This article analyzes how tweets posted from Donald Trump's Android device were significantly more negative and aggressive than those from his campaign's iPhone, which were more neutral and polished. The researchers used sentiment analysis to identify emotional differences between the two sets of tweet. I found this especially relevant to the discussion of inauthenticity in Chapter 6, because it shows that even when a social media account seems to belong to one person, multiple voices or personas may actually be behind it. This raises important ethical questions about authenticity and accountability online.

I think the story of Lonelygirl15 is particularly intriguing. At first, she shared her life on YouTube as an ordinary girl, and many people found her sincere and approachable, like a friend to confide in. But later, it was discovered that it was actually a performance, a web drama meticulously planned by actors and the production team. When the truth came out, the reaction of the audience was very strong - not only because they were deceived, but also because the feeling of "being understood" and "being connected" suddenly vanished. I can understand the emotion of betrayal because the trust we invest in the online world is, in essence, as precious as trust in real life. But looking back, I also think the phenomenon of Lonelygirl15 reminds us: Authenticity doesn't necessarily mean "complete truth". It can also be a more complex emotional experience. Even if it was a performance, the sense of companionship and resonance people gained while watching was also real. Perhaps this is the most contradictory and most human aspect of the internet age.

[f10] Regarding the “unidentified professor”: This report reveals how community skepticism (strange memorial service phone calls, no Arizona State University records) confirmed that @Sciencing_Bi was a McLaughlin scam and how the scam caused multiple harms: identity theft, community manipulation, and the discrediting of a genuine Title IX whistleblower. The name reference and institutional confirmation are powerful sources. Combined with BuzzFeed [f9] and The Verge [f13], this strengthens the timeline and mitigates the bias of a single source.

Deciding whether or not to go to college and get my bachelors was a very difficult decision, and ultimately I was influenced by my parents. My dad especially encouraged me to go to residential college and get my degree, which is also encouraged by the master narrative.

Emerging adulthood

How would this study and its findings look if conducted in relatively poorer areas of the country? What does youth culture think about post-secondary education in Alabama, Mississippi, West Virginia?

This story about Dr. McLaughlin and the fake @Sciencing_Bi account really shocked me. It made me realize how easily emotional trust can be built, and then betrayed, on social media. As someone who often reads personal stories online, I've never thought deeply about whether those stories could be fabricated. What feels most unethical here is not only the lying itself, but how it exploited people's empathy for marginalized identities and real victims of harassment. It's frightening that authenticity online now requires skepticism, and that our compassion can be used as a tool for manipulation.

This idea plays out often in short-form video content (such as TikTok, Youtube shorts, or Instagram reels). Creators like hayleyybaylee and janeinsane exemplify this idea. They made sarcastic and satirical videos that at first appear to be reality. Hayleyybaylee makes videos about a day in her life as a "billionaire's girlfriend," where she does absurd tasks around her fancy New York apartment dressed in full glam. Janeinsane makes videos about Utah moms. She exaggerates their stereotypical qualities (for instance, using Stanley cups). Both creators are playful and fun, and their audiences aren't mad that they aren't completely authentic. The purpose of their content isn't to inform or teach, it's simply to make others laugh. That is where it's excusable to let authenticity slide.

I can truly understand this statement. The feeling of being deceived is truly awful - not only because we were deceived, but also because we start to doubt our ability to make correct judgments. This reminds me of some "true stories" accounts I followed on social media earlier. Later, I discovered that they were actually fabricated. The sense of loss is deeper than just an information error. Perhaps the reason why we react so strongly to "falsehood" is that trust is an emotional investment for us. When others take advantage of this trust, we lose not only the authenticity of the information but also the sense of security between people.

Let’s answer this question: Performance itself does not compromise authenticity. As defined in this chapter, a connection is authentic when the service provided matches the actual service. If the audience can identify with a carefully crafted persona and feel the intimacy promised, the connection is authentic. If important facts are hidden (for example, through undisclosed sponsorships or deceptive practices), it becomes fake. In my research on the Bluesky bot, labeling posts as coming from a bot maintained trust and correlated the connection provided with reality.

Adler, Mortimer J. 1940. “How to Mark a Book.” Saturday Review of Literature 6: 250–52. https://www.unz.com/print/SaturdayRev-1940jul06-00011/ (January 11, 2023).

Annotations: https://via.hypothes.is/https://docdrop.org/download_annotation_doc/Adler---1940---How-to-Mark-a-Book-fehef.pdf

Annotations alternate: https://jonudell.info/h/facet/?user=chrisaldrich&max=100&exactTagSearch=true&expanded=true&url=https%3A%2F%2Fdocdrop.org%2Fdownload_annotation_doc%2FAdler---1940---How-to-Mark-a-Book-fehef.pdf

Prior [.pdf copy]9https://stevenson.ucsc.edu/academics/stevenson-college-core-courses/how-to-mark-a-book-1.pdf): - Annotations https://hypothes.is/users/chrisaldrich?q=url%3Ahttps%3A%2F%2Fstevenson.ucsc.edu%2Facademics%2Fstevenson-college-core-courses%2Fhow-to-mark-a-book-1.pdf<br /> - Alternate annotation link https://jonudell.info/h/facet/?user=chrisaldrich&max=100&exactTagSearch=true&expanded=true&url=https%3A%2F%2Fstevenson.ucsc.edu%2Facademics%2Fstevenson-college-core-courses%2Fhow-to-mark-a-book-1.pdf

Summary

• Marking a book helps in increasing "the most efficient kind of reading."
• The marked (pun intended) difference between physical vs. intellectual ownership of books
• 3 types of book owners:
  • 1. collector of wood pulp and ink
  • 1. one whose read most and dipped into some
  • 1. one who's annotated and sucked the marrow out of them
• Active reading (annotating and staying awake) and engaging deeply, arguing with, and questioning the author is the point of reading.
• A historical record of your active reading allows you to continue the conversation you've had with the author and yourself. (p12)
• Adler's method of reading and marking:
  1. Underlining major points of importance
  2. Vertical lines for emphasis
  3. Marginal marks (stars, asterisks, etc.) (10-20 per book) to indicate the most important statements in conjunction with dogearing these pages for making it easier to find them subsequently
  4. Numbers in the margin to sequence arguments
  5. Page numbers in the margin for linking ideas across pages, ostensibly for juxtaposing them later
  6. Circling key words or phrases (unsaid here, but this is helpful for indexing as well as helping one to come to terms with the author)
  7. Marginal writing for synopsis of sections as well as questions raised by the text; use of endpapers for a personal index of ideas presented chronologically throughout the book
• Objections to marking books:
  • Using scratch pad (or index cards, which he doesn't mention specifically, but which could be implied) so as not to destroy a precious or rare physical copy (this is a repetition from earlier in the article)
  • Marking slows you down. This is part of the point! Slowing down makes you engage with the author and get more out of the text.
  • You can't loan books because they contain your important thoughts which you don't want to give away (and lose the historical record of your thinking). Solution: Simply require friends to buy their own copy.

This practice is not too dissimilar to that used by zettelkasten practitioners (including Niklas Luhmann) who broadly used his bibliographic cards this way.

By separating his index and ideas from the book and putting them into a physical index, it makes them easier to juxtapose with other ideas over time rather than having them anchored directly to the book itself. For academics and researchers, this will tend to help save time from having to constantly retrieve these portions from individual books.

This poke at "fancy" bookplates is a rhetorical call back to those who would attempt to weakly show only physical and not intellectual ownership by "pasting his bookplate inside the cover."

Was "fancy thinking" used in a pejorative sense prior to this article? Are subsequent uses of it poking fun of the parallelism (to fancy bookplates) which Adler sets up here?

The making of a personal index is a first step in building a mesh of knowledge. In just a few years, Vannevar Bush will speak of "associative trails" a phrase he uses twice in "As We May Think" (The Atlantic, July 1945), but of potentially more import is his phrase "associative indexing" which lays way to either juxtaposing or linking two ideas (either similar or disjoint) together. It bears asking the question of of whether it's more valuable to index and juxtapose similar ideas or disjoint ideas which may more frequently lead to better, more useful, and more relevant and rich future ideas.

It affords an immediate step, however, to associative indexing, the basic idea of which is a provision whereby any item may be caused at will to select immediately and automatically another. This is the essential feature of the memex. The process of tying two items together is the important thing. Bush, Vannevar. 1945. “As We May Think.” The Atlantic 176: 101–8. https://www.theatlantic.com/magazine/archive/1945/07/as-we-may-think/303881/ (October 22, 2022). #

Adler creates a very specific and subtle definition of ownership as it applies to books.

It's not too dissimilar to Antoine de Saint-Exupéry's idea of ownership or love of people. "Unique to me in all the world"

Many have spoken of "books as wallpaper" or "intellectual furniture", but here Mortimer J. Adler goes beyond owning them solely as material culture, but turning them into intellectual and personal culture.

When they sit upon the shelf after being intellectually owned, they can serve as a mnemonic touchstone, which is a method of supercharging their value as lowly "decorative wallpaper", and instead making them living active, intellectual wallpaper.

:

The frontal lobe controls decisions making emotion and behavior damage here can change who a person is.

After the accident, Gage's personality changed. he became impulsive and had trouble controlling emotions.

A railroad worker who survived when an iron rod went through his skills in 1848

It helps produce speech. damage here makes it hard to talk or form words.

Vision and visual processing

Hearing,memory, and understanding language

Touch,pressure, and spatial sense

Reasoning, planning, movement. and speech

Midbrain- helps process movement, hearing, and vision

controls thinking, emotions,memory, and decision making

they take this matter very seriously china has this document in it goverment website

in china playbook

I really agree with this idea that critique should be two-way. In many classroom or work settings, feedback feels one-sided — someone tells you what’s wrong, and you just listen. But when designers explain their rationale, it opens up a more meaningful conversation. I found Ko’s framing useful because it reminds me that critique is about growth and understanding, not just judgment. It changes my perspective on feedback — instead of feeling defensive, I can see it as a collaborative dialogue to refine ideas together.

I think this section nails something that goes way beyond design. It’s just good communication in general. I agree that giving balanced feedback forces you to slow down and actually see what’s working instead of jumping straight to criticism. The “shit sandwich” rule might sound funny, but it’s surprisingly useful, especially in group projects where people can get defensive fast. It made me realize that how feedback is delivered can decide whether an idea improves or dies.

Was this really yesterday?

/hyperpost/🌐/🎭/gyuri/do/web/snarf/📅/2025/10/2/Metaphysics.of.Adjacency.html

self

view

perspectives viewpoints future perfect orientation

recontextualization

meaning/intent-fully named associative reticulate complexes spanning their own meaningful contexts or rather connected conPlexes

the Nameless Book of Heaven - myth metaphor and mystery

Its measure electrical activity. They use a array of electrodes around a person's head

Measures the blood flow and oxygen levels

Best showing for structure (anatomical detail)

once in the bloodstream the tracer's movement is monitored

a technique that takes a number of x-rays of a particular section of a person's body or brain

are we sure?

When Iago says "Call up her father," it is the beginning of his manipulation and how he gets others to be tools of his own cunning devices. On the surface, the line can be interpreted as simply an order, but in fact, it shows how cunning and devious Iago is. Instead of confronting Othello or Desdemona himself, he uses Roderigo to set things on fire for him. By waking Brabantio at midnight, Iago knows that he will be provoking anger, confusion, and fright, emotions that make humans vulnerable to manipulation. This is significant because it makes clear that Iago's evil does not result from aggression but from his ability to provoke reaction and manipulate feelings. He is aware of how to use timing and fright to make fighting break out in moments of peace, and this line alone is the start of that. It also shows one of the prevailing motifs of Othello: the idea that destruction is oftentimes started quietly in suggestion and manipulation and not in outright brutality. By having others "call up" the trouble for him, Iago gets to preserve his good name while the damage is being done, highlighting how risky lying can be when clothed in peaceful words.

See also the precursor of personal indexing which Mortimer J. Adler mentions in 1940: https://hypothes.is/a/cPcoAqhVEfC0rJOZ0Pm-8Q

academic brained? I don’t mean that too harshly but: social context matters!! people find formulas meaningful because they are literally performative not because of their novelty

Comment 2

Comment 1

Note 1

want it to be true

This goes back to the theme and discussion from last week on centering care and creating a brave space.

The story of Pheblas in “Death by Water” mirrors that of Ulysses in Tennyson’s poem under the same name. In both stories, the men are drawn to the water, to a life of adventure despite their old age. Ulysses, through all of his experiences is “a part of all that [he has] met,” existing as all prior parts of himself. Furthermore, as he lives his last days, thinking about how little of him there is left, he sees salvation in the “eternal silence” and seeks to follow this like a “sinking star.” The idea of a “sinking star” inverts the typical shooting star – a symbol of hope and wishes. The “sinking star” then becomes a symbol of inevitability. Additionally, in a story connected to the sea in all ways, the “sinking” also acts as a reference to drowning – sinking and dying in the exact place Ulysses yearns to return to. Similarly, in “Death by Water” “Phlebas the Phoenician” (which is a direct reference back to Madame Sosostris’s “drowned Phoenician Sailor”) dies in the water. The lines “As he rose and fell/He passed the stages of his age and youth/Entering the whirlpool” showcase a similar phenomenon to Ulysses, passing through all previous versions to himself. Even his rising and falling harks back to Ulysses’ sinking. The parallel between the two men, who were both once young and handsome, sacrificed to the water that once sustained them is an interesting image.

Is this working?

西班牙裔

eLife Assessment

This important manuscript evaluates how sample size and demographic balance of reference cohorts affect the reliability of normative models. The evidence supporting the conclusions is convincing, although some additional analysis and clarifications could improve the generalisability of the conclusions. This work will be of interest to clinicians and scientists working with normative models.

Reviewer #1 (Public review):

Summary:

Overall, this is a well-designed and carefully executed study that delivers clear and actionable guidance on the sample size and representative demographic requirements for robust normative modelling in neuroimaging. The central claims are convincingly supported.

Strengths:

The study has multiple strengths. First, it offers a comprehensive and methodologically rigorous analysis of sample size and age distribution, supported by multiple complementary fit indices. Second, the learning-curve results are compelling and reproducible and will be of immediate utility to researchers planning normative modelling projects. Third, the study includes both replication in an independent dataset and an adaptive transfer analysis from UK Biobank, highlighting both the robustness of the results and the practical advantages of transfer learning for smaller clinical cohorts. Finally, the clinical validation ties the methodological work back to clinical application.

Weaknesses:

There are two minor points for consideration:

(1) Calibration of percentile estimates could be shown for the main evaluation (similar to that done in Figure 4E). Because the clinical utility of normative models often hinges on identifying individuals outside the 5th or 95th percentiles, readers would benefit from visual overlays of model-derived percentile curves on the curves from the full training data and simple reporting of the proportion of healthy controls falling outside these bounds for the main analyses (i.e., 2.1. Model fit evaluation).

(2) The larger negative effect of left-skewed sampling likely reflects a mismatch between the younger training set and the older test set; accounting explicitly for this mismatch would make the conclusions more generalisable.

Reviewer #2 (Public review):

Summary:

The authors test how sample size and demographic balance of reference cohorts affect the reliability of normative models in ageing and Alzheimer's disease. Using OASIS-3 and replicating in AIBL, they change age and sex distributions and number of samples and show that age alignment is more important than overall sample size. They also demonstrate that models adapted from a large dataset (UK Biobank) can achieve stable performance with fewer samples. The results suggest that moderately sized but demographically well-balanced cohorts can provide robust performance.

Strengths:

The study is thorough and systematic, varying sample size, age, and sex distributions in a controlled way. Results are replicated in two independent datasets with relatively large sample sizes, thereby strengthening confidence in the findings. The analyses are clearly presented and use widely applied evaluation metrics. Clinical validation (outlier detection, classification) adds relevance beyond technical benchmarks. The comparison between within-cohort training and adaptation from a large dataset is valuable for real-world applications.

