Residential segregation makes students in low income families subject to a lower quality education than higher income families within public schools.

Income inequality, particularly amongst families, seems to directly influence education inequality. Lack of quality in education also directly impacts future income inequality in return.

To combat this problem, the government has implemented programs to increase low income families' buying power. However, as we've learned in this class, these programs haven't been the most effective.

Being in a academic environment as an average student in a low income family, according to studies, hinders kids' learning and academic performance. This can be attributed to teacher quality, the behaviors of peers, and the resources provided by the school the low income family student attends.

Educational gap has an especially crucial impact on skill attainment in the earlier stages of life, as kids in both low and high income families rely primarily on their families to have access to educational materials. Income directly impacts the educational materials these families can provide to their children, and to develop in the field of STEM, access to these materials is especially crucial.

Gap impacts academic preparedness for college, preventing low income family students from having the same collegiate opportunities as high income family students. This also can go on to impact skill attainment, putting low income family students at a disadvantage in terms of the job market.

Gap mentioned in the previous annotation is only increasing.

Residential segregation makes students in low income families subject to a lower quality education than higher income families within public schools.

Income inequality, particularly amongst families, seems to directly influence education inequality. Lack of quality in education also directly impacts future income inequality in return.

This metaphor means when you think of a rainy day, you compare it to a bad day ( in my opinion). It implies that rather than being put on by economic or political shortcomings, misery is cyclical and natural, like weather cycles ( meaning it will be over soon, it will pass)

The headline gives a sense of people to be scared about the recent government shutdown causing food assistance that people need to be cut (a reason to be fearful). The phrase positions families as defensive or under attack, highlighting a lack of security.

My Mamaw always said “We are from here”, I never really understood as a child because she never said more like a town or country. Later I learned that we are literally from America because we are Native Americans. Her mother, who would have been my Great Great Grandmother, was from a reservation if I remember my history correctly. The correlation about how she took care of nature and how I was taught to do the same is from our native roots. None of my other ancestors or family do this, which I find very strange that they do not have histories or traditions tied to the land.

This reminds me of my Gramma and my Mamaw, (her mom) who always took me on outings to the mountains, the beach, fishing and more because they both would always have a bag that we kept for trash. This bag was to make sure that we left the places we went to better than how we came across them. If that meant emptying our bag a couple of times then so be it but we never left trash if we came across something while we were out. My husband’s family was horrified that we picked up trash when we went out and just could not understand why we did. I taught this to my children and we all would forget sometimes to bring a bag then we would wash our finds that ended up in our pockets. It has become a bit of a laughing point because of all the little finds that come home with me. My son and I compare fishing hooks and lures that we find while fishing.

Learning to think outside of the box for research can really give different perspectives about any subject and can be so liberating to pick things you would not normally learn about. In class I try to have the students learn about things they are interested in but do not have experience with. This way the students are not relying on ideas or information that they already know and have to go outside the comfort zone. We have also been doing this in the foods class I teach and it gives us so much more diversity.

Recommend rewording

I appreciate these types of helpful tips!

I find having interspersed code like this in narrative descriptions sometimes hard to follow since I don't know what "largest_bg_to_client_zone" is meant to be. Would a reader/user of this manual be familiar with this code already?

some funky formatting going on here

Is this a commonly known term? I wasn't familiar with it so I looked it up

maybe "run validation here"? Otherwise it's unclear what "stop" this references

write out

Severe injury to the stomach and head generates the same monetary penalty, which indicates that they are seen as equal in terms of losses. However, for an injury to the head, there is no extra penalty required for physician's pay. This is troubling as a head injury of this severity would most certainly give cause for medical treatment to at the very least treat the wound (in the likely case that there were no treatments for psychological or neurological damages). Perhaps they assumed that the person would succumb to their wounds in this case, but taking that perspective makes it difficult to rationalize them as equivalent losses. It is likely that an injury to the ribs or stomach might also cause the death of the individual during this time but it is strange that there is an acknowledgement of an attempt for treatment.

Interesting shows a serious punishment for those who try to hide their crimes. Shows a likely level of leniency if one confessed to a crime and did not try and hide it.

Interesting how calling someone a harlot is a serious offense showing how important a women purity was in Frankish society.

Troubling I find it alarming that a woman is considered significantly less valuable if she can not bear children

interesting that a Roman who owns land or is a table companion of the king is worth 3 times as much as a roman who is not one of the aforementioned things.

Surprising. Why would the three men be required to pay 10 shillings each but if there are more than three they olny have to pay 5 each. The law makes no sense to me and is suprising.

With the punishments regarding children and women who are either able to or are currently rearing children being so much more severe compared to the average punishment for murdering a free man, it shows to me that the Franks valued children a great deal. It might be due to the ability to expand the kingdom if there are more people in it.

I find it surprising that calling a woman a harlot is considered to be a worse crime that calling someone a fox or a hare. I also am surprised that it does not have to be proven either.

I find this law surprising, as it seems that hiding someone or a body is deemed much worse than if you just were to have killed someone. The punishment is triple the cost of normal murder.

This law is interesting to me as it clearly shows how much the Franks valued the ability to bear children in women. Killing a woman who can still bear children is 3 times as costly compared to killing a woman who can no longer have any.

I find this interesting, as it shows a lack of equality among the Franks and the Romans. Given that these are Frankish laws, it makes sense that the penalty towards them would be less compared to that of the average free man.

It is interesting how the penalty for rape (of women) and assault and robbery (of men) has the same monetary penalty, which implies that these offenses were viewed as equal in terms of losses. In both cases, the way the law is written takes into account two primary factors: bodily harm and loss of property. I would assume that in the case of rape that the reason the penalty is the same as assault and robbery is that there is some implied economic value lost as a result of that offense.

Introduction to Functional Analysis<br /> by [[Casey Rodriguez]] via MIT Mathematics | MIT OpenCourseWare accessed on 2025-10-30T14:15:24

Introduction to Functional Analysis<br /> by [[Richard Melrose]] via | Mathematics | MIT OpenCourseWare<br /> accessed on 2025-10-30T14:14:14

L'ICANN s'occupe aussi d'attribuer les adresses IP à l'ensemble d'Internet en IPv4 ou IPv6 et d'assurer leurs fonctionnements.

Katie Nolan Destroys UNC’S Mike Lombardi Over His 'Cute' Typewriter Video<br /> by [[Stephen Douglas]] for Sports Illustrated<br /> accessed on 2025-10-30T13:53:59

https://www.berkley-fishing.com/collections/line-tools/products/line-counter

Off-label use of a line counter with typewriter ribbon length?

Off-label uses for linewinders as typewriter ribbon winders?

• https://www.walmart.com/ip/Fishing-Line-Winder-Spooler-Aluminum-Alloy-Spooling-Station-Adjustable-Rotating-Axis-Steel-Frame-Clamp-Workbench-Fishing-Line-Spooler-Machine-Multipl/17084510284?classType=REGULAR
• https://www.walmart.com/ip/Fishing-Line-Spoolers-Adjustable-Fishing-Line-Winders-Spoolers-Machine-Spinnings-Baitcasting-Reel-Spooling/17886252799?classType=VARIANT
• https://www.walmart.com/ip/Apooke-Spinning-Reel-Fishing-Line-Fishingline-Winder-Equipment-Winder-Spooler-Machine-with-Suction-Cup-Fishing-Line-Spooler/5648376830?classType=REGULAR

https://www.trianglesport.com/

Potential off-label use cases for their line winders with respect to spooling typewriter ribbon?

http://www.deansphotographica.com/machining/projects/springs/springs.html

I found this to be pretty messed up and definitely unethical. If we look at Kant's ideals of Deontology, this is unethical as it violates trust, free will and uses the personal data of users to further the gain of higher-ups managing the platform, at the cost of user autonomy.

this is very fascinating

I wish I could shout this from the rooftops. I personally know for a fact my anxiety is increased when I keep my nose stuck to my screen. Why is that? It's because of all the nonsense that is posted to public forums, it is because the horrible events are publicized more so than the good events. I have known several people who have taken a 'screen break' and come back from it so much healthier mentally, but get drug back into the same dark hole. As society, what would we do without technology and a screen? How different would YOUR life be if you came home from work and set your phone face down, and were just present in your home for the evening. Would your children be happier? Would you and your spouse bond more? I think it is something everyone should make a challenge to succeed.

While I can see and understand how it is against the law to continuously spam individuals, what would be a better way of collecting debt and advertising? I believe there are different 'levels' of spam, and it is hard to determine anymore what is truly spam and what is advertisement. Would there be a better way for us to sign up for emails, clubs, coupons, etc. without opening our lives to the chaos of spam? How would a company ensure to keep all of their clients information confidential to avoid them getting spammed? OR are we not realizing that bigger companies are selling our information under the table, and that is how spam becomes reality?

I highly agree with this. I am not an employer, however, I do occasionally am looking for childcare. I also breed miniature dachshunds, and will review a persons social media page before rehoming a puppy them. It is not to be judgmental, but to use it as a statement piece in my opinion. I personally keep most of my private life locked down for only personal friends and family to see, and I rarely post things publicly.

Standards baked into the assertions makes it possible to have apples-to-apples comparisons of courses and other programs regardless of what they are called.

