Chorus 2

This is one of the most famous lines from the song. It's the perfect mix of optimism and reality - saying that even in the good stuff there's something imperfect, something to remind you life isn't lawless. It's such a down-to-earth way of acknowledging that things aren't always as tight as they seem.

This lyric neglects to address a specific emotion to make room for a variety of emotions the listener may possibly be experiencing. This given space allows the listener to prescribe their own worry, sadness, anger, frustration, or other struggles to this lyric and relate. This simplicity and vagueness that creates space for listeners to relate is a common technique used throughout the song to make it more broadly accessible

Originally, the lyric was supposed to and with "piss off" instead of the final studio version as noted in the lyrics here, "get out." The phrasing was revised due to anxiety over the line hurting the track's air time on the radio. This anxiety is emblematic of a shift in the band's attitude towards radio since their carlier album, "Working Man's Dead That era of the band's career relished in counter-cultural ignorance of platforming their music through radio to achieve air-time revenue. Instead, they spread their music through live performances allowing fans to bootleg audio recordings.

Like the previous lyric "it's alright" these lines have the same function. This is the chorus of the song. The lyrics "I will get by" are general and simple. They are quintessential examples of the positive messaging caked in throughout the lyrics. No matter the struggle, it ensures the listener that the trouble will pass and their current worries will enter the realm of the past. It suggests the bare minimum of survival is enough, and makes the complicated stressful nature of life more simple and less existential. Those lyrics function therapeutically and appeal universally to an audience of listeners. The inclusiveness of the lyrics makes one ot the most memorable parts of the song appealing to a larger mainstream audience.

Chorus 1

Follow up on last post here

This positive message repeats itself throughout the song's lyrics. It effectively complements the joyful music that backs Jerry Garcia's vocals. The inherent positivity and happiness the project harbors arguably helped the Grateful Dead reach a broader audience of pop music consumers which made the record go platinum.

The "it's alright" lyric first appears here but regularly reoccurs throughout the song's lyrics. The structure of the line is clear and concise and uses a common phrase of reassurance. This lyric's repetition makes it a bigger takeaway after listening to the song. in reaches out to listeners and naturalizes their anxiety, stress, and struggles, it provides relief that alleviates some of the pressure felt by listeners. It also reinforces overarching themes of hopefulness and joy. The overall uplifting function of this lyric mimics pop music

These three lines establish a consistent meter of seven syllables per lyric and a consistent rhyme scheme stressing the "air" sound at the ending of each last word. This sonically makes the lyrics have more synchronicity and consistency and establishes an Inviting, accessible lyrical rhythm

The world feels fake. its like they are frustrated with all the surface level stuff, and the rawness of the world is no where to be found.

This line gives off the vibe of life being too scripted or fake. A painted sky-one that's supposed to be natural - feels artificial. it speaks to how things in the world can seem really constructed, especially when you're just going through the motions.

The clear juxtaposition of "early" and "late" in the song's first two lines establishes connectivity through generating a reciprocal This choice adds a palatable coherence and flow to hook listeners and encourage listening further into the song

Time is always moving, but here it feels like everything is out of sync. It hints at the feeling that no matter how hard you try, you're always behind.

Opening the song with a sense of earliness signifies dawn and the beginning of a new day. The following lyrics, "Paint by numbers morning sky,* "Dawn is breaking everywhere," and "Draw the curtains," clarity and emphasize morning imagery, newness, and a sense of rebirth. The choice to start the song like starting a new day employs chronology and fullness that makes the track more appealing.

This conclusive lyric sets up the ending chorus of the song and nicely wraps up the track in a satisfactory, full-circle fashion. The lyrics comment on a cohesive structure of the song that establishes a clear lyrical flow and chronology that is satisfactory for music listeners.

4th extended (modified) chorus

Chorus 3

Chorus 1

Chorus 2

The use of "we" stresses the innate collectivism the song harbors throughout its structure. The union of artist and audience further makes the song palatable and accessible for a broader universal audience beyond the band's regular returning listeners.

This lyric builds on the song's simplification of stress. Problems can seem big and overwhelming when one is "in the weeds," however the general use of reassuring language here adds to the song's "safe haven" environment that comforting lyrics create.

The lyric "and try to keep a little love" again stresses positivity and is a repetition of a previous lyric "and try to keep a little grace." This is another example of how lyrical repetition is used regularly throughout the song. It resembles similar lyrical trends in popular mainstream music that are musically satisfactory to the listener. "Love" a trope commonly sung about in popular music, is also integrated into the lyrics of the song.

The lyrical repetition emphasizes the overarching message of perseverance throughout the song. The original "I will get by" line switches the use of pronouns to "we" which is symbolic of the song's use of collectivism to make itself more accessible to its audience.

This lyric presents positive imagery of carrying on and persevering. It's emblematic of "choosing to be happy" and contributes to the song's overall positive messaging.

Trying to keep a little grace is emblematic of keeping one's "cool" and persevering through hard times. This message is emblematic of the song's uplifting purpose of stressing perseverance. This contributes to the song's positive messaging.

This alludes to the old military alphabet before it was changed in 1952 to what it is today.

The ABCs are a stand-in term for struggles that "we all must face". The song employs collectivism, uniting its listeners under their different struggles.

Danniel Integral

Together, each \(x \in \theta_0\) and each \(y \in \theta_1\) intersect, making the map surjective.

In term of congruence, it means \(I_i \vee I_j = R\), which make decomposition map for algebra with permutable congruence surjective

Mathematical Structures From Linear Algebra over Rings to Geometry with Sheaves

Todo bien con tu proyecto. Te quedo muy bien el video y la modelacion esta bastante bien logradad.. Mi pricipal observación tiene que ver con la documentación de tu implementación en R. Sólo me faltan los docstrings de cada función. En un futuro si te gustaria seguir desarrollando de forma mas formal, es necesario considerarlas. En general todo muy bien.

Sobre tu calificación. Según mis cuentas el total suma 92 para tu calificación final.

Definition: ideas are reoccurent, as meaningful today as when they were written

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？

松浦さんがすごい尊重してくれる部分

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eterogeneous means made up of different or dissimilar components, or having a non-uniform composition

"homogeneous" refers to a mixture where the composition is uniform throughout,

Maybe "with" should be "when"??

as in "adjustments are needed when ..."

几乎消除

this is a reason for the use of realistic evaluation

We should mention that the description can't serve as the accessible description for an image. That has to be linked to separately.

This section offers a very useful method in accessing research on topics using the reference section as articles and documents in that section will definitely contain information regarding the main topic and can help in branching out and broadening research. It can also be used as a great tool for cross referencing information.

Ok, good...well written. I wonder if there is perhaps more continuity in the two phases of residence cited? But, certainly the scale, at least, of the commissioned work in Milan was a major factor.... BTW, the "Sforza Horse" is a modern reconstruction and should be id'd as such

Zelfwaardering werkt in het brein een beetje zoals beloning. In een experiment maakten mensen een profiel en kregen ze likes als sociale beloning. In een MRI-scanner werd gekeken hoe hun brein reageerde. Als ze een like verwachtten en die ook kregen, zagen onderzoekers een reactie in het brein, net zoals bij beloningen zoals eten of geld. Dit heet een reward prediction error (RPE).

Zelfwaardering bleek samen te hangen met activiteit in een hersengebied dat te maken heeft met zelfbeeld (vmPFC/pgACC). Dit laat zien dat ons zelfbeeld beïnvloed kan worden door sociale feedback, zoals likes, net zoals hoe beloningen onze waardering voor iets beïnvloeden. Dus: zelfwaardering werkt in het brein vergelijkbaar met andere vormen van beloning.

Dit werkt als volgt: de aap voorspelt nu de beloning door het lichtje. Als hij exact krijgt wat hij verwacht, gebeurt er niets bijzonders. Maar als hij méér krijgt dan verwacht, is er een positieve verrassing (prediction error) en worden de neuronen actief. Als hij minder krijgt dan verwacht, of helemaal niets, dan reageren de neuronen juist minder.

Molecular findings:

Severe bleeding phenotype in homozygotes, alleviated bleeding phenotype in heterozygotes.

Difficult to classify based on the phenotype and plasma levels.

p.M771V variant shows elevated proVWF to mature VWF ratio, reduced levels of cleaved VWFpp.

p.M771V has potential to negatively influence VWFpp cleavage to contribute to decreased VWF processing in endothelial cells.

Other variants near p.M771 locus reported to disrupt the cleavage by furin. (Variants are at R763 and R760 respectively).

Other researchers have come across the p.M771 variant and reported it as well.

functional studies performed in cord blood derived ECFCs through adenine base editing and overexpressed in HEK293 cells.

Utilized Immunocytochemistry staining and confocal analysis

ELISA to test for secreted VWF in ECFCs

Multimer assay and densitometry graphs to view VWF multimer patterns

Western blotting to view proteolytic processing of VWF and VWFpp

Genomic sequencing to verify mutations.

Authors here used SpCas9-mediated base editing to mimic patient variant if primary ECFCs are not available or are difficult to culture to assist in improved characterization in a true endothelial background.

Disease: Von Willebrand Disease (VWD)

Patient(s): Found in 2 families

Variant: VWF NM_000552.5: c.2311A>G, p.(M771V Homozygous variant in exon 18 (VWF D' domain; 8 residues down from proteolytic VWFpp furin cleavage site)

Family: In family 1 there are 4 homozygous patients (2 male and 2 female), and one heterozygous patient (1 female). The affected females are denoted as person 1 and person 4 and the affected males are person 2 and person 3. There are three WT family members (1 female and 2 male), grandparents of these members are of unknown genotype including a daughter of an affected female and a WT male. Note here that in the family a p.R2663P variant has co-segregated with the above-mentioned variant but is not suspected to be the pathogenic driver of resulting bleeding tendency.

In family 2 the parents of the homozygous affected male are of unknown genotype. The affected male is denoted as person 7.

Phenotypes: Person 1- nose bleed, skin bleed, GI bleeding, oral cavity bleeds, Menorrhagia, muscle bleeding, and joint bleeding. Receives on-demand treatment for bleeding.

Person 2-Nose bleed, skin bleed, bleeding from small wounds, oral cavity bleeds, bleeding after tooth extraction, joint bleeding. Received prophylactic treatment, reduced to on-demand treatment after a few years.

Person 3-Nose bleed, skin bleed, oral cavity bleeds, bleeding after tooth extraction, muscle bleeding. Receives on-demand treatment for bleeding phenotype.

Person 4- Nose bleed, bleeding from small wounds, oral cavity bleed, bleeding after tooth extraction, joint bleeding. Received prophylactic treatment that was increased after her menarche.

Person 7- Nose bleed, oral cavity bleeds, bleeding after surgery or trauma, joint bleeding. Previously on prophylaxis, now managing bleeding with on-demand treatment.

Note that both the p.R2663P co-segregated variant and p.M771V variant are reported in NCBI dbSNP database but functional effect not yet established.

NGS confirmed the genotype of all study participants.

test

In this area it gives a interpretation how the comparison comes into play with anthropology using some aspects and gives an understanding from one society to another and the difference between many factors also including its approach

First one to annotate on appstream

test

AMBITIOUS thesis....too ambitious! Quite a lot of effort went into this project...but perhaps a narrower approach might have been a better approach...every aspect of Michelangelo's artistic output is really beyond the scope of a 4 page StoryMap...you touch on a number of important areas of Michelangelos' life and career....next tome pick one and do a deeper dive into the specific scholarship on the subject.

SEM cannot give you quantitative height information.

should be area!

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

OK, good. I think some more time spent on how precisely Manet's work differed from the typical Salon work might have been merited. The typical landscape scenes of the impressionists also don't really play a huge role in Manet's universe....so, where does that leave us? A painter preoccupied with the grand figure narratives of the past...Titian, Velasquez, Goya etc...socially connected to the avante-gard impressionists, nevertheless eschewing their most obvious stylistic concerns....a sui generis figure that is all but impossible to pigeonhole...for me Manet is the most compelling painter among the impressionists and their followers...the first modern artist in me estimation.

phenomenon

Date missing.

ohui

La explicacion es bastante clara y autoncontenida. Me gusta la aplicación y se ve que el proyecto da para extenderlo a juegos dinámicos.

No vi el video donde se presenta tu proyecto. Aún así creo que con lo que tienes alcanzas el 100. Buen trabajo

Will bring up in next all-team meeting, but we would like this to be an interactive multiple choice questionnaire. I will pass the necessary "feedback" portion on to Bob and Vanessa

In the H5P below, slide 3 of 4 has much smaller font than the rest.

For clarity, maybe "Keep in mind that a problem fosters an unfavorable situation that you want to change"

Last reviewed 3/19/2024 (v1.1 Update)

SITC continuously evaluates the field for emerging data and new FDA approvals. Updates to the recommendations, tables, treatment algorithms, and/or guideline text in this publication and made with the approval of the SITC NMSC CPG Expert Panel. More information on SITC Guidelines can be found at sitcancer.org/guidelines.

v1.1 Update Summary

• The FDA granted accelerated approval for retifanlimab (anti-PD-1 ICI) for the treatment of adult patients with metastatic or recurrent locally advanced MCC in March 2023. The NMSC CPG was updated in the following locations: Introduction, Merkel Cell Carcinoma, Recommended Immunotherapies for MCC, Figure 1 – FDA-Approved ICI agents for NMSC, Table 2 – NMSC Landmark Clinical Trial Data Leading to FDA Approvals for ICIs for MCC and Novel Strategies and Promising Future Directions. [Ref 177, 178]

• Data have been reported demonstrating efficacy with neoadjuvant anti-PD-1 ICI therapy prior to curative-intent surgery in patients with CSCC. Based on these practice-changing data, the NMSC CPG was updated in the following locations: Based on these practice-changing data, the NMSC CPG was updated in the following locations: Recommended Immunotherapies for CSCC, and Novel Strategies and Promising Future Directions for CSCC. [Ref 179]

• Data have been reported demonstrating efficacy combining an anti-PD-(L)1 ICI with an anti-CTLA-4 ICI for patients with advanced MCC. Based on these practice-changing data, the NMSC CPG was updated in the following locations: Merkel Cell Carcinoma, Recommended Immunotherapies for MCC, and Novel Strategies and Future Directions for CSCC. [Ref 180, 181]

See highlighted text for updated content and more detailed information.

Last Reviewed 3/15/2024 (v1.2 Update)

SITC continuously evaluates the field for practice-changing data and new FDA approvals. The information on this page provides a detailed overview of updates to the guideline content based on changes in the field. Updates to the guideline outlined below were made with the approval of SITC's Breast Cancer CPG Expert Panel. More information on SITC Guidelines can be found at sitcancer.org/guidelines.

v1.2 Update Summary

• The FDA granted accelerated approval for dostarlimab for the treatment of dMMR recurrent or advanced solid tumors (along with the VENTANA MMR RxDx Panel companion diagnostic to detect dMMR) that have progressed on or following prior treatment and who have no satisfactory alternative treatment options on August 17, 2021. Based on these approvals, the Breast Cancer CPG has been updated in the following locations: Immunotherapy with PD-(L)1 Inhibitors for the Treatment of Advanced/Metastatic Breast Cancer and Diagnostics and Biomarker Testing in Patients with Advanced/Metastatic Breast Cancer. [Ref 290]
• The FoundationOne CDx assay was approved as the companion diagnostic for identifying patients with MSI-H tumors for treatment with pembrolizumab in February 2022. Based on this approval, the Breast Cancer CPG has been updated in the following locations: Diagnostics and Biomarker Testing in Patients with Advanced/Metastatic Breast Cancer. [Ref 291]

See highlighted text for updated content and more detailed information.

https://www.liberation.fr/societe/en-direct-cyclone-chido-a-mayotte-il-faudra-etre-beaucoup-plus-dur-vis-a-vis-de-limmigration-des-comores-lance-retailleau-20241218_AHY5ULB74ZCN3FTUSFXHDDD5WQ/

Excelente. Jazmin. Me gusto tu trabajo. Tu calificacion final, por supuesto es un 100.

