Rainy conditions caused consistent delays in processing speed, showing that weather makes the brain slower at taking in and interpreting information. Participants were 126-139 ms slower during rain, showing a meaningful cognitive delay.

So the noble class defines what a standard variety means

Warm-weather SAD is less common and tends to show up as anxiety, agitation, insomnia, and weight loss, while winter SAD is more tied to fatigue, oversleeping, weight gain, low energy, and social withdrawal. Winter symptoms are driven by shorter days, cold weather, and reduced sunlight, but Dumler notes that extreme heat can increase agitation, especially for people on psychiatric medications that make temperature regulation harder.

It is important to note this sentence that is outlined because privacy is now implied. The question in this case is complicated by the definition of when the phone booth is a public versus private space. When the space is private it ensures that a person's privacy is protected under the Fourth Amendment.

Despite strong theoretical and empirical support for weather's influence on happiness, major uncertainties remain due to things such as differing weather factors, the variability of emotional responses across situations, the potential for daily activities to overshadow weather effects, and the scarcity of experimental research needed to establish clear casual relationships.

Diet can support well-being. By providing vitamin D through foods, especially in low-slight seasons, incorporating omega-3-rich fatty fish to reduce the risk of depression, eating fresh fruits and vegetables to boost happiness, and taking melatonin supplements when needed can support both mood and sleep regulation.

Serotonin, a key mood-regulating neurotransmitter, is strongly influenced by sunlight. Its production and receptor activity decrease in low-light conditions, contributing to winter drops in serotonin levels, and the development of SAD, with symptoms varying by age and gender. This demonstrates that reduced light exposure can trigger negative emotional states through lowered serotonin activity.

Melatonin, a light-sensitive hormone that rises in darkness and regulates the sleep wake cycle, also influences happiness by affecting key mood-related neurochemicals like serotonin and dopamine, and its balance can be disrupted by factors such as artificial light, caffeine, alcohol, and stress, highlighting its important role in both emotional regulation and overall well-being.

Favorable weather like sunshine, warmth, low humidity, and light winds encourage outdoor activity but also promote the formation and stagnation of pollutants, while less pleasant conditions like rain, cold, or strong winds tend to clean the air, with events like typhoons and heavy rainfall significantly improving air quality.

Past experiences with extreme weather damage and worries about future climate change can jointly reduce current well-being, showing that both lived trauma and ongoing uncertainty contribute to long-term emotional strain in an increasingly unpredictable climate.

EWEs like hurricanes and wildfires significantly harm emotional well-being, with studies showing that adolescents and adults exposed to disasters such as Hurricane Maria, Hurricane Katrina, and major wildfires experience heightened levels of depression, anxiety, and other mental health problems months after the events.

Extreme weather events (EWEs), made more frequent and severe due to global warming, create both direct impacts (like storms, floods, and heatwaves), and indirect impacts (such as degraded water quality and land-use changes), all of which interact with social and demographic factors to shape and often worsen mental health and overall well-being.

We don't know if these allegations are against him or his wife- this is the only article that highlights this though.

cut

Refers to the spaCy documentation, as at https://spacy.io/api/token

Desde que me lo presentaron, siempre me ha desagradado el Test Driven Design (TDD), pues me parecía absurdamente burocrático y contra flujo. Afortunadamente, gracias al podcast de Book Overflow, encontré un autor reconocido, John Ousterhout, creador de Tcl/Tk y "A Philosophy of software design", que comparte mi opinón respecto a escribir los test antes de escribir el código y dice que en el TDD no se hace diseño, sino que se depura el software hasta su existencia.

Mi enfoque, que podría llamarse Argumentative Driven Design o ADD es uno en el que el código se desarrolla para mostrar un argumento en favor de una hipótesis, y las pruebas de código se van creando en la medida en que uno necesita inspeccionar y manipular los objetos que dicho código produce.

En palabras práctica, esto quiere decir que los test y su configuración deberían hacerse cuando uno necesita hacer un "print" (para probar/inspeccionar/manipular un estado/elemento del sistema) y no antes, lo cual aumenta la utilidad, no interrumpe el flujo y responde preguntas similares a las de este apartado, respecto a de dónde provienen las pruebas y qué hacer con los resultados exitosos.

This should come in later, because it is confusing with the second one. The second one is mostly about this. When I read it so close to the beginning I was like oh wait, isn't this the other one?

like this

Too long, hard to read.

I think the main idea here is that it's not only technical, but you also get to be part of a community of like minded individuals.

Just mention the choice once, it could be more compact, from chatgpt: After Hours is choose-your-own-adventure: take one of two complementary courses, or both for the complete experience.

Love the electronic mischief

A little long to read, especially with after hours at the end I think

What does a true reconciliation look like in Canada? When all reserves have access to clean water. When indigenous communities living on reserves does not have to pay twice the price for their grocery items. When police officers stop targeting indigenous people and stop dealing with them with violence. When indigenous women are not targets of sex trafficking or image for sex exploitation and amusements. When settlers like white supremacy and other races stop claiming this land as “their” land and keep the indigenous communities in the backgrounds not as the “true owners” of this land. When indigenous children and youth are accepted for their cultural and spiritual beliefs and given and necessary support for their mental health supports in their communities. That is when the true reconciliation will start, and true acceptance will start to take place.

By using race as a tie breaker the court is actually discriminating on the bases of race which is what makes it unconstitutional. The intentions of the court from the outside seem to have good intentions but it is actually in violation of the exact concept that they are trying to "repair".

further reading idea

German

cartographic enterprise explanation and information

another approach to explain German WO

giving information for German specifically

finding time is one of the most challenging parts of the cycle. You have to make it a priotity!

You have to have the foundation and relationship for the conference to be successful.

I agree that most teachers need influence and impact, NOT power and control from their leadership!

this is important with the work I often do with teachers who speak english as a second language. We have to clarify and not make assumptions of understanding.

Listen more, Talk less!!!! THIS! Stop talking so much. I remind teachers of this with their students and I think it is a great reminder for us as mentors.

I love using video but so many people stuggle with this tool. Finding ways to make it fun is important and such a valuable tool

He remains present in the everyday world, in the earth beneath us.

The power of words.

Nature seems to dare him; playful challenge from the world.

Metaphor for universal connection and order in nature.

Indicates innocence or lack of awareness; the young men are passive or unconcerned with the observer.

The house represents social status and separation from nature.

Transpire = emerge or rise from.

Grass grows from the bodies of the dead.

Old terms for different ethnic and social groups: 1. Kanuck: Canadian 2. Tuckahoe: A term for people from the American South 3. Cuff: A racist old term for a Black man The point is that the grass grows the same for all.

A universal symbol; grass reveals the same truth everywhere, regardless of race or class.

A metaphor, grass as a divine token or reminder (“remembrancer”), as if left by God with His “name in the corners.”

Emphasizes the poet’s connection to the American land; a theme of belonging.

Throughput is easier to increase than raw speed/perf

after each signal phase you have to provide less and less information about the author/writer.

the

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

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

In this manuscript, the authors employed fast MAS NMR spectroscopy to investigate the gel aggregation of longer repeat (48×) RNAs, revealing inherent folding structures and interactions (i.e., G-quadruplex and duplex). The dynamic structure of the RNA gel was not resolved at high resolution, and only the structural features-namely, the coexistence of G-quadruplexes and duplexes-were inferred. The 1D and 2D NMR spectra were not assigned to specific atomic positions within the RNA, which makes it difficult to perform molecular dynamics (MD) modeling to elucidate the dynamic nature of the RNA gel. The following comments are provided for the authors' consideration:

Reviewer #1, Comment 1:

Figure 2E and Figure 3A: The data suggest that Ca²⁺ promotes stronger G-quadruplex formation within the RNA gel compared with Mg²⁺. This observation is somewhat puzzling, as Mg²⁺ is generally known to stabilize G-quadruplex structures. The authors should clarify this discrepancy.

__Response: __Mg2+ is also a stabilizer of double-stranded RNA. In most cases, Mg²⁺ stabilizes RNA duplexes more significantly than it stabilizes G-quadruplexes. When Mg2+ is removed and replaced for Ca2+, RNA duplex is destabilized more than G4 structures. We have added a clarification regarding that to the Conclusions section.

Reviewer #1, Comment 2:

Figures 2 and 3: The authors use the chemical shift at δN 144.1 ppm to distinguish between G-quadruplex and duplex structures. How was the reliability of this assignment evaluated? Chemical shifts of RNA atoms can be influenced by various factors such as intermolecular interactions, conformational stress, and local chemical environment, not only by higher-order structures. This point should be substantiated by citing relevant references or by analyzing additional RNA structures exhibiting δN 144.1 ppm signals using NMR spectroscopy.

Response: The assignment was made by comparing the chemical shifts with published data and by comparing the obtained spectra with existing datasets in the lab. We have added an explanation to the Results section and cited the literature. The 144.1 ppm was an illustrative value selected for guiding the discussion and we noted that it could sound too specific. We modified Figure 2 to outline the regions of chemical shifts in accordance with our interpretation of spectra.

Reviewer #1, Comment 3:

The authors state that "Our findings demonstrate that fast MAS NMR spectroscopy enables atomic-resolution monitoring of structural changes in GGGGCC repeat RNA of physiological lengths." This claim appears overstated, as no molecular model was constructed to define atomic coordinates based on NMR restraints.

Response: We agree and we have rewritten the conclusions to be more precise in wording. The new text does not mention “atomic-resolution” anymore.

Reviewer #1, Comment 4: Figure 3B: The experiment using nuclear extracts supplemented with Mg²⁺ to study RNA aggregation via 2D NMR may not accurately reflect intracellular conditions. It would be informative to perform a parallel experiment using nuclear extracts without additional Mg²⁺ to better simulate the native environment for RNA folding.

__Response: __We agree that we have not yet approached physiological conditions and that it would be interesting to obtain data for conditions at physiological Mg2+ concentrations in the range between 0.5 mM – 1 mM. The buffer of purchased nuclear extracts does not contain MgCl2, so some MgCl2 would still need to be added. In our opinion, nuclear extracts are actually not the optimal way to move forward, since they still differ from real in cell environment with the caveat that their composition is not well controlled. Full reconstitution with recombinant proteins might be a better approach because stoichiometry can be better regulated.

__Reviewer #1 (Significance (Required)): __ In this manuscript, the authors employed fast MAS NMR spectroscopy to investigate the gel aggregation of longer repeat (48×) RNAs, revealing inherent folding structures and interactions (i.e., G-quadruplex and duplex). The dynamic structure of the RNA gel was not resolved at high resolution, and only the structural features-namely, the coexistence of G-quadruplexes and duplexes-were inferred. The 1D and 2D NMR spectra were not assigned to specific atomic positions within the RNA, which makes it difficult to perform molecular dynamics (MD) modeling to elucidate the dynamic nature of the RNA gel.

Response: We agree that constraints for molecular dynamics cannot be derived from these data. The focus of this work is methodological: to demonstrate how 1H-15N 2D correlation spectra can be used to characterize G-G pairing in RNA gels directly. Such spectra could be used to study effects of small molecules or interacting proteins for example.

__Reviewer #2 (Evidence, reproducibility and clarity (Required)): __ The manuscript by Kragelj et al. has the potential to become a valuable study demonstrating the role and power of modern solid-state NMR spectroscopy in investigating molecular assemblies that are otherwise inaccessible to other structural biology techniques. However, due to poor experimental execution and incomplete data interpretation, the manuscript requires substantial revision before it can be considered for publication in any journal.

__Reviewer #2, Major Concern __Inspection of the analytical gels of the transcribed RNA clearly shows that the desired RNA product constitutes only about 10% of the total crude transcript. The RNA must therefore be purified, for example by preparative PAGE, before performing any NMR or other biophysical studies. As it stands, all spectra shown in the figures represent a combined signal of all products in the crude mixture rather than the intended 48 repeat RNA. Consequently, all analyses and conclusions currently refer to a heterogeneous mixture of transcripts rather than the specific target RNA.

Response: The estimate of 10% 48xG4C2 on the gel is an overstatement. While multiple bands are visible, they correspond to dimers or multimers of the 48xG4C2 RNA. Transcripts that are longer than 48xG4C2 cannot occur in our transcription conditions. Bands at lower masses than expected are folded RNA. The high repeat length and the presence of Mg²⁺ during transcription promote multimerization, which is not fully reversed by denaturation in urea. If shorter transcripts had arisen from early termination they would be still substantially longer than 24 repeats based of what is visible on the gel and would thus remain within the pathological length range. Therefore, the observed NMR spectra primarily report on 48 repeat lengths.

__Reviewer #2, Specific Comments 1: __The statements: "We show that a technique called NMR spectroscopy under fast Magic Angle Spinning (fast MAS NMR) can be used to obtain structural information on GGGGCC repeat RNAs of physiological lengths. Fast MAS NMR can be used to obtain structural information on biomolecules regardless of their size." on page 1 are not entirely correct. Firstly, not only fast MAS NMR but MAS NMR in general can provide structural information on biomolecules regardless of their size. Fast MAS primarily allows for ¹H-detected experiments, improves spectral resolution, and reduces the required sample amount. Conventional ¹³C-detected solid-state MAS NMR can provide very similar structural information. A more thorough review of relevant literature could help address this issue.

Response: We have clarified the distinction between MAS NMR and Fast MAS NMR in the introduction.

__Reviewer #2, Specific Comments 2: __Secondly, MAS NMR has already been applied to systems of comparable complexity - for instance, the (CUG)₉₇ repeat studied by the Goerlach group as early as 2005. That work provided a comprehensive structural characterization of a similar molecular assembly. The authors are strongly encouraged to cite these studies (e.g., Riedel et al., J. Biomol. NMR, 2005; Riedel et al., Angew. Chem., 2006).

Response: We added a mention of that study in the introduction.

Reviewer #2, Experimental Description 1: The experimental details are poorly documented and need to be described in sufficient detail for reproducibility. Specifically: 1. What was the transcription scale? What was the yield (e.g., xx mg RNA per 1 mL transcription reaction)?

Response: Between 3.5 mg and 4.5 mg per 10 ml transcription reaction. We’ve added this information to the methods.

Reviewer #2, Experimental Description 2: 2. Why was the transcription product not purified? Dialysis only removes small molecules, while all macromolecular impurities above the cutoff remain. What was the dialysis cutoff used?

