italics, as it is a foreign word. I don't think it is necessary to capitalize it

replace with: intonarumori

replace with: Russolo's

add full stop after Russolo

italics?

delete space after full stop

Would it be ok to refer to my book here, where I have argued this? See: Laura Scuriatti, Mina Loy's Critical Modernism (Gainesville: University Press of Florida, 2019), pp. 46-60.

italics except for FUTURISM

italics, except for LOVE

italics except for LOVE

italics, except for LOVE (twice)

italics, except NATURE

italics, except FUTURISM

everything in italics except: FUTURISM, LOVE, INMATES, MEN

everything in italics except FUTURISM and LOVE

everything in italics except LOVE

everything in italics except FUTURISM; replace Drags with: drags

draws and forward in italics

italics

italics

replace with: Nature

replace with: inconsummate

delete line break

replace with: have to

replace with: they

replace dash with hyphen

italics

replace with: now?

replace with: proto-poem

replace with: life-blood

replace with: Future

Replace with: A MAN

replace with: A MAN

This isn't in Crangle's edition. if we follow it, this should be deleted

replace with: continuing

replace with: to

italics

replace with: DIANA

replace with: By

replace with: holding

no line break before: tomorrow...

add line break before (turning the first three dots should be next to eyes, not in a new line

replace with: left-hand

replace with: Pass

add line break after full stop

replace hyphen with em-dash

add line break before: No use

delete: up

add line break before Those

no line break between first and dots

replace with: shoulders

replace hyphens with em-dashes

no line break after mud

line break after you....?

replace hyphen with em-dash

replace with: anxiously

replace with: LOONY

replace with: DIANA

the three dots should be next to know

replace hyphen with em-dash

replace with: DIANA

delete: he

line break after the bracket and three dots after pig

replace with: snoozily

replace with: DIANA

replace with: LOONY

replace with: PICKED PEOPLE

replace with: LOONY

indented in Crangle

replace with: DIANA

I would replace "he" with "Loony" to make the sentence clearer and more elegant

replace with: DIANA

replace with: bric-à-brac line should be indented (as in Crangle)

indented in Crangle

no line break between round and numbers

replace with: And

replace with: talk about something!

replace with: warm-footed

replace with: DIANA delete full stop

replace with: to

replace with: back) - the latter is an em-dash

replace with: DIANA's

line break after dots

delete full stop

the dots should be next to submerged

delete endnote

delete endnote

replace with: LOONY's

replace with dash

replace with: DIANA

the dots should be next to caress, not in a new line

Could we add a refence to my book here, where I write about this issue? Also, if we want to keep our initials for the notes, this would end with: (LS). The reference to my book is: See L. Scuriatti, Mina Loy's Critical Modernism (Gainesville: University of Florida Press, 2019), pp. 113-116.

add space. It should read: museums and

replace "souls to" with: soul's

replace with: china

replace "his" with: the

replace with: anticipate

replace "for autumn" with: for the autumn

replace with: "Has to have"

replace with: DIANA

replace with: Futurism

Replace with HOUSELESS LOONY

replace with LADY DIANA

no line break

italics

There does not seem to be a line break in Crangle's edition, but it is not clear. Is there a break in The Dial?

delete full stop

replace with FRIENDS

dots should be next to "you", and not in a new line

the three dots should not be on a new line, but next to frost

it should read "into the Arabic" - although it sounds wrong. Either we correct it and add [sic], or insert an excision sign to show that we have intervened onto the text

delete full stop

Italics. "With" should be "with"

italics

italics

delete "his"

delete commas. It should read: Ossy you know

delete comma

insert space between note and knows

hyphenate: steam-heating

delete is

italics

italics

Capitalized: People

Résumé de la vidéo [00:00:03][^1^][1] - [00:03:49][^2^][2]:

Cette vidéo présente un entretien pour un poste de directeur des opérations, qui s'avère être le rôle d'une mère. Les candidats réagissent à la description du poste, qui exige une disponibilité constante, aucune pause, et une multitude de compétences, sans salaire. La révélation que des milliards de personnes, les mères, occupent déjà ce poste, suscite admiration et gratitude.

Points forts: + [00:00:24][^3^][3] Description du poste * Intitulé : Directeur des opérations * Exige une grande mobilité et endurance * Travail debout et activité constante + [00:01:02][^4^][4] Horaires exigeants * 135 heures par semaine, potentiellement 24/7 * Pas de pauses, même pour manger * Nécessite d'excellentes compétences en négociation et relationnelles + [00:01:57][^5^][5] Sacrifices personnels * Pas de vie personnelle, pas de vacances * Charge de travail accrue pendant les fêtes * Attitude positive exigée en tout temps + [00:02:34][^6^][6] Aucune rémunération * Le poste ne prévoit aucun salaire * Réaction de surprise et d'incrédulité des candidats * Révélation que le poste est celui d'une mère

Résumé de la vidéo [00:02:40][^1^][1] - [00:03:49][^2^][2]:

La vidéo présente un entretien pour un poste de directeur des opérations, qui s'avère être une métaphore pour le rôle d'une mère. Les candidats sont choqués par les exigences extrêmes du poste, qui incluent une disponibilité 24/7, aucune pause, et des compétences dans divers domaines, le tout sans salaire. La révélation que des milliards de personnes, les mères, occupent déjà ce poste, suscite admiration et gratitude.

Points forts: + [00:02:40][^3^][3] Les exigences du poste * Disponibilité constante, sans pauses * Compétences en médecine, finance, et arts culinaires * Capacité à travailler dans un environnement chaotique + [00:03:01][^4^][4] La révélation sur le poste * Le poste est une métaphore pour le rôle de mère * Les mères rencontrent toutes les exigences sans salaire * Éveil de la reconnaissance pour le travail des mères + [00:03:15][^5^][5] Réactions émotionnelles * Les candidats expriment leur admiration pour les mères * Réflexion sur l'appréciation de leurs propres mères * Les mères sont célébrées pour leur dévouement inconditionnel

eLife assessment

This work identifies the molecular function of an orphan human transporter, SLC35G1, providing convincing but somewhat incomplete evidence that this protein is involved in intestinal citrate absorption. This work provides important insight into transporter function and human physiology.

A policy decision about how often we may have to go to the page allocator.

computes a several values for the allocator based on the size and flags of the allocator being created.

Depending on the CONFIG_SLUB_TINY should ther be an active slab for each CPU?

calculate the order (power of two number of pages) that each slab in this allocator should have.

eLife assessment

This is a valuable study on the diffusion rates of drug molecules in human-derived cells, highlighting that their diffusion behavior depends on their charged state. It proposes that blocking drug protonation enhances diffusion and fractional recovery, suggesting improved intracellular availability of weakly basic drugs. The correlation between pKa and intracellular diffusion is solid and well-supported, but the study would benefit from a more rigorous statistical treatment and a balanced comparison across different types of compounds. Despite these limitations, the findings are significant for drug design and understanding the biophysical behavior of small molecules in cells.

eLife assessment

This useful study draws on published single-cell and spatial transcriptomic data of colon cancer liver metastasis to clarify the pro- and anti-tumorigenic properties of NK cells. The authors discover increased GZMK+ resting NK cells in the tumor tissue and reduced abundance of KIR2DL4+ activated NK cells. However, the evidence is currently incomplete, as the models used to validate the hypothesis and claims are inadequate and lack necessary controls.

Congratulations, Arc motherfuckers with iPads: they finally... pushed the maximize button or whatever.

李沐 按照计算层的数量定义层数

High level membership description

delete

put a testimonial in this container

Change to high level membership description higher up

Differentiator - add container above about why this approach works

layout as columns?

move featured in banner

delete

lay out differently

proven system - banner

if

to what?

in banner

delete

a "magic" formula for computing the amount of memory per zone.

XD this sounds fun

Such is the risk of limiting beliefs.

"He who looks for external validation is not properly grounded in life." -- Marcus Aurelius (20 June 2024 future edit, this must be Epictetus)

In other words, do not care about what others think... Heed their advice, take it into account, but ultimately you must make the decision yourself.

YAML is not generally considered easily editable by humans

DataCite is not a data repository.

doing something that is different from what people consider to be normal or acceptable

doing something that is different from what people consider to be normal or acceptable

AIM for Climate Investments

PREPARE and VACS: $50 million announced at COP28

Agriculture Innovation Mission AIM for Climate

PREPARE and the Vision for Adapted Crops and Soils multi-donor funding platform

delete

and frustrated?

Banner

Pain point in container 1

Hyperlink

Love this!

delete

Plus

You said people join for community Highlight main benefit What's the final impact on the biz: growth?

Target customer pain point Desire Then introduce

nn.CrossEntropyLoss是PyTorch中的一个类，它实现了交叉熵损失函数。交叉熵损失函数常用于多分类问题，它可以度量模型的预测概率分布与真实概率分布之间的差异。

reduction='none'是一个参数，它指定了如何对每个样本的损失进行聚合。'none'表示不进行聚合，即返回一个损失值的向量，向量的每个元素对应一个样本的损失。其他可能的值包括'mean'（返回所有样本损失的平均值）和'sum'（返回所有样本损失的总和）。

在 train_ch3 → train_epoch_ch3 中内置优化器是 l.mean().backwar()

在这个例子中，我们选择'none'是因为我们想要在后续的计算中手动处理每个样本的损失，例如，我们可能想要计算每个样本损失的平均值，或者只关注损失最大的几个样本。

Résumé de la vidéo [00:00:05][^1^][1] - [00:22:49][^2^][2] : La vidéo présente une recherche-action intitulée "Lutter contre l'échec, repenser la relation pédagogique" menée à l'Université Saint-Louis. Elle aborde les défis de l'échec universitaire et les moyens de le surmonter en réévaluant les méthodes pédagogiques et en soutenant les étudiants de manière plus personnalisée.

Points forts : + [00:00:05][^3^][3] Contexte et objectifs de la recherche * Lancement de la recherche dans le cadre de la fusion des universités * Objectif de comprendre et d'adresser l'échec étudiant * Financement par l'université pour une approche collective + [00:08:01][^4^][4] Méthodologie et résultats préliminaires * Suivi d'une cohorte d'étudiants sur trois ans * Identification de profils d'étudiants et de leurs chances de réussite * Importance de l'adaptation des méthodes pédagogiques aux besoins des étudiants + [00:13:00][^5^][5] Profil des étudiants à l'entrée de l'université * Étudiants investis mais inégalement informés et préparés * Confiance en la capacité de réussir malgré des préparations diverses * Six profils d'étudiants identifiés avec des chances de réussite variables + [00:19:00][^6^][6] Expérience des étudiants à l'université * Difficultés d'adaptation à l'autonomie universitaire * Variabilité dans l'appréciation des méthodes d'enseignement * Nécessité d'ajuster les méthodes de travail pour la réussite

Résumé de la vidéo [00:22:51][^1^][1] - [00:44:26][^2^][2] : La vidéo aborde la recherche-action sur la lutte contre l'échec scolaire et la redéfinition de la relation pédagogique. Elle examine les expériences des étudiants durant le premier quadrimestre à l'université, leurs attentes envers les enseignants et l'institution, et l'impact des profils d'entrée sur la réussite académique.

Points forts : + [00:22:51][^3^][3] Expérience du premier quadrimestre * Transition difficile pour les étudiants * Questions sur les attentes et la manière de répondre à ces attentes * Gestion de l'autonomie et attentes envers les enseignants + [00:23:48][^4^][4] Rôle de l'enseignant * Doit être un expert et proche des étudiants * Importance de l'empathie et de l'accompagnement * Les cours doivent être utiles, structurés et bien soutenus + [00:27:00][^5^][5] Hétérogénéité des expériences étudiantes * Différences dans la façon de vivre l'expérience universitaire * Le profil d'entrée n'influence pas directement l'expérience universitaire * Importance de l'assistance et des ressources institutionnelles + [00:32:01][^6^][6] Facteurs de réussite académique * Objectifs académiques et heures de travail personnel * Impact des profils d'entrée sur la réussite * Nécessité de changer les paramètres de fonctionnement de l'université + [00:37:31][^7^][7] Satisfaction et sens dans l'expérience universitaire * Satisfaction non corrélée à la réussite * Les étudiants les plus satisfaits sont souvent parmi les profils les plus fragiles * Importance de la clarté des attentes et de la communication avec les étudiants + [00:42:07][^8^][8] Recommandations pour l'amélioration * Travailler en amont avec l'enseignement obligatoire * Informer sur l'hétérogénéité des publics * Intégrer des tests réflexifs dans le cursus académique

Résumé de la vidéo [00:44:28][^1^][1] - [01:05:38][^2^][2]:

La vidéo présente une recherche-action sur la lutte contre l'échec scolaire et la redéfinition de la relation pédagogique. Elle aborde les propositions pour améliorer le suivi des étudiants, l'importance de ne pas présumer de leur autonomie, et la nécessité de restructurer le calendrier universitaire.

Points saillants: + [00:44:28][^3^][3] Suivi individualisé des étudiants * Proposition de suivis universels mais personnalisés * Importance d'accompagner les étudiants vers l'autonomie * Nécessité d'évaluer la compréhension des consignes + [00:45:26][^4^][4] Apprentissage du travail personnel * Focalisation sur le travail personnel en dehors des cours * Proposition d'organiser des semaines types pour guider les étudiants * Importance de la quantité et de la qualité du travail personnel + [00:46:24][^5^][5] Réorganisation du cadrimestre * La première session arrive trop tard pour les étudiants * Proposition de découper le premier cadrimestre en deux parties * Importance des retours et feedbacks après les mini-sessions + [00:51:01][^6^][6] Débat politique sur l'éducation * Discussion entre journalistes et politiciens sur les thèmes abordés * Échanges sur l'encadrement des étudiants et l'orientation scolaire * Débat sur l'impact du décret paysage et le calendrier universitaire

Résumé de la vidéo [01:05:39][^1^][1] - [01:23:45][^2^][2]:

La vidéo présente un débat politique sur la recherche-action intitulée "Lutter contre l'échec, repenser la relation pédagogique". Les intervenants discutent des stratégies pour combattre les inégalités dans l'éducation, notamment en améliorant l'orientation des étudiants et en gérant mieux la transition entre l'enseignement secondaire et supérieur. Ils soulignent l'importance d'une analyse à long terme pour suivre les progrès des étudiants et abordent le rôle des facteurs socio-économiques dans la réussite éducative.