Ok, big subjects touched on in the project. Good effort to draw from secondary sources. ..but the reading, the bibliography is quite basic. Cassatt seems to be an independent figure asserting feminist ideas...the depiction of the traditional roles of women, child-rearing , needlepoint, celebration of motherhood, etc. can be seen as empowering, but it might also be argued that these self-same subjects could be viewed as safe, "domestic" subjects, only modestly nudging the liberation of women forward.<br /> I was very interested in her travels which may be another clue about her indepedence...but might also raise interesting questions about her social status...was she a feminist pioneer or rather a wealthy scioness free to move about without the hindrance of and obeisance to a husband. Perhaps she was both which would merit tconsideration

This is a lot to unpack...for example, wasn't much of her time spent in France and thus her paintings were on french "subjects" ?

Todo Bien David. Alcansaste sobradamente el 100 de calificación. Vamos por buen rumbo.

Imagen de cada personaje

Kraven Johnson clips

Bias By: Slant

Bias by: Opinion Statements Presented as Fact.

I think this is subjective bias because when they say it says make some sense can be seen as a way to put down the system.

I think this is fair and balanced because they are stating facts that they have noticed.

This is fair commentary because they are stating an unbiased fact

Hi Gabriel,

I just wanted to take a moment to share my thoughts on your project. I truly appreciate the effort and dedication you put into it, and I’m thrilled to say you achieved a perfect final grade of 100! Well done!

The bibliography is quite thin...the thesis, while informative barely gets at much that is not pretty introductory and the commentary on the works is perfunctory as well

EXPLAIN

ID: Orazio Gentileschi was an important painter in this period in Rome

Ok, theproject reads too much like a survey of life and career, which is good as far as it goes, but the problem with this is that it doesn't really get us much further down the road in our understanding of J.L.David,,,and the geographic connections line up with basic information as well.

Show casting" do you mean showcasing?

There is not a clear thesis in all of this introductory material, which is informative, but too general.

this shit oswsijoffewnjdfijowedsbjlifeasjzdfhdihfgweasfjuewejkbfhusahgfwygsudgysayugdwqgdgywjgdgjgjyedfgwsgdfjasghjhshdjksdcdkjcfsdfdsxdsakjd skibike sigma shsahshjashahjsahjahjshjajhashaahjsaajsjas kys nigger

test

If they're able to come home in March it will be a nine month trip for them. Almost a whole year in space instead of the 8 days planned

This isn't looking great for Boeing, who is already dealing with blowback from mechanical failures in their commercial airliners. The astronauts have taken the situation well because they are both from the Navy.

This is an interesting story. I found it cool that when searching for French news I stumbled upon a story that had two Americans at its' center because news in space is very relevant on an international level. Nine months in space is a pretty long time, basically an entire pregnancy term. It seems like the landscape for manufacturing at the level of space travel may need a shakeup and for competition to push the old manufacturers like Boeing to up their standards. The consequences can be massive.

mired

Failures

transported

Shipwrecked

Nasa Announced that it was postponing the Crew-10 launch until March at the earliest while they build a new ship.

Their ship named the Starliner departed back in September and it was decided that the astronauts would come home on a SpaceX Crew-10 Ship.

The two astronauts have been stuck in the International Space station since June due to mechanical faults on the spaceship that brought them there. Their names are Butch Wilmore and Suni Williams.

Voici un sommaire minuté des temps forts de la transcription :

• 0:38 - Introduction et salutations
• 1:03 - Sujet de la conférence : "Le scientifiquement prouvé" et "l'appel au complot", deux facettes d'une même méprise épistémique.
• 1:14 - Domaine de l'épistémologie et philosophie des sciences.
• 1:35 - Contexte de la rhétorique antagoniste entre "le scientifiquement prouvé" et "l'appel au complot".
• 2:42 - Objectif de la conférence : démontrer l'erreur épistémique commune aux deux extrêmes.
• 3:17 - Plan de la conférence en 4 parties.
• 3:55 - Cas d'étude : la découverte des satellites de Jupiter par Galilée en 5 étapes.
• 4:50 - Contexte de l'astronomie et cosmologie aristotélicienne.
• 6:20 - Importance du contexte pour fixer le champ des contre-possibilités à l'hypothèse de Galilée.
• 7:24 - Processus épistémique d'élimination des possibilités d'erreur.
• 11:35 - Définition de la connaissance comme processus d'élimination des contre-possibilités pertinentes au contexte.
• 12:55 - Présentation de la métathéorie contextuelle de la connaissance de David Lewis.
• 15:45 - Définition du "scientifiquement prouvé" et de "l'appel au complot".
• 18:50 - L'erreur épistémique commune aux deux faces : la nécessité d'éliminer TOUTES les contre-possibilités ?
• 21:57 - Appel aux communicateurs de la science à ne pas présenter la science comme infaillible.

Briefing Doc: Le scientifiquement prouvé et l'appel au complot, les deux faces d'une erreur épistémique Source: Conférence d'Olivier Sartenaer, professeur de philosophie des sciences, REC Toulouse.

Lien: https://www.youtube.com/watch?v=Yt8WjvthM_0

Thème principal: La conférence explore une erreur épistémologique commune aux arguments du « scientifiquement prouvé » et de l'« appel au complot ».

Idées et faits importants:

L'erreur épistémique: Croire que la connaissance scientifique nécessite l'élimination de toutes les contre-possibilités, qu'elles soient pertinentes ou non au contexte donné. Le "scientifiquement prouvé" prétend atteindre une vérité absolue en éliminant toutes les objections, même les plus improbables. L'appel au complot exploite cette faille en introduisant des contre-possibilités irrationnelles, sapant ainsi la validité de la connaissance scientifique. Le cas de Galilée: Sartenaer utilise la découverte des satellites de Jupiter par Galilée pour illustrer le processus de construction de la connaissance scientifique. Galilée a dû réfuter les contre-possibilités pertinentes au contexte de l'époque (cosmologie aristotélicienne), sans se soucier de complots imaginaires. Citation: « Connaître c'est éliminer les contre possibilités pertinentes et pertinentes au regard de quoi du contexte. » (12:35) Théorie contextualiste de la connaissance: Inspirée par David Lewis, cette théorie souligne l'importance du contexte pour déterminer la validité d'une affirmation de connaissance. Ce qui est considéré comme "connu" peut varier selon le contexte et les normes de vérité appliquées. Exemple: Savoir qu'on a deux mains est une évidence dans un contexte "mondain", mais devient sujet à caution dans un contexte philosophique qui introduit des scénarios sceptiques ("cerveau dans une cuve"). Conséquences pour la communication scientifique: La rhétorique du "scientifiquement prouvé" est contre-productive car elle renforce le complotisme. Citation: « Aussitôt que vous considérez la science comme infaillible vous ouvrez la porte à l'argument sceptique [...] vous tombez dans le piège cartésien. » (22:20) Les communicateurs scientifiques doivent insister sur le caractère faillible de la science, capable d'évoluer et de se corriger au fil des découvertes. Conclusion: La conférence met en garde contre une vision dogmatique de la science et plaide pour une communication plus nuancée qui reconnaisse ses limites tout en affirmant sa robustesse.

eLife Assessment

With compelling electrophysiological and behavioural evidence, this work establishes that the activity of insulin-producing cells (IPCs) depends on the nutritional state in Drosophila and that, like in mammals, there is also an incretin-like effect with IPCs responding to glucose feeding but not to glucose perfusion. Moreover, the authors demonstrate that DH44 neurons respond to glucose perfusion and, together with IPCs, modulate locomotor activity. This important study on the neuronal regulation of metabolic homeostasis will be of interest to both neuroscience and to medical research in diabetes.

Reviewer #1 (Public review):

Summary:

This study presents useful insights into the in vivo dynamics of insulin-producing cells (IPCs), key cells regulating energy homeostasis across the animal kingdom. The authors further provide compelling evidence using adult Drosophila melanogaster that IPCs, unlike neighboring DH44 cells, do not respond to glucose directly, but that glucose can indirectly regulate IPC activity after ingestion supporting an incretin-like mechanism in flies similar to mammals. The authors link decreased activity of IPCs to hyperactivity observed in starved flies, a locomotive behavior aimed to increase food search. Furthermore, the authors provide evidence that IPCs receive inhibitory inputs from Dh44 neurons, which are linked to increased locomotor activity.

This paper is of outstanding interest to scientists aiming to understand metabolic control of circuit dynamics, in particular for internal state-linked behaviors competing with the feeding state.

Strengths:

(1) By using whole cell patch clamp recording, the authors convincingly showed the activity pattern and regulation of IPCs and neighboring DH44 neurons under different feeding states and in various refeeding paradigms.<br /> (2) The paper provides compelling evidence that IPCs are not directly and acutely activated by glucose, but rather through a post-ingestive incretin-like mechanism. In addition, the authors show that Dh44 neurons located adjacent to the IPCs respond to bath application of nutritive sugars contrary to the IPCs.<br /> (3) The paper also provides useful data on the regulation of IPC activity by Dh44 neurons, which is useful to understand their regulation in vivo.

No major weaknesses remain in the revised version of this work.

Reviewer #2 (Public review):

Summary

In this study, Bisen et al. characterized the state-dependency of insulin-producing cells in the brain of Drosophila melanogaster. They successfully established that IPC activity is modulated by the nutritional state and age of the animal. Interestingly, they demonstrate that IPCs respond to the ingestion of glucose, rather than to perfusion with it, an observation reminiscent of the incretin effect in mammals. The study is well conducted and presented and the experimental data convincingly support the claims made.

Strengths

The study makes great use of the tools available in *Drosophila* research, demonstrating the effect that starvation and subsequent refeeding have on the physiological activity of IPCs as well as on the behavior of flies to then establish causal links by making use of optogenetic tools.<br /> It is particularly nice to see how the authors put their findings in context to published research and use for example TDC2 neuron activation or DH44 activity to establish baselines to relate their data to.

Reviewer #3 (Public review):

Although insulin release is essential in the control of metabolism, adjusted to nutritional state, and plays major roles in normal brain function as well as in aging and disease, our knowledge about the activity of insulin-producing (and releasing) cells (IPCs) in vivo in limited.

In this technically demanding study, IPC activity is studied in the Drosophila model system by fine in vivo patch clamp recordings with parallel behavioral analyses and various optogenetic as well as feeding manipulations.

The data provide compelling evidence that IPC activity is increased with a slow time course after feeding a high glucose diet. By contrast, IPC activity is not directly affected by rising blood glucose levels. This is reminiscent of the incretin effect known from vertebrates and points to a conserved mechanism in insulin production and release upon sugar feeding.

Moreover, the data confirm earlier studies that nutritional state strongly affects locomotion. Surprisingly, strong evidence shows that IPC activity makes only a negligible contribution to this. Instead, other modulatory neurons that are directly sensitive to blood glucose levels strongly affect locomotion. Together, these data reveal a network of multiple parallel and interacting neuronal layers to orchestrate the physiological, metabolic, and behavioral responses to the nutritional state. Together with the data from a previous study, this work sets the stage to dissect the architecture and function of this network.

Strengths:

State-of-the-art current clamp in situ patch clamp recordings in behaving animals are a demanding but powerful method to provide novel insight into the interplay of nutritional state, IPC activity, and locomotion. The patch clamp recordings and the parallel behavioral analyses are of high quality, as are the optogenetic manipulations. The data showing that starvation silences IPC activity in young flies (younger than 1 week) are excellent. The evidence for the claim that locomotor activity is not increased upon IPC activity but upon the activity of other blood glucose sensitive modulatory neurons (Dh44) is compelling, too. The study provides a great system to experimentally dissect the interplay of insulin production and release with metabolism, physiology, nutritional state, and behavior. Demonstrating the incretin effect in Drosophila provides novel experimental routes to further study it. During the revision process, compelling evidence has been added to underscore the incretin effect, the finding that IPCs themselves do not sense sugars, and that feeding a high sugar diet does not cause unspecific stress responses.

I found no more weaknesses: The authors have carefully addressed all of my previous critiques by adding compelling new data and carefully revising the text. This paper provides a prime example of how responsible authors can utilize this constructive (but relatively new) reviewing procedure to make a very good manuscript even better.

Author response:

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

Public Reviews: 

Reviewer #1 (Public Review): 

Summary: 

This study presents useful insights into the in vivo dynamics of insulin-producing cells (IPCs), key cells regulating energy homeostasis across the animal kingdom. The authors provide compelling evidence using adult Drosophila melanogaster that IPCs, unlike neighboring DH44 cells, do not respond to glucose directly, but that glucose can indirectly regulate IPC activity after ingestion supporting an incretin-like mechanism in flies, similar to mammals. The authors link the decreased activity of IPCs to hyperactivity observed in starved flies, a locomotive behavior aimed at increasing food search. 

Furthermore, there is supporting evidence in the paper that IPCs receive inhibitory inputs from Dh44 neurons, which are linked to increased locomotor activity. However, although the electrophysiological data underlying the dynamics of IPCs in vivo is compelling, the link between IPCs and other potential elements of the circuitry (e.g. octopaminergic neurons) regulating locomotive behaviors is not clear and would benefit from more rigorous approaches. 

This paper is of interest to cell biologists and electrophysiologists, and in particular to scientists aiming to understand circuit dynamics pertaining to internal state-linked behaviors competing with the feeding state, shown here to be primarily controlled by the IPCs. 

Strengths: 

(1) By using whole-cell patch clamp recording, the authors convincingly showed the activity pattern of IPCs and neighboring DH44 neurons under different feeding states. 

(2) The paper provides compelling evidence that IPCs are not directly and acutely activated by glucose, but rather through a post-ingestive incretin-like mechanism. In addition, the authors show that Dh44 neurons located adjacent to the IPCs respond to bath application of glucose contrary to the IPCs. 

(3) The paper provides useful data on the firing pattern of 2 key cell populations regulating foodrelated brain function and behavior, IPCs and Dh44 neurons, results which are useful to understand their in vivo function. 

Weaknesses: 

(1) The term nutritional state generally refers to the nutrients which are beneficial to the animal. In Figure 1, the authors showed that IPCs respond to glucose but not proteins. To validate the term nutritional state the authors could test the effect of a non-nutritive sugar (e.g. D-arabinose or L-Glucose) on the post-ingestive physiological responses of the IPCs.

We thank the referee for this insightful comment. Following their suggestion, we included two new experimental data sets, which we added to Figure 1: We show that IPCs do not respond to the non-nutritive sugar D-arabinose (Figure 1H). In order to further expand this data set and our conclusions, we additionally show that IPCs do respond to fructose – a second nutritive sugar in addition to glucose (Figure 1H). Together, these data sets permit the conclusion that IPCs are sensitive to the ingestion of nutritive sugars, and do not respond to ingestion of nonnutritive sugars or high protein diets. Thus, we validate the term nutritional state.

(2) It is difficult to grasp the main message from the figures in the result section as some figures have several results subsections referring to different points the authors want to make. The key results of a figure will be easier to understand if they are summarized in one section of the results. Alternatively, a figure can be split into 2 figures if there are several key messages in those figures, e.g. Figures 2 and 3.  

We appreciate this suggestion and have made several changes to our manuscript to add more clarity. Among other things, we have changed the order of data presentation in Figure 2, as suggested by the referee below, where we now start with the IPC activation data rather than the OAN activation. We also swapped the order of data presentation and split Figure S1 into Figures S1 & S2. Moreover, we re-arranged the panel order in supplementary figure S4. This significantly improved the flow of the results section. Since the figures the referee refers to contain comparative data, for example between diets (Figure 1) or neuron types (Figure 2), we prefer to keep these data sets together. However, we have carefully revised the results section to more clearly relate our statements to individual figure panels.

(3) The prime investigation of the paper is about the physiological response and locomotive behavioral readout linked to IPCs. The authors do not show a link between OANs and IPCs in terms of functional or behavioral readouts. In Figure 2 the authors first start with stating a link between OAN neurons and locomotion changes resulting from internal feeding states. The flow of the paper would be better if the authors focused on the effect of optogenetic activation of IPCs under different feeding states and their impact on fly locomotion. If the experiments done on optogenetic activation of OANs were to validate the experimental approach the data on OAN neurons is better suited for the supplement without the need of a subsection in the result section on the OANs.  