Response: RNA was purified using dialysis and phenol-chloroform precipitation. We have added the information about molecular weight cutoff for dialysis membranes to the methods.

Reviewer #2, Experimental Description 3: 3. How much RNA was used for each precipitation experiment? Were the amounts normalized? For example, if 10 mg of pellet were obtained, what fraction of that mass corresponded to RNA? Was this ratio consistent across all samples?

Response: In the test gel formations, we used 180.0 µg per condition. We used 108.0 µg of RNA for gelation test in the presence of nuclear extracts. We have not determined the water content in the gels. We added this information to methods and results section.

Reviewer #2, Experimental Description 4: 4. Why is there a smaller amount of precipitate when nuclear extract (NE) or CaCl₂ is added?

Response: The apparent difference in pellet size may reflect variations in water content rather than RNA quantity. While the Figure 1 might entice to directly compare pellet weights across different ion series tests, our primary goal was to determine the minimal divalent-ion concentrations required to reproducibly obtain gels. We have added a clarification in the Results section and in the Figure 1 caption regarding the comparability of conditions

Reviewer #2, Experimental Description 5: 5. The authors should describe NE addition in more detail: What is the composition of NE? What buffer was used (particularly Mg²⁺ and salt concentrations)? Was a control performed with NE buffer-type alone (without NE)?

Response: We have added the full description of NE buffer to the methods section. Its composition is: 40 mM Tris pH 8.0, 100 mM KCl, 0.2 mM EDTA, 0.5 mM PMSF, 0.5 mM DTT, 25 % glycerol. After mixing the nuclear extract with RNA, the target buffer was: 20 mM Tris pH 8.0, 90 mM KCl, 0.1 mM EDTA, 0.25 mM PMSF, 0.75 mM DTT, 12.5% glycerol, and 10 mM MgCl2.

We have not performed a control with NE buffer-type alone but we confirmed separately that glycerol does not affect gel formation.

Reviewer #2, Experimental Description 6: 6. How much pellet/RNA material was actually packed into each MAS rotor?

Response: Starting with a 5 mg pellet, we packed a rotor with a volume of 3 µl. We added this information to the methods section.

Reviewer #2, Additional Clarifications: P5. What is meant by "selective" in the phrase "We recorded a selective 1D-¹H MAS NMR spectrum of 48×G₄C₂ RNA gels"?

Response: That was a typo. We meant imino-selective. It is now corrected.

__Reviewer #2, Additional Clarifications: __ There are also several contradictions between statements in the text and the corresponding figures. For example: • Page 4: The authors write that "The addition of at least 5 mM Mg²⁺ was required for significant 48×G₄C₂ aggregation." However, Figure 1E shows significant aggregation already at 3 mM MgCl₂ (NE−), and in samples containing NE, aggregation appears even at 1 mM MgCl₂. Was aggregation already present in the sample containing NE but without any added MgCl₂?

Response: We changed text in the results section to more closely align with what’s depicted on the figure. There was some aggregation present in the nuclear extracts but it was of different quantity and quality. We clarified this in the results section.

__Reviewer #2 (Significance (Required)): __ The manuscript by Kragelj et al. has the potential to become a valuable study demonstrating the role and power of modern solid-state NMR spectroscopy in investigating molecular assemblies that are otherwise inaccessible to other structural biology techniques.

In its current form, tthe manuscript has significant experimental concerns - particularly the lack of RNA purification and inadequate description of materials and methods. The data therefore cannot support the conclusions presented. I recommend extensive revision and repetition of the experiments using purified RNA material before further consideration for publication.

__Response: __We’ve addressed the concerns about RNA purification within the response to the first comment (Major concern).

__Reviewer #3 (Evidence, reproducibility and clarity (Required)): __ This is an interesting manuscript reporting evidence for formation of both hairpins and G-quadruplexes within RNA aggregates formed by ALS expansion repeats (GGGGCC)n. This is in line with literature but never directly confirmed. Given the novelty of the method (NMR magic angle) and of the data (NMR on aggregate), I believe this manuscript should be considered for publication. I also trust the methods are appropriately reported and reproducible.

Below are my main points:

Major points:

__Reviewer #3, Comment 1: __ 1) RNA aggregation of the GGGGCCn repeat has been reported for expansion as short as 6-8 repeats (see Raguseo et al. Nat Commun 2023), so the authors might not see aggregation under the conditions they use for these shorter repeats but this can happen under physiological conditions . The ionic strengths and the conditions used can vary heavily the phase diagram and the authors therefore should tone down significantly their conclusions. They characterise one aggregate that is likely to contain both secondary structures under the conditions used (in terms of ion and pHs). However, it has been shown in Raguseo et al that aggregates can arise by both intermolecular G4s and hairpins (or a mixture of them) depending on the ionic conditions used. This means that what the authors report might not be necessarily relevant in cells, which should be caveated in the manuscript.

__Response: __We toned down our statements regarding aggregation of shorter repeats in the introduction. We added the citation to Raguseo et al. Nat Commun 2023, which indeed provides useful insights about aggregation of GGGGCC repeats. In Supplementary Figure 1, we had data on gel formation with 8x and 24x repeats which showed these repeat lengths form gels to some extent. We oversimplified our conclusion and said there were no aggregates which needs correction, especially considering other studies reported in the literature have observed in vitro aggregation of these repeat lengths. We modified the results section to reflect this nuance.

__Reviewer #3, Comment 2: __ 2) It would be important to perform perturbation experiments that might promote/disrupt formation of the G4 or hairpin and see if this affect RNA aggregation, which has been already reported by Raguseo et al, and wether this can be appreciated spectroscopically in their assay. This can be done by taking advantage of some of the experiments reported in the manuscript mentioned above, such as: PDS treatment (favouring monomolecular G4s and preventing aggregation), Li vs K treatment (favouring hairpin over G4s), NMM photo-oxidation (disassembling G4s) or addition of ALS relevant RNA binding proteins (i.e. TDP-43). Not all of these controls need to be performed but it would be good to reconcile how the fraction of G4 vs hairpin reflect aggregates' properties, since the authors offer such a nice technique to measure this.

Response: We appreciate the reviewer’s suggestions and we would be eager to do the perturbation experiments in the future. However, these experiments would require additional optimization and waiting for approval and availability of measurement time on a high-field NMR spectrometer. Given that the primary goal of this manuscript is reporting on the methodological approach, we think the current data adequately demonstrate the technique’s utility.

__Reviewer #3, Comment 3: __ 3) I disagree with the speculation of the monomolecular G4 being formed within the condensates, as the authors have no evidence to support this. It has been shown that n=8 repeat forms multimolecular G4s that are responsible of aggregation, so the authors need to provide direct evidence to support this hypothesis if they want to keep it in the manuscript, as it would clash with previous reports (Raguseo et al Nat Commun 2023)

Response: We agree that multimolecular G4s contribute to aggregation in our 48xG4C2 gels. We also realized, after reading this comment, that the original presentation of data and schematics may have unintentionally suggested the presence of monomolecular G4 in our RNA gels. To address this, we have added a clarification to the results section, we modified Figure 2 and 3, and we included a new Supplementary Figure 4. For clarification, both multimolecular and monomolecular G4s in model oligonucleotides produce imino 1H and 15N chemical shifts in the same region and cannot be distinguished by the experiments used in our study. Based on the observations reported in the literature, we believe that G4s in 48xG4C2 form primarily intermolecularly, although direct experimental proof is not available with the present data.

Minor points:

__Reviewer #3, Comment 4: __ 4) An obvious omission in the literature is Raguseo et al Nat Commun 2023, extensively mentioned above. Given the relevance of the findings reported in this manuscript for this study, this should be appropriately referenced for clarity.

Response: We’ve added the citation to Raguseo et al Nat Commun 2023 to the introduction where in vitro aggregation is discussed.

__Reviewer #3, Comment 5: __ 5) The schematic in Figure 3 is somehow confusing and the structures reported and how they relate to aggregate formation is not clear. Given that in structural studies presentation and appearance is everything, I would strongly recommend to the authors to improve the clarity of the schematic for the benefit of the readers.

Response: We thank you for your comment. We’ve modified the figure, and we hope it is now clearer.

Providing that the authors can address the criticisms raised, I would be supportive of publication of this fine study.

Reviewer #3 (Significance (Required)):

The main strength of this paper is to provide direct evidence of DNA secondary structure formation within aggregates, which is something that has not been done before. This is important as it reconcile with the relevance of hairpin formation for the disease (reported by Disney and co-workers) and the relevance of G4-formation in the process of aggregation through multimolecular G4-formation (reported by Di Antonio and co-workers). Given the significance of the findings in this context and the novelty of the method applied to the study of RNA aggregation, this reviewer is supportive for publication of this manuscript and of its relevance to the field. I would be, however, more careful in the conclusions reported and would add additional controls to strengthen the conclusions.

Response: We thank the reviewer for the comment. In the conclusion section, we have added a statement highlighting the potential roles of both double-stranded and G4 structures in gel formation, in line with what has been reported in previous studies.

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

This is an interesting manuscript reporting evidence for formation of both hairpins and G-quadruplexes within RNA aggregates formed by ALS expansion repeats (GGGGCC)n. This is in line with literature but never directly confirmed. Given the novelty of the method (NMR magic angle) and of the data (NMR on aggregate), I believe this manuscript should be considered for publication. I also trust the methods are appropriately reported and reproducible.

Below are my main points:

Major points:

1) RNA aggregation of the GGGGCCn repeat has been reported for expansion as short as 6-8 repeats (see Raguseo et al. Nat Commun 2023), so the authors might not see aggregation under the conditions they use for these shorter repeats but this can happen under physiological conditions . The ionic strengths and the conditions used can vary heavily the phase diagram and the authors therefore should tone down significantly their conclusions. They characterise one aggregate that is likely to contain both secondary structures under the conditions used (in terms of ion and pHs). However, it has been shown in Raguseo et al that aggregates can arise by both intermolecular G4s and hairpins (or a mixture of them) depending on the ionic conditions used. This means that what the authors report might not be necessarily relevant in cells, which should be caveated in the manuscript.

2) It would be important to perform perturbation experiments that might promote/disrupt formation of the G4 or hairpin and see if this affect RNA aggregation, which has been already reported by Raguseo et al, and wether this can be appreciated spectroscopically in their assay. This can be done by taking advantage of some of the experiments reported in the manuscript mentioned above, such as: PDS treatment (favouring monomolecular G4s and preventing aggregation), Li vs K treatment (favouring hairpin over G4s), NMM photo-oxidation (disassembling G4s) or addition of ALS relevant RNA binding proteins (i.e. TDP-43). Not all of these controls need to be performed but it would be good to reconcile how the fraction of G4 vs hairpin reflect aggregates' properties, since the authors offer such a nice technique to measure this.

3) I disagree with the speculation of the monomolecular G4 being formed within the condensates, as the authors have no evidence to support this. It has been shown that n=8 repeat forms multimolecular G4s that are responsible of aggregation, so the authors need to provide direct evidence to support this hypothesis if they want to keep it in the manuscript, as it would clash with previous reports (Raguseo et al Nat Commun 2023)

Minor points:

4) An obvious omission in the literature is Raguseo et al Nat Commun 2023, extensively mentioned above. Given the relevance of the findings reported in this manuscript for this study, this should be appropriately referenced for clarity.

5) The schematic in Figure 3 is somehow confusing and the structures reported and how they relate to aggregate formation is not clear. Given that in structural studies presentation and appearance is everything, I would strongly recommend to the authors to improve the clarity of the schematic for the benefit of the readers.

Providing that the authors can address the criticisms raised, I would be supportive of publication of this fine study.

Significance

The main strength of this paper is to provide direct evidence of DNA secondary structure formation within aggregates, which is something that has not been done before. This is important as it reconcile with the relevance of hairpin formation for the disease (reported by Disney and co-workers) and the relevance of G4-formation in the process of aggregation through multimolecular G4-formation (reported by Di Antonio and co-workers). Given the significance of the findings in this context and the novelty of the method applied to the study of RNA aggregation, this reviewer is supportive for publication of this manuscript and of its relevance to the field. I would be, however, more careful in the conclusions reported and would add additional controls to strengthen the conclusions.

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

The manuscript by Kragelj et al. has the potential to become a valuable study demonstrating the role and power of modern solid-state NMR spectroscopy in investigating molecular assemblies that are otherwise inaccessible to other structural biology techniques. However, due to poor experimental execution and incomplete data interpretation, the manuscript requires substantial revision before it can be considered for publication in any journal.

Major Concern

Inspection of the analytical gels of the transcribed RNA clearly shows that the desired RNA product constitutes only about 10% of the total crude transcript. The RNA must therefore be purified, for example by preparative PAGE, before performing any NMR or other biophysical studies. As it stands, all spectra shown in the figures represent a combined signal of all products in the crude mixture rather than the intended 48× repeat RNA. Consequently, all analyses and conclusions currently refer to a heterogeneous mixture of transcripts rather than the specific target RNA.

Specific Comments

The statements: "We show that a technique called NMR spectroscopy under fast Magic Angle Spinning (fast MAS NMR) can be used to obtain structural information on GGGGCC repeat RNAs of physiological lengths. Fast MAS NMR can be used to obtain structural information on biomolecules regardless of their size." on page 1 are not entirely correct. Firstly, not only fast MAS NMR but MAS NMR in general can provide structural information on biomolecules regardless of their size. Fast MAS primarily allows for ¹H-detected experiments, improves spectral resolution, and reduces the required sample amount. Conventional ¹³C-detected solid-state MAS NMR can provide very similar structural information. A more thorough review of relevant literature could help address this issue. Secondly, MAS NMR has already been applied to systems of comparable complexity - for instance, the (CUG)₉₇ repeat studied by the Goerlach group as early as 2005. That work provided a comprehensive structural characterization of a similar molecular assembly. The authors are strongly encouraged to cite these studies (e.g., Riedel et al., J. Biomol. NMR, 2005; Riedel et al., Angew. Chem., 2006).

