Author response:

Reviewer #1 (Public review):

Summary:

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

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

Strengths:

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

Weaknesses:

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

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

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

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

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

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

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

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

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

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

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

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

Reviewer #3 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

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

1 + 1ができないことで一時期問題になった

（計算機ではないから）

プロンプトで見返してやもう一度考えてと言うのは実はあんま効果ない？

勘違いすることをハルシネーションという

岩盤って意味らしい

Dit ontwerp vergelijkt groepen op basis van veronderstelde motivatie. Bijvoorbeeld, mensen die betrokken zijn bij een rechtszaak (litigation) worden vergeleken met mensen die dat niet zijn (nonlitigation), waarbij de eerste groep naar verwachting een bepaalde responsstijl vaker zal vertonen.

Interne validiteit: Zwak, omdat onderzoekers geen controle hebben over de toewijzing van groepen of gestandaardiseerde procedures.

Externe validiteit: Zwak tot matig, omdat de deelnemers zich vaak in echte situaties bevinden met belangrijke gevolgen. Deze gevolgen kunnen hun beslissingen beïnvloeden, maar er is geen onafhankelijke classificatie van responsstijlen, wat het testen van de effectiviteit bemoeilijkt. Eerdere empirische tests hebben zwakke resultaten opgeleverd.

Classificatie: Niet te testen, omdat het onmogelijk is om de nauwkeurigheid van de classificatie vast te stellen zonder de groepstoewijzing goed te kennen.

Kortom, dit ontwerp is beperkt in zijn bruikbaarheid vanwege een gebrek aan controle en classificatie.

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

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

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

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

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

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

Het doel van deze designs is om methoden te bieden waarmee onderzoekers en clinici beter kunnen begrijpen hoe mensen reageren, of ze symptomen eerlijk rapporteren of proberen te misleiden, en om dit op een wetenschappelijke manier te meten.

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

Dit verwijst naar meetinstrumenten die specifiek zijn ontworpen om te beoordelen of iemand doet alsof hij een bepaald syndroom heeft, zoals posttraumatische stressstoornis (PTSS) of depressie. Deze schalen zijn gericht op het identificeren van het simuleren van symptomen die bij een bepaald syndroom horen, en ze zijn minder algemeen dan bredere schalen voor malingering.

Dit punt suggereert dat wanneer malingering wordt vastgesteld, het betekent dat de persoon geen echte psychische of medische stoornis heeft. Malingering impliceert dat de persoon zijn symptomen opzettelijk vervalst voor persoonlijk gewin, waardoor de mogelijkheid van een authentieke, onderliggende stoornis wordt uitgesloten. Dit idee is echter omstreden, omdat malingering en echte stoornissen soms samen kunnen voorkomen.

Malingering is een rigide, herhalende stijl van reageren, gericht op misleiding.

Deze term verwijst naar de voordelen die iemand kan halen uit ziek zijn of symptomen vertonen, zoals medeleven of financiële compensatie. Hoewel deze term duidelijke definities heeft, is het probleem dat de betekenis verschilt tussen verschillende vakgebieden zoals de psychodynamische (psychologische voordelen), gedragsmatige (beloning door gedrag) en forensische (juridische voordelen) context. Dit kan tot verwarring leiden.

Dit verwijst naar het melden van een onverwacht hoog aantal klachten of symptomen, vooral bij uitgebreide vragenlijsten met meerdere schalen. Overrapportage is niet hetzelfde als feigning (bewust overdrijven of simuleren). De term is vaag omdat het zowel opzettelijke overdrijving als onbedoelde fouten kan betekenen, en daarom niet precies genoeg is om het gedrag te beschrijven.

Dit wordt soms ten onrechte gebruikt als vervanging voor malingering (het opzettelijk simuleren of overdrijven van klachten). Het probleem is dat deze term onduidelijk is, omdat wat "beste" inspanning is per persoon kan verschillen. Iemands inspanning kan worden beïnvloed door zowel interne factoren (zoals vermoeidheid) als externe omstandigheden (zoals stress). Het geeft dus geen duidelijk beeld van opzettelijk gedrag.

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

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

Deze benadering stelt dat bedrog alleen mag worden aangenomen wanneer er overtuigend bewijs is van misleiding, vooral in juridische situaties. Dit beschermt de cliënt tegen ongegronde beschuldigingen en zorgt ervoor dat beschuldigingen van bedrog goed onderbouwd zijn voordat ze worden gerapporteerd.

Deze hypothese stelt dat als er eenmaal bedrog wordt ontdekt bij een cliënt, het waarschijnlijk is dat er meer leugens in het verhaal zitten. Dit maakt dat de therapeut extra waakzaam moet zijn en alle vormen van misleiding moet documenteren, omdat de geloofwaardigheid van de cliënt in zijn geheel in twijfel wordt getrokken.

RIGHTTT

TRUEEE

the reader and author connection (found in memoirs, essays) vs the reader and the characters connection (found in historical accounts, reports, but most especially novels)

difficult questions concerning ethics of writing CNF

重要的是记忆勾子 也就是CUE

Résumé de la Vidéo

Cette vidéo présente le syndrome de la bande, un phénomène affectant les préadolescents et adolescents, qui croient que l'appartenance à un groupe offre une protection. Emmanuel Piqué discute des comportements douloureux engendrés par cette croyance, comme la quête désespérée d'acceptation et la tolérance de mauvais traitements pour ne pas être exclus. Il propose le concept de "cordon sanitaire" comme alternative, encourageant la création de liens protecteurs avec des individus bienveillants plutôt que de chercher l'approbation d'un groupe.

Points Forts: 1. Le syndrome de la bande [00:00:10][^1^][1] * Affecte les jeunes qui cherchent protection dans les groupes * Génère des comportements douloureux et de rejet * Basé sur une croyance délétère influencée par les médias 2. Les comportements problématiques [00:00:31][^2^][2] * Désir intense d'intégration dans des groupes non accueillants * Acceptation de mauvais traitements pour appartenir à un groupe * Exemple de Morgan, maltraitée par son groupe d'amies 3. Le cordon sanitaire [00:03:14][^3^][3] * Stratégie alternative pour créer des liens protecteurs * Choix des individus basé sur la gentillesse perçue * Encourage l'indépendance vis-à-vis des groupes problématiques

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

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

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

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

Reviewer #2 (Public review):

Summary:

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

Reviewer #3 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

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

http://mytypewriter.com/rubber-feet.aspx

Imi place foarte mult ideea.

Suggests rubber feet from http://mytypewriter.com/rubber-feet.aspx

Résumé de la Vidéo

Cette vidéo présente des conseils d'Emmanuel Piqué, thérapeute systémique, sur la manière d'aider un enfant rejeté par ses camarades à l'école. Il partage l'histoire de Mia, une fillette de 7 ans qui entretient une relation difficile avec une camarade, Jade. La vidéo explore les tentatives de la mère de Mia pour résoudre le problème et propose une approche narrative pour aider Mia à comprendre sa situation et à prendre des décisions éclairées concernant son amitié avec Jade.

Points Forts: 1. La problématique du rejet scolaire [00:00:00][^1^][1] * Emmanuel Piqué aborde le sujet du rejet scolaire * Il partage l'histoire de Mia, rejetée par une camarade * La mère de Mia est inquiète et cherche des solutions 2. Les tentatives de résolution [00:01:24][^2^][2] * La mère de Mia explique ce qu'est une amitié saine * Elle interroge Mia sur sa relation avec Jade * Elle a tenté de parler aux parents de Jade et à la direction de l'école 3. L'approche narrative [00:03:19][^3^][3] * Piqué suggère de raconter une histoire métaphorique à Mia * L'histoire vise à aider Mia à réfléchir sur sa situation * Il souligne l'importance du choix de Mia et du respect de sa décision

Résumé de la Vidéo

La vidéo présente une discussion avec Emmanuel Pic, fondateur des centres Chagrin Scolaire, qui explore les méthodes de l'école de Palo Alto pour transformer les relations dysfonctionnelles en fonctionnelles, en particulier dans le contexte de l'éducation et de la parentalité. Elle souligne l'importance de responsabiliser les enfants et les adolescents en leur permettant de gérer leurs propres apprentissages et de faire face aux conséquences de leurs choix.

Points Forts: 1. Introduction à Chagrin Scolaire [00:00:01][^1^][1] * Présentation d'Emmanuel Pic * Discussion sur le harcèlement scolaire * L'impact des centres Chagrin Scolaire 2. L'école de Palo Alto [00:01:46][^2^][2] * Fondements et philosophie * Approche de la communication et des relations * Importance de la responsabilisation 3. Méthodes thérapeutiques [00:03:33][^3^][3] * Techniques pour résoudre les problèmes * Exemples de situations familiales * Stratégies pour changer les comportements 4. Responsabilisation des enfants [00:09:00][^4^][4] * Encourager l'autonomie dans les devoirs * Conséquences de l'implication parentale * Importance de l'envie d'apprendre Résumé de la vidéo

Cette vidéo aborde la manière dont les étiquettes et les diagnostics affectent la perception et l'interaction avec les enfants, en particulier ceux ayant des besoins spéciaux. Elle souligne l'importance de voir au-delà des étiquettes pour reconnaître les ressources individuelles de chaque enfant. La vidéo discute également des défis auxquels les enseignants sont confrontés lorsqu'ils essaient de s'adapter à chaque élève tout en suivant le programme scolaire, et comment cela peut conduire à des situations paradoxales et à un sentiment d'échec.

