:

• "Take your résumé bulletpoints from this [insert poorly (over)worded job description] to this [provide your polished version of the bullet" with our "Line by Line Review"

• "Let us help you market yourself most effectively to give you an edge in this highly competitive market"

'she felt sick' has the same impact

tone is too chatty

if she's laughing so hard her eyes water, how is her face barely moving?

tone is off

italics - also we know that she can control this, so the sentence is redundant

'in the frenzy to escape'

'the smell of eggs now mixing with the stench of tobacco...'

'- which came from the trash, definitely - '

what do the italics symbolise here

they're on the wall? how?

i think this word offsets the tone

this is too long

this is too long and we lose track of the sentence

interesting name but runs the risk of sounding too 2016 YA

i'm not sure about this paragraph - i don't think the repetition is effective here, and 'not even actually' is clunky. i also don't like the italics

Below the laptop, there's supposed to be a line, "Plus, don't miss out on our exclusive resources and tools -- available now on Eddy!"

Text is supposed to be dark blue. "Get in touch with our team" is supposed to be underlined and bolded

Color should be dark blue (same as the "Grants Available" line

Color of the text here is supposed to be a lighter blue. See figma

(same comment for all the body text in this section)

There's supposed to be subheader text below this, but it doesn't show up here. See the figma file

Author response:

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

Reviewer #1 (Public Review):

Summary:

The extra macrochaetae (emc) gene encodes the only Inhibitor of DNA binding protein (Id protein) in Drosophila. Its best-known function is to inhibit proneural genes during development. However, the emc mutants also display nonproneural phenotypes. In this manuscript, the authors examined four non-proneural phenotypes of the emc mutants and reported that they are all caused by inappropriate non-apoptotic caspase activity. These non-neuronal phenotypes are: reduced growth of imaginal discs, increased speed of the morphogenetic furrow, and failure to specify R7 photoreceptor neurons and cone cells during eye development. Double mutants between emc and either H99 (which deletes the three pro-apoptotic genes reaper, grim, and hid) or the initiator caspase dronc suppress these mutant phenotypes of emc suggesting that the cell death pathway and caspase activity are mediating these emc phenotypes. In previous work, the authors have shown that emc mutations elevate the expression of ex which activates the SHW pathway (aka the Hippo pathway). One known function of the SHW pathway is to inhibit Yorkie which controls the transcription of the inhibitor of apoptosis, Diap1. Consistently, in emc clones the levels of Diap1 protein are reduced which might explain why caspase activity is increased in emc clones giving rise to the four non-neural phenotypes of emc mutants.

However, this increased caspase activity is not causing ectopic apoptosis, hence the authors propose that this is nonapoptotic caspase activity. In the last part of the manuscript, the authors ruled out that Wg, Dpp, and Hh signaling are the target of caspases, but instead identified Notch signaling as the target of caspases, specifically the Notch ligand Delta. Protein levels of Delta are increased in emc clones in an H99- and dronc-dependent manner. The authors conclude that caspase-dependent non-apoptotic signaling underlies multiple roles of emc that are independent of proneural bHLH proteins.

Strengths:

Overall, this is an interesting manuscript and the findings are intriguing. It adds to the growing number of non-apoptotic functions of apoptotic proteins and caspases in particular. The manuscript is well written and the data are usually convincingly presented.

Weaknesses:

(1)  One major concern I have is the observation by the authors in Figure 3C in which protein levels of Diap1 are still reduced in emc H99 double mutant clones. If Diap1 is still reduced in these clones, shouldn't caspases still be derepressed? Given that emc H99 double mutants rescue all emc phenotypes examined, the observation that Diap1 levels are still reduced in emc H99 clones is inconsistent with the authors' model. The authors need to address this inconsistency.

The effect of H99 emc clones on Diap1 protein levels is consistent with our conclusions.  The reviewer’s concern probably relates to previous work that shows that RHG proteins act by antagonizing DIAP1, so that Diap1 is epistatic to RHG (PMID:10481910), and that RHG proteins affect DIAP1 protein levels, and in particular that HID promotes DIAP1 ubiquitylation leading to its destruction (PMID:12021767).  First, epistasis means that in the absence of DIAP1, RHG levels do not affect cell survival.  DIAP1 protein is not absent in emc/emc eye clones, however, it is reduced.  It is not only possible but expected that RHG levels would affect survival when DIAP1 levels are only reduced.  Secondly, we did not see a difference in DIAP1 levels between H99/H99 clones and H99/+ cells within the same specimen, suggesting that rpr, grim and hid might not affect DIAP1 levels. It is possible that Hid protein only affects DIAP1 levels when overexpressed, as in the aforementioned paper (PMID:12021767), and that physiological RHG levels affect DIAP1 activity.  The H99 deficiency also eliminates Rpr and Grim, which may affect DIAP1 without ubiquitylating it. In our experiments, however, there are no cells completely wild type for the H99 region for comparison in the same specimen, so our results do not rule out the H99 deletion having a dominant effect on DIAP1 levels both inside and outside the clones.  What our data clearly showed is that emc affected DIAP1 levels independently of any potential RHG effect, and we hypothesized this was through diap1 transcription, because we showed previously that emc affects yki, a transcriptional regulator of the diap1 gene, but we have not demonstrated transcriptional regulation of diap1 directly in emc clones.  We modified the manuscript to better delineate these issues (lines 275-284).    

(2) Are Diap1 protein levels reduced in all emc clones, including clones anterior to the furrow? This is difficult to see in Figure 3B. it is also recommended to look in emc mosaic wing discs.

We now mention that DIAP1 levels were only reduced in  emc clones posterior to the morphogenetic furrow, not anterior to the morphogenetic furrow or in emc clones in wing imaginal discs (lines 284-5) and Figure 3 supplement 1.  

(3) The authors speculate that Delta may be a direct target of caspase cleavage (Figure 9B), but then rule it out for a good reason. However, I assume that the increased protein levels of Delta in emc clones (Figure 7) are the results of increased transcription. In that case, shouldn't caspases control the transcriptional machinery leading to Delta expression?

Thank you for suggesting that caspases control the transcription of Dl.  We added this possibility to the manuscript (lines 499-500).  At one time there was a Dl-LacZ transcriptional reporter, which would have made it straightforward to assess Dl transcription in emc clones, but this strain does not seem to exist now.  We have not attempted in situ hybridization to Dl transcripts in mosaic discs.  

(4) How does caspase activity in emc clones cause reduced growth? Is this also mediated through Delta signaling?

We do not know what is the caspase target responsible for reduced growth in wing discs.

(5) Figure 1M: Is there a similar result with emc dronc mosaics?

The emc dronc clones do not show as dramatic a growth advantage in a Minute background.  This is consistent with the smaller effect of emc dronc in the non-Minute background also (Figure 1N).  We mention this in the revised paper (lines 232-3).     

Reviewer #2 (Public Review):

Id proteins are thought to function by binding and antagonizing basic helix-loop-helix (bHLH) transcription factors but new findings demonstrate roles for emc including in tissues where no proneural (Drosophila bHLH) genes are known to function. The authors propose a new mechanism for developmental regulation that entails restraining new/novel non-apoptotic functions of apoptotic caspases.

Specifically, the data suggest that loss of emc leads to reduced expression of diap1 and increased apoptotic caspase activity, which does not induce apoptosis but elevates Delta expression to increase N activity and cause developmental defects. Indeed, many of the phenotypes of emc mutant clones can be rescued by a chromosomal deficiency that reduces caspase activation or by mutations in the initiator caspase Dronc. A related manuscript that shows that loss of emc results in increased da, linked previously to diap1 expression, provides supporting data. There is increasing appreciation that apoptotic caspases have non-apoptotic roles. This study adds to the emerging field and should be of interest to readers.

The data, for the most part, support the conclusions but I do have concerns about some of the data and the interpretations that should be addressed.

Reviewer #3 (Public Review):

The work extends earlier studies on the Drosophila Id protein EMC to uncover a potential pathway that explains several tissue-scale developmental abnormalities in emc mutants. It also describes a non-apoptotic role for caspases in cell biology.

Strengths:

The work adds to an emerging new set of functions for caspases beyond their canonical roles as cell death mediators. This novelty is a major strength as well as its reliance on genetic-based in vivo study. The study will be of interest to those who are curious about caspases in general.

Weaknesses:

The manuscript relies on imaging experiments using genetic mosaic imaginal discs. It is for the most part a qualitative analysis, showing representative samples with a small number of mutant clones in each. Although the senior author has a long track record of using experiments like this to rigorously discover regulatory mechanisms in this system, it is straightforward in 2023 to use Fiji and other image analysis tools to measure fluorescence. Such measurements could be done for all replicate clones of a given genotype as well as genetic control sampling. These could be presented in plots that would not only provide quantitative and statistical measurements, but will be more reader- friendly to those who are not fly people.

We added quantification of anti-Delta and anti-Diap1 levels to the manuscript (Figures 3E and 7E).  We agree that this facilitates statistical confirmation of the results and may be more accessible to non-experts.  We do have concerns that these quantifications might be given too much weight.  For example, we cannot measure the background level of anti-DIAP1 labeling by labeling diap1 null mutant cells, because such cells do not survive.  Although we measure ~20% reduction in emc clones in the eye disc, and none in the wing disc, both measures could be underestimates if some of the labeling is non-specific, as is very possible.  We discuss this in the Methods (lines 166-9).

Likewise, more details are needed to describe how clone areas were measured in Figure 1. Did they measure each clone and its twin spot, and then calculate the area ratio for each clone and its paired twin spot? This would be the correct way to analyze the data, yielding many independent measurements of the ratio. And doing so would obviate the need to log transform the data which is inexplicable unless they were averaging clones and twins within a disc and making replicates. More explanation is needed and if they indeed averaged, then they need to calculate the ratios pairwise for each clone and twin.

We added details of clone size measurements and analysis to the methods (lines 141-6).  Although it might be useful to compare individual clones and corresponding twin spots, the only rigorous way to associate individual clones with individual twin spots, or even to determine what is one clone and what is one twin spot, is to use recombination rates low enough that significantly less than one recombination occurs per disc.  This would require many more dissections and we did not do this.  We now clarify in the manuscript that the analysis is indeed based on the ratio of total area of clones and twin spots with replicates, and that Log-transformation is to improve the normality of the ratio data suitable for parametric significance testing, not because clones and twin spots were summed from each sample.  We consulted with a statistician over this approach.  

Reviewer #1 (Recommendations For The Authors):

Lines 319/320: "Frizzled-3 RFP expression was not changed in in emc clones (Figure 4A)". This was actually not shown in Fig 4A (in fact this result was not shown at all). Fig 4A shows the result for emc nkd3 which the authors incorrectly assigned to Figure 4B (line 324).

We apologize for labeling Figure 4A and 4B incorrectly.

The title of Figure 6 is inaccurate. The title does not indicate what is shown in this figure. A more accurate title would be: Notch activity and function in emc mutant clones.

We provided a new title for Figure 6. 

Reviewer #2 (Recommendations For The Authors):

There is no information on how reproducible the data is. How many discs were examined in each experiment and in how many technical or biological replicates? Can fluorescence signals be quantified within and outside the clones and presented to illustrate reproducibility and significance? This is especially needed for Fig 7, which shows key data that N ligand Delta is elevated in emc clones but dronc and H99 mutations rescue this phenotype. I can see that the Dl signal is brighter in the GFP- emc clone in Fig 7B but I can also see a brighter Dl signal in the small clone and perhaps also in the large clone in C. The difference between B and C could be simply disc-to-disc variation, which should be addressed with quantification and presentation of all data points.

We added the number of samples to each figure legend.  We quantified the fluorescence signals for Figures 3 and 7.  Quantification shows that the difference between 7B and 7C is highly significant, not disc to disc variation.

Fig 2B does not support the conclusion. It is supposed to show premature Sens expression and therefore abnormal morphogenetic furrow progression in emc clones. But the yellow arrow is pointing to GFP+ (wild type) cells and it is within this GFP+ region that most premature Sens expression is seen.

We relocated the arrows in Figure 2B to point precisely to the premature differentiation.  When the morphogenetic furrow is accelerated in emc mutant, GFP – tissue, it does not stop when wild type, GFP+ tissue is encountered again, it continues at a normal pace.  Accordingly, emc+ regions that are anterior to emc- regions can also experience accelerated differentiation (please see lines 594-8).

Fig 1 shows that while H99 deficiency restores the growth of emc clones to wild type level (Fig 1N), placing these in the Minute background made emc clones grow better than emc wild type but Minute neighbors (Fig 1M). The latter cells were nearly absent, suggesting elimination through cell competition. For the rest of the figures, some experiments are done in the Minute background (e.g., emc H99 clones in Fig 2D) while others are not in the Minute background (e.g., emc H99 clones in Fig 7D). Why the switch between backgrounds from experiment to experiment?

Figure 2D shows emc H99 clones in a Minute background so that it can be compared with panels 2A-C, which show clones of other genotypes in a Minute background.  These clones almost take over the eye disc.  In Figure 7D, it was important to show the Dl expression pattern in a substantial wild type region, which could only be shown using the non-Minute background.  We have no indication that a Minute background changes the properties of the nonMinute clone, other than allowing its greater growth.  

The first 3 paragraphs of the Introduction are overly detailed and read more like a review article. These could be made more concise to focus on the founding data for this manuscript, which are the published findings that emc mutations elevate ex expression (line 129) and that ex mutants show elevated diap1 expression (line 125). These do not show up until the very end of the Introduction.

We shortened the Introduction to focus more rapidly on the topics relevant to these experiments.

In several places, the space between the end of the sentence and the citation is missing (e.g., lines 57, 68, and 75).

The spacing of citations was fixed.

Line 247. 'morphogenetic furrow that found each ommatidia...' should use a word besides 'found.'

We corrected line 247.

Reviewer #3 (Recommendations For The Authors):

(1) The authors show that inhibiting caspases rescues the growth defect of emc clones. However, they did not find excessive TUNEL staining in emc clones that would explain why the clones would be so small - excessive cell death. How reliable was their tunel staining in being able to detect excessive apoptosis (only negative data was shown). Could they induce excessive cell death using radiation or some other means to ensure the assay is robust? If death is not occurring in emc clones, a deficiency worth addressing is that they do not discuss or explore how the caspases then inhibit clone growth. Is it expanded cell cycle times, or smaller cells?? And that phenotype does not fit with their end model of Delta being the only moderator of emc since it is not playing a significant role in tissue growth anterior to the furrow.One would assume using the commercial antibody against activated caspase would be another readout for emc clones and this would bolster their claim that excessive caspase activation occurs in the emc cells.

We have added Dcp1 staining in Figure 2 supplement 3 to show that TUNEL staining is reliable.

(2) Figure 3D has really large emc clones when GMR-Diap is present. But the large clones are anterior to the furrow where Diap would not be overexpressed. Is this just an unusual sample with a coincidentally big emc M+ clone? It speaks to my concerns about the qualitative nature of the data.