Strong start

Here we see the sentiment which led to the second war. Its honestly not at all surprising that they would fight again considering what the British were doing.

I like this paper. Its like the other side to Lin Zixu's formally addressed piece. Its always fun to see people outright talking crazy in old language.

Just big and greedy. Fr though the British had such a crazy entitled mindset. Is this what colonialism does to a nation?

I'm not exactly sure what this is referring to. Possibly the many wars between the British and other powers

Such crazy punishments for a war that was fought over literally dumping heroin. I get why the Chinese regard this as the beginning of the century of humiliation.

The situation becomes intense but he explains his point clearly. Through his words he attempts to establish that the colonists belong to a larger collective than their individual selves. Through his words he transforms the American Revolution into a worldwide fight for human rights although this vision did not fully apply to all residents of the colonies.

The line stood out to me because Paine directly challenges people who accept traditions without ever questioning their origins. The way people accept current rules and social norms because they have always existed aligns with the situation he describes.

Paine presents America as a sanctuary which protects liberty. The concept he presents in this work later evolved into on of the most common sayings “land of the free.” The ideas he presents in this work match current discussions about refugees and democratic societies.

Paine says everyone is created equal, but this makes me wonder how does this align with the reality that slavery still existed in the colonies? What voices are missing or ignored in this argument for equality?

This is Paine saying basically he knows that not everyone will be in his favor. Its not a normal thing for the people in this era and hes being different from the rest.

Paine explains that power becomes unlawful when it is abused throughout multiple generations. The British rule has caused enough suffering to colonists that they should now doubt its authority. The argument connects how American colonists viewed their rights regarding taxation and representation.

The sentence caught my attention because it indicates that people learn from personal experiences rather than through pure discussions. Paine recognizes that his logical arguments will not convince people until they witness the failures of the current system. Through this statement he demonstrates his awareness of how people naturally resist changes.

Paine sounds very egotistical here, makes me wonder what he would thing of modern day government? Would he see our big governing system as a threat or a protection of our rights? Which evil might be the right word for todays modern government.

Paine is saying that the Dutch Republic is proof that a country can survive without a king. This is was also at a time when a lot of people thought that's how people had stability. He is trying to tell them that self governing and independence is worth it and is possible.

Paine describes the escalating conflicts which eventually resulted in the American Revolution. The King actively supported policies which harmed the colonists after they had already demonstrated their opposition through protests against the Stamp Act and Intolerable Acts.

This means passing down the power through the bloodline. Sort of like when a king has a son, the next king in going to be the child.

This reminds me a lot of the original story of Justine Sacco's tweet. And as much as I vehemently disagree with Musk's views and rhetoric, him being open about the way the Twitter/X algorithm works is interesting to me. I previously said that social media doesn't benefit off of positive interactions, but rather being the paper on which arguments are written. And Musk outright states that, interaction, positive or negative, is interaction, and will boost content in that vein whether you like it or not, so as to try and elicit more participation from you.

A great combo of creativity and reframing. I sometimes do this in a smaller round and with digital tools.

I do like a good flip of perspective. This method could use a double flip, though. Before finding a solution, ask, what could make the situation worse?

I'm interested in knowing what colors are best to use in a mind map?

I do agree with this statement, as this can weigh as an opportunity to have a competitive advantage in your idea.

I would question the proper implementation of this activity within the brainstorming process. The article mentions how typical brainstorming sessions are dominated by extroverts, so I think that stereotype would seem into this technique as well. I think it would also discourage people if nobody puts a sticker on their sheet of paper. So I think this would work in already well established group of individuals.

I think this a cool idea! This taps into more of the process side of ideation, getting down to the nitty gritty of what is physically possible with an idea. I also think that this is a charming bridge between artistic people and structured "real life scenario" type of thinkers.

I love mind mapping, it is my go-to ideation technique! I love the drawing strategies listed, they are very creative!

How could you intentionally include those who may need more time to ideate (ex. those who lean more towards internal processing)?

The crazy eights activity seems useful, but I am would also go one step further and have the participants act in small teams, but while also avoiding co mingeling between teams. I think that ideas will come out in a "flow" that can chage direction based on things the participants may not even precieve. Seperating the teams would stop anyone person from influencing one another, even if it is accidental.

I was curious about how removing constraints affects the outcome of creativity. I have often heard that ilimitations breed creativity. When would it be most appropirate to use each method?

This makes me wonder-- does a pre-existing culture of collaboration need exist for this approach to work? What if this is done in a hostile/competitive culture-- how could ego come in to play?

This is such an interesting approach because it takes the pressure off of and takes the judgement out of generating a "good idea" and lets you think backward toward something that works

If I were a participant, I would feel overwhelmed having to share so much information in such a short time and then present it to my colleagues right after.

I love that! A lot of times, it’s hard to immediately come up with what we want, but it’s easy to know what we want to avoid.

The size of the paper also affects our thoughts; when I have a larger paper, the idea I get also becomes larger.

This is a very useful technique. When I think about a new idea, I have a habit of thinking from the goal, so if someone asks me this kind of question, it would increase my creative thoughts.

I love mind mapping, this is my favorite brainstorming idea. Writing down a problem is a good addition to where I would usually use a mind map and start with a goal and map out how to acheive that goal.

This sounds good in theory to me, I feel if everyone is working towards a central goal this should be presented as a group or on a white board so everyone is on the same page.

https://www.reddit.com/r/typewriters/comments/1oi6808/space_runs_too_fast_with_loud_buzzing_sound_plz/ reply via u/ahelper

https://old.reddit.com/r/typewriters/comments/1oincod/can_someone_tell_me_what_yearmodel_this_is/

https://www.reddit.com/r/typewriters/comments/1ojf7oe/oil_for_typewriter/

via /jbhusker<br /> SC 6 series part 1.

Understanding matrices as data structures makes linear algebra feel much more intuitive to me. Thinking of rows as observations and columns as variables helps explain why the column space represents all possible linear combinations of attributes, essentially the space where our predictions or projections live. It also connects nicely to concepts like eigenvalues and eigenvectors, where the directions of highest variance in the data correspond to the principal components or dominant eigenvectors of the covariance matrix.

I think what is meant by a good model also differs on what the goal of the model is. For functional modelling we might be more interested in the relationship between the attributes and how they relate to each other, while for prediction, having a model that can predict on unseen data would matter more than interpretability.

opinion

fact

opinion

I wonder how certain trends such as this one became a thing in the first place.

I don't know why it took me this long to understand the fact that culture is literally in everything. It seems so normal to me but it may seem weird to other people from different cultures.

This makes me think that anthropologists could have overlooked many things that contributed to different cultures. Something so obsolete could have been completely ignored.

I wonder how different cultures interpreted dreams. I also wonder how religion affected how they perceived their dreams.

I have always wondered where blinking originated, who was the first to do it?

Museums are filled with material culture.

This is very interesting, dreams mean so much more in other cultures.

The way people are raised can affect how they see the world.

Winking is so similar to blinking, its just using one eye, so its strange that we have to learn how to do it while we are born knowing how to blink.

This is a good way to view culture.

https://www.reddit.com/r/rs_x/comments/1npjd4t/if_i_may_be_so_bold/

These comments...

New jersey was seemingly becoming less blue that it had been in the past.

Though not physically on the ballot, trump is heavily influencing this election.

https://www.alibaba.com/product-detail/12-7mmx920M-Black-Inked-Ribbon-Inked_1600231049141.html

Bulk manufacturer of typewriter ribbon

This is a very interesting technique! I want to try this technique myself - I really like how it places a collaborative focus on ideation.

I have a personal belief that the answers to most questions are about 1 or 2 more "but why's" from finding a unique solution. This made me curious to see what other innovations could be uncovered if we just asked "why is it that way" to entrenced problems.

Do these wishes need to only be tangible? What about the impossible ideations?

I had a very similar activity in my summer courses called the "worst business pitch competition" and we tasked the students with who could come up with the worst idea that made some sense. This is useful as something to remember for the next time I conduct this task as I realized you need to pull the ideas back to reality for any use to be gained from them.

This is an intriguing scope. I'm interested to know the time frame it take some to think some of their ideas bad.

I can agree with this. I've done a kickstarter for some projects, and it led to me being able to build a community from my supporters.

This is a really cool idea to brainstorm because it helps challenge the status quo and makes you really look into aspects of an organization that are taken for granted

This makes me curious-- how does somebody know if they've questioned every aspect of their business? What if there are some features that are overlooked?

I agree with this idea, sometimes we can get some interesting ideas when we are relaxing.

I saw many brainstorming techniques set 20 to 30 as an ideal number, but what's the difference between 10 and 20 to 30?

I like this idea; questioning is the very first step to changing our perspectives. Without knowing the products deeply, we cannot acquire new ideas and insights.

I am not a huge fan of spending time creating bad ideas, maybe integrate rest breaks instead or something to get people refreshed.

I enjoyed this kind of thinking, every industry is tough to enter, but there usually are cracks within each industry where things can be improved. Looking at tings from this perspective allows you to find niche markets or discover a market that was alot bigger than you may have originally thought.

The best way to break down a prepositional phrase is it’s a enhancement to give addition to the sentence to achieve a better fulfillment of the sentence.