Points saillants : 1. Perception des enfants avec des besoins spéciaux [00:19:01][^1^][1] * Impact des étiquettes sur l'interaction * Importance de reconnaître les ressources des enfants * Effet des diagnostics sur les relations 2. Défis pour les enseignants [00:20:17][^2^][2] * Adaptation à divers besoins dans la classe * Gestion des attentes contradictoires * Sentiment d'échec face à l'impossibilité de répondre à tous les besoins 3. Conséquences des attentes sociétales [00:22:00][^3^][3] * Pression sur les enfants pour qu'ils s'adaptent au monde * Réalité de l'entreprise et attentes de performance * Nécessité d'aider les enfants à développer leurs propres ressources 4. Approche de l'école de Palo Alto [00:22:57][^4^][4] * Focus sur les relations et la résolution de problèmes * Expansion des centres 180 degrés en France * Difficultés rencontrées pour introduire des méthodes alternatives en France Résumé de la vidéo

Cette vidéo aborde la souffrance liée au harcèlement scolaire et les outils pour y faire face. L'oratrice discute de l'importance de la prévention et de l'intervention, soulignant que malgré les efforts, les statistiques sur le harcèlement restent alarmantes. Elle partage son expérience en tant que psycho-praticienne et auteure, mettant en lumière les stratégies pour aider les enfants et les adolescents à surmonter le harcèlement et à s'épanouir.

Points saillants : 1. Outils contre le harcèlement [00:38:35][^1^][1] * Importance des outils pour gérer la souffrance * Stratégies pour faire face au harcèlement collectif et individuel * Nécessité d'une action face aux statistiques stagnantes 2. Expérience professionnelle [00:39:16][^2^][2] * Transition de généraliste à spécialiste du harcèlement scolaire * Révélation de la fréquence du harcèlement à travers les consultations * Modélisation d'une méthode pour résoudre la souffrance scolaire 3. Publications et conseils [00:41:46][^3^][3] * Livres écrits pour aider les parents et les professionnels * Approche pragmatique et stratégique pour le bien-être des adolescents * Importance de l'éducation des parents pour soutenir les adolescents 4. La méthode de Palo Alto [00:44:22][^4^][4] * Utilisation de la méthode de Palo Alto pour introduire de la souplesse * Approche stratégique pour traiter le harcèlement et les conflits * Importance de la communication entre parents et enseignants

Résumé de la Vidéo

La partie 4 de la vidéo aborde les conférences revigorantes organisées pour célébrer les 10 ans de l'école de Palo Alto au service de l'enfant dyslexique. Ces micro-conférences, qui auront lieu à Paris le 14 juin, visent à partager des connaissances et des expériences pour aider au développement de l'enfant.

Points Forts : 1. Conférences revigorantes [00:58:34][^1^][1] * Une dizaine d'intervenants * Thème : l'école de Palo Alto au service de l'enfant dyslexique * Format : mini-conférences de dix minutes 2. Célébration des 10 ans [00:59:40][^2^][2] * Événement pour marquer une décennie * Objectif : partager des expériences et des connaissances * Importance de l'accessibilité et de la communication 3. Organisation et accès [00:59:27][^3^][3] * Les conférences seront filmées * Accessibles même pour ceux qui ne sont pas à Paris * Encouragement à la participation et à l'interaction 4. Conclusion et remerciements [01:00:09][^4^][4] * Appréciation pour l'engagement envers les enfants * Invitation à poser des questions et à interagir après l'événement * Reconnaissance de l'importance de l'accessibilité éducative

presuming competence, disabled agency

Typewriter Video Series - Episode 234: Royal QDL Spacing by [[Joe Van Cleave]]

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

eLife Assessment

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

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

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

Reviewer #2 (Public review):

Summary:

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

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

Weaknesses:

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

Reviewer #3 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

eLife Assessment

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

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

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

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

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

Overall:

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

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

Reviewer #3 (Public review):

Summary:

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

Strengths:

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

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

Rigorous analysis approaches are used throughout.

Weaknesses:

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

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

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

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

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

Weaknesses:

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

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

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

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

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

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

回帰問題で汎用的に使用されるメトリクス - MAE (Mean Absolute Error) - 予測値と実際の値の平均絶対誤差。値は0から無限大まで、値が小さいほどモデルの精度が高い。 - 回帰分析で使用され、予測誤差の平均を評価する。 - MSE (Mean Squared Error) - 予測値と実際の値の二乗誤差の平均。値は常に正で、値が小さいほどモデルの精度が高い。 - 回帰分析で使用され、モデルの予測精度を評価する。 - R2 (Coefficient of Determination) - 回帰モデルが従属変数の分散をどれだけ説明できるかを定量化する。値は1から-1の間で、1に近いほどモデルが良好であることを示す。0に近いとモデルがほとんど説明できないことを示す。 - 回帰分析で、モデルがどの程度データの変動を説明できるかを評価する。 - RMSE (Root Mean Squared Error) - 予測値と実際の値の二乗誤差の平方根。値は0から無限大まで、値が小さいほどモデルの精度が高い。 - 回帰分析で、特に大きな誤差や外れ値を評価するために使用される。

その他のメトリクス - Inference Latency - モデルのリアルタイム予測リクエストに対する応答時間を秒単位で測定。 - モデルの推論速度を評価し、特にエンドポイントでのリアルタイム推論において使用される。

1. Recall
2. 実際の正解の中で、どれだけ正しく予測できたかを示す。0から1の値を取り、1が最高。
3. バイナリ分類で、特に見逃し（False Negative）のコストが高い場合に重要。
4. Precision
5. 正しく予測した割合。0から1の値を取り、1が最高。
6. バイナリ分類で、特に誤検知（False Positive）のコストが高い場合に重要。
7. Accuracy
8. 正しく分類された項目の数の比率。0から1の値を取り、1が完全性を示す。
9. 一般的な分類タスクで使用される。
10. Balanced Accuracy
11. 正解率を、真陽性（TP）と真陰性（TN）をそれぞれ正規化した後に計算する。
12. 不均衡データセットでのバイナリ分類に使用される。
13. AUC
14. バイナリ分類モデルの性能を示す指標。ROC曲線の下の面積。1が最高。
15. バイナリ分類で、特に確率的な予測を評価するために使用される。
16. F1
17. PrecisionとRecallの調和平均。0から1の値を取り、1が最高の性能を示す。
18. バイナリ分類で、PrecisionとRecallのバランスを評価する。

多クラス分類用のメトリクス 1. F1 Macro - 各クラスのF1スコアを平均して計算する。0から1の値を取り、1が最高の性能を示す。 - 多クラス分類に使用され、各クラスに対するモデルの全体的な性能を評価する。 2. Precision Macro - 各クラスのPrecisionを計算し、それを平均して算出する。0から1の値を取り、1が最高の構成を示す。 - 多クラス分類で、モデルの全体的なPrecisionを評価する。 3. Recall Macro - 各クラスのRecallを計算し、それを平均して算出する。0から1の値を取り、1が最高のRecallを示す。 - 多クラス分類で、モデルの全体的なRecallを評価する。 4. LogLoss - 予測確率の品質を評価するメトリクス。値は0から無限大までで、0が完全予測を示す。 - バイナリおよび多クラス分類で、確率出力の精度を評価する。

教示なし学習 - IP Insights - IPv4アドレスの使用パターンを学習し、IPv4アドレスとユーザーIDやアカウント番号などのエンティティ巻の関係をとらる - 不正検出や異常な IPアドレスの使用パターンを特定するために使用 - K-Means - データ内の離散的なグループを見つけ、グループ内のメンバーが互いに似ており他のグループとは異なるようにする - クラスタリングに使用。データポイントをk個のグループ分けに使用 - Principal Component Analysis(PCA) - データセット内の次元を削減、データポイントを主成分に投影してできるだけ多くの情報や変動を保持する - 次元削減でデータの可視化やモデルの計算効率の向上に使用 - Random Cut Forest(RCP) - 規則的またはパターン化されたデータから逸脱する異常なデータポイントを検出する - 異常検知に使用され、特に異常なデータ点や外れ値を検出するタスクに適している

eLife Assessment

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

Reviewer #1 (Public review):

Summary:

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

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

Strengths:

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

Weaknesses:

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

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

Reviewer #2 (Public review):

Summary:

The authors sought to investigate the role of nociceptor neurons in the pathogenesis of pollution-mediated neutrophilic asthma.

Strengths:

The authors utilize TRPV1 ablated mice to confirm effects of intranasally administered QX-314 utilized to block sodium currents.

The authors demonstrate that via artemin, which is upregulated in alveolar macrophages in response to pollution, sensitizes JNC neurons thereby increasing their responsiveness to pollution. Ablation or inactivity of nociceptor neurons prevented the pollution induced increase in inflammation.

Weaknesses:

While neutrophilic, the model used doesn't appear to truly recapitulate a Th2/Th17 phenotype. No IL-17A is visible/evident in the BALF fluid within the model. (Figure 3F).

Unclear of the relevance of the RNAseq dataset, none of the identified DEGs were evaluated in the context of mechanism.

The authors overall achieved the aim of demonstrating that nociceptor neurons are important to the pathogenesis of pollution-exacerbated asthma. Their results support their conclusions overall, although there are ways the study findings can be strengthened. This work further evaluates how nociceptor neurons contribute to asthma pathogenesis important for consideration while proposing treatment strategies for undertreated asthma endotypes.

Reviewer #3 (Public review):

Asthma is a complex disease that includes endogenous epithelial, immune, and neural components that respond awkwardly to environmental stimuli. Small airborne particles with diameters in the range of 2.5 micrometers or less, so-called PM2.5, are generally thought to contribute to some forms of asthma. These forms of asthma may have increased numbers of neutrophils and/or eosinophils present in bronchoalveolar lavage fluid and are difficult to treat effectively as they tend to be poorly responsive to steroids. Here, Wang and colleagues build on a recent model that incorporated PM2.5 which was found to have a neutrophilic component. Wang altered the model to provide an extra kick via the incorporation of ovalbumin. Building on their prior expertise linking nociceptors and inflammation, they find that silencing TRPV1-expressing neurons either pharmacologically or genetically, abrogated inflammation and the accumulation of neutrophils. By examining bronchoalveolar lavage fluid, they found not only that levels of the number of cytokines were increased, but also that artemin, a protein that supports neuronal development and function, was elevated, which did not occur in nociceptor-ablated mice. They also found that alveolar macrophages exposed to PM2.5 particles had increased artemin transcription, suggesting a further link between pollutants, and immune and neural interactions.

