(proposition)al = statement

the word proposition in propositional knowledge means that it is stating a statement with truth or that holds value.

(post)erior= after the fact

the word post in posterior knowledge means that it is knowledge after. meaning knowledge after it has been experienced.

prior = before

meaning that priori knowledge is based on an knowledge that has not been experienced.

Only when the woman follows these customs/rules will other people say she is humble around other's. Pretty much being a slave and fearing everyone is an ordinary life for a woman during this time.

The three customs are degrading and disgusting.

this explains the anxiety

Even if these two people fail to perform to the Holy expectations set in place, by having God punish them it indirectly authorizes whatever Priests that are still unpunished to do as they please, even if what they are doing is 'wrong'. Cynical as it may be, it must be possible for Priests to do bad things without divine intervention, much like how 'disordered existence' is present elsewhere in life.

Direct Christology starts here

To understand how education can be part of the solution, we need to look at how both families and schools have played a role in the gap between the educational outcomes of wealthy and low-income children.

Every time I see a graph and read about it in the section below, it makes me wonder. How would this graph look like today Would it grow? Would it shirk? Would the graph have a negative impact or a positive impact on the low-income community?

As I am reading it bothers me the way that when a person tries to accomplish something or make their dream come true. They always move the finish line to make them work harder. There are times when they add requirements that the person doesn't have.

While I was reading this text it made me think was there a race that had higher high school graduates? If so, what race? Why specifically that race?

This reading makes me think, was it difficult to attend college? What did the collage application process look like? Was there many majors to choose from? Or was there a specific major that the student had chosen?

Use this for checking if can scrape data off site

based of the yes and no, it seems like having the entire length of the object is not the most effective method of dimensioning. Best to dimension each different shape to avoid clutterness

it's interesting because when you think about gayness and church, you don't really think about self-acceptance in this way. usually around me, it's because the church wasn't able to approve this part of your identity that some friends left the institution.

it seems that the rules in the American Catholic Church are more liberal than in Europe or at least in France ? with many differents schools of thought. and way more easier to create your own. this particularity seems to have a specific role in the high number of "gay-identified churches" at that time, in comparaison with other countries for exemple.

I agreed. In the city I live, Marseille, young queer people from different religions or spiritualities created a collective named Prière Queer Marseille, with the intention of rethinking rituals. But also because they are religious people who didn't find enough space in queer network to live their religious beliefs.

it is the very first time i heard about "gay-identified churches". does a movement like that exist in France as well ?

It’s tough because school expects parents to engage, but for some families, it’s not that simple. I'm pretty lucky since my mom participated my school life until high school. But I understand when parents are too busy working to make a living or feel disconnected from the school system, the student can end up feeling unsupported or alone in managing school challenges.

interesting quote!! I think this separates the morals of individuals, inherently it ethically makes sense to do good here to help others when it cannot hurt you

guilt should not be the deciding factor though, or the driving force behind doing something morally "good"

then why are people so hesitant to do it

obligation is an interesting perspective as well

people are sometimes in their own world so much so that they don't realize how well off they are and how capable they are of helping others

at what point though is that money "earned" and at what point should it be donated? where is the line? Because people who work hard SHOULD be able to use their money

people tend to blame the government or higher power, when in reality everyone could be contributing a little bit

argument to support claim

"to tell a lie or lies", "to conceal, hide, to be silent about" Truth is often hidden from people, some people are afraid of the truth. people hide the truth to protect other from being hurt by it, or because they wouldn't be able to accept it

understanding it as unlawful acts don't affirm the the system of law, but rather the system of law exists because there is such thing as unlawful acts

so they are dependent on one another/do not exist without each other

suggests that punitive behaviour is only punitive because it occurs in a socially constructed reality of law

it sucks because most of the help they need is neglected by their parents by their parents because of they don't have time. Depending on families they are very firm with the idea that disorders don't exist so they don't seek that help. Kids tend to be more impatient because they know the parents won't care. Personality takes a big role in these house holds in which the kids tend to want to speak loud almost yelling, attitude, and the need to want to hit.

These are the most common that i know and i can say that i've seen and experienced. Emotionally and social challenges, i know it causes fear of speaking up or feeling comfortable without feeling judged. In a household of stress you get used to keeping your words to yourself. So when it comes to speaking up in a school environment we always feel bad that we're saying the wrong things.

It's always so shocking when people come to visit and see the reality of what it is to live in LA. Throughout the years it gets more and more expensive to live hear. Rent, utilities, necessities, EVERYTHING. It feels like even to breathe you have to pay for air. Especially in single family household you see how the parent who stays with the kids goes through it all alone.

This is one of the main forms that when i think of poverty, situational poverty comes to mind. It comes from environmental issues, divorce, and health problems. I think in my community growing up this one has always been the one i'm exposed to the moest. It's a sad reality and i'll say myself that i grew up with divorced parents the question would be based off rent, when we would start school who would buy what.

To me, poverty means someone from a low income community who basically lives by day and parents live check to check. This falls under trying to figure out and have the stress of how will i get the money to pay rent, we have to make sure this food lasts the whole week or, i wonder if we will have enough to eat tomorrow. I was raised in the environment of when i wanted things i understood that i would not be able to get it even if it wasn't expensive that money is money i knew my family needed. As a teacher i see that theres families who recently got a home after being homeless.

Answering the question i honestly don't know how i would fully feel with my kids being in their class just because of the effort being phrased in the reading. As much as people try to justify as to why they give better education to those with higher income because they deserve it, it's still not justifiable. The sympathy is true because we try to be understanding that they have so much going on at home.

I feel like with this mentality it makes everything harder to want to make an effort with the students. You're basically ready to give up and giving u7p pn your own class. I can say i speak for myself too because with my students i can have although day and i'm just so ready to go home sometimes but i will drain myself to make myself present as much as i can. Relating to wanting to give up is more of my mentality being ill just let them do whatever.

It is completely understandable for Hawkins to feel frustrated but it's hard being able to avoid these situations because of the low income situations. I know i can speak for myself sometimes i would have to go to school late because there would be no one to give me a ride. As much as i loved school, yes home issues do tend to interfere and as much as i would try to focus too. The inappropriate behavior as much as it's wrong it sucks that. it comes from lack of attention.

.

John Milton (1608-1674) Background Birth and Education: Born in London to a prosperous family; well-educated by tutors and at prestigious colleges (funded by his father). Early Aspirations: Planned to become an Anglican priest; a great believer in Cromwell and the Commonwealth. Personal Life: Married three times; briefly imprisoned after the monarchy was restored in 1660. Health: Became totally blind by 1652; died of kidney failure in 1674. Literary Contributions Poetic Works: Known for Latin elegies and English odes, considered models of poetic beauty. Philosophical Bridge: His works connect the Renaissance and the Reformation. Major Political Writer: Advocated for the English Revolution and defended the execution of Charles I. Paradise Lost Publication: Released in 1667; considered Milton's masterpiece. Form: Written in blank verse (Miltonic verse) – unrhymed iambic pentameter. Content: An epic poem that retells the story of Adam and Eve, beginning with themes from Genesis. Purpose: Aims to "justify the ways of God to men." Epic Poem Characteristics Long Narrative: Tells a comprehensive story involving significant themes. Warfare or Supernatural Elements: Engages with grand themes of conflict and divine intervention. In Medias Res: Begins in the midst of action rather than at the start. Cultural Ideals: Reflects and expresses the ideals and traditions of the time. Themes and Analysis Paradise Lost can be read as a representation of the failure of the English Revolution, with characters embodying Milton's critiques of the Anglican Church and exploring profound theological and philosophical questions. These notes summarize Milton's life, literary contributions, and the significance of Paradise Lost.

Historical Background Conflicts and Civil War King vs. Parliament: Growing tensions lead to the English Civil War (1642–1648). Key Events: Capture and beheading of Charles I. Establishment of the Commonwealth (1649). Oliver Cromwell: Puritan leader; ruled with authoritarianism, suppressing arts and leisure activities. Metaphysical Poets Definition and Characteristics Metaphysical Poets: A group of poets in 17th century England known for blending intellect and emotion. Key Traits: Use of metaphysical conceits: Extended, inventive metaphors. Simple language: Direct and clear expression. Rapid thought association: Engages the reader. Telescoping of images: Compact and layered imagery. Key Metaphysical Poets John Donne (1572-1631) Background: Inherited wealth; known for a lifestyle of pleasures. Secret marriage led to imprisonment; later took Anglican orders. Poetic Categories: Youthful Love Lyrics: Romantic themes. Sacred Verses: Reflections on spirituality after personal loss. Style: Frequent use of conceits and dramatic contrasts. Sharp, direct language; often concludes with surprising ideas. Flexible forms that adapt to themes. Major Work: "The Flea"

Key Questions: How does the flea symbolize seduction? Why is killing the flea considered "three sins in killing three"? How does the speaker respond to the lady after the flea's death? How might the lady argue against the speaker's logic? Andrew Marvell (1621-1678) Background: Politically active; wrote pamphlets and satires. Friend of John Milton; known for a controversial, often conflicted character. Notable Work: "To His Coy Mistress" Themes: Carpe diem: Urgency of love and time. Structure: IF: Idealized love without time constraints. BUT: Reality of time's passage. SO: Urges immediate action in love. Key Questions:

What is the speaker's goal in the poem? Is he proposing marriage or simply urging for intimacy? What is the nature of his argument? How logical and effective is his reasoning? New Criticism and Metaphysical Poets Reevaluation: New Criticism revisits these poets 300 years later. T.S. Eliot's View: Describes metaphysical poets as intellectual, combining disparate ideas into cohesive works. Distinction between intellectual poets and reflective poets, emphasizing depth of thought.

John Donne Metaphysical Poetry: Donne is a key figure in metaphysical poetry, known for his use of metaphysical conceits—extended metaphors that draw surprising connections between unlike objects.

"Death Be Not Proud": This poem personifies Death, arguing against its power and instilling a sense of defiance towards mortality. Read here. Analysis.

"The Flea": In this seduction poem, the flea becomes a metaphor for the speaker's argument that the act of love is trivialized by its connection to death. The speaker argues that killing the flea would be equivalent to committing multiple sins, referencing the idea of three lives in one. After the flea is killed, the speaker shifts tactics, trying to convince the woman that her honor is unaffected. Read here. Analysis.

Andrew Marvell "To His Coy Mistress": This carpe diem poem presents a logical progression in three stanzas: a hypothetical "If," a contrasting "But," and a conclusion "So." Marvell explores themes of time and beauty, urging his mistress to seize the moment of love. Read here. Analysis.

John Milton Paradise Lost: Considered one of the greatest epic poems, it explores themes of disobedience, free will, and redemption. Milton's portrayal of Satan as the epic hero challenges traditional notions of heroism. The poem opens with a focus on humanity's fall and Milton's intent to "justify the ways of God to men." Read the excerpt here. Analysis.

These readings collectively offer insights into the themes of love, mortality, and the human condition, characteristic of 17th-century poetry.

You said: make notes ChatGPT said: ChatGPT Here are concise notes on each of the key readings and poets:

John Donne Metaphysical Poetry Founder of metaphysical poetry. Known for metaphysical conceits (extended, surprising metaphors). "Death Be Not Proud" Theme: Defiance against Death. Personification: Death is addressed directly, diminishing its power. Analysis: Challenges the fear of mortality. Read here. More analysis. "The Flea" Theme: Seduction and persuasion. Conceit: The flea symbolizes the union of the lovers' blood. Religious references: Killing the flea equates to three sins (symbolizing the Trinity). Shift in argument: After the flea is killed, the speaker argues that her honor remains intact. Read here. More analysis. Andrew Marvell "To His Coy Mistress" Theme: Carpe diem (seize the day). Structure: Stanza 1: "If" (idealized love). Stanza 2: "But" (the inevitability of time). Stanza 3: "So" (urge to act). Imagery: Explores themes of time and beauty. Argument: Urges the mistress to embrace love now. Read here. More analysis. John Milton Paradise Lost Epic poem: Focuses on Adam and Eve's disobedience. Satan as hero: Challenges traditional heroism. Themes: Free will, redemption, and the nature of evil. Purpose: To "justify the ways of God to men." Opening lines: Discuss the fall of man and the consequences of disobedience.

religion not entirely derived from economic structures- sometimes the inverse occurs

ethicality in the supernatural

rationalization of religiosity parallel to urban development

at this stage priesthood is utilized to prevent demons and ill omens

transcendence of farming leads to questions of meaning of life --> toward religion

middle and low class depends on rational work/labor that contrasts with existence of supernatural events- wtf was he talking ab previously

hierocracy crucial to caesaropapist andpersonally charismatic leader to offer legitimacy to political system

strongest arm of hierocratic development is theological thought and training

in tensions between political and hierocratic power- rarely a simple solution involving the victory of one or the other

rulers can use monks to influence masses but clash inevitably when rulers ask them to intervene in religious affairs

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

eLife Assessment

This important study explores the neural basis for a well known auditory illusion, often utilized in movie soundtracks, in which a sequence of two complex tones can be perceived as either rising or falling in pitch depending on the context in which they are presented. Convincing single-neuron data and analyses are presented to show that correlates of these pitch-direction changes are found in the ferret primary auditory cortex. While these findings provide an interesting link between cortical activity and perception, the manuscript could be clearer on the wider implications of the failure of traditional decoding models to account for these results.

Reviewer #1 (Public review):

Summary:

Previous work demonstrated a strong bias in the percept of an ambiguous Shepard tone as either ascending or descending in pitch, depending on the preceding contextual stimulus. The authors recorded human MEG and ferret A1 single-unit activity during presentation of stimuli identical to those used in the behavioral studies. They used multiple neural decoding methods to test if context-dependent neural responses to ambiguous stimulus replicated the behavioral results. Strikingly, a decoder trained to report stimulus pitch produced biases opposite to the perceptual reports. These biases could be explained robustly by a feed-forward adaptation model. Instead, a decoder that took into account direction selectivity of neurons in the population was able to replicate the change in perceptual bias.

Strengths:

This study explores an interesting and important link between neural activity and sensory percepts, and it demonstrates convincingly that traditional neural decoding models cannot explain percepts. Experimental design and data collection appear to have been executed carefully. Subsequent analysis and modeling appear rigorous. The conclusion that traditional decoding models cannot explain the contextual effects on percepts is quite strong.

Weaknesses:

Beyond the very convincing negative results, it is less clear exactly what the conclusion is or what readers should take away from this study. The presentation of the alternative, "direction aware" models is unclear, making it difficult to determine if they are presented as realistic possibilities or simply novel concepts. Does this study make predictions about how information from auditory cortex must be read out by downstream areas? There are several places where the thinking of the authors should be clarified, in particular, around how this idea of specialized readout of direction-selective neurons should be integrated with a broader understanding of auditory cortex.

Reviewer #2 (Public review):

Summary:

This is an elegant study investigating possible mechanisms underlying the hysteresis effect in the perception of perceptually ambiguous Shepard tones. The authors make a fairly convincing case that the adaptation of pitch direction sensitive cells in auditory cortex is likely responsible for this phenomenon.

Strengths:

The manuscript is overall well written. My only slight criticism is that, in places, particularly for non-expert readers, it might be helpful to work a little bit more methods detail into the results section, so readers don't have to work quite so hard jumping from results to methods and back.

The methods seem sound and the conclusions warranted and carefully stated. Overall I would rate the quality of this study as very high, and I do not have any major issues to raise.