Making Tab Stops for an LC Smith Typewriter : 3 Steps - Instructables<br /> by [[Instructables]]<br /> accessed on 2025-10-30T11:30:29

It’s crazy how intense this time was! Elite homes and temples were burned down, showing there was major social unrest. Between 850 and 900, the population dropped by 90%, but most people didn’t die—they just left the cities and moved elsewhere to start new lives.

It’s so interesting how old money systems worked! Even though people usually only minted pennies, the same value ratios lasted for centuries in currencies like the French livre, British pound, Italian lira, and Spanish libra.

It’s so smart how paper and credit payment systems made trade easier! Gold and silver were super heavy, so using paper or trust-based payments meant merchants could carry more goods instead of piles of metal coins.

I think it’s so cool how Charlemagne changed things after 802! Instead of people swearing loyalty to local lords, every man over twelve had to take an oath directly to him as emperor. It really showed how powerful and organized his rule was!

peergos

I like how Volk shows that learning doesn’t just happen in schools and that these families create their own literacy spaces at home too

https://discord.com/channels/639936208734126107/639938269030907914/1302694827682697330

Pelicram's Shellac Rescue Formula aka The Cowboy's Delight.

This will help bring back a deeper black color shellaced panels which have been yellowed and damaged by UV over the years. With enough elbow grease it will remove the old shellac completely but it takes a very long time and you're likely to damage any decals present on the panel. In most cases the procedure described below will be sufficient to restore the appearance to an acceptable level.

The recipe: - 70% Light machine oil. - 30% IPA (Isopropyl Alcohol) or White/Mineral Spirits.

Ideally use an oil that is dissolved into the IPA/Mineral Spirits, if they settle into separate layers make sure you shake the mixture thoroughly before applying.

Mix the oil and solvent in something like a dropper bottle or similar vessel for convenient application.

• Clean part with Fulgentin (Or general purpose cleaner of your choice) and wipe dry.
• Apply oil/ipa mix to part and rub in lightly with clean microfiber cloth or shop towel. Use plenty of the mix, it should not feel dry.
• Wipe with microfiber cloth after 15 minutes to get rid of any excess.
• Do not apply any kind of wax (like Renessaince Wax) afterwards, from my testing it will bring back the haziness.

https://discord.com/channels/639936208734126107/639938983220215828/1131197256691875881

The Marvelous Mrs. Maisel S5, E2

not annotatable

31 ene 2023 Catálogo: https://archivesetmanuscrits.bnf.fr/ark:/12148/cc60309f 30 oct 2025: Visor: https://gallica.bnf.fr/ark:/12148/btv1b53200091x

Visor: https://gallica.bnf.fr/ark:/12148/btv1b52505727p

Catálogo: https://archivesetmanuscrits.bnf.fr/ark:/12148/cc60312z Galería incompleto: https://mandragore.bnf.fr/recherche/avancee?searchData={%22formType%22:%22UD%22,%22formField%22:[{%22operator%22:null,%22value%22:%22Latin%202495B%22,%22exactValue%22:true,%22critere%22:%22UD_COTE%22}]}

Otra: https://portail.biblissima.fr/ark:/43093/mdatab86f068a79b2b088088f3f7ecf931feb5482e7c4

Catálogo: https://archivesetmanuscrits.bnf.fr/ark:/12148/cc60311q Visor: https://gallica.bnf.fr/ark:/12148/btv1b52526358t

I think the distinction between bilingualism and translanguaging is important. Knowing multiple languages and have a comprehensive, culminating usage of them is different. School environments often encourage the usage of one or the other, but rarely both. Honestly, I didn't even know what translanguaging was until I took Education classes at UCI, and I think that is all the more reason to spread awareness of its benefits and foster it in students.

Catálogo: https://archivesetmanuscrits.bnf.fr/ark:/12148/cc60311q Visor: https://gallica.bnf.fr/ark:/12148/btv1b52526358t

https://www.typewriters101.com/how-to-ship-a-typewriter.html

This passage shows how important trust and relationships are for undocumented students navigating in school. Those placed in advanced tracks had smaller classes and more access to teachers and counselors, which helped them feel safe enough to share their status and ask for help. It wasn’t just about academics, it was about being seen and supported. When students feel like someone genuinely cares, they’re more likely to open up and get the guidance they need. This reminds me how many school structures can either build or block those connections, and how much that matters for students facing extra challenges.

Undocumented students face extra challenges when trying to transfer from high school to college, especially without strong support from their schools. While other marginalized groups also struggle with access, undocumented students deal with even more barriers because they’re excluded from the federal and state aid. This makes it harder for them to apply to college or find scholarships. On top of that, their legal status adds stress that affects their well-being and ability to focus. It’s important for schools to step up and offer real support so all students aren’t left to figure it all out alone. Everyone deserves a fair chance to succeed.

This section highlights how undocumented students are blocked from key support programs like TRIO, things like work-study, even though they often meet the same criteria as students who qualify. These programs are meant to help low-income, first-gen, and minority students, but because they’re federally funded, undocumented students are left out. Which sucks terribly. That means they miss out on both financial help and the chance to build support systems on campus. It’s so frustrating to see how students who need the most help are often the ones with the fewest resources and support. This problem we have to address together.

This was interesting. Flor’s story shows how being undocumented can shape someone’s whole life, starting from school. She had to work from a young age to help her family, which made it hard to stay in school and feel supported. Looking back, she wishes someone had reached out and shown they had cared. Her words made me think about how many students feel alone and unseen, especially when they’re dealing with tough situations. As a future teacher, I want to help be that person who notices and supports students like Flor. Everyone deserves someone who believes in them and helps them see a beautiful future for themselves.

They began to provide features for creating compound documents to the web - user base at large.

self

https://www.reddit.com/r/typewriters/comments/1oblfvo/service_booklet_2/

See also Google Drive document with details.

https://typewriterdatabase.com/1926-mercedes-4.19461.typewriter<br /> "Steile Sierschrift" (Steep ornamental font) <br /> RaRo foundry mark 5 circle I<br /> paired with<br /> RaRo foundry mark 57 AR

https://typewriterdatabase.com/1926-mercedes-4.19461.typewriter via Kevin Stallaert

I think this is probably the root cause of a majority of financial struggles with young adults. It really is crazy just how much student tuition are now and going along with that just how much debt we obtain. I truly believe that student tuitions shouldn't be so expensive. I really don't understand what schools are doing with all of this money and don't think it's necessary how much they take with us.

I don't really understand the logic of this. The story a man told in this article before this said he tried to get a credit card so that he could build his credit score and apply for apartments but when the price is so high to obtain a card and there as so many fees and taxes it makes it really hard for young people to get one. This really makes me realize I need to start building my credit as fast as possible.

RR\ID Summary of Reviews: This preprint retrospectively investigates the relationship between SARS-Cov-2 genomic sequence data, patient clinical characteristics and infection persistence in 69 Californian patients. It is rated as strong/reliable by reviewers. The authors claim that certain populations may be more susceptible to persistent SARS-Cov-2 infection, and that persistent infections may have evolutionary trajectories that are different from circulating lineages. Reviewers highlight the thoroughness of the analysis of existing sequence data and the clear articulation of a framework for mining available public health data to probe the impact of viral mutations on pathogenicity. Reviewers recommend an analysis of the mutations viruses may have acquired before the initial sequencing, increased discussion of the novelty of the findings and of why persistent mutations did not spread further in the population. Further suggestions for improvement focus on the clarity of data representations.

Read directly in RR\ID: https://rrid.mitpress.mit.edu/pub/afgxepb7/release/1

Reviewer #1: Evidentiary Rating: Reliable

Written Review: The data from this manuscript largely support the stated conclusions. There is a thorough evaluation of existing SARS-CoV-2 sequence data to probe interesting questions, such as what conserved mutations (novel and known) were found in persistently infected patients from California, and what factors (age, sex, etc.) were associated with persistent infection versus acute infection.  One of the main strengths of this manuscript was providing a “framework” for mining public health data to ask important questions about viral mutation and association with pathogenicity. The authors do a nice job of describing their approach to this complicated topic and acknowledging limitations and potential improvements to this approach. Overall, the manuscript describes the use of public health data to monitor persistent infection and viral evolution. Some changes, as listed below, could be helpful in improving the manuscript: * Line 166-168, clarify in which direction the statistical significance was found. * The manuscript would benefit from more information on how the findings are different from other related, published manuscripts. * Information on conserved mutations in non-coding regions would be interesting. * Table 3 would benefit from listing references for the different “descriptions.” * Lines 164-166, list the ages for each fatal case * More discussion on how these persistent infections didn’t “spread” throughout the population would be informative.