There are substantial caveats that must be attached to the suggestions by the authors that targeting nociceptors might provide an approach to the treatment of neutrophilic airway inflammation in pollution-driven asthma in general and wildfire-associated respiratory problems in particular. These caveats include the uncertainty of the relevance of the conventional source of PM2.5, to pollution and asthma. According to the National Institute of Standards and Technology (NIST), the standard reference material (SRM) 2786 is a mix obtained from an air intake system in the Czech Republic. It is not clear exactly what is in the mix, and a recent bioRxiv preprint, https://www.biorxiv.org/content/10.1101/2023.08.18.553903v3.full.pdf reveals the presence of endotoxin. Care should thus be taken in interpreting data using particulate matter. Regarding wildfires, there is data that indicates that such exposure is toxic to macrophages. What impact might that then have on the production of cytokines, and artemin, in humans?

eLife Assessment

This important study presents the first identification of the odorant receptor for the trail pheromone in termites. The evidence supporting the conclusions is compelling, with state-of-the-art neurophysiological and genetic methods. The work will be of broad interest in multiple disciplines, such as entomology, chemical ecology, and sensory physiology.

Reviewer #1 (Public review):

Summary:

In their comprehensive analysis Diallo et al. deorphanise the first olfactory receptor of a non-hymenopteran eusocial insect - a termite and identified the well-established trail pheromone neocembrene as the receptor's best ligand. By using a large set of odorants the authors convincingly show that, as expected for a pheromone receptor, PsimOR14 is very narrowly tuned. While the authors first make use of an ectopic expression system, the empty neuron of Drosophila melanogaster, to characterise the receptor's responses, they next perform single sensillum recordings with different sensilla types on the termite antenna. By that, they are able to identify a sensillum that houses three neurons, of which the B neuron exhibits the narrow responses described for PsimOR14. Hence the authors do not only identify the first pheromone receptor in a termite but can even localize its expression on the antenna. The authors in addition perform a structural analysis to explain the binding properties of the receptor and its major and minor ligands (as this is beyond my expertise, I cannot judge this part of the manuscript). Finally, they compare expression patterns of ORs in different castes and find that PsimOR14 is more strongly expressed in workers than in soldier termites, which corresponds well with stronger antennal responses in the worker caste.

Strengths:

The manuscript is well-written and a pleasure to read. The figures are beautiful and clear. I actually had a hard time coming up with suggestions.

Weaknesses:

Whenever it comes to the deorphanization of a receptor and its potential role in behaviour (in the case of the manuscript it would be trail-following of the termite) one thinks immediately of knocking out the receptor to check whether it is necessary for the behaviour. However, I definitely do not want to ask for this (especially as the establishment of CRISPR Cas-9 in eusocial insects usually turns out to be a nightmare). I also do not know either, whether knockdowns via RNAi have been established in termites, but maybe the authors could consider some speculation on this in the discussion.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors performed the functional analysis of odorant receptors (ORs) of the termite Prorhinotermes simplex to identify the receptor of trail-following pheromone. The authors performed single-sensillum recording (SSR) using the transgenic Drosophila flies expressing a candidate of the pheromone receptor and revealed that PsimOR14 strongly responds to neocembrene, the major component of the pheromone. Also, the authors found that one sensillum type (S I) detects neocembrene and also performed SSR for S I in wild termite workers. Furthermore, the authors revealed the gene, transcript, and protein structures of PsimOR14, predicted the 3D model and ligand docking of PsimOR14, and demonstrated that PsimOR14 is higher expressed in workers than soldiers using RNA-seq for heads of workers and soldiers of P. simplex and that EAG response to neocembrene is higher in workers than soldiers. I consider that this study will contribute to further understanding of the molecular and evolutionary mechanisms of the chemoreception system in termites.

Strength:

The manuscript is well written. As far as I know, this study is the first study that identified a pheromone receptor in termites. The authors not only present a methodology for analyzing the function of termite pheromone receptors but also provide important insights in terms of the evolution of ligand selectivity of termite pheromone receptors.

Weakness:

As you can see in the "Recommendations to the Authors" section below, there are several things in this paper that are not fully explained about experimental methods. Except for this point, this paper appears to me to have no major weaknesses.

Reviewer #3 (Public review):

Summary:

Chemical communication is essential for the organization of eusocial insect societies. It is used in various important contexts, such as foraging and recruiting colony members to food sources. While such pheromones have been chemically identified and their function demonstrated in bioassays, little is known about their perception. Excellent candidates are the odorant receptors that have been shown to be involved in pheromone perception in other insects including ants and bees but not termites. The authors investigated the function of the odorant receptor PsimOR14, which was one of four target odorant receptors based on gene sequences and phylogenetic analyses. They used the Drosophila empty neuron system to demonstrate that the receptor was narrowly tuned to the trail pheromone neocembrene. Similar responses to the odor panel and neocembrene in antennal recordings suggested that one specific antennal sensillum expresses PsimOR14. Additional protein modeling approaches characterized the properties of the ligand binding pocket in the receptor. Finally, PsimOR14 transcripts were found to be significantly higher in worker antennae compared to soldier antennae, which corresponds to the worker's higher sensitivity to neocembrene.

Strengths:

The study presents an excellent characterization of a trail pheromone receptor in a termite species. The integration of receptor phylogeny, receptor functional characterization, antennal sensilla responses, receptor structure modeling, and transcriptomic analysis is especially powerful. All parts build on each other and are well supported with a good sample size.

Weaknesses:

The manuscript would benefit from a more detailed explanation of the research advances this work provides. Stating that this is the first deorphanization of an odorant receptor in a clade is insufficient. The introduction primarily reviews termite chemical communication and deorphanization of olfactory receptors previously performed. Although this is essential background, it lacks a good integration into explaining what problem the current study solves.

Selecting target ORs for deorphanization is an essential step in the approach. Unfortunately, the process of choosing these ORs has not been described. Were the authors just lucky that they found the correct OR out of the 50, or was there a specific selection process that increased the probability of success?

The authors assigned antennal sensilla into five categories. Unfortunately, they did not support their categories well. It is not clear how they were able to differentiate SI and SII in their antennal recordings.

The authors used a large odorant panel to determine receptor tuning. The panel included volatile polar compounds and non-volatile non-polar hydrocarbons. Usually, some heat is applied to such non-volatile odorants to increase volatility for receptor testing. It is unclear how it is possible that these non-volatile compounds can reach the tested sensilla without heat application.

eLife Assessment

This valuable study builds on previous work by the authors by presenting a potentially key method for correcting optical aberrations in GRIN lens-based micro endoscopes used for imaging deep brain regions. By combining simulations and experiments, the authors show that the obtained field of view is significantly increased with corrected, versus uncorrected microendoscopes. The evidence supporting the claims of the authors is solid, although some aspects of the manuscript should be clarified and missing information provided. Because the approach described in this paper does not require any microscope or software modifications, it can be readily adopted by neuroscientists who wish to image neuronal activity deep in the brain.

Reviewer #1 (Public review):

Summary:

Sattin, Nardin, and colleagues designed and evaluated corrective microlenses that increase the useable field of view of two long (>6mm) thin (500 um diameter) GRIN lenses used in deep-tissue two-photon imaging. This paper closely follows the thread of earlier work from the same group (e.g. Antonini et al, 2020; eLife), filling out the quiver of available extended-field-of-view 2P endoscopes with these longer lenses. The lenses are made by a molding process that appears practical and easy to adopt with conventional two-photon microscopes.

Simulations are used to motivate the benefits of extended field of view, demonstrating that more cells can be recorded, with less mixing of signals in extracted traces, when recorded with higher optical resolution. In vivo tests were performed in the piriform cortex, which is difficult to access, especially in chronic preparations.

The design, characterization, and simulations are clear and thorough, but not exhaustive (see below), and do not break new ground in optical design or biological application. However, the approach shows much promise, including for applications not mentioned in the present text such as miniaturized GRIN-based microscopes. Readers will largely be interested in this work for practical reasons: to apply the authors' corrected endoscopes.

Strengths:

The text is clearly written, the ex vivo analysis is thorough and well-supported, and the figures are clear. The authors achieved their aims, as evidenced by the images presented, and were able to make measurements from large numbers of cells simultaneously in vivo in a difficult preparation.

Weaknesses:

(1) The novelty of the present work over previous efforts from the same group is not well explained. What needed to be done differently to correct these longer GRIN lenses?

(2) Some strong motivations for the method are not presented. For example, the introduction (page 3) focuses on identifying neurons with different coding properties, but this can be done with electrophysiology (albeit with different strengths and weaknesses). Compared to electrophysiology, optical methods more clearly excel at genetic targeting, subcellular measurements, and molecular specificity; these could be mentioned. Another example, in comparing microfabricated lenses to other approaches, an unmentioned advantage is miniaturization and potential application to mini-2P microscopes, which use GRIN lenses.

(3) Some potentially useful information is lacking, leaving critical questions for potential adopters:

How sensitive is the assembly to decenter between the corrective optic and the GRIN lens? What is the yield of fabrication and of assembly?

Supplementary Figure 1: Is this really a good agreement between the design and measured profile? Does the figure error (~10 um in some cases on average) noticeably degrade the image? How do individual radial profiles compare to the presented means?<br /> What is the practical effect of the strong field curvature? Are the edges of the field, which come very close to the lens surface, a practical limitation?

The lenses appear to be corrected for monochromatic light; high-performance microscopes are generally achromatic. Is the bandwidth of two-photon excitation sufficient to warrant optimization over multiple wavelengths?

