The man is clearly jealous, even though there are two old friends catching up, he wants to make the conversation about himself.

The narrator treats the blind man as the "inspirational overcomers" trope: he completes an everyday task but he is in admiration at how this can be possible without being able to see.

Media expectations and stereotypes of the man.

He has never met someone that is blind before but has expectations for what he is going to look like / how he is going to act.

This comment by the narrator is used to lessen the man or make fun of him for not being able to see. He knows the side of the train will not make a difference to him, but asks it anyways.

The narrator has a predetermined vision of what the man "should" look like or what he is going to look like due to stereotypes, likely through the media.

Shows more jealousy towards the man and his wife's relationship with him.

The narrator shows pity, not for her death and what the blind man is going through, but for the blind man's wife and him not being able to understand why someone would date a blind man because they cannot be seen. This shows more of his ableist beliefs towards the man.

The man clearly has other prejudice and negative beliefs, not just ableist but also racist. Connects to a similar theme of the man seeing himself as the best and others as less than him.

This shows that the wife attempting to build some empathy from the narrator, but he does not seem to care for the man even while he is going through a tougher time.

Shows the narrator's belief that he is better than others. Even though this man is non-disabled, he still sees himself as more important and does not even give the man a name.

This shows how the narrator has some jealousy towards the man and his wife's relationship and is thrown off by the man.

Connects to the "bitter cripple" trope we discussed. The narrator makes his opinions based on what he sees in the media rather than his own experiences.

This line also shows the narrator's prejudice towards people with disabilities and makes it seem like he feels he is better than him for being nondisabled.

Refers to the man as "this blind man" showing that he only sees him for his disability, rather than him as individual. Shows his prejudice early on in the story.

this isn't active enough. suggest 'by actively making sense of those insights and deciding what to do next'

This is way too obvious. Perhaps the question could focus on the Growth element. "Which part of the SAG⇄E Insights framework focuses on recognising what you are learning over time — including what you learn from mistakes or challenges?

Growth is about recognising how you’re developing over time. This includes what you’re learning from mistakes, challenges, or things that didn’t go as planned, and what those experiences are teaching you about your learning and capabilities

These SAG prompts are contextualised to a learner doing a self-review. This is a nice way to introduce the insight-giving, less threatening. But it would be helpful to have a page that gives example prompts that show how the teacher will focus their insights, and to introduce prompts to shape peer-to-peer insights.

To date, I have visualised the framework with a simple left to right representation. This helps to preserve the back and forth dialogic function of the ⇄.

I have uploaded the image I use (graphically quite simple) to the files area of the Canvas sight. https://courses.ecu.edu.au/courses/64682/files?preview=11007312

The title of the diagram should either be the full name or (because the diagram includes the SAG⇄E part it can simply be titled 'Insights for Learning Framework'.

'your life after you graduate' We need to be cognisant that not all courses lead to a profession. University learning also influence their non-work lives too. This is particularly true of this framework which can shape how they interact with others regardless of context.

insights

offered each other some insights

this feels like an AI generated interpretation of 'engagement'. Suggested edit 'and how you actively make sense of those insights and decide what to do next'. This also introduces the shift from 'feedback' to 'insights' a key differentiator of this approach.

This sentence captures only a narrow subset of feedback literacy:

✔ Making sense of feedback ✔ Taking action based on feedback (implicitly, and framed narrowly as “improve your work”)

The dimensions of feedback literacy that are missing are: * Appreciating feedback as a valuable part of learning * Managing emotional responses * Seeking feedback proactively * Curating and connecting feedback across time * Using feedback to support identity development and capability narratives

We might not want to complicate this intro video, instead it would be helpful to have a page on Feedback literacy where this is expanded to include all of the dimensions and discussed further. This also lifts the importance and cognisant awareness of users of the module of Feedback Literacy as a construct (something to be working on).

self review or reflection

here it might be useful to use 'an assignment'.

'on your work' is more useful. One of the things we want to set up here is that feedback isn't just about assignment/assessments. The framework has been specifically designed to support insight giving in a variety of learning moments including work-in-development.

as this is the first time the referring to the framework I would (as the author of the framework) prefer that the full name be used. SAG⇄E Insights for Learning framework. I will make annotations throughout this module on how to correctly refer to the framework.

same as page title

This should probably be sources of feedback. For me type of feedback would typically refer to things like written comments, audio or video recording or verbal in person. The intro video quite correctly/and usefully identifies that what we want learners to recognise is that feedback/insights can come from several different sources. This ties in with how the framework is setup where the SAG portion is intended for the insight giver.

could the yellow colour and 'Question 1' confuse students as per previous quizzes and this is just a check-in?

** Global feedback: a lot of this section is about scheduling. There needs to be a section also on what if someone doesn't do their part; doesn't respond to emails; don't attend meetings etc.

We think this is the most important section - perhaps allude to it earlier? such as first module as a key element

This might be confusing for students, as the sentence suggests a list of phrases will follow, but none is provided until the content section below the paragraph. Suggest reference to below and rewording.

we don't think this term will make sense to students. Rethink in terms of the Charter - what do students do when students meet expectations and boundaries

Peer collaboration: The content of the module focuses on collusion; there’s another module for this. Could positives be highlighted/How to provide good peer feedback etc?

This is global feedback - students must acknowledge use of AI so we need one to acknowledge vids have been produced by AI. (Disclaimer at the beginning of each module will be fine).

Remove SAGE from this overview. We're not clear why there's such a focus on SAGE throughout - it makes sense in the feedback module but not teamwork module. We can't assume students will know what it is. Please include a sentence in the overview highlighting the importance of teamwork in terms of employability

Select one example you could keep as evidence of your communication or teamwork capability for the future.

the development of transferable capabilities.

The focus seems to be dividing tasks based on personalities. Maybe add feedback section on others’ roles/supporting others’? Maybe mention team reviewing work using rubric [template]? mentioned in next page?

Self-management isn't one of the capabilities listed for CTP - Collaboration and Communication would be more appropriate.

R0:

Reviewer #1: The review is important to improve outcomes on cholera surveillance and response. However, there are a number of critical issues that must be addressed to ensure the manuscript conforms to the standard of scientific writing and scoping review. 1. Certain sections were ommitted e.g Quality assessment and Data analysis 2. The roles of the authors in the scooping exercise also omitted 3. The results and discussion sections are mixed up. The authors began discussing the findings in the result.

Reviewer #2: Given the ongoing cholera pandemic and its recurrent outbreaks in sub-Saharan Africa, it is commendable that the authors undertook a comprehensive mapping of cholera research in Kenya. 1.For the search strategy, the query “cholera AND Kenya” across all databases is overly restrictive and likely excluded studies using alternative terminology such as “Vibrio cholerae”, “waterborne disease”, or “WASH-related cholera”. I would recommend providing the full keywords, filters and timelines used for each database, to help in reproducibility, as stated in the PRISMA-ScR Checklist (Item 8). 2.Please provide the last search date or timeframe. 3.The authors mentioned the systematic search of five databases, including Google Scholar, Web of Science, PubMed, Embase, and Scopus. However, in the PRISMA flow diagram (Figure 1), there is no data for Google Scholar. 4.The use of Rayyan is recognized. However, reviewer roles, conflict resolution, and data extraction validation are not stated. 5.The authors mentioned the inclusion of non-primary studies, such as reviews, but stated “ineligible study design” as a reason for exclusion in Figure 1. A clarification on this is could be beneficial. 6.For each included study, the authors should present the characteristics of the data charted with respective citations in a table. 7.In section 3.2, the authors provide an informative table which shows the geographic focus of the studies across multiple countries, including Kenya. For a scoping review centered on Kenya, a similar table or map that shows the distribution of studies/ data on the county-level could be added. 8.Themes such as mortality and risk factors of cholera could be explored and discussed further to strengthen the manuscript. 9.The Results-Discussion boundary seems blurred. Discussion begins to appear within “Future directions” paragraphs under each theme. I would recommend that the authors consolidate all “Future directions” into a single Discussion summarising what is known and unknown.

The process of creating or producing a response.

The act of searching for and finding relevant information from a large, external, and non-static data source (your knowledge base).

The integration step ensures the answer is grounded, meaning it is based on the verifiable facts provided in the retrieved context, not solely on the LLM's general, potentially outdated, or hallucinated pre-trained knowledge.

the context window is the finite amount of tokens (words or word fragments) an LLM can actively see and process in a single pass to generate a response.

This component acts as the translator that converts human-readable text (like a document chunk or a user's question) into a numerical vector (an embedding).

The raw Large Language Model (LLM) is stateless—it resets completely after every API call, possessing no inherent memory of past interactions. To create the "illusion of state" and maintain a conversation, the application developer must manually manage the entire dialogue history and all necessary context, packaging it into a new, comprehensive prompt that fits within the LLM's finite context window for every single turn.

The best way

Messages are the building blocks that allow LangChain to maintain the state of a conversation, which is necessary for multi-turn chat applications.

A Message doesn't just carry the raw text (content); it also carries crucial metadata (like the message type—HumanMessage, AIMessage, etc.) that tells the LLM how to interpret it.

Model Suggestion: The LLM's initial call returns an AIMessage containing the suggestion to use a specific tool (the tool_calls object).

Developer Action (Execution): The developer's code must intercept this message, parse the tool name and arguments, and manually execute the corresponding Python function.

Result Feedback: The developer must then package the output of the tool execution into a ToolMessage and send it back to the Model, along with the previous conversation history, for the Model to complete its final reasoning and generate the answer.

For most new projects focused on universality and best practice, init_chat_model() is the recommended and modern approach because it promotes provider agnosticism and reduces boilerplate code.

An Agent often requires multiple calls to the LLM (Thought, Action, Observation, Thought, etc.) to complete a task. Each call incurs cost and latency.1

When you bind tools directly to a Model, the model makes a single, stateless decision. It suggests the best tool for the immediate prompt and then stops.

The Agent, however, uses its loop (often ReAct: Reason, Act, Observe) to execute complex strategies

The difference lies in autonomy and execution flow: A Model with Tools (via direct binding/function calling) is a single, stateless step where the LLM merely suggests the best tool and its arguments, requiring the developer to manually execute the tool and initiate any subsequent calls. In contrast, an Agent with Tools leverages an Agent Executor to manage a dynamic, multi-step loop (e.g., ReAct), where the LLM acts as the planner, deciding which tool to call next, and the Executor automatically runs the tool, feeds the observation back to the model, and repeats the cycle until the complex, multi-step goal is autonomously achieved.

• Motivations and Realism: Define personal success (e.g., promotion, networking, paid speaking) and commit to the long-term effort—e.g., 7 years from first user group talk to international conference.
• Year 1 - Get Good: Craft a unique talk, deliver at local user groups/Meetups, iterate based on feedback (fix demos, slides, length), repeat multiple times.
• Year 2 - Get Seen: Submit to small community conferences (e.g., DDD events), network actively (pre-event dinners, stay engaged, follow up via chats/LinkedIn/email), secure video recordings for credibility.
• Year 3 - Get Accepted: Build talks and network, submit 2-3 focused abstracts to CfPs (use lists like codeasaur.us), leverage connections, keep content fresh, track rejection stats realistically.
• Year 4 - Get Bored (Sustain): Assess burnout, align with goals (fun, leads, pay), vary event types, always respect audience—stop or pivot if disinterested.
• Final Advice: Work hard, enjoy, teach valuably; offers abstract/slide reviews at dylanbeattie@gmail.com.