Reviewer #2: Evidentiary Rating: Strong

Written Review: This is an excellent manuscript.  I have a few suggestions that may make the manuscript more useful for the reader.  * Fig 2.  Please indicate which Omicron lineages the different Nextclade lineages represent (eg, BA.1). * It would be useful if there were a similarly styled graphic below the current figure which shows when the various nextclade clades were in circulation.  If I am not mistaken, some of the patient infections were not detected for the first time until a while after that clade had stopped circulating.  This would help in illustrating it for the reader. * The authors don’t make it easy to look up what the different convergent changes are other than the ones that are 3 or more times.  I would recommend adding all of the mutations to the main table that occurred at a position 2 or more times.   Alternatively, they could just adjust the table to make it so that it can be more easily sorted based on position.  Or they could add another column that lists how many times there were mutations at this position.  Any would work. * The authors only focus on the mutation that occurred between the first and last times it was sequenced.  I think it would be worthwhile to enumerate the consensus changes in the genome that differ from the closest ancestor on the phylogenetic tree.  In other words, what mutations were acquired before the virus was sequenced the first time.  There probably aren’t that many of these.

help people learn by focusing on understanding, using all language skills, rather than being language police

counters an SAE-only pedagogy,

mixing languages or dialects not as an error, but smart way to use full language toolkit,

diversity, "code-meshing, globalization challenge the premise SAE is the only legitimate academic register,

changing name doesn't solve problem, as long as standard tests use SAE that's what is focused on, meaning SAE is used as the real measure of success

treat home language as a temporary crutch, goal is to get them to SAE

schools openly forcing SAE as the main rule, grading and goals help to confuse speaking or using "correct" English with being smart or good at the subject

accomodations for English proficiency reaffirm SAE as the norm

fear of foreigners helped make SAE the official language in schools,

give SAE power by making it the official rule, makes other styles seem wrong, thus justifying exclusion from situations

policies range from actively attacking to passively doing nothing to help other language styles keeping SAE in charge

Hi what are y'all being for Halloween?

Interesante reflexion. @danicotillas mira esto

eLife Assessment

This study by Roseby and colleagues shows that region-specific mechanosensation - especially anterior-dorsal inputs - controls larval self-righting, and links this to Hox gene function in sensory neurons. The work is important for understanding how body plan cues shape sensorimotor behaviour, and the experimental toolkit will be of use to others. The strength of evidence is solid with respect to the assays developed and the involvement of the anterior region; it is incomplete with respect to dorso-ventral involvement in that region and the role of Hox genes in the process. These findings will be of broad interest to researchers studying neural circuits, developmental genetics, and the evolution of behaviour.

Reviewer #1 (Public review):

Summary:

Roseby and colleagues report on a body region-specific sensory control of the fly larval righting response, a body contortion performed by fly larvae to correct their posture when they find themselves in an inverted (dorsal side down) position. This is an important topic because of the general need for animals to move about in the correct orientation and the clever methodologies used in this paper to uncover the sensory triggers for the behavior. Several innovative methodologies are developed, including a body region-specific optogenetic approach along different axial positions of the larva, region-specific manipulation of surface contacts with the substrate, and a 'water unlocking' technique to initiate righting behaviors, a strength of the manuscript. The authors found that multidendritic neurons, particularly the daIV neurons, are necessary for righting behavior. The contribution of daIV neurons had been shown by the authors in a prior paper (Klann et al, 2021), but that study had used constitutive neuronal silencing. Here, the authors used acute inactivation to confirm this finding. Additionally, the authors describe an important role for anterior sensory neurons and a need for dorsal substrate contact. Conversely, ventral sensory elements inhibit the righting behavior, presumably to ensure that the ventral-side-down position dominates. They move on to test the genetic basis for righting behavior and, consistent with the regional specificity they observe, implicate sensory neuron expression of Hox genes Antennapedia and Abdominal-b in self-righting.

Strengths:

Strengths of this paper include the important question addressed and the elegant and innovative combination of methods, which led to clear insights into the sensory biology of self-righting, and that will be useful for others in the field. This is a substantial contribution to understanding how animals correct their body position. The manuscript is very clearly written and couched in interesting biology.

Limitations:

(1) The interpretation of functional experiments is complicated by the proposed excitatory and inhibitory roles of dorsal and ventral sensory neuron activity, respectively. So, while silencing of an excitatory (dorsal) element might slow righting, silencing of inputs that inhibit righting could speed the behavior. Silencing them together, as is done here, could nullify or mask important D-V-specific roles. Selective manipulation of cells along the D-V axis could help address this caveat.

(2) Prior studies from the authors implicated daIV neurons in the righting response. One of the main advances of the current manuscript is the clever demonstration of region-specific roles of sensory input. However, this is only confirmed with a general md driver, 190(2)80, and not with the subset-specific Gal4, so it is not clear if daIV sensory neurons are also acting in a regionally specific manner along the A-P axis.

(3) The manuscript is narrowly focused on sensory neurons that initiate righting, which limits the advance given the known roles for daIV neurons in righting. With the suite of innovative new tools, there is a missed opportunity to gain a more general understanding of how sensory neurons contribute to the righting response, including promoting and inhibiting righting in different regions of the larva, as well as aspects of proprioceptive sensing that could be necessary for righting and account for some of the observed effects of 109(2)80.

(4) Although the authors observe an influence of Hox genes in righting, the possible mechanisms are not pursued, resulting in an unsatisfying conclusion that these genes are somehow involved in a certain region-specific behavior by their region-specific expression. Are the cells properly maintained upon knockdown? Are axon or dendrite morphologies of the cells disrupted upon knockdown?

(5) There could be many reasons for delays in righting behavior in the various manipulations, including ineffective sensory 'triggering', incoherent muscle contraction patterns, initiation of inappropriate behaviors that interfere with righting sequencing, and deficits in sensing body position. The authors show that delays in righting upon silencing of 109(2)80 are caused by a switch to head casting behavior. Is this also the case for silencing of daIV neurons, Hox RNAi experiments, and silencing of CO neurons? Does daIII silencing reduce head casting to lead to faster righting responses?

(6) 109(2)80 is expressed in a number of central neurons, so at least some of the righting phenotype with this line could be due to silenced neurons in the CNS. This should at least be acknowledged in the manuscript and controlled for, if possible, with other Gal4 lines.

Other points

(7) Interpretation of roles of Hox gene expression and function in righting response should consider previous data on Hox expression and function in multidendritic neurons reported by Parrish et al. Genes and Development, 2007.

(8) The daIII silencing phenotype could conceivably be explained if these neurons act as the ventral inhibitors. Do the authors have evidence for or against such roles?

Reviewer #2 (Public review):

Summary

This work explores the relationship between body structure and behavior by studying self-righting in Drosophila larvae, a conserved behavior that restores proper orientation when turned upside-down. The authors first introduce a novel "water unlocking" approach to induce self-righting behavior in a controlled manner. Then, they develop a method for region-specific inhibition of sensory neurons, revealing that anterior, but not posterior, sensory neurons are essential for proper self-righting. Deep-learning-based behavioral analysis shows that anterior inhibition prolongs self-righting by shifting head movement patterns, indicating a behavioral switch rather than a mere delay. Additional genetic and molecular experiments demonstrate that specific Hox genes are necessary in sensory neurons, underscoring how developmental patterning genes shape region-specific sensory mechanisms that enable adaptive motor behaviors.

Strengths

The work of Roseby et al. does what it says on the tin. The experimental design is elegant, introducing innovative methods that will likely benefit the fly behavior community, and the results are robustly supported, without overstatement.

Weaknesses:

The manuscript is clearly written, flows smoothly, and features well-designed experiments. Nevertheless, there are areas that could be improved. Below is a list of suggestions and questions that, if addressed, would strengthen this work:

(1) Figure 1A illustrates the sequence of self-righting behavior in a first instar larva, while the experiments in the same figure are performed on third instar larvae. It would be helpful to clarify whether the sequence of self-righting movements differs between larval stages. Later on in the manuscript, experiments are conducted on first instar larvae without explanation for the choice of stage. Providing the rationale for using different larval stages would improve clarity.

(2) What was the genotype of the larvae used for the initial behavioral characterization (Figure 1)? It is assumed they were wild type or w1118, but this should be stated explicitly. This also raises the question of whether different wild-type strains exhibit this behavior consistently or if there is variability among them. Has this been tested?

(3) Could the observed slight leftward bias in movement angles of the tail (Figure 1I and S1) be related to the experimental setup, for example, the way water is added during the unlocking procedure? It would be helpful to include some speculation on whether the authors believe this preference to be endogenous or potentially a technical artifact.

(4) The genotype of the larvae used for Figure 2 experiments is missing.

(5) The experiment shown in Figure 2E-G reports the proportion of larvae exhibiting self-righting behavior. Is the self-righting speed comparable to that measured using the setup in Figure 1?

(6) Line 496 states: "However, the effect size was smaller than that for the entire multidendritic population, suggesting neurons other than the daIVs are important for self-righting". Although I agree that this is the more parsimonious hypothesis, an alternative interpretation of the observed phenomenon could be that the effect is not due to the involvement of other neuronal populations, but rather to stronger Gal4 expression in daIVs with the general driver compared to the specific one. Have the authors (or someone else) measured or compared the relative strengths of these two drivers?

(7) Is there a way to quantify or semi-quantify the expression of the Hox genes shown in Figure 6A? Also, was this experiment performed more than once (are there any technical replicates?), or was the amount of RNA material insufficient to allow replication?

(8) Since RNAi constructs can sometimes produce off-target effects, it is generally advisable to use more than one RNAi line per gene, targeting different regions. Given that Hox genes have been extensively studied, the RNAis used in Figure 6B are likely already characterized. If this were the case, it would strengthen the data to mention it explicitly and provide references documenting the specificity and knockdown efficiency of the Hox gene RNAis employed. For example, does Antp RNAi expression in the 109(2)80 domain decrease Antp protein levels in multidendritic anterior neurons in immunofluorescence assays?

