pendiente para su referencia

Verbindung

Ajouter la référence

They won’t be spending their money at her racist restaurant if they don’t like blacks there, and he’ll make sure to let everyone else know.

The day and people was really busy and they all calmed down and cooled off a little after everything was done

これも

Embeddingしベクトルに変換する

バッチ処理でEmbeddingし、ベクトルに変換する

Joe is a control freak due to his anxieties and insecurities. Now, he sees Janie's defiance of him, and the townspeople's ridicule, as an existential threat to his control. This brings his deep-seated fear of losing control back up to the forefront of his mind.

Reviewer #2 (Public review):

Summary:

Chen and colleagues conducted a cross-sectional longitudinal study, administering high-definition transcranial direct stimulation (HD-tDCS) targeting the left DLPFC to examine the effect of HD-tDCS on real-world procrastination behavior. They find that seven sessions of active neuromodulation to the left DLPFC elicited greater modulation of procrastination measures (e.g., task-execution willingness, procrastination rates, task aversiveness, outcome value) relative to sham. They show that HD-tDCS reduces task aversiveness and increases task-execution willingness on real-world tasks as quantified by intensive experience sampling methods, providing causal evidence for the role of DLPFC in modulating contextual features to delaying or completing one's goals.

Strengths:

• This is a well-designed protocol with rigorous administration of high-definition transcranial direct current stimulation across multiple sessions. The intensive experience sampling approach which probes and assesses self-relevant task goals is innovative and aims to address an important question regarding the specific role of DLPFC in modulating specific features of chronic procrastination behavior (e.g., task-execution willingness, task aversiveness).

• The quantification of task aversiveness through AUC metrics is a clever approach to account for the temporal dynamics of task aversiveness, which is notoriously difficult to quantify.

Weaknesses:

• While the findings that neurostimulation reduces procrastination behavior is compelling, there remain several alternative interpretations for these effects. For example, it could be that the task-execution willingness isn't increased per se, but rather that the goal completion becomes more valuable as participants learn from feedback or become more aware of their successful attainment of or failure to complete task goals. It is unclear whether the effects could be driven by improved working memory or attention to the reported tasks (and this limitation is addressed by the authors). In short, it is also difficult to examine the temporal dynamics of how these goals are selected across time.

• It is unclear whether the current evidence support long-retention of this neurostimulation intervention. The study includes one 6-month timepoint after the study to examine the long-term retention of the neural stimulation effect. Future studies that evaluate the long-term effects across multiple time points would strengthen the evidence for the robustness of this intervention.

Wow the grandmother wasn’t joking that she was going to die soon after Janie got married.

Animals know and can sense the hurricane happen and is real

The involvement of the fortune teller reflects the desperation of the modern era. People nowadays have lost faith in traditional religion so they have the urge to turn to a cheap spirituality and superstitions to find answers. It shows how low society now has fallen, like seeking guidance from a woman with so called "wicked pack with cards" instead of true wisdom of religion.

Dust usually symbolises physical death which is commonly known as "ashes to ashes," but in this line, it feels more like the insignificance of the human existence. The "fear" there is not just about dying but it also about the realization that life might not be nothing more than dry, empty dust without any higher spiritual meaning.

GREAT FIGURE!

This passage gives an important perspective on how social media can blur the boundaries between healthy emotional sharing and “trauma dumping.” It explains how parasocial relationships and large online audiences can make people overshare personal struggles without considering the impact on others. I think the passage is effective because it encourages people to think more carefully about online communication, empathy, and emotional boundaries.

This passage is powerful because it shows how social media algorithms can influence people’s emotions and behavior without them even realizing it. The example of Facebook’s experiment raises important ethical concerns about privacy, consent, and manipulation online. It also makes readers think more critically about how technology affects mental health and daily interactions.

I like this because it connects social anxiety to the mechanics of feeds and likes. You’re not just doing something, but you’re also pre-imagining how it will look online and how people will react. That constant “watching yourself being watched” seems like it could intensify anxiety and make it harder to just be present.

If they're saying that social media detoxes don't work, then what does? I personally feel like I am less connected to others when I am on social media because when I am with other people in real life I am on my phone more, and connecting with others who are in my life less. I think it's pretty disagreeable to say that being on social media means connection and not being on social media means isolation because I think the opposite is true considering that doomscrolling could take up the majority of someone's day.

Testing, testing

6:1is HUUUGE.

过于简单，但是经典，可以作为toy case使用，不可作为大case

考虑做生产时序建模与质量管理相关的研究

考虑与WM811K综合使用，产出复合故障诊断论文

CellProfiler pipelines.

Peer Review File for the first revision.

https://figshare.com/articles/online_resource/Cell_profiler_pipelines_and_reviewer_comments/31753408

https://k51qzi5uqu5dj8hkrwg9fd9g4khdcp2mu5koizmx610l7ov49536s8ldbq2nih.ipns.dweb.link/2026/03/26/indy.%F0%9F%8C%90.0/

http://webui.ipfs.io.ipns.localhost:8080/#/files/%F0%9F%92%BB/thinkpad/%F0%9F%A7%8A/me/%F0%9F%93%93

💻/thinkpad/🧊/me/📓/2026/03/26/indy.🌐.0/

Contrast ratio of 4.5:1 means that the foreground text colour should be at least 4.5 times brighter (or darker) than the background.

You can check on: https://webaim.org/resources/contrastchecker/

It had now been replicated in 💻/thinkpad/🧊/me/📓/2026/03/26

http://webui.ipfs.io.ipns.localhost:8080/#/files/%F0%9F%92%BB/thinkpad/%F0%9F%A7%8A/me/%F0%9F%93%93/2026/03/26/indy.%F0%9F%8C%90.0/

hello

/💻/thinkpad/🧊/♖/hyperpost/1/I/📓/2026/03/26/indy.🌐.0

indy.web.arcive)

indy.web.archive

teaching efficacy superflous

Acknowledges that queer people are not absent from the conversation or research but there is a default to cisheterosexuality as normative.

Addressing the lack of attention to sex, sexuality and gender identity in the original framing rhetorical listening

Remove the first alternative! Interferes with the next exploratory steps!

Not needed in this page at all since we re-direct user to a new Razorpay page

DOI: 10.1186/s40478-026-02240-y

Resource: (Sigma-Aldrich Cat# G3893, RRID:AB_477010)

Curator: @scibot

SciCrunch record: RRID:AB_477010

What is this?

DOI: 10.1186/s40478-026-02240-y

Resource: (EnCor Biotechnology Cat# MCA-5C10, RRID:AB_2572311)

Curator: @scibot

SciCrunch record: RRID:AB_2572311

What is this?

DOI: 10.1186/s40478-026-02240-y

Resource: RRID:AB_3678889

Curator: @scibot

SciCrunch record: RRID:AB_3678889

What is this?

DOI: 10.1186/s40478-026-02240-y

Resource: (EnCor Biotechnology Cat# CPCA-GFAP, RRID:AB_2109953)

Curator: @scibot

SciCrunch record: RRID:AB_2109953

What is this?

DOI: 10.1186/s40478-026-02240-y

Resource: (Enzo Life Sciences Cat# BML-FG6090-0100, RRID:AB_2050678)

Curator: @scibot

SciCrunch record: RRID:AB_2050678

What is this?

DOI: 10.1186/s40478-026-02240-y

Resource: RRID:AB_3678888

Curator: @scibot

SciCrunch record: RRID:AB_3678888

What is this?

DOI: 10.1186/s40478-026-02240-y

Resource: RRID:AB_3711244

Curator: @scibot

SciCrunch record: RRID:AB_3711244

What is this?

DOI: 10.1186/s40478-026-02240-y

Resource: (Agilent Cat# Z0334, RRID:AB_10013382)

Curator: @scibot

SciCrunch record: RRID:AB_10013382

What is this?

DOI: 10.1016/j.isci.2026.115716

Resource: (Cell Signaling Technology Cat# 3671, RRID:AB_330248)

Curator: @scibot

SciCrunch record: RRID:AB_330248

What is this?

DOI: 10.1016/j.isci.2026.115716

Resource: (Thermo Fisher Scientific Cat# A-11012, RRID:AB_2534079)

Curator: @scibot

SciCrunch record: RRID:AB_2534079

What is this?

DOI: 10.1016/j.isci.2026.115716

Resource: (Cell Signaling Technology Cat# 3629, RRID:AB_2249358)

Curator: @scibot

SciCrunch record: RRID:AB_2249358

What is this?

DOI: 10.1016/j.isci.2026.115716

Resource: (Cell Signaling Technology Cat# 2118, RRID:AB_561053)

Curator: @scibot

SciCrunch record: RRID:AB_561053

What is this?

DOI: 10.1016/j.isci.2026.115716

Resource: (Cell Signaling Technology Cat# 13166, RRID:AB_2798136)

Curator: @scibot

SciCrunch record: RRID:AB_2798136

What is this?

DOI: 10.1016/j.isci.2026.115716

Resource: (Cell Signaling Technology Cat# 93065, RRID:AB_2800199)

Curator: @scibot

SciCrunch record: RRID:AB_2800199

What is this?

DOI: 10.1016/j.isci.2026.115716

Resource: (Thermo Fisher Scientific Cat# A-11001, RRID:AB_2534069)

Curator: @scibot

SciCrunch record: RRID:AB_2534069

What is this?

DOI: 10.1016/j.isci.2026.115716

Resource: (Cell Signaling Technology Cat# 2500, RRID:AB_10839118)

Curator: @scibot

SciCrunch record: RRID:AB_10839118

What is this?

DOI: 10.1016/j.isci.2026.115716

Resource: (Cell Signaling Technology Cat# 6943, RRID:AB_10013641)

Curator: @scibot

SciCrunch record: RRID:AB_10013641

What is this?