Experimental Description

The experimental details are poorly documented and need to be described in sufficient detail for reproducibility. Specifically:

1. What was the transcription scale? What was the yield (e.g., xx mg RNA per 1 mL transcription reaction)?
2. Why was the transcription product not purified? Dialysis only removes small molecules, while all macromolecular impurities above the cutoff remain. What was the dialysis cutoff used?
3. How much RNA was used for each precipitation experiment? Were the amounts normalized? For example, if 10 mg of pellet were obtained, what fraction of that mass corresponded to RNA? Was this ratio consistent across all samples?
4. Why is there a smaller amount of precipitate when nuclear extract (NE) or CaCl₂ is added?
5. The authors should describe NE addition in more detail: What is the composition of NE? What buffer was used (particularly Mg²⁺ and salt concentrations)? Was a control performed with NE buffer-type alone (without NE)?

6. How much pellet/RNA material was actually packed into each MAS rotor? Additional Clarifications P5. What is meant by "selective" in the phrase "We recorded a selective 1D-¹H MAS NMR spectrum of 48×G₄C₂ RNA gels"? There are also several contradictions between statements in the text and the corresponding figures. For example:

7. Page 4: The authors write that "The addition of at least 5 mM Mg²⁺ was required for significant 48×G₄C₂ aggregation." However, Figure 1E shows significant aggregation already at 3 mM MgCl₂ (NE−), and in samples containing NE, aggregation appears even at 1 mM MgCl₂. Was aggregation already present in the sample containing NE but without any added MgCl₂?

Significance

The manuscript by Kragelj et al. has the potential to become a valuable study demonstrating the role and power of modern solid-state NMR spectroscopy in investigating molecular assemblies that are otherwise inaccessible to other structural biology techniques.

In its current form, tthe manuscript has significant experimental concerns - particularly the lack of RNA purification and inadequate description of materials and methods. The data therefore cannot support the conclusions presented. I recommend extensive revision and repetition of the experiments using purified RNA material before further consideration for publication.

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

In this manuscript, the authors employed fast MAS NMR spectroscopy to investigate the gel aggregation of longer repeat (48×) RNAs, revealing inherent folding structures and interactions (i.e., G-quadruplex and duplex).

The dynamic structure of the RNA gel was not resolved at high resolution, and only the structural features-namely, the coexistence of G-quadruplexes and duplexes-were inferred. The 1D and 2D NMR spectra were not assigned to specific atomic positions within the RNA, which makes it difficult to perform molecular dynamics (MD) modeling to elucidate the dynamic nature of the RNA gel. The following comments are provided for the authors' consideration:

1. Figure 2E and Figure 3A: The data suggest that Ca²⁺ promotes stronger G-quadruplex formation within the RNA gel compared with Mg²⁺. This observation is somewhat puzzling, as Mg²⁺ is generally known to stabilize G-quadruplex structures. The authors should clarify this discrepancy.
2. Figures 2 and 3: The authors use the chemical shift at δN 144.1 ppm to distinguish between G-quadruplex and duplex structures. How was the reliability of this assignment evaluated? Chemical shifts of RNA atoms can be influenced by various factors such as intermolecular interactions, conformational stress, and local chemical environment, not only by higher-order structures. This point should be substantiated by citing relevant references or by analyzing additional RNA structures exhibiting δN 144.1 ppm signals using NMR spectroscopy.
3. The authors state that "Our findings demonstrate that fast MAS NMR spectroscopy enables atomic-resolution monitoring of structural changes in GGGGCC repeat RNA of physiological lengths." This claim appears overstated, as no molecular model was constructed to define atomic coordinates based on NMR restraints.
4. Figure 3B: The experiment using nuclear extracts supplemented with Mg²⁺ to study RNA aggregation via 2D NMR may not accurately reflect intracellular conditions. It would be informative to perform a parallel experiment using nuclear extracts without additional Mg²⁺ to better simulate the native environment for RNA folding.

Significance

In this manuscript, the authors employed fast MAS NMR spectroscopy to investigate the gel aggregation of longer repeat (48×) RNAs, revealing inherent folding structures and interactions (i.e., G-quadruplex and duplex).

The dynamic structure of the RNA gel was not resolved at high resolution, and only the structural features-namely, the coexistence of G-quadruplexes and duplexes-were inferred. The 1D and 2D NMR spectra were not assigned to specific atomic positions within the RNA, which makes it difficult to perform molecular dynamics (MD) modeling to elucidate the dynamic nature of the RNA gel.

Bind parameters allow for different values while still showing as the same statement.

The query optimizer can't reason about what the output of the function may be.

I can include Garland-Thomson on how to be disabled: Most people do not know it!

this is an annotation

• Linux desktop market share has grown from 1.5% in 2020 to over 4% globally in 2024 and exceeds 5% in the US by 2025; including ChromeOS pushes it above 11%.
• Key drivers include Windows 10 end-of-support, Windows 11's unpopular changes (e.g., AI integration, interface shifts), and Zorin OS seeing 78% downloads from Windows users.
• Additional factors: better gaming via Steam/Proton, improved distro usability, hardware compatibility, privacy concerns, and EU digital sovereignty pushing governments to Linux alternatives like EU OS.
• Broader Linux dominance: Android (Linux-based) holds 72.55% global mobile share; US gov sites see 23% Linux traffic including Android/ChromeOS.

Hacker News Discussion

• Top comment praises KDE Plasma on Fedora over GNOME for Windows-like features, configurability (window rules, settings panel), and dev workflow superiority to WSL; prefers AMD GPUs.
• Distro recs: Pop!_OS for simplicity/accessibility, Kubuntu LTS for longevity (e.g., 5+ years on old hardware), atomic Fedora Kinoite for rollbacks.
• Pain points: Video editing lags (DaVinci Resolve codec issues, Kdenlive text woes), hardware glitches (Nvidia, fingerprints), weaker file pickers/accessibility vs Windows/macOS.
• Debates: CLI tools suffice for some; toxicity accusations fly; Linux dev admits accessibility gaps; Windows' telemetry/AI drives switches; market share undercounted by adblockers.

• CLAUDE.md is a special onboarding file to familiarize Claude (an AI code assistant) with your codebase.
• It should clearly outline the WHY (purpose of the project), WHAT (tech stack, project structure, key components), and HOW (development process, running tests, build commands) for Claude.
• The file helps Claude understand your monorepo or multi-application project and know where to look for things without flooding it with unnecessary details.
• Keep CLAUDE.md concise and focused; ideally, it should be under 300 lines, with many recommending less than 60 lines for clarity and relevance.
• Use progressive disclosure: point Claude to where to find further information rather than including all details upfront, avoiding overwhelming the model’s context window.
• Complement CLAUDE.md with tools like linters, code formatters, hooks, and slash commands to separate concerns like implementation and formatting.
• CLAUDE.md is a powerful leverage point for getting better coding assistance but must be carefully crafted—not auto-generated.
• The file should include core commands, environment setup, guidelines, and unexpected behaviors relevant to the repository.
• Encouraging Claude to selectively read or confirm files before use can help maintain focus during sessions.

Hacker News Discussion

• Users emphasized the benefit of explicit instruction patterns like "This is what I'm doing, this is what I expect," which improves monitoring and recovery from errors.
• Some commenters felt these markdown files had marginal gains and that model quality mattered more than the presence of CLAUDE.md.
• A few highlighted the importance of writing documentation primarily for humans rather than solely for LLMs.
• Discussion included anticipation of more stateful LLMs with better memory, which would impact how such onboarding files evolve.
• Recommendations included hierarchical or recursive context structures in CLAUDE.md for large projects, allowing a root file plus targeted sub-files.
• Comments supported having Claude address the user specifically to verify it is following instructions properly.
• Some users noted improvements in model adherence compared to past versions, making CLAUDE.md files more effective now.
• Practical tips were shared for managing large monorepos and integrating CLAUDE.md with version control status.

[INT] The new lawyer who is now renting the narrator's old Wall Street offices confirms that Bartleby is still on the premises, refusing any copying.

I appreciate the depth of research and clarity in your writing. rocket fortress

When I looked at the “Free Market” source in the bibliography, what surprised me was how neutral the Wikipedia definition sounded compared to how the term is used in real debates. The page describes the free market almost like a clean, ideal system where supply and demand naturally balance. But reading it after the chapter made me notice the gap between that ideal and the way companies like Meta actually operate. In theory, free markets are supposed to create competition and benefit consumers, but in reality we often see the opposite—big platforms trying to reduce competition and lock users in. It made me wonder whether the “free market” idea still makes sense in industries where network effects basically guarantee that only one or two companies will dominate.

Doctorow’s idea of enshittification—that platforms inevitably degrade as they shift value first toward users, then toward business partners, and finally toward extracting maximum profit for themselves—really stuck with me. It feels like an unusually clear description of how many social-media sites evolve over time.

test

do.how-innotation as incantation

If you can dream up and elaborate in digital form, in an info-morphic indyweb page, your tacit intent,

what you would like to experience to happen in a named context using using the tralmarks in line intentionally transparent notation that allows one to write to think and articulate yet to be real-ized digital experiences that can transmute formulative thinking, the butterflies into caterpillars of intentional formulations that progressively able to come to a lively digital experiences lively butterflies of digitial fwork flows to engage with and experience to realize yOur intent

name it, elaborate the implicate context, formulate eht you had in mind, write to think it through to the last detailed structures and inerpretation that would be required to accomplish what you had in mind do it progressively till it fully formed explicitly, spell it out, when you complete that you have the spell to invoke it

a practical magic incantation capable of invoking the named capability, process morphic transformation presentation to be expierice, to deliver what you spelled out, need whenever you need ot for your purposes

I was closiing stale windows. and one about do.how to add favicons

I used the notation '>do.how - favicon'

then did search for annotations that match that trailmark

and low behold I have a way of realling all my annotations marked as >do-how

Geat demonstration of the power of notation and its systematic use!

a way to leverage hypothesis as a platform for solcial netowrorked search/serendioity engine

All 1 needs is to adopt the notation! for it.

Yet another exemplar how Civilization can advance through notation

Reading about how Meta increases profit by getting more users, more user time, and more personal data makes me think about how invisible these incentives are in my own daily social-media habits. I notice that whenever I open Instagram “just for one minute,” the platform always manages to keep me scrolling. After learning that this isn’t accidental—but a direct way to maximize ad revenue—I feel more critical of the design decisions that shape my behavior. It makes me wonder: if a company’s fiduciary duty is to maximize profits, are they ever able to prioritize user well-being over engagement? Or is the system designed so that our attention will always be treated as the product rather than something that deserves protection?

Ezek helyes címei lásd fent

eLife Assessment

This is a valuable study of the physiological mechanisms promoting network activity during fever in the mouse neocortex. The supporting evidence is solid, and has improved with revision, along with increased clarity of presentation.

Reviewer #1 (Public review):

The paper by Chen et al describes the role of neuronal themo-TRPV3 channels in the firing of cortical neurons at fever temperature range. The authors began by demonstrating that exposure to infrared light increasing ambient temperature causes body temperature rise to fever level above 38{degree sign}C. Subsequently, they showed that at the fever temperature of 39{degree sign}C, the increased spike threshold (ST) increased in both populations (P12-14 and P7-8) of cortical excitatory pyramidal neurons (PNs). However, the spike number only decreased in P7-8 PNs, while it remained stable in P12-14 PNs at 39{degree sign}C. In addition, the fever temperature also reduced the late peak postsynaptic potential (PSP) in P12-14 PNs. The authors further characterized the firing properties of cortical P12-14 PNs, identifying two types: STAY PNs that retained spiking at 30{degree sign}C, 36{degree sign}C and 39{degree sign}C, and STOP PNs that stopped spiking upon temperature change. They further extended their and analysis and characterization to striatal medium spiny neurons (MSNs) and found that STAY MSNs and PNs shared same ST temperature sensitivity. Using small molecule tools, they further identified that themo-TRPV3 currents in cortical PNs increased in response to temperature elevation, but not TRPV4 currents. The authors concluded that during fever, neuronal firing stability is largely maintained by sensory STAY PNs and MSNs that express functional TRPV3 channels. Overall, this study is well designed and executed with substantial controls, some interesting findings and quality of data.

Comments on revisions:

My previous concerns have been addressed in this revised manuscript.

Reviewer #2 (Public review):

Summary:

The authors studied the excitability of layer 2/3 pyramidal neurons in response to layer four stimulation at temperatures ranging from 30 to 39{degree sign}C in P7-8, P12-P14, and P22-P24 animals. They also measure brain temperature and spiking in vivo in response to externally applied heat. Some pyramidal neurons continue to fire action potentials in response to stimulation at 39{degree sign}C and are referred to as "stay neurons." Stay neurons have unique properties, aided by the expression of the TRPV3 channel.

Strengths:

The authors focused on layer 2/3 neuronal excitability at three developmental stages: during the window of susceptibility to febrile seizures, before the window opens, and after it closes.

Electrophysiological experiments are rigorously performed and carefully interpreted.

The cellular electrophysiology is further confirmed. The authors compared the seizure susceptibility of TRPV3 knockout, heterozygous, and wild-type mice. EEG recording would have strengthened the study, but they are challenging in this age group.

Finally, the authors studied TRPV3 expression with immunohistochemistry.

Reviewer #3 (Public review):

Summary:

This important study combines in vitro and in vivo recording to determine how the firing of cortical and striatal neurons changes during a fever range temperature rise (37-40 oC). The authors found that certain neurons will start, stop, or maintain firing during these body temperature changes. The authors further suggested that the TRPV3 channel plays a role in maintaining cortical activity during fever.

Strengths:

The topic of how the firing pattern of neurons changes during fever is unique and interesting. The authors carefully used in vitro electrophysiology assays to study this interesting topic.

Weaknesses:

(1) In vivo recording is a strength of this study. However, data from in vivo recording is only shown in Fig 5A,B. This reviewer suggests the authors further expand on the analysis of the in vivo Neuropixels recording. For example, to show single spike waveforms and raster plots to provide more information on the recording. The authors can also separate the recording based on brain regions (cortex vs striatum) using the depth of the probe as a landmark to study the specific firing of cortical neurons and striatal neurons. It is also possible to use published parameters to separate the recording based on spike waveform to identify regular principal neurons vs fast-spiking interneurons. Since the authors studied E/I balance in brain slices, it would be very interesting to see whether the "E/I balance" based on the firing of excitatory neurons vs fast-spiking interneurons might be changed or not in the in vivo condition.

