Could the variance in years of meditation experience among participants influence the results, and how is this controlled?

Does this study distinguish between the neural effects of mindfulness meditation and focused-attention meditation, or is it treating all types similarly?

In this passage, he brings into light the pride, patriotism, and sense of gratitude, stating that it is for these reasons that such a day of commemorations regarding their nation's historical backdrop would be a motive for celebration and memory. He refers to the Declaration of Independence as "the ring-bolt to the chain of your nation's destiny," underlining the fundamental role this single document played in shaping the future of the nation. The "ring-bolt" metaphor follows the view that the Declaration serves as an anchor, one that is necessary in nature and principle to the country's identity and values. When he says, "the principles contained in that instrument are saving principles," he declares those ideas to be fundamental to the survival and growth of the nation. The call to "stand by those principles" is, therefore, a serious appeal to stand by freedom and justice, being true to them under all circumstances and against all opposition. This seriousness of the commitment is underlined with "at whatever cost," implying that such a defense could entail considerable sacrifice. This is a potent reminder of a greater good: responsibilities attendant on citizenship and the duty to nurture the ideals on which the nation was built.

In the selected passage, the speaker says, "Oppression makes a wise man mad," which can be taken to mean that to be consistently oppressed-not treated fairly-will drive even the most sensible person into frustration or anger. He calls the audience's "fathers" wise men; thus, their intelligence and astuteness could discern that a wrong was being committed against them. The term "restive" suggests a growing impatience and chafing under their oppression, underscoring their emotional reaction to such injustice at the hands of their masters. When he says they were "victims of grievous wrongs," he makes plain just how bad they suffered, grievously enough that such injustices could not conceivably be righted under the legal and political strictures of colonialism. The sentence "With brave men there is always a remedy for oppression" insinuates that where there is courage, something will be done about it, and it therefore signals some sort of turning point in their struggle. The last sentence shows that the idea of complete separation from British rule was born out of this oppression, signaling the birth of a revolutionary idea that would lead to independence. This is a passage epitomizing the transformation of frustration to a proactive movement for freedom.

In this text, he referred to July 2, 1776, when the Continental Congress passed an important decision that was to become the fate of the nation afterward. He noted that such a decision was controversial and did not find favor with people who loved comfort and stability more than the concept of freedom by noting the "dismay of the lovers of ease, and the worshipers of property.". The sentence "clothed that dreadful idea with all the authority of national sanction" would seem to invoke a sense of independence as a bold but terrifying concept, yet imbued with the weighty legitimacy of official ratification. By acknowledging that the resolutions today lack the same degree of "transparency," he suggests a sense of lucidity or determination in the choice of Congress that transcends those in comparison to more contemporary declarations, which perhaps are not as forthright or significant. He offers to read the resolution to remind his audience of what it presumes to be the foundational values of their nation, reinforcing the important connection that historical moment has with their present circumstances.

In the passage, the speaker establishes a contrast between the beliefs of their ancestors and those beliefs that do exist then concerning government. He said that these "fathers" did not consider government as infallible nor its actions as beyond question. On the contrary, these individuals felt empowered to challenge the British government regarding certain laws and policies they had defined as unjust. The speaker charged that he had indeed, by using terms such as "unjust, unreasonable, and oppressive," shown strong feelings of discontent to fuel the call for independence. He emphasized how ready they were to resist what was felt to be unfair treatment, giving reasons why it was not only proper but also necessary to stand up against such measures. He then identifies himself with their position when he declares his mind on those matters as being the same as theirs, which further cements him with the audience and, most importantly, their common legacy of resistance. The latter thus lays a potential foundation for discussing the necessity to challenge the authorities, to stand up and speak in support of justice.

In the following excerpt, he addresses the audience as "Fellow-citizens," which attaches a sense of unity to them and a shared identity. He does not go into great detail explaining the historical significance of the day; thus, it suggests that he wants to make his message simple. He mentions a momentous occasion 76 years in the past when citizens of America were British subjects, naming their colony status. The inclusion of "the style and title of your 'sovereign people'" is to constitute that the notion of American independence and self-governance had not been obtained. The phrase "under the British Crown" underlines the absence of autonomy in those times. Also, while saying, "your fathers esteemed the English Government as the home government," he presents early Americans as holding loyalty and attachment to England as their "fatherland." This sets up another argument about the American identity and its journey toward independence.

In this passage, the speaker identifies that Frederick Douglass intelligently addressed an audience that is closely tied to religion. Douglass succeeds in integrating America with religious ideas integration that few people would usually think of. Besides arousing sentiment within listeners of his speech, this tactic questions the belief system of those who opposed him to take an intellectual step back and feel the moral imperative of such a situation. Weaving these complex ideas together, Douglass showed that he was able not only to spur change but also make people think deeply; this again constitutes a strategic use of rhetoric in appealing to an audience.

Through this speech, he addresses the audience, showing them how very different it is from where he presently stands and where he was on the slave plantation from which he escaped. He speaks to them about the great distance and the numerous obstacles he had experienced in moving from such a hard place to where he is presently. He was surprised and thankful to stand in front of the audience today, reflecting that it is amazing how such hardships can be overcome. He shared his journey from painful experiences to a hopeful present.

However, they are attributable to a third party. So Tom's life wouldn't be meaningful for Tom, but it would be meaningful to the team making Tom's decisions. So for an objection to determinism, this example is invalid because determinism doesn't claim that our life choices are determined by virtue of another (who would be able to take responsibility), but by virtue of circumstance, which is void of responisbility.

If this is true, then technically we would all be controlled by scientists given the comparison. But if that were true that we were all controlled by scientists, it wouldn't really be us who considered the meaningfulness of life- it would be the scientists. I don't think the scientists would have us believe in the meaninglessness of their project as it would go against their best interest.

but, as suggested by Pisciotta, Tom's life isn't really HIS own, therefore the stipulation that his own view of HIS life is not sufficient is irrelevant because there's no life of his own to feel satisfied with.

VERY IMPORTANT PASSAGE

not because pride and satisfaction would be invalid due to determinism, but because these senses should be alotted elsewhere- the scientists.

What tools or neuroimaging methods are best suited for tracking these connectivity changes in future research? Should I mention the limitations of current technology in my paper?

Could focused-attention meditation be used as a tool to enhance learning or recovery in individuals with cognitive impairments? Should this angle be incorporated into my research?

Can I find other studies that explore the link between meditation practices and enhanced cognitive flexibility or attentional control?

Should I expand my research to include cognitive flexibility as a specific outcome of neuroplasticity through focused-attention meditation?

The reduction of the attentional blink after OM meditation suggests that meditation can improve the brain’s ability to engage and disengage from stimuli. This finding introduces a new angle on how meditation can train cognitive flexibility, which is crucial for neuroplasticity.

How can I incorporate this finding into my research question—should I compare neuroplastic changes at different stages of meditation (novice vs. expert)?

Does the reduction in brain activation at advanced stages imply that long-term practitioners experience deeper neuroplastic changes than novices, or does it suggest that the brain becomes more efficient with less effort?

If neuroplasticity through meditation is similar to other skill acquisitions, could the underlying neural mechanisms (e.g., the formation of new synaptic connections) overlap with non-meditative cognitive training methods?

How do the effects of Focused Attention (FA) meditation differ from Open Monitoring (OM) meditation in terms of brain plasticity? Should I narrow my research to one type, or explore both for comparison?

If the plan was adopted it would lead to the colonies practically being recolonized by england.

This is what critics want

This is the reason why

This is true broadly speaking, but not in every instance. There is no sound-image for words like 'the' or 'have'

I like the idea of learning as an apprenticeship

Makes me think of evidence based questioning and analysis.

As with anything, this practice will take time. Time on both the teachers and students. PATIENCE

Students often just want the outcome and can't explain their thinking or how the got there.

Not to simply give up! This is where we often get stuck.

oh remember everything we said were talking about? yeah we actually dgaf

realistically- monks can't be anti-economic for very long

monasticism has rational achievements despite charismatic and anti-rational views

attain salvation by preparing to take on the world

One type of monastic is rejecting the world

what do you do with the people in charismatic office who want to reject all mundane concerns?

inevitably- office charisma opposes personal charisma in order to maintain it's own power

church supports economically "weak" supported and championed against the economically powerful whom church can't control

hierocratic church regenerated through protection and way of life of officials and by providing an education that ensures officials power

four features of "church" 1. rise of professional priesthood away from world with official duties and distinctive way of life 2. unveirsal domination- not ethnically or familially limited 3. dogma and rites systemized through educational systems 4. needs compulsory org to separate charisma from persons administering it

Church is an office of charisma? Distributes the resource of eternal blessings for members born into it

caesaropapist isn't eradication of charismatic it's integration of them as part of state to serve state's purposes

priests sanction in exchange for ruler's support

priestly charisma compromises with secular power

hierocracy- instances in which separate religious body asserts body over king- prevent king from accumulation of all resources

instances like this lead to secular control of religious sectors

legitimized by priests whose expertise is in the will of God

if doesn't have or can't inherit charisma- power must be secured through other means like a priesthood (in case of not elected or selected leader)

A person's inherited charisma belongs to them in a way it doesn't for all leaders that are charismatic to some degree. Therefore, the toppling of this individual de legitimizes a lot more than just the one leader/

the monarch necessary in maintaining legitimacy of power structure even if they're not truly the ones ruling

money economy needed for modern bureaucracy

sometimes those who's office can be taken from them favored as the are more forced to abide by convention

for life positions in this instance still don't assign duty as officials "property"

use of popular vote - worse candidates

Political powers care about things other than technical competence.

laymen don't know what's needed as much as superior

starts off with appointments and moves to elections

less experienced developers accept more suggeted code (copilot) and benefit relatively versus more experienced developers. Suggesting that the set ways of experienced developers work against fully exploting code generation by genAI.

Conclusion: LLMs cover certain areas that were before solved by human brainpower on stack overflow, and that is measurable over a period of oct-2021 -> march 2023. Reddit does not show this decline and this is alledged due to social fabric on reddit.

I want to know if it is possible to extract claims from this paper automatically in a way that it integrates certain relations in a knowledge graph, utilizing a system that improves itself.

it sounds like this experiment was based primarily subjectively. I sometimes have an issue with this gap between the subjective and the objective conclusions which have been drawn from the experiment.

build-bridging as a service [!!!]

In this paragraph and elsewhere, the authors emphasize the need for "Alaska" to have greater sovereignty and also condemn environmental protection as an "injustice" against the people of the state. However, as our other comments indicate, issues of Native sovereignty, food security, Indigenous rights, and struggles for environmental justice are completely disregarded in the monolithic, pro-development vision advanced in this report.

In the Trump administration's rush to drill in the Arctic National Wildlife Refuge, science was often disregarded or manipulated (including in the BLM studies that culminated in the 2020 EIS). For excellent reporting on these issues during the Trump years, see Adam Federman's 2019 article in Politico: https://www.politico.com/interactives/2019/trump-science-alaska-drilling-rush/ And for a brief, longer-term perspective on science denialism in the Arctic Refuge debate (ranging from the Reagan era to the Trump era), see: https://time.com/6332304/arctic-refuge-drilling-distortions/

It should be noted that the history and impact of ANCSA are incredibly complex--both for the many different tribal nations in Alaska and for the unfolding of different environmental conflicts across the state. To begin, ANCSA explicitly sought to undermine the sovereignty of tribes by making newly-created Native corporations the beneficiaries of land and financial resources. ANCSA, as Thomas R. Berger explained in his important book Village Journey (1985), did not give title to Indigenous people but rather "placed their land at risk" (p. 6 https://www.google.com/books/edition/Village_Journey/2nzYGwAACAAJ?hl=en) As indicated in another comment on this page, the authors of this report demonstrate little knowledge or concern about the diverse tribal governments and Indigenous peoples of Alaska; rather, they want to lump them together as a monolithic entity. Alaska Natives, in pages of this report, do not seem to care about subsistence practices, cultural traditions, and food sovereignty. These claims also overlook the long and significant history of alliances and coalitions forged between Indigenous peoples, environmentalists, and other groups, including around such high-profile environmental debates such as the Arctic National Wildlife Refuge.

This is a call to repeal the 1971 Wild Free Roaming Horses and Burros Act and subsequent acts that prevented the slaughter of horses. It also flies in the face of several court cases that have deemed slaughter policies illegal under these laws. See https://www.blm.gov/learn/blm-library/subject-guides/wild-horse-and-burro-subject-guide/policy-and-legal-resources#:~:text=The%20Public%20Rangelands%20Improvement%20Act%20of%201978%20(PRIA)%20amended%20the,and%20Management%20Act%20of%201976 It is important to note here that Pendley is veiling this agenda carefully, under the guise of reform and ecological considerations.

We cannot find this study. We did find one by rangeland ecologists in that year, which recommends a different kind of study to determine if ecologies with an overabundance of wild horses can recover and how long that recover would take. See https://doi.org/10.1093/biosci/biz060.

This is an exaggeration. Congress passed the 1971 bill on the heels of overwhelming public support. The massive letter-writing campaign by school children alone was astounding. See https://www.oupress.com/9780806149271/the-size-of-the-risk/

Pendley offers no citation or other evidence tor this sweeping claim how westerners felt about the relocation ["All of them...appreciated"]. If this were an undergraduate history paper, such a claim would get red-marked as both unsubstantiated and highly exaggerated. As pointed out earlier, the relocation was highly disruptive for employees. As of August 2024, the Grand Junction office had 24 employees.

https://www.gjsentinel.com/news/blm-continues-staffing-up-western-hq/article_ea8b8430-5052-11ef-812d-2be536fa4557.html

Tribal-federal relationships at Bears Ears were not built up with opportunities but manipulated an undermined. If past is prologue, this is an empty statement. https://www.pbs.org/newshour/politics/native-american-tribes-call-trumps-revamp-of-tribal-advisory-commission-a-slap-in-the-face

It isn't at all clear where Pendley gets the idea that Congress set aside the 1002 areas "specifically for future oil and gas exploration and development." ANILCA, codified in 16 USC Section 3142 a reads: "The purpose of this section is to provide for a comprehensive and continuing inventory and assessment of the fish and wildlife resources of the coastal plain of the Arctic National Wildlife Refuge; an analysis of the impacts of oil and gas exploration, development, and production, and to authorize exploratory activity within the coastal plain in a manner that avoids significant adverse effects on the fish and wildlife and other resources." There has been legitimate debate about whether or not oil and gas development is possible without negative impact on fish and wildlife.

The implication in much of the discussion about Alaska (including the case of the Arctic National Wildlife Refuge) is that the story is one pitting environmentalists versus oil and gas drilling proponents--a narrative frame that overlooks the long history of Indigenous leadership and advocacy in this fights. In the case of the Arctic National Wildlife Refuge, for example, the Gwich'in Nation completely reframed the narrative, turning a traditional wilderness campaign into a transnational fight for Indigenous rights and environmental justice. For a brief history of the Gwich'in role in the Arctic Refuge struggle, see https://www.washingtonpost.com/outlook/2021/03/14/indigenous-advocacy-transformed-fight-over-oil-drilling-arctic-refuge/

The Biden Administration restored unprecedented cuts to national monuments from the previous administration, most famously with Bears Ears in Utah which has begun a comanagement arrangement with local tribes.

The reduction under Trump, which the Biden administration reversed, may have exceeded presidential authority. Litigation ended after these monuments' restoration but will likely start up again should a new conservative administration heed Pendley's call.

https://www.sltrib.com/news/environment/2022/06/22/utah-tribes-secure-co/

https://crsreports.congress.gov/product/pdf/R/R45718

There are important political disagreements over the development of federal fossil fuels, and these deserve attention. The Trump administration's rhetoric and executive orders were aimed at increasing fossil fuel development; the Biden administration's rhetoric and executive orders were aimed at reducing fossil fuel development. But it is important to qualify this with two important points: 1) the rate of fossil fuel extraction is driven more by the market economy, with prices determined by global markets, than by federal policy, and 2) lower fossil fuel prices on these global markets will disincentivize its extraction in the US, and likely bring current record levels of US oil and gas production down.

Like the National Park Service (below), the Bureau of Indian Affairs receives almost no attention in this chapter. Indigenous peoples appear in this document only when they desire oil and gas production. But the BIA has been active under Secretary Haaland--the first Native American to hold this position--in addressing past and contemporary injustices. Her leadership for understanding Boarding School history, along with other episodes of missing and murdered Indigenous Peoples, is especially noteworthy. Neglect of all these issues and endeavors is one more way that Project 2025 has utterly expunged the challenges of environmental justice from its radar screen. The momentum generated by Haaland would likely not continue under an Interior administration that follows Project 2025. https://www.doi.gov/priorities/strengthening-indian-country https://prospect.org/environment/2024-09-26-preserving-public-lands/

A more grounded and explanatory way of referring to the DOI's "deploying of wolves" is this: "restoring endangered species according to requirements in the Endangered Species Act."

This is grossly misleading, as it suggests the turn toward environmental values came primarily from the executive branch. Certainly, the Carter administration advanced an environmental agenda, but the administration was implementing statutes passed with broad bipartisan support in Congress and signed by Republican presidents Nixon and Ford. https://www.unmpress.com/9780826352965/nixon-and-the-environment/

This statement is oblique and justified by a discursive footnote about Wyoming. Interior continues to operate in a bipartisan manner consistent with the laws enacted by Congress pursuant to the powers granted it by the Property Clause. https://scholarlycommons.law.emory.edu/cgi/viewcontent.cgi?article=1453&context=elj What Pendley disagrees with is the primacy of federal power on federal lands over the interests of state and private landowners. In western states with a preponderance of public lands, this primacy of federal power over federal land can conflict with state, county, and private land interests. However, it does not abrogate the power of the federal government to make decisions about its property. (See https://crsreports.congress.gov/product/pdf/R/R44267).

This statement from Pendley's book obfuscates the original purpose of Interior. According to Robert M. Utley and Barry Mackintosh in their administrative history of the agency in 1989 (for Interior's 150th anniversary), the federal government reorganized the departments mentioned to better serve the nation's internal affairs (as opposed to foreign affairs). Congress also meant for Interior to address the organization of the newly acquired cession from Mexico by the Treaty of Guadalupe Hidalgo. The bill creating Interior had bipartisan support. Over time, its mission evolved to where it now stands, providing stewardship of the nation’s land and related resources. (See https://npshistory.com/publications/doi/dept-everything-else.pdf)

Pendley has had a long career in government and conservative legal activism. He served in Interior in both the Reagan and Trump Administrations and has served as president of Mountain States Legal Foundation, a conservative law firm that has been on the forefront for decades in undermining public lands protected and administered by the Department of the Interior.

Pendley wrote a fawning biography of Reagan called Sagebrush Rebel for Regenery Publishing. Support for the Sagebrush Rebellion undermines many of the responsibilities Interior safeguards.

He was acting director of the Bureau of Land Management in 2019-20 and was never confirmed by the Senate. A federal judge removed him for serving unlawfully as nominated (but unconfirmed) "acting" director for longer than 210 days. Pendley refused to follow the ruling.

Pendley has dismissed science about ozone depletion and climate change and has joked about killing endangered species. He has also mocked Indigenous beliefs about sacred sites.

https://www.regnery.com/9781621571568/sagebrush-rebel/ https://www.nytimes.com/2020/09/26/us/william-perry-pendley-blm.html https://www.theguardian.com/environment/2020/oct/10/william-perry-pendley-bureau-of-land-management-refuses-to-leave https://www.cnn.com/2019/10/08/politics/william-perry-pendley-blm-kfile/index.html

what is this??

they would be if determinist Tom was true since one could blame the third party

In the case of Determinist Tom, this would be invalid because since Tom's actions are being controlled by others, he has no real control over mending his past mistakes.

eLife Assessment

These authors present findings on the role of the sirtuins SIRT1 and SIRT3 during Salmonella Typhimurium infection. This valuable study increases our understanding of the mechanisms used by this pathogen to interact with its host and may have implications for other intracellular pathogens. The reviewers disagreed on the strength of the evidence to support the claims. Although one reviewer found the strength of the evidence convincing, the other found that it was incomplete, and that the main claims are only partially supported, as can be seen from the public reviews.