Points saillants: + [01:05:39][^3^][3] Propositions du PS pour l'éducation * Mettre en place un observatoire de la vie étudiante * Améliorer l'orientation et la transition entre les niveaux d'enseignement * Attaquer les déterminants socio-économiques de l'échec + [01:08:27][^4^][4] Approche du PTB sur la taille des classes * Réduire la taille des classes pour une meilleure attention individuelle * Proposer des classes de 15 à 17 élèves jusqu'à 8 ans et environ 20 élèves après * Lier la taille des classes à la diminution des inégalités et à la réussite + [01:12:01][^5^][5] Coût des études universitaires selon le PTB * Réduire les frais pour atténuer les inégalités * Proposer des repas à 2 € et diminuer le prix des côtes * Tendre vers la gratuité du minerval + [01:14:43][^6^][6] Formation des enseignants et assistants * Améliorer la formation des enseignants pour traiter les lacunes du secondaire * Utiliser les compétences des professeurs de promotion sociale * Assurer un suivi adéquat des étudiants pour leur réussite

Résumé de la vidéo [01:23:46][^1^][1] - [01:44:37][^2^][2] : La vidéo présente un débat politique sur la recherche-action intitulée "Lutter contre l'échec, repenser la relation pédagogique". Les intervenants discutent des moyens d'améliorer l'enseignement supérieur en Belgique, notamment en réformant le financement, en intégrant des tests d'orientation non contraignants et en renforçant l'aide à la réussite dans les cursus universitaires.

Points forts : + [01:23:46][^3^][3] Financement de l'enseignement supérieur * Nécessité de refinancer et d'ouvrir l'enveloppe budgétaire * Financement basé sur le nombre d'étudiants * Importance de la pédagogie dans l'orientation + [01:25:46][^4^][4] Tests d'orientation et aide à la réussite * Tests pour identifier les lacunes des étudiants * Aide à la réussite intégrée au cursus * Premier quadrimestre avec modules pédagogiques transversaux + [01:27:19][^5^][5] Conditions de vie des étudiants * Impact de la précarité étudiante sur la réussite * Propositions pour soutenir les étudiants financièrement * Sortie de l'enveloppe fermée pour réduire la concurrence entre établissements + [01:30:00][^6^][6] Évaluation des acquis de base et contrat d'aide à la réussite * Proposition d'évaluation obligatoire mais non contraignante en juillet * Contrat d'aide à la réussite axé sur la remédiation * Importance de l'orientation et de l'évaluation précoce pour la réussite + [01:34:19][^7^][7] Formation et encadrement pédagogique des enseignants * Nécessité de former les enseignants à la pédagogie * Coordination entre l'enseignement secondaire et supérieur * Inclusion et co-enseignement dans les classes précaires + [01:37:04][^8^][8] Vision des étudiants et utilisation des nouvelles technologies * Importance de la réflexivité et du regard critique des enseignants * Utilisation des outils contemporains comme chat GPT dans l'enseignement * Adaptation de l'enseignement aux enjeux actuels et aux besoins des étudiants

Résumé de la vidéo [01:44:39][^1^][1] - [02:04:25][^2^][2] : La vidéo aborde la recherche-action "Lutter contre l'échec, repenser la relation pédagogique" et discute des défis de l'enseignement supérieur, notamment la nécessité d'adapter les méthodes pédagogiques à l'hétérogénéité des classes et l'importance de la formation des enseignants. Elle souligne également le rôle de l'enseignement supérieur dans la promotion de la justice sociale et l'égalité des chances, ainsi que les implications des examens d'entrée et du financement sur la réussite des étudiants.

Points saillants: + [01:44:39][^3^][3] Défis de l'enseignement supérieur * Adaptation aux grandes classes hétérogènes * Formation continue des enseignants * Impact des examens d'entrée sur la diversité étudiante + [01:47:29][^4^][4] Justice sociale et égalité des chances * Rôle de l'enseignement supérieur dans la réduction des inégalités * Importance du soutien aux étudiants sans antécédents universitaires * Nécessité d'un financement adéquat pour l'enseignement + [01:49:00][^5^][5] Financement et réforme de l'enseignement * Débat sur le refinancement de l'enseignement supérieur * Réforme des rythmes académiques et de la pédagogie * Propositions pour améliorer l'orientation et l'aide à la réussite + [01:59:02][^6^][6] Innovations pédagogiques et accueil des étudiants * Nouvelles approches pour l'accueil et l'intégration des étudiants en première année * Importance de l'auto-réflexion et de la compréhension du sens de l'éducation universitaire * Initiatives pour renforcer l'aide à la réussite et l'engagement étudiant

Résumé de la vidéo [02:04:27][^1^][1] - [02:06:15][^2^][2] :

La vidéo présente une discussion sur la fondation d'une université, soulignant l'importance de la diversité dans son conseil et la nécessité de financements privés pour compléter les fonds publics. Elle met en avant la collaboration avec des entreprises privées pour renforcer la position de l'université dans la région bruxelloise.

Points forts : + [02:04:27][^3^][3] Diversité du conseil * Représentation de la diversité étudiante * Membres variés comme Joseph Chovanek et Akima d'Armouche + [02:04:50][^4^][4] Financements privés et regard extérieur * Apport de fonds complémentaires * Challenge des méthodes et positionnement régional + [02:05:28][^5^][5] Fondation de l'université * Projet aligné sur les objectifs inclusifs * Booster complémentaire au soutien public + [02:05:44][^6^][6] Convergence politique * Accord sur les besoins et envies malgré les différences * Motivation collective pour relever les défis

Résumé de la vidéo [00:00:04][^1^][1] - [00:24:22][^2^][2] : La vidéo présente une journée d'étude sur l'expérience des jeunes aidants et des jeunes endeuillés. Elle aborde le cadre du dispositif "La vie, la mort, on en parle", initié au printemps 2021, qui est un portail de ressources sur la mort et le deuil pour les enfants et adolescents. La vidéo met en lumière l'importance de la recherche et de l'éducation sur ces sujets délicats.

Points forts : + [00:00:04][^3^][3] Introduction de la journée d'étude * Remerciements et contexte du dispositif "La vie, la mort, on en parle" * Présentation des objectifs et du programme de la journée + [00:02:01][^4^][4] Développement du dispositif de recherche * Lancement d'un dispositif de recherche sur la confrontation des jeunes à la finitude * Études sur la scolarisation des jeunes en situation palliative et des jeunes orphelins + [00:06:07][^5^][5] Présentation des recherches en cours * Focus sur les jeunes aidants endeuillés et l'impact du deuil sur la scolarité * Projets de recherche futurs et collaborations + [00:14:44][^6^][6] Intervention de partenaires et spécialistes * Contributions de la Fondation SIRP et de l'association Jeune Aident Ensemble * Importance du soutien et de la reconnaissance des jeunes orphelins

Résumé de la vidéo [00:24:24][^1^][1] - [00:47:01][^2^][2] : La vidéo présente une journée d'étude sur le vécu des jeunes aidants et des jeunes endeuillés. Elle explore les défis auxquels sont confrontés les enfants et les parents après la perte d'un proche, notamment en termes de scolarité et de soutien social.

Points forts : + [00:24:24][^3^][3] Impact sur les parents et la scolarité * Les parents partagent leurs stratégies pour aider leurs enfants à l'école * Certains informent l'école, d'autres changent d'école ou de maison * Les enfants peuvent être distraits ou avoir des comportements modifiés + [00:27:30][^4^][4] Perspective des enfants et changements observés * Les enfants expriment le manque, la tristesse et le vide ressenti * La perte affecte leur attention, concentration et comportement à l'école * Certains enfants se renferment ou ont des difficultés à suivre les cours + [00:30:45][^5^][5] Camarades comme ressources * Les amis peuvent devenir un soutien émotionnel important * Les enfants partagent des expériences similaires et renforcent les liens * L'identification et la proximité avec les pairs qui ont vécu des pertes similaires + [00:34:11][^6^][6] Conséquences à long terme et rôle des enseignants * La mort d'un parent a un impact sur la réussite scolaire et professionnelle * Les enseignants peuvent se sentir mal à l'aise et démunis face à la mort * Il est crucial de soutenir l'enfant et de créer une relation de confiance avec le parent

Résumé de la vidéo [00:47:03][^1^][1] - [01:07:56][^2^][2]:

La vidéo aborde les besoins des jeunes aidants et des jeunes endeuillés, en mettant l'accent sur la méconnaissance de ces besoins dans différents contextes sociaux et l'importance de la discussion et de la reconnaissance pour répondre à ces besoins.

Points forts: + [00:47:03][^3^][3] Méconnaissance des besoins * Manque de connaissance des besoins des enfants * Nécessité de discussions entre famille, école et loisirs * Importance de partager et d'exprimer les besoins + [00:52:01][^4^][4] Expérience des enseignants avec les orphelins * Les enseignants décrivent l'expérience comme déstabilisante * Ambivalence émotionnelle et besoin d'ajustement relationnel * Impact marquant sur les enseignants et les élèves + [00:57:01][^5^][5] Étude sur les représentations des enseignants * Exploration des attitudes des enseignants envers les jeunes orphelins * Manque de préparation et de formation pour gérer le deuil * Importance du soutien des collègues et des ressources disponibles

Résumé de la vidéo [01:07:57][^1^][1] - [01:31:51][^2^][2] : La vidéo présente une journée d'étude sur le vécu des jeunes aidants et des jeunes endeuillés. Elle aborde l'importance de travailler avec les familles, en particulier avec le parent restant, pour mieux comprendre et accompagner les enfants endeuillés. Les intervenants discutent de l'impact du deuil sur le développement et l'éducation des enfants, ainsi que de la nécessité d'une approche plus dynamique et intégrée pour soutenir les enfants et les familles touchées par le deuil.

Points saillants: + [01:08:00][^3^][3] Travailler avec les familles * Nécessité de collaboration avec le parent restant * Comprendre les comportements des enfants endeuillés * Prévenir les répercussions, notamment scolaires + [01:10:14][^4^][4] Anticipation de la prise en charge * Importance de l'anticipation dans les soins palliatifs * Impact de la préparation sur le deuil des enfants * Différences selon le type de décès et l'anticipation psychique + [01:20:00][^5^][5] Développement et éducation des enfants * Intégration de la dynamique de développement dans l'approche * Impact du deuil sur la trajectoire éducative des enfants * Nécessité d'opérationnaliser le modèle théorique pour la pratique + [01:26:13][^6^][6] Influence du milieu social sur le deuil * Conséquences socioéconomiques du deuil sur les familles * Variabilité de l'impact du deuil selon le milieu social * Relation entre le deuil et les facteurs sociaux et économiques

Résumé de la vidéo [01:31:53][^1^][1] - [01:55:59][^2^][2]:

Cette partie de la vidéo aborde la gestion du deuil chez les jeunes, en particulier dans le contexte scolaire. Les intervenants discutent des défis rencontrés par les enseignants et les professionnels de la santé pour soutenir les élèves endeuillés, ainsi que de l'importance de la communication et du soutien familial.

Points forts: + [01:32:00][^3^][3] La communication familiale * L'importance de rassembler la famille pour discuter * Les mécanismes de protection peuvent entraver la communication * Les entretiens familiaux permettent d'aborder les non-dits + [01:34:07][^4^][4] L'utilisation de la littérature jeunesse * Peut servir de média pour initier la conversation sur le deuil * Utile pour les échanges entre élèves et enseignants * Ne suffit pas seul, mais aide à structurer la parole + [01:34:39][^5^][5] Le rôle des enseignants * Souvent les premiers à prendre en charge l'orphelinage * La bienveillance des enseignants est cruciale * La temporalité du deuil peut affecter les apprentissages + [01:37:26][^6^][6] Le deuil à l'école * Le deuil fait partie de la vie de l'école * Nécessité de sensibiliser et former le personnel * L'impact du deuil sur les apprentissages est significatif + [01:45:01][^7^][7] La professionnalisation du personnel * Formation sur l'accompagnement du deuil en milieu scolaire * Intégration de la thématique dans l'adaptation à l'emploi * Élargissement de la formation à d'autres membres de la communauté scolaire + [01:50:07][^8^][8] Évaluation des pratiques professionnelles * Évaluation exploratoire sur l'accompagnement des jeunes en deuil * Importance de la formation pour réduire l'appréhension * Proposition de groupes de parole pour les élèves endeuillés

Résumé de la vidéo [01:56:01][^1^][1] - [02:01:05][^2^][2]:

Cette partie de la vidéo aborde l'expérience des jeunes aidants et endeuillés, mettant en lumière l'importance de reconnaître et d'intégrer la mort et le deuil dans le contexte scolaire. L'intervenant souligne la nécessité de former et de sensibiliser les éducateurs pour mieux accompagner les élèves dans leur parcours scolaire et de vie.