The work convincingly shows that age alignment is crucial and that adapted models can reach good performance with fewer samples. However, some dataset-specific patterns (noted above) should be acknowledged more directly, and the practical guidance could be sharper.

Weaknesses:

The paper uses a simple regression framework, which is understandable for scalability, but limits generalization to multi-site settings where a hierarchical approach could better account for site differences. This limitation is acknowledged; a brief sensitivity analysis (or a clearer discussion) would help readers weigh trade-offs. Other than that, there are some points that are not fully explained in the paper:

(1) The replication in AIBL does not fully match the OASIS results. In AIBL, left-skewed age sampling converges with other strategies as sample size grows, unlike in OASIS. This suggests that skew effects depend on where variability lies across the age span.

(2) Sex imbalance effects are difficult to interpret, since sex is included only as a fixed effect, and residual age differences may drive some errors.

(3) In Figure 3, performance drops around n≈300 across conditions. This consistent pattern raises the question of sensitivity to individual samples or sub-sampling strategy.

(4) The total outlier count (tOC) analysis is interesting but hard to generalize. For example, in AIBL, left-skew sometimes performs slightly better despite a weaker model fit. Clearer guidance on how to weigh model fit versus outlier detection would strengthen the practical message.

(5) The suggested plateau at n≈200 seems context-dependent. It may be better to frame sample size targets in relation to coverage across age bins rather than as an absolute number.

Author response

We would like to thank the editors and two reviewers for the assessment and the constructive feedback on our manuscript, “Toward Robust Neuroanatomical Normative Models: Influence of Sample Size and Covariates Distributions”. We appreciate the thorough reviews and believe the constructive suggestions will substantially strengthen the clarity and quality of our work. We plan to submit a revised version of the manuscript and a full point-by-point response addressing both the public reviews and the recommendations to the authors. 

Reviewer 1. 

In revision, we plan to address the reviewer’s comments by: (i) strengthen the interpretation of model fit through reporting the proportion of healthy controls within and outside the extreme percentile bounds; (ii) adding age-resolved overlays of model-derived percentile curves compared to those from the full reference cohort for key sample sizes and regions; (iii) quantifying age-distribution alignment between train and test set; and (iv) summarizing model performance as a joint function of age-distribution alignment and sample size.

Reviewer 2. 

In the revised manuscript, we will (i) expand the Discussion to more clearly outline the trade-offs between simple regression frameworks and hierarchical models for normative modeling (e.g., scalability, handling of multi-site variation, computational considerations), and discuss alternative approaches and harmonization as important directions for multi-site settings; (ii) contextualize OASIS-3 vs AIBL differences by quantifying train– test age-alignment across sampling strategies and emphasize that skewness should be interpreted relative to the target cohort’s alignment rather than absolute numbers. (iii) reassess sex-imbalance effects by reporting expected age distributions per condition and re-evaluate sex effects while controlling for age; (iv) investigate the apparent dip at n≈300 dip by increasing sub-sampling seeds, testing neighboring sample sizes, and using an alternative age-binning scheme to clarify the observed artifact; (v) clarify potential divergence between tOC separation and global fit under discrepancies in demographic distributions and relate tOC to age-alignment distance; (vi)  reframe the sample-size guidance in terms of distributional alignment rather than an absolute n.

ეს სტატია შეაჯამებს შვეიცარიაში მიმდინარე ბიომრავალფეროვნების კრიზისს და იმ გამოწვევებს, რომლებსაც ქვეყანა ხვდება დაცული ტერიტორიების გაფართოებისას. შვეიცარიამ გაამახვილა ყურადღება გარემოს დაცვის აუცილებლობაზე,თუმცა ქვეყნის დაცული ტერიტორიების ფართობი არ აკმაყოფილებს საერთაშორისო მიზნებს. სტატია მიანიშნებს ,რომ შვეიცარიის დაცული ტერიტორიების ფართობი მხოლოდ 6.6%-დან 13.4% პროცენტამდე მერყეობს ,რაც დაბალია 2020 წლისთვის დაწესებულ 17%იან მიზანთან შედარებით . სტატიიის ერთ-ერთი მთავარი თემააა 2016 წელს უშედეგოდ დასრულებული PARC ADULA პროექტი , რომელიც მიზნად ისახავდა ბიომრავალფეროვნების დაცვას და ადგილობრივი ეკონომიკის გაძლიერებას. თუმცა, ადგილობრივმა მოსახლეობამ დაგმო პროექტი , ტრადიციული საქმიანობის ხელშეშლის მიზეზით, როგორიცაა ნადირობა და თევზაობა. სტატია ხაზს უსვამს, რომ შვეიცარიის პოლიტიკა ადგილობრივ თვითმმართველობების ავტონომიას პრიორიტეტს აძლევს , რაც ართლებს ზოგად-ეროვნულ ინიციატივებს, როგორიცაა დაცული ტერიოტრიების გაფართოება. სტატიის ბოლო ნაწილი გვეუბნება , რომ შვეიცარიის მიზანია დაცული ტერიტორების გაფართოება, რისთვისაც საჭიროა ადგილობრივი თემების მხარდაჭერა, ცნობიერების ამაღლება და ეროვნული ინტერესესბის დაცვა.

eLife Assessment

This important paper reports the development of proteins and small molecules that induce degradation of a clinically-relevant oncogenic transcription factor, LMO2. The findings provide a proof of concept that PROTAC-type chemicals can be developed against intrinsically disordered proteins. The methods provide a blueprint for rational design of PROTACs starting from intracellular antibody paratopes. Overall, the paper is supported by solid evidence and will be of interest to chemical biologists and cancer pharmacologists.

Reviewer #2 (Public review):

Summary:

Sereesongsaeng et al. aimed to develop degraders for LMO2, an intrinsically disordered transcription factor activated by chromosomal translocation in T-ALL. The authors first focused on developing biodegraders, which are fusions of an anti-LMO2 intracellular domain antibody (iDAb) with cereblon. Following demonstrations of degradation and collateral degradation of associated proteins with biodegraders, the authors proceeded to develop PROTACs using antibody paratopes (Abd) that recruit VHL (Abd-VHL) or cereblon (Abd-CRBN). The authors show dose-dependent degradation of LMO2 in LMO2+ T-ALL cell lines, as well as concomitant dose-dependent degradation of associated bHLH proteins in the DNA-binding complex. LMO2 degradation via Abd-VHL was also determined to inhibit proliferation and induce apoptosis in LMO2+ T-ALL cell lines.

Strengths:

The topic of degrader development for intrinsically disordered proteins is of high interest and the authors aimed to tackle a difficult drug target. The authors evaluated methods including the development of biodegraders, as well as PROTACs that recruit two different E3 ligases. The study includes important chemical control experiments, as well as proteomic profiling to evaluate selectivity.

Weaknesses:

Several weaknesses remain in this study:

(1) The overall degradation achieved is not highly potent (although important proof-of-concept);

(2) The mechanism of collateral degradation is not completely addressed. The authors acknowledge possible explanations, which would require mutagenesis and structural studies to further dissect;

(3) The proteomics experiments do not detect LMO2, which the authors attribute to its size, making it difficult to interpret.

Author response:

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

Reviewer #1 (Public review): 

Summary:

The authors describe the degradation of an intrinsically disordered transcription factor (LMO2) via PROTACs (VHL and CRBN) in T-ALL cells. Given the challenges of drugging transcription factors, I find the work solid and a significant scientific contribution to the field. 

Strengths: 

(1) Validation of LMO2 degradation by starting with biodegraders, then progressing to chemical degrades. 

(2)interrogation of the biology and downstream pathways upon LMO2 degradation (collateral degradation §

(3) Cell line models that are dependent/overexpression of LMO2 vs LMO2 null cell lines. 

(4) CRBN and VHL-derived PROTACs were synthesized and evaluated. 

Weaknesses: 

(1) The conventional method used to characterize PROTACs in the literature is to calculate the DC50 and Dmax of the degraders, I did not find this information in the manuscript. 

As noted in the reply to referee’s point 4 below, our first generation compounds are not highly potent. The DC₅₀ values have been computed specifically using Western blot reflected in the data shown in Fig. 2. The revised version Supplementary Fig. S3 shows these quantified Western blot data from a time course of treating KOPT-K1 cells with either Abd-CRBN and Abd-VHL, where the 24 hour blot data are shown in Figure 2, G and E, and the quantified data from each 24 hour treatment are quantified in Supplementary Fig. S3). With these data, the DC₅₀ values 9 μM for Abd-CRBN and 15 μM Abd-VHL), included in in the main text and the Supplementary Fig. S3 figure legend.

In addition, the loss of signal of the LMO2-Rluc reporter protein from PROTAC treated cells shown in Fig. 2M has been used to calculate a half-point of degradation; although strictly not DC₅₀, as it measures a reporter protein, this yielded values are 10 μM for Abd-CRBN and 9 μM Abd-VHL. 

(2) The proteomics data is not very convincing, and it is not clear why LMO2 does not show in the volcano plot (were higher concentrations of the PROTAC tested? and why only VHL was tested and not CRBN-based PROTAC?).

Due to the relatively small size of the LMO2 protein, it is challenging to produce enough unique peptides for reliable identification, especially to distinguish some proteins in the LMO2 complex.  

(3) The correlation between degradation potency and cell growth is not well-established (compare Figure 4C: P12-Ichikawa blots show great degradation at 24 and 48 hrs, but it is unclear if the cell growth in this cell line is any better than in PF-382 or MOLT-16) - Can the authors comment on the correlation between degradation and cell growth?  

In this study (Fig. 4) we did not aim to compare the effect of LMO2 loss on cell growth among LMO2 positive cells. Rather, we aimed to evaluate the LMO2 importance for cell growth in LMO2-expressing T-ALL cells compared to non-expressing cells and to correlate the loss of the protein with this effect on the cell growth. In addition, the treatment of cells with the LMO2 compounds did now show an effect to LMO2 negative cells until at least 48 hours of treatment indicating that low toxicity of our PROTAC compounds and providing correlation between LMO2 loss and cell growth. 

(4) The PROTACs are not very potent (double-digit micromolar range?) - can the authors elaborate on any challenges in the optimization of the degradation potency? 

The Abd methodology to use intracellular domain antibodies to screen for compounds that bind to intrinsically disordered proteins such as the LMO2 transcription factors offers a tractable approach to hard drug targets but, in so doing, creates challenging factors to improve the potency that are not the same as those targets for which structural data are available. LMO2 is an intrinsically disordered protein, for which soluble recombinant protein is not readily available to identify the binding pocket of compounds. The potency has so far been optimized solely based on the different moieties substituted in cell-based SAR studies (http://advances.sciencemag.org/cgi/content/full/7/15/eabg1950/DC1) and all new compounds were tested with BRET assays. Thus, currently optimization of the degradation potency (including properties such as improved solubility) for the LMO2-binding compounds relies on chemical modification the three areas of the compounds indicated in Fig. 2 B,C.  

(5) The authors mentioned trying six iDAb-E3 ligase proteins; I would recommend listing the E3 ligases tried and commenting on the results in the main text. 

The six chimaeric iDAb-E3 ligase proteins involved one anti-LMO2 iDAb and three different E3 ligase where either fused at the N- or the C-terminus of the VH (giving six protein formats). These six fusion proteins were described in the text referring to the degrader studies described in Supplementary Fig. 1. 

Reviewer #2 (Public review): 

Summary: 

Sereesongsaeng et al. aimed to develop degraders for LMO2, an intrinsically disordered transcription factor activated by chromosomal translocation in T-ALL. The authors first focused on developing biodegraders, which are fusions of an anti-LMO2 intracellular domain antibody (iDAb) with cereblon. Following demonstrations of degradation and collateral degradation of associated proteins with biodegraders, the authors proceeded to develop PROTACs using antibody paratopes (Abd) that recruit VHL (Abd-VHL) or cereblon (Abd-CRBN). The authors show dose-dependent degradation of LMO2 in LMO2+ T-ALL cell lines, as well as concomitant dose-dependent degradation of associated bHLH proteins in the DNA-binding complex. LMO2 degradation via Abd-VHL was also determined to inhibit proliferation and induce apoptosis in LMO2+ T-ALL cell lines. 

Strengths: 

The topic of degrader development for intrinsically disordered proteins is of high interest, and the authors aimed to tackle a difficult drug target. The authors evaluated methods, including the development of biodegraders, as well as PROTACs that recruit two different E3 ligases. The study includes important chemical control experiments, as well as proteomic profiling to evaluate selectivity. 

Weaknesses: 

The overall degradation is relatively weak, and the mechanism of potential collateral degradation is not thoroughly evaluated

The purpose of the study was to evaluate effects of LMO2 degraders. The mechanism of the observed collateral degradation could not be investigated directly within the scope of our study. In the main text, discussed two possible, not exclusive, explanations. One being that our work (and previously published, cited work) indicates that the DNA-binding bHLH proteins have relatively short half file (Supplementary Fig. S12) and may therefore be subject to normal turnover when the LMO2, which is in the complex, turns over. Further, the known structure of the LMO2-bHLH interactions (from Omari et al, doi: 10.1016/j.celrep.2013.06.008) was also examined for the location of lysines in the TAL1 & E47 partners (Supplementary Fig. S11). It is possible that their local association with the LMO2-E3-ligase complex created by the PROTAC interaction, could cause their concurrent degradation. Mutagenesis and structural analysis would be needed to establish this point.

In addition, experiments comparing the authors' prior work with their anti-LMO2 iDAb or Abl-L are lacking, which would improve our understanding of the potential advantages of a degrader strategy for LMO2.  

A major motivation behind developing the Antibody-derived (Abd) method to select compounds, which are surrogates of the antibody paratope, is because using iDAbs directly as inhibitors requires the development of delivery technologies for these macromolecules, as protein directly or as vectors or mRNA for their expression. Ultimately, high affinity anti-LMO2 iDAbs should directly be used as tractable inhibitors when delivery methods redeveloped. In the meantime, Abd compounds were envisaged as being surrogates suitable for development into reagents, and potentially drugs, by medicinal chemistry. We evaluated selected first generation LMO2-binding Abd compounds previously, finding their ability to interfere with LMO2-iDAb BRET signal to EC_max about 50% but these compounds do not have potency to have an effect on the interaction of LMO2 with a non-mutated iDAb (nM affinity). These data indicated that efficacy improvement for the PROTACs was needed. In addition, in the current study, we observed viability effects in T-ALL lines at high concentrations (20 μM) irrespective of LMO2 expression (Supplementary Fig. S 2A, B) These data indicated that efficacy improvement was needed and potentially converting the degraders (PROTACs) would add to in-cell potency. By adding the E3 ligase ligands, we found the toxicity of non-LMO2 expressing Jurkat was significantly reduced (Supplementary Fig. S 2E, F). 

Reviewer #2 (Recommendations for the authors): 

Suggestions for additional experiments: 

(1) The data presented is primarily focused on demonstrating targeted degradation of LMO2, with a focus on phenotypes such as proliferation and apoptosis. In this manuscript, there are limited comparative evaluations of anti-LMO2 iDAb or Abl-L to show the potential benefits of a degrader approach to their previously described work, as well as why targeted degradation is in fact, advantageous. For example, the authors' previous work has shown that anti-LMO2 iDAb inhibits tumor growth in a mouse transplantation model. Comparisons in vitro would be supportive of the importance of continued degrader optimization/development.  

we have previously shown that an anti-LMO2 scFv inhibits tumour growth in a mouse model but this work used an expressed scFv antibody that binds to LMO2 in nM range. The Abd compounds are much lower potency that the antibody and, because recombinant LMO2 is difficult to work with, we could only evaluate interactions of compounds with LMO2 in cell-based assays like BRET (LMO2-iDAb BRET). In this cell-based assay, the first generation Abd compounds do not have sufficient potency to block LMO2-iDAb interaction unless the affinity of the iDAb is reduced to sub-μM. The justification for proceeding on the degrader process rather than just using the protein-protein interaction (PPI) inhibition was based largely around the low potency of the first generation PPI compounds in cell assays and that incorporation protein degradation with PPI inhibition would enhance the efficacy.

