Nos invita a explorar nuestra humanidad desde una perspectiva relacional y afectiva. Somos más que individuos aislados; somos parte de una red de vida en constante movimiento.

El proceso de devenir (más-que) humano es relacional:

Aquí, se sugiere que nuestra humanidad no es un estado estático, sino un proceso en constante evolución. No somos simplemente humanos; estamos en un continuo de transformación. Esta transformación ocurre a través de nuestras relaciones con otros seres y con el entorno.

Csordas nos invita a reconocer nuestra corporeidad activa y a apreciar cómo nuestros cuerpos son más que meros receptáculos pasivos. Somos tejedores de hilos que nos conectan con el mundo y con los demás.

Bautista nos invita a pensar más allá de las divisiones y jerarquías impuestas por la colonización. La humanidad es un proceso compartido y colectivo que se nutre a través de nuestras relaciones y la valoración de la vida de los demás.

Ser humano es hacerse humano: Esta afirmación sugiere que la humanidad no es algo dado o estático, sino un proceso dinámico. Ser humano implica un continuo de transformación y construcción de nuestra identidad. No nacemos completamente humanos; nos convertimos en humanos a lo largo de nuestras vidas.

Es producir una relación: Aquí, Bautista enfatiza la importancia de las relaciones interpersonales. La humanidad se manifiesta en la forma en que nos relacionamos con los demás. Nuestra humanidad se construye a través de la conexión con otros seres humanos.

Reivindicando mi humanidad reivindicando la vida del otro: Esta parte es especialmente poderosa. Bautista nos insta a reconocer nuestra humanidad al mismo tiempo que reconocemos la humanidad de los demás. Al reivindicar la vida del otro, afirmamos nuestra propia humanidad. Esto implica solidaridad, empatía y responsabilidad hacia los demás.

Sintonizar emocionalmente con los sentimientos de otra persona y responder de manera comprensiva y sensible. Va más allá de simplemente entender lo que otros están sintiendo (empatía cognitiva); implica compartir, en cierta medida, las emociones de los demás y mostrar preocupación genuina por su bienestar. En otras palabras, la sintonía afectiva nos permite conectar de manera más profunda con los demás al estar verdaderamente presentes en sus experiencias emocionales.

Nuestra responsabilidad es crear espacios donde las voces se escuchen, las experiencias se validen y la humanidad se celebre. A través de este proceso, contribuimos a una sociedad más empática y comprensiva.

Facilitar la Expresión Creativa: Como investigadores, debemos crear un ambiente propicio para que las personas compartan sus experiencias y testimonios de manera creativa.

Atestiguar Narrativas como Experiencia de Humanización: Atestiguar significa ser testigo de las historias y experiencias de otros.

Ética y Cuidado: Como investigadores, debemos abordar esta responsabilidad con ética y cuidado.

La narrativa testimonial se utiliza para capturar las voces de quienes son testigos y narradores de acontecimientos históricos y políticos.

En América Latina, los testimonios han adquirido una impronta singular. Se centran en la perspectiva del actor, evidenciando el lugar político de su enunciación. Algunos desafíos en torno al uso de la narrativa testimonial en el enfoque cualitativo incluyen debates sobre el giro subjetivo y la fetichización del discurso testimonial.

I feel like the American Dream can be summarized to the idea that working hard in America will make you rich, or at least more than enough money to survive.

Traditional and heterosexual norms set the foundational understanding for many relationship-focused programmes. I think it's interesting to point out how these programs follow a Christian formula of forgiveness and confession. This is important to note so you can better understand how these shows frame the situation within each program and put certain expectations on how things should play out.

DOI: 10.1016/j.celrep.2021.108889

Resource: (R and D Systems Cat# AF3369, RRID:AB_2239879)

Curator: @Naa003

SciCrunch record: RRID:AB_2239879

What is this?

DOI: 10.1016/j.celrep.2021.108883

Resource: (Millipore Cat# MAB1910, RRID:AB_2082643)

Curator: @Naa003

SciCrunch record: RRID:AB_2082643

What is this?

I chose this section because I did not realize I had ADHD till I was an adult. Though my brother got diagnosed as a child, my symptoms were less obvious but are easy to point out in hindsight: I always had missing assignments, I couldn't bring myself to do homework but knew enough to get by, I hyperfocused on books, I was disorganized, and much more. Today, I have a lot of skills and coping strategies to manage my ADHD. This is important because as I go into the workforce, I will need to continue to work in a way that is most effective instead of "right"

Jesse, R., & Griffiths, R. R. (2014). Psilocybin research at Johns Hopkins: A 2014 report. Seeking the sacred with psychoactive substances: Chemical paths to spirituality and to god, 2, 29-43.

This book serves as a foundational component within a Construction Project Management degree program. It is designed to acquaint you with the essential principles of construction project management (CPM), its organizational context, and the multifaceted skill set integral to the field. When organized into modules and curated to offer a coherent pathway we aim to provide a comprehensive yet accessible exploration of CPM fundamentals. Please note that while this book is able to provide a broad overview of various topics we are only offering introductory insights into the profession in this text.

Don't start a sentence with "this book" twice in a row. See sticky note for suggested re-write.

Add punctuation to remain consistent with above numbering.

Welcome to Introduction to Construction Project Management! Here we will delve into the comprehensive life-cycle principles that shape the built environment spanning from inception and design to operational efficiency and eventual decommissioning. While our focus lies on construction and project management facets we aim to foster a holistic understanding of life-cycle concepts. All necessary resources are within this book and no additional materials are required. Join us as we blend expertise and practical insights to equip you to the challenges and opportunities ahead.

Те власти, с которыми мы уже давно сталкиваемся, никуда пока не ушли.

Those powers with which we have long struggled, have not gone anywhere yet.

Чтобы был какой-нибудь шанс на выживание и процветание (вместо застаивания и вымирание), народу пора взять свою судьбу в свои руки

In order to have a chance at survival and flourishing (instead of stagnation and dying out), ordinary people must take their own fate into their own hands

基于Llama3 和Groq 构建的AI新闻应用

收集ai

ds

Pero esto ha empezado a cambiar con la introducción de plataformas como Netflix, Spotify, Steam, etc

Quiénes y por qué obras??

This statement alone summarizes how I believe equity and inclusion should be approached. Now it is very common to see marked "safe spaces" and no judgement zones when in reality that should not be necessary. People should be accepting of others as a baseline and not as an addition to their character. This is important in any field, but I would like to highlight business since you will depend on positive interactions with other people to succeed in your career.

Typo

I like how the article connects long-term outcomes to the motivation behind our behavior. For instance, being intrinsically motivated to do certain tasks comes from a place of interest/passion, rather than a means to an end. I think a big takeaway here is to think about how our internships connect to our long-term professional goals, and whether or not those goals come from a place of passion or personal significance.

this link is broken as of 5-05-2024

I’ve seen it done before!

Very important.

Taking a picture of this!!!!

Sequence and pacing is important ive realized!

Tone is important!

I love trying to incorporate epiphany’s!

This is great!

Vivid description is the only way i like reading books!

I like this description!

Haven’t seen this triangle since middle school!

Rhetorical again.

HCCECR-Going to make this an acronym!

Dont over do it!

Now i get it.

Concrete info!

Never knew this!

Simplicity!!

RE-contextualizng

good to know!

Good to know!

Needed this!

Optional

Writing all of this down

Interested to know what this is!

Thats me!

You've referenced Part II twice. I suspect the first reference in this pair should be to Part I : https://doi.org/10.1002/sim.7993

Didn’t find that out till recently!

Agreed!

I paraphrase everything!!

How do you know its outdated?

Good evidence!

Foundation!

agreed

Great example!!

Writing this down in BOLD

counterarguments can make your argument stronger i’ve realized!

It’s weird how in different subjects a word can mean something completely different.

Hooks are crazy important!

great outline.

I need to search up this word!!

Organizing is so important!

Rhetoric seems to make its way into a lot of topics.

Had no idea that this is what it was called.

I think this is something my paper is missing. I seem to having trouble proposing a solution that works.

Making a note for when i write an argumentative essay

Writing this down!

I am going to do this!

great definition

Very important!

Never knew this.

Great example!

i relate ethos to emotions/morality

Pathos i think pathalogical emotion

logos i think logic

the examples above are very helpful.

Good to know!

Had no idea that these terms are rooted from Aristotle!

This is interesting, because recently I have seen a series of Instagram commercials on Youtube, depicting teenagers (mainly teenage girls). It makes me wonder how Meta is able to justify doing that, while knowing their platform is harmful for that demographic.

making a mental note

never knew that!?

writing this down!!

Great quote!

I am too familiar!!!!

Writing this down right now!!!!

Great example!!!

relate to this, this is brilliant.

This is the perfect definition of rhetoric.

Important, i’ve realized!

making a mental note!!!

Russian: Имя “Колетт”- одно из самых популярных и распространённых женских имён во Франции, как “Маша” в России. В этом случае его можно рассматривать как символ Франции и французской культуры. Фактическая отсылка- французская писательница, журналистка и актриса- мим Сидони- Габриель-Колетт (1873-1954). Происходит же имя от имени Николь- Ника- Богиня Победы. Во французском сознании- “побеждающие," “победа людей”. В свете этого, можно трактовать, как отсылку к людям страдающим от войны и людям, находящимся в заточении в России. Исторически русская и французская культуры тесно связаны и переплетены между собой. Имя Николя и Колетт часто встречается в русской литературе.

English: The name “Colette” is one of the most popular and common female names in France, like “Masha” in Russia. In this case, the name could be interpreted as a symbol of France and French culture. Colette could also be in reference to the French writer, journalist, mime, and actor, Sidonie-Gabrielle Colette (1873 - 1954). Her work highlighted female desires and lives, and she was known to have same-sex relations. The meaning of the name Colette is victorious, or people's victory. The name comes from the name feminine Nicole and further, the masculine, Greek name Nicolas. Additionally, Nike is the goddess of victory in Greek mythology. Perhaps this is in reference to the people suffering from the war and hope for those stuck in Russia. Throughout history, Russian and French culture were always connected somehow. This is intertwined within this name. The names Nicolas and Colette are often found in Russian literature.

https://www.smithsonianmag.com/history/colette-revolutionized-french-literature-with-her-frank-depictions-of-female-desire-180981513/

Russian: Кинжал (в античных и европейских трагедиях) как изящный и простой способ прекратить безумие( для тех , кто не способен его больше выносить). Стилет- узкий нож, используемый для нанесения колотых смертельных ран.

English: The dagger (in ancient and European tragedies) is a simple and elegant/graceful way to stop the madness (for those who can no longer bear it). A stiletto is a slender dagger, often used for stabbing.

Russian: лирическая героиня либо приглашает на собственный концерт, или просто на хороший концерт в новой реальности. Это можно рассматривать как метафору того, что жизнь продолжается.

English: Perhaps the literary hero is referring to her own concert. She is either inviting someone to her concert or to someone else's concert (inviting someone from her past life, people back in Russia, her fans). The concert could symbolize her new life, and that life goes on.

Russian: Может быть, однажды мы вернемся (в Россию)? Призрачная, зыбкая надежда. Далее в припеве музыка становится мрачно- насыщенной и переходит в жутковато- головокружительные звуки кружащейся карусели.

English: Maybe, one day we will return (…to Russia)? As terrible as things are right now, Zemfira still expresses a notion of returning. During this chorus, the music becomes more complex and feels like the dizzying turns of a carousel.

Russian: можно трактовать как принятие героем факта, что обратной дороги нет, возвращение невозможно, и поэтому больше уже не важно.

English: Perhaps Zemfira has let go of this yearning to return to Russia. There seems to be no hope, and so she accepting it.

Russian: Земфира курит десятилетиями. Она уже прожила долгую жизнь, выкурила бесконечное количество сигарет. Теперь ей надо начинать новую жизнь в новой стране.

English: Zemfira has had a smoking habit for decades. She has already lived for so long, smoked so many cigarettes. And yet, here she is in France, starting a new chapter; a new life in a new country.

Russian: Колетт юная девушка. Также как лирический герой Земфиры, которая только начинает её жизнь во Франции.

English: Colette is a young girl. Perhaps the literary hero of Zemfira is opening a new chapter in France; it is also young and just its beginning.

Russian: букет- символ симпатии

English: The bouquet is a symbol of sympathy and understanding.

Russian: Колетт- юная, светлая, полная надежд.

English: Colette is very young, innocent, and light.

Russian: Этот персонаж (имя) часто мелькает в таблоидах и жёлтой прессе.

**English: ** This figure/popular name is always written about in the papers/tabloids.

Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

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Reply to the reviewers

Reviewer #1 (Evidence, reproducibility and clarity (Required)):

Summary: In this paper, Dresselhaus et al (2023) investigate the possibility that known cargoes of extracellular vesicles (EVs) released at the Drosophila neuromuscular junction have cell-autonomous functions rather than functions specifically conferred as a condition of their release in EVs, in vivo. To do so, authors focus their studies on use of Tsg101-KD, a mutant of the ESCRT-I machinery, of the ESCRT EV biogenesis pathway, and are able to show that for some endogenously-expressed, fluorescently-tagged cargoes, fluorescence intensity in the pre-synaptic compartment is significantly elevated (Syt4 and Evi) and the postsynaptic intensity in the muscle is significantly decreased (Syt4, Evi, APP, and Nrg).