• Insomnia is a Common Problem: Insomnia is widespread, though increased awareness makes it seem like a modern issue. It's often triggered by stressful life events (e.g., divorce, job loss) [00:03:39], [00:21:03].
• The Danger of Counter-Intuitive Habits:
  • Do not try to "delete thoughts" when you can't sleep, as this is counterproductive [00:00:07], [00:28:34].
  • Do not mistake light sleep (N1) for being awake; people often underestimate their total sleep time [00:16:19].
  • Attempting to "catch up" on sleep (going to bed early, sleeping in) destroys the crucial sleep pressure (ciśnienie na sen) [00:22:39].
• Core Principles of Sleep Therapy (CBT-I):
  • Fixed Wake-up Time is Key: The most important rule is to maintain a consistent wake-up time, varying it by no more than an hour, even on weekends [00:25:55].
  • Sleep Restriction: If you spend 8.5 hours in bed but only sleep 6, the initial goal is to restrict your time in bed to 6 hours to build up sleep pressure [00:24:12], [00:26:15].
  • Stimulus Control: If you cannot fall asleep after 20 minutes (or wake up at 3 AM), get out of bed and engage in a calming, non-stressful activity (like watching a show or reading) until you feel intensely sleepy, then return to bed [00:29:09].
• Consequences of Sleep Deprivation:
  • Relationships: Lack of sleep increases the risk of divorce, reduces empathy, and makes couples argue worse [00:09:44], [00:10:14].
  • Health and Cognition: It weakens the immune system (doubling the risk of catching a cold) [00:11:37], drops testosterone levels (by 10-15%) [00:12:07], impairs strong willpower, leads to worse decision-making, and increases the craving for junk food [00:14:30], [00:31:32].
  • The "3 AM Brain" operates with low positive and high negative affect; do not make major life decisions or ruminate during this time [00:30:12], [00:30:48].
• Medication and Chronotypes:
  • Zolpidem (Z-drugs) is generally not recommended for primary insomnia, as its side effects (e.g., amnesia, accidents, increased depression/suicidal thoughts) can be more severe than the insomnia itself [00:36:49].
  • Chronotypes (larks vs. owls) are genetically determined; waking up at 5 AM is a development guru myth that can be harmful for "owls" [00:55:12], [00:56:44]. Owls and adolescents benefit from later school/work start times [00:59:21], [01:00:13].
• Managing Shift Work:
  • Shift work increases health and mental health risks [00:43:15].
  • Strategies: Nap for 1-1.5 hours before the night shift, use caffeine only in the first half of the night, eat well during the shift, and get a small nap (20 minutes) if possible [00:48:26], [00:49:55], [00:51:00].
  • Day sleep is always worse than night sleep; it's recommended to accept a shorter day sleep to get daylight exposure and physical activity later, which helps regulate the circadian rhythm [00:52:02], [00:52:48].
• Dreams: Dreams help the psyche process daily events and emotions [01:07:05]. If you have recurring nightmares, use Imagery Rehearsal Therapy (IRT): change the end of the nightmare, write down the positive new ending, and visualize it daily for 10-15 minutes [01:08:42].

• ZUS develops an advanced algorithm to help PIP target companies hiding employment contracts under B2B arrangements, replacing random inspections with risk-based targeting.
• The algorithm analyzes ZUS's vast payer data for patterns indicating fictitious self-employment, such as issuing only one monthly invoice to a single client.
• Other red flags include providing services to a former employer immediately after switching to B2B and continuous, stable cooperation resembling an employment relationship (fixed place, time, subordination).
• ZUS will provide PIP with a scored list of high-risk firms; data exchange with PIP and KAS is planned, initially manual, transitioning to automated interfaces.
• This addresses PIP's resource shortages, enabling precise controls instead of reactive or random ones, increasing detection of illegal practices for tax optimization.
• Implications for businesses: Higher risks for those using aggressive B2B optimizations, potentially disrupting models reliant on cheap labor disguised as self-employment.

Cool, a big database of storms!

Interesting claim

I know of UNC, but what's the other one?

I do wonder about this statement "our approach captures the most important parameters" - at least in their study.

@hychem95 @ebucholtz

I had saved df_activity as a csv and thought we should be getting smiles for that and not df, but maybe I misunderstood something, and we converted df to that after we had removed inconclusives, and the difference is not the size, but this has 0s and 1s. So I think we could have used either.

BUT, when I look at the csv I am seeing salts.

I am seeing two strategies, remove the salts, or just keep the largest fragement. Do you have input?

@hychem95 I am going through Ehren's notebook but can't comment on it as it is not in a jupyter book, but can here. Why are we doing this cids = df.cid.astype(int).tolist()

Shouldn't we be doing this? cids = df_activity.cid.astype(int).tolist()

All these 20th-century colonial experiences have more in common than we know!

The fucking BBC has done this, and even though they still let you subscribe via RSS, they have episodes available way earlier on their app.

Northeast Brazil national park, looks amazing

This term refers to the intense awe and wonder at the power, or complexity of the plant world. It goes beyond simply finding plants "beautiful" and is more like a sense of being overwhelmed, and feeling insignificant as one species amongst the vastness of nature and its different species.

CONNECTIONS TO RADICAL VS. REFORMISTS AND WHY FEMINISM MUST BE FOR ALL HOOKS' WORK

LIKE HOW IT WAS FOR BODIES

REMINDS ME OF POLI SCI

epistimic power

i THINK IT IS SO INTERESTING HOW THERE ARE SO MANY parallels BETWEEN PEOPLE AND NATURE

OKAY BUT WHY DOES THIS MATTER? PLANTS CANNOT TELL TIME

READING WITHIN THE GRAIN

LOOKING AT BODIES AS CULTRUAL TEXTS

i CAN RELATE also like talking about Western notions of productivity

ONLY PHYSICAL THOUGH AND REMINDS ME OF MY OWN INVISIBLE ONES

LIKE BODIES, COLOINALISM, ETC.

BODIES CONNECTION AND SIMONE

KIND OF LIKE THE DIVINE WORK

WHY USE QUEER THOUGH?

WHY USE QUEER THOUGH?

Like others.

LIKE BODIES AND HISTORIES OF COLONIALISM WHERE SETTLERS CAME IN AND RUINED THE NATIVE NATIONS ALREADY THERE THAT WERE AMERICAN OR CANADIAN

THIS IDEA REMINDS ME OF THE INVISIBLE GIRL READING

POSITIONALITY OR STANDPOINT?

THIS REMINDS ME OF THE WITCH

AND THIS IS USUALLY THOSE OF FEMINIST HISTORY

HOW?

METAPHOR FOR PEOPLE

I FEEL LIKE THIS MATTERS BUT DO NOT KNOW WHY/HOW

I FEEL LIKE THIS MATTERS BUT DO NOT KNOW WHY/HOW

ANGER ACCORDING TO HOOKS IS HOW RIGHT?

CONNECTS TO BODIES, BUT I DO NOT KNOW WHAT THE FIRST PART.

LIKE THE WITCH

THIS IS LIKE HISTORY IN GENERAL. I WONDER WHAT THIS MEANS BY HUMAN? IS IT WHO HISTORY SEES AS HUMAN OR IMPORTANT?

EPISTIMOLOGY

kinship structures

THIS IS REALLY IMPORTANT WHEN LOOKING AT WHAT COLONIALSIM IS

SETTLER COLONIALISM

epistemic powers & speaking in tounges

SUPER IMPORTANT

Like those of the body with people

Like the witch

find out more about this

This is why we MUST study colonialism when talking about feminism - maybe bring in definitions of feminism too

Epestemic Power.

SUPER IMPORTANT - could this connect to to Sara Ahmed?

Get over the fear of difference.

think of something for this

Women and ecofeminism

like how women were seen as weird things to study but really it all makes sense.

Sounds interesting, but what would that look like? - also this is why we have more than one feminism i feel like botany is a metaphor for feminism

THIS is why context and understanding the history is important.

This is also why it is important to why we have more than one kind of feminism.

does it though?

how though? I thought it was colonial.

This is very important

witch capitalism

Ecofeminism- this also reminds me of western v.s Indigenous Epistimoogy

Like the witch

scientific = masculine and also like the witch, also like private v.s public

without sacrificing the individual for the collective

Would this lead to reality fracture and silos? Does culture not need some shared context? how do you avoid failure at the other extreme?

The combination of ONT and Illumina is great - I wondered if you have found a tradeoff of trying to maximise finding diversity, i.e., reads that have differences, but also minimize retaining reads with sequencing errors that look artificially dissimilar. Presumably, walking the line between the two is critical to not over-inflating diversity estimates and retaining only confident 'true' standing diversity - I would love to know more about how you navigate this!

I believe that a profit-sharing model for the country of origin of biodiversity has to be central to the commodification of biological diversity. I am curious about a couple of practical aspects of your implementation of this. Firstly, how do you determine the 'value' and therefore the royalties associated with the point of use of data prior to commercialization (are there some minimum royalties that are immediately owed to the country of origin at the point of use?), and subsequently I couldn't find a description in the manuscript of what constitutes a royalty vs. profit from the use of a sequence. When you say that 100% royalties will go to the data source A when a natural sequence is used, how does this compare with the profit gleaned from products developed from that sequence? Without this clarity, it feels rather obtuse as to how much countries are truly being compensated (my impression is that 'royalties' models of compensation have rightly been long criticized in other sectors due to their opacity and underweighting of small to mid-size contributors).

Given the strength of biological foundation models will lie in their breadth of understanding, how do you balance sampling previously sparsely/unsampled environments (which presumably contribute substantially to new taxa/sequences) with less unique environments that exhibit more homogenous taxonomic diversity to get an idea of standing patterns of biological variation? I would imagine that capturing that standing variation is also an important component of understanding biology as a whole. Presumably, models will fail to generalize patterns and will overweight the prevalence of novelty in novel environments when they are more selectively sampled than other environments?

Increasing the breadth of sampling to this extent is fantastic. I was wondering whether you have an estimate of the increase in phylogenetic branch length across the data resulting from the addition of these additional taxa. I'm also curious as to whether these 'species' are all microbes or whether you also pick up DNA from macro-organisms, and if so, what the increase in 'traditionally' described species looks like compared to when you use OTUs?

Knowing your audience and speaking to their concerns is absolutely critical. This can then help to shape the other appeals you use in order to maximize their impact on your audience.

This is a very important factor as it ensures that your argument is heard at the most effective time. This helps to massively boost how receptive ones audience is to hearing your argument and message.

Logos is probably the most effective tool as it cites logic, statistics, and facts. This creates a mountain of evidence for one's own position which helps strengthen your argument.

Pathos is a very effective tool though it is more powerful on some people than it is on others. Additionally one must be careful that they use it to achieve the intended effect and not create side effects.

Does this show up for other people who use hypothes.is ?

for - Yuval Noah Harari - youtube - Big Think - Yuval Noah Harari - Why advanced societies fall for mass delusion

• SRG comment - mass delusion -
• A good talk that looks at the dangers posed by AI:
  • it can be considered a legal person and autonomously derail institutions and the legal system
  • it can amplify the harmful control of dictators and authoritarian leaders
  • information is not truth. Truth is a rare form of information and overloading information systems with fictions is a way for totalitarian leaders to gain mass following

for - progress trap - social media - misinformation - AI algorithms hijacking and pretending to be human

for - progress trap - AI - totalitarian government - can exploit for centralized, non-self-correcting control

for - totalitarian systems - a little truth plus a lot of fiction

for - quote - flooded with misinformation - Yuval Noah Harari - The more we flood the world with information, - unless we make the effort to construct institutions that invest in truth, - we'll be flooded by fiction, illusion, delusion and junk information.

for - comparison - information and truth - truth is a very special type of information

for - nationalism - role of - makes us care beyond a small dunbar number

for - comparison - self correcting mechanisms - science has - religions don't have

for - example - no self-correcting mechanism in religion - 10th commandment and slaves

for - AI - need for self-correcting institutions that regulate AI

for - progress trap - AI can become political lobbyist for increasing rights of AI

for - progress trap - AI as legal person (US Corporation) - richest person in the world could be an AI

for - AI - Alien Intelligence, not Artificial Intelligence - It is not an Artifact that we create and control

for - futures - AI - millions of AI bots making decisions about us

for - surveillance state - AI makes it possible

for - surveillance state - Yuvah Noah Harari

for - AI - AlphaGo - analogy - humans stuck on small island for 2,000 years

The full form where you can see that it is supposed to be "Rapid transit" will be more impactful to drive home the irony

the approach

Love the list. Very comprehensive and actionable. Like Ilya said, it would be better to make this a suggestion

More streamlined if what is a case study and why it is important come up front. Bullet points 2 to 5 can be added as links after that explanation rather than being upfront

A two line description of each article type upfront will make the website more scannable

A couple of lines saying that you are thrilled people are here to share contributions to make the world better would be helpful

It would be helpful to understand what CC4... means for me as a writer. And I also don't think this needs to be on the top. A dedicated section explaining how you pay people for contributions and how licensing works would be better.

I find this interesting because their way of getting rid of the debt and boasting the economy was to spend even more money and give out more loans to veterans.

How did the government let it get as bad as it did? Did they even know the signs were there and choose to ignore it resulting in it backfiring on the American people.

I personally can't imagine being a woman and being in the lower class in this time period especially in this time period. it's so fascinating how America treated everyone back then if you weren't really rich or a man.

undermining decentralization and

harming performance for legitimate browser users.

And some of the largest share of user agents we see are from

some of our best supported languages,

Go and NodeJS, and

as such should be easy candidates for migration to native retrieval.

Provide Sweep: Solving the DHT Bottleneck for Self-Hosting IPFS at Scale

/💻/asus/🧊/me/🏛️/2025/12/15/ann0tate

ann0tate~ipfs-shipyard-self.hosting-at.scale-provide.sweep

dweb.link

Directive set_fact is very useful for decomposing your tasks and sending proper input to them thus using handlers like functions with dynamic parameters. You can just register result of the set fact into reg-variable and then notify handler. It will be available at the handler like "{{ reg-variable.ansible_facts.one_fact }}".