We replaced Figure 3D with an example of smaller clones.  Nowhere have we suggested that  GMR-DIAP1 affects clone size.

(3) Figure 9B is very speculative and not appropriate since the authors have zero data to support that cleavage mechanism. It is fit for the next paper if the idea is correct. The panel should be removed.

We did not intend Figure 9B to imply that we think Dl itself is the relevant target of non-apoptotic caspases.  Since apparently we gave that impression, we removed this to a supplemental figure.  We still think it is worth showing that Dl does not contain predicted caspase sites expected to activate signaling. 

(4) Figure 9A could be made more clear. Their pathway represents the mutant cells in the mosaic disc. Why not also outline what you think is happening in the emc+ cells as well?

It is difficult to make a comparable diagram for normal cells, because none of this pathway happens in normal cells.  We modified the figure legend to indicate this (lines 677-8).

(5) The one emc ci clone they show spanning the furrow has a very non-continuous furrow advance phenotype. This is unlike the emc clones where the furrow advance is graded about the clone. And it resembles the SuH clones they show. This result and the synergistic effect on clone sizes they mention need more discussion and thought put into it. It argues ci is doing something with respect to emc action. loss of ci might not rescue size and furrow advance but actually, it makes it worse! This is interesting and might suggest an inhibitory role for ci in emc or a parallel role for ci in mediating growth and progression that is redundant with emc.

We agree that aspects of the emc ci phenotype are not clear.  We discuss this in the revised manuscript (lines 373-5).  

(6) Related to point 7, it is a weak argument for non-autonomy that graded furrow advance in emc clones is evidence for emc acting nonautonomously through Delta. Its weakness is combined with its lack of significance relative to the other findings. It should be deleted as should the SuH data.

We agree that the evidence that emc affects morphogenetic furrow progression non-autonomously is not compelling and have revised the manuscript to soften this conclusion (lines 426-7).  We do not want to remove this idea, because it does in fact have significance for other findings.  Specifically, it supports the idea that the emc effect in the morphogenetic furrow is due to trans-activation by Delta, whereas  the effect on R7 and cone cell differentiation is due to autonomous cis-inhibition.  We think this is important to keep in the paper.

Reviewer #1 (Public review):

Summary:

The extra macrochaetae (emc) gene encodes the only Inhibitor of DNA binding protein (Id protein) in Drosophila. Its best-known function is to inhibit proneural genes during development. However, the emc mutants also display non-proneural phenotypes. In this manuscript, the authors examined four non-proneural phenotypes of the emc mutants and reported that they are all caused by inappropriate non-apoptotic caspase activity. These non-neuronal phenotypes are: reduced growth of imaginal discs, increased speed of the morphogenetic furrow, and failure to specify R7 photoreceptor neurons and cone cells during eye development. Double mutants between emc and either H99 (which deletes the three pro-apoptotic genes reaper, grim, and hid) or the initiator caspase dronc suppress these mutant phenotypes of emc suggesting that the cell death pathway and caspase activity are mediating these emc phenotypes. In previous work, the authors have shown that emc mutations elevate the expression of ex which activates the SHW pathway (aka the Hippo pathway). One known function of the SHW pathway is to inhibit Yorkie which controls the transcription of the inhibitor of apoptosis, Diap1. Consistently, in emc clones the levels of Diap1 protein are reduced which might explain why caspase activity is increased in emc clones giving rise to the four non-neural phenotypes of emc mutants. However, this increased caspase activity is not causing ectopic apoptosis, hence the authors propose that this is non-apoptotic caspase activity. In the last part of the manuscript, the authors ruled out that Wg, Dpp, and Hh signaling are the target of caspases, but instead identified Notch signaling as the target of caspases, specifically the Notch ligand Delta. Protein levels of Delta are increased in emc clones in an H99- and dronc-dependent manner. The authors conclude that caspase-dependent non-apoptotic signaling underlies multiple roles of emc that are independent of proneural bHLH proteins.

Strengths:

Overall, this is an interesting manuscript and the findings are intriguing. It adds to the growing number of non-apoptotic functions of apoptotic proteins and caspases in particular. The manuscript is well written and the data are usually convincingly presented.

Weaknesses:

The authors have addressed all my concerns and questions.

Reviewer #2 (Public review):

Id proteins are thought to function by binding and antagonizing basic helix-loop-helix (bHLH) transcription factors but new findings demonstrate roles for emc including in tissues where no proneural (Drosophila bHLH) genes are known to function. The authors propose a new mechanism for developmental regulation that entails restraining new/novel non-apoptotic functions of apoptotic caspases.

Specifically, the data suggest that loss of emc leads to reduced expression of diap1 and increased apoptotic caspase activity, which does not induce apoptosis but elevates Delta expression to increase N activity and cause developmental defects. Indeed, many of the phenotypes of emc mutant clones can be rescued by a chromosomal deficiency that reduces caspase activation or by mutations in the initiator caspase Dronc. A related manuscript that shows that loss of emc results in increased da, linked previously to diap1 expression, provides supporting data. There is increasing appreciation that apoptotic caspases have non-apoptotic roles. This study adds to the emerging field and should be of interest to the readers.

The revised manuscript addresses my concerns from the first round of review.

Reviewer #3 (Public review):

The work extends earlier studies on the Drosophila Id protein EMC to uncover a potential pathway that explains several tissue-scale developmental abnormalities in emc mutants. It also describes a non-apoptotic role for caspases in cell biology.

Strengths:

The work adds to an emerging new set of functions for caspases beyond their canonical roles as cell death mediators. This novelty is a major strength as well as its reliance on genetic-based in vivo study. The study will be of interest to those who are curious about caspases in general.

Weaknesses:

The authors did an adequate job in dealing with the limitations of the reviewed preprint. Although they could have done more, they chose not to for reasons they adequately defended.

Author response:

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

Reviewer #1 (Public Review):

(1) This experiment sought to determine what effect congenital/early-onset hearing loss (and associated delay in language onset) has on the degree of inter-individual variability in functional connectivity to the auditory cortex. Looking at differences in variability rather than group differences in mean connectivity itself represents an interesting addition to the existing literature. The sample of deaf individuals was large, and quite homogeneous in terms of age of hearing loss onset, which are considerable strengths of the work. The experiment appears well conducted and the results are certainly of interest. I do have some concerns with the way that the project has been conceptualized, which I share below.

Thank you for acknowledging the strengths and novelty of our study. We have now addressed the conceptual issues raised; please see below in the specific comments.

(2) The authors should provide careful working definitions of what exactly they think is occurring in the brain following sensory deprivation. Characterizing these changes as 'largescale neural reorganization' and 'compensatory adaptation' gives the impression that the authors believe that there is good evidence in support of significant structural changes in the pathways between brain areas - a viewpoint that is not broadly supported (see Makin and Krakauer, 2023). The authors report changes in connectivity that amount to differences in coordinated patterns of BOLD signal across voxels in the brain; accordingly, their data could just as easily (and more parsimoniously) be explained by the unmasking of connections to the auditory cortex that are present in typically hearing individuals, but which are more obvious via MR in the absence of auditory inputs.

We thank the Reviewer for the suggestion to clarify and better support our stance regarding reorganization. We indeed believe that the adaptive changes in the auditory cortex in deafness represent real functional recruitment for non-auditory functions, even in the relatively limited large-scale anatomical connectivity changes. This is supported by animal works showing causal evidence for the involvement of deprived auditory cortices in non-auditory tasks, in a way that is not found in hearing controls (e.g., Lomber et al., 2010, Meredith et al., 2011, reviewed in Alencar et al., 2019; Lomber et al., 2020). Whether the word “reorganization” should be used is indeed debated recently (Makin and Krakauer, 2023). Beyond terminology, we do agree that the basis for the changes in recruitment seen in the brains of people with deafness or blindness is largely based on the typical anatomical connectivity at birth. We also agree that at the group level, there is poor evidence of large-scale anatomical connectivity differences in deprivation. However, we think there is more than ample evidence that the unmasking and more importantly re-weighting of non-dominant inputs gives rise to functional changes. This is supported by the relatively weaker reorganization found in late-onset deprivation as compared to early-onset deprivation. If unmasking of existing connectivity without any functional additional changes were sufficient to elicit the functional responses to atypical stimuli (e.g., non-visual in blindness and non-auditory in deafness), one would expect there to be no difference between early- and late-onset deprivation in response patterns. Therefore, we believe that the fact that these are based on functions with some innate pre-existing inputs and integration is the mechanism of reorganization, not a reason not to treat it as reorganization. Specifically, in the case of this manuscript, we report the change in variability of FC from the auditory cortex, which is greater in deafness than in typically hearing controls. This is not an increase in response per se, but rather more divergent values of FC from the auditory cortex, which are harder to explain in terms of ‘unmasking’ alone, unless one assumes unmasking is particularly variable. The mechanistic explanation for our findings is that in the absence of auditory input’s fine-tuning and pruning of the connectivity of the auditory cortex, more divergent connectivity strength remains among the deaf. Thus, auditory input not only masks non-dominant inputs but also prunes/deactivates exuberant connectivity, in a way that generates a more consistently connected auditory system. We have added a shortened version of these clarifications to the discussion (lines 351-372).

(3) I found the argument that the deaf use a single modality to compensate for hearing loss, and that this might predict a more confined pattern of differential connectivity than had been previously observed in the blind to be poorly grounded. The authors themselves suggest throughout that hearing loss, per se, is likely to be driving the differences observed between deaf and typically-hearing individuals; accordingly, the suggestion that the modality in which intentional behavioral compensation takes place would have such a large-scale effect on observed patterns of connectivity seems out of line.

Thank you for your critical insight regarding our rationale on modality use and its impact on connectivity patterns in the deaf compared to the blind. After some thought, we agree that the argument presented may not be sufficiently strong and could distract from the main findings of our study. Therefore, we have decided to remove this claim from our revised manuscript.

(4) The analyses highlighting the areas observed to be differentially connected to the auditory cortex and areas observed to be more variable in their connectivity to the auditory cortex seem somewhat circular. If the authors propose hearing loss as a mechanism that drives this variability in connectivity, then it is reasonable to propose hypotheses about the directionality of these changes. One would anticipate this directionality to be common across participants and thus, these areas would emerge as the ones that are differently connected when compared to typically hearing folks.

We are a little uncertain how to interpret this concern.  If the question was about the logic leading to our statement that variability is driven by hearing loss, then yes, we indeed were proposing hearing loss as a mechanism that drives this variability in connectivity to the auditory cortex; we regret this was unclear in the original manuscript. This logic parallels the proposal made with regard to the increased variability in FC in blindness; deprivation leads to more variable outcomes, due to the lack of developmental environmental constraints (Sen et al., 2022). Specifically, we first analyzed the differences in within-group variability between deaf and hearing individuals (Fig. 1A), followed by examining the variability ratio (Fig. 1B) in the same regions that demonstrated differences. The first analysis does not specify which group shows higher variability; therefore, the second analysis is essential to clarify the direction of the effect and identify which group, and in which regions, exhibits greater variability. We have clarified this in the revised manuscript (lines 125-127): “To determine which group has larger individual differences in these regions (Figure 1B), we computed the ratio of variability between the two groups (deaf/hearing) in the areas that showed a significant difference in variability (Figure 1A)”. Nevertheless, this comment can also be interpreted as predicting that any change in FC due to deafness would lead to greater variability. In this case, it is also important to mention that while we would expect regions with higher variability to also show group differences between the deaf and the hearing (Figure 2), our analysis demonstrates that variability is present even in regions without significant group mean differences. Similarly, many areas that show a difference between the groups in their FC do not show a change in variability (for example, the bilateral anterior insula and sensorimotor cortex). In fact, the correlation between the regions with higher FC variability (Figure 1A) and those showing FC group differences (Figure 2B) is significant but rather modest, as we now acknowledge in our revised manuscript (lines 324-328). Therefore, increased FC and increased variability of FC are not necessarily linked. 

(5) While the authors describe collecting data on the etiology of hearing loss, hearing thresholds, device use, and rehabilitative strategies, these data do not appear in the manuscript, nor do they appear to have been included in models during data analysis. Since many of these factors might reasonably explain differences in connectivity to the auditory cortex, this seems like an omission.

We thank the Reviewer for their comment regarding the inclusion of these variables in our manuscript. We have now included additional information in the main text and a supplementary table in the revised manuscript that elaborates further on the etiology of hearing loss and all individual information that characterizes our deaf sample. Although we initially intended to include individual factors (e.g., hearing threshold, duration of hearing aid use, and age of first use) in our models, this was not feasible for the following reasons: 1) for some subjects, we only have a level  of hearing loss rather than specific values, which we could not use quantitatively as a nuisance variable (it was typical in such testing to ascertain the threshold of loss as belonging to a deafness level, such as “profound” and not necessarily go into more elaborate testing to identify the specific threshold), and 2) this information was either not collected for the hearing participants (e.g., hearing threshold) or does not apply to them (e.g., age of hearing aid use), which made it impossible to use the complete model with all these variables. Modeling the groups separately with different variables would also be inappropriate. Last, the distribution of the values and the need for a large sample to rigorously assess a difference in variability also precluded sub-dividing the group to subgroup based on these values. 

Therefore, we opted for a different way to control for the potential influence of these variables on FC variability in the deaf. We tested the correlation between the FC from the auditory cortex and each of these parameters in the areas that showed increased FC in deafness (Figures 1A, B), to see if it could account for the increased variability. This ROI analysis did not reveal any significant correlations (all p > .05, prior to correction for multiple comparisons; see Figures S4, S5, and S6 for scatter plots). The maximal variability explained in these ROIs by the hearing factors was r2\=0.096, whereas the FC variability (Figure 1B) was increased by at least 2 in the deaf. Therefore, it does not seem like these parameters underlie the increased variability in deafness. To test if these variables had a direct effect on FC variability in other areas in the brain, we also directly computed the correlation between FC and each factor individually. At the whole-brain level, the results indicate a significant correlation between AC-FC and hearing threshold, as well as a correlation between AC-FC and the age of hearing aid use onset, but not for the duration of hearing aid use (Figure S3). While these may be interesting on their own, and are added to the revised manuscript, the regions that show significant correlations with hearing threshold and age of hearing aid use are not the same regions that exhibit FC variability in the deaf (Figures 1A, B).

Overall, these findings suggest that although some of these factors may influence FC, they do not appear to be the driving factors behind FC variability. Finally, in terms of rehabilitative strategies, only one deaf subject reported having received long-term oral training from teachers. This participant started this training at age 2, as now described in the participants’ section. We thank the reviewer for raising this concern and allowing us to show that our findings do not stem from simple differences ascribed to auditory experience in our participants. 