We agree with the reviewer’s suggestion and switched the order of the figure panels and text to aid the flow of the manuscript. We now show and discuss the IPC activation data first (Figure 2C-H) and OAN activation afterwards (Figure 2I-K). We did keep the OAN data in the main document, though, since that facilitates comparisons between the small effects of IPC activation and the large, well-established effects of OAN activation.

(4) Figure 2F shows that optogenetic activation of IPCs in fed flies does not influence their locomotor output. In the text, the conclusion linked to Figure 2F-H states that IPC activation reduces starvation-induced hyperactivity which is a statement more suited to Figure 2I-K. 

We edited the text accordingly.

(5) The authors show activation of Dh44 neurons leads to hyperpolarisation of the IPCs. What is the functional link between non-PI Dh44 neurons and the IPCs? Do IPCs express DH44R or is DH44 required for this effect on IPCs? Investigating a potential synaptic or peptidergic link between DH44 neurons and IPCs and its effect on behavior would benefit the paper, as it is so far not well connected. 

Although we have not performed any experiments dedicated to investigating the functional link between DH44Ns outside the PI and the IPCs in this study, there are two lines of evidence supporting that this connection is relatively direct. First, IPCs do express DH44R1 & R2, as we show in a parallel study in eLife (Held M, et al. ‘Aminergic and peptidergic modulation of Insulin-Producing Cells in Drosophila’. eLife. 2024;13. doi:10.7554/ELIFE.99548.1). Second, we performed functional connectivity experiments using a Leucokinin (LK) driver line in that paper. This driver line labels two pairs of non-PI DH44Ns in the VNC, which are DH44 and LK positive (Zandawala et al 2018). Activating that line leads to inhibition of IPCs, similar to the effect we observed here for DH44N activation. These two lines of evidence suggest that there could be a direct peptidergic connection between DH44+ neurons and IPCs. We have added a paragraph mentioning these experiments to our discussion:

‘Notably, the DH44^PINs express the DH44 peptide, as confirmed by anti-DH44 stainings(100). This also applies to a large fraction of neurons labelled in the broad DH44 driver line(100). However, a subset of neurons labelled in the broad line did not exhibit DH44 immunoreactivity(100), and might therefore not actually express the DH44 peptide. Hence, the inhibition of IPCs could be driven by neurons in the DH44 driver line that do not express DH44. A strong candidate for the inhibition are LK and DH44-positive neurons, which are labelled by the broad line(76). In a parallel study, we showed that LK-expressing neurons strongly inhibit IPCs(30), similar to the broad DH44 line used here. Furthermore, evidence from single-nucleus transcriptomic analysis shows that IPCs express DH44-R1 and DH44-R2 receptors(30). Therefore, it is possible that DH44Ns communicate with IPCs through a direct peptidergic connection. Notably, the inhibitory effect of non-PI DH44Ns on IPCs was very strong and fast, suggesting that a connection via classical synapses is more likely. Regardless, our results show that the glucose sensing DH44^PINs and IPCs act independently of each other.’

Reviewer #2 (Public Review): 

Summary: 

In this study, Bisen et al. characterized the state-dependency of insulin-producing cells in the brain of *Drosophila melanogaster*. They successfully established that IPC activity is modulated by the nutritional state and age of the animal. Interestingly, they demonstrate that IPCs respond to the ingestion of glucose, rather than to perfusion with it, an observation reminiscent of the incretin effect in mammals. The study is well conducted and presented and the experimental data convincingly support the claims made. 

Strengths: 

The study makes great use of the tools available in *Drosophila* research, demonstrating the effect that starvation and subsequent refeeding have on the physiological activity of IPCs as well as on the behavior of flies to then establish causal links by making use of optogenetic tools. 

It is particularly nice to see how the authors put their findings in context to published research and use for example TDC2 neuron activation or DH44 activity to establish baselines to relate their data to. 

Weaknesses: 

I find the inability of SD to rescue the IPC starvation effect in Figure 1G&H surprising, given that the fully fed flies were raised and kept on that exact diet. Did the authors try to refeed flies with SD for longer than 24 hours? I understand that at some point the age effect would also kick in and counteract potential IPC activity rescue. I think the manuscript would benefit if the authors could indicate the exact age of the SD refed flies and expand a bit on the discussion of that point.  

We have expanded the first paragraph of our discussion to tackle these questions, in particular the potential effect of aging, as suggested by the referee. We now also indicate the exact age of the flies. Moreover, we have conducted additional experiments in which we added either glucose or arabinose to our standard diet (Figure 1H). As we would have expected based on our hypothesis that the glucose concentration in our standard diet was too low to cause an increase in IPC activity after starvation, we find that feeding standard diet plus glucose increases IPC activity to the same level as glucose only, and that adding arabinose to the standard diet does not lead to increased IPC activity after starvation (Figure 1H).

The incretin-like effect is exciting and it will be interesting in the future to find out what might be the signal mediating this effect. It is interesting that IPCs in explants seem to be responsive to glucose. I think it would help if the authors could briefly discuss possible sources for the different findings between these in fact very different preparations. Could the the absence of the inhibitory DH44 feedback in the *ex-vivo* recordings for example play a role? 

We thank the referee for this interesting point and expanded our discussion accordingly. We included that, in particular in brain explants without a VNC, the inhibitory connection we describe might be absent, as the referee suggested: ‘Previous ex vivo studies suggested that IPCs, like pancreatic beta cells, sense glucose cell-autonomously(23,24). Consistent with this, we observed an increase in IPC activity after the ingestion of glucose (Figure 2B). However, IPC activity did not increase during the perfusion of glucose directly over the brain. Importantly, the fly preparations were kept alive for several hours allowing the glucose-rich saline to enter circulation and reach all body parts. Several factors may explain the difference between ex vivo and in vivo preparations. First, in ex vivo studies, certain regulatory feedback mechanisms present in vivo could be absent. For example, the strong inhibitory input IPCs receive from DH44Ns we found would likely be absent in brain explants without a VNC. A lack of inhibitory feedback might allow for more direct glucose sensing by IPCs ex vivo, whereas in vivo, the IPC response could be suppressed by more complex systemic feedback. Second, we attempted to use the intracellular saline formulation employed in a previous ex vivo study44. However, we observed that IPCs depolarized quickly using this saline, leading to unstable recordings that did not meet our quality standards for in vivo experiments. Another possible explanation for the lack of an effect of glucose might have been that the dominant circulating sugar in flies is trehalose(70,71) which is derived from glucose. When we extended our experiments, we found that trehalose perfusion did not affect IPC activity either, strengthening the idea that IPCs do not directly sense changes in hemolymph sugar levels. Therefore, our findings suggest that, similar to mammals, IPC activity and hence, insulin release, is not simply modulated by hemolymph sugar concentration in Drosophila.’ 

The incretin-like effect the authors observed seems to start only after 5h which seems longer than in mammals where, as far as I know, insulin peaks around 1h. Do the authors have ideas on how this timescale relates to ingestion and glucose dynamics in flies? 

We have now included the following section in the discussion to explicitly address the question of different activity dynamics in flies and mammals, but also the limitations of our electrophysiological approach in this regard: ‘We observed that IPC activity increased over a timescale of hours, which is longer compared to the fast insulin response in mammals, where insulin typically peaks within an hour of feeding(97). In flies, insulin levels rise within minutes of refeeding, followed by a drop after 30 min(20). Our experimental techniques limit our ability to capture these fast initial dynamics, since the preparation for intracellular recordings requires tens of minutes, so that we typically recorded IPC activity at least 20 min after the last food ingestion. Notably, studies in fasted mammals have shown that insulin peaks within minutes of refeeding, followed by a rapid decline, with levels stabilizing as feeding continues(98,99). We speculate a similar dynamic could be present in flies, but with our approach, we capture the steady-state reached tens of minutes after food ingestion rather than a potential initial peak.’ 

The authors mention "a decrease in the FV of IPC-activated starved flies even before the first optogenetic stimulation (Figure 2I),". Could this be addressed by running an experiment in darkness, only using the IR illumination of their behavioral assay? 

We thank the referee for pointing out this unexpected result. We discuss this in more detail in the new version of our manuscript and expand on the reasons for not performing these optogenetic activation experiments in the dark: First, the red LED required to activate CsChrimson triggers strong startle responses in dark-adapted flies, which mask other behavioral effects, in particular subtle ones such as those observed for IPCs. The startle response is much reduced when performing experiments under low background light conditions. Second, flies, at least in our hands, do not exhibit robust foraging behavior or starvation-induced hyperactivity in the dark, which is critical for our behavioral experiments. However, we also explain in our discussion that we believe the effect of background illumination is relatively small, since flies expressing CsChrimson in OANs or DH44Ns show comparable activity levels to controls. Hence, a part of this effect is likely attributable to leak currents induced by CsChrimson expression. We would like to point out though that we are careful in our description of the IPC effect on behavior, and focus on the fact that it is considerably smaller than the effects of other modulatory neurons (DH44Ns and OANs).

The authors show an inhibitory effect of DH44 neuron activation on IPC activity. They further demonstrate that DH44PI neurons are not the ones driving this and thus conclude that "...IPCs are inhibited by DH44Ns outside the PI.". As the authors mentioned the broad expression of the DH44-Gal4 line, can they be sure that the cells labeled outside the PI are actually DH44+? If so they should state this more clearly, if not they should adapt the discussion accordingly.   

We have substantially added to our discussion of this point, according to the referee’s great suggestion. In short, the broad line includes neurons that are DH44 positive and neurons that are not: ‘Notably, the DH44^PINs express the DH44 peptide, as confirmed by anti-DH44 stainings(100). This also applies to a large fraction of neurons labelled in the broad DH44 driver line(100). However, a subset of neurons labelled in the broad line did not exhibit DH44 immunoreactivity(100), and might therefore not actually express the DH44 peptide. Hence, the inhibition of IPCs could be driven by neurons in the DH44 driver line that do not express DH44.’

Reviewer #3 (Public Review): 

Although insulin release is essential in the control of metabolism, adjusted to nutritional state, and plays major roles in normal brain function as well as in aging and disease, our knowledge about the activity of insulin-producing (and releasing) cells (IPCs) in vivo is limited. 

In this technically demanding study, IPC activity is studied in the Drosophila model system by fine in vivo patch clamp recordings with parallel behavioral analyses and optogenetic manipulation. 

The data indicate that IPC activity is increased with a slow time course after feeding a high-glucose diet. By contrast, IPC activity is not directly affected by increasing blood glucose levels. This is reminiscent of the incretin effect known from vertebrates and points to a conserved mechanism in insulin production and release upon sugar feeding. 

Moreover, the data confirm earlier studies that nutritional state strongly affects locomotion. Surprisingly, IPC activity makes only a negligible contribution to this. Instead, other modulatory neurons that are directly sensitive to blood glucose levels strongly affect modulation. Together, these data indicate a network of multiple parallel and interacting neuronal layers to orchestrate the physiological, metabolic, and behavioral responses to nutritional state. Together with the data from a previous study, this work sets the stage to dissect the architecture and function of this network. 

Strengths: 

State-of-the-art current clamp in situ patch clamp recordings in behaving animals are a demanding but powerful method to provide novel insight into the interplay of nutritional state, IPC activity, and locomotion. The patch clamp recordings and the parallel behavioral analyses are of high quality, as are the optogenetic manipulations. The data showing that starvation silences IPC activity in young flies (younger than 1 week) are compelling. The evidence for the claim that locomotor activity is not increased upon IPC activity but upon the activity of other blood glucose-sensitive modulatory neurons (Dh44) is strong. The study provides a great system to experimentally dissect the interplay of insulin production and release with metabolism, physiology, and behavior. 

Weaknesses: 

Neither the mechanisms underlying the incretin effect, nor the network to orchestrate physiological, metabolic, and behavioral responses to nutritional state have been fully uncovered. Without additional controls, some of the conclusions would require significant downtoning. Controls are required to exclude the possibility that IPCs sense other blood sugars than glucose. The claim that IPC activity is controlled by the nutritional state would require that starvation-induced IPC silencing in young animals can be recovered by feeding a normal diet. At current firing in starvation, silenced IPCs can only be induced by feeding a high-glucose diet that lacks other important ingredients and reduces vitality. Therefore, feasible controls are needed to exclude that diet-induced increases in IPC firing rate are caused by stress rather than nutritional changes in normal ranges. The finding that refeeding starved flies with a standard diet had no effect on IPC activity but a strong effect on the locomotor activity of starved flies contradicts the statement that locomotor activity is affected by the same dietary manipulations that affect IPC activity. The compelling finding that starvation induces IPC firing would benefit from determining the time course of the effect. The finding that IPCs are not active in fed animals older than 1 week is surprising and should be further validated. 

We thank the referee for the thoughtful and constructive criticism of our experiments and conclusions. Below, we lay out how we tackled the individual points raised by the referee.

(1) ‘Controls are required to exclude the possibility that IPCs sense other blood sugars than glucose.’  

To address this point, we conducted experiments in which we perfused trehalose (Figure 3B), the main circulating hemolymph sugar in Drosophila and other insects. Our results clearly show that trehalose does not affect IPC activity upon perfusion, confirming our statements that IPCs do not sense key blood sugars directly.

(2) ‘Feasible controls are needed to exclude that diet-induced increases in IPC firing rate are caused by stress rather than nutritional changes in normal ranges’. 

We agree with the referee that this point was not completely fleshed out in our first submission. We have now performed additional experiments in which we added glucose (and fructose) to our standard diet (Figure 1H). Flies feeding on this diet received all necessary nutrients but still experienced high concentrations of sugars. The effects of high glucose in a standard diet background were indistinguishable from those of high glucose in agarose, confirming that the IPCs respond to sugar rather than stress. Another important observation in this context is that IPCs in flies kept on a high protein diet exhibited much lower spike rates than flies exhibiting the high glucose diet, even though they had a much shorter lifespan and therefore, presumably, experienced much higher stress levels (Figure 1H, Figure S1). These observations underline that stress is certainly not the primary factor here.

(3) ‘The finding that refeeding starved flies with a standard diet had no effect on IPC activity but a strong effect on the locomotor activity of starved flies contradicts the statement that locomotor activity is affected by the same dietary manipulations that affect IPC activity.’

We have revised the respective section of the results and discussion accordingly and are more careful and clearer in our interpretation of this behavioral dataset: ‘These results show that the locomotor activity was affected by the same dietary manipulations that had strong effects on IPC activity. However, IPC activity changes alone cannot explain the modulation of starvation-induced hyperactivity. On the one hand, high-glucose diets which drove the highest activity in IPCs were not sufficient to reduce locomotor activity back to baseline levels. On the other hand, refeeding flies with SD did not revert the effects of starvation on IPC activity (Figure 1H), but it was sufficient to reduce the locomotor activity below baseline levels (Figure 2B). This suggests that the modulation of starvation-induced hyperactivity is achieved by multiple modulatory systems acting in parallel.’

(4) ‘The compelling finding that starvation induces IPC firing would benefit from determining the time course of the effect.’

We followed the referee’s excellent suggestion and determined the time course of the starvation effect in three timesteps, similar to the experiments we did for refeeding (Figure 1G). In addition, we now also quantify the number of active IPCs (i.e., IPCs that fired at least one action potential during our five-minute analysis window), which further illustrates the dynamics of the starvation and refeeding effects. We find that the starvation effect is graded, and that IPC activity decreases with increasing starvation duration.

(5) ‘The finding that IPCs are not active in fed animals older than 1 week is surprising and should be further validated.’

To address the referee’s comment, we have added 14 new IPC recordings from flies in the 6–26-day range, such that we now have recordings from 9-14 IPCs for each age range (Figure S2B). They confirmed our previous analysis and strengthened the finding that IPC activity dramatically decreases after 8 days (on our standard diet). The total number of IPCs in this supplementary dataset was thus increased from 34 to 48.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors): 

(1) Do IPCs respond to glucose specifically after ingestion or generally to any other nutritive sugars? To tackle this question the IPC responses in starved flies can be recorded after refeeding flies with other nutritive sugars (fructose, sucrose). 

To address this important question, we have performed additional experiments in which we refed starved flies with fructose, as a nutritive sugar, and arabinose, as a non-nutritive sugar. As expected, IPCs responded to fructose but not arabinose and hence nutritive sugars in general. We describe and discuss these key results in the new version of our manuscript.

