Is that all it means? Usually when I see this term, it sounds like they mean cleaning out inactive contacts, not just those that have asked to be removed.

I mean, obviously you would remove those who ask to be removed... But it seems you would do so immediately, not "regularly" at some later time. I guess it depends how you implement your list system?

This makes a lot of sense if I think about it like this: Base notes are often warm, like the sun, sometimes leathery, often masculine, and floral heart notes, intoxicating, like the moon is said to be.

Résumé de la vidéo [00:00:01][^1^][1] - [00:20:21][^2^][2]:

Cette vidéo présente une discussion entre Adam Grant et Dan Ariely, économiste comportemental, sur la vérité honnête concernant la malhonnêteté. Ils explorent la fréquence de la malhonnêteté dans les organisations, l'impact des petits tricheurs par rapport aux grands, et comment les comportements négatifs peuvent devenir acceptables dans un environnement organisationnel. Ils discutent également du rôle de la direction, des lanceurs d'alerte et de l'importance d'un code de conduite clair pour prévenir la malhonnêteté.

Points forts: + [00:00:01][^3^][3] La malhonnêteté dans les organisations * Très commune, mais principalement de petits tricheurs * Les petits tricheurs ont un impact économique plus important que les grands + [00:03:45][^4^][4] Le rôle de la direction et des lanceurs d'alerte * La direction peut influencer le comportement organisationnel * Les lanceurs d'alerte sont souvent traités comme des outsiders + [00:06:20][^5^][5] La pente glissante de la malhonnêteté * Les grandes fraudes commencent souvent par de petits pas * Importance de reconnaître les premiers signes de comportement contraire à l'éthique + [00:13:01][^6^][6] L'importance d'un code de conduite clair * Un code de conduite précis aide les individus à distinguer le bien du mal * Les règles floues permettent une rationalisation plus facile de la malhonnêteté

Resources plays an important role in the backward design process. Quality resources can enhance student engagement and make teaching more effective.

The leaning activities suggested here give me more ideas for class activities I can use in my class.

Thinking about essential questions has always been my weakness. I was wondering if any of you have thought processes or strategies that could help a novice teacher identify essential questions for a course. How do you create a link between unit key questions and individual lesson essential questions?

I hadn't pictorially felt this before. But I've noticed both before and after learning about reverse design that it's easy for me to get so limited by what I set out to do in the beginning that I forget the reason and purpose for goal setting.

For my final project, the assessment is delivering a two-minute talk on the subject of the unit. This aligns with the three modes of communication: interpersonal, interpretive, and presentational.

Very good point! I found that the five "C" goals Communication, Cultures, Connections, Comparisons, and Communities) help me prioritize my teaching content.

I disagree with the author. Clothing topics can indeed be significant. Clothing integrates history and reflects cultural and social development. It serves as a comprehensive cultural carrier with distinct characteristics of time, nationality, region, customs, and artistry. Moreover, it acts as a cultural symbol that reflects the politics, economy, thought, morality, and faith of different times. In my AP Chinese course, I include a unit on traditional Chinese clothing to explore these rich cultural dimensions.

I teach AP Chinese Language and Culture. The course emphasizes communication, focusing on understanding and being understood by others, by applying interpersonal, interpretive, and presentational skills in real-life situations. It adheres to the standards described in the ACTFL Performance Descriptors for Language Learners.

Die bewusste Entscheidung der USA gegen eine entschlossene Klimapolitik Anfang der 90er Jahre läuft für Natur auf die spätere Isolierung der USA unter Trump hinaus. Man koppelt sich vom Rest der Erde ab. Damit wird das moderne Projekt, die gesamte Welt zu mobilisieren obsolet. Der Kapitalismus zieht sich auf ein, nach Möglichkeit durch Mauern gesichertes Territorium zurück und zerstört ohne Beschränkungen die Erde außerhalb diess Gebiets.

Der Begriff der Natur endpolitisiert die Ökologie, weil im neuzeitlichen Paradigma die Natur genau als der Teil der Realität definiert ist der sich jenseits des politischen Streits befindet. Über die Natur gibt es objektives Wissen, aber genau deshalb ist sie in politische Konflikte nicht direkt eingebunden.

Die Erde, der Platz, an dem wir leben, bestimmt einerseits unsere Identität immer mit. Andererseits ist auch alles, von dem wir leben, auf der Erde lokalisierbar. Beides fällt in der aktuellen Situation der übernutzung der Erde auseinander. Natur spricht davon, dass viele in der Gegenwart Pflicht nicht wissen, wo sie leben. Bei einer Lebensweise, wie die Erde nicht überfordert, musses dagegen für alle möglich sein, sich zu lokalisieren.

In der ökologischen Krisensituation wird der Boden neu politisiert, es entstehen neue emotionale und begriffliche Beziehungen zu ihm, er wird zum Katalysator von Konflikten, Dabei steht die Beziehung zum Boden im Widerspruch zur kapitalistischen Nutzung der Erde. Es geht hier nicht um eine rückwärtsgewandte Beziehung zum Boden im Sinne der Blut und Bodenpolitik der Nazis, sondern um die Entdeckung von etwas neuem und den Qualitäten von Erde und Boden, die bisher ignoriert wurden.

Latour spricht davon, dass wir es heute nicht mit einer extensiven, sondern einer intensiven Beziehung zur Erde zu tun haben. Sie befiehlt bezieht sich auf mehrere Dimensionen, von denen unser Leben direkt abhängt.

Zu Boden und Territorium besteht ein direkter und immer auch politischer Bezug. Es gibt erkennbare Rechte an ihnen Komma bzw fehlende Rechte sie sind aufgeteilt, und der Umgang muss mit ihnen muss geregelt werden. Heute geht es dabei nicht nur um die zu beherrschende oder nicht zu beherrschende Oberfläche sondern um die vieldimensionale Realität des Bodens, von dem wir abhängen. Wir bewegen uns auf dem Boden und alle politischen und wirtschaftlichen Beziehungen finden im geographischen Raum statt, haben für ihn Konsequenzen und sind oft direkt auf ihn bezogen.

Dieser Widerstand gegen den Kapitalismus wird dann zentral in dem Buch über die ökologische Klasse. offensichtlich gehört hier Polanyi auch zum Hintergrund.

Hier kommen die unterschiedlichen Positionen von Charbonnier und Latour sehr deutlich heraus. Charbonnier hält an der Idee einer gesteuerten und kontrollierten Modernisierung fest.

Ch 3: Coding Attention Mechanisms

https://taz.de/FFF-Aktivistin-ueber-Europawahl/!6015051/

https://www.nytimes.com/2024/06/17/climate/labor-unions-fema-disaster-relief.html

The slides seem to not be in the link anymore.

https://www.nytimes.com/2024/06/17/climate/labor-unions-fema-disaster-relief.html

Precisa de um rascunho mínimo

Que veux-tu dire par "genre"? Genre littéraire? Mais au paragraphe suivant tu parles plutôt de critère géorgaphiques.

Welcome ?

In OCRMyPDF v15.4.4, this doesn't seem the case. When using the --pdf-renderer hocr option explicitly: * I get a .hocr file in the temporary files folder; * I avoid segmentation errors when reading the file with Mozilla's PDF.js; * When text is selected using a PDF reader, I can see the OCRed text (instead of blank characters).

What about an hOCR file?

Probably an invisible font for invisible text overlay. Is it possible to use a non-glyphless font, yet invisible (i.e., it would only show when highlighting the text)?

The relationship that communities have with more-than-human elements characterised the very own ontology of these communities. Oslender analyse this...

Therefore, is it up to the PDF reader to determine what is a "word", a "line", a blank space, etc?

Résumé de la vidéo [00:00:00][^1^][1] - [00:18:57][^2^][2]:

Cette vidéo présente un colloque sur l'éducation, abordant les transformations du système scolaire français, l'impact de la massification scolaire sur la société, et les défis actuels de l'éducation. L'orateur discute des changements dans les carrières scolaires, l'orientation des élèves, et l'évolution des structures familiales. Il souligne l'importance croissante de l'éducation pour les parents diplômés et les conséquences sur les performances des élèves.

Points forts: + [00:01:01][^3^][3] Contexte du colloque * Suite à un cours sur l'éducation * Examen des transformations scolaires * Impact de la massification sur la société + [00:02:01][^4^][4] Transformations scolaires * Évolution des carrières scolaires * Question de l'orientation des élèves * Différences de compétences acquises + [00:03:00][^5^][5] Impact social de l'éducation * Augmentation de la monoparentalité * Corrélation entre éducation et conjugalité * Influence de l'éducation sur la fécondité + [00:05:26][^6^][6] Rôle des familles dans l'éducation * Importance de l'action éducative familiale * Inégalités de ressources et d'efficacité * Exigence accrue des parents diplômés + [00:07:00][^7^][7] Défis actuels de l'éducation * Chute des performances scolaires * Décalage entre évaluations et compétences réelles * Nécessité d'approches individuelles et contextuelles + [00:14:46][^8^][8] Focus sur les mathématiques * Préoccupation croissante pour le niveau en mathématiques * Impact potentiel sur la recherche scientifique * Importance des activités périscolaires et des compétitions

Résumé de la vidéo [00:00:00][^1^][1] - [00:18:57][^2^][2]:

Cette vidéo présente un colloque sur l'éducation, abordant les transformations du système scolaire français, l'impact de la massification scolaire sur la société, et les défis actuels de l'éducation. L'orateur discute des changements dans les carrières scolaires, l'orientation des élèves, et l'évolution des structures familiales. Il souligne l'importance croissante de l'éducation pour les parents diplômés et les conséquences sur les performances des élèves.

Points forts: + [00:01:01][^3^][3] Contexte du colloque * Suite à un cours sur l'éducation * Examen des transformations scolaires * Impact de la massification sur la société + [00:02:01][^4^][4] Transformations scolaires * Évolution des carrières scolaires * Question de l'orientation des élèves * Différences de compétences acquises + [00:03:00][^5^][5] Impact social de l'éducation * Augmentation de la monoparentalité * Corrélation entre éducation et conjugalité * Influence de l'éducation sur la fécondité + [00:05:26][^6^][6] Rôle des familles dans l'éducation * Importance de l'action éducative familiale * Inégalités de ressources et d'efficacité * Exigence accrue des parents diplômés + [00:07:00][^7^][7] Défis actuels de l'éducation * Chute des performances scolaires * Décalage entre évaluations et compétences réelles * Nécessité d'approches individuelles et contextuelles + [00:14:46][^8^][8] Focus sur les mathématiques * Préoccupation croissante pour le niveau en mathématiques * Impact potentiel sur la recherche scientifique * Importance des activités périscolaires et des compétitions

Manage files - you can now select multiple files

just make sure that you have moved cl11 into wvd infra and done that section of the conditional access lab, changed device settings to domain joined

2

1

You can certainly keep them out on shelves and rely on occasional dusting.

If they're in a dustier-than-typical room or you have compounding factors, like the presence of cats or dogs (like my German Shedder, I meant German Shepherd), and don't want to go the route of traditional fabric-like dust covers, you might consider doing a thicker plastic/acrylic cover which will give you a clear plastic layer of protection, but still show off your machines.

I live in Los Angeles and there are half a dozen places that do this sort of custom plastic work all the time for very reasonable rates. Searching for "plastic fabricator memorabilia case" along with variations of plastics (acrylic, lucite, plexiglass) should get you what you want locally. (Here's a few examples I've used in Los Angeles before to give you an idea: https://solterplastics.com/, https://www.plasticfactoryinc.com/, https://www.customacrylicproducts.com/, https://plexidisplays.com/). Search for something similar in your area for easier communication and cheaper pick up/shipping.

If you search around for companies that make plastic displays, particularly for memorabilia (baseball bats, baseball cards, etc.), you can have them design and make a custom sized clear plastic box/enclosure that will keep the dust and dirt out, but still allow you to see the machine inside. If done well it may actually make them appear more precious because you've taken the additional precaution.

Enclosed glass shelving is also a potential solution as well, but requires a larger investment and also requires more work to rotate machines out for regular use.

Most of my machines get daily use, so I'm not really using them for display or presentation purposes (except for one machine which sits on our library card catalog, but even then, it is frequently used as a standing desk, for occasional poetry by everyone in the family, or for guests who want to try their hand). I go through lots of index cards, so I'll usually temporarily protect against dust, dirt, and fur by slipping an index card on top of the hood or slightly into it to protect the segment.

But at the end of the day, as long as you haven't used WD-40 or some other lubricant on your segment and typebars (and what typewriter monster would do such barbaric things?), you should easily be able to go long periods between dustings and still have a highly functional machine. After all, who hasn't bought a machine full of dirt, dust, White Out, and eraser shavings/crumbs that still works like a dream?

It may bear brief mention for those who display their machines and forget, that you might also disengage the paper lock/paper release lever which will release the tension on your rubber rollers against the platen so that they don't go "flat" or become misshapen when not in use for long periods.

Expansion of https://hypothes.is/a/NjoVMA1REe-f47d0T4ZOkg

Reply to u/Styr0foam at https://www.reddit.com/r/typewriters/comments/1djgjv2/how_to_display_typewriters_properly/

対して　でしょうか

感想

項目　と　要素　の使い分けがよくわかりません。原文のitem と elementsに対応しているのはわかるのですが。

リストに入ったら要素、入る前は項目？

そうすると「リストを定義」項にある「リスト中の個々の項目」とか、「個々の要素を変更」項にある「変更したい項目のインデックス」が不思議な感じですね。

「削除した」がちょっとわかりにくく感じました

原文

If you want to work with an element that you're removing from the list,

代案

リストから削除する要素を使って作業したい

後ろから数えた要素　はいかがでしょうか

新しいプログラマーでも　はいかがでしょうか

空のリストを準備してから　はいかがでしょうか

typo できます

Literature is an art that must be studied and apply in order to achieve ans imptove our reading, listening and critical thinking skills.

https://www.liberation.fr/environnement/climat/yannick-jadot-sur-totalenergies-qui-aurait-pu-predire-que-le-senat-soit-aussi-severe-avec-une-multinationale-20240619_BAOX26RJENGCTF2LOUDHXW5RYY/

Epidemiology by Design

Applying Quantitative Bias Analysis to Epidemiologic Data

Author response:

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

eLife assessment:

The statistical analyses are incomplete.

I find that the eLife assessment mentions “statistical analyses are incomplete” while the reviewers mention that the data are compelling and results are technically solid. Besides all observations in the manuscript are presented with robust and established norms of statistical analysis. Therefore, I would kindly request to remove the statement as it undermines the article.

Public Reviews:

Reviewer #1 (Public Review):

Strengths:

The use of data from before COVID-19 is both a strength and a weakness. Because COVID had effects on vascular health and had higher death rates for groups with the comorbidities of interest here, it has likely shifted the demographics in ways that would shift the results in unpredictable ways if the analysis were repeated with current data. This can be a strength in providing a reference point for studying those changes as well as allowing researchers to study differences between regions without the complication of different public health responses adding extra variation to the data. On the other hand, it limits the usefulness of the data in research concerned with the current status of the various populations.

We completely agree with the observation, but were restricted as the purpose was to use the most robust and technically qualified data from GBD. The post COVID19 GBD data has not yet been released, but I am sure the observations made in the study can help in guiding the issues in the post COVID era too, because genetics is not going to change in these population groups.

However, we did highlight this aspect of COVID19 even in our original version and also in the revised version.

Reviewer #2 (Public Review):

Weaknesses:

The presentation is not focused. It would be better to include p-values and focus presentation on the main effects from the dataset analysis.