Reviewer #2 (Public review):

Dipasree Hajra et al demonstrated that Salmonella was able to modulate the expression of Sirtuins (Sirt1 and Sirt3) and regulate the metabolic switch in both host and Salmonella, promoting its pathogenesis. The authors found Salmonella infection induced high levels of Sirt1 and Sirt3 in macrophages, which were skewed toward the M2 phenotype allowing Salmonella to hyper-proliferate. Mechanistically, Sirt1 and Sirt3 regulated the acetylation of HIF-1alpha and PDHA1, therefore mediating Salmonella-induced host metabolic shift in the infected macrophages. Interestingly, Sirt1 and Sirt3-driven host metabolic switch also had an effect on the metabolic profile of Salmonella. Counterintuitively, inhibition of Sirt1/3 led to increased pathogen burdens in an in vivo mouse model. Overall, this is a well-designed study.

The revised manuscript has addressed all of the previous comments. The re-analysis of flow cytometry and WB data by authors makes the results and conclusion more complete and convincing.

Reviewer #3 (Public review):

Summary:

In this paper Hajra et al have attempted to identify the role of Sirt1 and Sirt3 in regulating metabolic reprogramming and macrophage host defense. They have performed gene knock down experiments in RAW macrophage cell line to show that depletion of Sirt1 or Sirt3 enhances the ability of macrophages to eliminate Salmonella Typhimurium. However, in mice inhibition of Sirt1 resulted in dissemination of the bacteria but the bacterial burden was still reduced in macrophages. They suggest that the effect they have observed is due to increased inflammation and ROS production by macrophages. They also try to establish a weak link with metabolism. They present data to show that the switch in metabolism from glycolysis to fatty acid oxidation is regulated by acetylation of Hif1a, and PDHA1.

Strengths:

The strength of the manuscript is that the role of Sirtuins in host-pathogen interactions have not been previously explored in-depth making the study interesting. It is also interesting to see that depletion of either Sirt1 or Sirt3 result in a similar outcome.

Weaknesses:

The major weakness of the paper is the low quality of data, making it harder to substantiate the claims. Also, there are too many pathways and mechanisms being investigated. It would have been better if the authors had focussed on either Sirt1 or Sirt3 and elucidated how it reprograms metabolism to eventually modulate host response against Salmonella Typhimurium. Experimental evidences are also lacking to prove the proposed mechanisms. For instance they show correlative data that knock down of Sirt1 mediated shift in metabolism is due to HIF1a acetylation but this needs to be proven with further experiments.

Author response:

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

Reviewer #2 (Public Review):

Dipasree Hajra et al demonstrated that Salmonella was able to modulate the expression of Sirtuins (Sirt1 and Sirt3) and regulate the metabolic switch in both host and Salmonella, promoting its pathogenesis. The authors found Salmonella infection induced high levels of Sirt1 and Sirt3 in macrophages, which were skewed toward the M2 phenotype allowing Salmonella to hyper-proliferate. Mechanistically, Sirt1 and Sirt3 regulated the acetylation of HIF-1alpha and PDHA1, therefore mediating Salmonella-induced host metabolic shift in the infected macrophages. Interestingly, Sirt1 and Sirt3-driven host metabolic switch also had an effect on the metabolic profile of Salmonella. Counterintuitively, inhibition of Sirt1/3 led to increased pathogen burdens in an in vivo mouse model. Overall, this is a well-designed study.

Comments on revised version:

The authors have performed additional experiments to address the discrepancy between in vitro and in vivo data. While this offers some potential insights into the in vivo role of Sirt1/3 in different cell types and how this affects bacterial growth/dissemination, I still believe that Sirt1/3 inhibitors could have some effect on the gut microbiota contributing to increased pathogen counts. This possibility can be discussed briefly to give a better scenario of how Sirt1/3 inhibitors work in vivo. Additionally, the manuscript would improve significantly if some of the flow cytometry analysis and WB data could be better analyzed.

We are highly grateful for your valuable and insightful comments. Thank you for appreciating the merit of our manuscript. As rightly pointed out by the eminent reviewer, we acknowledge the probable link of Sirtuin on gut microbiota and its effect on increased bacterial loads as indicated by previous literature studies (PMID: 22115311, PMID: 19228061). These reports suggested that a low dose of Sirt1 activator, resveratrol treatment in rats for 25 days treatment under 5% DSS induced colitis condition led to alterations in gut microbiota profile with increased lactobacilli and bifidobacteria alongside reduced abundance of enterobacteria. This study correlates with our study wherein we have detected enhanced Salmonella (belonging to Enterobacteriaceae family) loads under both Sirt1/3 in vivo knockdown condition or inhibitor-treated condition in C57BL/6 mice and reduced burden under Sirt-1 activator treatment SRT1720.

As per your valid suggestion, we have discussed this possibility in our discussion section. (Line- 541-548).

We have incorporated the suggestions for the improvement in the analysis of WB data and flow cytometry.

Reviewer #3 (Public Review):

Summary:

In this paper Hajra et al have attempted to identify the role of Sirt1 and Sirt3 in regulating metabolic reprogramming and macrophage host defense. They have performed gene knock down experiments in RAW macrophage cell line to show that depletion of Sirt1 or Sirt3 enhances the ability of macrophages to eliminate Salmonella Typhimurium. However, in mice inhibition of Sirt1 resulted in dissemination of the bacteria but the bacterial burden was still reduced in macrophages. They suggest that the effect they have observed is due to increased inflammation and ROS production by macrophages. They also try to establish a weak link with metabolism. They present data to show that the switch in metabolism from glycolysis to fatty acid oxidation is regulated by acetylation of Hif1a, and PDHA1.

Strengths:

The strength of the manuscript is that the role of Sirtuins in host-pathogen interactions has not been previously explored in-depth making the study interesting. It is also interesting to see that depletion of either Sirt1 or Sirt3 results in a similar outcome.

Weaknesses:

The major weakness of the paper is the low quality of data, making it harder to substantiate the claims. Also, there are too many pathways and mechanisms being investigated. It would have been better if the authors had focussed on either Sirt1 or Sirt3 and elucidated how it reprograms metabolism to eventually modulate host response against Salmonella Typhimurium. Experimental evidence is also lacking to prove the proposed mechanisms. For instance they show correlative data that knock down of Sirt1 mediated shift in metabolism is due to HIF1a acetylation but this needs to be proven with further experiments.

We appreciate the reviewer’s critical analysis of our work. In the revised manuscript, we aimed to eliminate the low-quality data sets and have tried to substantiate them with better and conclusive ones, as directed in the recommendations for the author section. We agree with the reviewer that the inclusion of both Sirtuins 1 and 3 has resulted in too many pathways and mechanisms and focusing on one SIRT and its mechanism of metabolic reprogramming and immune modulation would have been a less complicated alternative approach. However, as rightly pointed out, our work demonstrated the shared and few overlapping roles of the two sirtuins, SIRT1 and SIRT3, together mediating the immune-metabolic switch upon Salmonella infection. As per the reviewer’s suggestion, we have performed additional experiments with HIF-1α inhibitor treatment in our revised manuscript to substantiate our correlative findings on SIRT1-mediated regulation of host glycolysis (Fig.7G). We wanted to clarify our claim in this regard. Our results suggested that loss of SIRT1 function triggered increased host glycolysis alongside hyperacetylation of HIF-1α. HIF-1α is reported to be one of the important players in glycolysis regulation (Kierans SJ, Taylor CT. Regulation of glycolysis by the hypoxia-inducible factor (HIF): implications for cellular physiology. J Physiol. 2021;599(1):23-37. doi:10.1113/JP280572.) and additionally, SIRT1 has been shown to regulate HIF-1α acetylation status (Lim JH, Lee YM, Chun YS, Chen J, Kim JE, Park JW. Sirtuin 1 modulates cellular responses to hypoxia by deacetylating hypoxia-inducible factor 1 alpha. Mol Cell. 2010;38(6):864-878. doi:10.1016/j.molcel.2010.05.023.) Further, ectopic expression of SIRT1 has been demonstrated to reduce glycolysis by negatively regulating HIF-1α. (Wang Y, Bi Y, Chen X, et al. Histone Deacetylase SIRT1 Negatively Regulates the Differentiation of Interleukin-9-Producing CD4(+) T Cells. Immunity. 2016;44(6):1337-1349. doi:10.1016/j.immuni.2016.05.009). We have subsequently shown in Fig. 7G, that the increase in host glycolysis upon SIRT knockdown in the infected macrophages gets lowered upon HIF-1α inhibitor treatment, suggesting that one of the mechanisms of SIRT-mediated regulation of host glycolysis is via regulation of HIF-1α. However, this warrants further future mechanistic research.

Recommendations for the authors:

Reviewer #2 (Recommendations For The Authors):

(1) Figures 8I-S: are only viable cells used for analysis? Please provide gating strategy used for these analyses.

(2) Many changes seen in WB seem to be marginal. Since the authors used densitometric plot to quantify the band intensities, I expect these experiments were repeated at least three times. Please indicate the number of repeats. For instance, Figures 7C, 7I (UI SCR vs UI shSIRT3), 7J, show marginal changes or no changes. What do other WB images look like? Are they more convincing than the ones currently shown? Please provide them in the response letter.

(3) Figure 7C: label is a bit misleading. Please relabel the figure title to Acetylated HIF vs total levels

(4) Figure 7J: which band is AcPDHA1?

(1) We are highly apologetic for not clarifying our gating strategy for the analysis.

We initially gated the viable splenocyte population based on Forward scatter (FSC) and Side Scatter (SSC). This gated population was further subjected to gating based on cell FSC-H (height) versus FSC-A (area). Subsequently, the population was gated as per SSC-A and GFP (expressed by intracellular bacteria) based on the autofluorescence exhibited by the uninfected control (Fig. 8I-J).

Author response image 1.

UNINFECTED

Author response image 2.

VEHICLE CONTROL INFECTED

Author response image 3.

EX-527 INFECTED

Author response image 4.

3TYP INFECTED

Author response image 5.

SRT 1720 INFECTED

For gating different cell types such as F4/80 (PE) positive population in Fig. 8K-L, the viable cell population was gated based on SSC-A versus PE-A to gate the macrophage population. These macrophage populations were gated further based on GFP (Salmonella) + population to obtain the percentage of macrophage population harboring GFP+ bacteria. Similar strategies were followed for other cell types as depicted in Fig. 8M-S, Fig. S8.

(2) We agree with the reviewer’s concern with the marginal changes in the western blots (Figures 7C, 7I (UI SCR vs UI shSIRT3), 7J). As per the suggestions, we have provided the alternate blot images and have indicated the number of repeats in the manuscript. The alternate blot images are provided herewith:

Author response image 6.

Alternate blot images for Fig. 7B-C

Author response image 7.

Alternate blot images for Fig. 7I, J

(1) We are highly thankful to the reviewer for recommending this suggestion. We have made the necessary modifications of relabelling Fig. C to Acetylated HIF-1α over total HIF-1α as per the suggestion.

(2) 7J Acetylated PDHA1 has been duly pointed as per the suggestion. We are extremely apologetic for the inconvenience caused.

Author response image 8

Reviewer #3 (Recommendations For The Authors):

The authors have done some work to improve the manuscript. However, the data presented lacks clarity.

Fig 4B: I still do not see a change in Ac p65 in the less saturated blot. It looks reduced as the band is distorted. I am not sure how this could be quantified.

Fig S2 b-actin bands are hyper saturated, and it is not possible to decipher the knockdown efficiency. It is probably better to provide a ponceau staining similar to S2C. The band intensity values are out of place.

Fig 5F HADHA blot: Lane 1 expression appears to be significantly higher than lane 3, but the values mentioned do not match the intensity of the bands.

It is hard to interpret the authors' claim that the shift in metabolism is HIF1a-dependent.

Fig 7B: I would expect HIF1a acetylation to be increased in UI ShSIRT1 compared to UI SCR. The blot shows reduced HIF1a acetylation.

Fig 7D: SIRT1 immunoprecipitates with HIF1a equally under all conditions. Is this what the authors expect? Labelling of the blots are not clear. It looks like the bottom SIRT1 blot is from Beads IgG control.

Fig 7H: How does PDHA1 interact with SIRT3 so strongly in shSIRT3 cells (lane 2)?

Authors have mentioned in their response that a knockdown of 40% has been achieved in the uninfected but the blot does not reflect that. SIRT3 expression seems to be more in the knockdown.

Blots are also not labelled properly especially Input. The lanes are not marked.

We thank the reviewer for acknowledging the improvements in the revised version and for suggesting further clarifications and improvements.

We have tried to incorporate the specified modifications to the best of our abilities in the revised manuscript.

We are highly apologetic for the inconclusive blot image in the figure 4B. We have provided an alternative blot image with better clarity for Fig.4B used for quantification analysis.

Author response image 9.

As per the reviewer’s valuable suggestions, we have provided the ponceau image in the Fig. S2B.

We thank the reviewers for rightly pointing out the discrepancy in the band intensity quantification in the Fig. 5F. We have re-evaluated the intensities on imageJ and have provided with the correct band intensities. We are highly apologetic for the inaccuracies.

As per the reviewer’s previous suggestion, we have performed additional experiments with HIF-1α inhibitor treatment in our revised manuscript to substantiate our correlative findings on SIRT1-mediated regulation of host glycolysis (Fig.7G). We wanted to clarify our claim in this regard. Our results suggested that loss of SIRT1 function triggered increased host glycolysis alongside hyperacetylation of HIF-1α. HIF-1α is reported to be one of the important players of glycolysis regulation (Kierans SJ, Taylor CT. Regulation of glycolysis by the hypoxia-inducible factor (HIF): implications for cellular physiology. J Physiol. 2021;599(1):23-37. doi:10.1113/JP280572.) and additionally, SIRT1 has been shown to regulate HIF-1α acetylation status (Lim JH, Lee YM, Chun YS, Chen J, Kim JE, Park JW. Sirtuin 1 modulates cellular responses to hypoxia by deacetylating hypoxia-inducible factor 1alpha. Mol Cell. 2010;38(6):864-878. doi:10.1016/j.molcel.2010.05.023.) Further, ectopic expression of SIRT1 has been demonstrated to reduce glycolysis by negatively regulating HIF-1α. (Wang Y, Bi Y, Chen X, et al. Histone Deacetylase SIRT1 Negatively Regulates the Differentiation of Interleukin-9-Producing CD4(+) T Cells. Immunity. 2016;44(6):1337-1349. doi:10.1016/j.immuni.2016.05.009). We have subsequently shown in Fig. 7G, that the increase in host glycolysis upon SIRT knockdown in the infected macrophages gets lowered upon HIF-1α inhibitor treatment, suggesting that one of the mechanisms of SIRT-mediated regulation of host glycolysis is via regulation of HIF-1α. However, this warrants further future mechanistic research.

We agree with the reviewer’s claim of increased HIF-1α acetylation in the UI sh1 versus UI SCR. The apparent reduced acetylation depicted in UI sh1 in Fig. 7B could be attributed to lower HIF-1α levels in the UI sh1 compared to UI SCR. Therefore, we have provided an alternate blot image that been used for quantification in Fig. 7C (Author response image 6).

To answer the reviewer’s question in Fig. 7D, we have noticed more or less equal degree of immunoprecipitation of HIF-1α under pull down of HIF-1α in all the sample cohorts under conditions of SIRT1 inhibitor treatment. However, we have observed reduced interaction of HIF-1α with SIRT1 in the infected sample upon SIRT1 inhibitor treatment.

We thank the reviewers for suggesting improvements in the blot labelling and for raising this concern. We have corrected the blot labelling to avoid the previous confusion.

We appreciate the reviewer’s concern and therefore we have provided an alternate blot image for Fig. 7H which might address the previous stated concern wherein we have achieved an enhanced SIRT3 knockdown percentage.

We are extremely apologetic for the improper labelling of the Input blot with unmarked lanes. We have addressed this issue by labelling the lanes in the input section of the blots.

!

The document was written to tell the story of the life of Boston King, British Soldier who was enslaved by Americans and escaped multiple times

King could no longer handle the horrible conditions his master had him under so he felt that he needed to escape to a new master, which he also left after a short time and eventually got married and started a family.

King was a religious man, as many were back then. He used his religion for hope that things would become better from these bad times, in which they did.

Boston King was promised "great rewards" and received very little for his long walk to pass on the letter. He was promised things but in the end he didn't get his deserved reward.

American's were enslaving blacks, so they felt their only way of escape from slavery was joining the British. And with joining the British and not being slaves, it opened them up to better opportunities and not being stuck in an enslaved, poor quality life.

Boston King was part of the British Military and was enslaved by the Americans but escaped back to the British, This is his memoir which was written while he was still alive.

A "loyalist" is someone who loyally fought against the British, against the Americans. Boston King was a good soldier for the British.

......

The policy problem is that no state wants to host a depository for civilian nuclear waste. Nevada was chosen for political reasons and now that it's a swing state it is unrealistic to think that Yucca Mountain will be used. For how Yucca Mountain was selected and the problem of nuclear waste in general see: https://www.ucpress.edu/books/the-road-to-yucca-mountain/hardcover

It is commendable that this chapter actually suports the efforts of DOE to clean up the enormous environmental damage produced by decades of nuclear weapons production.

But: is this an effort to reclassify the waste so as to make the clean-up process cheaper? Is this just an example of cutting corners?

Having non-scientists direct scientific research is a troubling directive, but a strategy that crops up across many of the environmental plans of Project 2025.

Here the chapter ventures into outright climate denialism, beyond its usual approach of inattention or indifference. Decades of study have made clear both the disproportionately large contributions of the developed countries to the greenhouse emissions causing climate change https://www.cgdev.org/media/who-caused-climate-change-historically and the disproportionate burden that devleoping nations are already enduring from its consequences. https://www.usglc.org/blog/climate-change-and-the-developing-world-a-disproportionate-impact/

While support here for prevention of Uyghur forced labor may sound laudable, it runs against the grain of this chapter and Project 2025 in general, which purport to pare back or limit government. Except when they don't. This particular requirement is so anomalous that we suspect it may result from a personal favor or relationship.

This is consistent with the call to end funding for carbon capture utilization and storage (on p. 376) and the larger call for US energy policy to not subsidize any form of energy [implicitly: except for those already subsidized]. Since this chapter studiously avoids the manner in which oil and natural gas are currently subsidized, such points only further highlight the preference asserted here for the oil and gas industry, to the detriment of the nuclear power and coal industries as well as solar, wind, etc.

By picking winners, using government reshuffling to help force the nation's energy supply back into a single basket, fossil fuels, Project 2025 seems determine to undermine energy security and reliability.

One could argue that expanding renewable energy production helps achieve these goals, both of energy access and energy security.

big Jewish community is the focus of his travel, he doesnt notice other things, rest is fictious

Unprintable

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eLife Assessment

This valuable study proposes that protein secreted by colon cancer cells induces cells with Paneth-like properties that favor colon cancer metastasis. The evidence supporting the conclusions is solid but the study would benefit from more direct experiments to test the functional role of Paneth-like cells and to monitor metastasis from colon tumors. The work will be of interest to researchers studying colon cancer metastasis.