(2) In Figure 2, the x and y axes are not annotated on all subfigures, which might help improve clarity. 

We have annotated the subfigures as requested.

(3) In the discussion on page 9 ("...we observed an increase in IPC activity after the ingestion of glucose (Figure 2B)."), the authors refer to Figure 2B instead of 3C.

We have fixed this oversight.

Reviewer #2 (Recommendations For The Authors): 

Introduction 

I think it could be helpful for the reader if you would briefly state the number of IPCs and whether you are targeting all of them with Dilp2-Gal4. 

We included the numbers according to the suggestion. 14 IPCs are labeled in the driver line, and this is the number of IPCs commonly assumed to be present in the PI.

Figures 

In some Figures (for example 1D & E) the authors state the number of IPCs recorded (N) but not the number of animals used (n). This should be stated as the data from within an animal are dependent and might give insights about IPC heterogeneity. 

We have compiled tables for the supplementary material (Tables S5 & S6) in which we state the number of IPCs and DH44^PINs recorded and the number of different flies for each figure panel. We have recorded an average of 1.4 IPCs per fly (217 IPCs from 160 flies). We therefore expect the bias introduced by individual flies to be rather small. However, in our parallel study, we specifically investigate the heterogeneity of IPCs by maximizing the number of IPCs recorded per fly (Held M, et al. ‘Aminergic and peptidergic modulation of Insulin-Producing Cells in Drosophila’. eLife. 2024;13. doi:10.7554/ELIFE.99548.1). In the case of DH44PINs, we recorded 24 neurons in 21 flies – 1.1 neurons per fly.

- Figure 3D: There is some white visible among the cell bodies in the overlay. I assume this comes from projecting across layers rather than indicating DH44 - IPC overlap? It would help to explicitly state that. 

We have added a statement to the results section, in which we explain that most of the white is due to overlap in the z-projection rather than overlap in the driver lines. However, there are few cases (typically one to two cells per brain), in which neurons labeled by the DH44 line also stain positive for Dilp2, indicating they express both neuropeptides. We have added this information to the manuscript:  

Results: ‘DH44^PINs are anatomically similar to IPCs, and their cell bodies are located directly adjacent to those of IPCs in the PI, making them an ideal positive control for our experiments (Figure 3D). A small subset of DH44^PINs also expresses Dilp2(75), and our immunostainings confirmed colocalization of Dilp2 and DH44 in a single neuron (Figure 3D, white arrow).’

In figure caption: ‘UAS-myr-GFP was expressed under a DH44-GAL4 driver to label DH44 neurons. GFP was enhanced with anti-GFP (green), brain neuropils were stained with anti-nc82 (cyan), and IPCs were labelled using a Dilp2 antibody (magenta). White arrow indicates Dilp2 and DH44-GAL4 positive neuron. The other white regions in the image result from an overlap in z-projections between the two channels, rather than from antibody colocalization.’

- Figure 4I: One might get the impression that the fast onset peak of activity precedes the stimulation onset, using a thinner line width might help avoid that. 

This effect is due to a combination of using relatively heavy lines for clear visibility of the data and a gentle smoothing step (a 2s median filter, which corresponds to less than 1% of the 300s stimulation window) in our analysis of the behavioral data. However, inspection of the raw data clearly shows increases in velocity after the onset of the optogenetic activation. We clarified this in the figure caption: ‘Average FV across all DH44N activation trials based on two independent replications of the experiment in I. Note that the peak in average FV lies within the first frame of the stimulation window.’

- S3 panel letters do not match references in the text.

We fixed this oversight.

Formatting 

- Page 10: The paragraphs on the bottom of the page got switched around.

This has been fixed.

- Page 14: The first paragraph after the header "Free-walking assay" seems to be coming from elsewhere. 

We apologize for this slightly embarrassing mistake. We used our related bioRxiv preprint (Held et al.) as a template for formatting this paper, and accidentally left this part of the methods section in the manuscript. We have fixed this error in our resubmission.

Reviewer #3 (Recommendations For The Authors): 

Major suggestions: 

(1) The data show convincingly that IPC activity is decreased by starvation during the first week of adult life (Figures 1C and D). However, the conclusion that IPC activity is controlled by the nutritional state requires additional care. First, refeeding starved adult animals with a normal diet does not bring back normal IPC firing rates (Figure 1H). Therefore, IPC activity does not strictly follow changes in nutritional state, but IPCs are silenced by starvation. Second, from the second week of adult life on, IPCs are silent anyway, and thus unlikely responsive to changes in the nutritional state anymore (which might be different on a different standard diet?) The only effect of feeding on IPC activity is observed upon feeding starved, young animals with high glucose for 12-24 hrs (Figure 1G). However, it is not clear whether increased IPC firing is caused by the effects of high glucose on the nutritional state in a normal range, or because of diet-induced stress (the diet also severely shortens lifespan, Figure 1S). Does high glucose also increase IPC firing rate in young, fed animals? These would have strongly increased glucose concentrations but not suffer the stress of not getting any other nutrients. Such experiments would be required to make the statement that glucose feeding increases IPC firing rate. 

We have performed several experiments to address this criticism. First, we performed a time course analysis of the starvation effect. We show that the IPC activity reduction is graded, and that IPC activity declines already after two hours of starvation, a timepoint at which stress levels should still be relatively small (Figure 1G). Second, we refed flies with high glucose concentrations added to the standard diet (Figure 1H). This minimized any potential stress responses due to a lack in nutrients. Third, we now show that IPCs specifically respond to nutritive (glucose and fructose), but not to non-nutritive sugars (arabinose, Figure 1H). We believe that these data sets, in addition to the graded refeeding effect, make a strong case for the nutritional state dependent modulation of IPCs. 

(2) The testing of locomotor activity is well done, nicely recapitulates starvation-induced increases in locomotion, and adds interesting novel findings on refeeding with high glucose versus high protein diet. However, the statement that locomotor activity was affected by the same dietary manipulations that had strong effects on IPC activity does not reflect the data presented. Refeeding starved flies with a standard diet had no effect on IPC activity (Figure 1H) but a strong effect on locomotor activity of starved flies (a strong reduction, even stronger than high glucose diet, Figure 2B). 

We have revised the respective section of the results and discussion accordingly and are more careful and clearer in our interpretation of this behavioral dataset: ‘These results show that the locomotor activity was affected by the same dietary manipulations that had strong effects on IPC activity. However, IPC activity changes alone cannot explain the modulation of starvationinduced hyperactivity. On the one hand, high-glucose diets which drove the highest activity in IPCs were not sufficient to reduce locomotor activity back to baseline levels. On the other hand, refeeding flies with SD did not revert the effects of starvation on IPC activity (Figure 1H), but it was sufficient to reduce the locomotor activity below baseline levels (Figure 2B). This suggests that the modulation of starvation-induced hyperactivity is achieved by multiple modulatory systems acting in parallel.’

Related to points 1 and 2, a key statement that the results establish that IPC activity is controlled by the nutritional state requires care. What the data convincingly show is that IPC activity is near zero upon starvation. 

As described above, we have added several extensive data sets (fructose feeding, arabinose feeding, trehalose perfusion, starvation time course) to show that we indeed observe a nutritional state dependent modulation of IPCs and describe these new results in the results and discussion.

(3) The time course of nutritional state-dependent changes of IPC activity is claimed to be slow, several hours to days. Unless I have missed a figure, the underlying data are not presented (only for high glucose diet). It would be great if this could also be shown for a standard diet with higher glucose concentrations than the one used so that it rescues starvation-induced IPC silencing without shortening lifespan (if this is feasible?). The data showing starvation-induced IPC silencing are convincing, but, unless I have missed it, the time course has not been determined. It would be very nice to actually show this. Have different starvation times been tested in relation to IPC firing rate, and if yes, with what time resolution? Does IPC activity change already after 0.5 or 1 or a few hours of starvation? If starvation can silence IPCs faster than assumed, the nearzero IPC activity in animals older than a week could very well be caused by longer time intervals between meals. 

We have performed experiments to address both important points raised by the referee here. 1) We have added high glucose concentrations to our standard diet, and show that it has the same effect – a significant increase in IPC activity – as the high glucose diet (Figure 1H). 2) We have analyzed the time course of IPC activity reduction in response to starvation (Figure 1G). Indeed, we find that a few hours of starvation start reducing IPC activity. We discuss the possibility that reduced IPC activity in older flies could be due to reduced food intake: ‘One of our experiments demonstrated that IPC activity was heavily diminished in flies older than 10 days (Figure S2B). A possible explanation could be that flies feed less as they age. However, this only holds true for flies older than 14 days86. Therefore, reduced IPC activity in 10-11 day old flies is unlikely to result from reduced food intake and likely involves inhibition of insulin signaling.’

(4) The data on the proposed incretin effect are of high importance in potentially highlighting a highly conserved link between glucose ingestion and insulin release. An important control would be to test different sugars, such as trehalose, an important blood sugar of flies. If glucose is converted into trehalose and this is what IPCs sense, then perfusion of glucose has no effect. The fact fantastic experiments show that the DH44 neurons are sensitive to glucose perfusion does rule out that IPCs sense a different sugar. This would be very different from the incretin effect that requires additional hormones. In addition, as mentioned above, controls are required to show that high glucose affects IPCs as a nutrient and not as a stressor (see point 1), for example refeeding with a standard diet that contains a higher glucose concentration but does not reduce lifespan. Another great control to solidify the exciting claim on the incretin effect would be to knock out candidate Drosophila incretin hormones and test whether a high glucose diet stops increasing the IPC firing rate (although simpler controls might also do the job). 

We have performed the two key experiments suggested by the referee. 1) We perfused trehalose as the primary blood sugar of flies and showed that IPCs do not respond to trehalose perfusion (Figure 3B & C). This further strengthens the finding that IPC activity in flies shows an incretin-like effect. 2) We have added high concentrations of glucose to our standard diet to provide flies with a full diet that contains high glucose concentrations. IPC activity in these flies was indistinguishable from the activity in flies which consumed pure glucose diets. In contrast, IPC activity in flies kept on a high protein diet, which dramatically reduced lifespan, was very low. These results clearly show that higher IPC activity is not due to increased stress levels, but a function of nutritive sugar ingestion. We further validated this hypothesis by refeeding flies with fructose as a nutritive sugar, which increased IPC activity, and arabinose as a non-nutritive sugar, which did not affect IPC activity (Figure 1H).

Another point that might be relevant to this discussion is that IPC activity is almost entirely shut down during flight in Drosophila (which we showed in Liessem et al. 2023, Current Biology 33 (3), 449-463. e5). Several ‘stress hormones’ are released during flight, including octopamine. The fact that IPC activity is low in flying flies, starved flies, and flies kept on a pure protein diet (which all experience high stress levels), to us, very clearly suggests that stress is not the predominant factor here. We would also like to point out that, while the lifespan was reduced in flies kept on pure glucose diets, survival rates were at 100% until day 14, and we carried out our experiments on day 2 after starvation. Hence, these flies might not (yet) experience particularly high stress levels.

(5) The discussion relates the absence of IPC firing in animals older than 1 week to aging. However, given that the flies fed on a normal diet show the typical lifespan for Drosophila, a 10-dayold fly is still in its youth. Maybe flies at 10 days eat simply less and thus IPC spiking goes down as in starved flies, especially because the standard diet used contains low glucose. Do IPCs also become silent after a week if the animals are fed with a standard diet that contains a higher glucose concentration? Without additional controls, this part of the discussion is pretty speculative and should be revised. 

We agree with the reviewer, that it is not clear whether reduced IPC activity is a direct result of physiological changes that occur with aging, or an indirect effect of reduced food intake, which occur during aging. In both cases, in our view, it would be an age-related effect. Since this is a minor point of our manuscript, we decided not to perform additional experiments, other than significantly increasing the sample size for the aging data set already presented to shore up our findings (Figure S2B). We have, however, revisited the discussion of this point according to the referee’s suggestion: ‘One of our experiments demonstrated that IPC activity was heavily diminished in flies older than 10 days (Figure S2B). A possible explanation could be that flies feed less as they age. However, this only holds true for flies older than 14 days(85). Therefore, reduced IPC activity in 10-11 day old flies is unlikely to result from reduced food intake and likely involves inhibition of insulin signaling.’

Other suggestions: 

(6) For the mixed effects of octopamine and tyramine on larval locomotion that are referred to, it might be interesting to also look at Schützler et al 2019, PNAS because it shows that starvation activates TBH so that the octopamine to tyramine ratio is increased. 

We refer to Schützler et al. in the following paragraph of our discussion: ‘This intermittent locomotor arrest has been previously described in adult flies and is thought to be mediated by ventral unpaired median OANs, which have been suggested to suppress long-distance foraging behavior(69). Since these are not the only neurons we activate in the TDC2 line, we speculate that the stopping phenotype could also result from concerted effects of octopamine and tyramine modulating muscle contractions(65-67) and motor neuron excitability(68), as previously described in Drosophila larvae, or from OANs interfering with pattern generating networks in the ventral nerve cord (VNC) during longer activation(69).’  

(7) The reference list requires care. For example, reference 43 is identical to 67, reference 66 gives no information on incretin-like hormones in Drosophila as stated in the text 

We carefully double-checked our reference list and corrected the mistakes mentioned.

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Reply to the reviewers

We thank the reviewers for their insightful comments, and we address all their comments in the detailed point-by-point responses provided below.

Reviewer #1

__Evidence, reproducibility and clarity __

*In the manuscript entitled "Inhibition of glycolysis in tuberculosis-mediated metabolic rewiring reduces HIV-1 spread across macrophages", Vahlas and colleagues investigated the hypothesis that Mtb interferes with HIV-1 infection of human macrophages, as they represent a common target cell type. In particular, they observed that a conditioned medium generated from Mtb-infected macrophages (Mtb-CM) induces tunneling nanotubes (TNT) in HIV-infected macrophages thereby facilitating viral spreading. At the same time, Mtb-CM induced a glycolytic pathway leading to ATP accumulation in HIV-infected macrophages, an essential pathway for TNT induction whereas pharmacological interference with such a metabolic switch resulted in a reduced viral production.

Experimental approach: primary human monocytes differentiated into monocyte-derived macrophages (MDM) in the presence of a TB-dominated microenvironment (Mtb-CM). The intracellular rate of ATP production was evaluated by the Seahorse technology at day 3 of MDM differentiation. The measurements of basal extracellular acidification rate (ECAR) and basal oxygen consumption rate (OCR) were used to calculate ATP production rate from glycolysis (GlycoATP) and mitochondrial OXPHOS (MitoATP).*

* This is a well-conducted, innovative study exploring the interaction of two main human pathogens, i.e. Mtb and HIV, sharing macrophages as common target cell. The manuscript is clearly written and the conclusions and hypotheses are supported by experimental evidence. I have two general points that I encourage the authors to address.*

We thank the Reviewer for his/her valuable comments and address all provided comments below.

1. __ As mentioned in the Discussion, macrophage infection by HIV is characterized by the accumulation of preformed, infectious virions in VCC (Virus Containing Compartments) that can be pharmacologically modulated both in terms of accumulation and rapid release in the absence of cell cytopathicity. Although the modulation of VCC was not the objective of the present study, it would be important to discuss their role and their potential modulation by Mtb and/or metabolic modifications, if known.__ In the discussion, we mentioned that “In HIV-1 infected macrophages, ATP is also vital for the release of particles from virus-containing compartments (Graziano et al., 2015)”. Graziano et al. (PMID 26056317) showed that extracellular ATP favors the release of virions actively accumulating within the VCC of infected macrophages through its interaction with the P2X7 receptor. This study will be discussed more in detail in the revised version of the manuscript.

In addition, we fully agree with the reviewer that exploring potential modifications in the formation of virus containing compartments (VCC) following Mtb infection, CmMTB treatment or metabolic alterations is highly relevant. Importantly, VCCs are specific compartments in infected macrophages where new virions are generated and protected from the immune system and antiretroviral therapies. Interestingly, Siglec-1 was shown to be involved in VCC formation in infected macrophages (Jason E Hammonds et al., 2017; PMID 28129379), and we demonstrated that the level of expression of this lectin is increased in CmMTB-treated cells (Dupont et al., PMID: 32223897). We propose to perform new experiments during the revision process to look whether the formation of VCC is disturbed in CmMTB-treated macrophages upon HIV-1 infection, using the tetraspanin CD81 and/or Siglec-1 along with HIV-Gag to assess VCC formation (as in Reviewer Figure 1).