The significant p-values were restricted to public health data only to identify and distinguish differences in incidence, prevalence and mortality and how they differ across world populations. These differences have often been interpreted from socio-economic point of view, while our manuscript presents the reasons for differences for main condition (Stroke) and its comorbid condition among different ethnicities from a genetic perspective. This genetic perspective was further explored to identify unique ethnic specific variants and their patterns of linkage disequilibrium in distinguishing the phenotypic variations. Considering the quantum and diversity of data, both for public health and GWAS data, there can be several directions but for presentation we focused only on the most distinguishing and established phenotypic differences. I am sure this will open up avenues for several future investigations including COVID, as has been highlighted by the reviewers too. All observations were presented with robust and established norms of statistical analysis.

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

Thanks for the constructive observations on strengths and weaknesses of our manuscript. Interestingly, some of the weaknesses mentioned here also turns out to be the strength of the article. For example COVID19 has been mentioned by the reviewer as a driver to increase the mortality in some comorbid conditions and stroke. Firstly, I must clarify that, our data is from PreCOVID era and we indeed mention that in COVID era, COVID-19 might differentially impact the risk of stroke. Possibly this differential influence on the comorbidities of stroke, is likely to be influenced by its underlying genetics of stroke and its comorbidities.

I have tried to address the concerns raised by the reviewers, which ideally doesn’t impact the original manuscript. Statistical limitation has been commented pertaining to P-values, which has been clarified here. However, certain minor concerns such as abbreviations have been resolved in the revised manuscript. My response to weakness and reviewer’s comments are mentioned below.

Reviewer #1 (Public Review):

Strengths:

The data provided here will provide a foundation for a lot of future research into the causes of the observed correlations as well as whether the observed differences in comorbidities across regions have clinically relevant effects on risk management.

Weaknesses:

• As with any cross-national analysis of rates, the data is vulnerable to differences in classification and reporting across jurisdictions.

GBD data is the most robust and most comprehensive data resource which has been used and accepted globally in predicting the health metrics statistics.

GBD data indeed considers normalisations, regarding classification and reporting.

To the best of our knowledge this is the best available resource to consider all health metrics analysis.

• Furthermore, given the increased death rate from COVID-19 associated with many of these comorbid conditions and the long-term effects of COVID-19 infection on vascular health, it is expected that many of the correlations observed in this dataset will shift along with the shifting health of the underlying populations.

I must clarify that we have used data prior to COVID-19.

But yes the patterns after COVID19 will shift due to the impact of covid. This makes the study even more relevant as the comorbid conditions of stroke are also the risk drivers for COVID19 and mortality. This shift has been reported by some authors, which has been discussed in the discussion.

Therefore, understanding the genetic factors underlying stroke and its comorbid conditions might help in resolving how COVID19 might differentially impact on health outcome.

We did highlight this aspect of COVID19 even in our original version.

Introduction 1st para:

“It is the accumulated risk of comorbid conditions that enhances the risk of stroke further. Are these comorbid conditions differentially impacted by socio-economic factors and ethnogeographic factors. This was clearly evident in COVID era, when COVID-19 differentially impacted the risk of stroke, possibly due to its differential influence on the comorbidities of stroke.”

Discussion 3rd para:

“Studies reported reduction in life expectancy in 31 of 37 high-income countries, deduced to be due to COVID-191 . However, it would be unfair to ignore the comorbid conditions which could also be the critical determinants for reduced life expectancy in these countries.”

Recommendations For The Authors:

On page 5, the authors make a note about Africa and the Middle East having the highest ASMR for high SBP and comment about the relative populations of these regions. The populations of the regions are irrelevant to the rate.

Since the study is on comorbid factors of stroke and its impact on mortality therefore, relative burden seems critical. This has been further elaborated here to justify the comment, which indeed is an integral part of the original manuscript.

Paragraph referred – Results section 2:

“Ethno-regional differences in mortality and prevalence of stroke and its major comorbid conditions

We observed interesting patterns of ASMRs of stroke, its subtypes and its major comorbidities across different regions over the years as shown in figure 1a, table 1 and supplementary files S2 & S3. When assessed in terms of ranks, high SBP is the most fatal condition followed by IHD in all regions, except Oceania (OCE) where IHD and high SBP swap ranks. Africa (AFR; 206.2/100000, 95%UI 177.4-234.2) and Middle East (MDE; 198.6/100000, 95%UI 162.8-234.4) have the highest ASMR for high SBP, even though they rank as only the third and sixth most populous continents (fig. S2), respectively.”

On page 17, the authors are alarmed by a large ratio between prevalence rates and mortality rates for certain conditions. This is confusing since this indicates that these conditions are not as dangerous as the other conditions.

This has been further elaborated here to justify the comment, which indeed is an integral part of the original manuscript.

Paragraph referred – Discussion para 1:

“While the global stroke prevalence is nearly 15 times its mortality rate, prevalence of comorbid conditions such as high SBP, high BMI, CKD, T2D are alarmingly 150- to 500-fold higher than their mortality rates. These comorbid conditions can drastically affect the outcome of stroke.”

In Figure 4, the colors are not defined.

In Structure plot colours are assigned as per each K, it doesn’t directly refer to any population. But the plot distinguishes the stratification of populations as per K. Ramasamy, R.K., Ramasamy, S., Bindroo, B.B. et al. STRUCTURE PLOT: a program for drawing elegant STRUCTURE bar plots in user friendly interface. SpringerPlus 3, 431 (2014). https://doi.org/10.1186/2193-1801-3-431

Reviewer #2 (Public Review):

Strengths:

The idea is interesting and the data are compelling. The results are technically solid.

The authors identify specific genetic loci that increase the risk of a stroke and how they differ by region.

Weaknesses:

The presentation is not focused. It would be better to include p-values and focus presentation on the main effects of the dataset analysis.

I presume the comment is made with reference to results with significant p-values.

P-values are mentioned in the main text when referring to significant decrease or increase with respect to global rates and time e.g. P-values for comparison of a year 2019, are based on regional rates to global rates of 2019. Supplementary table S2a (mortality) and S3a (prevalence) e.g. P-values for comparison between year is based on 2019 rates to 2009 rates in Supplementary table S2b (mortality) and S3b (prevalence) e.g. P-values for proportional mortality and proportional prevalence in Supplementary table S4 and S5 is also based on global rates.

Recommendations For The Authors:

It would be better to minimize the use of acronyms. Often one has to go back to decipher what the acronym stands for. It is fine to have acronyms in figure legends, if necessary. However, at least for regions, please do not use acronyms.

In the revised version we have tried to minimise the Acronyms.

Removed the acronyms for regions and other places wherever possible however, the diseases acronyms have been maintained as per the GBD terms.

Please focus the presentation on the main results. Currently, the presentation wanders and repeats itself a lot.

Since the manuscript tries to address the global and regional rates of prevalence, mortality and its relationship to genetic correlates, it is difficult not to repeat the same to stress the significant observations coming out of different analysis methods. This might reflect on some amount of repetitiveness but the intention was to stress the significant observations.

I would also recommend acknowledging and discussing socioeconomic factors earlier in the manuscript.

Current mention happens in 3rd para of Discussion

“The changing dynamics of stroke or its comorbid conditions can be attributed to multitude of factors. Often global burden of stroke has been discussed from the point of view of socio-economic parameters. Studies indicate that half of the stroke-related deaths are attributable to poor management of modifiable risk factors 8,9. However, we observe that different socio-economic regions are driven by different risk factors.”

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eLife assessment

This fundamental study analyzes the roles of post-translational modifications of tubulin by generating a large panel of tubulin mutants and describing their effects on morphogenesis and function of sensory neurons in C. elegans. The work, which is of interest to all cell biologists, in particular researchers with an interest in the microtubule cytoskeleton and neurobiology, presents conclusions that are supported by solid evidence. Demonstrating that all introduced mutations have the intended consequences and exploring their direct effect on microtubules would further increase the impact of the work.

说白了，是利用图片进行分类的

Author response:

Reviewer #1 (Public Review):

Summary and Strengths:

The ability of Wolbachia to be transmitted horizontally during parasitoid wasp infections is supported by phylogenetic data here and elsewhere. Experimental analyses have shown evidence of wasp-to-wasp transmission during coinfection (eg Huigins et al), host to wasp transmission (eg Heath et al), and mechanical ('dirty needle') transmission from host to host (Ahmed et al). To my knowledge this manuscript provides the first experimental evidence of wasp to host transmission. Given the strong phylogenetic pattern of host-parasitoid Wolbachia sharing, this may be of general importance in explaining the distribution of Wolbachia across arthropods. This is of interest as Wolbachia is extremely common in the natural world and influences many aspects of host biology.

Weaknesses:

The first observation of the manuscript is that the Wolbachia strains in hosts are more closely related to those in their parasitoids. This has been reported on multiple occasions before, dating back to the late 1990s. The introduction cites five such papers (the observation is made in other studies too that could be cited) but then dismisses them by stating "However, without quantitative tests, this observation could simply reflect a bias in research focus." As these studies include carefully collected datasets that were analysed appropriately, I felt this claim of novelty was rather strong. It is unclear why downloading every sequence in GenBank avoids any perceived biases, when presumably the authors are reanalysing the data in these papers.

Thank you for bringing this to our attention, and we will make the necessary amendments in our revised manuscript.

I do not doubt the observation that host-parasitoid pairs tend to share related Wolbachia, as it is corroborated by other studies, the effect size is large, and the case study of whitefly is clearcut. It is also novel to do this analysis on such a large dataset. However, the statistical analysis used is incorrect as the observations are pseudo-replicated due to phylogenetic non-independence. When analysing comparative data like this it is essential to correct for the confounding effects of related species tending to be similar due to common ancestry. In this case, it is well-known that this is an issue as it is a repeated observation that related hosts are infected by related Wolbachia. However, the authors treat every pairwise combination of species (nearly a million pairs) as an independent observation. Addressing this issue is made more complex because there are both the host and symbiont trees to consider. The additional analysis in lines 123-124 (including shuffling species pairs) does not explicitly address this issue.

We concur with your observation regarding the non-independence of the data due to phylogenetic relationships. While common phylogenetic correction methods are indeed not directly applicable to wsp distances between species pairs, we are investigating the potential of phylogenetic mixed models to address this issue. We hope to include a revised analysis using this approach in our revised manuscript.

The sharing of Wolbachia between whitefly and their parasitoids is very striking, although this has been reported before (eg the authors recently published a paper entitled "Diversity and Phylogenetic Analyses Reveal Horizontal Transmission of Endosymbionts Between Whiteflies and Their Parasitoids"). In Lines 154-164 it is suggested that from the tree the direction of transfer between host and parasitoid can be inferred from the data. This is not obvious to me given the poor resolution of the tree due to low sequence divergence. There are established statistical approaches to test the direction of trait changes on a tree that could have been used (a common approach is to use the software BEAST).

Thank you for your insightful comments regarding the transfer direction of Wolbachia between whiteflies and their parasitoids. We acknowledge the concern about the resolution of the phylogenetic tree and the inference of the direction of Wolbachia transmission based on the available data. We considered the high infection frequency and obligate nature of Wolbachia in En. formosa, which exhibits a 100% infection rate, as a strong indicator that recent transmission of Wolbachia in this clade likely occurred from En. formosa to B. tabaci. We appreciate your recommendation and will ensure that our conclusions are supported by a more statistically sound approach. As you suggested, we will employ the software BEAST to rigorously test the direction of transmission, and we will revise our statements accordingly.

Reviewer #2 (Public Review):

The paper by Yan et al. aims to provide evidence for horizontal transmission of the intracellular bacterial symbiont Wolbachia from parasitoid wasps to their whitefly hosts. In my opinion, the paper in its current form consists of major flaws.

Weaknesses:

The dogma in the field is that although horizontal transmission events of Wolbachia occur, in most systems they are so rare that the chances of observing them in the lab are very slim.

For the idea of bacteria moving from a parasitoid to its host, the authors have rightfully cited the paper by Hughes, et al. (2001), which presents the main arguments against the possibility of documenting such transmissions. Thus, if the authors want to provide data that contradict the large volume of evidence showing the opposite, they should present a very strong case.

In my opinion, the paper fails to provide such concrete evidence. Moreover, it seems the work presented does not meet the basic scientific standards.

We are grateful for your critical perspective on our work. Nonetheless, we are confident in the credibility of our findings regarding the horizontal transmission of Wolbachia from En. formosa to B. tabaci. Our study has documented this phenomenon through phylogenetic tree analyses, and we have further substantiated our observations with rigorous experiments in both cages and petri dishes. The horizontal transfer of Wolbachia was confirmed via PCR, with the wsp sequences in B. tabaci showing complete concordance with those in En. formosa. Additionally, we utilized FISH, vertical transmission experiments, and phenotypic assays to demonstrate that the transferred Wolbachia could be vertically transmitted and induce significant fitness cost in B. tabaci. All experiments were conducted with strict negative controls and a sufficient number of replicates to ensure reliability, thereby meeting basic scientific standards. The collective evidence we present points to a definitive case of Wolbachia transmission from the parasitoid En. formosa to the whitefly B. tabaci.

My main reservations are:

• I think the distribution pattern of bacteria stained by the probes in the FISH pictures presented in Figure 4 looks very much like Portiera, the primary symbiont found in the bacterium of all whitefly species. In order to make a strong case, the authors need to include Portiera probes along with the Wolbachia ones.

We are very grateful for your critical evaluation regarding the specificity of FISH in our study. We assure the reliability of our FISH results based on several reasons.

1) We implemented rigorous negative controls which exhibited no detectable signal, thereby affirming the specificity of our hybridization. 2) The central region of the whitefly nymphs is a typical oviposition site for En. formosa. Post-parasitism, we observed FISH signals around the introduced parasitoid eggs, distinct from bacteriocyte cells which are rich in endosymbionts including Portiera (FIG 3e-f). This observation supports the high specificity of our FISH method. 3) In the G3 whiteflies, we detected the presence of Wolbachia in bacteriocytes in nymphs and at the posterior end of eggs in adult females (FIG 4). This distribution pattern aligns with previously reported localizations of Wolbachia in B. tabaci (Shi et al., 2016; Skaljac et al., 2013). Furthermore, the distribution of Wolbachia in the whiteflies does indeed exhibit some overlap with that of Portiera (Skaljac et al., 2013; Bing et al., 2014). 4) The primers used in our FISH assays have been widely cited (Heddi et al., 1999) and validated in studies on B. tabaci and other systems (Guo et al., 2018; Hegde et al., 2024; Krafsur et al., 2020; Rasgon et al., 2006; Uribe-Alvarez et al., 2019; Zhao et al., 2013). Taking all these points into consideration, we stand by the reliability of our FISH results.

References:

Bing XL, Xia WQ, Gui JD, Yan GH, Wang XW, Liu SS. 2014. Diversity and evolution of the Wolbachia endosymbionts of Bemisia (Hemiptera: Aleyrodidae) whiteflies. Ecol Evol, 4(13): 2714-37.

Guo, Y, Hoffmann, AA, Xu, XQ, Zhang X, Huang HJ, Ju JF, Gong JT, Hong XY. 2018. Wolbachia-induced apoptosis associated with increased fecundity in Laodelphax striatellus (Hemiptera: Delphacidae). Insect Mol Biol, 27: 796-807.

Heddi A, Grenier AM, Khatchadourian C, Charles H, Nardon P. 1999. Four intracellular genomes direct weevil biology: Nuclear, mitochondrial, principal endosymbiont, and Wolbachia. Proc Natl Acad Sci USA, 96: 6814-6819.

Hegde S, Marriott AE, Pionnier N, Steven A, Bulman C, Gunderson E, et al. 2024. Combinations of the azaquinazoline anti-Wolbachia agent, AWZ1066S, with benzimidazole anthelmintics synergise to mediate sub-seven-day sterilising and curative efficacies in experimental models of filariasis. Front Microbiol, 15: 1346068.