(9) In addition to increasing self-righting time, does Antp downregulation also affect head casting behavior or head movement speed? A more detailed behavioral characterization of this genetic manipulation could help clarify how closely it relates to the behavioral phenotypes described in the previous experiments.

(10) Does down-regulation of Antp in the daIV domain also increase self-righting time?

Author response:

We are very pleased to hear the overall positive views and constructive criticisms of eLife Editors and Reviewers on our work. In particular, we appreciate their global assessment that the work is important for understanding how body plan cues shape sensorimotor behavioural patterns, that the strength of evidence is solid, and their views that our experimental toolkit will be useful to others. We also very much appreciate eLife’s assessment that our findings will be of broad interest to researchers studying neural circuits, developmental genetics, and the evolution of behaviour.

Regarding Reviewer 1, we thank them for their positive comments on the value of our study, highlighting that our paper addresses an important question using an elegant and innovative combination of methods, which leads to clear insights into the sensory biology of self-righting, which they consider shall be useful for others in the field. We are also very pleased to hear that they consider that our study makes a substantial contribution to understanding how animals correct their body position and that the manuscript is very clearly written and couched in interesting biology. In a revised version of the manuscript, we will consider some of the interesting points raised by Rev1, including the possibility of conducting new experiments using neuronal subset-specific Gal4s, to establish whether daIV sensory neurons are also acting in a regionally specific manner along the A-P axis.

Turning to the comments by Rev2, we are grateful to them for considering that our experimental design is elegant, and that it introduces innovative methods that will likely benefit the fly behavior community, and the results are robustly supported. In connection to other comments, in a revised manuscript we will consider addressing the question of whether normal levels of expression of the Hox gene Antennapedia within the daIV domain are essential for self-righting. We will also seek to add technical replicates to our Hox expression molecular analysis, amend typos and incorporate several of the constructive corrections mentioned.

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

Learn more at Review Commons

Reply to the reviewers

The authors do not wish to provide a response at this time.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

Referee #3

Evidence, reproducibility and clarity

Summary:

In the manuscript "Nucleosome positioning shapes cryptic antisense transcription", Kok and colleagues perform a characterization of nucleosome remodeling factors in S. pombe by assaying the impact of their deletion on antisense transcription and nucleosome organization. They find that deletion of Hrp3 leads to up-regulation of antisense RNA transcripts as well as disruption of phased nucleosomes in gene bodies. The authors then establish a catalogue of antisense transcripts in S. pombe using long read RNA sequencing, which they use to analyze the relationship between nucleosome positioning and antisense transcription. Through this analysis, they associate nucleosome positioning with the initiation of antisense transcription and conclude that nucleosome positioning within gene bodies represses cryptic antisense transcription. They further support this observation by showing that the up-regulated genes in the Hrp3 knock-out are enriched for genes usually expressed in meiosis, which in S. pombe often occur as nested transcripts in reverse orientation. Using growth assays under various stress conditions, the authors narrow down the domain responsible for the phenotype to the C-terminal CHCT domain. To address how Hrp3 gains specificity, they perform an in-silico interaction prediction screen to identify Prf1 as a putative interactor of the CHCT domain. Using recombinant expression in bacteria followed by pulldowns from lysates, they confirm the interaction and introduce point mutants that abolish the interaction. The authors then link the interaction with Prf1 to transcriptional elongation, where they observe a correlation between Hrp3 presence and chromatin marks of transcription elongation, especially H2BK119ub, which is also reduced in the Hrp3 knockout. They further demonstrate that both gene body nucleosome phasing and antisense transcription are similarly affected in the prf1 knockout as well as the hrp1-hrp3-prf1 triple knock-out cells, which indicates that they affect the same pathway.

Major comments:

The manuscript is well-written and the claims are generally supported by the data. The authors demonstrate scientific rigor through comprehensive experiments using single and double knockouts. I have three main comments that can be addressed through additional analysis and limited experimentation:

1. The authors use the terms "Prf1" and "Paf1 complex" interchangeably multiple times in the manuscript (eg. Line 296). However, the experimental data presented only demonstrate a connection between Prf1 and Hrp3. Furthermore, published literature establishes that Prf1 and Paf1 represent distinct entities in S. pombe (Mbogning et al., 2013, PLoS Genetics 9(3): e1004029). The authors should clarify this distinction and use consistent, accurate terminology throughout the text. Reference: Mbogning, J., et al. (2013). The PAF Complex and Prf1/Rtf1 Delineate Distinct Cdk9-Dependent Pathways Regulating Transcription Elongation in Fission Yeast. PLoS Genetics, 9(3), e1004029. https://doi.org/10.1371/journal.pgen.1004029

2. The authors demonstrate that Hrp3 limits antisense promoter usage; however, the analysis lacks characterization of sequence composition, promoter classes (TATA-box versus TATA-less), or identification of enriched transcription factor motifs near these sites. A more thorough bioinformatic analysis would strengthen the paper and potentially reveal interesting biology, as the effect may be specific to certain transcription factors or promoter architectures.

3. The Hrp3-Prf1 interaction is demonstrated solely through recombinant overexpression and pulldown assays, which carries the risk of detecting non-physiological interactions. While the authors use mutations to verify pulldown specificity, in vivo evidence for this interaction is absent. Given that the authors cite a recent preprint demonstrating sophisticated techniques to show S. cerevisiae Chd1-Prf1 interactions, I presume standard approaches such as co-immunoprecipitation followed by mass spectrometry or Western blot were attempted. Even negative results from such experiments should be reported, as readers will likely question the physiological relevance of the interaction. Additionally, establishing the hierarchy between Hrp3, Prf1, and H2BK119Ub is crucial. While the authors show that Hrp3 ChIP-seq signal correlates with gene expression levels, the proposed Prf1-Hrp3 interaction raises questions about recruitment specificity and hierarchy. The authors mention in lines 344-345: "...the CHCT domain of Hrp3 is critical for its association with transcription elongation along the gene body..." which requires support from experimental data. Testing Hrp3 ChIP-seq in Prf1-depleted conditions would clarify how specificity is achieved and substantiate the functional importance of this interaction. As the authors have all the required strains I would estimate around 1.5-2 months for data generation and analysis.

4. [Optional] Based on strucutre predictions the authors suggest that the interaction of of CHD1 and RTF1 is conserved in arabidopsis and mouse. This should be further supported by pulldown assays and also the pre-print (Reference nr. 99) should be cited as they show similar results using yeast-tow-hybrid assays

Minor comments:

1. Figure 1B: Grouping individual panels according to different paralog groups would make the figure more accessible.

2. Figure 1D: The display of antisense transcription is not accessible. Perhaps boxplots, like those in Figures 2B and 5D, would be easier to read.

3. Line 335: The transition is abrupt and would benefit from additional explanation. Why do the authors use Rtf1 instead of Prf1 here? Consistent nomenclature would improve clarity.

4. Line 352: For the phrase "significant loss," please provide a statistical test or omit the word "significant."

5. Figure 7F: The model presented in panel F suggests that there are two parallel routes that lead to nucleosome phasing; however, the authors state in the text (lines 363-364): "further supporting the idea that Hrp3 and Prf1 act together in the same pathway to control antisense transcription." The model and the text should align better.

Significance

• In the study, the authors establish Hrp3, one of the fission yeast CHD1 remodelers, as a crucial regulator of antisense transcription within gene bodies, which they link to both fitness penalties and the regulation of genes typically expressed during meiosis. They further link the recruitment of Hrp3 at gene bodies to transcriptional elongation, which provides an interesting model for how antisense transcription is prevented in actively transcribed regions of the genome.

• The study is overall very well executed and controlled and provides strong evidence for connecting Hrp3 with the repression of antisense transcription using adequate experiments and technologies. This provides novel insights into a widespread phenomenon present in many organisms. A point that needs further improvement is the suggested physical link between Hrp3 and Prf1. Despite potentially being challenging to address using molecular biology techniques, the authors can further improve the study by dissecting the genetic hierarchy of Hrp3 and Prf1 using accessible tools. This study will be of interest to a broad audience in basic research as it addresses the broad question of how antisense transcription is repressed and provides mechanistic insights into this process. Consequently, this study will be relevant for the broader field of transcriptional regulation and could provide entry points for studying the role of CHD remodelers in other organisms.

• Field of expertise: chromatin biology, small RNA mediated heterochromatin formation

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

Referee #2

Evidence, reproducibility and clarity

Kok et al. report on the role of the chromatin remodelers Hrp1 and Hrp3 in maintaining nucleosome positioning and preventing antisense transcription in Schizosaccharomyces pombe. As commented below, the main criticism of the manuscript is that the first half describes results that are very similar to those already reported by several other laboratories. Therefore, the main novel aspect of the work is the interaction between Hrp3 and the Prf1 subunit of the PAF complex.