Reviewer Figure 1: VCC formation in multinucleated HIV-1 infected macrophages. Human macrophages were infected with HIV-1 (NLAd8-VSVG, 3 days) and stained with HIV-gag and CD81 to stain the VCC.

__ Understanding the purpose of using a VSV-g based infection system, nonetheless it would be important to know whether metabolic modulation does affect CD4 and CCR5 expression on MDM and its consequence for their susceptibility to HIV infection, in addition to the effects on TNT formation and viral transfer between cells.__

We appreciate this comment. The reviewer correctly understands that we used VSVG pseudotyped virus in this study to eliminate the effect of metabolic modulation on the expression of HIV entry receptors and potentially on virus entry. It has been previously demonstrated in CD4 T cells that the nutrient modulation does not affect HIV entry when the Blam-Vpr assay is used (Clerc et al., 2019, PMID 32373781, supplemental Figure 6).

In addition, as demonstrated in our earlier work (Souriant et al. Cell Reports, 2019), CmMTB treatment increases the levels of both CD4 and CCR5 on the surface of macrophages. However, it does not impact HIV entry, as shown using the same Blam-Vpr assay. Therefore, the exacerbation of HIV-1 infection in the TB-environment is not a consequence of increased viral entry. This will be clarified in the revised version of the manuscript.

As suggested by the reviewer, we will also conduct new experiments during the revision process. Specifically, we will assess the levels of entry receptors using flow cytometry analysis and measure virus entry using the Blam-Vpr fusion assay in CmMTB-treated cells, with or without Oxamate treatment (to inhibit glycolysis).

Specific points:

1. __ "TB-PE" (pleural effusion) is neither specified in the Results nor in the Methods sections.__ We thank the reviewer for pointing out this omission. TB-PE refers to pleural effusions from TB patients, a term we had previously defined only in the introduction and figure legends. We will ensure that this definition is explicitly stated in the Result and Methods sections of the revised manuscript.

__ Figure 3A does not seem to display cell viability, but rather HIV Gag expression by IFA. __

Indeed, there is an error in the text regarding cell viability. Cell viability following drug treatments was assessed by flow cytometry, as shown in Figure S2C. In Figure 3A, we included nuclear staining (in addition to HIV Gag) to confirm that cell density is not affected. This will be corrected in the revised manuscript. Additionally, we will perform F-actin staining to evaluate cell morphology and further confirm that all key parameters, i.e., viability, cell density, and cell morphology, are unaffected by the drugs used in Figure 3.

Furthermore, Figure 3C indicates Gag expression, not "HIV infection" (see page 8, Results).

We thank the reviewer for helping us to clarify this issue. In Figure 3C, the term “infection index” refers to the percentage of HIV Gag-positive cells resulting from productive infection. This is calculated as the total number of nuclei in HIV Gag-stained cells divided by the total number of nuclei, multiplied by 100, as described in the Methods section.

We have previously used this method to estimate the HIV infection rate in our published studies (Souriant et al., 2019; Dupont et al., 2020; Mascarau et al., 2023). To further improve the clarity and interpretation of the figure, we will include a clear definition of the infection index in the figure legend in the revised version of the manuscript.

Significance

The paper addresses a poorly explored area, i.e. the interaction of Mtb and HIV during infection of macrophages. The authors focused on a specific aspect of such an interaction (I,e, the modulation of nanotubes formation and transfer of virions to target cells), but their results can be extrapolated in a broader context, particularly if the authors will be willing to address my general questions. Although specific in its experimental approach, the implication of the study will be of interest to a general audience.

We appreciate this positive comment.__ __

Reviewer #2

__Evidence, reproducibility and clarity __

The current work is based on previous observations that the abundance of lung macrophages is augmented in NHPs with active TB and exacerbated in those coinfected with SIV (Dupont et al., 2022; Dupont et al., 2020; Souriant et al., 2019). Further work with these TB-induced immunomodulatory macrophages demonstrated an increased susceptibility to HIV-1 replication and spread via the formation of tunneling nanotubes (TNTs), (Souriant et al., 2019). In the present manuscript, the authors connected these findings with the metabolic state of macrophages (glycolysis vs OXPHOS). Using a range of metabolic inhibitors coupled with seahorse assays and microscopy confirmed the role of Mtb-induced glycolytic shift in inducing the formation of TNTs and the spread of HIV. The work is well-planned and executed. However, the study is mainly correlative without any molecular insights. The knowledge generated is important and valuable for future studies to understand the molecular players in regulating immunometabolism during HIV-TB coinfection.

We thank the Reviewer for his/her valuable comments, and we address all provided comments below.

Major Comments:

There are conflicting reports about Mtb's impact on macrophage ECAR and OXPHOS, which authors have acknowledged. Therefore, including OCR and ECAR plots along with the glycoATP and MitoATP data will be useful. Similarly, OCR/ECAR plots without any conditioned medium should be included to clarify the role of Mtb infection on OCR/ECAR.

In this manuscript, we evaluated the intracellular rate of ATP production in macrophages (day 3 of differentiation) treated with either cmCTR or cmMTB using Seahorse technology. Measurements of extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), both before and after the addition of oligomycin (an ATP synthase inhibitor), were used to calculate the contributions of glycolysis (GlycoATP, Figure 1B) and mitochondrial OXPHOS (MitoATP, Figure S1C) to total ATP production (Figure 1A).

We agree with the reviewer that displaying basal OCR/ECAR plots (bioenergetic profiles) would help characterize the overall energy phenotypes of macrophages. These graphs will be prepared and included in Figure S1. Furthermore, we will enhance the discussion and interpretation of these findings in the Results section of the revised manuscript.

As suggested, we will also assess ATP production using Seahorse technology for control cells (day 3 differentiated in RPMI) and provide OCR/ECAR plots for these new experiments.

__Fig 2G image is not convincing. While HIF1 alpha seems more in the nucleus, the overall morphology of the cell is more compact. Additional verification is needed. __

Regarding the specific comment on Fig. 2G, the reviewer is correct that the morphology of CmMTB-treated cells differs from that of CmCTR-treated cells. We have previously shown that CmMTB-treated macrophages display an M(IL-10) phenotype, characterized by a CD16+CD163+MerTK+PD-L1+ signature, morphological changes (cells appear rounder and form more TNTs), nuclear translocation of phosphorylated STAT3, and increased susceptibility to Mtb or HIV-1 infection (Dupont et al., 2022; Dupont et al., 2020; Lastrucci et al., 2015; Souriant et al., 2019).

As shown in Figure 2H, HIF1-α is predominantly cytoplasmic in most control cells, whereas an increased number of cells with nuclear HIF-1α staining were observed in CmMTB-treated cells. To quantify this observation, we manually assessed the ratio of HIF-1α signal intensity between the nucleus and cytoplasm in over 50 cells from three different donors. This methodology was not adequately explained in the Methods section and will be clarified in the revised manuscript. We also propose to include more representative images of HIF-1α-stained cells to support these findings.

Furthermore, genetic evidence is required in order to confirm if HIF1 alpha is the primary regulator of glycolytic shift by cmMTB/PE-TB, leading to more HIV dissemination by the TNT formation.

We fully agree that further experiments are essential to formally demonstrate that HIF-1α activation is responsible for the observed increase in HIV-1 infection and TNT formation in CmMTB-treated cells. To address this hypothesis, we propose conducting key experiments during the revision process

We will first use pharmacological approaches to modulate HIF-1α levels, as described in our recent publication (Maio et al., eLife, PMID 38922679). Specifically, we will test the HIF-1α inhibitor PX-478 as well as dimethyloxalylglycine (DMOG), a compound that stabilizes HIF-1α expression. These drugs will be applied 24h prior to HIV-1 infection in CmMTB-treated cells, and we will quantify HIV-1 infection and TNT formation on day 6 using immunofluorescence (IF).

In parallel, though technically challenging, we will attempt to reduce HIF-1α expression (and consequently its activity) in primary human monocytes using a siRNA-mediated depletion approach. This method has been successfully employed in our previous studies to target STAT3, STAT1 and Siglec-1 (Dupont et al., 2020; Lastrucci et al., 2015; Dupont et al., 2022). Under these conditions, we will measure HIV-1 infection and TNT formation on day 6 by IF.

Also, the authors have used only one tool to measure HIV levels -microscopy. While important, another method for verifying findings is needed. This is important as the effect of inhibitors (UK5099) is marginal.

In the present manuscript, we assess HIV-1 infection levels using two methods: microscopy (Figure 3 and 4I) and flow cytometry (Figure S2H-I). To address the reviewer’s comment, we propose to complement our current analysis of HIV-1 infection by evaluating HIV-1 replication through the measurement of HIV-p24 release in the supernatant of CmMTB-treated macrophages following drug treatments, as previously performed (Dupont et al., 2020; Souriant et al., 2019; Dupont et al., 2022; Mascarau et al., 2024; Raynaud-Messina et al., 2018).

Regarding the slight increase of HIV-1 infection (Gag expression by IF, Figure 3A) upon UK5099 treatment, we appreciate the reviewer’s valuable observation. Enhancing glycolysis levels remains a considerable challenge in studies targeting metabolic pathways, as most approaches focus on inhibiting glycolysis. However, in our study, the effect UK5099 on HIV-1 infection is reproducible and statistically significant, as demonstrated by analyzes of data from more than ten donors using IF (Figure 3C) and eight donors by flow cytometry (Figure S2H-I).

We acknowledge that the specific image provided in Fig. 3A for the UK5099 condition may not be the most representative and could cause confusion. To address this, we will replace the current image with a more representative one in the revised version of the manuscript.

Authors have used oxamate to inhibit glycolysis. Inhibition of LDH could lead to inhibition of NAD/NADH regeneration, thereby slowing down glycolysis. However, lack of lactate could have wide-ranging influence on cells as lactate could regulate several post-translational modifications, including lactylation. While the authors argued against using 2-DG, several findings confirm the glycolysis inhibitory potential of 2-DG when infected with Mtb. This should be included.

We understand the reviewer’s comment regarding the glucose analog 2-DG, which is widely used to inhibit glycolysis. Notably, recent studies have used it to show that glycolytic activity is critical for reactivating HIV-1 in macrophage reservoirs (Real et al., 2022, PMID 36220814).

In our study, we did not initially use 2-DG because it also inhibits glucose contribution to OXPHOS, making it challenging to distinguish between the roles of glycolysis and OXPHOS in macrophages (Wang et al., Cell Metabolism, PMID 30184486). Unlike Oxamate or GSK 2837, which specifically target LDHA, 2-DG does not exclusively affect glycolysis. Furthermore, inhibiting glucose metabolism with 2-DG is expected to yield similar results to glucose deprivation, as demonstrated in Figures 3H-K.

To address this, we propose conducting the suggested experiments using 2-DG in CmMTB-treated macrophages during the revision process. This will allow to assess their susceptibility to HIV-1 under this treatment. We will subsequently discuss the effects of 2-DG and integrate these results into the revised version of the manuscript.

A standard glycolytic function test (glucose, oligomycin and 2-DG injection) should be performed to assess the effect of TB-PE and cmMTB on the macrophages directly.

We appreciate the reviewer’s comment and will address it by testing the ability of CmMTB to alter the glycolytic activity of macrophages using the Seahorse Glycolytic Rate Assay. This assay, a refined version of the classical Seahorse Glycolysis Stress Test (see https://www.agilent.com/en/products/cell-analysis/glycolysis-assays-using-cell-analysis-technology), relies on an algorithm that generates the Proton Efflux Rate (PER), providing a robust quantitative measurement of glycolytic function. PER is directly correlated with lactate accumulation, enabling us to calculate glycolytic parameters that will complement our existing assays aimed at characterizing the glycolytic pathway in CmMTB-treated macrophages. We plan to perform these measurements and include the results in Figure 2.

__ Depriving glucose is not the best way to show the effect of glucose on HIV infection and MGC formation, as it can affect other aspects of cellular physiology, such as redox and bioenergetics. Instead, the use of galactose in place of glucose would generate ATP only by ____OXPHOS. Some key experiments should be repeated using galactose as a sole C source.__

We agree with this comment. In M2 macrophages, it has been shown that both glucose deprivation (as demonstrated in this study, Figure 3H-K) and glucose substitution with galactose (Wang et al., Cell Metabolism, PMID 30184486) effectively suppress glycolytic activity. Galactose must first be metabolized by the Leloir pathway before entering glycolysis, resulting in a significant reduction in glycolytic flux.

As suggested by the reviewer, we will complement our study by using galactose as the carbon source instead of glucose in a new set of experiments during the revision process.

__ UK5099 and oxamate nuclei seem smaller and less bright compared to the control. Images between control and UK5099 appear marginally different (non-significant).__

Figure 3A may not clearly convey that the nuclei are unaffected by the treatment. To address this, we will adjust the images, particularly the DAPI staining settings, to ensure accurate interpretation.

Regarding the slight effect of UK5099 treatment on Gag expression (infection index), as discussed above, this effect is reproducible and significant. We will replace the current image in Figure 3A with a more representative one.

The overall impact of the study is limited as the authors provide no evidence on the mechanism of how glycolysis induces TNT formation, which needs to be more characterized.

We fully agree that understanding how glycolysis induces tunneling nanotubes (TNTs) is a crucial and challenging question. This challenge stems from the incomplete understanding of the molecular mechanisms underlying TNT formation and the contradictory results reported across different cell types.

In our study, we demonstrated that inhibiting glycolysis—using Oxamate, GSK, or glucose deprivation—reduces TNT formation, whereas promoting glycolysis with UK5099 enhances their formation. We discuss in the manuscript that glycolysis likely provides the energy required for actin cytoskeletal rearrangements, which are essential for TNT formation.

Moreover, ATP plays a critical role in supporting cellular functions depending on actin remodeling, such as cell migration and the epithelial-to-mesenchymal transition (DeWane et al., 2021, PMID__33558441).__

To try to investigate the molecular mechanisms underlying TNT formation in our model, we propose the following experiments during the revision process:

• HIF1-α and TNT formation: IF staining of HIF1-α will be performed to correlate TNT formation with the level of HIF1-α nuclear translocation (as quantified in Figure 2I). This experiment aims to demonstrate a link between HIF1-α activation and TNT formation.
• Effect of HIF1-α inhibition: TNT formation will be quantified upon inhibiting HIF1-α activity using pharmacological approaches and/or siRNA-mediated gene silencing in HIV-1-infected CmMTB-treated cells.
• GLUT-1 focalization and TNT formation: To establish a connection between glycolysis and TNT formation, we will localize the primary glucose transporter GLUT-1 in relation to TNTs in CmMTB-treated macrophages. This approach builds on previous work on microvilli, which are F-actin structures with similarities to TNTs (Hexige et al., 2015, PMID: 25561062). Confocal or super-resolution microscopy will be employed to determine whether GLUT-1 accumulates at specific TNT sites. Through these experiments, we aim to provide deeper insights into the role of glycolysis in TNT formation.

__Minor comments:

The manuscript does not clearly show how the total ATP was calculated from the ATP rate assay.__

We will ensure that the method for calculating total ATP is explicitly described in the Methods section of the revised manuscript. __ In figure 1 (and everywhere else) the units on the y-axis should be corrected to [pmol/min] instead of pmol and the Seahorse profiles should mention whether the axis represents OCR or ECAR.__

The reviewer is correct. The axes in the relevant figures for ATP rate results (Figure 1A, B, C, D and Figure S1A, B, C) will be revised in the updated version of the manuscript.

The authors have called the macrophages highly glycolytic in first set of results which is misleading. Although the glycoATP contribution is increasing, overall ATP production is still majorly through oxidative phosphorylation (70% vs 25%).

We fully agree with the reviewer’s comment. As mentioned in the Result section “Approximately 90% of ATP production in macrophages differentiated with cmCTR came from OXPHOS; this parameter was reduced to 70% when conditioned with cmMTB (Figure 1E-F).” CmMTB and TB-PE drive macrophages toward an M2/M(IL-10) phenotype (Lastrucci et al. 2015), and based on the extensive literature on metabolism of anti-inflammatory M2 macrophages, this phenotype primarily relies on OXPHOS and fatty acid oxidation (for review see Biswas and Mantovani, Cell Metabolism, 2012).