Weaknesses:

I think this study is about as good as it can be with the current state of the art. Generally speaking, one has to bear in mind that this is an observational, rather than an interventional study, and therefore only able to identify plausible candidate mechanisms rather than making definitive identifications. However, the study nevertheless represents a significant advance over the current state of knowledge, and about as good as it can be with the techniques that are currently widely available.

Author response:

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

Reviewer #1 (Public Review):

Summary:

Previous work demonstrated a strong bias in the percept of an ambiguous Shepard tone as either ascending or descending in pitch, depending on the preceding contextual stimulus. The authors recorded human MEG and ferret A1 single-unit activity during presentation of stimuli identical to those used in the behavioral studies. They used multiple neural decoding methods to test if context-dependent neural responses to ambiguous stimulus replicated the behavioral results. Strikingly, a decoder trained to report stimulus pitch produced biases opposite to the perceptual reports. These biases could be explained robustly by a feed-forward adaptation model. Instead, a decoder that took into account direction selectivity of neurons in the population was able to replicate the change in perceptual bias.

Strengths:

This study explores an interesting and important link between neural activity and sensory percepts, and it demonstrates convincingly that traditional neural decoding models cannot explain percepts. Experimental design and data collection appear to have been executed carefully. Subsequent analysis and modeling appear rigorous. The conclusion that traditional decoding models cannot explain the contextual effects on percepts is quite strong.

Weaknesses:

Beyond the very convincing negative results, it is less clear exactly what the conclusion is or what readers should take away from this study. The presentation of the alternative, "direction aware" models is unclear, making it difficult to determine if they are presented as realistic possibilities or simply novel concepts. Does this study make predictions about how information from auditory cortex must be read out by downstream areas? There are several places where the thinking of the authors should be clarified, in particular, around how this idea of specialized readout of direction-selective neurons should be integrated with a broader understanding of auditory cortex.

While we have not used the term "direction aware", we think the reviewer refers generally to the capability of our model to use a cell's direction selectivity in the decoding. In accordance with the reviewer's interpretation, we did indeed mean that the decoder assumes that a neuron does not only have a preferred frequency, but also a preferred direction of change in frequency (ascending/descending), which is what we use to demonstrate that the decoding in this way aligns with the human percept. We have adapted the text in several places to clarify this, in particular expanding the description in the Methods substantially.

Reviewer #2 (Public Review):

The authors aim to better understand the neural responses to Shepard tones in auditory cortex. This is an interesting question as Shepard tones can evoke an ambiguous pitch that is manipulated by a proceeding adapting stimulus, therefore it nicely disentangles pitch perception from simple stimulus acoustics.

The authors use a combination of computational modelling, ferret A1 recordings of single neurons, and human EEG measurements.

Their results provide new insights into neural correlates of these stimuli. However, the manuscript submitted is poorly organized, to the point where it is near impossible to review. We have provided Major Concerns below. We will only be able to understand and critique the manuscript fully after these issues have been addressed to improve the readability of the manuscript. Therefore, we have not yet reviewed the Discussion section.

Major concerns

Organization/presentation

The manuscript is disorganized and therefore difficult to follow. The biggest issue is that in many figures, the figure subpanels often do not correspond to the legend, the main body, or both. Subpanels described in the text are missing in several cases.

We have gone linearly through the text and checked that all figure subpanels are referred to in the text and the legend. As far as we can tell, this was already the case for all panels, with the exception of two subpanels of Fig. 5.

Many figure axes are unlabelled.

We have carefully checked the axes of all panels and all but two (Fig. 5D) were labeled. As is customary, certain panels inherit the axis label from a neighboring panel, if the label is the same, e.g. subpanels in Fig. 6F or Fig. 5E, which helps to declutter the figure. We hope that with this clarification, the reviewer can understand the labels of each panel.

There is an inconsistent style of in-text citation between figures and the main text. The manuscript contains typos and grammatical errors. My suggestions for edits below therefore should not be taken as an exhaustive list. I ask the authors to consider the following only a "first pass" review, and I will hopefully be able to think more deeply about the science in the second round of revisions after the manuscript is better organized.

While we are puzzled by the severity of issues that R2 indicates (see above, and R3 qualifies it as "well written", and R1 does not comment on the writing negatively), we have carefully gone through all specific issues mentioned by R2 and the other reviewers. We hope that the revised version of the paper with all corrections and clarifications made will resolve any remaining issues.

Frequency and pitch

The terms "frequency" and "pitch" seem to be used interchangeably at times, which can lead to major misconceptions in a manuscript on Shepard tones. It is possible that the authors confuse these concepts themselves at times (e.g. Fig 5), although this would be surprising given their expertise in this field. Please check through every use of "frequency" and "pitch" in this manuscript and make sure you are using the right term in the right place. In many places, "frequency" should actually be "fundamental frequency" to avoid misunderstanding.

Thanks for pointing this out. We have checked every occurrence and modified where necessary.

Insufficient detail or lack of clarity in descriptions

There seems to be insufficient information provided to evaluate parts of these analysis, most critically the final pitch-direction decoder (Fig 6), which is a major finding. Please clarify.

Thanks for pointing this out. We have extended the description of the pitch-direction decoder and highlighted its role for interpreting the results.

Reviewer #3 (Public Review):

Summary:

This is an elegant study investigating possible mechanisms underlying the hysteresis effect in the perception of perceptually ambiguous Shepard tones. The authors make a fairly convincing case that the adaptation of pitch direction sensitive cells in auditory cortex is likely responsible for this phenomenon.

Strengths:

The manuscript is overall well written. My only slight criticism is that, in places, particularly for non-expert readers, it might be helpful to work a little bit more methods detail into the results section, so readers don't have to work quite so hard jumping from results to methods and back.

Following this excellent suggestion, we have added more brief method sketches to the Results section, hopefully addressing this concern.

The methods seem sound and the conclusions warranted and carefully stated. Overall I would rate the quality of this study as very high, and I do not have any major issues to raise.

Thanks for your encouraging evaluation of the work.

Weaknesses:

I think this study is about as good as it can be with the current state of the art. Generally speaking, one has to bear in mind that this is an observational, rather than an interventional study, and therefore only able to identify plausible candidate mechanisms rather than making definitive identifications. However, the study nevertheless represents a significant advance over the current state of knowledge, and about as good as it can be with the techniques that are currently widely available.

Thanks for your encouraging evaluation of our work. The suggestion of an interventional study has also been on our minds, however, this appears rather difficult, as it would require a specific subset of cells to be inhibited. The most suitable approach would likely be 2p imaging with holographic inhibition of a subset of cells (using ArchT for example), that has a preference for one direction of pitch change, which should then bias the percept/behavior in the opposite direction.

Reviewer #1 (Recommendations For The Authors):

MAJOR CONCERNS

(1) What is the timescale used to compute direction selectivity in neural tuning? How does it compare to the timing of the Shepard tones? The basic idea of up versus down pitch is clear, the intuition for the role of direction tuning and its relation to stimulus dynamics could be laid out more clearly. Are the authors proposing that there are two "special" populations of A1 neurons that are treated differently to produce the biased percept? Or is there something specific about the dynamics of the Shepard stimuli and how direction selective neurons respond to them specifically? It would help if the authors could clarify if this result links to broader concepts of dynamic pitch coding in general or if the example reported here is specific (or idiosyncratic) to Shepard tones.

We propose that the findings here are not specific to Shepard tones. To the contrary, only basic properties of auditory cortex neurons, i.e. frequency preference, frequency-direction (i.e. ascending or descending) preference, and local adaptation in the tuning curve, suffice. Each of these properties have been demonstrated many times before and we only verified this in the lead-up to the results in Fig. 6. While the same effects should be observable with pure tones, the lack of ambiguity in the perception of direction of a frequency step for pure tone pairs, would make them less noticeable here. Regarding the time-scale of the directional selectivity, we relied on the sequencing of tones in our paradigm, i.e. 150 ms spacing. The SSTRFs were discretized at 50 ms, and include only the bins during the stimulus, not during the pause. The directional tuning, i.e. differences in the SSTRF above and below the preferred pitchclass for stimuli before the last stimulus, typically extended only one stimulus back in time. We have clarified this in more detail now, in particular in the added Methods section on the directional decoder.

(2) (p. 9) "weighted by each cell's directionality index ... (see Methods for details)" The direction-selective decoder is interesting and appears critical to the study. However, the details of its implementation are difficult to locate. Maybe Fig. 6A contains the key concepts? It would help greatly if the authors could describe it in parallel with the other decoders in the Methods.

We have expanded the description of the decoder in the Methods as the reviewer suggests.

LESSER CONCERNS

p. 1. (L 24) "distances between the pitch representations...." It's not obvious what "distances" means without reading the main paper. Can some other term or extra context be provided?

We have added a brief description here.

p. 2. (L 26) "Shepard tones" Can the authors provide a citation when they first introduce this class of stimuli?

Citation has been added.

p. 3 (L 4) "direction selective cells" Please define or provide context for what has a direction. Selective to pitch changes in time?

Yes, selective to pitch changes in time is what is meant. We have further clarified this in the text.

p. 4 (L 9-19). This paragraph seems like it belongs in the Introduction?

Given the concerns raised by R2 about the organization of the manuscript we prefer to keep this 'road-map' in the manuscript, as a guidance for the reader.

p. 4 (L 32) "majority of cells" One might imagine that the overlap of the bias band and the frequency tuning curve of individual neurons might vary substantially. Was there some criterion about the degree of overlap for including single units in the analysis? Does overlap matter?

We are not certain which analysis the reviewer is referring to. Generally, cells were not excluded based on their overlap between a particular Bias band and their (Shepard) tuning curve. There are several reasons for this: The bias was located in 4 different, overlapping Shepard tone regions, and all sounds were Shepard tones. Therefore, all cells overlapped with their (Shepard) tuning curve with one or multiple of the Biases. For decoding analysis, all cells were included as both a response and lack of a response is contributing to the decoding. If the reviewer is referring only to the analysis of whether a cell adapts, then the same argument applies as above, i.e. this was an average over all Bias sequences, and therefore every responding cell was driven to respond by the Bias, and therefore it was possible to also assess whether it adapted its response for different positions inside the Bias. We acknowledge that the limited randomness of the Bias sequences in combination with the specific tuning of the cells could in a few cases create response patterns over time that are not indicative of the actual behavior for repeated stimulation, however, since the results are rather clear with 91% of cells adapting, we do not think this would significantly change the conclusions.

p. 5 (L 17) "desynchronization ... behaving conditions" The logic here is not clear. Is less desynchronization expected during behavior? Typically, increased attention is associated with greater desynchronization.

Yes, we reformulated the sentence to: While this difference could be partly explained by desynchronization which is typically associated with active behavior or attention [30], general response adaptation to repeated stimuli is also typical in behaving humans [31].

p. 7 (L 5) "separation" is this a separation in time?

Yes, added.

p. 7 (L 33) "local adaptation" The idea of feedforward adaptation biasing encoding has been proposed before, and it might be worth citing previous work. This includes work from Nelken specifically related to SSA. Also, this model seems similar to the one described in Lopez Espejo et al (PLoS CB 2019).

Thanks for pointing this out. We think, however, that neither of these publications suggested this very narrow way of biasing, which we consider biologically implausible. We have therefore not added either of these citations.

p. 11 (L. 17) The cartoon in Fig. 6G may provide some intuition, but it is quite difficult to interpret. Is there a way to indicate which neuron "votes" for which percept?

This is an excellent idea, and we have added now the purported perceptual relation of each cell in the diagram.

p. 12 (L. 8). "classically assumed" This statement could benefit from a citation. Or maybe "classically" is not the right word?

We have changed 'classically' to 'typically', and now cite classical works from Deutsch and Repp. We think this description makes sense, as the whole concept of bistable percepts has been interpreted as being equidistant (in added or subtracted semitone steps) from the first tone, see e.g. Repp 1997, Fig.2.

p. 12 (L. 12) "...previous studies" of Shepard tone percepts? Of physiology?

We have modified it to 'Relation to previous studies of Shepard tone percepts and their underlying physiology", since this section deals with both.

p. 12 (L. 25) "compatible with cellular mechanisms..." This paragraph seems key to the study and to Major Concern 1, above. What are the dynamics of the task stimuli? How do they compare with the dynamics of neural FM tuning and previously reported studies of bias? And can the authors be more explicit in their interpretation - should direction selective neurons respond preferentially to the Shepard tone stimuli themselves? And/or is there a conceptual framework where the same neurons inform downstream percepts of both FM sweeps and both normal (unbiased) and biased Shepard tones?

The reviewer raises a number of different questions, which we address below:

- Dynamics of the task stimuli in relation to previously reported cellular biasing: The timescales tested in the studies mentioned are similar to what we used in our bias, e.g. Ye et al 2010 used FM sweeps that lasted for up to 200ms, which is quite comparable to our SOA of 150ms.

- Preferred responses to Shepard tones: no, we do not think that there should be preferred responses to Shepard tones, but rather that responses to Shepard tones can be thought of as the combined responses to the constituent tones.

- Conceptual framework where the same neurons inform about FM sweeps and both normal (unbiased) and biased Shepard tones: Our perspective on this question is as follows: To our knowledge, the classical approach to population decoding in the auditory system, i.e. weighted based on preferred frequency, has not been directly demonstrated to be read out inside the brain, and certainly not demonstrated to be read out in only this way in all areas of the brain that receive input from the auditory cortex. Rather it has achieved its credibility by being linked directly with animal performance or match with the presented stimuli. However, these approaches were usually geared towards a representation that can be estimated based on constituent frequencies. Additional response properties of neurons, such as directional selectivity have been documented and analyzed before, however, not been used for explaining the percept. We agree that our use of this cellular response preference in the decoding implicitly assumes that the brain could utilize this as well, however, this seems just as likely or unlikely as the use of the preferred frequency of a neuron. Therefore we do not think that this decoding is any more speculative than the classical decoding. In both cases, subsequent neurons would have to implicitly 'know' the preference of the input neuron, and weigh its input correspondingly.

We have added all the above considerations to the discussion in an abbreviated form.

p. 15 (L. 15). Is there a citation for the drive system?

There is no publication, but an old repository, where the files are available, which we cite now: https://code.google.com/archive/p/edds-array-drive/

p. 16 (L. 24) "position in an octave" It is implied but not explicitly stated that the Shepard tones don't contain the fundamental frequency. Can the authors clarify the relationship between the neural tuning band and the bands of the stimulus. Did a single stimulus band typically fall in a neuron's frequency tuning curve? If not 1, how many?

Yes, it is correct that the concept of fundamental frequency does not cleanly apply to Shepard tones, because it is composed of octave spaced pure tones, but the lowest tone is placed outside the hearing range of the animal and amplitude envelope (across frequencies). Therefore one or more constituent tones of the Shepard tone can fall into the tuning curve of a neuron and contribute to driving the neuron (or inhibiting it, if they fall within an inhibitory region of the tuning curve). The number of constituent tones that fall within the tuning curve depends on the tuning width of the neurons. The distribution of tuning widths to Shepard tones is shown in Fig. S1E, which indicated that a lot of neurons had rather narrow tuning (close to the center), but many were also tuned widely, indicated that they would be stimulated by multiple constituent tones of the Shepard tone. As the tuning bandwidth (Q30: 30dB above threshold) of most cortical neurons in the ferret auditory cortex (see e.g. Bizley et al. Cerebral Cortex, 2005, Fig.12) is below 1, this means that typically not more than 1 tone fell into the tuning curve of a neuron. However, we also observed multimodal tuning-curves w.r.t. to Shepard tones, which suggests that some neurons were stimulated by more than 2 or more constituent tones (again consistent with the existence of more broadly tuned neurons (see same citation). We have added this information partly to the manuscript in the caption of Fig. S1E.

p. 17 (L. 32). "Fig 4" Correct figure ref? This figure appears to be a schematic rather than one displaying data.