Specific points:

1. The articles of Hennig et al. (2012), Pointner et al. (2012) and Shim et al. (2012) are cited in the manuscript (line 119, Refs. 61-63) only as a confirmation of the minor effect of the absence of Hrp1 on nucleosome positioning and antisense expression. However, these three articles reached the same conclusion as Kok et al. that the absence of Hrp3 in S. pombe causes severe, genome-wide loss of nucleosome positioning and overexpression of antisense transcripts, whereas the absence of Hrp1 has a much weaker effect. These results were also discussed in a short review article (Touat-Todeschini et al. EMBO J. 2012. 31: 4371). Although Kok et al. analysed transcription at a higher resolution and mapped transcription initiation using Pro-Seq (Figures 1, 2 and 3), their results do not add much to what was already reported in these previous studies.

2. Several sites in the manuscript state that Hrp3 belongs to the SWI/SNF family of chromatin remodelers (for example, line 92). However, Hrp3 is a member of the CHD family, whose members have a very different structure and function (see, for example, Clapier et al. 2017. Nat Rev Mol Cell Biol 18: 407; Paliwal et al. 2024 TIGs 41:236).

3. The authors should indicate where the nucleosome remodelling activity of some of the proteins in Figure 1A like Irc20, Rrp1, Rrp2 and Mot1) has been reported.

4. The analysis of nucleosome positioning by aggregating thousands of genes, such as those shown in Figure 1B, has low resolution and can only detect gross alterations affecting many genes. Nevertheless, several mutants, such as swr1∆ and rrp1∆, also exhibit altered nucleosomal profiles in Figure 1B. In other cases, the occupancy of the first and second nucleosomes after the TSS is reduced relative to the wild type. Therefore, it cannot be concluded that "nucleosome arrays in wild type and most remodeller mutant cells were highly ordered and regular" (line 105).

5. Although it was previously reported that hrp3∆ mutants overexpress antisense transcripts (see point 1 above), it is unclear how this finding is represented in Figure 1D. Similarly, it not clear either why antisense transcription is undetectable in hrp1∆ relative to WT in Figure 1D, yet significantly higher than in WT in Figures 2B, 3A and 3B. Furthermore, sense transcription in the single and double mutants is comparable to WT in Figure 2A, yet much higher in Figure S3B.

6. Figure S3C claims that antisense transcription is higher in genes with greater nucleosome disruption in the double mutant hrp1∆hrp3∆. However, without a quantitative analysis, it is difficult to discern any significant differences in the degree of disruption across the four quartiles of antisense expression.

7. Figures 3D and S4C show that the TSS of antisense transcription colocalizes with a region resistant to MNase that is at least 300 bp wide. This size does not correspond to that occupied by a nucleosome and contrasts with the expected size of the four nucleosome peaks downstream from it.

8. In relation to the previous point, Figure S4C (bottom) shows that the centre of the region above the TSS is slightly displaced in the three mutants. This displacement corresponds to an increase in the G+C content of approximately 1.5% (Figure S4C top), equivalent to an increase of less than 2.5 Gs and Cs every 150 bp of nucleosomal DNA. Without some cause and effect experiments, it is difficult to attribute a functional significance to such a tiny difference. How repetitive is this difference in biological replicates?

9. The authors should also explain how the position of the dyads was estimated in the double mutant hrp1∆hrp3∆ in Figure S4B. The severe loss of nucleosomal positioning suggests that the dyads occupy different positions in different cells within the same population. While most of the remaining figures show data for the three mutants, this figure shows results for the double hrp1∆hrp3∆ mutant only.

10. Figures 3G and 3H show the analysis of the promoter activity of some regions upstream from antisense transcripts, achieved by replacing the endogenous ura4 gene promoter with these regions. This analysis lacks negative controls showing the level of transcription in the recipient strain following the removal of the endogenous ura4 promoter and its replacement for genomic regions not associated with the initiation of antisense transcription in the mutants. Furthermore, transcription should be measured by quantitative PCR of the ura4 mRNA rather than by the more indirect method of measuring OD600 in 384-well plates (line 708).

11. Figure F4 suggests that Hrp3 may regulate the expression of genes specific to meiosis by showing an anticorrelation between the expression levels of Hrp3 and a selection of genes that are upregulated during meiosis (MUGs) 5 hours after the onset of meiosis. While this is an interesting possibility, it will remain speculative until it is demonstrated that the level of Hrp3 protein is reduced at the same stage of meiosis, and that MUG overexpression is associated with reduced nucleosomal occupancy adjacent to their TSS at that stage.

12. The experiments in Figures 5 and 6, which describe the interaction between the Hpr3-specific CHCT domain and the Prf1 protein, are interesting and represent the main element of novelty of the manuscript. However, this interaction in figure 6D and 6E should be confirmed in vivo.

13. Kok et al. indicate that the triple prf1∆ hrp1∆ hrp3∆ mutant exhibits stronger growth defects than the single prf1∆ mutant. However, Figure S9F shows that no growth is detectable in the single prf1∆ mutant, a phenotype that cannot be exacerbated in the triple mutant. Perhaps the use of a prf1 mutant showing a less severe phenotype migh help.

Significance

As indicated in point 1, the first half of the manuscript describes results that are very similar to those already reported in the literature.

The interaction between Hrp3 and the Prf1 subunit is new and interesting, and could lead to further research and a new manuscript.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

Referee #1

Evidence, reproducibility and clarity

This is an excellent study that leverages the chromatin biology of Schizosaccharomyces pombe to uncover the central role of CHD1-family remodelers in maintaining nucleosome organisation and suppressing cryptic transcription. The work is carefully executed. In short, the authors show that Hrp3 is the primary CHD1-family remodeler responsible for maintaining nucleosome organisation over gene bodies. This represses antisense transcription from cryptic promoters in gene bodies. They provide evidence that Hrp3 is repressed in meiosis to allow the induction of meiotic genes. They further identified that a conserved domain, the CHCT domain of Hrp3, is essential for its interaction with Prf1 (PAF complex subunit), which is critical for the chromatin organisation in gene bodies. This manuscript is of excellent quality and is an important contribution towards understanding how transcription initiation is repressed within gene bodies. I have small comments and suggestions for clarification.

Minor comments:

• The study demonstrates that Hrp3 represses antisense transcription at meiotic genes, showing that Hrp3 is reduced in meiosis, which could facilitate the induction of meiotic genes. Is there a phenotype in the hrp3Δ or the hrp1Δ hrp3Δ mutant in relation to meiosis? E.g. do these strains enter meiosis uncontrolled?

• Figure 3C - ORC4 Locus TSS presentation. The presented data do not show a well-defined TSS on the sense strand. For reference, it would be useful to show that sense TSS is not altered between the different strains.

• The study focuses on antisense cryptic transcription, which is relatively easy to measure by RNA-seq. Often, however, cryptic transcription can also occur in the sense direction in gene bodies. Do the authors also find evidence of cryptic sense transcription in gene bodies (based on TSS-seq data)? This could be useful for completeness to report, as this could lead to aberrant protein-coding isoforms.

• The manuscript alternates between "Prf1" (S. pombe) and "RTF1" (other eukaryotes). This is at times confusing. I recommend consistent use of gene nomenclature.

• The authors show epistatic interaction for nucleosome spacing in Figure 7D for the prf1Δ and hrp1Δ hrp3Δ prf1Δ strains. It would be informative to have the hrp1Δ hrp3Δ data also included in Figure 7D, like in the other figure panels.

Significance

This is an excellent study that leverages the chromatin biology of Schizosaccharomyces pombe to uncover the central role of CHD1-family remodelers in maintaining nucleosome organisation and suppressing cryptic transcription. This manuscript is of excellent quality and is an important contribution towards understanding how transcription initiation is repressed within gene bodies.

I am an expert on transcription regulation and noncoding transcription.

This just ignited rage in me- as a woman with young children and who has been told by peers and family members that staying home with the kids would be better for their 'development'. Societies views towards women's place in the workforce are still sexist and a battle women face every day.

These are interesting questions because as discussed in this article, even with new data collection, and the intention is there to create a diverse data set, if it is people from a majority group collecting the data, often unknowingly a diverse group of people are left out of the collection. This is in reference to the facial recognition example. Moreover, I would ask, 'Who does the work (and who is pushed out) AND how can we actively find ways to figure out the groups being pushed out- because sometimes our own unconscious bias won't be able to come up with that answer ourselves.

These restrictions do persist today; between transportation barriers and computer literacy barriers alone. I have seen first hand several people unable to vote during the last town election because they were unable to get to the fire hall where voting was taking place. And for many, voting online requires one on one help with the technology; something not everyone has.

YAY!!! Developmental in a good context. But also YAY for C-R-A. Hmm. Can my thing be doing this for higher levels?

Yup. Important stuff.

Okay, there's sort of an implied both/and here.

Big Honking Red Flag. Let's Jump right on the Either Or BOAT!!!! TRADITIONAL REFORM I bet one is bad!!!

Okay let's see if/how they use this to defend eliminating developmental education courses. <br /> Hey, at least they acknowledge it instead of syaing it's all about anxiety.

Can I highlight this in SEVEN COLORS ????

OK, in the context of the 'tough decisions' eh? Good to recognize we need to deal with that instead of pretending everybody will have the resources to do it right.