Reviewer #2 (Public Review):

(1) The paper has two main merits. Firstly, it documents a new and important characteristic of the re-organization of the brains of the deaf, namely its variability. The search for a welldefined set of functions for the deprived auditory cortex of the deaf has been largely unsuccessful, with several task-based approaches failing to deliver unanimous results. Now, one can understand why this was the case: most likely there isn't a fixed one well-defined set of functions supported by an identical set of areas in every subject, but rather a variety of functions supported by various regions. In addition, the paper extends the authors' previous findings from blind subjects to the deaf population. It demonstrates that the heightened variability of connectivity in the deprived brain is not exclusive to blindness, but rather a general principle that applies to other forms of deprivation. On a more general level, this paper shows how sensory input is a driver of the brain's reproducible organization.

We thank the Reviewer for their observations regarding the merits of our study. We appreciate the recognition of the novelty in documenting the variability of brain reorganization in deaf individuals. 

(2) The method and the statistics are sound, the figures are clear, and the paper is well-written. The sample size is impressively large for this kind of study.

We thank the Reviewer for their positive feedback on the methodology, statistical analysis, clarity of figures, and the overall composition of our paper. We are also grateful for the acknowledgment of our large sample size, which we believe significantly strengthens the statistical power and the generalizability of our findings.

(3) The main weakness of the paper is not a weakness, but rather a suggestion on how to provide a stronger basis for the authors' claims and conclusions. I believe this paper could be strengthened by including in the analysis at least one of the already published deaf/hearing resting-state fMRI datasets (e.g. Andin and Holmer, Bonna et al., Ding et al.) to see if the effects hold across different deaf populations. The addition of a second dataset could strengthen the evidence and convincingly resolve the issue of whether delayed sign language acquisition causes an increase in individual differences in functional connectivity to/from Broca's area. Currently, the authors may not have enough statistical power to support their findings.

We thank the Reviewer for their constructive suggestion to reinforce the robustness of our findings. While we acknowledge the potential value of incorporating additional datasets to strengthen our conclusions, the datasets mentioned (Andin and Holmer, Bonna et al., Ding et al.) are not publicly available, which limits our ability to include them in our analysis. Additionally, datasets that contain comparable groups of delayed and native deaf signers are exceptionally rare, further complicating the possibility of their inclusion. Furthermore, to discern individual differences within these groups effectively, a substantially larger sample size is necessary. As such, we were unfortunately unable to perform this additional analysis. This is a challenge we acknowledge in the revised manuscript (lines 442-445), especially when the group is divided into subcategories based on the level of language acquisition, which indeed reduces our statistical power. We have however, now integrated the individual task accuracy and reaction time parameters as nuisance variables in calculating the variability analyses; all the results are fully replicated when accounting for task difficulty. We also report that there was no group difference in activation for this task between the groups which could affect our findings. 

We would like to note that while we would like to replicate these findings in an additional cohort using resting-state, we do not anticipate the state in which the participants are scanned to greatly affect the findings. FC patterns of hearing individuals have been shown to be primarily shaped by common system and stable individual features, and not by time, state, or task (Finn et al., 2015; Gratton et al., 2018; Tavor et al., 2016). While the task may impact FC variability, we have recently shown that individual FC patterns are stable across time and state even in the context of plasticity due to visual deprivation (Amaral et al., 2024). Therefore, we expect that in deafness as well there should not be meaningful differences between resting-state and task FC networks, in terms of FC individual differences. That said, we are exploring collaborations and other avenues to access comparable datasets that might enable a more powerful analysis in future work. This feedback is very important for guiding our ongoing efforts to verify and extend our conclusions.

(4) Secondly, the authors could more explicitly discuss the broad implications of what their results mean for our understanding of how the architecture of the brain is determined by the genetic blueprint vs. how it is determined by learning (page 9). There is currently a wave of strong evidence favoring a more "nativist" view of brain architecture, for example, face- and object-sensitive regions seem to be in place practically from birth (see e.g. Kosakowski et al., Current Biology, 2022). The current results show what is the role played by experience.

We thank the Reviewer for highlighting the need to elaborate on the broader implications of our findings in relation to the ongoing debate of nature vs. nurture. We agree that this discussion is crucial and have expanded our manuscript to address this point more explicitly. We now incorporate a more detailed discussion of how our results contribute to understanding the significant role of experience in shaping individual neural connectivity patterns, particularly in sensory-deprived populations (lines 360-372).

Reviewer #3 (Public Review):

Summary:

(1) This study focuses on changes in brain organization associated with congenital deafness. The authors investigate differences in functional connectivity (FC) and differences in the variability of FC. By comparing congenitally deaf individuals to individuals with normal hearing, and by further separating congenitally deaf individuals into groups of early and late signers, the authors can distinguish between changes in FC due to auditory deprivation and changes in FC due to late language acquisition. They find larger FC variability in deaf than normal-hearing individuals in temporal, frontal, parietal, and midline brain structures, and that FC variability is largely driven by auditory deprivation. They suggest that the regions that show a greater FC difference between groups also show greater FC variability.

Strengths:

-  The manuscript is well written.

-  The methods are clearly described and appropriate.

-  Including the three different groups enables the critical contrasts distinguishing between different causes of FC variability changes.

-  The results are interesting and novel.

We thank the Reviewer for their positive and detailed feedback. Their acknowledgment of the clarity of our methods and the novelty of our results is greatly appreciated.

Weaknesses:

(2) Analyses were conducted for task-based data rather than resting-state data. It was unclear whether groups differed in task performance. If congenitally deaf individuals found the task more difficult this could lead to changes in FC.

We thank the Reviewer for their observation regarding possible task performance differences between deaf and hearing participants and their potential effect on the results. Indeed, there was a difference in task accuracy between these groups. To account for this variation and ensure that our findings on functional connectivity were not confounded by task performance, we now included individual task accuracy and reaction time as nuisance variables in our analyses. This approach allowed us to control for any performance differences. The results now presented in the revised manuscript account for the inclusion of these two nuisance variables (accuracy and reaction time) and completely align with our original conclusions, highlighting increased variability in deafness, which is found in both the entire deaf group at large, as well as when equating language experience and comparing the hearing and native signers. The correlation between variability and group differences also remains significant, but its significance is slightly decreased, a moderate effect we acknowledge in the revised manuscript (see comment #4). The differences between the delayed signers and native signers are also retained (Figure 3), now aligning better with language-sensitive regions, as previously predicted. The inclusion of the task difficulty predictors also introduced an additional finding in this analysis, a significant cluster in the right aIFG. Therefore, the inclusion of these predictors reaffirms the robustness of the conclusions drawn about FC variability in the deaf population.

We would like to note that while we would like to replicate these findings in an additional cohort using resting-state if we had access to such data, we do not anticipate the state in which the participants are scanned to greatly affect the findings. FC patterns of hearing individuals have been shown to be primarily shaped by common system and stable individual features, and not by time, state, or task (Finn et al., 2015; Gratton et al., 2018; Tavor et al., 2016). While the task may impact FC variability, we have recently shown that individual FC patterns are stable across time and state even in the context of plasticity due to visual deprivation (Amaral et al., 2024). Therefore, we expect that in deafness as well there should not be meaningful differences between resting-state and task FC networks, in terms of FC individual differences. We have also addressed this point in our manuscript (lines 442-451).

(3) No differences in overall activation between groups were reported. Activation differences between groups could lead to differences in FC. For example, lower activation may be associated with more noise in the data, which could translate to reduced FC.

We thank the reviewer for noting the potential implications of overall activation differences on FC. In our analysis of the activation for words, we found no significant clusters showing a group difference between the deaf and hearing participants (p < .05, cluster-corrected for multiple comparisons) - we also added this information to the revised manuscript (lines 542-544). This suggests that the differences in FC observed are not confounded by variations in overall brain activation between the groups under these conditions.

(4) Figure 2B shows higher FC for congenitally deaf individuals than normal-hearing individuals in the insula, supplementary motor area, and cingulate. These regions are all associated with task effort. If congenitally deaf individuals found the task harder (lower performance), then activation in these regions could be higher, in turn, leading to FC. A study using resting-state data could possibly have provided a clearer picture.

We thank the Reviewer for pointing out the potential impact of task difficulty on FC differences observed in our study. As addressed in our response to comment #2, task accuracy and reaction times were incorporated as nuisance variables in our analysis. Further, these areas showed no difference in activation between the groups (see response to comment #3 above). Notably, the referred regions still showed higher FC in congenitally deaf individuals even when controlling for these performance differences. Additionally, these findings are consistent with results from studies using resting-state data in deaf populations, further validating our observations. Specifically, using resting-state data, Andin & Holmer (2022), have shown higher FC for deaf (compared to hearing individuals) from auditory regions to the cingulate cortex, insular cortex, cuneus and precuneus, supramarginal gyrus, supplementary motor area, and cerebellum. Moreover, Ding et al. (2016) have shown higher FC for the deaf between the STG and anterior insula and dorsal anterior cingulated cortex. This suggests that the observed FC differences are likely reflective of genuine neuroplastic adaptations rather than mere artifacts of task difficulty. Although we wish we could augment our study with resting-state data analyzed similarly, we could not at present acquire or access such a dataset. We acknowledge this limitation of our study (lines 442-451) in the revised manuscript and intend to confirm that similar results will be found with resting state data in the future.

(5) The correlation between the FC map and the FC variability map is 0.3. While significant using permutation testing, the correlation is low, and it is not clear how great the overlap is.

We acknowledge that the correlation coefficient of 0.3, while statistically significant, indicates a moderate overlap. It's also worth noting that, using our new models that include task performance as a nuisance variable, this value has decreased somewhat, to 0.24 (which is still highly significant). It is important to note that the visual overlap between the maps is not a good estimate of the correlation, which was performed on the unthresholded maps, to estimate the link not only between the most significant peaks of the effects, but across the whole brain patterns. This correlation is meant to suggest a trend rather than a strong link, but especially due to its consistency with the findings in blindness, we believe this observation merits further investigation and discussion. As such, we kept it in the revised manuscript while moderating our claims about its strength.

Reviewer #1 (Recommendations For The Authors):

(1) Page 4: Does auditory cortex FC variability..." FC is not yet defined.

Corrected, thanks.

(2) Page 4: "It showed lower variability..." What showed this?

Clarified, thanks.

(3) Page 11: "highlining the importance" should read "highlighting the importance".

Corrected, thanks.

(4) Page 11: Do you really mean to suggest functional connectivity does not vary as a function of task? This would not seem well supported.

We do not suggest that FC doesn’t vary as a function of task, and have revised this section (lines 447-451). 

(5) Page 12: "there should not to be" should read "there should not be".

Corrected, thanks.

(6) Page 12: "and their majority" should read "and the majority".

Corrected, thanks.

Reviewer #2 (Recommendations For The Authors):

Major

(1) Although this is a lot of work, I nonetheless have another suggestion on how to test if your results are strong and robust. Perhaps you could analyze your data using an ROI/graph-theory approach. I am not an expert in graph theory analysis, but for sure there is a simple and elegant statistic that captures the variability of edge strength variability within a population. This approach could not only validate your results with an independent analysis and give the audience more confidence in their robustness, but it could also provide an estimate of the size of the effect size you found. That is, it could express in hard numbers how much more variable the connections from auditory cortex ROI's are, in comparison to the rest of the brain in the deaf population, relative to the hearing population.

We thank the Reviewer for suggesting the use of graph theory as a method to further validate our findings. While we see the potential value in this approach, we believe it may be beyond the scope of the current paper, and merits a full exploration of its own, which we hope to do in the future.  However, we understand the importance of showing the uniqueness of the connectivity of the auditory cortex ROI as compared to the rest of the brain. So, in order to bolster our results, we conducted an additional analysis using control regions of interest (ROIs). Specifically, we calculated the inter-individual variability using all ROIs from the CONN Atlas (except auditory and language regions) as the control seed regions for the FC. We showed that the variability of connectivity from the auditory cortex is uniquely more increased on deafness, as compared to these control ROIs (Figure S1). This additional analysis supports the specificity of our findings to the auditory cortex in the deaf population. We aim to integrate more analytic approaches, including graph theory methods, in our future work.

Minor

(1) Some citations display the initial of the author in addition to the last name, unless there is something I don't know about the citation system, the initial shouldn't be there.

This is due to the citation style we're using (APA 7th edition, as suggested by eLife), which requires including the first author's initials in all in-text citations when citing multiple authors with the same last name.  

Reviewer #3 (Recommendations For The Authors):

(1) I recommend that the authors provide behavioral data and results for overall neural activation.

Thanks. We have added these to the revised manuscript. Specifically, we report that there was no difference in the activation for words (p < .05, cluster-corrected for multiple comparisons) between the deaf and hearing participants. Further, we report the behavioral averages for accuracy and reaction time for each group, and have now used these individual values explicitly as nuisance variables in the revised analyses.

(2) For the correlation between FC and FC variability, it seemed a bit odd that the permuted data were treated additionally (through Gaussian smoothing). I understand the general logic (i.e., to reintroduce smoothness), but this approach provides more smoothing to the permutation than the original data. It is hard to know what this does to the statistical distribution. I recommend using a different approach or at least also reporting the p-value for non-smoothed permutation data.

In response to this suggestion and to ensure transparency in our results, we have now included also the p-value for the non-smoothed permutation data in our revised manuscript (still highly significant; p < .0001). Thanks for this proposal.

(3) For the map comparison, a plot with different colors, showing the FC map, the FC variability map, and one map for the overlap on the same brain may be helpful.

We thank the Reviewer for their suggestion to visualize the overlap between the maps. However, we performed the correlation analysis using the unthresholded maps, as mentioned in the methods section of our manuscript, specifically to estimate the link not only between the most significant peaks of the effects, but across the whole brain patterns. This is why the maps displayed in the figures, which are thresholded for significance, may not appear to match perfectly, and may actually obscure the correlation across the brain. This methodological detail is crucial for interpreting the relationship and overlap between these maps accurately but also explains why the visualization of the overlap is, unfortunately, not very informative.

eLife Assessment

This study presents valuable data on the increase in individual differences in functional connectivity with the auditory cortex in individuals with congenital/early-onset hearing loss compared to individuals with normal hearing. The evidence supporting the study's claims is convincing, although additional work using resting-state functional connectivity and further links to how the results align with the underlying biology could have further strengthened the study. The work will be of interest to neuroscientists working on brain plasticity and may have implications for the design of interventions and compensatory strategies.

Reviewer #1 (Public review):

This experiment sought to determine what effect congenital/early-onset hearing loss (and associated delay in language onset) has on the degree of inter-individual variability in functional connectivity to the auditory cortex. Looking at differences in variability rather than group differences in mean connectivity itself represents an interesting addition to the existing literature. The sample of deaf individuals was large, and quite homogeneous in terms of age of hearing loss onset, which are considerable strengths of the work. The experiment appears well conducted and the results are certainly of interest.