In addition, the viability experiments are also very short-term; is there a reason why the authors did not carry out these experiments for 3-5 days to fully understand the impacts on proliferation? 

In Supplementary Fig. S5, we did show assays up to 3 days. In KOPT-K1 (LMO2+), the LMO2 levels were reduced during the time course of this assay (from a single compound dose at time zero) (Supplementary Fig S 5A, B). We also show CellTitreGlo assays up to 3 days and, with these second generation compounds, we observed sustained effects on KOPT-K1 (LMO2+) but low non-DMSO toxicity in Jurkat (LMO2-) (revised version Supplementary (Fig S5 C, D).

(2) The potential mechanism of collateral degradation is interesting and important in evaluating the on-target responses and consequences of degrading LMO2. At this time, the data supporting collateral degradation is limited and would be strengthened by showing that it is not due to a change in mRNA levels and not due to complex dissociation. Overall, the kinetics and depth of loss of complex members such as E47 in Figure 3 appear more substantial than LMO2 itself, and as presented, collateral degradation is not effectively demonstrated. In addition, to aid in the readers' assessments, additional background and references around the roles of TAL1 and E47 would be helpful. For example, structurally, where do they (and other associated proteins that are not degraded) fit in the complex? 

We have responded above in relation to the Public Review Comments and note that a structure of the complex was in submitted version (now revised version Supplementary Fig. S11). 

(3) In Figure 1A, the blots show decreased levels of endogenous CRBN with iDAB-CRBN. Is this a known consequence of this approach in these cell lines? Does the partial recovery of endogenous CRBN in KOPTK1 cells have any indication of iDAB-CRBN levels? 

We cannot be sure why the endogenous level of CRBN decreases in doxycycline treated cells. It has been shown (DOI:10.1371/journal.pone.0064561) that doxycycline used in the inducible expression system (and its derivatives), such as the lentivirus we used, has an effect to gene expression patterns, which can be increase or decrease expression. Although the published study did not examine CRBN expression, the effect might explain the CRBN expression decrease on doxycycline addition and remains the same level after that. 

(4) In Figure S7, the authors do not fully explain the results and why there is minimal rescue with epoxomicin (S7A) or MLN4924 (S7J). This could indicate an alternative mechanism of degradation and loss at play, given the lack of rescue. Can the authors comment on this discrepancy, and have they looked autophagy inhibitor or other agents to achieve the chemical rescue? 

In the experiments such as in revised version Supplementary Fig. S6, we used KOPT-K1 cells with a single concentration of the inhibitors and the cells may less susceptible to the epoxomicin (0.8 μM) but lenalidomide and free thalidomide restored the LMO2 levels fully. In the main text Fig. 3D, we also showed that including epoxomicin and thalidomide with the Abd-CRBN in KOPT-K1 and CCRF-CEM restore LMO2 levels, supporting the conclusion that the main mechanism of degradation is through ubiquitination proteosomal route.

(5) For the proteomics data, it would be helpful to have the proteins in yellow highlighted to have them noted in 5D and 5E. In addition, can the authors comment on why LMO2 or their collateral targets are not confirmed in the table? Furthermore, 5C is difficult to interpret; if there are no significantly changing proteins in the Jurkat cells, why are there pathways that are identified? 

As mentioned in reply to referee 1, due to the relatively small size of the LMO2 protein, it is challenging to produce enough unique peptides for reliable identification, especially to distinguish some proteins in the LMO2 complex where expression levels are low.

This confuses me because at the time they had slaves yet he is saying all men are originally equal, so then why do they have slaves if they are equal? I also wonder if he just included this because it would make him appeal to more people as to get as many people on his side as possible.

Paine's goal in saying this is to get the colonist to rally together and to unite as one against Britain. He is saying how the king is a tyrant and is slandering him pretty much so he can get people to join him.

He is telling the colonist to get ready and to be excited for the new world when they break free from Britain. He is saying that it will be a safe haven for freedom. The fugitive means freedom and he is telling the colonist to prepare to have freedom.

This paper is written for the people of the colonies to raise up against the Brits. His goal is also to give them a spark to push for independence. Through this paper he tries to give them some hope and reasons as to why they should break away.

Historical Context- He is talking to the colonist about how because of there being kings they caused wars by being too greedy. His goal in saying this is to turn the colonist against having a king and breaking away from the kings rule in Britain.

A Tumult is loud and confused noise, especially one caused by a large amount of people.

I have a question about this paper as to what he means there were no kings in the beginning? Because if they are christians like I thought the British all were then wouldn't he believe that Jesus was the king of men?

This was a weird word to me but it means to root out and destroy completely.

The Author of this document is Thomas Paine, it was written in 1776. This document is important because it was a very huge part of the colonies actually splitting from Britian.

Not sure we need this separate page. The map can be embedded at the bottom of the Repository page.

Add 3 sections which link to other pages: 1. Publish Open Access 2. Share your Work in Purdue e-Pubs/ the Repository 3. Publish with Libraries

"ua maila" is translated "it is calm." Not sure how this was arrived at. To me, it looks like

"In the stillness of the birds, [because it has just] rained."

2.

Human Needs Theory directly conflicts with Survival of the fittest as it argues that aggression is not stemmed from innate human behavior, but rather, they stem from social circumstances that do not satisfy human needs.

eLife Assessment

This valuable study concerns a model for transgenerational epigenetic inheritance, the learned avoidance by C. elegans of the PA14 pathogenic strain of Pseudomonas aeruginosa. A recent study questioned whether transgenerational inheritance in this paradigm lacks robustness. The authors of this study have worked independently of the group that reported the original phenomenon and also independently of the group that challenged the original report. With solid data, this study independently validates findings previously reported by the Murphy group, confirming that the paradigm is reproducible elsewhere. The reviewers also appreciated the information on reagent sources used by different groups. The present study is therefore of broad interest to anyone studying genetics, epigenetics, or learned behavior.

Reviewer #1 (Public review):

Summary:

The manuscript addresses the discordant reports of the Murphy (Moore et al., 2019; Kaletsky et al., 2020; Sengupta et al., 2024) and Hunter (Gainey et al., 2025) groups on the existence (or robustness) of transgenerational epigenetic inheritance (TEI) controlling learned avoidance of C. elegans to Pseudomonas aeruginosa. Several papers from Colleen Murphy's group describe and characterize C. elegans transgenerational inheritance of avoidance behaviour. In the hands of the Murphy group, the learned avoidance is maintained for up to four generations, however, Gainey et al. (2025) reported an inability to observe inheritance of learned avoidance beyond the F1 generation. Of note, Gainey et al used a modified assay to measure avoidance, rather than the standard assay used by the Murphy lab. A response from the Murphy group suggested that procedural differences explained the inability of Gainey et al.(2025) to observe TEI. They found two sources of variability that could explain the discrepancy between studies: the modified avoidance assay and bacterial growth conditions (Kaletsky et al., 2025). The standard avoidance assay uses azide as a paralytic to capture worms in their initial decision, while the assay used by the Hunter group does not capture the worm's initial decision but rather uses cold to capture the location of the population at one point in time.

In this short report, Akinosho, Alexander, and colleagues provide independent validation of transgenerational epigenetic inheritance (TEI) of learned avoidance to P. aeruginosa as described by the Murphy group by demonstrating learned avoidance in the F2 generation. These experiments used the protocol described by the Murphy group, demonstrating reproducibility and robustness.

Strengths:

Despite the extensive analyses carried out by the Murphy lab, doubt may remain for those who have not read the publications or for those who are unfamiliar with the data, which is why this report from the Vidal-Gadea group is so important. The observation that learned avoidance was maintained in the F2 generation provides independent confirmation of transgenerational inheritance that is consistent with reports from the Murphy group. It is of note that Akinosho, Alexander et al. used the standard avoidance assay that incorporates azide, and followed the protocol described by the Murphy lab, demonstrating that the data from the Moore and Kaletsky publications are reproducible, in contrast to what has been asserted by the Hunter group.

Comments on revised version:

I am happy with the responses to reviews.

Reviewer #2 (Public review):

Summary:

The manuscript "Independent validation of transgenerational inheritance of learned pathogen avoidance in C. elegans" by Akinosho and Vidal-Gadea offers evidence that learned avoidance of the pathogen PA14 can be inherited for at least two generations. In spite of initial preference for the pathogen when exposed in a 'training session', 24 hours of feeding on this pathogen evoked avoidance. The data are robust, replicated in 4 trials, and the authors note that diminished avoidance is inherited in generations F1 and F2.

Strengths:

These results contrast with those reported by Gainey et al, who only observed intergenerational inheritance for a single generation. Although the authors' study does not explain why Gainey et el fail to reproduce the Murphy lab results, one possibility is that a difference in a media ingredient could be responsible.

Comments on revised version:

The responses to the reviewer comments appear reasonable for the most part.

Reviewer #3 (Public review):

Summary:

This short paper aims to provide an independent validation of the transgenerational inheritance of learned behaviour (avoidance) that has been published by the Murphy lab. The robustness of the phenotype has been questioned by the Hunter lab. In this paper, the authors present one figure showing that transgenerational inheritance can be replicated in their hands. Overall, it helps to shed some light on a controversial topic.

Strengths:

The authors clearly outline their methods, particularly regarding the choice of assay, so that attempting to reproduce the results should be straightforward. It is nice to see these results repeated in an independent laboratory.

Comments on revised version:

I'm happy with the response to reviewers.

Author response

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

Reviewer #1 (Public Review):

Confirmation of daf-7::GFP data and inheritance beyond F2

Reviewer suggested confirming daf-7::GFP molecular marker data and testing inheritance beyond the F2 generation to further strengthen the findings.

We agree these experiments would provide valuable mechanistic insights into the molecular basis of transgenerational inheritance. However, our study was specifically designed as a reproducibility study focusing on the central controversy regarding F2 inheritance (Gainey et al. vs. Murphy lab findings). The daf-7::GFP molecular marker experiments, while important for understanding mechanisms, represent a different research question requiring extensive additional resources and expertise beyond the scope of this validation study. Our primary goal was to provide independent confirmation of the disputed F2 inheritance using standardized behavioral assays. It is our hope that future work will pursue these important mechanistic validations.

"Exhaustive attempts" language

Reviewer disagreed with characterizing Gainey et al.'s efforts as "exhaustive attempts" since they modified the original protocol.

We revised this statement in the Results and Discussion to more accurately reflect the experimental situation: "In contrast, Gainey et al. (2025), representing the Hunter group, reported that while parental and F1 avoidance behaviors were evident, transgenerational inheritance was not reliably observed beyond the F1 generation under their experimental conditions."

Importance of sodium azide

Reviewer suggested including more discussion about the recent findings on the importance of sodium azide in the assay, referencing the Murphy group's response paper.

We have prominently highlighted the critical role of sodium azide in our Introduction with strengthened language that emphasizes its importance for resolving the scientific controversy: "Critically, Kaletsky et al. (2025) demonstrated that omission of sodium azide during scoring can completely abolish detection of inherited avoidance, revealing that this key methodological difference may explain the conflicting results between laboratories. The use of sodium azide to immobilize worms at the moment of initial bacterial choice appears essential for capturing the inherited behavioral response. These findings highlight how seemingly minor methodological variations can dramatically impact detection of transgenerational inheritance and underscore the need for independent replication using standardized protocols."

Protocol fidelity statement

Reviewer requested a more direct statement clarifying that we followed the Murphy group protocol, noting that we made some modifications.

We followed the core Murphy lab protocol with two evidence-based optimizations that preserve the essential experimental elements: 1) We used 400 mM sodium azide instead of 1 M based on preliminary data showing the higher concentration caused premature paralysis before worms could make behavioral choices, and 2) We used liquid NGM buffer instead of M9 to maintain chemical consistency with the solid NGM plates used for worm culture, minimizing potential osmotic stress. These modifications improved experimental reliability while maintaining the critical components: sodium azide immobilization, bacterial lawn density standardization (OD₆₀₀ = 1.0), and synchronized scoring conditions that are essential for detecting inherited avoidance.

Overstated dilution claim

Reviewer noted that the statement about "gradual decrease" in avoidance strength was overstated and didn't reflect the actual data presented in the manuscript.

We removed this statement.

Environmental variables phrasing

Reviewer found the sentence about environmental variables unclear, noting that Gainey et al. didn't actually acknowledge variability but saw it as indicating error or stochastic processes.

We refined this statement for greater precision and clarity: "This underscores the assay's sensitivity to environmental variables, such as synchronization method and bacterial lawn density. This highlights the importance of consistency across experimental setups and support the view that context-dependent variation may underlie previously reported discrepancies."

Reviewer #2 (Public Review):

Reagent sourcing

Reviewer suggested listing the sources of media ingredients with company names and catalog numbers, as this might be important for reproducibility.

To ensure complete reproducibility, we created a comprehensive Table S3 listing all reagents, suppliers, and catalog numbers used in our experiments. This detailed information enables exact replication of our experimental conditions and addresses potential variability that might arise from different reagent sources between laboratories.

Reviewer #3 (Public Review):

Raw data transparency

Reviewer noted that while a spreadsheet with choice assay results was provided, the individual raw data from assays was not included, which would be helpful for assessing sample sizes.

We now provide complete experimental transparency through Table S2, which contains individual choice indices from all 138 assays conducted across four independent trials. This comprehensive dataset allows full assessment of our experimental outcomes, statistical robustness, and reproducibility while enabling other researchers to perform independent statistical analyses.

F1/F2 assay disparity

Reviewer questioned whether the higher number of F2 assays compared to F1 represented truly independent assays, asking if multiple F2 assays were performed from offspring of one F1 plate (which would not represent independent assays).

We clarified this important statistical consideration in Methods (Transgenerational Testing): "Each behavioral assay was conducted using animals from a biologically independent growth plate. While F2 plates were derived from pooled embryos from multiple F1 parents, each assay represents an independent biological replicate with no reuse of animals across assays. F2 assays (n=45) exceeded F1 assays (n=20) due to PA14-induced fecundity reduction in trained worms, limiting the number of viable F1 progeny. The higher number of F2 assays reflects the greater reproductive success of healthy F1 animals and provides additional statistical power for population-level behavioral comparisons." We also enhanced our Controls section to clarify that "Our experimental design employed population-level comparisons across generations using unpaired statistical analyses, with no attempt to track individual lineages across generations."

Methodological variations overstatement

Reviewer felt the Introduction overstated the findings by suggesting the authors "address potential methodological variations," when they only used one assay setup throughout.

We have corrected the Introduction to accurately reflect our study design and scope: "Here, we adapted the protocol established by the Murphy group, maintaining the critical use of sodium azide to paralyze worms at the time of choice, to test whether parental exposure to PA14 elicits consistent avoidance in subsequent generations. Our study specifically focuses on the transmission of learned avoidance through the F2 generation, beyond the intergenerational (F1) effect, because this is where divergence between published studies begins."

Reviewer #1 (Recommendations for the authors):

Worm numbers

Reviewer noted that information about the number of worms used should be included in the training and choice assay methods section rather than separated.

We clarified worm numbers and sample sizes in the Methods (Controls and Additional Considerations): "Each individual assay averaged 62 ± 43 animals (range: 15-150 worms per assay), with a total of 138 assays conducted across four independent experimental trials. The variation in worm numbers per assay reflects natural variation in worm recovery and immobilization efficiency during choice assays. We conducted an average of 8.5 assays per condition during each of the four replicates."

Figure 1 legend and consistency

Reviewer identified several issues: inconsistent terminology ("treated" vs "trained"), incorrect statistical test naming, missing p-value annotations, and need for consistency between figure and legend. We have systematically addressed all figure consistency and statistical annotation issues:

Replaced inconsistent "treated" terminology with "trained" throughout

Corrected the statistical test description to accurately reflect our analysis: "Kruskal-Wallis oneway ANOVA followed by Dunn's post hoc" which properly corresponds to the statistical tests detailed in Table S1

Added explicit p-value annotations in the figure legend: "*p<0.05, **p<0.01 means and SEM shown (see Table S1 for statistics and Table S2 for raw data)"

Ensured consistent terminology between figure and legend

NGM vs. M9 buffer

Reviewer questioned whether we used NGM buffer or M9 buffer for washing steps, noting that NGM isn't usually referred to as "buffer."