We note that throughout our study, we detected endogenous Nrg with a well-characterized monoclonal antibody, not a fluorescent tag. We and others previously demonstrated that endogenous Nrg detected by this antibody is trafficked from neurons into EVs, using the same pathways as other EV cargoes such as Syt4, APP and Evi (Blanchette et al., 2022; Enneking et al., 2013; Walsh et al., 2021). Thus, the EV trafficking phenotypes in our study are consistent across fluorescently tagged cargo (endogenous knockin for Syt4 and GAL4/UAS-driven for APP and Evi), as well as for untagged, endogenous Nrg, thus controlling for effects of either overexpression or tagging.

These findings suggest that these cargoes become trapped in the endosomal system (colocalizing with early, late, and recycling endosomal compartments), rather than undergoing secretion in EVs targeting post-synaptic muscle and glia as usual. This phenotype is recapitulated for select cargoes using mutants of both early and late components of ESCRT pathway machinery. They further characterize the Tsg101 mutant, demonstrating co-occurrence of an autophagic flux defect, but as the cargo phenotype is present without induction of the autophagic flux defect for their Hrs mutants, authors suggest the overlapping role of Tsg101 in autophagy is independent of its role in the ESCRT pathway/ EV secretion. Subsequently, they use previously defined functional phenotypes of the Evi (number of active zones, number of boutons, number of developmentally-arrested ghost boutons) and Syt-4 (number of transient ghost boutons and mEJPs) cargoes to show a minimal dependence on cargo delivery via ESCRT-derived EVs for these cargoes to carry out their synaptic growth and plasticity functions in vivo. However, it should be notes that for Evi/ Wg cargo, there is a slight increase in developmentally-arrested ghost boutons suggesting the cargo may not be entirely independent of EV-mediated cargo delivery. Finally, authors express an anti-GFP proteasome-directed nanobody using motor neuron or muscle-specific drivers and find that Syt4-GFP cargo doesn't enter muscle cytoplasm as fluorescence is maintained and cargo is not degraded by the muscle proteasome. While authors suggest this as evidence of EV-mediated transfer for cargo proteostasis, it is not explicitly shown that Syt4 cargo is, in fact, trafficked and degraded by the lysosome or hypothesized how Syt4 function or post-synaptic localization may be carried out independently of EVs.

We have added new data showing that Syt4 is taken up by glial and muscle phagocytosis (Fig. 7), and included in the discussion several possible interpretations for how Syt4 activity is carried out independently of its traffic into EVs. Indeed we believe it is more likely to function in the presynaptic neuron rather than the postsynaptic muscle.

Major comments:

R1.1 It is difficult to evaluate the findings of this study without knowing the extent of ESCRT pathway impairment. Please provide data quantifying the degree of knockdown/ mutant expression for each ESCRT component (i.e., western blot)

To address the reviewer’s request to specifically measure the degree of knockdown in the RNAi lines, we tested all available reagents. Unfortunately no Drosophila Tsg101 antibody exists and we did not receive a reply to our requests for a Shrub antibody. An Hrs antibody exists, but we found that none of three available Hrs RNAi lines depleted Hrs signal, or caused a phenotype similar to the HrsD28 point mutant, suggesting that they are not effective at knocking down the protein. Therefore, we were unable to specifically measure the level of depletion in motor neurons for RNAi of Tsg101, Shrub, or Hrs.

However, we can make a strong argument that our knockdowns were sufficiently effective to answer the questions in our study. We used RNAi as only one of several complementary tools to manipulate ESCRT function (i.e. we also used loss-of-function mutants (HrsD28/Deficiency) and dominant negative mutants (Vps4DN)). These mutants caused a comparable and severe loss of EVs to RNAi (Fig 2): therefore the extent of depletion in the RNAi experiments was sufficient to cause a similarly severe phenotype as genomic or DN mutations, meeting the definition of a bona fide loss-of-function. We also know, since we used these complementary strategies, that the phenotypes we observe are very unlikely to be due to off-target effects of the RNAi.

More importantly, what is directly relevant for our subsequent functional experiments is to know the extent of EV depletion, which we have explicitly measured throughout the paper. It is unclear what additional insights would be gained by knowing whether the strong Tsg101 and Shrub RNAi phenotypes are due to incomplete versus complete knockdown, given that we do measure the extent of EV depletion under these conditions. Further, we note that tsg101 null mutants die as first instar larvae (Moberg et al., 2005), raising the possibility that a more complete knockdown in neurons would be lethal early in development and make our study impossible. Indeed HrsD28 is an early stop that preserves the VHS and FYVE domains but truncates the C-terminal ⅔ of the protein. Its (occasional) survival to third instar indicates that it may be a severe hypomorph rather than a null.

We have added a sentence in the text (p12 line 21-25) to clarify that we do not know the exact extent of knockdown for our RNAi experiments, but that by genetic definitions, they meet the criteria of a loss-of-function manipulation.

R1.2 Loss of ESCRT machinery likely disrupts the release of small EVs to a significant extent; however, the authors do not show that EV release is entirely lost, only that 1) cargoes are backed up in the endosomal system due to endosomal dysfunction and 2) fluorescence of cargoes in the postsynaptic compartment is diminished. To claim that ESCRT-derived EVs with the relevant cargoes are lost, the authors should perform immunogold labelling with TEM. This would provide direct evidence that the cargoes examined here are packaged in ILVs, and that the ILVs are of a size (~50-150nm) consistent with exosomes (which should really be referred to as small extracellular vesicles (sEVs) per the minimal information for studies of extracellular vesicles (MISEV 2018 [https://doi.org/10.1080/20013078.2018.1535750]) Additionally, EM would show the loss of cargo packaging and provide information about where these cargoes localize in the presence of ESCRT mutants/loss-of-function.

EM (including some limited immunoEM) studies requested by Reviewer 1 have previously been performed in this system by us and by the Budnik and Verstreken labs (Koles et al., 2012; Korkut et al., 2009; Korkut et al., 2013; Lauwers et al., 2018; Walsh et al., 2021). MVBs at the NMJ contain ~50-100 nm ILVs, and can often be seen proximal to or fusing with the plasma membrane. Mutants such as Hsp90 that block this fusion also block EV release, arguing that these MVBs are the source of EV (Lauwers et al., 2018). By immunoEM, the EV cargo Evi localizes to MVBs (Koles et al., 2012). ~50-200 nm structures containing immunogold against Evi were also observed in the subsynaptic reticulum between the neuron and the muscle, as well as in membrane compartments in the muscle cytoplasm (Koles et al., 2012; Korkut et al., 2009). Thus, the criteria requested by the reviewer have previously been established in this system.

In response to the reviewer’s request to show that these structures are altered in ESCRT mutants, we attempted immunoEM experiments in the Tsg101KD condition. However, similar to the previously published results (Koles et al., 2012; Korkut et al., 2009), immunoEM in thick tissue such as Drosophila larval fillets is quite challenging, and we found it very difficult to retain immunogenicity together with excellent fixation and preservation of membrane structures, such that we could rigorously measure compartment morphology and size. Even if we did achieve good structural preservation, exosomes are ambiguous in complex membrane-rich tissues, since cross-sections through the extensively infolded muscle membrane (e.g. see Fig 3B) are very similar in size to EVs.

As an alternative and more robust approach, we used STED microscopy, with a resolution of ~50nm, where we could conduct a rigorous and properly powered study of directly labeled EV cargoes (New data in Fig. S1). We show that postsynaptic Nrg and APP-GFP are found in structures with a mean diameter of ~125 nm, consistent with small EVs or exosomes, and these are strongly depleted in the Tsg101KD animals (to similar levels as antibody background far from the site of EV accumulation), as expected. Note that we are able to detect particles significantly smaller than 125 nm in the distribution, suggesting that the resolution of our system is sufficient to measure EV width.

We also note that several of these cargoes are detected via an intracellular tag (Syt4, APP, Evi) or antibody against an intracellular domain (Nrg), so by topology they must be membrane-bound in the EVs rather than cleaved from the cell surface. We and others have previously shown that this postsynaptic signal is entirely derived from the presynaptic neuron, by using neuronal UAS-expression of a tagged protein, by neuronal RNAi of the endogenous gene, or by the tissue-specific tagging approach in the current manuscript (Fig. S4). We have also previously shown that these puncta contain the tetraspanin Sunglasses (CG12143/Tsp42Ej), which is an EV marker (Walsh et al., 2021). We have added new data to our manuscript (Fig. S1A) to show that neuronally-derived tetraspanin EVs are depleted in upon Tsg101KD. Therefore, the reviewer’s point “2) fluorescence of cargoes in the postsynaptic compartment is diminished.” is the most direct and sensitive test of trans-synaptic cargo transfer, and is the precise parameter that we are trying to manipulate to test the functions of this transfer.

We believe that light microscopy showing loss of presynaptically-derived cargoes in the postsynaptic region is the best and most direct argument for loss of EV secretion, compared to the ambiguity of EM. It is also exactly the method that led to the proposal for the signaling function of EVs in previous work, which our current manuscript is revisiting. We are now using improved tests of that original hypothesis by examining it in light of additional membrane trafficking mutants (and finding that it no longer holds up). Overall, given the preponderance of evidence from the preceding literature and our studies indicating that (1) these cargoes are indeed in EVs and (2) we see a strong enough depletion of transsynaptic transfer to challenge the hypothesis that EVs serve signaling functions (see R1.3 response below), we are reluctant to spend more time attempting immunoEM which is not likely to resolve membrane structures.

To address the point of EV terminology used in our manuscript, we think it is very unlikely that the postsynaptic structures are not exosomes. The criteria defined by MISEV for exosomes is that they are endosomally-derived from MVBs, ideally with the EV “caught in the act of release” upon fusion with the plasma membrane. As noted above, cargoes such as Syt4 and Evi are observed by immunoEM in MVBs, and these can be found in the process of fusing with the plasma membrane (i.e. caught in the act of release) (Koles et al., 2012; Korkut et al., 2009; Korkut et al., 2013; Lauwers et al., 2018). Mutants that block MVB fusion also block EV release at the NMJ (Lauwers et al., 2018). These EVs require ESCRT for their formation and are trapped in endosomes rather than the plasma membrane upon ESCRT depletion (this study). They depend on multiple components of the endosomal system (Rab GTPases, retromer) for their formation (Koles et al., 2012; Walsh et al., 2021). Taken together, it seems to us that there is sufficient data to argue that these are exosomes. However, as the reviewers requested, we have called them EVs in the revised paper (and only suggest they are exosomes in the discussion).

R1.3 Other biogenesis pathways utilize multivesicular bodies to generate EVs, most prominently the nSMase2/ceramide synthesis pathway (which operates in an ESCRT-independent manner). It is possible that this pathway compensates when there are defects in the canonical ESCRT pathway. Thus, it is imperative for the authors to show that the cargo secretion no longer occurs in the presence of ESCRT mutations/loss-of-function. The authors should also use nSMase2 pathway mutants to see if the phenotypes in cargo trafficking (i.e., pre/ post-synaptic protein levels) are recapitulated.

The reviewer asked us to show that cargo secretion does not occur in the ESCRT mutants. We reiterate that at the limits of detection of our assay, we see a very strong depletion of secretion__, and that EV cargo levels are not distinguishable from background (__Figure S1). Perhaps Reviewer 1’s concern is that since it would never be possible to show that we have depleted EVs completely (i.e. below the level of detection of our assays), that it is not possible to challenge the hypothesis that EV traffic is required for the proposed signaling functions of EVs. Indeed, they mention in their overall assessment “as it is unknown if minor sources of cargo+ EVs are sufficient in maintaining functional phenotype”. We do have some information on this, as described in the manuscript (p3 lines 41-43; p7 lines 25-31; p11 lines 27-30) and as follows: The critical argument against this concern is that other trafficking mutants with residual levels of EVs (rab11 or nwk) do show loss of signaling function (Blanchette et al., 2022; Korkut et al., 2013). Therefore residual EVs, even at the lower level of detection of our assay, are not enough to support signaling. The main difference is that in nwk and rab11 mutants the levels of the cargo in the donor presynaptic neuron are also strongly depleted, unlike in the ESCRT mutants. This strongly suggests that the cargoes are signaling from the presynaptic compartment, rather than in EVs. We have added the nwk mutant to show this baseline in Figure 2A,D. Similarly, our new results showing that hrs mutants retain Wg signaling while Tsg101 mutants do not, despite a similar degree of EV depletion (new data with more cargoes in Figure 2A-F), argues that residual EVs do not account for the lack of disruption of signaling. Finally, we have been transparent in our discussion that trace amounts of EVs could still exist, including by alternative pathways, but are unlikely to provide function (p11 lines 25-33).

We agree that it might be an interesting future mechanistic direction to ask if the SMase pathway works with or in parallel to the ESCRT pathway (both have been suggested in the literature). However, we do not believe that this is essential for the current work: The SMase pathway is unlikely to be “compensating”, since EVs are already very strongly depleted with ESCRT disruption alone. We also note that SMase depletion may also affect other trafficking pathways (Back et al., 2018; Choezom and Gross, 2022; Niekamp et al., 2022), and therefore might not provide any clarifying information if it did disrupt signaling. In summary, we believe the depletion we see in single ESCRT mutants is sufficient to (1) establish the role of ESCRT in EV traffic in this system, and (2) test the role of transsynaptic transfer in signaling functions of cargoes.