Reviewer #2 (Public review):

Summary:

This study focuses on changes in brain organization associated with congenital deafness. The authors investigate differences in functional connectivity (FC) and differences in the variability of FC. By comparing congenitally deaf individuals to individuals with normal hearing, and by further separating congenitally deaf individuals into groups of early and late signers, the authors can distinguish between changes in FC due to auditory deprivation and changes in FC due to late language acquisition. They find larger FC variability in deaf than normal-hearing individuals in temporal, frontal, parietal, and midline brain structures, and that FC variability is largely driven by auditory deprivation. They suggest that the regions that show a greater FC difference between groups also show greater FC variability.

Strengths:

The manuscript is well-written, and the methods are clearly described and appropriate. Including the three different groups enables the critical contrasts distinguishing between different causes of FC variability changes. The results are interesting and novel.

Weaknesses:

Analyses were conducted for task-based data rather than resting-state data. The authors report behavioral differences between groups and include behavioral performance as a nuisance regressor in their analysis. This is a good approach to account for behavioral task differences, given the data. Nevertheless, additional work using resting-state functional connectivity could remove the potential confound fully.

The authors have addressed my concerns well.

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a lot of good business people seem to reject the church- is it in this rejection from Church and tradition that they are successful?

capitalist spirit- re-organization and re-orientation of every part of business to increase profit beyond sustainability or expectation

capitalist behavior with traditionalist forms of business- non challenging nature

don't need to be capitalist entrepreneur to put forth "capitalist spirit"

Economic enterprise doesn't equal capitalism

mindset of capitalism cannot be perpetuated by certain economic conditions- higher or lower wages- must be a long re-education on a mass scale

capitalism has biggest beef with traditionalism- which Weber will specify

there's always been randoms who only want to make money

ethical duty to do job in a capitalist society

money making as expression of virtue

closely related to religious ideas

Weird paradox- the making of money is the end goal as opposed to what can be produced from said money

values are beyond egocentric

Western capitalism distinct in its ethical coloring and purpose of daily life

because the aspects of modern life would not fare with the old protestants- we can't look for a sort of materialistic or non materialistic way of living

why are those who rejected wordliness also wealthy?

protestants are maybe now seen as indulgent albeit hardworking but that wasn't always the case- used to have very sober character

So now we have to look at the difference in religions

usually minority groups are the ones driving peculiar economic activities but not the case with the Catholics

beyond just a class or wealth issues- Protestants more likely to acquire skilled labour and administrative positions in factories than Catholics

Stratifications in inherited wealth from Protestants and Catholics can't account for the differences in education

misconception that protestant reformation brought about this separation of church and state that allowed for capitalist entrepreneurs to flourish but historically, that isn't the case

What do we want to know? Why are the business leaders, owners of capital, and more technically trained people all protestant?

find where legal system and formal perfection of West came from

what social structures fed into this use of technical knowledge in the west?

West had technical utilization of scientific knowledge

Concerned with development of western bourgeois class and what makes it unique, related but different from capitalistic organization of labor,

rational organization of capitalist enterprise result of: 1. separation of business from household 2. rational book-keeping

capitalistic economic action- exchange with the expectation of profit

So do most other mothers thus showing how stupid the concept of racism is.

The fact the spainards take women as slaves shows their cruelty and how contradictory they are to being "civilized"

A detailed potryal of the awfulness of both sides of the atlantic slave trade where tribal leaders sold slaves to white europeans

Could be a double meaning for converting the slaves to christianity?

Does the setting also make this american literature then (takes place in south america)

Then does this not kind of invalidate the whole concept of slavery and racism the spainards participate in?

The identity of the american is a lot less descriptive then the spainard, possible eluding to the concept of the melting pot

Same ethnicity as past writers like De la Casas

could this mean the captian or the actual ship itself?

who?

outing the foreshadowing?

wow the imagery here with this simile is very intriguing

a lot of detail about this ship man.....

oh.

Kind of hospitality like

guidance

Wondering if he's still referencing the boat or an actual figure of a woman

perception

Human caregivers are more reliable than robots caregivers because robots might malfunction. Then, the patient would have a hard time doing tasks temporarily without the robot. This would also lead to the costs of fixing the robot.

This could be one reason that the robot in Robot & Frank was faceless, and why most robots in current development that I have seen do not have a animal-like or human-like face.

This building of genuine connections and relationships is likely going to be the most difficult to emulate using robots. Perhaps human care could be more focused on this aspect of caregiving if robots were to take care of the menial aspects.

Even though they do very demanding work, caregivers are not usually compensated enough for the work they do. Removing this could be one of the main benefits of using technology to assist or replace this work.

Test

Hippocampus atrophy → physiological inability to dampen the disease

The queerness of color is important because it can explain the difference between our worlds.

Purpose: This part of the podcast as well as the whole podcast is mainly entertainment with sprinkles of information when it comes to the success of Bad bunny. Though they are providing facts and data the nature of the podcast is for the viewers to find entertainment in talking about music artists and their journey.

Audio: They used background music to set the atmosphere for the views for the rest of the podcast. The music gives the viewers excited to listed to the conversation of this topic which is music. At the end of the podcast they play a Bad Bunny song which gives the fans of this artist some enjoyment in listening to the song and shows others who don't know who he is a taste of his music.

UTF-8

RPC and security. Kerberos V5 will get used.

At long last she has maybe reached her finally hope of getting out of that bird suit. This conveys that sometimes you have to accept the fate whether it turns out good or bad.

forcces them to kill yourself

blind to the danger, almost as if a sense of fear eludes them.

Margaret Atwood is referencing the song of a siren and how it is so irresistible it led sailors to their death.

the entire piece, specifically this sentence, is an example of the overall theme of the piece, which is also the repeated refrain of "clearly expressing something" and the consideration of the content expressed. The structure of this sentence presents concrete ideas but in a difficult-to-digest form (long run-on sentence, basic/filler words repeated often) Is the content about clearly expressing something being clearly expressed? if someone is clearly expressing something in a way that is not clear, does that take away the something of the thing expressed?

the humanity/the struggle makes him more human, more convincingly great

I have not been here; I would like to see these murals.

Value of knowledge in a zettelkasten as a function of reference(use/look up) frequency; links to other ideas; ease of recall without needing to look up (also a measure of usefulness); others?

Define terms and create a mathematical equation of stocks and flows around this system of information. Maybe "knowledge complexity" or "information optimization"? see: https://hypothes.is/a/zejn0oscEe-zsjMPhgjL_Q

takes into account the value of information from the perspective of a particular observer<br /> relative information value

cross-reference: Umberto Eco on no piece of information is superior: https://hypothes.is/a/jqug2tNlEeyg2JfEczmepw

Inspired by idea in https://hypothes.is/a/CdoMUJCYEe-VlxtqIFX4qA

inspired by, but definitely not take from as not in evidence

Many people have "daily notes" and "project notes" in what they consider to be their zettelkasten workflow. These can be thought of as subcategories of reference notes (aka literature notes, bibliographic notes). The references in these cases are simply different sorts of material than one would traditionally include in this category. Instead of indexing the ideas within a book or journal article, you're indexing what happened to you on a particular day (daily notes) or indexing ideas or progress on a particular project (project notes). Because they're different enough in type and form, you might keep them in their own "departments" (aka folders) within your system just the same way that with enough material one might break out their reference notes to separate books from newspapers, journal articles, or lectures.

In general form and function they're all broadly serving the same functionality and acting as a ratchet and pawl on the information that is being collected. They capture context; they serve as reminder. The fact that some may be used less or referred to less frequently doesn't make them necessarily less important

I think social media affects relationship maintenance in some positive and some negative ways; positive in that partners are able to maintain intimacy over longer distances or time periods and communication is made easier, but negative in that the expectation of communication can make expectations of a relationship difficult. I know in my relationship, we had to manage our expectations around the other’s texting habits because it was upsetting both of us at the frequency of communication, but only because the world had made constant availability the norm.

It’s interesting that this preference is getting more and more distinct, rather than less pronounced. In a progressive society that is trying to reduce the amount of gender stereotypes that are displayed in our society, this one doesn’t seem to be one that’s very challenged. I know for myself, I definitely didn’t want to be approaching people when I was romantically engaging with new others, and my now-boyfriend is the one that reached out to me first. I don’t think female-initiating dances would work anymore either, as it’s clear that society dictates that men should be pursuing women. Why hasn’t this standard gone away when so many others have?

This speed dating experiment is really interesting, as I’ve heard of all of these studies that show that men value attractiveness and women value economic status, and that that supports an evolutionary hypothesis. It’s intriguing that a more ecologically valid approach shows us that these findings aren’t necessarily true, and that men and women aren’t very different in what we desire in a partner when in a real situation.

Sounds like you're doing what Mortimer J. Adler and Charles van Doren would call "inspectional reading" and outlining the space of your topic. This is both fine and expected. You have to start somewhere. You're scaffolding some basic information in a new space and that's worthwhile. You're learning the basics.

Eventually you may come back and do a more analytical read and/or cross reference your first sources with other sources in a syntopical read. It's at these later two levels of reading where doing zettelkasten work is much more profitable, particularly for discerning differences, creating new insights, and expanding knowledge.

If you want to think of it this way, what would a kindergartner's zettelkasten contain? a high school senior? a Ph.D. researcher? 30 year seasoned academic researcher? Are the levels of knowledge all the same? Is the kindergartner material really useful to the high school senior? Probably not at all, it's very basic. As a result, putting in hundreds of atomic notes as you're scaffolding your early learning can be counter-productive. Read some things, highlight them, annotate them. You'll have lots of fleeting notes, but most of them will seem stupidly basic after a month or two. What you really want as main notes are the truly interesting advanced stuff. When you're entering a new area, certainly index ideas, but don't stress about capturing absolutely everything until you have a better understanding of what's going on. Then bring your zettelkasten in to leverage yourself up to the next level.

• Adler, Mortimer J. “How to Mark a Book.” Saturday Review of Literature, July 6, 1940. https://www.unz.com/print/SaturdayRev-1940jul06-00011/
• Adler, Mortimer J., and Charles Van Doren. How to Read a Book: The Classical Guide to Intelligent Reading. Revised and Updated edition. 1940. Reprint, Touchstone, 2011.

reply to u/jack_hanson_c at https://old.reddit.com/r/Zettelkasten/comments/1g9dv9b/is_scoping_the_subject_a_counterzettelkasten/

thesis restated!

Thesis?

Thesis?

I wasn't really sure how the encryption process worked so this was interesting to read. The number of times I've forgotten my password at work and had to call for assistance the fact that there was a special line devoted just for that tells you how important it is to control this information and what it locks away.

I've often wondered what hackers could and could not do and in a lot of ways I'm sure there's no limitation to what havoc they can cause. A few companies I've worked for were very strict about opening emails that only came from the company itself. Reading all of the ways hackers can work around things makes you feel really vulnerable.

this adds code tags which could prevent content from being displayed for some Safari users (this is the case when the tags are wrapped around H5P activities)

replaced screenshot

redid the screenshot (without text)

Finally some serious attention to Native American interests and rights. But the overriding concern here seems to be enabling these groups, as well, to step up extraction from public lands.

Pendley's proposals would effectively dismantle decades of agency efforts to implement Congressional mandates by executive fiat, without consulting Congress. Challenges to many of these proposals are likely to succeed in court.

Pretty much all manual typewriters use 1/2" (12.7mm) wide ribbon which is most of what you're probably going to find in the marketplace.

The thing that changes from machine to machine is is the potentially proprietary spools and those are usually specific to how the ribbion auto switch is effectuated. Most ribbon comes with small grommets about 10-12 inches from the ends as many machines need this to trigger the switch over. If the plastic spools you purchase don't work with your particular machine you simply spend a minute or two to hand wind it onto your existing original (metal) spools and go from there.

There are lots of videos on YouTube showing how to hand wind ribbon onto a machine. Sarah has a pretty reasonable one: https://www.youtube.com/watch?v=up412FjTEkw

Even Tom Hanks has a ribbon changing video... https://www.youtube.com/watch?v=GBbsNKaVAB0

Incidentally, if your seller specifies them, the Underwoods take Group 9 (GR9) spools. Likely not helpful or illustrative for you, but certainly interesting from a historical perspective, Ted Munk has a catalog of Typewriter Ribbon varieties Offered by Underwood in 1956: https://munk.org/typecast/2020/08/23/typewriter-ribbon-varieties-offered-by-underwood-in-1956/

reply to u/prettiestGOAT at https://old.reddit.com/r/typewriters/comments/1g8z0fm/can_anyone_help_id_this_underwood_typewriter_and/

very abstract imagery, very intuitively written. Reminds me of poetry I've written that made sense to me but I've scrapped because it would be gibberish to others, but it's interesting to be a reader and to try and unpack the speaker's garbled thoughts.

Chris M. recommends to use a layered system for music categorization:

• Layer 1) Genres / Subgenres
• Layer 2) Energy
• Layer 3) Vibe

Genre itself is the main overall (and broad) genre. Subgenres are tag-like and related to when you want to play it more granularly.

Energy is a measurement of the average energy of the song.

Vibes refer to the emotions and memories it brings up to you and potentially others you play it for. Some questions he asks: - 1) How does it make me feel? - 2) What does it remind me of? - 3) Where would I play it? - 4) When would I play it? - 5) Why would I play it? - 6) Who would I play it for?

When looking for songs in the library, it's very important to answer a few questions to filter. Not just to save storage space, but also to ensure the quality of one's library.

Chris M. recommends a SHORT LIST... Music you come across that you like and think about downloading, you put in there. Then wait for 24h before listening again to it. Finally, ask 3 questions before deciding to add it: - 1) Do I still like it? - 2) Would I play it out? - 3) Would I pay money for it?

It reflects the multiplicity of the role that teachers play not only in the classroom, but also in the personal and social life of students. This focus on student equality and respect ensures that everyone feels included in the class and, hence, creates a climate of confidence and self-esteem. Standards in core subjects are essential, but so too is a commitment to life skills like the appreciation of diversity and respect. These lessons run far beyond the school gates and nurture children to be well-intentioned and compassionate citizens.

p7?

p6

p5 (in reference to p4)

p4 (CONT)

p4

p3?

Chris M. suggests to start building a playlist with the end in mind. This is logical because it's easier to backtrack transitions then to do it forward.

Edit: This he suggested in a different video, not this one.

Playing into the idea of transitions... Perhaps it's useful to keep small playlists with only a handful of songs (3-5) that are PERFECT together. This can be used as a sort of repository for the creation of larger playlists later to save time.

Transitions he mentions: - A) Instant RAGE Slower Song -> Instant Drop Instant Drop = a song that immediately pulls up the speed. - B) Slow Down, Speed Up Hype song with a gradual slow down, leading into an immediate speed up. Kick or beat drop from track 2. - C) Vibez to Vibez Track 1 to Track 2 while remaining energy (energy the same) but switching the genres. - D) Get up and Dance Weird ending that you CAN'T dance to leading into something that you HAVE to dance to. - E) The SLOW DOWN Song pace doesn't matter. Slow Ending to a Slow Beginning. - F) The Lit Switch Lead one LIT song seamlessly into another LIT song, regardless of genre. It maintains hype level. - G) Is that the same X Have a similar sounding X playing at the end of track 1 and at the beginning of track 2... X can be anything, for example an instrument/guitar. - H) Weird BUT Effective Track 2 starts with a sound effect and you use that to transition seamlessly with track 1. - I) The Dip/The Rip Track A with decent pace to Track B (slowest song of the 3) to Track C which starts slow but picks up the pace again Can be reversed into the hip... Then the middle song is the fastest song.