Points forts: + [01:56:01][^3^][3] L'importance de la formation * Nécessité d'augmenter les compétences * Importance de la persévérance malgré les défis financiers * Réinscription du programme dans le plan académique + [01:57:02][^4^][4] La mort et le deuil à l'école * Reconnaissance de la mort comme sujet scolaire * Importance de la sensibilisation et de la formation des éducateurs * Intégration de la mort dans le parcours de vie des élèves + [01:58:02][^5^][5] L'égalité des chances et l'inclusion * Inscription du programme dans les orientations ministérielles * Promotion de l'école inclusive pour tous les enfants * Accès à une scolarité diversifiée pour tous + [01:59:01][^6^][6] Sensibilisation des chefs d'établissement * Difficulté à engager les chefs d'établissement * Importance de la politique d'établissement pour la santé * Déploiement potentiel dans d'autres académies + [02:00:04][^7^][7] Accompagnement des élèves endeuillés * Mise en place de moyens supplémentaires pour l'accompagnement * Liaison avec les équipes des lycées pour un suivi continu * Objectif d'un accompagnement pérenne pour les élèves

Résumé de la vidéo [00:00:04][^1^][1] - [00:23:49][^2^][2]:

Cette vidéo présente la deuxième partie d'une journée d'étude sur l'expérience des jeunes aidants et des jeunes endeuillés. Elle se concentre sur les défis et les impacts de ces rôles sur le bien-être et la scolarité des jeunes.

Points forts: + [00:00:04][^3^][3] Définition et rôle des jeunes aidants * Un jeune aidant est un enfant ou adolescent qui aide régulièrement un membre de sa famille souffrant d'une maladie ou d'un handicap. * Ils peuvent effectuer des tâches ménagères, administratives, ou fournir un soutien moral et des soins personnels. * L'aide apportée est évaluée sur un continuum de faible à très importante. + [00:04:37][^4^][4] Évolution de la reconnaissance des jeunes aidants en France * La prise de conscience en France a commencé en 2014, avec des initiatives clés et la création de l'association nationale Jeunes Aidants Ensemble. * En 2019, le gouvernement a identifié les jeunes aidants dans la stratégie "Agir pour les aidants". + [00:07:05][^5^][5] Recherche sur les jeunes aidants * Un programme de recherche vise à identifier les jeunes aidants en France, étudier les facteurs de protection et de vulnérabilité, et développer des interventions pour les aider. * Des études ont été menées pour comprendre les caractéristiques, les besoins et les difficultés des jeunes aidants. + [00:20:26][^6^][6] Conséquences sur la santé et la scolarité * Les jeunes aidants rapportent une moins bonne qualité de vie et ont souvent eux-mêmes des problèmes de santé. * Ils ont tendance à redoubler plus souvent et à choisir des formations à distance pour rester proches de leurs proches aidés.

Résumé de la vidéo [00:21:00][^1^][1] - [01:40:17][^2^][2]:

Cette partie de la vidéo se concentre sur les jeunes aidants en France, leur identification, les défis auxquels ils sont confrontés et les efforts pour développer des interventions de soutien. La présentation souligne l'importance de reconnaître et d'accompagner ces jeunes, qui fournissent souvent des soins à un membre de la famille souffrant de maladie ou de handicap.

Points forts: + [00:21:00][^3^][3] Recherche sur les jeunes aidants * Identification des jeunes aidants en France * Étude des facteurs de protection et de vulnérabilité * Développement d'interventions pour les soutenir + [00:41:00][^4^][4] Limites de la recherche actuelle * Échantillon limité ne permettant pas de généraliser * Manque d'informations sur le contexte scolaire des jeunes + [00:57:00][^5^][5] Expérience dans une école d'ingénieurs * Mise en place d'entretiens pour comprendre les besoins des étudiants endeuillés * Adaptation des aménagements scolaires en fonction des situations individuelles + [01:18:00][^6^][6] Difficultés systémiques dans l'éducation * L'épuisement des jeunes aidants affecte leur capacité à s'engager dans les démarches administratives * Besoin d'un soutien pour naviguer dans les processus institutionnels

Résumé de la vidéo [01:21:00][^1^][1] - [01:23:09][^2^][2]:

Cette partie de la vidéo se concentre sur les résultats d'une étude qualitative exploratoire en psychologie, nommée "jadisp", qui examine la perception des interférences entre le processus de deuil et la scolarité chez les jeunes aidants. L'étude a été soutenue financièrement par la Fondation Osirp et vise à comprendre les conséquences de l'apport des jeunes aidants, en particulier sur leur scolarité.

Points forts: + [01:21:00][^3^][3] Présentation de l'étude jadisp * Recherche qualitative exploratoire en psychologie * Soutien financier de la Fondation Osirp * Focus sur les jeunes aidants et le deuil + [01:22:18][^4^][4] Définition des jeunes aidants * Clarification sur l'apport des jeunes aidants * Conséquences multiples, y compris sur la scolarité * Importance de la prise en compte de ces incidences + [01:22:55][^5^][5] Résultats de l'étude * Interférences entre deuil et scolarité * Perception des jeunes aidants sur ces interférences * Objectif de mieux comprendre et accompagner les jeunes aidants

1 стадия изучения 2 влюбленность подкреплять стабильность. и классное в 1 момент нужно сделать легкую яму 3.начало любви

The lopsided asymmetry here is charring. Imagine putting in time like that, where responses are instantly generated. You'll feel a) that this is worth something because you spent time on this, and we equate such investment in others with depth, but here there's no other b) there will always be a response by generated personas, and you will feel a likely 'social' pressure to respond in kind.

These descriptions are very uncanny valley. Imagine a community where each AI friend has its own unique digital life, ready to share moments, create memories, post images just like real friends Butterflies is more than just a social network; it’s a fresh approach to connection Imagine a place where every friend understands you perfectly,

eLife assessment

This important study examines the relationship between expiratory airflow and vocal pitch in adult mice during the production of ultrasonic vocalizations and also identifies a molecularly defined population of brainstem neurons that regulates mouse vocal production across development. The evidence supporting the study's conclusions that expiratory airflow shapes vocal pitch and that these brainstem neurons preferentially regulate expiratory airflow is novel and compelling. This work will be of interest to neuroscientists working on mechanisms and brainstem circuits that regulate vocal production and vocal-respiratory coordination.

Reviewer #1 (Public Review):

Summary:

In this important work, the authors propose and test a model for the control of murine ultrasonic vocalizations (USV) in which two independent mechanisms involving changes in laryngeal opening or airflow control vocal tone. They present compelling experimental evidence for this dual control model by demonstrating the ability of freely behaving adult mice to generate vocalizations with various intonations by modulating both the breathing pattern and the laryngeal muscles. They also present novel evidence that these mechanisms are encoded in the brainstem vocalization central neural pattern generator, particularly in the component in the medulla called the intermediate reticular oscillator (iRO). The results presented clearly advance understanding of the developmental nature of the iRO, its ability to intrinsically generate and control many of the dynamic features of USV, including those related to intonation, and its coordination with/control of expiratory airflow patterns. This work will interest neuroscientists investigating the neural generation and control of vocalization, breathing, and more generally, neuromotor control mechanisms.

Strengths:

Important features and novelty of this work include:

(1) The study employs an effective combination of anatomical, molecular, and functional/ behavioral approaches to examine the hypothesis and provide novel data indicating that expiratory airflow variations can change adult murine USV's pitch patterns.

(2) The results significantly extend the authors' previous work that identified the iRO in neonatal mice by now presenting data that functionally demonstrates the existence of the critical Penk+Vglut2+ iRO neurons in adult mice, indicating that the iRO neurons maintain their function in generating vocalization throughout development.

(3) The results convincingly demonstrate that the iRO neurons encode and can generate vocalizations by modulating both breathing and the laryngeal muscles.

(4) The anatomical mapping and tracing results establish an important set of input and output circuit connections to the iRO, including input from the vocalization-promoting subregions of the midbrain periaqueductal gray (PAG), as well as output axonal projections to laryngeal motoneurons, and to the respiratory rhythm generator in the preBötzinger complex.

(5) These studies advance the important concept that the brainstem vocalization pattern generator integrates with the medullary respiratory pattern generator to control expiratory airflow, a key mechanism for producing various USV types characterized by different pitch patterns.

Weaknesses:

A limitation is that the cellular and circuit mechanisms by which the vocalization pattern generator integrates with the respiratory pattern generator to control expiratory airflow has not been fully worked out, requiring future studies.

Reviewer #2 (Public Review):

Summary:

Both human and non-human animals modulate the frequency of their vocalizations to communicate important information about context and internal state. While regulation of the size of the laryngeal opening is a well-established mechanism to regulate vocal pitch, the contribution of expiratory airflow to vocal pitch is less clear. To consider this question, this study first characterizes the relationship between the dominant frequency contours of adult mouse ultrasonic vocalizations (USVs) and expiratory airflow using whole-body plethysmography. The authors also include data from a single mouse that combines EMG recordings from the diaphragm and larynx with plethysmography to provide evidence that the respiratory central pattern generator can be re-engaged to drive "mini-breaths" that occur during the expiratory phase of a vocal breath. Next, the authors build off of their previous work characterizing intermediate reticular oscillator (iRO) neurons in mouse pups to establish the existence of a genetically similar population of neurons in adults and show that artificial activation of iRO neurons elicits USV production in adults. Third, the authors examine the acoustic features of USV elicited by optogenetic activation of iRO and find that a majority of natural USV types (as defined by pitch contour) are elicited by iRO activation and that these artificially elicited USVs are more likely than natural USVs to be marked by positive intonation (positive relationship between USV dominant frequency and expiratory airflow).

Strengths:

Strengths of the study include the novel consideration of expiratory airflow as a mechanism to regulate vocal pitch and the use of intersectional methods to identify and activate the iRO in adult mice. The establishment of iRO neurons as a brainstem population that regulates vocal production across development is an important finding.

Weaknesses:

The conclusion that the respiratory CPG is re-engaged during "mini-breaths" throughout a given vocal breath would be strengthened by including analyses from more than one mouse.

Author response:

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

In the revised manuscript we have included an additional study that significantly contributes to the conclusions and models of the original version. Briefly, Figure 3 now describes our characterization of the diaphragm and laryngeal muscle activities (electromyography, EMG) during endogenous vocalizations. These EMGs also serve as representations of the brainstem breathing central pattern generator (CPG) inspiratory and post-inspiratory generating neurons, respectively. In our original submission, we found that many of the vocalizations had changes in pitch that mirrored the change in expiratory airflow (we termed positive intonation), and we proposed that the coordination of breathing muscles (like the inspiratory muscles) and larynx patterned this. This mechanism is akin to our findings for how neonatal cries are rhythmically timed and produced (Wei et al. 2022). The newly presented EMG data re-inforces this idea. We found that for vocalizations with positive intonation, the inspiratory diaphragm muscle has an ectopic burst(s) of activity during the expiration phase which corresponds to a decrease in airflow and pitch, and this is followed by laryngeal muscle activity and increased pitch. This can be cycled throughout the expiration to produce complex vocalizations with oscillations in pitch. A basal breath is hardwired for the laryngeal muscle activity to follow the diaphragm, so the re-cycling of this pattern nested within an expiration (a ‘mini-breath’ in a ‘breath’) demonstrates that the vocalization patterning system engages the entire breathing CPG. This contrasts with the canonical model that activity of the laryngeal premotor neurons control all aspects of producing / patterning vocalizations. Furthermore, this mechanism is exactly how the iRO produces and patterns neonatal vocalizations (Wei et al. 2022) and motivates the likely use of the iRO in adult vocalizations.

Response to recommendations for the authors:

Reviewer #1:

(1) The authors should note in the Discussion that the cellular and circuit mechanisms by which the vocalization pattern generator integrates with the respiratory pattern generator to control expiratory airflow have not been fully worked out, requiring future studies.

This was noted in the discussion section “The iRO likely patterns intonation for endogenous phonation”.

(2) Please change the labeling of the last supplemental figure to Figure Supplemental 5.

Thank you for identifying this.

Reviewer #2:

Major concerns

(1) While it is true that modulation of activity in RAm modulates the laryngeal opening, this statement is an incomplete summary of prior work. Previous studies (Hartmann et al., 2020; Zhang et al., 1992, 1995) found that activation of RAm elicits not just laryngeal adduction but also the production of vocal sounds, albeit vocal sounds that were spectrally dissimilar from speciestypical vocalizations. Moreover, a recent study/preprint that used an activity-dependent labeling approach in mice to optogenetically activate RAm neurons that were active during USV production found that re-activation of these neurons elicits USVs that are acoustically similar to natural USVs (Park et al., 2023). While the authors might not be required to cite that recent preprint (as it is not yet peer-reviewed), the fact that activation of RAm elicits vocal sounds is clear evidence that its effects go beyond modulating the size of the laryngeal opening, as this alone would not result in sound production (i.e., RAm activation must also recruit expiratory airflow). The authors should include these relevant studies in their Introduction. Moreover, the rationale for the model proposed by the authors (that RAm controls laryngeal opening whereas iRO controls expiratory airflow) is unclear with regard to these prior studies. The authors should include a discussion of how these prior findings are consistent with their model (as presented in the Introduction, as well as in Figure 4 and relevant Discussion) that RAm modulates the size of laryngeal opening but not expiratory airflow.

An introduction and discussion of the Veerakumar et. al. 2023 and Park et. al. 2024 manuscripts describing RAm in mice has now been included.

The iRO serves to coordinate the breath airflow and laryngeal adduction to produce sound and the intonation within it that mirrors the breath airflow. This occurs because the iRO can control the breathing CPG (synaptic input to the preBötC inspiratory pacemaker) and is premotor to multiple laryngeal muscles (Wei et. al. 2022). The modulation of the expiratory airflow is by inducing momentary contraction of the diaphragm (via excitation of the preBötC) which opposes (a.k.a. slows) expiration. This change in flow results in a decrease in pitch (Fig. 3 in the revised manuscript, Wei et. al. 2022).

It is our understanding that the basic model for RAm evoked USVs is that RAm evokes laryngeal adduction (and presumed abdominal expiratory muscle activation) and this activity is momentarily stopped during the breath inspiration by inhibition from the preBötC (Park et. al. 2024). So, in this basic model, any change in pitch and expiratory airflow would be controlled by tuning RAm activity (i.e., extent of laryngeal adduction). In this case, the iRO induced inspiratory muscle activity should not occur during expiration, which is not so (Fig. 3). Note, the activity of abdominal expiratory muscles during endogenous and RAm evoked USVs has not been characterized, so the contribution of active expiration remains uncertain. This is an important next step.