(2) The author should propose a potential mechanism for how TRPV3 helps to maintain cortical activity during fever. Would calcium influx-mediated change of membrane potential be the possible reason? Making a summary figure to put all the findings into perspective and propose a possible mechanism would also be appreciated.

(3) The author studied P7-8, P12-14, and P20-26 mice. How do these ages correspond to the human ages? it would be nice to provide a comparison to help the reader understand the context better.

Comments on revisions:

In this revised version, the authors nicely addressed my critiques. I have no more comments to make.

Author response:

The following is the authors’ response to the original reviews

Public Reviews:

Reviewer #1 (Public review):

The paper by Chen et al describes the role of neuronal themo-TRPV3 channels in the firing of cortical neurons at a fever temperature range. The authors began by demonstrating that exposure to infrared light increasing ambient temperature causes body temperature to rise to a fever level above 38{degree sign}C. Subsequently, they showed that at the fever temperature of 39{degree sign}C, the spike threshold (ST) increased in both populations (P12-14 and P7-8) of cortical excitatory pyramidal neurons (PNs). However, the spike number only decreased in P7-8 PNs, while it remained stable in P12-14 PNs at 39 degrees centigrade. In addition, the fever temperature also reduced the late peak postsynaptic potential (PSP) in P12-14 PNs. The authors further characterized the firing properties of cortical P12-14 PNs, identifying two types: STAY PNs that retained spiking at 30{degree sign}C, 36{degree sign}C, and 39{degree sign}C, and STOP PNs that stopped spiking upon temperature change. They further extended their analysis and characterization to striatal medium spiny neurons (MSNs) and found that STAY MSNs and PNs shared the same ST temperature sensitivity. Using small molecule tools, they further identified that themo-TRPV3 currents in cortical PNs increased in response to temperature elevation, but not TRPV4 currents. The authors concluded that during fever, neuronal firing stability is largely maintained by sensory STAY PNs and MSNs that express functional TRPV3 channels. Overall, this study is well designed and executed with substantial controls, some interesting findings, and quality of data. Here are some specific comments:

(1) Could the authors discuss, or is there any evidence of, changes in TRPV3 expression levels in the brain during the postnatal 1-4 week age range in mice?

This is an excellent question. To our knowledge, no published studies have documented changes in TRPV3 expression in the mouse brain during the first to fourth postnatal weeks. Research on TRPV3 expression has primarily relied on RT-PCR analysis of RNA from dissociated adult brain tissue (Jang et al., 2012; Kumar et al., 2018), largely due to the limited availability of effective antibodies for brain sections at the time. Furthermore, the Allen Brain Atlas does not provide data on TRPV3 expression in the developing or postnatal brain. To address this gap, we performed immunohistochemistry to examine TRPV3 expression at P7,

P14, and P21 (Figure 7). To confirm specificity, the TRPV3 antibody was co-incubated with a TRPV3 blocker (Figure 7A, top row, right panel). While immunohistochemistry is semiquantitative, we observed a trend toward increased TRPV3 expression in the cortex, striatum, hippocampus, and thalamus from P7 to P14.

(2) Are there any differential differences in TRPV3 expression patterns that could explain the different firing properties in response to fever temperature between the STAY- and STOP neurons?

This is another excellent question, and we plan to explore it in the future by developing reporter mice for TRPV3 expression and viral tools that leverage endogenous TRPV3 promoters to drive a fluorescent protein, enabling monitoring of cells with native TRPV3 expression. To our knowledge, such tools do not currently exist. Creating them will be challenging, as it requires identifying promoters that accurately reflect endogenous TRPV3 expression.

We have not yet quantified TRPV3 expression in STOP and STAY neurons. However, our analysis of evoked spiking at 30, 36, and 39 °C suggests that TRPV3 may mark a population of cortical pyramidal neurons that tend to remain active (“STAY”) as temperatures increase. While we have not directly compared TRPV3 expression between STAY and STOP neurons at feverrange temperatures, intracellular blockade of TRPV3 with forsythoside B (50 µM) significantly reduced the proportion of STAY neurons (Figure 9B). Consistently, spiking was also significantly reduced in Trpv3⁻/⁻ mice (Figure 10D).

In our immunohistochemical analysis, TRPV3 was detected in L4 barrels and in L2/3, where we observed a patchy distribution with some regions showing more intense staining (Figure 7B). It is possible that cells with higher TRPV3 levels correspond to STAY neurons, while those with lower levels correspond to STOP neurons. As we develop tools to monitor activity based on endogenous TRPV3 levels, we anticipate gaining deeper insight into this relationship.

(3) TRPV3 and TRPV4 can co-assemble to form heterotetrameric channels with distinct functional properties. Do STOP neurons exhibit any firing behaviors that could be attributed to the variable TRPV3/4 assembly ratio?

There is some evidence that TRPV3 and TRPV4 proteins can physically associate in HEK293 cells and native skin tissues (Hu et al., 2022).TRPV3 and TRPV4 are both expressed in the cortex (Kumar et al., 2018), but it remains unclear whether they are co-expressed and coassembled to form heteromeric channels in cortical excitatory pyramidal neurons. Examination of the I-V curve from HEK cells co-expressing TRPV3/4 heteromeric channels shows enhanced current at negative membrane potentials (Hu et al., 2022).

Currently, we cannot characterize cells as STOP or STAY and measure TRPV3 or TRPV4 currents simultaneously, as this would require different experimental setups and internal solutions. Additionally, the protocol involves a sequence of recordings at 30, 36, and 39°C, followed by cooling back to 30°C and re-heating to each temperature. Cells undergoing such a protocol will likely not survive till the end.

In our recordings of TRPV3 currents, which likely include both STOP and STAY cells, we do not observe a significant current at negative voltages, suggesting that TRPV3/4 heteromeric channels may either be absent or underrepresented, at least at a 1:1 ratio. However, the possibility that TRPV3/4 heteromeric channels could define the STOP cell population is intriguing and plausible.

(4) In Figure 7, have the authors observed an increase of TRPV3 currents in MSNs in response to temperature elevation?

We have not recorded TRPV3 currents in MSNs in response to elevated temperatures. Please note that the handling editor gave us the option to remove these data from the paper, and we elected to do so to develop them as a separate manuscript.

(5) Is there any evidence of a relationship between TRPV3 expression levels in D2+ MSNs and degeneration of dopamine-producing neurons?

This is an interesting question, though it falls outside our current research focus in the lab. A PubMed search yields no results connecting the terms TRPV3, MSNs, and degeneration. However, gain-of-function mutations in TRPV4 channel activity have been implicated in motor neuron degeneration (Sullivan et al., 2024) and axon degeneration (Woolums et al., 2020). Similarly, TRPV1 activation has been linked to developmental axon degeneration (Johnstone et al., 2019), while TRPV3 blockade has shown neuroprotective effects in models of cerebral ischemia/reperfusion injury in mice (Chen et al., 2022).

The link between TRPV activation and cell degeneration, however, may not be straightforward. For instance, TRPV1 loss has been shown to accelerate stress-induced degradation of axonal transport from retinal ganglion cells to the superior colliculus and to cause degeneration of axons in the optic nerve (Ward et al., 2014). Meanwhile, TRPV1 activation by capsaicin preserves the survival and function of nigrostriatal dopamine neurons in the MPTP mouse model of Parkinson's disease (Chung et al., 2017).

(6) Does fever range temperature alter the expressions of other neuronal Kv channels known to regulate the firing threshold?

This is an active line of investigation in our lab. The results of ongoing experiments will provide further insight into this question.

Reviewer #2 (Public review):

Summary:

The authors study the excitability of layer 2/3 pyramidal neurons in response to layer four stimulation at temperatures ranging from 30 to 39 Celsius in P7-8, P12-P14, and P22-P24 animals. They also measure brain temperature and spiking in vivo in response to externally applied heat. Some pyramidal neurons continue to fire action potentials in response to stimulation at 39 C and are called stay neurons. Stay neurons have unique properties aided by TRPV3 channel expression.

Strengths:

The authors use various techniques and assemble large amounts of data.

Weaknesses:

(1) No hyperthermia-induced seizures were recorded in the study.

The goal of this manuscript is to uncover age-related physiological changes that enable the brain to maintain function at fever-range temperatures, typically 38–40°C. Febrile seizures in humans are also typically induced within this temperature range. Given this context, we initially did not examine hyperthermia-induced seizures. However, as requested, we assessed the effects of reduced Trpv3 expression on hyperthermia-induced seizures in WT(Trpv3^+/+), heterozygous (Trpv3^+/-), and homozygous knockout (Trpv3^-/-) P12 pups. Please see figure 10.

While T_b at seizure onset and the rate of T_b increase leading to seizure were not significantly different among genotypes, the time to seizure from the point of loss of postural control (LPC), defined as collapse and failure to maintain upright posture, was significantly longer in Trpv3^+/- and Trpv3^-/- mice. Together, these results indicate that reduced TRPV3 function enhances resistance to seizure initiation and/or propagation under febrile conditions, likely by decreasing neuronal depolarization and excitability.

(2) Febrile seizures in humans are age-specific, extending from 6 months to 6 years. While translating to rodents is challenging, according to published literature (see Baram), rodents aged P11-16 experience seizures upon exposure to hyperthermia. The rationale for publishing data on P7-8 and P22-24 animals, which are outside this age window, must be clearly explained to address a potential weakness in the study.

As requested, we have added an explanation in the “Introduction” for our rationale in including age ranges that flank the period of susceptibility to hyperthermia-induced seizures (see lines 80–100). In summary, we emphasize that this design provides negative controls, allowing us to determine whether the changes observed in the P12–14 window are specific to this developmental period.

(3) Authors evoked responses from layer 4 and recorded postsynaptic potentials, which then caused action potentials in layer 2/3 neurons in the current clamp. The post-synaptic potentials are exquisitely temperature-sensitive, as the authors demonstrate in Figures 3 B and 7D. Note markedly altered decay of synaptic potentials with rising temperature in these traces. The altered decays will likely change the activation and inactivation of voltage-gated ion channels, adjusting the action potential threshold.

The activation and inactivation of voltage-gated ion channels can modulate action potential threshold. Indeed, we have identified channels that contribute to the temperature-induced increase in spike threshold, including BK channels and Scn2a. However, Figure 4B represents a cell with no inhibition at 39°C, and thus the observed loss of the late postsynaptic potential (PSP). This primarily contributes to the prolonged decay of the synaptic potentials. By contrast, cells in which inhibition is retained, when exposed to the same thermal protocol, do not exhibit such extended decay.

(4) The data weakly supports the claim that the E-I balance is unchanged at higher temperatures. Synaptic transmission is exquisitely temperature-sensitive due to the many proteins and enzymes involved. A comprehensive analysis of spontaneous synaptic current amplitude, decay, and frequency is crucial to fully understand the effects of temperature on synaptic transmission.

We did not intend to imply that E-I balance is generally unchanged at higher temperatures. Our statements specifically referred to observations in experiments conducted during the P20–26 age range in cortical pyramidal neurons. We are conducting a parallel line of investigation examining the differential susceptibility of E-I balance across age and temperature, and we have observed age- and temperature-dependent effects. Recognizing that our earlier wording may have been misleading, we have removed this statement from the manuscript.

(5) It is unclear how the temperature sensitivity of medium spiny neurons is relevant to febrile seizures. Furthermore, the most relevant neurons are hippocampal neurons since the best evidence from human and rodent studies is that febrile seizures involve the hippocampus.

Thank you for the opportunity to provide clarification. The goal of this manuscript is to uncover age-related physiological changes that enable the brain to maintain stable, non-excessive neuronal firing at fever-range temperatures (typically 38–40°C). We hypothesize that these changes are a normal part of brain development, potentially explaining why most children do not experience febrile seizures. By understanding these mechanisms, we may identify points in the process that are susceptible to dysfunction, due to genetic mutations, developmental delays, or environmental factors, which could provide insight into the rare cases when seizures occur between 2–5 years of age.

Our aim was not to establish a link between medium spiny neuron (MSN) function and febrile seizures. MSNs were included in this study as a mechanistic comparison because they represent a non-pyramidal, non-excitatory neuronal subtype, allowing us to assess whether the physiological changes observed in L2/3 excitatory pyramidal neurons are unique to these cells. Please note that the handling editor gave us the option to remove these data from the manuscript, and we chose to do so, developing these findings into a separate manuscript.

(6) TRP3V3 data would be convincing if the knockout animals did not have febrile seizures.

We find that approximately equal numbers of excitatory neurons either start or stop firing at fever-range temperatures (typically 38–40 °C). Neurons that continue to fire (“STAY” cells), thus play a key role in maintaining stable, non-excessive network activity. While future studies will examine the mechanisms driving some neurons to initiate spiking, our findings suggest that a reduction in the number of STAY cells could influence more subtle aspects of seizure dynamics, such as time to onset, by decreasing overall network excitability. We assessed the effects of reduced Trpv3 expression on hyperthermia-induced seizures in WT(Trpv3^+/+), heterozygous (Trpv3^+/-), and homozygous knockout (Trpv3^-/-) P12 pups. As you stated, these mice have hyperthermic seizures, however, we noted that the time to seizure from the point of loss of postural control (LPC), defined as collapse and failure to maintain upright posture, was significantly longer in Trpv3^+/- and Trpv3^-/- mice. Normally, seizures happen shortly after this point, but notably, Trpv3^-/- mice took twice as long to reach seizure onset compared with wildtype mice. In an epileptic patient, this increased time may be sufficient for a caretaker to move the patient to a safer location, reducing the risk of injury during the seizure.

Consistent with findings that TRPV3 blockade using 50 µM forsythoside B reduces spiking in cortical L2/3 pyramidal neurons, we observed significantly reduced spiking in Trpv3^-/- mice as well (Figure 10D). Analysis of postsynaptic potentials in these neurons showed that, in WT mice, PSP amplitude increased with temperature elevation into the febrile range, whereas this temperature-dependent depolarization was absent in Trpv3^-/- mice (Figure 10E). Together, these results indicate that reduced TRPV3 function enhances resistance to seizure initiation and/or propagation under febrile conditions, likely by decreasing neuronal depolarization and excitability.