Reviewer #1 (Public review):

Summary:

The authors addressed the influence of DKK2 on colorectal cancer (CRC) metastasis to the liver using an orthotopic model transferring AKP-mutant organoids into the spleens of wild-type animals. They found that DKK2 expression in tumor cells led to enhanced liver metastasis and poor survival in mice. Mechanistically, they associate Dkk2-deficiency in donor AKP tumor organoids with reduced Paneth-like cell properties, particularly Lz1 and Lyz2, and defects in glycolysis. Quantitative gene expression analysis showed no significant changes in Hnf4a1 expression upon Dkk2 deletion. Ingenuity Pathway Analysis of RNA-Seq data and ATAC-seq data point to a Hnf4a1 motif as a potential target. They also show that HNF4a binds to the promoter region of Sox9, which leads to LYZ expression and upregulation of Paneth-like properties. By analyzing available scRNA data from human CRC data, the authors found higher expression of LYZ in metastatic and primary tumor samples compared to normal colonic tissue; reinforcing their proposed link, HNF4a was highly expressed in LYZ+ cancer cells compared to LYZ- cancer cells.

Strengths:

Overall, this study contributes a novel mechanistic pathway that may be related to metastatic progression in CRC.

Weaknesses:

The main concerns are related to incremental gains, missing in vivo support for several of their conclusions in murine models, and missing human data analyses.

Main comments

Novelty:<br /> The authors previously described the role of DKK2 in primary CRC, correlating increased DKK2 levels to higher Src phosphorylation and HNF4a1 degradation, which in turn enhances LGR5 expression and "stemness" of cancer cells, resulting in tumor progression (PMID: 33997693). A role for DKK2 in metastasis has also been previously described (sarcoma, PMID: 23204234)

Mouse data:<br /> (a) The authors analyzed liver mets, but the main differences between AKT and AKP/Dkk2 KO organoids could arise during the initial tumor cell egress from the intestinal tissue (which cannot be addressed in their splenic injection model), or during pre-liver stages, such as endothelial attachment. While the analysis of liver mets is interesting, given that Paneth cells play a role in the intestinal stem cell niche, it is questionable whether a study that does not involve the intestine can appropriately address this pathway in CRC metastasis.<br /> (b) The overall number of Paneth cells found in the scRNA-seq analysis of liver mets was low (17 cells, Fig.3), and assuming that these cells are driving the differences seems somewhat far-fetched.<br /> (c) Fig. 6 suggests a signaling cascade in which the absence of DKK2 leads to enhanced HNF4A expression, which in turn results in reduced Sox9 expression and hence reduced expression of Paneth cell properties. It is therefore crucial that the authors perform in vivo (splenic organoid injection) loss-of-function experiments, knockdown of Sox9 expression in AKP organoids, and Sox9 overexpression experiments in AKP/Dkk2 KO organoids to demonstrate Sox9 as the central downstream transcription factor regulating liver CRC metastasis.<br /> (d) Given the previous description of the role of DKK2 in primary CRC, it is important to define the step of liver metastasis affected by Dkk2 deficiency in the metastasis model. Does it affect extravasation, liver survival, etc.?

Human data:<br /> Can the authors address whether the expression of Dkk2 changes in human CRC and whether mutations in Dkk2 as correlated with metastatic disease or CRC stage?

Bioinformatic analysis<br /> GEO repositories remain not open (at the time of the re-review) and SRA links for raw data are still unavailable. Without access to raw data, it is not possible to verify the analyses or fully assess the results. A part of the article was made by re-analyzing public data so the authors should make even the raw available and not just the count tables

Reviewer #2 (Public review):

Summary:

The authors propose that DKK2 is necessary for the metastasis of colon cancer organoids. They then claim that DKK2 mediates this effect by permitting the generation of lysozyme-positive Paneth-like cells within the tumor microenvironmental niche. They argue that these lysozyme-positive cells have Paneth-like properties in both mouse and human contexts. They then implicate HNF4A as the causal factor responsive to DKK2 to generate lysozyme-positive cells through Sox9.

Strengths:

The use of a genetically defined organoid line is state-of-the-art. The data in Figure 1 and the dependence of DKK2 for splenic injection and liver engraftment, as well as the long-term effect on animal survival, are interesting and convincing. The rescue using DKK2 administration for some of their phenotype in vitro is good. The inclusion and analysis of human data sets help explore the role of DKK2 in human cancer and help ground the overall work in a clinical context.

Remaining Weaknesses after revision:

(1) The authors have effectively explained the regulation of HNF4A at both mRNA and protein levels. To further strengthen their findings, I recommend using CRISPR technology to generate DKK2 and HNF4A double knockout organoids. This approach would allow the authors to investigate whether the AKP liver metastasis is restored in the double knockout condition. Such an experiment would provide more direct evidence that HNF4A protein stabilization is the crucial mechanism for liver metastasis suppression following DKK2 knockout.

Author response:

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

Public Reviews: 

Reviewer #1 (Public Review): 

Summary: 

The authors addressed the influence of DKK2 on colorectal cancer (CRC) metastasis to the liver using an orthotopic model transferring AKP-mutant organoids into the spleens of wild-type animals. They found that DKK2 expression in tumor cells led to enhanced liver metastasis and poor survival in mice. Mechanistically, they associate Dkk2-deficiency in donor AKP tumor organoids with reduced Paneth-like cell properties, particularly Lz1 and Lyz2, and defects in glycolysis. Quantitative gene expression analysis showed no significant changes in Hnf4a1 expression upon Dkk2 deletion. Ingenuity Pathway Analysis of RNA-Seq data and ATAC-seq data point to a Hnf4a1 motif as a potential target. They also show that HNF4a binds to the promoter region of Sox9, which leads to LYZ expression and upregulation of Paneth-like properties. By analyzing available scRNA data from human CRC data, the authors found higher expression of LYZ in metastatic and primary tumor samples compared to normal colonic tissue; reinforcing their proposed link, HNF4a was highly expressed in LYZ+ cancer cells compared to LYZ- cancer cells. 

Strengths: 

Overall, this study contributes a novel mechanistic pathway that may be related to metastatic progression in CRC. 

Weaknesses: 

The main concerns are related to incremental gains, missing in vivo support for several of their conclusions in murine models, and missing human data analyses. Additionally, methods and statistical analyses require further clarification. 

Main comments: 

(1) Novelty 

The authors previously described the role of DKK2 in primary CRC, correlating increased DKK2 levels to higher Src phosphorylation and HNF4a1 degradation, which in turn enhances LGR5 expression and "stemness" of cancer cells, resulting in tumor progression (PMID: 33997693). A role for DKK2 in metastasis has also been previously described (sarcoma, PMID: 23204234). 

(2) Mouse data 

a) The authors analyzed liver mets, but the main differences between AKT and AKP/Dkk2 KO organoids could arise during the initial tumor cell egress from the intestinal tissue (which cannot be addressed in their splenic injection model), or during pre-liver stages, such as endothelial attachment. While the analysis of liver mets is interesting, given that Paneths cells play a role in the intestinal stem cell niche, it is questionable whether a study that does not involve the intestine can appropriately address this pathway in CRC metastasis. 

We value the reviewer’s comment that the splenic injection model cannot represent metastasis from the primary tumors, intravasation and extravasation. Therefore, we performed the orthotopic transplantation of AKP and KO organoids into the colon directly then, tested metastasis of cancer.

Author response image 1.

Primary tumor formation and liver metastasis by orthotopic transplantation of AKP or KO colon cancer organoids. 6-8 week-old male C57BL/6J mice were treated with 2.5% DSS dissolved in drinking water for 5 days, followed by regular water for 2 days to remove gut epithelium. After recovery with the regular water, the colon was flushed with 1000 μl of 0.1% BSA in PBS. Then, 200,000 dissociated organoid cells in 200 μl of 5% Matrigel and 0.1% BSA in PBS were instilled into the colonic luminal space. After infusion, the anal verge was sealed with Vaseline. 8 weeks after transplantation, the mice were sacrificed to measure primary tumor formation and liver metastasis.

As a result, 4 out 6 mice in the control group successfully formed colorectal primary tumors whereas only 2 out 6 mice showed primary tumor formation in the KO group (Author response image 1A). The size of tumors was reduced by about half (10-12 mm to 5-7 mm). Only one AKP mouse developed metastasized nodules in the liver (Author response image 1B). Next, to measure the circulating tumor cells, we harvested at least 500 ul of bloods from the portal vein and then analyzed tdTomato-positive tumor cells (Author response image 2). Flow cytometry analysis of PBMCs showed the presence of tdTomatohiCD45- cells as well as tdTomatomidCD45+ cells in 2 out of 6 AKP mice, while no tdTomato-positive cells were observed in the PBMCs of KO organoid-transplanted mice.

Due to the limited numbers of mice showed primary and metastatic tumor formation, we cannot provide a statistic analysis of DKK2-mediated metastasis. However, our revised data indicate a trend that DKK2 KO reduced primary tumor formation, the number of circulating tumor cells and liver metastasis. This trend is consistent with our previous report in the iScience paper, which showed that DKK2 KO reduced AOM/DSS-induced polyp formation about 60 % and decreased metastasis in the splenic injection model system in this manuscript. Further studies are necessary to confirm this trend and to provide the underlying mechanisms of intravasation and extravasation of circulating tumor cells.

Author response image 2.

Flow cytometry analysis of tdTomato+ circulating colon tumor cells in PBMCs. PBMCs were harvested via the portal vein after euthanasia. CD45 and tdTomato were analyzed by flow cytometry.

b) The overall number of Paneth cells found in the scRNA-seq analysis of liver mets was strikingly low (17 cells, Figure 3), and assuming that these cells are driving the differences seems somewhat far-fetched. Adding to this concern is inappropriate gating in the flow plot shown in Figure 6. This should be addressed experimentally and in the interpretation of data. 

We appreciate for reviewer’s comments to clarify this point. Since the number of LYZ+ cells is low in our scRNA-seq analysis, we performed flow cytometry in Figure 6H showing the clear population expressing LYZ in the same splenic injection model of metastasis. Figure 6H is a representative image of triplicates for each group and we performed this experiment three times, independently. As suggested, we changed the graph format and updated the gating and statistical analysis in Fig 6H and 6I. This in vivo result confirmed our in vitro data showing that DKK2 KO reduced LYZ+ cells while increase the HNF4α1 proteins.

c) Figures 3, 5, and 6 show the individual gene analyses with unclear statistical data. It seems that the p-values were not adjusted, and it is unclear how they reached significance in several graphs. Additionally, it was not stated how many animals per group and cells per animal/group were included in the analyses. 

In Fig. 3, mouse scRNA-seq data were generated from pooled cancer samples from 5 animals per group. The Wilcoxon signed-rank test was performed for each gene and/or regulon activity. Since multiple testing adjustments were not performed, a p-value adjustment is neither needed nor applicable..

In Fig. 5, human data were analyzed. Cells from the same sample are dependent, but differential gene expression (DEG) analysis typically calculates statistics under the assumption that they are independent. This assumption may explain the low p-values observed in our data. To address this issue, we applied pseudobulk DEG analysis to our human single-cell data. Even after correcting for statistical error, we confirmed that the genes of interest still exhibited significantly different expression patterns (Author response image 3).

Author response image 3.

Pseudobulk DEG analysis confirmed the differential expression genes of interest.

In Fig.6H-6I, the number of animals per group is provided in the figure legend.

d) Figure 6 suggests a signaling cascade in which the absence of DKK2 leads to enhanced HNF4A expression, which in turn results in reduced Sox9 expression and hence reduced expression of Paneth cell properties. It is therefore crucial that the authors perform in vivo (splenic organoid injection) loss-of-function experiments, knockdown of Sox9 expression in AKP organoids, and Sox9 overexpression experiments in AKP/Dkk2 KO organoids to demonstrate Sox9 as the central downstream transcription factor regulating liver CRC metastasis. 

Sox9 is a well-established marker gene for Paneth cell formation in the gut. Therefore, overexpression or knockout of the Sox9 gene would result in either an increase or decrease in Paneth cells in the organoids. We believe that the suggested experiments fall outside the scope of this manuscript. Instead, we demonstrated the change in the Paneth cell differentiation marker, Sox9, in the presence or absence of DKK2.

e) Given the previous description of the role of DKK2 in primary CRC, it is important to define the step of liver metastasis affected by Dkk2 deficiency in the metastasis model. Does it affect extravasation, liver survival, etc.? 

We appreciate the reviewer’s insights and perspectives. Regarding liver survival, it is well known that stem cell niche formation is a critical step for the outgrowth of metastasized cancer cells (Fumagalli et al. 2019, Cell Stem Cell). LYZ+ Paneth cells are recognized as stem cell niche cells in the intestine, and human scRNA-seq data have shown that LYZ+ cancer cells express stem cell niche factors such as Wnt and Notch ligands. To determine whether LYZ+ cancer cells act as stem cell niche cells, we performed confocal microscopy to assess whether LYZ+ cancer cells express WNT3A and DLL4 in AKP organoids (Author response image 4). The results show that LYZ labeling co-localizes with DLL4 and WNT3A expression, while the organoid reporter tdTomato is evenly distributed. Additionally, our in vitro and in vivo data indicate that DKK2 deficiency leads to a reduction of LYZ+ cancer cells, which may contribute to stem cell niche formation. Based on these findings, we propose that DKK2 is an essential factor for stem cell niche formation, which is required for cancer cell survival in the liver during the early stages of metastasis. Although our revised data confirmed the trend that DKK2 deficiency decreases liver metastasis, we have not yet determined whether DKK2 is involved in extravasation. This research topic should be addressed in future studies.

Author response image 4.

Confocal microscopy analysis for lysozyme (LYZ) and Paneth cell-derived stem cell niche factors, WNT3A and DLL4 in AKP colon cancer organoids.

The method is described in the supplemental information. The list of antibodies used: DLL4 (delta-like 4) Polyclonal Antibody (Invitrogen, PA5-85931), WNT3A Polyclonal Antibody (Invitrogen, PA5-102317), Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488 (Invitrogen, A-11008), Anti-Lysozyme C antibody (H-10, Santacurz, sc-518083), Goat anti-Mouse IgM (Heavy chain) Secondary Antibody, Alexa Fluor™ 647 (Invitrogen, A-21238).

(3) Human data 

Can the authors address whether the expression of Dkk2 changes in human CRC and whether mutations in Dkk2 as correlated with metastatic disease or CRC stage? 

The human data were useful in identifying the presence of LYZ+ cancer cells with Paneth cell properties. However, due to the limited number of late-stage patient samples with high DKK2 expression, the results were not statistically significant. Nevertheless, the trend suggests a positive correlation between DKK2 expression and the malignant stage of CRC.

(4) Bioinformatic analysis 

The authors did not provide sufficient information on bioinformatic analyses. The authors did not include information about the software, cutoffs, or scripts used to make their analyses or output those figures in the manuscript, which challenges the interpretation and assessment of the results. Terms like "Quantitative gene expression analyses" (line 136) "visualized in a Uniform Approximation and Projection" (line 178) do not explain what was inputted and the analyses that were executed. There are multiple forms to align, preprocess, and visualize bulk, single cell, ATAC, and ChIP-seq data, and depending on which was used, the results vary greatly. For example, in the single-cell data, the authors did not inform how many cells were sequenced, nor how many cells had after alignment and quality filtering (RNA count, mt count, etc.), so the result on Paneth+ to Goblet+ percent in lines 184 and 185 cannot be reached because it depends on this information. The absence of a clustering cutoff for the single-cell data is concerning since this greatly affects the resulting cluster number (https://www.nature.com/articles/s41592-023-01933-9). The authors should provide a comprehensive explanation of all the data analyses and the steps used to obtain those results. 

We apologize for the insufficient information. Below, we provide detailed information on the data analyses, which are also available in the GEO database (Bulk RNA-seq: GSE157531, ATAC-seq: GSE157529, ChIP-seq: GSE277510). Methods are updated in the current version of supplemental information.

(5) Clarity of methods and experimental approaches 

The methods were incomplete and they require clarification. 

We’ve updated our methods as requested by the reviewer.

Reviewer #2 (Public Review): 

Summary: 

The authors propose that DKK2 is necessary for the metastasis of colon cancer organoids. They then claim that DKK2 mediates this effect by permitting the generation of lysozyme-positive Paneth-like cells within the tumor microenvironmental niche. They argue that these lysozyme-positive cells have Paneth-like properties in both mouse and human contexts. They then implicate HNF4A as the causal factor responsive to DKK2 to generate lysozyme-positive cells through Sox9. 

Strengths: 

The use of a genetically defined organoid line is state-of-the-art. The data in Figure 1 and the dependence of DKK2 for splenic injection and liver engraftment, as well as the long-term effect on animal survival, are interesting and convincing. The rescue using DKK2 administration for some of their phenotype in vitro is good. The inclusion and analysis of human data sets help explore the role of DKK2 in human cancer and help ground the overall work in a clinical context. 

Weaknesses: 

In this work by Shin et al., the authors expand upon prior work regarding the role of Dickkopf-2 in colorectal cancer (CRC) progression and the necessity of a Paneth-like population in driving CRC metastasis. The general topic of metastatic requirements for colon cancer is of general interest. However, much of the work focuses on characterizing cell populations in a mouse model of hepatic outgrowth via splenic transplantation. In particular, the concept of Paneth-like cells is primarily based on transcriptional programs seen in single-cell RNA sequencing data and needs more validation. Although including human samples is important for potential generality, the strength could be improved by doing immunohistochemistry in primary and metastatic lesions for Lyz+ cancer cells. Experiments that further bolster the causal role of Paneth-like CRC cells in metastasis are needed. 

Recommendations for the Authors:

Reviewing Editor (Recommendations for the Authors): 

Here we note several key concerns with regard to the main conclusions of the paper. Additional experiments to directly address these concerns would be required to substantially update the reviewer evaluation. 

(1) Demonstration of a causal role of Paneth-like cells in CRC metastasis, for example by sorting the Paneth-like cells - either by the markers they identified in the subsequent single cell or by scatter - to establish whether the frequency of the Paneth-like cells in a culture of organoids is directly correlated with tumorigenicity and engraftment. 

We sincerely appreciate the reviewing editor’s comment. First, as previously reported (Shin et al., iScience 2021), there is no difference in proliferation between WT and KO during in vitro organoid culture or in vivo colitis-induced tumors. However, DKK2 deficiency led to morphological changes, which we analyzed using bulk RNA-seq. As described in the manuscript, Paneth cell marker genes, such as Lysozymes and defensins, were significantly reduced in DKK2 KO AKP organoids.

Due to the nature of these markers, it is technically challenging to isolate live LYZ+ cancer cells. To address this issue in the future, we plan to develop organoids that express a reporter gene specific for Paneth cells. In this manuscript, we demonstrated a correlation between DKK2 and the formation of LYZ+ cancer cells. In both the splenic injection model (Fig. 1) and the orthotopic transplantation model (Fig. R1-R2), we observed that transplantation of cancer organoids with reduced numbers of LYZ+ cells (KO organoids) led to decreased metastatic tumor formation. The number of LYZ+ cells in KO-transplanted mice remained low in liver metastasized tumor nodules (Fig. 6H-I6). Immunohistochemistry further confirmed that LYZ+ cancer cells were barely detectable in KO samples (Author response image 5). These data suggest that DKK2 is essential for the formation of LYZ+ cancer cells, which are necessary for outgrowth following metastasis.

Author response image 5.