Krafsur AM, Ghosh A, Brelsfoard CL. 2020. Phenotypic response of Wolbachia pipientis in a cell-free medium. Microorganisms, 8: 1060.

Rasgon JL, Gamston, CE, Ren X. 2006. Survival of Wolbachia pipientis in cell-free medium. Appl Environ Microbiol, 72: 6934-6937.

Shi P, He Z, Li S, An X, Lv N, Ghanim M, Cuthbertson AGS, Ren SX, Qiu BL. 2016. Wolbachia has two different localization patterns in whitefly Bemisia tabaci AsiaII7 species. PLoS One, 11: e0162558.

Skaljac M, Zanić K, Hrnčić S, Radonjić S, Perović T, Ghanim M. 2013. Diversity and localization of bacterial symbionts in three whitefly species (Hemiptera: Aleyrodidae) from the east coast of the Adriatic Sea. Bull Entomol Res, 103(1): 48-59.

Uribe-Alvarez C, Chiquete-Félix N, Morales-García L, Bohórquez-Hernández A, Delgado-Buenrostro N L, Vaca L, et al. 2019. Wolbachia pipientis grows in Saccharomyces cerevisiae evoking early death of the host and deregulation of mitochondrial metabolism. MicrobiologyOpen, 8: e00675.

Zhao DX, Zhang XF, Chen DS, Zhang YK, Hong XY, 2013. Wolbachia-host interactions: Host mating patterns affect Wolbachia density dynamics. PLoS One, 8: e66373.

• If I understand the methods correctly, the phylogeny presented in Figure 2a is supposed to be based on a wide search for Wolbachia wsp gene done on the NCBI dataset (p. 348). However, when I checked the origin of some of the sequences used in the tree to show the similarity of Wolbachia between Bemisia tabaci and its parasitoids, I found that most of them were deposited by the authors themselves in the course of the current study (I could not find this mentioned in the text), or originated in a couple of papers that in my opinion should not have been published to begin with.

We appreciate your meticulous examination of the sources for our sequence data. All the sequences included in our phylogenetic analysis were indeed downloaded from the NCBI database as of July 2023. The sequences used to illustrate the similarity of Wolbachia between B. tabaci and its parasitoids include those from our previously published study (Qi et al., 2019), which were sequenced from field samples. Additionally, some sequences were also obtained from other laboratories (Ahmed et al., 2009; Baldo et al., 2006; Van Meer et al., 1999). We acknowledge that in our prior research (Qi et al., 2019), the sequences were directly submitted to NCBI and, regrettably, we did not update the corresponding publication information after the article were published. It is not uncommon for sequences on NCBI, with some never being followed by a published paper (e.g., FJ710487- FJ710511 and JF426137-JF426149), or not having their associated publication details updated post-publication (for instance, sequences MH918776-MH918794 from Qi et al., 2019, and KF017873-KF017878 from Fattah-Hosseini et al., 2018). We recognize that this practice can lead to confusion and apologize for the oversight in our work.

References:

Ahmed MZ, Shatters RG, Ren, SX, Jin GH, Mandour NS, Qiu BL. 2009. Genetic distinctions among the Mediterranean and Chinese populations of Bemisia tabaci Q biotype and their endosymbiont Wolbachia populations. J Appl Entomol, 133: 733-741.

Baldo L, Hotopp JCD, Jolley KA, Bordenstein SR, Biber SA, Choudhury RR, et al. 2006. Multilocus sequence typing system for the endosymbiont Wolbachia pipientis. Appl Environ Microbiol, 72: 7098-110.

Fattah-Hosseini S, Karimi J, Allahyari H. 2014. Molecular characterization of Iranian Encarsia formosa Gahan populations with natural incidence of Wolbachia infection. J Entomol Res Soc, 20: 85–100.

Qi LD, Sun JT, Hong XY, Li YX. 2019. Diversity and phylogenetic analyses reveal horizontal transmission of endosymbionts between whiteflies and their parasitoids. J Econ Entomol, 112(2): 894-905.

Van Meer MM, Witteveldt J, Stouthamer R. 1999. Phylogeny of the arthropod endosymbiont Wolbachia based on the wsp gene. Insect Mol Biol, 8: 399-408.

• The authors fail to discuss or even acknowledge a number of published studies that specifically show no horizontal transmission, such as the one claimed to be detected in the study presented.

Thank you for bringing this to our attention. We will address and discuss the published studies that report no evidence of horizontal transmission, as you've highlighted, in the revised version of our manuscript.

Reviewer #3 (Public Review):

This is a very ordinary research paper. The horizontal of endosymbionts, including Wolbachia, Rickettsia etc. has been reported in detail in the last 10 years, and parasitoid vectored as well as plant vectored horizontal transmission is the mainstream of research. For example, Ahmed et al. 2013 PLoS One, 2015 PLoS Pathogens, Chiel et al. 2014 Enviromental Entomology, Ahmed et al. 2016 BMC Evolution Biology, Qi et al. 2019 JEE, Liu et al. 2023 Frontiers in Cellular and Infection Microbiology, all of these reported the parasitoid vectored horizontal transmission of endosymbiont. While Caspi-Fluger et al. 2012 Proc Roy Soc B, Chrostek et al. 2017 Frontiers in Microbiology, Li et al. 2017 ISME Journal, Li et al. 2017 FEMS, Shi et al. 2024 mBio, all of these reported the plant vectored horizontal transmission of endosymbiont. For the effects of endosymbiont on the biology of the host, Ahmed et al. 2015 PLoS Pathogens explained the effects in detail.

Thank you very much for your insightful comments and for highlighting the relevant literature in the field of horizontal transmission of endosymbionts, including Wolbachia and Rickettsia. After careful consideration of the studies you have mentioned, we believe that our work presents significant novel contributions to the field. 1) Regarding the parasitoid-mediated horizontal transmission of Wolbachia, most of the cited articles, such as Ahmed et al. 2013 in PLoS One and Ahmed et al. 2016 in BMC Evolutionary Biology, propose hypotheses but do not provide definitive evidence. The transmission of Wolbachia within the whitefly cryptic species complex (Ahmed et al. 2013) or between moths and butterflies (Ahmed et al. 2016) could be mediated by parasitoids, plants, or other unknown pathways. 2) Chiel et al. (2014 in Environmental Entomology reported “no evidence for horizontal transmission of Wolbachia between and within trophic levels” in their study system. 3) The literature you mentioned about Rickettsia, rather than Wolbachia, indirectly reflects the relative scarcity of evidence for Wolbachia horizontal transmission. For example, the evidence for plant-mediated transmission of Wolbachia remains isolated, with Li et al. 2017 in The ISME Journal being one of the few reports supporting this mode of transmission. 4) While the effects of endosymbionts on their hosts are not the central focus of our study, the effects of transgenerational Wolbachia on whiteflies are primarily demonstrated to confirm the infection of Wolbachia into whiteflies. Furthermore, the effects we report of Wolbachia on whiteflies are notably different from those reported by Ahmed et al. 2015 in PLoS Pathogens, likely due to different whitefly species and Wolbachia strains. 6) More importantly, our study reveals a mechanism of parasitoid-mediated horizontal transmission of Wolbachia that is distinct from the mechanical transmission suggested by Ahmed et al. 2015 in PLoS Pathogens. Their study implies transmission primarily through host-feeding contamination, without the need for Wolbachia to infect the parasitoid, suggesting host-to-host transmission at the same trophic level. In contrast, our findings demonstrate transmission from parasitoids to hosts through unsuccessful parasitism, which represents cross-trophic level transmission. To our knowledge, this is the first experimental evidence that Wolbachia can be transmitted from parasitoids to hosts. We believe these clarifications and the novel insights provided by our research contribute valuable knowledge to the field.

References:

Ahmed MZ, De Barro PJ, Ren SX, Greeff JM, Qiu BL. 2013. Evidence for horizontal transmission of secondary endosymbionts in the Bemisia tabaci cryptic species complex. PLoS One, 8: e53084.

Ahmed MZ, Li SJ, Xue X, Yin XJ, Ren SX, Jiggins FM, Greeff JM, Qiu BL. 2015. The intracellular bacterium Wolbachia uses parasitoid wasps as phoretic vectors for efficient horizontal transmission. PLoS Pathog, 10: e1004672.

Ahmed MZ, Breinholt JW, Kawahara AY. 2016. Evidence for common horizontal transmission of Wolbachia among butterflies and moths. BMC Evol Biol, 16: 118. doi.org/10.1186/s12862-016-0660-x.

Caspi-Fluger A, Inbar M, Mozes-Daube N, Katzir N, Portnoy V, Belausov E, Hunter MS, Zchori-Fein E. 2012. Horizontal transmission of the insect symbiont Rickettsia is plant-mediated. Proc Biol Sci, 279(1734): 1791-6.

Chiel E, Kelly SE, Harris AM, Gebiola M, Li X, Zchori-Fein E, Hunter MS. 2014. Characteristics, phenotype, and transmission of Wolbachia in the sweet potato whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae), and its parasitoid Eretmocerus sp. nr. emiratus (Hymenoptera: Aphelinidae). Environ Entomol, 43(2): 353-62.

Chrostek E, Pelz-Stelinski K, Hurst GDD, Hughes GL. 2017. Horizontal transmission of intracellular insect symbionts via plants. Front Microbiol, 8: 2237.

Li SJ, Ahmed MZ, Lv N, Shi PQ, Wang XM, Huang JL, Qiu BL. 2017. Plantmediated horizontal transmission of Wolbachia between whiteflies. ISME J, 11: 1019-1028.

Li YH, Ahmed MZ, Li SJ, Lv N, Shi PQ, Chen XS, Qiu BL. 2017. Plant-mediated horizontal transmission of Rickettsia endosymbiont between different whitefly species. FEMS Microbiol Ecol, 93(12). doi: 10.1093/femsec/fix138.

Liu Y, He ZQ, Wen Q, Peng J, Zhou YT, Mandour N, McKenzie CL, Ahmed MZ, Qiu BL. 2023. Parasitoid-mediated horizontal transmission of Rickettsia between whiteflies. Front Cell Infect Microbiol, 12: 1077494. DOI: 10.3389/fcimb.2022.1077494

Qi LD, Sun JT, Hong XY, Li YX. 2019. Diversity and phylogenetic analyses reveal horizontal transmission of endosymbionts between whiteflies and their parasitoids. J Econ Entomol, 112: 894-905.

Shi PQ, Wang L, Chen XY, Wang K, Wu QJ, Turlings TCJ, Zhang PJ, Qiu BL. 2024. Rickettsia transmission from whitefly to plants benefits herbivore insects but is detrimental to fungal and viral pathogens. mBio, 15(3): e0244823.

Weaknesses:

In the current study, the authors downloaded the MLST or wsp genes from a public database and analyzed the data using other methods, and I think the authors may not be familiar with the research progress in the field of insect symbiont transmission, and the current stage of this manuscript lacking sufficient novelty.

We appreciate your critical perspective on our study. However, we respectfully disagree with the viewpoint that our manuscript lacks sufficient novelty.

Résumé de la vidéo [00:00:00][^1^][1] - [00:22:50][^2^][2]:

Cette vidéo est un webinaire intitulé "La participation en pratiques" qui explore l'importance de la participation active des populations dans les politiques publiques, en particulier dans le domaine de la santé et de l'action sociale. Il met en lumière les différentes formes et intensités de participation, ainsi que les défis et les stratégies pour une participation efficace.

Points forts: + [00:00:00][^3^][3] Introduction et bienvenue * Présentation du webinaire et instructions pour les participants * Annonce de l'enregistrement du webinaire pour partage ultérieur + [00:01:03][^4^][4] Allocution de Marie Persiani * Importance de la participation des populations * Reconnaissance des savoirs d'expérience des acteurs et des populations + [00:05:40][^5^][5] Présentation par Émilie Feriel * Méthodologie et contexte de la capitalisation des expériences de participation * Discussion sur l'évolution de la participation dans les politiques publiques + [00:13:18][^6^][6] Présentation de Timothé Decluse * Explication de la capitalisation des expériences et son contexte national * Importance de partager les savoirs issus de l'expérience pratique

Résumé de la vidéo [00:22:52][^1^][1] - [00:46:39][^2^][2] : La vidéo présente un webinaire sur la participation en pratiques, mettant en lumière l'importance de la capitalisation des expériences dans divers projets sociaux et de santé publique. Elle aborde les méthodes de capitalisation, l'engagement des participants et l'impact sur les politiques et stratégies.

Points forts : + [00:22:52][^3^][3] L'objectif de la capitalisation * Sert à la réflexion pédagogique et à l'amélioration des pratiques * Informe sur la participation et soutient la mise en œuvre des politiques * Engage la collaboration avec le domaine de la recherche + [00:25:59][^4^][4] La commission de recrutement participative * Implique les résidents dans le processus de recrutement * Favorise l'autodétermination et la participation active * Permet une évaluation et un retour d'expérience constructifs + [00:35:08][^5^][5] Le projet de jardin partagé * Encourage la cohésion sociale et le bien-être des participants * Intègre la permaculture et la bienveillance dans les activités * Offre des opportunités d'apprentissage et de partage au sein de la communauté

Résumé de la vidéo [00:46:42][^1^][1] - [01:11:52][^2^][2]:

Cette vidéo présente un webinaire sur la participation citoyenne dans divers projets sociaux en France. Elle met en lumière l'importance de l'entraide, de l'expérience partagée et de la gouvernance participative dans le domaine de la santé mentale et de l'insertion sociale.

Points forts: + [00:46:42][^3^][3] Présentation de l'UDAF 52 * Introduction du dispositif participatif "Peridance" * Utilisation de l'expérience vécue comme outil de partage et de rétablissement * Focus sur l'entraide pour les personnes isolées avec des expériences rares en santé mentale + [00:52:05][^4^][4] Gouvernance participative * Changement des codes et normes dans une organisation pyramidale * Prises de décisions collectives et ascendantes * Horizontalité dans la distribution des rôles et l'organisation du travail + [00:56:38][^5^][5] Contrat social multipartite * Renforcement de l'intégration sociale et de la participation citoyenne * Méthode participative et collaborative "Spirale" du Conseil de l'Europe * Focus sur le bien-être individuel et collectif + [01:09:36][^6^][6] Le village de l'insertion * Dispositif expérimental pour les personnes en grande marginalité * Hébergement pérenne dans un environnement à basse exigence * Construction des règles avec les habitants pour favoriser l'insertion sociale

Résumé de la vidéo [01:11:55][^1^][1] - [01:35:36][^2^][2] : La vidéo présente un webinaire sur la participation citoyenne dans le cadre d'un projet de village d'insertion. Elle aborde les méthodes de sélection des résidents, l'établissement d'un conseil de village, et l'importance de l'appropriation des lieux par les habitants. Le projet vise à intégrer des personnes marginalisées dans la communauté en leur offrant un espace de vie adapté et en les impliquant dans la gestion du village.