The distinguisher between a great playlist and a normal playlist is the flow between each songs. In other words, the transitions.

Excellent video on the creation of playlists.

This basically stated that America had exclusive power of Latin American countries

The Boxer Rebellion was a Chinese revolt against outward interaction with foreign nations. America smothered the flame of uprising because they wanted cheap trade with China.

the U.S. "dealt the final blow"

Wilson attacked Veracruz without the permission of congress

Trade of loans for authority over Latin America

Gave America the right to aquire lands with guano deposits

Mahan believed in overseas conquest which impacted Roosevelt's motivation to intervene in foreign places

Author response:

Reviewer #1 (Public Review):

Summary

The authors asked if parabrachial CGRP neurons were only necessary for a threat alarm to promote freezing or were necessary for a threat alarm to promote a wider range of defensive behaviors, most prominently flight.

Major Strengths of Methods and Results

The authors performed careful single-unit recording and applied rigorous methodologies to optogenetically tag CGRP neurons within the PBN. Careful analyses show that single-units and the wider CGRP neuron population increases firing to a range of unconditioned stimuli. The optogenetic stimulation of experiment 2 was comparatively simpler but achieved its aim of determining the consequence of activating CGRP neurons in the absence of other stimuli. Experiment 3 used a very clever behavioral approach to reveal a setting in which both cue-evoked freezing and flight could be observed. This was done by having the unconditioned stimulus be a "robot" traveling along a circular path at a given speed. Subsequent cue presentation elicited mild flight in controls and optogenetic activation of CGRP neurons significantly boosted this flight response. This demonstrated for the first time that CGRP neuron activation does more than promote freezing. The authors conclude by demonstrating that bidirectional modulation of CGRP neuron activity bidirectionally aTects freezing in a traditional fear conditioning setting and aTects both freezing and flight in a setting in which the robot served as the unconditioned stimulus. Altogether, this is a very strong set of experiments that greatly expand the role of parabrachial CGRP neurons in threat alarm.

We would like to sincerely thank the reviewer for the positive and insightful comments on our work. We greatly appreciate the acknowledgment of our new behavioral approach, which allowed us to observe a dynamic spectrum of defensive behaviors in animals. Our use of the robot-based paradigm, which enables the observation of both freezing and flight, has been instrumental in expanding our understanding of how parabrachial CGRP neurons modulate diverse threat responses. We are pleased that the reviewer found this methodological innovation to be a valuable contribution to the field.

Weaknesses

In all of their conditioning studies the authors did not include a control cue. For example, a sound presented the same number of times but unrelated to US (shock or robot) presentation. This does not detract from their behavioral findings. However, it means the authors do not know if the observed behavior is a consequence of pairing. Or is a behavior that would be observed to any cue played in the setting? This is particularly important for the experiments using the robot US.

We appreciate the reviewer’s insightful comment regarding the absence of a control cue in our conditioning studies. First, we would like to mention that, in response to the Reviewer 3, we have updated how we present our flight data by following methods from previously published papers (Fadok et al., 2017; Borkar et al., 2024). Instead of counting flight responses, we calculated flight scores as the ratio of the velocity during the CS to the average velocity in the 7 s before the CS on the conditioning day (or 10 s for the retention test). This method better captures both the speed and duration of fleeing during CS. With this updated approach, we observed a significant difference in flight scores between the ChR2 and control groups, even during conditioning, which may partly address the reviewer’s concern about whether the observed behavior is a consequence of CS-US pairing.

However, we agree with the reviewer that including an unpaired group would provide stronger evidence, and in response, we conducted an additional experiment with an unpaired group. In this unpaired group, the CS was presented the same number of times, but the robot US was delivered randomly within the inter-trial interval. The unpaired group did not exhibit any notable conditioned freezing or flight responses. We believe that this additional experiment, now reflected in Figure 3, further strengthens our conclusion that the fleeing behavior is driven by associative learning between the CS and US, rather than a reaction to the cue itself.

The authors make claims about the contribution of CGRP neurons to freezing and fleeing behavior, however, all of the optogenetic manipulations are centered on the US presentation period. Presently, the experiments show a role for these neurons in processing aversive outcomes but show little role for these neurons in cue responding or behavior organizing. Claims of contributions to behavior should be substantiated by manipulations targeting the cue period.

We appreciate the reviewer’s constructive comments. We would like to emphasize that our primary objective in this study was to investigate whether activating parabrachial CGRP neurons—thereby increasing the general alarm signal—would elicit different defensive behaviors beyond passive freezing. To this end, we focused on manipulating CGRP neurons during the US period rather than the cue period.

Previous studies have shown that CGRP neurons relay US signals, and direct activation of CGRP neurons has been used as the US to successfully induce conditioned freezing responses to the CS during retention tests (Han et al., 2015; Bowen et al., 2020). In our experiments, we also observed that CGRP neurons responded exclusively to the US during conditioning with the robot (Figure 1F), and stimulating these neurons in the absence of any external stimuli elicited strong freezing responses (Figure 2B). These findings, collectively, suggest that activation of CGRP neurons during the CS period would predominantly result in freezing behavior.

Therefore, we manipulated the activity of CGRP neurons during the US period to examine whether adjusting the perceived threat level through these neurons would result in diverse dfensive behaivors when paired with chasing robot. We observed that enhancing CGRP neuron activity while animals were chased by the robot at 70 cm/s made them react as if chased at a higher speed (90 cm/s), leading to increased fleeing behaviors. While this may not fully address the role of these neurons in cue responding or behavior organizing, we found that silencing CGRP neurons with tetanus toxin (TetTox) abolished fleeing behavior even when animals were chased at high speeds (90 cm/s), which usually elicits fleeing without CGRP manipulation (Figure 5). This supports the conclusion that CGRP neurons are necessary for processing fleeing responses.

In summary, manipulating CGRP neurons during the US period was essential for effectively investigating their role in adjusting defensive responses, thereby expanding our understanding of their function within the general alarm system. We hope this clarifies our experimental design and addresses the concern the reviewer has raised.

Appraisal

The authors achieved their aims and have revealed a much greater role for parabrachial CGRP neurons in threat alarm.

Discussion

Understanding neural circuits for threat requires us (as a field) to examine diverse threat settings and behavioral outcomes. A commendable and rigorous aspect of this manuscript was the authors decision to use a new behavioral paradigm and measure multiple behavioral outcomes. Indeed, this manuscript would not have been nearly as impactful had they not done that. This novel behavior was combined with excellent recording and optogenetic manipulations - a standard the field should aspire to. Studies like this are the only way that we as a field will map complete neural circuits for threat.

We sincerely thank the reviewer for their positive and encouraging comments. We are grateful for the acknowledgment of our efforts in employing a novel behavioral paradigm to study diverse defensive behaviors. We are pleased that our work contributes to advancing the understanding of neural circuits involved in threat responses.

Reviewer #3 (Public Review):

Strengths:

The study used optogenetics together with in vivo electrophysiology to monitor CGRP neuron activity in response to various aversive stimuli including robot chasing to determine whether they encode noxious stimuli diTerentially. The study used an interesting conditioning paradigm to investigate the role of CGRP neurons in the PBN in both freezing and flight behaviors.

Weakness:

The major weakness of this study is that the chasing robot threat conditioning model elicits weak unconditioned and conditioned flight responses, making it diTicult to interpret the robustness of the findings. Furthermore, the conclusion that the CGRP neurons are capable of inducing flight is not substantiated by the data. No manipulations are made to influence the flight behavior of the mouse. Instead, the manipulations are designed to alter the intensity of the unconditioned stimulus.

We sincerely thank the reviewer for the thoughtful and constructive comments on our manuscript. In response to this feedback, we revisited our analysis of the flight responses and compared our methods with those used in previous literatures examining similar behaviors.

We reviewed a study investigating sex differences in defensive behavior using rats (Gruene et al., 2015). In that study, the CS was presented for 30 s, and active defensive behvaior – referred to as ‘darting’ – was quantified as ‘Dart rate (dart/min)’. This was calculated by doubling the number of darts counted during the 30-s CS presentation to extrapolate to a per-min rate. The highest average dart rate observed was approximatley 1.5. Another relevant studies using mice quantified active defensive behavior by calculating a flight score—the ratio of the average speed during each CS to the average speed during the 10 s pre-CS period (Fadok et al., 2017; Borkar et al., 2024). This method captures multiple aspects of flight behavior during CS presentation, including overall velocity, number of bouts, and duration of fleeing. Moreover, it accounts for each animal’s individual velocity prior to the CS, reflecting how fast the animals were fleeing relative to their baseline activity.

In our original analysis, we quantified flight responses by counting rapid fleeing movements, defined as movements exceeding 8 cm/s. This approach was consistent with our previous study using the same robot paradigm to observe unique patterns of defensive behavior related to sex differences (Pyeon et al., 2023). Based on our earlier findings, where this approach effectively identified significant differences in defensive behaviors, we believed that this method was appropriate for capturing conditioned flight behavior within our specific experimental context. However, prompted by the reviewer's insightful comments, we recognized that our initial method might not fully capture the robustness of the flight responses. Therefore, we re-analyzed our data using the flight score method described by Fadok and colleagues, which provides a more sensitive measure of fleeing during the CS.

Re-analyzing our data revealed a more robust flight response than previously reported, demonstrating that additional CGRP neuron stimulation promoted flight behavior in animals during conditioning, addressing the concern that the data did not substantiate the role of CGRP neurons in inducing flight. In addition, we would like to emphasize the findings from our final experiment, where silencing CGRP neurons, even under high-threat conditions (90 cm/s), prevented animals from exhibiting flight responses. This demonstrates that CGRP neurons are necessary in influencing flight responses.

We have updated all flight data in the manuscript and revised the relevant figures and text accordingly. We appreciate the opportunity to enhance our analysis. The reviewer's insightful observation led us to adopt a better method for quantifying flight behavior, which substantiates our conclusion about the role of CGRP neurons in modulating defensive responses.

Borkar, C.D., Stelly, C.E., Fu, X., Dorofeikova, M., Le, Q.-S.E., Vutukuri, R., et al. (2024). Top- down control of flight by a non-canonical cortico-amygdala pathway. Nature 625(7996), 743-749.

Bowen, A.J., Chen, J.Y., Huang, Y.W., Baertsch, N.A., Park, S., and Palmiter, R.D. (2020). Dissociable control of unconditioned responses and associative fear learning by parabrachial CGRP neurons. Elife 9, e59799.

Fadok, J.P., Krabbe, S., Markovic, M., Courtin, J., Xu, C., Massi, L., et al. (2017). A competitive inhibitory circuit for selection of active and passive fear responses. Nature 542(7639), 96-100.

Gruene, T.M., Flick, K., Stefano, A., Shea, S.D., and Shansky, R.M. (2015). Sexually divergent expression of active and passive conditioned fear responses in rats. Elife 4, e11352.

Han, S., Soleiman, M.T., Soden, M.E., Zweifel, L.S., and Palmiter, R.D. (2015). Elucidating an a_ective pain circuit that creates a threat memory. Cell 162(2), 363-374.

Pyeon, G.H., Lee, J., Jo, Y.S., and Choi, J.-S. (2023). Conditioned flight response in female rats to naturalistic threat is estrous-cycle dependent. Scientific Reports 13(1), 20988.

Knower / observer not separate from the universe, not outside the system boundary Vgl [[Systems convening landscape als macroscope 20230906115130]] where the convener is integral part of it too, not an external change agent.

we don't allow any additional software. security software is just one example. please clarify

please remove this bullet point.. we decided to only limit it to Red Hat compatibility list. VMware for example is on it, so VMware is okay for us

Apa?

apa?

eLife Assessment

So et al. present an optimized protocol for single-nuclei RNA sequencing of adipose tissue in mice, ensuring better RNA quality and nuclei integrity. The authors use this protocol to explore the cellular landscape in both lean and diet-induced obese mice, identifying a dysfunctional hypertrophic adipocyte subpopulation linked to obesity. The data analyses are solid, and the findings are supported by the evidence presented. This study provides valuable information for the field of adipose tissue biology and will be particularly helpful for researchers using single-nuclei transcriptomics in various tissues.

Reviewer #1 (Public review):

Summary:

This manuscript from So et al. describes what is suggested to be an improved protocol for single-nuclei RNA sequencing (snRNA-seq) of adipose tissue. The authors provide evidence that modifications to the existing protocols result in better RNA quality and nuclei integrity than previously observed, with ultimately greater coverage of the transcriptome upon sequencing. Using the modified protocol, the authors compare the cellular landscape of murine inguinal and perigonadal white adipose tissue (WAT) depots harvested from animals fed a standard chow diet (lean mice) or those fed a high-fat diet (mice with obesity).

Strengths:

Overall, the manuscript is well written, and the data are clearly presented. The strengths of the manuscript rest in the description of an improved protocol for snRNA-seq analysis. This should be valuable for the growing number of investigators in the field of adipose tissue biology that are utilizing snRNA-seq technology, as well as those other fields attempting similar experiments with tissues possessing high levels of RNAse activity.

Moreover, the study makes some notable observations that provide the foundation for future investigation. One observation is the correlation between nuclei size and cell size, allowing for the transcriptomes of relatively hypertrophic adipocytes in perigonadal WAT to be examined. Another notable observation is the identification of an adipocyte subcluster (Ad6) that appears "stressed" or dysfunctional and likely localizes to crown-like inflammatory structures where pro-inflammatory immune cells reside.

Weaknesses:

Analogous studies have been reported in the literature, including a notable study from Savari et al. (Cell Metabolism). This somewhat diminishes the novelty of some of the biological findings presented here. This is deemed a minor criticism as the primary goal is to provide a resource for the field.

Reviewer #2 (Public review):

Summary:

In the present manuscript So et al describe an optimized method for nuclei isolation and single nucleus RNA sequencing (snRNA-Seq), which they use to characterize cell populations in lean and obese murine adipose tissues.

Strengths:

The detailed description of the protocol for single-nuclei isolation incorporating VRC may be useful to researchers studying adipose tissues, which contain high levels of RNAses.

While the majority of the findings largely confirm previous published adipose data sets, the authors present a detailed description of a mature adipocyte sub-cluster that appears to represent stressed or dying adipocytes present in obesity, and which is better characterized using the described protocol.

Weaknesses:

The use of VRC to enhance snRNA-seq has been previously published in other tissues, somewhat diminishing the novelty of this protocol.