R1.4 The authors' findings support that cargo trafficking is affected by widespread endosomal dysfunction but doesn't cleanly prove that 1) synaptic sEV release is lost and 2) that cargo-specific sEVs are lost. As previously mentioned, loss of cargo+ ILVs in MVEs by TEM could demonstrate this, but another useful approach would be to include in vitro Drosophila primary neuronal culture/ EV isolation and mass spec/proteomic characterization studies as proof of concept. According to widely agreed upon guidelines in the EV field, the authors should directly characterize their EV population to show 1) the appropriate size distribution associated with exosomes/sEVs, 2) the presence of traditional EV markers (i.e., tetraspanins), 3) changes in overall EV count by ESCRT mutants, and 4) decreased levels of cargo(es) of interest in the presence of ESCRT mutants/loss-of-function. In vitro experiments would be particularly helpful for quantifying the degree of loss of cargo-specific EVs with each ESCRT mutant. These experiments could also investigate the possibility that cargoes are secreted in nSMase2/ Ceramide-derived EVs, by showing that EV cargo levels are unaffected in nSMase mutants.

Our data already show loss of cargo-specific EVs, defined by puncta of several independent specific cargoes in the extraneuronal space and postsynaptic muscle. To further substantiate this, we have directly characterized our EV population and shown a distribution of ~125 nm extraneuronal structures containing the transmembrane cargoes Nrg and APP (by STED) as well as Evi, Syt4 and the EV marker tetraspanin (by confocal microscopy). This addresses the (1) size distribution, (2) EV marker and (3) count criteria. All these markers (cargoes and tetraspanins) are severely depleted from the postsynaptic area in the ESCRT mutants, satisfying the (4) decreased levels criteria. As noted above, we and others have repeatedly demonstrated that these postsynaptic puncta are derived from neurons, and since we are detecting the intracellular domain in all cases, must be membrane-bound. Others have previously shown by EM that several of these markers are surrounded by membrane and derived from neuronal MVBs (see R1.2). Note that we do not believe that ESCRT mutants must necessarily cleanly show enlarged endosomes without ILVs or a class E vps compartment - instead stalled endosomes appear to be targeted for autophagy in heterogeneous intermediates (Fig 3).

We do not believe that turning to a heterologous system (e.g. cultured primary Drosophila neurons, which do not even form functional synapses) is usefully translatable to results in neurons in vivo. Data from our lab and many other systems has shown that EV biogenesis and release pathways are highly cell-type specific (p9 lines 8-12), and also differ in different regions of neurons (eg synapses vs soma) (Blanchette and Rodal, 2020). Further, keeping the experimental setup of the original for EV signaling hypothesis is a prerequisite for our improved tests of this hypothesis. We do note that APP, Evi and Syt4 have been demonstrated by us and others to be released from Drosophila S2 cells in EVs defined by differential centrifugation, sucrose gradient buoyancy, electron microscopy and mass spectrometry (Koles et al., 2012; Korkut et al., 2009; Korkut et al., 2013; Walsh et al., 2021). However even if we did measure the precise change in EV number and cargoes upon ESCRT manipulation in these heterologous cells, it would not allow us to conclude that the same quantitative change was happening in the motor neurons of interest in vivo, which is the information we need to conduct our tests of cargo signaling function. All we would learn is whether ESCRT was required in that cell type, which would not be informative for our study.

We appreciate that EV researchers working in cell culture systems often use a set of approaches including bulk isolation, EM, and mass spectrometry. Our system does not allow for these approaches, but provides complementary strengths of single EV characterization, in vivo relevance with functional assays, and a wealth of genetic tools. MISEV itself states that it does not provide a set of agreed-upon rules that can be applied generically to any experiment. We agree with the MISEV statement that we should use the best available assays for the system under investigation.

R1.5 During functional tests of Evi+ motor neurons lacking generation of Evi+ EVs, there is a slight defect observed, namely the increased formation of developmentally arrested ghost boutons when Evi secretion in sEVs is lost. As mentioned, Evi is a transporter of Wg and it is possible for Wg to be transmitted between cells via normal diffusion. Thus, some basal levels of Wg may be reaching the muscle when its transfer via sEVs is abolished, and these basal levels may be sufficient to phenocopy the WT in the number of active zones and boutons. Is it possible that this element of Evi/ Wg function is dose-dependent and thus reliant on the extra Evi/ Wg transferred via sEVs? If possible, the authors should use a Wnt-signaling pathway reporter (i.e., fluorescently tagged Beta-Catenin) to measure the levels of Wnt signaling activity in the muscle when Evi/Wg+ EVs are present vs. abolished. If the degree of Wnt signaling (readout would be intensity of fluorescent reporter) is decreased without Evi+ sEVs, there may be a dose-dependent response. Otherwise, please more clearly disclose the partial loss of Evi function without Evi+ sEVs or state the intact function of Evi without sEVs as speculative.

We agree that Wg is likely to be reaching the muscle in the absence of Evi exosomes via conventional secretory mechanisms, and have conducted new experiments to test this hypothesis (Fig. 5). In Drosophila muscles, Wg does not signal via a conventional b-catenin pathway. Instead, neuronally-derived Wg activates cleavage of its receptor Fz2, resulting in translocation of a Fz2 C-terminal fragment into the nucleus (Mathew et al., 2005; Mosca and Schwarz, 2010). We did attempt to directly measure Wg (using antibodies or knockins) and though we were able to detect a specific presynaptic signal, the background noise throughout the postsynaptic muscle was too high for a sensible quantification. In response to the reviewer’s question and also R2.6), we collaborated with the laboratory of Timothy Mosca to test Fz2 nuclear import in Tsg101 and Hrs mutants (new Figure 5F-G). Strikingly, we found that Hrs mutants, despite being extremely sickly, have normal nuclear import of Frizzled. We also confirmed that Hrs mutants have dramatically depleted levels of all EV cargoes examined, including Evi (Figure 2A-F). On the other hand we found that Tsg101 knockdowns have dramatically reduced Wg signaling (and a concomitant defect in postsynaptic development). We do not rule out (but think it is unlikely) that very small amounts of EVs could be present in hrs but not tsg101 mutants. A more parsimonious interpretation is that additional membrane trafficking defects in the Tsg101 mutants (which are beyond the scope of this study to explore in detail) block an alternative mode of Wg release, perhaps conventional secretion. The fact that Hrs mutants, despite showing similar depletion of Evi EVs, do not have a signaling defect strongly argues that EV release per se is not required for Wg signaling.

R1.6 To support the authors' hypothesis that Syt4 transmission via EVs is a proteostatic mechanism, the authors should determine whether Syt4 cargo localizes to lysosomal compartments in muscle, glia, or both. Otherwise, the proteostatic degradation of Syt4 via EVs is speculative.

Our data suggest that EVs serve as one of several parallel proteostatic mechanisms for presynaptic cargoes. We have added new data to the manuscript to emphasize the advance our work makes in our understanding of these mechanisms, and have emphasized this in the discussion on p 11-12, lines 46-5).

1. Degradation of neuronally derived EVs in glia and muscles. Previous work has shown that EV cargoes such as Evi can be found in compartments in the muscle cytoplasm, and that a-HRP-positive puncta are taken up and degraded by glial and muscle phagocytosis (Fuentes-Medel et al., 2009). These a-HRP-positive structures, despite colocalizing with EV cargoes Syt4, Nrg and APP (Walsh et al., 2021), were not previously connected to EVs. We have added new data showing that muscle or glial-specific RNAi of the phagocytic receptor Draper leads to the accumulation of EVs containing Syt4 (new Figure 7G-H)). Together with our finding (Figure 7A-F) that Syt4 is not significantly detected in the muscle cytoplasm, these results indicate that the main destination for transynaptic transfer is phagocytosis by the recipient cell. We have not been able to convincingly detect EV cargoes in the endolysosomal system of muscles, even in mutants disrupting lysosomal traffic, likely because the small number of EVs released by neurons (even over days of development) are drastically diluted in the much larger muscle cell.
2. Compensatory endosomophagy in the neuron. __When EV release is blocked in Hrs or Tsg101 mutants, we observe an induction of autophagy in the neuron (__Figure 3B, E-G). However, in the absence of ESCRT manipulation, autophagy mutants do not accumulate EVs (Figure 3C,D. S2H-I). This suggests that autophagy is a compensatory mechanism that is induced in the absence of EV release.
3. Retrograde transport to cell bodies: We previously found that disruption of neuronal dynactin leads to accumulation EV cargoes in presynaptic terminals (Blanchette et al., 2022), suggesting that retrograde transport is a mechanism for removal of these cargoes from synapses. Interestingly, EV release is not increased in these conditions, indicating that the retrogradely transported compartment represents a late endosome without ILVs, or an MVB that cannot fuse with the plasma membrane.

   R1.7 Please discuss alternate modes of cargo transfer from the presynaptic compartment to the postsynaptic compartment that may be utilized when EV-mediated transfer is abolished (i.e., cytonemes or tunneling nanotubules).

We have added these possibilities to the discussion (p11 line 31), though we note that we do not observe any such structures, or indeed any Syt4 in the muscle cytoplasm, and there is no current evidence for such transsynaptic structures in this system. Conventional secretion of Wg into the extracellular space and signaling through its transmembrane receptor Frizzled2 can account for Wg signaling in the absence of exosomes.

R1.8 OPTIONAL: Investigate the mechanism of Syt4+ sEV fusion with the postsynaptic compartment (direct fusion with the plasma membrane, receptor-mediated fusion, endocytosis and unpacking, or endocytosis and degradation).

We note that the Budnik lab has already shown that HRP-positive EVs released by NMJs are taken up by glia and muscles (Fuentes-Medel et al., 2009), and we have added data showing that this also applies for Syt4 (Fig. 7). Our data are not consistent with Syt4 fusing with recipient cell membranes or entering the muscle cytoplasm. Further investigation of this mechanism is beyond the scope of this project.

Given that several fundamental questions have yet to be answered regarding the biogenesis pathways and machinery utilized for EV-mediated cargo secretion, and the necessity for further TEM studies and/or work with primary cultures to characterize ILVs and EVs, >6 months is estimated to perform the necessary experiments that may require learning/ optimizing new systems.

Minor comments:

R1.9 Please clarify the choice of using Tsg101 KD in place of mutants of other ESCRT machinery (i.e., Hrs). Especially as when the Tsg101 mutant was characterized, you found major defects in autophagic flux that were not present for HrsD28/Df.

Tsg101 RNAi was selected since it provides a neuron-autonomous knockdown, eliminating the complications of mutant effects in other tissues. These animals are also relatively healthy as third instar larvae compared to genomic mutants tsg1012 (L1 lethal) and HrsD28 or motor-neuron driven Vps4DN (where L3 larvae are rare). This made it easier to recover enough larvae to properly power experiments, and alleviated concerns that general sickness is contributing to the phenotype (though note that neuronal Tsg101KD does result in pupal lethality). Finally, we were unable to effectively knock down Hrs by RNAi (see R1.1). To extend our studies beyond Tsg101, we have included additional experiments in the revised manuscript showing that HrsD28 animals, despite being quite unhealthy, still retain Syt4-dependent functional plasticity (See R2.5 and R3.4) and Wg signaling.

R1.10 Please clarify why the specific method in experiment in Fig. 4E-J was chosen. As Syt4 is a transmembrane protein, is likely undergoes degradation via the lysosome, like other membrane-bound proteins. Is it known whether the proteasome-directed nanobody is sufficient to pull Syt4 from membrane-bound compartments to undergo degradation in the proteasome? Would it make more sense to use a lysosome-directed nanobody?

The GFP tag on Syt4 is cytosolic rather than lumenal. Our data show that when we express the proteosome-directed nanobody presynaptically, it efficiently degrades membrane-associated Syt4-GFP (Fig. 7B). Therefore we expect that this tool should be similarly effective on membrane-associated Syt4-GFP if it were exposed to the muscle cytoplasm. We have confirmed that it is effective in the muscle against DLG-GFP (Fig. S5A)

R1.11 Please provide further methodological information regarding the sample preparation for live imaging of axons to generate kymographs found in Fig. S3.

Additional details have been provided on p14 lines 10-24 and p15 lines 31-37.

R1.12 In Figure 1I and 1J, include representative image and quantification of Syt4-GFP pre- and post-synaptic intensity for HrsD28/Df for consistency with ShrubKD and Vps4DN in Figure 1K-P.

We generated and tested HrsD28; Syt4-GFP (Fig 2A,D), and HrsD28; Evi-GFP strains (Fig 2B-E). All EV cargoes exhibited a dramatic post-synaptic depletion in Hrs mutants, similar to the other ESCRT manipulations.

R1.13 In Figure 2H, please provide a cell type marker or HRP mask with a merged image for image clarity.

This image shows neuronal cell bodies in the ventral ganglion, which are densely packed relative to each other. The cell type specificity is provided by the motor neuron driver. We did not use a cell type marker or individually mask cells for analysis, but instead quantified intensity over the whole field of view. We can manually trace cell bodies in this image if requested, but it would not represent our ROI for analysis.