This "register-notify" construct works for any task but not every task returns the output you wish to see at handlers.

You can also add vars: for the block: these vars will be available for all tasks of the block. But you can't add the same listen to all tasks at the block level: you'll have to add it on a per-task basis.

In recent versions loop is more preferred than with_items but the idea is the same: you can use handlers via notify to trigger multiple actions per item in the loop. You just need to place same listen value to all needed handlers to run them all. These handlers can be even decomposed into different files if you want. Just include them in the main file to actually register them.

Истинное обучение невозможно, если искусственный интеллект выполняет работу вместо студента. Автор подчеркивает, что для усвоения знаний необходима активная умственная деятельность – осмысление и понимание материала.

В абзаце подчеркивается потенциальная опасность чрезмерного использования искусственного интеллекта. Исследователи из MIT Media Lab, хотя и признают, что их работа пока предварительная, предупреждают о возможности ухудшения когнитивных способностей и критического мышления из-за излишней зависимости от ИИ.

ИИ должен усиливать мышление человека, а не управлять им — иначе теряется критическое мышление и креативность.

Здесь делается акцент на процессе обучения, а не на результате. Это напрямую связано с образовательным контекстом: ИИ опасен не сам по себе, а при подмене учебного процесса готовыми ответами.

Так как ИИ выглядит как легкий и доступный инструмент для пользования, особенно важно сохранять холодную голову и рассудительность при его использовании.

Стоит помнить, что ИИ лишь анализирует и создает выжимку на базовом уровне, и у него пока нет возможности предлагать решения, сравнимые с человеческими

Результат от обучения возможен лишь при корректной работе с ИИ, как с помощником.

eLife Assessment

This is an overall compelling set of findings on the role of centrally produced estrogens in the control of behaviors in male medaka. The significance of the findings rests on the revealed potential mechanism between brain derived estrogens modulating social behaviors in males , supported by the analysis of multiple transgenic lines. The evidence for the broader claim is incomplete since it has not been extended to female medaka, and further experimentation would be necessary to fully validate the conclusions on the role of brain-derived estrogens. Nonetheless, the findings have led to important hypotheses on the hormonal control of behaviors in teleosts that can be tested further.

Reviewer #1 (Public review):

Summary:

This research group has consistently performed cutting-edge research aiming to understand the role of hormones in the control of social behaviors, specifically by utilizing the genetically-tractable teleost fish, medaka, and the current work is no exception. The overall claim they make, that estrogens modulate social behaviors in males is supported, with important caveats. For one, there is no evidence these estrogens are generated by "neurons" as would be assumed by their main claim that it is NEUROestrogens that drive this effect. While indeed the aromatase they have investigated is expressed solely in the brain, in most teleosts, brain aromatase is only present in glial cells (astrocytes, radial glia). The authors should change this description so as not to mislead the reader. Below I detail more specific strengths and weaknesses of this manuscript.

Strengths:

Excellent use of the medaka model to disentangle the control of social behavior by sex steroid hormones

The findings are strong for the most part because deficits in the mutants are restored by the molecule (estrogens) that was no longer present due to the mutation

Presentation of the approach and findings are clear, allowing the reader to make their own inferences and compare them with the authors'

Includes multiple follow-up experiments, which leads to tests of internal replication and an impactful mechanistic proposal

Findings are provocative not just for teleost researchers, but for other species since, as the authors point out, the data suggest mechanisms of estrogenic control of social behaviors may be evolutionary ancient

Weaknesses:

The experimental design for studying aggression in males has flaws, but it appears a typical resident-intruder type assay is not appropriate for medaka. seems other species may be better for studying aggression in teleosts.

Reviewer #3 (Public review):

Summary:

Taking advantage of the existence in fish of two genes coding for estrogen synthase, the enzyme aromatase, one mostly expressed in the brain (Cyp19a1b) and the other mostly found in the gonads (Cyp19a1a), this study investigates the role of brain-derived estrogens in the control of sexual and aggressive behavior in male medaka. The constitutive deletion of Cyp19a1b, confirmed by the ablation of its transcript, markedly reduced brain estrogen content. This effect is accompanied by reduced sexual and aggressive behavior and reduced expression of the transcripts coding for androgen receptors (AR), ara and arb, in brain regions involved in social behavior regulation. Both AR expression and aspects of social behaviors were restored by adult treatment with estrogens, providing some support for a role of aromatization. Expression analysis of AR isoforms and behavior of mutants of estrogen receptors (ER) indicates that these effects are likely mediated by the activation of the esr1 and esr2a isoforms. Together, these results provide valuable insights into the role of brain-derived estrogens in social behavior in fish.

Strengths:

This study evaluates the role of brain "specific" Cyp19a1 in the social behavior in male teleost fish, which as a taxon are more abundant and yet proportionally less studied that the most common birds and rodents. Therefore, evaluating the generalizability of results from higher vertebrates is important. The study suggests that, as opposed to mammals, the facilitatory role of brain-derived estrogens on mating and aggression is limited to adulthood.

Results obtained from multiple mutant lines converge to show that estrogens most likely synthesized in the brain drives aspects of male sexual behavior.

The comparative discussion of the age-dependent abundance of brain aromatase in fish vs mammals and its role in organization vs activation is important beyond the study of the targeted species.

Weaknesses:

Most experiments are weakly powered (low sample size).

The variability of the mRNA content for a same target gene between experiments (genotype comparison vs E2 treatment comparison) raises questions about the reproducibility of the data (apparent disappearance of genotype effect).

Conclusions :

Overall, the present study provides convincing evidence for a facilitatory role of estrogens originating from the brain on sexual behavior and aggressive behavior in male medaka. The role of specific estrogen receptor isoforms underlying the expression of androgen receptors is supported by converging evidence from multiple mutant lines.

Author response:

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

Public Reviews:

Reviewer #1 (Public Review):

Summary:

This research group has consistently performed cutting-edge research aiming to understand the role of hormones in the control of social behaviors, specifically by utilizing the genetically-tractable teleost fish, medaka, and the current work is no exception. The overall claim they make, that estrogens modulate social behaviors in males and females is supported, with important caveats. For one, there is no evidence these estrogens are generated by "neurons" as would be assumed by their main claim that it is NEUROestrogens that drive this effect. While indeed the aromatase they have investigated is expressed solely in the brain, in most teleosts, brain aromatase is only present in glial cells (astrocytes, radial glia). The authors should change this description so as not to mislead the reader. Below I detail more specific strengths and weaknesses of this manuscript.

We thank the reviewer for this positive evaluation of our work and for the helpful comments and suggestions. Regarding the concern that the term “neuroestrogens” may be misleading, we addressed this in the previous revision by consistently replacing it throughout the manuscript with “brain-derived estrogens” or “brain estrogens.”

In addition, the following sentence was added to the Introduction (line 61): “In teleost brains, including those of medaka, aromatase is exclusively localized in radial glial cells, in contrast to its neuronal localization in rodent brains (Forlano et al., 2001; Diotel et al., 2010; Takeuchi and Okubo, 2013).”

Strenghth:

Excellent use of the medaka model to disentangle the control of social behavior by sex steroid hormones 

The findings are strong for the most part because deficits in the mutants are restored by the molecule (estrogens) that was no longer present due to the mutation 

Presentation of the approach and findings are clear, allowing the reader to make their own inferences and compare them with the authors' 

Includes multiple follow-up experiments, which leads to tests of internal replication and an impactful mechanistic proposal 

Findings are provocative not just for teleost researchers, but for other species since, as the authors point out, the data suggest mechanisms of estrogenic control of social behaviors may be evolutionary ancient 

We thank the reviewer again for their positive evaluation of our work.

Weakness:

As stated in the summary, the authors are attributing the estrogen source to neurons and there isn't evidence this is the case. The impact of the findings doesn't rest on this either

As mentioned above, we addressed this in the previous revision by replacing “neuroestrogens” with “brain-derived estrogens” or “brain estrogens” throughout the manuscript. In addition, the following sentence was added to the Introduction (line 61): “In teleost brains, including those of medaka, aromatase is exclusively localized in radial glial cells, in contrast to its neuronal localization in rodent brains (Forlano et al., 2001; Diotel et al., 2010; Takeuchi and Okubo, 2013).”

The d4 versus d8 esr2a mutants showed different results for aggression. The meaning and implications of this finding are not discussed, leaving the reader wondering

This comment is the same as one raised in the first review (Reviewer #1’s comment 2 on weaknesses), which we already addressed in our initial revision. For the reviewer’s convenience, we provide the response below:

Line 300: As the reviewer correctly noted, circles were significantly reduced in mutant males of the Δ8 line, whereas no significant reduction was observed in those of the Δ4 line. However, a tendency toward reduction was evident in the Δ4 line (P = 0.1512), and both lines showed significant differences in fin displays. Based on these findings, we believe our conclusion that esr2a^−/− males exhibit reduced aggression remains valid. To clarify this point and address potential reader concerns, we have revised the text as follows: “esr2a^−/− males exhibited significantly fewer fin displays (P = 0.0461 and 0.0293 for Δ8 and Δ4 lines, respectively) and circles (P = 0.0446 and 0.1512 for Δ8 and Δ4 lines, respectively) than their wild-type siblings (Fig. 5L; Fig. S8E), suggesting less aggression” was edited to read “esr2a^−/− males from both the Δ8 and Δ4 lines exhibited significantly fewer fin displays than their wild-type siblings (P = 0.0461 and 0.0293, respectively). Circles followed a similar pattern, with a significant reduction in the Δ8 line (P = 0.0446) and a comparable but non-significant decrease in the Δ4 line (P =0.1512) (Figure 5L, Figure 5—figure supplement 3E), showing less aggression.”

Lack of attribution of previous published work from other research groups that would provide the proper context of the present study

This comment is also the same as one raised in the first review (Reviewer #1’s comment 3 on weaknesses). In our previous revision, in response to this comment, we cited the relevant references (Hallgren et al., 2006; O’Connell and Hofmann, 2012; Huffman et al., 2013; Jalabert et al., 2015; Yong et al., 2017; Alward et al., 2020; Ogino et al., 2023) in the appropriate sections. We also added the following new references and revised the Introduction and Discussion accordingly:

(2) Alward BA, Laud VA, Skalnik CJ, York RA, Juntti SA, Fernald RD. 2020. Modular genetic control of social status in a cichlid fish. Proceedings of the National Academy of Sciences of the United States of America 117:28167–28174. DOI: https://doi.org/10.1073/pnas.2008925117

(39) O’Connell LA, Hofmann HA. 2012. Social status predicts how sex steroid receptors regulate complex behavior across levels of biological organization. Endocrinology 153:1341–1351. DOI:https://doi.org/10.1210/en.2011-1663

(54) Yong L, Thet Z, Zhu Y. 2017. Genetic editing of the androgen receptor contributes to impaired male courtship behavior in zebrafish. Journal of Experimental Biology 220:3017–3021.DOI:https://doi.org/10.1242/jeb.161596

There are a surprising number of citations not included; some of the ones not included argue against the authors' claims that their findings were "contrary to expectation"

In our previous revision, we cited the relevant references (Hallgren et al., 2006; O’Connell and Hofmann, 2012; Huffman et al., 2013; Jalabert et al., 2015) in the Introduction. We also revised the text to remove phrases such as “contrary to expectation” and “unexpected.”

The experimental design for studying aggression in males has flaws. A standard test like a residentintruder test should be used.

Following this comment, we have attempted additional aggression assays using the resident-intruder paradigm. However, these experiments did not produce consistent or interpretable results. As noted in our previous revision, medaka naturally form shoals and exhibit weak territoriality, and even slight differences in dominance between a resident and an intruder can markedly increase variability, reducing data reliability. Therefore, we believe that the approach used in the present study provides a more suitable assessment of aggression in medaka, regardless of territorial tendencies. We will continue to explore potential refinements in future studies and respectfully ask the reviewer to evaluate the present work based on the assay used here.

While they investigate males and females, there are fewer experiments and explanations for the female results, making it feel like a small addition or an aside

While we did not adopt this comment in our previous revision, we have carefully reconsidered the reviewers’ feedback and have now decided to remove the female data. This change allows us to present a more focused and cohesive story centered on males. The specific revisions are outlined below:

Abstract

Line 25: The text “, thereby revealing a previously unappreciated mode of action of brain-derived estrogens. We additionally show that female fish lacking Cyp19a1b are less receptive to male courtship and conversely court other females, highlighting the significance of brain-derived estrogens in establishing sex-typical behaviors in both sexes.” has been revised to “. Taken together, these findings reveal a previously unappreciated mode of action of brain-derived estrogens in shaping male-typical behaviors.”