GRIN lenses are often used to access a 3D volume by scanning in z (including in this study). How does the corrective lens affect imaging performance over the 3D field of view?

(4) The in vivo images (Figure 7D) have a less impressive resolution and field than the ex vivo images (Figure 4B), and the reason for this is not clear. Given the difference in performance, how does this compare to an uncorrected endoscope in the same preparation? Is the reduced performance related to uncorrected motion, field curvature, working distance, etc? Regarding Figure 7, there is no analysis of the biological significance of the calcium signals or even a description of where olfactory stimuli were presented. The timescale of jGCaMP8f signals in Figure 7E is uncharacteristically slow for this indicator (compared to Zhang et al 2023 (Nature)), though perhaps this is related to the physiology of these cells or the stimuli.

(5) The claim of unprecedented spatial resolution across the FOV (page 18) is hard to evaluate and is not supported by references to quantitative comparisons. The promises of the method for future studies (pages 18-19) could also be better supported by analysis or experiment, but these are minor and to me, do not detract from the appeal of the work.

(6) The text is lengthy and the material is repeated, especially between the introduction and conclusion. Consolidating introductory material to the introduction would avoid diluting interesting points in the discussion.

Reviewer #2 (Public review):

In this manuscript, the authors present an approach to correct GRIN lens aberrations, which primarily cause a decrease in signal-to-noise ratio (SNR), particularly in the lateral regions of the field-of-view (FOV), thereby limiting the usable FOV. The authors propose to mitigate these aberrations by designing and fabricating aspherical corrective lenses using ray trace simulations and two-photon lithography, respectively; the corrective lenses are then mounted on the back aperture of the GRIN lens.

This approach was previously demonstrated by the same lab for GRIN lenses shorter than 4.1 mm (Antonini et al., eLife, 2020). In the current work, the authors extend their method to a new class of GRIN lenses with lengths exceeding 6 mm, enabling access to deeper brain regions as most ventral regions of the mouse brain. Specifically, they designed and characterized corrective lenses for GRIN lenses measuring 6.4 mm and 8.8 mm in length. Finally, they applied these corrected long micro-endoscopes to perform high-precision calcium signal recordings in the olfactory cortex.

Compared with alternative approaches using adaptive optics, the main strength of this method is that it does not require hardware or software modifications, nor does it limit the system's temporal resolution. The manuscript is well-written, the data are clearly presented, and the experiments convincingly demonstrate the advantages of the corrective lenses.

The implementation of these long corrected micro-endoscopes, demonstrated here for deep imaging in the mouse olfactory bulb, will also enable deep imaging in larger mammals such as rats or marmosets.

Reviewer #3 (Public review):

Summary:

This work presents the development, characterization, and use of new thin microendoscopes (500µm diameter) whose accessible field of view has been extended by the addition of a corrective optical element glued to the entrance face. Two micro endoscopes of different lengths (6.4mm and 8.8mm) have been developed, allowing imaging of neuronal activity in brain regions >4mm deep. An alternative solution to increase the field of view could be to add an adaptive optics loop to the microscope to correct the aberrations of the GRIN lens. The solution presented in this paper does not require any modification of the optical microscope and can therefore be easily accessible to any neuroscience laboratory performing optical imaging of neuronal activity.

Strengths:

(1) The paper is generally clear and well-written. The scientific approach is well structured and numerous experiments and simulations are presented to evaluate the performance of corrected microendoscopes. In particular, we can highlight several consistent and convincing pieces of evidence for the improved performance of corrected micro endoscopes:<br /> a) PSFs measured with corrected micro endoscopes 75µm from the centre of the FOV show a significant reduction in optical aberrations compared to PSFs measured with uncorrected micro endoscopes.<br /> b) Morphological imaging of fixed brain slices shows that optical resolution is maintained over a larger field of view with corrected micro endoscopes compared to uncorrected ones, allowing neuronal processes to be revealed even close to the edge of the FOV.<br /> c) Using synthetic calcium data, the authors showed that the signals obtained with the corrected microendoscopes have a significantly stronger correlation with the ground truth signals than those obtained with uncorrected microendoscopes.

(2) There is a strong need for high-quality micro endoscopes to image deep brain regions in vivo. The solution proposed by the authors is simple, efficient, and potentially easy to disseminate within the neuroscience community.

Weaknesses:

(1) Many points need to be clarified/discussed. Here are a few examples:

a) It is written in the methods: « The uncorrected microendoscopes were assembled either using different optical elements compared to the corrected ones or were obtained from the corrected probes after the mechanical removal of the corrective lens. »<br /> This is not very clear: the uncorrected microendoscopes are not simply the unmodified GRIN lenses?

b) In the results of the simulation of neuronal activity (Figure 5A, for example), the neurons in the center of the FOV have a very large diameter (of about 30µm). This should be discussed. Also, why is the optical resolution so low on these images?

c) It seems that we can't see the same neurons on the left and right panels of Figure 5D. This should be discussed.

d) It is not very clear to me why in Figure 6A, F the fraction of adjacent cell pairs that are more correlated than expected increases as a function of the threshold on peak SNR. The authors showed in Supplementary Figure 3B that the mean purity index increases as a function of the threshold on peak SNR for all micro endoscopes. Therefore, I would have expected the correlation between adjacent cells to decrease as a function of the threshold on peak SNR. Similarly, the mean purity index for the corrected short microendoscope is close to 1 for high thresholds on peak SNR: therefore, I would have expected the fraction of adjacent cell pairs that are more correlated than expected to be close to 0 under these conditions. It would be interesting to clarify these points.

e) Figures 6C, H: I think it would be fairer to compare the uncorrected and corrected endomicroscopes using the same effective FOV.

f) Figure 7E: Many calcium transients have a strange shape, with a very fast decay following a plateau or a slower decay. Is this the result of motion artefacts or analysis artefacts? Also, the duration of many calcium transients seems to be long (several seconds) for GCaMP8f. These points should be discussed.

g) The authors do not mention the influence of the neuropil on their data. Did they subtract the neuropil's contribution to the signals from the somata? It is known from the literature that the presence of the neuropil creates artificial correlations between neurons, which decrease with the distance between the neurons (Grødem, S., Nymoen, I., Vatne, G.H. et al. An updated suite of viral vectors for in vivo calcium imaging using intracerebral and retro-orbital injections in male mice. Nat Commun 14, 608 (2023). https://doi.org/10.1038/s41467-023-36324-3; Keemink SW, Lowe SC, Pakan JMP, Dylda E, van Rossum MCW, Rochefort NL. FISSA: A neuropil decontamination toolbox for calcium imaging signals. Sci Rep. 2018 Feb 22;8(1):3493. doi: 10.1038/s41598-018-21640-2. PMID: 29472547; PMCID: PMC5823956)<br /> This point should be addressed.

h) Also, what are the expected correlations between neurons in the pyriform cortex? Are there measurements in the literature with which the authors could compare their data?

(2) The way the data is presented doesn't always make it easy to compare the performance of corrected and uncorrected lenses. Here are two examples:

a) In Figures 4 to 6, it would be easier to compare the FOVs of corrected and uncorrected lenses if the scale bars (at the centre of the FOV) were identical. In this way, the neurons at the centre of the FOV would appear the same size in the two images, and the distances between the neurons at the centre of the FOV would appear similar. Here, the scale bar is significantly larger for the corrected lenses, which may give the illusion of a larger effective FOV.

b) In Figures 3A-D it would be more informative to plot the distances in microns rather than pixels. This would also allow a better comparison of the micro endoscopes (as the pixel sizes seem to be different for the corrected and uncorrected micro endoscopes).

(3) There seems to be a discrepancy between the performance of the long lenses (8.8mm) in the different experiments, which should be discussed in the article. For example, the results in Figure 4 show a considerable enlargement of the FOV, whereas the results in Figure 6 show a very moderate enlargement of the distance at which the person's correlation with the first ground truth emitter starts to drop.

a) There is also a significant discrepancy between measured and simulated optical performance, which is not discussed. Optical simulations (Figure 1) show that the useful FOV (defined as the radius for which the size of the PSF along the optical axis remains below 10µm) should be at least 90µm for the corrected microendoscopes of both lengths. However, for the long microendoscopes, Figure 3J shows that the axial resolution at 90µm is 17µm. It would be interesting to discuss the origin of this discrepancy: does it depend on the microendoscope used? Are there inaccuracies in the construction of the aspheric corrective lens or in the assembly with the GRIN lens? If there is variability between different lenses, how are the lenses selected for imaging experiments?

Reviewer #2 (Public review):

Summary:

This manuscript builds on the authors' 2020 study by combining tissue expansion with light sheet microscopy to quantify the organism-wide spatial distribution of various cell types in the planarian.

Strengths:

(1) The quantification of cell types as a function of animal size and regeneration stages could be a useful resource for the planarian research community.

(2) The high-quality images can help clarify some anatomical structures within the planarian tissues.

Weaknesses:

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

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

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

A few major issues with the claims:

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

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

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

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

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

Reviewer #3 (Public review):

Summary:

In this manuscript, the authors apply tissue expansion and tiling light sheet microscopy to study allometric growth and regeneration in planaria. They developed image analysis pipelines to help them quantify different neuronal subtypes and muscles in planaria of different sizes and during regeneration. Among the strengths of this work, the authors provide beautiful images that show the potential of the approaches they are taking and their ability to quantify specific cell types in relatively large numbers of whole animal samples. Many of their findings confirm previous results in the literature, which helps validate the techniques and pipelines they have applied here. Among their new observations, they find that the body wall muscles at the anterior and posterior poles of the worm are organized differently and show that the muscle pattern in the posterior head of beta-catenin RNAi worms resembles the anterior muscle pattern. They also show that glial cell processes appear to be altered in beta-catenin or insulin receptor-1 RNAi worms. Weaknesses include some over-interpretation of the data and lack of consideration or citation of relevant previous literature, as discussed below.