Reviewer #3 (Public review):

Summary:

This important study combines in vitro and in vivo recording to determine how the firing of cortical and striatal neurons changes during a fever range temperature rise (37-40 oC). The authors found that certain neurons will start, stop, or maintain firing during these body temperature changes. The authors further suggested that the TRPV3 channel plays a role in maintaining cortical activity during fever.

Strengths:

The topic of how the firing pattern of neurons changes during fever is unique and interesting. The authors carefully used in vitro electrophysiology assays to study this interesting topic.

Weaknesses:

(1) In vivo recording is a strength of this study. However, data from in vivo recording is only shown in Figures 5A,B. This reviewer suggests the authors further expand on the analysis of the in vivo Neuropixels recording. For example, to show single spike waveforms and raster plots to provide more information on the recording. The authors can also separate the recording based on brain regions (cortex vs striatum) using the depth of the probe as a landmark to study the specific firing of cortical neurons and striatal neurons. It is also possible to use published parameters to separate the recording based on spike waveform to identify regular principal neurons vs fast-spiking interneurons. Since the authors studied E/I balance in brain slices, it would be very interesting to see whether the "E/I balance" based on the firing of excitatory neurons vs fast-spiking interneurons might be changed or not in the in vivo condition.

As requested, we have included additional analyses and figures related to the in vivo recording experiments in Figure 5. Specifically, we added examples of multiunit and single-spike waveforms, as well as autocorrelation histograms (ACHs). ACHs were used because raster plots of individual single units would not be very informative given the long recording period. Additionally, Figure 5F was also aimed to replace raster plots as it helps to track changes in the firing rate of a single neurons over time.

Additionally, all recordings were conducted in the cortex at a depth of ~1 mm from the surface, and no recordings were performed in the striatum. Based on the reviewing editor’s suggestions, we decided to remove the striatal data from the manuscript and develop this aspect of the project for a separate publication.

Lastly, we used published parameters to classify recordings based on spike waveform into putative regular principal neurons and interneurons. To clarify this point, we have now included descriptions that were previously listed only in the “Methods” section into the “Results” section as well.

The paragraph below from the methods section describes this procedure.

“Following manual curation, based on their spike waveform duration, the selected single units (n= 633) were separated into putative inhibitory interneurons and excitatory principal cells (Barthóet al., 2004). The spike duration was calculated as the time difference between the trough and the subsequent waveform peak of the mean filtered (300 – 6000 Hz bandpassed) spike waveform. Durations of extracellularly recorded spikes showed a bimodal distribution (Hartigan’s dip test; p < 0.001) characteristic of the neocortex with shorter durations corresponding to putative interneurons (narrow spikes) and longer durations to putative principal cells (wide spikes). Next, k-means clustering was used to separate the single units into these two groups, which resulted in 140 interneurons (spike duration < 0.6 ms) and 493 principal cells (spike duration > 0.6 ms), corresponding to a typical 22% - 78% (interneuron – principal) cell ratio”.

As suggested, we calculated the E/I balance using the average firing rates of excitatory and inhibitory neurons in the in vivo condition. Our analysis revealed that the E/I balance remained unchanged (see Author response image 1). Nonetheless, following the option provided by the reviewing editor, we have chosen to remove the statement referencing E/I balance from the manuscript.

Author response image 1.

(2) The author should propose a potential mechanism for how TRPV3 helps to maintain cortical activity during fever. Would calcium influx-mediated change of membrane potential be the possible reason? Making a summary figure to put all the findings into perspective and propose a possible mechanism would also be appreciated.

Thank you for your helpful suggestion. In response, we have included a summary figure (Figure 11) illustrating the hypothesis described in the Discussion section. We agree with your assessment that Trpv3 most likely contributes to maintaining cortical activity during fever by promoting calcium influx and depolarizing the membrane potential.

(3) The author studied P7-8, P12-14, and P20-26 mice. How do these ages correspond to the human ages? it would be nice to provide a comparison to help the reader understand the context better.

Ideally, the mouse to human age comparison should depend on the specific process being studied. Per your suggestion, we have added additional references in the Introduction (Dobbing and Sands, 1973; Baram et al., 1997; Bender et al., 2004) to help readers better understand the correspondence between mouse and human ages.

Recommendations for the authors:

Reviewer #2 (Recommendations for the authors):

(3) Perform I-F curves to study the intrinsic properties of layer 2/3 neurons without the confound of evoked responses.

We performed F-I curve analyses (Figures 2H–I), as suggested by Reviewer 2, to study intrinsic properties of L2/3 neurons without evoked responses. Although rheobase increased at 39 °C compared to 30 °C, consistent with findings such as depolarized spike threshold and reduced input resistance, the mean number of spikes across current steps did not differ.

Reviewer #3 (Recommendations for the authors):

Some statistical descriptions are not clearly stated. For example, what statistical methods were used in Fig 2E? The effect size in Fig 2D seems to be quite small. The authors are advised to consider "nested analysis" to further increase the rigor of the analysis. Does each dot mean one neuron? Some of the data points might not be totally independent. The author should carefully check all figures to make sure the stats methods are provided for each panel.

We apologize for not including statistical details in Figure 2E. We have now added this information and verified that statistical descriptions are provided in all figure legends. In Figure 2D, each dot represents a cell, with measurements taken from the same cell at 30°C, 36°C, and 39°C. Given this design, the appropriate test is a one-way repeated-measures ANOVA.

eLife Assessment

This study provides a useful investigation of human-AI interaction and decision-making, using both behavioral and electrophysiological measures. However, the theoretical framework and experimental design are incomplete, with an unclear task structure and feedback implementation limiting interpretability. With these issues addressed, the work could make a significant contribution to understanding human-AI collaboration.

Reviewer #1 (Public review):

Summary:

In the study by Roeder and colleagues, the authors aim to identify the psychophysiological markers of trust during the evaluation of matching or mismatching AI decision-making. Specifically, they aim to characterize through brain activity how the decision made by an AI can be monitored throughout time in a two-step decision-making task. The objective of this study is to unfold, through continuous brain activity recording, the general information processing sequence while interacting with an artificial agent, and how internal as well as external information interact and modify this processing. Additionally, the authors provide a subset of factors affecting this information processing for both decisions.

Strengths:

The study addresses a wide and important topic of the value attributed to AI decisions and their impact on our own confidence in decision-making. It especially questions some of the factors modulating the dynamical adaptation of trust in AI decisions. Factors such as perceived reliability, type of image, mismatch, or participants' bias toward one response or the other are very relevant to the question in human-AI interactions.

Interestingly, the authors also question the processing of more ambiguous stimuli, with no real ground truth. This gets closer to everyday life situations where people have to make decisions in uncertain environments. Having a better understanding of how those decisions are made is very relevant in many domains.

Also, the method for processing behavioral and especially EEG data is overall very robust and is what is currently recommended for statistical analyses for group studies. Additionally, authors provide complete figures with all robustness evaluation information. The results and statistics are very detailed. This promotes confidence, but also replicability of results.

An additional interesting method aspect is that it is addressing a large window of analysis and the interaction between three timeframes (evidence accumulation pre-decision, decision-making, post-AI decision processing) within the same trials. This type of analysis is quite innovative in the sense that it is not yet a standard in complex experimental designs. It moves forward from classical short-time windows and baseline ERP analysis.

Weaknesses:

This manuscript raises several conceptual and theoretical considerations that are not necessarily answered by the methods (especially the task) used. Even though the authors propose to assess trust dynamics and violations in cooperative human-AI teaming decision-making, I don't believe their task resolves such a question. Indeed, there is no direct link between the human decision and the AI decision. They do not cooperate per se, and the AI decision doesn't seem, from what I understood to have an impact on the participants' decision making. The authors make several assumptions regarding trust, feedback, response expectation, and "classification" (i.e., match vs. mismatch) which seem far stretched when considering the scientific literature on these topics.

Unlike what is done for the data processing, the authors have not managed to take the big picture of the theoretical implications of their results. A big part of this study's interpretation aims to have their results fit into the theoretical box of the neural markers of performance monitoring.

Overall, the analysis method was very robust and well-managed, but the experimental task they have set up does not allow to support their claim. Here, they seem to be assessing the impact of a mismatch between two independent decisions.

Nevertheless, this type of work is very important to various communities. First, it addresses topical concerns associated with the introduction of AI in our daily life and decisions, but it also addresses methodological difficulties that the EEG community has been having to move slowly away from the static event-based short-timeframe analyses onto a more dynamic evaluation of the unfolding of cognitive processes and their interactions. The topic of trust toward AI in cooperative decision making has also been raised by many communities, and understanding the dynamics of trust, as well as the factors modulating it, is of concern to many high-risk environments, or even everyday life contexts. Policy makers are especially interested in this kind of research output.

Reviewer #2 (Public review):

Summary:

The authors investigated how "AI-agent" feedback is perceived in an ambiguous classification task, and categorised the neural responses to this. They asked participants to classify real or fake faces, and presented an AI-agent's feedback afterwards, where the AI-feedback disagreed with the participants' response on a random 25% of trials (called mismatches). Pre-response ERP was sensitive to participants' classification as real or fake, while ERPs after the AI-feedback were sensitive to AI-mismatches, with stronger N2 and P3a&b components. There was an interaction of these effects, with mismatches after a "Fake" response affecting the N2 and those after "Real" responses affecting P3a&b. The ERPs were also sensitive to the participants' response biases, and their subjective ratings of the AI agent's reliability.

Strengths:

The researchers address an interesting question, and extend the AI-feedback paradigm to ambiguous tasks without veridical feedback, which is closer to many real-world tasks. The in-depth analysis of ERPs provides a detailed categorisation of several ERPs, as well as whole-brain responses, to AI-feedback, and how this interacts with internal beliefs, response biases, and trust in the AI-agent.

Weaknesses:

There is little discussion of how the poor performance (close to 50% chance) may have affected performance on the task, such as by leading to entirely random guessing or overreliance on response biases. This can change how error-monitoring signals presented, as they are affected by participants' accuracy, as well as affecting how the AI feedback is perceived.

The task design and performance make it hard to assess how much it was truly measuring "trust" in an AI agent's feedback. The AI-feedback is yoked to the participants' performance, agreeing on 75% of trials and disagreeing on 25% (randomly), which is an important difference from the framing provided of human-AI partnerships, where AI-agents usually act independently from the humans and thus disagreements offer information about the human's own performance. In this task, disagreements are uninformative, and coupled with the at-chance performance on an ambiguous task, it is not clear how participants should be interpreting disagreements, and whether they treat it like receiving feedback about the accuracy of their choices, or whether they realise it is uninformative. Much greater discussion and justification are needed about the behaviour in the task, how participants did/should treat the feedback, and how these affect the trust/reliability ratings, as these are all central to the claims of the paper.

There are a lot of EEG results presented here, including whole-brain and window-free analyses, so greater clarity on which results were a priori hypothesised should be given, along with details on how electrodes were selected for ERPs and follow-up tests.

Reviewer #3 (Public review):

The current paper investigates neural correlates of trust development in human-AI interaction, looking at EEG signatures locked to the moment that AI advice is presented. The key finding is that both human-response-locked EEG signatures (the CPP) and post-AI-advice signatures (N2, P3) are modulated by trust ratings. The study is interesting, however, it does have some clear and sometimes problematic weaknesses:

(1) The authors did not include "AI-advice". Instead, a manikin turned green or blue, which was framed as AI advice. It is unclear whether participants viewed this as actual AI advice.

(2) The authors did not include a "non-AI" control condition in their experiment, such that we cannot know how specific all of these effects are to AI, or just generic uncertain feedback processing.

(3) Participants perform the task at chance level. This makes it unclear to what extent they even tried to perform the task or just randomly pressed buttons. These situations likely differ substantially from a real-life scenario where humans perform an actual task (which is not impossible) and receive actual AI advice.

(4) Many of the conclusions in the paper are overstated or very generic.

Author response:

A major point all three reviewers raise is that the ‘human-AI collaboration’ in our experiment may not be true collaboration (as the AI does not classify images per se), but that it is only implied. The reviewers pointed out that whether participants were genuinely engaged in our experimental task is currently not sufficiently addressed. We plan to address this issue in the revised manuscript by including results from a brief interview we conducted after the experiment with each participant, which asked about the participant’s experience and decision-making processes while performing the task. Additionally, we also measured the participants’ propensity to trust in AI via a questionnaire before and after the experiment. The questionnaire and interview results will allow us to more accurately describe the involvement of our participants in the task. Additionally, we will conduct additional analyses of the behavioural data (e.g., response times) to show that participants genuinely completed the experimental task. Finally, we will work to sharpen our language and conclusions in the revised manuscript, following the reviewers’ recommendations.

Reviewer #1:

Summary:

In the study by Roeder and colleagues, the authors aim to identify the psychophysiological markers of trust during the evaluation of matching or mismatching AI decision-making. Specifically, they aim to characterize through brain activity how the decision made by an AI can be monitored throughout time in a two-step decision-making task. The objective of this study is to unfold, through continuous brain activity recording, the general information processing sequence while interacting with an artificial agent, and how internal as well as external information interact and modify this processing. Additionally, the authors provide a subset of factors affecting this information processing for both decisions.

Strengths:

The study addresses a wide and important topic of the value attributed to AI decisions and their impact on our own confidence in decision-making. It especially questions some of the factors modulating the dynamical adaptation of trust in AI decisions. Factors such as perceived reliability, type of image, mismatch, or participants' bias toward one response or the other are very relevant to the question in human-AI interactions.

Interestingly, the authors also question the processing of more ambiguous stimuli, with no real ground truth. This gets closer to everyday life situations where people have to make decisions in uncertain environments. Having a better understanding of how those decisions are made is very relevant in many domains.

Also, the method for processing behavioural and especially EEG data is overall very robust and is what is currently recommended for statistical analyses for group studies. Additionally, authors provide complete figures with all robustness evaluation information. The results and statistics are very detailed. This promotes confidence, but also replicability of results.

An additional interesting method aspect is that it is addressing a large window of analysis and the interaction between three timeframes (evidence accumulation pre-decision, decision-making, post-AI decision processing) within the same trials. This type of analysis is quite innovative in the sense that it is not yet a standard in complex experimental designs. It moves forward from classical short-time windows and baseline ERP analysis.