R1.14 In Figure 4B, please provide quantification for the differences between 1) WT Mock and Tsg101 MOCK and 2) WT Stim and Tsg101KD Stim to show that upon stimulation, WT and Tsg101 undergo the same increase in the number of ghost boutons/ NMJ in Muscle 4.

We have added these statistical comparisons to the graph (Fig. 6B)

R1.15 In Figure 3 G and H, use consistent scale bars to compare between temperatures.

We have removed the Shrub data at 20º as it did not provide additional insight to the manuscript.

Reviewer #1 (Significance (Required)):

General assessment (Strengths):

-Use of Drosophila NMJ model system consistent with others in the field and exceptional harnessing of genetic tools for mutations across the ESCRT pathway (-0, -I, -III, etc.) -Identification of ESCRT pathway mutants that do not deplete pre-synaptic cargo levels but generate endosomal dysfunction, indicative of a possible decrease in secretion of cargoes via EVs -Implementing functional characterization of Evi/ Wg and Syt4 cargoes, consistent with previous work in the field; highly reproducible

-Sufficiently thorough investigation of the cross-regulation of autophagy and EV biogenesis by Tsg101

General assessment (Weaknesses):

-Lack of investigation of known ESCRT-independent pathways/ genes involved in the generation of sEVs (i.e., nSMase2/ Ceramide) especially as it is unknown if minor sources of cargo+ EVs are sufficient in maintaining functional phenotype

See R1.3 for comments on this point

-Lack of sEV characterization and validation of EVs derived from mutant

We have added STED data to measure EV size, and described the challenges in EV membrane measurements by EM in the in vivo system.

-Does not show the loss of cargoes of interest on EVs from mutants other than through back-up of cargoes in the presynaptic endocytic pathway (Rab7, Rab5, Rab11)

We strongly disagree with this comment. We have explicitly measured the loss of numerous cargoes in postsynaptic structures that have been rigorously established to be EVs in this and previous publications. Our findings are not limited to back-up of presynaptic structures.

-Lack of rigorous investigation of the claim that Evi and Syt4 are released via EVs for proteostatic means is missing. Authors should demonstrate the degradation of EV cargoes by recipient cells (either muscle OR glia)

We have added new data and discussion on multiple and compensatory proteostatic pathways.

-If EV-mediated cargo transfer is not required, authors should investigate alternate modes of cargo transfer more rigorously (i.e., diffusion of Wg, suggest/ test hypotheses for mechanism of Syt4 function or transfer).

We have included discussion of alternate modes of transfer for Wg (i.e. conventional secretion). By contrast, for Syt4 we believe it is acting in the donor cell without transfer, and have included alternate interpretations of the previous literature that had suggested its function in muscles.

Advance: -Compared with other recent in vivo studies of EVs where donor EVs are loaded with a cargo, such as Cre, which uniquely identifies recipient cells through Cre recombination-mediated expression of a fluorescent reporter (Zomer et al 2015, Cell), this study relies on the readout of fluorescently tagged cargo in the recipient cells to represent transfer via EVs. While numerous studies in the Drosophila field focus on the same small set of known EV cargoes at the NMJ (Koles et al., 2012; Gross et al., 2012; Korkut et al., 2013; Korkut et al., 2009; Walsh et al., 2021), there is a noticeable lack of EV characterization based on MISEV (i.e. TEM of EVs, size distribution, enrichment of well-known EV markers [https://doi.org/10.1080/20013078.2018.1535750]) that would significantly strengthen the work and make it more widely accepted in the EV field.

As mentioned above, many of these criteria (including EV size and enrichment of known EV markers) are already established in the previous literature for this system. As requested, we have also added similar data to our revised manuscript.

-In this study, the use of ESCRT machinery mutants is proven as a new technical method in delineating the role of EV cargoes in cell-autonomous versus EV-dependent functions. This is the first study, to my knowledge, that has leveraged mutants from both early and late ESCRT complexes for the study of EVs in Drosophila. Additionally, the finding that some cargoes may be able to carry out their signaling functions, independent of transfer via EVs, provides key mechanistic insight into one possible role of EVs as proteostatic shuttles for cargo. This work also begins to address a fundamental question in the field, which is to delineate roles that EVs actually carry out in physiological conditions, compared to the many roles that have been shown possible in vitro.

We appreciate the reviewer’s insight into the impact of our work.

Audience: -Basic research (endosomal biology, ESCRT pathway, cell signaling, neurodevelopment)

-Specialized (Drosophila, Neurobiology; Extracellular Vesicles)

-This article will be of interest to basic scientists in the field of endosomal trafficking and extracellular vesicle biology as well as though studying the nervous system in Drosophila melanogaster. As the field of extracellular vesicle biology has broad implications in the spread of pathogenic cargoes in cancer and neurodegenerative disease, the basic biology associated with EVs has some translational relevance.

Expertise (Keywords):

-ESCRT and nSMase2 EV biogenesis pathways

-EV characterization in vitro/ live imaging studies

-EV release and uptake

-Neuronal and glial cell biology

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

This manuscript addresses the role of exosome secretion in neuromuscular junction development in Drosophila, a system that has been proposed to depend on exosomes. In particular, delivery of Wingless via exosomes has been proposed to promote structural organization of the synapse. Previously, however, the studies that proposed this model targeted the cargoes themselves, rather than targeting exosome biogenesis or secretion. In this new study, exosome biogenesis is targeted via knockdown of the ESCRT components Hrs, TSG101, and Chmp4. The authors find that some previously ascribed functions are not inhibited by these knockdowns. In particular, formation of active zones, as defined by BRP-positive puncta (total and per micrometer), and total bouton numbers. It does look like there is a partial defect in BRP-positive puncta per micrometer, but it is not significant. For ghost bouton formation, there is a similar increase in evi-mutant and ESCRT-KD NMJs (with some subtle differences depending on abdominal segment and temperature). They also examine the role of Syt4, which has been proposed to be transferred from nerve to muscle cells at the junction and to regulate mEJP frequency after stimulation. They found no difference in mEJP frequency after stimulation between WT and TSG101-KD animals, although they did not have a positive control with inhibition of Syt4. They did do an elegant experiment to demonstrate that most of extracellularly transferred Syt4 does not reach the muscle cytoplasm. Overall, it is an interesting paper, mostly well controlled and rigorous, and well-written. It is an important contribution to the EV and NMJ fields. The data should provoke reconsideration of some of the functions that were previously ascribed to exosome transfer at the NMJ. However, I do think that there are some overly strong statements and the functions of the exosomes at the synapse were quite narrowly examined. For example, the title of the paper is pretty strong and the abstract does not say which functions were or were not affected by TSG101 KD. There are also a couple of experiments that would enhance the manuscript. Some specific suggestions are below:

R2.1 Title: "ESCRT disruption provides evidence against signaling functions for synaptic exosomes" seems a bit broad -- only evi/Wg and Syt4 functions were examined at NMJ synapses, not all signaling functions of all exosomes at all synapses. Something like, "ESCRT disruption provides evidence against signaling functions for exosome-carried evi/Wg and Syt4 at the neuromuscular junction" seems a bit more reasonable.

We are open to changing the title to: “ESCRT disruption provides evidence against transsynaptic signaling functions for some extracellular vesicle cargoes” though we prefer to leave it as is since “provides evidence against” is already fairly understated.

__ __R2.2 Abstract: the description of the actual data is very little, just one sentence saying that "many" of the signaling functions are retained with ESCRT depletion. I think a bit more focus on the actual data is warranted.

We have edited the abstract to include more detail on the signaling phenotypes.

__

__R2.3 Results section:

Fig 3: What does A2 and A3 mean for the graphs in c,d,e, g, h? Please specify in figure legend.

We have described in the figure legends that A2 and A3 refer to specific abdominal segments in the larvae.

R2.4 The sentence "Further, active zones in Tsg101KD appeared morphologically normal by TEM (Fig.2B)." is confusing to me. What do you mean by that? Are you referring to the following two sentences about feathery DLG and SSR? But the feathery DLG I presume is in Fig 3, where that staining is. And I also don't know what feathery DLG means -- it should be pointed out in the appropriate image.

Presynaptic active zones are defined by an electron-dense T-shaped pedestal at sites of synaptic vesicle release, and can be seen in the TEM in what is now Figure 3B, marked as AZ. We have also labeled AZ by immunofluorescence (Fig. 5A) and they appear normal.

By contrast, Dlg primarily labels the postsynaptic apparatus associated with the infoldings of the muscle membrane. In control animals, Dlg immunostaining is relatively tightly and smoothly clustered within ~1µm of the presynaptic neuron. By contrast, in Evi mutants, there are wisps of Dlg-positive structures extending from the bouton periphery. We have added arrows in what is now Fig. 5C to indicate the feathery structures.

R2.5 Fig 4 addresses Syt4 function. However, there is no positive control inhibiting Syt4 to see if there is a change. Just comparison of WT and TSG101. It seems like this positive control is in order.

We have added the positive control (Fig. 6E-F) reproducing the previously reported result that Syt4 mutants lack the high-frequency stimulation-induced increase in mEPSP frequency (HFMR). We have also added new data on HrsD28 genomic mutants. Despite the fact that few of these larvae survive and they are quite unhealthy, they still exhibit robust HFMR, similar to the Tsg101KD larvae, strongly supporting our hypothesis.

R2.6 Discussion: I think some discussion of what ghost boutons are and what the possible significance is of the evi and ESCRT mutant phenotype of enhanced ghost bouton formation

We have added more discussion on the ghost bouton phenotype (p11 lines 5-14), especially in light of our new findings that Hrs and Tsg101 mutants may distinguish alternative modes of Wg secretion (see R1.5)

R2.7 Also, in the Discussion, it is mentioned that Wg probably gets secreted in the ESCRT mutants -- presumably this accounts for the discrepancy between evi mutants and the ESCRT mutants. An experiment to actually test this would greatly enhance the manuscript.

We have added this experiment as addressed in R1.5

Reviewer #2 (Significance (Required)):

Overall, it is an interesting paper, mostly well controlled and rigorous, and well-written. It is an important contribution to the EV and NMJ fields. The data should provoke reconsideration of some of the functions that were previously ascribed to exosome transfer at the NMJ. However, I do think that there are some overly strong statements and the functions of the exosomes at the synapse were quite narrowly examined. For example, the title of the paper is pretty strong and the abstract does not say which functions were or were not affected by TSG101 KD.

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

Dresselhaus et al. investigates signaling functions for synaptic exosomes at the Drosophila NMJ. Exosomes are widely seen in vivo and in vitro. They are clearly sufficient to induce signaling responses in vitro, but whether they normally fulfill signaling functions in vivo has not been rigorously addressed. The authors make use of several mutants that block exosome release to test whether exosome release is important for two distinct signaling pathways: the Evi/Wg pathway and the Syt4 signaling pathway. Both pathways have been implicated in neuron to muscle signaling. Surprisingly, the authors find scant evidence that exosome release is required for either pathway. They convincingly show that knockdown of Tsg101 (an ESCRT-I component) does not phenocopy many synaptic phenotypes of either wg or syt4. Instead, they propose that in vivo, exosomes may serve as a proteostatic mechanism, as a mechanism for the neuron to dispose of unwanted/damaged proteins.

Specific comments are below:

R3.1 Loss of Tsg101 has been linked to upregulated MAPK stress signaling pathways and autophagy. Thus, it's possible that activating such compensatory mechanisms in Tsg101 knockdown animals could mask phenotypes associated with specific loss of EV cargoes such as Wg or Syt4. Indeed, the authors demonstrate that loss of Tsg101 and Hrs have very different effects on synaptic autophagy. To provide additional evidence that Wg or Syt4 signaling is independent of EV release, it would be good to check for wg/syt4 phenocopy in additional ESCRT complex mutants. I understand they did a bit with Shrub knockdown at low temperature in Figure 3, but the temperature-dependence of the ghost bouton phenotype clouds the interpretation. Could the authors try a motorneuron driver with a more restricted phenotype to overcome the lethality issues, or alternatively use one of their other ESCRT component mutants? This is obviously the central claim of the manuscript, and it would be strengthened by carrying out phenotypic analysis in mutants other than the Tsg101 RNAi line.

As noted for R2.5, we have added HFMR experiments for the HrsD28 genomic mutant, and found that despite being very unhealthy, they exhibit robust HFMR similar to Tsg101KD. We also confirmed dramatic depletion of Syt4 EVs in the HrsD28 mutant. Thus, the preserved Syt4 signaling function in ESCRT mutants with depleted EV Syt4 is not restricted to Tsg101, and does not depend on the co-occurring autophagy phenotype.

R3.2 In Figure 1, the authors show that neuronal Tsg101 RNAi dramatically reduces "postsynaptic" levels of exosome cargoes at the L3 stage to argue that exosome release is blocked in this mutant. While this seems very likely at the L3 stage, it is unclear when Tsg101 levels are reduced and thus when exosome release is impaired in this background. This is important because we don't know when these signaling pathways act. For example, it is possible that the critical period for Wg and Syt4 signaling is during the L1 stage, and that Tsg101 knockdown is incomplete at that stage. It is important to assay exosome release at earlier larval stage, particularly when RNAi is the method used to reduce gene function.