Strengths:

This method of tissue expansion will be useful for researchers interested in studying this experimental animal. The authors provide high-quality images that show the utility of this technique. Their analysis pipeline permits them to quantify cell types in relatively large numbers of whole animal samples.

The authors provide convincing data on changes in total neurons and neuronal sub-types in different-sized planaria. They report differences in body wall muscle pattern between the anterior and posterior poles of the planaria, and that these differences are lost when a posterior head forms in beta-catenin RNAi planaria. They also find that glial cell projections are reduced in insulin receptor-1 RNAi planaria.

Weaknesses:

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

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

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

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

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

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

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

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

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

eLife Assessment

The study provides valuable insight into the biological significance of SARS-CoV-2 by using a series of computational analyses of viral proteins. While evidence is solid, it is obscured by a lack of clarity about the objectives of the analyses and in the overall writing of the article. The study will be impactful to the researchers in the field but will benefit from improved presentation.

Reviewer #1 (Public review):

Summary:

Park et al. conducted various analyses attempting to elucidate the biological significance of SARS-CoV-2 mutations. However, the study lacks a clear objective. The specific goals of the analyses in each subsection are unclear, as is how the results from these subsections are interconnected. Compiling results from unrelated analyses into a single paper can be confusing for readers. Clarifying the objective and narrowing down the topics would make the paper's purpose clearer.

The logic of the study is also unclear. For instance, the authors developed an evaluation score, APESS, for analyzing viral sequences. Although they state that the APESS score correlates with viral infectivity, there is no explanation in the results section about why this is the case.

The structure of the paper should be reconsidered.

Reviewer #2 (Public review):

Summary:

The authors have developed a machine learning tool AIVE to predict the infectivity of SARS-CoV-2 variants and also a scoring metric to measure infectivity. A large number of virus sequences were used with a very detailed analysis that incorporates hydrophobic, hydrophilic, acid, and alkaline characteristics. The protein structures were also considered to measure infectivity and search for core mutations. The study especially focused on the S protein of SARS-CoV-2. The contents of this study would be of interest to many researchers related to this area and the web service would be helpful to easily analyze such data without in-depth bioinformatics expertise.

Strengths:

- Analysis of large-scale data.

- Experimental validation on a partial set of searched mutations.

- A user-friendly web-based analysis platform that is made public.

Weaknesses:

- Complexity of the research.

eLife Assessment

This important paper employs multiple experimental approaches and presents evidence that changes in membrane voltage directly affect ERK signaling to regulate cell division. This result is relevant because it supports an ion channel-independent pathway by which changes in membrane voltage can affect cell growth. The reviewers point out that some experimental results and interpretations are compelling, but the strength of evidence is incomplete and additional experiments are needed to rule out other possible interpretations of the data.

Reviewer #1 (Public review):

Summary:

This is a contribution to the field of developmental bioelectricity. How do changes of resting potential at the cell membrane affect downstream processes? Zhou et al. reported in 2015 that phosphatidylserine and K-Ras cluster upon plasma membrane depolarization and that voltage-dependent ERK activation occurs when constitutive active K-RasG12V mutants are overexpressed. In this paper, the authors advance the knowledge of this phenomenon by showing that membrane depolarization up-regulates mitosis and that this process is dependent on voltage-dependent activation of ERK. ERK activity's voltage-dependence is derived from changes in the dynamics of phosphatidylserine in the plasma membrane and not by extracellular calcium dynamics.

Strengths:

Bioelectricity is an important field for areas of cell, developmental, and evolutionary biology, as well as for biomedicine. Confirmation of ERK as a transduction mechanism, and a characterization of the molecular details involved in control of cell proliferation, is interesting and impactful.

Weaknesses:

The functional cell division data need to be stronger. They show that increasing K+ increases proliferation and argue that since a MEK inhibitor (U0126) reduces proliferation in K+ treated cells, K+ induces cell division via ERK. But I don't see statistics to show that the rescue is significant, and I don't see a key U0126-only control. If the U0126 alone reduces proliferation, the combined effect wouldn't prove much.

Also, unless I'm missing something, it looks like every sample in their control has exactly the same number of mitotic cells. I understand that they are normalizing to this column, but shouldn't they be normalizing to the mean, with the independent values scattering around 1? It doesn't seem like it can be paired replicates since there are 6 replicates in the control and 4 replicates in one of the conditions?

Reviewer #2 (Public review):

Sasaki et al. use a combination of live-cell biosensors and patch-clamp electrophysiology to investigate the effect of membrane potential on the ERK MAPK signaling pathway, and probe associated effects on proliferation. This is an effect that has long been proposed, but convincing demonstration has remained elusive, because it is difficult to perturb membrane potential without disturbing other aspects of cell physiology in complex ways. The time-resolved measurements here are a nice contribution to this question, and the perforated patch clamp experiments with an ERK biosensor are fantastic - they come closer to addressing the above difficulty of perturbing voltage than any prior work. It would have been difficult to obtain these observations with any other combination of tools.

However, there are still some concerns as detailed in specific comments below:

Specific comments:<br /> (1) All the observations of ERK activation, by both high extracellular K+ and voltage clamp, could be explained by cell volume increase (more discussion in subsequent comments). There is a substantial literature on ERK activation by hypotonic cell swelling (e.g. https://doi.org/10.1042/bj3090013, https://doi.org/10.1002/j.1460-2075.1996.tb00938.x, among others). Here are some possible observations that could demonstrate that ERK activation by volume change is distinct from the effects reported here:<br /> (i) Does hypotonic shock activate ERK in U2OS cells?<br /> (ii) Can hypotonic shock activate ERK even after PS depletion, whereas extracellular K+ cannot?<br /> (iii) Does high extracellular K+ change cell volume in U2OS cells, measured via an accurate method such as fluorescence exclusion microscopy?<br /> (iv) It would be helpful to check the osmolality of all the extracellular solutions, even though they were nominally targeted to be iso-osmotic.

(2) Some more details about the experimental design and the results are needed from Figure 1:<br /> (i) For how long are the cells serum-starved? From the Methods section, it seems like the G1 release in different K+ concentration is done without serum, is this correct? Is the prior thymidine treatment also performed in the absence of serum?<br /> (ii) There is a question of whether depolarization constitutes a physiologically relevant mechanism to regulate proliferation, and how depolarization interacts with other extracellular signals that might be present in an in vivo context. Does depolarization only promote proliferation after extended serum starvation (in what is presumably a stressed cell state)? What fraction of total cells are observed to be mitotic (without normalization), and how does this compare to the proliferation of these cells growing in serum-supplemented media? Can K+ concentration tune proliferation rate even in serum-supplemented media?

(3) In Figure 2, there are some possible concerns with the perfusion experiment:<br /> (i) Is the buffer static in the period before perfusion with high K+, or is it perfused? This is not clear from the Methods. If it is static, how does the ERK activity change when perfused with 5 mM K+? In other words, how much of the response is due to flow/media exchange versus change in K+ concentration?<br /> (ii) Why do there appear to be population-average decreases in ERK activity in the period before perfusion with high K+ (especially in contrast to Fig. 3)? The imaging period does not seem frequent enough for photobleaching to be significant.

(4) Figure 3 contains important results on couplings between membrane potential and MAPK signaling. However, there are a few concerns:<br /> (i) Does cell volume change upon voltage clamping? Previous authors have shown that depolarizing voltage clamp can cause cells to swell, at least in the whole-cell configuration: https://www.cell.com/biophysj/fulltext/S0006-3495(18)30441-7 . Could it be possible that the clamping protocol induces changes in ERK signaling due to changes in cell volume, and not by an independent mechanism?<br /> (ii) Does the -80 mV clamp begin at time 0 minutes? If so, one might expect a transient decrease in sensor FRET ratio, depending on the original resting potential of the cells. Typical estimates for resting potential in HEK293 cells range from -40 mV to -15 mV, which would reach the range that induces an ERK response by depolarizing clamp in Fig. 3B. What are the resting potentials of the cells before they are clamped to -80 mV, and why do we not see this downward transient?

(5) The activation of ERK by perforated voltage clamp and by high extracellular K+ are each convincing, but it is unclear whether they need to act purely through the same mechanism - while additional extracellular K+ does depolarize the cell, it could also be affecting function of voltage-independent transporters and cell volume regulatory mechanisms on the timescales studied. To more strongly show this, the following should be done with the HEK cells where there is already voltage clamp data:<br /> (i) Measure resting potential using the perforated patch in zero-current configuration in the high K+ medium. Ideally this should be done in the time window after high K+ addition where ERK activation is observed (10-20 minutes) to minimize the possibility of drift due to changes in transporter and channel activity due to post-translational regulation.<br /> (ii) Measure YFP/CFP ratio of the HEK cells in the high K+ medium (in contrast to the U2OS cells from Fig. 2 where there is no patch data).<br /> (iii) The assertion that high K+ is equivalent to changes in Vmem for ERK signaling would be supported if the YFP/CFP change from K+ addition is comparable to that induced by voltage clamp to the same potential. This would be particularly convincing if the experiment could be done with each of the 15 mM, 30 mM, and 145 mM conditions.

(6) Line 170: "ERK activity was reduced with a fast time course (within 1 minute) after repolarization to -80 mV." I don't see this in the data: in Fig. 3C, it looks like ERK remains elevated for > 10 min after the electrical stimulus has returned to -80 mV

Reviewer #3 (Public review):

Summary:

This paper demonstrates that membrane depolarization induces a small increase in cell entry into mitosis. Based on previous work from another lab, the authors propose that ERK activation might be involved. They show convincingly using a combination of assays that ERK is activated by membrane depolarization. They show this is Ca2+ independent and is a result of activation of the whole K-Ras/ERK cascade which results from changed dynamics of phosphatidylserine in the plasma membrane that activates K-Ras. Although the activation of the Ras/ERK pathway by membrane depolarization is not new, linking it to an increase in cell proliferation is novel.