DOI: 10.1016/j.isci.2026.115716

Resource: (Cell Signaling Technology Cat# 2541, RRID:AB_2174466)

Curator: @scibot

SciCrunch record: RRID:AB_2174466

What is this?

DOI: 10.1016/j.isci.2026.115716

Resource: (Cell Signaling Technology Cat# 3528, RRID:AB_2160882)

Curator: @scibot

SciCrunch record: RRID:AB_2160882

What is this?

DOI: 10.1016/j.isci.2026.115716

Resource: (Sigma-Aldrich Cat# V9131, RRID:AB_477629)

Curator: @scibot

SciCrunch record: RRID:AB_477629

What is this?

DOI: 10.1016/j.isci.2026.115716

Resource: RRID:AB_2291558

Curator: @scibot

SciCrunch record: RRID:AB_2291558

What is this?

DOI: 10.1016/j.isci.2026.115716

Resource: (BD Biosciences Cat# 610467, RRID:AB_2128060)

Curator: @scibot

SciCrunch record: RRID:AB_2128060

What is this?

DOI: 10.1016/j.isci.2026.115716

Resource: (Cell Signaling Technology Cat# 8556, RRID:AB_10891442)

Curator: @scibot

SciCrunch record: RRID:AB_10891442

What is this?

DOI: 10.1016/j.isci.2026.115716

Resource: (Jackson ImmunoResearch Labs Cat# 111-035-144, RRID:AB_2307391)

Curator: @scibot

SciCrunch record: RRID:AB_2307391

What is this?

DOI: 10.1016/j.isci.2026.115716

Resource: (Cell Signaling Technology Cat# 4848, RRID:AB_10694415)

Curator: @scibot

SciCrunch record: RRID:AB_10694415

What is this?

DOI: 10.1016/j.isci.2026.115716

Resource: (Jackson ImmunoResearch Labs Cat# 115-035-068, RRID:AB_2338505)

Curator: @scibot

SciCrunch record: RRID:AB_2338505

What is this?

DOI: 10.1016/j.isci.2026.115716

Resource: RRID:AB_3698765

Curator: @scibot

SciCrunch record: RRID:AB_3698765

What is this?

DOI: 10.1016/j.isci.2026.115716

Resource: (Sigma-Aldrich Cat# A5979, RRID:AB_476749)

Curator: @scibot

SciCrunch record: RRID:AB_476749

What is this?

remove the TOC on top of the page, move to left nav bar below

Local Loop solves the payment gridlock.

- dill green

アニミズム擁護？

Kamenetz’s “Snapchat dysmorphia” – when individuals opt for cosmetic surgery so as to resemble their filtered self-portraits – illustrates how the harms identified by the CIDER analysis can be demonstrated. Users are seeing a manipulated version of what they may believe is an improved version of themselves because of the way in which the filters were built. At no point during the design process does this premise get questioned. Likewise, there is no even distribution of the costs associated with the premises (i.e., those who already have body-image issues or suffer from depression will experience the negative consequences far greater than others). This relates closely to the chapter’s assertion that increasing accessibility to a tool known to produce harm is a morally wrongful action.

In this article they explain that the research on teen's mental health on facebook and instagram are not as accurate since they are self reported and teen's already have an impression that social media can be harmful. They also explain the mixed feelings of social media because studies show that social media is harmful but there are also studies that show that social media is beneficial.

This article breaks down what the new 988 number actually does and why it matters beyond “just another hotline.” It shows how 988 is supposed to fix a big problem with mental health crises going through 911, where people often end up in chaotic ERs or in dangerous encounters with police instead of getting real support. What I found most useful is how it connects the simple idea of a 3 digit number to the huge amount of work behind the scenes: building up local and backup call centers, funding more staff, and creating mobile crisis teams so someone can actually show up and help in person.

This article discussed director Bo Burnham's own issues growing up with anxiety and how they influenced him to make the film "Eighth Grade". Burnham explains that though he did not notice his anxiety in middle school, when the film takes place, it started to affect him starting in his sophomore year. He describes the feeling of pressure to perform because of his popularity gained from YouTube. He then realized that teen girls around the ages of 13-14 related to his feelings, which inspired him to create the film.

This short podcast talks about sharing mental health issues on the internet, and poses questions how memes about mental health can trivialize it, and how posting about mental health can affect your opportunities down the line. It also talks about how you can find support from peers when you post online. One thing I think the story fails to include is the comments you are inviting when you post about your personal mental health online. While you can find support for mental health online, your peers are not professionals, and many are not kind. When you post about your personal mental health online, you invite all sorts of comments, including ones that are hurtful and might make things worse. I personally feel like social media, while it could be helpful for finding support in different communities, shouldn't be used as a reliable outlet for your struggles. People should go to their friends, families, and therapists, and find support in real life first and foremost.

I can't really say that I really like this article- especially item 1, which basically states that one shouldn't blame oneself for their social media addiction because larger, systemics forces are the ones truly driving it. Granted, the part about the systemic forces is absolutely true, but that doesn't mean one should abandon all personal responsibility from the equation. As someone who probably uses too much social media, and is actively trying to reduce their yield, I don't think I would've made progress if I didn't feel bad about myself at least a bit. Of course, one shouldn't overdo and think they're a defective person for not being able to "detox" immediately (again, there were systemic forces working against you) but you shouldn't fall into idea that it isn't, at least, partially one's own fault and therefore within one's personal control.

indy.clip-web.of.documents,bookburners.paradise,internet.archive,digital.vellum,emulate.everything

using inbrowser.link

using dweb.link

https://bafybeibltifg56ikknfje35vf2eul33jzvy2jxopehqse2h2rm2ku5p4ju.ipfs.dweb.link/?filename=IPFS.Alphw.Why.we%3Bmust~YouTube%20Transcript%20Generator%20%EF%BD%9C%20Extract%20%26%20Download%20Video%20Transcripts%20(5_5_2026%203%EF%BC%9A11%EF%BC%9A50%20PM).html

The emphasis by the author of this chapter to avoid generalization when talking about neurotypes, as well as her encouragement to "look at specific traits," shares some similarities with what Socrates said about writing in his Phaedrus (Socrates was concerned that writing addressed all people and nobody in particular, as a result, writing could never be responsive to the person who was reading). Labels for categories, such as "Autism," or "ADHD," work similarly to how writing works, they reduce a spectrum to a single target audience. As a result, using the CIDER approach to break down design into its various components, while looking at each component separately (i.e., trait-specific), can be seen as asking designers to design dialogically rather than categorically. Rather than asking "what needs do autistic users have?", the question would ask something similar to "how does somebody who has a specific sensitivity to motion experience things?". This chapter makes an epistemological claim (that is, a claim about knowledge) quietly, specifically, the author believes that good design, just as good rhetorical communication should, is dependent upon understanding specifics rather than relying on generalizations.

La Peregrina tiene prisa y una cita que no puede revelar. El reloj y el nombre del lugar crean una atmósfera de misterio que invita a preguntarse quién es realmente este personaje y qué la espera en el Rabión

I think that social media has been bad for my mental health when I compare myself to others online, but good for my mental health find I find content that is motivating. I think that although social media platforms can play a part in the mental health of their users, I also think that the content users view/post can play a critical role as well.

I have noticed that social media can have major effects on my mental health. An example of the bad impact that social media can have is cyberbullying. I know I have experienced people on social media using anonymous accounts to harass me, which can influence my self-esteem. However, one positive impact of social media is its ability to connect people. I feel my mood is boosted when I can easily share funny things or text my friends on social media.

I honestly think that the things games do of "You've been online for [insert number] hours; maybe you should take a break" so that users are more self-conscious about the time they spend on social media. I at least think it would work for me, because sometimes, while using social media for a long time, I check the time, realize how much time I've spend on social media, and then I promptly log off.

Interesting data- I have a couple of technical comments/questions. 1. How many cells, and thus EVs, were needed for the lipodomics and proteomics analyses? This information was not included. 2. You compared the B-EV lipodome to the whole cell lipodome and conclude that the B-EV lipodome is most like the plasma membrane. But the whole cell lysate would contain the average of all membranes not just plasma membrane. Can you comment on this?

Make this as a best practices section for higher SR.

ok

People twist it

This whole paragraph is about how he thinks America is just being a hypocrite

He states the obvious about what is making him disappointed.

America gaslights itself to believe in it does know bad or makes mistakes.

Maybe a little bit of this style

Type of leadership style I like

Throughout the Rogers, page they put black subheadings to indicate a separate sections of the page. This is good accessibility, because the text is large, bold and readable. Furthermore because of the layout it helps manage the information presented.

This annotation is primarily for the French option, which is great if you need it. However the implementation of this tool is quite poor and flawed from my view. The tool is regulated in the top corner and in small text resulting in poor visibility. Furthermore, in terms of languages it is limited only have two options of French and English. Keeping in mind Rogers is a national communication service provider in Canada. The tool should have more language options due to the fact that Canada is a country where citizens speak multiple languages, where English or French may not be a persons first language. Overall if Rogers wants to properly serve the people the tool needs to be more flexible, and visible on the website.

This accessibility feature is using identifiable images to indicate the topic for the page link. For example the laptop icon indicating the internet offers Rogers provided. Furthermore, when you however over the icon it highlights the box and underlines the hyperlink. Reinforcing the idea of the selection of the items to the use. However, one flaw of this feature is the font of the text not being bold enough. Due to the fact, someone who struggles with reading words may struggle the front chosen. Overall looking at the accessibility feature this is an effective tool with potential tweaks for improvements.

I am highlighting the slideshow feature of what Rogers has to offer. This tool is effective due to the interactivity of the slide web-based element. In terms of the fact the user has the option to scroll throughout using the bubbles or the arrows, Furthermore, the slide has a feature which you can pause it from moving on, Allowing the user to take in information at their own pace or have more accessibility to click on whatever is shown to the user. Overall, an effective tool for showcasing information to a user!