The snRNA-seq data sets presented in this manuscript, when compared with numerous previously published single-cell analysis of adipose tissue, represent an incremental contribution. The nuclei-isolation protocol may represent an improvement in transcriptional analysis for mature adipocytes, however other stromal populations may be better sequenced using single intact-cell cytoplasmic RNA-Seq.

Reviewer #3 (Public review):

The authors aimed to improve single-nucleus RNA sequencing (snRNA-seq) to address current limitations and challenges with nuclei and RNA isolation quality. They successfully developed a protocol that enhances RNA preservation and yields high-quality snRNA-seq data from multiple tissues, including a challenging model of adipose tissue. They then applied this method to eWAT and iWAT from mice fed either a normal or high-fat diet, exploring depot-specific cellular dynamics and gene expression changes during obesity. Their analysis included subclustering of SVF cells and revealed that obesity promotes a transition in APCs from an early to a committed state and induces a pro-inflammatory phenotype in immune cells, particularly in eWAT. In addition to SVF cells, they discovered six adipocyte subpopulations characterized by a gradient of unique gene expression signatures. Interestingly, a novel subpopulation, termed Ad6, comprised stressed and dying adipocytes with reduced transcriptional activity, primarily found in eWAT of mice on a high-fat diet. Overall, the methodology is sound, and the data presented supports the conclusions drawn. Further research based on these findings could pave the way for potential novel interventions in obesity and metabolic disorders, or for similar studies in other tissues or conditions.

Strengths:

The authors have presented a compelling set of results. They have compared their data with two previously published datasets and provide novel insight into the biological processes underlying mouse adipose tissue remodeling during obesity. The results are generally consistent and robust. The revised Discussion is comprehensive and puts the work in the context of the field.

Weaknesses:

• The adipose tissues were collected after 10 weeks of high-fat diet treatment, lacking the intermediate time points for identifying early markers or cell populations during the transition from healthy to pathological adipose tissue.<br /> • The expansion of the Ad6 subpopulation in obese iWAT and gWAT is interesting. The author claims that Ad6 exhibited a substantial increase in eWAT and a moderate rise in iWAT (Figure 4C). However, this adipocyte subpopulation remains the most altered in iWAT upon obesity. Could the authors elaborate on why there is a scarcity of adipocytes with ROS reporter and B2M in obese iWAT?<br /> • While the study provides extensive data on mouse models, the potential translation of these findings to human obesity remains uncertain.

Revised version: The authors have properly revised the paper in response to the above questions, and I have no other concerns.

Author response:

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

Public Reviews: 

Reviewer #1 (Public Review): 

Summary: 

This manuscript from So et al. describes what is suggested to be an improved protocol for single-nuclei RNA sequencing (snRNA-seq) of adipose tissue. The authors provide evidence that modifications to the existing protocols result in better RNA quality and nuclei integrity than previously observed, with ultimately greater coverage of the transcriptome upon sequencing. Using the modified protocol, the authors compare the cellular landscape of murine inguinal and perigonadal white adipose tissue (WAT) depots harvested from animals fed a standard chow diet (lean mice) or those fed a high-fat diet (mice with obesity). 

Strengths: 

Overall, the manuscript is well-written, and the data are clearly presented. The strengths of the manuscript rest in the description of an improved protocol for snRNA-seq analysis. This should be valuable for the growing number of investigators in the field of adipose tissue biology that are utilizing snRNA-seq technology, as well as those other fields attempting similar experiments with tissues possessing high levels of RNAse activity. 

Moreover, the study makes some notable observations that provide the foundation for future investigation. One observation is the correlation between nuclei size and cell size, allowing for the transcriptomes of relatively hypertrophic adipocytes in perigonadal WAT to be examined. Another notable observation is the identification of an adipocyte subcluster (Ad6) that appears "stressed" or dysfunctional and likely localizes to crown-like inflammatory structures where proinflammatory immune cells reside. 

Weaknesses:  

Analogous studies have been reported in the literature, including a notable study from Savari et al. (Cell Metabolism). This somewhat diminishes the novelty of some of the biological findings presented here. Moreover, a direct comparison of the transcriptomic data derived from the new vs. existing protocols (i.e. fully executed side by side) was not presented. As such, the true benefit of the protocol modifications cannot be fully understood. 

We agree with the reviewer’s comment on the limitations of our study. Following the reviewer's suggestion, we performed a new analysis by integrating our data with those from the study by Emont et al. Please refer to the Recommendation for authors section below for further details.

Reviewer #2 (Public Review):

Summary: 

In the present manuscript So et al utilize single-nucleus RNA sequencing to characterize cell populations in lean and obese adipose tissues. 

Strengths: 

The authors utilize a modified nuclear isolation protocol incorporating VRC that results in higherquality sequencing reads compared with previous studies. 

Weaknesses:  

The use of VRC to enhance snRNA-seq has been previously published in other tissues. The snRNA-seq snRNA-seq data sets presented in this manuscript, when compared with numerous previously published single-cell analyses of adipose tissue, do not represent a significant scientific advance. 

Figure 1-3: The snRNA-seq data obtained by the authors using their enhanced protocol does not represent a significant improvement in cell profiling for the majority of the highlighted cell types including APCs, macrophages, and lymphocytes. These cell populations have been extensively characterized by cytoplasmic scRNA-seq which can achieve sufficient sequencing depth, and thus this study does not contribute meaningful additional insight into these cell types. The authors note an increase in the number of rare endothelial cell types recovered, however this is not translated into any kind of functional analysis of these populations. 

We acknowledge the reviewer's comments on the limitations of our study, particularly the lack of extension of our snRNA-seq data into functional studies of new biological processes. However, this manuscript has been submitted as a Tools and Resources article. As an article of this type, we provide detailed information on our snRNA-seq methods and present a valuable resource of high-quality mouse adipose tissue snRNA-seq data. In addition, we demonstrate that our improved method offers novel biological insights, including the identification of subpopulations of adipocytes categorized by size and functionality. We believe this study offers powerful tools and significant value to the research community.

Figure 4: The authors did not provide any evidence that the relative fluorescent brightness of GFP and mCherry is a direct measure of the nuclear size, and the nuclear size is only a moderate correlation with the cell size. Thus sorting the nuclei based on GFP/mCherry brightness is not a great proxy for adipocyte diameter. Furthermore, no meaningful insights are provided about the functional significance of the reported transcriptional differences between small and large adipocyte nuclei. 

To address the reviewer's point, we analyzed the Pearson correlation coefficient for nucleus size vs. adipocyte size and found R = 0.85, indicating a strong positive correlation. In addition, we performed a new experiment to determine the correlation between nuclear GFP intensity and adipocyte nucleus size, finding a strong correlation with R = 0.91. These results suggest that nuclear GFP intensity can be a strong proxy for adipocyte size. Furthermore, we performed gene ontology analysis on genes differentially regulated between large and small adipocyte nuclei. We found that large adipocytes promote processes involved in insulin response, vascularization and DNA repair, while inhibiting processes related to cell migration, metabolism and the cytoskeleton. We have added these new data as Figure 4E, S6E, S6G, and S6H (page 11)

Figure 5-6: The Ad6 population is highly transcriptionally analogous to the mAd3 population from Emont et al, and is thus not a novel finding. Furthermore, in the present data set, the authors conclude that Ad6 are likely stressed/dying hypertrophic adipocytes with a global loss of gene expression, which is a well-documented finding in eWAT > iWAT, for which the snRNA-seq reported in the present manuscript does not provide any novel scientific insight. 

As the reviewer pointed out, a new analysis integrating our data with the previous study found that Ad3 from our study is comparable to mAd3 from Emont et al. in gene expression profiles. However, significant discrepancies in population size and changes in response to obesity were observed, likely due to differences in technical robustness. The dysfunctional cellular state of this population, with compromised RNA content, may have hindered accurate capture in the previous study, while our protocol enabled precise detection. This underscores the importance of our improved snRNA-seq protocol for accurately understanding adipocyte population dynamics. We have revised the manuscript to include new data in Figure S7 (page 14).

Reviewer #3 (Public Review): 

Summary:  

The authors aimed to improve single-nucleus RNA sequencing (snRNA-seq) to address current limitations and challenges with nuclei and RNA isolation quality. They successfully developed a protocol that enhances RNA preservation and yields high-quality snRNA-seq data from multiple tissues, including a challenging model of adipose tissue. They then applied this method to eWAT and iWAT from mice fed either a normal or high-fat diet, exploring depot-specific cellular dynamics and gene expression changes during obesity. Their analysis included subclustering of SVF cells and revealed that obesity promotes a transition in APCs from an early to a committed state and induces a pro-inflammatory phenotype in immune cells, particularly in eWAT. In addition to SVF cells, they discovered six adipocyte subpopulations characterized by a gradient of unique gene expression signatures. Interestingly, a novel subpopulation, termed Ad6, comprised stressed and dying adipocytes with reduced transcriptional activity, primarily found in eWAT of mice on a high-fat diet. Overall, the methodology is sound, the writing is clear, and the conclusions drawn are supported by the data presented. Further research based on these findings could pave the way for potential novel interventions in obesity and metabolic disorders, or for similar studies in other tissues or conditions. 

Strengths:  

• The authors developed a robust snRNA-seq technique that preserves the integrity of the nucleus and RNA across various tissue types, overcoming the challenges of existing methods. 

• They identified adipocyte subpopulations that follow adaptive or pathological trajectories during obesity. 

• The study reveals depot-specific differences in adipose tissues, which could have implications for targeted therapies. 

Weaknesses: 

• The adipose tissues were collected after 10 weeks of high-fat diet treatment, lacking the intermediate time points for identifying early markers or cell populations during the transition from healthy to pathological adipose tissue. 

We agree with the reviewers regarding the limitations of our study. To address the reviewer’s comment, we revised the manuscript to include this in the Discussion section (page 17).  

• The expansion of the Ad6 subpopulation in obese iWAT and gWAT is interesting. The author claims that Ad6 exhibited a substantial increase in eWAT and a moderate rise in iWAT (Figure 4C). However, this adipocyte subpopulation remains the most altered in iWAT upon obesity. Could the authors elaborate on why there is a scarcity of adipocytes with ROS reporter and B2M in obese iWAT?

We observed an increase in the levels of H2DCFA reporter and B2M protein fluorescence in adipocytes from iWAT of HFD-fed mice, although this increase was much less compared to eWAT, as shown in Figure 6B (left panel). These increases in iWAT were not sufficient for most cells to exceed the cutoff values used to determine H2DCFA and B2M positivity in adipocytes during quantitative analysis. We have revised the manuscript to clarify these results (page 13).

• While the study provides extensive data on mouse models, the potential translation of these findings to human obesity remains uncertain. 

To address the reviewer’s point, we expanded our discussion on the differences in adipocyte heterogeneity between mice and humans. We attempted to identify human adipocyte subclusters that resemble the metabolically unhealthy Ad6 adipocytes found in mice in our study; however, we did not find any similar adipocyte types. It has been reported that human adipocyte heterogeneity does not correspond well to that of mouse adipocytes (Emont et al. 2022). In addition, the heterogeneity of human adipocyte populations is not reproducible between different studies (Massier et al. 2023). Interestingly, this inconsistency is unique to adipocytes, as other cell types in adipose tissues display reproducible sub cell types across species and studies (Massier et al. 2023). Our findings indicate that adipocytes may exhibit a unique pathological cellular state with significantly reduced RNA content, which may contribute to the poor consistency in adipocyte heterogeneity in prior studies with suboptimal RNA quality. Therefore, using a robust method to effectively preserve RNA quality may be critical for accurately characterizing adipocyte populations, especially in disease states. It may be important to test in future studies whether our snRNA-seq protocol can identify consistent heterogeneity in adipocyte populations across different species, studies, and individual human subjects. We have revised the manuscript to include this new discussion (page 17).

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors): 

Suggested points to address: 

(1) The authors suggest that their improved protocol for maintaining RNA/nucleus integrity results in a more comprehensive analysis of adipose tissue heterogeneity. The authors compare the quality of their snRNA-seq data to those generated in prior studies (e.g., Savari et al.). What is not clear is whether additional heterogeneity/clusters can be observed due directly to the protocol modifications. A direct head-to-head comparison of the protocols executed in parallel would of course be ideal; however, integrating their new dataset with the corresponding data from Savari et al. could help address this question and help readers understand the benefits of this new protocol vs. existing protocols. 

The data from Savari et al. are of significantly lower quality, likely because they were generated using earlier versions of the 10X Genomics system, and this study lacks iWAT data. To address the reviewer’s point, we instead integrated our data with those from the other study by Emont et al. (2022), which used comparable tissue types and experimental systems. The integrated analysis confirmed the improved representation of all cell types present in adipose tissues in our study, with higher quality metrics such as increased Unique Molecular Identifiers (UMIs) and the number of genes per nucleus. These results indicate that our protocol offers significant advantages in generating a more accurate representation of each cell type and their gene expression profiles. New data are included in Figure S2 (page 7).

(2) The exact frequency of the Ad6 population in eWAT of mice maintained on HFD is a little unclear. From the snRNA-seq data, it appears that roughly 47% of the adipocytes are in this "stressed state." In Figure 6, it appears that greater than 75% of the adipocytes express B2M (Ad6 marker) and greater than 75% of adipocytes are suggested to be devoid of measurable PPARg expression. The latter seems quite high as PPARg expression is essential to maintain the adipocyte phenotype. Is there evidence of de-differentiation amongst them (i.e. acquisition of progenitor cell markers)? Presenting separate UMAPs for the chow vs. HFD state may help visualize the frequency of each adipocyte population in the two states. Inclusion of the stromal/progenitor cells in the visualization may help understand if cells are de-differentiating in obesity as previously postulated by the authors. Related to Point # 1 above, is this population observed in prior studies and at a similar frequency?

To address the reviewer’s point, we analyzed the expression of adipocyte progenitor cell (APC) markers, such as Pdgfra, in the Ad6 population. We did not detect significant expression of APC markers, suggesting that Ad6 does not represent dedifferentiating adipocytes. Instead, they are likely stressed and dying cells characterized by an aberrant state of transcription with a global decline.

When integrating our data with the datasets by Emont et al., we observed an adipocyte population in the previous study, mAd3, comparable to Ad6 in our study, with similar marker gene expression and lower transcript abundance. However, the population size of mAd3 was much smaller than that of Ad6 in our data and did not show consistent population changes during obesity. This discrepancy may be due to different technical robustness; the dysfunctional cellular state of this population, with its severely compromised RNA contents, may have made it difficult to accurately capture using standard protocols in the previous study, while our protocol enabled robust and precise detection. We added new data in Figure S6I and S7 (page 14) and revised the Discussion (page 17).

Additional points  

(1) The authors should be cautious in describing subpopulations as "increasing" or "decreasing" in obesity as the data are presented as proportions of a parent population. A given cell population may be "relatively increased." 

To address the reviewer's point, we revised the manuscript to clarify the "relative" changes in cell populations during obesity in the relevant sections (pages 8, 9, 10, 11, and 15).