It is therefore logical that overall ATP production in these cells remains predominantly through OXPHOS. However, we observe a significant decrease in OXPHOS activity following CmMTB treatment, alongside a marked increase in glycolysis (Figure 1).

Referring to CmMTB-treated macrophages as highly glycolytic was inaccurate, indeed, and this terminology will be corrected, with a clearer explanation provided in the revised manuscript.

Fig 3: Why does the HIV gag protein signal appear as irregular large spots?

In Figure 3A, the resolution used is sufficient to quantify the number of cells positive for HIV Gag (and thus the infection index). However, it does not allow for detailed examination of the intracellular localization of Gag as “spots”. The reviewer is correct that, within macrophages, the Gag signal often appears as large and intense cytoplasmic “spots” corresponding to the VCC, as illustrated in Reviewer Figure 1 in response to Reviewer 1.

__Referees cross-commenting:

I agree with the reviewer# 1 assessment. However, I feel that mechanistically paper could be improved and by performing more experiments.__

We fully agree that additional experiments are essential to improve the manuscript. We will address all comments and perform the experiments suggested by Reviewer 2, particularly to better characterize the metabolic state of our cells, provide evidence for the role of glycolysis in HIV-1 exacerbation, and further elucidate the mechanism by which glycolysis induces TNT formation.

Significance

The knowledge generated is important and valuable for future studies to understand the molecular players in regulating immunometabolism during HIV-TB coinfection.

We appreciate this positive comment.

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Referee #2

Evidence, reproducibility and clarity

The current work is based on previous observations that the abundance of lung macrophages is augmented in NHPs with active TB and exacerbated in those coinfected with SIV (Dupont et al., 2022; Dupont et al., 2020; Souriant et al., 2019). Further work with these TB-induced immunomodulatory macrophages demonstrated an increased susceptibility to HIV-1 replication and spread via the formation of tunneling nanotubes (TNTs), (Souriant et al., 2019). In the present manuscript, the authors connected these findings with the metabolic state of macrophages (glycolysis vs OXPHOS). Using a range of metabolic inhibitors coupled with seahorse assays and microscopy confirmed the role of Mtb-induced glycolytic shift in inducing the formation of TNTs and the spread of HIV. The work is well-planned and executed. However, the study is mainly correlative without any molecular insights. The knowledge generated is important and valuable for future studies to understand the molecular players in regulating immunometabolism during HIV-TB coinfection.

Major Comments

There are conflicting reports about Mtb's impact on macrophage ECAR and OXPHOS, which authors have acknowledged. Therefore, including OCR and ECAR plots along with the glycoATP and MitoATP data will be useful. Similarly, OCR/ECAR plots without any conditioned medium should be included to clarify the role of Mtb infection on OCR/ECAR.

Fig 2G image is not convincing. While HIF1 alpha seems more in the nucleus, the overall morphology of the cell is more compact. Additional verification is needed. Furthermore, genetic evidence is required in order to confirm if HIF1 alpha is the primary regulator of glycolytic shift by cmMTB/PE-TB, leading to more HIV dissemination by the TNT formation.

Also, the authors have used only one tool to measure HIV levels -microscopy. While important, another method for verifying findings is needed. This is important as the effect of inhibitors (UK5099) is marginal.

Authors have used oxamate to inhibit glycolysis. Inhibition of LDH could lead to inhibition of NAD/NADH regeneration, thereby slowing down glycolysis. However, lack of lactate could have wide-ranging influence on cells as lactate could regulate several post-translational modifications, including lactylation. While the authors argued against using 2-DG, several findings confirm the glycolysis inhibitory potential of 2-DG when infected with Mtb. This should be included.

A standard glycolytic function test (glucose, oligomycin and 2-DG injection) should be performed to assess the effect of TB-PE and cmMTB on the macrophages directly.

Depriving glucose is not the best way to show the effect of glucose on HIV infection and MGC formation, as it can affect other aspects of cellular physiology, such as redox and bioenergetics. Instead, the use of galactose in place of glucose would generate ATP only by OXPHOS. Some key experiments should be repeated using galactose as a sole C source.

UK5099 and oxamate nuclei seem smaller and less bright compared to the control. Images between control and UK5099 appear marginally different (non-significant).

The overall impact of the study is limited as the authors provide no evidence on the mechanism of how glycolysis induces TNT formation, which needs to be more characterized.

Minor comments:

The manuscript does not clearly show how the total ATP was calculated from the ATP rate assay.

In figure 1 (and everywhere else) the units on the y-axis should be corrected to [pmol/min] instead of pmol and the Seahorse profiles should mention whether the axis represents OCR or ECAR.

The authors have called the macrophages highly glycolytic in first set of results which is misleading. Although the glycoATP contribution is increasing, overall ATP production is still majorly through oxidative phosphorylation (70% vs 25%).

Fig 3: Why does the HIV gag protein signal appear as irregular large spots?

Referees cross-commenting

I agree with the reviewer# 1 assessment. However, i feel that mechanistically paper could be improved and by performing more experiments.

Significance

The knowledge generated is important and valuable for future studies to understand the molecular players in regulating immunometabolism during HIV-TB coinfection.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

Referee #1

Evidence, reproducibility and clarity

In the manuscript entitled "Inhibition of glycolysis in tuberculosis-mediated metabolic rewiring reduces HIV-1 spread across macrophages", Vahlas and colleagues investigated the hypothesis that Mtb interferes with HIV-1 infection of human macrophages, as they represent a common target cell type. In particular, they observed that a conditioned medium generated from Mtb-infected macrophages (Mtb-CM) induces tunneling nanotubes (TNT) in HIV-infected macrophages thereby facilitating viral spreading. At the same time, Mtb-CM induced a glycolytic pathway leading to ATP accumulation in HIV-infected macrophages, an essential pathway for TNT induction whereas pharmacological interference with such a metabolic switch resulted in a reduced viral production.

Experimental approach: primary human monocytes differentiated into monocyte-derived macrophages (MDM) in the presence of a TB-dominated microenvironment (Mtb-CM). The intracellular rate of ATP production was evaluated by the Seahorse technology at day 3 of MDM differentiation. The measurements of basal extracellular acidification rate (ECAR) and basal oxygen consumption rate (OCR) were used to calculate ATP production rate from glycolysis (GlycoATP) and mitochondrial OXPHOS (MitoATP).

This is a well-conducted, innovative study exploring the interaction of two main human pathogens, i.e. Mtb and HIV, sharing macrophages as common target cell. The manuscript is clearly written and the conclusions and hypotheses are supported by experimental evidence. I have two general points that I encourage the authors to address.

1. As mentioned in the Discussion, macrophage infection by HIV is characterized by the accumulation of preformed, infectious virions in VCC (Virus Containing Compartments) that can be pharmacologically modulated both in terms of accumulation and rapid release in the absence of cell cytopathicity. Although the modulation of VCC was not the objective of the present study, it would be important to discuss their role and their potential modulation by Mtb and/or metabolic modifications, if known.
2. Understanding the purpose of using a VSV-g based infection system, nonetheless it would be important to know whether metabolic modulation does affect CD4 and CCR5 expression on MDM and its consequence for their susceptibility to HIV infection, in addition to the effects on TNT formation and viral transfer between cells.

Specific points:

1. "TB-PE" (pleural effusion) is neither specified in the Results nor in the Methods sections.
2. Figure 3A does not seem to display cell viability, but rather HIV Gag expression by IFA. Furthermore, Figure 3C indicates Gag expression, not "HIV infection" (see page 8, Results).

Significance

The paper addresses a poorly explored area, i.e. the interaction of Mtb and HIV during infection of macrophages. The authors focused on a specific aspect of such an interaction (I,e, the modulation of nanotubes formation and transfer of virions to target cells), but their results can be extrapolated in a broader context, particularly if the authors will be willing to address my general questions.

Although specific in its experimental approach, the implication of the study will be of interest to a general audience.

eLife Assessment

Although others have proposed that OHC electromotility subserves cochlear amplification by acting as a "fluid pump", and evidence for this has been found using electrical stimulation of excised cochleae, this fundamental study substantially advances our understanding of cochlear homeostasis. This is the first report to test the pumping effect in vivo and consider its implications for cochlear homeostasis and drug delivery. The manuscript provides compelling evidence for OHC-based fluid flow within the cochlea.

Reviewer #1 (Public review):

Summary:

The authors test the "OHC-fluid-pump" hypothesis by assaying the rates of kainic acid dispersal both in quiet and in cochleae stimulated by sounds of different levels and spectral content. The main result is that sound (and thus, presumably, OHC contractions and expansions) result in faster transport along the duct. OHC involvement is corroborated using salicylate, which yielded results similar to silence. Especially interesting is the fact that some stimuli (e.g., tones) seem to provide better/faster pumping than others (e.g., noise), ostensibly due to the phase profile of the resulting cochlear traveling-wave response.

Strengths:

The experiments appear well controlled and the results are novel and interesting. Some elegant cochlear modeling that includes coupling between the organ of Corti and the surrounding fluid as well as advective flow supports the proposed mechanism.

The current limitations and future directions of the study, including possible experimental tests, extensions of the modeling work, and practical applications to drug delivery, are thoughtfully discussed.

Reviewer #2 (Public review):

Although recent cochlear micromechanical measurements in living animals have shown that outer hair cells drive broadband vibration of the reticular lamina, the role of this vibration in cochlear fluid circulation remains unclear. The authors hypothesized that motile outer hair cells facilitate cochlear fluid circulation. To test this, they investigated the effects of acoustic stimuli and salicylate on kainic acid-induced changes in the cochlear nucleus activities. The results reveal that low-frequency tones accelerate the effect of kainic acid, while salicylate reduces the impact of acoustic stimuli, indicating that outer hair cells actively drive cochlear fluid circulation.

The major strengths of this study lie in its high significance and the synergistic use of both electrophysiological recording and computational modeling. Recent in vivo observations of the broadband reticular lamina vibration challenge the traditional view of frequency-specific cochlear amplification. Furthermore, there is currently no effective noninvasive method to deliver the drugs or genes to the cochlea. This study addresses these important questions by observing outer hair cells' roles in the cochlear transport of kainic acid. The author utilized a well-established electrophysiological method to produce valuable new data and a custom-developed computational model to enhanced the interpretation of their experimental results.

The authors successfully validated their hypothesis, showing through the experimental and modeling results that active outer hair cells enhance cochlear fluid circulation in the living cochlea.

These findings have significant implications for advancing our understanding of cochlear amplification and offer promising clinical applications for treating hearing loss by accelerating cochlear drug delivery.

Reviewer #3 (Public review):

Summary:

This study reveals that sound exposure enhances drug delivery to the cochlea through the non-selective action of outer hair cells. The efficiency of sound-facilitated drug delivery is reduced when outer hair cell motility is inhibited. Additionally, low-frequency tones were found to be more effective than broadband noise for targeting substances to the cochlear apex. Computational model simulations support these findings.

Strengths:

The study provides compelling evidence that the broad action of outer hair cells is crucial for cochlear fluid circulation, offering a novel perspective on their function beyond frequency-selective amplification. Furthermore, these results could offer potential strategies for targeting and optimizing drug delivery throughout the cochlear spiral.

Weaknesses:

The primary weakness of this paper lies in the surgical procedure used for drug administration through the round window. Opening the cochlea can alter intracochlear pressure and disrupt the traveling wave from sound, a key factor influencing outer hair cell activity. However, the authors do not provide sufficient details on how they managed this issue during surgery. Additionally, the introduction section needs further development to better explain the background and emphasize the significance of the work.

Author response:

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

Reviewer #1 (Public review):

Summary:

The authors test the "OHC-fluid-pump" hypothesis by assaying the rates of kainic acid dispersal both in quiet and in cochleae stimulated by sounds of different levels and spectral content. The main result is that sound (and thus, presumably, OHC contractions and expansions) result in faster transport along the duct. OHC involvement is corroborated using salicylate, which yielded results similar to silence. Especially interesting is the fact that some stimuli (e.g., tones) seem to provide better/faster pumping than others (e.g., noise), ostensibly due to the phase profile of the resulting cochlear traveling-wave response.

Strengths:

The experiments appear well controlled and the results are novel and interesting. Some elegant cochlear modeling that includes coupling between the organ of Corti and the surrounding fluid as well as advective flow supports the proposed mechanism.

The current limitations and future directions of the study, including possible experimental tests, extensions of the modeling work, and practical applications to drug delivery, are thoughtfully discussed.

Weaknesses:

Although the authors provide compelling evidence that OHC motility can usefully pump fluid, their claim (last sentence of the Abstract) that wideband OHC motility (i.e., motility in the "tail" region of the traveling wave) evolved for the purposes of circulating fluid---rather then emerging, say, as a happy by-product of OHC motility that evolved for other reasons---seems too strong.

We adjusted our tone to be less assertive.

Our measurements and simulations coherently suggest that active outer hair cells in the tail region of cochlear traveling waves drive cochlear fluid circulation.

Reviewer #2 (Public review):

Although recent cochlear micromechanical measurements in living animals have shown that outer hair cells drive broadband vibration of the reticular lamina, the role of this vibration in cochlear fluid circulation remains unknown. The authors hypothesized that motile outer hair cells may facilitate cochlear fluid circulation. To test this hypothesis, they investigated the effects of acoustic stimuli and salicylate, an outer hair cell motility blocker, on kainic acid-induced changes in the cochlear nucleus activities. The results demonstrated that acoustic stimuli reduced the latency of the kainic acid effect, with low-frequency tones being more effective than broadband noise. Salicylate reduced the effect of acoustic stimuli on kainic acid-induced changes. The authors also developed a computational model to provide a physical framework for interpreting experimental results. Their combined experimental and simulated results indicate that broadband outer hair cell action serves to drive cochlear fluid circulation.

The major strengths of this study lie in its high significance and the synergistic use of electrophysiological recording of the cochlear nucleus responses alongside computational modeling. Cochlear outer hair cells have long been believed to be responsible for the exceptional sensitivity, sharp tuning, and huge dynamic range of mammalian hearing. However, recent observations of the broadband reticular lamina vibration contradict widely accepted view of frequency-specific cochlear amplification. Furthermore, there is currently no effective noninvasive method to deliver the drugs or genes to the cochlea, a crucial need for treating sensorineural hearing loss, one of the most common auditory disorders. This study addresses these important questions by observing outer hair cells' roles in the cochlear transport of kainic acid. The well-established electrophysiological method used to record cochlear nucleus responses produced valuable new data, and the custom-developed developed computational model greatly enhanced the interpretation of the experimental results.

The authors successfully tested their hypothesis, with both the experimental and modeling results supporting the conclusion that active outer hair cells can enhance cochlear fluid circulation in the living cochlea.

The findings from this study can potentially be applied for treating sensorineural hearing loss and advance our understanding of how outer hair cells contribute to cochlear amplification and normal hearing.

Reviewer #3 (Public review):

Summary:

This study reveals that sound exposure enhances drug delivery to the cochlea through the nonselective action of outer hair cells. The efficiency of sound-facilitated drug delivery is reduced when outer hair cell motility is inhibited. Additionally, low-frequency tones were found to be more effective than broadband noise for targeting substances to the cochlear apex. Computational model simulations support these findings.

Strengths:

The study provides compelling evidence that the broad action of outer hair cells is crucial for cochlear fluid circulation, offering a novel perspective on their function beyond frequency-selective amplification. Furthermore, these results could offer potential strategies for targeting and optimizing drug delivery throughout the cochlear spiral.

Weaknesses:

The primary weakness of this paper lies in the surgical procedure used for drug administration through the round window. Opening the cochlea can alter intracochlear pressure and disrupt the traveling wave from sound, a key factor influencing outer hair cell activity. However, the authors do not provide sufficient details on how they managed this issue during surgery. Additionally, the introduction section needs further development to better explain the background and emphasize the significance of the work.