Histology of Lysozyme positive cells in metastasized tumor nodules in liver of colon cancer organoid transplanted mice. Immunohistochemistry of Lysozyme positive Paneth-like cells cells in liver metastasized colon cancer (Upper panels, DAB staining). Identification of tumor nodules by H&E staining (lower panels, Scale bar = 100 μm). Magnified tumor nodules are shown in the 2nd and 3rd columns (Scale bar = 25 μm). Arrows indicate Lysozyme positive Paneth like cells in tumor epithelial cells. Infiltration of Lysozyme positive myeloid cells is detected in both AKP and KO tumor nodules. AKP: Control colon cancer organoids carrying mutations in Apc, Kras and Tp53 genes. KO: Dkk2 knockout colon cancer organoids

(2) Further characterization of Lyz+/Paneth-like cells to further the authors' argument for the unique function that they have in their tumor model. Specifically, do the cells with Paneth-like cells secrete Wnt3, EGF, Notch ligand, and DII4 as normal Paneth cells do? 

We appreciate the reviewing editor’s comment. In response, we performed confocal microscopy analysis to examine the protein levels of LYZ, Wnt3A, and DLL4 in AKP colon cancer organoids (Author response image 4). The data presented above show that LYZ+ cancer cells express both Wnt3A and DLL4, suggesting that LYZ+ colon cancer cells may function similarly to Paneth cells, which are stem cell niche cells. Furthermore, using the Panglao database, we demonstrated that LYZ+/Paneth-like cells exhibit typical Paneth cell properties in human scRNA-seq data (Fig. 4 and Fig. 5). These findings suggest that LYZ+ colon cancer cells possess Paneth cell properties.

(3) Experiments to test metastasis, ideally from orthotopic colonic tumors, to ensure phenotypes aren't restricted to the splenic model of hepatic colonization and outgrowth used at present. 

We are in agreement with the reviewing editor and reviewers, which is why we conducted the orthotopic transplantation experiment. However, we encountered challenges in establishing this model effectively. After multiple trials, we observed that many mice did not form primary tumors, and the variability, particularly in metastasis, was difficult to control. Only a few AKP-transplanted mice developed liver metastasis. The representative revision data have been provided above. Nevertheless, we believe that this model needs further improvement and optimization to reliably study metastasis originating from primary tumors.

(4) To generalize claims to human cancer, the authors should test whether loss of DKK2 impacts LYZ+ cancer cells in human organoids and affects their engraftment in immunodeficient mice compared to control. Another more correlative way to validate the LYZ+ expression in human colon cancer would be to stain for LYZ in metastatic vs. primary colon cancer, expecting metastatic lesions to be enriched for LYZ+ cells. 

We agree with your point, and this will be addressed in future studies.

(5) Clarifying inconsistencies regarding effect of DKK2 loss on HNF4A (Figure 1E vs Figure 6I). 

In Figure 1 E, we measured the mRNA levels of HNF4A in metastasized foci by qPCR while in Figure 6I, we measured the protein level of HNF4A by flow cytometry. Recent studies, including our previous report, have shown that HNF4A protein levels are regulated by proteasomal degradation mediated by pSrc (Mori-Akiyama et al. 2007, Gastroenterology, Bastide et al. 2007, Journal of Cell Biology, Shin et al. 2021 iScience). Consequently, while the mRNA levels remained unchanged in Fig. 1E, we observed a reduction of HNF4A protein levels in Figure 6I.

(6) Addressing concerns about statistics and reporting as outlined by Reviewer 1. 

Thank you very much for your assistance in improving our manuscript. The updates have been incorporated as detailed above.

These are the central reviewer concerns that would require additional experimentation to update the editorial summary. Other concerns should be addressed in a revision response but do not require additional experimentation. 

Reviewer #1 (Recommendations For The Authors): 

Specific comments: 

• Do Dkk2-KO organoids grow normally?

Yes, in vitro.

Since the authors reported on the effects of Dkk2 in the induction/maintenance of the Paneth cell niche, changes in AKP organoid numbers of growth rate between Dkk2-WT and KO would be an expected outcome. 

Disruption of Paneth cell formation in normal organoids is expected to alter growth. However, DKK2 KO in colon cancer organoids with mutations in the Apc, Kras, and Tp53 genes exhibits growth rates and organoid sizes similar to those of WT AKP controls. In contrast to in vitro observations, we observed a significant reduction in metastasized tumor growth in vivo. Further analyses of factors derived from LYZ+ cancer cells will help address the discrepancy in DKK2's absence between in vitro and in vivo conditions.

• Figure 1: 

- Panel C: The legend indicates what c.p. stands for.

c.p.m. stands for count per minutes for in vivo imaging analysis. This has been updated in the Figure legend.

- Panel E: Please comment on the possible underlying reasons for the lack of change in HNF4a1 levels. 

This has been updated in response to the reviewing editor’s comment (5) above.

- Panel E: Number of mice from which isolated cancer nodules were harvested. 

Total mice per group were 5. This has been updated in the legend.

• Figure 2: 

- Suggestion: Panel A should be presented in Figure 1 since Dkk2 KO organoids are already used in Figure 1. 

We added this to present the recovery of DKK2 by adding recombinant DKK2 proteins in Fig.2.

- Panel B: Please explain why these genes are marked in blue. 

It has been described in the legend. “Paneth cell marker genes are highlighted as blue circles (AKP=3 and KO=5 biological replicates were analyzed).”

• Figure 3: 

- Indicate the number of cells recovered from AKP vs. KO mice (since liver metastasis was already reduced in KO mice). This should be shown in a UMAP. 

- Panel A: 4th line in the pathways, correct "Singel" typo. 

We appreciate your correction. It has been fixed.

- Panel A: There are multiple versions of PanglaoDB with different markers; a list of all that was used to determine cell type should be provided. 

- Panel C: Bar value for the WNT pathway is not displayed, and there is no legend to indicate the direction of the analysis (that is, AKPvsKO or KOvsAKP). 

It is KOvsAKP, described in the figure legend.

- Panel C: Ingenuity pathway analysis is not a good tool to look at this type of result because it does not include the gene fold changes in the analysis, so it only provides a Z-score of the presence of that pathway and not the degree it is increased or fold changes - recommend substituting any type of GSEA analysis, such as fgsea. -o Panel D: the term "Patient" to refer to mice is confusing. Use "Mice" or "Treatment" or "Condition" instead. 

Corrected

- Panel D: Information about the number of mice per group, cells per animal (or liver let) used, and additional clarification about the statistical analysis used is required, as differences shown in this panel appear subtle given the standard variation in each group. Box plots need to show individual/raw values. 

• Figure 4: 

- Panel E: It would be helpful to show the cutoff lines for the Paneth cell score and Lyz expression in the graphs. 

It has been updated in response to the reviewer’s request.

• Figure 5: 

- Panel B: again, information about the number of "patients" or cells used and clarification about the statistical analysis used is required as the display of data generates concerns about the distribution within groups. Box plots need to show individual/raw values

It has been updated in response to the reviewer’s request.

• Figure 6: 

- Panel A: Add a legend to inform the direction of the process (e.g., red, activation, blue, repression). We noticed the Yap1 bar data had no color. Is there a reason for that? Please explain this point in the revised manuscript. 

Red color added for the Yap1.

- Panel A: Ingenuity pathway analysis is not a good tool to look at this type of results because it does not include the gene Foldchanges in the analysis, so it only provides a Z-score of the presence of that pathway and not the degree it is increased or not. I recommend substituting any type of GSEA analysis, such as fgsea. 

- Panels A&B: Again, only p-value scores were provided, while fold changes are necessary to define the ratio of presence increase of normal vs. AKP. 

- Panel D: No raw or pre-processed ChIP-seq data was provided. Additionally, please indicate exactly the genome location (it seems the image was edited from a raw made on UCSC genome browser-it should be remade by adding coordinates and other important information (genes around, epigenetic, etc.). 

- Panel H/I: Flow cytometry gating is inappropriate, as its catching cells are negative for LYZ in both AKP and KO cells, resulting in an overestimation of the number of Lyz cells. Gating should specifically select very few LYZ-positive cells in the top/left quadrant. 

The updates have been made, and the statistical data have been re-analyzed.

- Panel J: Information about the number of animals/organoids or cells used and clarification about the statistical analysis used is required, as the display of data generates concerns about the distribution within groups. Box plots need to show individual/raw values. 

• Overall: 

- A supplementary table with all the sequenced libraries and their depth, read length/cell count should be provided.

All of the information is now available in the GEO database. We used previously published human epithelial datasets for human single cell analysis (Joanito*, Wirapati*, Zhao*, Nawaz* et al, Nat Genetics, 2022, PMID: 35773407).

- The Hallmark Geneset used is very broad, and the authors should confirm the results on GO bp. 

Using Gene Ontology biological processes (GO bp), we observed that glycolysis-related genes were enriched in our newly described cell population, although the adjusted p-value did not exceed 0.05.

Author response image 6

GSEA with GOBP pathway highlighted glycoprotein and protein localization to extracellular region, both of which are related Paneth cell functions. Paneth cells secrete α-defensins, angiogenin-4, lysozyme and secretory phospholipase A2. The enriched glycoprotein process and protein localization not extracellular region reflect the characteristics of Paneth cells. 

- qPCR is not a good way to confirm sequencing results; while PCR data is pre-normalized, sequencing is normalized only after quantification, so results on 6 E and F should be shown on the sequencing data. 

The expression level of Sox9 is relatively low. In our bulk RNA-seq data, the averages for Sox9 in AKP versus DKK2 KO are 28.2 and 25.1, respectively. While there is a similar trend, the difference is not statistically significant in this dataset, and we did not include an experimental group for reconstitution. Therefore, we conducted qPCR experiments for the reconstitution study by adding recombinant DKK2 (rmDKK2) protein to the culture. Furthermore, it is well established that Sox9 is an essential transcription factor for the formation of LYZ+ Paneth cells. Based on this, we assessed the levels of LYZ and Sox9 using qPCR and confocal microscopy in the presence or absence of DKK2.

• Edits in the text: 

- There are several typographical errors. Specific suggestions are provided below. 

- Line 43: "Chromatin immunoprecipitation followed by sequencing analysis," state analysis of what cells before continuing with "revealed..." revealed... 

- Line 77: Recent findings have identified 

- Line 138: were reduced in KO tumor samples à rephrase to clarify "KO-derived liver tumors" 

- Line 167: Recombinant mouse DKK2 protein treatment in KO organoids partially rescued this effect. Add "partially" since adding rmDkk2 didn't fully restore Lyz1 and Lyz2 levels. 

- Line 185-187: the authors should not reference Figure 6 because it has not been introduced yet. 

- Line 198-199: The authors claimed a correlation between Dkk2 expression and Lgr5 expression; however, the graph presented in Figure 3B does not indicate this. The R-value was 0.11, which does not indicate a correlative expression between these genes. 

- Line 232-233: the authors need to show any connection to Dkk2 gene expression in human samples in order to draw that conclusion. 

- Line 294: expression, leading to the formation 

- Line 347: Wnt ligand (correct Wng typo) 

We have modified our manuscript in accordance with the reviewer’s suggestions.

Reviewer #2 (Recommendations For The Authors): 

Specific criticisms/suggestions: 

Author claim 1: Dkk2 is necessary for liver metastasis of colon cancer organoids. <br /> This model is one of hepatic colonization and eventual outgrowth and not metastasis. Metastasis is optimally assessed using autochthonous models of cancer generation, with the concomitant intravasation, extravasation, and growth of cancer cells at the distant site. The authors should inject their various organoids in an orthotopic colonic transplantation assay, which permits the growth of tumors in the colon, and they can then identify metastasis in the liver that results from that primary cancer lesion (i.e., to better model physiologic metastasis from the colon to liver). 

The data of orthotopic colonic transplantation data has been provided above (Author response images 1 and 2).

Author claim 2: DKK2 is required for the formation of lysozyme-positive cells in colon cancer. 

It would greatly strengthen the authors' claim if supraphysiologic or very high amounts of DKK2 enhance CRC organoid line engraftment ( i.e., the specific experiment being pre-treatment with high levels of DKK2 and immediate transplantation to see a number of outgrowing clones). If DKK2 is causal for the engraftment of the tumors, increased DKK2 should enhance their capacity for engraftment. 

Paneth cells have physical properties permitting sorting and are readily identifiable on flow cytometry. The authors should demonstrate increased tumorigenicity and engraftment by sorting the Paneth-like cells-either by the markers they identified in the subsequent single cell or by scatter to establish whether the frequency of the Paneth-like cells in a culture of organoids is directly correlated with engraftment potential. 

Further characterization of the Paneth-like cells would help further the authors' argument for the unique function that they have in their tumor model. Specifically, do the cells with Paneth-like cells secrete Wnt3, EGF, Notch ligand, and DII4 as normal Paneth cells do? Immunofluorescence, sorting, or western blots would all be reasonable methods to assess protein levels in the sorted population. 

This has been performed and provided above (Author response images 1 and 3)

Author claim 3: Lyzosome (LYZ)+ cancer cells exhibit Paneth cell properties in both mouse and human systems. 

For the claim to be general to human cancer, the author should demonstrate that loss of DKK2 impacts LYZ+ cancer cells in human organoids and affects their engraftment in immunodeficient mice compared to control. Another more correlative way to validate the LYZ+ expression in human colon cancer would be to stain for LYZ in metastatic vs. primary colon cancer, expecting metastatic lesions to be enriched for LYZ+ cells. 

The claims on the metabolic function of Paneth-like cells need more clarification. Do the cancer cells with Paneth features have a distinct metabolic profile compared to the other cell populations? The authors should address this through metabolic characterization of isolated LYZ+ cells with Seahorse or comparison of Dkk2 KO to WT organoids (i.e., +/-LYZ+ cancer cell population). 

To address this question, we need to develop organoids with a Paneth cell reporter gene. We appreciate the reviewer’s comment, and this should be pursued in future studies.

Author claim 4: HNF4A mediates the formation of Lysozyme (Lyz)-positive colon cancer cells by DKK2. 

The authors implicate HNF4A and Sox9 as causal effectors of the Paneth-like cell phenotype and subsequent metastatic potential. There appears to be some discordance regarding the effect of DKK2 loss on HNF4A. In Figure 1E, the authors show that gene expression in metastatic colon cancer cells for HNF4A in DKK2 knockout vs AKP control is insignificant. However, in Figure 6I, there is a highly significant difference in the number of HNF4A positive cells, more than a 3-fold percentage difference, with a p-value of <0.0001. If there is the emergence of a rare but highly expressing HNF4A cell type that on aggregate bulk expression leads to no difference, but sorts differentially, why is it not identified in the single-cell data set? These data together are highly inconsistent with regards to the effect of DKK2 on HNF4A and require clarification. 

Previous studies have demonstrated that HNF4A is regulated by proteasomal degradation mediated by pSrc. As a result, the mRNA level of HNF4A remains unchanged, while the protein level is significantly reduced in colon cancer cells. DKK2 KO leads to decreased Src phosphorylation, resulting in the recovery of HNF4A protein levels. This explains why HNF4A cannot be detected in scRNA-seq datasets, which measure mRNA. We have shown this in our previous report. In this manuscript, based on ChIP-seq data using an anti-HNF4A monoclonal antibody, as well as confocal microscopy and qPCR data for the Sox9 gene, we propose that HNF4A acts as a regulator of cancer cells exhibiting Paneth cell properties.

由于不同“一带一路”国家的气候、地貌、文化、生活习惯、经济条件和农村生活污水质量不同，其农村生活污水处理应遵循以下原则：1 .选择最简单、抗冲击负荷能力强、顺应性稳定的工艺。农村地区日污水量较小，水量主要分布在早上、中午和晚上，日变化系数为5-10 （Wang, 2018）。根据水量、水质变化大的特点，在处理农村污水时应优先采用抗冲击负荷能力强的工艺。2。选择无电或能耗少、运行成本低的节能工艺。农村污水处理厂的运行成本高，规模小，单位能耗高，对于经济条件薄弱的农村地区来说是难以承受的（Zheng et al., 2020），这使得这些设施在后期由于缺乏资金而容易出现运行失败。iii选择操作简单、维护少的工艺。农村地区广泛分布着各种农村污水站，缺乏污水处理专业人员进行操作和维护。因此，在农村污水处理中，操作维护简单、无需频繁进行工艺测试或调整的设施是必不可少的（Wang, 2021a, 2021b）。iv.选择有利于资源利用的进程。污水资源化利用是指污水经过无害化处理后达到一定的水质标准，可以作为再生水用于居民生活、生态供水、农业灌溉等，替代常规水资源，或者从污水中提取其他资源和能源。对增加水资源供给，缓解灌溉水短缺，保障水生态安全具有重要意义。五、因地制宜进行调整。农村污水处理工艺选择前应进行实地调查。根据接收水的功能要求，综合考虑农村经济条件、基础设施完备状况、自然环境、农村用水习惯、用水量、常住人口等因素，确定适合本地区的处理工艺。周边工厂、养殖场的气候条件、污水状况及最终排水目的地

自然生物处理是利用自然生态或人工生态功能对污水进行净化的工艺技术。污水是通过土壤或人工载体的理化作用、自然生物的净化功能、水生植物的截留吸收等方式净化的。它具有净化效率高、运行维护成本低、管理水平低等特点，得到了广泛的应用。人工湿地和稳定塘是农村污水处理中常用的两种处理方法。

与城市相比，村镇人口分布分散，地形复杂，缺乏完整的管网，使得污水收集和处理更加困难（Deng & Wheatley, 2016）。由于世界各地农村地区在地理、气候、生态条件的多样性下，经济社会条件、人口因素、风俗习惯等方面存在很大差异，生活污水的质量和数量也不尽相同。此外，由于选择不同的处理工艺作为污水处理终端，可能导致基础设施成本、处理效果、运维管理的复杂性等方面存在较大差异。显然，应充分考虑当地情况，采用处理效果好、能耗低、运维管理简单、排放达标的可靠、安全的污水处理工艺，而不应采用单一的污水处理工艺。

与美国和日本相比，中国农村污水处理在21世纪初起步较晚，经历了婴儿期、发展状态和快速发展阶段。第一阶段，婴儿期（2005-2008）。中国开始高度重视农村环境保护，并尝试通过制定政策来引导产业发展。第二阶段，发展阶段（2009-2015）。政府以政策研讨、资金支持、示范基地建设为重点.第三阶段，快速发展阶段（2016 -）。这是一个政策机制完善、区域综合服务蓬勃发展的阶段。

管理政策是解决生态环境问题的动力。但我国农村污水的管理还没有城市污水的管理发达。美国和日本是最早研究农村污水管理的国家之一。中国作为发展中国家和世界第二大经济体，在农村污水治理方面也积累了丰富的经验

农村生活污水未经有效处理/回收而排放后，会造成一系列环境风险，包括(A)污染饮用水源。将未经处理的生活污水或低于标准的污水排入地表水，可能污染饮用水源，造成疾病传播。(b)土壤和地下水污染。在地下水位较低的地区，未经有效处理的农村生活污水可能引发地下水污染的高风险，如大肠杆菌超标。(c)水生生态系统平衡受到破坏，水生生物的稳定性和多样性减少，影响鱼类的生存和渔业生产。(d)又黑又臭的水。农村生活污水中的有机物持续发酵，会产生硫化氢、硫醇、氨等恶臭物质，以及硫化铁、硫化锰等黑色物质，产生黑臭水，丧失功能，影响景观和人体健康。(e)水体富营养化。水生生物的过度繁殖会导致水体透明度和溶解氧迅速下降，污染物指标急剧增加，水质恶化。(f)滋生蚊子和苍蝇。生活污水的积累为蚊子和苍蝇等害虫的繁殖提供了温床

由于经济和技术限制，“一带一路”发展中国家农村地区的生活污水处理率较低。经济增长良好的华东、东南沿海发达地区率先开展农村污水处理，投入巨资进行污水处理，实现了农村环境处理基础设施完备，农村污水处理全覆盖。相反，中西部地区由于农村环境治理基础设施投入不足以及生活习惯和自然条件的影响，农村污水处理率较低