Results

Line 88: The text “Loss of cyp19a1b function in these fish was verified by measuring brain and peripheral levels of sex steroids. As expected, brain estradiol-17β (E2) in both male and female homozygous mutants (cyp19a1b^−/−) was significantly reduced to 16% and 50%, respectively, of the levels in their wild-type (cyp19a1b^+/+) siblings (P = 0.0037, males; P = 0.0092, females) (Fig. 1, A and B). In males, brain E2 in heterozygotes (cyp19a1b^−/−) was also reduced to 45% of the level in wild-type siblings (P = 0.0284) (Fig. 1A), indicating a dosage effect of cyp19a1b mutation. In contrast, peripheral E2 levels were unaltered in both cyp19a1b^−/− males and females (Fig. S1, C and D), consistent with the expected functioning of Cyp19a1b primarily in the brain. Strikingly, brain levels of testosterone, as opposed to E2, increased 2.2-fold in cyp19a1b^−/− males relative to wild-type siblings (P = 0.0006) (Fig. 1A). Similarly, brain 11KT levels in cyp19a1b^−/− males and females increased 6.2- and 1.9-fold, respectively, versus wild-type siblings (P = 0.0007, males; P = 0.0316, females) (Fig. 1, A and B). These results show that cyp19a1b-deficient fish have reduced estrogen levels coupled with increased androgen levels in the brain, confirming the loss of cyp19a1b function. They also suggest that the majority of estrogens in the male brain and half of those in the female brain are synthesized locally in the brain. In addition, peripheral 11KT levels in cyp19a1b^−/− males and females increased 3.7- and 1.8-fold, respectively (P = 0.0789, males; P = 0.0118, females) (Fig. S1, C and D), indicating peripheral influence in addition to central effects.” has been revised to “Loss of cyp19a1b function in these fish was verified by measuring brain and peripheral levels of sex steroids in males. As expected, brain estradiol-17β (E2) in homozygous mutants (cyp19a1b^−/−) was significantly reduced to 16% of the levels in wild-type (cyp19a1b^+/+) siblings (P = 0.0037) (Figure 1A). Brain E2 in heterozygotes (cyp19a1b^+/−) was also reduced to 45% of wild-type levels (P = 0.0284) (Figure 1A), indicating a dosage effect of the cyp19a1b mutation. In contrast, peripheral E2 levels were unaltered in cyp19a1b^−/− males (Figure 1B), consistent with the expected functioning of Cyp19a1b primarily in the brain. Strikingly, brain testosterone levels, as opposed to E2, increased 2.2-fold in cyp19a1b^−/− males relative to wild-type siblings (P = 0.0006) (Figure 1A). Similarly, brain 11KT levels increased 6.2-fold (P = 0.0007) (Figure 1A). These results indicate that cyp19a1b-deficient males have reduced estrogen coupled with elevated androgen levels in the brain, confirming the loss of cyp19a1b function. They also suggest that the majority of estrogens in the male brain are synthesized locally in the brain. Peripheral 11KT levels also increased 3.7-fold in cyp19a1b^−/− males (P = 0.0789) (Figure 1B), indicating peripheral influence in addition to central effects.”

Line 211: “expression of vt in the pNVT of cyp19a1b^−/− males was significantly reduced to 18% as compared with cyp19a1b^+/+ males (P = 0.0040), a level comparable to that observed in females” has been revised to “expression of vt in the pNVT of cyp19a1b^−/− males was significantly reduced to 18% as compared with cyp19a1b^+/+ males (P = 0.0040).”

The subsection entitled “cyp19a1b-deficient females are less receptive to males and instead court other females,” which followed line 311, has been removed.

Discussion

The two paragraphs between lines 373 and 374, which addressed the female data, have been removed.

Materials and methods

Line 433: “males and females” has been changed to “males”.

Line 457: “focal fish” has been changed to “focal male”.

Line 458: “stimulus fish” has been changed to “stimulus female”.

Line 458: “Fig. 6, E and F, ” has been deleted.

Line 460: “; wild-type males in Fig. 6, A to C” has been deleted.

Line 466: The text “The period of interaction/recording was extended to 2 hours in tests of courtship displays received from the stimulus esr2b-deficient female and in tests of mating behavior between females, because they take longer to initiate courtship (12). In tests using an esr2b-deficient female as the stimulus fish, where the latency to spawn could not be calculated because these fish were unreceptive to males and did not spawn, the sexual motivation of the focal fish was instead assessed by counting the number of courtship displays and wrapping attempts in 30 min. The number of these mating acts was also counted in tests to evaluate the receptivity of females. In tests of mating behavior between two females, the stimulus female was marked with a small notch in the caudal fin to distinguish it from the focal female.” has been revised to “In tests using an esr2b-deficient female as the stimulus fish, the latency to spawn could not be calculated because the female was unreceptive to males and did not spawn. Therefore, the sexual motivation of the focal male was assessed by counting the number of courtship displays and wrapping attempts in 30 min. To evaluate courtship displays performed by stimulus esr2bdeficient females toward focal males, the recording period was extended to 2 hours, as these females take longer to initiate courtship (Nishiike et al., 2021). In all video analyses, the researcher was blind to the fish genotype and treatment.”

Line 499: “brains dissected from males and females of the cyp19a1b-deficient line (analysis of ara, arb, vt, gal, npba, and esr2b) and males of the esr1-, esr2a-, and esr2b-deficient lines” has been revised to “male brains from the cyp19a1b-deficient line (analysis of ara, arb, vt, and gal) and from the esr1-, esr2a-, and esr2b-deficient lines.”

Line 504: “After color development for 15 min (gal), 40 min (npba), 2 hours (vt), or overnight (ara, arb, and esr2b)” has been revised to “After color development for 15 min (gal), 2 hours (vt), or overnight (ara and arb).”

Line 516: “Thermo Fisher Scientific, Waltham, MA” has been changed to “Thermo Fisher Scientific” to avoid redundancy.

Line 565: The subsection entitled “Measurement of spatial distances between fish” has been removed.

Line 585: “6/10 cyp19a1b^+/+, 3/10 cyp19a1b^+/−, and 6/10 cyp19a1b^−/− females were excluded in Fig. 6B;” has been deleted.

References

The following references have been removed:

Capel B. 2017. Vertebrate sex determination: evolutionary plasticity of a fundamental switch. Nature Reviews Genetics 18:675–689. DOI: https://doi.org/10.1038/nrg.2017.60

Hiraki T, Nakasone K, Hosono K, Kawabata Y, Nagahama Y, Okubo K. 2014. Neuropeptide B is femalespecifically expressed in the telencephalic and preoptic nuclei of the medaka brain. Endocrinology 155:1021–1032. DOI: https://doi.org/10.1210/en.2013-1806

Juntti SA, Hilliard AT, Kent KR, Kumar A, Nguyen A, Jimenez MA, Loveland JL, Mourrain P, Fernald RD. 2016. A neural basis for control of cichlid female reproductive behavior by prostaglandin F2α. Current Biology 26:943–949. DOI: https://doi.org/10.1016/j.cub.2016.01.067

Kimchi T, Xu J, Dulac C. 2007. A functional circuit underlying male sexual behaviour in the female mouse brain. Nature 448:1009–1014. DOI: https://doi.org/10.1038/nature06089

Kobayashi M, Stacey N. 1993. Prostaglandin-induced female spawning behavior in goldfish (Carassius auratus) appears independent of ovarian influence. Hormones and Behavior 27:38–55.

DOI:https://doi.org/10.1006/hbeh.1993.1004

Liu H, Todd EV, Lokman PM, Lamm MS, Godwin JR, Gemmell NJ. 2017. Sexual plasticity: a fishy tale. Molecular Reproduction and Development 84:171–194. DOI: https://doi.org/10.1002/mrd.22691

Munakata A, Kobayashi M. 2010. Endocrine control of sexual behavior in teleost fish. General and Comparative Endocrinology 165:456–468. DOI: https://doi.org/10.1016/j.ygcen.2009.04.011

Nugent BM, Wright CL, Shetty AC, Hodes GE, Lenz KM, Mahurkar A, Russo SJ, Devine SE, McCarthy MM. 2015. Brain feminization requires active repression of masculinization via DNA methylation. Nature Neuroscience 18:690–697. DOI: https://doi.org/10.1038/nn.3988

Shaw K, Therrien M, Lu C, Liu X, Trudeau VL. 2023. Mutation of brain aromatase disrupts spawning behavior and reproductive health in female zebrafish. Frontiers in Endocrinology 14:1225199.

DOI:https://doi.org/10.3389/fendo.2023.1225199

Stacey NE. 1976. Effects of indomethacin and prostaglandins on the spawning behaviour of female goldfish. Prostaglandins 12:113–126. DOI: https://doi.org/10.1016/s0090-6980(76)80010-x

Figure 1

Panel B, which originally showed steroid levels in female brains, has been replaced with steroid levels in the periphery of males, originally presented in Figure S1, panel C. Accordingly, the legend “(A and B) Levels of E2, testosterone, and 11KT in the brain of adult cyp19a1b^+/+, cyp19a1b^+/−, and cyp19a1b^−/− males (A) and females (B) (n = 3 per genotype and sex).” has been revised to “(A, B) Levels of E2, testosterone, and 11KT in the brain (A) and periphery (B) of adult cyp19a1b^+/+, cyp19a1b^+/−, and cyp19a1b^−/− males (n = 3 per genotype).”

Figure 3

The female data have been deleted from Figure 3. The revised Figure 3 is presented.

The corresponding legend text has been revised as follows:

Line 862: “males and females (n = 4 and 5 per genotype for males and females, respectively)” has been changed to “males (n = 4 per genotype)”.

Line 864: “males and females (n = 4 except for cyp19a1b^+/+ males, where n = 3)” has been changed to “males (n = 3 and 4, respectively)”.

Figure 6

Figure 6 and its legend have been removed.

Figure 1—figure supplement 1

Panel C, showing male data, has been moved to Figure 1B, as described above, while panel D, showing female data, has been deleted. The corresponding legend “(C and D) Levels of E2, testosterone, and 11KT in the periphery of adult cyp19a1b^+/+, cyp19a1b^+/−, and cyp19a1b^−/− males (C) and females (D) (n = 3 per genotype and sex). Statistical differences were assessed by Bonferroni’s post hoc test (C and D). Error bars represent SEM. *P < 0.05.” has also been removed.

Line 804: Following this change, the figure title has been updated from “Generation of cyp19a1bdeficient medaka and evaluation of peripheral sex steroid levels” to “Generation of cyp19a1b-deficient medaka.”

The statistics comparing "experimental to experimental" and "control to experimental" isn't appropriate 

This comment is the same as one raised in the first review (Reviewer #1’s comment 7 on weaknesses), which we already addressed in our initial revision. For the reviewer’s convenience, we provide the response below:

The reviewer raised concerns about the statistical analysis used for Figures 4C and 4E, suggesting that Bonferroni’s test should be used instead of Dunnett’s test. However, Dunnett’s test is commonly used to compare treatment groups to a reference group that receives no treatment, as in our study. Since we do not compare the treated groups with each other, we believe Dunnett’s test is the most appropriate choice.

Line 576: The reviewer’s concern may have arisen from the phrase “comparisons between control and experimental groups” in the Materials and methods. We have revised it to “comparisons between untreated and E2-treated groups in Figure 4C and D” for clarity.

Reviewer #3 (Public Review):

Summary:

Taking advantage of the existence in fish of two genes coding for estrogen synthase, the enzyme aromatase, one mostly expressed in the brain (Cyp19a1b) and the other mostly found in the gonads (Cyp19a1a), this study investigates the role of brain-derived estrogens in the control of sexual and aggressive behavior in medaka. The constitutive deletion of Cyp19a1b markedly reduced brain estrogen content in males and to a lesser extent in females. These effects are accompanied by reduced sexual and aggressive behavior in males and reduced preference for males in females. These effects are reversed by adult treatment with supporting a role for estrogens. The deletion of Cyp19a1b is associated with a reduced expression of the genes coding for the two androgen receptors, ara and arb, in brain regions involved in the regulation of social behavior. The analysis of the gene expression and behavior of mutants of estrogen receptors indicates that these effects are likely mediated by the activation of the esr1 and esr2a isoforms. These results provide valuable insight into the role of estrogens in social behavior in the most abundant vertebrate taxon, however the conclusion of brain-derived estrogens awaits definitive confirmation.

We thank this reviewer for their positive evaluation of our work and comments that have improved the manuscript.

Strength:

Evaluation of the role of brain "specific" Cyp19a1 in male teleost fish, which as a taxon are more abundant and yet proportionally less studied that the most common birds and rodents. Therefore, evaluating the generalizability of results from higher vertebrates is important. This approach also offers great potential to study the role of brain estrogen production in females, an understudied question in all taxa.

Results obtained from multiple mutant lines converge to show that estrogen signaling, likely synthesized in the brain drives aspects of male sexual behavior.