This is one of the more common accessibility tools implemented recently throughout different companies. This being, getting a chat bot to help aid you with your inquires. This tool can allow users to sort through pages of information. As well as provide an option for additional support if needed. However, this tool is dependent on the user and the keyword they use. Furthermore, with this change, it has been the replacement for human interaction, In some cases the thing that would aid you the most depending on the issues of the user. Overall would say this is good, but in specific cases.

Sdvsfvasfv

Introspective: This passage stood out to me because it uses a simple image to explain something difficult. I also liked the comparison between the mind and a steady flame cause it emphasizes how peaceful and focused the mind can become through meditation.

pictures all over. likely scam.

Introspective: This passage stood out to me because it presents the Divine as active in the world rather than distant.

Introspective: I feel like I relate to this passage because it feels very realistic.

Theological: This description of Krishna’s cosmic form reflects the power and majesty of the Divine.

Theological: This passage reminds me of the Upanishads because the Divine is described as existing throughout all creation. This also reflects the idea that Brahman exists in everything.

Theological: This passage reflects the Divine as eternal and beyond time.

Psychoanalytic: This passage reflects bhakti-yoga because the focus is on devotion and love toward the Divine.

eLife Assessment

This study provides direct and compelling evidence that lamellipodial protrusions dynamically adjust Arp2/3 complex incorporation in response to mechanical counterforces, while also modulating cellular responsiveness to upstream signals like Rac GTPase. By combining endogenous labeling, live-cell imaging, and optogenetic signaling activation, the work demonstrates how adhesion state and physicochemical perturbations reproducibly alter branched actin organization, offering a fundamental advance over previous works. The findings deliver significant insights that will resonate broadly with cell biologists and biochemists studying actin dynamics and mechanotransduction.

Reviewer #1 (Public review):

Summary:

This is an interesting study describing intensity changes of lamellipodial Arp2/3 complex incorporation dependent on the substratum the cells are spreading on (PLL vs fibronectin), but also on manipulation of either contractility or osmotic pressure or even external mechanical load exerted onto cells, e.g., by increasing medium viscosity. The authors use quite fancy cell systems for their studies, first of all, a CRISPR-engineered fibroblast cell line in which both endogenous loci of the Arp2/3 complex subunit Arpc2 are tagged with mScarlet, but at the same time, conditionally removable using tamoxifen. These lines, optionally also harboring Pxn-GFP and Lifeact-miRFP670, have previously been described by the authors (Chandra et al, 2022, PMID: 34861242). In addition, they use cells allowing local photoactivation of Rac signalling through a Tiam1 activation module combined with Halo-tagged Arpc2, apparently stably co-expressed in tamoxifen-treated Arpc2-KO fibroblasts. These cells may or may not have been published previously.

Overall, the study provides convincing evidence that Arp2/3 complex accumulation in the lamellipodium negatively correlates with its width and perhaps the mechanical load these actin networks are exposed to at the leading edge membrane, shown initially through allowing cells to spread on substrates in which the formation of integrin-based adhesions is poor (PLL) or stimulated (through fibronectin). In the latter case, lamellipodia are comparably narrow, perhaps reasonably well clutched, and thus feel sufficient counter-force at the leading edge membrane to build a dense, Arp2/3-dependent actin network. Albeit interesting and important to show as the authors did, these results are not entirely surprising given the literature published on actin remodeling in cells in conditions similar to those used by the authors (i.e., on PLL). Thus, the results should be better embedded into the context of this previous literature to more precisely reveal which aspects are new and interesting and which ones are more or less intuitive and expected.

However, the authors also show yet another result, which is quite spectacular indeed, revealing dramatic local protrusion of a Rac-dependent lamellipodium on PLL only in the presence of methylcellulose, but not on PLL alone. Although the authors cannot fully explain the mechanisms causing these results, they are thought-provoking and will certainly stimulate future, relevant research on this topic and new insights. Altogether, I think this is an interesting study that can be shared rapidly, given that the authors provide more experimental detail and transparency concerning their used cell model systems. Aside from a few other suggestions for amendments and corrections, I would also recommend citing classical literature that has provided the basis for the interpretation of the results shown here, as specified below.

Specific criticism and comments:

(1) I feel the paper is interesting for actin remodeling and Arp2/3 complex aficionados, but quite difficult to read and to understand in places for non-experts in the field, so I think the text requires more detailed explanation of specific terms, model systems used, and overall correction of either grammatical or semantic errors, or colloquial language.

(2) In general, I think the characterization of Arp2/3 complex incorporation into the lamellipodia of cells spreading on PLL versus FN is interesting, as it has not been done previously in such a systematic fashion to my knowledge. However, I think the authors could emphasize better how this relates to previously established structural features of actin filament networks, published on PLL. So more than 3 decades ago, Hotchin & Hall published clear evidence that starved fibroblasts can only form focal complexes or adhesions downstream of PDGF or LPA-stimulation if seeded on FN, but not on PLL (see Figure 1 in PMID: 8557752). Around the same time, Flinn and Ridley showed this virtual absence of classical, Rac-dependent focal complexes to be accompanied by the formation of beautiful, broad lamellipodia (see Fig. 1A in PMID: 8743960), which only formed in the absence of excess RhoA activity and thus contractility by the way (see also below). A few years later, Small et al summarized all these phenotypes in a comprehensive review and also showed that cells on PLL (similar to the rapidly migrating keratocytes) combined large, flat lamellipodia with tiny, nascent adhesions scattered throughout these structures (see Figure 2 in PMID: 10047522). These authors also noted that the sole inhibitor-mediated reduction of contractility could switch FN-phenotypes with narrow, ruffling lamellipodia and peripheral focal complexes back to a PLL-type phenotype of broad lamellipodia (see Figure 1 in PMID: 10047522). In the following decade then, different labs (Verkhovsky, Bershadsky, Vavylonis, Watanabe et al) showed beautiful phase contrast or fluorescence movies illustrating that the broad lamellipodial phenotype of cells plated on PLL was accompanied by low frequency membrane ruffling and instead a rapid, continuous rearward flow of continuously assembling actin filament networks, partly also directly shown with actin networks labeled with both LifeAct and Arp2/3 complex subunits (see e.g. PMIDs 18800171 and 22500749). In Alexandrova et al, 2008 (PMID 18800171), authors showed that the formation of adhesions in spreading cells triggers the transition from fast to slow flow (which is of course relevant to the current study and conclusions), whereas Ryan et al, 2012 (PMID 22500749) already established the broad incorporation of actin and Arp2/3 complex into the very broad lamellipodia formed on PLL by Xenopus fibroblasts and the rapid flow of both components from distal to proximal lamellipodial regions. None of these seminal studies has been cited, although they are highly relevant for the interpretation and conclusions of the results presented. I would strongly recommend specifically referring to these studies, as this will actually support the conclusions and interpretations drawn.

(3) On the subject of literature, on the second page of the intro, end of 2nd paragraph, the authors describe Rac signaling to Arp2/3 complex through WRC considered essential for Arp2/3-mediated actin assembly at lamellipodial leading edges, but aside from one of their own papers cite none of the seminal studies by Insall, Scita, Stradal, Rottner, Bogdan labs having published seminal aspects on this pathway.

Considering the rapid F-actin flow in lamellipodia, obviously accompanied by admittedly sparse but continuous Arp2/3 complex incorporation, it is not so surprising that the latter will be obligatory here, and also the accumulation of its prominent activator WRC, as well as the branch stabilizer cortactin. Thus, the data described on page 3 of the Results section could also be framed in the context of all this previously published knowledge, providing a more comprehensive and realistic view of the relevance and novelty of the described data.

(4) In the abstract, the authors state in the context of the force-feedback mechanism established in vitro for the formation of Arp2/3 complex-dependent actin networks that "this phenomenon has not been explored through the examination of real-time responses of endogenous actin networks in cells". In my view, this is not correct, as in their prominent Cell paper, the Sixt laboratory has done exactly that (Mueller et al, 2017, PMID: 18800171). Although Mueller et al have not looked at Arp2/3 complex dynamics as far as I recall, they have still connected the extent and hence intensity of actin networks at the leading edges of keratocyte lamellipodia with the forces exerted onto them, including direct experimental manipulation of those forces. Although the study has been cited in an independent context, this point should be made clear, and the corresponding sentence in the abstract should be amended.

(5) One point that struck me a little bit was the authors' detailed description of cell spreading on PLL and the quite strong variability of Arp2/3 incorporation dependent on the timing after spreading (as for instance the very strong and quite narrow Arp2/3 leading edge intensity at 2 hours post-seeding in Figure 3S2D). In the authors' view, they have worked with a very clean system, as they emphasized to even have eliminated the FN-locus in their cells, excluding the secretion of endogenous FN (PMID: 34861242), but how about ECM components potentially present in serum, such as, for instance, vitronectin? Indeed, it looks like the authors have done all experiments in the presence of 10% serum as far as I can see, although most of the classical PLL-experiments mentioned above have been performed with starved cells in the absence of serum. I think it would generate a more complete picture of the phenotypes and results as compared to the literature if the authors performed a subset of the key experiments on PLL without serum. I don't think the starving of cells as such is important and could be counteracted by simply lamellipodia-inducing growth factors adding into the spreading medium, traditionally perhaps PDGF or EGF (dependent on the receptor distribution of those fibroblasts), but the absence of serum would have two advantages: it would not only exclude any potential impact of serum-containing ECM components, but also alleviate the hyperstimulation of the Rho-pathway through LPA-bound BSA, the major serum-protein, which has previously been shown to counteract the "undisturbed" formation of PLL-type lamellipodia (see Figure 1B in Flinn & Ridley, PMID: 8743960).