We have prominently featured and thoroughly clarified our rationale for using liquid NGM buffer in the Methods (Synchronization of Worms section). The explanation now appears upfront in the methods: "We used liquid NGM buffer instead of M9 buffer (as specified in the original Murphy protocol) to maintain chemical consistency with the solid NGM culture plates. This modification minimizes potential osmotic stress since liquid NGM matches the pH (6.0) and ionic composition of the growth medium, whereas M9 buffer has a different pH (7.0) and ionic profile." We provide detailed chemical differences and explain that this modification maintains consistency with culture conditions while preserving essential experimental procedures.

Grammar/typos

Reviewer noted that the manuscript needed thorough proofreading to address grammatical errors and typographical mistakes.

We have conducted comprehensive proofreading and editing throughout the manuscript to resolve grammatical and typographical errors. Specific improvements include: clarified sentence structure in the Introduction and Results sections, corrected technical terminology consistency, improved figure legend clarity, and enhanced overall readability while maintaining scientific precision.

Sodium azide concentration

Reviewer noted that our sodium azide concentration differed from the Moore paper and requested comment on this difference.

We have included explicit justification for our sodium azide concentration choice in the Methods (Training and Choice Assay): "We used 400 mM sodium azide rather than the 1 M concentration reported by Moore et al. (2019) because preliminary trials showed that higher concentrations caused premature paralysis before worms could reach either bacterial spot, potentially biasing choice measurements. The 400 mM concentration provided sufficient immobilization while preserving the behavioral choice window."

Reviewer #2 (Recommendations for the authors):

Comparative reagent analysis

Reviewer suggested creating a supplemental table comparing reagent sources between our study, Gainey et al., and Murphy et al., proposing that media ingredient differences might explain the discrepancies.

While direct reagent comparison between laboratories was beyond the scope of this validation study, we recognize this as an important consideration for understanding experimental variability. Our comprehensive reagent sourcing information (Table S3) provides the foundation for future comparative studies. We encourage collaborative efforts to systematically compare reagent sources across laboratories, as media component differences could contribute to the experimental variability observed between research groups. Such analyses would be valuable for establishing standardized protocols across the field.

Conclusion

We hope that these revisions satisfactorily address the reviewers’ concerns. We believe these improvements significantly strengthened the manuscript's contribution to resolving this important scientific controversy.

We thank the reviewers again for their invaluable insights and constructive feedback, which have substantially improved the quality and impact of our work.

Niet in college's, wel vaak in tentamen

Needs to be daily research about bullying!

Top management team

3:1 en dus niet 3:1.5! Volgens het artikel zelfs eerder 2,9:1

Hoort bij fase 3; onthouden daar kan een vraag over komen

Uit mijn ervaringen vragen ze niet meer dan dit: Strength-based performance appraisal (SBPA) -> focuses on identifying, appreciating, and developing employee’s qualities in line with the company goals. (future use and development of employee strengths). The SBPA is aimed to overcome the pitfalls of the traditional performance appraisal exercise. Negative and positive feedback

Over de 'pitfall' zit bijna altijd een vraag, maar meer weten is altijd beter.

Zat vorig jaar in tentamen !

What you should consider when evaluating your resources

Some questions to keep in mind when evaluating your sources

test komentáře

Get with a reference librarian if and when you have trouble with search strategies.

Examples of secondary sources

Examples of primary sources

what would atatürk do?

foreign agents law

!

SPREAD the european values

Before Gezi Protest??

is the exergy balance still part of the scope, from what I understood an energy balance is performed. And the atomic balance ? to confirm

Het denken ontwikkelt zich in een sociaal-culturele context.

Sensorimotorische fase (0-2 jaar). - Gedachte = openlijke fysieke acties - Alles in het heden.

Preoperationele fase (2-7 jaar). - Denken verder dan hier en nu - Smboliseren van afwezige gebeurtenissen en objecten --> representational insight + "pretend play". - Perceptuele centratie --> langer is ook ouder. + - Egocentriciteit --> ik heb een broertje, dus iedereen heeft een broertje.

Concreet operationele fase (7-11 jaar). - Concrete operaties --> nadenken over de omkeerbare gevolgen van acties (gebaseerd op ervaringen) - Decentratie = kind snapt in deze fase de conservatietaak (smal glas = breed glas) - Perspectief van ander

Formeel operationele fase (11-16 jaar). - Abstract denken

Piaget = belang van de fysieke omgeving.

Vygotsky = belang van de sociale omgeving.

Cognitief psychologisch perspectief = informatieverwerking.

Westerse culturen: meer analystisch (individuele motivatie). Dus bij creatieve problemen oplossen: meer abstract.

Niet-westerse culturen: meer holistisch (context motivatie). Dus bij creatieve problemen oplossen: meer functioneel.

Juiste afleidingen: - Affirm the atecedent = C is een A --> C is een B. - Deny the consequent = C is niet B --> C is niet A

Onjuiste afleidingen: - Deny the antecedent = C is niet A --> C is niet B - Affirming the consequent = C is een B --> C is een A

Een syllogisme is valide als de conclusie noodzakelijkerwijs volgt uit de premissen.

Een syllogisme is waar als het syllogisme valide is en alle premissen waar zijn.

Logisch gokwerk: ik heb 10 vingers, mijn buurman heeft 10 vingers, mijn ouders hebben 10 vingers.

--> Hypothese: alle mensen hebben 10 vingers.

Logisch bewijs

Gyuri Web Snarf 2025-10-2

exp - lore - eriment - perience

the info-morphic-communication flow

Let the spice of Life flow

O dedilhar das cordas do violão.

denoting the first, original, or typical form of something.

extremely high

test

eLife Assessment

This paper presents a valuable theory and analysis of the role of neurogenesis and inhibitory plasticity in the drift of neural representations in the olfactory system. For one of the findings, regarding the impact of neurogenesis on the drift, the evidence remains incomplete. The reason lies in the differences in variability/drift of the mitral/tufted cell responses observed in the model compared to experimental observations, where these responses remain stable over extended time scales.

Reviewer #1 (Public review):

Summary:

The authors build a network model of the olfactory bulb and the piriform cortex and use it to run simulations and test their hypotheses. Given the model's settings, the authors observe drift across days in the responses to the same odors of both the mitral/tufted cells, as well as of piriform cortex neurons. When representing the M/T and PCx responses within a lower-dimensional space, the apparent drift is more prominent in the PCx, while the M/T responses appear in comparison more stable. The authors further note that introducing spike-time dependent plasticity (STDP) at bulb synapses involving abGCs slows down the drift in the PCx representations, and further link this to the observation that repeated exposure to the same odorant slows down drift in the piriform cortex.

The model is clearly explained and relies on several assumptions and observations:

(1) Random projections of MTC from the olfactory bulb to the piriform cortex, random intra-piriform connectivity, and random piriform to bulb connectivity.

(2) Higher dimensionality of piriform cortex representations compared to M/T responses, which enables superior decoding of odor identity in the piriform cortex.

(3) Spike time-dependent plasticity (STDP) at synapses involving the abGCs.

The authors address an open topical problem, and the model is elegant in its simplicity. I have however, several major concerns with the hypotheses underlying the model and with its biological plausibility.

Concerns:

(1) In their model, the authors propose that MTC remain stable at the population level, despite changes in individual MTC responses.

The authors cite several experimental studies to support their claims that individual MTC responses to the same odors change (some increase, some decrease) across days. Interpreting the results of these studies must, however, take into account the variability of M/T responses across odor presentation repeats within the same session vs. across sessions. In the Shani-Narkiss et al., Frontiers in Neural Circuits, 2023 study referenced, a large fraction of the variability across days in M/T responses is also observed across repeats to the same odorant in the same session (Shani-Narkiss et al., Figure 4), while the authors have M/T responses in the same session that are highly reproducible. This is an important point to consider and address, since it constrains how much of the variability in M/T responses can be attributed to adult neurogenesis in the olfactory bulb versus to other networks' inhibitory mechanisms, which do not rely on neurogenesis. In the authors' model, the variability in M/T responses observed across days emerges as a result of adult-born neurogenesis, which does not need to be the main source of variability observed in imaging experiments (Shani-Narkiss et al., Figure 4).

Another study (Kato et al., Neuron, 2012, Figure 4) reported that mitral cell responses to odors experienced repeatedly across 7 days tend to sparsen and decrease in amplitude systematically, while mitral cell responses to the same odor on day 1 vs. day 7 when the odor is not presented repeatedly in between seem less affected (although the authors also reported a decrease in the CI for this condition). As such, Kato et al. mostly report decreases in mitral cell odor responses with repeated odor exposure at both the individual and population level, and not so much increases and decreases in the individual mitral cell responses, and stability at the population level.

(2) In Figure 1, a set of GCs is killed off, and new GCs are integrated in the network as abGC. Following the elimination of 10% of GCs in the network, new cells are added and randomly assigned synaptic weights between these abGCs and MTC, GCs, SACs, and top-down projections from PCx. This is done for 11 days, during which time all GCs have gone through adult neurogenesis.

Is the authors' assumption here that across the 11 days, all GCs are being replaced? This seems to depart from the known biology of the olfactory bulb granule cells, i.e., GCs survive for a large fraction of the animal's life.

(3) The authors' model relies on several key assumptions: random projections of MTC from the olfactory bulb to the piriform cortex, random intra-piriform connectivity, and random piriform to bulb connectivity. These assumptions are not necessarily accurate, as recent work revealed structure in the projections from the olfactory bulb to the piriform cortex and structure within the piriform cortex connectivity itself (Fink et al., bioRxiv, 2025; Chae et al., Cell, 2022; Zeppilli et al., eLife, 2021).

How do the results of the model relating adult neurogenesis in the bulb to drift in the piriform cortex representations change when considering an alternative scenario in which the olfactory bulb to piriform and intra-piriform connectivity is not fully distributed and indistinguishable from random, but rather is structured?

(4) I didn't understand the logic of the low-dimensional space analysis for M/T cells and piriform cortex neurons (Figures 2 & 3). In the authors' model, the full-ensemble M/T responses are reorganized over time, presumably due to the adult-born neurogenesis. Analyzing a lower-dimensional projection of the ensemble trajectories reveals a lower degree of re-organization. This is the same for the piriform cortex, but relatively, the piriform ensembles displayed in a low-dimensional embedding appear to drift more compared to the M/T ensembles.

This analysis triggers a few questions: which representation is relevant for the brain function - the high or the low-dimensional projection? What fraction of response variance is included in the low-dimensional space analysis? How did the authors decide the low-dimensional cut-off? Why does STDP cause more drift in piriform cortex ensembles vs. M/T ensembles? Is this because of the assumed higher dimensionality of the piriform cortex representations compared to the mitral cells?

(5) Could the authors comment whether STDP at abGC synapses and its impact on decreasing drift represent a new insight, and also put it into context? Several studies (e.g., Lledo, Murthy, Komiyama groups) reported that abGC integrates in the network in an activity-dependent manner, and not randomly, and as such stabilizes the active neuronal responses, which is consistent with the authors' report.

Related, I couldn't find through the manuscript which synapses involving abGCs they focus on, or what is the relative contribution of the various plastic synapses shown in the cartoon from Figure 4 A1 (circles and triangles).

6) The study would be strengthened, in my opinion, by including specific testable predictions that the authors' models make, which can be further food for thought for experimentalists.<br /> How does suppression of adult-born neurogenesis in the OB impact the stability of mitral cell odor responses? How about piriform cortex ensembles?

Reviewer #2 (Public review):

Summary:

The authors address a critical problem in olfactory coding. It has long been known that adult neurogenesis, specifically in the form of adult-born granule cells that embed into the existing inhibitory networks on the olfactory bulb, can potentially alter the responses of Mitral/Tufted neurons that project activity to the Piriform Cortex and to other areas of the brain. Fundamentally, it would seem that these granule cells could alter the stability of neural codes in the OB over time. The authors develop a spiking network model to explore how stability can be achieved both in the OB over time and in the PC, which receives inputs. The model recapitulates published activity recordings of M/T cells and shows how activity in different M/T cells from the same glomerulus shifts over time in ways that, in spite of the shift, preserve population/glomerular level codes. However, these different M/T cells fan out onto different pyramidal cells of the PC, which gives rise to instability at that level. STDP then, is necessary to maintain stability at the PC level as long as odor environments remain constant. These results may also apply to a similar neurogenesis-based change in the Dentate Gyrus, which generates instability in CA1/3 regions of the hippocampus

Strengths:

A robust network model that untangles important, seemingly contradictory mechanisms that underlie olfactory coding.

Weaknesses:

The work is a significant contribution to understanding olfactory coding. But the manuscript would benefit from a brief discussion of why neurogenesis occurs in the first place - e.g., injury, ongoing needs for plasticity, and adapting to turnover of ORNs. There is literature on this topic. It seems counterintuitive to have a process in the MOB (and for that matter in the DG) that potentially disrupts the ability to generate stable codes both in the MOB and PC, and in particular a disruption that requires two different mechanisms - multiple M/T cells per glomerulus in the MOB and STDP in the PC - to counteract.

Given that neurogenesis has an important function, and a mechanism is in place to compensate for it in the MOB, why would it then be disrupted in fan-out projections to the PC? The answer may lie in the need for fan-out projections so that pyramidal neurons in the PC can combinatorially represent many different inputs from the MOB. So something like STDP would be needed to maintain stability in the face of the need for this coding strategy.

This kind of discussion, or something like it, would help readers understand why these mechanisms occur in the first place. It is interesting that PC stability requires that odor environments be stable, and that this stability drives PC representational stability. This result suggests experimental work to test this hypothesis. As such, it is a novel outcome of the research.

Reviewer #3 (Public review):

Summary

The authors set out to explore the potential relationship between adult neurogenesis of inhibitory granule cells in the olfactory bulb and cumulative changes over days in odor-evoked spiking activity (representational drift) in the olfactory stream. They developed a richly detailed spiking neuronal network model based on Izhikevich (2003), allowing them to capture the diversity of spiking behaviors of multiple neuron types within the olfactory system. This model recapitulates the circuit organization of both the main olfactory bulb (MOB) and the piriform cortex (PCx), including connections between the two (both feedforward and corticofugal). Adult neurogenesis was captured by shuffling the weights of the model's granule cells, preserving the distribution of synaptic weights. Shuffling of granule cell connectivity resulted in cumulative changes in stimulus-evoked spiking of the model's M/T cells. Individual M/T cell tuning changed with time, and ensemble correlations dropped sharply over the temporal interval examined (long enough that almost all granule cells in the model had shuffled their weights). Interestingly, these changes in responsiveness did not disrupt low-dimensional stability of olfactory representations: when projected into a low-dimensional subspace, population vector correlations in this subspace remained elevated across the temporal interval examined. Importantly, in the model's downstream piriform layer, this was not the case. There, shuffled GC connectivity in the bulb resulted in a complete shift in piriform odor coding, including for low-dimensional projections. This is in contrast to what the model exhibited in the M/T input layer. Interestingly, these changes in PCx extended to the geometrical structure of the odor representations themselves. Finally, the authors examined the effect of experience on representational drift. Using an STDP rule, they allowed the inputs to and outputs from adult-born granule cells to change during repeated presentations of the same odor. This stabilized stimulus-evoked activity in the model's piriform layer.