We appreciate the constructive appraisal of our work.

Weaknesses:

R1.1. This manuscript raises several conceptual and theoretical considerations that are not necessarily answered by the methods (especially the task) used. Even though the authors propose to assess trust dynamics and violations in cooperative human-AI teaming decision-making, I don't believe their task resolves such a question. Indeed, there is no direct link between the human decision and the AI decision. They do not cooperate per se, and the AI decision doesn't seem, from what I understood to have an impact on the participants' decision making. The authors make several assumptions regarding trust, feedback, response expectation, and "classification" (i.e., match vs. mismatch) which seem far stretched when considering the scientific literature on these topics.

This issue is raised by the other reviewers as well. The reviewer is correct in that the AI does not classify images but that the AI response is dependent on the participants’ choice (agree in 75% of trials, disagree in 25% of the trials). Importantly, though, participants were briefed before and during the experiment that the AI is doing its own independent image classification and that human input is needed to assess how well the AI image classification works. That is, participants were led to believe in a genuine, independent AI image classifier on this experiment.

Moreover, the images we presented in the experiment were taken from previous work by Nightingale & Farid (2022). This image dataset includes ‘fake’ (AI generated) images that are indistinguishable from real images.

What matters most for our work is that the participants were truly engaging in the experimental task; that is, they were genuinely judging face images, and they were genuinely evaluating the AI feedback. There is strong indication that this was indeed the case. We conducted and recorded brief interviews after the experiment, asking our participants about their experience and decision-making processes. The questions are as follows:

(1) How did you make the judgements about the images?

(2) How confident were you about your judgement?

(3) What did you feel when you saw the AI response?

(4) Did that change during the trials?

(5) Who do you think it was correct?

(6) Did you feel surprised at any of the AI responses?

(7) How did you judge what to put for the reliability sliders?

In our revised manuscript we will conduct additional analyses to provide detail on participants’ engagement in the task; both in the judging of the AI faces, as well as in considering the AI feedback. In addition, we will investigate the EEG signal and response time to check for effects that carry over between trials. We will also frame our findings more carefully taking scientific literature into account.

Nightingale SJ, and Farid H. "AI-synthesized faces are indistinguishable from real faces and more trustworthy." Proceedings of the National Academy of Sciences 119.8 (2022): e2120481119.

R1.2. Unlike what is done for the data processing, the authors have not managed to take the big picture of the theoretical implications of their results. A big part of this study's interpretation aims to have their results fit into the theoretical box of the neural markers of performance monitoring.

We indeed used primarily the theoretical box of performance monitoring and predictive coding, since the make-up of our task is similar to a more classical EEG oddball paradigm. In our revised manuscript, we will re-frame and address the link of our findings with the theoretical framework of evidence accumulation and decision confidence.

R1.3. Overall, the analysis method was very robust and well-managed, but the experimental task they have set up does not allow to support their claim. Here, they seem to be assessing the impact of a mismatch between two independent decisions.

Although the human and AI decisions are independent in the current experiment, the EEG results still shed light on the participant’s neural processes, as long as the participant considers the AI’s decision and believes it to be genuine. An experiment in which both decisions carry effective consequences for the task and the human-AI cooperation would be an interesting follow-up study.

Nevertheless, this type of work is very important to various communities. First, it addresses topical concerns associated with the introduction of AI in our daily life and decisions, but it also addresses methodological difficulties that the EEG community has been having to move slowly away from the static event-based short-timeframe analyses onto a more dynamic evaluation of the unfolding of cognitive processes and their interactions. The topic of trust toward AI in cooperative decision making has also been raised by many communities, and understanding the dynamics of trust, as well as the factors modulating it, is of concern to many high-risk environments, or even everyday life contexts. Policy makers are especially interested in this kind of research output.

Reviewer #2:

Summary:

The authors investigated how "AI-agent" feedback is perceived in an ambiguous classification task, and categorised the neural responses to this. They asked participants to classify real or fake faces, and presented an AI-agent's feedback afterwards, where the AI-feedback disagreed with the participants' response on a random 25% of trials (called mismatches). Pre-response ERP was sensitive to participants' classification as real or fake, while ERPs after the AI-feedback were sensitive to AI-mismatches, with stronger N2 and P3a&b components. There was an interaction of these effects, with mismatches after a "Fake" response affecting the N2 and those after "Real" responses affecting P3a&b. The ERPs were also sensitive to the participants' response biases, and their subjective ratings of the AI agent's reliability.

Strengths:

The researchers address an interesting question, and extend the AI-feedback paradigm to ambiguous tasks without veridical feedback, which is closer to many real-world tasks. The in-depth analysis of ERPs provides a detailed categorisation of several ERPs, as well as whole-brain responses, to AI-feedback, and how this interacts with internal beliefs, response biases, and trust in the AI-agent.

We thank the reviewer for their time in reading and reviewing our manuscript.

Weaknesses:

R2.1. There is little discussion of how the poor performance (close to 50% chance) may have affected performance on the task, such as by leading to entirely random guessing or overreliance on response biases. This can change how error-monitoring signals presented, as they are affected by participants' accuracy, as well as affecting how the AI feedback is perceived.

The images were chosen from a previous study (Nightingale & Farid, 2022, PNAS) that looked specifically at performance accuracy and also found levels around 50%. Hence, ‘fake’ and ‘real’ images are indistinguishable in this image dataset. Our findings agree with the original study.

Judging based on the brief interviews after the experiment (see answer to R.1.1.), all participants were actively and genuinely engaged in the task, hence, it is unlikely that they pressed buttons at random. As mentioned above, we will include a formal analysis of the interviews in the revised manuscript.

The response bias might indeed play a role in how participants responded, and this might be related to their initial propensity to trust in AI. We have questionnaire data available that might shed light on this issue: before and after the experiment, all participants answered the following questions with a 5-point Likert scale ranging from ‘Not True’ to ‘Completely True’:

(1) Generally, I trust AI.

(2) AI helps me solve many problems.

(3) I think it's a good idea to rely on AI for help.

(4) I don't trust the information I get from AI.

(5) AI is reliable.

(6) I rely on AI.

The propensity to trust questionnaire is adapted from Jessup SA, Schneider T R, Alarcon GM, Ryan TJ, & Capiola A. (2019). The measurement of the propensity to trust automation. International Conference on Human-Computer Interaction.

Our initial analyses did not find a strong link between the initial (before the experiment) responses to these questions, and how images were rated during the experiment. We will re-visit this analysis and add the results to the revised manuscript.

Regarding how error-monitoring (or the equivalent thereof in our experiment) is perceived, we will analyse interview questions 3 (“What did you feel when you saw the AI response”) and 6 (“Did you feel surprised at any of the AI responses”) and add results to the revised manuscript.

The task design and performance make it hard to assess how much it was truly measuring "trust" in an AI agent's feedback. The AI-feedback is yoked to the participants' performance, agreeing on 75% of trials and disagreeing on 25% (randomly), which is an important difference from the framing provided of human-AI partnerships, where AI-agents usually act independently from the humans and thus disagreements offer information about the human's own performance. In this task, disagreements are uninformative, and coupled with the at-chance performance on an ambiguous task, it is not clear how participants should be interpreting disagreements, and whether they treat it like receiving feedback about the accuracy of their choices, or whether they realise it is uninformative. Much greater discussion and justification are needed about the behaviour in the task, how participants did/should treat the feedback, and how these affect the trust/reliability ratings, as these are all central to the claims of the paper.

In our experiment, the AI disagreements are indeed uninformative for the purpose of making a correct judgment (that is, correctly classifying images as real or fake). However, given that the AI-generated faces are so realistic and indistinguishable from the real faces, the correctness of the judgement is not the main experimental factor in this study. We argue that, provided participants were genuinely engaged in the task, their judgment accuracy is less important than their internal experience when the goal is to examine processes occurring within the participants themselves. We briefed our participants as follows before the experiment:

“Technology can now create hyper-realistic images of people that do not exist. We are interested in your view on how well our AI system performs at identifying whether images of people’s faces are real or fake (computer-generated). Human input is needed to determine when a face looks real or fake. You will be asked to rate images as real or fake. The AI system will also independently rate the images. You will rate how reliable the AI is several times throughout the experiment.”

We plan to more fully expand the behavioural aspect and our participants’ experience in the revised manuscript by reporting the brief post-experiment interview (R.1.1.), the propensity to trust questionnaire (R.2.1.), and additional analyses of the response times.

There are a lot of EEG results presented here, including whole-brain and window-free analyses, so greater clarity on which results were a priori hypothesised should be given, along with details on how electrodes were selected for ERPs and follow-up tests.

We chose the electrodes mainly to be consistent across findings, and opted to use central electrodes (Pz and Fz), as long as the electrode was part of the electrodes within the reported cluster. We can in our revised manuscript also report on the electrodes with the maximal statistic, as part of a more complete and descriptive overview. We will also report on where we expected to see ERP components within the paper. In short, we did expect something like a P3, and we did also expect to see something before the response what we call the CPP. The rest of the work was more exploratory, with a more careful expectation that bias would be connected to the CPP, and the reliability ratings more to the P3; however, we find the opposite results. We will include this in our revised work as well.

We selected the electrodes primarily to maintain consistency across our findings and figures, and focused on central electrodes (Pz and Fz), provided they fell within the reported cluster. In the revised manuscript, we will also report the electrodes showing the maximal statistical effects to give a more complete and descriptive overview. Additionally, we will report where we expected specific ERP components to appear. In brief, we expected to see a P3 component post AI feedback, and a pre-response signal corresponding to the CPP. Beyond these expectations, the remaining analyses were more exploratory. Although we tentatively expected bias to relate to the CPP and reliability ratings to the P3, our results showed the opposite pattern. We will clarify this in the revised version of the manuscript.

Reviewer #3:

The current paper investigates neural correlates of trust development in human-AI interaction, looking at EEG signatures locked to the moment that AI advice is presented. The key finding is that both human-response-locked EEG signatures (the CPP) and post-AI-advice signatures (N2, P3) are modulated by trust ratings. The study is interesting, however, it does have some clear and sometimes problematic weaknesses:

(1) The authors did not include "AI-advice". Instead, a manikin turned green or blue, which was framed as AI advice. It is unclear whether participants viewed this as actual AI advice.

This point has been raised by the other reviewers as well, and we refer to the answers under R1.1., and under R2.1. We will address this concern by analysing the post-experiment interviews. In particular, questions 3 (“What did you feel when you saw the AI response”), 4 (“Did that change during the trials?”) and 6 (“Did you feel surprised at any of the AI responses”) will give critical insight. As stated above, our general impression from conducting the interviews is that all participants considered the robot icon as decision from an independent AI agent.

(2) The authors did not include a "non-AI" control condition in their experiment, such that we cannot know how specific all of these effects are to AI, or just generic uncertain feedback processing.

In the conceptualization phase of this study, we indeed considered different control conditions for our experiment to contrast different kinds of feedback. However, previous EEG studies on performance monitoring ERPs have reported similar results for human and machine supervision (Somon et al., 2019; de Visser et al., 2018). We therefore decided to focus on one aspect (the judgement of observation of an AI classification), also to prevent the experiment from taking too long and risking that participants would lose concentration and motivation to complete the experiment. Comparing AI vs non-AI feedback, is still interesting and would be a valuable follow-up study.

Somon B, et al. "Human or not human? Performance monitoring ERPs during human agent and machine supervision." NeuroImage 186 (2019): 266-277.

De Visser EJ, et al. "Learning from the slips of others: Neural correlates of trust in automated agents." Frontiers in human neuroscience 12 (2018): 309.

(3) Participants perform the task at chance level. This makes it unclear to what extent they even tried to perform the task or just randomly pressed buttons. These situations likely differ substantially from a real-life scenario where humans perform an actual task (which is not impossible) and receive actual AI advice.

This concern was also raised by the other two reviewers. As already stated in our responses above, we will add results from the post-experiment interviews with the participants, the propensity to trust questionnaire, and additional behavioural analyses in our revised manuscript.

Reviewer 1 (R1.3) also brought up the situation where decisions by the participant and the AI have a more direct link which carries consequences. This will be valuable follow-up research. In the revised manuscript, we will more carefully frame our approach.

(4) Many of the conclusions in the paper are overstated or very generic.

In the revised manuscript, we will re-phrase our discussion and conclusions to address the points raised in the reviewer’s recommendations to authors.

eLife Assessment

This important study provides convincing evidence that envelope-carrying Ty3/gypsy retrotransposons (errantiviruses) are ancient, widespread, and actively expanding across nearly all major animal phyla. Using comprehensive phylogenetic and AlphaFold2-based structural analyses, the authors show that these elements independently acquired membrane fusion proteins early in metazoan evolution, likely predating the bilaterian-non-bilaterian split. While some aspects could be more clearly contextualized and explained better, the work offers insights into the deep evolutionary roots of retroelement-envelope associations and the origins of retroviruses.

Reviewer #1 (Public review):

Summary:

This manuscript provides a comprehensive systematic analysis of envelope-containing Ty3/gypsy retrotransposons (errantiviruses) across metazoan genomes, including both invertebrates and ancient animal lineages. Using iterative tBLASTn mining of over 1,900 genomes, the authors catalog 1,512 intact retrotransposons with uninterrupted gag, pol, and env open reading frames. They show that these elements are widespread-present in most metazoan phyla, including cnidarians, ctenophores, and tunicates-with active proliferation indicated by their multicopy status. Phylogenetic analyses distinguish "ancient" and "insect" errantivirus clades, while structural characterization (including AlphaFold2 modeling) reveals two major env types: paramyxovirus F-like and herpesvirus gB-like proteins. Although bot envelope types were identified in previous analyses two decades ago, the evolutionary provenance of these envelope genes was almost rudimentary and anecdotal (I can say this because I authored one of these studies). The results in the present study support an ancient origin for env acquisition in metazoan Ty3/gypsy elements, with subsequent vertical inheritance and limited recombination between env and pol domains. The paper also proposes an expanded definition of 'errantivirus' for env-carrying Ty3/gypsy elements outside Drosophila.

Strengths:

(1) Comprehensive Genomic Survey:<br /> The breadth of the genome search across non-model metazoan phyla yields an impressive dataset covering evolutionary breadth, with clear documentation of search iterations and validation criteria for intact elements.