The comparative discussion of the age-dependent abundance of brain aromatase in fish vs mammals and its role in organization vs activation is important beyond the study of the targeted species.  - The authors have made important corrections to tone down some of the conclusions which are more in line with the results. 

We thank the reviewer again for their positive evaluation of our work and the revisions we have made.

weaknesses:

No evaluation of the mRNA and protein products of Cyp19a1b and ESR2a are presented, such that there is no proper demonstration that the mutation indeed leads to aromatase reduction. The conclusion that these effects dependent on brain derived estrogens is therefore only supported by measures of E2 with an EIA kit that is not validated. No discussion of these shortcomings is provided in the discussion thus further weakening the conclusion manuscript.

In response to this and other comments, we have now provided direct validation that the cyp19a1b mutation in our medaka leads to loss of function. Real-time PCR analysis showed that cyp19a1b transcript levels in the brain were reduced by approximately half in cyp19a1b^+/− males and were nearly absent in cyp19a1b^−/− males, consistent with nonsense-mediated mRNA decay

In addition, AlphaFold 3-based structural modeling indicated that the mutant Cyp19a1b protein lacks essential motifs, including the aromatic region and heme-binding loop, and exhibits severe conformational distortion (see figure; key structural features are annotated as follows: membrane helix (blue), aromatic region (red), and heme-binding loop (orange)). 

Results:

Line 101: The following text has been added: “Loss of cyp19a1b function was further confirmed by measuring cyp19a1b transcript levels in the brain and by predicting the three-dimensional structure of the mutant protein. Real-time PCR revealed that transcript levels were reduced by half in cyp19a1b^+/− males and were nearly undetectable in cyp19a1b^−/− males, presumably as a result of nonsense-mediated mRNA decay (Lindeboom et al., 2019) (Figure 1C). The wild-type protein, modeled by AlphaFold 3, exhibited a typical cytochrome P450 fold, including the membrane helix, aromatic region, and hemebinding loop, all arranged in the expected configuration (Figure 1—figure supplement 1C). The mutant protein, in contrast, was severely truncated, retaining only the membrane helix (Figure 1—figure supplement 1C). The absence of essential domains strongly indicates that the allele encodes a nonfunctional Cyp19a1b protein. Together, transcript and structural analyses consistently demonstrate that the mutation generated in this study causes a complete loss of cyp19a1b function.”

Materials and methods

Line 438: A subsection entitled “Real-time PCR” has been added. The text of this subsection is as follows: “Total RNA was isolated from the brains of cyp19a1b^+/+, cyp19a1b^+/−, and cyp19a1b^−/− males using the RNeasy Plus Universal Mini Kit (Qiagen, Hilden, Germany). cDNA was synthesized with the SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA). Real-time PCR was performed on the LightCycler 480 System II using the LightCycler 480 SYBR Green I Master (Roche Diagnostics). Melting curve analysis was conducted to verify that a single amplicon was obtained in each sample. The β-actin gene (actb; GenBank accession number NM_001104808) was used to normalize the levels of target transcripts. The primers used for real-time PCR are shown in Supplementary file 2.”

Line 448: A subsection entitled “Protein structure prediction” has been added. The text of this subsection is as follows: “Structural predictions of Cyp19a1b proteins were conducted using AlphaFold 3 (Abramson et al., 2024). Amino acid sequences corresponding to the wild-type allele and the mutant allele generated in this study were submitted to the AlphaFold 3 prediction server. The resulting models were visualized with PyMOL (Schrödinger, New York, NY), and key structural features, including the membrane helix, aromatic region, and heme-binding loop, were annotated.”

References

The following two references have been added:

Abramson J, Adler J, Dunger J, Evans R, Green T, Pritzel A, Ronneberger O, Willmore L, Ballard AJ, Bambrick J, Bodenstein SW, Evans DA, Hung CC, O'Neill M, Reiman D, Tunyasuvunakool K, Wu Z, Žemgulytė A, Arvaniti E, Beattie C, Bertolli O, Bridgland A, Cherepanov A, Congreve M, CowenRivers AI, Cowie A, Figurnov M, Fuchs FB, Gladman H, Jain R, Khan YA, Low CMR, Perlin K, Potapenko A, Savy P, Singh S, Stecula A, Thillaisundaram A, Tong C, Yakneen S, Zhong ED, Zielinski M, Žídek A, Bapst V, Kohli P, Jaderberg M, Hassabis D, Jumper JM. 2024. Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature 630:493–500. DOI: https://doi.org/10.1038/s41586-024-07487-w

Lindeboom RGH, Vermeulen M, Lehner B, Supek F. 2019. The impact of nonsense-mediated mRNA decay on genetic disease, gene editing and cancer immunotherapy. Nature Genetics 51:1645–1651.DOI:https://doi.org/10.1038/s41588-019-0517-5

Figure 1

The real-time PCR results described above have been incorporated in Figure 1, panel C, with the corresponding legend provided below (line 788).

(C) Brain cyp19a1b transcript levels in cyp19a1b^+/+, cyp19a1b^+/−, and cyp19a1b^−/− males (n = 6 per genotype). Mean value for cyp19a1b^+/+ males was arbitrarily set to 1.

The subsequent panels have been renumbered accordingly. The entirety of the revised Figure 1.

Figure 1—figure supplement 1

The AlphaFold 3-generated structural models described above have been incorporated in Figure 1— figure supplement 1, panel C, with the corresponding legend provided below (line 811).

(C) Predicted three-dimensional structures of wild-type (left) and mutant (right) Cyp19a1b proteins. Key structural features are annotated as follows: membrane helix (blue), aromatic region (red), and heme-binding loop (orange).

The entirety of the revised Figure 1—figure supplement 1 is presented

The information on the primers used for real-time PCR has been included in Supplementary file 2.

The functional deficiency of esr2a was already addressed in the previous revision. For clarity, we have reproduced the relevant information here.

A previous study reported that female medaka lacking esr2a fail to release eggs due to oviduct atresia (Kayo et al., 2019, Sci Rep 9:8868). Similarly, in this study, some esr2a-deficient females exhibited spawning behavior but were unable to release eggs, although the sample size was limited (Δ8 line: 2/3; Δ4 line: 1/1). In contrast, this was not observed in wild-type females (Δ8 line: 0/12; Δ4 line: 0/11). These results support the effective loss of esr2a function. To incorporate this information into the manuscript, the following text has been added to the Materials and methods (line 423): “A previous study reported that esr2a-deficient female medaka cannot release eggs due to oviduct atresia (Kayo et al., 2019). Likewise, some esr2a-deficient females generated in this study, despite the limited sample size, exhibited spawning behavior but were unable to release eggs (Δ8 line: 2/3; Δ4 line: 1/1), while such failure was not observed in wild-type females (Δ8 line: 0/12; Δ4 line: 0/11). These results support the effective loss of esr2a function.”

Most experiments are weakly powered (low sample size).

This comment is essentially the same as one raised in the first review (Reviewer #3’s comment 7 on weaknesses). We acknowledge the reviewer’s concern that the histological analyses were weakly powered due to the limited sample size. In our earlier revision, we responded as follows:

Histological analyses were conducted with a relatively small sample size, as our previous experience suggested that interindividual variability in the results would not be substantial. Since significant differences were detected in many analyses, further increasing the sample size was deemed unnecessary.

The variability of the mRNA content for a same target gene between experiments (genotype comparison vs E2 treatment comparison) raises questions about the reproducibility of the data (apparent disappearance of genotype effect).

This comment is the same as one raised in the first review (Reviewer #3’s comment 8 on weaknesses), which we already addressed in our initial revision. For the reviewer’s convenience, we provide the response below:

As the reviewer pointed out, the overall area of ara expression is larger in Figure 2J than in Figure 2F. However, the relative area ratios of ara expression among brain nuclei are consistent between the two figures, indicating the reproducibility of the results. Thus, this difference is unlikely to affect the conclusions of this study.

Additionally, the differences in ara expression in pPPp and arb expression in aPPp between wild-type and cyp19a1b-deficient males appear less pronounced in Figures 2J and 2K than in Figures 2F and 2H. This is likely attributable to the smaller sample size used in the experiments for Figures 2J and 2K, resulting in less distinct differences. However, as the same genotype-dependent trends are observed in both sets of figures, the conclusion that ara and arb expression is reduced in cyp19a1b-deficient male brains remains valid.

Conclusions:

Overall, the claims regarding role of estrogens originating in the brain on male sexual behavior is supported by converging evidence from multiple mutant lines. The role of brain-derived estrogens on gene expression in the brain is weaker as are the results in females. 

We appreciate the reviewer’s positive evaluation of our findings on male behavior. The concern regarding the role of brain-derived estrogens in gene expression has been addressed in our rebuttal, and the female data have been removed so that the analysis now focuses on males. The specific revisions for removing the female data are described in Response to reviewer #1’s comment 6 on weaknesses.

Recommendations For The Authors:

Reviewer #1 (Recommendations For The Authors):

The manuscript is improved slightly. I am thankful the authors addressed some concerns, but for several concerns the referees raised, the authors acknowledged them yet did not make corresponding changes to the manuscript or disagreed that they were issues at all without explanation. All reviewers had issues with the imbalanced focus on males versus females and the male aggression assay. Yet, they did not perform additional experiments or even make changes to the framing and scope of the manuscript. If the authors had removed the female data, they may have had a more cohesive story, but then they would still be left with inadequate behavior assays in the males. If the authors don't have the time or resources to perform the additional work, then they should have said so. However, the work would be incomplete relative to the claims. That is a key point here. If they change their scope and claims, the authors avoid overstating their findings. I want to see this work published because I believe it moves the field forward. But the authors need to be realistic in their interpretations of their data. 

In response to this and related comments, we have removed the female data and focused the manuscript on analyses in males. The specific revisions are described in Response to reviewer #1’s comment 6 on weaknesses. Additionally, we have validated that the cyp19a1b mutation in our medaka leads to loss of function (see Response to reviewer #3’s comment 1 on weaknesses), which further strengthens the reliability of our conclusions regarding male behavior.

I agree with the reviewer who said we need to see validation of the absence of functional cyp19a1 b in the brain. However, the results from staining for the protein and performing in situ could be quizzical. Indeed, there aren't antibodies that could distinguish between aromatase a and b, and it is not uncommon for expression of a mutated gene to be normal. One approach they could do is measure aromatase activity, but they are *sort of* doing that by measuring brain E2. It's not perfect, but we teleost folks are limited in these areas. At the very least, they should show the predicted protein structure of the mutated aromatase alleles. It could show clearly that the tertiary structure is utterly absent, giving more support to the fact that their aromatase gene is non-functional. 

As noted above, we have further validated the loss of cyp19a1b function by measuring cyp19a1b transcript levels in the brain and predicting the three-dimensional structure of the mutant protein. These analyses confirmed that cyp19a1b function is indeed lost, thereby increasing the reliability of our conclusions. For further details, please refer to Response to reviewer #3’s comment 1 on weaknesses.

With all of this said, the work is important, and it is possible that with a reframing of the impact of their work in the context of their findings, I could consider the work complete. I think with a proper reframing, the work is still impactful. 

In accordance with this feedback, and as described above, we have reframed the manuscript by removing the female data and focusing exclusively on males. This revision clarifies the scope of our study and reinforces the support for our conclusions. For further details, please refer to Response to reviewer #1’s comment 6 on weaknesses.

(1) Clearly state in the Figure 1 legend that each data point for male aggressive behaviors represents the total # of behaviors calculated over the 4 males in each experimental tank.

In response to this comment, we have revised the legend of Figure 1K (line 797). The original legend, “(K) Total number of each aggressive act observed among cyp19a1b^+/+, cyp19a1b^+/−, or cyp19a1^−/− males in the tank (n = 6, 7, and 5, respectively),” has been updated to “(K) Total number of each aggressive act performed by cyp19a1b^+/+, cyp19a1b^+/−, and cyp19a1b^−/− males. Each data point represents the sum of acts recorded for the 4 males of the same genotype in a single tank (n = 6, 7, and 5 tanks, respectively).” This clarifies that each data point reflects the total behaviors of the 4 males within each tank.

(2) The authors wrote under "Response to reviewer #1's major comment "...the development of male behaviors may require moderate neuroestrogen levels that are sufficient to induce the expression of ara and arb, but not esr2b, in the underlying neural circuitry": "This may account for the lack of aggression recovery in E2-treated cyp19a1b-deficient males in this study.".

What is meant by the latter statement? What accounts for the lack of aggression? The lack of increase in esr2b? Please clarify. 

Line 365: In response to this comment, “This may account for the lack of aggression recovery in E2treated cyp19a1b-deficient males in this study.” has been revised to “Considering this, the lack of aggression recovery in E2-treated cyp19a1b-deficient males in this study may be explained by the possibility that the E2 dose used was sufficient to induce not only ara and arb but also esr2b expression in aggression-relevant circuits, which potentially suppressed aggression.”