Strengths

This paper suggests a link between adult neurogenesis in the olfactory bulb and representational drift in the piriform cortex. Using an elegant spiking network that faithfully recapitulates the basic physiological properties of the olfactory stream, the authors tackle a question of longstanding interest in a creative and interesting manner. As a purely theoretical study of drift, this paper presents important insights: synaptic turnover of recurrent inhibitory input can destabilize stimulus-evoked activity, but only to a degree, as representations in the bulb (the model's recurrent input layer) retain their basic geometrical form. However, this destabilized input results in profound drift in the model's second (piriform) layer, where both the tuning of individual neurons and the layer's overall functional geometry are restructured. This is a useful and important idea in the drift field, and to my knowledge, it is novel. The bulb is not the only setting where inhibitory synapses exhibit turnover (whether through neurogenesis or synaptic dynamics), and so this exploration of the consequences of such plasticity on drift is valuable. The authors also elegantly explore a potential mechanism to stabilize representations through experience, using an STDP rule specific to the inhibitory neurons in the input layer. This has an interesting parallel with other recent theoretical work on drift in the piriform (Morales et al., 2025 PNAS), in which STDP in the piriform layer was also shown to stabilize stimulus representations there. It is fascinating to see that this same rule also stabilizes piriform representations when implemented in the bulb's granule cells.

The authors also provide a thoughtful discussion regarding the differential roles of mitral and tufted cells in drift in piriform and AON and the potential roles of neurogenesis in archicortex.

In general, this paper puts an important and much-needed spotlight on the role of neurogenesis and inhibitory plasticity in drift. In this light, it is a valuable and exciting contribution to the drift conversation.

Weaknesses

I have one major, general concern that I think must be addressed to permit proper interpretation of the results.

I worry that the authors' model may confuse thinking on drift in the olfactory system, because of differences in the behavior of their model from known features of the olfactory bulb. In their model, the tuning of individual bulbar neurons drifts over time. This is inconsistent with the experimental literature on the stability of odor-evoked activity in the olfactory bulb.

In a foundational paper, Bhalla & Bower (1997) recorded from mitral and tufted cells in the olfactory bulb of freely moving rats and measured the odor tuning of well-isolated single units across a five-day interval. They found that the tuning of a single cell was quite variable within a day, across trials, but that this variability did not increase with time. Indeed, their measure of response similarity was equivalent within and across days. In what now reads as a prescient anticipation of the drift phenomenon, Bhalla and Bower concluded: "it is clear, at least over five days, that the cell is bounded in how it can respond. If this were not the case, we would expect a continual increase in relative response variability over multiple days (the equivalent of response drift). Instead, the degree of variability in the responses of single cells is stable over the length of time we have recorded." Thus, even at the level of single cells, this early paper argues that the bulb is stable.

This basic result has since been replicated by several groups. Kato et al. (2012) used chronic two-photon calcium imaging of mitral cells in awake, head-fixed mice and likewise found that, while odor responses could be modulated by recent experience (odor exposure leading to transient adaptation), the underlying tuning of individual cells remained stable. While experience altered mitral cell odor responses, those responses recovered to their original form at the level of the single neuron, maintaining tuning over extended periods (two months). More recently, the Mizrahi lab (Shani-Narkiss et al., 2023) extended chronic imaging to six months, reporting that single-cell odor tuning curves remained highly similar over this period. These studies reinforce Bhalla and Bower's original conclusion: despite trial-to-trial variability, olfactory bulb neurons maintain stable odor tuning across extended timescales, with plasticity emerging primarily in response to experience. (The Yamada et al., 2017 paper, which the authors here cite, is not an appropriate comparison. In Yamada, mice were exposed daily to odor. Therefore, the changes observed in Yamada are a function of odor experience, not of time alone. Yamada does not include data in which the tuning of bulb neurons is measured in the absence of intervening experience.)

Therefore, a model that relies on instability in the tuning of bulbar neurons risks giving the incorrect impression that the bulb drifts over time. This difference should be explicitly addressed by the authors to avoid any potential confusion. Perhaps the best course of action would be to fit their model to Mizrahi's data, should this data be available, and see if, when constrained by empirical observation, the model still produces drift in piriform. If so, this would dramatically strengthen the paper. If this is not feasible, then I suggest being very explicit about this difference between the behavior of the model and what has been shown empirically. I appreciate that in the data there is modest drift (e.g., Shani-Narkiss' Figure 8C), but the changes reported there really are modest compared to what is exhibited by the model. A compromise would be to simply apply these metrics to the model and match the model's similarity to the Shani-Narkiss data. Then the authors could ask what effect this has on drift in piriform.

The risk here is that people will conclude from this paper that drift in piriform may simply be inherited from instability in the bulb. This view is inconsistent with what has been documented empirically, and so great care is warranted to avoid conveying that impression to the community.

Major comments (all related to the above point)

(1) Lines 146-168: The authors find in their model that "individual M/T cells changed their responses to the same odor across days due to adult-neurogenesis, with some cells decreasing the firing rate responses (Fig.2A1 top) while other cells increased the magnitude of their responses (Fig. 2A2 bottom, Fig. S2)" they also report a significant decrease in the "full ensemble correlation" in their model over time. They claim that these changes in individual cell tuning are "similar to what has been observed by others using calcium imaging of M/T cell activity (Kato et al., 2012 and Yamada et al., 2017)" and that the decrease in full ensemble correlation is "consistent with experimental observations (Yamada et al., 2017)." However, the conditions of the Kato and Yamada experiments that demonstrate response change are not comparable here, as odors were presented daily to the animals in these experiments. Therefore, the changes in odor tuning found in the Kato and Yamada papers (Kato Figure 4D; Yamada Figure 3E) are a function of accumulated experience with odor. This distinction is crucial because experience-induced changes reflect an underlying learning process, whereas changes that simply accumulate over time are more consistent with drift. The conditions of their model are more similar to those employed in other experiments described in Kato et al. 2012 (Figure 6C) as well as Shani-Narkiss et al. (2023), in which bulb tuning is measured not as a function of intervening experience, but rather as a function of time (Kato's "recovery" experiment). What is found in Kato is that even across two months, the tuning of individual mitral cells is stable. What alters tuning is experience with odor, the core finding of both the Kato et al., 2012 paper and also Yamada et al., 2017. It is crucial that this is clarified in the text.

(2) The authors show that in a reduced-space correlation metric, the correlation of low-dimensional trajectories "remained high across all days"..."consistent with a recent experimental study" (Shani-Narkiss et al., 2023). It is true that in the Shani-Narkiss paper, a consistent low-dimensional response is found across days (t-SNE analysis in Shani-Narkiss Figure 7B). However, the key difference between the Shani-Narkiss data and the results reported here is that Shani-Narkiss also observed relative stability in the native space (Shani-Narkiss Figure 8). They conclude that they "find a relatively stable response of single neurons to odors in either awake or anesthetized states and a relatively stable representation of odors by the MC population as a whole (Figures 6-8; Bhalla and Bower, 1997)." This should be better clarified in the text.

(3) In the discussion, the authors state that "In the MOB, individual M/T cells exhibited variable odor responses akin to gain control, altering their firing rate magnitudes over time. This is consistent with earlier experimental studies using calcium-imaging." (L314-6). Again, I disagree that these data are consistent with what has been published thus far. Changes in gain would have resulted in increased variability across days in the Bhalla data. Moreover, changes in gain would be captured by Kato's change index ("To quantify the changes in mitral cell responses, we calculated the change index (CI) for each responsive mitral cell-odor pair on each trial (trial X) of a given day as (response on trial X - the initial response on day 1)/(response on trial X + the initial response on day 1). Thus, CI ranges from −1 to 1, where a value of −1 represents a complete loss of response, 1 represents the emergence of a new response, and 0 represents no change." Kato et al.). This index will capture changes in gain. However, as shown in Figure 4D (red traces), Figure 6C (Recovery and Odor set B during odor set A experience and vice versa), the change index is either zero or near zero. If the authors wish to claim that their model is consistent with these data, they should also compute Kato's change index for M/T odor-cell pairs in their model and show that it also remains at 0 over time, absent experience.

eLife Assessment

This valuable study compares auditory cortex responses to sounds and cochlear implant stimulation measured with surface electrode grids in rats. Beyond the reduced frequency resolution of cochlear implants observed previously, this study suggests key discrepancies between neuronal representations of cochlear stimulations and natural sounds. However, the evidence for this potentially interesting result is incomplete because there is a lack of evidence for the effectiveness of the comparison method. This study is of interest to researchers in the auditory neuroscience field and clinicians implementing treatments with cochlear implants.

Reviewer #1 (Public review):

Summary:

This manuscript addresses an important question: whether cortical population codes for cochlear-implant (CI) stimulation resemble those for natural acoustic input or constitute a qualitatively different representation. The authors record intracranial EEG (µECoG) responses to pure tones in normal-hearing rats and to single-channel CI pulses in bilaterally deafened, acutely implanted rats, analysing the data with ERP/high-gamma measures, tensor component analysis (TCA), and information-theoretic decoding. Across several readouts, the acoustic condition supports better single-trial stimulus classification than the CI condition. However, stronger decoding does not, on its own, establish that the acoustic responses instantiate a "richer" cortical code, and the evidence for orderly spatial organisation is not compelling for CI, and is also less evident than expected for normal-hearing, given prior knowledge. The overall narrative is interesting, but at present, the conclusions outpace the data because of statistical, methodological, and presentation issues.

Strengths:

The study poses a timely, clinically relevant question with clear implications for CI strategy. The analytical toolkit is appropriate: µECoG captures mesoscale patterns; TCA offers a transparent separation of spatial and temporal structure; and mutual-information decoding provides an interpretable measure of single-trial discriminability. Within-subject recordings in a subset of animals, in principle, help isolate modality effects from inter-animal variability. Where analyses are most direct, the acoustic condition yields higher single-trial decoding accuracy, which is a meaningful and clearly presented result.

Weaknesses:

Several limitations constrain how far the conclusions can be taken. Parts of the statistical treatment do not match the data structure: some comparisons mix paired and unpaired animals but are analysed as fully paired, raising concerns about misestimated uncertainty. Methodological reporting is incomplete in places; essential parameters for both acoustic and electrical stimulation, as well as objective verification of implantation and deafening, are not described with sufficient detail to support confident interpretation or replication. Figure-level clarity also undermines the message. In Figure 2, non-significant slopes for CI, repeated identification of a single "best channel," mismatched axes, and unclear distinctions between example and averaged panels make the assertion of spatial organisation unconvincing; importantly, the normal-hearing panels also do not display tonotopy as clearly as expected, which weakens the key contrast the paper seeks to establish. Finally, the decoding claims would be strengthened by simple internal controls, such as within-modality train/test splits and decoding on raw ERP/high-gamma features to demonstrate that poor cross-modal transfer reflects genuine differences in the underlying responses rather than limitations of the modelling pipeline.

Reviewer #2 (Public review):

Summary:

This article reports measurements of iEEG signals on the rat auditory cortex during cochlear implant or sound stimulation in separate groups of rats. The observations indicate some spatial organization of cochlear implant stimuli, but that is very different from cochlear implants.

Strengths:

The study includes interesting analyses of the sound and cochlear implant representation structure based on decoders.

Weaknesses:

The observation that responses to cochlear implant stimulation (stimulation) are spatially organized is not new (e.g., Adenis et al. 2024).

The claim that spatial and temporal dimensions contribute information about the sound is also not new; there is a large literature on this topic. Moreover, the results shown here are extremely weak. They show similar levels of information in the spatial and temporal dimensions, and no synergy between the two dimensions. This is however, likely the consequence of high measurement noise leading to poor accuracy in the information estimates, as the authors state.

The main claim of the study - the mismatch between cochlear implant and sound representation - is not supported. The responses to each modality are measured in different animals. The authors do not show that they actually can compare representations across animals (e.g., for the same sounds). Without this positive control, there is no reason to think that it is possible to decode from one animal with a decoder trained on another, and the negative result shown by the authors is therefore not surprising.

Reviewer #3 (Public review):

Summary:

Through micro-electroencephalography, Hight and colleagues studied how the auditory cortex in its ensemble responds to cochlear implant stimulation compared to the classic pure tones. Taking advantage of a double-implanted rat model (Micro-ECoG and Cochlear Implant), they tracked and analyzed changes happening in the temporal and spatial aspects of the cortical evoked responses in both normal hearing and cochlear-implanted animals. After establishing that single-trial responses were sufficient to encode the stimuli's properties, the authors then explored several decoder architectures to study the cortex's ability to encode each stimulus modality in a similar or different manner. They conclude that a) intracranial EEG evoked responses can be accurately recorded and did not differed between normal hearing and cochlear-implanted rats; b) Although coarsely spatially organized, CI-evoked responses had higher trial-by-trial variability than pure tones; c) Stimulus identity is independently represented by temporal and spatial aspect of cortical representations and can be accurately decoded by various means from single trials; d) and that Pure tones trained decoder can't decode CI-stimulus identity accurately.

Strength:

The model combining micro-eCoG and cochlear implantation and the methodology to extract both the Event Related Potentials (ERPs) and High-Gammas (HGs) is very well designed and appropriately analyzed. Likewise, the PCA-LDA and TCA-LDA are powerful tools that take full advantage of the information provided by the cortical ensembles.

The overall structure of the paper, with a paced and exhaustive progress through each step and evolution of the decoder, is very appreciable and easy to follow. The exploration of single-trial encoding and stimulus identity through temporal and spatial domains is providing new avenues to characterize the cortical responses to CI stimulations and their central representation. The fact that single trials suffice to decode the stimulus identity regardless of their modality is of great interest and noteworthy. Although the authors confirm that iEEG remains difficult to transpose in the clinic, the insights provided by the study confirm the potential benefit of using central decoders to help in clinic settings.

Weaknesses:

The conclusion of the paper, especially the concept of distinct cortical encoding for each modality, is unfortunately partially supported by the results, as the authors did not adequately consider fundamental limitations of CI-related stimulation.

First, the reviewer assumed that the authors stimulated in a Monopolar mode, which, albeit being clinically relevant, notoriously generates a high current spread in rodent models. Second, comparing the averaged BF maps for iEEG (Figure 2A, C), BFs ranged from 4 to 16kHz with a predominance of 4kHz BFs. The lack of BFs at higher frequencies hints at a potential location mismatch between the frequency range sampled at the level of the cortex (low to medium frequencies) and the frequency range covered by the CI inserted mostly in the first turn-and-a-half of the cochlea (high to medium frequencies). Looking at Figure 2F (and to some extent 2A), most of the CI electrodes elicited responses around the 4kHz regions, and averaged maps show a predominance of CI-3-4 across the cortex (Figure 2C, H) from areas with 4kHz BF to areas with 16kHz BF. It is doubtful that CI-3-4 are located near the 4kHz region based on Müller's work (1991) on the frequency representation in the rat cochlea.

Taken together with the Pearsons correlations being flat, the decoder examples showing a strong ability to identify CI-4 and 3 and the Fig-8D, E presenting a strong prediction of 4kHz and 8kHz for all the CI electrodes when using a pure tone trained decoder, it is possible that current spread ended stimulating indistinctly higher turns of the cochlea or even the modiolus in a non-specific manner, greatly reducing (or smearing) the place-coding/frequency resolution of each electrode, which in turn could explain the coarse topographic (or coarsely tonotopic according to the manuscript) organization of the cortical responses. Thus, the conclusion that there are distinct encodings for each modality is biased, as it might not account for monopolar smearing. To that end, and since it is the study's main message and title, it would have benefited from having a subgroup of animals using bipolar stimulations (or any focused strategy since they provide reduced current spread) to compare the spatial organization of iEEG responses and the performances of the different decoders to dismiss current spread and strengthen their conclusion.

Nevertheless, the reviewer wants to reiterate that the study proposed by Hight et al. is well constructed, relevant to the field, and that the overall proposal of improving patient performances and helping their adaptation in the first months of CI use by studying central responses should be pursued as it might help establish new guidelines or create new clinical tools.

This statement accurately reveals that the core dilemma of cross-cultural education lies in the conflicting interactions between teachers and students caused by cultural differences. This conflict stems from differing understandings of how authority is expressed. Different cultures define respect differently. In African American culture, direct instruction is viewed as a sign of responsibility and care; whereas, middle-class white culture emphasizes equality through negotiation. Educational equity cannot be achieved solely through resource investment; it also requires the deconstruction of cultural power relations. If teachers fail to reflect on their own cultural assumptions about authority, any reform of teaching methods will likely be ineffective.