Definition is standard issue stuff ;)

eLife Assessment

This important study uses single-neuron Patch-seq RNA sequencing to investigate the process by which RNA editing can produce protein diversity and regulate function in various cellular contexts. The computational analyses of the data collected are convincing, and from an analytical standpoint, this paper is a notable advance in seeking to provide a biological context for massive amounts of data in the field. The study would be of interest to biologists looking at the effects of RNA editing in the diversification of cellular behaviour.

Reviewer #1 (Public review):

The importance of RNA editing in producing protein diversity is a widespread process that can regulate how genes function in various cellular contexts. Despite the importance of the process, we still lack a thorough knowledge of the profile of RNA editing targets in known cells. Crane and colleagues take advantage of a recently acquired scRNAseq database for Drosophila type Ib and Is larval motoneurons and identify the RNA editing landscape that differs in those cells. They find both canonical (A --> I) and non-canonical sites and characterize the targets, their frequencies, and determine some of the "rules" that influence RNA editing. They compare their database with existing databases to determine a reliance on the most well-known deaminase enzyme ADAR, determine the activity-dependence of editing profiles, and identify editing sites that are specific to larval Drosophila, differing from adults. The authors also identify non-canonical editing sites, especially in the newly appreciated and identified regulator of synaptic plasticity, Arc1.

The paper represents a strong analysis of recently made RNAseq databases from their lab and takes a notable approach to integrate this with other databases that have been recently produced from other sources. One of the places where this manuscript succeeds is in a thorough approach to analyzing the considerable amount of data that is out there regarding RNAseq in these differing motoneurons, but also in comparing larvae to adults. This is a strong advance. It also enables the authors to begin to determine rules for RNA editing. From an analytical standpoint, this paper is a notable advance in seeking to provide a biological context for massive amounts of data in the field. Further, it addresses some biological aspects in comparing WT and adar mutants to assess one potential deaminase, addresses activity-dependence, and begins to reveal profiles of canonical and non-canonical editing.

Reviewer #2 (Public review):

Summary:

The study uses single-neuron Patch-seq RNA sequencing in two subgroups of Drosophila larval motoneurons (1s and 1b) and identifies 316 high-confidence canonical mRNA edit sites, which primarily (55%) occur in the coding regions of the mRNAs (CDS). Most of the canonical mRNA edits in the CDS regions include neuronal and synaptic proteins such as Complexin, Cac, Para, Shab, Sh, Slo, EndoA, Syx1A, Rim, RBP, Vap33, and Lap, which are involved in neuronal excitability and synaptic transmission. Of the 316 identified canonical edit sites, 60 lead to missense RNAs in a range of proteins (nAChRalpha5, nAChRalpha6, nAChRbeta1, ATPalpha, Cacophony, Para, Bsk, Beag, RNase Z) that are likely to have an impact on the larval motoneurons' development and function. Only 27 sites show editing levels higher than 90% and a similar editing profile is observed between the 1s and 1b motoneurons when looking at the number of edit sites and the fraction of reads edited per cell, with only 26 RNA editing sites showing a significant difference in the editing level. The variability of edited and unedited mRNAs suggests stochastic editing. The two subsets of motoneurons show many noncanonical editing sites, which, however, are not enriched for neuron-specific genes, therefore causing more silent changes compared to canonical editing sites. Comparison of the mRNA editing sites and editing rate of the single neuron Patch-seq RNA sequencing dataset to three other RNAseq datasets, one from same stage larval motoneurons and two from adult heads nuclei, show positive correlations in editing frequencies of CDS edits between the patch-sec larval 1b + 1s MNs and all other three datasets, with stronger correlations for previously annotated edits and weaker correlations for unannotated edits. Several of the identified editing targets are only present in the single neuron Patch-seq RNA sequencing dataset, suggesting cell-type-specific or developmental-specific editing. Editing appears to be resistant to changes in neuronal activity as only a few sites show evidence of being activity-regulated.

Strengths:

The study employs GAL4 driver lines available in the Drosophila model to identify two subtypes of motoneurons with distinct biophysical and morphological features. In combination with single-neuron Patch-seq RNA sequencing, it provides a unique opportunity to identify RNA editing sites and rates specific to specific motoneuron subtypes. The RNA seq data is robustly analysed, and high-confidence mRNA edit sites of both canonical and noncanonical RNA editing are identified.

The mRNA editing sites identified from the single neuron Patch-seq RNA sequencing data are compared to editing sites identified across other RNAseq datasets collected from animals at similar or different developmental stages, allowing for the identification of editing sites that are common to all or specific to a single dataset.

Weaknesses:

Although the analysed motoneurons come from two distinct subtypes, it is unclear from how many Drosophila larvae the motoneurons were collected and from which specific regions along the ventral nerve cord (VNC). Therefore, the study does not consider possible differences in editing rate between samples from different larvae that could be in different active states or neurons located at different regions of the VNC, which would receive inputs from slightly different neuronal networks.

The RNA samples include RNAs located both in the nucleus and the cytoplasm, introducing a potential compartmental mismatch between the RNA and the enzymes mediating the editing, which could influence editing rate. Similarly, the age of the RNAs undergoing editing is unknown, which may influence the measured editing rates.

Reviewer #3 (Public review):

Summary:

The study consists of extensive computational analyses of their previously released Patch-seq data on single MN1-Ib and MNISN-Is neurons. The authors demonstrate the diversity of A>I editing events at single-cell resolution in two different neuronal cell types, identifying numerous A>I editing events that vary in their proportion, including those that cause missense mutations in conserved amino acids. They also consider "noncanonical" edits, such as C>T and G>A, and integrate publicly available data to support these analyses.

In general, the study contains a valuable resource to assess RNA editing in single neurons and opens several questions regarding the diversity and functional implications of RNA editing at single-cell resolution. The conclusions from the study are generally supported by their data; however, the study is currently based on computational predictions and would therefore benefit from experimentation to support their hypotheses and demonstrate the effects of the editing events identified on neuronal function and phenotype.

Strengths:

The study uses samples that are technically difficult to prepare to assess cell-type-specific RNA editing events in a natural model. The study also uses public data from different developmental stages that demonstrate the importance of considering cell type and developmental stage-specific RNA regulation. These critical factors, particularly that of developmental timing, are often overlooked in mechanistic studies.

Extensive computational analysis, using public pipelines, suitable filtering criteria, and accessible custom code, identifies a number of RNA editing events that have the potential to impact conserved amino acids and have subsequent effects on protein function. These observations are supported through the integration of several public data sets to investigate the occurrence of the edits in other data sets, with many identified across multiple data sets. This approach allowed the identification of a number of novel A>I edits, some of which appear to be specific to this study, suggesting cell/developmental specificity, whilst others are present in the public data sets but went unannotated.

The study also considers the role of Adar in the generation of A>I edits, as would be expected, by assessing the effect of Adar expression on editing rates using public data from adar mutant tissue to demonstrate that the edits conserved between experiments are mainly Adar-sensitive. This would be stronger if the authors also performed Patch-seq experiments in adar mutants to increase confidence in the identified edit sites.

Weaknesses:

Whilst the study makes interesting observations using advanced computational approaches, it does not demonstrate the functional implications of the observed editing events. The functional impact of the edits is inferred from either the nature of the change to the coding sequence and the amino acid conservation, or through integration of other data sets. Although these could indeed imply function, further experimentation would be required to confirm such as using their Alphafold models to predict any changes in structure. This limitation is acknowledged by the authors, but the overall strength of the interpretation of the analysis could be softened to represent this.

The study uses public data from more diverse cellular populations to confirm the role of Adar in introducing the A>I edits. Whilst this is convincing, the ideal comparison to support the mechanism behind the identified edits would be to perform patch-seq experiments on 1b or 1s neurons from adar mutants. However, although this should be considered when interpreting the data, these experiments would be a large amount of work and beyond the scope of the paper.

By focusing on the potential impact of editing events that cause missense mutations in the CDS, the study may overlook the importance of edits in noncoding regions, which may impact miRNA or RNA-binding protein target sites. Further, the statement that noncanonical edits and those that induce silent mutations are likely to be less impactful is very broad and should be reconsidered. This is particularly the case when suggesting that silent mutations may not impact the biology. Given the importance of codon usage in translational fidelity, it is possible that silent mutations induced by either A>I or noncanonical editing in the CDS impact translation efficiency. Indeed, this could have a greater impact on protein production and transcript levels than a single amino acid change alone.

Author response:

Reviewer #1:

Indicated the paper provided a strong analysis of RNAseq databases to provide a biological context and resource for the massive amounts of data in the field on RNA editing. The reviewer noted that future studies will be important to define the functional consequences of the individual edits and why the RNA editing rules we identified exist. We address these comments below.

(1) The reviewer wondered about the role of noncanonical editing to neuronal protein expression.