We have conducted this experiment. We noted accumulation of cargoes in Tsg101KD L1 larvae, indicating that the RNAi is effective early in development. However, we do not find many EVs in either wild-type or Tsg101KD first instar larvae (red is a-HRP, green is Syt4-GFP). This argues that it is unlikely that EV-mediated signaling has a critical period earlier in development. It is likely that the accumulation of EVs that we observe trapped in the muscle membrane reticulum in third instar larvae were laid down over days or hours of development. We do not propose to include these data in the manuscript unless the editors and reviewers prefer that we do so.

R3.3 If the Syt4 and Evi exosomes do not serve major signaling roles and are in fact neuronal waste, it seems likely they are phagocytosed by glia. Are levels of non-neuronal Syt4/Evi levels increased when glial phagocytosis in blocked (eg in draper mutants)?

As mentioned above, the Budnik lab previously showed that uptake and degradation of postsynaptic a-HRP-positive structures depends on glial and muscle phagocytosis.a-HRP recognizes a number of neuronally-derived glycoproteins (Snow et al., 1987). Though the Budnik lab had not previously linked these structures to EVs, we do know that they very strongly colocalize with known EV cargoes and depend on the exact same membrane traffic machinery for release, arguing that some a-HRP antigen proteins are also EV cargoes (Blanchette et al., 2022). To close this loop. we have added data showing that Syt4-positive EVs also depend on Draper for their clearance (Fig 7).

R3.4 For the HFMR experiment, it would be good to see the syt4-dependent phenotype as a positive control.__ __

As mentioned for R2.5, we have added the Syt4 positive control (Figure 6E,F), which fails to show HFMR as expected.

.__ __R3.5 In the abstract, the authors state that, "the cargoes are likely to function cell autonomously in the motorneuron". Isn't it alternatively possible that these proteins (wg in particular) could signal to the muscle in a non-exosome dependent pathway?

Yes, we believe that Wg is likely released by another mechanism (perhaps conventional secretion). As noted for R1.5 and R2.6, we have added new data in Fig. 5 showing that Frizzled nuclear import IS NOT disrupted in Hrs mutants, despite dramatic loss of Evi EVs. Interestingly Frizzled nuclear import (and postsynaptic development) IS altered in neuronal Tsg101KD larvae, which disrupt additional membrane trafficking pathways beyond EV release (see Fig. 3). This is particularly interesting in light of the normal Syt4 signaling in Tsg101KD larvae, and supports the hypothesis that Syt4 can function without leaving the neuron, while Wg must be released, albeit not via Hrs-dependent EV formation. Another (less parsimonious) interpretation is that very small amounts of Wg release in the Hrs mutant are sufficient to promote Frizzled nuclear import.

Reviewer #3 (Significance (Required)):

This is an important paper that is well-organized and logically presented. It makes a clear and largely compelling case against major signaling roles for exosomes at this synapse. The authors should be commended for publishing this work, which demands a re-evaluation of proposed key roles for exosomes at the fly NMJ. Given the intense interest in exosomes in neurobiology, this paper will be of great interest to neuronal cell biologists working across systems.

We thank the reviewer for their appreciation of the impact of our work on the field.

Back, M.J., H.C. Ha, Z. Fu, J.M. Choi, Y. Piao, J.H. Won, J.M. Jang, I.C. Shin, and D.K. Kim. 2018. Activation of neutral sphingomyelinase 2 by starvation induces cell-protective autophagy via an increase in Golgi-localized ceramide. Cell Death Dis. 9:670.

Blanchette, C.R., and A.A. Rodal. 2020. Mechanisms for biogenesis and release of neuronal extracellular vesicles. Curr Opin Neurobiol. 63:104-110.

Blanchette, C.R., A.L. Scalera, K.P. Harris, Z. Zhao, E.C. Dresselhaus, K. Koles, A. Yeh, J.K. Apiki, B.A. Stewart, and A.A. Rodal. 2022. Local regulation of extracellular vesicle traffic by the synaptic endocytic machinery. J. Cell Biol. 10.1083/jcb.202112094.

Choezom, D., and J.C. Gross. 2022. Neutral sphingomyelinase 2 controls exosome secretion by counteracting V-ATPase-mediated endosome acidification. J Cell Sci. 135.

Enneking, E.M., S.R. Kudumala, E. Moreno, R. Stephan, J. Boerner, T.A. Godenschwege, and J. Pielage. 2013. Transsynaptic coordination of synaptic growth, function, and stability by the L1-type CAM Neuroglian. PLoS Biol. 11:e1001537.

Fuentes-Medel, Y., M.A. Logan, J. Ashley, B. Ataman, V. Budnik, and M.R. Freeman. 2009. Glia and muscle sculpt neuromuscular arbors by engulfing destabilized synaptic boutons and shed presynaptic debris. PLoS Biol. 7:e1000184.

Koles, K., J. Nunnari, C. Korkut, R. Barria, C. Brewer, Y. Li, J. Leszyk, B. Zhang, and V. Budnik. 2012. Mechanism of evenness interrupted (Evi)-exosome release at synaptic boutons. J Biol Chem. 287:16820-16834.

Korkut, C., B. Ataman, P. Ramachandran, J. Ashley, R. Barria, N. Gherbesi, and V. Budnik. 2009. Trans-synaptic transmission of vesicular Wnt signals through Evi/Wntless. Cell. 139:393-404.

Korkut, C., Y. Li, K. Koles, C. Brewer, J. Ashley, M. Yoshihara, and V. Budnik. 2013. Regulation of postsynaptic retrograde signaling by presynaptic exosome release. Neuron. 77:1039-1046.

Lauwers, E., Y.C. Wang, R. Gallardo, R. Van der Kant, E. Michiels, J. Swerts, P. Baatsen, S.S. Zaiter, S.R. McAlpine, N.V. Gounko, F. Rousseau, J. Schymkowitz, and P. Verstreken. 2018. Hsp90 Mediates Membrane Deformation and Exosome Release. Mol Cell. 71:689-702 e689.

Mathew, D., B. Ataman, J. Chen, Y. Zhang, S. Cumberledge, and V. Budnik. 2005. Wingless signaling at synapses is through cleavage and nuclear import of receptor DFrizzled2. Science. 310:1344-1347.

Moberg, K.H., S. Schelble, S.K. Burdick, and I.K. Hariharan. 2005. Mutations in erupted, the Drosophila ortholog of mammalian tumor susceptibility gene 101, elicit non-cell-autonomous overgrowth. Dev Cell. 9:699-710.

Mosca, T.J., and T.L. Schwarz. 2010. The nuclear import of Frizzled2-C by Importins-beta11 and alpha2 promotes postsynaptic development. Nat Neurosci. 13:935-943.

Niekamp, P., F. Scharte, T. Sokoya, L. Vittadello, Y. Kim, Y. Deng, E. Sudhoff, A. Hilderink, M. Imlau, C.J. Clarke, M. Hensel, C.G. Burd, and J.C.M. Holthuis. 2022. Ca(2+)-activated sphingomyelin scrambling and turnover mediate ESCRT-independent lysosomal repair. Nat Commun. 13:1875.

Snow, P.M., N.H. Patel, A.L. Harrelson, and C.S. Goodman. 1987. Neural-specific carbohydrate moiety shared by many surface glycoproteins in Drosophila and grasshopper embryos. J Neurosci. 7:4137-4144.

Trajkovic, K., C. Hsu, S. Chiantia, L. Rajendran, D. Wenzel, F. Wieland, P. Schwille, B. Brugger, and M. Simons. 2008. Ceramide triggers budding of exosome vesicles into multivesicular endosomes. Science. 319:1244-1247.

Walsh, R.B., E.C. Dresselhaus, A.N. Becalska, M.J. Zunitch, C.R. Blanchette, A.L. Scalera, T. Lemos, S.M. Lee, J. Apiki, S. Wang, B. Isaac, A. Yeh, K. Koles, and A.A. Rodal. 2021. Opposing functions for retromer and Rab11 in extracellular vesicle traffic at presynaptic terminals. J Cell Biol. 220:e202012034.

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Referee #3

Evidence, reproducibility and clarity

Dresselhaus et al. investigates signaling functions for synaptic exosomes at the Drosophila NMJ. Exosomes are widely seen in vivo and in vitro. They are clearly sufficient to induce signaling responses in vitro, but whether they normally fulfill signaling functions in vivo has not been rigorously addressed. The authors make use of several mutants that block exosome release to test whether exosome release is important for two distinct signaling pathways: the Evi/Wg pathway and the Syt4 signaling pathway. Both pathways have been implicated in neuron to muscle signaling. Surprisingly, the authors find scant evidence that exosome release is required for either pathway. They convincingly show that knockdown of Tsg101 (an ESCRT-I component) does not phenocopy many synaptic phenotypes of either wg or syt4. Instead, they propose that in vivo, exosomes may serve as a proteostatic mechanism, as a mechanism for the neuron to dispose of unwanted/damaged proteins.

Specific comments are below:

Loss of Tsg101 has been linked to upregulated MAPK stress signaling pathways and autophagy. Thus, it's possible that activating such compensatory mechanisms in Tsg101 knockdown animals could mask phenotypes associated with specific loss of EV cargoes such as Wg or Syt4. Indeed, the authors demonstrate that loss of Tsg101 and Hrs have very different effects on synaptic autophagy. To provide additional evidence that Wg or Syt4 signaling is independent of EV release, it would be good to check for wg/syt4 phenocopy in additional ESCRT complex mutants. I understand they did a bit with Shrub knockdown at low temperature in Figure 3, but the temperature-dependence of the ghost bouton phenotype clouds the interpretation. Could the authors try a motorneuron driver with a more restricted phenotype to overcome the lethality issues, or alternatively use one of their other ESCRT component mutants? This is obviously the central claim of the manuscript, and it would be strengthened by carrying out phenotypic analysis in mutants other than the Tsg101 RNAi line.

In Figure 1, the authors show that neuronal Tsg101 RNAi dramatically reduces "postsynaptic" levels of exosome cargoes at the L3 stage to argue that exosome release is blocked in this mutant. While this seems very likely at the L3 stage, it is unclear when Tsg101 levels are reduced and thus when exosome release is impaired in this background. This is important because we don't know when these signaling pathways act. For example, it is possible that the critical period for Wg and Syt4 signaling is during the L1 stage, and that Tsg101 knockdown is incomplete at that stage. It is important to assay exosome release at earlier larval stage, particularly when RNAi is the method used to reduce gene function.

If the Syt4 and Evi exosomes do not serve major signaling roles and are in fact neuronal waste, it seems likely they are phagocytosed by glia. Are levels of non-neuronal Syt4/Evi levels increased when glial phagocytosis in blocked (eg in draper mutants)?

For the HFMR experiment, it would be good to see the syt4-dependent phenotype as a positive control.

In the abstract, the authors state that, "the cargoes are likely to function cell autonomously in the motorneuron". Isn't it alternatively possible that these proteins (wg in particular) could signal to the muscle in a non-exosome dependent pathway?

Significance

This is an important paper that is well-organized and logically presented. It makes a clear and largely compelling case against major signaling roles for exosomes at this synapse. The authors should be commended for publishing this work, which demands a re-evaluation of proposed key roles for exosomes at the fly NMJ. Given the intense interest in exosomes in neurobiology, this paper will be of great interest to neuronal cell biologists working across systems.

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Referee #2

Evidence, reproducibility and clarity

This manuscript addresses the role of exosome secretion in neuromuscular junction development in Drosophila, a system that has been proposed to depend on exosomes. In particular, delivery of Wingless via exosomes has been proposed to promote structural organization of the synapse. Previously, however, the studies that proposed this model targeted the cargoes themselves, rather than targeting exosome biogenesis or secretion. In this new study, exosome biogenesis is targeted via knockdown of the ESCRT components Hrs, TSG101, and Chmp4. The authors find that some previously ascribed functions are not inhibited by these knockdowns. In particular, formation of active zones, as defined by BRP-positive puncta (total and per micrometer), and total bouton numbers. It does look like there is a partial defect in BRP-positive puncta per micrometer, but it is not significant. For ghost bouton formation, there is a similar increase in evi-mutant and ESCRT-KD NMJs (with some subtle differences depending on abdominal segment and temperature). They also examine the role of Syt4, which has been proposed to be transferred from nerve to muscle cells at the junction and to regulate mEJP frequency after stimulation. They found no difference in mEJP frequency after stimulation between WT and TSG101-KD animals, although they did not have a positive control with inhibition of Syt4. They did do an elegant experiment to demonstrate that most of extracellularly transferred Syt4 does not reach the muscle cytoplasm. Overall, it is an interesting paper, mostly well controlled and rigorous, and well-written. It is an important contribution to the EV and NMJ fields. The data should provoke reconsideration of some of the functions that were previously ascribed to exosome transfer at the NMJ. However, I do think that there are some overly strong statements and the functions of the exosomes at the synapse were quite narrowly examined. For example, the title of the paper is pretty strong and the abstract does not say which functions were or were not affected by TSG101 KD. There are also a couple of experiments that would enhance the manuscript. Some specific suggestions are below:

Title: "ESCRT disruption provides evidence against signaling functions for synaptic exosomes" seems a bit broad -- only evi/Wg and Syt4 functions were examined at NMJ synapses, not all signaling functions of all exosomes at all synapses. Something like, "ESCRT disruption provides evidence against signaling functions for exosome-carried evi/Wg and Syt4 at the neuromuscular junction" seems a bit more reasonable.