农村污水排放与气候条件、经济发展水平密切相关，具有显著的地域性特征。与东南地区的居民不同，西北地区的农村居民受干旱气候的影响，生活用水量较少，特别是洗碗频率较低，污水排放量较少。

农村生活污水受日常生活、周期性人口流动等因素的影响，其数量和质量波动较大。农村人口流动性大，节假日返乡人数多，生活污水排放量明显增加。农村生活污水排放量存在明显的季节变化。由于夏季居民洗浴需求大，导致洗漱污水排放量增加，因此污水排放主要表现为水量大、浓度低

村地区的生活污水通常采用以下处理方法。（一）杠杆化处理。城市污水集中收集系统附近的村庄，由市政和企业污水管网处理；（ii）独立处理。经济和管理发达的农村地区自行建立生活污水处理系统（如综合处理设备、人工湿地等），经处理后排放污水；（三）简单处理或直接排放。

农村居民居住区域分散，生活污水排放区域范围广，污水收集管网不完善，难以集中处理，是重要的水源污染源。

大部分“一带一路”国家都是发展中国家，农村人口多，生活污水排放总量大。中国有63.4万个行政村和2.2万个镇，年生活污水排放量超过200亿吨，占全国总量的一半以上。

人类粪便、食物残渣和含磷洗涤剂是生活污水中总磷的主要来源。与氮一样，磷是生物生长的必需元素，也是水体养分污染的指标(Powers et al., 2016；Withers et al., 2009)。水中过量的氮和磷可能导致藻类过度繁殖，引起富营养化。由于生物除磷效率不够稳定，化学除磷工艺存在操作维护困难、价格昂贵等缺陷，磷处理一直是农村生活污水处理的难题。

生活污水（洗澡间、洗衣房、厨房、厕所）的冲洗是农村地区生活污水的主要来源，其比例与气候条件、生活水平和生活习惯有关。根据污水的水质，生活污水（洗浴、洗衣、厨房、厕所）被归类为灰水，而由排泄物和粪便冲洗产生的冲厕所污水被称为黑水（Paulo et al., 2013）。与灰水相比，黑水的污染物浓度较高，不易处理，但作为一种潜在的资源，具有很高的资源利用价值。

例如，中国企业在“一带一路”沿线国家开展了多项环保配套项目，中国制造的水处理厂已大量出口到东南亚、中亚、中东、非洲等地区。集污水预处理、生物处理、沉淀、消毒于一体的一体化污水处理设备，具有结构紧凑、占地面积小、施工周期短、处理效率高、经济合理等特点，特别适用于没有管网的农村地区分散式生活污水处理。

this is how pure legal theory separates law as ought from the analogy to law as a norm (like morality as a norm)

this is how law still has an element of its ideological past, in that it law is understood as a norm (something we 'ought' to follow)

무한한 flow 에 대해서 시간 역학을 적용해 계산한다는 것으로 이해 했다는데 제시 된 식이 어떤 방식으로 유한하게 계산이 되는지 설명해 주실 수 있나요?

eLife Assessment

This useful study presents a novel microscopy technique called "Expansion Tiling Light Sheet Microscopy" and an accompanying computational pipeline for the faster collection and analysis of 3D volumetric images in animals like planarians. This approach produces beautiful 3D microscropy images and is solid on a technical level. However, due to the use of antibody reagents that visualize many – but not all – neurons and muscle subtypes, the evidence for the biological conclusions in this study remains incomplete. With the claims appropriately contextualized, this paper will be of interest to cell biologists working on imaging and analyzing whole animals.

Reviewer #1 (Public review):

Summary:

The planarian flatworm Schmidtea mediterranea is widely used as a model system for regeneration because of its remarkable ability to regenerate its entire body plan from very small fragments of tissue, including the complete and rapid regeneration of the CNS. Prior to this study, analysis of CNS regeneration in planaria has mostly been performed on a gross anatomical level. Despite its simplicity compared to vertebrates, the CNS of many invertebrates, including planaria, is nonetheless complex, intricate, and densely packed. Some invertebrate models allow the visualization of individual cellular components of the CNS using transgenic techniques. Until transgenesis becomes commonplace in planaria, the visualization and analysis of detailed CNS anatomy must rely on alternate approaches in order to capitalize on the immense promise of this system as a model for CNS regeneration. Another challenge for the study of the CNS more broadly is how to perform imaging of a complete CNS on a reasonable timescale such that multiple individuals per experimental condition can be imaged.

Strengths:

In this report, Lu et al. describe a careful and detailed analysis of the planarian neuroanatomy and musculature in both the homeostatic and regenerating contexts. To improve the effective resolution of their imaging, the authors optimized a tissue expansion protocol for planaria. Imaging was performed by light sheet microscopy, and the resulting optical sections were tiled to reconstruct whole worms. Labelled tissues and cells were then segmented to allow quantification of neurons and muscle fibers, as well as all cells in individual worms using a DNA dye. The resulting workflow can produce highly detailed and quantifiable 3D reconstructions at a rate that is fast enough to allow the analysis of large numbers of animals.

Weaknesses:

Lu et al. use their workflow to visualize RNA expression of five enzymes that are each involved in the biosynthetic pathway of different neurotransmitters/modulators, namely chat (cholinergeric), gad (GABAergic), tbh (octopaminergic), th (dopaminergic), and tph (serotonergic). In this way, they generate an anatomical atlas of neurons that produce these molecules. Collectively these markers are referred to as the "neuronpool." They overstate when they write, "The combination of these five types of neurons constitutes a neuron pool that enables the labeling of all neurons throughout the entire body." This statement does not accurately represent the state of our knowledge about the diversity of neurons in S. mediterranea. There are several lines of evidence that support the presence of glutamatergic and glycinergic neurons, including the following. The glutamate receptor agonists NMDA and AMPA both produce seizure-like behaviors in S. mediterranea that are blocked by the application of glutamate receptor antagonists MK-801 and DNQX (which antagonize NMDA and AMPA glutamate receptors, respectively; Rawls et al., 2009). scRNA-Seq data indicates that neurons in S. mediterranea express a vesicular glutamate transporter, a kainite-type glutamate receptor, a glycine receptor, and a glycine transporter (Brunet Avalos and Sprecher, 2021; Wyss et al., 2022). Two AMPA glutamate receptors, GluR1 and GluR2, are known to be expressed in the CNS of another planarian species, D. japonica (Cebria et al., 2002). Likewise, there is abundant evidence for the presence of peptidergic neurons in S. mediterranea (Collins et al., 2010; Fraguas et al., 2012; Ong et al., 2016; Wyss et al., 2022; among others) and in D. japonica (Shimoyama et al., 2016). For these reasons, the authors should not assume that all neurons can be assayed using the five markers that they selected. The situation is made more complex by the fact that many neurons in S. mediterranea appear to produce more than one neurotransmitter/modulator/peptide (Brunet Avalos and Sprecher, 2021; Wyss et al., 2022), which is common among animals (Vaaga et al., 2014; Brunet Avalos and Sprecher, 2021). However the published literature indicates that there are substantial populations of glutamatergic, glycinergic, and peptidergic neurons in S. mediterranea that do not produce other classes of neurotransmission molecule (Brunet Avalos and Sprecher, 2021; Wyss et al., 2022). Thus it seems likely that the neuronpool will miss many neurons that only produce glutamate, glycine or a neuropeptide.

The authors use their technique to image the neural network of the CNS using antibodies raised vs. Arrestin, Synaptotagmin, and phospho-Ser/Thr. They document examples of both contralateral and ipsilateral projections from the eyes to the brain in the optic chiasma (Figure 1C-F). These data all seem to be drawn from a single animal in which there appears to be a greater than normal number of nerve fiber defasciculatations. It isn't clear how well their technique works for fibers that remain within a nerve tract or the brain. The markers used to image neural networks are broadly expressed, and it's possible that most nerve fibers are too densely packed (even after expansion) to allow for image segmentation. The authors also show a close association between estrella-positive glial cells and nerve fibers in the optic chiasma.

The authors count all cell types, neuron pool neurons, and neurons of each class assayed. They find that the cell number to body volume ratio remains stable during homeostasis (Figure S3C), and that the brain volume steadily increases with increasing body volume (Figure S3E). They also observe that the proportion of neurons to total body cells is higher in worms 2-6 mm in length than in worms 7-9 mm in length (Figure 2D, S3F). They find that the rate at which four classes of neurons (GABAergic, octopaminergic, dopaminergic, serotonergic) increase relative to the total body cell number is constant (Figure S3G-J). They write: "Since the pattern of cholinergic neurons is the major cell population in the brain, these results suggest that the above observation of the non-linear dynamics between neurons and cell numbers is likely from the cholinergic neurons." This conclusion should not be reached without first directly counting the number of cholinergic neurons and total body cells. Given that glutamatergic, glycinergic, and peptidergic neurons were not counted, it also remains possible that the non-linear dynamics are due (in part or in whole) to one or more of these populations.

The authors next assayed the production of different classes of neurons in regenerating post-pharyngeal tail fragments. At 14 dpa, they find significantly reduced proportions of octopaminergic, GABAergic, and dopaminergic neurons in these regenerated animals (Figure 3K). Given that these three neuron classes are primarily found in the brain region (Figure S2A), this suggests that the brains of these animals may not have finished regenerating by 14 dpa.

The authors next applied their imaging and segmentation technique to the musculature using the 6G10 antibody. They find that the body wall muscle fibers from the dorsal and ventral body walls integrate differently at the anterior end (to form a cobweb-like arrangement) compared to the posterior end (Figure 4I). They knock down β-catenin in regenerating head anterior fragments and find that the resulting double-headed worms produce a cobweb-like arrangement at both ends (Figure 4J).

RNAi knockdown of inr-1 is known to produce mobility defects and have elongated bodies relative to control animals (Lei et al., 2016; Figure S6A). To understand the nature of these defects, the authors image the muscle of inr-1 RNAi animals and find increased circular body wall muscle fibers on both dorsal and ventral sides, while β-catenin RNAi animals have increased longitudinal muscle fibers on the dorsal side (Figure 6C). The inr-1 RNAi animals also have reduced cholinergic neurons (Figure S6B), and ectopic expression of the GABAergic marker gad in the periphery (Figure S6B). Lastly the authors simultaneously image muscle and estrella-positive glia and find that these glia lack their typically elaborate stellate morphology in inr-1 RNAi animals (Figure 6E, S6E-K). The combination of this muscle, neuronal, and glial defects may account for the mobility defects observed in inr-1 RNAi worms.

Author response:

Reviewer #1 (Public review):

Lu et al. use their workflow to visualize RNA expression of five enzymes that are each involved in the biosynthetic pathway of different neurotransmitters/modulators, namely chat (cholinergeric), gad (GABAergic), tbh (octopaminergic), th (dopaminergic), and tph (serotonergic). In this way, they generate an anatomical atlas of neurons that produce these molecules. Collectively these markers are referred to as the "neuronpool." They overstate when they write, "The combination of these five types of neurons constitutes a neuron pool that enables the labeling of all neurons throughout the entire body." This statement does not accurately represent the state of our knowledge about the diversity of neurons in S. mediterranea. There are several lines of evidence that support the presence of glutamatergic and glycinergic neurons, including the following. The glutamate receptor agonists NMDA and AMPA both produce seizure-like behaviors in S. mediterranea that are blocked by the application of glutamate receptor antagonists MK-801 and DNQX (which antagonize NMDA and AMPA glutamate receptors, respectively; Rawls et al., 2009). scRNA-Seq data indicates that neurons in S. mediterranea express a vesicular glutamate transporter, a kainite-type glutamate receptor, a glycine receptor, and a glycine transporter (Brunet Avalos and Sprecher, 2021; Wyss et al., 2022). Two AMPA glutamate receptors, GluR1 and GluR2, are known to be expressed in the CNS of another planarian species, D. japonica (Cebria et al., 2002). Likewise, there is abundant evidence for the presence of peptidergic neurons in S. mediterranea (Collins et al., 2010; Fraguas et al., 2012; Ong et al., 2016; Wyss et al., 2022; among others) and in D. japonica (Shimoyama et al., 2016). For these reasons, the authors should not assume that all neurons can be assayed using the five markers that they selected. The situation is made more complex by the fact that many neurons in S. mediterranea appear to produce more than one neurotransmitter/modulator/peptide (Brunet Avalos and Sprecher, 2021; Wyss et al., 2022), which is common among animals (Vaaga et al., 2014; Brunet Avalos and Sprecher, 2021). However the published literature indicates that there are substantial populations of glutamatergic, glycinergic, and peptidergic neurons in S. mediterranea that do not produce other classes of neurotransmission molecule (Brunet Avalos and Sprecher, 2021; Wyss et al., 2022). Thus it seems likely that the neuronpool will miss many neurons that only produce glutamate, glycine or a neuropeptide.

In response to your comments, we agree that our initial statement regarding the "neuron pool" overstated the extent of neuronal coverage provided by the five selected markers. We have revised the sentence as “The combination of these five types of neurons constitutes a neuron pool that enables the labeling of most of the neurons throughout the entire body, including the eyes, brain, and pharynx”. 

Furthermore, we chose the five neurotransmitter systems (cholinergic, GABAergic, octopaminergic, dopaminergic, and serotonergic) based on their well-characterized roles in planarian neurobiology and the availability of reliable markers. However, we acknowledge the limitations of this approach and recognize that it does not encompass all neuron types, particularly those involved in glutamatergic, glycinergic, and peptidergic signaling, which have been documented in S. mediterranea. We will also add the content about other neuron types in our revised manuscript “Additionally, there is considerable diversity among glutamatergic, glycinergic, and peptidergic neurons in planarians. Many neurons in S. mediterranea express more than one neurotransmitter or neuropeptide, which adds further complexity to the system.”

The authors use their technique to image the neural network of the CNS using antibodies raised vs. Arrestin, Synaptotagmin, and phospho-Ser/Thr. They document examples of both contralateral and ipsilateral projections from the eyes to the brain in the optic chiasma (Figure 1C-F). These data all seem to be drawn from a single animal in which there appears to be a greater than normal number of nerve fiber defasciculatations. It isn't clear how well their technique works for fibers that remain within a nerve tract or the brain. The markers used to image neural networks are broadly expressed, and it's possible that most nerve fibers are too densely packed (even after expansion) to allow for image segmentation. The authors also show a close association between estrella-positive glial cells and nerve fibers in the optic chiasma. 

Thank you for your detailed feedback. While we did not perform segmentation of all neuron fibers, we were able to segment more isolated fibers that were not densely packed within the neural tracts. We use 120 nm resolution to segment neurons along the three axes. Our data show the presence of both contralateral and ipsilateral projections of visual neurons. Although Figure 1C-F shows data from one planarian, we imaged three independent specimens to confirm the consistency of these observations. In the revised manuscript, we will include a discussion on the limitations of TLSM in reconstructing neural networks, particularly when it comes to resolving fibers within densely packed regions of the nerve tracts.

The authors count all cell types, neuron pool neurons, and neurons of each class assayed. They find that the cell number to body volume ratio remains stable during homeostasis (Figure S3C), and that the brain volume steadily increases with increasing body volume (Figure S3E). They also observe that the proportion of neurons to total body cells is higher in worms 2-6 mm in length than in worms 7-9 mm in length (Figure 2D, S3F). They find that the rate at which four classes of neurons (GABAergic, octopaminergic, dopaminergic, serotonergic) increase relative to the total body cell number is constant (Figure S3G-J). They write: "Since the pattern of cholinergic neurons is the major cell population in the brain, these results suggest that the above observation of the non-linear dynamics between neurons and cell numbers is likely from the cholinergic neurons." This conclusion should not be reached without first directly counting the number of cholinergic neurons and total body cells. Given that glutamatergic, glycinergic, and peptidergic neurons were not counted, it also remains possible that the non-linear dynamics are due (in part or in whole) to one or more of these populations. 

We have removed the statement "Since the pattern of cholinergic neurons is the major cell population in the brain, these results suggest that the above observation of the non-linear dynamics between neurons and cell numbers is likely from the cholinergic neurons." We changed this statement into “These results suggest that the above observation of the non-linear dynamics between neurons and cell numbers is not likely from the octopaminergic, GABAergic, dopaminergic and serotonergic neurons. Since our neuron pool may not include glutamatergic, glycinergic, and peptidergic neurons, we would like to add the possibility that the non-linear dynamics may be from cholinergic neurons or other neurons not included in our staining.”

Reviewer #2 (Public review): 

Weaknesses: 

(1) The proprietary nature of the microscope, protected by a patent, limits the technical details provided, making the method hard to reproduce in other labs. 

Thank you for your comment. We understand the importance of reproducibility and transparency in scientific research. We would like to point out that the detailed design and technical specifications of the TLSM are publicly available in our published work: Chen et al., Cell Reports, 2020. Additionally, the protocol for C-MAP, including the specific experimental steps, is comprehensively described in the methods section of this paper. We believe that these resources should provide sufficient information for other labs to replicate the method.

(2) The resolution of the analyses is mostly limited to the cellular level, which does not fully leverage the advantages of expansion microscopy. Previous applications of expansion microscopy have revealed finer nanostructures in the planarian nervous system (see Fan et al. Methods in Cell Biology 2021; Wang et al. eLife 2021). It is unclear whether the current protocol can achieve a comparable resolution. 

Thank you for raising this important point. The strength of our C-MAP protocol lies in its fluorescence-protective nature and user convenience. Notably, the sample can be expanded up to 4.5-fold linearly without the need for heating or proteinase digestion, which helps preserve fluorescence signals. In addition, the entire expansion process can be completed within 48 hours. While our current analysis focused on cellular-level structures, our method can achieve comparable or better resolution and we will add this information in the revised manuscript.

(3) The data largely corroborate past observations, while the novel claims are insufficiently substantiated. 

A few major issues with the claims: 

(4) Line 303-304: While 6G10 is a widely used antibody to label muscle fibers in the planarian, it doesn't uniformly mark all muscle types (Scimone at al. Nature 2017). For a more complete view of muscle fibers, it is important to use a combination of antibodies targeting different fiber types or a generic marker such as phalloidin. This raises fundamental concerns about all the conclusions drawn from Figures 4 and 6 about differences between various muscle types. Additionally, the authors should cite the original paper that developed the 6G10 antibody (Ross et al. BMC Developmental Biology 2015). 

We appreciate the reviewer’s insightful comments and acknowledge that 6G10 does not uniformly label all muscle fiber types. We agree that this limitation should be recognized in the interpretation of our results. we will revise the manuscript to explicitly state the limitations of using 6G10 alone for muscle fiber labeling and highlight the need for additional markers. We would also clarify that the primary objective of our study was not to distinguish all muscle fiber types but rather to demonstrate the application of our 3D tissue reconstruction method in addressing traditional research questions. Nonetheless, we agree that expanding the labeling strategy in future studies would allow for a more thorough investigation of muscle fiber diversity. We will ensure all citations are properly revised and updated in our next version.

(5) Lines 371-379: The claim that DV muscles regenerate into longitudinal fibers lacks evidence. Furthermore, previous studies have shown that TFs specifying different muscle types (DV, circular, longitudinal, and intestinal) both during regeneration and homeostasis are completely different (Scimone et al., Nature 2017 and Scimone et al., Current Biology 2018). Single-cell RNAseq data further establishes the existence of divergent muscle progenitors giving rise to different muscle fibers. These observations directly contradict the authors' claim, which is only based on images of fixed samples at a coarse time resolution. 

Thank you for your valuable feedback. Our intent was not to suggest that DV muscles regenerate into longitudinal fibers. Our observations focused on the wound site, where DV muscle fibers appear to reconnect, and longitudinal fibers, along with other muscle types, gradually regenerate to restore the structure of the injured area. We will revise the relevant sections of the manuscript to clarify this dynamic process more accurately.

(6) Line 423: The manuscript lacks evidence to claim glia guide muscle fiber branching. 