Thanks for pointing this out, changed to Fig. 5.

p. 18 (L. 25). "assign a pitchclass" Can the authors refer to a figure illustrating this process?

Added.

p. 19 (L. 17). Is mu the correct symbol?

Thanks. We changed it to phi_i, as in the formula above.

p. 19 (L 19). "convolution" in time? Frequency?

Thanks for pointing this out, the term convolution was incorrect in this context. We have replaced it by "weighted average" and also adapted and simplified the formula.

p. 19 (L 25) "SSTRF" this term is introduced before it is defined. Also it appears that "SSTRF" and "STRF" are sometimes interchanged.

Apologies, we have added the definition, and also checked its usage in each location.

p. 23 (Fig 2) There is a mismatch between panel labels in the figure and in the legend. Bottom right panel (B3), what does time refer to here?

Thanks for pointing these out, both fixed.

p. 24 (L 23) "shifts them away" away from what?

We have expanded the sentence to: "After the bias, the decoded pitchclass is shifted from their actual pitchclass away from the biased pitchclass range ... "

p. 25 (L 7) "individual properties" properties of individual subjects?

Thanks for pointing this out, the corresponding sentence has been clarified and citations added.

p. 26 (L 20) What is plotted in panel D? The average for all cells? What is n?

Yes, this is an average over cells, the number of cells has now been added to each panel.

p. 28 (L 3) How to apply the terms "right" "right" "middle" to the panel is not clear. Generally, this figure is quite dense and difficult to interpret.

We have changed the caption of Panel A and replaced the location terms with the symbols, which helps to directly relate them to the figure. We have considered different approaches of adding or removing content from the figure to help make it less dense, but that all did not seem to help. For lack of better options we have left it in its current form.

MINOR/TYPOS

p. 3 (L 1) "Stimulus Specific Adaptation" Capitalization seems unnecessary

Changed.

p. 4 (L 14) "Siple"

Corrected.

p. 9 (L 10) "an quantitatively"

Corrected

p. 9 (L 20) "directional ... direction ... directly ... directional" This is a bit confusing as directseems to mean several different things in its different usages.

We have gone through these sentences, and we think the terms are now more clearly used, especially since the term 'direction' occurs in several different forms, as it relates to different aspects (cells/percept/hypothesis). Unfortunately, some repetition is necessary to maintain clarity.

Reviewer #2 (Recommendations For The Authors):

Detailed critique

Stimuli

It would be very useful if the authors could provide demos of their stimuli on a website. Many readers will not be familiar with Shepard tones and the perceptual result of the acoustical descriptions are not intuitive. I ended up coding the stimuli myself to get some intuition for them.

We have created some sample tones and sequences and uploaded them with the revision as supplementary documents.

Abstract

P1 L27 'pitch and...selective cells' - The authors haven't provided sufficient controls to demonstrate that these are "pitch cells" or "selective" to pitch direction. They have only shown that they are sensitive to these properties in their stimuli. Controls would need to be included to ensure that the cells aren't simply responding to one frequency component in the complex sound, for example. This is not really critical to the overall findings, but the claim about pitch "selectivity" is not accurate.

Fair point. We have removed the word 'selective' in both occurrences.

Introduction

P2 L14-17: I do not follow the phonetic example provided. The authors state that the second syllable of /alga/ and /arda/ are physically identical, but how is this possible that ga = da? The acoustics are clearly different. More explanation is needed, or a correction.

Apologies for the slightly misleading description, it has now been corrected to be in line with the original reference.

P2,L26-27: Should the two uses of "frequency" be "F0" and "pitch" here? The tones are not separated in frequency by half and octave, but "separated in [F0]" by half an octave, correct? Their frequency ranges are largely overlapping. And the second 'frequency', which refers to the percept, should presumably be "pitch".

Indeed. This is now corrected.

P3 L2-6: Unclear at this point in the manuscript what is the difference between the 3 percepts mentioned: perceived pitch-change direction, Shepard tone pitches, and "their respective differences". (It becomes clear later, but clarification is needed here).

We have tried a few reformulations, however, it tends to overload the introduction with details. We believe it is preferable to present the gist of the results here, and present the complete details later in the MS.

P3 L6-7 What does it mean that the MEG and single unit results "align in direction and dynamics"? These are very different signals, so clarification is needed.

We have phrased the corresponding sentence more clearly.

Results

Throughout: Choose one of 'pitch class', 'pitchclass', or 'pitch-class' and use it consistently.

Done.

P4L12 - would be helpful at this point to define 'repulsive effect'

We have added another sentence to clarify this term.

P4, L14 "simple"

Done

P4, L12 - not clear here what "repulsive influence" means

See above.

P4, L17 - alternative to which explanation? Please clarify. In general, this paragraph is difficult to interpret because we do not yet have the details needed to understand the terms used and the results described. In my opinion, it would be better to omit this summary of the results at the very beginning, and instead reveal the findings as they come, when they can be fully explained to the Reader.

We agree, but we also believe that a rather general description here is useful for providing a roadmap to the results. However, we have added a half-sentence to clarify what is meant by alternative.

P4 L30 - text says that cells adapt in their onset, sustained and offset responses, but only data for onset responses are shown (I think - clarification needed for fig 2A2). Supp figure shows only 1 example cell of sustained and offset, and in fact there is no effect of adaptation in the sustained response shown there.

Regarding the effect of adaptation and whether it can be discerned from the supplementary figure: the shown responses are for 10 repetitions of one particular Bias sequence. Since the response of the cell will depend on its tuning and the specific sequence of the Shepard tones in this Bias, it is not possible to assess adaptation for a given cell. We assess the level of adaptation, by averaging all biases (similar to what is shown in Fig. 2A2) per cell, and then fit an exponential to it, separately by response type. The step direction of the exponential, relative to the spontaneous rate is then used to assess the kind of adaptation. The vast majority of cells show adaptation. We have added this information to the Methods of the manuscript.

P4, L32 - please state the statistical test and criterion (alpha) used to determine that 91% of cells decreased their responses throughout the Bias sequence. Was this specifically for onset responses?

Thanks for pointing this out, test and p-value added. Adaptation was observed for onset, sustained and offset responses, in all cases with the vast majority showing an adapting behavior, although the onset responses were adapting the most.

P4 L36 - "response strength is reduced locally". What does "locally" mean here? Nearby frequencies?

We have added a sentence here to clarify this question.

Figure 1 - this appears to be the wrong version of the figure, as it doesn't match the caption or results text. It's not possible to assess this figure until these things are fixed. Figure 1A schematic of definition of f(diff) does not correspond to legend definition.

As far as we can tell, it is all correct, only the resolution of the figure appears to be rather low. This has been improved now.

Fig 2 A2 - is this also onset responses only?

Yes, added to the caption.

Fig 2 A3 - add y-axis label. The authors are comparing a very wide octave band (5.5 octaves) to a much narrower band (0.5 octaves). Could this matter? Is there something special about the cut-off of 2.5 octaves in the 2 bands, or was this an arbitrary choice?

Interesting question.... essentially our stimulus design left us only with this choice, i.e. comparing the internal region of the bias with the boundary region of the bias, i.e. the test tones. The internal region just corresponds to the bias, which is 5 st wide, and therefore the range is here given as 2.5 st relative to its center, while the test tones are at the boundary, as they are 3 st from the center. The axis for the bias was mislabelled, and has now been corrected. The y-axis label is matched with the panel to the left, but has now been added to avoid any confusion.

Fig 2A4 - does not refer to ferret single unit data, as stated in the text (p5L8). Nor does supp Fig2, as stated. Also, the figure caption does not match the figure.

Apologies, this was an error in the code that led to this mislabelling. We have corrected the labels, which also added back the recovery from the Bias sequence in the new Panel A4.

P5 l9 - Figure 3 is not understandable at this point in the text, and should not be referred to here. There is a lot going on in Fig 3, and it isn't clear what you are referring to.

Removed.

P5 L12 - by Fig 2 B1, I assume you mean A4? Also, F2B1 shows only 1 subject, not 2.

Yes, mislabeled by mistake, and corrected now.

Fig2B2 -What is the y-axis?

Same as in the panel to its left, added for clarity.

Stimuli: why are tones presented at a faster rate to ferrets than to humans?

The main reason is that the response analysis in MEG requires more spacing in time than the neuronal analysis in the ferret brain.

P5 L6 - there is no Fig 5 D2? I don't think it is a good idea to get the reader to skip so far ahead in the figures at this stage anyway, even if such a figure existed. It is confusing to jump around the manuscript

Changed to 'see below'

P5 L8 - There is no Figure 2A4, so I don't know whether this time constant is accurate.

This was in reference to a panel that had been removed before, but we have added it back now.

P5 L16: "in humans appears to be more substantial (40%) than for the average single units under awake conditions". One cannot directly compare magnitude of effects in MEG and single unit signals in this way and assume it is due to behavioural state. You are comparing different measures of neural activity, averaged over vastly different numbers of numbers, and recorded from different species listening to different stimuli (presentation rates).

Yes, that's why the next sentence is: "However, comparisons between the level of adaptation in MEG and single neuron firing rates may be misleading, due to the differences in the signal measured and subsequent processing.", and all statements in the preceding sentences are phrased as 'appears' and 'may'. We think we have formulated this comparison with an appropriate level of uncertainty. Further, the main message here is that adaptation is taking place in both active and passive conditions.

P5 L25 -I do not see any evidence regarding tuning widths in Fig s2, as stated in the text.

Corrected to Fig. S1.

P5 l26 - Do not skip ahead to Fig 5 here. We aren't ready to process that yet.

OK, reference removed.

P5 l27 - Do you mean because it could be tuning to pitch chroma, not height?

Yes, that is a possible interpretation, although it could also arise from a combination of excitatory and inhibitory contributions across multiple octaves.

P5 l33 - remove speculation about active vs passive for reasons given above.

Removed.

P6L2-6 'In the present...5 semitone step' - This is an incorrect interpretation of the minimal distance hypothesis in the context of the Shepard tone ambiguity. The percept is ambiguous because the 'true' F0 of the Shepard tones are imperceptibly low. Each constituent frequency of a single tone can therefore be perceived either as a harmonic of some lower fundamental frequency or as an independent tone. The dominant pitch of the second tone in the tritone pair may therefore be biased to be perceived at a lower constituent frequency (when the bias sequence is low) or at a higher constituent frequency (when the bias sequence is high). The text states that the minimal distance hypothesis would predict that an up-bias would make a tritone into a perfect fourth (5 semitones). This is incorrect. The MDH would predict that an up-bias would reduce the distance between the 1st tone in the ambiguous pair and the upper constituent frequency of the 2nd tone in the pair, hence making the upper constituent frequency the dominant pitch percept of the 2nd tone, causing an ascending percept.

The reviewer here refers to a “minimal distance hypothesis”, which without a literature reference,is hard for us to fully interpret. However, some responses are given below:

- "The percept is ambiguous because the 'true' F0 of the Shepard tones are imperceptibly low." This statement appears to be based on some misconception: due to the octave spacing (rather than multiple/harmonics of a lowest frequency), the Shepard tones cannot be interpreted as usual harmonic tones would be. It is correct that the lowest tone in a Shepard tone is not audible, due to the envelope and the fact that it could in principle be arbitrarily small... hence, speaking about an F0 is really not well-defined in the case of a Shepard tone. The closest one could get to it would be to refer to the Shepard tone that is both in the audible range and in the non-zero amplitude envelope. But again, since the envelope is fading out the highest and lowest constituent tones, it is not as easy to refer to the lowest one as F0 (as it might be much quieter than the next higher constituent.

- "The dominant pitch of the second tone in the tritone pair may therefore be biased to be perceived at a lower constituent frequency (when the bias sequence is low) or at a higher constituent frequency (when the bias sequence is high)." This may relate to some known psychophysics, but we are unable to interpret it with certainty.

- "The text states that the minimal distance hypothesis would predict that an up-bias would make a tritone into a perfect fourth (5 semitones). This is incorrect." We are unsure how the reviewer reaches this conclusion.

- "The MDH would predict that an up-bias would reduce the distance between the 1st tone in the ambiguous pair and the upper constituent frequency of the 2nd tone in the pair, hence making the upper constituent frequency the dominant pitch percept of the 2nd tone, causing an ascending percept." Again, in the absence of a reference to the MDH, we are unsure of the implied rationale. We agree that this is a possible interpretation of distance, however, we believe that our interpretation of distance (i.e. distances between constituent tones) is also a possible interpretation.

Fig 4: Given that it comes before Figure 3 in the results text, these should be switched in order in the paper.

Switched.

PCA decoder: The methods (p18) state that the PCA uses the first 3 dimensions, and that pitch classes are calculated from the closest 4 stimuli. The results (P6), however, state that the first 2 principal components are used, and classes are computed from the average of 10 adjacent points. Which is correct, or am I missing something?

Thanks for pointing this out, we have made this more concrete in the Methods to: "The data were projected to the first three dimensions, which represented the pitch class as well as the position in the sequence of stimuli (see Fig. 43A for a schematic). As the position in the Bias sequence was not relevant for the subsequent pitch class decoding, we only focussed on the two dimensions that spanned the pitch circle." Regarding the number of stimuli that were averaged: this might be a slight misunderstanding: Each Shepard tone was decoded/projected without averaging. However, to then assign an estimated pitch class, we first had to establish an axis (here going around the circle), where each position along the axis was associated with a pitch class. This was done by stepping in 0.5 semitone steps, and finding the location in decoded space that corresponded to the median of the Shepard tones within +/- 0.25st. To increase the resolution, this circular 'axis' of 24 points was then linearly interpolated to a resolution of 0.05st. We have updated the text in the Methods accordingly. The mentioning of 10 points for averaging in the Results was correct, as there were 240 tones in all bias stimuli, and 24 bins in the pitch circle. The mentioning of an average over 4 tones in the Methods was a typo.

Fig 3A: axes of pink plane should be PC not PCA

Done.

Fig 3B: the circularity in the distribution of these points is indeed interesting! But what do the authors make of the gap in the circle between semitones 6-7? Is this showing an inherent bias in the way the ambiguous tone is represented?

While we cannot be certain, we think that this represents an inhomogeneous sampling from the overall set of neural tuning preferences, and that if we had recorded more/all neurons, the circle would be complete and uniformly sampled (which it already nearly is, see Fig.4C, which used to be Fig. 3C).

Fig 3B (lesser note): It'd be preferable to replace the tint (bright vs. dark) differentiation of the triangles to be filled vs. unfilled because such a subtle change in tint is not easily differentiable from a change in hue (indicating a different variable in this plot) with this particular colour palette

We have experimented with this suggestion, and it didn't seem to improve the clarity. However, we have changed the outline of the test-pair triangles to white, which now visually separates them better.

P6 l32 - Please indicate if cross-validation was used in this decoder, and if so, what sort. Ideally, the authors would test on a held-out data set, or at least take a leave-one-out approach. Otherwise, the classifier may be overfit to the data, and overfitting would explain the exceptional performance (r=.995) of the classifier.