Strengths

A major strength of the study is the use of different techniques - live imaging with ERK reporters, as well as Western blotting to demonstrate ERK activation as well as different methods for inducing membrane depolarization. They also use a number of different cell lines. Via Western blotting the authors are also able to show that the whole MAPK cascade is activated.

Weaknesses

A weakness of the study is the data in Figure 1 showing that membrane depolarization results in an increase of cells entering mitosis. There are very few cells entering mitosis in their sample in any condition. This should be done with many more cells to increase confidence in the results. The study also lacks a mechanistic link between ERK activation by membrane depolarization and increased cell proliferation.

The authors did achieve their aims with the caveat that the cell proliferation results could be strengthened. The results for the most part support the conclusions.

This work suggests that alterations in membrane potential may have more physiological functions than action potential in the neural system as it has an effect on intracellular signalling and potentially cell proliferation.

Kasten, R. M. “First Aid for Typewriters.” Popular Science Monthly, May 1941.

Don't oil the segment.

Interesting that Kasten recommends against oiling the carriage rails of portables and standard machines which run on ball bearings.

dry cleaning solvents in 1941 were likely Varsol or Stoddard's Formula.

compare to trichloroethane<br /> https://hypothes.is/a/EyBIAFXAEe-AcP-Atlj_aQ

Note discontinuation of carbon tetrachloride<br /> https://hypothes.is/a/bfdi_I90Ee-OQLN0HpsE7Q

toothpick as a typewriter tool

测试

https://en.wikipedia.org/wiki/Carbon_tetrachloride

Carbon tetrachloride or carbon tet is a non-flammable, dense, colorless liquid which was often used as a cleaning agent in the mid 1900s, but was phased out due to safety and environmental concerns. High exposure can affect the central nervous system and cause damage to the liver and kidneys. Prolonged exposure can be fatal.

Glen, John M. (1996). Highlander: No Ordinary School, 2nd ed. Knoxville: University of Tennessee Press. p. 138.

fdsf

Hints for a Happy Typewriter<br /> Bryan Kravitz, Nancy Gorrell, 1983<br /> https://typewriterdatabase.com/1983-Hints4HappyTypewriter.index.manual

Some good, basic home care and use from 1983. Home mechanics in 2024 are probably capable of a bit more without the backstop of a typewriter mechanic.

This guide suggest the use of solvents like alcohol or trichloroethane for cleaning type slugs and internals. Note that trichloroethane manufacture and use has diminished significantly since 1996 when it was identified by the Montreal Protocol as a contributor to ozone depletion.

need explanation...

This is such an important concept

What is "targeting" in this context?

This is the very conflation that zionist make in their representation of israel as the expression of all "jewish collectivity." Are zionists therefore in violation of IHRA?

The author of this "working definition," Kenneth Stern, has made plain that the legal formalization of IHRA is an "attack on academic freedom and free speech," and that IHRA should never have been adopted as "campus hate speech code."

Illustrations, not prescriptions as these examples came to be used.

What is the actual purpose of this qualifier? Who decides what kind or class of criticism is similar to another? Leveled by whom?

The organization or the definition?

Why "any other country"? Not all countries are genocidal, settler-colonial, apartheid states + the biggest recipient of U.S. military aid. Israel and the U.S. are acting in ways not replicated by any other states currently, so deserve singling out.

Hints at a claim that you don’t need to be Jewish to be affected by antisemitism. E.g. https://x.com/Almuraqiba/status/1814577232192417842

What does this mean/refer to? Again, significant lack of clarity, concreteness, specificity (a particular problem for anything posing as a definition).

See "Hate with Dylan Rodriguez" for a critique of hate as a concept that is used to hijack anti-racist policies and struggles https://criticalzionismstudies.org/2024/05/21/hate-with-dylan-rodriguez

What might these be?

This is dog-whistle Islamophobia - "religious extremism" as a cause of Jew-hatred is an animating tenet of anti-Muslim racism, particularly since the War on Terror.

While a legitimate example of antisemitism, this example has been used to shield Israel and Zionists from criticism of their influence of these institutions. E.g. On p.3 of the Academic Engagement Network's 2022 Guide and Resource Book (https://academicengagement.org/2022-guide/), the BDS movement is described in these terms: "...the BDS movement's vehement anti-Zionist and anti-Israel rhetoric, campaigns, and programming often end up trafficking in centuries-old conspiracies, tropes, and canards about Jewish power, greed, and undue influence."

Or may not be? How else, then, might it be expressed? There is a lot of hedging here and, for a definition, a real lack of specificity and clarity.

The framing offers another way to put forward the case for the “collective Jew.”

This framing continues the harmful Zionist logic that exceptionalizes the Nazi holocaust of Jews as a single most horrible atrocity while denying the magnitude and significance of other genocides and atrocities. It also resists a structural analysis of colonialism, imperialism, and racism that would illuminate connections between the atrocities committed by Nazis and those perpetrated by Zionists and other colonial powers/regimes.

Note that antisemitism is defined as an apprehension, not a material system or structure of oppression and exploitation. a liberal conceptualization of racism as prejudice, bias, or stereotyping simply.

Up until recently, the legislative Jewish caucus in California had the Israeli flag as its banner image. how is such an image supposed to be read?

Makes self-determination synonymous with statehood which is 1) an absurd proposition and 2) ignores the fact that Zionism denies Palestinian self-determination in any form. It also presumes that describing a state as racist threatens its existence. if that were the case, no modern nation-state would exist.

Which? Lack of specificity

E.g., by censoring inquiry into racist policies implemented by Israel, even though other "liberal" states are subject to such inquiry, ironically by describing such inquiry antisemitic specifically when it is directed toward Israel? in other words, this principle actually requires that a double standard be applied -- in favor of Israel.

While a legitimate example of antisemitism, this accusation has been hurled to discredit investigative accounts of actual war crimes committed by Israel. * E.g. “Israel accused the ICJ of blood libel” (https://www.theguardian.com/world/2023/dec/29/south-africa-accuses-israel-of-committing-genocide-in-gaza); “Tales of infanticide have stoked hatred of Jews for centuries. They echo still today” (https://www.theguardian.com/commentisfree/2024/oct/06/tales-of-infanticide-have-stoked-hatred-of-jews-for-centuries-they-echo-still-today).

This "non-legally binding working definition" has been pushed as both legally binding and authoritative.

Ignores the fact that Israel is not a democracy.

Refuses to name racism.

This sentence highlights the importance of focusing your research by narrowing your topic, which helps you find more specific and relevant information for a deeper, more effective project.

This sentence stresses the importance of choosing the right keywords when conducting online searches, as they help you find the most relevant information for your research.

good lab thing for study

I like the ideas of "to do" tables. I'd put them at the front instead though. Also maybe include fewer detail to de-densify the whole operation.

Lab report requirements will be interlaced throughout the assignment, with a comprehensive check off table at the end.

CUT

Maybe we write the graphing for them or otherwise simplify this sign off, but this "do X task and plot it" style of sign off doesn't always serve a learning objective and generally takes a lot of time.

These would all be separated into their own code blocks with function requirements and demo instructions in text blocks above

This sign off was a pain. It would have been easier to check if students made their robot do a little show.

Sign off 3: * Demonstrate servo_jp() by moving between two joint positions * Demonstrate interpolate_jp() by moving between the same two positions over 10 seconds * Interpolate between XXXX and YYYY over 20 seconds while printing out measured_js * Interpolate between YYYY and XXXX over 20 seconds while printing out setpoint_js * Interpolate between XXXX and YYYY over 20 seconds while printing out goal_js

Completely out of place! This should come BEFORE the instructions on what functions to write. Students should be following these directions WHILE they write those functions. I'm moving this to sign-off 2 territory.

This sign off needs to change a bit. I want this to become a "git practical".

Engagement marker: Student A: Add a comment to lab_1.mlx, and push it to a new branch

Students B&C: Pull student A's code, and show what happens to your file when you switch branches

This sign off is perfect as is--maybe it's so simple it's not even necessary. Run the provided code, show me that your robot moves in an arc, that's all I need to see.

Honestly I don't even know if there are any comprehension questions I can add that align with the LOs of the course. This is just a logistical step.

I might upgrade this "important" to a "warning: do NOT modify these files unless told". We're reworking the files to "accident-proof" the robots a bit, and we'd rather students not disable our protections.

Pre-labs are going to be a big part of at least one or two of these rewritten labs. Might be worth having a section dedicated to reminding students what they did (or should have done).

Ok, this is where the procedure actually begins. Little bit deceiving that it's signified with some bold + underline 12pt text and not a proper heading.

Important information. I'd be more likely to read it if it were a figure caption

This paragraph is redundant and tautological

No, this is not important. If students are messing with the file these IDs are used in (even in the current version of the lab), they've seriously screwed up.

This lab talks a lot of talk about teamwork, but is absolutely toothless when it comes to measuring student achievement in it! Major alignment issue.

I like this. The rewrite will have different objectives, but likely a similar objectives section.

Good! This is how I'd introduce the lab.

In re: "Team programming": This LO needs to be HAMMERED wayyyyy harder than it's being treated by this lab doc. We need to introduce measurable objectives (e.g. commit quotas, branch requirements, reflections on who did what that reference commit IDs).

This is essentially a list of all course objectives. I agree that this should be presented in the first lab to give a roadmap, but this list doesn't provide me with much information about how these topics relate to one another and why they are in this course together.

Overall comments: This section is all over the place. We bounce from "what we will be doing in this lab" to "specific robot details" to "litany of course objectives; most of which are not addressed in this lab"

This paragraph gives high level lab goals. Unfortunately, it fails to highlight any learning objective of the course! The rewrite will have a paragraph that serves the same purpose, but it will have a higher focus on LOs.