Points forts : + [01:12:01][^3^][3] Sélection des résidents * Critères basés sur l'adhésion aux structures classiques * Choix de personnes à la rue depuis longtemps * Importance de la projection personnelle dans le village + [01:13:21][^4^][4] Création du conseil de village * Objectif de définir les règles de vie commune * Participation active des futurs habitants * Évolution des règles selon les besoins des résidents + [01:16:08][^5^][5] Appropriation des lieux * Liberté de personnalisation des modules d'habitation * Renommage du village par ses habitants * Objectif de déstigmatisation et d'ouverture sur le quartier + [01:17:02][^6^][6] Activités ouvertes à la ville * Organisation d'événements comme la fête de la musique * Invitation des habitants du quartier à participer * Présentation du village à travers des activités conviviales + [01:22:00][^7^][7] Perspectives d'avenir * Souhait de pérenniser le village * Proposition de solutions alternatives en cas de fermeture * Adaptation aux projets individuels des résidents + [01:26:17][^8^][8] Transition vers une organisation horizontale * Efforts pour intégrer les résidents dans les décisions * Importance du soutien institutionnel pour la participation * Réalisation d'actions concrètes comme le jardin participatif

Résumé de la vidéo [01:35:38][^1^][1] - [01:58:27][^2^][2]:

Cette partie du webinaire aborde les pratiques de participation dans la mise en œuvre de projets, soulignant l'importance de définir les objectifs et les limites de la participation dès le début. Les intervenants discutent des outils formels, de l'importance du soutien institutionnel, des défis de financement, et de l'adaptation de la participation à l'environnement du projet. Ils mettent également en évidence le rôle des compétences professionnelles et des participants, ainsi que les effets positifs de la participation sur les individus et les collectivités.

Points forts: + [01:35:38][^3^][3] Définition des objectifs de participation * Importance de fixer les contours de la participation en amont * Nécessité d'intégrer les participants dans le processus de définition * Établissement des limites de décision pour éviter les désillusions + [01:37:43][^4^][4] Soutien institutionnel et financements * Culture de participation au sein des structures * Sensibilisation des supérieurs et soutien essentiel à la pérennisation * Coûts associés à la participation, souvent sous-estimés + [01:39:59][^5^][5] Adaptation de la participation à l'environnement * Flexibilité nécessaire face à l'imprévisibilité de la participation * Levée des contraintes institutionnelles pour favoriser l'expérimentation * Importance du partenariat et de la coordination pour mobiliser les publics + [01:43:19][^6^][6] Compétences des professionnels et des publics * Posture des professionnels favorisant l'horizontalité des relations * Formation et accompagnement des participants pour renforcer leur engagement * Effets positifs sur le bien-être, l'estime de soi et le sentiment d'appartenance

I agree in principle with the idea "you are given back to you", but I wouldn't call that individuzation, much less becoming OVER-individuated. If anything, I find the effects of platforms today, namely TikTok, only intensifies the problem of "mass society" and the need to fit in even more. I guess I am open to the idea of an individual, through the processes expalined by the author, being strongly encouraged, or even forced, to rely and over-express a specific trait or opinion they find to be binding them to a group. So, in conclusion, we have the problem of mass society preventing us from showing our individualities because of peer pressure and desire to stay connected on social media, while simultaneously overly relying on some part, or parts, of our personality to, yet again, connect and fit in, but this time around in a specific group or subsection of said mass society. I would argue that this syntetic and overexxagerated development of the persona is still a part of the whole depersonalization process, with just the mechanism to get there a bit different.

I was already chuckling at the first sentence, but then came the second one...

While I know this to be true, even if judging just by personal user epxerience, I cannot help but wonder about recent changes in the algorithm, and the fact that we see less and less of our friends pop up on our feeds. For exemple, it happens more and more often now that I find out my friends posted on social media, only when I go specifically to check their personal profiles.

The definition itselfof an echo chamber...

Was not what the previous paragraph meant rather the publicization of our intimate discourse?

I find it interesting that we usally think of Facebook, and social platforms in general, changing how, whilst this article examines the change in why we talk about politics.

typo? を

「最初のループ」の意図が伝わりにくいと思いました。

代案

最初はエイリアンが画面の右端からはみ出るようなループでやってみて、

必ずしもはみ出るとは限らない（1つ手前のエイリアンの後ろの空白が１匹未満のときは２匹分の幅を取ると条件を満たす）ので気になりました。

原文のit would place the final alienの語感だと、「はみ出る可能性があります」でしょうか。

サンプルコードが先に提示されているので「試してみます」はちょっと違和感があります

原文 A first attempt at defining this loop might look like this:

直訳

このループを定義する最初の取り組みはこんな風になるでしょう

代案

最初に思いつくのはこういうループ定義でしょう。

to offset the first alien in the fleet from the left edge of the screen. とあるので左端ではないと思います。

代案

画面の左端からこの幅だけ空けた位置に

訳としては合っていると思いますが、わかりにくいです。

ポイントはwithout overcrowdingですよね。詰め込みすぎてない＝適切な数である、と考えました

代案（ちょっと意訳） ゲーム画面上部をちょうどよい数のエイリアンで埋める必要があります。

最後の2行のコードの

平仮名が多くて読みにくい気がしたので、「この後さらに」または「このあと更に」の方がいいと思います。

原文を読むと「current_x」と「ある最大値(some maximum value)」を比較するという意味のようですが、この訳だと意味を汲み取りにくく感じました。「ある」はなくして「current_x と最大値を比較します。」の方が意味が通りやすい気がします。

他の項目では「。」はないのでトルでよさそうです

A ênfase que tem sido colocada na sustentabilidade, tanto no que respeita ao desenvolvimento da sociedade e da economia, como da educação e do uso das tecnologias digitais, nomeadamente por parte de organismos internacionais de referência, é digna de destaque. Podemos verificar isto mesmo na Agenda 2030 da Organização das Nações Unidas (ONU, 2016), pela definição de 17 objetivos que definem as prioridades e aspirações para o desenvolvimento sustentável a nível global, bem como no Relatório da UNESCO (2022) “Reimaginar nossos futuros juntos. Um novo contrato social para a educação”, onde se apela a um novo contrato social para a educação, fundamentado nos direitos humanos e na ação coletiva para a paz, a justiça e a sustentabilidade. Ainda a OECD (2023), recomenda aos decisores políticos e às instituições educativas o desenvolvimento de uma visão estratégica e coordenação de políticas, visando a integração sustentada das tecnologias digitais na educação.

Grief beyond human

Kaeda

sea and narrator

Former village being transcorporeal

coral colony

Asimov in his fiction was fascinated by the advent of statistics, so let's not read things into it wrt predicting futures. We know that's not what statistics do, and still are committed to the waving of invisible hands like it's the 18th century. Connecting this to memetics is likely a fruitless path.

Journal appeared 1997-2005 http://pcp.vub.ac.be/jom-emit/past.html

You can only output three words in a line

In this comprehensive guide to milk delivery app development, we will explore the different categories and benefits of developing a milk delivery app. We will also explore the key features, complete development process, and tips for successfully launching a SaaS-based Milk Delivery App Solution.

This blog post will discuss the 10 Top Milk Delivery Apps Globally that are transforming and revolutionizing the dairy industry. We will examine their salient characteristics, business strategies, and all-embracing online milk delivery business value propositions.

Keith Richards

https://www.keithrichards.com/

https://www.youtube.com/watch?v=hOZjbLuf04Q&t=9s

https://www.youtube.com/watch?v=c6p7ZnflDMI

https://www.youtube.com/watch?v=rTiMgce-23c

https://www.youtube.com/watch?v=W5WgattbjlY

Fusion Body Art Product Review – Rainbow Cakes

• En édition du magasin, je ne peux pas modifier les infos du magasin (adresse, teléphones, description ...)
• Il manque une indication qu'on se trouve sur une page d'édition
• Je ne vois pas comment publier mes modifications ou les annuler
• je ne peux pas modifier le tri des produits / les réorganiser

Lors de l'ajout d'un produit le style du titre n'est pas correct (pas de noir pur, pas la bonne typo) prix : ce n'est pas un champ "number" description : proposer une description basique en placeholder ex "pain de tradition avec des graines de lin"

Chores?!

...I s2g.

eLife assessment

This study presents a valuable syngeneic zebrafish model for studying glioblastoma and will be of interest to neuro-oncologists and cancer biologists. Using a feasible in vivo model to study the tumour microenvironment, cell/cell interaction, and immunity, the data are compelling, and opens up new lines of inquiries for future investigation on the impact of efferocytosis on tumor progression and cell of origin in this model as well as assessments of drug resistance mechanisms, using inhibitors to MAPK , Akt and/or mTOR pathway.

Reviewer #1 (Public Review):

Summary:

The authors have developed a zebrafish model of glioblastoma and characterized this, with a particular focus on the role of recruited myeloid cells in the tumours. Microglia/macrophages in the tumours are proposed to have an inflammatory phenotype and are engaged in phagocytosis. Knockout of Irf7 and Irf8 genes enhanced tumour initiation. Depleting mature myeloid cell types with chlodronate also enhanced tumour initiation. It is proposed that in early stage tumours, microglia/macrophages have tumour suppressive activity.

Strengths:

The authors have generated a novel glioblastoma model in zebrafish. Two key strengths of the zebrafish model are that early stage tumours can be studied and in vivo visualization can be readily performed. The authors show video of microglia/macrophages adopting the ameboid phenotype in tumours (as is observed in human tumours) and engaging in phagocytosis. Video 1 was very impressive in my opinion and shows the model is a very useful tool to study microglia/macrophage:glioblastoma cell interactions. The irf7/irf8 knockdown and the chlodronate experiments are consistent with a role for mature myeloid cells in suppressing tumour initiation, suggesting that the model may also be very valuable in understanding immune surveillance in glioblastoma initiation.

Weaknesses:

EGFRvIII is mainly associated with the classical subtype, so the mesenchymal subtype might be unexpected here. This could be commented on. Some more histologic characterization of the tumours would be helpful. Are they invasive, do larger tumours show necrosis and microvascular proliferation? This would help with understanding the full potential of the new model. Current thinking in established human glioblastoma is that the M1/M2 designations for macrophages are not relevant, with microglia macrophage populations showing a mixture of pre- and anti-inflammatory features. Ideally there would be a much more detailed characterization of the intratumoral microglia/macrophage population here, as single markers can't be relied upon. Phagocytosis could have antitumour effects through removal of live cancer cells, or could be cancer promoting if apoptotic cancer cells are being rapidly cleared with concomitant activation of an immunosuppressive phenotype in the phagocytes (i.e. efferocytosis). It may be possible to distinguish between these two types of phagocytosis experimentally. Do the irf7/8 and chlodronate experiments distinguish between effects on microglia/macrophages and dendritic cells?

Update: The more detailed description of the tumour histology is very interesting and the authors have addressed my previous concerns nicely.

Reviewer #2 (Public Review):

Summary:

Glioblastoma is a common primary brain cancer, that is difficult to treat and has a low survival rate. The lack of genetically tractable and immunocompetent vertebrate animal model has prevented discovery of new therapeutic targets and limited efforts for screening of pharmaceutical agents for the treatment of the disease. Here Weiss et al., express oncogenic variants frequently observed in human glioblastoma within zebrafish lacking the tumor suppressor TP53 to generate a patient-relevant in vivo model. The authors demonstrate that loss of TP53 and overexpression of EGFR, PI3KCA, and mScarlet (p53EPS) in neural progenitors and radial glia leads to visible fluorescent brain lesions in live zebrafish. The authors performed RNA expression analysis that uncovered a molecular signature consistent with human mesenchymal glioblastoma and identified gene expression patterns associated with inflammation. Live imaging revealed high levels of immune cell infiltration and associations between microglia/macrophages and tumor cells. To define functional roles for regulators of inflammation on specific immune-related responses during tumorigenesis, transient CRISPR/Cas9 gene targeting was used to disrupt interferon regulator factor proteins and showed Inflammation-associated irf7 and irf8 are required to inhibit p53EPS tumor formation. Further, experiments to deplete the macrophages using clodronate liposomes suggest that macrophages contribute to the suppression of tumor engraftment following transplantation. The authors' conclusions are supported by the data and the experiments are thoroughly controlled throughout. Taken together, these results provide new insights into the regulation of glioblastoma initiation and growth by the surrounding microenvironment and provide a novel in vivo platform for the discovery of new molecular mechanisms and testing of therapeutics.

Strengths/Weaknesses:

The authors convincingly show that co-injection of activated human EGFRviii, PI3KCAH1047R, and mScarlet into TP53 null zebrafish promotes formation of fluorescent brain lesions and glioblastoma-like tumor formation. The authors include histological characterization of the tumors, as well as quantifications of p-ERK and p-AKT staining to highlight increased activation of the MAPK/AKT signaling pathways in their tumor model.

The authors use a transplantation assay to further test the tumorigenic potential of dissociated cells from glial-derived tumors in the context of specific manipulations of the tumour microenvironment.

The authors nicely show high levels of immune cell infiltration and associations between microglia/macrophages and tumor cells. Quantification of the emergence of macrophages over time in relation to tumor initiation and growth is provided and supports the observations of tumor suppressive activity of the phagocytes. The authors also attempt to delineate if other leukocyte populations are involved and observe tumor formation without significant infiltration of neutrophils.

The authors provide evidence for key genetic regulators of the local microenvironment, showing increased p53EPS tumor initiation following Ifr7 gene knock-down and loss of irf7 expression in the TME.

Author response:

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

Reviewer #1:

“EGFRvIII is mainly associated with the classical subtype, so the mesenchymal subtype might be unexpected here. This could be commented on.” 

We acknowledge that EGFRvIII is most often associated with the classical subtype of glioblastoma and agree that mesenchymal subtype classification may be unexpected given the use of her4.1:EGFRvIII as a driver in our model. We would like to highlight the fact that our brain tumors do also express certain markers associated with the classical subtype including neural precursor and neural stem cell markers like sox2, ascl1b, and gli2 (Supplementary Fig 4, 5; Supplementary Table 1-3). However, our transcriptomic data was not found to significantly enrich for classical subtype gene expression, compared to normal brains. This could be due to a significant contribution of normal brain tissue to our analyses (bulk tumor burdened brains were harvested for RNA sequencing), as well as the significant contribution of mesenchymal subtype signatures and/or inflammatory gene expression in our brain tumor-positive samples. Because signatures associated with inflammation consist of some of the most highly upregulated genes in our samples, this could potentially dilute out and/or lessen alterative subtype and/or signature gene expression. Importantly, it is now widely appreciated that patient tumors simultaneously consist of heterogenous tumor cells reflecting multiple molecular subtypes (Couturier et al., 2020; Darmanis et al., 2017; Neftel et al., 2019), providing glioblastoma with a high level of phenotypic plasticity. We also demonstrate that the contribution of additional drivers not always present with EGFRvIII in patient glioblastoma enhances primary brain tumors in vivo. This result is consistent with more aggressive glioblastomas seen in patients with EGFRvIII variants and TP53 loss-of-function mutations (Ruano et al., 2009). It will therefore be interesting in the future to consider how single or multiple driver mutations contribute to subtype-specific gene expression in our model, as well as histopathology, relative to patients. We have included some of these discussion points to our revised manuscript.     

“Some more histologic characterization of the tumors would be helpful. Are they invasive, do larger tumors show necrosis and microvascular proliferation? This would help with understanding the full potential of the new model.”

We have updated our manuscript to include more histolopathological characterization and images (Supplementary Fig 2).

“Current thinking in established glioblastoma is that the M1/M2 designations for macrophages are not relevant, with microglia macrophage populations showing a mixture of pre- and anti-inflammatory features. Ideally, there would be a much more detailed characterization of the intratumoral microglia/macrophage population here, as single markers can’t be relied upon.”

We performed additional gene set enrichment analyses (GSEA) using our sequencing datasets and compared p53EPS gene expression to M1/M2 macrophage expression signatures and expression signatures from MCSF-stimulated macrophages at early and late (M2 polarized) time-points. From this analysis, we detected enrichment for markers of both pro- and antiinflammatory features, however, with stronger and significant enrichment for gene expression signatures associated with classical pro-inflammatory M1 macrophages. We have included these GSEA plots and gene set enrichment lists as supplementary materials (Supplementary Fig 6, Supplementary Table 6). We also performed GSEA against a broad curated set of immunologic gene sets (C7: immunologic signature gene sets, Molecular Signatures Database, (Liberzon et al., 2011)) and have included the list of signatures and enrichment scores as a supplementary table (Supplementary Table 6). 