We will remove this statement from the revised version. Instead, we will focus on describing our observations of the connections between glial cells and muscle fibers.

(7) Lines 432/478: The conclusion about neuronal and muscle guidance on glial projections is similarly speculative, lacking functional evidence. It is possible that the morphological defects of estrella+ cells after bcat1 RNAi are caused by Wnt signaling directly acting on estrella+ cells independent of muscles or neurons. 

We understand that this approach is insufficient and we will revise the manuscript to more clearly state the limitations of our data. We will describe our observations as preliminary and suggest that further experiments are required.

(8) Finally, several technical issues make the results difficult to interpret. For example, in line 125, cell boundaries appear to be determined using nucleus images; in line 136, the current resolution seems insufficient to reliably trace neural connections, at least based on the images presented. 

We use two setups for imaging cells and neuron projections. For cellular resolution imaging, we utilized a 1× air objective with a numerical aperture (NA) of 0.25 and a working distance of 60 mm (OLYMPUS MV PLAPO). The voxel size used was 0.8×0.8×2.5 µm3. This configuration resulted in a resolution of 2×2×5 µm3 and a spatial resolution of 0.5×0.5×1.25 µm3 with 4× isotropic expansion. Alternatively, for sub-cellular imaging, we employed a 10×0.6 SV MP water immersion objective with 0.8 NA and a working distance of 8 mm (OLYMPUS). The voxel size used in this configuration was 0.26×0.26×0.8 µm3. As a result of this configuration, we achieved a resolution of 0.5×0.5×1.6 µm3 and a spatial resolution of 0.12×0.12×0.4 µm3 with a 4.5× isotropic expansion. The higher resolution achieved with sub-cellular imaging allows us to observe finer structures and trace neural connections.

Regarding your question about cell boundaries, we will revise the manuscript to specify that the boundaries we identified are those of each nucleus, rather than entire cells. This distinction will be made clear in the revised version.

Reviewer #3 (Public review): 

Weaknesses: 

(1) The work would have been strengthened by a more careful consideration of previous literature. Many papers directly relevant to this work were not cited. Such omissions do the authors a disservice because in some cases, they fail to consider relevant information that impacts the choice of reagents they have used or the conclusions they are drawing. 

For example, when describing the antibody they use to label muscles (monoclonal 6G10), they do not cite the paper that generated this reagent (Ross et al PMCID: PMC4307677), and instead, one of the papers they do cite (Cebria 2016) that does not mention this antibody. Ross et al reported that 6G10 does not label all body wall muscles equivalently, but rather "predominantly labels circular and diagonal fibers" (which is apparent in Figure S5A-D of the manuscript being reviewed here). For this reason, the authors of the paper showing different body wall muscle populations play different roles in body patterning (Scimone et al 2017, PMCID: PMC6263039, also not cited in this paper) used this monoclonal in combination with a polyclonal antibody to label all body wall muscle types. Because their "pan-muscle" reagent does not label all muscle types equivalently, it calls into question their quantification of the different body wall muscle populations throughout the manuscript. It does not help matters that their initial description of the body wall muscle types fails to mention the layer of thin (inner) longitudinal muscles between the circular and diagonal muscles (Cebria 2016 and citations therein). 

Ipsilateral and contralateral projections of the visual axons were beautifully shown by dye-tracing experiments (Okamoto et al 2005, PMID: 15930826). This paper should be cited when the authors report that they are corroborating the existence of ipsilateral and contralateral projections. 

Thank you for your feedback. We will incorporate these citations and clarifications into the revised manuscript. We acknowledge the limitations of this approach and recognize that it does not encompass all neuron types, particularly those involved in glutamatergic, glycinergic, and peptidergic signaling. We will also add the content about other neuron types in our revised version.

(2) The proportional decrease of neurons with growth in S. mediterranea was shown by counting different cell types in macerated planarians (Baguna and Romero, 1981; https://link.springer.com/article/10.1007/BF00026179) and earlier histological observations cited there. These results have also been validated by single-cell sequencing (Emili et al, bioRxiv 2023, https://www.biorxiv.org/content/10.1101/2023.11.01.565140v). Allometric growth of the planaria tail (the tail is proportionately longer in large vs small planaria) can explain this decrease in animal size. The authors never really discuss allometric growth in a way that would help readers unfamiliar with the system understand this. 

Thank you for your feedback. We will incorporate these citations and clarifications into the revised manuscript.

(3) In some cases, the authors draw stronger conclusions than their results warrant. The authors claim that they are showing glial-muscle interactions, however, they do not provide any images of triple-stained samples labeling muscle, neurons, and glia, so it is impossible for the reader to judge whether the glial cells are interacting directly with body wall muscles or instead with the well-described submuscular nerve plexus. Their conclusion that neurons are unaffected by beta-cat or inr-1 RNAi based on anti-phospho-Ser/Thr staining (Fig. 6E) is unconvincing. They claim that during regeneration "DV muscles initially regenerate into longitudinal fibers at the anterior tip" (line 373). They provide no evidence for such switching of muscle cell types, so it is unclear why they say this. 

We acknowledge that some of our conclusions were overclaimed given the current data, and we appreciate the opportunity to clarify and refine these claims in the revised manuscript. Regarding the statement that "DV muscles initially regenerate into longitudinal fibers at the anterior tip" (line 373), as addressed in our previous response, this phrasing was unclear. Our intent was not to imply that DV muscles switch into longitudinal fibers. Instead, we observed that muscle fibers reconnect at the wound site, with longitudinal fibers and other muscle types gradually restoring the structure. We will revise this section to better describe the dynamic changes observed during regeneration.

(4) The authors show how their automated workflow compares to manual counts using PI-stained specimens (Figure S1T). I may have missed it, but I do not recall seeing a similar ground truth comparison for their muscle fiber counting workflow. I mention this because the segmented image of the posterior muscles in Figure 4I seems to be missing the vast majority of circular fibers visible to the naked eye in the original image. 

Thank you for raising this important point. We will include a ground truth comparison of our automated muscle fiber counting with manual counts in the supplementary figures. Regarding the observation of missing circular fibers in Figure 4I, we agree that the segmentation appears to have missed a significant number of circular fibers in this particular image. This may have been due to limitations in the current parameters of the segmentation algorithm, especially in distinguishing fibers in regions of varying intensity or overlap. We are revisiting the segmentation parameters to improve the accuracy of detecting circular fibers, and we will provide an updated version of Figure 4I in the revised manuscript.

(5) It is unclear why the abstract says, "We found the rate of neuron cell proliferation tends to lag..." (line 25). The authors did not measure proliferation in this work and neurons do not proliferate in planaria. 

Thank you for bringing this to our attention. What we intended to convey was the increase in neuron number during homeostasis. We will revise the abstract to avoid this mistake in this context and instead describe it as the increase in neuron numbers due to progenitor cell differentiation during homeostasis.

(6) It is unclear what readers are to make of the measurements of brain lobe angles. Why is this a useful measurement and what does it tell us? 

The measurement of brain lobe angles is intended to provide a quantitative assessment of the growth and morphological changes of the planarian brain during regeneration. Additionally, the relevance of brain lobe angles has been explored in previous studies, such as Arnold et al., Nature, 2016, further supporting its use as a meaningful parameter.

(7) The authors repeatedly say that this work lets them investigate planarians at the single-cell level, but they don't really make the case that they are seeing things that haven't already been described at the single-cell level using standard confocal microscopy. 

Thank you for your comment. We agree that single-cell level imaging has been previously achieved in planarians using conventional confocal microscopy. However, our goal was to extend the application of expansion microscopy by combining C-MAP with tiling light sheet microscopy (TLSM), which allows for faster and high-resolution 3D imaging of whole-mount planarians. This combination offers several key advantages over traditional confocal microscopy. For example, it enables high-throughput imaging across entire organisms with a level of detail and speed that is not easily achieved using confocal methods. This approach allows us to investigate the planarian nervous system at multiple developmental and regenerative stages in a more comprehensive manner, capturing large-scale structures while preserving fine cellular details. The ability to rapidly image whole planarians in 3D with this resolution provides a more efficient workflow for studying complex biological processes. We believe this distinction is significant and represents an advance over previous methods. We will clarify this point in the manuscript to better distinguish our approach from standard techniques.

Pulpa nekrozu ve kalan ürünler, iltihap apikal foramen, lateral kanallar, dentin kanalları veya iatrojenik yollar yoluyla periodonsiyuma iletilebilir.

Pulpa ile periodontal membran arasındaki yakın anatomik ilişkiler, iltihabın bu bölgeler arasında da geçebileceğini düşündürmektedir.

Endodontik tedavi sırasında anatomik apeksten 1,5 mm koronal olarak çalışmamızın sebebi (özellikle vital dişlerde) pulpa ile doğrudan ilişkide olan PDL'yi tahrip ederek bu bölgede patolojiye yol açmamaktır.

Apikal foramenin boyutu daha büyüktür, bu da patolojinin geçişi açısından daha risklidir

Boyut olarak en büyük bağlantı yolu

Servikal bölgede mine-sement birleşiminin olmadığı durumlar

eLife Assessment

This study provides a valuable perspective on visual cortex architecture by identifying two cortical gradients that change across the lifespan and have distinct functional and structural features. The first gradient captures well-mapped variations in cortical thickness and myelination markers from early sensory to higher-order cortex, while the second gradient shows divergence in these measures with a more localized structure, notably predicting a previously unknown cluster of visual field maps in the anterior temporal lobe. The large-scale lifespan data are compelling, but the evidence overall is incomplete with key questions around methodical checks and implementation, the standard of evidence for the new visual maps, and how the gradient model relates to sharp tissue boundaries parcellating the cortex.

Reviewer #1 (Public review):

Summary:

The manuscript uses large-scale existing datasets that span almost the full range of human life (5-100 years) to identify two distinct architectural cortical gradients within the visual cortex. These gradients are distinct: in one, cytoarchitecture and myeloarchitecture converge and in the other, they diverge. The authors tested whether these gradients mapped onto known functional properties of the visual cortex, as well as accounting for visual behaviours that are impacted throughout the lifespan. The manuscript also reports the identification of a hitherto unknown cluster of visual field maps in the anterior temporal lobe.

Strengths:

A major strength of the current manuscript is the use of large-scale measurements of human brain structure throughout the lifespan, courtesy of the Human Connectome Project Initiative. The scope of this cross-sectional analysis would be rare, if not impossible to achieve through an individual project.

The approach employed holds promise for assessing the link between large-scale anatomical gradients in the brain and functional/behavioural properties. The current manuscript focuses on the visual cortex but the approach could easily be implemented across the brain in general.

Weaknesses:

While the evidence in favour of the two gradients largely supports the claims, the evidence for a new visual field map cluster in the anterior temporal lobe falls short of the level used historically when identifying visual field maps in the visual cortex and is, at present, not convincing.

More specifically, the progressions of polar angle within the putative anterior lobe cluster are highly variable across subjects. Few subjects have convincing polar angle reversals at either the horizontal or vertical meridians. In other cases, a putative border is shown that spans different polar angles, which does not align with the accepted definitions for visual field maps in the cortex.

Reviewer #2 (Public review):

Summary:

The authors used large MRI data sets of the Human Connectome Project (HCP) and also conducted additional pRF analyses to describe the structural architecture of the human visual cortex in reference to its functional features. By conducting a PCA, they identify 2 components that explain around 50% of the variance, the driven by a positive co-variance between cortical thickness and T1/T2 ratio, the second by their negative co-variance. The first PC spans most early visual cortex and hence shows a relation to pRF size when taking both early and late visual areas into account. The second is more variable in location and does not relate to pRF size or visual hierarchy. The relationship between these two gradients to cell body density using the BigBrain is explored.

Strengths:

The authors make an attempt to describe the overall architectural features of the cortex and link it to features of functional representations, and the underlying histology, using different sets of datasets and methods, including histology. They highlight that investigating the structural architecture of the cortex provides important information on their intrinsic organization and common features.

Weaknesses:

The neurobiological model does not take into consideration present knowledge about the microstructural organization of the visual system. This limits the way the results are interpreted correctly. Critical information on the layer-specific myeloarchitecture and cytoarchitecture (and their relation to cortical thickness), as explored for example by Sereno et al. 2013 Cereb Cortex, is missing. There is no information given with respect to how different visual areas differ in their microstructural profile. It is also not mentioned that cortical parcellation is indeed characterized by sharp boundaries between areas, rather than structural gradients, so it remains unclear why focusing on a gradient is of interest. The authors cite the parcellation atlas by Glasser et al. 2016, but do not discuss the rationale of this publication, which was not the definition of gradients, but the definition of sharp boundaries for cortex parcellation. Indeed (as explained below), the results of the authors seem to a large extent to be driven by cortex parcellation, but instead of acknowledging this fact, the authors write (line 179) that "we hypothesize that these local deviations from the canonical thickness and density of cortex underlie the finer-scale division of visual cortex into categorically distinct regions. That is, does the realization of the cortex into distinct regions involve these regions becoming more distinct from a prototypical cortical sheet (i.e., gradient 1)?" - While the first sentence is reasonable, the second sentence is pure speculation ignoring present knowledge on cortical parcellation of this area according to which there is no "prototypical cortical sheet", but each area has its distinct microstructural profile.

Instead of building on present, detailed knowledge of brain anatomy and in-vivo cortex parcellation of the visual system and its known relation to visual maps, the authors focus on two metrics of cortex architecture (mean T1/T1 over depth and cortical thickness), and conduct a PCA to explore their shared variance. It needs to be clarified if the PCA was conducted correctly. There is no mention of standardizing the variables, which could bias the results. In addition, in a PCA, all possible features are categorized as vector components, and those are scanned through the samples, hence, one such analysis per vertex. But the authors write "in which participants are features and cortical vertices are samples" and "the thickness and tissue density maps were concatenated". This needs clarification. The architecture of the PCA should be visualized better.

Because the PCA only contains two features, PC1 is driven by the positive relationship between cortical thickness and mean T1/T2, whereas PC2 is driven by their negative relationship. Because in the early visual cortex, cortical thickness and mean T1/T2 correlate positively, it naturally follows that PC1 relates to pRF size (but mediated by the actual cortex parcellation). However, it is unclear why this insight is interesting. I also do not share the view that "these findings demonstrate that gradient 1 acts as a global gradient enveloping the entire visual cortex (...) while gradient 2 acts as a local gradient specific to individual visual streams". I think this relationship between cortical thickness and T1/T2 ratio does not have much to do with local and global gradients. But if so, stronger arguments as to why this should be the case should be presented.

What the authors make of this result (particularly the discussion starting line 366) is not clear to me. I cannot follow the line of argumentation, which in my view is too far away from the data.

I'm a firm believer in the fact that every single person is entitled to be the sexual creature they want to be, as long as it doesn't cross the line and hurt another, break a crime, or lie to gain access to. Of course, the culture of sex had to change when the nature of sexual creativity and wants themselves changed. As we evolve, culture evolves with and within us. Wanting to feel good or become sexually close or aware is expected, especially since we, as humans, have little knowledge of what it is that truly makes us humans. We need to be taught Anatomy far past eyes, ears, nose, and so on. How can we understand how our culture truly changes if we do not understand why we need to change.

I find it absolute magic that we can gather in groups of anything we want with the digital dawn of AI's, "friends", "likes","pages", and such we are able to.

Has an ABABCC rhyme scheme that is consistent throughout the entire poem.

scrap value = the estimated worth of an asset at the end of its useful life.

Very Informative

Lombard and Vera have a disagreement during a seemingly peaceful conversation, and based on the subsequent text, Vera appears to be quite convinced of her suspicions.

Why does General MacArthur say that everyone is waiting for the end while also stating that Vera won't understand this as a form of release?

In the current situation with multiple deaths, people have different perspectives on the issues at hand. However, some have also identified the killer's tricks, revealing the killer's talent for confusion and malicious enjoyment.

Mikroorganizmaların ve toksik ürünlerin periodontal membrandan geçmesine neden olur

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

Die CO2-Emissionen durch Waldbrände haben seit 2001 um 60% zugenommen, wie sich aus einer neuen Studie ergibt. Den größten Anteil daran haben die borealen Wälder Kanadas und Sibiriens. Sie gehören zu einem Typ von Wäldern, der besonders schlecht an die globale Erhitzung angepasst ist. Die beobachteten Emissionen durch Waldbrände machen unwahscheinlicher, dass Wälder in Zukunft von Menschen emittiertes CO2 reduzieren können. https://www.nytimes.com/2024/10/17/climate/carbon-fires-forests-global-warming.html

Studie: https://www.science.org/doi/10.1126/science.adl5889

https://theconversation.com/extreme-wildfires-have-doubled-in-just-20-years-heres-the-science-233375

Hier ist die Beschriftung des Codes verrückt. Punkt 4 und 5 weisen auf die falschen Zeilen im Code.

Shall we add some screenshots here?

Remove this line and add this line: In some very special cases, the Percentage fulfilled (%) may not display data as you expect. For example, you use Card to display the overall Percentage fulfilled (%) for a period, in this case the Percentage fulfilled (%) will be a sum instead of an average.

When the effort type is FTE, the data of Demand, Fulfilled Demand or Unmet Demand may vary while the selected time period varies. Take an example, if a position (as each position has a role, role represents position in this new data source) has 2 FTEs of Demand in August and September, but no demand in both July and October, you will see the position has 2 FTEs of Demand if the time period is from August to September, but you will see approximately 1 FTEs of Demand for the position if you change the period from July to October. If you think FTE is not so straightforward, try another two effort types Person Days or Hours. The Demand will not change in above case if the effort type is Person Days or Hours.

If a team has multiple levels sub-teams, only the current team and its direct sub team are displayed, allocations for level three and higher sub-teams are rolled up to their level two ancestral team.

eLife Assessment

The authors present three valuable transgenic models carrying three representative exon deletions of the dystrophin gene. The findings are supported by rigorous biochemical assays and state-of-the-art microscopy methods, although the evidence, while overall solid, is only partially developed, and some points could be improved.

Reviewer #1 (Public review):

Summary:

In this article the authors described mouse models presenting with backer muscular dystrophy, they created three transgenic models carrying three representative exon deletions: ex45-48 del., ex45-47 19 del., and ex45-49 del.. This article is well written but needs improvement in some points.

Strengths:

This article is well written. The evidence supporting the authors' claims is robust, though further implementation is necessary. The experiments conducted align with the current state-of-the-art methodologies.

Weaknesses:

This article does not analyze atrophy in the various mouse models. Implementing this point would improve the impact of the work

Reviewer #2 (Public review):

Miyazaki et al. established three distinct BMD mouse models by deleting different exon regions of the dystrophin gene, observed in human BMD. The authors demonstrated that these models exhibit pathophysiological changes, including variations in body weight, muscle force, muscle degeneration, and levels of fibrosis, alongside underlying molecular alterations such as changes in dystrophin and nNOS levels. Notably, these molecular and pathological changes progress at different rates depending on the specific exon deletions in the dystrophin gene. Additionally, the authors conducted extensive fiber typing, revealing a site-specific decline in type IIa fibers in BMD mice, which they suggest may be due to muscle degeneration and reduced capillary formation around these fibers.

Strengths:

The manuscript introduces three novel BMD mouse models with different dystrophin exon deletions, each demonstrating varying rates of disease progression similar to the human BMD phenotype. The authors also conducted extensive fiber typing across different muscles and regions within the muscles, effectively highlighting a site-specific decline in type IIa muscle fibers in BMD mice.

Weaknesses:

The authors have inadequate experiments to support their hypothesis that the decay of type IIa muscle fibers is likely due to muscle degeneration and reduced capillary formation. Further investigation into capillary density and histopathological changes across different muscle fibers is needed, which could clarify the mechanisms behind these observations.