(6) Regarding the scanning EM-images shown in the Supplement, currently called Figure 3S2A and -B (in the text erroneously termed Figures 3S1A and-B, see above). I am not sure how representative these individual EM-images of the cell plated on PLL are, given the data of rapid rearward flow of actin and Arp2/3 complex subunits, at least at early stages of spreading. Again, the classical literature on PLL-type lamellipodia and, in particular, previously published movies of such lamellipodia suggest broad lamellipodia with few ruffles, and the opposite with cells plated on FN. So in this context, the scanning EM-data shown on both PLL and FN do neither fit the authors' own data very well nor the literature, and I would recommend making sure that the individual cells selected were (i) correctly annotated and (ii) representative of a specific time point of spreading actually fitting the previously described data.

(7) It also surprised me to see that the authors describe the spreading process on PLL to actually be much slower than on FN (see Figure 3S2C - in the text Figure 3S1C). It is tempting to speculate that this might change if plating the cells in serum-free medium, as traditionally, full spreading and lamellipodia formation downstream of PDGF-stimulation (at least in 3T3 fibroblasts) is described to occur in the range of 10-30 minutes at maximum, and not several hours as shown here. This point could also be considered, or at least discussed.

(8) The movies are of very high quality and beautiful to look at, but it would help the reader to get a bit more information in the legends (like the meaning of the time-stamps, which will display elapsed time in minutes:seconds I assume, but this info is missing from the legends as far as I can see. Also, it would help the reader to better mark in the movies when a specific treatment kicks in. For instance, in movie 10, the legend states treatment starts at 10:00 (minutes:seconds?), but it would help very much if the authors could paste the term "blebbistatin" directly into the movie, beginning with the frame of treatment start.

Reviewer #2 (Public review):

The authors work with endogenously labeled Arp2/3 complexes in mouse fibroblast cell lines plated on surfaces coated with fibronectin or poly-L-lysine. They observe increased retrograde flow, but decreased actin and Arp2/3 densities, in the absence of integrin-based adhesions. Interestingly, they further find that an increase in branching density can be achieved in the absence of adhesion by a diverse set of perturbations, including blebbistatin, physical compression under agarose, and methylcellulose-mediated increases in extracellular viscosity. Although all of these conditions are likely to have pleiotropic effects on cell physiology and signaling, one plausible common denominator is that they promote cell spreading and may thereby increase membrane tension.

This study addresses a question of broad interest. The relationship between protrusive actin assembly, resisting forces, and membrane tension has received considerable attention in recent years (for a recent overview, see PMID: 38991476). Earlier work established that branched actin networks can respond to force by increasing network density in vitro (PMID: 26771487; PMID: 35748355), and pioneering work from the Sixt laboratory showed that keratocyte lamellipodia adapt to resisting forces by increasing actin density in cells (PMID: 28867286). Against that background, the manuscript contains novel and insightful observations. At the same time, the current version would be strengthened by a more rigorous mechanistic analysis and by clearer reporting of experimental systems and statistics.

Major points:

(1) Engagement with prior work on membrane tension and protrusion.

The relationship between protrusive actin assembly and membrane tension is a subject of major current interest (PMID: 38991476), and it is unfortunate that the authors do not engage more fully with seminal prior work on this subject. In particular, work from the Weiner laboratory showed that membrane tension can act as an inhibitor of cell protrusion and branched actin assembly, at least in some cell types (PMID: 22265410; PMID: 37311454). In addition, a membrane-tension-sensitive signaling pathway involving PLD2 and mTORC2 has been proposed to mediate this negative feedback (PMID: 27280401). These findings appear, at least at first glance, to contrast with the model advanced here, in which elevated membrane tension is associated with increased branching density. A more explicit discussion of these findings and of the apparent differences between systems would be essential. Testing the relevance of some of the proposed negative-feedback regulators, for example, mTORC2 or PLD2, under at least some conditions expected to increase membrane tension would substantially strengthen the manuscript.

(2) The central assumption regarding membrane tension should be tested directly.

Part of the model put forward by the authors rests on the assumption that most of the perturbations used to promote cell spreading, with the exception of hyperosmotic treatment, also increase membrane tension. This is a testable hypothesis. Multiple mechanical and optical methods have been established for this purpose, including tether pulling, micropipette aspiration, and fluorescent membrane-tension probes. Directly measuring membrane tension under at least a subset of the key perturbations would significantly strengthen the manuscript.

(3) WAVE and cortactin localization should be quantified.

The claim that WAVE and cortactin localization are independent of fibronectin-integrin engagement (Figure 2A-B) deserves to be established quantitatively. I appreciate that some variability is expected because these experiments use exogenous fluorescently tagged constructs, but the current presentation relies too heavily on representative kymographs. Quantitative analysis would make this conclusion more convincing.

(4) The interpretation of the increased-viscosity experiments needs stronger physical justification.

I am aware of the recent high-profile work showing that elevated extracellular viscosity can promote migration (PMID: 36323783), and the present manuscript is clearly supporting this. However, the physical basis for this perturbation is neither well reasoned nor explained clearly enough here. The authors use 0.6% methylcellulose of the 1500 cP grade (the relevant viscosity of the final medium should be stated explicitly btw!). Estimating the added viscosity at 7 cP = 0.007 Pa·s (up from 1 to 8 cP), one can formulate the rough back-of-the-envelope calculation for the added viscous stress:

delta τ = delta η v/h

where τ= viscous stress (Pa = pN/µm²), η = viscosity, v= protrusion speed, h = characteristic shear length scale. For cells protruding at 1 um/min, this resistance will be 0.00001-0.001 Pa. Even if the cells would protrude 100 times faster, the resistance would not exceed one pascal! Hence, the added bulk viscous stress opposing protrusion at this viscosity appears negligible relative to the known force-generating capacity of lamellipodia. This does not invalidate the biological phenotype, but it does suggest that the interpretation should be much more careful.

(5) Cell lines and experimental systems are insufficiently described.

Most biological experiments in this manuscript appear to have been performed in engineered mouse fibroblast lines, but the Methods do not provide sufficient clarity about which specific cell lines were used in which experiments. More concerning, the manuscript refers inconsistently to the base model as both a mouse dermal fibroblast line and MEFs, while the only clearly distinct named line appears to be JR20 fibroblasts used for traction-force microscopy. Along similar lines, the Arp2/3 knockout cells in Figure 2 are not adequately explained in the Results, Methods, or figure legends, regarding how these cells were generated or how the knockout was validated. The authors only later note in the Discussion that these conditional knockouts were described in an earlier paper. In general, the manuscript would benefit from much more explicit reporting of which cell line or derivative was used in each experiment.

(6) Some experiments and quantifications appear to suffer from limited replication.

For example, the optogenetic Rac activation experiment in Figure 2E appears to have been performed possibly only for a single cell per condition, since the raw intensity traces are shown without clear indicators of variability. If that reading is correct, this is below the standard typically expected for mechanistic support and seriously reduces confidence in the strength of this particular conclusion.

(7) Statistical reporting needs clarification.

Although the Methods state that the graphs show 95% confidence intervals, the manuscript does not clearly define the underlying statistical unit for many quantified datasets. In several figures, sample sizes are reported as numbers of cells pooled across only two or three independent experiments, but it is not clear whether the authors performed statistical analyses on pooled single-cell measurements or on experiment-level means. The authors should explicitly state for each quantified panel what n represents, what the error bars denote, which statistical test was used, and whether the analyses were performed on per-cell values or on independent experimental replicates.

(8) The Discussion is rather expansive relative to the amount of experimental evidence presented.

Parts of the Discussion feel more speculative and interpretive than necessary, and the manuscript would be strengthened by focusing the Discussion more tightly on the principal findings, limitations, and immediate implications of the work.

Reviewer #3 (Public review):

Summary:

Butler et al. investigated how different force mechanisms influence Arp2/3-related branched actin networks at the leading edge of lamellipodial protrusions in mouse dermal fibroblasts. In particular, their study aimed at characterizing the specific contribution and interplay between load force and adhesion signaling on the regulation of branched actin networks in live-cell experiments using endogenously one-labeled Arp2/3 subunit. A key finding of their work is that by plating fibroblasts on two different substrates supporting or not integrin engagement, they observe striking differences in branched network architectures that cannot be explained solely by integrin signaling. Instead, several of their results point to mechanical feedback resulting from changes in membrane tension during spreading, regulating the density of branched actin networks. Finally, by modifying the extracellular viscosity, the authors suggest that the stress generated at the actin-membrane interface would play a key role in regulating branched actin density in protrusions.

Major Strengths:

(1) The combination of methods used in this paper (endogenous labeling of Arp2/3, Arp2/3 genetic knockout, optogenetic activation of Rac) provides a unique opportunity to monitor spatial and temporal reorganization of endogenous branched networks generated by Arp2/3 in live cells in response to different biochemical and mechanical manipulations.

(2) The authors provide a deep characterization of the actin-network organization and dynamics observed when plating cells on different substrates, engaging or not integrins (Figure 1 and associated supplementary: intensity and width of the signal in protrusions, retrograde flow, incorporation of actin to the edge, nascent focal adhesions), which serves as a strong basis to build the rest of the paper. They also offer a comprehensive analysis of the different parameters that could explain the lack of dense branched actin network at the leading edge of fibroblasts grown on PLL-coated surfaces (they exclude the contribution of reduced branch nucleation by NPF or insufficient branch stabilization in Figure 2, the insufficient integrin-mediated signaling activating NPF in Figure 2).