(2) Robust Phylogenetic Inference:<br /> The use of maximum likelihood trees on both pol and env domains, with thorough congruence analysis, convincingly separates ancient from lineage-specific elements and demonstrates co-evolution of env and pol within clades.

(3) Structural Insights:<br /> AlphaFold2-based predictions provide high-confidence structural evidence that both env types have retained fusion-competent architectures, supporting the hypothesis of preserved functional potential.

(4) Novelty and Scope:<br /> The study challenges previous assumptions of insect-centric or recent env acquisition and makes a compelling case for a Pre-Cambrian origin, significantly advancing our understanding of animal retroelement diversity and evolution. THIS IS A MAJOR ADVANCE.

(5) Data Transparency:<br /> I appreciate that all data, code, and predicted structures are made openly available, facilitating reproducibility and future comparative analyses.

Major Weaknesses

(1) Functional Evidence Gaps:<br /> The work rests largely on sequence and structure prediction. No direct expression or experimental validation of envelope gene function or infectivity outside Drosophila is attempted, which would be valuable to corroborate the inferred roles of these glycoproteins in non-insect lineages. At least for some of these species, there are RNA-seq datasets that could be leveraged.

(2) Horizontal Transfer vs. Loss Hypotheses:<br /> The discussion argues primarily for vertical inheritance, but the somewhat sporadic phylogenetic distributions and long-branch effects suggest that loss and possibly rare horizontal events may contribute more than acknowledged. Explicit quantitative tests for horizontal transfer, or reconciliation analyses, would strengthen this conclusion. It's also worth pointing out that, unlike retrotransposons that can be found in genomes, any potential related viral envelopes must, by definition, have a spottier distribution due to sampling. I don't think this challenges any of the conclusions, but it must be acknowledged as something that could affect the strength of this conclusion

(3) Limited Taxon Sampling for Certain Phyla:<br /> Despite the impressive breadth, some ancient lineages (e.g., Porifera, Echinodermata) are negative, but the manuscript does not fully explore whether this reflects real biological absence, assembly quality, or insufficient sampling. A more systematic treatment of negative findings would clarify claims of ubiquity. However, I also believe this falls beyond the scope of this study.

(4) Mechanistic Ambiguity:<br /> The proposed model that env-containing elements exploit ovarian somatic niches is plausible but extrapolated from Drosophila data; for most taxa, actual tissue specificity, lifecycle, or host interaction mechanisms remain speculative and, to me, a bit unreasonable.

Minor Weaknesses:

(1) Terminology and Nomenclature:<br /> The paper introduces and then generalizes the term "errantivirus" to non-insect elements. While this is logical, it may confuse readers familiar with the established, Drosophila-centric definition if not more explicitly clarified throughout. I also worry about changes being made without any input from the ICTV nomenclature committee, which just went through a thorough reclassification. Nevertheless, change is expected, and calling them all errantiviruses is entirely reasonable.

(2) Figures and Supplementary Data Navigation:<br /> Some key phylogenies and domain alignments are found only in supplementary figures, occasionally hindering readability for non-expert audiences. Selected main-text inclusion of representative trees would benefit accessibility.

(3) ORF Integrity Thresholds:<br /> The cutoff choices for defining "intact" elements (e.g., numbers/placement of stop codons, length ranges) are reasonable but only lightly justified. More rationale or sensitivity analysis would improve confidence in the inclusion criteria. For example, how did changing these criteria change the number of intact elements?

(4) Minor Typos/Formatting:<br /> The paper contains sporadic typographical errors and formatting glitches (e.g., misaligned figure labels, unrendered symbols) that should be addressed.

Reviewer #2 (Public review):

Summary:

The authors first surveyed metazoan genomes to identify homologs of Drosophila errantiviruses and classified them into two groups, "insect" and "ancient" elements, supporting the hypothesis of an early evolutionary origin for these retrotransposons. They subsequently identified two distinct types of envelope proteins, one resembling the glycoprotein F of paramyxoviruses and the other akin to the glycoprotein B of herpesviruses. Despite differences in their primary amino acid sequences, these proteins display notable structural similarity in their predicted domain architectures. The congruence between the phylogenies of the envelope and pol genes further supports the ancient origin of the envelope genes, challenging earlier hypotheses that proposed recent recombination events with baculoviruses. Additional analysis of the Pol "bridge region" corroborated the divergence among these elements, consistent with a pattern of limited cross-species recombination. Finally, by comparing these elements with non-envelope-containing Gypsy retrotransposons, the authors concluded that errantiviruses originated from multiple elements independently.

Strengths:

The conclusions of this study are based on a comprehensive collection of errantiviruses identified across a wide range of metazoan genomes. These findings are further supported by multiple lines of evidence, including phylogenetic congruence and the diverse evolutionary origins of envelope genes. AlphaFold2-assisted protein domain structure analyses also provided key insights into the characterization of these elements. Together, these results present a compelling case that errantiviruses arose independently through multiple evolutionary events, extending well beyond previous hypotheses.

Weaknesses:

It would be beneficial to emphasize in the Abstract the potential impact of this work by more clearly articulating the current knowledge gap in the field. While the second paragraph of the Introduction briefly touches on this point, highlighting the broader significance in the Abstract would better capture readers' interest. Additionally, some methodological choices would benefit from clearer justification and explanation. For instance, in Figure 6, the selection of the bridge region/RNase H domain is not explicitly explained, leaving the rationale for its choice unclear. As a minor point, some figure labels and texts are too small and difficult to read, and improving their legibility would enhance overall clarity.

Reviewer #3 (Public review):

Summary and Significance:

In this work, Cary and Hayashi address the important question of when, in evolution, certain mobile genetic elements (Ty3/gypsy-like non-LTR retrotransposons) associated with certain membrane fusion proteins (viral glycoprotein F or B-like proteins), which could allow these mobile genetic elements to be transferred between individual cells of a given host. It is debated in the literature whether the acquisition of membrane fusion proteins by non-LTR retrotransposons is a rather recent phenomenon that separately occurred in the ancestors of certain host species or whether the association with membrane fusion proteins is a much more ancient one, pre-dating the Cambrian explosion. Obviously, this question also touches upon the origin of the retroviruses, which can spread between individuals of a given host but seem restricted to vertebrates. Based on convincing data, Cary and Hayashi argue that an ancient association of non-LTR retrotransposons with membrane fusion proteins is most probable.

Strengths:

The authors take the smart approach to systematically retrieve apparently complete, intact, and recently functional Ty3/gypsy-like non-LTR retrotransposons that, next to their characteristic gag and pol genes, additionally carry sequences that are homologous to viral glycoprotein F (env-F) or viral glycoprotein B (env-B). They then construct and compare phylogenetic trees of the host species and individual encoded proteins and protein domains, where 3D-structure calculations and other features explain and corroborate the clustering within the phylogenetic trees. Congruence of phylogenetic trees and correlation of structural features is then taken as evidence for an infrequent recombination and a long-term co-evolution of the reverse transcriptase (encoded by the pol gene) and its respective putative membrane fusion gene (encoded by env-F or env-B). Importantly, the env-F and env-B containing retrotransposons do not form a monophyletic group among the Ty3/gypsy-like non-LTR retrotransposons, but are scattered throughout, supporting the idea of an originally ancient association followed by a random loss of env-F/env-B in individual branches of the tree (and rather rare re-associations via more recent recombinations).

Overall, this is valuable, stimulating, and important work of general and fundamental interest, but still also somewhat incompletely explored, imprecisely explained, and insufficiently put into context for a more general audience.

Weaknesses:

Some points that might be considered and clarified:

(1) Imprecise explanations, terms, and definitions:

It might help to add a 'definitions box' or similar to precisely explain how the authors decided to use certain terms in this manuscript, and then use these terms consistently and with precision.

a) In particular, these are terms such as 'vertebrate retrovirus' vs 'retrovirus' vs 'endogenized retrovirus' vs 'endogenous retrovirus' vs 'non-LTR retrotransposon' and 'Ty3/gypsi-like retrotransposon' vs 'Ty3/gypsy retrotransposon' vs 'errantivirus'.

b) The comment also applies to the term 'env' used for both 'env-F' and 'env-B', where often it remains unclear which of the two protein types the authors refer to. This is confusing, particularly in the methods, where the search for the respective homologs is described.

c) Other examples are the use of the entire pol gene vs. pol-RT for the definition of the Ty3/gypsy clade and for the generation of phylogenetic trees (Methods and Figure S1), and the names for various portions of pol that appear without prior definition or explanation (e.g., 'pro' in Figure 1A, 'bridge' in Figure S1C, 'the chromodomain' in the text and Figure 7).

d) It is unclear from the main text which portions of pol were chosen to define pol-RT and why. The methods name the 'palm-and-fingers', 'thumb', and 'connections' domains to define RT. In the main text, the 'connection' domain is called 'tether' and is instead defined as part of the 'bridge' region following RT, which is not part of RT.

(2) Insufficient broader context:

a) The introduction does not state what defines Ty3/gypsy non-LTR retrotransposons as compared to their closest relatives (Ty1/copia retrotransposons, BEL/pao retrotransposons, vertebrate retroviruses). This makes it difficult to judge the significance and generality of the findings.

b) The various known compositions of Ty3/gypsi-like retrotransposons are not mentioned and explained in the introduction (open reading frames, (poly-)proteins and protein domains, and their variable arrangement, enzymatic activities, and putative functions), and the distribution of Ty3/gypsi-like retrotransposons among eukaryotes remains unclear. The introduction does not mention that Ty3/gypsi-like retrotransposons apparently are absent from vertebrates, and Figure 7 is not very clear about whether or not it includes sequences from plants ('Chromoviridae').

c) The known association of Ty3/gypsi-like retrotransposons from different metazoan phyla with putative membrane fusion proteins (env-like) genes is mentioned in the introduction, but literature information, whether such associations also occur in the context of other retrotransposons (e.g., Ty1/ copia or BEL/pao), is not provided. The abstract is somewhat misleading in this respect. Finally, the different known types of env-like genes are not mentioned and explained as part of the introduction ('env-f', 'env-B', 'retroviral env', others?)

d) Some key references and reviews might be added:

- Pelisson, A. et al. (1994) https://www.embopress.org/doi/abs/10.1002/j.1460-2075.1994.tb06760.x<br /> (next to Song et al. (1994), for the identification of env in Ty3/gypsy)

- Boeke, J.D. et al. (1999)<br /> In Virus Taxonomy: ICTV VIIth report. (ed. F.A. Murphy),. Springer-Verlag, New York.<br /> (cited by Malik et al. (2000) - for the definition and first use of the term 'errantivirus')

- Eickbush, T.H. and Jamburuthugoda, V.K. (2008) https://doi.org/10.1016/j.virusres.2007.12.010<br /> (on the classification of retrotransposons and their env-like genes)

- Hayward, A. (2017) https://doi.org/10.1016/j.coviro.2017.06.006<br /> (on scenarios of env acquisition)

(3) Incomplete analysis:

a) Mobile genetic elements are sometimes difficult to assemble correctly from short-read sequencing data. Did the authors confirm some of their newly identified elements by e.g., PCR analysis or re-identification in long-read sequencing data?

b) The authors mention somewhat on the side that there are Ty3/gypsy elements with a different arrangement (gag-env-pol instead of gag-pol-env). Why was this important feature apparently not used and correlated in the analysis? How does it map on the RT phylogenetic tree? Which type of env is found with either arrangement? Is there evidence for a loss of env also in the case of gag-env-pol elements?

c) Sankey plots are insufficiently explained. How would inconsistencies between trees (recombinations) show up here? Why is there no Sankey plot for the analysis of env-B in Figure 5?

d) Why are there no trees generated for env-F and env-B like proteins, including closely related homologous sequences that do NOT come from Ty3/gypsy retrotransposons (e.g., from the eukaryotic hosts, from other types of retrotransposons (Ty1/copia or BEL/pao), from viruses such as Herpesvirus and Baculovirus)? It would be informative whether the sequences from Ty3/gypsy cluster together in this case.

e) Did the authors identify any other env-like ORFs (apart from env-F and env-B) among Ty3/gypsy retrotransposons? Did they identify other, non-env-like ORFs that might help in the analysis? It is not quite clear from the methods if the searches for env-F and env-B - containing Ty3/gypsy elements were done separately and consecutively or somehow combined (the authors generally use 'env', and it is not clear which type of protein this refers to).

f) Why was the gag protein apparently not used to support the analysis? Are there different, unrelated types of gag among non-LTR retrotransposons? Does gag follow or break the pattern of co-evolution between RT and env-F/env-B?

g) Data availability. The link given in the paper does not seem to work (https://github.com/RippeiHayashi/errantiviruses_2025/tree/main). It would be useful for the community to have the sequences of the newly identified Ty3/gypsy retrotransposons listed readily available (not just genome coordinates as in table S1), together with the respective annotations of ORFs and features.

Author response:

We appreciate thorough and highly valuable feedback from the reviewers. We will take their suggestions on board and prepare a revised manuscript focusing on the following points:

(1) As reviewers pointed out, we did not evaluate horizontal transfer events of env-containing Ty3/gypsy elements. We consistently observed that elements found in the same phylum/class/superfamily cluster together in the POL phylogenetic tree, suggesting an ancient acquisition of env to the Ty3/gypsy elements—separation should not be as clear as we observed should they had been frequently gained from animals across different phylum/class/superfamilies. However, this does not exclude more recent horizontal transfer events that may occur between closely related species. We will perform gene-tree species-tree reconciliation analyses in clades that have enough elements and represented species to estimate the frequency of horizontal transfer events.

(2) We did not find env-containing Ty3/gypsy elements in some animal phyla such as Echinodermata and Porifera, but this could be due to the quality or number of available genome assemblies as reviewers suggested. To address this, we will mine GAG-POL gypsy elements in the genomes that were devoid of GAG-POL-ENV elements and compare their abundance with other genomes that carry GAG-POL-ENV elements. If GAG-POL gypsy elements were similarly abundantly identified, that would indicate that the observed absence of GAG-POL-ENV elements is not due to poor quality of genome assemblies.

(3) We will include F-type and HSV-gB type ENV proteins from known viruses in the phylogenetic analysis to investigate their ancestry and potential recombination events with env-containing Ty3/gypsy elements.