Abstract: the description of the actual data is very little, just one sentence saying that "many" of the signaling functions are retained with ESCRT depletion. I think a bit more focus on the actual data is warranted.

Results section: Fig 3: What does A2 and A3 mean for the graphs in c,d,e, g, h? Please specify in figure legend.

The sentence "Further, active zones in Tsg101KD appeared morphologically normal by TEM (Fig. 2B)." is confusing to me. What do you mean by that? Are you referring to the following two sentences about feathery DLG and SSR? But the feathery DLG I presume is in Fig 3, where that staining is. And I also don't know what feathery DLG means -- it should be pointed out in the appropriate image.

Fig 4 addresses Syt4 function. However, there is no positive control inhibiting Syt4 to see if there is a change. Just comparison of WT and TSG101. It seems like this positive control is in order. Discussion: I think some discussion of what ghost boutons are and what the possible significance is of the evi and ESCRT mutant phenotype of enhanced ghost bouton formation

Also, in the Discussion, it is mentioned that Wg probably gets secreted in the ESCRT mutants -- presumably this accounts for the discrepancy between evi mutants and the ESCRT mutants. An experiment to actually test this would greatly enhance the manuscript.

Significance

Overall, it is an interesting paper, mostly well controlled and rigorous, and well-written. It is an important contribution to the EV and NMJ fields. The data should provoke reconsideration of some of the functions that were previously ascribed to exosome transfer at the NMJ. However, I do think that there are some overly strong statements and the functions of the exosomes at the synapse were quite narrowly examined. For example, the title of the paper is pretty strong and the abstract does not say which functions were or were not affected by TSG101 KD.

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Referee #1

Evidence, reproducibility and clarity

Summary:

In this paper, Dresselhaus et al (2023) investigate the possibility that known cargoes of extracellular vesicles (EVs) released at the Drosophila neuromuscular junction have cell-autonomous functions rather than functions specifically conferred as a condition of their release in EVs, in vivo. To do so, authors focus their studies on use of Tsg101-KD, a mutant of the ESCRT-I machinery, of the ESCRT EV biogenesis pathway, and are able to show that for some endogenously-expressed, fluorescently-tagged cargoes, fluorescence intensity in the pre-synaptic compartment is significantly elevated (Syt4 and Evi) and the postsynaptic intensity in the muscle is significantly decreased (Syt4, Evi, APP, and Nrg). These findings suggest that these cargoes become trapped in the endosomal system (colocalizing with early, late, and recycling endosomal compartments), rather than undergoing secretion in EVs targeting post-synaptic muscle and glia as usual. This phenotype is recapitulated for select cargoes using mutants of both early and late components of ESCRT pathway machinery. They further characterize the Tsg101 mutant, demonstrating co-occurrence of an autophagic flux defect , but as the cargo phenotype is present without induction of the autophagic flux defect for their Hrs mutants, authors suggest the overlapping role of Tsg101 in autophagy is independent of its role in the ESCRT pathway/ EV secretion. Subsequently, they use previously defined functional phenotypes of the Evi (number of active zones, number of boutons, number of developmentally-arrested ghost boutons) and Syt-4 (number of transient ghost boutons and mEJPs) cargoes to show a minimal dependence on cargo delivery via ESCRT-derived EVs for these cargoes to carry out their synaptic growth and plasticity functions in vivo. However, it should be notes that for Evi/ Wg cargo, there is a slight increase in developmentally-arrested ghost boutons suggesting the cargo may not be entirely independent of EV-mediated cargo delivery. Finally, authors express an anti-GFP proteasome-directed nanobody using motor neuron or muscle-specific drivers and find that Syt4-GFP cargo doesn't enter muscle cytoplasm as fluorescence is maintained and cargo is not degraded by the muscle proteasome. While authors suggest this as evidence of EV-mediated transfer for cargo proteostasis, it is not explicitly shown that Syt4 cargo is, in fact, trafficked and degraded by the lysosome or hypothesized how Syt4 function or post-synaptic localization may be carried out independently of EVs.

Major comments:

• It is difficult to evaluate the findings of this study without knowing the extent of ESCRT pathway impairment. Please provide data quantifying the degree of knockdown/ mutant expression for each ESCRT component (i.e., western blot)
• Loss of ESCRT machinery likely disrupts the release of small EVs to a significant extent; however, the authors do not show that EV release is entirely lost, only that 1) cargoes are backed up in the endosomal system due to endosomal dysfunction and 2) fluorescence of cargoes in the postsynaptic compartment is diminished. To claim that ESCRT-derived EVs with the relevant cargoes are lost, the authors should perform immunogold labelling with TEM. This would provide direct evidence that the cargoes examined here are packaged in ILVs, and that the ILVs are of a size (~50-150nm) consistent with exosomes (which should really be referred to as small extracellular vesicles (sEVs) per the minimal information for studies of extracellular vesicles (MISEV 2018 [https://doi.org/10.1080/20013078.2018.1535750])
• Additionally, EM would show the loss of cargo packaging and provide information about where these cargoes localize in the presence of ESCRT mutants/loss-of-function.
• Other biogenesis pathways utilize multivesicular bodies to generate EVs, most prominently the nSMase2/ceramide synthesis pathway (which operates in an ESCRT-independent manner). It is possible that this pathway compensates when there are defects in the canonical ESCRT pathway. Thus, it is imperative for the authors to show that the cargo secretion no longer occurs in the presence of ESCRT mutations/loss-of-function. The authors should also use nSMase2 pathway mutants to see if the phenotypes in cargo trafficking (i.e., pre/ post-synaptic protein levels) are recapitulated.
• The authors' findings support that cargo trafficking is affected by widespread endosomal dysfunction but doesn't cleanly prove that 1) synaptic sEV release is lost and 2) that cargo-specific sEVs are lost. As previously mentioned, loss of cargo+ ILVs in MVEs by TEM could demonstrate this, but another useful approach would be to include in vitro Drosophila primary neuronal culture/ EV isolation and mass spec/proteomic characterization studies as proof of concept. According to widely agreed upon guidelines in the EV field, the authors should directly characterize their EV population to show 1) the appropriate size distribution associated with exosomes/sEVs, 2) the presence of traditional EV markers (i.e., tetraspanins), 3) changes in overall EV count by ESCRT mutants, and 4) decreased levels of cargo(es) of interest in the presence of ESCRT mutants/loss-of-function. In vitro experiments would be particularly helpful for quantifying the degree of loss of cargo-specific EVs with each ESCRT mutant. These experiments could also investigate the possibility that cargoes are secreted in nSMase2/ Ceramide-derived EVs, by showing that EV cargo levels are unaffected in nSMase mutants.
• During functional tests of Evi+ motor neurons lacking generation of Evi+ EVs, there is a slight defect observed, namely the increased formation of developmentally arrested ghost boutons when Evi secretion in sEVs is lost. As mentioned, Evi is a transporter of Wg and it is possible for Wg to be transmitted between cells via normal diffusion. Thus, some basal levels of Wg may be reaching the muscle when its transfer via sEVs is abolished, and these basal levels may be sufficient to phenocopy the WT in the number of active zones and boutons. Is it possible that this element of Evi/ Wg function is dose-dependent and thus reliant on the extra Evi/ Wg transferred via sEVs? If possible, the authors should use a Wnt-signaling pathway reporter (i.e., fluorescently tagged Beta-Catenin) to measure the levels of Wnt signaling activity in the muscle when Evi/Wg+ EVs are present vs. abolished. If the degree of Wnt signaling (readout would be intensity of fluorescent reporter) is decreased without Evi+ sEVs, there may be a dose-dependent response. Otherwise, please more clearly disclose the partial loss of Evi function without Evi+ sEVs or state the intact function of Evi without sEVs as speculative.
• To support the authors' hypothesis that Syt4 transmission via EVs is a proteostatic mechanism, the authors should determine whether Syt4 cargo localizes to lysosomal compartments in muscle, glia, or both. Otherwise, the proteostatic degradation of Syt4 via EVs is speculative.
• Please discuss alternate modes of cargo transfer from the presynaptic compartment to the postsynaptic compartment that may be utilized when EV-mediated transfer is abolished (i.e., cytonemes or tunneling nanotubules).
• OPTIONAL: Investigate the mechanism of Syt4+ sEV fusion with the postsynaptic compartment (direct fusion with the plasma membrane, receptor-mediated fusion, endocytosis and unpacking, or endocytosis and degradation).
• Given that several fundamental questions have yet to be answered regarding the biogenesis pathways and machinery utilized for EV-mediated cargo secretion, and the necessity for further TEM studies and/or work with primary cultures to characterize ILVs and EVs, >6 months is estimated to perform the necessary experiments that may require learning/ optimizing new systems.

Minor comments:

• Please clarify the choice of using Tsg101 KD in place of mutants of other ESCRT machinery (i.e., Hrs). Especially as when the Tsg101 mutant was characterized, you found major defects in autophagic flux that were not present for HrsD28/Df.
• Please clarify why the specific method in experiment in Fig. 4E-J was chosen. As Syt4 is a transmembrane protein, is likely undergoes degradation via the lysosome, like other membrane-bound proteins. Is it known whether the proteasome-directed nanobody is sufficient to pull Syt4 from membrane-bound compartments to undergo degradation in the proteasome? Would it make more sense to use a lysosome-directed nanobody?
• Please provide further methodological information regarding the sample preparation for live imaging of axons to generate kymographs found in Fig. S3.
• In Figure 1I and 1J, include representative image and quantification of Syt4-GFP pre- and post-synaptic intensity for HrsD28/Df for consistency with ShrubKD and Vps4DN in Figure 1K-P.
• In Figure 2H, please provide a cell type marker or HRP mask with a merged image for image clarity.
• In Figure 4B, please provide quantification for the differences between 1) WT Mock and Tsg101 MOCK and 2) WT Stim and Tsg101KD Stim to show that upon stimulation, WT and Tsg101 undergo the same increase in the number of ghost boutons/ NMJ in Muscle 4.
• In Figure 3 G and H, use consistent scale bars to compare between temperatures.

Significance

General assessment (Strengths):

• Use of Drosophila NMJ model system consistent with others in the field and exceptional harnessing of genetic tools for mutations across the ESCRT pathway (-0, -I, -III, etc.)
• Identification of ESCRT pathway mutants that do not deplete pre-synaptic cargo levels but generate endosomal dysfunction, indicative of a possible decrease in secretion of cargoes via EVs
• Implementing functional characterization of Evi/ Wg and Syt4 cargoes, consistent with previous work in the field; highly reproducible
• Sufficiently thorough investigation of the cross-regulation of autophagy and EV biogenesis by Tsg101

General assessment (Weaknesses):

• Lack of investigation of known ESCRT-independent pathways/ genes involved in the generation of sEVs (i.e., nSMase2/ Ceramide) especially as it is unknown if minor sources of cargo+ EVs are sufficient in maintaining functional phenotype
• Lack of sEV characterization and validation of EVs derived from mutant
• Does not show the loss of cargoes of interest on EVs from mutants other than through back-up of cargoes in the presynaptic endocytic pathway (Rab7, Rab5, Rab11)
• Lack of rigorous investigation of the claim that Evi and Syt4 are released via EVs for proteostatic means is missing. Authors should demonstrate the degradation of EV cargoes by recipient cells (either muscle OR glia)
• If EV-mediated cargo transfer is not required, authors should investigate alternate modes of cargo transfer more rigorously (i.e., diffusion of Wg, suggest/ test hypotheses for mechanism of Syt4 function or transfer).

Advance:

• Compared with other recent in vivo studies of EVs where donor EVs are loaded with a cargo, such as Cre, which uniquely identifies recipient cells through Cre recombination-mediated expression of a fluorescent reporter (Zomer et al 2015, Cell), this study relies on the readout of fluorescently tagged cargo in the recipient cells to represent transfer via EVs. While numerous studies in the Drosophila field focus on the same small set of known EV cargoes at the NMJ (Koles et al., 2012; Gross et al., 2012; Korkut et al., 2013; Korkut et al., 2009; Walsh et al., 2021), there is a noticeable lack of EV characterization based on MISEV (i.e. TEM of EVs, size distribution, enrichment of well-known EV markers [https://doi.org/10.1080/20013078.2018.1535750]) that would significantly strengthen the work and make it more widely accepted in the EV field.
• In this study, the use of ESCRT machinery mutants is proven as a new technical method in delineating the role of EV cargoes in cell-autonomous versus EV-dependent functions. This is the first study, to my knowledge, that has leveraged mutants from both early and late ESCRT complexes for the study of EVs in Drosophila. Additionally, the finding that some cargoes may be able to carry out their signaling functions, independent of transfer via EVs, provides key mechanistic insight into one possible role of EVs as proteostatic shuttles for cargo. This work also begins to address a fundamental question in the field, which is to delineate roles that EVs actually carry out in physiological conditions, compared to the many roles that have been shown possible in vitro.