(2) The authors should also be cautious in ascribing "function" to adipocyte populations based solely on their expression signatures. Statements such as those in the abstract, "...providing novel insights into the mechanisms orchestrating adipose tissue remodeling during obesity..." should probably be toned down as no such mechanism is truly demonstrated. 

To address the reviewer's point, we revised the manuscript by removing or replacing the indicated terms or phrases with more suitable wording in the appropriate sections (page 2, 10, 12, 14)

Reviewer #3 (Recommendations For The Authors): 

(1) The authors might consider expanding a discussion on the potential implications of their findings, especially the newly identified adipocyte subpopulations and depot-specific differences for human studies. 

To address the reviewer’s point, we attempted to identify human adipocyte subclusters that resembled our dysfunctional Ad6 adipocytes in mice; however, we did not find any similar adipocyte types. It has been reported that human adipocyte heterogeneity does not correspond well to that of mouse adipocytes (Emont et al. 2022). In addition, the heterogeneity of human adipocyte populations is not reproducible between different studies (Massier et al. 2023). Interestingly, this inconsistency is unique to adipocytes, as other cell types in adipose tissues display reproducible sub cell types across species and studies (Massier et al. 2023). Our findings indicate that adipocytes may exhibit a unique pathological cellular state with significantly reduced RNA content, which may contribute to the poor consistency in adipocyte heterogeneity in prior studies with suboptimal RNA quality. Therefore, using a robust method to effectively preserve RNA quality may be critical for accurately characterizing adipocyte populations, especially in disease states. It may be important to test in future studies whether our snRNA-seq protocol can identify consistent heterogeneity in adipocyte populations across different species, studies, and individual human subjects. We have revised the manuscript to include this new discussion (page 17)

(2) typo: "To generate diet-induced obesity models". 

We revised the manuscript to correct it.

How does the practice of self-disclosure evolve over time in relationships, and what impact does this evolution have on the overall emotional intimacy and trust between partners?

Author response:

Reviewer #1 (Public Review):

The authors examined the hypothesis that plasma ApoM, which carries sphingosine-1-phosphate (S1P) and activates vascular S1P receptors to inhibit vascular leakage, is modulated by SGLT2 inhibitors (SGLTi) during endotoxemia. They also propose that this mechanism is mediated by SGLTi regulation of LRP2/ megalin in the kidney and that this mechanism is critical for endotoxin-induced vascular leak and myocardial dysfunction. The hypothesis is novel and potentially exciting. However, the author's experiments lack critical controls, lack rigor in multiple aspects, and overall does not support the conclusions.

Thank you for these comments. We have now directly addressed this hypothesis by using proximal tubule-specific inducible megalin/Lrp2 knockout mice, which remains an innovative hypothesis about how SGLT2i can reduce vascular leak.

Reviewer #2 (Public Review):

Apolipoprotein M (ApoM) is a plasma carrier for the vascular protective lipid mediator sphingosine 1-phospate (S1P). The plasma levels of S1P and its chaperones ApoM and albumin rapidly decline in patients with severe sepsis, but the mechanisms for such reductions and their consequences for cardiovascular health remain elusive. In this study, Ripoll and colleagues demonstrate that the sodium-glucose co-transporter inhibitor dapagliflozin (Dapa) can preserve serum ApoM levels as well as cardiac function after LPS treatment of mice with diet-induced obesity. They further provide data to suggest that Dapa preserves serum ApoM by increasing megalin-mediated reabsorption of ApoM in renal proximal tubules and that ApoM improves vascular integrity in LPS treated mice. These observations put forward a potential therapeutic approach to sustain vascular protective S1P signaling that could be relevant to other conditions of systemic inflammation where plasma levels of S1P decrease. However, although the authors are careful with their statements, the study falls short of directly implicating megalin in ApoM reabsorption and of ApoM/S1P depletion in LPS-induced cardiac dysfunction and the protective effects of Dapa.

The observations reported in this study are exciting and potentially of broad interest. The paper is well written and concise, and the statements made are mostly supported by the data presented. However, the mechanism proposed and implied is mostly based on circumstantial evidence, and the paper could be substantially improved by directly addressing the role of megalin in ApoM reabsorption and serum ApoM and S1P levels and the importance of ApoM for the preservation for cardiac function during endotoxemia. Some observations that are not necessarily in line with the model proposed should also be discussed.

The authors show that Dapa preserves serum ApoM and cardiac function in LPS-treated obese mice. However, the evidence they provide to suggest that ApoM may be implicated in the protective effect of Dapa on cardiac function is indirect. Direct evidence could be sought by addressing the effect of Dapa on cardiac function in LPS treated ApoM deficient and littermate control mice (with DIO if necessary).

The authors also suggest that higher ApoM levels in mice treated with Dapa and LPS reflect increased megalin-mediated ApoM reabsorption and that this preserves S1PR signaling. This could be addressed more directly by assessing the clearance of labelled ApoM, by addressing the impact of megalin inhibition or deficiency on ApoM clearance in this context, and by measuring S1P as well as ApoM in serum samples.

Methods: More details should be provided in the manuscript for how ApoM deficient and transgenic mice were generated, on sex and strain background, and on whether or not littermate controls were used. For intravital microscopy, more precision is needed on how vessel borders were outland and if this was done with or without regard for FITC-dextran. Please also specify the type of vessel chosen and considerations made with regard to blood flow and patency of the vessels analyzed. For statistical analyses, data from each mouse should be pooled before performing statistical comparisons. The criteria used for choice of test should be outlined as different statistical tests are used for similar datasets. For all data, please be consistent in the use of post-tests and in the presentation of comparisons. In other words, if the authors choose to only display test results for groups that are significantly different, this should be done in all cases. And if comparisons are made between all groups, this should be done in all cases for similar sets of data.

Thank you for these comments. We have now tested the direct role of Lrp2 with respect to SGLT2i in vivo and in vitro, and our study now shows that Lrp2 is required for the effect of dapagliflozin on ApoM. ApoM deficient and transgenic mice were previously described and published by our group (PMID: 37034289) and others (PMID: 24318881), and littermate controls were used throughout our manuscript. We agree that the effect on cardiac function is likely indirect in these models, and as yet we do not have the tools in the LPS model to separate potential endothelial protective vs cardiac effects. In addition, since the ApoM knockout has multiple abnormalities that include hypertension, secondary cardiac hypertrophy, and an adipose/browning phenotype, all of which may influence its response to Dapa in terms of cardiac function, these studies will be challenging to perform and will require additional models that are beyond the scope of this manuscript.

For intravital microscopy, vessel borders were outlined blindly without regard for FITC-dextran. We believe it is important to show multiple blood vessels per mouse since, as the reviewer points out, there is quite a bit of vessel heterogeneity. These tests were performed in the collaborator’s laboratory, and data analysis was blinded, and the collaborator was unaware of the study hypothesis at the time the measurements were performed and analyzed. They have previously reported this is a valid method to show cremaster vessel permeability (PMID: 26839042).

We have updated our methods section and updated the figure legends to clearly indicate the statistical analyses we used. For 2 group comparison we used student’s t-test, and for multiple groups one-way ANOVA with Sidak's correction for multiple comparisons was used throughout the paper when the data are normally distributed, and Kruskal-Wallis was used when the data are not normally distributed.

Reviewer #3 (Public Review):

The authors have performed well designed experiments that elucidate the protective role of Dapa in sepsis model of LPS. This model shows that Dapa works, in part, by increasing expression of the receptor LRP2 in the kidney, that maintains circulating ApoM levels. ApoM binds to S1P which then interacts with the S1P receptor stimulating cardiac function, epithelial and endothelial barrier function, thereby maintaining intravascular volume and cardiac output in the setting of severe inflammation. The authors used many experimental models, including transgenic mice, as well as several rigorous and reproducible techniques to measure the relevant parameters of cardiac, renal, vascular, and immune function. Furthermore, they employ a useful inhibitor of S1P function to show pharmacologically the essential role for this agonist in most but not all the benefits of Dapa. A strength of the paper is the identification of the pathway responsible for the cardioprotective effects of SGLT2is that may yield additional therapeutic targets. There are some weaknesses in the paper, such as, studying only male mice, as well as providing a power analysis to justify the number of animals used throughout their experimentation. Overall, the paper should have a significant impact on the scientific community because the SGLT2i drugs are likely to find many uses in inflammatory diseases and metabolic diseases. This paper provides support for an important mechanism by which they work in conditions of severe sepsis and hemodynamic compromise.

Thank you for these comments.

Author response:

Reviewer #1 (Public Review):

This paper proposes a novel framework for explaining patterns of generalization of force field learning to novel limb configurations. The paper considers three potential coordinate systems: cartesian, joint-based, and object-based. The authors propose a model in which the forces predicted under these different coordinate frames are combined according to the expected variability of produced forces. The authors show, across a range of changes in arm configurations, that the generalization of a specific force field is quite well accounted for by the model.

The paper is well-written and the experimental data are very clear. The patterns of generalization exhibited by participants - the key aspect of the behavior that the model seeks to explain - are clear and consistent across participants. The paper clearly illustrates the importance of considering multiple coordinate frames for generalization, building on previous work by Berniker and colleagues (JNeurophys, 2014). The specific model proposed in this paper is parsimonious, but there remain a number of questions about its conceptual premises and the extent to which its predictions improve upon alternative models.

A major concern is with the model's premise. It is loosely inspired by cue integration theory but is really proposed in a fairly ad hoc manner, and not really concretely founded on firm underlying principles. It's by no means clear that the logic from cue integration can be extrapolated to the case of combining different possible patterns of generalization. I think there may in fact be a fundamental problem in treating this control problem as a cue-integration problem. In classic cue integration theory, the various cues are assumed to be independent observations of a single underlying variable. In this generalization setting, however, the different generalization patterns are NOT independent; if one is true, then the others must inevitably not be. For this reason, I don't believe that the proposed model can really be thought of as a normative or rational model (hence why I describe it as 'ad hoc'). That's not to say it may not ultimately be correct, but I think the conceptual justification for the model needs to be laid out much more clearly, rather than simply by alluding to cue-integration theory and using terms like 'reliability' throughout.

We thank the reviewer for bringing up this point. We see and treat this problem of finding the combination weights not as a cue integration problem but as an inverse optimal control problem. In this case, there can be several solutions to the same problem, i.e., what forces are expected in untrained areas, which can co-exist and give the motor system the option to switch or combine them. This is similar to other inverse optimal control problems, e.g. combining feedforward optimal control models to explain simple reaching. However, compared to these problems, which fit the weights between different models, we proposed an explanation for the underlying principle that sets these weights for the dynamics representation problem. We found that basing the combination on each motor plan's reliability can best explain the results. In this case, we refer to ‘reliability’ as execution reliability and not sensory reliability, which is common in cue integration theory. We have added further details explaining this in the manuscript.

“We hypothesize that this inconsistency in results can be explained using a framework inspired by an inverse optimal control framework. In this framework the motor system can switch or combine between different solutions. That is, the motor system assigns different weights to each solution and calculates a weighted sum of these solutions. Usually, to support such a framework, previous studies found the weights by fitting the weighed sum solution to behavioral data (Berret, Chiovetto et al. 2011). While we treat the problem in the same manner, we propose the Reliable Dynamics Representation (Re-Dyn) mechanism that determines the weights instead of fitting them. According to our framework, the weights are calculated by considering the reliability of each representation during dynamic generalization. That is, the motor system prefers certain representations if the execution of forces based on this representation is more robust to distortion arising from neural noise. In this process, the motor system estimates the difference between the desired generalized forces and generated generalized forces while taking into consideration noise added to the state variables that equivalently define the forces.”

A more rational model might be based on Bayesian decision theory. Under such a model, the motor system would select motor commands that minimize some expected loss, averaging over the various possible underlying 'true' coordinate systems in which to generalize. It's not entirely clear without developing the theory a bit exactly how the proposed noise-based theory might deviate from such a Bayesian model. But the paper should more clearly explain the principles/assumptions of the proposed noise-based model and should emphasize how the model parallels (or deviates from) Bayesian-decision-theory-type models.

As we understand the reviewer's suggestion, the idea is to estimate the weight of each coordinate system based on minimizing a loss function that considers the cost of each weight multiplied by a posterior probability that represents the uncertainty in this weight value. While this is an interesting idea, we believe that in the current problem, there are no ‘true’ weight values. That is, the motor system can use any combination of weights which will be true due to the ambiguous nature of the environment. Since the force field was presented in one area of the entire workspace, there is no observation that will allow us to update prior beliefs regarding the force nature of the environment. In such a case, the prior beliefs might play a role in the loss function, but in our opinion, there is no clear rationale for choosing unequal priors except guessing or fitting prior probabilities, which will resemble any other previous models that used fitting rather than predictions.

Another significant weakness is that it's not clear how closely the weighting of the different coordinate frames needs to match the model predictions in order to recover the observed generalization patterns. Given that the weighting for a given movement direction is over- parametrized (i.e. there are 3 variable weights (allowing for decay) predicting a single observed force level, it seems that a broad range of models could generate a reasonable prediction. It would be helpful to compare the predictions using the weighting suggested by the model with the predictions using alternative weightings, e.g. a uniform weighting, or the weighting for a different posture. In fact, Fig. 7 shows that uniform weighting accounts for the data just as well as the noise-based model in which the weighting varies substantially across directions. A more comprehensive analysis comparing the proposed noise-based weightings to alternative weightings would be helpful to more convincingly argue for the specificity of the noise-based predictions being necessary. The analysis in the appendix was not that clearly described, but seemed to compare various potential fitted mixtures of coordinate frames, but did not compare these to the noise-based model predictions.

We agree with the reviewer that fitted global weights, that is, an optimal weighted average of the three coordinate systems should outperform most of the models that are based on prediction instead of fitting the data. As we showed in Figure 7 of the submitted version of the manuscript, we used the optimal fitted model to show that our noise-based model is indeed not optimal but can predict the behavioral results and not fall too short of a fitted model. When trying to fit a model across all the reported experiments, we indeed found a set of values that gives equal weights for the joints and object coordinate systems (0.27 for both), and a lower value for the Cartesian coordinate system (0.12). Considering these values, we indeed see how the reviewer can suggest a model that is based on equal weights across all coordinate systems. While this model will not perform as well as the fitted model, it can still generate satisfactory results.

To better understand if a model based on global weights can explain the combination between coordinate systems, we perform an additional experiment. In this experiment, a model that is based on global fitted weights can only predict one out of two possible generalization patterns while models that are based on individual direction-predicted weights can predict a variety of generalization patterns. We show that global weights, although fitted to the data, cannot explain participants' behavior. We report these new results in Appendix 2.