Indeed, the role of noncanonical editing has been poorly studied compared to the more common A-to-I ADAR-dependent editing. Most non-canonical coding edits we found actually caused silent changes at the amino acid level, suggesting evolutionary selection against this mechanism as a pathway for generating protein diversity. As such, we suspect that most of these edits are not altering neuronal function in significant ways. Two potential exceptions to this were non-canonical edits that altered conserved residues in the synaptic proteins Arc1 and Frequenin 1. The C-to-T coding edit in the activity-regulated Arc1 mRNA that encodes a retroviral-like Gag protein involved in synaptic plasticity resulted in a P124L amino acid change (see Author response image 1 panel A below). ~50% of total Arc1 mRNA was edited at this site in both Ib and Is neurons, suggesting a potentially important role if the P124L change alters Arc1 structure or function. Given Arc1 assembles into higher order viral-like capsids, this change could alter capsid formation or structure. Indeed, P124 lies in the hinge region separating the N- and C-terminal capsid assembly regions (panel B) and we hypothesize this change will alter the ability of Arc1 capsids to assemble properly. We plan to experimentally test this by rescuing Arc1 null mutants with edited versus unedited transgenes to see how the previously reported synaptic phenotypes are modified. We also plan to examine the ability of the change to alter Arc1 capsid assembly in a collaboration using CyroEM.

Author response image 1.

A. AlphaFold predictions of Drosophila Arc1 and Frq1 with edit site noted. B. Structure of the Drosophila Arc1 capsid. Monomeric Arc1 conformation within the capsid is shown on the right with the location of the edit site indicated.

The other non-canonical edit (G-to-A) that stood out was in Frequenin 1 (Frq1), a multi-EF hand containing Ca²⁺ binding protein that regulates synaptic transmission, that resulted in a G2E amino acid substitution (location within Frq1shown in panel A above). This glycine residue is conserved in all Frq homologs and is the site of N-myristoylation, a co-translational lipid modification to the glycine after removal of the initiator methionine by an aminopeptidase. Myristoylation tethers Frq proteins to the plasma membrane, with a Ca²⁺-myristoyl switch allowing some family members to cycle on and off membranes when the lipid domain is sequestered in the absence of Ca²⁺. Although the G2E edit is found at lower levels (20% in Ib MNs and 18% in Is MNs), it could create a pool of soluble Frq1 that alters it’s signaling. We plan to functionally assay the significance of this non-canonical edit as well. Compared to edits that alter amino acid sequence, determining how non canonical editing of UTRs might regulate mRNA dynamics is a harder question at this stage and will require more experimental follow-up.

(2) The reviewer noted the last section of the results might be better split into multiple parts as it reads as a long combination of two thoughts.

We agree with the reviewer that the last section is important, but it was disconnected a bit from the main story and was difficult for us to know exactly where to put it. All the data to that point in the paper was collected from our own PatchSeq analysis from individual larval motoneurons. We wanted to compare these results to other large RNAseq datasets obtained from pooled neuronal populations and felt it was best to include this at the end of the results section, as it no longer related to the rules of RNA editing within single neurons. We used these datasets to confirm many of our edits, as well as find evidence for some developmental and neuron-specific cell type edits. We also took advantage of RNAseq from neuronal datasets with altered activity to explore how activity might alter the editing machinery. We felt it better to include that data in this final section given it was not collected from our original PatchSeq approach.

Reviewer #2:

Noted the study provided a unique opportunity to identify RNA editing sites and rates specific to individual motoneuron subtypes, highlighting the RNAseq data was robustly analyzed and high-confidence hits were identified and compared to other RNAseq datasets. The reviewer provided some suggestions for future experiments and requested a few clarifications.

(1) The reviewer asked about Figure 1F and the average editing rate per site described later in the paper.

Indeed, Figure 1F shows the average editing rate for each individual gene for all the Ib and Is cells, so we primarily use that to highlight the variability we find in overall editing rate from around 20% for some sites to 100% for others. The actual editing rate for each site for individual neurons is shown in Figure 4D that plots the rate for every edit site and the overall sum rate for that neuron in particular.

(2) The reviewer also noted that it was unclear where in the VNC the individual motoneurons were located and how that might affect editing.

The precise segment of the larvae for every individual neuron that was sampled by Patch-seq was recorded and that data is accessible in the original Jetti et al 2023 paper if the reader wants to explore any potential anterior to posterior differences in RNA editing. Due to the technical difficulty of the Patch-seq approach, we pooled all the Ib and Is neurons from each segment together to get more statistical power to identify edit sites. We don’t believe segmental identify would be a major regulator of RNA editing, but cannot rule it out.

(3) The reviewer also wondered if including RNAs located both in the nucleus and cytoplasm would influence editing rate.

Given our Patch-seq approach requires us to extract both the cytoplasm and nucleus, we would be sampling both nuclear and cytoplasmic mRNAs. However, as shown in Figure 8 – figure supplement 3 D-F, the vast majority of our edits are found in both polyA mRNA samples and nascent nuclear mRNA samples from other datasets, indicating the editing is occurring co-transcriptionally and within the nucleus. As such, we don't think the inclusion of cytoplasmic mRNA is altering our measured editing rates for most sites. This may not be true for all non-canonical edits, as we did see some differences there, indicating some non-canonical editing may be happening in the cytoplasm as well.

Reviewer #3:

indicated the work provided a valuable resource to access RNA editing in single neurons. The reviewer suggested the value of future experiments to demonstrate the effects of editing events on neuronal function. This will be a major effort for us going forwards, as we indeed have already begun to test the role of editing in mRNAs encoding several presynaptic proteins that regulate synaptic transmission. The reviewer also had several other comments as discussed below.

(1) The reviewer noted that silent mutations could alter codon usage that would result in translational stalling and altered protein production.

This is an excellent point, as silent mutations in the coding region could have a more significant impact if they generate non-preferred rare codons. This is not something we have analyzed, but it certainly is worth considering in future experiments. Our initial efforts are on testing the edits that cause predictive changes in presynaptic proteins based on the amino acid change and their locale in important functional domains, but it is worth considering the silent edits as well as we think about the larger picture of how RNA editing is likely to impact not only protein function but also protein levels.

(2) The reviewer noted future studies could be done using tools like Alphafold to test if the amino acid changes are predicted to alter the structure of proteins with coding edits.

This is an interesting approach, though we don’t have much expertise in protein modeling at that level. We could consider adding this to future studies in collaboration with other modeling labs.

(3) The reviewer wondered if the negative correlation between edits and transcript abundance could indicate edits might be destabilizing the transcripts.

This is an interesting idea, but would need to be experimentally tested. For the few edits we have generated already to begin functionally testing, including our published work with editing in the C-terminus of Complexin, we haven’t seen a change in mRNA levels causes by these edits. However, it would not be surprising to see some edits reducing transcript levels. A set of 5’UTR edits we have generated in Syx1A seem to be reducing protein production and may be acting in such a manner.

(4) The reviewer wondered if the proportion of edits we report in many of the figures is normalized to the length of the transcript, as longer transcripts might have more edits by chance.

The figures referenced by the reviewer (1, 2 and 7) show the number of high-confidence editing sites that fall into the 5’ UTR, 3’ UTR, or CDS categories. Our intention here was to highlight that the majority of the high confidence edits that made it through the stringent filtering process were in the coding region. This would still be true if we normalized to the length of the given gene region. However, it would be interesting to know if these proportions match the expected proportions of edits in these gene regions given a random editing rate per gene region length across the Drosophila genome, although we did not do this analysis.    

(5) The reviewer noted that future studies could expand on the work to examine miRNA or other known RBP binding sites that might be altered by the edits.

This is another avenue we could pursue in the future. We did do this analysis for a few of the important genes encoding presynaptic proteins (these are the most interesting to us given the lab’s interest in the synaptic vesicle fusion machinery), but did not find anything obvious for this smaller subset of targets.

(6) The reviewer suggested sequence context for Adar could also be investigated for the hits we identified.

We haven’t pursued this avenue yet, but it would be of interest to do in the future. In a similar vein, it would be informative to identify intron-exon base pairing that could generate the dsDNA template on which ADAR acts.

(7) The reviewer noted the disconnect between Adar mRNA levels and overall editing levels reported in Figure 4A/B.

Indeed, the lack of correlation between overall editing levels and Adar mRNA abundance has been noted previously in many studies. For the type of single cell Patch-seq approach we took to generate our RNAseq libraries, the absolute amount of less abundant transcripts obtained from a single neuron can be very noisy. As such, the few neurons with no detectable Adar mRNA are likely to represent that single neuron noise in the sampling. Per the reviewer’s question, these figure panels only show A-to-I edits, so they are specific to ADAR.

(8) The reviewer notes the scale in Figure 5D can make it hard to visualize the actual impact of the changes.

The intention of Figure 5D was to address the question of whether sites with high Ib/Is editing differences were simply due to higher Ib or Is mRNA expression levels. If this was the case, then we would expect to see highly edited sites have large Ib/Is TPM differences. Instead, as the figure shows, the vast majority of highly-edited sites were in mRNAs that were NOT significantly different between Ib and Is (red dots in graph) and are therefore clustered together near “0 Difference in TPMs”. TPMs and editing levels for all edit sites can be found in Table 1, and a visualization of these data for selected sites is shown in Figure 5E.

Include a reference & description of the figure to give it context

recommend "non" over "un"

Add "Python" as well, since in 2.3 Python is listed, along with R and Quarto

Efficiency, scale, and impact are nested within the power center of a collaborative effort.

Difficult to assess (and impossible at scale) is proving to be a myth. It's doable, we just have to want to.

Embed, identify, assess. Not enough to have content there; need to validate proficiency. ALSO on this page: partnerships (for application practice of skills) and also the free, statewide learning module...good admissions play on the data.