Reviewer #3 (Public review):

Summary:

Large Language Models have revolutionized Artificial Intelligence and can now match or surpass human language abilities on many tasks. This has fueled interest in cognitive neuroscience in exposing representational similarities between Language Models and brain recordings of language comprehension. The current study breaks from this mold by: (1) Systematically identifying sentence structures for which brain and Large Language Model representations diverge. (2) Demonstrating that brain representations for these sentences can be better accounted for by a model structured by the semantic roles of words in the sentence. As such, the study may now fuel interest in characterizing how Large Language Models and brain representations differ, which may prompt new, more brain-like language models.

Strengths:

(1) This study presents a bold and solid challenge to a literature trend that has touted similarities between Transformer models and human cognition based on representational correlations with brain activity. This challenge is substantiated by identifying sentences for which brain and model representations of sentences diverge and explaining those divergences using models structured by semantic roles/syntax.

(2) This study conducts a rigorous pre-registered analysis of a comprehensive selection of the state-of-the-art Large Language Models, on a controlled sentence comprehension fMRI dataset. The analysis is conducted within a Representation Similarity framework to support similarity comparisons between graph structures and brain activity without needing to vectorize graphs. Transformer models are predicted and shown to diverge from brain representations on subsets of sentences with similar word-level content but different sentence structures.

(3) The study introduces a 7T fMRI sentence comprehension dataset and accompanying human sentence similarity ratings, which may be a fruitful resource for developing more human-like language models. Unlike other model-based sentence datasets, the relation between grammatical structure and word-level content is controlled, and subsets of sentences for which models and brains diverge are identified.

Weaknesses:

(1) The interpretation of findings is nuanced. Although Transformers underperform as brain models on the critical subsets of controlled sentences, a Transformer outperforms all other models when evaluated on the union of all sentences when both word-level content and structure vary. Transformers also yield equivalent or better models of human behavioral data. Thus, although Transformers have demonstrable flaws as human models, which are pinpointed here, in the general case, (some) Transformers are more human-like than the other models considered.

(2) There may be confounds between the critical sentence structure manipulations and visual representations of sentence stimuli. This is inconvenient because activation in brain regions that process semantics tends to partially correlate with visual cortex representations, and computational models tend to reflect the number of words/tokens/elements in sentences. Although the study commendably controls for confounds associated with sentence length, there could still be residual effects that remain. For instance, the Graph model correlates most strongly with the visual cortex despite these sentence length controls.

(3) Sentence similarity computations are emphasized as the basis for unifying comparative analyses of graph structures and vector data. A strength of this approach is that correlation is not always the ideal similarity metric. However, a weakness is that similarity computations are not unified across models. This has practical consequences here because different similarity metrics applied to the same model produce positive or negative correlations with brain data.

Author response:

We thank the reviewers for their insightful comments on our manuscript. Here we briefly highlight our responses to several issues raised by reviewers, and also provide a summary of planned changes to be made with the next draft.

Reviewer 1:

(1) The reviewer questions the rationale for averaging sentence embeddings across different models. However, our method involves computing correlations separately for each model, then averaging the correlations. We also report model correlations for each model separately in Fig S2. We will clarify this in our revised manuscript.

(2) We agree with the reviewer that including a context-free grammar model as a comparison would be informative. We will incorporate this in the revised manuscript.

(3) The reviewer raises questions about the low correlation between behavioural and brain similarities. While the behavioural judgements are made by different participants and involve a different task than the neuroimaging results, nonetheless we agree the difference is surprising and warrants more detailed consideration. We will provide additional discussion of the relationship between behavioural judgements and brain data in the revised manuscript.

(4) The reviewer suggests contrasting our models with a ‘semantic ground truth’, as in our design matrix shown in Fig 1. While our design matrix served as the basis for constructing a set of stimuli with systematic modifications, we respectfully suggest that it should not be regarded as a ‘semantic ground truth’. In particular, sentence pairs within each category will not have the same degrees of semantic similarity since the words and context differ across sentences in a graded manner. Furthermore, while we anticipated ‘different’ sentence pairs would be less similar than ‘swapped’ sentence pairs, and that within each of the six block diagonals the ‘modified’ or ‘substituted’ sentence pairs would be the most similar, we did not have any prediction about the magnitude of these differences. Our goal was to construct a set of sentence pairs which spanned a range of semantic similarities, and allowed for dissociation between lexical similarity and overall similarity in meaning. The design matrix is not intended to represent a ‘ground truth’ that human judgements or brain representations would be expected to conform with.

(5) In the revised draft we will modify the location of Fig. 5 so that it flows better with the text.