“Phagocytosis could have anti-tumor effects through removal of live cancer cells or could be cancer-promoting if apoptotic cells are being rapidly cleared with concomitant activation of an immunosuppressive phenotype in the phagocytes (ie. efferocytosis).” 

We looked at efferocytosis-associated gene expression in our sequencing dataset (124 “efferocytosis” genes, GeneCards), and while we detected upregulation of certain genes associated with efferocytosis in p53EPS brains, we did not detect significant enrichment for the entire gene set. Furthermore, we did not detect up-regulation of key efferocytosis receptors including Axl and Tyro3 (Supplementary Table 1, 2), compared to normal brains. While efferocytosis may contribute to tumor growth and evolution, this GSEA combined with our functional data supporting an inhibitory role for phagocytes in p53EPS tumor initiation and engraftment following transplantation (Fig 4, Fig 5, Supplementary Fig 7), suggests that efferocytosis is not a major driver of tumor formation in our model. However, how efferocytosis affects tumor progression in our model and/or relapse following therapy will be an interesting feature to explore in the future using temporal manipulations of phagocytes and/or treatments with chemical inhibitors.

Author response image 1.

Gene Set Enrichment Analysis (GSEA) for efferocytosis-associated gene expression (124 “efferocytosis” genes in GeneCards) in tp53EPS tumor brains, compared to normal zebrafish brains.

Normalized enrichment score (NES) and p-value are indicated. 

“Do the irf7/8 and chlodronate experiments distinguish between effects on microglia/macrophages and dendritic cells?”

In addition to microglia/macrophages, the IRF8 transcription factor has been shown to control survival and function of dendritic cells (Sichien et al., 2016). Chlodronate treatments are also used to deplete both macrophages and dendritic cells in vivo. Therefore, we cannot distinguish the effects of these manipulations in our experiments and have updated our manuscript throughout to reflect this.     

Reviewer #2:

“The authors state that oncogenic MAPK/AKT pathway activation drives glial-derived tumor formation. It would be important to include a wild-type or uninjected control for the pERK and pAKT staining shown in Fig1 I-K to aid in the interpretation of these results. Likewise, quantification of the pERK and pAKT staining would be useful to demonstrate the increase over WT, and would also serve to facilitate comparison with the similar staining in the KPG model (Supp Fig 2D).”

We have updated Fig 1 and Supplementary Fig 3D (formerly Fig 2D), to include histology from tumor-free uninjected control animals, as well as quantifications of p-ERK and p-AKT staining to highlight increased MAPK/AKT signaling pathway activation in our tumor model.  

“The authors use a transplantation assay to further test the tumorigenic potential of dissociated cells from glial-derived tumors. Listing the percentage of transplants that generate fluorescent tumor would be helpful to fully interpret these data. Additionally, it was not clear based on the description in the results section that the transplantation assay was an “experimental surrogate” to model the relapse potential of the tumor cell. This is first mentioned in the discussion. The authors may consider adding a sentence for clarity earlier in the manuscript as it helps the reader better understand the logic of the assay.” 

We have clarified in the text the percentage of transplants that generated fluorescent tumor (1625%, n=3 independent screens). This is also represented in Fig 5C,D. We also added text when introducing the transplantation assay, explaining that transplantation is frequently used as an experimental surrogate to assess relapse potential, and that our objective was to assess tumor cell propagation in the context of specific manipulations within the TME.  

“The authors nicely show high levels of immune cell infiltration and associations between microglia/macrophages and tumor cells. However, a quantification of the emergence of macrophages over time in relation to tumor initiation and growth would provide significant support to the observations of tumor suppressive activity of the phagocytes. Along these lines, the inclusion of a statement about when leukocytes emerge during normal development would be informative for those not familiar with the zebrafish model.”

In zebrafish, microglia colonize the neural retina by 48 hpf, and the optic tectum by 84 hpf (Herbomel et al., 2001), prior to when we typically observe lesions in our p53EPS brains. To validate the emergence of microglia prior to tumor formation in p53EPS, we have now used live confocal imaging through the brains of uninjected control and p53EPS injected zebrafish at 5, 7 and 9 dpf. As expected, microglia were present throughout the cephalic region and in the brain at 5 dpf (120 hpf). At this stage, p53EPS injected zebrafish brains displayed mosaic cellular expression of her4.1:mScarlet; however, cells were sparse and diffuse, and no large intensely fluorescent tumor-like clusters were detected at this stage (n=12/12 tumor negative). At 7 dpf, microglia were observed in the brains of control and p53EPS zebrafish; however, at this stage we detected clusters of her4.1:mScarlet+ cells (n=5/9), indicative of tumor formation. Lesions were found to be surrounded and/or infiltrated by mpeg:_EGFP+ microglia. Finally, at 9 dpf _her4.1:mScarlet+ expression became highly specific to tumor lesions, and these lesions were associated with _mpeg:_EGFP+ microglia/macrophages (n=8/8 of tumor-positive zebrafish). These descriptions along with representative images has been added to Figure 3.

“From the data provided in Figure 4G and Supp Fig 7b, the authors suggest that “increased p53EPS tumor initiation following Irf gene knock-down is a consequence of irf7 and irf8 loss-of-function in the TME.” Given the importance of the local microenvironment highlighted in this study, spatial information on the form of in situ hybridization to identify the relevant location of the expression change would be important to support this conclusion.”

We performed fluorescent in situ hybridization (using HCR RNA-FISH, Molecular Instruments) on whole mount control and irf7 CRISPR-injected p53EPG animals (her4.1:EGFRvIII +her4.1:PI3KCAH1047R + her4.1:GFP, GFP was used in this case because of probe availability).

Representative confocal projections through tumors, as well as single optical sections are presented and discussed in Figure 4, highlighting the location of irf7 expression change following gene knock-down. We found significant irf7 signal in and surrounding p53EPS tumors at early stages of tumor formation_. This expression was reduced and/or lost following _irf7 CRISPR gene targeting, consistent with RT-PCR data (Supplementary Fig 7).          

“The authors used neutral red staining that labels lysosomal-rich phagocytes to assess enrichment at the early stages of tumor initiation. The images in Figure 3 panel A should be labeled to denote the uninjected controls to aid in the interpretation of the data. In Supplemental Figure 6, the neutral red staining in the irf8 CRISPR-injected larvae looks to be increased, counter to the quantification. Can the authors comment if the image is perhaps not representative?”

We have updated Figure 3 and Supplementary Figure 6 to aid in the interpretation of our results. In Fig 3A, we used tumor-negative controls from our injected cohorts. This was done to control for exogenous transgene presence and/or over-expression prior to (or in the absence of) malignant transformation. In Supplementary Fig 6, our images are representative, but we have now used unprocessed images with arrowheads to highlight neutral-red positive foci for clarity. In our original manuscript the images contained software generated markers, which could have obscured and/or confused the neutral red staining we were trying the highlight.    

Recommendations For the Authors:

Reviewer #1: 

“The PI 3-kinase does a lot more than just activating mTOR and Akt – I would suggest modifying that sentence in the introduction.”

We have adjusted text in the introduction to reflect the broad role for PI3K signaling.

Reviewer #2:

“In Supplemental Fig 1, it would be helpful for the authors to provide a co-stain, such as DAPI to label all nuclei, which would allow the reader to assess the morphology of the cells in the context of the surrounding tissue.”

We have included brightfield images in Supplementary Fig 1, that together with her4.1:mScarlet fluorescence, should help readers assess tumor location and morphology in the context of surrounding tissue. Tumor cell morphology at high-resolution can be visualized in Fig 3, Movie 1 and Movie 2.

“The authors state that oncogenic MAPK/AKT pathway activation drives glial-derived tumor formation. The authors may consider testing if the addition of an inhibitor of MAPK signaling may prevent or decrease the formation of glial-derived tumors in this context to further support their results.” 

To further assess the role for MAPK activation, we decided to test the effect of 50uM AZD6244 MAPK inhibitor following transplantation of dissociated primary p53EPS cells into syngeneic CG1 strain zebrafish embryos, similar to as previously described (Modzelewska et al., 2016). Following 5 days of drug treatments, we did not detect significant differences in tumor engraftment or in tumor size between DMSO control and AZD6244-treated cohorts, suggesting that MAPK inhibition is not sufficient to prevent p53EPS engraftment and growth in our model. In the future, assessments of on-target drug effects, possible resistance mechanisms, and/or testing MAPK inhibitors in combination with other targeted agents including Akt and/or mTOR inhibitors (Edwards et al., 2006; McNeill et al., 2017; Schreck et al., 2020) will enhance our understanding of potential therapeutic strategies.

Author response image 2.

Dorsal views of 8 dpf zebrafish larvae engrafted with her4.1:mScarlet+ p53EPS tumor cells following treatment from 3-8dpf with 0.1% DMSO (control) or 50uM AZD6244. Tumor cell injections were performed at 2 dpf into syngeneic CG1 strain embryos. The percentage of total animals with persisting engraftment following drug treatments, as well as tumor size (microns squared, quantified using Carl Zeiss ZEN software) are shown for control and AZD6244 treated larvae. 

“Have the authors tested if EGFR and PI3KCA driven by other neural promoters produce similar results, or not? This would help support the specificity of her4.1 neural progenitors and glia as the cell of origin in this model.”

At this time, we have not tested other neural promoters. However, previous reports describe a zebrafish zic4-driven glioblastoma model with mesenchymal-like gene expression (Mayrhofer et al., 2017), supporting neural progenitors as a cell of origin. In the future it will be interesting to test sox2, nestin, and gfap promoters to further define and support her4.1-expressing neural progenitors and glia as the cell of origin in our model.

“Other leukocyte populations, such as neutrophils, can also respond to inflammatory cues. Can the authors comment if neutrophils are also observed in the TME?”

We performed initial assessments of neutrophils in the TME using our expression datasets as well as her4.1:EGFRvIII + her4.1:PI3KCAH1047R co-injection into Tg(mpx:EGFP) strain zebrafish. We observed tumor formation without significant infiltration of mpx:EGFP+ neutrophils. Future investigations will be important to assess differences in the contributions of different myeloidderived lineages in the TME of p53EPS, as well as how heterogeneity may be altered depending on different oncogenic drivers and/or stage of tumor progression, as seen in human glioblastoma (Friedmann-Morvinski and Hambardzumyan, 2023). We have added text in the disscussion section of our manuscript to indicate the possibility of neutrophils and/or other immune cell types contributing to p53EPS tumor biology. 

Author response image 3.

Control-injected tumornegative and tumor-positive Tg(mpx:EGFP) zebrafish at 10 dpf. Tg(mpx:EGFP) strain embryos were injected at the one-cell stage with her4.1:EGFRvIII + her4.1:PI3KCAH1047R + her4.1:mScarlet.

“It is not clear if the transcriptomics data has been deposited in a publicly available database, such as the Gene Expression Omnibus (GEO). Sharing of these data would be a benefit to the field and facilitate use in other studies.”

We have uploaded all transcriptomic data to GEO under accession GSE246295.

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Author response:

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

Reviewer #1 (Recommendations For The Authors):

(1) Original blots in Figures 2E and 2H should be shown as well as the quantification of miR-182-5p overexpression in HepG2 cells. miR-182-5p expression in T2D patients was 2.3-fold higher than ND patients. The lack of insights into the degree of miR-182-5p overexpression precluded proper interpretation of the data presented.

Thank you very much for these comments. We now include the original uncut blots and relevant bands (new supplementary figure 3A) as well as the quantification of miR-182-5p expression in mimic-treated HepG2 cells in the supplement (new supplementary figure 2).

(2) What are the upstream transcriptional regulators of miR-182-5p?

To the best of our knowledge the upstream transcriptional regulators of miR-182-5p are currently unknown.

(3) What's the purpose of the weight cycling cohort? Figure 3A only showed that miR-182-5p expression was highly correlated to body weight, but the cohort can not explain why the human cohort has different miR-182-5p expression. GTT and ITT data are lacking for this cohort and thus cannot demonstrate a causal link between insulin sensitivity and miR-182-5p. The lack of histological evidence cannot show the relationship between NAFLD and miR-182-5p.

The purpose of the weight cycling cohort was to demonstrate that miR-182-5p is dynamically altered and that it can be reversed to almost control levels by weight loss. Thereby we validate in mice that obesity is associated with miR-182-5p upregulation (HFD group without intervention) and we propose that the adverse effects of increased miR-182-5p in obesity might be reversible by weight loss.  We did not perform ITTs and GTTs in this weigh cycling cohort because the HFD-model in C57BL/6 mice is well established and it can be assumed that glucose- and insulin-tolerance deteriorated during HFD feeding (doi.org/10.1038/oby.2007.608; doi:10.1007/978-1-61779-430-8_27 and improved after weight loss (doi:10.1038/s41598-023-40514-w). To corroborate this assumption, we provide plasma insulin along with as other important metabolic marker of the weight cycling model in supplemental figure 5A.

(4) Loss-of-function of miR-182-5p and/or gain-of-function of Lrp6 in vivo or in vitro would clarify the importance of the miR-182-5p-Lrp6 axis and provide more direct evidence for its potential as a therapeutic target.

We absolutely agree with the reviewer that loss of miR-182 and gain of LRP6 function experiments are missing. However, we provide miR-182 gain of function experiments that impressively show increased liver triglycerides after only seven days of miR-182 overexpression. Because these in vivo data are only short-term, we stated our conclusions carefully and point out that we do not have evidence for a direct involvement of miR-182-5p in insulin signaling. We are now planning follow-up studies in which miR-182-5p will be overexpressed and also antagonized for a longer time. However, for the timeframe of this revision process these extensive studies are not feasible and we ask the reviewer for his/her understanding.

(5) The schematic summary is too complex and includes too many assumptions to faithfully represent the data shown in this study.

We agree, the schematic summary is very complex. Therefore we simplified the upper part (new figure 5) and only focused on the clearly regulated genes and main pathways.

Reviewer #2 (Recommendations For The Authors):

(1) Although lots of microarray analyses were performed in this study, the authors didn't systemically investigate the function of miR-182 in T2DM or NAFLD. The current data provided in this manuscript may only support that miR-182 is involved in the homeostasis of glucose or insulin.

We thank the reviewer for this comment and agree that the nature of or data is mostly correlative. We tried to overcome this by performing mechanistic in vitro data. Because overexpression of miR-182-5p decreases inulin signaling in vitro and induces hyperinsulinemia in vivo we still strongly believe that miR-182-5p is highly relevant for the homeostasis of glucose and insulin.

(2) The authors used miRNA mimics to overexpress miR-182 in mice. How to emphasize the target specificity in the liver? Normally, adeno-associated virus 8 (AAV8) is used to specifically target the liver.

Tail vein injections as used in our experimental set-up are known to deliver compounds directly to the liver via the portal vein. For modulation of microRNAs in the liver it is an established technique to deliver mimics (or inhibitors) via the tail vein (doi:10.1007/978-1-62703-435-7_18; doi: 10.1089/10430349950017734). To account for off-target effects we quantified miR-182-5p and target gene expression in spleen and heart. Although miR-182-5p concentrations in mimic treated mice were strongly increased in these tissues, expression in the liver was still highest (new supplementary figure 6A).

(3) The HE and Oil red staining of the mouse liver should be shown in miR-182-5p overexpressing mice compared with the control mice, which could provide a more intuitive view of the fat content in the mouse liver.