(3) After having ruled out the influence of adhesion signaling in the regulation of branched actin-network density at the leading edge of the cells, the authors demonstrate that the enrichment of Arp2/3 at the leading edge is evolving together with cell spreading, suggesting a possible role of membrane tension in the process (Figure 3 and associated supplementary). To prove their point, they tested numerous methods to promote adhesion-independent cell spreading (Figures 4 to 6), while describing well the limitations of each of these techniques. These methods included promoting rapid spreading on PLL-coated substrate using blebbistatin or physical compression under agarose, and finally increasing extracellular viscosity by treating cells with methylcellulose. All of these treatments led to very consistent results upon the increase in membrane tension, supporting the idea of membrane tension controlling the branched actin organization of cells. This conclusion was further supported by an experiment (Figure 4 S1) in which a hyper-osmotic shock was performed, increasing the actin-membrane interface stress while keeping the spreading area of cells, which led to a drastic increase in Arp2/3 density at the protrusions.

(4) By activating Rac optogenetically in cells plated on PLL treated with methylcellulose (Figure 8), the authors observe the formation of robust protrusions enriched in Arp2/3, showing that increased extracellular viscosity can bypass the requirement for ECM proteins to activate protrusion driven by signaling.

Weaknesses:

(1) Although the lamellipodial architecture in cells plated on PLL appears very different from the one developed by cells grown on fibronectin (Figure 1, wider and less homogenous), the branched network is still present, and one may wonder how these differences can affect the functionality of the lamellipodia (for example, by measuring the impact on migration in 2D and 3D systems).

(2) To explain the differences observed in the branched actin networks developed by cells on PLL and FN, the authors envision several hypotheses, among which signaling factors or branched-promoting factors would be decreased in the absence of integrin adhesions. They could have, in addition, assessed actin network dynamics and turnover (we could imagine that competition between Arp2/3- and non-Arp2/3- driven structures could be different in the presence or absence of adhesions, the competition being nicely visible from Figure 2B and 2C, where, in the absence of Arp2/3, cells form prominent filopodia).

(3) All of the methods used to apply physical forces on barbed ends have their own caveats and alter not only membrane tension (but the limitations are discussed in the paper). The paper may have benefited from micropatterning the cells to either reduce or force the spreading of cells in a controlled fashion. In addition, the conclusions on levels of interface stress between plasma-membrane and the barbed-ends of actin lamellipodial networks rely on an estimate of the effect of perturbations rather than on actual measurements of these stress levels.

Likely impact of the work on the field, and the utility of the methods and data to the community:

Although the finding that branched actin networks respond to the application of physical force by increasing their density was already known from previous in vitro studies, this paper offers a detailed and compelling characterization of the reorganization of endogenously labelled branched actin networks upon different mechanical perturbations. In addition to showing the effect of increased extracellular viscosity on promoting branched actin network densification in the absence of ECM, this paper sheds new light on the interplay between signaling and mechanics in regulating protrusion and spreading. While the authors show that both signaling and mechanical feedback are important regulators of branched actin regulation and cell spreading, they demonstrate that optogenetic Rac activation is not sufficient to trigger branch network formation in the absence of sufficient mechanical support. They thus propose that biochemical signaling would act at a different level than mechanics by promoting protrusion persistence and coherence. This work will therefore impact the field of cell biology in offering a new perspective to understand the interplay between mechanical and biochemical feedback in 2D and 3D migration. It may also have broader implications as the formation of branched actin networks under the regulation by mechanical loads has been shown to be involved in other processes such as endocytosis.

Psychoanalytic: This passage reflects jnana-yoga cause it focuses on wisdom/self-realization.

Psychoanalytic: This specific passage helped me understand karma-yoga as acting without attachment to the results.

eLife Assessment

In this study, Yuan and colleagues perform transcriptomic and epigenomic experiments to study open chromatin regions and transcripts that change upon larval settlement in the sponge Amphimedon. The authors present compelling evidence to show that sponge larvae prepare for receiving an environmental cue (sunset) by extensively modifying their chromatin accessibility in the vicinity of genes that are going to be regulated during metamorphosis. The study represents a fundamental advance in understanding the fine genetic control of larval settlement and has significance beyond the immediate field of sponge larval biology.

Reviewer #1 (Public review):

Summary:

Yuan and colleagues present a thorough study of gene activation before and during metamorphosis in sponge larvae, combining in-depth analyses of staged transcriptomes and chromatin accessibility profiling (ATACseq). Amongst several very interesting findings, the study reveals that the acquisition of settlement competence, which arises in response to decreasing light at sunset, is characterized by changes in chromatin accessibility that anticipate strong transcriptional shifts occurring as metamorphosis starts. Another notable finding is a set of transcription factors amongst the genes strongly up-regulated at the onset of metamorphosis. In addition, larvae exposed to constant light, a condition that stalls metamorphosis, were found to activate metabolic pathways that are not normally expressed in swimming larvae. Together, the findings provide a rare level of understanding into how environmental conditions can promote deployment of alternative developmental programs in planktonic larvae.

Strengths:

This is a very comprehensive, well-documented and rigorous study of a phenomenon of wide interest. It will inspire researchers working on other species to look for similar, environmentally-driven "anticipatory" epigenetic mechanisms. It also provides a wealth of detailed information on genes, notably transcription factors, that are candidates for involvement in regulating specific metamorphosis transitions - and beyond. The data presented here are thus undoubtedly a rich and valuable resource.

Weaknesses:

I see no significant weaknesses; however, the documentation of the data is very compressed, with all the findings contained in 4 multi-panel figures with succinct legends. It is not always straightforward to connect the conclusion statements in the text to the figures. Although the relevant data is available in supplementary files, I would appreciate more help in navigating the data to assess the support for key conclusions, if possible, illustrating each text conclusion explicitly in the main figures.

Reviewer #2 (Public review):

Summary:

It is demonstrated that sponge larvae prepare for receiving the environmental cue (sunset) by extensively modifying their chromatin accessibility in the vicinity of genes that are going to be regulated during metamorphosis, in the absence of large gene expression changes. This program can be offset by modifying the cue (making light constant), leading to a novel molecular state.

Strengths:

This is a top-notch study of a key lifecycle transition in an organism of great phylogenetic importance, involving concurrent gene expression and chromatic accessibility profiling (to the best of my knowledge, this has never been done in non-bilaterians and likely anywhere outside Vertebrata). The result is highly non-trivial. There is also an additional experiment modifying the key environmental cue (constant light), adding additional insight.

Weaknesses:

I have only a couple of suggestions.

(1) Not all new pre-emptively opened OCR regions are associated with genes that are going to be regulated during metamorphosis. Is their association with such genes statistically significant? (Fisher's exact test?)

(2) Re: extended discussion on possible reasons for activation of specific transcription factor families. I feel it is not terribly useful since it is hardly more than guesswork. The authors should consider condensing this part to better emphasize the major (and most unexpected) large-scale regulation patterns.

(3) Re: enrichment analysis based on significant genes (Figure 1H): Even though it is a common practice, there is nuance: as we all know very well, many genes pass a significance threshold not because they are highly differentially regulated (i.e., show large fold-change), but because they are more abundantly expressed overall and so the statistical power for them is greater. A good example is ribosomes - before we realized what was happening, they would show up as enriched in almost every experiment of ours, which was not very useful since their fold-change was quite trivial. I see the authors have ribosome enrichment too, and I suspect there are a few more functional groups that made it because they tend to express highly on average. Ideally, we want to see what is enriched among highly regulated genes, not among abundantly expressed genes. Because of this we moved to compute enrichment based only on fold-change, using the GO_MWU package (https://github.com/z0on/GO_MWU). I suggest authors give it a shot, to see if the enrichment results become more interpretable. GO_MWU is also very powerful to analyze enrichment in WGCNA modules, in case the authors want to try that.

Reviewer #3 (Public review):

Summary:

In their manuscript, Huifang Yan and colleagues perform RNA-seq (CEL-seq) and ATAC-seq experiments to profile the transcriptome and chromatin accessibility of sponge larvae across larval competence, settlement and early postlarval development. Amphimedon, the sponge species that they use, is amenable to lab experiments and can therefore be a convenient model for experimenting with this otherwise difficult to assay ecological parameters and cues. They had previously observed that light conditions (diminished light) at sunset are critical for larvae to enter a pre-settlement stage and prime them for settlement and metamorphosis. In this paper, they report that these conditions induce a gain of accessibility in many genes, including transcription factors, and that altering these conditions by providing continuous light at sunset affects this reprogramming event.

Strengths:

The above is a very interesting observation, one that the authors speculate could have a broader significance and be a theme in many more larvae. I agree with the authors that this is an important finding, and I think that the paper will be interesting for a broad readership. If this is the case, the authors open up a new theme of chromatin regulation, extensively studied in mammalian contexts, but severely understudied in pretty much every other context.

Weaknesses:

I think, however, that their paper often reports the data in a difficult-to-follow way, and that other sorts of analyses would have made the results more accessible for a broad readership. Here, I present some suggestions that the authors might want to take into account to improve their results.

eLife Assessment

This large-scale comparative study of odorant receptor (OR) genes across more than 100 insect species, combining sequence- and structure-based approaches, aims to explore the evolution of this large gene family involved in the detection of odorant signals by olfactory neurons. This useful work uncovers a structural feature unique to the odorant receptor co-receptor Orco that reduces ligand binding affinity. However, the strength of evidence is incomplete: the pipeline for in silico identification of odorant receptor genes lacks validation through comparison with known odorant receptor repertoires from previously studied species, and claims regarding odor response spectra, evolutionary, and ecological interpretations are not fully supported by the analyses.

Reviewer #1 (Public review):

Objectives of the study and impact of the work:

The authors of this article primarily aim to reconstruct the evolutionary history of the insect odorant receptor (OR) family, which is responsible for the detection of odorant signals by olfactory neurons. Due to the lack of phylogenetic signal present in the sequences of this multigene family, which evolves very rapidly, phylogenetic analyses have so far never made it possible to precisely retrace how ORs diversified prior to the appearance of present-day insect orders, and what the drivers of this diversification were. For example, one may suspect that the adaptation of ORs to odors emitted by plants constituted a critical step in insect evolution during the "angiosperm terrestrial revolution," which occurred at the end of the Cretaceous, but nothing currently allows this to be asserted.