(6) We agree that the discussion of the differences between brain regions could be expanded. We will include this in the revised version of our manuscript. The reviewer questions our inclusion of the simple-average and group-average RSA analysis as they show similar results. We included both analyses in line with our preregistration, and also because we believe the fact that two distinct approaches to analyzing the data yield similar results strengthens our conclusions.

(7) We believe that the grid-like pattern in the RSA results is an important unexpected finding that warrants discussion in the main manuscript.

Reviewer 2:

(1) The reviewer argues that our stimuli do not fully control for lexical content across conditions, and that a more appropriate paradigm may be to utilise minimal pairs in which only a single variable of interest (such as sentence structure) is modified. We agree that most of our sentence pairs do not constitute minimal pairs, however this was not our objective. Our study design aimed to synthesise traditional minimal pair approaches with more recent research paradigms using naturalistic stimuli. As such, we selected stimuli which are more complex and contain more variable features than traditional minimal pair studies, but which also are tailored to highlight differences which are of particular theoretical interest. Because we are interested in comparing the effects of multiple sentence elements and semantic roles, a systematic pairwise comparison of minimal pairs is not necessarily optimal. Instead, we designed our stimuli to leverage the advantage of fMRI in that we can measure the brain representations corresponding to each sentence, and hence can conduct a full series of pairwise comparisons of sentence representations. Most of these comparisons will not be between minimal pairs, but we selected sentences so as to provide a range of semantic similarities (low to high), while also providing for semantic contrasts of theoretical interest (such as the ‘swapped’ and ‘substituted’ sentence pairs). We do not claim this approach to be universally superior to a minimal pair approach, but we do believe our novel approach provides additional insights and a new perspective on semantic representation relative to minimal pair studies. We will add additional detail in the revised manuscript providing additional explanation for how stimuli were chosen, and contrasting this with minimal pair approaches.

(2) The reviewer notes that low RSA correlations do not imply that transformers fail to encode syntactic information. We acknowledge this in our discussion (page 10), where we also highlight that our focus is not on whether transformers encode such information, but rather what transformer representations can tell us about how sentence structure is represented in the brain. Our results indicate that transformer embeddings do not have the same geometric properties as brain representations of sentence meaning, at least for certain types of sentences where lexical information is insufficient to determine overall meaning. The reviewer also notes that transformer embeddings are highly anisotropic, however we adjust for this by normalising each feature as discussed on page 14. Finally, the reviewer notes that the transformers we examine differ in architecture and training objectives. This is not critical for our study because we are not seeking to determine which architecture or training objectives are best. Our goal is simply to compare a range of approaches and see which, if any, have similar sentence representations to those formed by the brain. In fact, our results indicate that architecture and training regime make relatively little difference for our stimuli.

(3) The reviewer argues that RSA correlations do not measure the extent to which a model encodes syntactic information. This is very similar to the previous point. We do not claim that our results show that transformers do not encode syntactic information. Rather, our claim is that sentence embeddings derived from transformers have different geometric properties to brain representations, and that brain representations are better described by models explicitly representing key semantic roles. From this we conclude that, at least for the sentences we present, the brain is highly sensitive to semantic roles in a way that transformer representations are not (at least to the same extent). We also respectfully disagree with the reviewer’s suggestions that sentence length and orthographic or lexical similarities may drive model correlations with brain activity. As we discuss on page 19, we explicitly control for differences in sentence length when computing correlations. Our process for constructing our sentence set also controls for lexical similarity by generating pairs of sentences with all or mostly the same words but different orderings. We did not explicitly address orthographic similarity, but this will be strongly correlated with lexical similarity.

Reviewer 3:

(1) The reviewer emphasises the need for nuance in our conclusions, given that some of the transformers achieve higher correlations when assessed over the full set of sentences. We agree with this comment, and will modify the discussion section in the revised manuscript to address this point. Having said that, we would like to note one of the disadvantages of transformers as a model of mind or brain representations is that they are largely a ‘black box’ whose workings are poorly understood. One advantage of hybrid models like our simple semantic role model is that they can be much easier to interpret, thereby enabling them to be used to determine which features are most important for brain representations of sentence meaning, and what mechanisms are used to combine individual words into a full sentence. Given their relative simplicity and interpretability, we believe hybrid models have considerable value as scientific tools, even in cases where they achieve comparable correlations to transformers. We will highlight this issue more clearly in our revised manuscript.

(2) The reviewer notes that despite our existing controls, residual confounds of sentence length may remain. We agree that this is a potential issue, and will add discussion to the revised manuscript. We also will present further supplementary analyses which we believe indicate that sentence length effects do not drive our main results. At the same time, we believe the fact that our results are robust to simultaneously controlling for sentence length and the ‘minimum length effect’ (Fig. S5) indicates they are not primarily driven by sentence length effects.