Unfortunately the livers were flash frozen and not optimally prepared for later histological analyses. Nevertheless, we performed H&E stainings in all livers and provide representative HE stainings of two control and two miR-182-mimic treated mice (new supplementary figure 5D). The increase hepatic lipid content is clearly visible in the H&E staining of miR-182-mimic treated mice and supports our previous findings of increased hepatic triglycerides (Figure 4H). Due to the freezing process, livers were damaged and Oil red staining was impossible.

(4) After overexpression of miR-182-5p in mice, the serum insulin levels were increased. Does miR-182-5p affect insulin resistance in mice? The insulin tolerance test (ITT) experiment needs to be performed.

We thank the reviewer for this comment. Indeed, the performance of an ITT would have clarified the effects of miR-182 on insulin tolerance best. Because we did not see differences in the GTT after treating mice acutely with the miR-182 mimic we decided to not perform the ITT in this short-term. The increased fasting serum levels after miR-182-5p mimic treatment (Fig. 4G) suggest that rather insulin sensitivity than insulin secretion is disturbed by miR-182-5p. We are aware, that in future experiments mice should be treated for a longer period with miR-182-5p mimics and that an ITT should be performed in these more chronic studies.

(5) In Figure 2H, the author measured the level of p-Akt/Akt to indicate the effect of miR-182-5p on insulin resistance in HepG2 cells. It is best to provide the western blotting results of p-AKT and t-AKT after HepG2 cells are treated with or without insulin.

We now provide the full blots for all western blotting experiments as new supplemental figure 3B. The HepG2 cells were stimulated with 20 nM insulin 10 min before harvest as described in 2.11 and consequently Akt and p-Akt were quantified. We did not analyze Akt and p-Akt without stimulation because Akt is rarely phosphorylated in the basal non-insulin stimulated state.

(6) This study suggests that miR-182-5p may promote insulin resistance and hyperinsulinemia by downregulating LRP6. Nevertheless, to confirm this conclusion, we suggest you transfect miR-182-5p after downregulating the level of LRP6 with its siRNA for further validation.

Because miR-182-5p targets LRP6 as we have validated by luciferase-assays, LRP6 levels are already low after miR-182-5p overexpression. Thus, the additional downregulation of LRP6 by other means (such as siRNAs) does not make sense in our opinion.

(7) The author described that serum miR-182-5p was neither altered in T2D nor correlated with hepatic miR-182-5p expression, so is it suitable as the biomarker of T2D?

Yes, as the reviewer stated correctly, serum concentrations of miR-182-5p were not related to its liver concentrations or the type 2 diabetic state. We therefore suggest that circulating miR-182-5p levels are not a suitable biomarker for T2D. We clarified this in the discussion.

(8) What are the changes in fasting blood glucose levels in HFD, HC, and YoYo mouse models? Is there a correlation between miR-182-5p level and fasting blood glucose level in T2D patients and mouse models?

Unfortunately, we did not measure the fasting blood glucose levels in this mouse model and therefore cannot answer this question. However, we provide the fasting insulin levels of our mouse models and their positive correlations with miR-182-5p (Fig. 3D and Suppl.Fig. 5D). In T2D humans, hepatic miR-182-5p correlates positively with fasting glucose (Fig. 2B).

(9) The capitalization of the letters in "STrengthening the Reporting of OBservational studies in Epidemiology" should be checked. What does the "Among these is miRNAs miR-182-5p" mean? Please clarify it.

The “STrengthening the Reporting of OBservational studies in Epidemiology “ report form is abbreviated as “STROBE” list. We this capitalized the letters that are used to build the abbreviation.

“Among these is miRNAs miR-182-5p” is a typo for which we apologize. It should mean “Among these conserved miRNAs is miR-182-5p.” We corrected this error.

Reviewer #3 (Recommendations For The Authors):

(1) The functional importance of miR-182 on gene expression is not rigorously tested.

(A) Many of the target genes in Fig. 1C and Fig. 3 are controlled by multiple factors that are known to be increased with obesity (e.g., lipogenic genes are increased by hyperinsulinemia), making it likely that their association with miR-182 is correlative rather than a consequence of miR-182 increases.

We thank the reviewer for this comment and agree that miR-182 is not the only factor regulating the here investigated genes. We rather propose, that miR-182 could be an additional upstream regulator that holds the potential to modify entire pathways of insulin signaling and lipogenesis. However, miR-182 should be not viewed as an on/off-switch as it likely plays a modulating role. Although, our in vivo data stemming from humans and mice are correlative we believe that the in vitro data derived in HepG2 cells clearly show a causal role for miR-182-5ß in decreasing LRP6 and insulin signaling, indicated by lower AKT phosphorylation after miR-182-5p overexpression.

(B) 500-fold overexpression of miR-182 does not significantly change gene expression. The authors need to knockdown miR-182 in mice and then feed them a chow versus high-fat diet. If miR-182 is a significant regulator of these genes, the effects of the diet will be blunted.

We thank the reviewer for the constructive criticism and agree that an optimal experiment would be to antagonize miR-182-5p in mice to rescue glucose and lipid metabolism. There here presented in vivo upregulation of miR-182-5p was a proof-of-concept study to confirm our hypothesis in a reasonable timeframe. We are aware, that follow-up studies are needed, and we are now planning studies in which miR-182-5p will be overexpressed and also antagonized for a longer time. However, for the timeframe of this revision process these extensive studies are not feasible and we ask the reviewer for his/her understanding. 

(2) It has previously been shown that miR-182 is in a polycistrionic microRNA locus that is activated directly by SREBP-2. Is this also true in humans? If so, this would indicate that miR-182 is a marker of SREBP activity. How does the nuclear active form of SREBP1 and SREBP2 change in the human livers and HFD-fed mice?

We thank the reviewer for this very interesting question. Suitable experiments to investigate if miR-182-5p is activated by SREBF would be EMSAs or ChIPs. Unfortunately we have only frozen protein lysate of the human livers left in which such experiments cannot be performed. We agree that this should be prioritizes in the future.

(3) Similarly, to test the role of LRP6 in mediating the effects of miR-182, the authors should compare the effects of miR-182 overexpression in the presence and absence of LRP6.

Because miR-182-5p targets LRP6 as we have validated by luciferase-assays, LRP6 levels are already low after miR-182-5p overexpression. Thus, the additional downregulation of LRP6 by other means (such as siRNAs) does not make sense in our opinion.

(4) The methods are a bit confusing. The authors state that "we applied a logistic regression analysis for the 594 mature miRNAs using the NAFLD activity score (NAS) as a cofactor to exclude any bias by hepatic fat content, lobular inflammation, and fibrosis." However, they later showed that miR-182 levels are correlated with NAS. Please clarify.

We excluded NAFLD explicitly as driving factor for the association to T2D by including a surrogate (the NAFLD activity score) as cofactor. It is well known that NAFLD and T2D are indeed likely associated to each other. Since not all our included individuals with T2D have NAFLD and vice versa, a second correlation with NAS revealed also that a high NAS is associated with higher expression of miR-182.

(5) Does two-fold overexpression of miR-182 (which mimics the effects of HFD) have any effect on chow-fed mice?

This is a very interesting question that we unfortunately cannot answer right now. We are planning further mouse studies in which we will include a chow-fed mice as controls.

eLife assessment

Building on on the observation of an increase in miR-182-5p in diabetic patients, the authors investigated the role of miR-182-5p and its target gene LRP6 in dysregulated glucose tolerance and fatty acid metabolism in obese type 2 diabetics. The use of human livers complemented by supporting data in mice and cells are strengths, but the evidence presented remains incomplete. The findings provide valuable insights into the role of miRNAs in the regulation of liver metabolism and insulin sensitivity in individuals with diabetes and fatty liver disease.

Reviewer #1 (Public Review):

Summary:

This study demonstrated a novel exciting link between conserved miRNA-target axis of miR-182-Lrp6 in liver metabolism which causatively contributes to type 2 diabetes and NAFLD in mice and, potentially, humans.

Strengths:

The direct interaction and inhibition of Lrp6 by miR-182 is convincingly shown. The effects of miR-182-5p on insulin sensitivity are also credible for the in vivo and in vitro gain-of-function experiments.

Weaknesses:

However, the DIO cohorts lack key assays for insulin sensitivity such as ITT or insulin-stimulated pAKT, as well as histological evidence to support their claims and strengthen the link between miR-182-5p and T2D or NAFLD. Besides, the lack of loss-of-function experiments limits its aptitude as potential therapeutic target.

Reviewer #2 (Public Review):

Summary:

In this study, Christin Krause et al mapped the hepatic miRNA-transcriptome of type 2 diabetic obese subjects, identified miR-182-5p and its target genes LRP6 as potential drivers of dysregulated glucose tolerance and fatty acid metabolism in obese T2-diabetics.

Strengths:

This study contains some interesting findings and are valuable for the understanding of key regulatory role of miRNAs in the pathogenesis of T2D.

Weaknesses:

The authors didn't systemically investigate the function of miR-182 in T2DM or NAFLD.

Reviewer #3 (Public Review):

Summary:

In this manuscript, Krause and colleagues identify miR-182 as diabetes-associated microRNA: miR-182 is increased in bariatric surgery patients with versus without T2D; miR-182 was the only microRNA associated with three metabolic traits; miR-182 levels were associated with increased body weight in mice under different dietary manipulations; overexpression in Hep-G2 led to a decrease in LRP6; and overexpression in HFD fed mice led to increased insulin and liver TG. The manuscript provides a potentially useful resource of microRNA expression in human livers, though the functional importance of miR-182 remains unclear.

Strengths:

The use of human tissues and good sample sizes is strong.

Weaknesses:

The study remains primarily correlative; the in vivo overexpression is non-physiological; and the mechanisms by which miR-182 exerts its effects are not rigorously tested.

found on 6/19/2024

eLife assessment

This study provides a fundamental advance in palaeontology by reporting the fossils of a new invertebrate, Beretella spinosa, and inferring its relationship with already described species. The analysis placed the newly described species in the earliest branch of moulting invertebrates. The study, supported by convincing fossil observation, hypothesizes that early moulting invertebrate animals were not vermiform.

Reviewer #1 (Public Review):

Summary:

Wang and co-workers characterise the fossil of Beretella spinosa from the early Cambrian, Yanjiahe Formation, South China. Combining morphological analyses with phylogenetic reconstructions, the authors conclude that B. spinosa is closely related to Saccorhytus, an enigmatic fossil recently ascribed to Ecdysozoa, or moulting animals, as an extinct "basal" lineage. Finding additional representatives of the clade Saccorhytida strengthens the idea that there existed a diversity of body plans previously underappreciated in Ecdysozoa, which may have implications for our understanding of the earliest steps in the evolution of this major animal group.

Strengths:

I'm not a paleobiologist; therefore, I cannot give an expert opinion on the descriptions of the fossils. However, the similarities with Saccorhytus seem evident, and the phylogenetic reconstructions are adequate. Evolutionary interpretations are generally justified, and the consolidation of Saccorhytida as the extinct sister lineage to extant Ecdysozoans will have significant implications for our understanding of this major animal clade.

Weaknesses:

While I generally agree with the author's interpretations, the idea of Saccorhytida as a divergent, simplified off-shot is slightly contradictory with a probably non-vermiform ecdysozoan ancestor. The author's analyses do not discard the possibility of a vermiform ecdysozoan ancestor (importantly, Supp Table 4 does not reconstruct that character), and outgroup comparison with Spiralia (and even Deuterostomia for Protostomia as a whole) indicates that a more or less anteroposteriorly elongated (i.e., vermiform) body is likely common and ancestral to all major bilaterian groups, including Ecdysozoa. Indeed, Figure 4 b depicts the potential ancestor as a "worm". The authors argue that the simplification of Saccorhytida from a vermiform ancestor is unlikely "because it would involve considerable anatomical transformations such as the loss of vermiform organization, introvert and pharynx in addition to that of the digestive system". However, their data support the introvert as a specialisation of Scalidophora (Fig. 4a and Supp Table 4), and a pharyngeal structure cannot be ruled out in Saccorhytida. Likewise, loss of an anus is not uncommon in Bilateria. Moreover, this can easily become a semantics discussion (to what extent can an animal be defined as "vermiform"? Where is the limit?). Therefore, I suggest to leave the evolutionary scenario more open. Supporting Saccorhytida as a true group at the early steps of Ecdysozoa evolution is important and demonstrates that animal body plans are more plastic than previously appreciated. However, with the current data, it is unlikely that Saccorhytida represents the ancestral state for Ecdysozoa (as the authors admit), and a vermiform nature is not ruled out (and even likely) in this animal group. Suggesting that the ancestral Ecdysozoan might have been small and meiobenthic is perhaps more interesting and supported by the current data (phylogeny and outgroup comparison with Spiralia).

Reviewer #2 (Public Review):

Summary:

This work provides important anatomical features of a new species from the Lower Cambrian, which helps advance our understanding of the evolutionary origins of animal body plans. The authors interpreted that the new species possessed a bilateral body covered with cuticular polygonal reticulation and a ventral mouth. Based on cladistic analyses using maximum likelihood, Bayesian, and parsimony, the new species was placed, along with Saccorhytus, in a sister-group ("Saccorhytida") of the Ecdysozoa. The phylogenetic position of Saccorhytida suggests a new scenario of the evolutionary origin of the crown ecdysozoan body plan.

Strengths:

Although the new species reported in this paper show strange morphologies, the interpretation of anatomical features was based on detailed observations of multiple fossil specimens, thereby convincing at the moment. Morphological data about fossil taxa in the Ediacaran and Early Cambrian are quite important for our understanding of the evolution of body plans (and origins of phyla) in paleontology and evolutionary developmental biology, and this paper represents a valuable contribution to such research fields.

Weaknesses:

The preservations of the specimens, in particular on the putative ventral side, are not good, and the interpretation of the anatomical features need to be tested with additional specimens in future. The monophyly of Cycloneuralia (Nematoida + Scalidophora) was not necessarily well-supported by cladistic analyses (Supplementary Figures 7-9), and the evolutionary scenario (Fig. 4) also need to be tested in future works. On the other hand, the revised version provides important contributions from currently available data, and the above-mentioned problems should be studied in a separate paper in future.

Author response:

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

Public reviews:

Reviewer 1:

Weaknesses:

While I generally agree with the author's interpretations, the idea of Saccorhytida as a divergent, simplified off-shot is slightly contradictory with a probably non-vermiform ecdysozoan ancestor. The author's analyses do not discard the possibility of a vermiform ecdysozoan ancestor (importantly, Supplementary Table 4 does not reconstruct that character),

Saccorhytids are only known from the early Cambrian and their unique morphology has no equivalent among any extinct or extant ecdysozoan groups. This prompted us to consider them as a possible dead-end evolutionary off-shot. The nature of the last common ancestor of ecdysozoan (i.e. an elongated worm-like or non-vermiform animal with capacities to renew its cuticle by molting) remains hypothetical. At present, palaeontological data do not allow us to resolve this question. The animal in Fig. 4b at the base of the tree is supposed to represent an ancestral soft-bodied form with no cuticle from which ecdysozoan evolved via major innovations (cuticular secretion and ecdysis). Its shape is hypothetical as indicated by a question mark. Our evolutionary model is clearly intended to be tested by further studies and hopefully new fossil discoveries.