There are very nice examples, notably in Drosophilids, derived from comparisons between closely related species and documenting mechanisms of OR adaptation to certain signals. However, what the authors attempt to do in this work is to produce a macroevolutionary analysis at the scale of insects as a whole, based almost exclusively on bioinformatic analyses. To do this, they annotated OR genes in about one hundred insect species and developed pipelines for analyzing sequence similarity, structural similarity, and functional similarity, the latter being estimated through a molecular docking approach. An important feature in the evolution of insect ORs is the emergence of a unique co-receptor, called Orco, which appears to be an OR that has lost the ability to bind odorants. In addition to the large-scale bioinformatic analysis, the authors also aim to explore more specifically the factors that favored the emergence of Orco and the selective advantage conferred by the existence of OR-Orco complexes.

Given the importance of odorant receptors in insect biology and in their adaptation to different environments and lifestyles, retracing their evolutionary history is indeed a major question in evolutionary biology. In principle, this type of work therefore has the potential to become a reference in the field and to provide a basis for significant scientific advances.

Major strengths and weaknesses:

The sampling chosen for collecting OR sequences is very impressive, with more than 100 insect families represented, covering most of the major orders. This sampling appears appropriate for the question being addressed. The analysis pipeline used to collect the sequences makes sense, relying on homology-based annotation tools coupled with a structure-based filter. Nevertheless, one can note aberrant numbers of ORs for certain species (much lower than reality), which indicates that the pipeline probably did not function correctly for all genomes. In the absence of a validation step comparing the results with already known OR repertoires, it is difficult to estimate the overall quality of the data. The authors chose to apply a fairly stringent filter on sequence quality (based on predicted 3D structure), which reduces the number from 14,000 to 9,000. This choice seems logical given the subsequent use of these data, but it inevitably leads to data loss. The fact that some OR genes may be missing and that the total number may not be exact for each species is not prohibitive for studying the evolution of the family at a broad scale; however, it calls into question certain results that rely on this total number, such as the correlation between the number of ORs and genome size, lifestyle, and diet.

From the dataset collected, the authors attempted to categorize ORs in several ways, starting with the reconstruction of sequence similarity networks. The approach is interesting, but in the end, the results do not seem to be sufficiently exploited, and it is not obvious what the advantage of this approach is compared with the "classical" phylogenetic approach, which generally fails to reveal homology relationships between ORs from species belonging to different insect orders. Here again, the majority of the clusters identified are "order-specific," and when this is not the case, the authors did not attempt to exploit the results. For example, clusters SeqC26 or SeqC28, which appear to be shared by many insects, are potentially very interesting. It might have been relevant to combine this similarity-based clustering approach with phylogenetic reconstructions within each shared cluster.

The clustering based on structure also leads to the identification of a majority of "order-specific" clusters, but once again, the clusters shared by several orders are not truly exploited, which does not provide new insight into the evolution of ORs. However, the authors highlight a group of ORs in flies that appear to possess an unusual intracellular region. This is interesting, although it is a result more relevant to OR structure than to their evolution. The function of these ORs in Drosophila melanogaster, if it is known, is not discussed.

The analysis of structural diversity then leads the authors to focus on the Orco co-receptors, which are characterized by modifications of the binding pocket and the emergence of an extracellular loop that could explain the loss of the ability to bind odorant molecules. This part, which relies on in vitro experiments, is interesting and constitutes the most striking result of the study, which could in itself have been the subject of a separate manuscript. However, the molecular dynamics modelling does not add anything in the way it is conducted (5 ns is too short).

The rest of the manuscript is based on the prediction of OR response spectra using molecular docking. The work that has been carried out is extremely substantial, and the objective of linking clusters based on sequence similarity or 3D structural similarity with functional categories is entirely relevant. Nevertheless, I see two major problems with this in silico functional analysis:

(1) The docking score threshold used was chosen thoughtfully, which is very good, and according to the calculation performed, should ensure a true positive rate of more than 20%, which is excellent in such a docking analysis. But in the absence of functional validation, this 20% true positive rate is not sufficient to extrapolate OR function as the authors do in the remainder of the manuscript. The risk of error remains too high to compare in such detail the function of ORs from insects with different lifestyles or diets.

(2) The six functional clusters identified are only slightly different from one another, with similar detection of all chemical families except acids and amines (which was expected, given that these families are a priori detected by IRs rather than ORs). This shows that even though the approach is relevant and deserves to be tested, it cannot be used to establish a link between groups/lineages of ORs and response spectra at the scale of insects as a whole. This is reflected in the final analysis by the fact that there is no visible link between sequence or structural clusters and functional clusters. Given the uncertainty surrounding the docking results, the entire subsequent analysis of the relationship between the Binding Breadth Index and ecological variables is highly questionable.

Finally, the evolutionary analysis proposed to conclude that the work suffers from an incorrect interpretation: ORs of non-holometabolous insects cannot be considered equivalent to those of species that existed before the Permian-Triassic extinction. The fact that a locust or a cockroach has more narrowly tuned ORs than holometabolous insects does not mean that this was also the case for ancestral insects. To advance this type of conclusion, it would be necessary to conduct a phylogenetic analysis and reconstruct ancestral states, which is not the case here.

In summary, despite the large number of analyses performed, the authors do not succeed in achieving the stated objective of reconstructing the evolutionary history of insect ORs, and the results obtained do not sufficiently support the conclusions regarding the links between OR repertoires and environment or lifestyle.

Reviewer #2 (Public review):

The remarkable evolvability of the olfactory system enables animals to rapidly adapt to dynamic and chemically complex environments. Over the past two decades, substantial effort has been devoted to uncovering the evolutionary principles that drive the diversification of odorant receptors (ORs), yielding key insights into the forces shaping their striking variability in both vertebrates and insects. In this manuscript, Zhang and colleagues analyze the OR repertoires of over 100 insect species, leveraging sequence and structural similarity to infer patterns of gene family evolution within this diverse and ecologically important clade. By integrating sequence-based and structure-based comparisons, their study builds on a compelling and recently emerging line of research made possible by the advent of AlphaFold, which has previously clarified the phylogenetic relationship between insect Ors and the gustatory receptor gene family and revealed the unexpectedly deep evolutionary origins of this ancient structural fold.

Applying this approach to a large set of ORs derived from species throughout the insect phylogeny, the authors confirm many previously reported patterns of OR evolution. Unfortunately, the way these results are presented lacks clarity in what is already known from previous work in the field versus what is a novel finding based on the analysis of this dataset.

It is unclear how complete the odorant receptor sets are. I recommend benchmarking the pipeline by comparing its output to a gold standard and a frequently vetted complete OR set, such as that of Robertson and Wanner 2006 or similar.

Using their structural clustering approach, the authors identify a structural feature mostly unique to the OR co-receptor ORco, a beta-sheet in EL2, which they functionally show reduces odorant binding affinity - a key aspect of ORco, which does not bind ligands in the ancestral ligand-binding site. This is a particularly strong part of the manuscript, since the authors support their in silico-derived hypothesis with functional data.

Lastly, in an attempt to assess the relationship between sequence identity and structure on one hand and function on the other, the authors perform an in silico structure prediction and chemical docking analysis. As it stands, this part is on the more speculative side since the docking approach has not been verified with available functional datasets.

eLife Assessment

The study presents useful findings on the behavioral effects of nicotine exposure, suggesting the Drosophila larva as a potential model organism for studying underlying neural circuits. However, the evidence supporting the claims of the authors is incomplete and would benefit from more rigorous analysis and explanations. The study falls short of identifying the neural mechanisms and is therefore of interest to those with an interest in pharmacology and behavior.

Reviewer #1 (Public review):

Summary:

Dancausse et al. investigate behavioral responses to nicotine exposure in Drosophila larvae. They discover that high concentrations of nicotine lead to less movement and twitching, which recover slowly after several hours. Exposure to lower concentrations, however, increases locomotion and leads to hyperactive behavior. The authors also perform pharmacological and genetic manipulations to address the role of dopamine for these behavioral changes. Additionally, they test the role of MB intrinsic neurons by genetic silencing. Both Dopamine and MB manipulations affect responses to nicotine exposure. Finally, they investigate how larvae respond to repeated exposures to nicotine and find that they do not habituate. Additionally, repeated exposure to nicotine leads to a preference towards higher concentrations in a gradient assay.

Strengths:

The authors use rigorous behavioral analysis and discover interesting concentration and experience-dependent effects of nicotine exposure on locomotion in fly larvae, which will be worth investigating in the future to decipher the underlying neural mechanism.

Weaknesses:

As the manuscript currently stands, the results of genetic manipulations are hard to interpret and rather inconclusive. The genetic manipulations have been performed using broadly expressing genetic driver lines, which weakens the conclusions drawn by the authors. Thus, no specific neural populations or brain regions have been discovered, and there is little insight into the underlying neural mechanism.

Based on gradient experiments, the authors suggest that fly larvae could serve as a model organism for addiction. This claim is quite strong, but no control experiments are shown for shorter exposure or a single exposure with a longer resting period before the gradient test. To compare this to addiction-like behaviors, more control experiments should be performed.

The authors should clarify better how experiments were performed in Materials and Methods. Generally, the authors perform novel behavioral analysis, which is not explained in enough detail. The nicotine concentration that has been used for most experiments is this a relevant concentration comparable to other studies? This information would be useful to put into context with other findings.