“To better understand if a model based on global weights can explain the combination between coordinate systems, we perform an additional experiment. We used the idea of experiment 3 in which participants generalize learned dynamics using a tool. That is, the arm posture does not change between the training and test areas. In such a case, the Cartesian and joint coordinate systems do not predict a shift in generalized force pattern while the object coordinate system predicts a shift that depends on the orientation of the tool. In this additional experiment, we set a test workspace in which the orientation of the tool is 90° (Appendix 2- figure 1A). In this case, for the test workspace, the force compensation pattern of the object based coordinate system is in anti-phase with the Cartesian/joint generalization pattern. Any globally fitted weights (including equal weights) can produce either a non-shifted or 90° shifted force compensation pattern (Appendix 2- figure 1B). Participants in this experiment (n=7) showed similar MPE reduction as in all previous experiments when adapting to the trigonometric scaled force field (Appendix 2- figure 1C). When examining the generalized force compensation patterns, we observed a shift of the pattern in the test workspace of 14.6° (Appendix 2- figure 1D). This cannot be explained by the individual coordinate system force compensation patterns or any combination of them (which will always predict either a 0° or 90° shift, Appendix 2- figure 1E). However, calculating the prediction of the Re-Dyn model we found a predicted force compensation pattern with a shift of 6.4° (Appendix 2- figure 1F). The intermediate shift in the force compensation pattern suggests that any global based weights cannot explain the results.”

With regard to the suggestion that weighting is changed according to arm posture, two of our results lower the possibility that posture governs the weights:

(1) In experiment 3, we tested generalization while keeping the same arm posture between the training and test workspaces, and we observed different force compensation profiles across the movement directions. If arm posture in the test workspaces affected the weights, we would expect identical weights for both test workspaces. However, any set of weights that can explain the results observed for workspace 1 will fail to explain the results observed in workspace 2. To better understand this point we calculated the global weights for each test workspace for this experiment and we observed an increase in the weight for the object coordinates system (0.41 vs. 0.5) and a reduction in the weights for the Cartesian and joint coordinates systems (0.29 vs. 0.24). This suggests that the arm posture cannot explain the generalization pattern in this case.

(2) In experiments 2 and 3, we used the same arm posture in the training workspace and either changed the arm posture (experiment 2) or did not change the arm posture (experiment 3) in the test workspaces. While the arm posture for the training workspace was the same, the force generalization patterns were different between the two experiments, suggesting that the arm posture during the training phase (adaptation) does not set the generalization weights.

Overall, this shows that it is not specifically the arm posture in either the test or the training workspaces that set the weights. Of course, all coordinate models, including our noise model, will consider posture in the determination of the weights.

Reviewer #2 (Public Review):

Leib & Franklin assessed how the adaptation of intersegmental dynamics of the arm generalizes to changes in different factors: areas of extrinsic space, limb configurations, and 'object-based' coordinates. Participants reached in many different directions around 360{degree sign}, adapting to velocity-dependent curl fields that varied depending on the reach angle. This learning was measured via the pattern of forces expressed in upon the channel wall of "error clamps" that were randomly sampled from each of these different directions. The authors employed a clever method to predict how this pattern of forces should change if the set of targets was moved around the workspace. Some sets of locations resulted in a large change in joint angles or object-based coordinates, but Cartesian coordinates were always the same. Across three separate experiments, the observed shifts in the generalized force pattern never corresponded to a change that was made relative to any one reference frame. Instead, the authors found that the observed pattern of forces could be explained by a weighted combination of the change in Cartesian, joint, and object-based coordinates across test and training contexts.

In general, I believe the authors make a good argument for this specific mixed weighting of different contexts. I have a few questions that I hope are easily addressed.

Movements show different biases relative to the reach direction. Although very similar across people, this function of biases shifts when the arm is moved around the workspace (Ghilardi, Gordon, and Ghez, 1995). The origin of these biases is thought to arise from several factors that would change across the different test and training workspaces employed here (Vindras & Viviani, 2005). My concern is that the baseline biases in these different contexts are different and that rather the observed change in the force pattern across contexts isn't a function of generalization, but a change in underlying biases. Baseline force channel measurements were taken in the different workspace locations and conditions, so these could be used to show whether such biases are meaningfully affecting the results.

We agree with the reviewer and we followed their suggested analysis. In the following figure (Author response image 1) we plotted the baseline force compensation profiles in each workspace for each of the four experiments. As can be seen in this figure, the baseline force compensation is very close to zero and differs significantly from the force compensation profiles after adaptation to the scaled force field.

Author response image 1.

Baseline force compensation levels for experiments 1-4. For each experiment, we plotted the force compensation for the training, test 1, and test 2 workspaces.

Experiment 3, Test 1 has data that seems the worst fit with the overall story. I thought this might be an issue, but this is also the test set for a potentially awkwardly long arm. My understanding of the object-based coordinate system is that it's primarily a function of the wrist angle, or perceived angle, so I am a little confused why the length of this stick is also different across the conditions instead of just a different angle. Could the length be why this data looks a little odd?

Usually, force generalization is tested by physically moving the hand in unexplored areas. In experiment 3 we tested generalization using a tool which, as far as we know, was not tested in the past in a similar way to the present experiment. Indeed, the results look odd compared to the results of the other experiments, which were based on the ‘classic’ generalization idea. While we have some ideas regarding possible reasons for the observed behavior, it is out of the scope of the current work and still needs further examination.

Based on the reviewer’s comment, we improved the explanation in the introduction regarding the idea behind the object based coordinate system

“we could represent the forces as belonging to the hand or a hand-held object using the orientation vector connecting the shoulder and the object or hand in space (Berniker, Franklin et al. 2014).” The reviewer is right in their observation that the predictions of the object-based reference frame will look the same if we change the length of the tool. The object-based generalized forces, specifically the shift in the force pattern, depend only on the object's orientation but not its length (equation 4).

The manuscript is written and organized in a way that focuses heavily on the noise element of the model. Other than it being reasonable to add noise to a model, it's not clear to me that the noise is adding anything specific. It seems like the model makes predictions based on how many specific components have been rotated in the different test conditions. I fear I'm just being dense, but it would be helpful to clarify whether the noise itself (and inverse variance estimation) are critical to why the model weights each reference frame how it does or whether this is just a method for scaling the weight by how much the joints or whatever have changed. It seems clear that this noise model is better than weighting by energy and smoothness.

We have now included further details of the noise model and added to Figure 1 to highlight how noise can affect the predicted weights. In short, we agree with the reviewer there are multiple ways to add noise to the generalized force patterns. We choose a simple option in which we simulate possible distortions to the state variables that set the direction of movement. Once we calculated the variance of the force profile due to this distortion, one possible way is to combine them using an inverse variance estimator. Note that it has been shown that an inverse variance estimator is an ideal way to combine signals (e.g., Shahar, D.J. (2017) https://doi.org/10.4236/ojs.2017.72017). However, as we suggest, we do not claim or try to provide evidence for this specific way of calculating the weights. Instead, we suggest that giving greater weight to the less variable force representation can predict both the current experimental results as well as past results.

Are there any force profiles for individual directions that are predicted to change shape substantially across some of these assorted changes in training and test locations (rather than merely being scaled)? If so, this might provide another test of the hypotheses.

In experiments 1-3, in which there is a large shift of the force compensation curve, we found directions in which the generalized force was flipped in direction. That is, clockwise force profiles in the training workspace could change into counter-clockwise profiles in the test workspace. For example, in experiment 2, for movement at 157.5° we can see that the force profile was clockwise for the training workspace (with a force compensation value of 0.43) and movement at the same direction was counterclockwise for test workspace 1 (force compensation equal to -0.48). Importantly, we found that the noise based model could predict this change.

Author response image 2.

Results of experiment 2. Force compensation profiles for the training workspace (grey solid line) and test workspace 1 (dark blue solid line). Examining the force nature for the 157.5° direction, we found a change in the applied force by the participants (change from clockwise to counterclockwise forces). This was supported by a change in force compensation value (0.43 vs. -0.48). The noise based model can predict this change as shown by the predicted force compensation profile (green dashed line).

I don't believe the decay factor that was used to scale the test functions was specified in the text, although I may have just missed this. It would be a good idea to state what this factor is where relevant in the text.

We added an equation describing the decay factor (new equation 7 in the Methods section) according to this suggestion and Reviewer 1 comment on the same issue.

Reviewer #3 (Public Review):

The author proposed the minimum variance principle in the memory representation in addition to two alternative theories of the minimum energy and the maximum smoothness. The strength of this paper is the matching between the prediction data computed from the explicit equation and the behavioral data taken in different conditions. The idea of the weighting of multiple coordinate systems is novel and is also able to reconcile a debate in previous literature.

The weakness is that although each model is based on an optimization principle, but the derivation process is not written in the method section. The authors did not write about how they can derive these weighting factors from these computational principles. Thus, it is not clear whether these weighting factors are relevant to these theories or just hacking methods. Suppose the author argues that this is the result of the minimum variance principle. In that case, the authors should show a process of how to derive these weighting factors as a result of the optimization process to minimize these cost functions.

The reviewer brings up a very important point regarding the model. As shown below, it is not trivial to derive these weights using an analytical optimization process. We demonstrate one issue with this optimization process.

The force representation can be written as (similar to equation 6):

We formulated the problem as minimizing the variance of the force according to the weights w:

In this case, the variance of the force is the variance-covariance matrix which can be minimized by minimizing the matrix trace:

We will start by calculating the variance of the force representation in joints coordinate system:

Here, the force variance is a result of a complex function which include the joints angle as a random variable. Expending the last expression, although very complex, is still possible. In the resulted expression, some of the resulted terms include calculating the variance of nested trigonometric functions of the random joint angle variance, for example:

In the vast majority of these cases, analytical solutions do not exist. Similar issues can also raise for calculating the variance of complex multiplication of trigonometric functions such as in the case of multiplication of Jacobians (and inverse Jacobians)

To overcome this problem, we turned to numerical solutions which simulate the variance due to the different state variables.

In addition, I am concerned that the proposed model can cancel the property of the coordinate system by the predicted variance, and it can work for any coordinate system, even one that is not used in the human brain. When the applied force is given in Cartesian coordinates, the directionality in the generalization ability of the memory of the force field is characterized by the kinematic relationship (Jacobian) between the Cartesian coordinate and the coordinate of interest (Cartesian, joint, and object) as shown in Equation 3. At the same time, when a displacement (epsilon) is considered in a space and a corresponding displacement is linked with kinematic equations (e.g., joint displacement and hand displacement in 2 joint arms in this paper), the generated variances in different coordinate systems are linked with the kinematic equation each other (Jacobian). Thus, how a small noise in a certain coordinate system generates the hand force noise (sigma_x, sigma_j, sigma_o) is also characterized by the kinematics (Jacobian). Thus, when the predicted forcefield (F_c, F_j, F_o) was divided by the variance (F_c/sigma_c^2, F_j/sigma_j^2, F_o/sigma_o^2, ), the directionality of the generalization force which is characterized by the Jacobian is canceled by the directionality of the sigmas which is characterized by the Jacobian. Thus, as it has been read out from Fig*D and E top, the weight in E-top of each coordinate system is always the inverse of the shift of force from the test force by which the directionality of the generalization is always canceled.

Once this directionality is canceled, no matter how to compute the weighted sum, it can replicate the memorized force. Thus, this model always works to replicate the test force no matter which coordinate system is assumed. Thus, I am suspicious of the falsifiability of this computational model. This model is always true no matter which coordinate system is assumed. Even though they use, for instance, the robot coordinate system, which is directly linked to the participant's hand with the kinematic equation (Jacobian), they can replicate this result. But in this case, the model would be nonsense. The falsifiability of this model was not explicitly written.

As explained above, calculating the variability of the generalized forces given the random nature of the state variable is a complex function that is not summarized using a Jacobian. Importantly the model is unable to reproduce or replicate the test force arbitrarily. In fact, we have already shown this (see Appendix 1- figure 1), where when we only attempt to explain the data with either a single coordinate system (or a combination of two coordinate systems) we are completely unable to replicate the test data despite using this model. For example, in experiment 4, when we don’t use the joint based coordinate system, the model predicts zero shift of the force compensation pattern while the behavioral data show a shift due to the contribution of the joint coordinate system. Any arbitrary model (similar to the random model we tested, please see the response to Reviewer 1) would be completely unable to recreate the test data. Our model instead makes very specific predictions about the weighting between the three coordinate systems and therefore completely specified force predictions for every possible test posture. We added this point to the Discussion

“The results we present here support the idea that the motor system can use multiple representations during adaptation to novel dynamics. Specifically, we suggested that we combine three types of coordinate systems, where each is independent of the other (see Appendix 1- figure 1 for comparison with other combinations). Other combinations that include a single or two coordinate system can explain some of the results but not all of them, suggesting that force representation relies on all three with specific weights that change between generalization scenarios.”

Deze criteria helpen neuropsychologen om op een betrouwbare manier te beoordelen of verschillende gegevens samenhangend zijn, een proces dat ook wel een coherentieanalyse wordt genoemd. De "zeven C's" helpen de onderzoeker om een goed beeld te krijgen van de klinische situatie van de patiënt.

Continuïteit: Dit betekent dat de symptomen van de patiënt zich ontwikkelen op een manier die je zou verwachten op basis van wat wetenschappelijk bekend is over de aandoening. Bijvoorbeeld, als een aandoening volgens de wetenschap verergert na verloop van tijd, dan moet dit ook bij de patiënt zichtbaar zijn.

Consistentie van presentatie over tijd: Dit verwijst naar hoe stabiel of consistent de symptomen van de patiënt zijn. De manier waarop de patiënt zijn of haar klachten presenteert, zou in de loop van de tijd niet drastisch moeten veranderen zonder duidelijke reden.

Congruentie: Dit betekent dat de verschillende aspecten van de klachten en het klinische beeld van de patiënt met elkaar moeten kloppen. Verschillende symptomen of gedragingen moeten logisch in elkaar passen en elkaar niet tegenspreken.

Compliance (medewerking aan behandeling): Dit kijkt naar hoe goed de patiënt de behandeling opvolgt. Als een patiënt zich niet aan het voorgeschreven behandelplan houdt, kan dit invloed hebben op de klinische uitkomst en moet dit worden meegewogen in de beoordeling.

Causaliteit: Hier wordt gekeken of de aandoening daadwerkelijk de oorzaak is van de klachten van de patiënt, of dat er een andere of aanvullende oorzaak kan zijn die de symptomen beter verklaart.

Comorbiditeiten: Dit verwijst naar het controleren of er andere aandoeningen of stoornissen (comorbiditeiten) aanwezig zijn die de klachten van de patiënt kunnen verklaren. Deze kunnen soms de hoofdklacht verergeren of de symptomen verwarrend maken.

Culturele factoren: Culturele achtergronden kunnen invloed hebben op hoe een patiënt zijn of haar klachten presenteert. Het is belangrijk om te overwegen of de cultuur van de patiënt een rol speelt in hoe de symptomen worden ervaren en gecommuniceerd.

After WW2, communism is seen as the biggest threat to Western Society.

Version 3 of this preprint has been peer-reviewed and recommended by Peer Community in Genomics.<br /> See the peer reviews and the recommendation.