Cross-validation was not used, as the purpose of the decoder is here to create a standard against which to compare the biased responses in the ambiguous pair, which were not used for training of the decoder. We agree that if we instead used a cross-validated decoder (which would only apply to the local average to establish the pitch class circle) the correlation would be somewhat lower, however, this is less relevant for the main question, i.e. the influence of the Bias sequence on the neural representation of the ambiguous pair. We have added this information to the corresponding section.

Fig 3D: I understood that these pitch classifications shown by the triangles were carried out on the final ambiguous pair of stimuli. I thought these were always presented at the edges of the range of other stimuli, so I do not follow how they have so many different pitchclass values on the x-axis here.

There were 4 Biases, centered at 0,3,6 or 9 semitones, and covering [-2.5,2.5]st relative to this center. Therefore the edges of the bias ranges (3st away from their centers) happen to be the same as the centers, e.g. for the Bias centered at 3, the ambiguous pair would be a 0-6 or 6-0 step. Therefore there are 4 locations for the ambiguous tones on the x-axis of Fig. 4D (previously 3D).

Figure 4: This demonstration of the ambiguity of Shepard pairs may be misleading. The actual musical interval is never ambiguous, as this figure suggests. Only the ascending vs descending percept is ambiguous. Therefore the predictions of the ferret A1 decoding (Fig 3D) and the model in Fig 5 are inconsistent with perception in two ways. One (which the authors mention) is the direction of the bias shift (up vs down). Another (not mentioned here) is that one never experiences a shift in the shepard tone at a fraction of a semitone - the musical note stays the same, and changes only in pitch height, not pitch chroma.

We are unsure of the reviewer’s direction with this question. In particular the second point is not clear to us: "...one (who?) never (in this experiment? in real life?) experiences a bias shift in the Shepard tone at a fraction of a semitone" (why is this relevant in the current experiment?). Pitch chrome would actually be a possible replacement for pitch class, but somehow, the previous Shepard tone literature has referred to it as pitch class.

P7 l12 - omit one 'consequently'

Changed to 'Therefore'.

P7 l24 - I encourage the authors to not use "local" and "global" without making it clear what space they refer to. One tends to automatically think of frequency space in the auditory system, but I think here they mean f0 space? What is a "cell close to the location of the bias"? Cells reside in the brain. The bias is in f0 space. The use of "local" and "global" throughout the manuscript is too vague.

Agreed, the reference here was actually to the cell's preferred pitch class, not its physical location (which one might arguably be able to disambiguate, given the context). We have changed the wording, and also checked the use of global/local throughout the manuscript. The main use of 'global/local' is now in reference to the range of adaptation, and is properly introduced on first mention.

P7 L26 -there is no Fig 5D1. Do you mean the left panel of 5D?

Thanks. Changed.

FigS3 is referred to a lot on p7-8. Should this be moved to the main text?

The main reason why we kept it in the supplement is that it is based on a more static model, which is intended to illustrate the consequences of different encoding schemes. In order to not confuse the reader about these two models, we prefer to keep it in the supplement, which - for an online journal - makes little difference since the reader can just jump ahead to this figure in the same way as any other figure.

Fig 5C, D - label x-axis.

Added.

Fig 5E - axis labels needed. I don't know what is plotted on x and y, and cannot see red and green lines in left plot

Thanks for noticing this, colors corrected, axes labeled.

Page 8 L3-15 - If I follow this correctly, I think the authors are confusing pitch and frequency here in a way that is fundamental to their model. They seem to equate tonotopic frequency tuning to pitch tuning, leading to confused implications of frequency adaptation on the F0 representation of complex sounds like Shepard tones. To my knowledge, the authors do not examine pure tone frequency tuning in their neurons in this study. Please clarify how you propose that frequency tuning like that shown in Fig 5A relates to representation of the F0 of Shepard tones. Or...are the authors suggesting these neural effects have little to do with pitch processing and instead are just the result of frequency tuning for a single harmonic of the Shepard tones?

We agree that it is not trivial to describe this well, while keeping the text uncluttered, in particular, because often tuning properties to stimulus frequency contribute to tuning properties of the same neuron for pitch class, although this can be more or less straightforward: specifically, for some narrowly tuned cells, the Shepard tuning is simply a reflection of their tuning to a single octave range of the constituent tones (see Fig. S1). For more broadly tuned cells, multiple constituent tones will contribute to the overall Shepard tuning, which can be additive, subtractive, or more complex. The assumption in our approach is that we can directly estimate the Shepard tuning to evaluate the consequence for the percept. While this may seem artificial, as Shepard tones do not typically occur in nature, the same argument could be made against pure tones, on which classical tuning curves and associated decodings are often based. Relating the Shepard tuning to the classical tuning would be an interesting study in itself, although arguably relating the tuning of one artificial stimulus to another. Regarding the terminology of pitch, pitch class and frequency: The term pitch class is commonly used in the field of Shepard tones, and - as we indicated in the beginning of the results: "the term pitch is used interchangeably with pitch class as only Shepard tones are considered in this study". We agree that the term pitch, which describes the perceptual convergence/construction of a tone-height from a range of possible physical stimuli, needs to be separated from frequency as one contributor/basis for the perception of a pitch. However, we think that the term pitch can - despite its perceptual origin - also be associated with neuron/neural responses, in order to investigate the neural origin of the pitch percept. At the same time, the present study is not targeted to study pitch encoding per se, as this would require the use of a variety of stimuli leading to consistent pitch percepts. Therefore, pitch (class) is here mainly used as a term to describe the neural responses to Shepard tones, based on the previous literature, and the fact that Shepard tones are composite stimuli that lead to a pitch percept. The last sentence has been added to the manuscript for clarity.

P7-9: I wasn't left with a clear idea of how the model works from this text. I assume you have layers of neurons tuned to frequency or f0 (based on the real data?), which are connected in some way to produce some sort of output when you input a sound? More detail is needed here. How is the dynamic adaptation implemented?

The detailed description of the model can be found in the Methods section. We have gone through the corresponding paragraph and have tried to clarify the description of the model by introducing a high-level description and the reference to the corresponding Figure (Fig. 5A) in the Results.

Fig6A: Figure caption can't be correct. In any case, these equations cannot be understood unless you define the terms in them.

We have clarified the description in the caption.

Fig 6/directionality analysis: Assuming that the "F" in the STRFs here is Shepard tone f0, and not simple frequency?

We have changed the formula in the caption and the axis labels now.

Fig 6C - y-axis values

In the submission, these values were left out on purpose, as the result has an arbitrary scale, but only whether it is larger or smaller than 0 counts for the evaluation of the decoded directionality (at the current level of granularity). An interesting refinement would be to relate the decoded values to animal performance. We have now scaled the values arbitrarily to fit within [-1,1], but we would like to emphasize that only their relative scale matters here, not their absolute scale.

Fig 6E - can't both be abscissa (caption). I might be missing something here, but I don't see the "two stripes" in the data that are described in the caption.

Thank you. The typo is fixed. The stripes are most clearly visible in the right panel of Fig. 6E, red and blue, diagonally from top left to bottom right.

Fig 6G -I have no idea what this figure is illustrating.

This panel is described in the text as follows: "The resulting distribution of activities in their relation to the Bias is, hence, symmetric around the Bias (Fig. 6G). Without prior stimulation, the population of cells is unadapted and thus exhibits balanced activity in response to a stimulus. After a sequence of stimuli, the population is partially adapted (Fig. 6G right), such that a subsequent stimulus now elicits an imbalanced activity. Translated concretely to the present paradigm, the Bias will locally adapt cells. The degree of adaptation will be stronger, if their tuning curve overlaps more with the biased region. Adaptation in this region should therefore most strongly influence a cell’s response. For example, if one considers two directional cells, an up- and a down-selective cell, cocentered in the same frequency location below the Bias, then the Bias will more strongly adapt the up-cell, which has its dominant, recent part of the SSTRF more inside the region of the Bias (Fig. 6G right). Consistent with the percept, this imbalance predicts the tone to be perceived as a descending step relative to the Bias. Conversely, for the second stimulus in the pair, located above the Bias, the down-selective cells will be more adapted, thus predicting an ascending step relative to the previous tone."

I might be just confused or losing steam at this point, but I do not follow what has been done or the results in Fig 6 and the accompanying text very well at all. Can this be explained more clearly? Perhaps the authors could show spike rate responses of an example up-direction and down-direction neuron? Explain how the decoder works, not just the results of it.

We agree that we are presenting something new here. However, it is conceptually not very different from decoding based on preferred frequencies. We have attempted to provide two illustrations of how the decoder works (Fig. 6A) and how it then leads to the percept using prototypical examples of cellular SSTRFs (Fig. 6G). We have added a complete, but accessible description to the Methods section. Showing firing rates of neurons would unfortunately not be very telling, given the usual variability in neural response and the fact that our paradigm did not have a lot of repetitions (but instead a lot of conditions), which would be able to average out the variability on a single neuron level.

Discussion - I do not feel I can adequately critique the author's interpretation of the results until I understand their results and methods better. I will therefore save my critique of the discussion section for the next round of revisions after they have addressed the above issues of disorganization and clarity in the manuscript.

We hope that the updated version of the manuscript provides the reviewer now with this possibility.

Methods

P15L7 - gender of human subjects? Age distribution? Age of ferrets?

We have added this information.

P16L21 - What is the justification for randomizing the phase of the constituent frequencies?

The purpose of the randomization was to prevent idiosyncratic phase relationships for particular Shepard tones, which would depend in an orderly fashion on the included base-frequencies if non-randomized, and could have contributed to shaping the percept for each Shepard tone in a way that was only partly determined by the pitch class of the Shepard tone. Added to the section.

P17L6 - what are the 2 randomizations? What is being randomized?

Pitch classes and position in the Bias sequence. Added to the section.

P16 Shepard Tuning section - What were the durations of the tones and the time between tones within a trial?

Thanks, added!

Equations - several undefined terms in the equations throughout the manuscript.

Thanks. We have gone through the manuscript and all equations and have introduced additional definitions where they had been missing.

Reviewer #3 (Recommendations For The Authors):

P3L10: "passive" and "active" conditions come totally out of the blue. Need introducing first. (Or cut. If adaptation is always seen, why mention the two conditions if the difference is not relevant here?)

We have added an additional sentence in the preceding paragraph, that should clarify this. The reason for mentioning it is that otherwise a possible counter-argument could be made that adaptation does not occur in the active condition, which was not tested in ferrets (but presents an interesting avenue for future research).

P3L14 "siple" typo

Corrected.

P4L1 "behaving humans" you should elaborate just a little here on what sort of behavior the participants engaged in.

Thanks for pointing this out. We have clarified this by adding an additional sentence directly thereafter.

P4 adaptation: I wonder whether it would be useful to describe the Bias condition a bit more here before going into the observations. The reader cannot know what to expect unless they jump ahead to get a sense of what the Bias looks like in the sense of how many stimuli are in it, and how similar they are to each other. Observations such as "the average response strength decreases as a function of the position in the Bias sequence" are entirely expected if the Bias is made up of highly repetitive material, but less expected if it is not. I appreciate that it can be awkward to have Methods after Results, but with a format like that, the broad brushstroke Methods should really be incorporated into the Results and only the tedious details should be reserved for the Methods to avoid readers having to jump back and forth.

Agreed, we have inserted a corresponding description before going into the details of the results.

Related to this (perhaps): Bottom of P4, top of P5: "significantly less reduced (33%, p=0.0011, 2 group t-test) compared to within the bias (Fig. 2 A3, blue vs. red), relative to the first responses of the bias" ... I am at a loss as to what the red and blue symbols in Fig 2 A3 really show, and I wonder whether the "at the edges" to "within the Bias" comparison were to make sense if at this stage I had been told more about the composition of the Bias sequence. Do the ambiguous ('target') tones also occur within the Bias? As I am unclear about what is compared against what I am also not sure how sound that comparison is.

We have added an extended description of the Bias to the beginning of this section of the manuscript. For your reference: the Shepard tones that made up the ambiguous tones were not part of the Bias sequence, as they are located at 3st distance from the center of the Bias (above and below), while the Bias has a range of only +/- 2.5st.

Fig 2: A4 B1 B2 labels should be B1 B2 B3

Corrected.

Fig 2 A2, A3: consider adjusting y-axis range to have less empty space above the data. In A3 in particular, the "interesting bit" is quite compressed.

Done, however, while still matching the axes of A2 and A3 for better comparability.

I am under the strong impression that the human data only made it into Fig 2 and that the data from Fig 3 onwards are animal data only. That is of course fine (MEG may not give responses that are differentiated enough to perform the sort of analyses shown in the later figures. But I do think that somewhere this should be explicitly stated.

Yes, the reviewer's observation is correct. The decoding analyses could not be conducted on the human MEG data and was therefore not further pursued. Its inclusion in the paper has the purpose of demonstrating that even in humans and active conditions, the local adaptation is present, which is a key contributor to the two decoding models. We now state this explicitly when starting the decoding analysis.

P5L2 "bias" not capitalized. Be consistent.

All changed to capitalized.

P5L8 reference to Fig 2 A4: something is amiss here. From legend of Fig 2 it seems clear that panel A4 label is mislabeled B1. Maybe some panels are missing to show recovery rates?

Apologies for this residual text from a previous version of the manuscript. We have gone through all references and corrected them.

P6L7 comma after "decoding".

Changed.

Fig 3, I like this analysis. What would be useful / needed here though is a little bit more information about how the data were preprocessed and pooled over animals. Did you do the PCA separately for each animal, then combine, or pool all units into a big matrix that went into the PCA? What about repeat, presentations? Was every trial a row in the matrix, or was there some averaging over repeats? (In fact, were there repeats??)

Thanks for bringing up these relevant aspects, which were partly insufficiently detailed in the manuscript. Briefly, cells were pooled across animals and we only used cells that could meaningfully contribute to the decoding analysis, i.e. had auditory responses and different responses to different Shepard tones. Regarding the responses, as stated in the Methods, "Each stimulus was repeated 10 times", and we computed average responses across these repetitions. Single trials were not analyzed separately. We have added this information in the Methods, and refer to it in the Results.

Also, there doesn't appear to be a preselection of units. We would not necessarily expect all cortical neurons to have a meaningful "best pitch" as they may be coding for things other than pitch. Intuitively I suspect that, perhaps, the PCA may take care of that by simply not assigning much weight to units that don't contribute much to explained variance? In any event I think it should be possible, and would be of some interest, to pull out of this dataset some descriptive statistics on what proportion of units actually "care about pitch" in that they have a lot (or at least significantly more than zero) of response variance explained by pitch. Would it make sense to show a distribution of %VE by pitch? Would it make sense to only perform the analysis in Fig 3 on units that meet some criterion? Doing so is unlikely to change the conclusion, but I think it may be useful for other scientists who may want to build on this work to get a sense of how much VE_pitch to expect.

We fully agree with the reviewer, which is why this information is already presented in Supplementary Fig.1, which details the tuning properties of the recorded neurons. Overall, we recorded from 1467 neurons across all ferrets, out of which 662 were selected for the decoding analysis based on their driven firing rate (i.e. whether they responded significantly to auditory stimulation) and whether they showed a differential response to different Shepard tones The thresholds for auditory response and tuning to Shepard tones were not very critical: setting the threshold low, led to quantitatively the same result, however, with more noise. Setting the thresholds very high, reduced the set of cells included in the analysis, and eventually that made the results less stable, as the cells did not cover the entire range of preferences to Shepard tones. We agree that the PCA based preprocessing would also automatically exclude many of the cells that were already excluded with the more concrete criteria beforehand. We have added further information on this issue in the Methods section under the heading 'Unit selection'.