These two paragraphs introduce the robotic arm used in the class. A little more detail than is truly necessary.

The sentence expresses a student's frustration about having to include other people's ideas before sharing their own opinion, asking why they can't just skip that and state their view directly.

This sentence emphasizes that skilled writers use certain techniques automatically, which help them clearly express complex ideas in their writing.

I think this helps remind the reader what the main goal of what they are reading is and makes the writing feel more structured, clear, and complete.

I am writing this comment after finishing the chapter. I really like that it started with this story; it's a brilliant way to represent the idea. I also believe that if Dr. X had summarized the opposing views early on, the presentation would have been more compelling.

It's interesting how unsell this advice is, but I think starting with summarizing the other views is received best by people with the other view.

Not exactly related, but I noticed that in some classes, the instructor starts the semester without laying the groundwork for what we will be discussing. They dive deep into the topic, leaving many students confused and unable to understand what is being said. It's important to write in a way that everyone can understand, even if it means starting with small concepts. This approach can help readers conduct their own research on the topic and return with a better background for understanding the material.

Dont want to track peak velocity, not interested

Refuses to name racism.

The original case?

Explain how Snow learned about the agent, the host, and the environment. The Agent: The white "Rice" particles that he identified in the water but also humans stool The Host: Humans who harbored the disease Environment: Using the same contaminated water source

He found the source, the particles that were also found in the stools of the victims

How he came to the conclusion about how it spread

He assumed this was the cause of the disease

The agent: the cause of the disease

The host: the organisms that harbors the disease

The environment: the factors that cause or allow the transmission of disease [not just the physical environment, but also the social environment (actions/behaviors of people)]

Outbreak of chloera caused the investigation

理解递归程序：头脑这样想： - 用“树”的方式理解递归过程，构建出脑海的递归树，把这个问题套上去 - 确定边界条件 - 大问题 --> 小问题 ：自上而下

迭代会好理解些： - 循环终止条件 - 自下而上

Résumé de la vidéo [00:00:00][^1^][1] - [00:10:23][^2^][2]:

Cette vidéo explore comment les adolescentes YouTubeuses mettent en scène leur féminité en ligne. Elle présente les recherches de Claire Balle, sociologue, sur les pratiques numériques des jeunes filles sur YouTube.

Points forts : + [00:00:00][^3^][3] Développement de l'identité féminine * Affirmation identitaire en ligne * Étude des vidéos de filles et garçons * Importance des vidéos "je suis bizarre" et "anti-boyfriend tag" + [00:02:47][^4^][4] Proximité et sociabilité * Partage d'expériences personnelles * Attente de soutien des abonnés * Mention fréquente d'autres YouTubeuses + [00:04:46][^5^][5] Utilisation de l'intimité * Validation de l'identité par les pairs * Différences de genre dans l'expression de l'intimité * Sexualité et honte corporelle chez les filles + [00:06:30][^6^][6] Caractéristiques féminines involontaires * Manies et habitudes perçues comme féminines * Exigences dans le domaine amoureux * Perfectionnisme et propreté + [00:07:52][^7^][7] Dramatisation et standardisation * Effets de dramatisation pour représenter la féminité * Standardisation des modes de présentation * Influence des médias et réseaux sociaux

note for week 3 presentation

非常好的问题。让我为您详细解释这些概念。

1. 最优化问题：

最优化问题是指在给定的约束条件下，寻找可能解中的最佳解（最大值或最小值）。例如： - 寻找最短路径 - 最大化利润 - 最小化成本

1. 最优子结构：

最优子结构是指问题的最优解包含其子问题的最优解。换句话说，可以通过组合子问题的最优解来构造原问题的最优解。

特点： - 问题可以被分解为更小的子问题 - 子问题的最优解能够用来构造原问题的最优解

例子：最短路径问题 如果从A到C的最短路径经过B，那么A到B的这段路径一定是A到B的最短路径，B到C的这段路径一定是B到C的最短路径。

1. 无后效性：

无后效性是指某阶段的状态一旦确定，则此后过程的演变不再受此前各状态及决策的影响。

特点： - 当前状态仅与前一个状态有关，与更早的状态无关 - **未来的决策只依赖于当前状态，不依赖于如何达到当前状态 ** 例子：0-1背包问题 在考虑是否将第i个物品放入背包时，只需要考虑当前背包的剩余容量和第i个物品的价值，而不需要关心背包中已经放入了哪些物品。