This analysis reveals a deeper challenge to educational equity that is even well-intentioned teachers can inadvertently marginalize minority students if they lack the skills to engage in culturally responsive teaching. Therefore, educational reform must prioritize cultural inclusion, not simply resource allocation.

This statement profoundly reveals the core mechanism of cultural conflict in education, pointing out that when there are significant differences between school culture and family culture, teachers may misjudge students' abilities, intentions or talents due to differences in language use and interaction patterns. This statement reveals that the core challenge of educational equity lies in whether schools can truly "see" and respect students' cultural backgrounds. If educators measure students solely against the prevailing culture, differences become "deficiencies." However, if they are viewed as resources, cultural conflict can be transformed into opportunities for educational innovation. Ultimately, the mission of education is not to eliminate differences, but to build a shared space for growth within them.

Hanno Berger, Steueranwalt

Part of an experiment

eLife Assessment

This important study uses simultaneous EEG and fMRI recordings to shed light on the relationship between alpha and gamma oscillations and specific cortical layers. The sophisticated methodology provides solid evidence for correlations between oscillatory power and the strength and contents of fMRI signals in different cortical layers, though some caveats remain. This paper will be of interest to neuroscientists studying the role and mechanisms of alpha and gamma oscillations.

Reviewer #1 (Public review):

In this manuscript, Clausner and colleagues use simultaneous EEG and fMRI recordings to clarify how visual brain rhythms emerge across layers of early visual cortex. They report that gamma activity correlates positively with feature-specific fMRI signals in superficial and deep layers. By contrast, alpha activity generally correlated negatively with fMRI signals, with two higher frequencies within the alpha reflecting feature-specific fMRI signals. This feature-specific alpha code indicates an active role of alpha oscillations in visual feature coding, providing compelling evidence that the functions of alpha oscillations go beyond cortical idling or feature-unspecific suppression.

The study is very interesting and timely. Methodologically, it is state-of-the-art. The findings on a more active role of alpha activity that goes beyond the classical idling or suppression accounts are in line with recent findings and theories. In sum, this paper makes a very nice contribution. I still have a few comments that I outline below, regarding the data visualization, some methodological aspects, and a couple of theoretical points.

(1) The authors put a lot of effort into the figure design. For instance, I really like Figure 1, which conveys a lot of information in a nice way. Figures 3 and 4, however, seem overengineered, and it takes a lot of time to distill the contents from them. The fact that they have a supplementary figure explaining the composition of these figures already indicates that the authors realized this is not particularly intuitive. First of all, the ordering of the conditions is not really intuitive. Second, the indication of significance through saturation does not really work; I have a hard time discerning the more and less saturated colors. And finally, the white dots do not really help either. I don't fully understand why they are placed where they are placed (e.g., in Figure 3). My suggestion would be to get rid of one of the factors (I think the voxel selection threshold could go: the authors could run with one of the stricter ones, and the rest could go into the supplement?) and then turn this into a few line plots. That would be so much easier to digest.

(2) The division between high- and low-frequency alpha in the feature-specific signal correspondence is very interesting. I am wondering whether there is an opposite effect in the feature-unspecific signal correspondence. Would the high-frequency alpha show less of a feature-unspecific correlation with the BOLD?

(3) In the discussion (line 330 onwards), the authors mention that low-frequency alpha is predominantly related to superficial layers, referencing Figure 4A. I have a hard time appreciating this pattern there. Can the authors provide some more information on where to look?

(4) How did the authors deal with the signal-to-noise ratio (SNR) across layers, where the presence of larger drain veins typically increases BOLD (and thereby SNR) in superficial layers? This may explain the pattern of feature-unspecific effects in the alpha (Figure 3). Can the authors perform some type of SNR estimate (e.g., split-half reliability of voxel activations or similar) across layers to check whether SNR plays a role in this general pattern?

(5) The GLM used for modelling the fMRI data included lots of regressors, and the scanning was intermittent. How much data was available in the end for sensibly estimating the baseline? This was not really clear to me from the methods (or I might have missed it). This seems relevant here, as the sign of the beta estimates plays a major role in interpreting the results here.

(6) Some recent research suggests that gamma activity, much in contrast to the prevailing view of the mechanism for feedforward information propagation, relates to the feedback process (e.g., Vinck et al., 2025, TiCS). This view kind of fits with the localization of gamma to the deep layer here?

(7) Another recent review (Stecher et al., 2025, TiNS) discusses feature-specific codes in visual alpha rhythms quite a bit, and it might be worth discussing how your results align with the results reported there.

Reviewer #2 (Public review):

The authors address a long-standing controversy regarding the functional role of neural oscillations in cortical computations and layer-specific signalling. Several studies have implicated gamma oscillations in bottom-up processing, while lower-frequency oscillations have been associated with top-down signalling. Therefore, the question the authors investigate is both timely and theoretically relevant, contributing to our understanding of feedforward and feedback communication in the brain. This paper presents a novel and complicated data acquisition technique, the application of simultaneous EEG and fMRI, to benefit from both temporal and spatial resolution. A sophisticated data analysis method was executed in order to understand the underlying neural activity during a visual oddball task. Figures are well-designed and appropriately represent the results, which seem to support the overall conclusions. However, some of the claims (particularly those regarding the contribution of gamma oscillations) feel somewhat overstated, as the results offer indeed some significant evidence, but most seem more like a suggestive trend. Nonetheless, the paper is well-written, addresses a relevant and timely research question, introduces a novel and elegant analysis approach, and presents interesting findings. Further investigation will be important to strengthen and expand upon these insights.

One of the main strengths of the paper lies in the use of a well-established and straightforward experimental paradigm (the visual oddball task). As a result, the behavioural effects reported were largely expected and reassuring to see replicated. The acquisition technique used is very novel, and while this may introduce challenges for data analysis, the authors appear to have addressed these appropriately.

Later findings are very interesting, and mainly in line with our current understanding of feedback and feedforward signalling. However, the layer weight calculation is lacking in the manuscript. While it is discussed in the methods, it would help to briefly explain in the results how these weights are calculated, so that the reader can better follow what is being interpreted.

Line 104 states there is one virtual channel per hemisphere for low and high frequencies. It may be helpful to include the number of channels (n=4) in the results section, as specified in the methods. Also, this raises the question of whether a single virtual channel (i.e., voxel) provides sufficient information for reproducibility.

One area that would benefit from further clarification is the interpretation of gamma oscillations. The evidence for gamma involvement in the observed effects appears somewhat limited. For example, no significant gamma-related clusters were found for the feature-unspecific BOLD signal (Figure 2). Significant effects emerged only when the analysis was restricted to positively responding voxels, and even then, only for the contrast between EEG-coherent and EEG-incoherent conditions in the feature-specific BOLD response. It remains unclear how to interpret this selective emergence of gamma-related effects. Given previous literature linking gamma to feedforward processing, one might expect more robust involvement in broader, feature-unspecific contrasts. The current discussion presents the gamma-related findings with some confidence, and the manuscript would benefit from a more nuanced reflection on why these effects may not have appeared more broadly. The explanation provided in line 230, that restricting the analysis to positively responding voxels may have increased the SNR, is reasonable, but it may not fully account for the absence of gamma effects in V1's feature-unspecific response. Including the actual beta values from Figure 4 in the legend or main text would also help readers better assess the strength and specificity of the reported effects.

Relating to behavioural findings for underlying neural activity, could the authors test on a trial-by-trial basis how behavioural performance relates to the BOLD signal / oscillatory activity change? Line 305 states that "Since behavioural performance in the present study was consistently high at 94% on average and participants were instructed to respond quickly to potential oddball stimuli, a higher alpha frequency might reflect a more successful stimulus encoding and hence faster and more accurate behavioural performance." Also, this might help to relate the findings to the lower vs upper alpha functionality difference.

In Figure 4, the EEG alpha specificity plot shows relatively large error bars, and there is visible overlap between the lower and upper alpha in both congruent and incongruent conditions. While upper alpha shows a positive slope across conditions and lower alpha remains flat, the interaction appears to be driven by the change from congruent to incongruent in upper alpha. It is worth clarifying whether the simple effects (e.g., lower vs upper within each condition) were tested, given the visual similarity at the incongruent condition. Overall, the significant interaction (p < 0.001, FDR-corrected) is consistent with diverging trends, but a breakdown of simple effects would help interpret the result more clearly. Was there a significant difference between lower and upper alpha in congruent or incongruent conditions?

Overall, this study provides a valuable contribution to the literature on oscillatory dynamics and laminar fMRI, though some interpretations would benefit from further clarification or qualification.

Reviewer #3 (Public review):

Summary:

Clausner et al. investigate the relationship between cortical oscillations in the alpha and gamma bands and the feature-specific and feature-unspecific BOLD signals across cortical layers. Using a well-designed stimulus and GLM, they show a method by which different BOLD signals can be differentiated and investigated alongside multiple cortical oscillatory frequencies. In addition to the previously reported positive relationship between gamma and BOLD signals in superficial layers, they show a relationship between gamma and feature-specific BOLD in the deeper layers. Alpha-band power is shown to have a negative relationship with the negative BOLD response for both feature-specific and feature-unspecific contrasts. When separated into lower (8-10Hz) and upper (11-13Hz) alpha oscillations, they show that higher frequency alpha showed a significantly stronger negative relationship with congruency, and can therefore be interpreted as more feature-specific than lower frequency alpha.

Strengths:

The use of interleaved EEG-fMRI has provided a rich dataset that can be used to evaluate the relationship of cortical layer BOLD signals with multiple EEG frequencies. The EEG data were of sufficient quality to see the modulation of both alpha-band and gamma-band oscillations in the group mean VE-channel TFS. The good EEG data quality is backed up with a highly technical analysis pipeline that ultimately enables the interpretation of the cortical layer relationship of the BOLD signal with a range of frequencies in the alpha and gamma bands. The stimulus design allowed for the generation of multiple contrasts for the BOLD signal and the alpha/gamma oscillations in the GLM analysis. Feature-specific and unspecific BOLD contrasts are used with congruently or incongruently selected EEG power regressors to delineate between local and global alpha modulations. A transparent approach is used for the selection of voxels contributing to the final layer profiles, for which statistical analysis is comprehensive but uses an alternative statistical test, which I have not seen in previous layer-fMRI literature.

A significant negative relationship between alpha-band power and the BOLD signal was seen in congruently (EEGco) selected voxels (predominantly in superficial layers) and in feature-contrast (EEGco-inco) selected (superficial and deep layers). When separated into lower (8-10Hz) and upper (11-13Hz) alpha oscillations, they show that higher frequency alpha showed a significantly stronger negative relationship with congruency than lower frequency alpha. This is interpreted as a frequency dissociation in the alpha-BOLD relationship, with upper frequency alpha being feature-specific and lower frequency alpha corresponding to general modulation. These results are a valuable addition to the current literature and improve our current understanding of the role of cortical alpha oscillations.

There is not much work in the literature on the relationship between alpha power and the negative BOLD response (NBR), so the data provided here are particularly valuable. The negative relationship between the NBR and alpha power shown here suggests that there is a reduction in alpha power, linked to locally reduced BOLD activity, which is in line with the previously hypothesized inhibitory nature of alpha.

Weaknesses:

It is not entirely clear how the draining vein effect seen in GE-BOLD layer-fMRI data has been accounted for in the analysis. For the contrast of congruent-incongruent, it is assumed that the underlying draining effect will be the same for both conditions, and so should be cancelled out. However, for the other contrasts, it is unclear how the final layer profiles aren't confounded by the bias in BOLD signal towards the superficial layers. Many of the profiles in Figure 3 and Figure 4A show an increased negative correlation between alpha power and the BOLD signal towards the superficial layers.

When investigating if high alpha (8-10 Hz) and low alpha (11-13 Hz) are two different sources of alpha, it would be beneficial to show if this effect is only seen at the group level or can be seen in any single subjects. Inter-subject variability in peak alpha power could result in some subjects having a single low alpha peak and some a single high alpha peak rather than two peaks from different sources.

The figure layout used to present the main findings throughout is an innovative way to present so much information, but it is difficult to decipher the main findings described in the text. The readability would be improved if the example (Appendix 0 - Figure 1) in the supplementary material is included as a second panel inside Figure 3, or, if this is not possible, the example (Appendix 0 - Figure 1) should be clearly referred to in the figure caption.

eLife Assessment

This important study shows that visual search for upright and rotated objects is affected by rotating participants in a VR and gravitational reference frame. However, the evidence supporting this conclusion is incomplete, given the authors' use of normalized response time and the assumption that object recognition across rotations requires mental rotation.

Reviewer #1 (Public review):

Summary:

The current study sought to understand which reference frames humans use when doing visual search in naturalistic conditions. To this end, they had participants do a visual search task in a VR environment while manipulating factors such as object orientation, body orientation, gravitational cues, and visual context (where the ground is). They generally found that all cues contributed to participants' performance, but visual context and gravitational cues impacted performance the most, suggesting that participants represent space in an allocentric reference frame during visual search.

Strengths:

The study is valuable in that it sheds light on which cues participants use during visual search. Moreover, I appreciate the use of VR and precise psychophysical predictions (e.g., slope vs. intercept) to dissociate between possible reference frames.

Weaknesses:

It's not clear what the implications of the study are beyond visual search. Moreover, I have some concerns about the interpretation of Experiment 1, which relies on an incorrect interpretation of mental rotation. Thus, most of the conclusions rely on Experiment 2, which has a small sample size (n = 10). Finally, the statistical analyses could be strengthened with measures of effect size and non-parametric statistics.

Reviewer #2 (Public review):

Summary:

This paper addresses an interesting issue: how is the search for a visual target affected by its orientation (and the viewer's) relative to other items in the scene and gravity? The paper describes a series of visual search tasks, using recognizable targets (e.g., a cat) positioned within a natural scene. Reaction times and accuracy at determining whether the target was present or absent, trial-to-trial, were measured as the target's orientation, that of the context, and of the viewer themselves (via rotation in a flight simulator) were manipulated. The paper concludes that search is substantially affected by these manipulations, primarily by the reference frame of gravity, then visual context, followed by the egocentric reference frame.

Strengths:

This work is on an interesting topic, and benefits from using natural stimuli in VR / flight simulator to change participants' POV and body position.

Weaknesses:

There are several areas of weakness that I feel should be addressed.

(1) The literature review/introduction seems to be lacking in some areas. The authors, when contemplating the behavioral consequences of searching for a 'rotated' target, immediately frame the problem as one of rotation, per se (i.e., contrasting only rotation-based explanations; "what rotates and in which 'reference frame[s]' in order to allow for successful search?"). For a reader not already committed to this framing, many natural questions arise that are worth addressing.

1a) Why do we need to appeal to rotation at all as opposed to, say, familiarity? A rotated cat is less familiar than a typically oriented one. This is a long-standing literature (e.g., Wang, Cavanagh, and Green (1994)), of course, with a lot to unpack.

1b) What are the triggers for the 'corrective' rotation that presumably brings reference frames back into alignment? What if the rotation had not been so obvious (i.e. for a target that may not have a typical orientation, like a hand, or a ball, or a learned, nonsense object?) or the background had not had such clear orientation (like a cluttered non-naturalistic background of or a naturalistic backdrop, but viewed from an unfamiliar POV (e.g., from above) or a naturalistic background, but not all of the elements were rotated)? What, ultimately, is rotated? The entire visual field? Does that mean that searching for multiple targets at different angles of rotation would interfere with one another?

1c) Relatedly, what is the process by which the visual system comes to know the 'correct' rotation? (Or, alternatively, is 'triggered to realize' that there is a rotation in play?) Is this something that needs to be learned? Is it only learned developmentally, through exposure to gravity? Could it be learned in the context of an experiment that starts with unfamiliar stimuli?