(3) The reviewer notes that the method for computing similarities differs between the vector-based (mean and transformer) models, and the hybrid and syntax-based models, thereby potentially adding an additional confound to our results. We agree that this is a potential limitation, and our correlations should always be understood as applying to a model paired with a similarity metric. However, we believe that this is mostly unavoidable when comparing different formalisms. An alterative approach of first embedding a graph into a vector and then training an encoding model on the graph embeddings has a similar limitation of being dependent not just on the graph representation, but also on the way it was embedded into a vector and the way the encoding model was trained. Arguably this process is more opaque than similarity methods, since it is unclear to what extent the graph embeddings preserve the logic and properties of a graph-based representation. Further, it not clear whether there is any single method which can overcome the difficulty of comparing distinct formalisms for representing semantics. The reviewer also highlights how the correlations measured for the syntax model differ greatly depending on whether the Smatch or WWLK similarity metrics are used. We believe this highlights the need for careful examination of commonly used graph similarity metrics, as has been noted in previous research. We will include additional discussion of this issue in our revised manuscript.

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eLife Assessment

This useful study introduces a computational pipeline for designing RNA in situ fluorescence hybridization probes that could improve the sensitivity and specificity of RNA detection in cells. While the approach is novel and the preliminary data suggestive, the evidence supporting a clear advantage over existing probe design strategies is incomplete. The work will be of interest to researchers developing or using molecular tools for imaging RNA in cells.

Reviewer #1 (Public review):

The authors describe a new computational pipeline designed to identify smFISH probes with improved RNA detection compared to preexisting approaches. smFISH is a powerful and relatively straightforward technique to detect single RNAs in cells at subcellular resolution, which is critical for understanding gene expression regulation at the RNA level. However, existing methods for designing smFISH oligos suffer from several limitations, including off-target binding that produces high background signals, as well as a restricted number of probes that are sufficiently specific to target shorter-than-average mRNAs. To address these challenges, the authors developed TrueProbes, a computational method that aims to minimize off-target-mediated background fluorescence.

Overall, the study addresses a technically relevant problem. If improved, this would allow researchers to study gene expression regulation more effectively using single-molecule FISH. However, based on the current presentation of data, it is not yet clear that TrueProbes offers significant advantages over preexisting pipelines. In the following section, I describe some concerns, which should be adequately addressed.

Major Comments:

(1) The manuscript currently presents only one example in which different pipelines were tested to generate probes (targeting ARF4). While the images suggest that both TrueProbes and Stellaris outperform the other pipelines, the comparison is potentially misleading because the number of probes used differs substantially. I recommend that the authors include at least three independent examples in which an equal number of probes are designed across pipelines, so that signal-to-noise can be assessed in a controlled and comparable way. This would allow the probe number to be held constant while directly evaluating performance.

(2) It is also unclear how many biological replicates were performed for the ARF4 experiments. If only a single replicate was included, it is difficult to conclude that TrueProbes consistently outperforms other pipelines in a robust and reproducible manner. I suggest the authors include data from at least three biological replicates with appropriate statistical analysis, and ideally extend this to additional smFISH targets as outlined in Comment 1.

(3) No controls are presented to demonstrate that the TrueProbes-designed smFISH spots are specifically detecting ARF4. The current experiment primarily measures signal-to-noise, but it remains possible that some detected spots do not correspond to ARF4 mRNAs. Since one of the major criteria used by TrueProbes is to limit cross-hybridization, the authors should perform ARF4 knockdown experiments and demonstrate that nearly all ARF4 smFISH signal is lost. A similar approach should be applied to the additional examples recommended in Comment 1.

(4) In the limitations of the study, the authors note that "RNA secondary and tertiary structures are not included, which may lead to inaccuracies if binding sites are structurally occluded." However, I am not convinced that this is a true limitation, since formamide in the smFISH protocol should denature secondary structures and allow oligo access to the RNA. I recommend that the authors comment on this point and clarify whether secondary structure poses a practical limitation in smFISH probe design.

(5) The authors also correctly acknowledge in their limitations that "RNA-protein interactions, which can modulate accessibility of the transcript, are not modeled." I suggest referencing relevant studies on this issue, particularly Buxbaum et al. (2014, Science), which would provide important context.