让我们通过一个简单的例子来说明这些概念：

问题：计算从1到n的最大连续子数组和。

```python def max_subarray_sum(arr): n = len(arr) dp = [0] * n dp[0] = arr[0]

for i in range(1, n):
    dp[i] = max(arr[i], dp[i-1] + arr[i])

return max(dp)

示例

arr = [-2, 1, -3, 4, -1, 2, 1, -5, 4] print(max_subarray_sum(arr)) # 输出: 6 ```

在这个例子中：

1. 最优化问题：我们在寻找最大的连续子数组和。

2. 最优子结构：

3. 子问题：对于每个位置i，我们考虑以i结尾的最大子数组和。

4. 最优解构造：原问题的最优解是所有子问题最优解中的最大值。

5. 无后效性：

6. 在计算dp[i]时，我们只需要知道dp[i-1]和arr[i]，不需要知道之前的具体决策。

7. 未来的决策不会影响当前的决策。

8. 重叠子问题：

9. 在计算过程中，我们重复使用了之前计算的结果（dp[i-1]）。

通过理解这些特性，我们可以更好地识别和解决适合使用动态规划的问题。这些特性使得我们能够将复杂问题分解为更小的子问题，并有效地重用子问题的解来构建最终的最优解。

您提出了一个很好的问题。我来解释一下动态规划和分治算法的区别，并通过一个简单的例子来说明。

动态规划和分治算法的主要区别：

1. 子问题的依赖性：
2. 动态规划：子问题之间通常是相互依赖的。

3. 分治算法：子问题通常是独立的。

4. 重叠子问题：

5. 动态规划：存在大量重叠子问题。

6. 分治算法：子问题通常不重叠。

7. 解决方式：

8. 动态规划：自底向上或自顶向下（带备忘录）解决。
9. 分治算法：通常自顶向下递归解决。

让我们通过斐波那契数列的计算来举例说明：

斐波那契数列定义：F(n) = F(n-1) + F(n-2)，其中 F(0) = 0, F(1) = 1

1. 分治算法实现：

python def fibonacci_divide_conquer(n): if n <= 1: return n return fibonacci_divide_conquer(n-1) + fibonacci_divide_conquer(n-2)

这个实现会导致大量重复计算。例如，计算 F(5) 时：

F(5) ├── F(4) │ ├── F(3) │ │ ├── F(2) │ │ │ ├── F(1) │ │ │ └── F(0) │ │ └── F(1) │ └── F(2) │ ├── F(1) │ └── F(0) └── F(3) ├── F(2) │ ├── F(1) │ └── F(0) └── F(1)

可以看到，F(3)、F(2)等被重复计算了多次。

1. 动态规划实现：

python def fibonacci_dp(n): if n <= 1: return n dp = [0] * (n + 1) dp[1] = 1 for i in range(2, n + 1): dp[i] = dp[i-1] + dp[i-2] return dp[n]

这个实现避免了重复计算。我们只计算每个子问题一次，并将结果存储在 dp 数组中。

通过这个例子，我们可以看到：

1. 子问题依赖：F(n) 依赖于 F(n-1) 和 F(n-2)。
2. 重叠子问题：在分治方法中，较小的斐波那契数被重复计算多次。
3. 动态规划的优势：通过存储中间结果，避免了重复计算，大大提高了效率。

这就是为什么在处理具有重叠子问题的问题时，动态规划通常比分治算法更有效。动态规划利用了问题的这种结构，通过存储和重用子问题的解来提高效率。

reply to u/ArousedByApostasy at https://old.reddit.com/r/Zettelkasten/comments/1g8diq4/any_books_about_how_someone_used_zettelkasten_to/

If you're suffering from the delusion (and many do) that Zettelkasten is only about Luhmann and his own writing and 4-5 recent books on the topic, you're only lacking creativity and some research skills. Seemingly Luhmann has lots of good PR, particularly since 2013, but this doesn't mitigate the fact that huge swaths of the late 1800s to the late 1900s are chock-a-block full of books produced by these methods. Loads of examples exist under other names prior to that including florilegia, commonplace books, the card system, card indexes, etc.

Your proximal issue is that the scaffolding used to write all these books is generally invisible because authors rarely, if ever, talk about their methods and as a result, they're hard to "see". This doesn't mean that they don't exist.

I've got a list of about 50+ books about the topic of zettelkasten or incredibly closely related methods dating back to 1548 if you want to peruse some: https://www.zotero.org/groups/4676190/tools_for_thought/collections/V9RPUCXJ/tags/note%20taking%20manuals/items/F8WSEABT/item-list

There are a variety of examples of people's note collections that you can see in various media and compare to their published output. I've collected several dozens of examples, many of which you can find here: https://boffosocko.com/research/zettelkasten-commonplace-books-and-note-taking-collection/

Interesting examples to get you started:

• Vladimir Nabokov's estate published copies of his index cards for the novel The Original of Laura which you can purchase and read in its index card format. You can find a copy of his index card diary as Insomniac Dreams from Princeton University Press: https://press.princeton.edu/books/paperback/9780691196909/insomniac-dreams
• S.D. Goitein - researchers on the Cairo Geniza still use his note collection to produce new scholarship; though he had 1/3 the number of note cards compared to Luhmann, his academic writing output was 3 times larger. If you dig around you can find a .pdf copy of his collection of almost 30,000 notes and compare it to his written work.
• There's a digitized collection of W. Ross Ashby's notes (in notebook and index card format) which you can use to cross reference his written books and articles. https://ashby.info/
• Wittgenstein had a well-known note collection which underpinned his works (as well as posthumous works). See: Wittgenstein, Ludwig. Zettel. Edited by Gertrude Elizabeth Margaret Anscombe and Georg Henrik von Wright. Translated by Gertrude Elizabeth Margaret Anscombe. Second California Paperback Printing. 1967. Reprint, Berkeley and Los Angeles, California: University of California Press, 2007.
• Roland Barthes had a significant collection from which he both taught and wrote; His notes following his mother's death can be read in the book Morning Diary which were published as index card-based notes.
• The Marbach exhibition in 2013 explored six well-known zettelkasten (including Luhmann's): Gfrereis, Heike, and Ellen Strittmatter. Zettelkästen: Maschinen der Phantasie. 1st edition. Marbach am Neckar: Deutsche Schillerges, 2013. https://www.amazon.de/-/en/Heike-Gfrereis/dp/3937384855/.
• Philosopher John Locke wrote a famous treatise on indexing commonplace books which underlay his own commonplacing and writing work: Locke, John, 1632-1704. A New Method of Making Common-Place-Books. 1685. Reprint, London, 1706. https://archive.org/details/gu_newmethodmaki00lock/mode/2up.
• Historian Jacques Barzun, a professor, dean and later provost at Columbia, not only wrote dozens of scholarly books, articles, and essays out of his own note collection, but also wrote a book about some of the process in a book which has over half a dozen editions: Barzun, Jacques, and Henry F. Graff. The Modern Researcher. New York, Harcourt, Brace, 1957. http://archive.org/details/modernreseracher0000unse. In his private life, he also kept a separate shared zettelkasten documenting the detective fiction which he read and was a fan. From this he produced A Catalogue of Crime: Being a Reader's Guide to the Literature of Mystery, Detection, and Related Genres (with Wendell Hertig Taylor). 1971. Revised edition, Harper & Row, 1989: ISBN 0-06-015796-8.
• Erasmus, Agricola, and Melanchthon all wrote treatises which included a variation of the note taking methods which were widely taught in the late 1500s at universities and other schools.
• The Jonathan Edwards Center at Yale has a digitized version of his note collection called the Miscellanies that you can use to cross reference his written works.
• A recent example I've come across but haven't mentioned to others until now is that of Barrett Wendell, a professor at Harvard in the late 1800s, taught composition using a zettelkasten or card system method.
• Director David Lynch used a card index method for writing and directing his movies based on the method taught to him by Frank Daniel, a dean at the American Film Institute.
• Mortimer J. Adler et al. created a massive group zettelkasten of western literature from which they wrote volumes 2 and 3 (aka The Syntopicon) of the Great Books of the Western World. See: https://forum.zettelkasten.de/discussion/2623/mortimer-j-adlers-syntopicon-a-topically-arranged-collaborative-slipbox
• Before he died, historian Victor Margolin made a YouTube video of how he wrote the massive two volume World History of Design which included a zettelkasten workflow: https://www.youtube.com/watch?v=Kxyy0THLfuI
• Martin Luther King, Jr. kept a zettelkasten which is still extant and might allow you to reference his notes to his written words.
• The Brothers Grimm used a zettelkasten method (though theirs was slips nailed to a wall) to create The Deutsches Wörterbuch (The German Dictionary that preceeded the Oxford Dictionary). The DWB was begun in 1838 by Jacob Grimm and Wilhelm Grimm who worked on it through the letter F prior to their deaths. The dictionary project was ended in 1961 after 123 years of work which resulted in 16 volumes. A further 17th source volume was released in 1971.
• Here's an interesting video of Ryan Holliday's method condensed over time: https://www.youtube.com/watch?v=dU7efgGEOgk
• Because Halloween is around the corner, I'll even give you a published example of death by zettelkasten described by Nobel Prize winner Anatole France in one of his books: https://boffosocko.com/2022/10/24/death-by-zettelkasten/

If you dig in a bit you can find and see the processes of others like Anne Lamott, Gottfried Wilhelm Leibniz, Bob Hope, Michael Ende, Twyla Tharp, Kate Grenville, Marcel Mauss, Claude Lévi-Strauss, Phyllis Diller, Carl Linnaeus, Beatrice Webb, Isaac Newton, Harold Innis, Joan Rivers, Umberto Eco, Georg Christoph Lichtenberg, Raymond, Llull, George Carlin, and Eminem who all did variations of this for themselves for a variety of output types.

These barely scratch the surface of even Western intellectual history much less other cultures which have broadly similar methods (including oral cultures). If you do a bit of research into any major intellectual, you're likely to uncover a similar underlying method of work.

While there are some who lionize Luhmann, he didn't invent or even perfect these methods, but is just a drop of water in a vast sea of intellectual history.

And how did I write this short essay response? How do I have all these examples to hand? I had your same question years ago and read and researched my way into an answer. I have both paper and digital zettelkasten from which to query and write. I don't count my individual paper slips of which there are over 15,000 now, but my digital repository is easily over 20,000 (though only 19K+ are public).

I hope you manage to figure out some version of the system for yourself and manage to create something interesting and unique out of it. It's not a fluke and it's not "just a method for writing material about zettelkasten itself".

mutualism

interesting -- not the history of the fact of homosexuality, but the identity category

sometimes acquired status is official (run for office for position) and can reflect old patriarchies that trying to come back

social position of official is stronger where there's a demand for trained experts- a training that only the wealthy can afford

loyalty to office = loyalty to an impersonal and functional purpose as opposed to a person

activities are official duties

regulation not derived from individual judgement but from accordance with rules

training in field of specialization- increasingly important

items of official different from org.

monocratically organized hierarchy- lower offices supervised by higher ones and can be appealed to them

state = bureaucratic agency private = bureaucratic enterprise

methodical provisions for rules and for getting employees

officials distributing duties does it in a stable way and according to the rules

necessary regular activities are official duties

Modern bureaucracy means...

I don't know a significant amount about care homes in Japan, however, in America they are a bit infamous for poor care. There are a host of reasons for why this occurs. This makes me wonder if Americans would opt for robots over humans.

Is there a way to address this issue in the robots without trashing them? Is this a training Ai issue? Either way, this must be addressed.

I have always found it interesting that the government has a say/controls specifically how elders are treated in a family. In our western culture there is no specific requirement for how elders are cared for.

How is it looking now? What impact did COVID have on this timeline?

If the cost of robot care is similar to an elders home, what would people choose?

Would these have to be robots? They could be simple machines. It may even be easier for elders to accept that way.

good

Is this correct? Are these now scalaras rather than vectors?

The declaration of Independence in 1776. Politicians and the seeking eyes of the people and their freedom. July 4th 1776, Thomas Jefferson and many other leaders of the colonies got together and signed the document. It was signed and ratified in the Pennsylvania state house. A lot of people often ask questions about the Declaration of independence. I wonder where the document is? I also wonder about how the document being a piece of paper is held and preserved. The declaration document was ratified to grant freedom to the people and formally introduce equality. To separate from Great Britian and to form their own civilization. The audience they were reaching was Great Britian.

is it only for the summary cube? or also the pnl cube

really intend "glob"

Confused because the sample file listed does not match the columns here... what is listed here is a subset.

formula that shows the ratio of elements present in the compound

Also known as "NOAA Research," this office maintains NOAA's "Climate.gov" website, with such "alarmist" features as a "global climate dashboard" charting rising CO2 and other greenhouse gases, sea levels, and ocean heat, as well as declining sea ice and mountain glaciers. https://www.climate.gov/about.

The Trump administration let this portal go unfunded and at one point shut it down. The suppression of talk about climate change on this and other agency portals during these years, documented by EDGI, was far-reaching. Climate researchers had to alter webpages, for instance, by changing "climate change" to "climate," apparently taken as less politically charged. https://envirodatagov.org/wp-content/uploads/2019/07/New_Digital_Landscape_EDGI_July_2019.pdf

Trump's proposed funding cuts for this agency averaged a whopping 35.7% over the first three years. However, Congress pushed back hard, and actually authorized increases in it budget (8.5%) and staff (3.5%).<br /> https://envirodatagov.org/embattled-landscape-series-part-2b-the-declining-capacity-of-federal-environmental-science/

Trump's average proposed budget cuts for his first three years for the Fish and Wildlife Service were slightly higher than those for NOAA itself--more than 18%. However, FWS fared much better in Congress, actually increasing its budget by 2% (adjusting for inflation). It nevertheless lost 5.8% of its staff. https://envirodatagov.org/embattled-landscape-series-part-2b-the-declining-capacity-of-federal-environmental-science/

Trump tried to cut this service's budget by an average of 15.9% over his first years, but thanks to Congress its staff and budget barely budged. https://envirodatagov.org/embattled-landscape-series-part-2b-the-declining-capacity-of-federal-environmental-science/

For Project 2025, data supporting the reality of anthropogenic climate change appears biased by definition, since it takes sides in an imagined "climate debate" among scientists. This is a throwback to an earlier version of climate denialism. https://allianceforscience.org/blog/2024/01/new-analysis-exposes-shifts-in-climate-denial-tactics-on-youtube/

Trump's average proposed budget cuts for this service averaged a more modest 15% for his first three years. But through Congressional action, the inflation-adjusted budget fell over 40% and the staff over 10% during his time in office. https://envirodatagov.org/embattled-landscape-series-part-2b-the-declining-capacity-of-federal-environmental-science/

Here's a belated recognition that the data used by commercial operations is indeed from the NWS. What it would mean for the NWS to also "fully commercialize its forecasting operations" is not at all clear.

A claim that seems to disregard how so much today's commercial services actually hinge on federal weather data. As AccuWeather chief executive Steven R. Smith put it: "NOAA’s 'foundational data' helps inform AccuWeather’s own forecasting software, artificial intelligence and meteorologists." Moreover, “it has never been our goal to take over the provision of all weather information.” https://www.washingtonpost.com/weather/2024/07/22/project-2025-weather-service-trump/