Audience:

• Basic research (endosomal biology, ESCRT pathway, cell signaling, neurodevelopment)
• Specialized (Drosophila, Neurobiology; Extracellular Vesicles)
• This article will be of interest to basic scientists in the field of endosomal trafficking and extracellular vesicle biology as well as though studying the nervous system in Drosophila melanogaster. As the field of extracellular vesicle biology has broad implications in the spread of pathogenic cargoes in cancer and neurodegenerative disease, the basic biology associated with EVs has some translational relevance.

Expertise (Keywords):

• ESCRT and nSMase2 EV biogenesis pathways
• EV characterization in vitro/ live imaging studies
• EV release and uptake
• Neuronal and glial cell biology

This interaction was really interesting because of I know that girls are more often commented on their appearance. Meanwhile boys are not praised on appearance as much. However the difference was how appearance is even judged on their school work like handwriting or formatting. While I knew this to be true in physical appearance, the way in which their intellectual work is judged was surprising.

This is interesting because students would likely benefit from receiving support and discussion from their teachers. It makes me wonder if teachers maybe scared from sharing their criticism of their students work. And if they are scared, how has and can it affect her students work. In other words, how is it impacting their ability to improve or understand material in the long term if they are not given criticism or little praise from their teachers.

It is interesting to know how girls approach certain classroom behaviors, and how the Sadkers defined these interactions as "girl pause". In other words, this form of acting sis on the basis of ones gender and impacts ones own identity on being able to create their identity. But these means that for educators they would be tasked with creating a certain environment that asks for collective contribution from students.

This point makes me think about how teachers and educators are working to try to improve the level of comfort in this class, and ensure how girls, minorities, and new English speakers are able to participate in a class. While this is a significant role for teachers to tackle, but is necessary for them to address issues that could determine a students ability to develop confidence and academic skills.

I clicked on this link because it's something that I've been thinking about doing. I feel the motivations section captures some of reasons why I want to take a break from social media. Increasing mindfulness, and reconnecting with nature are two strong reasons.

When discussing the effects of social media, the benefits of it are usually not talked about. I think finding an online community is a way to further propel your passions with those with similar interests as you. The community can be a stepping stone leading to in real life event planning, or a place to work together thousands of miles away.

It was important for the author to include this portion of the implications of immortalizing homosexuality, especially in schools because that is where students begin to foster their own ideas and identities. But what is important to point to is how ethnicity or race effects gender and sexuality identity. For communities of color, it is more difficult for young adults to share their identity with their family because of their histories where anything but being heterosexual would cost ones life. This is not to say that it is not prevalent in white populations, however people of color are increasingly at risk of night finding the right resources or support because they are historically underserved.

One experience that dissimilar to the sexualize policing girls in high school both Latinas and Black girls were instantly impacted by this in school. During the hottest days of the year, where our school was located would be in 90s to 110s, and many girls would wear tank tops, shorts, skirts, and other pieces. But many of us would be dress coded because it was "showing too much skin". Our school had bad air conditioning and old classrooms, and with little ventilation being in class would be uncomfortable. However the same was not said to our male counterparts who had shorts. This demonstrated how the dress coding was because we were being sexualized and therefore targeted. The only time were we supported was when a few teachers posted on their doors they would not be taking part in dress code because it was targeting and sexualizing girls.

This becomes even more true once in middle or high school as girls of color are affected by discriminations of their bodies. But the persistence of these practices are created to uplift male and/or white dominated spaces. In other words, aptriahcal systems commonly target girls and women of color as uncontrollable and can only be stopped by a white man. As a result, the controlling of Latina, Black, Asian, and Indigenous bodies are learned by men who teach one another to stereotype girls and women.

The study's focus of Latinas at SCHS to understand their schooling and mechanisms throughout high school is interesting because of the growing popualtion of Latinx students across the country. By focusing on Latinas, it targets both gender, ethnicity, and at time immigration status. More importantly, the focus on gender helps identify how gender roles and forms of oppression begin in public schooling that continue into adulthood. As explained by the author, commonly are sexualized. This creates danger for Latinas, and of course Black, Asian, and Indigenous girls who are sexualized on the basis os stereotypes because of their skin color and connotations.

These tactics are common in schools with predominantly Black and Latino schools in low income communities. As result, many of these students are constrained to the life to prison like schools. This is because of attempts to control crime however, this socializes students who are commonly targeted as being guilty of crimes to be socialized into that role. Instead of providing the right and sufficient resources to improve attendance, or completion of k-12 the they are targeted.

Résumé de la vidéo [00:00:10][^1^][1] - [00:32:18][^2^][2] : Cette vidéo présente une conférence de Jérôme Lang sur le choix social computationnel. Il aborde l'intersection de l'intelligence artificielle, des sciences informatiques, de l'économie et de la théorie du choix social. Lang discute des problèmes de prise de décision collective, de la manipulation de vote, de la démocratie liquide et de l'agrégation des croyances.

Points forts : + [00:00:10][^3^][3] Introduction au choix social computationnel * Présentation de Jérôme Lang et de son parcours multidisciplinaire * Aperçu de la théorie du choix social et de ses applications + [00:07:07][^4^][4] Contribution de l'informatique et de l'IA * Impact de l'informatique sur la résolution de problèmes complexes en choix social * Nouveaux paradigmes et objets d'étude introduits par l'IA + [00:08:14][^5^][5] Démocratie liquide * Explication du concept de démocratie liquide et de ses avantages * Discussion sur les problèmes algorithmiques liés à la démocratie liquide + [00:15:13][^6^][6] Choix social épistémique et crowdsourcing * Application de l'agrégation des croyances au crowdsourcing * Utilisation des votes pour estimer la vérité sur le monde réel + [00:22:55][^7^][7] Vote itératif * Présentation du vote itératif et de son potentiel pour améliorer le bien-être social * Exemple de manipulation de vote pour atteindre un consensus + [00:31:19][^8^][8] Budget participatif * Introduction au concept de budget participatif * Discussion sur la manière dont les votes peuvent influencer la sélection des projets Résumé de la vidéo [00:32:20][^1^][1] - [01:03:36][^2^][2] : La vidéo présente une conférence de Jérôme Lang sur le choix social computationnel, abordant des sujets tels que le budget participatif, la sélection de comités diversifiés et les systèmes de vote. Lang discute des méthodes de vote et de sélection, des problèmes algorithmiques sous-jacents et de leur impact sur la prise de décision collective dans divers contextes.

Points forts : + [00:32:20][^3^][3] Le budget participatif à Paris * Utilisation du vote par approbation jusqu'en 2020 * Les citoyens votaient pour des projets dans leur arrondissement et pour tout Paris * Discussion sur l'efficacité de l'algorithme glouton par rapport à une approche globale + [00:38:14][^4^][4] La sélection de comités * Les défis de la création de comités équilibrés avec diverses contraintes * Exploration des méthodes pour atteindre des proportions cibles ou respecter des contraintes strictes * Exemples de la sélection de comités dans un contexte académique + [00:44:43][^5^][5] Les appariements stables * Présentation du problème de l'appariement stable entre médecins et hôpitaux * Discussion sur l'existence d'appariements stables et leur importance dans les systèmes d'affectation * Examen de la manière dont les préférences des parties influencent la stabilité des appariements + [00:51:15][^6^][6] Les plateformes de vote en ligne * Comparaison entre Doodle et la plateforme de vote en ligne développée par l'Université de Grenoble * Avantages de l'utilisation de plateformes sans publicité et axées sur la recherche pour les décisions collectives

Résumé de la vidéo [00:00:07][^1^][1] - [00:21:52][^2^][2]:

Maël PEGNY discute de l'importance pour les scientifiques sociaux de comprendre le calcul et l'informatique, en particulier dans le contexte de l'apprentissage automatique et de la modélisation des thèmes. Il explore les problèmes génériques liés à l'abstraction, à la validation intuitive des résultats et à la pluralité des formalisations dans la recherche.

Points forts: + [00:00:07][^3^][3] L'importance de la compréhension du calcul * La nécessité pour les scientifiques sociaux de comprendre les outils informatiques * La distinction entre spécification et implémentation * L'impact de l'abstraction sur l'interprétation des calculs + [00:05:03][^4^][4] L'intuition et la formalisation * La traduction des problèmes intuitifs en problèmes formels * La spécification comme guide pour l'exécution des méthodes computationnelles * La validation des résultats computationnels par rapport à l'intuition initiale + [00:11:15][^5^][5] La pluralité des formalisations * Les différentes manières de formaliser un problème intuitif * L'interprétation de la pluralité comme clarification conceptuelle ou échec méthodologique * Les décisions pragmatiques prises lors de la formalisation + [00:15:36][^6^][6] Les concepts opaques et la méthodologie * La difficulté de définir des concepts intuitifs comme les thèmes de recherche * La comparaison entre l'intuition et la formalisation * La question de savoir si la philosophie des sciences a pleinement reconnu ces problèmes méthodologiques Résumé de la vidéo [00:21:53][^1^][1] - [00:44:03][^2^][2] : La vidéo présente une conférence de Maël PEGNY sur l'importance de la compréhension des calculs en sciences sociales, en particulier en ce qui concerne le machine learning et la métaprogrammation. PEGNY discute des défis liés à la formalisation des problèmes intuitifs et à la spécification des calculs, soulignant les ambiguïtés terminologiques et les difficultés d'interprétation. Il explore également la notion de métriques de performance en machine learning et leur rôle dans la formalisation partielle des intuitions de départ.

Points saillants : + [00:22:00][^3^][3] Défis de la formalisation * Difficulté à caractériser les actions sans formalisation explicite * Débat sur la terminologie appropriée pour décrire les processus + [00:23:01][^4^][4] Métaprogrammation en machine learning * Processus de programmation d'un algorithme qui génère un autre programme * Importance de comprendre les calculs sous-jacents à la fois de l'algorithme d'apprentissage et du modèle résultant + [00:24:53][^5^][5] Rôle des métriques de performance * Les métriques guident l'apprentissage et optimisent la performance * Elles constituent une forme de formalisation partielle des intuitions + [00:37:04][^6^][6] Identification des thèmes de recherche * Approche intuitive et non systématique pour déterminer les thèmes * Comparaison entre les résultats intuitifs et ceux générés par le machine learning Résumé de la vidéo [00:44:05][^1^][1] - [01:04:55][^2^][2]:

La vidéo présente une conférence de Maël PEGNY sur l'importance de la compréhension des méthodes computationnelles en sciences sociales. Il discute des défis liés à l'analyse de données et à l'interprétation des résultats obtenus par machine learning, soulignant les limites des approches traditionnelles et la nécessité d'adapter les méthodes statistiques aux complexités des données sociales.

Points forts: + [00:44:05][^3^][3] Choix de la base de données * Importance de la sélection des données * Différences entre autodescription et publications + [00:45:35][^4^][4] Problèmes d'interprétation * Difficultés d'interprétation des résultats * Nécessité d'ajuster les stop words en NLP + [00:48:04][^5^][5] Pluralité des formalisations * Comparaison de différentes méthodes d'analyse * Impact de la complexité des modèles sur l'interprétation + [00:57:52][^6^][6] Épistémologie des statistiques * Questionnement sur la nouveauté des problèmes posés par le machine learning * Pertinence des traditions statistiques pour comprendre l'apprentissage automatique Résumé de la vidéo 01:04:57 - 01:09:09 : La partie 4 de la vidéo aborde la nécessité pour les scientifiques sociaux de comprendre les calculs dans le contexte de l'apprentissage profond et de la statistique. Elle explore le choc que le succès de l'apprentissage profond a provoqué chez les statisticiens et comment cela a conduit à une réévaluation des fondements de la théorie statistique.

Points forts : + [01:05:00][^1^][1] Surprise des statisticiens * L'apprentissage profond a surpris les statisticiens * Reconsidération des fondements de la théorie statistique * Recherche d'une compréhension théorique de l'apprentissage profond + [01:05:49][^2^][2] Nouveautés dues à l'échelle de calcul * Changements qualitatifs avec l'augmentation de la masse des données * Nouveautés apparaissent avec le calcul à grande échelle * Importance de la modélisation formelle des problèmes + [01:07:37][^3^][3] Différenciation des enjeux de formalisation * Distinction entre mathématiques, logique et informatique théorique * La formalisation en informatique diffère de celle en mathématiques * La tradition de méthodes formelles rigoureuses est peu représentée en machine learning

Résumé de la vidéo [00:00:06][^1^][1] - [00:31:01][^2^][2] : La vidéo présente une conférence de Benedict Eastaugh sur la théorie du choix social dans les sociétés infinies. Il explore le paradoxe de Condorcet et les conditions d'Arrow pour une fonction de bien-être social, ainsi que les possibilités offertes par les sociétés infinies pour résoudre ces paradoxes.