Reviewer #2 (Public review):

Summary:

CNS function relies on a balance of excitatory and inhibitory activity. Use of addictive stimulants such as nicotine results in a chronic imbalance of these activities, and often this activity acts through dopamine pathways. To address how stimulants cause dysfunctional signaling in the DA neurotransmitter system and how this impacts neural circuit activity and behavior, the authors of this study begin to establish Drosophila larvae as a model for studying nicotine exposure.

They focus on three questions:<br /> (1) In what ways does nicotine-driven hyperactivation modulate behavior?<br /> (2) What roles do neural circuits play in these responses?<br /> (3) What are the mechanisms of drug dependence and addiction-like plasticity?

To this end, the authors use high-resolution behavioral, genetic, and pharmacological methods.

The authors show that exposure to nicotine alters the behavioral repertoire of larval Drosophila, with effects that are long-lasting (hours) and dose-dependent. Most of the study uses a 5-minute exposure to "moderate" levels of nicotine because this dosage produces the greatest potentiation of larval crawling speed. Concomitant with increases in crawling speed, they find alterations in other behavioral parameters-crawl "efficiency" and turn rate are reduced; whereas head swings are faster and more likely to be accepted. They find that reducing the activity of dopaminergic neurons reverses the valence of behavioral change upon exposure to nicotine. For example, crawling speed is decreased upon nicotine exposure in a Ple>Kir2.1 manipulation in comparison to controls. Moreover, they demonstrate that the effect of nicotine on the quantified set of behaviors depends on dopamine signaling. Beyond implicating dopamine signaling, they implicate the mushroom body, and particularly the gamma-neurons, in mediating exposure to nicotine.

The authors further probe how nicotine exposure alters larval behavior. First, they determine what happens to crawling speed with multiple exposures, finding sustained higher crawling speeds relative to controls. Second, as a model for addition-like behavior, they examine larval behavior on a nicotine gradient after repeated nicotine exposure. The data in Figure 7D are particularly compelling, showing that after nicotine exposure, larvae prefer high concentrations of nicotine.

Strengths:

In a concise set of experiments, the authors demonstrate a nicotine-induced behavioral change, its interaction with a neurotransmitter system, and a locus of action within the CNS. Thus, the authors set the stage for the use of Drosophila larvae as a model to better understand addiction-related behaviors.

Weaknesses:

This is a clear advance for the field of larval neurogenetics, but the extent to which it changes the way we think about nicotine exposure more generally is less clear. Nonetheless, the authors clearly achieved the goal they set out to attain.

Reviewer #3 (Public review):

Summary:

Dancausse et al. examine behavioral responses to nicotine administration in larvae. The study first distinguishes between spasms and extreme hyperexcitability elicited at high doses from a hyperactivity state triggered at lower (~1 mM feeding) doses. They then focus on the hyperactivity state and examine if dopaminergic neuron function is involved (via transgenic and pharmacological manipulations). Next, the role of the Mushroom body, a site of integration in the larval brain, is interrogated. In these studies, the authors use multiple approaches to draw complementary conclusions. The last section examines the effect of repeated nicotine exposure and of nicotine preference following repeated exposure. The findings are foundational for future studies looking to use Drosophila larvae as a system to study nicotine addiction.

Strengths:

Overall, I think the study is of broad importance. The neurogenetics community gets valuable insight into how ACh excitation interplays with DA signaling to regulate movement. For the addiction community, the work describes a valuable system to further interrogate genetic and environmental factors potentially driving addiction under well-controlled conditions. The quantitative analysis is generally well done, and the use of multiple experimental strategies to buttress conclusions is commendable.

Weaknesses:

(1) Conceptual point. Insects use ACh as the primary excitatory neurotransmitter, with nAChRs broadly expressed, while vertebrates use Glutamate in this role. (Arguably, nicotine expression in tobacco plants evolved as an insecticide, broadly disrupting the central excitatory neurotransmitter.) In vertebrates, central ACh neurons are relatively sparse - primarily originating from the basal forebrain.

Based on these distinctions, it is important to consider/contrast nicotine-driven hyperexcitation from other methods to produce broad hyperexcitation (e.g., inhibition of GABA, high K+, elevated temperature, etc). Many of these methods to induce hyperexcitability would also modulate DA circuitry.

A discussion of the role of ACh in insect vs. vertebrate brains is necessary to interpret the experimental design and findings with regard to addiction. These points should be addressed in the intro and discussion.

(2) (Figure 1) Relatedly, how do the behaviors elicited in Figure 1B (30 or 60 mM) compare to the convulsions described following electroshock stimulation to induce a seizure? My suspicion is that you're essentially triggering a seizure (or seizures) in these larvae.

(3) (Figure 4) Is a statistical analysis between the CS, Ple>Kir, Ple, and Kir locomotion at baseline done? Presumably, these manipulations would alter the intrinsic activity levels of the larvae?

(4) (General quantitative question) How do the parameters co-vary across individuals following nicotine admin? Crawl speed and peristalsis frequency are analyzed. Turning doesn't seem to be considered. Do individuals that show large increases in velocity also show the largest reductions in turn rate? Are these relations preserved following the DA metabolism and MB function interventions?

(5) (Discussion / general question) Beyond DA, other monoamines are involved in regulating larval locomotion - OA and TA are a clear example from Fox et al. (2006). Could the authors comment on whether they would expect similar findings in other neurotransmitter systems or if these neurotransmitter systems are involved in the ACh -> DA interplay studied here?

(6) (Discussion) Following the establishment of nicotine preference, do larvae exhibit signs of 'withdrawal' or changes in baseline behavior when deprived of nicotine? For example, in Figure 6, does the speed following nic administration ever 'go below' the H2O line?

Author response:

We appreciate the extremely helpful feedback from the reviewers and editors for our manuscript. We are happy that the reviewers have appreciated what we are doing here, performing the initial work that should set the stage with Drosophila larva as a model for hyperactive stimulant response. Every comment is certainly addressable within a reasonably short time period and we look forward to improving our paper in an upcoming revision.

We have some confusion about the “fundamental issue” of using nicotine, as we see the excitation as the fundamental effect we are studying, but we can continue to discuss and clarify this.

We plan to make significant edits to our introduction and background sections to better frame the goals of the work, and will clarify and expand on our methods, and more carefully make any claims about neural mechanisms.

露台

垂柳

巨型拱形纪念建筑

拆除

宴会厅

Ziervogel, G. (2019). Unpacking the Cape Town drought: Lessons learned. WIREs Water, 6(5), e1354. https://doi.org/10.1002/wat2.1354

Venter, Z. S., Shackleton, C. M., Van Staden, F., Selomane, O., & Masterson, V. A. (2020). Green apartheid: Urban green infrastructure remains unequally distributed across income and race geographies in South Africa. Stockholm Resilience Centre. https://www.stockholmresilience.org/publications/publications/2021-01-07-green-apartheid-urban-green-infrastructure-remains-unequally-distributed-across-income-and-race-geographies-in-south-africa.html

Ziervogel, G. (2019). Unpacking the Cape Town drought: Lessons learned. WIREs Water, 6(5), e1354. https://doi.org/10.1002/wat2.1354

McCauley, M. (2019). Water rights: Day Zero. UCLA School of Law. https://law.ucla.edu/sites/default/files/PDFs/Academics/McCauley-Water%20Rights%20and%20Day%20Zero.pdf

McCauley, M. (2019). Water rights: Day Zero. UCLA School of Law. https://law.ucla.edu/sites/default/files/PDFs/Academics/McCauley-Water%20Rights%20and%20Day%20Zero.pdf

McCauley, M. (2019). Water rights: Day Zero. UCLA School of Law. https://law.ucla.edu/sites/default/files/PDFs/Academics/McCauley-Water%20Rights%20and%20Day%20Zero.pdf

World Energy Council. (2019). Drought in South Africa: Extreme weather conditions (September 2019). https://www.worldenergy.org/assets/downloads/Drought_South_Africa_-Extreme_weather_conditions_SEP2019.pdf

Venter, Z. S., Shackleton, C. M., Van Staden, F., Selomane, O., & Masterson, V. A. (2020). Green apartheid: Urban green infrastructure remains unequally distributed across income and race geographies in South Africa. Stockholm Resilience Centre. https://www.stockholmresilience.org/publications/publications/2021-01-07-green-apartheid-urban-green-infrastructure-remains-unequally-distributed-across-income-and-race-geographies-in-south-africa.html

World Energy Council. (2019). Drought in South Africa: Extreme weather conditions (September 2019). https://www.worldenergy.org/assets/downloads/Drought_South_Africa_-Extreme_weather_conditions_SEP2019.pdf

World Energy Council. (2019). Drought in South Africa: Extreme weather conditions (September 2019). https://www.worldenergy.org/assets/downloads/Drought_South_Africa_-Extreme_weather_conditions_SEP2019.pdf

Macrotrends. (n.d.). South Africa GDP (gross domestic product). https://www.macrotrends.net/global-metrics/countries/zaf/south-africa/gdp-gross-domestic-product

Macrotrends. (n.d.). South Africa GDP (gross domestic product). https://www.macrotrends.net/global-metrics/countries/zaf/south-africa/gdp-gross-domestic-product

U.S. Department of Agriculture, Forest Service. “Record of Decision: Arizona Snowbowl Facilities Improvements Final EIS and Forest Plan Amendment #21.” 2005.

Rodgers, Dennis, and Bruce O’Neill. “Infrastructural Violence: Introduction to the Special Issue.” Ethnography 13, no. 4 (December 2012): 401–412.

Dunstan, Adam. “Victims of ‘Adaptation’: Climate Change, Sacred Mountains, and Perverse Resilience.” Journal of Political Ecology 26, no. 1 (2019): 704–719.

U.S. Department of Agriculture, Forest Service. “Final Environmental Impact Statement for Arizona Snowbowl Facilities Improvements, Volume 2: Response to Comments on the Draft Environmental Impact Statement.” 2005.

Arizona Department of Water Resources. “Surface Water Overview.” Accessed April 13, 2026.