1d) Why the appeal to natural images? I appreciate any time a study can be moved from potentially too stripped-down laboratory conditions to more naturalistic ones, but is this necessary in the present case? Would the pattern of results have been different if these were typical laboratory 'visual search' displays of disconnected object arrays?

1e) How should we reconcile rotation-based theories of 'rotated-object' search with visual search results from zero gravity environments (e.g., for a review, see Leone (1998))?

1f) How should we reconcile the current manipulations with other viewpoint-perspective manipulations (e.g., Zhang & Pan (2022))?

(2) The presentation/interpretation of results would benefit from more elaboration and justification.

2a) All of the current interpretations rely on just the RT data. First, the RT results should also be presented in natural units (i.e., seconds/ms), not normalized. As well, results should be shown as violin plots or something similar that captures distribution - a lot of important information is lost when just presenting one 'average' dot across participants. More fundamentally, I think we need to have a better accounting for performance (percent correct or d') to help contextualize the RT results. We should at least be offered some visualization (Heitz, 2014) of the speed accuracy trade-off for each of the conditions. Following this, the authors should more critically evaluate how any substantial SAT trends could affect the interpretation of results.

2b) Unless I am missing something, the interpretation of the pattern of results (both qualitatively and quantitatively in their 'relative weight' analysis) relies on how they draw their contrasts. For instance, the authors contrast the two 'gravitational' conditions (target 0 deg versus target 90 deg) as if this were a change in a single variable/factor. But there are other ways to understand these manipulations that would affect contrasts. For instance, if one considers whether the target was 'consistent' (i.e., typically oriented) with respect to the context, egocentric, and gravitational frames, then the 'gravitational 0 deg' condition is consistent with context, egocentric view, but inconsistent with gravity. And, the 'gravitational 90 deg' condition, then, is inconsistent with context, egocentric view, but consistent with gravity. Seen this way, this is not a change in one variable, but three. The same is true of the baseline 0 deg versus baseline 90 deg condition, where again we have a change in all three target-consistency variables. The 'one variable' manipulations then would be: 1) baseline 0 versus visual context 0 (i.e., a change only in the context variable); 2) baseline 0 versus egocentric 0 (a change only in the egocentric variable); and 3) baseline 0 versus gravitational 0 (a change only in the gravitational variable). Other contrasts (e.g., gravitational 90 versus context 90) would showcase a change in two variables (in this case, a change in both context and gravity). My larger point is, again, unless I am really missing something, that the choice of how to contrast the manipulations will affect the 'pattern' of results and thereby the interpretation. If the authors agree, this needs to be acknowledged, plausible alternative schemes discussed, and the ultimate choice of scheme defended as the most valid.

2c) Even with this 'relative weight' interpretation, there are still some patterns of results that seem hard to account for. Primarily, the egocentric condition seems hard to account for under any scheme, and the authors need to spend more time discussing/reconciling those results.

2d) Some results are just deeply counterintuitive, and so the reader will crave further discussion. Most saliently for me, based on the results of Experiment 2 (specifically, the fact that gravitational 90 had better performance than gravitational 0), designers of cockpits should have all gauges/displays rotate counter to the airplane so that they are always consistent with gravity, not the pilot. Is this indeed a fair implication of the results?

2e) I really craved some 'control conditions' here to help frame the current results. In keeping with the rhetorical questions posed above in 1a/b/c/d, if/when the authors engage with revisions to this paper, I would encourage the inclusion of at least some new empirical results. For me the most critical would be to repeat some core conditions, but with a symmetric target (e.g. a ball) since that would seem to be the only way (given the current design) to tease out nuisance confounding factors such as, say, the general effect of performing search while sideways (put another way, the authors would have to assume here that search (non-normalized RT's and search performance) for a ball-target in the baseline condition would be identical to that in the gravitational condition.)

Reviewer #3 (Public review):

The study tested how people search for objects in natural scenes using virtual reality. Participants had to find targets among other objects, shown upright or tilted. The main results showed that upright objects were found faster and more accurately. When the scene or body was rotated, performance changed, showing that people use cues from the environment and gravity to guide search.

The manuscript is clearly written and well designed, but there are some aspects related to methods and analyses that would benefit from stronger support.

First, the sample size is not justified with a power analysis, nor is it explained how it was determined. This is an important point to ensure robustness and replicability.

Second, the reaction time data were processed using different procedures, such as the use of the median to exclude outliers and an ad hoc cut-off of 50 ms. These choices are not sufficiently supported by a theoretical rationale, and could appear as post-hoc decisions.

Third, the mixed-model analyses are overall well-conducted; however, the specification of the random structure deserves further consideration. The authors included random intercepts for participants and object categories, which is appropriate. However, they did not include random slopes (e.g., for orientation or set size), meaning that variability in these effects across participants was not modelled. This simplification can make the models more stable, but it departs from the maximal random structure recommended by Barr et al. (2013). The authors do not explicitly justify this choice, and a reviewer may question why participant-specific variability in orientation effects, for example, was not allowed. Given the modest sample sizes (20 in Experiment 1 and 10 in Experiment 2), convergence problems with more complex models are likely. Nonetheless, ignoring random slopes can, in principle, inflate Type I error rates, so this issue should at least be acknowledged and discussed.

This sentence reveals the deep dilemma that black women face in the process of developing their identity: even if they live in black communities and have more social support, they still cannot escape the influence of systematic devaluation and social prejudice, especially economic disadvantages, which will exacerbate this oppression.

This sentence not only describes the laws of psychological development, but also points to the core of social fairness. Only when every teenager is no longer forced to defend themselves or doubt themselves because of their racial identity can true identity freedom be achieved.

This statement highlights the dynamic nature of racial identity development. Natural integration in childhood and group differentiation in adolescence are not contradictory, but rather strategies individuals use at different stages to cope with social pressure. The core task of the education system is to recognize the importance of racial identity while avoiding essentializing it into rigid labels. Instead, it supports adolescents in their exploration and self-definition.

eLife Assessment

This study provides important results with regard to the ongoing debate of the relationship between internalizing psychopathology and learning under uncertainty. The methods and analyses are solid, and the results are backed by a large sample size, yet the study could still benefit from a more detailed discussion about the difference in experimental design and analysis compared to previous studies. If these concerns are addressed, this study would be of interest to researchers in clinical and computational psychiatry for the behavioral markers of psychopathological symptoms.

Reviewer #1 (Public review):

The authors conducted a series of experiments using two established decision-making tasks to clarify the relationship between internalizing psychopathology (anxiety and depression) and adaptive learning in uncertain and volatile environments. While prior literature has reported links between internalizing symptoms - particularly trait anxiety - and maladaptive increases in learning rates or impaired adjustment of learning rates, findings have been inconsistent. To address this, the authors designed a comprehensive set of eight experiments that systematically varied task conditions. They also employed a bifactor analysis approach to more precisely capture the variance associated with internalizing symptoms across anxiety and depression. Across these experiments, they found no consistent relationship between internalizing symptoms and learning rates or task performance, concluding that this purported hallmark feature may be more subtle than previously assumed.

Strengths:

(1) A major strength of the paper lies in its impressive collection of eight experiments, which systematically manipulated task conditions such as outcome type, variability, volatility, and training. These were conducted both online and in laboratory settings. Given that trial conditions can drive or obscure observed effects, this careful, systematic approach enables a robust assessment of behavior. The consistency of findings across online and lab samples further strengthens the conclusions.

(2) The analyses are impressively thorough, combining model-agnostic measures, extensive computational modeling (e.g., Bayesian, Rescorla-Wagner, Volatile Kalman Filter), and assessments of reliability. This rigor contributes meaningfully to broader methodological discussions in computational psychiatry, particularly concerning measurement reliability.

(3) The study also employed two well-established, validated computational tasks: a game-based predictive inference task and a binary probabilistic reversal learning task. This choice ensures comparability with prior work and provides a valuable cross-paradigm perspective for examining learning processes.

(4) I also appreciate the open availability of the analysis code that will contribute substantially to the field using similar tasks.

Weakness:

(1) While the overall sample size (N = 820 across eight experiments) is commendable, the number of participants per experiment is relatively modest, especially in light of the inherent variability in online testing and the typically small effect sizes in correlations with mental health traits (e.g., r = 0.1-0.2). The authors briefly acknowledge that any true effects are likely small; however, the rationale behind the sample sizes selected for each experiment is unclear. This is especially important given that previous studies using the predictive inference task (e.g., Seow & Gillan, 2020, N > 400; Loosen et al., 2024, N > 200) have reported non-significant associations between trait anxiety symptoms and learning rates.

(2) The motivation for focusing on the predictive inference task is also somewhat puzzling, given that no cited study has reported associations between trait anxiety and parameters of this task. While this is mitigated by the inclusion of a probabilistic reversal learning task, which has a stronger track record in detecting such effects, the study misses an opportunity to examine whether individual differences in learning-related measures correlate across the two tasks, which could clarify whether they tap into shared constructs.

(3) The parameterization of the tasks, particularly the use of high standard deviations (SDs) of 20 and 30 for outcome distributions and hazard rates of 0.1 and 0.16, warrants further justification. Are these hazard rates sufficiently distinct? Might the wide SDs reduce sensitivity to volatility changes? Prior studies of the circle version of this predictive inference task (e.g., Vaghi et al., 2019; Seow & Gillan, 2020; Marzuki et al., 2022; Loosen et al., 2024; Hoven et al., 2024) typically used SDs around 12. Indeed, the Supplementary Materials suggest that variability manipulations did not seem to substantially affect learning rates (Figure S5)-calling into question whether the task manipulations achieved their intended cognitive effects.

(4) Relatedly, while the predictive inference task showed good reliability, the reversal learning task exhibited only "poor-to-moderate" reliability in its learning-rate estimates. Given that previous findings linking anxiety to learning rates have often relied on this task, these reliability issues raise concerns about the robustness and generalizability of conclusions drawn from it.

(5) As the authors note, the study relies on a subclinical sample. This limits the generalizability of the findings to individuals with diagnosed disorders. A growing body of research suggests that relationships between cognition and symptomatology can differ meaningfully between general population samples and clinical groups. For example, Hoven et al. (2024) found differing results in the predictive inference task when comparing OCD patients, healthy controls, and high- vs. low-symptom subgroups.

(6) Finally, the operationalization of internalizing symptoms in this study appears to focus on anxiety and depression. However, obsessive-compulsive disorder is also generally considered an internalizing disorder, which presents a gap in the current cited literature of the paper, particularly when there have been numerous studies with the predictive inference task and OCD/compulsivity (e.g., Vaghi et al., 2019; Seow & Gillan, 2020; Marzuki et al., 2022; Loosen et al., 2024; Hoven et al., 2024), rather than trait anxiety per se.

Overall:

Despite the named limitations, the authors have done very impressive work in rigorously examining the relationship between anxiety/internalizing symptoms and learning rates in commonly used decision-making tasks under uncertainty. Their conclusion is well supported by the consistency of their null findings across diverse task conditions, though its generalizability may be limited by some features of the task design and its sample. This study provides strong evidence that will guide future research, whether by shifting the focus of examining dysfunctions of larger effect sizes or by extending investigations to clinical populations.

Reviewer #2 (Public review):

Summary:

In this work, the authors recruited a large sample of participants to complete two well-established paradigms: the predictive inference task and the volatile reversal learning task. With this dataset, they not only replicated several classical findings on uncertainty-based learning from previous research but also demonstrated that individual differences in learning behavior are not systematically associated with internalizing psychopathology. These results provide valuable large-scale evidence for this line of research.

Strengths:

(1) Use of two different tasks.

(2) Recruitment of a large sample of participants.

(3) Inclusion of multiple experiments with different conditions, demonstrating strong scientific rigor.

Weaknesses:

Below are questions rather than 'weaknesses':

(1) This study uses a large human sample, which is a clear strength. However, was the study preregistered? It would also be useful to report a power analysis to justify the sample size.

(2) Previous studies have tested two core hypotheses: (a) that internalizing psychopathology is associated with overall higher learning rates, and (b) that it is associated with learning rate adaptation. In the first experiment, the findings seem to disconfirm only the first hypothesis. I found it unclear how, in the predator task, participants were expected to adjust their learning rate to adapt to volatility. Could the authors clarify this point?

(3) According to the Supplementary Information, Model 13 showed the best fit, yet the authors selected Model 12 due to the larger parameter variance in Model 13. What would the results of Model 13 look like? Furthermore, do Models 12 and 13 correspond to the optimal models identified by Gagne et al. (2020)? Please clarify.

(4) In the Discussion, the authors addressed both task reliability and parameter reliability. However, the term reliability seems to be used differently in these two contexts. For example, good parameter recovery indicates strong reliability in one sense, but can we then directly equate this with parameter reliability? It would be helpful to define more precisely what is meant by reliability in each case.

(5) The Discussion also raises the possibility that limited reliability may represent a broader challenge facing the interdisciplinary field of computational psychiatry. What, in the authors' view, are the key future directions for the field to mitigate this issue?

Sociometer-theorie van Leary = zelfrespect (gevoel van goedkeuring, acceptatie en sympathie voor zichzelf) komt voort uit een interne meter die acceptatie en afwijzing in een sociale omgeving meet.

We construeren een zelfperceptie op basis van een zelfvergelijking met een referentiegroep. --> Big-fish-in-small-pond effect = studenten die op de middelbare de beste waren, zijn dat op de uni niet meer --> daling zelfvertrouwen.

Positieve illusory bias = volwassenen overschatten hun eigen capaciteiten.

Attitude = elke overtuiging/ mening die een beoordelend element bevat. Expliciete attidudes = bewuste, verbaal benoembare beoordelingen (langzaam, overwegen, prefrontale cortex) Impliciete attitudes = onbewuste, gemanifesteerd in automatische mentale associaties (snel, emotioneel, limbisch systeem --> impliciete associatietests: objecten sneller geclassificeerd als ze al sterk met elkaar geassocieerd zijn.

• Preconventioneel
• Straf vermijden
• Individueel voordeel
• Conventioneel
• Andere plezieren
• Autoriteiten bepalen wat goed en slecht is
• Post-conventioneel
• Maatschappij en individu beschermen
• Universele principes

Adolescentie = overgang van 'kind' naar 'volwassene'.

Verschuiving van focus op ouders naar leeftijdsgenoten --> vrienden kiezen met dezelfde interesses en gedrag.

Conformiteit = ze willen eruit en ze willen zich gedragen als leeftijdsgenoten.

Piaget = morele ontwikkeling --> kinderen leren met elkaar over het maken van sociale regels.

Vygotsky = impulscontrole (volgen van sociale regels) --> om mee te doen moet je je wel aan de regels houden.

Gemengde leeftijden = weinig competitie, leermomenten voor jongere (profiteren van kennis ouderen) en oudere (versterken kennis door onderwijzen) kinderen.

Sekseverschillen = - Fysieke en mentale gezondheid: meer agressie bij jongens, meisjes meer mentale stoornissen. - Fysieke ontwikkeling: jongens zijn actiever. - Cognitieve ontwikkeling: meisjes hebben betere taalvaardigheid - Sociaal-emotionele ontwikkeling: verschillen in risicogedrag, speelgoedkeuze en speelstijl.

! Ouders behandelen hun kinderen vanaf dag 1 gebaseerd op gender: meer praten met meisjes en meer stoeien met jongens

Kinderen beseffen al vroeg dat (1) ze een biologisch geslacht hebben en (2) dat dit geslacht altijd zal blijven bestaan. Ze vormen zichzelf actief volgens het concept van gender in een bepaalde cultuur.

Rond 8-11 jaar: hoogtepunt van de neiging om jongens (grote, hiërarchisch georganiseerde groepen + competitieve spellen + pesten/ opscheppen + koning van de heuvel) en meisjes (kleinere, meer intieme groepen + coöperatief spel + subtielere competitie + touwtje springen) te scheiden.