P9 "tones This" missing period.

Changed.

P10L17 comma after "analysis"

Changed.

My goal in this class is to walk away with a new understanding of how to learn new languages. I really love this piece of advice as i believe that being surrounded by people who speak that language natively is the best way to learn. I also think that this mindset can be applied to all aspects of life.

I truly believe texting is one of the worst ways to communicate. Tone is completely lost over text and it can lead to so much drama between people. It is so much easier to communicate on a call or in person where we can hear their tone of voice.

Culture is an interesting thing because culture can be found in the masses, and culture can be found within friend groups. Mass culture could look like how it is typically unexpectable to film someone in public. Then culture among friend groups could be inside jokes, clothes we collectively like, a shared music taste, etc.

multiple sources

source

word choice similar to pathos

no leeway

More numbers & facts

using sources to back argument

most society against marginalization

no one wants to be left out

Unjust invokes emotion

I like this definition a lot but i think it is also important to include social practices. I think one aspect of culture involves common practices, whether that be religion or what time you show up to an interview. I know a lot of families that say "if you aren't early your late" while others don't see value in showing up early to events.

This seems like the most beneficial form of technological assistance for the elderly with the least amount of drawbacks.

eLife Assessment

This revised version of the study is important, showing that age-related gut microbiota modulate uric acid metabolism through the NLRP3 inflammasome pathway and thereby regulate susceptibility to age-related gout. Several experimental approaches (mechanistic insights) and methods (data quality) are still incomplete. If strengthened, this paper would be of broad interest to researchers working on gout and the microbiota.

Reviewer #2 (Public review):

Summary:

The revised manuscript presents interesting findings on the role of gut microbiota in gout, focusing on the interplay between age-related changes, inflammation, and microbiota-derived metabolites, particularly butyrate. The study provides valuable insights into the therapeutic potential of microbiota interventions and metabolites for managing hyperuricemia and gout. While the authors have addressed many of the previous concerns, a few areas still require clarification and improvements to strengthen the manuscript's clarity and overall impact.

(1) While the authors mention that outliers in the data do not affect the conclusions, there remains a concern about the reliability of some figures (e.g., Figure 2D-G). It is recommended to provide a more detailed explanation of the statistical analysis used to handle outliers. Additionally, the clarity of the Western blot images, particularly IL-1β in Figure 3C, should be improved to ensure clear and supportive evidence for the conclusions.<br /> (2) The manuscript raises a key question about why butyrate supplementation and FMT have different effects on uric acid metabolism and excretion. While the authors have addressed this by highlighting the involvement of multiple bacterial genera, it is still recommended to expand on the differences between these interventions in the discussion, providing more mechanistic insights based on available literature.<br /> (3) It is noted that IL-6 and TNF-α results in foot tissue were requested and have been added to supplementary material. However, the main text should clearly reference these additions, and the supplementary figures should be thoroughly reviewed for consistency with the main findings. The use of abbreviations (e.g., ns for no significant difference) and labeling should also be carefully checked across all figures.<br /> (4) The manuscript presents butyrate as a key molecule in gout therapy, yet there are lingering concerns about its central role, especially given that other short-chain fatty acids (e.g., acetic and propionic acids) also follow similar trends. The authors should consider further acknowledging these other SCFAs and discussing their potential contribution to gout management. Additionally, the rationale for focusing primarily on butyrate in subsequent research should be made clearer.<br /> (5) The full-length uncropped Western blot images should be provided as requested, to ensure transparency and reproducibility of the data.<br /> (6) Despite the authors' revisions, several references still lack page numbers. Please ensure that all references are properly formatted, including complete page ranges.<br /> The manuscript has improved with the revisions made, particularly regarding clarifications on experimental design and the inclusion of supplementary data. However, some concerns about data quality, mechanistic insights, and clarity in the figures remain. Addressing these points will enhance the overall impact of the work and its potential contribution to the understanding of the gut microbiome in gout and hyperuricemia. A final revision, with careful attention to both major and minor points, is highly recommended before resubmission.

Reviewer #1 (Public review):

Summary:

In their manuscript the authors report that fecal transplantation from young mice into old mice alleviates susceptibility to gout. The gut microbiota in young mice is found to inhibit activation of the NLRP3 inflammasome pathway and reduce uric acid levels in the blood in the gout model.

Strengths:

They focused on the butanoate metabolism pathway based on the results of metabolomics analysis after fecal transplantation and identified butyrate as the key factor in mitigating gout susceptibility. In general, this is a well-performed study.

Weaknesses:

The discussion on the current results and previous studies regarding the effect of butyrate on gout symptoms is insufficient. The authors need to provide a more thorough discussion of other possible mechanisms and relevant literature.

Author response:

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

Reviewer #3 (Public Review):

Some critical comments are provided below：

(1) The data quality still needs to be improved. There are many outliers in the experimental data shown in some figures, e.g. Figure 2D-G. The presence of these outliers makes the results unreliable. The author should thoroughly review the data analysis in the manuscript. In addition, a couple of western blot bands, such as IL-1β in Figure 3C, are not clear enough, please provide clearer western blot results again to support the conclusion.

Following our comparative analysis, we have determined that these data do not affect our conclusions. Moreover, our experimental design included a total of six mice per group, with all mouse samples being subjected to testing.

(2) As shown in Figure 1G-I, foot thickness and IL-1β content in foot tissues of the Aged+Abx group were significantly reduced, but there was no difference in serum uric acid level. In addition, the Abx-untreated group should be included at all ages.

Thank you for your comment. We have included this data in Supplemental Material 4.

(3) Since FMT (Figure 4) and butyrate supplementation (Figure 8) have different effects on uric acid synthesis enzyme and excretion, different mechanisms may lie behind these two interventions. Transplantation with significantly enriched single strains from young mice, such as Bifidobacterium and Akkermansia, is the more reliable approach to reveal the underlying mechanism between gut microbiota and gout.

Thank you for your comment. Due to the involvement of multiple bacterial genera in gout and hyperuricemia, and the practical challenge of testing all strains, our focus shifted to the functional implications and metabolism of the microbiota. Experimental validation confirmed that butyrate exerts a dual-therapeutic effect in mitigating gout and hyperuricemia.

(4) In Figure 2F, the results showed the IL-1β, IL-6, and TNF-α content in serum, which was inconsistent with the authors' manuscript description (Line 171).

Thank you for your comment. The modifications to the results have been implemented.

(5) Figures 2F-H duplicate Supplementary Figures S1B-D. The authors should prepare the article more carefully to avoid such mistakes.

Thank you for your comment. We have corrected it in the manuscript.

(6) In lines 202-206, the authors stated that the elevated serum uric acid levels in the Young+Old or Young+Aged groups, but there is no difference in the results shown in Figure 4A.

Thank you for your comment. We have corrected it in the manuscript.

(7) Please visualize the results in Table 2 in a more intuitive manner.

The results have been presented in Table 2 with a more intuitive visual format. The detailed information is presented in Supplement 4.

(8) The heatmap in Figure 7A cannot strongly support the conclusion "the butyric acid content in the faeces of Young+PBS group was significantly higher than that in the Aged+PBS group". The author should re-represent the visual results and provide a reasonable explanation. In addition, please provide the ordinate unit of Supplementary Figure 7A-H.

Thank you for your comment. Figure 7A and Supplementary Figure 7A-H together illustrate "the butyric acid content in the faeces of Young+PBS group was significantly higher than that in the Aged+PBS group", and the specific units of short-chain fatty acids have been annotated in the manuscript.

(9) Uncropped original full-length western blot should be provided.

Thank you for your comment. We have made relevant notes in the paper.

Reviewer #1 (Recommendations For The Authors):

Gout, a prevalent form of arthritis among the elderly, exhibits an intricate relationship with age and gut microbiota. The authors found that gut microbiota plays a crucial role in determining susceptibility to age-related gout. They observed that age-related gut microbiota regulated the activation of the NLRP3 inflammasome pathway and modulated uric acid metabolism. "Younger" microbiota has a positive impact on the gut microbiota structure of old or aged mice, enhancing butanoate metabolism and butyric acid content. Finally, they found butyric acid exerts a dual effect, inhibiting inflammation in acute gout and reducing serum uric acid levels. This work's insights emphasize the potential of "young" gut microbiome in mitigating senile gout. The whole study was interesting, but there were some minor errors in the overall writing of the paper. The author should carefully check the spelling of the words in the text and the case consistency of the group names.

Questions:

(1) Line 118, line 142, and elsewhere 24 months in the same format as before.

Thank you for your comment. We have corrected it in the manuscript.

(2) Lines 123, Old and Aged group should be a complex number.

Thank you for your suggestion. We have corrected it in the manuscript.

(3) Why does line 133 mention the use of ABX? Please add a brief explanation.

Thank for your suggestion. The aim of utilizing ABX is to construct the linkage between gut microbiota, age, and gout.

(4) Lines 172-175, the description of TNF does not match the description of the result figure, may be the picture placement error, please correct this.

Thank you for your careful review. The error has been corrected and the accurate result has been inserted into the original manuscript.

(5) Lines183-185 and lines193-lines195, Pro-Caspase-1 and Pro-IL activate excess write.

Thank you for your careful review. We have corrected the error at the original location.

(6) Line 400, the text should not be written as increased.

Thank you for your careful review. We have corrected the error at the original location.

(7) "ns" needs to be added in the legend to indicate that there is no significant difference.

Thank you for your careful review. We have corrected the error at the original location.

(8) Lines 1080-1084 "Old or Aged control group and the old or aged group", group names should be case-sensitive.

Thank you for your suggestion. We have made the correct modification to the group names.

(9) Lines 1072-1073, "Representative western blot images of foot tissue NLRP3 pathways proteins" add band density.

Thank you for your suggestion. We have corrected the error on lines 1072-1073 of the article.

Reviewer #2 (Recommendations For The Authors):

Specific comments:

(1) In Figures 1G-H, the Aged+PBS group with antibiotic treatment shows a significant reduction in foot swelling and IL-1β compared to the Young+PBS and Old+PBS groups. The authors state that age-related changes in the gut microbiota exacerbate gout. However, why does only the Aged+PBS group improve with antibiotic treatment? It seems that butyrate alone cannot explain this phenomenon.

We utilize antibiotics for treatment in order to establish the relationship between gut microbiota, age, and gout. Different age groups are directly given antibiotics for treatment. We found that after clearing the gut microbiota and then stimulating with MSU, the trend of inflammation factors changing with age disappears.

(2) In Figure 2, the fecal transplantation from young mice improved the infiltration of inflammatory cells and inflammatory cytokines in the Old and Aged groups. However, in Supplementary Figure 1A, there is no improvement observed in the percentage of foot swelling. Is it appropriate to conclude that inflammation was improved even though foot swelling was not suppressed?

Although we did not observe changes in the swelling of the mice's feet, there were changes in the inflammatory cell infiltration and inflammation factors in the slices. We rely on a comprehensive assessment of various indicators to determine whether the inflammatory condition has improved or worsened.

(3) In line #249, the authors state that "the fecal microbiota from mice in the young group promotes uric acid elimination, inhibits reabsorption, and may contribute to the integrity of the intestinal barrier structure." However, Supplementary Figure 3F-H shows no significant alterations in Occludin and ZO-1 mRNA expression levels among all groups. Therefore, it is difficult to conclude that the fecal microbiota from the young group promotes the integrity of the intestinal barrier structure. A functional barrier assay, such as oral administration of FITC-dextran, would be necessary to verify the authors' conclusion.

In Supplementary Figure 3F-H, we observed that the mRNA expression of Occludin and ZO-1 increased but showed no significant difference. However, after the elderly mice were transplanted with the intestinal microbiota of young mice, the mRNA expression of JAMA showed a significant upward trend. Additionally, due to the scarcity of old mice, we were unable to perform the oral administration of FITC-dextran. However, we supplemented with immunohistochemical slices of Zo-1 and Occludin to support our viewpoint.

(4) In Figure 4, when comparing the young+PBS group with the old+PBS or aged+PBS groups, there are hardly any differences in the proteins involved in uric acid synthesis (ADA, GDA, XOD) or the genes involved in uric acid transport (URAT1, GLUT9, OAT1, OTA3, ABCG2). Since no changes in uric acid synthesis or transport pathways are observed with aging, it is questionable to conclude that fecal transplantation from young mice improves these pathways and lowers blood uric acid levels.

In the calculation process, we used different age groups of the control group as references, instead of directly using young mice. We then compared the data of mice of different ages, and the results are in Supplementary Material 4.

(5) In line 276, the authors describe "the Young +Old and Young+Aged groups tended to be closer to the Old+PBS and Aged+PBS groups, and the Old+Young and Aged+young groups tended to be closer to the Young+PBS group (Figure 5D)". Please conduct a statistical analysis.

(6) In line 298, the authors hypothesize that butyrate might be the key molecule responsible for controlling gout, as Bifidobacterium and Akkermansia were abundant in the Young group, and the butyrate pathway was prominent. However, neither Bifidobacterium nor Akkermansia are butyrate-producing bacteria. Thus, the conclusion appears to be biased toward butyrate, raising questions about this interpretation.

Upon comparison, we discovered other bacteria genera that produce butyrate, such as Lachnoclostridium. Additionally, literature (PMID:38126785, 26420851) reports have indicated that Bifidobacteria combined with other genera can enhance the production of butyrate. Meanwhile, Akkermansia, particularly the species Akkermansia muciniphila, has been found to confer several beneficial traits, as evidenced by preclinical studies. These traits include promoting the growth of butyrate-producing bacteria through the production of acetate, which leads to a decrease in the loss of the colonic bilayer and subsequent reduction in inflammation (PMID:35468952). Based on the predicted results of microbiome functions, we observed that the Butanoate_metabolism of the microbiota in young mice and the elderly mice recipients of young mouse microbiota was enhanced. Considering that Lachnoclostridium can produce butyrate, and that Bifidobacteria and Akkermansia can promote the production of butyrate by the intestinal microbiota, we speculated that butyrate might play a role in gout and hyperuricemia.

(7) In Supplementary Figure 7, acetic acid and propionic acid also show the same behavior as butyric acid. It is possible that these metabolites may also affect the development of gout.

Thank you for your suggestion. Indeed, Figure 7 does show a similar trend for acetic and propionic acids as for butyric acid. However, considering the predictive data of microbial function and the non-targeted metabolomic data, there is an enhancement of Butanoate_metabolism in both young mice and elderly mice receiving young mouse intestinal microbiota transplants. Therefore, we prioritized butyrate as the subject of our study. Due to the scarcity of elderly mice, we are unable to conduct subsequent experiments with acetic and propionic acids, which is one of the limitations of this study. This work will be addressed in our follow-up research.

(8) In Figure 6, the secondary bile acid biosynthesis pathway was also changed. However, there is little mention of secondary bile acid in the discussion section. Please carefully discuss other possibilities besides butyrate.

Thank you for your suggestion. We have incorporated a discussion about secondary bile acids into the relevant section of our manuscript.

(9) In line #330, the authors state, 'the metabolites identified as showing differential abundance between the groups were enriched in the butanoate metabolism pathway (Figure 6A-D).' However, there does not appear to be much difference in the butanoate metabolism pathway. Specifically, in Figure 6C, the butanoate metabolism pathway in the Old group does not differ from that in the Young group. Please explain in more detail whether the butanoate metabolism pathway is relevant in the Old group.

The metabolites identified as showing differential abundance between the groups were enriched in the butanoate metabolism pathway. The differential metabolites are enriched in the butyrate metabolism pathway; however, the non-targeted metabolomics did not reveal the extent of their enrichment.