…and outgroup comparison with Spiralia (and even Deuterostomia for Protostomia as a whole) indicates that a more or less anteroposteriorly elongated (i.e., vermiform) body is likely common and ancestral to all major bilaterian groups, including Ecdysozoa. Indeed, Figure 4b depicts the potential ancestor as a "worm". The authors argue that the simplification of Saccorhytida from a vermiform ancestor is unlikely "because it would involve considerable anatomical transformations such as the loss of vermiform organization, introvert, and pharynx in addition to that of the digestive system". However, their data support the introvert as a specialisation of Scalidophora (Figure 4a and Supplementary Table 4), and a pharyngeal structure cannot be ruled out in Saccorhytida. Likewise, loss of an anus is not uncommon in Bilateria. Moreover, this can easily become a semantics discussion (to what extent can an animal be defined as "vermiform"? Where is the limit?).

We agree that “worm” and “vermiform” are ill-defined terms. They are widely used in various palaeontological and biological papers to describe elongated tubular animals such as edydsozoans and annelids (see Giribet and Edgecombe 2017; popular textbook written by Nielsen 2012; Schmit-Rhaesa 2013; Brusca et al. 2023; Giribet and Edgecombe 2020). Very few other animals are termed “worms”. Changes have been made in the text to solve this semantic problem, for example in the abstract where we added (i.e elongated and tubular) to better define what we mean by “vermiform”.

Priapulid worms or annelids are examples of extremely elongated, tubular animals. In saccorhytids, the antero-posterior elongation is present (as it is in the vast majority of bilaterians) but extremely reduced, Saccorhytus and Beretella having a sac-like or beret-shape, respectively. That such forms may have derived from elongated, tubular ancestors (e.g. comparable with present-day priapulid worms) would require major anatomical transformations that have no equivalent among modern animals. We agree that further speculation about the nature of these transformations is unnecessary and should be deleted simply because the nature of these ancestors is purely hypothetical. We also agree that the loss of anus and the extreme simplification of the digestive system is common among extant bilaterians. In Figure 4b, the hypothetical pre-ecdysozoan animal is slightly elongated (along its antero-posterior axis) but in no way comparable with a very elongated and cylindrical ecdysozoan worm (e.g. extant or extinct priapulid).

Therefore, I suggest to leave the evolutionary scenario more open. Supporting Saccorhytida as a true group at the early steps of Ecdysozoa evolution is important and demonstrates that animal body plans are more plastic than previously appreciated. However, with the current data, it is unlikely that Saccorhytida represents the ancestral state for Ecdysozoa (as the authors admit), and a vermiform nature is not ruled out (and even likely) in this animal group. Suggesting that the ancestral Ecdysozoan might have been small and meiobenthic is perhaps more interesting and supported by the current data (phylogeny and outgroup comparison with Spiralia).

We agree to leave the evolutionary scenario more open, especially the evolutionary process that gave rise to Saccorhytida. Again, we know nothing about the morphology of the ancestral ecdysozoan (typically the degree of body elongation, whether it had a differentiated introvert or not, whether it had a through gut or not). In Fig.4, the ancestral ecdysozoan is supposed to have evolved from a soft-bodied epibenthic animal through key innovations such as the secretion of a cuticle and ecdysis. It is a hypothesis that needs to be tested by further studies and fossil discoveries. Speculations concerning the process through which saccorhytids may have arisen have been deleted.

Reviewer 2:

Weaknesses:

The preservations of the specimens, in particular on the putative ventral side, are not good, and the interpretation of the anatomical features needs to be tested with additional specimens in the future. The monophyly of Cycloneuralia (Nematoida + Scalidophora) was not necessarily well-supported by cladistic analyses, and the evolutionary scenario (Figure 4) also needs to be tested in future works.

Yes, we agree that the animal described in our manuscrip remains enigmatic (e.g. the natures of its internal organs, its lifestyle, etc..). Whereas the dorsal side of the animal is well documented (consistent pattern of pointed sclerites), uncertainties remain concerning its ventral anatomy (typically the mouth location and shape). Additional better-preserved specimens will hopefully provide the missing information. Concerning Cycloneuralia, their monophyly is generally better supported by analyses based on morphological characters than in molecular phylogenies.

Reviewer 3:

Weaknesses:

I, as a paleontology non-expert, experienced several difficulties in reading the manuscript. This should be taken into consideration when assuming a wide range of readers including non-experts.

We have ensured that the text is comprehensible to biologists. The main results are summarized in relatively simple diagrams (e.g. Fig. 4) that can be understood by non-specialized readers. We are aware that technical descriptive terms may appear obscure to non-specialists. We can hardly avoid them in the descriptive parts. However, our figures (e.g. SEM images and 3D-reconstruction) are clear enough to give the reader a clear idea of the morphology of Beretella.

Recommendations for the authors:

All three reviewers appreciate the discovery and found the merit of publishing this manuscript. They also raised some concerns about the data presentation. The authors are requested to perform no additional analysis but to go through all the reviewer comments and rebut or intake them in revising the manuscript.

Reviewer 1:

- Line 41: comma after "ecdysozans".

OK, done.

- Formatting style: add a space before references.

OK, done.

- Line 169: B. spinosa in italics

OK, done.

- Line 157: could the "relatively large opening" in the flattened ventral side of a mouth (even when altered by the fossilisation process)?

Most bilaterians have a mouth. There is no opening on the relatively well-preserved dorsal side of Beretella, that could be interpreted as a mouth. In contrast the flattened ventral side often show a depressed area that could potentially bear a mouth. This ventral area is often pushed in and poorly preserved. The cuticle of this ventral side might have been relatively thinner, perhaps more flexible than that of the dorsal one (with strong sclerites). These differences might explain why the possible oral area is poorly preserved.

- Line 178: "position of the mouth"

OK, done.

- Line 219: "These sclerites, unknown..."

OK, done.

- Line 282: update reference formatting

OK, done.

- Line 298: remove reference to Supplementary Table 4, as it does not refer to the possible vermiform nature of the last common ecdysozoan ancestor?

OK, done.

- Figure 4a: change "paired legs" for "paired appendages"?

OK, done.

- Supplementary Table 4: For TGE and Introvert, the state 0 (absent) should be in bold and underlined (as it is the most likely state).

OK, done.

Reviewer 2:

Line 25: "from the early Cambrian" should be changed into "from the lower Cambrian"

OK, done.

Line 126: The range of maximum length should be reported in µm (rather than mm) just like those of maximum width and height.

OK, done.

Lines 191-192: Please recheck the figure panels of Saccorhytus (Supplementary Figure 4c) and scalidophoran worm (Supplementary Figure 4d). Perhaps, the former should refer to Figure 4d, and the latter to Figure 4c?

OK, done.

Lines 239 and 241: "1" and "2" appear to stand for citations (the other journal style), but I am not certain what they are.

To avoid confusing, we replace ‘1’ and ‘2’ by ‘i’ and ‘ii’.

Figures 3d and 4a: "Cycloneuralia" should be included in the phylogenetic trees.

OK, done.

Figure 3: The caption for the panel d is redundant. It should be changed into, for example, "Phylogenetic tree obtained from cladistic analyses using maximum likelihood (IQTREE)."

OK, done.

Supplementary Figures 6-9: In the captions, more detailed explanations of the results (for example, "50% majority rule consensus of XXX trees" and "strict consensus of all 4 most-parsimonious trees") should be provided.

OK, done.

Supplementary Figures 8 and 9: The caption explains that Cycloneuralia is resolved as a paraphyletic group, but it is not certain because Nematoida, Scalidophora, and Panarthropoda are resolved in a polytomy.

We changed the sentence into:

“Note that Cycloneuralia does not appear as a monophyletic clade”

Reviewer 3:

Line 25 'tiny' - I suggest giving an absolute measure of the size.

We add ‘maximal length 3 mm’.

Line 29 'both forms' - This is hard to follow by a non-expert. Can this be replaced with 'fossil species'?

OK, done.

Line 32 'dead-end' - Is this word necessary? I suggest to skip this word, as it is obvious that this lineage is extinct.

OK, done.

Lines 80, 94, and 172 'Remarks' - I, as a palaeontology non-expert, cannot get this manuscript structure with a repetition of this same section title.

Our systematic descriptions follow the standard rules in palaeontology.

Line 119 - I could not get what this 'Member 5' that was not introduced earlier means.

In Stratigraphy, ‘member’ is a lithostratigraphic subdivision (a Formation is usually subdivided into several Members).

Lines 104, 105, 417, ... - The name of the organization or database hosting these IDs (CUB.... and ELIXX....) should also be supplied.

OK, done.

Lines 341 and 361 - These two Figures (Figures 1 and 2) have the same caption (with an addition to the one for Figure 1). There should be a distinction based on what is presented in each figure.

We corrected the caption of Figure 2 and wrote the following: ‘Beretella spinosa gen. et sp. nov.’.

Line 362-367 - There is no guide about what the individual figure panels (e.g., Figure 2g, 2h, and 2i) show in detail. This guide should be supplied. This also applies to Figure 3a-c - are they anterolateral (a), dorsal (b), and posterolateral (c) views? It is better to write clearly in this way.

OK, done.

Figure 3d - The color contrast is not sufficient, and this figure does not look reader-friendly. Plus, the division into Cycloneuralia and Panarthropoda is indicated above the tree, but it is not clear what range of lineages these clades include. For example, is Pliciloricidae included in Cycloneuralia? Also, is Collinsium included in Panarthropoda? This figure looks quite unreliable, and it should be easy to fix.

OK, done.

Line 277 legend of Figure 3 - Including the parenthesis only with the program name (IQTREE) is not useful at all. Isn't it enough to describe it in Methods?

OK, done. We remove (IQTREE).

Line 380 legend of Figure 3 - I could not get where 'thicker bars' are.

Known fossil record indicated by thicker vertical bars. We added “vertical”.

Line 453 - Give full names of the methods, maximum parsimony, and maximum-likelihood.

OK, done.

Line 489 - State clearly what 'the recent paper' means.

Replace ‘recent’ by ‘present’.

eLife assessment

The authors report that the neurohormone, bursicon, and its receptor, play a role in regulating aspects of the seasonal polyphenism of the bug, Cacopsylla chinensis. This important study shows that low temperature activates the bursicon signaling pathway during the transition from the summer to the winter form and that it affects cuticle pigment and chitin content, and cuticle thickness. In addition, the authors show that the microRNA miR-6012 targets the bursicon receptor, thereby modulating the function of the bursicon signaling pathway. The study's solid set of experiments and results reveal a role of bursicon signaling in regulating features of polyphenism related to the exoskeleton. Nevertheless, they only incompletely substantiate the authors' claims about the regulation of polyphenism itself.

Joint Public Review:

Summary:

Bursicon is a key hormone regulating cuticle tanning in insects. While the molecular mechanisms of its function are rather well studied--especially in the model insect Drosophila melanogaster, its effects and functions in different tissues are less well understood. Here, the authors show that bursicon and its receptor play a role in regulating aspects of the seasonal polyphenism of Cacopsylla chinensis. They found that low temperature treatment activated the bursicon signaling pathway during the transition from summer form to winter form and affect cuticle pigment and chitin content, and cuticle thickness. In addition, the authors show that miR-6012 targets the bursicon receptor, CcBurs-R, thereby modulating the function of bursicon signaling pathway in the seasonal polyphenism of C. chinensis. This discovery expands our knowledge of the roles of neuropeptide bursicon action in arthropod biology.

However, the study falls short of its claim that it reveals the molecular mechanisms of a seasonal polyphenism. While cuticle tanning is an important part of the pear psyllid polyphenism, it is not the equivalent of it. First, there are other traits that distinguish between the two morphs, such as ovarian diapause (Oldfield, 1970), and the role of bursicon signaling in regulating these aspects of polyphenism were not measured. Thus, the phenotype in pear psyllids, whereby knockdown bursicon reduces cuticle tanning seems to simply demonstrate the phenotypes of Drosophila mutants for bursicon receptor (Loveall and Deitcher, 2010, BMC Dev Biol) in another species (Fig. 2I, 4H). Second, the study fails to address the threshold nature of cuticular tanning in this species, although it is the threshold response (specifically, to temperature and photoperiod) that distinguishes this trait as a part of a polyphenism. Whereas miR-6012 was found to regulate bursicon expression, there no evidence is provided that this microRNA either responds to or initiates a threshold response to temperature. In principle, miR-6012 could regulate bursicon whether or not it is part of a polyphenism. Thus, the impact of this work would be significantly increased if it could distinguish between seasonal changes of the cuticle and a bona fide reflection of polyphenism.

Strengths:

This study convincingly identifies homologs of the genes encoding the bursicon subunits and its receptor, showing an alignment with those of another psyllid as well as more distant species. It also demonstrates that the stage- and tissue-specific levels of bursicon follow the expected patterns, as informed by other insect models, thus validating the identity of these genes in this species. They provide strong evidence that the expression of bursicon and its receptor depend on temperature, thereby showing that this trait is regulated through both parts of the signaling mechanism.

Several parallel measurements of the phenotype were performed to show the effects of this hormone, its receptor, and an upstream regulator (miR-6012), on cuticle deposition and pigmentation (if not polyphenism per se, as claimed). Specifically, chitin staining and TEM of the cuticle qualitatively show difference between controls and knockdowns, and this is supported by some statistical tests of quantitative measurements (although see comments below). Thus, this study provides strong evidence that bursicon and its receptor play an important role in cuticle deposition and pigmentation in this psyllid.

The study identified four miRNAs which might affect bursicon due to sequence motifs. By manipulating levels of synthetic miRNA agonists, the study successfully identified one of them (miR-6012) to cause a cuticle phenotype. Moreover, this miRNA was localized (by FISH) to the cuticle, body-wide. To our knowledge, this is the first demonstrated function for this miRNA, and this study provides a good example of using a gene of known function as an entry point to discovering others influencing a trait. Thus, this finding reveals another level of regulation of cuticle formation in insects.

Weaknesses:

(1) The introduction to this manuscript does not accurately reflect progress in the field of mechanisms underlying polyphenism (e.g., line 60). There are several models for polyphenism that have been used to uncover molecular mechanisms in at least some detail, and this includes seasonal polyphenisms in Hemiptera. Therefore, the justification for this study cannot be predicated on a lack of knowledge, nor is the present study original or unique in this line of research (e.g., as reviewed by Zhang et al. 2019; DOI: 10.1146/annurev-ento-011118-112448). The authors are apparently aware of this, because they even provide other examples (lines 104-108); thus the introduction seems misleading as framed.

(2) The data in Figure 2H show "percent of transition." However, the images in 2I show insects with tanned cuticle (control) vs. those without (knockdown). Yet, based on the description of the Methods provided, there appears to be no distinction between "percent of transition" and "percent with tanning defects". This an important distinction to make if the authors are going to interpret cuticle defects as a defect in the polyphenism. Furthermore, there is no mention of intermediate phenotypes. The data in 2H are binned as either present or absent, and these are the phenotypes shown in 2I. Was the phenotype really an all-or-nothing response? Instead of binning, which masks any quantitative differences in the tanning phenotypes, the authors should objectively quantify the degree of tanning and plot that. This would show if and to what degree intermediate tanning phenotypes occurred, which would test how bursicon affects the threshold response. This comment also applies to the data in Figures 4G and 6G. Since cuticle tanning is present in more insect than just those with seasonal polyphenism, showing how this responds as a threshold is needed to make claims about polyphenism.

(3) This study also does not test the threshold response of cuticle phenotypes to levels of bursicon, its receptor, or miR-6012. Hormone thresholds are the most widespread and, in most systems where polyphenism has been studied, the defining characteristic of a polyphenism (e.g., Nijhout, 2003, Evol Dev). Quantitative (not binned) measurements of a polyphenism marker (e.g., chitin) should be demonstrated to result as a threshold titer (or in the case of the receptor, expression level) to distinguish defects in polyphenism from those of its component trait.