We have now included a discussion of this topic which emphasizes that iRO and RAm likely have reciprocal interactions (supported by the evidence of this anatomical structure). These interactions would explain why excitation of either group can evoke USVs and, perhaps, the extent that either group contributes to a USV explains how the pitch / airflow changes. An important future experiment will be to determine the sufficiency of each site in the absence of the other.

(2) The authors provide evidence that the relationship between expiratory airflow and USV pitch is variable (sometimes positive, sometimes negative, and sometimes not related). While the representative spectrograms clearly show examples of all three relationship types, no statistical analyses are included to evaluate whether the relationship between expiratory airflow and USV pitch is different than what one would expect by chance. For example, if USV pitch were actually unrelated to expiratory airflow, one might nonetheless expect spurious periods of positive and negative relationships. The lack of statistical analyses to explicitly compare the observed data to a null model makes it difficult to fully evaluate to what extent the evidence provided by the authors supports their claims.

We have now included two null distributions and compared our observed correlation values to these. The two distributions were created by taking each USV / airflow pair and randomly shuffling either the normalized USV pitch values (pitch shuffled) or the normalized airflow values (airflow shuffled) to simulate the distribution of data should no relationship exist between the USV pitch and airflow.

(3) The relationship between expiratory airflow and USV pitch comes with two important caveats that should be described in the manuscript. First, even in USV types with an overall positive relationship between expiratory airflow and pitch contour, the relationship appears to be relative rather than absolute. For example, in Fig. 2E, both the second and third portions of the illustrated two-step USV have a positive relationship (pitch goes down as expiratory airflow goes down). Nonetheless, the absolute pitch of the third portion of that USV is higher than the second portion, and yet the absolute expiratory airflow is lower. The authors should include an analysis or description of whether the relationship between expiratory airflow and USV pitch is relative vs.

absolute during periods of 'positive intonation'.

The relationship between pitch and airflow is relative and this in now clarified in the text. To determine this, we visualized the relationship between the two variables by scatterplot for each of the USVs syllables and, as the reviewer notes, a given airflow cannot predict the resulting frequency and vice versa.

(4) A second important caveat of the relationship between expiratory airflow and USV pitch is  that changes in expiratory airflow do not appear to account for the pitch jumps that characterize mouse USVs (this lack of relationship also seems clear from the example shown in Fig. 2E). This caveat should also be stated explicitly.

The pitch jumps do not have a corresponding fluctuation in airflow, and this is now stated in the results and discussion.

(5) The authors report that the mode of relationship between expiratory airflow and USV pitch (positive intonation, negative intonation, or no relationship) can change within a single USV. Have the authors considered/analyzed whether the timing of such changes in the mode of relationship coincides with pitch jumps? Perhaps this isn’t the case, but consideration of the question would be a valuable addition to the manuscript.

We analyzed a subset of USVs with pitch jumps that were defined by a change >10 kHz, at least 5ms long, and had one or two jumps. The intonation relationships between the sub-syllables within a USV type were not stereotyped as evidenced by the same syllable being composed of combinations of both modes.

(6) The authors incorrectly state that PAG neurons important for USV production have been localized to the ventrolateral PAG. Tschida et al., 2019 report that PAG-USV neurons are located predominantly in the lateral PAG and to a lesser extent in the ventrolateral PAG (see Fig. 5A from that paper). The finding that iRO neurons receive input from VGlut2+ ventrolateral PAG neurons represents somewhat weak evidence that these neurons reside downstream of PAG-USV neurons. This claim would be strengthened by the inclusion of FOS staining (following USV production), to assess whether the Vglut+ ventrolateral PAG neurons that provide input to iRO are active in association with USV production.

This comment correctly critiques that our PAG à iRO tracing does not demonstrate that the labeled PAG neurons are sufficient nor necessary for vocalization. Directly demonstrating that activation and inhibition the PAG-iRO labeled neurons ectopically drives or prevents endogenous USVs is an important next step. While FOS implies this connectivity, it does not definitely establish it and so this experiment is impacted by some of the caveats of our tracing (e.g. PAG neurons that drive sniffing might be erroneously attributed to vocalization).

Our reading of the literature could not identify an exact anatomical location within the mouse PAG and this site appears to vary within a study and between independent studies (like within and between Tschida et. al. 2019 and Chen et. al. 2021). The labeling we observed aligns with some examples provided in these manuscripts and with the data reported for the retrograde tracing from RAm (Tschida et al 2019).

(7) In Figure S5A, the authors show that USVs are elicited by optogenetic activation of iRO neurons during periods of expiration. In that spectrogram, it also appears that vocalizations were elicited during inspiration. Are these the broadband vocalizations that the authors refer to in the Results? Regardless, if optogenetic activation of iRO neurons in some cases elicits vocalization both during inspiration and during expiration, this should be described and analyzed in the manuscript.

The sound observed on the spectrogram during inspiration is an artefact of laser evoked head movements that resulted in the fiber cable colliding with the plethysmography chamber. In fact, tapping an empty chamber yields the same broad band spectrogram signal. The evoked USV or harmonic band vocalization is distinct from this artefact and highlighted in pink.

(8) Related to the comment above, the authors mention briefly that iRO activation can elicit broadband vocalizations, but no details are provided. The authors should provide a more detailed account of this finding.

The broadband harmonic vocalizations we sometimes observe upon optogenetic stimulation of AAV-ChR2 expressing iRO neurons are akin to those previously described within the mouse vocal repertoire (see Grimsley et. al .2011). We have added this citation and mentioned this within the text. 

(9) The effects of iRO stimulation differ in a couple of interesting ways from the effects of PAGUSV activation. Optogenetic activation of PAG-USV neurons was not found to entrain respiration or to alter the ongoing respiratory rate and instead resulted in the elicitation of USVs at times when laser stimulation overlapped with expiration. In contrast, iRO stimulation increases and entrains respiratory rate, increases expiratory and inspiratory airflow, and elicits USV production (and also potentially vocalization during inspiration, as queried in the comment above). It would be informative for the authors to add some discussion/interpretation of these differences.

We have added a section of discussion to describe the how these different results may be explained by the iRO being a vocal pattern generator versus the PAG as a ‘gating’ signal to turn on the medullary vocalization patterning system (iRO and RAm). See discussion section ‘The iRO likely patterns intonation for endogenous phonation’.

(10) The analysis shown in Fig. 4D is not sufficient to support the author’s conclusion that all USV types elicited by iRO activation are biased to have more positive relationships between pitch and expiratory airflow. The increase in the relative abundance of down fm USVs in the opto condition could account for the average increase in positive relationship when this relationship is considered across all USV types in a pooled fashion. The authors should consider whether each USV type exhibits a positive bias. Although such a comparison is shown visually in Fig. 4G, no statistics are provided. All 7 USV types elicited by optogenetic activation of iRO should be considered collectively in this analysis (rather than only the 5 types currently plotted in Fig. 4G).

In the original submission the statistical analysis of r values between opto and endogenous conditions was included in the figure legend (‘panels E-G, two-way ANOVA with Sidak’s post-hoc test for two-way comparisons was used; all p-values > 0.05), and this has not changed in the revised manuscript. We have now provided the suggested comparison of opto vs endogenous USVs without down fm (Fig. 5D). This positive shift in r is statistically significant (…).

(11) The evidence that supports the author’s model that iRO preferentially regulates airflow and that RAm preferentially regulates laryngeal adduction is unclear. The current study finds that activation of iRO increases expiratory (and inspiratory) airflow and also elicits USVs, which means that iRO activation must also recruit laryngeal adduction to some extent. As the authors hypothesize, this could be achieved by recruitment of RAm through iRO’s axonal projections to that region.

Note, it is more likely that iRO is directly recruiting laryngeal adduction as they are premotor to multiple laryngeal muscles like the thyroarytenoid and cricothyroid (Wei et. al. 2022). The ‘Discussion’ now includes our ideas for how the iRO and RAm likely interact to produce vocalizations.

In the recent preprint from Fan Wang’s group (Park et al., 2023), those authors report that RAm is required for USV production in adults, and that activation of RAm elicits USVs that appear species-typical in their acoustic features and elicits laryngeal adduction (assessed directly via camera). Because RAm activation elicits USVs, though, it must by definition also recruits expiratory airflow. Can the authors add additional clarification of how the evidence at hand supports this distinction in function for iRO vs RAm?

See response to ‘Major Concern #1”.

Minor concerns 

(1) The authors might consider modifying the manuscript title. At present, it primarily reflects the experiments in Figure 2.

We have provided a title that we feel best reflects the major point of the manuscript. We hope that this simplicity enables it to be recognized by a broad audience of neuroscientists as well as specialists in vocalization and language.

(2) The statement in the abstract that "patterns of pitch are used to create distinct 'words' is somewhat unclear. Distinct words are by and large defined by combinations of distinct phonemes. Are the authors referring to the use of "tonemes" in tonal languages? If so, a bit more explanation could be added to clarify this idea. This minor concern includes both the Abstract, as well as the first paragraph of the Introduction.

We have clarified this line in the abstract to avoid the confusing comparison between mouse vocalizations and human speech. In the introduction we have expanded our explanation to clarify that variations in pitch are a component of spoken language that add additional meaning and depth to the underlying, phonemic structure. 

(3) Multiple terms are used throughout the manuscript to refer to expiratory airflow: breath shape (in the title), breath pattern, deviations in exhalation, power of exhalation, exhalation strength, etc. Some of these terms are vague in meaning, and a consolidation of the language would improve the readability of the abstract and introduction.

We have chosen a smaller selection of descriptive words to use when describing these breath features.

(4) Similarly, "exhalation" and "expiration" are both used, and a consistent use of one term would help readability.

See point 3.

(5) In a couple of places in the manuscript, the authors seem to state that RAm contains both laryngeal premotor neurons as well as laryngeal motor neurons. This is not correct to our knowledge., but if we are mistaken, we would ask that the authors add the relevant references that report this finding.

It is our understanding that the RAm is defined as the anatomical region consistent with the murine rostral and caudal ventral respiratory groups composed of multiple premotor neuron pools to inspiratory, expiratory, laryngeal, and other orofacial muscles. This is supported by neurons within RAm that reflect multiple phases of the inspiratory and expiratory cycle (Subramanian et. al. 2018) and excitation of sub-regions within RAm modulating multiple parts of the breathing control system (Subramanian et. al. 2018 and Subramanian 2009). Rabies tracing of the various premotor neurons which define the anatomical region of RAm in the mouse shows that they surround the motor neurons in the loose region of the nucleus ambiguus (the anatomical location of RAm) for multiple muscles of the upper airway system, such as the thyroarytenoid (Wu et. al. 2017, Dempsey et. al. 2021 and Wei et. al. 2022). Given that the name RAm reflects a broad anatomical location, we have used it to describe both the premotor and motor neurons embedded within it. We have now clarified this in the text.

(6) The statistical analysis applied in Figure 1C is somewhat confusing. The authors show two distributions that appear different but report a p-value of 0.98. Was the analysis performed on the mean value of the distributions for each animal, the median, etc.? If each animal has two values (one for USV+ breaths and one for USV- breaths), why not instead compare those with a paired t-test (or Wilcoxon rank sign)? Additional information is needed to understand how this analysis was performed.

The original manuscript version used a two-way anova to compare the normalized histogram of instantaneous frequency for breaths with (USV+) or without (USV-) for each animal (first factor: USV+/-, second factor: Frequency). The p-value for the first factor (USV) was 0.98 showing no statistically significant effect of USV on the distribution of the histogram.

For simplicity, we have instead performed the analysis as suggested and include a bar graph. This analysis shows that the instantaneous frequency of USV breaths is, in fact, statistically significantly lower than those without USVs. We have updated the figure legend and text to reflect this.

(7) The use of the word "syllable" to describe parts of a USV that are produced on a single breath may be confusing to some scientists working on rodent USVs. The term 'syllable' is typically used to describe the entirety of a USV, and the authors appear to use the term to describe parts of a USV that are separated by pitch jumps. The authors might consider calling these parts of USVs "sub-syllables".

We have clarified these descriptions throughout the text. We now refer to the categories as ‘syllable types’, define ‘syllables’ as ‘a continuous USV event’ with no more than 20ms of silence within and finally ‘sub-syllables’ to refer to components of the syllable separated by jumps in frequency (but not gaps in time).

(8) In Figure S3, final row, the authors show a USV produced on a single breath that contains two components separated by a silent period. This type of bi-syllabic USV may be rare in adults and is similar to what the authors showed in their previous work in pups (multiple USVs produced on a single expiration, separated by mini-inspirations). One might assume that the appearance of such USVs in pups and their later reduction in frequency represents a maturation of vocalrespiratory coordination. Nonetheless, the appearance of bi-syllabic USVs has not been reported in adult mice to our knowledge, and the authors might consider further highlighting this finding.

We were also struck by the similarity of these USVs to our study in neonates and such types of similarities sparked an interest in the role of the iRO in patterning adult USVs. We now include a description of the presence and abundance of bi- and tri-syllablic calls observed in our recordings to highlight this finding.

(9) Figure 4 is referenced at the end of the second Results section, but it would seem that the authors intended to reference Figure 2. 

For simplicity we included some of the referenced data within Fig. S5. We appreciate the recommendation.

(10) In the optogenetic stimulation experiments, the authors should clarify why bilateral stimulation was applied. Was unilateral stimulation ineffective or less effective? The rationale provided for the use of bilateral stimulation (to further localize neural activation) is unclear.

The iRO is bilateral and, we presume, functions similarly. So, we attempted to maximally stimulate the system. We have clarified this in the methods.

(11) Figure Supplemental '6' should be '5'.

Thanks!

(12) Last sentence of the Introduction: "Lasty" should be "lastly".

Thanks!