Author response:

Reviewer #1 (Public review):

Summary:

In this article the authors described mouse models presenting with backer muscular dystrophy, they created three transgenic models carrying three representative exon deletions: ex45-48 del., ex45-47 19 del., and ex45-49 del. This article is well written but needs improvement in some points.

Strengths:

This article is well written. The evidence supporting the authors' claims is robust, though further implementation is necessary. The experiments conducted align with the current state-of-the-art methodologies.

Weaknesses:

This article does not analyze atrophy in the various mouse models. Implementing this point would improve the impact of the work

We thank the reviewer for their constructive suggestions and comments on this work. Muscle hypertrophy is shown with growth in dystrophin-deficient skeletal muscle in mdx mice; thus, we did not pay attention to the factors associated with muscle atrophy in BMD mice. As the reviewer suggested, the examination of the association between type IIa fiber reduction and muscle atrophy is important, and the result is considered to be helpful in resolving the cause of type IIa fiber reduction in BMD mice.

Thus, we are planning to:

(1) Evaluate the cross-sectional areas (CSA) of muscles and compare them with the changes in the proportion of type IIa fibers.

(2) Evaluate the expression levels of Murf1 and Atrogin1 as markers of muscle atrophy using RT-PCR.

Reviewer #2 (Public review):

Summary

Miyazaki et al. established three distinct BMD mouse models by deleting different exon regions of the dystrophin gene, observed in human BMD. The authors demonstrated that these models exhibit pathophysiological changes, including variations in body weight, muscle force, muscle degeneration, and levels of fibrosis, alongside underlying molecular alterations such as changes in dystrophin and nNOS levels. Notably, these molecular and pathological changes progress at different rates depending on the specific exon deletions in the dystrophin gene. Additionally, the authors conducted extensive fiber typing, revealing a site-specific decline in type IIa fibers in BMD mice, which they suggest may be due to muscle degeneration and reduced capillary formation around these fibers.

Strengths:

The manuscript introduces three novel BMD mouse models with different dystrophin exon deletions, each demonstrating varying rates of disease progression similar to the human BMD phenotype. The authors also conducted extensive fiber typing across different muscles and regions within the muscles, effectively highlighting a site-specific decline in type IIa muscle fibers in BMD mice.

Weaknesses:

The authors have inadequate experiments to support their hypothesis that the decay of type IIa muscle fibers is likely due to muscle degeneration and reduced capillary formation. Further investigation into capillary density and histopathological changes across different muscle fibers is needed, which could clarify the mechanisms behind these observations.

We thank the reviewer for these positive comments and the very important suggestion about type IIa fiber reduction and capillary change around muscle fibers in BMD mice. From the results of the cardiotoxin-induced muscle degeneration and regeneration model, type IIa and IIx fibers showed delayed recovery compared with that of type-IIb fibers. However, this delayed recovery of type IIa and IIx could not explain the cause of the selective muscle fiber reduction limited to type IIa fibers in BMD mice. Therefore, we considered vascular dysfunction as the reason for the selective type IIa fiber reduction, and we found morphological capillary changes from a “ring pattern” to a “dot pattern” around type IIa fibers in BMD mice. However, the association between selective type IIa fiber reduction and the capillary change around muscle fibers in BMD mice remains unclear due to the lack of information about capillaries around type IIx and IIb fibers. The reviewer pointed out this insufficient evaluation of capillaries around other muscle fibers (except for type IIa fibers), and this suggestion is very helpful for explaining the association between selective type IIa fiber reduction and vascular dysfunction in BMD mice.

Thus, we are planning to:

(1) Evaluate the changes in capillary formation around other muscle fibers, except for type IIa fibers (e.g., type IIx and IIb fibers).

(2) Evaluate the endothelial area around other muscle fibers, except for type IIa fibers.

eLife Assessment

This study analyzes a cohort of small intestine neuroendocrine tumors, and the description of tumor-intrinsic programs that govern such rare cancers is felt to be valuable. The methods are for the most part felt to be solid, however, some broad concerns were raised that the possible separation of samples by a program may be impacted by fresh versus frozen sequencing. Similarly, the heterogeneity of the SiNET tumor microenvironment is unclear given a mixing of subtypes and the proliferation of NE and immune cells in SiNET could be influenced by technical factors. Recommendations were made to extend these data with other published datasets of SiNET tumors, expanding technical clarity and details, and validating findings using cell lines/PDX if available.

Reviewer #1 (Public review):

Summary:

The authors have assembled a cohort of 10 SiNET, 1 SiAdeno, and 1 lung MiNEN samples to explore the biology of neuroendocrine neoplasms. They employ single-cell RNA sequencing to profile 5 samples (siAdeno, SiNETs 1-3, MiNEN) and single-nuclei RNA sequencing to profile seven frozen samples (SiNET 4-10).

They identify two subtypes of siNETs, characterized by either epithelial or neuronal NE cells, through a series of DE analyses. They also report findings of higher proliferation in non-malignant cell types across both subtypes. Additionally, they identify a potential progenitor cell population in a single-lung MiNEN sample.

Strengths:

Overall, this study adds interesting insights into this set of rare cancers that could be very informative for the cancer research community. The team probes an understudied cancer type and provides thoughtful investigations and observations that may have translational relevance.

Weaknesses:

The study could be improved by clarifying some of the technical approaches and aspects as currently presented, toward enhancing the support of the conclusions:

(1) Methods: As currently presented, it is possible that the separation of samples by program may be impacted by tissue source (fresh vs. frozen) and/or the associated sequencing modality (single cell vs. single nuclei). For instance, two (SiNET1 and SiNET2) of the three fresh tissues are categorized into the same subtype, while the third (SiNET9) has very few neuroendocrine cells. Additionally, samples from patient 1 (SiNET1 and SiNET6) are separated into different subtypes based on fresh and frozen tissue. The current text alludes to investigations (i.e.: "Technical effects (e.g., fresh vs. frozen samples) could also impact the capture of distinct cell types, although we did not observe a clear pattern of such bias."), but the study would be strengthened with more detail.

(2) Results:<br /> Heterogeneity in the SiNET tumor microenvironment: It is unclear if the current analysis of intratumor heterogeneity distinguishes the subtypes. It may be informative if patterns of tumor microenvironment (TME) heterogeneity were identified between samples of the same subtype. The team could also evaluate this in an extension cohort of published SiNET tumors (i.e. revisiting additional analyses using the SiNET bulk RNAseq from Alvarez et al 2018, a subset of single-cell data from Hoffman et al 2023, or additional bulk RNAseq validation cohorts for this cancer type if they exist [if they do not, then this could be mentioned as a need in Discussion])

(3) Proliferation of NE and immune cells in SiNETs: The observed proliferation of NE and immune cells in SiNETs may also be influenced by technical factors (including those noted above). For instance, prior studies have shown that scRNA-seq tends to capture a higher proportion of immune cells compared to snRNA-seq, which should be considered in the interpretation of these results. Could the team clarify this element?

(4) Putative progenitors in mixed tumors: As written, the identification of putative progenitors in a single lung MiNEN sample feels somewhat disconnected from the rest of the study. These findings are interesting - are similar progenitor cell populations identified in SiNET samples? Recognizing that ideally additional validation is needed to confidently label and characterize these cells beyond gene expression data in this rare tumor, this limitation could be addressed in a revised Discussion.

Reviewer #2 (Public review):

Summary:

The research identifies two main SiNET subtypes (epithelial-like and neuronal-like) and reveals heterogeneity in non-neuroendocrine cells within the tumor microenvironment. The study validates findings using external datasets and explores unexpected proliferation patterns. While it contributes to understanding SiNET oncogenic processes, the limited sample size and depth of analysis present challenges to the robustness of the conclusions.

Strengths:

The studies effectively identified two subtypes of SiNET based on epithelial and neuronal markers. Key findings include the low proliferation rates of neuroendocrine (NE) cells and the role of the tumor microenvironment (TME), such as the impact of Macrophage Migration Inhibitory Factor (MIF).

Weaknesses:

However, the analysis faces challenges such as a small sample size, lack of clear biological interpretation in some analyses, and concerns about batch effects and statistical significance.

Reviewer #3 (Public review):

Summary:

In this study, the authors set out to profile small intestine neuroendocrine tumors (siNETs) using single-cell/nucleus RNA sequencing, an established method to characterize the diversity of cell types and states in a tumor. Leveraging this dataset, they identified distinct malignant subtypes (epithelial-like versus neuronal-like) and characterized the proliferative index of malignant neuroendocrine cells versus non-malignant microenvironment cells. They found that malignant neuroendocrine cells were far less proliferative than some of their non-malignant counterparts (e.g., B cells, plasma cells, epithelial cells) and there was a strong subtype association such that epithelial-like siNETs were linked to high B/plasma cell proliferation, potentially mediated by MIF signaling, whereas neuronal-like siNETs were correlated with low B/plasma cell proliferation. The authors also examined a single case of a mixed lung tumor (neuroendocrine and squamous) and found evidence of intermediate/mixed and stem-like progenitor states that suggest the two differentiated tumor types may arise from the same progenitor.

Strengths:

The strengths of the paper include the unique dataset, which is the largest to date for siNETs, and the potentially clinically relevant hypotheses generated by their analysis of the data.

Weaknesses:

The weaknesses of the paper include the relatively small number of independent patients (n = 8 for siNETs), lack of direct comparison to other published single-cell NET datasets, mixing of two distinct methods (single-cell and single-nucleus RNA-seq), lack of direct cell-cell interaction analyses and spatially-resolved data, and lack of in vitro or in vivo functional validation of their findings.

The analytical methods applied in this study appear to be appropriate, but the methods used are fairly standard to the field of single-cell omics without significant methodological innovation. As the authors bring forth in the Discussion, the results of the study do raise several compelling questions related to the possibility of distinct biology underlying the epithelial-like and neuronal-like subtypes, the origin of mixed tumors, drivers of proliferation, and microenvironmental heterogeneity. However, this study was not able to further explore these questions through spatially-resolved data or functional experiments.

Author response:

Reviewer #1 (Public review):

Summary:

The authors have assembled a cohort of 10 SiNET, 1 SiAdeno, and 1 lung MiNEN samples to explore the biology of neuroendocrine neoplasms. They employ single-cell RNA sequencing to profile 5 samples (siAdeno, SiNETs 1-3, MiNEN) and single-nuclei RNA sequencing to profile seven frozen samples (SiNET 4-10).

They identify two subtypes of siNETs, characterized by either epithelial or neuronal NE cells, through a series of DE analyses. They also report findings of higher proliferation in non-malignant cell types across both subtypes. Additionally, they identify a potential progenitor cell population in a single-lung MiNEN sample.

Strengths:

Overall, this study adds interesting insights into this set of rare cancers that could be very informative for the cancer research community. The team probes an understudied cancer type and provides thoughtful investigations and observations that may have translational relevance.

Weaknesses:

The study could be improved by clarifying some of the technical approaches and aspects as currently presented, toward enhancing the support of the conclusions:

(1) Methods: As currently presented, it is possible that the separation of samples by program may be impacted by tissue source (fresh vs. frozen) and/or the associated sequencing modality (single cell vs. single nuclei). For instance, two (SiNET1 and SiNET2) of the three fresh tissues are categorized into the same subtype, while the third (SiNET9) has very few neuroendocrine cells. Additionally, samples from patient 1 (SiNET1 and SiNET6) are separated into different subtypes based on fresh and frozen tissue. The current text alludes to investigations (i.e.: "Technical effects (e.g., fresh vs. frozen samples) could also impact the capture of distinct cell types, although we did not observe a clear pattern of such bias."), but the study would be strengthened with more detail.

We thank the reviewer for the thoughtful and constructive review. Due to the difficulty in obtaining enough SiNET samples, we used two platforms to generate data - single cell analysis of fresh samples, and single nuclei analysis of frozen samples. We opted to combine both sample types in our analysis while being fully aware of the potential for batch effects. We therefore agree that this is a limitation of our work, and that differences between samples should be interpreted with caution.

Nevertheless, we argue that the two SiNET subtypes that we have identified are very unlikely to be due to such batch effect. First, the epithelial SiNET subtype was not only detected in two fresh samples but also in one frozen sample (albeit with relatively few cells, as the reviewer correctly noted). Second, and more importantly, the epithelial SiNET subtype was also identified in analysis of an external and much larger cohort of bulk RNA-seq SiNET samples that does not share the issue of two platforms (as seen in Fig. 2f). Moreover, the proportion of samples assigned to the two subtypes is similar between our data and the external data. We therefore argue that the identification of two SiNET subtypes cannot be explained by the use of two data platforms. However, we agree that the results should be further investigated and validated by future studies, as is often done in research on rare tumors.

The reviewer also commented that two samples from the same patient which were profiled by different platforms (SiNET1 and SiNET6) were separated into different subtypes. We would like to clarify that this is not the case, since SiNET6 was not included in the subtype analysis due to too few detected Neuroendocrine cells, and was not assigned to any subtype, as noted in the text and as can be seen by its exclusion from Figure 2 where subtypes are defined. We apologize that our manuscript may have gave the wrong impression about SiNET6 classification (it is labeled in Fig. 4a in a misleading manner). In the revised manuscript, we will correct the labeling in Fig. 4a and clarify that SiNET is not assigned to any subtype. We will further acknowledge the limitation of the two platforms and the arguments in favor of the existence of two SiNET subtypes.

(2) Results:<br /> Heterogeneity in the SiNET tumor microenvironment: It is unclear if the current analysis of intratumor heterogeneity distinguishes the subtypes. It may be informative if patterns of tumor microenvironment (TME) heterogeneity were identified between samples of the same subtype. The team could also evaluate this in an extension cohort of published SiNET tumors (i.e. revisiting additional analyses using the SiNET bulk RNAseq from Alvarez et al 2018, a subset of single-cell data from Hoffman et al 2023, or additional bulk RNAseq validation cohorts for this cancer type if they exist [if they do not, then this could be mentioned as a need in Discussion])

We agree that analysis of an independent cohort will assist in defining the association between TME and the SiNET subtype. However, the sample size required for that is significantly larger than the data available. In the revised manuscript we will note that as a direction for future studies.

(3) Proliferation of NE and immune cells in SiNETs: The observed proliferation of NE and immune cells in SiNETs may also be influenced by technical factors (including those noted above). For instance, prior studies have shown that scRNA-seq tends to capture a higher proportion of immune cells compared to snRNA-seq, which should be considered in the interpretation of these results. Could the team clarify this element?

We agree that different platforms could affect the observed proportions of immune cells, and more generally the proportions of specific cell types. However, the low proliferation of Neuroendocrine cells and the higher proliferation of immune cells (especially B cells, but also T cells and macrophages) is consistently observed in both platforms, as shown in Fig. 4a, and therefore appears to be reliable despite the limitations of our work. We will clarify this consistency in the revised manuscript. 

(4) Putative progenitors in mixed tumors: As written, the identification of putative progenitors in a single lung MiNEN sample feels somewhat disconnected from the rest of the study. These findings are interesting - are similar progenitor cell populations identified in SiNET samples? Recognizing that ideally additional validation is needed to confidently label and characterize these cells beyond gene expression data in this rare tumor, this limitation could be addressed in a revised Discussion.

We agree with this comment and will add the need for additional validation for this finding in the revised Discussion.

Reviewer #2 (Public review):

Summary:

The research identifies two main SiNET subtypes (epithelial-like and neuronal-like) and reveals heterogeneity in non-neuroendocrine cells within the tumor microenvironment. The study validates findings using external datasets and explores unexpected proliferation patterns. While it contributes to understanding SiNET oncogenic processes, the limited sample size and depth of analysis present challenges to the robustness of the conclusions.

Strengths:

The studies effectively identified two subtypes of SiNET based on epithelial and neuronal markers. Key findings include the low proliferation rates of neuroendocrine (NE) cells and the role of the tumor microenvironment (TME), such as the impact of Macrophage Migration Inhibitory Factor (MIF).

Weaknesses:

However, the analysis faces challenges such as a small sample size, lack of clear biological interpretation in some analyses, and concerns about batch effects and statistical significance.

Reviewer #3 (Public review):

Summary:

In this study, the authors set out to profile small intestine neuroendocrine tumors (siNETs) using single-cell/nucleus RNA sequencing, an established method to characterize the diversity of cell types and states in a tumor. Leveraging this dataset, they identified distinct malignant subtypes (epithelial-like versus neuronal-like) and characterized the proliferative index of malignant neuroendocrine cells versus non-malignant microenvironment cells. They found that malignant neuroendocrine cells were far less proliferative than some of their non-malignant counterparts (e.g., B cells, plasma cells, epithelial cells) and there was a strong subtype association such that epithelial-like siNETs were linked to high B/plasma cell proliferation, potentially mediated by MIF signaling, whereas neuronal-like siNETs were correlated with low B/plasma cell proliferation. The authors also examined a single case of a mixed lung tumor (neuroendocrine and squamous) and found evidence of intermediate/mixed and stem-like progenitor states that suggest the two differentiated tumor types may arise from the same progenitor.

Strengths:

The strengths of the paper include the unique dataset, which is the largest to date for siNETs, and the potentially clinically relevant hypotheses generated by their analysis of the data.

Weaknesses:

The weaknesses of the paper include the relatively small number of independent patients (n = 8 for siNETs), lack of direct comparison to other published single-cell NET datasets, mixing of two distinct methods (single-cell and single-nucleus RNA-seq), lack of direct cell-cell interaction analyses and spatially-resolved data, and lack of in vitro or in vivo functional validation of their findings.

The analytical methods applied in this study appear to be appropriate, but the methods used are fairly standard to the field of single-cell omics without significant methodological innovation. As the authors bring forth in the Discussion, the results of the study do raise several compelling questions related to the possibility of distinct biology underlying the epithelial-like and neuronal-like subtypes, the origin of mixed tumors, drivers of proliferation, and microenvironmental heterogeneity. However, this study was not able to further explore these questions through spatially-resolved data or functional experiments.

RIGHTTT

TRUEEE

Het known-groups comparison design is lastig intern te controleren om de volgende redenen:

Geen willekeurige toewijzing: In tegenstelling tot simulatiestudies worden de groepen in een known-groups comparison niet willekeurig toegewezen aan experimentele condities. In plaats daarvan worden deelnemers geselecteerd op basis van reeds bestaande groepen, zoals mensen die als malingerers of als mensen met een echte stoornis zijn geclassificeerd. Omdat onderzoekers geen controle hebben over welke mensen in welke groep terechtkomen, hebben ze minder invloed op de omstandigheden waarin die groepen worden bestudeerd.

Afhankelijk van bestaande klinische of forensische data: Dit ontwerp vertrouwt sterk op de classificaties die eerder door deskundigen zijn gemaakt, zoals het identificeren van malingerers of mensen met authentieke stoornissen. Als deze initiële classificaties onjuist of onnauwkeurig zijn, kan dat de interne validiteit van de studie aantasten. De onderzoekers hebben weinig controle over hoe deze groepen in het verleden zijn vastgesteld, wat kan leiden tot fouten in de groepstoewijzing.

Beperkte controle over variabelen: Omdat het onderzoek plaatsvindt in een real-world setting (zoals een kliniek of rechtbank), is er minder controle over andere factoren die invloed kunnen hebben op het gedrag van de deelnemers. Dit kunnen variabelen zijn zoals de ernst van de stoornis, persoonlijke omstandigheden of de specifieke juridische situatie. Deze variabelen kunnen moeilijk worden gestandaardiseerd in een known-groups comparison, wat de controle en nauwkeurigheid van het onderzoek beïnvloedt.