This revision clarifies that, while moderate brain estrogen levels are sufficient to promote male behaviors via induction of ara and arb, the E2 dose used in this study may have additionally induced esr2b in circuits relevant to aggression, potentially underlying the lack of aggression recovery.

(3) This is a continuation of my comment/concern directly above. If the induction of ara and arb aren't enough, then how can, as the authors state, androgen signaling be the primary driver of these behaviors? 

In response to this follow-up comment, we would like to clarify that, as described above, the lack of aggression recovery in E2-treated cyp19a1b-deficient males is not due to insufficient induction of ara and arb, but instead is likely because esr2b was also induced in aggression-relevant circuits, which may have suppressed aggression. Therefore, the concern that androgen signaling cannot be the primary driver of these behaviors is not applicable.

(4) The authors' point about sticking with the terminology for the ar genes as "ara" and "arb" is not convincing. The whole point of needing a change to match the field of neuroendocrinology as a whole (that is, across all vertebrates) is researchers, especially those with high standing like the Okubo group, adopt the new terminology. Indeed, the Okubo group is THE leader in medaka neuroendocrinology. It would go a long way if they began adopting the new terminology of "ar1" and "ar2". I understand this may be laborious to a degree, and each group can choose to use their terminology, but I'd be remiss if I didn't express my opinion that changing the terminology could help our field as a whole. 

We sincerely appreciate the reviewer’s thoughtful comments regarding nomenclature consistency in vertebrate neuroendocrinology. We understand the motivation behind the suggestion to adopt ar1 and ar2. However, we consider the established nomenclature of ara and arb to be more appropriate for the following reasons.

First, adopting the ar1/ar2 nomenclature would introduce a discrepancy between gene and protein symbols. According to the NCBI International Protein Nomenclature Guidelines (Section 2B.Abbreviations and symbols;

https://www.ncbi.nlm.nih.gov/genbank/internatprot_nomenguide/), the ZFIN Zebrafish Nomenclature Conventions (Section 2. PROTEINS:https://zfin.atlassian.net/wiki/spaces/general/pages/1818394635/ZFIN+Zebrafish+Nomenclature+Con ventions), and the author guidelines of many journal

(e.g.,https://academic.oup.com/molehr/pages/Gene_And_Protein_Nomenclature), gene and protein symbols should be identical (with proteins designated in non-italic font and with the first letter capitalized). Maintaining consistency between gene and protein symbols helps avoid unnecessary confusion. The ara/arb nomenclature allows this, whereas ar1/ar2 does not.

Second, the two androgen receptor genes in teleosts are paralogs derived from the third round of wholegenome duplication that occurred early in teleost evolution. For such duplicated genes, the ZFIN Zebrafish Nomenclature Conventions (Section 1.2. Duplicated genes) recommend appending the suffixes “a” and “b” to the approved symbol of the human or mouse ortholog. This convention clearly indicates that these genes are whole-genome duplication paralogs and provides an intuitive way to represent orthologous and paralogous relationships between teleost genes and those of other vertebrates. As a result, it has been widely adopted, and we consider it logical and beneficial to apply the same principle to androgen receptors.

In light of these considerations, we respectfully maintain that the ara/arb nomenclature is more suitable for the present manuscript than the alternative ar1/ar2 system.

(5) In the discussion please discuss these potentially unexpected findings.

(a) gal was unaffected in female cyp19a1 mutants, but they exhibit mating behaviors towards females. Given gal is higher in males and these females act like females, what does this mean about the function of gal/its utility in being a male-specific marker (is it one??)? 

(b) esr2b expression is higher in female cyp19a1 mutants. this is unexpected as well given esr2b is required for female-typical mating and is higher in females compared to males and E2 increases esr2b expression. please explain...well, what this means for our idea of what esr2b expression tell us. 

We thank the reviewer for the insightful comments. As the female data have been removed from the manuscript, discussion of these findings in female cyp19a1b mutants is no longer necessary.

Reviewer #3 (Recommendations For The Authors):

The authors have addressed a number of answers to the reviewer's comments, notably they provided missing methodological information and rephrased the text. However, the authors have not addressed the main issues raised by the reviewers. Notably, it is regrettable that the reduced amount of brain aromatase cannot be confirmed, this seems to be the primary step when validating a new mutant. Even if protein products of the two genes may not be discriminated (which I can understand), it should be possible to evaluate the expression of a common messenger and/or peptide and confirm that aromatase expression is reduced in the brain. Since Cyp19a1b is relatively more abundant in the brain Cyp19a1a, this would strengthen the conclusion and provide confidence that the mutant indeed does silence aromatase expression in the brain. Although these short comings are acknowledged in the rebuttal letter, this is not mentioned in the discussion. Doing so would make the manuscript more transparent and clearer. 

As noted in Response to reviewer #3’s comment 1 on weaknesses, we have validated the loss of Cyp19a1b function by measuring its transcript levels in the brain and predicting the three-dimensional structure of the mutant protein. These analyses confirmed that Cyp19a1b function is indeed lost, thereby increasing the reliability of our conclusions.

FigS1 - panels C&D please indicate in which tissue were hormones measured. Blood?

We thank the reviewer for pointing this out. In our study, “peripheral” refers to the caudal half of the body excluding the head and visceral organs, not blood. Accordingly, we have revised the figure legend and the description in the Materials and Methods section as follows:

Legend for Figure 1B (line 787) now reads: “Levels of E2, testosterone, and 11KT in the brain (A) and peripheral tissues (caudal half of the body) (B) of adult cyp19a1b^+/+, cyp19a1b^+/−, and cyp19a1b^−/− males (n = 3 per genotype).”

Materials and methods (line 431): The sentence “Total lipids were extracted from the brain and peripheral tissues (from the caudal half) of” has been revised to “Total lipids were extracted from the brain and from peripheral tissues, specifically the caudal half of the body excluding the head and visceral organs, of.”

Additional Alterations:

We have reformatted the text and supporting materials to comply with the journal’s Author Guidelines. The following changes have been made:

(1) Figures and supplementary files are now provided separately from the main text.

(2) The title page has been reformatted without any changes to its content.

(3) In-text citations have been changed from numerical references to the author–year format.

(4) Figure labels have been revised from “Fig. 1,” “Fig. S1,” etc., to “Figure 1,” “Figure 1—figure supplement 1,” etc.

(5) Table labels have been revised from “Table S1,” etc., to “Supplementary file 1,” etc.

(6) Line 324: The typo “is” has been corrected to “are”.

(7) Line 382: The section heading “Materials and Methods” has been changed to “Materials and methods” (lowercase “m”).

(8) Line 383: The Key Resources Table has been placed at the beginning of the Materials and methods section.

(9) Line 389: The sentence “Sexually mature adults (2–6 months) were used for experiments, and tissues were consistently sampled 1–5 hours after lights on.” has been revised to “Sexually mature adults (2–6 months) were used for experiments and assigned randomly to experimental groups. Tissues were consistently sampled 1–5 hours after lights on.”

(10)  Line 393: The sentence “All fish were handled in accordance with the guidelines of the Institutional Animal Care and Use Committee of the University of Tokyo.” has been removed.

(11)  Line 589: The following sentence has been added: “No power analysis was conducted due to the lack of relevant data; sample size was estimated based on previous studies reporting inter-individual variation in behavior and neural gene expression in medaka.”

(12)  Line 598: The reference list has been reordered from numerical sequence to alphabetical order by author.

(13)  In the figure legends, notations such as “A and B” have been revised to “A, B.”

app discovery

History of discovery of Rosetta Stone

(...on July 15, 1799, during Napoleon Bonaparte’s Egyptian campaign, a French soldier Pierre Bouchard discovered a black basalt slab inscribed with ancient writing near the town of Rosetta, about 35 miles east of Alexandria. The irregularly shaped stone contained fragments of passages written in three different scripts: Greek, Egyptian hieroglyphics and Egyptian demotic. The ancient Greek on the Rosetta Stone told archaeologists that it was inscribed by priests honoring the king of Egypt, Ptolemy V, in the second century B.C. More startlingly, the Greek passage announced that the three scripts were all of identical meaning. The artifact thus held the key to solving the riddle of hieroglyphics, a written language that had been “dead” for nearly 2,000 years.) For rest of the article click Rosetta Stone Found

Why is Rosetta Stone important? Click Importance of Rosetta Stone

eLife Assessment

This paper describes a useful Bayesian model to estimate the probabilities of access, use, and use given access of insecticide-treated bed nets (ITNs), by using sub-national cross-sectional survey data and the annual number of ITNs received at the country level. The authors provide convincing evidence to support their modeling approach, which could be enhanced by more validation and exploration of model assumptions.

Reviewer #1 (Public review):

Summary:

This paper provides a novel method to improve the accuracy of predictions of the impact of ITN strategies, by using sub-national estimates of the duration of ITN access and use over time from cross-sectional survey data and annual country ITNs received.

Strengths:

The approach is novel, makes use of available data, and has considered all of the relevant components of ITN distributions.

Weaknesses:

(1) The main message of the paper was not very clear, and did not seem to fit the title. The title focuses on sub-national tailoring of ITN, but the abstract did not feature results directly about SNT. It was not very clear what the main result of the paper was - there are several ITN observations in the results and discussion. Most did not seem to be directly about SNT, but rather sub-national differences in use and access were accounted for in the analyses. It was not clear if the same conclusions would be reached without accounting for sub-national differences, but the estimates and predictions could be expected to be more accurate.

(2) Some of the results seemed to me to be apparent even without a modelling exercise (eg high coverage could not be maintained between campaigns, use would be higher with 2-yearly distributions rather than 3-yearly) or were not in themselves new insights (eg estimates of the duration of use). It would be helpful to clearly state what the novel results are in the abstract, the first paragraph of the discussion and the conclusions, and to make sure that the title is consistent.

(3) On L236, the link to SNT is stated: "the models indicate trends that can support sub-national tailoring of ITNs". They could indeed, but SNT itself is not done in this paper. It seems to be about improving sub-national predictions of the impact of single ITN strategies, by taking into account sub-national variation in access and use duration. This is useful, and the model developed has novel aspects.

(4) Individual countries may have records on when nets were distributed to the regions rather than needing to use the annual country number of nets together with the DHS data. It could be helpful to say what the analysis steps would be in that case.

(5) There were several assumptions that needed to be made in building the model. There is some validation of the timing of the distributions (L633 "verified where possible through discussion with interested parties nationally and internationally") and the fit of estimated access and use to survey data, and agreement between predictions of prevalence and MAP estimates. It would be helpful to say which assumptions are important for the results (and would be key knowledge gaps) and which would not make a difference. It might be possible to validate the net timing model using a country where net distributions are known reasonably well.

(6) What was assumed about what happens to old nets after a mass campaign was not clear. This assumption is likely to affect the predictions of access for the biennial distributions.

(7) L312 and elsewhere: That use given access declines with net age is plausible. However, I wondered if this could be partly a consequence of the assumptions in the model (eg the two exponential decays for access and use, the possible assumption that new nets displace the current ones when there is a mass campaign).

(8) The Methods section on Estimating historical use and access seemed to be aimed at readers familiar with formulae, but I think it could lose other interested readers. It could be useful to explain a little more about what is happening at each step and also why.

(9) The model was fitted to MAP estimates of PfPR2-10, which themselves come from a model. It may be that there is different uncertainty in the MAP estimates for different regions. I couldn't see this on the graph, but maybe the uncertainty is small. Was this taken into account in the fitting?

(10) Was uncertainty from each estimated component integrated into the other components?

(11) Eyeballing Figure 2 (Burkina Faso), there is a general pattern of decline in all the regions, some differences between the regions and some differences in how well the model fits between the regions. If possible, it could be helpful to say how much better the fit was when using region-specific compared to countrywide parameter values for access and use, and how different the results would be.

(12) The question of moving from a campaign every three to every two years may not be the most pertinent question in the current funding landscape. I realise that a paper is in development for a long time, but it would be helpful to comment on what else the model could be used for when fewer rather than more nets are likely to be available.

Reviewer #2 (Public review):

Summary:

The authors design a custom Bayesian model to estimate the probabilities of access, use and use given access of insecticide-treated nets in six African countries, providing sub-national estimates and inferring the average duration of ITN use and access. An individual-based model was employed to simulate malaria epidemics and estimate the effectiveness of different ITN distribution strategies. The study finds that the mean probability of use or access did not reach 80% (a universal coverage formely targeted by WHO) for any of the regions, even for biennial campaigns, demonstrates that switching from triennial to biennial distribution campaigns increases population use by 7.9%, and evaluates the impact of employing more efficient ITNs on P. falciparum prevalence.