Comments on revisions:

Thank you for addressing the comments and concerns. The author has responded to all points thoroughly and clarified them well. However, please include the key points from the responses to the comments (Introduction ((3), (5)) and Results ((5)) into the manuscript. While the explanations in the response letter are reasonable, the current descriptions in the manuscript may limit the reader's understanding. Expanding on these points in the Introduction, Results, or Discussion sections would enhance clarity and comprehensiveness.

Introduction (3): As inner-ear fluid homeostasis is maintained locally, longitudinal electro-chemical gradients, including the endocochlear potential, may vary along the cochlear length (Schulte and Schmiedt 1992; Sadanaga and Morimitsu 1995; Hirose and Liberman 2003).

Introduction (5): We do not want to distract the readers from the primary message by discussing different drug delivery methods into the inner ear. This paper is regarding active outer hair cells’ new role as the title suggests. An extensive discussion of drug delivery can confuse the theme of this work.

Results (5): High frequencies were not tested because they would not affect drug delivery to the apex of the cochlea (i.e., the traveling waves stop near the CF location.)

Not sure we need to mention file.path actually - here::here() is now pretty common so I don't think it matters to know the base solution

I would remove this, seems a bit out of scope and let's try not to overload them with informations

https://taz.de/Klimaschuetzer-zu-Wahlprogrammen/!6052182/

I will remove this now!

Comment on same text after its changed!

This is Page Note example!

They do deserve attention!

Der Weltbiodiversitätsrat IPBES fordert in zwei unmittelbar hintereinander publizierten Berichten, dem „Nexus Report“ und dem „Transformative Change Report“, ein radikale Transformation des bestehenden Wirtschaftssystems, um Kipppunkte nicht zu überschreiten und die miteinander zusammenhängenden ökologisch-sozialen Krisen zu bekämpfen https://www.lapresse.ca/actualites/environnement/2024-12-18/crise-de-la-biodiversite/un-rapport-choc-propose-de-reformer-le-capitalisme.php

Zum Transformative Change Report: https://www.ipbes.net/transformative-change/media-release

Zum Nexus Report: https://www.ipbes.net/nexus/media-release

wel zijn beïnvloed sociale steun & sociale steun beïnvloed welzijn

scron= minachting & gratitude = dankbaarheid

Zelfstigma is het proces waarbij een persoon negatieve stereotyperingen, vooroordelen, en stigma's uit de maatschappij over zichzelf internaliseert.

eLife Assessment

The manuscript presents valuable findings of bone remodeling under chronic unpredictable mild stress (CUMS). This is an interesting work on mental stress on bone health and osteoporosis, and the authors offer solid evidence of decreased bone mass mediated by miR-335-3p/Fos signaling in osteoclasts that are involved in the induction of bone loss caused by CUMS. This revised version provided new data that improved the quality of the manuscript and addressed the reviewers' concerns.

Reviewer #1 (Public review):

I have reviewed the manuscript "Psychological stress disturbs bone metabolism via miR-335-3p/Fos signaling in osteoclast" with interest. The described findings are relevant and useful for daily practice in periodontology. The paper is concise, professionally written, and easy to read. In this study, Jiayao et al. revealed the role of miR-335-3p in psychological stress-induced osteoporosis. CUMS mice were constructed to observe the femur phenotype, osteoclasts were identified as the main research object, and miRNA-seq was used to find the key miRNAs linking the brain and peripheral tissues. This study showed that miR-335-3p expression was simultaneously reduced in murine NAC, serum, and bone under psychological stress. The miR-335-3p/Fos/NFATC1 signaling pathway was validated in osteoclasts to reveal the potential mechanism of enhanced osteoclast activity under psychological stress. This study, from a new perspective of miRNAs, indicates a possible cause of disturbed bone metabolism due to psychological stress and may suggest a new approach to treating osteoporosis.

Reviewer #2 (Public review):

Zhang et al. established chronic unpredictable mild stress (CUMS) mouse model, which displayed osteoporosis phenotype, suggesting a potential correlation between psychological stress and bone metabolism. They found that miRNA candidate miR-335-3p is downregulated in the long bone of CUMS mice through microRNA sequencing experiments and qRT-PCR. They further demonstrated that miR-335-3p attenuates osteoclast activity via inhibiting Fos signaling, which can induce NFATC1 expression and regulate osteoclast activity.

My concerns have been addressed. And the quality of the manuscript is improved significantly.

Author response:

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

Public Reviews:

Reviewer #1 (Public Review):

I have reviewed, with interest, the manuscript "Psychological stress disturbs bone metabolism via miR-335-3p/Fos signaling in osteoclast". The described findings are relevant and useful for daily practice in periodontology. The paper is concise, professionally written, and easy to read. In this study, Jiayao et al. revealed the role of miR-335-3p in psychological stress-induced osteoporosis. CUMS mice were constructed to observe the femur phenotype, osteoclasts were identified as the primary research object, and miRNA-seq was used to find the key miRNAs linking the brain and peripheral tissues. This study showed that the expression of miR-335-3p was simultaneously reduced in mice's NAC, serum, and bone under psychological stress. The miR-335-3p/Fos/NFATC1 signaling pathway was validated in osteoclasts to reveal the potential mechanism of enhanced osteoclast activity under psychological stress. From a new perspective of miRNAs, this study indicates a possible cause of disturbed bone metabolism due to psychological stress and may suggest a new approach to treating osteoporosis.

We thank this reviewer for the instructive suggestions and encouragement.

Reviewer #2 (Public Review):

Zhang et al. established chronic unpredictable mild stress (CUMS) mouse model, which displayed osteoporosis phenotype, suggesting a potential correlation between psychological stress and bone metabolism. They found that miRNA candidate miR-335-3p is downregulated in the long bone of CUMS mice through microRNA sequencing and qRT-PCR experiments. They further demonstrated that miR-335-3p attenuates osteoclast activity via inhibiting Fos signaling, which can induce NFATC1 expression and regulate osteoclast activity.

Strengths:

The authors established CUMS mouse model and confirmed the osteoporosis phenotype through careful characterization of bone and analysis of osteoclast activity. They performed microRNA sequencing to identify the miRNA candidate regulating the bone loss in the CUMS mouse model. They also validated the expression of miR-335-3p and interfered with the function of miR-335-3p through an in vitro assay. Overall, the findings from this study provide important hints for the correlation between psychological stress and bone metabolism.

We thank this reviewer for the comprehensive summary and positive comment on our work.

Weakness:

The data provided by the authors are preliminary, especially the mechanistic insight, which needs to be enhanced. The authors have shown that miR-335-3p expression was altered in the CUMS mouse model and the change of its expression regulated osteoclast activity. The validation should be conducted in vivo, and the mechanism behind this should be investigated further.

We thank the reviewer’s important insight on the need for further in vivo validation of the role of miR-335-3p. Therefore, we designed and produced Antagomir-335-3p (antagonist) and Agomir-335-3p (agonist). Then, we injected them into the body through the tail vein for about 2 months and observed the bone phenotype in each group of mice. The results suggested that the decrease of miR-335-3p in vivo could lead to bone loss, which was consistent with our in vitro validation results (Figure 5H-I).

Reviewing Editor:

Method

(1) Bone histomorphometric analysis following ASBMR's guidelines Bone histomorphometric analysis of bone formation and bone resorption: The authors should follow ASBMR's guidelines for bone histomorphometry (PMCID: PMC3672237 and PMID: 3455637) to perform standard analyses of histomorphometry, rather than selected areas. They should also clearly describe a software used and define the areas analyzed.

We carefully re-analyzed bone histomorphometry according to ASBMR guidelines and combine this with our own understanding. At the same time, we improved the description of micro-CT and histological analysis in the method. If there is still any lack of standardization, we would be grateful for any constructive suggestions to improve this.

(2) Osteoclast cultures require nuclear staining to demonstrate multinucleated Trap positive cells.

We used the RAW264.7, a mouse macrophage-like cell line, for in vitro culture and induced its differentiation towards osteoclasts. Successfully induced osteoclasts showed enlarged cytoplasm and multinucleated fusion. Tartrate-resistant acid phosphatase (Trap) is the signature enzyme of osteoclasts. It can bind to the chromogen to exhibit a mauve color, based on the principle of azo-coupled immunohistochemistry. At the same time, small and rounded nuclei fused show a lighter color (author response image 1, yellow arrows). We attempted to stain the nuclei with hematoxylin based on this. However, it was unable to further distinguish the contours of the nuclei clearly due to the similar color to the Trap positive signals. Besides, many other scholars have assessed osteoclast activity in vitro experiments based solely on the results of Trap staining (area and number) (Cheng et al., 2022; Li et al., 2019; Ma et al., 2021; Zhong et al., 2023). Nevertheless, in the immunofluorescence staining of osteoclasts, the nuclei were labeled using a Hochest antibody to reflect the multinucleated fusion of osteoclasts (Figure 5G).  

(3) Osteoclast pit assays should be carried out to necessarily demonstrate the change of osteoclast resorption ability caused by miR-335-3p.

We added osteoclast pit assays to validate the role of miR-335-3p on osteoclast resorptive capacity (Figure 5D-E).

(4) Serum ELISA assay should be done to examine the global change of bone remodeling in the CUMS mice to assess bone formation and bone resorption that will support their claim.

We performed additional tests on serum concentrations of R-hydroxy glutamic acid protein (BGP), TRAP, Cathepsin K (CTSK), parathyroid hormone (PTH), calcium (CA), phosphate (P) in control and CUMS mice, which could better reflect the global change of bone remodeling in the CUMS mice (Figure 3— figure supplement 1).

(5) miR-RNA-seq: A labeled volcano plot should be used to replace the present one to show significant changes in differential gene expression.

We appreciate this great suggestion. We replaced the volcano plot that showed significant changes in differential gene expression (Figure 4B). We also uploaded the raw data to the GEO database (GSE253504), making the results clearer and more accessible.

Discussion

The authors should discuss previous works on the influences of hormones from the brain on chronic stress-induced bone loss and an association of these influences with their findings.

The discussion on the relationship between the bone metabolism regulation of both hormones and miR-335-3p in psychological stress was added in the second and fifth paragraphs of the discussion. To conclude, on the one hand, brain-derived and blood-transported miR-335-3p regulate bone metabolism synergistically. On the other hand, it exerted a more direct influence on bone under psychological stress.

Language

The language of the MS should be improved.

The manuscript has been carefully edited by a professional proofreader.

Reviewer #1 (Recommendations For The Authors):

(1) Figure 1F: The exact meaning of the Waveform Graph shown at left needs to be clarified for the not-so-experienced reader.

We added the more detailed meaning of the Waveform Graph in figure legends (Figure legend 1F).

(2) Is the concomitant increase in osteogenic and osteoblastic activity in this study consistent with that seen in similar disease studies? This could be added to the discussion.

In the fifth paragraph of the discussion section, we present the alterations of osteogenic and osteoblastic activity observed in other studies that are similar to ours. We also had a detailed discussion based on these observations.

(3) Figure 6A: Please highlight the key information to visualize the potential linkage among miR-335-3p, Fos, and osteoclast.

We highlighted the crucial linkage among miR-335-3p, Fos, and osteoclast with red arrows (Figure 6A)

4) Figure 6E: The specific area of the selected comparison needs to be clarified. Please add white dotted lines and lettering T (trabecular bone) and GP (growth plate) for the not-so-experienced reader. This will provide some orientation.

We used white dotted lines as well as letters to label the tissue in immunofluorescence staining images (Figure 6E).

(5) Line 350: "NAC derived and blood-trans, Ported miR-335-3p". There is a grammatical error. Please conduct general proofreading of the text and writing style.

Thank you for pointing this out. We have corrected this grammatical error, and we also checked the full text to correct similar errors.

Reviewer #2 (Recommendations For The Authors):

(1) miR-335-3p was downregulated in the femur in the CUMS mice. The possible mechanism for this outcome should be further discussed. In Figure 4B, the Volcano plot showed that only a few miRNA were differentially expressed between the control and CUMS mice. How do the authors explain this?

The chronic unpredictable mild stress (CUMS) model was constructed using normal mice. As the name of the model suggests, the stimulus is mild and does not cause developmental damage or teratogenic effects in mice. Conversely, CUMS has the potential to result in the chronic pathological conditions. Besides, in miRNA sequencing results from other tissues with similar models to ours, the number of differential miRNAs is also around a few dozen (Ma et al., 2019).

(2) The authors have demonstrated that miR-335-3p inhibits osteoclast differentiation based on an in vitro assay in Figure 5; however, an in vivo experiment is required to provide more solid evidence.

We strongly agree that in vivo experimental validation would bring more convincing results to this study. Therefore, we designed and produced Antagomir-335-3p (antagonist) and Agomir-335-3p (agonist), which were injected into mice via the tail vein every five days. Samples were collected at one and two months following the injection. We found that sustained two-month injections of antagomir could significantly lead to bone loss in mice (Figure 5H-I), which is consistent with our in vitro validation results.

However, the Agomir-miR-335-3p group did not exhibit a notable enhancement of bone mass. This may be attributed to the fact that the 11-week-old normal mice selected for this study were in their prime and did not have strong osteoclastic activity in vivo. Therefore, the osteoclastic inhibition of Agomir-335-3p could not be demonstrated.

In addition, no significant difference was seen one month after the injection. The main reason may be that the time is too short. On the one hand, the drug we injected was RNA preparation. They lacked stability resulting in poor delivery efficiency, which took some time to take effect. On the other hand, bone remodeling is also a time-consuming process.

(3) FOS and NFATC1 should be expressed in the nuclei of the cells, therefore, the quality of the images needs to be improved.

NFATC1 is a T-cell-activating nuclear factor that is activated in the nucleus to regulate the transcription of a variety of osteoclast-related genes, including ACP5, MMP9, etc. FOS could bind and interact with NFATC1, resulting in nuclear translocation and transcription activated. This could promote the differentiation and maturation of osteoclasts. They are both synthesized and processed in the cytoplasm and eventually enter the nucleus to perform their functions. Therefore, they are expressed in both the nucleus and the cytoplasm (Deng et al., 2022; Hounoki et al., 2008; Li et al., 2022).

In Figure 5G, we labeled cell nuclei with HOCHEST antibody with blue fluorescence, and more co-localized signals of nuclei (blue), FOS (red), and NFATC1 (green) were seen in the Inhibitor-miR-335-3p group, whereas the opposite result was observed in the Mimic-miR-335-3p group. These results indicated that inhibited miR-335-3p could promote osteoclast differentiation in vitro.

(4) The expression of FOS was elevated in CUMS group in Figure 6E; however, its mRNA level was unchanged, as shown in Figure 6 supplement; what is the explanation for this? How do the authors claim FOS is the downstream target if its mRNA expression is not impacted by CUMS?

The results demonstrated that miR-335-3p targeted binding to the mRNA of Fos did not result in mRNA degradation. Instead, this binding interferes with the protein translation process, which ultimately leads to the reduction of FOS protein.

(5) What would be the bone phenotype if a FOS inhibitor was injected into the control and CUMS mice? It is important to examine FOS function through an in vivo context.

The regulatory role of FOS for osteoclasts has been validated in numerous articles, both in vivo and in vitro(Aikawa et al., 2008; Cao et al., 2023; Cheng et al., 2022). For example, Aikawa et al. designed a small-molecule inhibitor of c-Fos/activator protein-1 (AP-1) using three-dimensional (3D) pharmacophore modeling, which helped verify the effect of FOS on osteoclasts in vivo(Aikawa et al., 2008).

We also strongly agree that in vivo injection of inhibitors of FOS, especially in CUMS mice, could further substantiate the role of miR-335-3p in osteoclasts under psychological stress. However, the study was constrained by the unavailability of commercially viable, efficacious small molecule inhibitors of FOS. In the future, we plan to design more precise therapeutic targets for psychological stress induced osteoporosis based on existing research ideas.