(13) There are two references for Hage et al., 2009. These should be distinguished as 2009a and 2009b for clarity.

Thanks!

please bold prompt

Author response:

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

We thank the reviewers and editor for their careful review of our work. We believe the resulting manuscript is much stronger. We agree with the comments made by Reviewer #2 regarding additional histology and neuronal data analysis, which will be presented in subsequent work.

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

Reviewer 1 (Public Weaknesses):

It was not always clear what the lesion size was. This information is important for future applica- tions, for example, in the visual cortex, where neurons are organized in retinotopy patterns.

We thank the reviewer for this feedback. While there is some variation in lesion volume for a given parameter set, we have added more details of the volumes of lesions created in our testing (Fig. 4 and Fig. 5).

It would be helpful if the author could add some discussion about whether and how this method could be used in other types of array/multi-contact electrodes, such as passive neuropixels, S- probes, and so on. In addition, though an op-amp was used in the design, it would still be helpful if the author could provide a recommended range for the impedance of the electrodes.

We thank the reviewer for this suggestion. We have both added a demonstration of use in a differ- ent multielectrode probe type (with a U-probe) in Fig. 8, and we have added a discussion about which types of multielectrode probes would be suitable on Page 15, Line 420.

“We demonstrated that our electrolytic lesioning technique works with a linear multicontact probe by testing with a U-Probe in ex vivo rabbit cortex. There are no particular limitations that would prevent our specific electrolytic lesioning technique and device from working with any passive multielectrode probe. The main requirements for use are that the probe has two electrodes that can directly (via whatever necessary adapters) connect to the lesioning device, such that arbitrary current can be passed into them as the anode and cathode. This would limit use of probes, like Neuropixels, where the on-chip acquisition and digitization circuitry generally precludes direct connection to electrodes [1], [2]. The impedance of the multielectrode probe should not be an issue, due to the use of an op amp. We showed use  with a Utah array (20-800 kΩ) and a U-Probe (1-1.5 MΩ). The specific op amp used here has a voltage range of ± 450 V, which assuming a desired output of 150 µA of current would limit electrode impedance to 6 MΩ. Though a different op amp could easily be used to accommodate a higher electrode impedance, it is unlikely that this would be necessary, since most electrodes have impedances between 100 kΩ to 1 MΩ [3].”

Reviewer 2 (Public Weaknesses):

In many of the figures, it is not clear what is shown and the analysis techniques are not well described.

We thank the reviewer for this feedback. We hope that our edits to both the figures and the text have improved clarity for readers.

The flexibility of lesioning/termination location is limited to the implantation site of the multielec- trode array, and thus less flexible compared to some of the other termination methods outlined in Appendix 2.

We thank the reviewer for this point. You are right that the lesioning location is limited to the multielectrode array’s implantation site, while other methods in Appendix 2 do not require prox- imity of the lesion location and the electrophysiology recording site. However, we believe that the closeness of the lesioning location to the microelectrode array is a strength - guaranteeing record- ings from the perilesional area - even with the small negative of reduced flexibility. Multielectrode arrays can be implanted in many areas of cortex. If one wanted to study distal effects of a lesion, additional electrophysiology probes could be implanted to record from those areas. We have noted this on Page 3, Line 117.

“While the link between the lesion location and the multielectrode location technically con- strains the lesion to an area of cortex in which a multielectrode array could be implanted, we see the connection as a positive, because it ensures recording some neuroelectrophysiology from the perilesional area in which recovery is hypothesized to occur (see Appendix 1Data Availabilityappendix.41).”

Although the extent of the damage created through the Utah array will vary based on anatomical structures, it is unclear what is the range of lesion volumes that can be created with this method, given a parameter set. It was also mentioned that they performed a non-exhaustive parameter search for the applied current amplitude and duration (Table S1/S2) to generate the most suitable lesion size but did not present the resulting lesion sizes from these parameter sets listed. Moreover, there’s a lack of histological data suggesting that the lesion size is precise and repeatable given the same current duration/amplitude, at the same location.

We thank the reviewer for this thoughtful feedback. We have added figures (Figs. 4 and 5), where we show the relationship between estimated lesion volume and the current amplitude and duration parameters. These figures include more data from the tests in Supplementary File 1 and Supplementary File 2. While there is some variation in lesion volume for a given current amplitude and duration, there is still a clear relationship between the parameters and lesion volume.

It is unclear what type of behavioral deficits can result from an electrolytic lesion this size and type (∼3 mm in diameter) in rhesus macaques, as the extent of the neuronal loss within the damaged parenchyma can be different from past lesioning studies.

While we appreciate the reviewer’s interest in the behavioral deficits associated with our lesions in rhesus macaques, reporting these falls beyond the scope of this manuscript. Future work will explore the behavioral deficits associated with these lesions

The lesioning procedure was performed in Monkey F while sedated, but no data was presented for Monkey F in terms of lesioning parameters, lesion size, recorded electrophysiology, histological, or behavioral outcomes. It is also unclear if Monkey F was in a terminal study.

We apologize for not being more explicit about the parameters used for the lesion in Monkey F. We have added this in Results on Page 5, Line 209 and in Methods on Page 19, Line 586.

“After this validation and refinement, one proof-of-concept lesion (150 µA direct current passed through adjacent electrodes for 45 seconds) was performed in an in vivo sedated rhe- sus macaque (Monkey F) in order to validate the safety of the procedure.”

“This lesion was created by applying 150 µA of direct current to two adjacent electrodes in the microelectrode array for 45 seconds.”

We also clarified the parameters used for the other lesions in Monkeys H and U in Results on Page 7, Line 233 and in Methods on Page 19, Line 586.

“In all of the fourteen lesions across two awake-behaving rhesus macaques (150 µA direct current passed through adjacent electrodes for 30 or 45 seconds (30s for Monkey U and 45s for Monkey H, except lesion H200120 which was for 50 seconds)), the current source worked as expected, providing a constant current throughout the duration of the procedure.”

“In these lesions, 150 µA of direct current was applied to two adjacent electrodes in the mi- croelectrode array for 30 or 45 seconds (30s for Monkey U, 45s for Monkey H), except in lesion H200120 where current was applied for 50 seconds.”

Monkey F was euthanized shortly after the lesion, so we now mention this on Page 19, Line 583.

“Based on this, and a lack of physiological signs of pain from the anaesthetized pig studies, a lesion was performed on a sedated rhesus macaque who was subsequently euthanized due to unrelated health complications (Monkey F; 16 year-old adult, male rhesus macaque) in order to further verify safety before use in awake-behaving rhesus.”

Because Monkey F was sedated and then euthanized shortly after, there is no behavioral data. As the lesion in sedated Monkey F was used to validate the safety of the procedure, any further data and analysis fall beyond the scope of this manuscript.

As an inactivation method, the electrophysiology recording in Figure 5 only showed a change in pairwise comparisons of clustered action potential waveforms at each electrode (%match) but not a direct measure of neuronal pre and post-lesioning. More evidence is needed to suggest robust neuronal inactivation or termination in rhesus macaques after electrolytic lesioning. Some exam- ples of this can be showing the number of spike clusters identified each day, as well as analyzing local field potential and multi-unit activity.

The reviewer has pointed out some short comings of the original analysis, which we believe have since been addressed with the revised analysis. LFP and spiking activity are functional measures that are more ambiguous in terms of loss and are also the subject of another manuscript currently under revision.

The advantages over recently developed lesioning techniques are not clear and are not discussed.

We thank the reviewer for noting this. We have added a section, also responding to their later request for us to compare our work to Khateeb et al. 2022, by adding a section to the Discussion on Page 16, Line 434.

“Perhaps the most unique advantage of our technique in comparison with other existing inactivation methods lies in Design Consideration #1: stable electrophysiology pre- and post-inactivation (Appendix 1Data Availabilityappendix.41). While several methods exist that allow for localization and size control of the inactivation (Design Consideration #2) and cross compatibility across regions and species (Design Consideration #3), few have achieved compatibility with stable electrophysiology. For example, some studies record electrophysiology only after the creation of the lesion, preventing comparison with baseline neuronal activity [4]. One recent study, Khateeb, et al., 2022, developed an inactivation method that is effectively combined with stable electrophysiology by creating photothrombotic lesions through a chronic cranial window integrated with an electrocorticography (ECoG) array [5], which may be appropriate for applications where local field potential (LFP) recording is sufficient. This approach has trade-offs with regards to the three design considerations presented in Appendix 1Data Availabilityappendix.41.

While Khateeb, et al., present a toolbox with integrated, stable electrophysiology from an ECoG array pre- and post- inactivation (Design Consideration #1), it demonstrated recordings from an ECoG array with limited spatial resolution. While a higher density ECoG array that would provide higher spatial resolution could be used, increasing the density of opaque electrodes might occlude optical penetration and constrain photothrombotic lesions. Further, ECoG arrays are limited to recording LFP, not electrophysiology at single neuron resolution, potentially missing meaningful changes in the neuronal population activity after lesioning. Khateeb, et al., demonstrated localization and control the size of inactivation (Design Consideration #2). In this manuscript, we have shown that the amount and duration of direct current are significant determinants of lesion size and shape, while with photothrombotic lesions, light intensity and aperture diameter are the significantly relevant parameters. One potential advantage of photothrombotic approaches is the use of optical tools to monitor anatomical and physiological changes after lesioning through the cranial window, though the research utility of this monitoring remains to be demonstrated.

Although the method presented by Khateeb, et al., shows some cross-compatibility (Design Consideration #3), it has greater limitations in comparison with the method presented here. For example, while Khateeb, et al., notes that the approach could be adapted for use in smaller organisms, no modification is needed for use in other species with this work’s approach–so long as a multielectrode probe is implantable. In this manuscript we demon- strate electrolytic lesioning spanning two multielectrode probes across rabbits, pigs, sheep, and rhesus macaques, and our same device could be easily used with other smaller species, like rats, in which multielectrode probes have been successfully implanted [6]. Further, the approach in Khateeb, et al., is limited to superficial brain structures, due to the need for opti- cal accessibility. As noted, fiber optics could allow access to deeper structures, which would bring associated additional tissue damage, but deeper structure lesioning was not demon- strated. In contrast, the approach presented here can be used in any region of cortex in which a multielectrode probe can be implanted, which, depending on the probe used, does not limit it to surface structures. For example, we demonstrated use of our lesioning tech- nique with a linear U-probe (Fig. 8figure.caption.25), which could be used to reach deeper layers of cortex or specific deep cortical structures. In both techniques, the location of the lesion is tied to the location of the electrophysiology (for Khateeb et al., wherever the cra- nial window and ECoG array are; for this technique, wherever the multielectrode probe has been implanted), which ensures that the electrophysiology will include recordings from the perilesional area. Neither work addresses the potential of their technique to induce chronic post-lesion behavioral effects, which is a key goal for future work.”

There is a lack of quantitative histological analysis of the change in neuronal morphology and loss.

We appreciate the reviewer’s desire for a quantitative histological analysis, however this falls out- side of the scope of this manuscript. We are not attempting to make strong claims about the number of neurons lost through lesioning or thoroughly characterize morphological changes in the neurons. The histology is intended to show that lesioning did lead to a loss of neurons, but the precise num- ber of neurons lost is neither in scope nor is likely to be highly conserved across lesions.

There is a lack of histology data across animals and on the reliability of their lesioning techniques across animals and experiments.

We thank the reviewer for this point. As stated above, we have now added Fig. 4 and Fig. 5, which includes volume estimates based on the histology from more of our ex vivo and in vivo testing across animals.

There is a lack of data on changes in cortical layers and structures across the lesioning and non- lesioning electrodes.

We acknowledge that the histology does not have the level of detail that is expected from many modern studies. However, the goal here was dramatically different: we sought to calibrate a novel lesion device, ensure it’s safe use in large mammals (specifically, non-human primates) and pro- vide estimates of the lesion size to compare with the literature. The extent of histology that could be performed and the tools available to us prevent such an in depth analysis. We can say based on shank length of the Utah arrays used and known anatomy that we have affected layer 2/3 and maybe a bit of layer 4.

Reviewer 1 (Recommendations For The Authors):

Figure 5b. It would be helpful if the author could plot the delta match separately for the lesion elec- trodes, near neighbor electrodes, and far neighbors. This would help understand the lesion effect, specifically whether the effect is selective (e.g., more potent for the lesion and adjacent electrodes.)

The fact that neuron loss is not particularly selective can already be seen in the spike waveform plots, arranged spatially on the array. Plenty of clear change is observed far from the lesion elec- trodes (marked with black dots) as well as nearby. We have made mention of this localized non- specificity in the main text and have ensured to remphasize in the figure legened. While a nice suggestion, we currently don’t feel this result rises to the level of a figure given it is not highly specific spatially.

Reviewer 2 (Recommendations For The Authors):

Overall the quality of the paper, the figures and the analysis used could be significantly improved. There is a lack of scientific rigor in the presentation of figures and analysis techniques. It is not clear what the authors are trying to communicate through the figures and their choice of figures to show is confusing (see below).

We thank the reviewer for their pointed critiques and believe we have addressed their concerns with many changes to the text, a revamped waveforms analysis, and both the expansion and addition of results.

The neurophysiology data shown doesn’t suggest neuronal loss, it only shows change which needs strong control data to show it is due to a lesion.

As detailed below, we have presented a revised analysis that provides this control. While the reviewer is right to point out we can distinguish actual neuron loss from neuron silencing, we be- lieve the new analysis rigorously indicates new rates of sample turnover beyond those expected from healthy state.

The histology figure should be replaced with a high-quality representation without folds.

We understand the reviewer’s suggestion. While ideally we would have many histology slices from each lesion, due to cost, we were only able to collect one histology slice per lesion. The folds were introduced by the company that performed the H&E staining, and we unfortunately cannot remove the folds. Therefore, despite the folds, this is the best and only image from this lesion. We hope that the markings on the figure and the comment in the caption is sufficient to explain to readers that the folds are not a result of the lesion but instead a result of the histology process.