Benneyworth, G. C. (2019). Land, labour, war and displacement: A history of four black concentration camps in the South African War (1899–1902). Historia, 64(2), 1–20. https://doi.org/10.17159/2309-8392/2019/v64n2a1 https://www.scielo.org.za/scielo.php?pid=S0018-229X2019000200001&script=sci_arttext

Venter, Z. S., Shackleton, C. M., Van Staden, F., Selomane, O., & Masterson, V. A. (2020). Green apartheid: Urban green infrastructure remains unequally distributed across income and race geographies in South Africa. Stockholm Resilience Centre. https://www.stockholmresilience.org/publications/publications/2021-01-07-green-apartheid-urban-green-infrastructure-remains-unequally-distributed-across-income-and-race-geographies-in-south-africa.html

Wood, L. (2015). The environmental impacts of colonialism [Honors thesis, Bridgewater State University]. Virtual Commons – Bridgewater State University. https://vc.bridgew.edu/cgi/viewcontent.cgi?params=%2Fcontext%2Fhonors_proj%2Farticle%2F1131%2F&path_info=Wood.pdf

Wood, L. (2015). The environmental impacts of colonialism [Honors thesis, Bridgewater State University]. Virtual Commons – Bridgewater State University. https://vc.bridgew.edu/cgi/viewcontent.cgi?params=%2Fcontext%2Fhonors_proj%2Farticle%2F1131%2F&path_info=Wood.pdf

Wood, L. (2015). The environmental impacts of colonialism [Honors thesis, Bridgewater State University]. Virtual Commons – Bridgewater State University. https://vc.bridgew.edu/cgi/viewcontent.cgi?params=%2Fcontext%2Fhonors_proj%2Farticle%2F1131%2F&path_info=Wood.pdf

BBC. The Boer Wars. BBC History. https://www.bbc.co.uk/history/british/victorians/boer_wars_01.shtml

Benneyworth, G. C. (2019). Land, labour, war and displacement: A history of four black concentration camps in the South African War (1899–1902). Historia, 64(2), 1–20. https://doi.org/10.17159/2309-8392/2019/v64n2a1 https://www.scielo.org.za/scielo.php?pid=S0018-229X2019000200001&script=sci_arttext

BBC. The Boer Wars. BBC History. https://www.bbc.co.uk/history/british/victorians/boer_wars_01.shtml

Hereford, Richard. “The Effects of Surface Runoff on Hart Prairie from Arizona Snow Bowl Facilities, Coconino National Forest.” Heal the Peaks Campaign, 2022.

Rodgers, Dennis, and Bruce O’Neill. “Infrastructural Violence: Introduction to the Special Issue.” Ethnography 13, no. 4 (December 2012): 401–412.

Dunstan, Adam. “Victims of ‘Adaptation’: Climate Change, Sacred Mountains, and Perverse Resilience.” Journal of Political Ecology 26, no. 1 (2019): 704–719.

Navajo Nation v. United States Forest Service, 535 F.3d 1058 (9th Cir. 2008) (en banc).

Navajo Nation Office of the President and Vice President, and Navajo Nation Office of the Speaker. “Joint Press Release on Hopi Settlement Agreement with the City of Flagstaff.” March 8, 2016.

U.S. Department of Agriculture, Forest Service. “Final Environmental Impact Statement for Arizona Snowbowl Facilities Improvements, Volume 2: Response to Comments on the Draft Environmental Impact Statement.” 2005.

U.S. Department of Agriculture, Forest Service. “Final Environmental Impact Statement for Arizona Snowbowl Facilities Improvements, Volume 1.” 2005.

American Indian Religious Freedom Act of 1978. Pub. L. No. 95-341, 92 Stat. 469. Codified at 42 U.S.C. § 1996.

Wilson v. Block, 708 F.2d 735 (D.C. Cir. 1983).

Hamilton, Lawrence. “Sacred vs Profane: Conflict over the San Francisco Peaks.” Mountain Research and Development 26, no. 4 (November 2006): 366–367.

U.S. Department of Agriculture, Forest Service. “Final Environmental Impact Statement for Arizona Snowbowl Facilities Improvements, Volume 2: Response to Comments on the Draft Environmental Impact Statement.” 2005.

Glowacka, Maria, Dorothy Washburn, and Justin Richland. “Nuvatukya’ovi, San Francisco Peaks: Balancing Western Economies with Native American Spiritualities.” Current Anthropology 50, no. 4 (August 2009): 547–561.

Glowacka, Maria, Dorothy Washburn, and Justin Richland. “Nuvatukya’ovi, San Francisco Peaks: Balancing Western Economies with Native American Spiritualities.” Current Anthropology 50, no. 4 (August 2009): 547–561.

Glowacka, Maria, Dorothy Washburn, and Justin Richland. “Nuvatukya’ovi, San Francisco Peaks: Balancing Western Economies with Native American Spiritualities.” Current Anthropology 50, no. 4 (August 2009): 547–561.

Glowacka, Maria, Dorothy Washburn, and Justin Richland. “Nuvatukya’ovi, San Francisco Peaks: Balancing Western Economies with Native American Spiritualities.” Current Anthropology 50, no. 4 (August 2009): 547–561.

Arizona Department of Water Resources. “Surface Water Overview.” Accessed April 13, 2026.

Vanderwarker, Amy. “Water and Environmental Justice.” Chap. 3 in A Twenty-First Century U.S. Water Policy, by Juliet Christian-Smith, Peter H. Gleick, Heather Cooley, Lucy Allen, Amy Vanderwarker, and Kate A. Berry. New York: Oxford University Press, 2012.

Estes, Nick. Our History Is the Future: Standing Rock Versus the Dakota Access Pipeline, and the Long Tradition of Indigenous Resistance. New York: Verso, 2019.

Börk, Karrigan, and Sonya Ziaja. “Amoral Water Markets?” Georgetown Law Journal 111, no. 6 (June 2023): 1335–1405.

Chatterjee, Pratap. “The Gold Rush Legacy: Greed, Pollution and Genocide.” Earth Island Journal 13, no. 2 (1998): p. 26

U.S. Department of Agriculture, Forest Service. “Record of Decision: Arizona Snowbowl Facilities Improvements Final EIS and Forest Plan Amendment #21.” 2005.

National Ski Areas Association and Brendle Group. “Climate Smart Snowmaking Fact Sheet.” 2023.

Note that it looks like there is an r value > 1; there isn't. It is just from the binning and dodged bars for males and females. If we needed to use this figure in a manuscript, I would clean up the axis limits.

Reminder of how to use these comments here!

U.S. Department of Agriculture, Forest Service. “Record of Decision: Arizona Snowbowl Facilities Improvements Final EIS and Forest Plan Amendment #21.” (2005): 9-17.

Hamilton, Lawrence. “Sacred vs Profane: Conflict over the San Francisco Peaks.” Mountain Research and Development 26, no. 4 (November 2006): 366–367.

Arizona Snowbowl. “About the Mountain.” Accessed April 13, 2026.

add our instagram link https://www.instagram.com/linean_co/

remove section

remove

remove

remove this section

increase the font size 4 sizes bigger

cut the gap of blank space above this by half

• Considerando que - conforme estipula o § 18 do art. 40 - a contribuição, de aposentados e pensionistas, para o RPPS somente incide sobre o valor que exceder o teto do RGPS, havendo déficit atuarial, poderá incidir contribuição sobre aposentadoria e pensões que superarem o valor do salário-mínimo.

• Não confundir com a contribuição extraordinária, visto que essa, enquanto novo tributo, somente terá lugar acaso a medida de ampliação da base de cálculo, consistente em tributar o valor que supere o salário-mínimo, for insuficiente.

Helping my kids with homework.

interesting

Texas General Land Office. State of Texas Action Plan for Disaster Recovery: Hurricane Harvey. Austin: Texas General Land Office, 2017. Revised through Substantial Amendment 13, 2023.

Texas General Land Office. State of Texas Action Plan for Disaster Recovery: Hurricane Harvey. Austin: Texas General Land Office, 2017. Revised through Substantial Amendment 13, 2023.

Despart, Zach. "How a Small Texas Town Got More Hurricane Harvey Recovery Money Than All of Houston." Houston Chronicle, 2021.

Billings, Stephen, Emily Gallagher, and Lowell Ricketts. "Let the Rich Be Flooded: The Distribution of Financial Aid and Distress after Hurricane Harvey." Journal of Financial Economics 146, no. 2 (2022): 797–819.

Rodgers, Dennis, and Bruce O'Neill. "Infrastructural Violence: Introduction to the Special Issue." Ethnography 13, no. 4 (2012): 401–412.

Rodgers, Dennis, and Bruce O'Neill. "Infrastructural Violence: Introduction to the Special Issue." Ethnography 13, no. 4 (2012): 401–412.

Chakraborty, Jayajit, Timothy W. Collins, and Sara E. Grineski. "Exploring the Environmental Justice Implications of Hurricane Harvey Flooding in Greater Houston, Texas." American Journal of Public Health 109, no. 2 (2019): 244–250.

A manera de conclusión, considero que el texto busca explicar cómo las dictaduras militares, las transiciones democráticas y el neoliberalismo estuvieron muy relacionados en América Latina. También comprendí que los autores critican que la democracia se haya reducido muchas veces sólo al hecho de votar, sin resolver problemas como la pobreza, la desigualdad y la falta de participación social, lo cual limita a una verdadera democracia. En general, el texto me ayudó a entender que los cambios políticos y económicos de la región no ocurrieron por separado, sino como parte de un mismo proceso histórico.

Una verdadera democracia debe incluir no sólo derechos políticos, sino también justicia social y reducción de la desigualdad.

La idea de que la riqueza “se derramaría” hacia toda la sociedad no produjo los resultados esperados.