Reviewer #2 (Public review):

Summary:

Hughes et al present a new single-molecule RNA fluorescence in situ hybridization (smFISH) probe design software, termed "TrueProbes" in this manuscript. They claim that all existing smFISH (and variants) probe design software packages have limitations that ultimately impact experimental performance. The author's claim to address the majority of these limitations in TrueProbes by introducing multiple computational steps to ensure high-quality probe design. The manuscript's goal is clear, and the authors provide some evidence by designing and targeting one gene. Overall, the manuscript lacks rigorous evidence to support the claims, does not demonstrate its suitability for a variety of smFISH-type experiments, and some of the provided quantification data are unclear. While TrueProbes clearly has potential, more data is required, or the authors should tone down the claims.

Strengths:

(1) The problem is well-articulated in the abstract and the introduction.

(2) Figures 3 and 4 follow a consistent color scheme where each probe design method has its own color, which helps the reader visually compare methods.

(3) The authors compared multiple probe design software packages both computationally and experimentally.

(4) TrueProbes does produce visually and quantitatively better results when compared to 2 of the 4 existing smFISH probe design packages (Paintshop and MERFISH panel designer).

(5) The authors introduce a comprehensive steady-state thermodynamic model to help optimally guide probe design.

Weaknesses:

(1) The abstract describes the problem well and introduces the solution (the TrueProbes software), but fails to provide specific ways in which the TrueProbes software performs better. The authors state that "...[TrueProbes] consistently outperformed alternatives across multiple computational metrics and experimental validation assays", but specific, quantitative evidence of improved performance would strengthen the statement.

(2) The text claims that TrueProbes outperforms all other probe design software, but Figure 3 indicates that TrueProbes has neither the greatest number of on-target binding nor the lowest number of off-target binding. The data in Figure 3 does not support the claims made in the text. Specifically, the authors claim that "RNA FISH Experimental Results Demonstrate that Off Target and Binding Affinity Inclusive Probe Design Improve RNA FISH Signal Discrimination" (lines 217-218). However, despite their claim that Stellaris and Oligostan-HT produce more off-target probes when evaluated with the TrueProbes framework, the experiment results are nearly identical. The authors should consider modifying their claims or performing new experiments that more clearly demonstrate their claims.

(3) The bar graphs in Figure 3 do not seem to agree with the probability graphs in Figure 4. For example, Figure 3 indicates that Stellaris probes have higher off-target binding than TrueProbes; however, in Figure 4, their probability graphs lie almost on top of each other.

(4) The authors performed validation for only one gene (ARF4), because "...it had the highest gene expression (in TPM units) and the fewest isoforms among all candidate genes for the Jurkat cell line" (lines 176-177). While the results do look good, this is a minimal use case and does not really showcase the power of their method. One experiment that could be helpful would be two-color (or more) smFISH in tissue, where the chances for off-target binding contributing to higher errors are much greater than in an adherent cell line.

(5) A common strategy for both smFISH and highly multiplexed methods is to use secondary DNA oligos with dye molecules instead of direct conjugation. Given that this is a primary design goal of PaintSHOP and the Zhuang lab's MERFISH probe design code, it would be helpful to demonstrate that TrueProbes can design a two-layer probe strategy for high-quality RNA-FISH labeling.

(6) The authors claim, "For every probe set, TrueProbes can simulate expected smRNA FISH outcomes including optimal probe, RNA, and salt concentrations and optionally account for probe secondary structure, hybridization temperature, multiple targets, fluorophore choice, DNA, nascent RNA, and photon count statistics (Figures S2A, S2B). The model can be used to generate predictions for temperature and cell line sensitivity, multi-target discrimination, multiple fluorophore colocalization; when provided transcript expression levels and probe/background intensity, it can start to generate predictions for spot intensity, background, signal to noise ratio, and false negative rates (Figure S2C)." (lines 156-163). Figure S2 is a flow chart and does not provide evidence for any of these items. The authors should provide evidence for these claims, either as a figure or an example script in their software repository. If that is not possible, then it should be removed.

(7) All thermodynamic equations are performed at steady state. The authors do not justify this assumption, and there is no discussion of the potential impacts of either low molecule numbers or violations of the well-mixed assumption. Can the authors please include a discussion on the potential impacts non non-steady state dynamics?

Reviewer #3 (Public review):

Summary:

This manuscript introduces a new platform termed "TrueProbes" for designing mRNA FISH probes. In comparison to existing design strategies, the authors incorporate a comprehensive thermodynamic and kinetic model to account for probe states that may contribute to nonspecific background. The authors validate their design pipeline using Jurkat cells and provide evidence of improved probe performance.

Strengths:

A notable strength of TrueProbes is the consideration of genome-wide binding affinities, which aims to minimize off-target signals. The work will be of interest to researchers employing mRNA FISH in certain human cell lines.