Points forts : + [00:00:06][^3^][3] Le paradoxe de Condorcet * Examine la règle de la majorité et ses limites * Présente un scénario où la règle de la majorité mène à une contradiction * Souligne le besoin de conditions supplémentaires pour les fonctions d'agrégation + [00:07:20][^4^][4] Les conditions d'Arrow * Discute de l'unanimité, de l'indépendance des alternatives irrélevantes et de la non-dictature * Explique le théorème d'impossibilité d'Arrow et ses implications * Mentionne le théorème de possibilité de Fishburn pour les sociétés infinies + [00:14:02][^5^][5] Modèles de sociétés infinies * Présente trois types de sociétés infinies : continues, infinies dans le temps et conditionnées par des états possibles * Questionne la pertinence et l'applicabilité de ces modèles * Aborde les défis liés à la prise de décision sociale dans ces contextes + [00:20:52][^6^][6] La non-calculabilité des fonctions de bien-être social * Examine la nature non-constructive des preuves et des fonctions résultantes * Discute de la relation entre les axiomes non-constructifs et le théorème de possibilité de Fishburn * Explore les implications pour les scénarios électoraux et les sociétés infinies Résumé de la vidéo [00:31:04][^1^][1] - [01:03:23][^2^][2]:

La deuxième partie de la vidéo se concentre sur la théorie du choix social dans les sociétés infinies, en utilisant le cadre formel de l'arithmétique du second ordre. Elle aborde les défis de la cardinalité et de l'effectivité, propose des solutions pour modéliser les sociétés comptables et examine les théorèmes de Kerman-Sunderman, Arrow et Fishburne dans ce contexte.

Points forts: + [00:31:04][^3^][3] Cadre formel et reverse mathematics * Introduction à l'arithmétique du second ordre et à la notion de contre-exemple récursif * Lien entre les mathématiques inversées et la théorie de la calculabilité + [00:35:08][^4^][4] Défis de la théorie du choix social pour les sociétés comptables * Problèmes de cardinalité avec un nombre infini de votants * Nécessité de conditions affaiblies pour satisfaire les théorèmes d'Arrow dans le cas fini + [00:45:00][^5^][5] Construction d'une société comptable * Définition des votants, des alternatives et des profils dans le cadre comptable * Conditions pour qu'une collection détermine une société, en termes de mesurabilité et de richesse combinatoire + [00:51:03][^6^][6] Théorèmes dans le cadre comptable * Preuve du théorème d'Arrow pour les sociétés finies dans RCA 0 * Discussion sur le théorème de possibilité de Fishburne et son statut non constructif Résumé de la vidéo [01:03:27][^1^][1] - [01:04:52][^2^][2]:

La partie 3 de la vidéo aborde la théorie du choix social dans les sociétés infinies, en se concentrant sur les résultats de manipulabilité et d'impossibilité dans les systèmes de vote, ainsi que sur les théorèmes de Gibbard-Satterthwaite et d'Arrow.

Points forts: + [01:03:27][^3^][3] Choix social et médicaments * Discussion sur l'indexation des résultats en fonction de conditions * Questionnement sur la plausibilité des états infinis + [01:04:10][^4^][4] Résultats de manipulabilité et théorèmes d'impossibilité * Mention des théorèmes de Gibbard-Satterthwaite et d'Arrow * Analyse des cas infinis et résultats similaires à la compréhension arithmétique + [01:04:45][^5^][5] Nature non constructive des résultats * Évocation de la non-constructivité et de la non-calculabilité * Clôture de la discussion par des remerciements

Résumé de la vidéo [00:00:06][^1^][1] - [00:27:33][^2^][2]:

Célia Zolynski discute de la transition du calcul du sujet à son empowerment dans le contexte de l'intelligence artificielle (IA) et de la régulation des plateformes. Elle aborde les enjeux de la régulation de l'IA, l'importance de protéger les droits fondamentaux face aux risques potentiels, et propose des pistes pour renforcer le pouvoir d'action des utilisateurs.

Points forts: + [00:00:06][^3^][3] Introduction et contexte * Présentation de Célia Zolynski et de son domaine d'expertise en droit du numérique * Importance de la régulation des plateformes et des systèmes d'IA + [00:03:06][^4^][4] Régulation des droits fondamentaux et IA * Approche actuelle de la régulation visant à protéger les droits fondamentaux * Nécessité d'une approche complémentaire pour renforcer le pouvoir d'action des utilisateurs + [00:10:01][^5^][5] Le règlement sur l'intelligence artificielle (AI Act) * Discussion sur le AI Act proposé par la Commission européenne * Classification des risques et mécanismes de mise en conformité + [00:17:01][^6^][6] Empowerment de l'utilisateur final * Stratégies pour améliorer la compréhension et l'interaction des utilisateurs avec les systèmes d'IA * Importance de la transparence et de la jouabilité pour l'empowerment Résumé de la vidéo [00:27:34][^1^][1] - [00:53:16][^2^][2]:

La vidéo présente une conférence de Célia Zolynski, qui aborde le sujet de l'impact des systèmes algorithmiques sur les individus et la société. Elle souligne l'importance de comprendre, débattre et potentiellement contester les contraintes techniques imposées par ces systèmes, en particulier dans le contexte des ressources humaines et de la reconnaissance faciale.

Points forts: + [00:27:34][^3^][3] Comprendre et débattre des contraintes techniques * Nécessité de réflexion collective sur l'utilisation des algorithmes * Importance de la formation pour un débat éclairé * Propositions de la Commission nationale consultative des droits de l'homme + [00:30:10][^4^][4] Droit de contester les décisions algorithmiques * Reconnaissance du droit de contestation en droit des données personnelles * Discussion sur l'accès aux preuves et la présomption de non-discrimination * Importance de la défense des libertés et droits fondamentaux + [00:34:18][^5^][5] Actions collectives pour la défense des droits * Nécessité de procédures pour organiser des actions collectives * Importance des organismes de promotion de l'égalité et de la défense des droits * Propositions pour une régulation plus humaniste de la technique + [00:36:01][^6^][6] Droit au paramétrage des systèmes algorithmiques * Proposition d'un droit de paramétrage pour l'utilisateur * Impact sur les algorithmes de recommandation et les assistants vocaux * Vision d'une interaction plus humaniste entre l'homme et la machine Résumé de la vidéo [00:53:17][^1^][1] - [01:10:10][^2^][2]:

La vidéo présente une discussion approfondie sur le Digital Markets Act (DMA) et son impact sur les grandes plateformes numériques, en particulier en ce qui concerne l'accès aux données et le contrôle du marché. Célia Zolynski explore les défis liés à la qualification des bases de données en tant qu'infrastructures essentielles et les obligations imposées aux acteurs dominants pour promouvoir l'innovation et la concurrence loyale.

Points saillants: + [00:53:17][^3^][3] Le marché numérique et le DMA * Analyse du rôle des grandes plateformes dans l'organisation du marché * Discussion sur les pratiques déloyales et l'effet de verrouillage du marché * Importance de l'accès aux données pour la concurrence et l'innovation + [00:57:03][^4^][4] Paramètres de l'utilisateur et contenu * Exploration de la possibilité pour les utilisateurs de contrôler l'affichage du contenu * Importance du design et de la facilité de modification des paramètres * Réflexion sur les paramètres utiles et déterminants pour les utilisateurs + [01:02:00][^5^][5] Fatigue du consentement et design * Discussion sur la fatigue du consentement et l'importance d'un design encourageant l'interaction * Idées pour préconfigurer les choix des utilisateurs concernant les cookies * Défis liés à l'équilibre entre préinstallation et contrôle utilisateur + [01:07:02][^6^][6] Complexité réglementaire et accès au marché * Analyse de la complexité des textes réglementaires et de leur impact sur les PME * Approche asymétrique imposant plus d'obligations aux grands acteurs * Objectif de ne pas créer de barrières réglementaires à l'entrée sur le marché

Boldfff

In den USA besteht seit langem eine Netzwerk, in dem PR-Firmen mit Unternehmen der Fossilindustrie zusammenarbeiten, Dabei werden u.a. durch gefakete Kampagnen und die Gründung von Umweltgruppen die eigentlichen Stakeholder der Desinformation versteckt. In einem Hearing eines Unterausschusses des Repräsentantenhauses wurden die Praktiken von PR-Firmen aufgedeckt. https://www.desmog.com/2022/09/14/house-committee-pr-climate-disinformation/

Risk factors of afib

The teachers insights into the interactions between boys and girls, and how gender is practiced in classrooms can be troubling. This is because interactions could either be addressed or need to be retaught in order to change see harassment as rites of passage. This is because students would likely replicate these interactions and learned behaviors once older. If unlearned, it would increase and continue normalized behavior of intimate partner violence and aggressiveness.

Verantwortliche der großen US-Fossilunternehmen haben vertraulich die öffentlichen Statements der Firmen zur Dekarbonisierung relativiert und in ihrer Öffentlichkeitsarbeit gegen die von ihnen selbst offiziell vertretene Politik agiert. Das geht aus Dokumenten hervor, deren Herausgabe der Ausschuss des Repräsentantenhauses für Oversight und Reform erzwungen hatte. https://www.nytimes.com/2022/09/14/climate/oil-industry-documents-disinformation.html

discusses the varied meanings of the "Middle Passage" in the Atlantic slave trade. For slave ship captains and crews, it was a segment of the triangular trade route involving goods and enslaved Africans. However, for enslaved Africans, the Middle Passage referred to the arduous ocean journey lasting up to six months aboard slave ships. This journey was the second leg of their trip, following an overland trek to coastal slave-trading factories in Africa and preceding their arrival in the Americas for forced labor. The passage highlights the different experiences and hardships endured by those involved in the slave trade.

describes a significant development in the legal status of Black individuals in English colonies during the 1660s. New laws established lifelong enslavement for people of African descent in places like Virginia and Barbados, leading to the permanent deprivation of freedom and the creation of distinct legal categories for enslaved Africans. These laws solidified strict racial barriers, elevating skin color from a superficial difference to a defining marker of a profound racial division between white and Black populations.

This passage highlights the economic insignificance of North American colonies compared to Caribbean sugar islands within the British Empire. Despite this, the colonies were intricately connected to larger Atlantic trade networks, emphasizing the emergence of a complex Atlantic World that linked Europe, Africa, and the Americas.

:)

In Chile wurde ein progressiver Verfassungsentwurf abgelehnt -– auch aufgrund einer massiven, von Superreichen finanzierten Desinformationskampagne. https://www.theguardian.com/commentisfree/2022/sep/06/chile-new-constitution-reject-pinochet

https://www.youtube.com/watch?v=V4jd6s4Q6TY

So that's where the min-max number nonsense is coming from...

Wrapper classes provide a way to use primitive data types as objects.

Useful when the programmer can only use objects and is restricted from utilising the primitive types as they are.

now also 5D

https://actu.fr/societe/cela-pourrait-meme-devenir-dangereux-interdire-totalement-les-ecrans-aux-enfants-fausse-bonne-idee_61016758.html

UNETR là một kiến trúc mạng nơ-ron kết hợp giữa Vision Transformer (ViT) và 3D convolutions. Dưới đây là các điểm quan trọng về kiến trúc này: 1. Vision Transformer (ViT):

• UNETR là một phiên bản tổng quát của ViT cho 3D convolutions.
• Nó thay thế 3D convolutions trong phần mã hóa bằng multi-head self-attention.

1. Chuyển đổi dữ liệu đầu vào:

   • Dữ liệu đầu vào 3D được chia thành các patch không giao nhau với kích thước 16x16x16.
   • Sau đó, dữ liệu được chiếu vào không gian nhúng (768 chiều) bằng một lớp tuyến tính và kết hợp với positional embedding.
   • Dữ liệu sau đó được xử lý bởi một encoder multi-head self-attention.

Attention U-Net là một kiến trúc mạng nơ-ron mở rộng từ kiến trúc cơ bản U-Net bằng cách thêm một attention gate vào phần giải mã (decoder). Dưới đây là các điểm quan trọng về kiến trúc này: 1. Attention Gate:

• Attention gate được áp dụng trên feature map từ phần mã hóa (encoder) trước khi nối chúng trong khối giải mã (decoder)
• Nó học xem các vùng của feature map từ encoder quan trọng nhất, dựa vào ngữ cảnh của feature map từ khối giải mã trước đó.
• Attention gate thực hiện việc nhân feature map từ encoder với trọng số tính toán. Các giá trị trọng số nằm trong khoảng (0, 1) và biểu thị mức độ chú ý mà mạng nơ-ron đang trả cho mỗi pixel

1. Kiến trúc của Attention Gate:

   • Đầu vào (input features) được nhân với trọng số chú ý (attention weights) (α)
   • Để tính α, input features và feature map từ encoder cùng được biến đổi bằng tích chập 1x1x1, sau đó cộng lại.
   • Tiếp theo, áp dụng hàm kích hoạt ReLU và một lớp tích chập 1x1x1 khác.
   • Cuối cùng, trọng số chú ý được nâng tỷ lệ với phương pháp trilinear interpolation.

Looks complex. Requires online SI OP. Sub-optimal UX.

ALSO YEAAAHH

YEAH

PULLING WORDS OUT OF MY BRAIN BROTHER!!!!!

RELEVANT TO MY THESISSSS!~!!!!!!!!!!!

This is why we required shared conceptualizations of effectiveness and an agreement over sources of evidence

VERY RELEVANT TO MY THESIS

ALSO VERY RELEVANT TO MY THESIS

Language is a clear demonstration of EBM-logic

Why is it strongly activating instead of deactivating

Potential challenge for my argument: narrative research is not well-defined