(10) In Figure 7, the authors measured the levels of short-chain fatty acids in the Young and Aged groups. They found butyrate in the feces of mice in the Young group was higher than that in the Aged group. However, I wonder whether the Old group also had low levels of butyrate or not.

In the experiment, we selected three representative groups to verify the hypothesis that butyrate may play a significant role in gout and hyperuricemia. Subsequently, we found that supplementing 18-month-old and 24-month-old mice with butyrate indeed reduced blood uric acid levels and alleviated gout symptoms. Since 18-month-old mice are difficult to obtain, we only conducted microbiome sequencing and non-targeted metabolomic analysis.

Minor issues:

(11) In line 74, what does MSU stand for? Please describe the abbreviation.

In line 74, MSU refers to Monosodium urate crystals.

(12) In line 136, please insert a space between "IL-1β" and "and".

Thank you for your suggestion. We have corrected the error of the article.

(13) In line 570, please describe the method of butyrate administration and also correct the grammatical errors.

Thank you for your suggestion. We have corrected the error of the article.

(14) Change the title of x axis in Figure 2F-H, "Serum ~" to "Peritoneal fluid ~", according to the legend.

Thank you for your suggestion. We have corrected this error in the manuscript.

(15) In line 302, "succinates" should be "butyric acid or butyrate".

Thank you for your suggestion. We have corrected this error in the manuscript.

Reviewer #3 (Recommendations For The Authors):

(1) The authors showed the results of IL-1β levels in foot tissues in Figure 1C and Figure 1H, and serum IL-1β, IL-6, and TNF-α levels in Figure 2F-H. Could the authors also provide the results of IL-6 and TNF-α in foot tissue in Figure 1?

Thank you for your suggestion. We have added the results of of IL-6 and TNF-α in foot tissue in supplementary material 4.

(2) There are some errors in the reference citation format, such as missing page numbers.

Thank you for your careful review. We have revised the references in our manuscript.

(3) There are too many writing errors in the manuscript, which greatly affect the understanding of the text. The manuscript must be carefully revised to improve its readability. It's recommended that a professional English writer or native speaker proofread the paper before submission. Some errors, but not limited to these errors, are listed below.

a. Line 107: The abbreviation for "short-chain fatty acid" should be SCFA, not SFCA.

Thank you for your careful review. We have corrected this error in the manuscript.

b. Line 136: There is a missing space between IL-1β and and. B.

Thank you for your careful review. We have corrected this error in the manuscript.

c. Line 145, the phrase "on gout on gout", and line 471, "that transplantation" are repeated.

Thank you for your careful review. We have corrected this error in the manuscript.

d. Line 152: "Age+PBS" should be "Aged+PBS".

Thank you for your careful review. We have corrected this error in the manuscript.

e. In Figure 1e, "Aded+PBS" should be "Aged+PBS".

Thank you for your careful review.  We have corrected the error in Figure 1e.

f. Line 152: The phrase "by via" is repeated.

Thank you for your suggestion. We have deleted the phrase "by via" in line 152.

g. "16S rDNA" in line 92 is inconsistent with the "16S rRNA" in line 652.

Thank you for your suggestion. We have revised the error in the manuscript to maintain consistency in professional terminology.

Observation:Every monopoly and all exclusive privileges are granted at the expense of the public which is out to receive a fair equivalent. The many millions which this act proposes to bestow on the stockholders of an existing bank must directly or indirectly out of the earnings of the American Citzens.

Interpretation: That every monopoly and all exclusive privileges are granted at the expense of the public and which receive a fair equivalent. Many millions of this act proposes to bestow stockholders of their existing bank must directly or indirectly out of the earning of the American People.

Observation: It appears that more than a fourth part of the stocks is held by foreigners and the residue is held by a few hundred of our citzens.

Interpretation: That the fourth part of stocks is held by foreigners and the residue is held by a few hundred of citzens.

Which grid would this be?

The effects of WW2 drastically affected POC by increasing rights and recognizing equality in the workplace, military, and society.

This is a prime example of how family education plays a role in child education. Based on their academic experiences, Alexander's parents are able to guide him on the path to being successful and obtaining a stable job. Their guidance plays a crucial role in helping him take the right steps towards a successful and abundant future. I remember feeling so lost when applying to colleges as a senior in high school. And while my parents both went to college, they were unsure of how to help me. They were unfamiliar with the common app and the UC application portals. I understand how difficult it can be to prepare for college without the guidance of your parents or another caregiving figure.

Observation: Thousands of men who have money claim a superiority over others, which poverty is a crime.

Interpretation: Thousands of men who have money claim a superiority over others such as man that are in poverty which is a crime and those that are guilty of it deserve not the rights of men.

Observation: That the Proprietors pay their men only in such money as they have to receive but that not the case and it is false because very many men in this city who draws from banks, on paydays and bankable money then run to exchange unbankable, unlawful money to pay their men with because they can make few bucks by the operation .

Interpretation: That the Proprietors pay their men only in such money as they have to receieve because it false since many men in this city chose to draw from banks on paydays and bankable money to exchange unbankable, unlawful money to their men with because they can make money.

This tool will allow students to put in audio descriptions and the tool will be able to find videos and photos they will need.

I would love to see what the 87% of the people think. I hope they find it useful and helpful.

Hopefully in 4 years, this Ai is a mastered tool to use. I will be able to help students with learning disabilities while also helping the other students.

This new tool helps students with speech problems to learn. Researchers are trying to teach Ai how to respond to different voices and different or hard to understand speeches. These students will be able to talk into Ai and Ai will be able to understand them and then help them with whatever they need.

This allows blind or low vision students to still learn and be a part of the class. These students can put an image into Ai and Ai will explain what the picture looks like.

Students with learning disabilities should use AI and be able to use it more than the other students. This can help those students correct and make sure words are correct. They could also use it for searching new ideas and outlines.

cause for infectious disease

eLife Assessment

This article describes a novel mechanism allows Drosophila to combat enteric pathogens while also preserving the beneficial indigenous microbiota. The authors provide compelling evidence that oral infection of Drosophila larvae by pathogenic bacteria activate a valve that traps the intruders in the anterior midgut, allowing them to be killed by antimicrobial peptides. This is an important finding revealing a new mechanism of host defense in the gut of insects.

Reviewer #1 (Public review):

Tleiss et al. demonstrate that while commensal Lactiplantibacillus plantarum freely circulate within the intestinal lumen, pathogenic strains such as Erwinia carotovora or Bacillus thuringiensis are blocked in the anterior midgut where they are rapidly eliminated by antimicrobial peptides. This sequestration of pathogenic bacteria in the anterior midgut requires the Duox enzyme in enterocytes, and both TrpA1 and Dh31 in enteroendocrine cells. This effect induces muscular muscle contraction, which is marked by the formation of TARM structures (thoracic ary-related muscles). This muscle contraction-related blocking happens early after infection (15mins). On the other side, the clearance of bacteria is done by the IMD pathway possibly through antimicrobial peptide production while it is dispensable for the blockage. Genetic manipulations impairing bacterial compartmentalization result in abnormal colonization of posterior midgut regions by pathogenic bacteria. Despite a functional IMD pathway, this ectopic colonization leads to bacterial proliferation and larval death, demonstrating the critical role of bacteria anterior sequestration in larval defense.

In general, this fundamentally important study reveals unique mechanisms in the gut immunity of Drosophila larvae. It also describes a previously understudied structure, TARM, which may play a crucial role in this process. This significant work substantially advances our understanding of pathogen clearance by identifying a new mode of pathogen eradication from the insect gut. The evidence supporting the authors' claims is compelling, and the study opens new avenues for future research in gut immunity.

Reviewer #2 (Public review):

Summary:

This article describes a novel mechanism of host defense in the gut of Drosophila larvae. Pathogenic bacteria trigger the activation of a valve that blocks them in the anterior midgut where they are subjected to the action of antimicrobial peptides. In contrast, beneficial symbiotic bacteria do not activate the contraction of this sphincter and can access the posterior midgut, a compartment more favorable to bacterial growth.

Strengths:

The authors decipher the underlying mechanism of sphincter contraction, revealing that ROS production by Duox activates the release of DH31 by enteroendocrine cells that stimulate visceral muscle contractions. Use of mutations affecting the Imd pathway or lacking antimicrobial peptides reveals their contribution to pathogen elimination in the anterior midgut.

Weaknesses:

The mechanism allowing the discrimination between commensal and pathogenic bacteria remains unclear.

Author response:

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

Reviewer #1 (Public Review):

Tleiss et al. demonstrate that while commensal Lactiplantibacillus plantarum freely circulate within the intestinal lumen, pathogenic strains such as Erwinia carotovora or Bacillus thuringiensis are blocked in the anterior midgut where they are rapidly eliminated by antimicrobial peptides. This sequestration of pathogenic bacteria in the anterior midgut requires the Duox enzyme in enterocytes, and both TrpA1 and Dh31 in enteroendocrine cells. This effect induces muscular muscle contraction, which is marked by the formation of TARM structures (thoracic ary-related muscles). This muscle contraction-related blocking happens early after infection (15mins). On the other side, the clearance of bacteria is done by the IMD pathway possibly through antimicrobial peptide production while it is dispensable for the blockage. Genetic manipulations impairing bacterial compartmentalization result in abnormal colonization of posterior midgut regions by pathogenic bacteria. Despite a functional IMD pathway, this ectopic colonization leads to bacterial proliferation and larval death, demonstrating the critical role of bacteria anterior sequestration in larval defense.

This important work substantially advances our understanding of the process of pathogen clearance by identifying a new mode of pathogen eradication from the insect gut. The evidence supporting the authors' claims is solid and would benefit from more rigorous experiments.

(1) The authors performed the experiments on Drosophila larvae. I wonder whether this model could extend to adult flies since they have shown that the ROS/TRPA1/Dh31 axis is important for gut muscle contraction in adult flies. If not, how would the authors explain the discrepancy between larvae and adults?

We have linked the adult phenotype to the larval model to explore the ROS/TrpA1/Dh31 axis in both contexts.  As highlighted in the discussion, however, there are key behavioral differences between larvae and adult flies. Unlike larvae, which remain in the food environment, adult flies have the ability to move away. This difference could impact the relevance of gut muscle contraction and bacterial clearance mechanisms between the two stages. Specifically, in larvae, the rapid ejection of gut contents due to muscle contraction poses a unique risk: larvae may inadvertently re-ingest the expelled material within minutes, which could influence their immune defenses. We have clarified this distinction and our hypothesis in the final section of the discussion, as it emphasizes the adaptive nature of this mechanism in larvae.

(2) The authors performed their experiments and proposed the models based on two pathogenic bacteria and one commensal bacterial at a relatively high bacterial dose. They showed that feeding Bt at 2X1010 or Ecc15 at 4X108 did not induce a blockage phenotype. 

I wonder whether larvae die under conditions of enteric infection with low concentrations of pathogenic bacteria. 

To address this, we have provided new data (Movie 5), in which larvae were fed a lower dose of Bt-GFP at 1.3 × 10^10 CFU/mL. In this video, we observe that when larvae ingest fewer bacteria, no blockage occurs, and the bacteria are able to reach the posterior midgut. As the bacterial load is lower, the fluorescence signal is weaker, but the movie clearly shows the excretion of bacteria. Importantly, under these conditions, no larval death was observed. These findings suggest that below a certain bacterial threshold, the pathogenicity is insufficient to: (1) trigger the blockage response, and (2) kill the larvae. In such cases, bacteria are likely eliminated through normal peristaltic movements rather than through the blockage mechanism described in our study.

If larvae do not show mortality, what is the mechanism for resisting low concentrations of pathogenic bacteria? 

As mentioned in our previous response, we hypothesize that the larvae’s ability to resist low concentrations of pathogenic bacteria is likely due to being below the threshold of virulence. At lower bacterial doses, the pathogenic load is insufficient to trigger the blockage mechanism or cause larval death. In these cases, it is probable that classical peristaltic movements of the gut efficiently eliminate the bacteria, preventing them from colonizing the posterior midgut or causing significant harm. Thus, the larvae rely on standard gut motility and immune mechanisms, rather than the blockage response, to clear lower doses of bacteria.

Why is this model only applied to high-dose infections? 

The reason this model primarily applies to high-dose infections is that lower concentrations of pathogenic bacteria do not trigger the blockage mechanism. As we mentioned in the manuscript, for low bacterial concentrations, where the GFP signal remains detectable, wild-type larvae are still able to resist live bacteria in the posterior part of the intestine.

Regarding the bacterial doses used in our experiments, it's important to clarify that we calculate the bacterial load based on colony-forming units (CFU). In our setup, there are approximately 5 × 10^4 CFU per midgut. For each experiment, we prepare 500 µl of contaminated medium containing 4 × 10^10 CFU. Fifty larvae are placed into this 500 µl of medium, meaning each larva ingests around 5 × 10^4 CFU within one hour of feeding.

This leads us to two key points:

(1) Continuous feeding might trigger the blockage response even at lower doses, as extended exposure to bacteria could lead to higher accumulation within the gut.

(2) Other defense mechanisms, such as the production of reactive oxygen species (ROS) or classical peristaltic movements, could be sufficient to eliminate lower bacterial doses (around 10^3 CFU or below).

We also refer to the newly provided Movie 5, where larvae fed with Bt-GFP at 1.3 × 10^10 CFU/mL show no blockage at low ingestion levels and successfully eliminate the bacteria.

(3) The authors claim that the lock of bacteria happens at 15 minutes while killing by AMPs happens 6-8 hours later. 

Our CFU data indicate that it’s after 4 to 6 hours that the quantity of bacteria decreases. We fixed this in the text.

What happened during this period? 

During the 4 to 6-hour period, several defense mechanisms are activated. ROS play a bacteriostatic and bacteriolytic role, helping to control bacterial growth. Concurrently, the IMD pathway is activated, leading to the transcription, translation, and secretion of antimicrobial peptides. These AMPs exert both bacteriostatic and bacteriolytic effects, contributing to the eventual clearance of the pathogenic bacteria.

More importantly, is IMD activity induced in the anterior region of the larval gut in both Ecc15 and Bt infection at 6 hours after infection? 

We have provided new data (Supplementary Figure 6) that includes RT-qPCR analysis of the whole larval gut in wt, TrpA1- and Dh31- genetic background after feeding with Lp, Ecc15, Bt, or yeast only. We monitored the expression of three different AMP-encoding genes and found that while AMP expression varied depending on the food content, there were no significant differences between the genotypes tested.

Additionally, we included new imaging data (Supplementary Figure 11) from AMP reporter larvae (Dpt-Cherry) fed with fluorescent Lp or Bt. In larvae infected with Bt, which is blocked in the anterior part of the gut, the dpt gene is predominantly induced in this region, indicating strong IMD pathway activity in response to Bt infection. Conversely, in larvae fed with Lp-GFP, the Dpt-Cherry reporter shows weak expression in the anterior midgut, and is barely detectable in the posterior midgut where Lp-GFP establishes itself. This aligns with previous findings by Bosco-Drayon et al. (2012), which demonstrated low AMP expression in the posterior midgut due to the presence of negative regulators of the IMD pathway, such as amidases and Pirk.