Should this be + or *? From the description of indicator \(\textbf{1}{s_t=s}\) and in the proof of Proposition 2.1, it seems be the latter.

add https://www.intel.com/content/www/us/en/developer/articles/technical/accelerating-language-model-inference-on-your-pc.html

Files modified after 30/09/2025

/hyperpost/🌐/🎭/gyuri/📓/2025/10/list-files.html

indirect meewerken aan aanmaak celmembraan, door aanmaak lipiden (steroiden enzo)

eLife Assessment

This valuable study describes MerQuaCo, a computational and automatic quality control tool for spatial transcriptomics datasets. The authors have collected a remarkable number of tissues to construct the main algorithm. The compelling strength of the evidence is demonstrated through a combination of empirical observations, automated computational approaches, and validation against existing software packages. MerQuaCo will interest researchers who routinely perform spatial transcriptomic imaging (especially MERSCOPE), as it provides an imperfection detector and quality control measures for reliable and reproducible downstream analysis.

Reviewer #1 (Public review):

Summary:

The authors present MerQuaCo, a computational tool that fills a critical gap in the field of spatial transcriptomics: the absence of standardized quality control (QC) tools for image-based datasets. Spatial transcriptomics is an emerging field where datasets are often imperfect, and current practices lack systematic methods to quantify and address these imperfections. MerQuaCo offers an objective and reproducible framework to evaluate issues like data loss, transcript detection variability, and efficiency differences across imaging planes.

Strengths:

(1) The study draws on an impressive dataset comprising 641 mouse brain sections collected on the Vizgen MERSCOPE platform over two years. This scale ensures that the documented imperfections are not isolated or anecdotal but represent systemic challenges in spatial transcriptomics. The variability observed across this large dataset underscores the importance of using sufficiently large sample sizes when benchmarking different image-based spatial technologies. Smaller datasets risk producing misleading results by over-representing unusually successful or unsuccessful experiments. This comprehensive dataset not only highlights systemic challenges in spatial transcriptomics but also provides a robust foundation for evaluating MerQuaCo's metrics. The study sets a valuable precedent for future quality assessment and benchmarking efforts as the field continues to evolve.

(2) MerQuaCo introduces thoughtful metrics and filters that address a wide range of quality control needs. These include pixel classification, transcript density, and detection efficiency across both x-y axes (periodicity) and z-planes (p6/p0 ratio). The tool also effectively quantifies data loss due to dropped images, providing tangible metrics for researchers to evaluate and standardize their data. Additionally, the authors' decision to include examples of imperfections detectable by visual inspection but not flagged by MerQuaCo reflects a transparent and balanced assessment of the tool's current capabilities.

Weaknesses:

(1) The study focuses on cell-type label changes as the main downstream impact of imperfections. Broadening the scope to explore expression response changes of downstream analyses would offer a more complete picture of the biological consequences of these imperfections and enhance the utility of the tool.

(2) While the manuscript identifies and quantifies imperfections effectively, it does not propose post-imaging data processing solutions to correct these issues, aside from the exclusion of problematic sections or transcript species. While this is understandable given the study is aimed at the highest quality atlas effort, many researchers don't need that level of quality to compare groups. It would be important to include discussion points as to how those cut-offs should be decided for a specific study.

(3) Although the authors demonstrate the applicability of MerQuaCo on a large MERFISH dataset, and the limited number of sections from other platforms, it would be helpful to describe its limitations in its generalizability.

Reviewer #2 (Public review):

Summary:

The authors present MerQuaCo, a computational tool for quality control in image-based spatial transcriptomic, especially MERSCOPE. They assessed MerQuaCo on 641 slides that are produced in their institute in terms of the ratio of imperfection, transcript density, and variations of quality by different planes (x-axis).

Strengths:

This looks to be a valuable work that can be a good guideline of quality control in future spatial transcriptomics. A well-controlled spatial transcriptomics dataset is also important for the downstream analysis.

Weaknesses:

The results section needs to be more structured.

Reviewer #3 (Public review):

Summary:

MerQuaCo is an open-source computational tool developed for quality control in image-based spatial transcriptomics data, with a primary focus on data generated by the Vizgen MERSCOPE platform. The authors analyzed a substantial dataset of 641 fresh-frozen adult mouse brain sections to identify and quantify common imperfections, aiming to replace manual quality assessment with an automated, objective approach, providing standardized data integrity measures for spatial transcriptomics experiments.

Strengths:

The manuscript's strengths lie in its timely utility, rigorous empirical validation, and practical contributions to methodology and biological discovery in spatial transcriptomics.

Weaknesses:

While MerQuaCo demonstrates utility in large datasets and cross-platform potential, its generalizability and validation require expansion, particularly for non-MERSCOPE platforms and real-world biological impact.

eLife Assessment

This study provides a valuable contribution to spatial transcriptomics by introducing MerQuaCo, a computational tool for standardizing quality control in image-based spatial transcriptomics datasets. The tool addresses the lack of consensus in the field and provides robust metrics to identify and quantify common imperfections in datasets. The work is supported by an impressive dataset and compelling analyses, and will be of significant interest to researchers focused on data reproducibility and downstream analysis reliability in spatial transcriptomics.

Reviewer #1 (Public review):

The authors present MerQuaCo, a computational tool that fills a critical gap in the field of spatial transcriptomics: the absence of standardized quality control (QC) tools for image-based datasets. Spatial transcriptomics is an emerging field where datasets are often imperfect, and current practices lack systematic methods to quantify and address these imperfections. MerQuaCo offers an objective and reproducible framework to evaluate issues like data loss, transcript detection variability, and efficiency differences across imaging planes.

Strengths

(1) The study draws on an impressive dataset comprising 641 mouse brain sections collected on the Vizgen MERSCOPE platform over two years. This scale ensures that the documented imperfections are not isolated or anecdotal but represent systemic challenges in spatial transcriptomics. The variability observed across this large dataset underscores the importance of using sufficiently large sample sizes when benchmarking different image-based spatial technologies. Smaller datasets risk producing misleading results by over-representing unusually successful or unsuccessful experiments. This comprehensive dataset not only highlights systemic challenges in spatial transcriptomics but also provides a robust foundation for evaluating MerQuaCo's metrics. The study sets a valuable precedent for future quality assessment and benchmarking efforts as the field continues to evolve.

(2) MerQuaCo introduces thoughtful metrics and filters that address a wide range of quality control needs. These include pixel classification, transcript density, and detection efficiency across both x-y axes (periodicity) and z-planes (p6/p0 ratio). The tool also effectively quantifies data loss due to dropped images, providing tangible metrics for researchers to evaluate and standardize their data. Additionally, the authors' decision to include examples of imperfections detectable by visual inspection but not flagged by MerQuaCo reflects a transparent and balanced assessment of the tool's current capabilities.

Comments on revisions:

All previous concerns have been fully addressed. The revised manuscript presents a robust, well-documented, and user-friendly tool for quality control in image-based spatial transcriptomics, a rapidly advancing area where objective assessment tools are urgently needed.

Reviewer #3 (Public review):

Summary:

MerQuaCo is an open-source computational tool developed for quality control in image-based spatial transcriptomics data, with a primary focus on data generated by the Vizgen MERSCOPE platform. The authors analyzed a substantial dataset of 641 fresh-frozen adult mouse brain sections to identify and quantify common imperfections, aiming to replace manual quality assessment with an automated, objective approach, providing standardized data integrity measures for spatial transcriptomics experiments.

Strengths:

The manuscript's strengths lie in its timely utility, rigorous empirical validation, and practical contributions to methodology and biological discovery in spatial transcriptomics.

Weaknesses:

While MerQuaCo demonstrates utility in large datasets and cross-platform potential, its generalizability and validation are currently limited by the availability of sufficient datasets from non-MERSCOPE platforms and non-brain tissues. The evaluation of data imperfections' impact on downstream analyses beyond cell typing (e.g., differential expression, spatial statistics, and cell-cell interactions) is also constrained by space and scope. However, these represent valuable directions for future work as more datasets become available.

Author response:

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

Reviewer #1 (Public review):

The authors present MerQuaCo, a computational tool that fills a critical gap in the field of spatial transcriptomics: the absence of standardized quality control (QC) tools for image-based datasets. Spatial transcriptomics is an emerging field where datasets are often imperfect, and current practices lack systematic methods to quantify and address these imperfections. MerQuaCo offers an objective and reproducible framework to evaluate issues like data loss, transcript detection variability, and efficiency differences across imaging planes.

Strengths:

(1) The study draws on an impressive dataset comprising 641 mouse brain sections collected on the Vizgen MERSCOPE platform over two years. This scale ensures that the documented imperfections are not isolated or anecdotal but represent systemic challenges in spatial transcriptomics. The variability observed across this large dataset underscores the importance of using sufficiently large sample sizes when benchmarking different image-based spatial technologies. Smaller datasets risk producing misleading results by over-representing unusually successful or unsuccessful experiments. This comprehensive dataset not only highlights systemic challenges in spatial transcriptomics but also provides a robust foundation for evaluating MerQuaCo's metrics. The study sets a valuable precedent for future quality assessment and benchmarking efforts as the field continues to evolve.

(2) MerQuaCo introduces thoughtful metrics and filters that address a wide range of quality control needs. These include pixel classification, transcript density, and detection efficiency across both x-y axes (periodicity) and z-planes (p6/p0 ratio). The tool also effectively quantifies data loss due to dropped images, providing tangible metrics for researchers to evaluate and standardize their data. Additionally, the authors' decision to include examples of imperfections detectable by visual inspection but not flagged by MerQuaCo reflects a transparent and balanced assessment of the tool's current capabilities.

Weaknesses:

(1) The study focuses on cell-type label changes as the main downstream impact of imperfections. Broadening the scope to explore expression response changes of downstream analyses would offer a more complete picture of the biological consequences of these imperfections and enhance the utility of the tool.

Here, we focused on the consequences of imperfections on cell-type labels, one common use for spatial transcriptomics datasets. Spatial datasets are used for so many other purposes that there are almost endless ways in which imperfections could impact downstream analyses. It is difficult to see how we might broaden the scope to include more downstream effects, while providing enough analysis to derive meaningful conclusions, all within the scope of a single paper. Existing studies bring some insight into the impact of imperfections and we expect future studies will extend our understanding of consequences in other biological contexts.

(2) While the manuscript identifies and quantifies imperfections effectively, it does not propose post-imaging data processing solutions to correct these issues, aside from the exclusion of problematic sections or transcript species. While this is understandable given the study is aimed at the highest quality atlas effort, many researchers don't need that level of quality to compare groups. It would be important to include discussion points as to how those cut-offs should be decided for a specific study.

Studies differ greatly in their aims and, as a result, the impact of imperfections in the underlying data will differ also, preventing us from offering meaningful guidance on how cut-offs might best be identified. Rather, our aim with MerQuaCo was to provide researchers with tools to generate information on their spatial datasets, to facilitate downstream decisions on data inclusion and cut-offs.

(3) Although the authors demonstrate the applicability of MerQuaCo on a large MERFISH dataset, and the limited number of sections from other platforms, it would be helpful to describe its limitations in its generalizability.

In figure 9, we addressed the limitations and generalizability of MerQuaCo as best we could with the available datasets. Gaining deep insight into the limitations and generalizability of MerQuaCo would require application to multiple large datasets and, to the best of our knowledge, these datasets are not available.

Reviewer #2 (Public review):

The authors present MerQuaCo, a computational tool for quality control in image-based spatial transcriptomic, especially MERSCOPE. They assessed MerQuaCo on 641 slides that are produced in their institute in terms of the ratio of imperfection, transcript density, and variations of quality by different planes (x-axis).

Strengths:

This looks to be a valuable work that can be a good guideline of quality control in future spatial transcriptomics. A well-controlled spatial transcriptomics dataset is also important for the downstream analysis.

Weaknesses:

The results section needs to be more structured.

We have split the ‘Transcript density’ subsection of the results into 3 new subsections.

Reviewer #3 (Public review):

MerQuaCo is an open-source computational tool developed for quality control in imagebased spatial transcriptomics data, with a primary focus on data generated by the Vizgen MERSCOPE platform. The authors analyzed a substantial dataset of 641 freshfrozen adult mouse brain sections to identify and quantify common imperfections, aiming to replace manual quality assessment with an automated, objective approach, providing standardized data integrity measures for spatial transcriptomics experiments.

Strengths:

The manuscript's strengths lie in its timely utility, rigorous empirical validation, and practical contributions to methodology and biological discovery in spatial transcriptomics.

Weaknesses:

While MerQuaCo demonstrates utility in large datasets and cross-platform potential, its generalizability and validation require expansion, particularly for non-MERSCOPE platforms and real-world biological impact.

We agree that there is value in expanding our analyses to non-Merscope platforms, to tissues other than brain, and to analyses other than cell typing. The limiting factor in all these directions is the availability of large enough datasets to probe the limits of MerQuaCo. We look forward to a future in which more datasets are available and it’s possible to extend our analyses

Reviewer #1(Recommendation for the Author):

(1) To better capture the downstream impacts of imperfections, consider extending the analysis to additional metrics, such as specificity variation across cell types, gene coexpression, or spatial gene patterning. This would deepen insights into how these imperfections shape biological interpretations and further demonstrate the versatility of MerQuaCo.

These are compelling ideas, but we are unable to study so many possible downstream impacts in sufficient depth in a single study. Insights into these topics will likely come from future studies.

(2) In Figure 7 legend, panel label (D) is repeated thus panels E-F are mislabelled. 

We have corrected this error.

(3) Ensure that the image quality is high for the figures. 

We will upload Illustrator files, ensuring that images are at full resolution.

Reviewer #2 (Recommendation for the Author):

(1) A result subsection "Transcript density" looks too long. Please provide a subsection heading for each figure. 

We have split this section into 3 with new subheadings.

(2) The result subsection title "Transcript density" sounds ambiguous. Please provide a detailed title describing what information this subsection contains. 

We have renamed this section ‘Differences in transcript density between MERSCOPE experiments’.

Minor: 

(1) There is no explanation of the black and grey bars in Figure 2A.

We have added information to the figure legend, identifying the datasets underlying the grey and black bars.

(2) In the abstract, the phrase "High-dimension" should be "High-dimensional". 

We have changed ‘high-dimension’ to ‘high-dimensional’.

(3) In the abstract, "Spatial results" is an unclear expression. What does it stand for? 

We have replaced the term ‘spatial results’ with ‘the outputs of spatial transcriptomics platforms’.

Reviewer #3 (Recommendation for the Author):

(1) While the tool claims broad applicability, validation is heavily centered on MERSCOPE data, with limited testing on other platforms. The authors should expand validation to include more diverse platforms and add a small analysis of non-brain tissue. If broader validation isn't feasible, modify the title and abstract to reflect the focus on the mouse brain explicitly.

We agree that expansion to other platforms is desirable, but to the best of our knowledge sufficient datasets from other platforms are not available. In the abstract, we state that ‘… we describe imperfections in a dataset of 641 fresh-frozen adult mouse brain sections collected using the Vizgen MERSCOPE.’

(2) The impact of data imperfections on downstream analysis needs a more comprehensive evaluation. The authors should expand beyond cluster label changes to include a) differential expression analysis with simulated imperfections, b) impact on spatial statistics and pattern detection, and c) effects on cell-cell interactions. 

Each of these ideas could support a substantial study. We are unable to do them justice in the limited space available as an addition to the current study.

(3) The pixel classification workflow and validation process need more detailed documentation. 

The methods and results together describe the workflow and validation in depth. We are unclear what details are missing.

(4) The manuscript lacks comparison to existing. QC pipelines such as Squidpy and Giotto. The authors should benchmark MerQuaCo against them and provide integration options with popular spatial analysis tools with clear documentation.

To the best of our knowledge, Squidpy and Giotto lack QC benchmarks, certainly of the parameters characterized by MerQuaCo. Direct comparison isn’t possible.

this except seems both literal and symbolic. the writer when referring to "stich the cut" seems genuine and literal and also while proceeding to stich the wound they want all their memories and life experience to be put back as such.

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