(4) Cuticle issue:<br /> (a) Unlike Fig. 6D and F, Figs. 2D and F do not correspond to each other. Especially the lack and reduction of chitin in ds-a+b! By fluorescence microscopy there is hardly any signal, whereas by TEM there is a decent cuticle. Additionally, the dsGFP control cuticle in 2D is cut obliquely with a thick and a thin chitin layer. This is misleading.<br /> (b) In Figs. 2F and 3F, the endocuticle appears to be missing, a portion of the procuticle that is produced post-molting. As tanning is also occurring post-molting, there seems to be a general problem with cuticle differentiation at this time point. This may be a timing issue. Please clarify.<br /> (c) To provide background information, it would be useful analyze cuticle formation in the summer and winter morphs of controls separately by light and electron microscopy. More baseline data on these two morphs is needed.<br /> (d) For the TEM study, it is not clear whether the same part of the insect's thorax is being sectioned each time, or if that matters. There is not an obvious difference in the number of cuticular layers, but only the relative widths of those layers, so it is difficult to know how comparable those images are. This raises two questions that the authors should clarify. First, is it possible that certain parts of the thoracic cuticle, such as those closer to the intersegmental membrane, are naturally thinner than other parts of the body? Second, is the tanning phenotype based on the thickness or on the number of chitin layers, or both? The data shown later in Figure 4I, J convincingly shows that the biosynthesis pathway for chitin is repressed, but any clarification of what this might mean for deposition of chitin would help to understand the phenotypes reported. Also, more details on how the data in Fig. 2G were collected would be helpful. This also goes for the data in Fig. 4 (bursicon receptor knockdowns).

(5) Tissue issue:<br /> The timed experiments shown in all figures were done in whole animals. However, we know from Drosophila that Bursicon activity is complex in different tissues. There is, thus, the possibility, that the effects detected on different days in whole animals are misleading because different tissues--especially the brain and the epidermis, may respond differentially to the challenge and mask each other's responses. The animal is small, so the extraction from single tissue may be difficult. However, this important issue needs to be addressed.

(6) No specific information is provided regarding the procedure followed for the rescue experiments with burs-α and burs-β (How were they done? Which concentrations were applied? What were the effects?). These important details should appear in the Materials and Methods and the Results sections.

(7) Pigmentation<br /> (a) The protocol used to assess pigmentation needs to be validated. In particular, the following details are needed: Were all pigments extracted? Were pigments modified during extraction? Were the values measured consistent with values obtained, for instance, by light microscopy (which should be done)?<br /> (b) In addition, pigmentation occurs post-molting; thus, the results could reflect indirect actions of bursicon signaling on pigmentation. The levels of expression of downstream pigmentation genes (ebony, lactase, etc) should be measured and compared in molting summer vs. winter morphs.

(8) L236: "while the heterodimer protein of CcBurs α+β could fully rescue the effect of CcBurs-R knockdown on the transition percent (Figure 4G 4H)". This result seems contradictory. If CcBurs-R is the receptor of bursicon, the heterodimer protein of CcBurs α+β should not be able to rescue the effect of CcBurs-R knockdown insects. How can a neuropeptide protein rescue the effect when its receptor is not there! If these results are valid, then the CcBurs-R would not be the (sole) receptor for CcBurs α+β heterodimer. This is a critical issue for this manuscript and needs to be addressed (also in L337 in Discussion).

(9) Fig. 5D needs improvement (the magnification is poor) and further explanation and discussion. mi6012 and CcBurs-R seem to be expressed in complementary tissues--do we see internal tissues also (see problem under point 2)? Again, the magnification is not high enough to understand and appreciate the relationships discussed.

(10) The schematic in Fig. 7 is a useful summary, but there is a part of the logic that is unsupported by the data, specifically in terms of environmental influence on cuticle formation (i.e., plasticity). What is the evidence that lower temperatures influence expression of miR-6012? The study measures its expression over life stages, whether with an agonist or not, over a single temperature. Measuring levels of expression under summer form-inducing temperature is necessary to test the dependence of miR-6012 expression on temperature. Otherwise, this result cannot be interpreted as polyphenism control, but rather the control of a specific trait.

eLife assessment

This paper addresses a question regarding the low overlap between genetic variants linked to human complex diseases and variants linked to differences in gene expression. Some of the analyses supporting the main claims are convincing, and the key conclusions are valuable and of interest to readers in the fields of human genetics and functional genomics. However, chromatin accessibility QTL (caQTL) also carry the limitation of not identifying the genes that directly mediate the influence on disease phenotypes.

Reviewer #1 (Public Review):

Most human traits and common diseases are polygenic, influenced by numerous genetic variants across the genome. These variants are typically non-coding and likely function through gene regulatory mechanisms. To identify their target genes, one strategy is to examine if these variants are also found among genetic variants with detectable effects on gene expression levels, known as eQTLs. Surprisingly, this strategy has had limited success, and most disease variants are not identified as eQTLs, a puzzling observation recently referred to as "missing regulation".

In this work, Jeong and Bulyk aimed to better understand the reasons behind the gap between disease-associated variants and eQTLs. They focused on immune-related diseases and used lymphoblastoid cell lines (LCLs) as a surrogate for the cell types mediating the genetic effects. Their main hypothesis is that some variants without eQTL evidence might be identifiable by studying other molecular intermediates along the path from genotype to phenotype. They specifically focused on variants that affect chromatin accessibility, known as caQTLs, as a potential marker of regulatory activity.

The authors present data analyses supporting this hypothesis: several disease-associated variants are explained by caQTLs but not eQTLs. They further show that although caQTLs and eQTLs likely have largely overlapping underlying genetic variants, some variants are discovered only through one of these mapping strategies. Notably, they demonstrate that eQTL mapping is underpowered for gene-distal variants with small effects on gene expression, whereas caQTL mapping is not dependent on the distance to genes. Additionally, for some disease variants with caQTLs but no corresponding eQTLs in LCLs, they identify eQTLs in other cell types.

Altogether, Jeong and Bulyk convincingly demonstrate that for immune-related diseases, discovering the missing disease-eQTLs requires both larger eQTL studies and a broader range of cell types in expression assays. It remains to be seen what fractions of the missing disease-eQTLs will be discovered with either strategy and whether these results can be extended to other diseases or traits.

It should be noted that the problem of "missing regulation" has been investigated and discussed in several recent papers, notably Umans et al., Trends in Genetics 2021; Connally et al., eLife 2022; Mostafavi et al., Nat. Genet. 2023. The results reported by Jeong and Bulyk are not unexpected in light of this previous work (all of which they cite), but they add valuable empirical evidence that mostly aligns with the model and discussions presented in Mostafavi et al.

Reviewer #2 (Public Review):

Summary:

eQTLs have emerged as a method for interpreting GWAS signals. However, some GWAS signals are difficult to explain with eQTLs. In this paper, the authors demonstrated that caQTLs can explain these signals. This suggests that for GWAS signals to actually lead to disease phenotypes, they must be accessible in the chromatin. This implies that for GWAS signals to translate into disease phenotypes, they need to be accessible within the chromatin.

However, fundamentally, caQTLs, like GWAS, have the limitation of not being able to determine which genes mediate the influence on disease phenotypes. This limitation is consistent with the constraints observed in this study.

(1) For reproducibility, details are necessary in the method section.

- Details about adding YRI samples in ATAC-seq: For example, how many samples are there, and what is used among public data? There is LCL-derived iPSC and differentiated iPSC (cardiomyocytes) data , not LCL itself. How does this differ from LCL, and what is the rationale for including this data despite the differences?

- caQTL is described as having better power than eQTL despite having fewer samples. How does the number of ATAC peaks used in caQTL compare to the number of gene expressions used in eQTL?

- Details about RNA expression data: In the method section, it states that raw data (ERP001942) was accessed, and in data availability, processed data (E-GEUV-1) was used. These need to be consistent.

How many samples were used (the text states 373, but how was it reduced from the original 465, and the total genotype is said to be 493 samples while ATAC has n=100; what are the 20 others?), and it mentions European samples, but does this exclude YRI?

(2) Experimental results determining which TFs might bind to the representative signals of caQTL are required.

(3) It is stated that caQTL is less tissue-specific compared to eQTL; would caQTL performed with ATAC-seq results from different cell types, yield similar results?

Could be used to argue that by supporting civilians that they could avoid harmful livelihoods.

Dim your high-beam headlights to low beams within 500 feat of a vehicle coming toward you or within 300 feet the vehicle you're following

Illegal: driving using only parking lights

Use it: -Too dark to see from 1000 feet away - Beginning 30 minutes after sunset - Until 30 minutes before sunset - Adverse weather- use low beam lights - Conditions such as clouds, dust, smoke or fog -Tunnels and mountain roads - Road signs - Help other see your vehicle

Avoid collisions and alert oncoming traffic on narrow mountain roads where you cant see 200 feet ahead

-100 feet before you turn - Before every lane change - Five seconds before you change lanes on a freeway - Before pulling next to the curb -Almost through the intersection

Signal to turn, change lanes, slow down or stop

Arm pointing to the left is a signal for left turn

Arm pointing up signals right turn

Arm pointing down signals slow or stop

12 o'clock position

When you're turning while backing up

When the operator requires you to remove a hand from the steering wheel

Method when you turn at low speeds, park, or need to recover from a skid

1. 8 and 4 o'clock
2. Grasp the opposite side by reaching across the steering wheel
3. Let go of the steering wheel
4. Reach across the arm still holding the wheel, grip the wheel, and pull up

https://www.thephilosopher1923.org/post/the-new-basics-politics

https://pierrecharbonnier.substack.com/p/la-transition-mission-impossible

Make the Pointing glove equivalent in size to the other hands above. Its too big.

Ensure number doesn't split over 2 lines

In our menu, you'll find these P's celebrated throughout and discover our obsession with pasta, pizza, fresh ingredients, local produce, and the perfect pizza dough. In everything we do, we aim for 'Mwah Perfetto!'

This key needs to be added like sticky navigation with the menu. So it seen where ver you are on the page. New icons have been supplied to cover the various spice levels (Mild-, Spicy and Hot)

Fairs and Festivals in Hiawassee, North Georgia

Линеек нет. Цвет-то для комментариев автоматически появляется? Если тут нет, то никак и не сделать? Ниже в аналогичных (кажется) случаях комментарии выделены цветом.

А тут никакой фрагмент не пропущен? Мне как будто звена не хватает.

Во втором заголовке радоволнами вместо радиоволнами

Una lectura muy interesante, lo que más me llamo la atención es el tema de las fichas en Mesopotamia que representaban bienes y los números, se iban haciendo más complejos a medida que avanzaba la civilización y por ello se dejó de utilizar.

La historia de la escritura es tan impresionante como conocer como ha ido evolucionando con el tiempo y ver la creatividad de los humanos para crear los sistemas de escritura y como fueron evolucionando con el tiempo, es por ello que es fundamental para la comunicación y conocimientos en los seres humanos. Algo que me llamo bastante la atención fue de la escritura cuneiforme, dónde se utiliza el estilete redondeado y se imprimía sobre la arcilla para grabar números y era un método de contabilidad, debido que se grababan los números. Después se utilizó un estilete afilado donde se indicaba que se estaba contando, al final estos modelos de estiletes fueron reemplazados por un estilete que tenía forma de cuña fue ahí que la escritura evolucione de a poco y para beneficio de la humanidad.

La escritura sumeria fue tan útil que otros pueblos de la región, como los acadios, eblaítas, hititas y ugaríticos, la adoptaron para sus propios idiomas. Esto demuestra la importancia y el impacto que tuvo la escritura sumeria en el desarrollo de la comunicación escrita en el antiguo Medio Oriente.

Me parecio muy intersante...Debido a que es muy importante que nosostros como docentres aprendamos el verdadero significado de como surgio la escritura debido a que se sustenta con simbolos y formas de los cuales las personas en la antiguedad se comunicaban.

El texto trata de la encuesta de hábitos lectores fue realizada en el 2021 a personas de 5 años en adelante con la finalidad de indagar y determinar los hábitos, prácticas y consumos culturales de la población.

"area"

physical health - whole foods tour - brenda turner - nutritionist

softap 配网的设备发现方式，通过ssid名称

Includes all no gender left behind

测试

此处需要修改为2018年

High bar to match!

Completely fucking false. Lazy avoidant pussy rhetoric from an ignorant coward

DOI: 10.1016/j.celrep.2021.108899

Resource: (Sigma-Aldrich Cat# I8140, RRID:AB_1163661)

Curator: @Naa003

SciCrunch record: RRID:AB_1163661

What is this?

DOI: 10.1016/j.celrep.2021.108875

Resource: (BDSC Cat# 8760,RRID:BDSC_8760)

Curator: @Naa003

SciCrunch record: RRID:BDSC_8760

What is this?

DOI: 10.1371/journal.pgen.1010289

Resource: (BDSC Cat# 32185,RRID:BDSC_32185)

Curator: @DavidDeutsch

SciCrunch record: RRID:BDSC_32185

What is this?

DOI: 10.1371/journal.pgen.1010289

Resource: BDSC_24464

Curator: @DavidDeutsch

SciCrunch record: RRID:BDSC_24464

What is this?

DOI: 10.1371/journal.pgen.1010289

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @DavidDeutsch

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1083/jcb.202209052

Resource: (BDSC Cat# 7063,RRID:BDSC_7063)

Curator: @DavidDeutsch

SciCrunch record: RRID:BDSC_7063

What is this?

DOI: 10.1083/jcb.202209052

Resource: (BDSC Cat# 7062,RRID:BDSC_7062)

Curator: @DavidDeutsch

SciCrunch record: RRID:BDSC_7062

What is this?

DOI: 10.1083/jcb.202209052

Resource: BDSC_65844

Curator: @DavidDeutsch

SciCrunch record: RRID:BDSC_65844

What is this?

DOI: 10.1083/jcb.202209052

Resource: (BDSC Cat# 6836,RRID:BDSC_6836)

Curator: @DavidDeutsch

SciCrunch record: RRID:BDSC_6836

What is this?

DOI: 10.1083/jcb.202209052

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @DavidDeutsch

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1080/19336934.2022.2149209

Resource: BDSC_86328

Curator: @DavidDeutsch

SciCrunch record: RRID:BDSC_86328

What is this?

DOI: 10.1080/19336934.2022.2149209

Resource: (BDSC Cat# 43642,RRID:BDSC_43642)

Curator: @DavidDeutsch

SciCrunch record: RRID:BDSC_43642

What is this?

DOI: 10.1080/19336934.2022.2149209

Resource: (BDSC Cat# 4517,RRID:BDSC_4517)

Curator: @DavidDeutsch

SciCrunch record: RRID:BDSC_4517

What is this?

DOI: 10.1080/19336934.2022.2149209

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @DavidDeutsch

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1186/s40478-022-01476-8

Resource: RRID:BDSC_53331

Curator: @DavidDeutsch

SciCrunch record: RRID:BDSC_53331

What is this?

DOI: 10.1186/s40478-022-01476-8

Resource: RRID:BDSC_27570

Curator: @DavidDeutsch

SciCrunch record: RRID:BDSC_27570

What is this?

DOI: 10.1186/s40478-022-01476-8

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @DavidDeutsch

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.3389/fimmu.2022.1040510

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @DavidDeutsch

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.3389/fimmu.2022.1040510

Resource: (BDSC Cat# 30142,RRID:BDSC_30142)

Curator: @DavidDeutsch

SciCrunch record: RRID:BDSC_30142

What is this?

DOI: 10.3389/fimmu.2022.1040510

Resource: (BDSC Cat# 55707,RRID:BDSC_55707)

Curator: @DavidDeutsch

SciCrunch record: RRID:BDSC_55707

What is this?