Strengths:

The authors developed a data-driven model that accounts for data collection imperfections and sources of uncertainty while differentiating between ITN use and access. They developed a methodology to infer the timing of a mass campaign from publicly available data instead of assuming fixed dates. The probability of use given access allows for determining the regions where ITN distribution is least effective. This work can help better inform future interventions by identifying regions where increasing mass campaign frequency or employing better ITNs are most effective. Finally, in addition to insights on ITN access and use for the six countries analyzed, the paper contributes a methodological framework that can likely be extended to other countries.

Weaknesses:

Since the models employed are rather complex, the description of the methodology may be hard to follow for most readers. In addition, the models assume many hypotheses, including:

(1) Exponential decay of ITN use/access.

(2) The decay rates for the probability of the ITN repelling and killing a mosquito are the same.

(3) Given a time instant, all individuals in the same administrative unit and have the same probability of using a net;

(4) ITN use/access decay models do not depend on the distribution strategy (e.g. bienal vs trienal distribution).

(5) The Bayesian model assumes some narrow prior distributions.

The impact of these hypotheses on the estimated parameters is not explored in the paper, and no sensitivity analyses are performed, although some limitations are discussed.

Author response:

We would like to thank both reviewers for taking the time to review the manuscript in detail. Your comments have been extremely useful and constructive. A revised version of the manuscript will seek to address the weaknesses raised, clarifying the reasons for the assumptions made, the impact they have and how they influence the policy implication of the work. We will clarify the language to differentiate the work from the standard sub-national tailoring which is typically conducted to support National Malaria Programmes and emphasise why our mechanistic model can provide greater information than simple summary statistics.

what´s the purpose of this button?

eLife Assessment

The authors provide a useful integrated analytical approach to investigating MASLD focused on diverse multiomic integration methods. The strength of evidence for this new resource is solid, as analyses highlight the importance of previously-described pathophysiologic processes, as well as unveil several new mechanisms as key features of MASLD in obese patients.

Reviewer #1 (Public review):

Summary:

Metabolic dysfunction-associated steatotic liver disease (MASLD) ranges from simple steatosis, steatohepatitis, fibrosis/cirrhosis, and hepatocellular carcinoma. In the current study, the authors aimed to determine the early molecular signatures differentiating patients with MASLD associated fibrosis from those patients with early MASLD but no symptoms. The authors recruited 109 obese individuals before bariatric surgery. They separated the cohorts as no MASLD (without histological abnormalities) and MASLD. The liver samples were then subjected to transcriptomic and metabolomic analysis. The serum samples were subjected to metabolomic analysis. The authors identified dysregulated lipid metabolism, including glyceride lipids, in the liver samples of MASLD patients compared to the no MASLD ones. Circulating metabolomic changes in lipid profiles slightly correlated with MASLD, possibly due to the no MASLD samples derived from obese patients. Several genes involved in lipid droplet formation were also found elevated in MASLD patients. Besides, elevated levels of amino acids, which are possibly related to collagen synthesis, were observed in MASLD patients. Several antioxidant metabolites were increased in MASLD patients. Furthermore, dysregulated genes involved in mitochondrial function and autophagy were identified in MASLD patients, likely linking oxidative stress to MASLD progression. The authors then determined the representative gene signatures in the development of fibrosis by comparing this cohort with the other two published cohorts. Top enriched pathways in fibrotic patients included GTPas signaling and innate immune responses, suggesting the involvement of GTPas in MASLD progression to fibrosis. The authors then challenged human patient derived 3D spheroid system with a dual PPARa/d agonist and found that this treatment restored the expression levels of GTPase-related genes in MASLD 3D spheroids. In conclusion, the authors suggested the involvement of upregulated GTPase-related genes during fibrosis initiation.

Concerns from first round of review:

(1) A recent study, via proteomic and transcriptomic analysis, revealed that four proteins (ADAMTSL2, AKR1B10, CFHR4 and TREM2) could be used to identify MASLD patients at risk of steatohepatitis (PMID: 37037945). It is not clear why the authors did not include this study in their comparison.

(2) The authors recruited 109 patients but only performed transcriptomic and metabolomic analysis in 94 liver samples. Why did the authors exclude other samples?

(3) The authors mentioned clinical data in Table 1 but did not present the table in this manuscript.

(4) The generated metabolomic data could be a very useful resource to the MASLD community. However, it is very confusing how the data was generated in those supplemental tables. There is no clear labeling of human clinical information in those tables. Also, what do those values mean in columns 47-154? This reviewer assumed that they are the raw data of metabolomic analysis in plasma samples. However, without clear clinical information in these patients, it is impossible that any scientist can use the data to reproduce the authors' findings.

(5) In Fig. 5B, the authors excluded the steatosis and fibrosis overlapped genes. Steatosis and fibrosis specific genes could simply reflect the outcomes rather than causes. In this case, the obtained results might not identify the gene signatures related to fibrosis initiation.

(6 In Fig. 6D, the authors used 3D liver spheroid to validate their findings. However, there is no images showing the 3D liver spheroid formation before and after PPARa/d agonist treatment. It is not clear whether the 3D liver spheroid was successfully established.

(7) The authors suggested that targeting LX-2 cells with Rac1 and Cdc42 inhibitors could reduce collagen production. Did the authors observe these two genes upregulated in mRNA and protein expression levels in their cohort when compared MASLD patients with and without fibrosis?

(8) Did the authors observe that the expression levels of Rac1 and Cdc42 are correlated with fibrosis progression in MASLD patients?

(9) Other studies have revealed several metabolite changes related to MASLD progression (PMID: 35434590, PMID: 22364559). However, the authors did not discuss the discrepancies between their findings with the previous studies.

Significance:

Overall, the current study might provide some new resources regarding transcriptomic and metabolomic data derived from obese patients with and without MASLD. The MASLD research community will be interested in the resource data.

Comments on revised version:

Thank you for the authors' responses to my concerns. I do not have any further comments.

Reviewer #2 (Public review):

In this paper, Kaldis and collaborators investigate the molecular heterogeneity of a 109 morbidly obese patient cohort, focusing on liver transcriptomics and metabolomics analysis from liver and serum. The main finding (i.e. upregulation of GTPase-coding genes) was validated in spheroids and a human HSC cell line. As these proteins are involved in critical cellular functions related to metabolism and cytoskeleton dynamics, these findings shed light on their involvement in human liver pathology which so far has been poorly (or even not) documented to date. This is an interesting addition to the current knowledge about chronic liver pathology and warranting further in-depth molecular investigations to address molecular mechanisms of action (cellular specificity, GTPase-driven pathways...).

Strengths:

Using a well-characterized patient cohort of moderate size, the study provide transcriptomic and metabolomic data of high quality with adequate statistical corrections which are a very useful resource for the community. Mechanistic experiments usefully hint at novel druggable targets in the early steps of fibrosis, hence probably in hepatic stellate cell activation.

Weaknesses:

Cross comparisons with other cohorts is informative but of limited interest due to patient classification issues, inherent to histological staging practices. The lack of correlation between transcriptomic and metabolomic data is deceptive but expected due to the systemic nature of metabolomic analysis and was also observed in recently published papers.

Comments on revised version:

I have no further comment about this amended version, aside from suggesting to add (if known) the time at which biopsies were collected. Time-of-day is an important yet often overlooked parameter of gene expression variation, and along the same line, the imposed fasting to bariatric surgery patients is also a matter of variation of gene expression and of metabolite abundance. It is hoped that future investigations will more precisely characterize the role of the newly identified targets in MASLD.

Reviewer #3 (Public review):

Summary:

Metabolic dysfunction associated liver disease (MASLD) describes a spectrum of progressive liver pathologies linked to life style-associated metabolic alterations (such as increased body weight and elevated blood sugar levels), reaching from steatosis over steatohepatitis to fibrosis and finally end stage complications, such as liver failure and hepatocellular carcinoma. Treatment options for MASLD include diet adjustments, weight loss, and the receptor-β (THR-β) agonist resmetirom, but remain limited at this stage, motivating further studies to elucidate molecular disease mechanisms to identify novel therapeutic targets.

In their present study, the authors aim to identify early molecular changes in MASLD linked to obesity. To this end, they study a cohort of 109 obese individuals with no or early-stage MASLD combining measurements from two anatomic sides: 1. bulk RNA-sequencing and metabolomics of liver biopsies, and 2. metabolomics from patient blood. Their major finding is that GTPase-related genes are transcriptionally altered in livers of individuals with steatosis with fibrosis compared to steatosis without fibrosis.

Comments from the first round of review:

(1) Confounders (such as (pre-)diabetes)

The patient table shows significant differences in non-MASLD vs. MASLD individuals, with the latter suffering more often from diabetes or hypertriglyceridemia. Rather than just stating corrections, subgroup analyses should be performed (accompanied with designated statistical power analyses) to infer the degree to which these conditions contribute to the observations. I.e., major findings stating MASLD-associated changes should hold true in the subgroup of MASLD patients without diabetes/of female sex and so forth (testing for each of the significant differences between groups).

Post-rebuttal update: The authors have performed the requested sub-group analysis and find the gene signatures hold for the non-diabetic sub-cohort, but not the diabetic subgroup. They denote a likely interaction between fibrosis and diabetes, that was not corrected for in the original analysis.

(2) External validation

Additionally, to back up the major GTPase signature findings, it would be desirable to analyze an external dataset of (pre)diabetes patients (other biased groups) for alternations in these genes. It would be important to know if this signature also shows in non-MASLD diabetic patients vs. healthy patients or is a feature specific to MASLD. Also, could the matched metabolic data be used to validate metabolite alterations that would be expected under GTPase-associated protein dysregulation?

Post-rebuttal update: The authors confirm that with the present data, insulin resistance cannot be fully ruled out as a confounder to the GTP-ase related gene signature. They however plan future mouse model experiments to study whether the GTPase-fibrosis signature differs in diabetic vs. non-diabetic conditions.

(3).3D liver spheroid MASH model, Fig. 6D/E

This 3D experiment is technically not an external validation of GTPase-related genes being involved in MASLD, since patient-derived cells may only retain changes that have happened in vivo. To demonstrate that the GTPase expression signature is specifically invoked by fibrosis the LX-2 set up is more convincing, however, the up-regulation of the GTPase-related genes upon fibrosis induction with TGF-beta, in concordance with the patient data, needs to be shown first (qPCR or RNA-seq). Additionally, the description of the 3D model is too uncritical. The maintenance of functional PHHs is a major challenge (PMID: 38750036, PMID: 21953633, PMID: 40240606, PMID: 31023926). It cannot be ruled out that their findings are largely attributable to either 1) the (other present) mesenchymal cells (i.e., mesenchyme-derived cells, such as for example hepatic stellate cells, not to be confused with mesenchymal stem cells, MSCs), or 2) related to potential changes in PHHs in culture, and these limitations need to be stated.

Post-rebuttal update: To address the concern of other cells than hepatocytes contributing to the observed effects in culture, the authors performed TGF-beta treatment in independent mono-cultures (Figure R4): LX-2 and hepatocytes, and the spheroid system. Surprisingly, important genes highlighted in Figure 6E for the spheroid system (RAB6A, ARL4A, RAB27B, DIRAS2) are all absent from this qPCR(?) validation experiment. The authors evaluate instead RAC1, RHOU, VAV1, DOCK2, RAB32. ­In spheroids, RHOU and RAB32 are down-regulated with TGF-B. In hepatocytes DOCK2 and RAC seemed up-regulated. They find no difference in these genes in LX-2 cells. Surprisingly, ACTA2 expression values are missing for LX-2 cells. Together, it is hard to judge which individual cell type recapitulates the changes observed in patients in this validation experiment, as the major genes called out in Figure 6E are not analyzed.

Unfortunately, the 3D liver spheroid model used (as presente­d in PMID39605182) lacks important functional validation tests of maintained hepatocyte identity in culture (at the very least Albumin expression and secretion plus CYP3A4 assay). This functional data (acquired at the time point in culture when the RNA expression analysis in 6E was performed) is indispensable prior to stating that mature hepatocytes cause the observed effects.

(4) Novelty / references

Similar studies that also combined liver and blood lipidomics/metabolomics in obese individuals with and without MASLD (e.g. PMID 39731853, 39653777) should be cited. Additionally, it would benefit the quality of the discussion to state how findings in this study add new insights over previous studies, if their findings/insights differ, and if so, why.

Post-rebuttal update: The authors have included the studies into their discussion.

Have to adjust the picture to not mention the pandas module

Ooooh, but the game was not for them. I wonder how Nintendo does its testing...

remove "years" from axis text to make the plot less clumpy

collect axis text and title

collect strip text

black english develeped because of need, history and survival

Debates about black english are about power

The arguement on the legitimacy of black english is rooted in american history

I tried out some of the Wexford cards today. They're generally the same exact thickness and general quality as the Amazon Basics and the Oxford branded cards I've got. They're probably closer in quality to Amazon Basics than the Oxford which are a bit "tighter weave". I've got almost a dozen different brands, perhaps one of these days I'll set up my microscope along with a camera and do photos of the differences in paper quality.