The authors suggest that this lesioning method will be compatible with any available multielec- trode probe theoretically. Since all testing was done with a Utah array, it will be helpful to add an explanation about potential constraints that will make a given array compatible with this method.

We thank the reviewer for this suggestion. As stated above, we have both added a demonstration of use in a different multielectrode probe type (with a U-probe) in Fig. 8, and we have added a discussion about which types of multielectrode probes would be suitable on Page 15, Line 420.

The authors should cite and discuss previous studies using electrolytic lesioning in awake-behaving animals to study the causal connection between the brain and behavior. (One example study: Morissette MC, Boye SM. Electrolytic lesions of the habenula attenuate brain stimulation reward. Behavioural brain research. 2008 Feb 11;187(1):17-26.)

We thank the reviewers for this suggestion. We have added a mention of existing electrolytic le- sioning studies on Page 2, Line 88.

“Prior termination studies mostly measure behavioral output, with no simultaneous measures of neuronal activity during the behavior, impairing their ability to provide insight into the causal connection between the brain and behavior [7]–[11], or with no baseline (i.e., pre- lesion) measures of neuronal activity [4].”

The authors should compare their technique with other recent lesioning studies in primates (e.g. Khateeb et al, 2022)

We again thank the reviewer for this point. Specifically not mentioning Khateeb et al. 2022 was a submission error on our part; we cited the paper in Appendix 2 in the version uploaded to the eLife submission portal, but we had uploaded the version prior to citing it to bioRxiv. We have combined addressing this with addressing a previous comment, as mentioned above, with a section in the Discussion on Page 16, Line 434.

In Appendix 2, the authors suggest that a major limitation of optogenetics and chemogenetic in- activation methods is the lack of rhesus-compatible constructs. However, several viral constructs have successful implementation in rhesus monkeys so far (e.g. Galvan A, Stauffer WR, Acker L, El-Shamayleh Y, Inoue KI, Ohayon S, Schmid MC. Nonhuman primate optogenetics: recent advances and future directions. Journal of Neuroscience. 2017 Nov 8;37(45):10894-903; Tremblay et al, Neuron 2020)

We thank the reviewer for pointing us to these papers. We have added a more thorough description of what we meant by lack of rhesus-compatible constructs in that Appendix.

“However, other challenges exist with using optogenetics as an inactivation method in nonhu- man primates, including difficulty reliably affecting behavior [12]. While several constructs for rhesus macaques have been developed [13], [14], reports of successfully inducing be- havioral effects have a small effect size and are less numerous than might be expected [12], and several null results have been published [15]–[17]. Other remaining challenges include the need to develop a head-mounted, battery powered light delivery system for multi-day delivery of light and difficulty integrating illumination with simultaneous chronic neuro- electrophysiology.”

For Figure 5b, only pairwise comparison results from monkey U (L11-14) are shown. It is unclear why such results from monkey H were shown in Figure 5a but not in 5b.

We thank the reviewer for pointing out this unconventional one monkey result. As described in the original submission, we previously omitted Monkey H from the analysis in Figure 5b (now Figure 7) since some of the lesions were closely spaced together, preventing well defined pre- and post- lesion rates of turnover. Never-the-less we have included Monkey H in all the revised analysis and believe even the less cleanly separated data shows useful indications of neuron loss or silencing evoked by the lesion.

Behavioral data (during a motor task) from the awake behaving monkeys (U and H) would greatly strengthen the claim that this lesioning method is capable of creating a behavioral effect and can be adopted to study the relationship between neural function and behavior outcomes.

While we are grateful for the reviewer’s interest in the application of our lesioning technique to studies involving behavior, a behavioral analysis of the effects of our electrolytic lesions falls be- yond the scope of this Tools and Resources manuscript. We would also like to point out that we do not claim that we have achieved a behavioral deficit in this manuscript.

Figure 2 would benefit from an illustration of the Utah array placement and the location of the sites used for lesioning. The authors can either overlay the illustrations on the current ex-vivo and histology images or create a separate schematic to demonstrate that for the readers. Also, Figure 2B needs to be replaced with one without the folds to avoid confusion for the readers.

We have added Figure 2 - figure supplement 1, which shows both the location within the Utah array of the two electrodes used to create the lesions as well as the relative size of the surface area of the lesion and the array. Unfortunately, as the lesion was created under the array, the exact location of the array relative to the lesion is unknown.

As mentioned above, Figure 2B is the only histological image from that lesion. We hope that the markings in the image as well as the caption sufficiently explain that the folds are unrelated to the lesion itself.

Figure 3, the conical region is not well delineated. Data across animals and lesion volume with respect to different parameters should be included.

We have included a supplemental figure, Figure 3 - figure supplement 1, where we have used a dashed white line to clearly indicate the area of damaged parenchyma, in case it was not clear in Figure 3a. We have also added volume estimates from lesions across animals and different param- eters. The ex vivo estimates are shown in Figure 4 and the in vivo estimates are shown in Figure 5.

Figure 4: it is not clear what is being communicated, and where the voltage traces are from.

We thank the reviewer for noting this confusion. We have added some lines in the text to explain what the voltage traces show, both in the caption to Fig. 6 and in the text on Page 7, Line 238.

“Traces only capture the values while the lesioning device was turned on (45 seconds for most lesions and 50 seconds for lesion H200120). A) Voltage traces. Discontinuity at the beginning of the traces indicates transient voltages that were too rapid to be captured by the voltmeter, lasting between 0.13 and 0.33 s. The fluctuating voltages, especially the rapid in- crease in voltage at the beginning of lesioning, emphasize the importance of using a current source to deliver consistent amounts of current into the brain.”

“The voltage across the microelectrode array fluctuated much more than the current did, em- phasizing that we made the correct choice in using a current source to ensure delivery of consistent amounts of current into the brain (Fig. 6figure.caption.19).”

Figure 5: why did the authors choose to use matching units as a measure of the lesion? It is surprising that there are still units on the location that the authors claim to be a lesion. To clarify that it would be helpful to show the location of the lesion in Figure 4a. Also, what can we conclude about the lesion induction when we see units on the lesion electrode? The change in unit match shows that there is a change in the network (although the authors need to show control for that so we know those changes don’t happen due to natural dynamics). It is not clear what is the time duration for pre-pre and post-post (i.e. minutes, seconds, hours). Do these comparisons come from the same time frame or are they coming from two fragments of time for both pre and post- conditions?

Aside from post-mortem histology and tissue assays, there is no good way to confirm neuron loss with chronically implanted electrode arrays in nonhuman primates. Waveforms were chosen as they are the one readily isolated physical measure of the system we are injuring. Although functional measures of activity could indicate neuron loss (topic of following papers), there are many conceivable changes in firing rate patterns that could manifest spuriously as loss, making the estimation of loss even more ambiguous and challenging this way.

We believe the new Figure 7 will make the procedure much more clear, while also providing the control requested by the reviewer, illustrating that new statistical categories of altered waveforms emerge during a lesion, beyond those associated with typical changes in waveform composition within multi-unit recordings seen during recording sample turnover fom healthy animals. We further note that by confining this analysis to four day spans at most, we have limited the impact of daily sample turnover described in the literature (Gallego, 2020).

The time duration for pre-session versus pre-session (pre-post and post-post), is some multiple of the approximate 24 hours between each daily recording session. Therefore, since restricting our- selves to four days separation, between 24 and 96 hours. Spikes are sampled from successful trial periods (so on the order of seconds, compiled into minutes across the whole recording session). Although already described in the main text, these points have been reemphasized in the figure legend.

CNO (line 931) needs to be explained.

We thank the reviewer for this point. We have defined CNO and its relevance in Appendix 2.

“Additionally, chronic inactivation over days may be logistically challenging, as the half life of clozapine N-oxide (CNO, a ligand used to activate DREADD receptors) is on the order of hours.”

eLife assessment

This paper reports a valuable new method for creating localized damage to candidate brain regions for functional and behavioral studies. The authors present solid support for their ability to create long-term local lesions with mm spatial resolution. The paper is likely to be of broad interest to brain researchers working to establish causal links between neural circuits and behavior.

Reviewer #1 (Public Review):

In the paper, the authors illustrated a novel method for Electrolytic Lesioning through a microelectronics array. This novel lesioning technique is able to perform long-term micro-scale local lesions with a fine spatial resolution (mm). In addition, it allows a direct comparison of population neural activity patterns before and after the lesions using electrophysiology. This new technique addresses a recent challenge in the field and provides a precious opportunity to study the natural reorganization/recovery at the neuronal population level after long-term lesions. It will help discover new causal insights investigating the neural circuits controlling behavior.

Comments on revised version:

We appreciate the revisions made by the authors in response to our comments on the previous version of their manuscript. They carefully addressed the majority of the concerns and performed additional experiments. The new figure illustrating the lesion volume as a function of electrolytic lesioning parameters provides a valuable reference for future experiments. In addition, the latest results on different versions of passive multielectrode probes, U-probe, demonstrate that the technique is applicable beyond the specific technical setup they employ. Overall, we believe that the revised manuscript is significantly improved.

Reviewer #2 (Public Review):

This work by Bray et al. presented a customized way to induce small electrolytic lesions in the brain using chronically implanted intracortical multielectrode arrays. This type of lesioning technique has the benefit of high spatial precision and low surgical complexity while allowing simultaneous electrophysiology recording before, during, and after the lesion induction. The authors have validated this lesioning method with a Utah array, both ex vivo and in vivo using pig models and awake-behaving rhesus macaques. Given its precision in controlling the lesion size, location, and compatibility with multiple animal models and cortical areas, the authors believe this method can be used to study cortical circuits in the presence of targeted neuronal inactivation or injury and to establish causal relationships before behavior and cortical activity.

Strengths:

- Overall the techniques, parameters, and data analysis methods are better described in the revised version.

- The authors added the section "Relationship Between Applied Current and Lesion Volume" as well as Figure 4 and 5 to address our comments regarding parameter testing. Multiple combinations of current amplitude and duration were tested and the induced lesion volumes were estimated, providing a better picture of why certain parameters were chosen for in vivo studies.

- The authors added Figure 7 which addressed our comment "more evidence is needed to suggest robust neuronal inactivation or termination in rhesus macaques after electrolytic lesioning." They went into more details to explain the observed changes in pairwise comparisons of spike waveforms (difference in projected radii). Particularly in Fig 7C, they identified a new cluster from the pre-post lesioning group, which effectively represented neuronal loss from the<br /> recorded population.

- The authors discussed their method in the context of other literature and stating its strength and limitation.

Major comments:

-The lack of histology limits the validation of lesion induction, ideally cell loss and neuronal loss in vivo needs to be quantified. In addition based on the lack of access to histology, it is not clear how the lesion volumes are calculated which also impacts the scientific rigor of the work. The authors mention that layers 2/3 and maybe 4 have been impacted. The lack of information on the extent of the lesion severely limits the use of their technique for neuroscience experiments.

-The lack of histology in combination with behavioral measures still limits the impact of the paper in the context of NHP research.

- Figure 5 involves fitting an exponential model to the generated lesion volume given the applied current amplitude and duration. However, the data from ex vivo sheep and pig cortex with the same current amplitude & three durations showed very large variability in lesion volume at Time = 2min (larger than the difference from 2 to ~2.2min). Very limited data points exist for the other two parameter combinations. These may suggest that the exponential fit is not the best model in this scenario.

- Regarding the comment on neuronal inactivation, the authors still did not show any evidence of single unit activity loss or changes in local field potential/multi-unit activity from the region being lesioned.

- Regarding this comment "The lesioning procedure was performed in Monkey F while sedated, but no data was presented for Monkey F in terms of lesioning parameters, lesion size, recorded electrophysiology, histological, or behavioral outcomes. It is also unclear if Monkey F was in a terminal study" the authors explained that "a lesion was performed on a sedated rhesus macaque (monkey F) who was subsequently euthanized due to unrelated health complications, in order to further verify safety before use in awake-behaving rhesus" but still no histology data is shown regarding monkey F to demonstrate this verification. Given that NHPs are highly valuable resources, it's important to make use of all collected data and to show that the induced lesion is comparable to those in the pig cortex.

Reviewer #1 (Public Review):

This paper describes "Ais", a new software tool for machine-learning-based segmentation and particle picking of electron tomograms. The software can visualise tomograms as slices and allows manual annotation for the training of a provided set of various types of neural networks. New networks can be added, provided they adhere to a Python file with an (undescribed) format. Once networks have been trained on manually annotated tomograms, they can be used to segment new tomograms within the same software. The authors also set up an online repository to which users can upload their models, so they might be re-used by others with similar needs. By logically combining the results from different types of segmentations, they further improve the detection of distinct features. The authors demonstrate the usefulness of their software on various data sets. Thus, the software appears to be a valuable tool for the cryo-ET community that will lower the boundaries of using a variety of machine-learning methods to help interpret tomograms.

eLife assessment

This work describes a new software platform for machine-learning-based segmentation of and particle-picking in cryo-electron tomograms. The program and its corresponding online database of trained models will allow experimentalists to conveniently test different models and share their results with others. The paper provides solid evidence that the software will be valuable to the community.

Reviewer #2 (Public Review):

Summary:

Last et al. present Ais, a new deep learning-based software package for the segmentation of cryo-electron tomography data sets. The distinguishing factor of this package is its orientation to the joint use of different models, rather than the implementation of a given approach. Notably, the software is supported by an online repository of segmentation models, open to contributions from the community.

The usefulness of handling different models in one single environment is showcased with a comparative study on how different models perform on a given data set; then with an explanation of how the results of several models can be manually merged by the interactive tools inside Ais.

The manuscripts present two applications of Ais on real data sets; one is oriented to showcase its particle-picking capacities on a study previously completed by the authors; the second one refers to a complex segmentation problem on two different data sets (representing different geometries as bacterial cilia and mitochondria in a mouse neuron), both from public databases.