Geen manipulatiecontroles: In simulatiestudies kunnen onderzoekers manipulatiecontroles gebruiken om te controleren of deelnemers zich op de juiste manier aan de condities houden (bijvoorbeeld of ze daadwerkelijk proberen te simuleren). In een known-groups comparison is dit niet mogelijk, omdat de groepen op basis van bestaande kenmerken worden vergeleken en er geen directe experimentele manipulatie plaatsvindt.

Zwakke externe validiteit. Mensen worden aangewezen en geïnstrueerd om te gaan malingeren, maar je weet niet of ze minder hun best doen dan dat ze doen in real life als ze bijvoorbeeld zouden malingeren. De omstandigheden waarin deelnemers zich bevinden zijn kunstmatig en missen vaak de emotionele, juridische of financiële druk die mensen in de werkelijkheid kunnen ervaren. In echte situaties, zoals in een rechtszaak of bij een klinisch consult, zijn de gevolgen van malingering vaak veel serieuzer, wat het gedrag van mensen beïnvloedt.

Dit betekent dat bij malingering (opzettelijk simuleren of overdrijven van symptomen), slechts een klein deel van het gedrag zichtbaar is, terwijl het grootste deel verborgen blijft.

Feigning is het opzettelijk fabriceren of overdrijven van symptomen, zonder aannames te doen over de motieven van de persoon. Psychologische tests kunnen worden gebruikt om feigning te identificeren.

Veelgemaakte fout: Te specifiek beschrijven van hoe iemand reageert, zelfs als de gegevens niet duidelijk of tegenstrijdig zijn. Aanbeveling: Werk in twee stappen: eerst algemeen, dan specifiek. Begin met een algemene beschrijving (bijv. "onbetrouwbare bron"). Als dat klopt, kijk dan of er genoeg gegevens zijn om meer gedetailleerd te worden.

Reviewer #1 (Public review):

Summary:

This study evaluates whether species can shift geographically, temporally, or both ways in response to climate change. It also teases out the relative importance of geographic context, temperature variability, and functional traits in predicting the shifts. The study system is large occurrence datasets for dragonflies and damselflies split between two time periods and two continents. Results indicate that more species exhibited both shifts than one or the other or neither, and that geographic context and temp variability were more influential than traits. The results have implications for future analyses (e.g. incorporating habitat availability) and for choosing winner and loser species under climate change. The methodology would be useful for other taxa and study regions with strong community/citizen science and extensive occurrence data.

Strengths:

This is an organized and well-written paper that builds on a popular topic and moves it forward. It has the right idea and approach, and the results are useful answers to the predictions and for conservation planning (i.e. identifying climate winners and losers). There is technical proficiency and analytical rigor driven by an understanding of the data and its limitations.

Weaknesses:

(1) The habitat classifications (Table S3) are often wrong. "Both" is overused. In North America, for example, Anax junius, Cordulia shurtleffii, Epitheca cynosura, Erythemis simplicicollis, Libellula pulchella, Pachydiplax longipennis, Pantala flavescens, Perithemis tenera, Ischnura posita, the Lestes species, and several Enallagma species are not lotic breeding. These species rarely occur let alone successfully reproduce at lotic sites. Other species are arguably "both", like Rhionaeschna multicolor which is mostly lentic. Not saying this would have altered the conclusions, but it may have exacerbated the weak trait effects.

(2) The conservative spatial resolution (100 x 100 km) limits the analysis to wide-ranging and generalist species. There's no rationale given, so not sure if this was by design or necessity, but it limits the number of analyzable species and potentially changes the inference.

(3) The objective includes a prediction about generalists vs specialists (L99-103) yet there is no further mention of this dichotomy in the abstract, methods, results, or discussion.

(4) Key references were overlooked or dismissed, like in the new edition of Dragonflies & Damselflies model organisms book, especially chapters 24 and 27.

Reviewer #2 (Public review):

Summary:

This paper explores a highly interesting question regarding how species migration success relates to phenology shifts, and it finds a positive relationship. The findings are significant, and the strength of the evidence is solid. However, there are substantial issues with the writing, presentation, and analyses that need to be addressed. First, I disagree with the conclusion that species that don't migrate are "losers" - some species might not migrate simply because they have broad climatic niches and are less sensitive to climate change. Second, the results concerning species' southern range limits could provide valuable insights. These could be used to assess whether sampling bias has influenced the results. If species are truly migrating, we should observe northward shifts in their southern range limits. However, if this is an artifact of increased sampling over time, we would expect broader distributions both north and south. Finally, Figure 1 is missed panel B, which needs to be addressed.

Reviewer #3 (Public review):

Summary:

In their article "Range geographies, not functional traits, explain convergent range and phenology shifts under climate change," the authors rigorously investigate the temporal shifts in odonate species and their potential predictors. Specifically, they examine whether species shift their geographic ranges poleward or alter their phenology to avoid extreme conditions. Leveraging opportunistic observations of European and North American odonates, they find that species showing significant range shifts also exhibited earlier phenological shifts. Considering a broad range of potential predictors, their results reveal that geographical factors, but not functional traits, are associated with these shifts.

Strengths:

The article addresses an important topic in ecology and conservation that is particularly timely in the face of reports of substantial insect declines in North America and Europe over the past decades. Through data integration the authors leverage the rich natural history record for odonates, broadening the taxonomic scope of analyses of temporal trends in phenology and distribution to this taxon. The combination of phenological and range shifts in one framework presents an elegant way to reconcile previous findings improving our understanding of the drivers of biodiversity loss.

Weaknesses:

The introduction and discussion of the article would benefit from a stronger contextualization of recent studies on biological responses to climate change and the underpinning mechanism.

The presentation of the results (particularly in figures) should be improved to address the integrative character of the work and help readers extract the main results. While the writing of the article is generally good, particularly the captions and results contain many inconsistencies and lack important detail. With the multitude of the relationships that were tested (the influence of traits) the article needs more coherence.

Primordialism is the idea that nations or ethnic identities are fixed, natural, and ancient.

eLife Assessment

The study by Chen and Phillips provides evidence for a dynamic switch in the small RNA repertoire of the Argonaute protein NRDE-3 during embryogenesis in C. elegans. The work is supported by solid experimental data, although some conclusions regarding the functional role of specific RNA granules remain uncertain. Nevertheless, this study offers valuable insights into RNA regulation and developmental biology, with broader implications for understanding small RNA pathways in other systems.

Reviewer #1 (Public review):

Summary:

Chen and Phillips describe the dynamic appearance of cytoplasmic granules during embryogenesis analogous to SIMR germ granules, and distinct from CSR-1-containing granules, in the C. elegans germline. They show that the nuclear Argonaute NRDE-3, when mutated to abrogate small RNA binding, or in specific genetic mutants, partially colocalizes to these granules along with other RNAi factors, such as SIMR-1, ENRI-2, RDE-3, and RRF-1. Furthermore, NRDE-3 RIP-seq analysis in early vs. late embryos is used to conclude that NRDE-3 binds CSR-1-dependent 22G RNAs in early embryos and ERGO-1-dependent 22G RNAs in late embryos. These data lead to their model that NRDE-3 undergoes small RNA substrate "switching" that occurs in these embryonic SIMR granules and functions to silence two distinct sets of target transcripts - maternal, CSR-1 targeted mRNAs in early embryos and duplicated genes and repeat elements in late embryos.

Strengths:

The identification and function of small RNA-related granules during embryogenesis is a poorly understood area and this study will provide the impetus for future studies on the identification and potential functional compartmentalization of small RNA pathways and machinery during embryogenesis.

Weaknesses:

(1) While the authors acknowledge the following issue, their finding that loss of SIMR granules has no apparent impact on NRDE-3 small RNA loading puts the functional relevance of these structures into question. As they note in their Discussion, it is entirely possible that these embryonic granules may be "incidental condensates." It would be very welcomed if the authors could include some evidence that these SIMR granules have some function; for example, does the loss of these SIMR granules have an effect on CSR-1 targets in early embryos and ERGO-1-dependent targets in late embryos?

(2) The analysis of small RNA class "switching" requires some clarification. The authors re-define ERGO-1-dependent targets in this study to arrive at a very limited set of genes and their justification for doing this is not convincing. What happens if the published set of ERGO-1 targets is used? Further, the NRDE-3 RIP-seq data is used to conclude that NRDE-3 predominantly binds CSR-1 class 22G RNAs in early embryos, while ERGO-1-dependent 22G RNAs are enriched in late embryos. a) The relative ratios of each class of small RNAs are given in terms of unique targets. What is the total abundance of sequenced reads of each class in the NRDE-3 IPs? b) The "switching" model is problematic given that even in late embryos, the majority of 22G RNAs bound by NRDE-3 is in the CSR-1 class (Figure 5D). c) A major difference between NRDE-3 small RNA binding in eri-1 and simr-1 mutants appears to be that NRDE-3 robustly binds CSR-122G RNAs in eri-1 but not in simr-1 in late embryos. This result should be better discussed.

(3) Ultimately, if the switching is functionally important, then its impact should be observed in the expression of their targets. RNA-seq or RT-qPCR of select CSR-1 and ERGO-1 targets should be assessed in nrde-3 mutants during early vs late embryogenesis.

Reviewer #2 (Public review):

Summary:

NRDE-3 is a nuclear WAGO-clade Argonaute that, in somatic cells, binds small RNAs amplified in response to the ERGO-class 26G RNAs that target repetitive sequences. This manuscript reports that, in the germline and early embryos, NRDE-3 interacts with a different set of small RNAs that target mRNAs. This class of small RNAs was previously shown to bind to a different WAGO-clade Argonaute called CSR-1, which is cytoplasmic, unlike nuclear NRDE-3. The switch in NRDE-3 specificity parallels recent findings in Ascaris where the Ascaris NRDE homolog was shown to switch from sRNAs that target repetitive sequences to CSR-class sRNAs that target mRNAs.

The manuscript also correlates the change in NRDE-3 specificity with the appearance in embryos of cytoplasmic condensates that accumulate SIMR-1, a scaffolding protein that the authors previously implicated in sRNA loading for a different nuclear Argonaute HRDE-1. By analogy, and through a set of corelative evidence, the authors argue that SIMR foci arise in embryogenesis to facilitate the change in NRDE-3 small RNA repertoire. The paper presents lots of data that beautifully documents the appearance and composition of the embryonic SIMR-1 foci, including evidence that a mutated NRDE-3 that cannot bind sRNAs accumulates in SIMR-1 foci in a SIMR-1-dependent fashion.

Weaknesses:

The genetic evidence, however, does not support a requirement for SIMR-1 foci: the authors detected no defect in NRDE-3 sRNA loading in simr-1 mutants. Although the authors acknowledge this negative result in the discussion, they still argue for a model (Figure 7) that is not supported by genetic data. My main suggestion is that the authors give equal consideration to other models - see below for specifics.

Reviewer #3 (Public review):

Summary:

Chen and Phillips present intriguing work that extends our view on the C. elegans small RNA network significantly. While the precise findings are rather C. elegans specific there are also messages for the broader field, most notably the switching of small RNA populations bound to an argonaute, and RNA granules behavior depending on developmental stage. The work also starts to shed more light on the still poorly understood role of the CSR-1 argonaute protein and supports its role in the decay of maternal transcripts. Overall, the work is of excellent quality, and the messages have a significant impact.

Strengths:

Compelling evidence for major shift in activities of an argonaute protein during development, and implications for how small RNAs affect early development. Very balanced and thoughtful discussion.

Weaknesses:

Claims on col-localization of specific 'granules' are not well supported by quantitative data.

eLife Assessment

This study presents valuable findings on changes in neuronal alpha activity elicited by prolonged pain in healthy human participants. The evidence supporting the claims of the authors, however, is incomplete and would benefit from clarifications of analytical strategies, additional statistical analyses, and shaping of the interpretations. With the methodological and interpretative parts strengthened, the work will be of interest to neuroscientists investigating the brain mechanisms of pain to identify new approaches to pain treatment

Reviewer #1 (Public review):

Summary:

Furman et al. reanalyze data from a previous study and investigate alterations of peak alpha frequency (PAF) and alpha power (AP) in the context of prolonged pain with electroencephalography (EEG). Using two experimental pain models (phasic and capsaicin heat pain), they set out to clarify if previously reported changes in alpha activity in chronic pain can already be observed during prolonged pain in healthy human participants. They conclude that PAF is reliably slowed, and AP reliably decreased in response to prolonged pain. From the patterns of their findings, they furthermore deduce that AP changes indicate the presence of ongoing pain while PAF changes reflect pain-associated states like sensitization which can outlast ongoing pain percepts and indicate a potential for experiencing pain. Lastly, they conclude that the reported changes in alpha activity are likely due to specific power decreases in the faster alpha range between 10 and 12 Hz and discuss potential clinical implications of their findings in terms of risk biomarkers and early pain interventions.

Strengths:

The study focuses on a timely topic with potential implications for chronic pain diagnosis and treatment, an area that urgently needs new approaches. The addressed questions nicely build upon and extend the previous work of the authors. The analyzed data set is comprehensive including two different prolonged pain paradigms, two visits following the same experimental procedures, and a total sample size of n = 61 participants. Thereby, it enabled internal replications of findings across both paradigms and visits, which is important to confirm the consistency of findings.

Weaknesses:

One overarching difficulty is the high number of analyses presented by the authors. They were in part developed "on the go", are not always easy to follow, and sidetrack the reader from the main findings. Only a minor part of the analyses is described in the methods section, while many analyses are outlined within the results, the supplementary material, and/or figure legends. In addition, a range of purely descriptive findings are displayed. Overall, the manuscript would clearly benefit from a more streamlined and consistent presentation of the applied methods and results.

Concerning the main findings, the presented evidence for a slowing of PAF and a reduction of AP in the context of both phasic and capsaicin heat pain and across both visits is convincing. The location of the peak of the effect at left frontocentral areas, however, remains puzzling. The authors convincingly show that the effect cannot be explained by activity related to the pain rating procedure and provide evidence that an effect of the same direction can also observed at corresponding electrodes contralateral to pain stimulation. However, further reasons are not discussed.

The conclusion that PAF slowing might be more related to pain-associated states like sensitization rather than the presence of ongoing pain is deduced from a continued slowing of PAF after capsaicin-induced pain has subsided, while AP goes back to baseline values. Although this speculation is interesting, the readers should be aware that this dissociation was unexpected and resulted in changes in the main a-priori-defined statistical contrasts presented in the methods section. Further replications in future studies are needed to strengthen this finding.

The last conclusion made by the authors is that the observed changes in alpha activity are caused by specific changes in the faster alpha range and are the least convincing. If I understand correctly, the only presented statistical evidence corroborating this conclusion is based on the single selected electrode C3 shown in Figure 5 A, D, and E. With the remaining parts of Figure 5 and Figure 6, differences are discussed but Figures do not include statistical results. Unless the discussed findings are backed up more clearly, the degree of mechanistic conclusions concerning the 10-12 Hz power changes throughout the title, abstract, and main manuscript and in relation to the multiple oscillators model seems not justified.

Lastly, it is important to note that the current manuscript was published as a preprint in 2021. Thus, the cited literature still needs to be updated, and the present findings need to be integrated with the work published since. For example, a recent systematic review on potential M/EEG-based biomarkers of chronic pain (Zebhauser et al., 2023, Pain) revealed that previous evidence concerning changes of alpha activity in chronic pain is much less consistent than currently outlined in the manuscript.

Overall:

All in all, the presented findings extend previous knowledge concerning the role of alpha activity in pain and thus represent a valuable contribution towards a better understanding of the mechanisms of pain and potential new treatment targets.

Reviewer #2 (Public review):

Summary:

This study investigated the modulation of alpha oscillations, specifically peak alpha frequency (PAF) and alpha power, during prolonged pain. The findings suggest that the alpha rhythm consists of multiple, independent oscillators, and suggest that the modulation of a "fast" oscillator may represent a promising therapeutic target for ongoing pain management.

Strengths:

EEG data were collected from a relatively large sample of participants, and the experiment was conducted using two prolonged pain models - phasic heat pain and capsaicin heat pain - at two separate testing visits approximately 8 weeks apart. The study produced reliable results across different pain models and at different testing intervals.

Weaknesses:

There are discrepancies between the results and their interpretation, indicating a need for more appropriate data analyses. Additionally, the experimental design does not adequately control for the potential time effects, which cannot be ruled out as a confounding factor.

Reviewer #3 (Public review):

Summary:

Furman et al. investigated how exposure to prolonged pain impacts human alpha oscillations recorded by electroencephalography (EEG). Two experimental models of prolonged pain were employed in healthy participants, phasic heat pain (PHP) and capsaicin heat pain (CHP). 61 participants completed two identical study visits separated by at least 8 weeks. Peak alpha frequency was reliably slowed by exposure to prolonged pain, whereas overall alpha power was reliably reduced. Both effects appeared to reflect a specific decrease in higher frequency (10-12Hz) alpha activity. The authors suggest that slowing of alpha oscillations is a reliable neural correlate of pain exposure and that manipulation of alpha activity may hold promise for treating chronic pain.

Strengths:

The study uses a within-participants design to show that exposure to pain is associated with acute changes in both the power and frequency of alpha oscillations.

By employing two experimental models of pain exposure and two separate testing visits, the authors were able to show that the effects of pain exposure on alpha activity are replicable across models and time.

Rigorous analysis approaches are used throughout.

Weaknesses:

No a priori power analysis is presented and (due to exclusions) most of the analyses conducted included (sometimes considerably) fewer participants than the overall sample size.

It is not clear whether the power and frequency changes represent two sides of the same coin or whether they reflect distinct mechanisms. The authors suggest in the manuscript that both effects may be explained by decreased power in 'fast' (8-12 Hz) alpha activity, but at other times interpret the effects to potentially represent distinct mechanisms. It would be useful for the authors to further clarify their thoughts on this point.

The statistical significance of some of the effects was dependent on analysis choices such as the exact frequency range chosen to identify alpha peaks.

No control condition was used, and I was left wondering if the effects would be specific to painful stimuli, or would also see the same effects for pleasant or neutral somatosensory stimuli?

eLife Assessment

The study is a valuable addition to the field, showing how particulate matter may be acting via nociceptor neurons towards neutrophilic asthma exacerbations. The solid evidence for the role of a nociceptive pathway in model systems is relevant to human asthma in its current form but would be further strengthened by mechanistic insights. This would be particularly relevant to further translational research towards blocking the exacerbating effect of air pollution on asthma.

Reviewer #1 (Public review):

Summary:

In the presented study, the authors aim to explore the role of nociceptors in the fine particulate matter (FPM) mediated Asthma phenotype, using rodent models of allergic airway inflammation. This manuscript builds on previous studies, and identify transciptomic reprogramming and an increased sensitivity of the jugular nodose complex (JNC) neurons, one of the major sensory ganglion for the airways, on exposure to FPM along with Ova during the challenge phase. The authors then use OX-314 a selectively permeable form of lidocaine, and TRPV1 knockouts to demonstrate that nociceptor blocking can reduce airway inflammation in their experimental setup.

The authors further identify the presence of Gfra3 on the JNC neurons, a receptor for the protein Artemin, and demonstrate their sensitivity to Artmein as a ligand. They further show that alveolar macrophages release Artemin on exposure to FPM.

Strengths:

The study builds on results available from multiple previous work, and presents important results which allow insights into the mixed phenotypes of Asthma seen clinically. In addition, by identifying the role of nociceptors, they identify potential therapeutic targets which bear high translational potential.

Weaknesses:

While the results presented in the study are highly relevant, there is a need for further mechanistic dissection to allow better inferences. Currently certain results seem assocaitive. Also, certain visualisations and experimental protocols presented in the manuscript need careful assessment and interpretation.

While Asthma is a chronic disease, the presented results are particularly important to explore Asthma exacerbations in response to acute expsoure to air pollutants. This is relevant in today's age of increasing air pollution and increasing global travel.