Are they mostly expressed in the anterior midgut in both bacterial infections? Several papers have shown quite different IMD activity patterns in the Drosophila gut. Zhai et al. have shown that in adult Drosophila, IMD activity was mostly absent in the R2 region as indicated by dpt-lacZ. Vodovar et al. have shown that the expression of dpt-lacZ is observable in proventriculus while Pe is not in the same region. Tzou et al. showed that Ecc15 infection induced IMD activity in the anterior midgut 24 hours after infection. 

Based on our new data (Supplementary Figure 11), we observe that Dpt-RFP expression is primarily localized in the anterior midgut and likely in the beginning of acidic region in larvae infected with Bt, Ecc and Lp. 

Using TrpA1 and Dh31 mutants, the authors found both Ecc15 and Bt in the posterior midgut. Why are they not evenly distributed along the gut? 

We observe that bacteria are not evenly distributed along the gut in wild-type larvae as well, with LP. This suggests that the transit time in the anterior part of the gut may be relatively short due to active peristaltism, which would make this region function as a "checkpoint" for bacteria that are not supposed to be blocked. Indeed, we confirmed that peristaltism is active during our intoxication experiments, which could explain the rapid movement of bacteria through the anterior midgut.

In contrast, bacteria tend to remain longer in the posterior midgut, which corresponds to the absorptive functions of intestinal cells in this region. This would explain why we observe more bacteria in the posterior midgut for Lp in control larvae and for Ecc15 and Bt in the TrpA1- and Dh31- mutants. Although a few bacteria are still found in the anterior midgut, they are consistently in much lower numbers compared to the posterior, as shown in Figures 1A and 3A of our manuscript.

Last but not least, does the ROS/TrpA1/Dh31 axis affect AMP expression?

We investigated whether the ROS/TrpA1/Dh31 axis influences AMP expression by performing RT-qPCR on the whole gut of larvae in wild-type, TrpA1-, and Dh31- genetic backgrounds. Larvae were fed with Lp, Ecc, Bt, or yeast (new data: Supplementary Figure 6). We monitored the expression of three different AMP-encoding genes and found that while AMP expression varied depending on the food content, there were no significant differences in AMP expression between the different genotypes.

Additionally, we provide imaging data from AMP reporter larvae (pDpt-Cherry) fed with fluorescent Lp or Bt (new data: Supplementary Figure 11). These results further confirm that the ROS/TrpA1/Dh31 axis does not significantly affect AMP expression in our experimental conditions.

(4) The TARM structure part is quite interesting. However, the authors did not show its relevance in their model. Is this structure the key-driven force for the blocking phenotype and killing phenotype? 

We agree that the TARM structures are a fascinating aspect of this study and acknowledge the interest in their potential role in the blocking and killing phenotypes. While we are keen to explore the specific contributions of these structures during bacterial intoxication, the current genetic tools available for manipulating TARMs target both TARM T1 and T2 simultaneously, as demonstrated by Bataillé et al., 2020 (Fig. 2). Of note, these muscles are essential for proper gut positioning in larvae, and their absence leads to significant defects in food intake and transit, which would confound the results of our intoxication experiments (see Fig. 6 from Bataillé et al., 2020).

Therefore, while TARMs are likely involved in these processes, the current limitations in selectively targeting them prevent us from definitively testing their role in bacterial blocking and killing at this stage. We hope to address this in future studies as more refined genetic tools become available.

Is the ROS/TrpA1/Dh31 axis required to form this structure?

To determine whether the ROS/TrpA1/Dh31 axis is required for the formation of TARM structures, we examined larval guts from control, TrpA1-, and Dh31- mutant backgrounds. Our new data (Supplementary Figure 8) show that the TARM T2 structures are still present in the mutants, indicating that the formation of these structures does not depend on the ROS/TrpA1/Dh31 axis.

Reviewer #2 (Public Review):

This article describes a novel mechanism of host defense in the gut of Drosophila larvae. Pathogenic bacteria trigger the activation of a valve that blocks them in the anterior midgut where they are subjected to the action of antimicrobial peptides. In contrast, beneficial symbiotic bacteria do not activate the contraction of this sphincter, and can access the posterior midgut, a compartment more favorable to bacterial growth.

Strengths:

The authors decipher the underlying mechanism of sphincter contraction, revealing that ROS production by Duox activates the release of DH31 by enteroendocrine cells that stimulate visceral muscle contractions. The use of mutations affecting the Imd pathway or lacking antimicrobial peptides reveals their contribution to pathogen elimination in the anterior midgut.

Weaknesses:

The mechanism allowing the discrimination between commensal and pathogenic bacteria remains unclear.

Based on our findings, we hypothesize that ROS play a crucial role in this discrimination process, with uracil release by pathogenic or opportunistic bacteria potentially serving as a key signal.

To test whether uracil could trigger this discrimination, we conducted experiments where Lp was supplemented with uracil. However, our results show that uracil supplementation alone was not sufficient to induce the blockage response (new data: Supplementary Figure 5). This suggests that while uracil may be a factor in bacterial discrimination, it is likely not the sole trigger, and additional bacterial factors or signals may be required to activate the blockage mechanism. 

The use of only two pathogens and one symbiotic species may not be sufficient to draw a conclusion on the difference in treatment between pathogenic and symbiotic species.

To address this concern, we performed additional intoxication experiments using Escherichia coli OP50, a bacterium considered innocuous and commonly used as a standard food source for C. elegans in laboratory settings. The results, presented in our updated data (new data: Fig 1B), show that E. coli OP50, despite being from the same genus as Ecc, does not trigger the blockage response. This further supports our conclusion that the gut’s discriminatory mechanism is specific to pathogenic bacteria, and not merely based on bacterial genus.

We can also wonder how the process of sphincter contraction is affected by the procedure used in this study, where larvae are starved. Does the sphincter contraction occur in continuous feeding conditions? Since larvae are continuously feeding, is this process physiologically relevant?

In our intoxication protocol, the larvae are exposed to contaminated food for 1 hour, during which the blockage ratio is quantified. Since this period involves continuous feeding with the contaminated food, we do not consider the larvae starved during the quantification process. Our observations show differences in the blockage response depending on the bacterial contaminant and the genetic background of the host. Additionally, we were able to trigger the blocking phenomenon using exogenous hCGRP.

Regarding the experimental setup for movie observations, it is true that larvae are immobilized on tape in a humid chamber, which is not a fully physiological context. However, in the new movie we provide (Movie 3), co-treatment with fluorescent Dextran (Red) and fluorescent Bt (Green) shows that both are initially blocked, followed by the posterior release of Dextran once the bacterial clearance begins.

Furthermore, to address the question of continuous exposure, we extended the exposure period to 20 hours instead of 1 hour. Even after prolonged exposure, we observed that pathogens are still blocked in the anterior part of the gut (new data: Supplementary Figure 2B). This supports the physiological relevance of the sphincter contraction and its ability to function under continuous feeding conditions.

Reviewer #1 (Recommendations For The Authors):

(1) The authors performed the experiments on Drosophila larvae. I wonder whether this model could extend to adult flies since they have shown that the ROS/TRPA1/Dh31 axis is important for gut muscle contraction in adult flies. If not, how would the authors explain the discrepancy between larvae and adults?

We link the adult phenotype to the one we describe in larvae in order to have the candidate approach toward the ROS/TrpA1/Dh31 axis. As we already mention in the discussion, while larvae stay in the food, adult flies can go away. If larvae eject their gut content, they may ingest it within minutes. We clarify our idea in the last part of the discussion.

(2) The authors performed their experiments and proposed the models based on two pathogenic bacteria and one commensal bacterial at a relatively high bacterial dose. They showed that feeding Bt at 2X1010 or Ecc15 at 4X108 did not induce a blockage phenotype. 

I wonder whether larvae die under conditions of enteric infection with low concentrations of pathogenic bacteria. 

Video provided with Bt-GFP 1.3 10^10 CFU/mL (new data: Movie 5). When larvae eat less, there is no blockage and bacteria can reach the posterior midgut. Note that the fluorescence is weak due to the low amount of bacteria ingested. The movie shows an excretion of the bacteria. There is also no death of the larvae. Together these results suggest that below a given threshold, the virulence of the bacteria is too weak to i) trigger a blockage and 2/ kill the larva. The bacteria are likely eliminated through classical peristaltism.

If larvae do not show mortality, what is the mechanism for resisting low concentrations of pathogenic bacteria? 

Maybe we are below the threshold of virulence. See our response just above.

Why is this model only applied to high-dose infections? 

As mentioned in the manuscript, lower concentrations do not trigger the blockage and for lower concentrations with a GFP signal still detectable, wild-type animals resist the presence of live-bacteria within the posterior part of the intestine.

About the doses, the CFU should be considered. Indeed, there are around 5.10^4 CFU per midgut. In our experimental procedure we calculate the amount of bacteria for 500 µl of contaminated medium (i.e. 4.10^10 CFU/500µl of medium). Then around 50 larvae were deposited in the 500µl of contaminated media. In this condition, one larva ingests 5.10^4 CFU. Moreover, larvae are only fed for 1h. 

So 1/ continuous feeding may also trigger locking even at lower doses and 2/ the other mechanisms of defenses (such as ROS) or peristalsis may be sufficient to eliminate lower doses (i.e. 10^3 CFU or below). See the new movie 5 we provide with Bt-GFP 1.3 10^10 CFU/mL

(3) The authors claim that the lock of bacteria happens at 15 minutes while killing by AMPs happens 6-8 hours later. 

Our CFU data indicate that it’s after 4 to 6 hours that the quantity of bacteria decreases. We fixed this in the text.

What happened during this period? 

ROS activity (bacteriostatic and bacteriolytic), IMD activation, AMP transcription, translation, secretion and bacteriostatic as well as bacteriolytic activity.

More importantly, is IMD activity induced in the anterior region of the larval gut in both Ecc15 and Bt infection at 6 hours after infection? 

We provide new data for larval whole gut RT-qPCR data in wt, TrpA1- and Dh31- genetic background fed with Lp or Ecc or Bt or yeast only (new data: SUPP6). We monitored 3 different AMP-encoding genes and found differences related to the food content, but no differences between genotypes. In addition, we provide images from AMP reporter animals (Dpt-Cherry) fed with fluorescent Lp or Bt (new data: SUPP11) showing that with Bt blocked in the anterior part of the intestine, the dpt gene is mainly induced in this area. Note that in the larva infected with Lp-GFP, the Dpt-Cherry reporter is weakly expressed in the anterior midgut. In the posterior midgut, the place where Lp-GFP is established, Dpt-Cherry is barely detectable. This observation is in line with the previous observation made by Bosco-Drayon et al., (2012) demonstrating the low level of AMP expression in the posterior midgut due to the expression of the IMD negative regulators such as amidases and pirk. In the larva infected with Bt-GFP, note the obvious expression of DptCherry in the anterior midgut colocalizing with the bacteria (new data: SUPP11).

Are they mostly expressed in the anterior midgut in both bacterial infections? Several papers have shown quite different IMD activity patterns in the Drosophila gut. Zhai et al. have shown that in adult Drosophila, IMD activity was mostly absent in the R2 region as indicated by dpt-lacZ. Vodovar et al. have shown that the expression of dpt-lacZ is observable in proventriculus while Pe is not in the same region. Tzou et al. showed that Ecc15 infection induced IMD activity in the anterior midgut 24 hours after infection. 

In ctrl animals fed Bt, Ecc and Lp we see Dpt-RFP in anterior midgut and likely in the beginning of acidic region. See the new data: SUPP11 images provided for the previous remark.

Using TrpA1 and Dh31 mutants, the authors found both Ecc15 and Bt in the posterior midgut. Why are they not evenly distributed along the gut? 

Same is true with Lp in wt; not evenly distributed. As if the transit time in the anterior part is very short due to peristaltism which would fit for a check point area if you’re not supposed to be blocked. Indeed, peristaltism is active during our intoxications. Then, it stays longer in the posterior part, fitting with the absorptive skills of the intestinal cells in this area. With Lp in ctrl or Ecc and Bt in TrpA1- and Dh31- mutants, there are always a few in the anterior midgut but always much less compared to the posterior. See our figure 1A and 3A.

Last but not least, does the ROS/TrpA1/Dh31 axis affect AMP expression?

We provide larval whole gut RT-qPCR data in wt, TrpA1- and Dh31- genetic background fed with Lp or Ecc or Bt or yeast only (new data: SUPP6). We monitored 3 different AMPencoding genes and found differences related to the food content, but no differences between genotypes. In addition, we provide images from AMP reporter animals (pDptCherry) fed with fluorescent Lp or Bt, (new data: SUPP11).

(4) The TARM structure part is quite interesting. However, the authors did not show its relevance in their model. Is this structure the key-driven force for the blocking phenotype and killing phenotype? 

Indeed, we would like to explore the roles of these structures and the putative requirement upon bacterial intoxication using some driver lines developed by the team that studied these muscles in vivo. However, the genetic tools currently available will target TARMsT1 and T2 at the same time. See Fig 2 form Bataillé et al, . 2020. Moreover, these TARMs are, at first, crucial for the correct positioning of the gut within the larvae and their absence lead to a global food intake and transit defect that will bias the outcomes of our intoxication protocol (see fig 6 from Bataillé et al,. 2020).

Is the ROS/TrpA1/Dh31 axis required to form this structure?

We provide images of larval guts from ctrl, TrpA1 and Dh31 mutants demonstrating the presence of the TARMs T2 structures despite the mutations (new data: SUPP8). In addition, we provide representative movies of peristalsis in intestines of Dh31 mutants fed or not with Ecc to illustrate that muscular activity is not abolished (new data: Movie 9 and Movie 10).

Minor points:

(1) Why not use the Pros-Gal4/UAS-Dh31 strain in Figure 3B in addition to hCGRP?

We opted for exogenous hCGRP addition because it allowed us precise timing control over Dh31 activation. Overexpression of Dh31 from embryogenesis or early larval stages could have significant and unintended effects on intestinal physiology, potentially confounding the results. While temporal control using TubG80ts could be an alternative, our focus was on identifying the specific cells responsible for the phenomenon.

To achieve this, we perturbed Dh31 production via RNAi, specifically targeting a limited number of enteroendocrine cells (EECs) using the DJ752-Gal4 driver, as described by Lajeunesse et al., 2010. Our new data (Supplementary Figure 4) demonstrate that Dh31 expression in this subset of cells is indeed necessary for the blockage phenomenon.

(2) Section title (line 287) refers to mortality, but no mortality data is in the figure.

We agree that the title referenced mortality, whereas no mortality data was presented in this section. We have updated the title to better reflect the data discussed in this part of the manuscript.

(3) It may be better to combine ROS-related contents in the same figure.

While it is technically feasible to consolidate the ROS-related content into one figure, doing so would require splitting essential data, such as the Gal4 controls for the RNAi assays and parts of the survival phenotype data. We believe that the current structure of the study, which first explores the molecular aspects of the phenomenon and then demonstrates its relevance to the animal’s survival, provides a clearer and more logical flow. For these reasons, we prefer to maintain the current figure layout.

Reviewer #2 (Recommendations For The Authors):

Major recommendation

(1) Other wild-type backgrounds should be added (including the w Drosdel background of the AMP14 deficient flies) to check the robustness of the phenotype.

To address the concern regarding the robustness of the phenotype across different wildtype backgrounds, we have tested additional genetic backgrounds, including w1, the isogenized w1118 and Oregon animals. 

The results (new data: Figure 1C) demonstrate that Lp is able to transit freely to the posterior part of the intestine in all backgrounds, while Ecc and Bt are blocked in the anterior part. These findings confirm the robustness of the phenotype across different wildtype strains.