The software described in the paper is compactly documented on its website, additionally providing links to some YouTube videos (less than an hour in total) where the authors videocapture and comment on major workflows.

In short, the manuscript describes a valuable resource for the community of tomography practitioners.

Strengths:

A public repository of segmentation models; easiness of working with several models and comparing/merging the results.

Weaknesses:

A certain lack of concretion when describing the overall features of the software that differentiate it from others.

Reviewer #3 (Public Review):

Summary:

In this manuscript, Last and colleagues describe Ais, an open-source software package for the semi-automated segmentation of cryo-electron tomography (cryo-ET) maps. Specifically, Ais provides a graphical user interface (GUI) for the manual segmentation and annotation of specific features of interest. These manual annotations are then used as input ground-truth data for training a convolutional neural network (CNN) model, which can then be used for automatic segmentation. Ais provides the option of several CNNs so that users can compare their performance on their structures of interest in order to determine the CNN that best suits their needs. Additionally, pre-trained models can be uploaded and shared to an online database.

Algorithms are also provided to characterize "model interactions" which allows users to define heuristic rules on how the different segmentations interact. For instance, a membrane-adjacent protein can have rules where it must colocalize a certain distance away from a membrane segmentation. Such rules can help reduce false positives; as in the case above, false negatives predicted away from membranes are eliminated.

The authors then show how Ais can be used for particle picking and subsequent subtomogram averaging and for the segmentation of cellular tomograms for visual analysis. For subtomogram averaging, they used a previously published dataset and compared the averages of their automated picking with the published manual picking. Analysis of cellular tomogram segmentation was primarily visual.

Strengths:

CNN-based segmentation of cryo-ET data is a rapidly developing area of research, as it promises substantially faster results than manual segmentation as well as the possibility for higher accuracy. However, this field is still very much in the development and the overall performance of these approaches, even across different algorithms, still leaves much to be desired. In this context, I think Ais is an interesting package, as it aims to provide both new and experienced users with streamlined approaches for manual annotation, access to a number of CNNs, and methods to refine the outputs of CNN models against each other. I think this can be quite useful for users, particularly as these methods develop.

Weaknesses:

Whilst overall I am enthusiastic about this manuscript, I still have a number of comments:

On page 5, paragraph 1, there is a discussion on human judgement of these results. I think a more detailed discussion is required here, as from looking at the figures, I don't know that I agree with the authors' statement that Pix2pix is better. I acknowledge that this is extremely subjective, which is the problem. I think that a manual segmentation should also be shown in a figure so that the reader has a better way to gauge the performance of the automated segmentation.

On page 7, the authors mention terms such as "emit" and "absorb" but never properly define them, such that I feel like I'm guessing at their meaning. Precise definitions of these terms should be provided.

For Figure 3, it's unclear if the parent models shown (particularly the carbon model) are binary or not. The figure looks to be grey values, which would imply that it's the visualization of some prediction score. If so, how is this thresholded? This can also be made clearer in the text.

Figure 3D was produced in ChimeraX using the hide dust function. I think some discussion on the nature of this "dust" is in order, e.g. how much is there and how large does it need to be to be considered dust? Given that these segmentations can be used for particle picking, this seems like it may be a major contributor to false positives.

Page 9 contains the following sentence: "After selecting these values, we then launched a batch particle picking process to determine lists of particle coordinates based on the segmented volumes." Given how important this is, I feel like this requires significant description, e.g. how are densities thresholded, how are centers determined, and what if there are overlapping segmentations?

The FSC shown in Figure S6 for the auto-picked maps is concerning. First, a horizontal line at FSC = 0 should be added. It seems that starting at a frequency of ~0.045, the FSC of the autopicked map increases above zero and stays there. Since this is not present in the FSC of the manually picked averages, this suggests the automatic approach is also finding some sort of consistent features. This needs to be discussed.

Page 11 contains the statement "the segmented volumes found no immediately apparent false positive predictions of these pores". This is quite subjective and I don't know that I agree with this assessment. Unless the authors decide to quantify this through subtomogram classification, I don't think this statement is appropriate.

In the methods, the authors note that particle picking is explained in detail in the online documentation. Given that this is a key feature of this software, such an explanation should be in the manuscript.

eLife assessment

In this study, camera trapping and species distribution models are used to show that human disturbance in mountain forests in the eastern Himalayas pushes medium-sized and large mammal species into narrower habitat space, thus increasing their co-occurrence. While the collected data provide a useful basis for further work, the study presents incomplete evidence to support the claim that increased co-occurrence may indicate positive interactions between species.

Reviewer #1 (Public Review):

Summary:

This study examines the spatial and temporal patterns of occurrence and the interspecific associations within a terrestrial mammalian community along human disturbance gradients. They conclude that human activity leads to a higher incidence of positive associations.

Strengths:

The theoretical framework of the study is brilliantly introduced. Solid data and sound methodology. This study is based on an extensive series of camera trap data. Good review of the literature on this topic.

Weaknesses:

The authors do not delve into the different types of association found in the study. A more ecological perspective explaining why certain species tend to exhibit negative associations and why others show the opposite pattern (and thus, can be used as indicator species) is missing. Also, the authors do not clearly distinguish between significant (true) non-random associations and random associations.

Anthropogenic pressures can shape species associations by increasing spatial and temporal co-occurrence, but above a certain threshold, the positive influence of human activity in terms of species associations could be reverted. This study can stimulate further work in this direction.

Reviewer #2 (Public Review):

Summary:

This study analyses camera trapping information on the occurrence of forest mammals along a gradient of human modification of the environment. The key hypotheses are that human disturbance squeezes wildlife into a smaller area or their activity into only part of the day, leading to increased co-occurrence under modification. The method used is joint species distribution modelling (JSDM).

Strengths:

The data source seems to be very nice, although since very little information is presented, this is hard to be sure of. Also, the JSDM approach is, in principle, a nice way of simultaneously analysing the data.

Weaknesses:

The manuscript suffers from a mismatch of hypotheses and methods at two different levels.

(1) At the lower level, we would need to better understand what the individual species do and "like" (their environmental niche).

(2) The hypothesis clearly asks for an analysis of the statistical interaction between human disturbance and co-occurrence. Yet, the study is not set up in a way to test this directly.

The hypotheses point towards presenting the spatial and the temporal niche, and how it changes, species for species, under human disturbance. To this, one could then add the layer of interspecific associations.

The change in activity and space use could be analysed by looking at the activity times and spatial distribution directly. If biotic interactions change along the disturbance gradient, then observed data are already the outcome of such changed interactions. We thus cannot use the data to infer them! But we can show, for each species, that the habitat preferences change along the disturbance gradient - or not, as the case may be.

The per-species models are simplistic: the predictors are only linear, and there are no statistical interactions. It is unclear how spatial autocorrelations of residuals were treated, although they form the basis for the association analysis. Why are times of day and day of the year not included as predictors IN INTERACTION with niche predictors and human disturbance, since they represent the temporal dimension on which niches are hypothesised to change?

The discussion has little to add to the results. The complexity of the challenge (understanding a community-level response after accounting for species-level responses) is not met, and instead substantial room is given to general statements of how important this line of research is. What is the advance in ecological understanding at the community level?

Author response:

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

Reviewer #1 (Public Review):

Summary:

This study examines the spatial and temporal patterns of occurrence and the interspecific associations within a terrestrial mammalian community along human disturbance gradients. They conclude that human activity leads to a higher incidence of positive associations.

Strengths:

The theoretical framework of the study is brilliantly introduced. Solid data and sound methodology. This study is based on an extensive series of camera trap data. Good review of the literature on this topic.

Weaknesses:

The authors use the terms associations and interactions interchangeably.

This is not the case. In fact, we state specifically that "... interspecific associations should not be directly interpreted as a signal of biotic interactions between pairs of species…" However, co-occurrence can be an important predictor of likely interactions, such as competition and predation. We stand by our original text.

It is not clear what the authors mean by "associations". A brief clarification would be helpful.

Our specific definition of what is meant here by spatial association can be found in the Methods section. To clarify, the calculation of the index of associations is based on the covariance for the two species of the residuals (epsilon) after consideration of all species-specific response to known environmental covariates. These covariances are modelled to allow them to vary with the level of human disturbance, measured as human presence and human modification. After normalization, the final index of association is a correlation value that varies between -1 (complete disassociation) and +1 (complete positive association).

Also, the authors do not delve into the different types of association found in the study. A more ecological perspective explaining why certain species tend to exhibit negative associations and why others show the opposite pattern (and thus, can be used as indicator species) is missing.

Suggesting the ecological underpinnings of the associations observed here would mainly be speculation at this point, but the associations demonstrated in this analysis do suggest promising areas for the more detailed research suggested.

Also, the authors do not distinguish between significant (true) non-random associations and random associations. In my opinion, associations are those in which two species co-occur more or less than expected by chance. This is not well addressed in the present version of the manuscript.

Results were considered to be non-random if correlation coefficients (for spatial association) or overlap (for temporal association) fell outside of 95% Confidence Intervals. This is now stated clearly in the Methods section.  In Figure 3—figure supplement 1-3 and Figure 4—figure supplement 1-3, p<0.01 levels are also presented.

The obtained results support the conclusions of the study.

Anthropogenic pressures can shape species associations by increasing spatial and temporal co-occurrence, but above a certain threshold, the positive influence of human activity in terms of species associations could be reverted. This study can stimulate further work in this direction.

Reviewer #2 (Public Review):

Summary:

This study analyses camera trapping information on the occurrence of forest mammals along a gradient of human modification of the environment. The key hypotheses are that human disturbance squeezes wildlife into a smaller area or their activity into only part of the day, leading to increased co-occurrence under modification. The method used is joint species distribution modelling (JSDM).

Strengths:

The data source seems to be very nice, although since very little information is presented, this is hard to be sure of. Also, the JSDM approach is, in principle, a nice way of simultaneously analysing the data.

Weaknesses:

The manuscript suffers from a mismatch of hypotheses and methods at two different levels.

(1) At the lower level, we first need to understand what the individual species do and "like" (their environmental niche). That information is not presented, and the methods suggest that the representation of each species in the JSDM is likely to be extremely poor.

The response of each species to the environmental covariates provides a window into their environmental niche, encapsulated in the beta coefficients for each environmental covariate. This information is presented in Figure 2.

(2) The hypothesis clearly asks for an analysis of the statistical interaction between human disturbance and co-occurrence. Yet, the model is not set up this way, and the authors thus do a lot of indirect exploration, rather than direct hypothesis testing.

Our JSDM model is set up specifically to examine the effect of human disturbance on co-occurrence, after controlling for shared responses to environmental variables.  It directly tests the first hypothesis, since, if increase in indices of human disturbance had not tended to increase the measured spatial correlations between species as detected by the model, we would have rejected our stated hypothesis that human modification of habitats results in increased positive spatial associations between species.

Even when the focus is not the individual species, but rather their association, we need to formulate what the expectation is. The hypotheses point towards presenting the spatial and the temporal niche, and how it changes, species for species, under human disturbance. To this, one can then add the layer of interspecific associations.

Examining each species one by one and how each one responds to human disturbance would miss the effects of any meaningful interactions between species.  The analysis presented provides a means to highlight associations that would have been overlooked.  Future research could go on to analyze the strongest associations in the community and the strongest effects of human disturbance so as to uncover the underlying interactions that give rise to them and the mechanisms of human impact.  We believe that this will prove to be a much more productive approach than trying to tackle this problem species by species and pair by pair.

The change in activity and space use can be analysed much simpler, by looking at the activity times and spatial distribution directly. It remains unclear what the contribution of the JSDM is, unless it is able to represent this activity and spatial information, and put it in a testable interaction with human disturbance.

The topic is actually rather complicated. If biotic interactions change along the disturbance gradient, then observed data are already the outcome of such changed interactions. We thus cannot use the data to infer them! But we can show, for each species, that the habitat preferences change along the disturbance gradient - or not, as the case may be.

Then, in the next step, one would have to formulate specific hypotheses about which species are likely to change their associations more, and which less (based e.g. on predator-prey or competitive interactions). The data and analyses presented do not answer any of these issues.

We suggest that the so-called “simpler” approach described above is anything but simple, and this is precisely what the Joint Species Distribution Model improves upon.  As pointed out in the Introduction, simply examining spatial overlap is not enough to detect a signal of meaningful biotic interaction, since overlap could be the result of similar responses to environmental variables.  With the JSDM approach, this would not be considered a positive association and would then not imply the possible existence of meaningful interaction.

Another more substantial point is that, according to my understanding of the methods, the per-species models are very inappropriate: the predictors are only linear, and there are no statistical interactions (L374). There is no conceivable species in the world whose niche would be described by such an oversimplified model.

While interaction terms can be included in the JSDM, this would considerably increase the complexity of the models.  In previous work, we have found no strong evidence for the importance of interaction terms and they do not improve the performance of the models.

We have no idea of even the most basic characteristics of the per-species models: prevalences, coefficient estimates, D2 of the model, and analysis of the temporal and spatial autocorrelation of the residuals, although they form the basis for the association analysis!

The coefficient estimates for response to environmental variables used in the JSDM are provided in Figure 2 and Figure 2—source data 1.

Why are times of day and day of the year not included as predictors IN INTERACTION with niche predictors and human disturbance, since they represent the temporal dimension on which niches are hypothesised to change?

Also, all correlations among species should be shown for the raw data and for the model residuals: how much does that actually change and can thus be explained by the niche models?

The discussion has little to add to the results. The complexity of the challenge (understanding a community-level response after accounting for species-level responses) is not met, and instead substantial room is given to general statements of how important this line of research is. I failed to see any advance in ecological understanding at the community level.

We agree that the community-level response to human disturbance is a complex topic, and we believe it is also a very important one.  This research and its support of the spatial compression hypothesis, while not providing definitive answers to detailed mechanisms, opens up new lines of inquiry that makes it an important advance.  For example, the strong effects of human disturbance on certain associations that were detected here could now be examined with the kind of detailed species by species and pair by pair analysis that this reviewer appears to demand.