Weaknesses:

However, in my view, the experimental validation is not sufficient to justify the broad claims of the platform. Given the number of assumptions in the model, additional experimental comparisons across probe design methods, ideally targeting transcripts with different expression levels, would be necessary to establish the general superiority of this approach.

Author response:

Reviewer #1 (Public Review):

The authors describe a new computational pipeline designed to identify smFISH probes with improved RNA detection compared to preexisting approaches. smFISH is a powerful and relatively straightforward technique to detect single RNAs in cells at subcellular resolution, which is critical for understanding gene expression regulation at the RNA level. However, existing methods for designing smFISH oligos suffer from several limitations, including off-target binding that produces high background signals, as well as a restricted number of probes that are sufficiently specific to target shorter-than-average mRNAs. To address these challenges, the authors developed TrueProbes, a computational method that aims to minimize off-target-mediated background fluorescence.

Overall, the study addresses a technically relevant problem. If improved, this would allow researchers to study gene expression regulation more effectively using single-molecule FISH. However, based on the current presentation of data, it is not yet clear that TrueProbes offers significant advantages over preexisting pipelines. In the following section, I describe some concerns, which should be adequately addressed.

Major Comments:

(1) The manuscript currently presents only one example in which different pipelines were tested to generate probes (targeting ARF4). While the images suggest that both TrueProbes and Stellaris outperform the other pipelines, the comparison is potentially misleading because the number of probes used differs substantially. I recommend that the authors include at least three independent examples in which an equal number of probes are designed across pipelines, so that signal-to-noise can be assessed in a controlled and comparable way. This would allow the probe number to be held constant while directly evaluating performance.

This is an important observation. We have already addressed this issue in Figures 3E-G and Supplementary Figure 4E-G, where we plotted the number of OFF-targets for each ON-target probe. If we select longer genes to ensure an equal number of designed probes with strong signals, we will still end up with the same number of ON-target probes. Consequently, Figures 3B-D and 3E-G would show similar trends, albeit with different values on the y-axis. Additionally, we will conduct an analysis using Stellaris at its highest probe design stringency setting to compare the software under its strictest design conditions. Additional experiments are outside the scope of the current manuscript.

(2) It is also unclear how many biological replicates were performed for the ARF4 experiments. If only a single replicate was included, it is difficult to conclude that TrueProbes consistently outperforms other pipelines in a robust and reproducible manner. I suggest the authors include data from at least three biological replicates with appropriate statistical analysis, and ideally extend this to additional smFISH targets as outlined in Comment 1.

Three biological replicates were utilized for the ARF4 experiments. As stated in the original submission, the average data from all three replicates is presented in Figure 4, while the data for each individual replicate can be found in Figure S5. Statistical analyses were conducted for both the pooled data in Figure 4 and the individual data in Figure S5. The results of all statistical calculations are detailed in Supplemental Table 1. We will update the text to clearly indicate the number of biological replicates and the outcomes of the statistical analysis.

(3) No controls are presented to demonstrate that the TrueProbes-designed smFISH spots are specifically detecting ARF4. The current experiment primarily measures signal-to-noise, but it remains possible that some detected spots do not correspond to ARF4 mRNAs. Since one of the major criteria used by TrueProbes is to limit cross-hybridization, the authors should perform ARF4 knockdown experiments and demonstrate that nearly all ARF4 smFISH signal is lost. A similar approach should be applied to the additional examples recommended in Comment 1.

Thank you for your suggestion. Currently, we lack the expertise in our lab to conduct such experiments, so they are beyond the scope of this manuscript. However, we will create additional supplementary figures to demonstrate that the likelihood of false positives is low, based on the assumption that current publicly available BLAST algorithms, genome annotations, and reference transcription expression data are accurate.

We will include a comparison in our supplementary materials showing the off-target RNA that can bind the highest number of probes simultaneously for each software. Additionally, we will perform a correlation analysis to illustrate the relationship between spot intensity for different software and the number of probes they design. This will help us estimate how the number of probes bound to RNA correlates with expected spot intensity ranges.

Using this information, along with autofluorescence background intensity measurements from no-probe controls, we will estimate the minimum number of probes that need to bind to targets to be detected as single spots. If this minimum is higher than the maximum number of simultaneous off-target probe bindings, we anticipate that the detected spot signal will primarily reflect ARF4 rather than other transcripts.

(4) In the limitations of the study, the authors note that "RNA secondary and tertiary structures are not included, which may lead to inaccuracies if binding sites are structurally occluded." However, I am not convinced that this is a true limitation, since formamide in the smFISH protocol should denature secondary structures and allow oligo access to the RNA. I recommend that the authors comment on this point and clarify whether secondary structure poses a practical limitation in smFISH probe design.