The Wexfords have some of the more textured feel of any I've seen out there. They have the standard red top line, but the rest of the lines seem almost grayish or nearly black compared to most other cards which have a medium to lighter blue coloring. The Wexfords also have an only very slightly thinner than usual 1/4" spacing. (It measures out to 0.2375 inches between lines rather than the typical 0.25" most American cards would have. This nets out to be a 6mm line which makes me think it's a more European/Japanese/Chinese card than an American one despite the 4x6" dimensions.)

reviewer challneges wheter respecting language

school norm conflict with african american cultural practices

language affects identitiy and family relationships

language value depends on who is speaking it

english only polices can affect students

testing bias makes bilingual kids seem behind

Synthèse sur l'Optimisation des Plaques de Gélatine DIY

Résumé Exécutif

Ce document de synthèse détaille les problèmes, solutions et innovations présentés dans le contexte de la fabrication et de l'utilisation de plaques d'impression à la gélatine faites maison (DIY).

L'analyse révèle trois problèmes majeurs avec les recettes traditionnelles :

un séchage prématuré de la peinture, des risques significatifs liés à l'utilisation d'alcool, et des réactions chimiques indésirables avec la peinture acrylique.

La solution centrale est l'adoption d'une nouvelle recette "sobre", qui élimine complètement l'alcool et le remplace par du propylène glycol.

Ce changement résout non seulement le risque d'incendie et les problèmes d'irritation, mais améliore également de manière significative la rétention d'eau de la plaque, prévenant ainsi le séchage de la peinture.

Parallèlement, de nouveaux protocoles de maintenance sont introduits, notamment une "routine de soins" en deux étapes (nettoyage et hydratation) pour préserver la surface de la plaque et inhiber la croissance microbienne.

Les recommandations de stockage ont été révisées pour préconiser un contenant hermétique, en conjonction avec cette nouvelle routine.

Enfin, des outils et méthodologies de précision sont proposés, comme le passage à des mesures en grammes et le lancement d'un "Calculateur de Recette 2.0".

Cet outil en ligne permet de personnaliser les recettes en fonction de la taille de la plaque et de la force (valeur de Bloom) de la gélatine.

Le document aborde également la cause du "caillage" de la peinture acrylique—un environnement acide—et fournit une solution de neutralisation à base de bicarbonate de soude.

--------------------------------------------------------------------------------

1. Problèmes Identifiés avec la Recette Originale

L'analyse de la recette originale de la plaque de gélatine DIY a mis en évidence plusieurs problèmes récurrents rencontrés par les utilisateurs, transformant parfois l'expérience d'impression en un processus frustrant.

1.1. Le Problème de la "Plaque Assoiffée"

Le problème le plus courant est celui d'une plaque qui sèche trop rapidement, rendant la peinture quasi impossible à retirer.

• Cause principale : Un surdosage de gélatine ou l'utilisation d'une gélatine à haute valeur de Bloom. Une telle plaque n'est pas entièrement saturée en eau et devient "très, très assoiffée".

• Mécanisme : La plaque de gélatine sèche aspire instantanément l'eau contenue dans la peinture acrylique. Les pigments adhèrent alors de manière presque irréversible à la surface.

• Conséquence : La plaque se comporte comme un "aimant suceur de peinture acrylique" plutôt que comme une surface de transfert antiadhésive.

• Analogies : L'auteure compare ce phénomène aux premières crêpes que l'on jette, expliquant que la plaque a besoin de "s'échauffer", c'est-à-dire de se saturer en eau, avant de fonctionner correctement.

1.2. Les Risques et Inconvénients de l'Alcool

L'alcool, ingrédient clé de l'ancienne recette pour réduire l'aspect collant et améliorer la conservation, présente deux inconvénients majeurs.

• Risque d'incendie : L'utilisation d'alcool (isopropylique, dénaturé, ou à haute teneur) lors du chauffage du mélange présente un risque réel d'incendie.

Une utilisatrice nommée Rita a d'ailleurs connu un tel incident, ce qui a été un catalyseur pour le changement de recette.

• Irritation : Les vapeurs d'alcool peuvent irriter les yeux et les voies respiratoires des utilisateurs.

• Déshydratation de la plaque : L'alcool contribue significativement à la déshydratation de la plaque sur le long terme.

L'auteure fait une analogie avec la "sensation de Sahara dans la bouche" après une soirée arrosée pour illustrer cet effet.

1.3. Comportement Anormal de la Peinture Acrylique

Certains utilisateurs ont rapporté un comportement "super étrange" de la peinture acrylique, qui se met à cailler ou à se décomposer sur la plaque.

• Cause : Un environnement acide (pH bas).

• Origine du problème : L'ajout d'acides comme le jus de citron ou l'acide citrique dans le mélange, souvent dans le but d'agir comme conservateur.

• Effet : Dans un milieu fortement acide, le système liant de la peinture acrylique peut se rompre, provoquant son caillage.

La peinture adhère alors davantage au rouleau qu'à la plaque elle-même.

2. La Nouvelle Recette "Sobre" : La Solution Centrale

Pour remédier à ces problèmes, la recette a été entièrement reformulée, la modification la plus importante étant le retrait de l'alcool, qualifiant la nouvelle plaque de "sobre".

2.1. Le Remplacement de l'Alcool par le Propylène Glycol

L'alcool est remplacé par le propylène glycol, décrit comme le "partenaire parfait" de la glycérine.

• Propriétés : Le propylène glycol appartient chimiquement à la famille des alcools, mais il est beaucoup moins volatil, ne s'évapore quasiment pas et présente un risque d'incendie significativement plus faible dans des conditions de cuisine normales.

• Bénéfices dans la recette :

◦ Stabilité : Il aide à rendre la plaque plus ferme et stable sans lui "voler toute son eau".  

◦ Rétention d'humidité : Il aide la plaque à rester flexible, à moins rétrécir et à conserver son humidité, ce qui garantit de belles impressions.   

◦ Conservation : Il contribue à ralentir la croissance des bactéries et des moisissures, agissant comme un agent de conservation.

• Conclusion de l'auteure : "Si je devais choisir entre 'Brûle bien' et 'Imprime bien'... je suis assez sûre que vous choisirez la plaque qui imprime parfaitement plutôt que le feu d'artifice dans la cuisine."

2.2. Expérimentations avec des Plaques "Fusion"

Des tests ont été menés sur des plaques "fusion" combinant les propriétés de la gélatine et d'agents gélifiants à base de plantes. Ces versions semblent résoudre nativement le problème de séchage de la peinture.

• Ingrédients testés :

◦ Gomme de xanthane   

◦ Konjac (ou glucomannane) : L'agent actif de la farine de konjac, connu pour son pouvoir gélifiant et épaississant extrême.

• Résultats préliminaires : Les plaques fusion semblent libérer plus de peinture sur le papier, laissant moins de résidus sur la surface. Les tests sont jugés "très prometteurs".

• Note : Une exploration plus approfondie de ces hydrogels est prévue dans une future vidéo.

3. Nouveaux Protocoles de Maintenance, de Stockage et de Réparation

La nouvelle approche s'accompagne de protocoles mis à jour pour entretenir, stocker et même réparer les plaques.

3.1. Réhydratation d'une Plaque Sèche

Une plaque devenue trop sèche peut être "ramenée à la vie" sans être refondue.

• Méthode : Un "bain" d'eau. La plaque est immergée dans l'eau pendant une durée allant de 3 à 48 heures, voire plus, jusqu'à ce qu'elle absorbe l'eau nécessaire et augmente de volume.

• Alternative : Si un contenant adapté n'est pas disponible, la surface peut être vaporisée d'eau, recouverte de papier essuie-tout humide et enveloppée dans un film plastique.

3.2. Nettoyage des Anciennes Couches de Peinture

Une découverte notable a été faite pour enlever les couches de peinture tenaces : La colle artisanale simple à base d'eau (colle blanche universelle) s'est avérée extrêmement efficace pour décoller les anciennes couches de peinture séchée de la surface de la plaque.

3.3. Nouvelle "Routine de Soins"

Un protocole de soins post-impression, comparé à une routine de soins pour la peau, est désormais recommandé pour préserver la plaque.

1. Nettoyage Doux : Vaporiser un spray nettoyant sur la plaque, essuyer avec un chiffon doux pour enlever les résidus de peinture.

2. Rinçage : Repasser sur la surface avec de l'eau claire pour éliminer tout tensioactif résiduel.

3. Hydratation et Protection : Masser une petite quantité d'un spray de soin sur la surface.

Les recettes pour ces sprays sont les suivantes :

Spray

Ingrédients (en grammes)

Instructions

Spray Nettoyant

- 500g Eau<br>- 2g Savon neutre<br>- 1g Alcool (pour dissoudre)<br>- 1g Huile essentielle (Arbre à thé ou Clou de girofle, optionnel)

Dissoudre l'huile essentielle dans l'alcool, ou directement dans le savon. Mélanger tous les ingrédients et verser dans un flacon pulvérisateur.

Spray de Soin

- 200g Eau<br>- 2g Huile pour bébé (huile minérale)<br>- 1g Huile essentielle d'arbre à thé<br>- 1g Huile essentielle de clou de girofle

Mélanger tous les ingrédients. Agiter vigoureusement avant chaque utilisation car le mélange est biphasique (l'huile se sépare de l'eau).

Le spray de soin laisse un "film protecteur huileux très fin" qui protège contre le dessèchement et rend la surface moins accueillante pour les microbes grâce aux propriétés des huiles essentielles.

3.4. Recommandations de Stockage Mises à Jour

• Ancienne recommandation (pour les plaques avec alcool) : Ne pas stocker dans un contenant hermétique les premiers jours pour permettre à l'humidité de s'échapper et éviter un "microclimat tropical" propice aux moisissures.

• Nouvelle recommandation (pour les plaques "sobres" avec routine de soin) : Stocker dans un contenant hermétique dès le début.

Cette approche est jugée optimiste pour minimiser la perte d'eau, les précautions étant prises par la routine de soin antimicrobienne.

4. Outils et Méthodologies de Précision

Pour améliorer la fiabilité et la reproductibilité des résultats, de nouvelles méthodologies ont été introduites.

4.1. Passage aux Mesures en Grammes

Toutes les nouvelles recettes sont désormais formulées en grammes plutôt qu'en unités de volume.

• Raison : La précision est cruciale, en particulier avec les agents gélifiants végétaux où "un demi-gramme de plus ou de moins peut déjà faire une énorme différence".

• Avantage pratique : Il devient très simple de calculer la perte d'eau lors de la refonte d'une plaque.

Il suffit de peser la plaque usagée, de comparer son poids au poids total initial des ingrédients, et d'ajouter la différence en eau lors de la refonte pour la restaurer à son état optimal.

4.2. Le Calculateur de Recette 2.0

https://ashrey.com/diy-gel-plate/

Un nouvel outil en ligne, le "Calculateur de Recette 2.0", a été développé.

• Fonctionnalités :

◦ Fonctionne entièrement en grammes.  

◦ Prend en compte la force de la gélatine (valeur de Bloom).   

◦ Permet de dimensionner les recettes précisément à la taille de plaque souhaitée.   

◦ Offre le choix entre différents types de plaques : standard, plus souple, ou la version expérimentale "fusion" avec hydrogel.

• Disponibilité : L'outil est accessible sur le site web de l'auteure. Le calculateur classique (en unités métriques et impériales) reste également disponible.

5. Diagnostic et Solution pour le Caillage de la Peinture

Le mystère du comportement anormal de la peinture acrylique a été résolu.

• Diagnostic : La peinture acrylique n'aime pas les environnements acides. Un pH bas provoque son caillage et la rupture de son système liant.

• Action à éviter : Ne pas ajouter d'acides (jus de citron, acide citrique) comme conservateurs dans le mélange de la plaque de gélatine.

• Solution de Réparation ("Fix d'Urgence") : Pour une plaque déjà acide, il est possible de neutraliser sa surface.

1. Préparer une solution alcaline douce : Dissoudre 2 à 3 grammes de bicarbonate de soude (disponible sous des noms comme "Kaisernatron" en Allemagne) dans 1 litre d'eau.  

2. Appliquer : Verser ou vaporiser la solution sur la surface de la plaque.  

3. Attendre : Laisser agir pendant 30 à 60 secondes. 

4. Essuyer : Sécher la plaque, puis la nettoyer à nouveau avec de l'eau propre ou une lingette pour bébé.  

5. Répéter si nécessaire jusqu'à ce que la plaque fonctionne correctement.