Reviewer #1 (Recommendations For The Authors):

L27 indicates instead of "idicates".

We thank the reviewer for catching that error.

L64 I would refer to potential interactions or just associations. It is always hard to provide evidence for the existence of true interactions.

We have revised to “potential interactions” to qualify this statement.

L69 Suggestion: distort instead of upset.

We thank the reviewer for catching that error.

L70-71 Here, authors use the term associations. Please, be consistent with the terminology throughout the manuscript.

We thank the reviewer for raising this important point.  The term “co-occurrence” appears to be used inconsistently in the literature, so we have tried to refer to it only when referencing the work of us. For us, co-occurrence means “spatial overlap” without qualification as to whether it is caused by interaction or simply by similar responses to environmental factors (see Blanchet et al. 2020, Argument 1). In our view, interactions refer to biotic effects like predation, competition, commensalism, etc., while associations are the statistical footprint of these processes.   In keeping with this understanding, in Line 73, we changed "association" to the stronger word "interaction," but in Line 76, we keep the words "spatiotemporal association", which is presumed to be the result of those interactions. In Line 91, we have changed “interactions” to “associations,” as we do not believe interactions were demonstrated in that study. 

L76 "Species associations are not necessarily fixed as positive or negative..." This sentence is misleading. I would say that species associations can vary across time and space, for instance along an environmental gradient.

We thank the reviewer for pointing out the potential for confusion.  In Line 79, we have changed as suggested.

L78 "Associations between free-ranging species are especially context-dependent" Loose sentence. Please, explain a bit further.

We have changed the sentence to be more specific; ”Interactions are known to be context-dependent; for example, gradients in stress are associated with variation in the outcomes of pairwise species interactions.”

L83-85 This would be a good place to introduce the 'stress gradient' hypothesis, which has also been applied to faunal communities in a few studies. According to this hypothesis, the incidence of positive associations should increase as environmental conditions harden.

In our review of the literature, we find that the stress gradient hypothesis is somewhat controversial and does not receive strong support in vertebrates.  We have added the phrase “…the controversial stress-gradient hypothesis predicts that positive associations should increase as environmental conditions become more severe…”

L86-88 Well, overall, the number of studies examining spatiotemporal associations in vertebrates is relatively small. That is, bird associations have not received much more attention than those of mammals. I find this introductory/appealing paragraph a bit rough. I think the authors can do better and find a better justification for their work.

We thank the reviewer for the comments.  We have rewritten the paragraph extensively to make it clearer and to provide a stronger justification for the study.

L106 "[...] resulting in increased positive spatial associations between species" I'd say that habitat shrinking would increase the level of species clustering or co-occurrence, but in my opinion, not necessarily the incidence of positive associations. It is not clear to me if the authors use positive associations as a term analogous to co-occurrence.

We thank the reviewer for raising this very important distinction.  Habitat shrinking would increase levels of species co-occurrence, but this is not particularly interested.  We wanted to test whether there were effects on species interactions, as revealed by associations.  We find that the terms association and co-occurrence are used somewhat loosely in the literature and so have made some new effort to clarify and systematize this in the manuscript.  For example, there appear to be a differences in the way “co-occurrence” is used in Boron 2023 and in Blanchet 2020. We do not use the term "positive spatial association" as analogous to "spatial co-occurrence.". Spatial co-occurrence, which for us has the meaning of spatial overlap, could simply be the result of similar reactions to environmental co-variates, not reflecting any biotic interaction. Joint Species Distribution Models enable the partitioning of spatial overlap and segregation into that which can be explained by responses to known environmental factors, and that which cannot be explained and thus might be the result of biotic interactions.  It is only the latter that we are calling spatial association, which can be positive or negative.   These associations may be the statistical footprint of biotic interactions.

Results:

Difference between random and non-random association patterns. It is not clear to me if the reported associations are significant or not. The authors only report the sign of the association (either positive or negative) but do not clarify if these associations indicate that two species coexist more or less than expected by chance. In my opinion, that is the difference between true ecological associations (e.g., via facilitation or competition effects) and random co-existence patterns. This is paramount and should be addressed in a new version of the manuscript.

This information is provided in Figure 3—figure supplement 1,2,3 and Figure 4—figure supplement 1,2,3.  This is referenced in the text as follows, “… correlation coefficients for 18 species pairs were positive and had a 95 % CI that did not overlap zero, and the number increased to 65 in moderate modifications but dropped to 29 at higher modifications" and so on. This criterion for significance (ie., greater than expected by chance) is now stated at the end of the Materials and methods.  In Figure 3—figure supplement 1,2,3 and Figure 4—figure supplement 1,2,3, those correlations that were significant at p<0.01 are also shown.

I am also missing a more ecological explanation for the observed findings. For instance, the top-ranked species in terms of negative associations is the red fox, whereas the muntjac seems to be the species whose presence can be used as an indicator for that of other species. What are the mechanisms underlying these patterns? Do red foxes compete for food with other species? Do the species that show positive associations (red goral, muntjac) have traits or a diet that are more different from those of other species? More discussion on these aspects (role of traits and the trophic niche) would be necessary to better understand the obtained results.

The purpose of this paper was to test the compression hypotheses, and we have tried to keep that as the focus.  However, the analysis does open up interesting lines of inquiry for future research to decipher the details of the interactions between species and the mechanisms by which human disturbance facilitates or disrupts these interactions. The reviewer raises some interesting possibilities, but at this point, any discussion along these lines would be largely speculation and could lengthen the paper without great benefit. 

Reviewer #2 (Recommendations For The Authors):

The manuscript should be accompanied by all data and code of analysis.

All data and RScripts have been made available in Science Data Bank: https://doi.org/10.57760/sciencedb.11804.

The sentence "not much is known" is weak: it suggests the authors did not bother to quantify what IS known, and simply waved any previous knowledge aside. Surely we have some ideas about who preys on whom, and which species have overlapping resource requirements (e.g., due to jaw width). For those, we would expect a particularly strong signal, if the association is indeed indicative of interactions.

We believe that the reviewer is referring to the statement in Line 90-92 about the lack of understanding of the resilience of terrestrial mammal associations to human disturbance.  We have added a reference to one very recent publication that addresses the issue (Boron et al., 2023), but otherwise we stand by our statement. We have, however, added a qualifier to make it clear that we did indeed look for previous knowledge; "However, a review of the literature indicates that ...."

Figures:

Fig. 1. This reviewer considers that this is too trivial and should be deleted.

This is a graphical statement of the hypotheses and may be helpful to some readers.

Fig. 2. Using points with error bars hides any potential information.

Done as suggested.

That only 4 predictors are presented is unacceptably oversimplified.

Only 4 predictors are included because, in previous work, we found that adding additional predictors or interactions did little to improve the model’s performance (Li et al. 2018, 2021 and 2022) and could lead to over-fitting.

Fig. 5. and 6. aggregate extremely strongly over species; it remains unclear which species contribute to the signal, and I guess most do not.

The number of detection events presented in Table 1 should help to clarify the relative contribution of each species to the data presented in Figures 5 and 6.

This reviewer considers that the introduction 'oversells' the paper.

L55: can you give any such "unique ecological information"

L60: Lyons et al. (Kathleen is the first name) has been challenged by Telford et al. (2016 Nature) as methodologically flawed.

The first name has been deleted.  The methodological flaw has to do with interpretation of the fossil record and choice of samples, not with the need to partition shared environmental preferences and interactions.

L61 contradicts line 64: Blanchet et al. (2022, specifying some arguments from Dormann et al. 2018 GEB) correctly point out that logically one cannot infer the existence or strength from co-occurrence data. It is thus wrong to then claim (citing Boron et al.) that such data "convey key information about interactions". The latter statement is incorrect. A tree and a beetle can have extremely high association and nothing to do with each other. Association does not mean anything in itself. When two species are spatially and temporally non-overlapping, they can exhibit perfect "anti-association", yet, by the authors' own definition, cannot interact.

We believe that the reviewer’s concerns arise from a misunderstanding of how we use the term association.  In our usage, an association is not the same as co-occurrence or overlap, which may simply be the result of shared responses to environmental variables.  The co-occurring tree and beetle would not be found to have any association in our analysis, only shared environmental sensitivities.  In contrast, associations can be the statistical footprint of interactions, and would be overlaid onto any overlap due to similar responses to the environment.  In the case of negative associations, such as might be the result of competitive exclusion or avoidance of predators, the two species would share environmental responses but show lower than expected spatial overlap.  Even though they might be only rarely found in the same vicinity, they would indeed be interacting when they were together.

Joint Species Distribution Models "allow the partitioning of the observed correlation into that which can be explained by species responses to environmental factors... and that which remains unexplained after controlling for environmental effects and which may reflect biotic interactions." (Garcia Navas et al. 2021). It is the latter that we are calling “associations.”

L63: Gilbert reference: Good to have a reference for this statement.

This point is important, but the reviewer’s comments below have made it clear that it is even more important to point out that strong interactions should be expected to lead to significant associations.  We have added a statement to clarify this.

L70-72: Incorrect, interactions play a role, not associations (which are merely statistical).

In this, we agree, and we have revised the statement to refer to interactions, not associations. In our view, an interaction is a biological phenomenon, while an association is the resulting statistical signal that we can detect.

L75: Associations tell us nothing, only interactions do. Since these can not be reliably inferred, this statement and this claim are wrong.

We thank the reviewer for raising this point, but we beg to disagree. Strong interactions should be expected to lead to significant associations that can be detected in the data. Associations, which can be measured reliably, are the evidence of potential interactions, and hence associations can tell us a great deal.  We have added a note to this effect after the Gilbert reference above to clarify this point.

However, we do accept that associations must be interpreted with caution. As Blanchet et al. 2020 explain, " …the co-occurrence signals (e.g. a significant positive or negative correlation value) estimated from these models could originate from any abiotic factors that impact species differently. Therefore, this correlation cannot be systematically interpreted as a signal of biotic interactions, as it could instead express potential non-measured environmental drivers (or combinations of them) that influence species distribution and co-distribution.”  Or alternatively an association could be the result of interaction with a 3rd species. 

L87: Regarding your claim, how would you know you DO understand? For that, you need to formulate an expectation before looking at the data and then show you cannot show what you actually measure. (Jaynes called this the "mind-projection fallacy".)

We are not sure if the reviewer is criticizing our paper or the entire field of community ecology.  Perhaps it is the statement that “….resilience of interspecific spatiotemporal associations of terrestrial mammals to human activity remains poorly understood….”  Since we are confident that the reviewer believes that mammals do interact, we guess that it is the term “association” that is questioned.  We have revised this to “…the impacts of human activity on interspecific interactions of terrestrial mammals remains poorly understood…” 

In this particular case, we did formulate an expectation before looking at the data, in the form of the two formal hypotheses that are clearly stated in the Introduction and illustrated in Figure 1. If the hypotheses had not been supported, then we would have accepted that we do not understand. But as the data are consistent with the hypotheses, we submit that we do understand a bit more now.

eLife assessment

This study provides a valuable resource by thoroughly benchmarking multiple sequencing-based tRNA quantification methods. The suggested best practice is supported by solid evidence from in silico experiments in multiple scenarios. The major weakness of the manuscript is the incomplete validation of newly generated experimental datasets.

Reviewer #1 (Public Review):

Summary:

In the manuscript titled "Benchmarking tRNA-Seq quantification approaches by realistic tRNA-Seq data simulation identifies two novel approaches with higher accuracy," Tom Smith and colleagues conducted a comparative evaluation of various sequencing-based tRNA quantification methods. The inherent challenges in accurately quantifying tRNA transcriptional levels, stemming from their short sequences (70-100nt), extensive redundancy (~600 copies in human genomes with numerous isoacceptors and isodecoders), and potential for over 100 post-transcriptional chemical modifications, necessitate sophisticated approaches. Several wet-experimental methods (QuantM-tRNA, mim-tRNA, YAMAT, DM-tRNA, and ALL-tRNA) combined with bioinformatics tools (bowtie2-based, SHRiMP, and mimseq) have been proposed for this purpose. However, their practical strengths and weaknesses have not been comprehensively explored to date. In this study, the authors systematically assessed and compared these methods, considering factors such as incorrect alignments, multiple alignments, misincorporated bases (experimental errors), truncated reads, and correct assignments. Additionally, the authors introduced their own bioinformatic approaches (referred to as Decision and Salmon), which, while not without flaws (as perfection is unattainable), exhibit significant improvements over existing methods.

Strengths:

The manuscript meticulously compares tRNA quantification methods, offering a comprehensive exploration of each method's relative performance using standardized evaluation criteria. Recognizing the absence of "ground-truth" data, the authors generated in silico datasets mirroring common error profiles observed in real tRNA-seq data. Through the utilization of these datasets, the authors gained insights into prevalent sources of tRNA read misalignment and their implications for accurate quantification. Lastly, the authors proposed their downstream analysis pipelines (Salmon and Decision), enhancing the manuscript's utility.

Weaknesses:

As discussed in the manuscript, the error profiles derived from real-world tRNA-seq datasets may still harbor biases, as reads that failed to "align" in the analysis pipelines were not considered. Additionally, the authors did not validate the efficacy of their "best practice" pipelines on new real-world datasets, preferably those generated by the authors themselves. Such validation would not only confirm the improvements but also demonstrate how these pipelines could alter biological interpretations.<br /> Because tRNA-sequencing methods have not been widely used (compared to mRNA-seq), many readers would not be familiar with the characteristics of different methods introduced in this study (QuantM-tRNA, mim-tRNA, YAMAT, DM-tRNA, and ALL-tRNA; bowtie2-based, SHRiMP, and mimseq; what are the main features of "Salmon?"). The manuscript will read better when the basic features of these methods are described in the manuscript, however brief.