Thank you for pointing this out. We will revise the manuscript to clarify: "We did not include RNA secondary and tertiary structures in the model because the use of formamide in RNA-FISH experiments denatures these structures, allowing oligonucleotides to access the RNA."

(5) The authors also correctly acknowledge in their limitations that "RNA-protein interactions, which can modulate accessibility of the transcript, are not modeled." I suggest referencing relevant studies on this issue, particularly Buxbaum et al. (2014, Science), which would provide important context.

Thank you for highlighting the literature that supports this limitation. We will include Buxbaum et al. (2014, Science) and additional studies that discuss how RNA-protein interactions can affect RNA-FISH experiments.

Reviewer #2 (Public review):

Summary:

Hughes et al present a new single-molecule RNA fluorescence in situ hybridization (smFISH) probe design software, termed "TrueProbes" in this manuscript. They claim that all existing smFISH (and variants) probe design software packages have limitations that ultimately impact experimental performance. The author's claim to address the majority of these limitations in TrueProbes by introducing multiple computational steps to ensure high-quality probe design. The manuscript's goal is clear, and the authors provide some evidence by designing and targeting one gene. Overall, the manuscript lacks rigorous evidence to support the claims, does not demonstrate its suitability for a variety of smFISH-type experiments, and some of the provided quantification data are unclear. While TrueProbes clearly has potential, more data is required, or the authors should tone down the claims.

We appreciate the reviewer’s thoughtful feedback. We will revise the text to ensure that all claims are backed by computational or experimental evidence. For claims that do not have supporting results, we will relocate them to the discussion section as potential future extensions. Since our probe design is open access, both we and the community can further develop our codes as needed.

Strengths:

(1) The problem is well-articulated in the abstract and the introduction.

(2) Figures 3 and 4 follow a consistent color scheme where each probe design method has its own color, which helps the reader visually compare methods.

(3) The authors compared multiple probe design software packages both computationally and experimentally.

(4) TrueProbes does produce visually and quantitatively better results when compared to 2 of the 4 existing smFISH probe design packages (Paintshop and MERFISH panel designer).

(5) The authors introduce a comprehensive steady-state thermodynamic model to help optimally guide probe design.

We like to thank the reviewer for pointing out the strength of the manuscript.

Weaknesses:

(1) The abstract describes the problem well and introduces the solution (the TrueProbes software), but fails to provide specific ways in which the TrueProbes software performs better. The authors state that "...[TrueProbes] consistently outperformed alternatives across multiple computational metrics and experimental validation assays", but specific, quantitative evidence of improved performance would strengthen the statement.

Thank you for acknowledging the clarity of the abstract and introduction. We will revise the abstract to provide more specific details on how TrueProbes outperforms other software. Additionally, we will include specific computational and experimental metrics that demonstrate TrueProbes' improved performance compared to other software.

(2) The text claims that TrueProbes outperforms all other probe design software, but Figure 3 indicates that TrueProbes has neither the greatest number of on-target binding nor the lowest number of off-target binding. The data in Figure 3 does not support the claims made in the text. Specifically, the authors claim that "RNA FISH Experimental Results Demonstrate that Off Target and Binding Affinity Inclusive Probe Design Improve RNA FISH Signal Discrimination" (lines 217-218). However, despite their claim that Stellaris and Oligostan-HT produce more off-target probes when evaluated with the TrueProbes framework, the experiment results are nearly identical. The authors should consider modifying their claims or performing new experiments that more clearly demonstrate their claims.

In Figure 3, we aim to convey two main points. 

The first point is to compare the number of ON-target probes designed by each software using their most stringent design criteria (Figure 3A). Currently, we are using a medium strict design criterion for Stellaris (level 3). As shown in the new supplementary figure XX, when we apply the most stringent design criteria for Stellaris (level 5), the number of ON-target probes decreases to XX probes. This clearly indicates that, based on theoretical calculations, TrueProbes can design more probes than any of its competitors.

The second point is to compare the number of OFF-targets produced by each probe design. To illustrate this, we used two different metrics. In Figures 3B-D, we compare the total number of probes bound to OFF-target RNA. However, since each software generates a different number of ON-target probes, the number of OFF-targets may vary simply due to the differences in ON-target probe counts. Therefore, we introduced a second metric to compare OFF-targets. In Figures 3E-G, we present the number of OFF-targets normalized by the number of ON-targets. Using this metric, TrueProbes shows the lowest number of OFF-targets. We will updat the manuscript to clarify this point.