Reference

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Li, Y., Yang, C., Jia, K., Wang, J., Wang, J., Ming, R., Xu, T., Su, X., Jing, Y., Miao, Y., Liu, C., & Lin, N. (2022). Fengshi Qutong capsule ameliorates bone destruction of experimental rheumatoid arthritis by inhibiting osteoclastogenesis. Journal of Ethnopharmacology, 282, 114602. https://doi.org/10.1016/j.jep.2021.114602

Li, Z., Huang, J., Wang, F., Li, W., Wu, X., Zhao, C., Zhao, J., Wei, H., Wu, Z., Qian, M., Sun, P., He, L., Jin, Y., Tang, J., Qiu, W., Siwko, S., Liu, M., Luo, J., & Xiao, J. (2019). Dual Targeting of Bile Acid Receptor-1 (TGR5) and Farnesoid X Receptor (FXR) Prevents Estrogen-Dependent Bone Loss in Mice. Journal of Bone and Mineral Research : the Official Journal of the American Society For Bone and Mineral Research, 34(4), 765-776. https://doi.org/10.1002/jbmr.3652

Ma, K., Zhang, H., Wei, G., Dong, Z., Zhao, H., Han, X., Song, X., Zhang, H., Zong, X., Baloch, Z., & Wang, S. (2019). Identification of key genes, pathways, and miRNA/mRNA regulatory networks of CUMS-induced depression in nucleus accumbens by integrated bioinformatics analysis. Neuropsychiatric Disease and Treatment, 15, 685-700. https://doi.org/10.2147/NDT.S200264

Ma, Q., Liang, M., Wu, Y., Luo, F., Ma, Z., Dong, S., Xu, J., & Dou, C. (2021). Osteoclast-derived apoptotic bodies couple bone resorption and formation in bone remodeling. Bone Research, 9(1), 5. https://doi.org/10.1038/s41413-020-00121-1

Zhong, L., Lu, J., Fang, J., Yao, L., Yu, W., Gui, T., Duffy, M., Holdreith, N., Bautista, C. A., Huang, X., Bandyopadhyay, S., Tan, K., Chen, C., Choi, Y., Jiang, J. X., Yang, S., Tong, W., Dyment, N., & Qin, L. (2023). Csf1 from marrow adipogenic precursors is required for osteoclast formation and hematopoiesis in bone. eLife, 12. https://doi.org/10.7554/eLife.82112

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eLife Assessment

This study presents an advance in efforts to use histone post-translational modification (PTM) data to model gene expression and predict epigenetic editing activity. Such models are broadly useful to the research community, especially ones that can model epigenetic editing activity, which is novel; additionally, the authors have nicely integrated datasets across cell types into their model. The work is mostly solid, but it would be strengthened by performing rigorous comparisons to existing methods that predict gene expression from PTM data and from additional model validation beyond dCas9-p300 based perturbations.

Reviewer #1 (Public review):

Batra, Cabrera and Spence et al. present a model which integrates histone posttranslational modification (PTM) data across cell models to predict gene expression with the goal of using this model to better understand epigenetic editing. This gene expression prediction model approach is useful if a) it predicts gene expression in specific cell lines b) it predicts expression values rather than a rank or bin, c) if it helps us to better understand the biology of gene expression or d) it helps us to understand epigenome editing activity. Problematically for point a) and b) it is easier to directly measure gene expression than to measure multiple PTMs and so the real usefulness of this approach mostly relates to c) and d).

Other approaches have been published that use histone PTM to predict expression (e.g. PMID 27587684, 36588793). Is this model better in some way? No comparisons are made although a claim is made that direct comparisons are difficult. I appreciate that the authors have not used the histone PTM data to predict gene expression levels of an "average cell" but rather that they are predicting expression within specific cell types or for unseen cell types. Approaches that predict expression levels are much more useful whereas some previous approaches have only predicted expressed or not expressed or a rank order or bin-based ranking. The paper does not seem to have substantial novel insights into understanding the biology of gene expression.

The approach of using this model to predict epigenetic editor activity on transcription is interesting and to my knowledge novel although only examined in the context of a p300 editor. As the author point out the interpretation of the epigenetic editing data is convoluted by things like sgRNA activity scoring and to fully understand the results likely would require histone PTM profiling and maybe dCas9 ChIP-seq for each sgRNA which would be a substantial amount of work.

Furthermore from the model evaluation of H3K9me3 is seems the model is performing modestly for other forms of epigenetic or transcriptional editing- e.g. we know for the best studied transcriptional editor which is CRISPRi (dCas9-KRAB) that recruitment to a locus is associated with robust gene repression across the genome and is associated with H3K9me3 deposition by recruitment of KAP1/HP1/SETDB1 (PMID: 35688146, 31980609, 27980086, 26501517).

One concern overall with this approach is that dCas9-p300 has been observed to induce sgRNA independent off target H3K27Ac (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8349887/ see Figure S5D) which could convolute interpretation of this type of experiment for the model.

Reviewer #2 (Public review):

Summary:

The authors build a gene expression model based on histone post-translational modifications, and find that H3K27ac is correlated with gene expression. They proceed to perturb H3K27ac at 13 gene promoters in two cell types, and measure gene expression changes to test their model.

Strengths:

The combination of multiple methods to model expression, along with utilizing 6 histone datasets in 13 cell types allowed the authors to build a model that correlates between 0.7-0.79 with gene expression. They use dCas9-p300 fusions to perturb H3K27ac and monitor gene expression to test their model. Ranked correlations of the HEK293 data showed some support for the predictions after perturbation of H3K27ac.

Weaknesses:

The perturbation of 5 genes in K562 with perturb-seq data shows a modest correlation of ~0.5 and isn't included in the main figures. The authors are then left to speculate reasons why the outcome of epigenome editing doesn't fit their predictions, which highlights the limited value in the current version of this method.

As mentioned before, testing genes that were not expressed being most activated by dCas9-p300 weaken the correlations vs. looking at a broad range of different gene expression as the original model was trained on.<br /> If the authors want this method to be used to predict outcomes of epigenome editing, expanding to dCas9-KRAB and other CRISPRa methods (SAM and VPR) would be useful. Those datasets are published and could be analyzed for this manuscript.<br /> The authors don't compare their method to other prediction methods.

Author response:

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

Reviewer #1 (Public Review):

Batra, Cabrera, Spence et al. present a model which integrates histone posttranslational modification (PTM) data across cell models to predict gene expression with the goal of using this model to better understand epigenetic editing. This gene expression prediction model approach is useful if a) it predicts gene expression in specific cell lines b) it predicts expression values rather than a rank or bin, c) it helps us to better understand the biology of gene expression, or d) it helps us to understand epigenome editing activity. Problematically for points a) and b) it is easier to directly measure gene expression than to measure multiple PTMs and so the real usefulness of this approach mostly relates to c) and d).

We thank the reviewer for their comment and we agree that directly measuring gene expression (e.g., by performing RNA-seq) is easier than performing multiple PTMs in a new cell line. We designed our approach keeping in mind that the primary use case is to understand how epigenome editing would affect gene expression.

Other approaches have been published that use histone PTM to predict expression (e.g. 27587684, 36588793). Is this model better in some way? No comparisons are made. The paper does not seem to have substantial novel insights into understanding the biology of gene expression. The approach of using this model to predict epigenetic editor activity on transcription is interesting and to my knowledge novel but I doubt given the variability of the predictions (Figures 6 and S7&8) that many people will be interested in using this in a practical sense. As the authors point out, the interpretation of the epigenetic editing data is convoluted by things like sgRNA activity scoring and to fully understand the results likely would require histone PTM profiling and maybe dCas9 ChIP-seq for each sgRNA which would be a substantial amount of work.

We thank the reviewer for this insightful comment. We have included citations for a series of papers (PMIDs: 27587684, 30147283, 36588793) that performed gene expression prediction using histone PTM data. However, each of these methods perform classification of gene expression as opposed to predicting the actual gene expression value via regression. Additionally, the referenced studies all work with Roadmap Epigenomics read depth data as opposed to p-values obtained from the ENCODE pipelines, making it difficult to make direct comparisons.

We outline in the Discussion section that by creating a comprehensive dataset of epigenome editing outcomes, which include quantification of histone PTMs before and after in situ perturbations, will improve our understanding of the effects of dCas9-p300 on gene expression and assist in the design of gRNAs for achieving fine-tuned control over gene expression levels. 

Furthermore from the model evaluation of H3K9me3 it seems the model is not performing well for epigenetic or transcriptional editing- e.g. we know for the best studied transcriptional editor which is CRISPRi (dCas9-KRAB) that recruitment to a locus is associated with robust gene repression across the genome and is associated with H3K9me3 deposition by recruitment of KAP1/HP1/SETDB1 (PMID: 35688146, 31980609, 27980086, 26501517). However, it seems from Figures 2&4 that the model wouldn't be able to evaluate or predict this.

We thank the reviewer for their comment. We have included a supplementary figure, Figure 4 – figure supplement 1, that quantifies how sensitive the trained gene expression model is to perturbations in H3K9me3. Indeed our data suggests that the model predictions are sensitive to perturbations in H3K9me3. For instance, there is a clear decrease and a gradual increase as the position where the perturbation is performed moves from upstream to downstream of the TSS. Additionally, the magnitude of the predicted fold-change is a function of how much the H3K9me3 is perturbed and hence the magnitude of change would be even higher if the perturbation magnitude is increased. However, this precise magnitude is hard to estimate In the absence of experimental perturbation data for H3K9me3.

The model seems to predict gene expression for endogenous genes quite well although the authors sometimes use expression and sometimes use rank (e.g. Figure 6) - being clearer with how the model predicts expression rather than using rank or fold change would be very useful.

We thank the reviewer for this important suggestion. We have added text in the revised manuscript to clarify that the model predicts gene expression values, which can be interpreted as rank or fold change, depending on the use case.

One concern overall with this approach is that dCas9-p300 has been observed to induce sgRNA-independent off-target H3K27Ac (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8349887/ see Figure S5D) which could convolute interpretation of this type of experiment for the model.

This is an excellent point and indeed, we and others have observed that dCas9-p300 can result in off-target H3K27ac levels (both increased and suppressed) across the genome. However, p300 is one of the few known proteins that can catalyze H3K27ac in the human genome, and H3K27ac remains a proxy for active genomic regulatory elements. Nevertheless, dCas9-p300 off target activity could certainly convolute our approach. We have included language to address this caveat in our discussion. Interestingly, even though dCas9-p300 (and other epigenome editing enzymes) can lead to off-target chromatin modifications, these effects often occur without coincident disruptions to the transcriptome. This suggests that many chromatin modifications, while “supportive” or “instructive” of/for transcription, may be insufficient (either alone or in the context of dCas9-based fusions) for transcriptional effects.

Figure 2

It seems this figure presents known rather than novel findings from the authors' description. Please comment on whether there are any new findings in this figure. Please comment on differences in patterns of repressive and activating histone PTMs between cell lines (e.g. H1-Esc H3K27me3 green 25-50% is more enriched than red 0-25%).

Thank you for pointing out this issue. We have revised the text in both the Results and Discussion sections to better articulate that the goal of this figure is to validate the hypothesis that there are consistent patterns of histone PTMs with respect to gene expression across different human cell types.

In Figure 2, which illustrates the raw histone marks data, the non-monotonic behavior of H3K27me3 in H1-hESC cells is indicative of a real biological phenomenon. This interpretation is supported by the relatively low Pearson correlation for the H3K27me3 mark observed in these cells, as documented in Figure 1b of another study: https://www.biorxiv.org/content/10.1101/2024.03.29.587323v1.

Figure 3&4

There are a number of approaches including DeepChrome and TransferChrome that predict endogenous gene expression from histone PTMs. I appreciate that the authors have not used the histone PTM data to predict gene expression levels of an "average cell" but rather that they are predicting expression within specific cell types or for unseen cell types. But from what is presented it isn't clear that the author's model is better or enabling beyond other approaches. The authors should show their model is better than other approaches or make clear why this is a significant advance that will be enabling for the field. For example is it that in this approach they are actually predicting expression levels whereas previous approaches have only predicted expressed or not expressed or a rank order or bin-based ranking?

We thank the reviewer for this comment. We have added text to clarify the difference between our approach and existing approaches. There are two key differences between our model and other approaches. First, the gene expression model that we have trained here predicts gene expression values instead of gene expression levels as either high or low. Second, we have trained our models on ENCODE p-value data instead of read depths obtained from the Roadmap Epigenomics Consortium.

Figure 5

From the methods, it seems gene activation is measured by qpcr in hek293 transfected with individual sgRNAs and dCas9-p300. The cells aren't selected or sorted before qPCR so how are we sure that some of the variability isn't due to transfection efficiency associated with variable DNA quality or with variable transfection efficiency?

This is a good question. All DNA preps were generated using high-quality reagents and consistent protocols. In addition, the only variable that changed with respect to transfection efficiency was the gRNA-encoding vector used in qPCR assays. We have added new data which demonstrates that transfection efficiency is shared across experiments (Figure 5 – figure supplement 1). We have also added additional experimental data as well as computational analysis analyzing a new dCas9-p300 based Perturb-seq dataset to the manuscript (Figure 6 – figure supplement 1), which use lentiviral transduction and RNA-seq as readouts and thus, are buffered against the variances mentioned by the Reviewer.

Figure 6

The use of rank in 6D and 6E is confusing. In 6D a higher rank is associated with higher expression while in 6E a higher rank seems to mean a lower fold change e.g. CYP17A1 has a low predicted fold-change rank and qPCR fold-change rank but in Figure 5 a very high qPCR fold change. Labeling this more clearly or explaining it in the text further would be useful.

We thank the reviewer for their suggestion. We have made relevant changes to the caption of Figure 6 to clarify this.

Reviewer #2 (Public Review):

Summary:

The authors build a gene expression model based on histone post-translational modifications and find that H3K27ac is correlated with gene expression. They proceed to perturb H3K27ac at 8 gene promoters, and measure gene expression changes to test their model.

Strengths:

The combination of multiple methods to model expression, along with utilizing 6 histone datasets in 13 cell types allowed the authors to build a model that correlates between 0.7-0.79 with gene expression. This group also utilized a tool they are experts in, dCas9-p300 fusions to perturb H3K27ac and monitor gene expression to test their model. Ranked correlations showed some support for the predictions after the perturbation of H3K27ac.

Weaknesses:

The perturbation of only 8 genes, and the only readout being qPCR-based gene expression, as opposed to including H3K27ac, weakened their validation of the computational model. Likewise, the use of six genes that were not expressed being most activated by dCas9-p300 might weaken the correlations vs. looking at a broad range of different gene expressions as the original model was trained on.

We thank the reviewer for their comments. We have added additional experimental data as well as computational analysis analyzing a new dCas9-p300 based Perturb-seq dataset to the manuscript. We observe that the models we have developed are able to predict the fold-change rank across genes reasonably well (Figure 6 – figure supplement 1), similar to what we observe in Figure 6E.

Reviewer #1 (Recommendations For The Authors):

The authors should comment on how their model is different from or better than other models that use histone PTM data to predict gene expression.

We thank the reviewer for this insightful suggestion. We have included citations for a series of papers (PMIDs: 27587684, 30147283, 36588793) that performed gene expression prediction using histone PTM data. However, each of these methods perform classification of gene expression as opposed to predicting the actual gene expression value via regression. Additionally, the referenced studies all work with Roadmap Epigenomics read depth data as opposed to p-values obtained from the ENCODE pipelines, making it difficult to make direct comparisons.

The authors need to make clear whether their model will apply to other common epigenetic or transcriptional editors such as CRISPRi/H3K9me3 which is widely used.

In this study, we focus on the histone changes induced by p300. However, future studies may use the framework described in our manuscript and apply it to other transcriptional editors as well.

The authors need to be clearer about where they are predicting expression and where they are using rank. Ideally, show both.

We thank the reviewer for this important suggestion. We have added text in the revised manuscript to clarify that the model predicts gene expression values, which can be interpreted as rank or fold change, depending on the use case.

The authors should ideally show a case where they use the model to make a prediction of genes that can and can not be activated by dCas9-p300 or other epigenetic editors and then prove this with experiments.

Thank you for the excellent suggestion. While it is indeed relevant, exploring this would extend beyond the scope of our current study. We consider it a valuable topic for future research.

Reviewer #2 (Recommendations For The Authors):

The y-axis in 5C needs to be labeled. The authors state it is "relative mRNA" but these numbers correlated with fold changes shown in Table S2.

We have clarified the definition of the Y-axis in the caption for Figure 5C.

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Some notes on blocking for different purposes in GEMM operations.

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eLife Assessment