(2) Although we recognize that this may be limited by the number of GFP-expressing species, other commensal and pathogenic bacteria should be tested in this assay (e.g. E. faecalis and Acetobacter).

We performed new intoxication experiments using Escherichia coli OP50, a wellestablished innocuous bacterial strain. The data, presented in Figure 1B (new data), show that E. coli OP50, despite being from the same genus as Ecc, does not trigger the blockage response. This further supports our hypothesis that the blockage phenomenon is specific to pathogenic bacteria and not simply related to the bacterial genus.

(3) It is important to test whether sphincter closure also occurs in continuous feeding conditions. This does not mean repeating all the experiments but just shows that this mechanism can take place in conditions where larvae are kept in a vial with food.

While the movies we provide involve larvae immobilized on tape in a humid chamber, which is not a fully physiological context, we now provide new data (Movie 3) showing that, after co-treatment with fluorescent Dextran (Red) and fluorescent Bt (Green), both substances are initially blocked in the anterior midgut. Later, the dextran is released posteriorly once bacterial clearance has begun.

Additionally, we extended the feeding period in our experiments from 1 hour to 20 hours to simulate more continuous exposure to contaminated food. Even under these prolonged conditions, we observed that pathogens are still blocked in the anterior part of the gut (new data: Supplementary Figure 2B). This confirms that the sphincter mechanism can function in continuous feeding conditions as well.

(4) What are the molecular determinants discriminating innocuous from pathogenic bacteria? Addressing this point will increase the impact of the article. The fact that Relish mutants have normal valve constriction suggests that peptidoglycan recognition is not involved. Is there a sensing of pathogen virulence factors? 

Our data suggest that uracil could be a key molecular determinant in discriminating between innocuous and pathogenic bacteria, as previously described by the W-J Lee team in several studies on adult Drosophila. However, in our experiments, exogenous uracil addition using the blue dye protocol (Keita et al., 2017) did not induce any significant changes in the larvae. Similarly, uracil supplementation in adult flies failed to trigger the Ecc expulsion and gut contraction phenotype, as reported by Benguettat et al., 2018. 

To further investigate this, we tested the addition of uracil during Lp-GFP intoxication. In these experiments, we did not observe any blockage of Lp (new data: Supplementary Figure 5). These results suggest that uracil might not be the sole trigger for the blockage response, or we may not be providing uracil exogenously in the most effective way. Alternatively, there could be other pathogen-specific virulence factors that contribute to this discrimination mechanism.

To address this question, the authors should infect larvae with Ecc15 evf- mutants or Ecc15 lacking uracil production. 

Thank you for your suggestion to use Ecc15 evf- mutants or Ecc15 lacking uracil production to explore the role of uracil in bacterial discrimination. While we have provided some data using uracil supplementation (new data: Supplementary Figure 5), we agree that testing mutants like PyrE would be an important next step. Unfortunately, we currently lack access to fluorescent PyrE or Ecc15 evf- mutants.

We are planning to address this by developing a new protocol involving fluorescent beads alongside bacteria. This approach will allow us to test several bacterial strains in parallel and better define the size threshold of the valve. However, we do not have the relevant data yet, but this will be a key focus of our future work.

Similarly, does feeding heat-killed Ecc15 or Bt induce sequestration in the anterior midgut (larvae may be fed dextran-FITC at the same time to track bacteria)?

Unfortunately, in our attempts to test heat-killed or ethanol-killed fluorescent Ecc15 for these experiments, we encountered an issue: while we were able to efficiently kill the bacteria, we lost the GFP signal required to track their position in the gut. This made it challenging to assess whether sequestration in the anterior midgut occurs with non-viable bacteria.

Is uracil or Bt toxin feeding sufficient to induce valve closure? 

As previously mentioned, uracil is a strong candidate for bacterial discrimination, and we have tested its role by adding exogenous uracil during Lp-GFP intoxication. However, in these experiments, Lp was not blocked (new data: Supplementary Figure 5). This suggests that uracil alone may not be sufficient to induce valve closure, or it may not be the only factor involved. It is also possible that our method of exogenous uracil supplementation may not be effectively mimicking the endogenous conditions.

Regarding Bt, we used vegetative cells without Cry toxins in our experiments. Cry toxins are only produced during sporulation and are enclosed in crystals within the spore. The Bt strain we used, 4D22, has been deleted for the plasmids encoding Cry toxins. As a result, there were no Cry toxins present in the Bt-GFP vegetative cells used in our assays. This has been clarified in the Materials and Methods section of the manuscript.

Would Bleomycin induce the same phenotype? 

Indeed, Bleomycin, as well as paraquat, has been shown to damage the gut and trigger intestinal cell proliferation in adult Drosophila through mechanisms involving TrpA1. Testing whether Bleomycin induces a similar phenotype in larvae would indeed be interesting.

However, one challenge we face in our intoxication protocol is that larvae tend to stop feeding when chemicals are added to their food mixture. We encountered similar difficulties in our DTT experiments, which were challenging to set up for this reason. Consequently, we aim to avoid approaches that might impair the general feeding activity of the larvae, as it can significantly affect the outcomes of our experiments.

Could this process of sphincter closure be more related to food poisoning?

If gut damage were the primary trigger for sphincter closure, we would indeed expect the blockage phenomenon to occur later following bacterial exposure. However, in our experiments, we observe the blockage occurring early after bacterial contact, suggesting that damage may not be the main trigger for this response.

That said, we have not yet tested bacterial mutants lacking toxins, nor have we tested a direct damaging agent such as Bleomycin, as proposed. These would be valuable future experiments to explore the potential role of gut damage more thoroughly in this process.

(5) Is Imd activation normal in trpA1 and DH31 mutants? The authors could use a diptericin reporter gene to check if Diptericin is affected by a lack of valve closure in trpA1.

To address this, we performed RT-qPCR on whole larval guts from wt, TrpA11 and Dh31KG09001 genetic background. Larvae were fed with Lp, Ecc, Bt or yeast only (new data: SUPP6). We monitored the expression of three different AMP-encoding genes and found that while AMP expression varied depending on the food content, there were no significant differences in AMP expression between the genotypes.

Additionally, we provide imaging data from AMP reporter animals (pDpt-Cherry) in a wildtype background, fed with fluorescent Lp or Bt (new data: Supplementary Figure 11). These images also support the conclusion that Diptericin expression is not significantly affected by a lack of valve closure in trpA1 and Dh31 mutants.

(6) Are the 2-6 DH31 positive cells the same cells described by Zaidman et al., Developmental and Comparative Immunology 36 (2012) 638-647.

The cells identified as hemocytes in the midgut junctions by Zaidman et al. are likely the same cells we describe in our study, as they are located in the same region and are Dh31 positive. We have added a reference to this paper and included lines in the manuscript acknowledging this connection.

Although confirming whether these cells are Hml+, Dh31+, and TrpA1+ would clarify their exact identity, this falls outside the scope of our current study. However, the possibility that these cells play a role in physical barrier immunity and also possess a hemocyte identity is indeed intriguing, and we hope future research will explore this further.

Minor points

(1) The mutations should be appropriately labelled with the allele name.

This has been fixed in the main text, in Fig Legends, and in figures. 

(2) Line 230-231: the sentence is unclear to me.

We simplified the sentence and do not refer to the expulsion in larvae.

(3) Discussion: although the discussion is already a bit long, it would be interesting to see if this process is likely to happen/has been described in other insects (mosquito, Bactrocera, ...).

We reviewed the available literature but were unable to find specific examples describing the blockage phenomenon in other insects. Most studies we found focused on symbiotic bacteria rather than pathogenic or opportunistic bacteria. However, as mentioned in our manuscript, the anterior localization of opportunistic or pathogenic bacteria has been observed in Drosophila by independent research groups.

(4) Line 546: add the Caudal Won-Jae Lee paper to state the posterior midgut is less microbicidal.

We added the reference at the right place, mentioning as well that it concerns adults. 

(5)  Figure 6 indicates what the cells are, shown by the arrow.

The sentence ‘the arrows point to TARMs’ is present in the legend of Fig6.

(6) Does the sphincter closure depend on hemocytes?

As mentioned above, the cells we identify as TrpA1+ in the midgut junction may be the same cells described by Zaidman et al., 2012, and earlier by Lajeunesse et al., 2010. Inactivating hemocytes using the Hml-Gal4 driver may also affect these Dh31+ cells, as they share similarities with hemocytes, as pointed out by Zaidman et al. However, distinguishing between hemocytes and Dh31+/TrpA1+ cells would require a genetic intersectional approach, which is beyond the scope of our current study.

Nevertheless, the possibility that these cells play a dual role in immunity (through blockage) and share characteristics with hemocytes while functioning as enteroendocrine cells (EECs) is quite intriguing and deserves further exploration in future studies.

Evidence of syncretism? I cannot think of an instance where this may be permissible in Abrahamic religions

Intrusive Japanese colonial government interference in local festival

This might be the future of education, and I am glad that teachers and educators are already on board for it.

Teachers and educators should be informed on what Ai is and how it works. This will prevent students to walk all over the teachers.

This definitely true. Teachers should definitely use the detecting app though. The teachers can see how much of AI the students used.

Surprising!! I thought teachers would not like it.

Teachers and educators should take advantage of Ai in this sense. Students love to take brain breaks and play games.

This was defiantly needed. I am glad that researchers thought of this and did it.

Ai should not be in the same category as a human or animal. It is not living.

Yes, this is so true. That is why we should have people overseeing these systems.

They should create these committees, and they should put them to use. People should be watching Ai closely so it doesn't become what people thinks it's going to be.

This is the best way to explain what AI is. Ai responds to us with the best answer that they think or have researched.

This could be true but also not be true. AI could take over and become the "new" humans, it might run our jobs. Ai will always stay on the computers.

This is just the new technology. Students are also getting smarter with AI. AI allows you to search ideas and examples for them

I don't think AI alone will do this. I think technology has already accomplished that.

This is definitely a worry that most schools systems have. Even without Ai, students would easily cheat but with AI its more easily too. Educators and teachers should encourage students to only to use AI when they are stuck or need some ideas.

This is a good idea. Parents are already busy; they should be able to get help with letters. Forming a letter for the schools can be difficult.

This was really good research that the AI department had to do. Before I read why I thought the educators probably would not like it. It does make sense that educators and parents are scared of it.

The people that created AI make is easy and assessable for the users that need it.

the term is defined as fear of new things or experiences. From my understanding of reading an article on this, it is a form of anxiety that can manifest in individuals as an aversion to trying new foods, or adapting to changes in their environment. In psychological research, neophobia is often studied in the context of behavioral ecology, particularly in relation to food choices and habitat selection. Animals that exhibit neophobia may avoid unfamiliar food sources, impacting their foraging behavior and overall survival

Colour temperature refers to the perceived warmth or coolness of light, measured in Kelvins (K). Warmer color temperatures, typically around 2700K to 3000K, emit a yellowish hue, while cooler temperatures (5000K and above) produce a bluish light. Research indicates that different color temperatures can influence animal behavior, including foraging and roosting patterns in bats. In New Zealand, the common use of cooler light sources in urban environments may have implications for bat ecology, potentially affecting their activity levels and interactions with prey. This research contributes to understanding how artificial lighting impacts nocturnal wildlife and informs strategies for minimizing light pollution

Ultrasonic acoustic monitoring is a method that employs microphones capable of detecting sounds above the frequency of human hearing, typically above 20kHz. This technology is crucial in studying bat populations, as many species, including long-tailed bats (Chaerephon pumilus), communicate and navigate using echolocation, producing ultrasonic calls that can inform researchers about their behavior, habitat use, and population dynamics.

what?

The next passage demonstrates that the quality of selflessness, whereby the signers of the Declaration of Independence placed the interests of their country above their own, is noble indeed. He says, "This is not the highest form of human excellence," thereby allowing that there could be virtues that rise above patriotism but still insisting that this particular virtue is rare and commendable. The phrase "ought to command respect" really drives this view home that such selflessness deserves the respect of others. The speaker then further emphasizes the idea of sacrifice when he says that one who can "intelligently lay down his life for his country" possesses a character that "it is not in human nature to despise." What this suggests is that, at the level where a man acts out bravery and commitment to a cause, the status in society of that individual is raised to the level of one worthy of honor and respect. What this text does, above all, is appeal to the patriotic virtue of self-sacrifice for the greater good.

In the following passage, he addresses his audience as his "Fellow Citizens," creating an atmosphere of great rapport, thus defining them by identity. He begins by showing respect to the "fathers of this republic" because the signers of the Declaration of Independence were brave and great. The phrase "great enough to give fame to a great age" infers that they gained not only personal fame but also gave much to the turning point of an age. He makes it a point to mention that it is rare for a nation to have such a number of "truly great men" together, which gives precedence over the normalcy of such a historical context. He readily admits that his own view of these figures is "not, certainly, the most favorable" and thus hints at a critical stance that may arise from his experiences or views on their actions. He then concludes, though, by saying he cannot consider their "great deeds" with anything other than admiration, showing that despite whatever critiques he may have, he realizes the deep impact their contributions had in founding the nation. This passage balances respect with critical awareness in appreciating historical figures.

Here, the speaker provides an image of marine imagery that reflects urgency and risk for the nation's state. He mentions "dark and threatening clouds" and "heavy billows, like mountains," which portend difficulties and barriers that might undermine the country's stability. "Huge forms of flinty rocks" speaks to the possible dangers should the country be led off course from its founding precepts. He further drives this point home with the phrase "that bolt drawn, that chain broken, and all is lost," indicating that to abandon these principles would prove disastrous. He then begs the audience to "cling to this day—cling to it, and to its principles," where he uses steadfastness and commitment, comparing the resolve of the people to a "storm-tossed mariner" desperately reaching for any hold on safety during a perilous night at sea. This metaphor shows that there is a dire need for unity and determination in surmounting the impending turbulent task.

Here, the speaker provides an image of marine imagery that reflects urgency and risk for the nation's state. He mentions "dark and threatening clouds" and "heavy billows, like mountains," which portend difficulties and barriers that might undermine the country's stability. "Huge forms of flinty rocks" speaks to the possible dangers should the country be led off course from its founding precepts. He further drives this point home with the phrase "that bolt drawn, that chain broken, and all is lost," indicating that to abandon these principles would prove disastrous. He then begs the audience to "cling to this day—cling to it, and to its principles," where he uses steadfastness and commitment, comparing the resolve of the people to a "storm-tossed mariner" desperately reaching for any hold on safety during a perilous night at sea. This metaphor shows that there is a dire need for unity and determination in surmounting the impending turbulent task.

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.

In the case of Tom, someone fulfills this requirement that isn't Tom.

This may very well be true, but his example does not fully exhibit this since there will always be an agent responsibly for actions, just may be the actions of others.

what about committing good deeds by accident? What about cases of a martyr- someone (uncontrollably dies due to intervention by another, and this wrongful death caused a huge civil rights movement- example George Floyd. Despite what anyones political values may be, it's undeniable the the death of George Floyd sparked the reimergence and growth of the BLM movement. Floyd had no control over the factors surrounding his death, yet I think anyone would consider this life meaningful in a sense.

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 steps could future research take to ensure these findings are more conclusive, and could additional longitudinal studies help?

Could the role of the right hippocampus in prospective memory be a key factor in how meditation impacts cognitive functions?

Are the anatomical templates used in MEG source reconstruction robust enough to capture individual variations in meditators' brain structures?

How does MEG's temporal resolution compare to fMRI in identifying neuroplasticity changes from meditation?

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?

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.

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)

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.

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