Regarding the experiments and their comparison to theoretical calculations: The theoretical calculations consider only the reference DNA and RNA genomes along with the oligonucleotide sequences for the probes. We then use a thermodynamic model to identify ON- and OFF-targets. Thus, these theoretical calculations represent an upper bound on the maximum possible number of ON-targets and the minimum number of OFF-targets. All other design software evaluated in this manuscript relies on the same or less reference data and makes certain assumptions. None of these methods quantitatively compare their computational designs with experimental results; they simply design probes based on unverified assumptions, conduct experiments, and present spot data to conclude that their probe designs are effective.

We will update the manuscript to clarify the goals of the theoretical model and its relationship to the experiments. Future work will be necessary to enhance our theoretical model to fully account for additional aspects of RNA-FISH experiments (e.g., formaldehyde crosslinking, hybridization conditions, washing steps) to better predict the experimental data shown in Figure 4. We will also adjuste our claims to accurately reflect the current capabilities of our theoretical framework and its relation to experimental outcomes.

(3) The bar graphs in Figure 3 do not seem to agree with the probability graphs in Figure 4. For example, Figure 3 indicates that Stellaris probes have higher off-target binding than TrueProbes; however, in Figure 4, their probability graphs lie almost on top of each other.

The predictions in Figure 3 regarding the number of probe off-target binding events, based on reference gene expression data, do not necessarily encompass all the information required to predict RNA-FISH signal intensity. Therefore, these predictions should not be expected to translate directly into the experimental results shown in Figure 4, particularly concerning the background signal.

While our software aims to minimize off-target probe binding, this does not automatically lead to a reduction in off-target background signal. Numerous other factors influence the spot background and overall signal-to-noise ratio (SNR) performance, beyond just probe-target binding interactions. Although we strive to minimize off-target background through probe binding, this approach is not designed to directly predict the SNR. Extending the computational analysis of probe binding dynamics to RNA-FISH signal intensity dynamics is beyond the scope of this study.

We have revised our text to clearly separate computational results from experimental results into two distinct sections. We will use different terminology to describe the outcomes of computational performance versus experimental performance, reducing potential confusion between these two aspects. Additionally, we will clarify our conceptual overview in Figure 1 regarding traditional probe design limitations related to sensitivity and specificity. We will specify how the signal from the number of probes bound to ON-target RNA, relative to those bound to OFF-targets and cellular autofluorescence, translates—either linearly or non-linearly—into the signal-to-noise ratio.

(4) The authors performed validation for only one gene (ARF4), because "...it had the highest gene expression (in TPM units) and the fewest isoforms among all candidate genes for the Jurkat cell line" (lines 176-177). While the results do look good, this is a minimal use case and does not really showcase the power of their method. One experiment that could be helpful would be two-color (or more) smFISH in tissue, where the chances for off-target binding contributing to higher errors are much greater than in an adherent cell line.

Thank you for highlighting these valuable experiments. Currently, our lab lacks the expertise to generate tissue samples beyond culturing cells. Additionally, implementing a two-color probe design in tissues containing different cell types with unknown expression levels presents further challenges. Due to these limitations, designing and conducting two-color experiments in tissue samples is beyond the scope of the current manuscript, but we plan to pursue this in the future.

(5) A common strategy for both smFISH and highly multiplexed methods is to use secondary DNA oligos with dye molecules instead of direct conjugation. Given that this is a primary design goal of PaintSHOP and the Zhuang lab's MERFISH probe design code, it would be helpful to demonstrate that TrueProbes can design a two-layer probe strategy for high-quality RNA-FISH labeling.

Thank you for bringing this to our attention. TrueProbes is currently designed and tested specifically for primary smRNA-FISH probes. Our focus is on demonstrating a new approach to designing these probes without the added complexities of secondary probes and multiplexing. Future work will expand on this foundation to incorporate secondary probe detection and transcript multiplexing.

(6) The authors claim, "For every probe set, TrueProbes can simulate expected smRNA FISH outcomes including optimal probe, RNA, and salt concentrations and optionally account for probe secondary structure, hybridization temperature, multiple targets, fluorophore choice, DNA, nascent RNA, and photon count statistics (Figures S2A, S2B). The model can be used to generate predictions for temperature and cell line sensitivity, multi-target discrimination, multiple fluorophore colocalization; when provided transcript expression levels and probe/background intensity, it can start to generate predictions for spot intensity, background, signal to noise ratio, and false negative rates (Figure S2C)." (lines 156-163). Figure S2 is a flow chart and does not provide evidence for any of these items. The authors should provide evidence for these claims, either as a figure or an example script in their software repository. If that is not possible, then it should be removed.