(4) Wherever relevant, we will clarify the terms using in the manuscript, provide rationale to our selection of POL domains used for structural and phylogenetic analyses, improve accessibility of figures, touch on gypsy elements in vertebrates, and make sure all concepts covered in the results are sufficiently introduced in the introduction.

Shakespeare’s marriage to Anne Hathaway and separation while he worked in London shows how playwrights had to balance family with career, even in the 1500s. It’s interesting how personal life may have influenced his plays.

Shakespeare is called the most famous English writer, but it’s interesting that we know so little about his life. This makes me think about how historical records shape our understanding of authors and their work.

I appreciate how the chapter points out that audiences in drama are part of the experience. Soliloquies, direct address, and even chaotic audience reactions (like the tomato story!) show how live performance creates a different kind of energy than reading. Drama is interactive in a way other genres aren’t.

This section shows why dialogue is the heart of drama. Because the narrator isn’t describing thoughts or backstory, the characters’ words and actions must reveal everything: personality, conflict, emotion, setting, and theme. It makes sense why playwrights must choose every line carefully there’s no internal monologue to fill in gaps.

I like the comparison between dialogue in novels and plays. Novels describe actions clearly, but in drama, actors interpret the stage directions and express action physically. This highlights how performance plays a huge role in meaning. The same written line could feel completely different depending on the actor’s delivery.

The explanation of acts and scenes shows how drama is organized differently than fiction. Plays have to consider practical limits like intermissions and stage transitions because they are performed live. This makes me appreciate how much planning goes into shaping each act around a key moment in the plot.

The breakdown of Aristotle’s tragic structure makes it easier to understand how tragedies create emotional responses. The idea of anagnorisis (recognition) followed by peripeteia (reversal of fortune) shows how tragedy builds tension and then releases it. This helps explain why tragedies feel so intense the protagonist realizes the truth too late.

The chapter emphasizes plot as the most important part of drama, and I like how it connects this to fiction. What stands out is the idea that plot structure isn’t fixed playwrights can shape events however they want as long as it creates an emotional effect. This reminds me that drama depends on intention more than a rigid formula.

The connection between Greek drama and religious worship is fascinating. It shows how early drama wasn’t just entertainment, but a civic and spiritual activity.

I find it interesting how the chapter says plays and novels are similar in plot and character, but playwrights have to communicate everything visually. This reminds me that drama relies more on performance than description.

How might capitalism look different (in theory or practice) if “free market” were more tightly regulated — for example, with labor protections, antitrust enforcement, or community ownership models? Does that still count as capitalism?

This quote shows that writing is about planning who will see it and how they will react to it. It taught me to think more carefully about my audience and purpose before creating a project so my message becomes clearer and more effective.

This quote stood out because it connects multimodal composing to jobs and careers. It made me realize that this type of writing is not just a class assignment but a skill used in real life. Many careers require people to use communication, technology, and visuals together, so learning this now helps students be ready for the future.

This shows where the idea of the five communication modes came from. The New London Group changed how education sees writing by explaining that meaning is created in many ways, not just through text. This helped me understand that multimodal composing has academic value and is not just something for social media.

This quote helped me understand that a “text” does not only mean a written paper. It includes things like videos, posts, and podcasts. This made me realize that I already interact with multimodal texts every day because I use social media and watch videos.

important

example of incorporating other languages into academic settings

important

language is malleable

Showcase for linguistic differences in a STEM based environment.

Promotes interaction between ethnic backgrounds.

Interesting argument for how English and math can be related.

Introduces language's role in education

Another promoter for code meshing, and cultural significance.

Euro-American representations of language.

More avocation for language spread.

Good writing point.

Another view of code meshing and an advocate of it.

Different kinds of code meshing and switching.

Racism in education, and introduces race into education.

Credibility

use introduction for essay, interesting way to showcase language diversity

this selection reflects on the different contexts of different communications

This quote speaks to acknowledging the variety of ways people communicate and the importance of it

someone's writing they are also communicating an essential part of themselves

New media forces us to rethink old theories.

Shift: audience is now real and responsive.

Print limits real-time interaction.

Classic idea: audience = mental construct.

Purpose of the article.

Returns to the central problem of identifying audience.

Emphasizing writing’s importance today.

Shows the article links audience to thinking and motivation.

Asking the core question about audience.

The author ends by reminding us that journalists will always have to rely on assumptions because audiences are too big and too complex to understand.

This helps explain why so many people avoid the news, there are easier, more entertaining choices everywhere.

This explains why people keep going back to the same news sources even when there are tons of options.

This highlights how journalists underestimate readers and don’t expect them to participate.

This reveals the bias built into newsrooms and why many communities feel misrepresented.

The author is saying journalists can’t actually see their audience anymore, so they have to guess who they’re writing for.

I didn't know that about Germany. Hopefully America can work through this.

There needs to be stronger leadership from scientists. People are more willing to support action when they trust the messenger and understand the reasoning behind the message.

This is an interesting point. To make progress on climate change, we have to start somewhere. That means offering a better alternative to what people are already doing, not just asking them to change out of principle. We definitely need a starting point though- some first step that shows people a clearer, more sustainable path forward. Problem is getting everyone to work together.

That is a crazy statistic. I didn't realize how bad the Irish famine was.

let's update this to 10000 USD

This article talks about the potential that A. I can discuss society and delve into how AI will affect the future. He targets A. I with an analogy about how A.I. in the future is like a consulting firm, which I found very interesting.

I think my views have changed in terms of how brutal social media can be and how challenging it is to monitor the content that gets posted. For example, I recall from a previous chapter about how the employees of some social media companies are scared of the things they had to see and take down on social media.

Students are more likely to learn a language style better if the teacher is more involved in supporting the students culture as a collective class instead of singling out individuals one at a time. The group setting and speaking practice helps everyone ( SAE speakers and HCE speakers) learn more and engage in their own experiences.

culturally congruent style was more effective

equality results in more cooperation and learning

the goal of KEEP research program

Involvement in facilitated direct questions has been led to more successful reading and participation in patterns for talk stories.

comprehension = relatively the same for stories in SE But HCE performed better for stories in HCE

results of reaction-timed comprehension test

BEV speakers picked up on contextual clues more than SE speakers

Def. Pedagogical: "Relating to teaching" (Oxford).

Def. interlocutors: ": one who takes part in dialogue or conversation" (Merriam Webster). Its not about how fully someone in a conversation understands the content but it is more important how they respond to the content.

"painfully obvious" Def. systematically: "a fixed plan or system" (Oxford).

AA children were considered to have less comprehension of the language

what's clear to some is unclear to others

little misunderstandings lead to large confusion

Def. "facility in using two dialects of the same language the teaching of Standard English to pupils who normally use a nonstandard dialect" (Merriam Webster).

Resistance is neccessary

Educational change could be maintenance of minorities

no real issues, if the communication is still effective

Solution

Def. "Creole, originally, any person of European (mostly French or Spanish) or African descent born in the West Indies or parts of French or Spanish America (and thus naturalized in those regions rather than in the parents’ home country). The term has since been used with various meanings, often conflicting or varying from region to region" (Brittanica)

Article Goal: insight to difficulties encountered by SESD students. This relates to BEING AWARE of the privileges that are in written work. This article is specific to the state of written work 36 years ago, so it is eye opening to what has changed and what has remained the same.

Def. Socioeconomic status: "Encompasses not only income but also educational attainment, occupational prestige, and subjective perceptions of social status and social class. SES encompasses quality-of-life attributes and opportunities afforded to people within society and is a consistent predictor of a vast array of psychological outcomes." Adapted from the APA Dictionary of Psychology

privileges vs non / the people who have a second dialect tend to do worse on their academics... Why?

Def. Prescriptivism: " the belief that there are correct and wrong ways to use language..." (Cambridge Dictionary) The black and white thinking is what breaks resistance and those who do not want to speak a specific way or write in SE.

teachers have different perspectives

results

the types of teachers

Long rejects absolute, harsh terms like "right" and "wrong" or "correct" and "incorrect" , suggesting "Better" is a more productive word for teachers. Instead of relying on rigid rules, he proposes a two-part test for evaluating disputed grammar: (1) an audience-based question ("how will the construction in question look to those who are going to see it?") and (2) a systems-based question ("how does it fit into the system of contemporary English?"). This approach shifts the goal from following abstract precepts to making effective rhetorical and systematic choices.

Long claims that the term "prescriptivism" is a "relatively new" and "derogatory word for standardization". It suggests that the academic rejection of "prescriptivism" may be a rejection of the idea of standardization itself, rather than just a rejection of specific "improper" rules.

Instead of absolute terms like "correct" or "incorrect," Long suggests a more practical evaluation. He asks writers to consider two key factors: the audience's perception and whether the language "fit[s] into the system of contemporary English". This shifts the goal from following rules to making effective rhetorical choices.

Long directly warns instructors about the negative impact of focusing on error. He states that "teachers of composition should be cautious in correcting student grammar because it is quite possible to overcorrect and make students feel insecure or alienated. This highlights the potential harm of prioritizing grammatical "correctness" over a student's confidence and the development of their authentic voice.

Long directly warns instructors about the negative impact of focusing on error. He states that "teachers of composition should be cautious in correcting student grammar because it is quite possible to overcorrect and make students feel insecure or alienated. This highlights the potential harm of prioritizing grammatical "correctness" over a student's confidence and the development of their authentic voice.

Long argues against a punitive model of grammar instruction. He claims grammar "should be taught positively, as an account of the structure of the language, not negatively, as an account of ways in which people often produce poorly constructed sentences". This supports a shift: instead of using grammar rules to punish student errors, educators should use grammar as a tool for linguistic understanding and analysis.

Long claims that the term "prescriptivism" is a "relatively new" and "derogatory word for standardization". It suggests that the academic rejection of "prescriptivism" may be a rejection of the idea of standardization itself, rather than just a rejection of specific "improper" rules.

Instead of simply enforcing "correctness," Lisabeth proposes that educators should "honor students' own language resources". Her practical solution involves a shift in teaching: rather than using a guide like The Elements of Style as a rulebook, teachers should assign it as an object for "rhetorical analysis". This, combined with reading "vernacular poetry and fiction" , helps students gain a "critical understanding of the language of power".

Lisabeth highlights the influence of this ideology by pointing out that The Elements of Style is the "number one most-assigned textbook" in the Open Syllabus Project. She argues that this widespread use, often by "well-meaning professors," shows how deeply these "racist dispositions" are "baked into American educational culture"

Citing Asao Inoue, Lisabeth claims that "all grading and assessment... that uphold singular, dominant standards that are racist and White Supremacist when used uniformly". She calls the popular belief that mastering Standard English will help students overcome social barriers the "American meritocratic myth", suggesting it's a false promise.

Lisabeth argues that modern style guides are not descended from pedagogical textbooks but from 19th-century "conversation" and etiquette guides. These guides were not about teaching language but about mapping "social, racial and gendered territory". She uses the metaphor of "racialized parlor and kitchen geography" to argue that style guides are designed to separate "correct" language (the parlor) from other "overreaching" vernaculars (the kitchen) .

Instead of simply enforcing "correctness," Lisabeth proposes that educators should "honor students' own language resources". Her practical solution involves a shift in teaching: rather than using a guide like The Elements of Style as a rulebook, teachers should assign it as an object for "rhetorical analysis". This, combined with reading "vernacular poetry and fiction" , helps students gain a "critical understanding of the language of power"

Lisabeth’s central argument is that popular, prescriptive style guides like The Elements of Style are "emblematic of the under-interrogated systemic racism of standardized English". She argues that classrooms using these guides are participating in "constructing English as White property" and implicitly underwriting "White Supremacist language" by valuing it as the only correct form.

The massive influence of this ideology by pointing out that The Elements of Style is the "number one most-assigned textbook" in the Open Syllabus Project. She argues that this widespread use, often by "well-meaning professors," shows how deeply these "racist dispositions" are "baked into American educational culture".

Citing Asao Inoue, Lisabeth claims that "all grading and assessment... that uphold singular, dominant standards that are racist and White Supremacist when used uniformly". She calls the popular belief that mastering Standard English will help students overcome social barriers the "American meritocratic myth", suggesting it's a false promise.

Lisabeth argues that modern style guides are not descended from pedagogical textbooks but from 19th-century "conversation" and etiquette guides. These guides were not about teaching language but about mapping "social, racial and gendered territory". Guides are designed to separate "correct" language (the parlor) from other "overreaching" vernaculars (the kitchen)

explain your reasoning of your ideas connecting them with your sources

Phonology is the study of how different speech sounds are organized and used in languages

The standard and conventional spelling system of writing a language.

Hello! I really appreciated this work. The parameter–sensitivity analysis is especially nice for connecting the experimental results to elucidate the role of mother–daughter asymmetry.

One question I had: you mention that the cln3Δwhi5Δ mutant shows reduced mother–daughter asymmetry compared to what the pure timer model predicts, which seems like an interesting discrepancy. Do you have any hypotheses about what biological coordination or parameter correlations might explain this?

I also wondered whether the microscopy data used for the mother–daughter asymmetry analysis might be made publicly available? I think it would be a valuable resource for others in the field.

AI helped with planning, drafting, and organizing content.

Limitations in AI output. Overreliance on AI or lack of critical engagement. Some assignments were difficult to complete effectively with AI support.

Highlights the need for AI literacy, critical thinking, and instructional support; informs educational AI design.

Students expected AI to act as a multi-tasking writing assistant, virtual tutor, and digital peer.

Investigates students’ perceptions and experiences with Generative AI (GenAI)–assisted academic writing using a ChatGPT4-embedded writing system.

goals - global governanve reform to improve emerging country participation,imporve global south corperation for inclusive and sustainable govenemnt

the expasion is as a result of the west not resposnidng to low ncome countries needs. "The west is arrogant towards the needs of the global south.

this

Daisy and Gatsby liked each other and they cared for each other.

Also serves to highlight the value of English proficiency in the world particularly for Russian students.

Demonstrates the value of English proficiency in learning and professional settings.

Emphasizes the importance of the teaching of English and English understanding as times change and English grows in use across the world.

this is mostly for fiction but reading helps people socially, emotionally, and helps to process information. The more we read the better we can communicate with one another.