The supplemental information of the article will be updated to include figures that illustrate predictions for each capability currently offered by TrueProbes, along with the scripts used to generate these predictions. Any capabilities that do not have corresponding scripts will be removed from this section and instead referred to as potential improvements or future additions to the TrueProbes framework in the discussion section.

(7) All thermodynamic equations are performed at steady state. The authors do not justify this assumption, and there is no discussion of the potential impacts of either low molecule numbers or violations of the well-mixed assumption. Can the authors please include a discussion on the potential impacts non non-steady state dynamics?

Thermodynamic equations are calculated at steady state because RNA-FISH hybridization reactions typically last from eight to twenty hours. This duration allows probes adequate time to localize to their targets and reach binding equilibrium, based on current estimates of DNA oligonucleotide association and dissociation rate constants. We will address the potential violation of the well-mixed assumption in the assumptions and limitations section, specifically discussing how RNA localization can affect the spatial distribution of both on-target and off-target probes within cells, which may disrupt the well-mixed condition.

Low molecule numbers are not a significant concern, as probe DNA oligonucleotide concentrations in RNA-FISH protocols are much higher than the number of transcripts present in cells, by several orders of magnitude.

The assumptions and limitations section will be revised to clearly state: “Probe hybridization reactions were computed at steady state because most RNA-FISH protocols utilize probe hybridization incubation steps lasting over eight hours, which should provide sufficient time to reach equilibrium based on current estimates of forward and reverse reaction rate constants. Predictions from the equilibrium model may be less accurate for RNA-FISH experiments with shorter hybridization times, where non-steady state dynamics can result in different transient outcomes depending on the duration of hybridization.”

Reviewer #3 (Public review):

Summary:

This manuscript introduces a new platform termed "TrueProbes" for designing mRNA FISH probes. In comparison to existing design strategies, the authors incorporate a comprehensive thermodynamic and kinetic model to account for probe states that may contribute to nonspecific background. The authors validate their design pipeline using Jurkat cells and provide evidence of improved probe performance.

Strengths:

A notable strength of TrueProbes is the consideration of genome-wide binding affinities, which aims to minimize off-target signals. The work will be of interest to researchers employing mRNA FISH in certain human cell lines.

Weaknesses:

However, in my view, the experimental validation is not sufficient to justify the broad claims of the platform. Given the number of assumptions in the model, additional experimental comparisons across probe design methods, ideally targeting transcripts with different expression levels, would be necessary to establish the general superiority of this approach.

We will revise our text to make our claims more specific and clearer, avoiding overgeneralizations and ensuring that all claims are adequately supported by the data we present.

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self-create/curate/document coevolve info structures and affordances (infordances) pertaining to the autopoietic boostrapping/development of hyperpost seeding the IndyWeb, bythe indy.player/actor 🎭/gyuri/ within the Web Hosted directory for the Peergos account: ♖/hyperpost

created: 2025-09-22

updated 202510-09

eLife Assessment

This set of experiments provides a valuable finding regarding the need for prior inhibitory training to recruit the infralimbic cortex in extinction learning. The multiple clever behavioral designs supply converging lines of evidence in a compelling manner, but several issues, such as the group sizes and appropriate analysis of data, render the overall strength of support incomplete. With these issues resolved, this manuscript will be of interest to behavioral neuroscientists, especially those interested in learning & memory and/or cortical function.

Reviewer #1 (Public review):

Summary:

The manuscript reports a series of experiments designed to test whether optogenetic activation of infralimbic (IL) neurons facilitates extinction retrieval and whether this depends on animals' prior experience. In Experiment 1, rats underwent fear conditioning followed by either one or two extinction sessions, with IL stimulation given during the second extinction; stimulation facilitated extinction retrieval only in rats with prior extinction experience. Experiments 2 and 3 examined whether backward conditioning (CS presented after the US) could establish inhibitory properties that allowed IL stimulation to enhance extinction, and whether this effect was specific to the same stimulus or generalized to different stimuli. Experiments 5 - 7 extended this approach to appetitive learning: rats received backward or forward appetitive conditioning followed by extinction, and then fear conditioning, to determine whether IL stimulation could enhance extinction in contexts beyond aversive learning and across conditioning sequences. Across studies, the key claim is that IL activation facilitates extinction retrieval only when animals possess a prior inhibitory memory, and that this effect generalizes across aversive and appetitive paradigms.

Strengths:

(1) The design attempts to dissect the role of IL activity as a function of prior learning, which is conceptually valuable.

(2) The experimental design of probing different inhibitory learning approaches to probe how IL activation facilitates extinction learning was creative and innovative.

Weaknesses:

(1) Non-specific manipulation.

ChR2 was expressed in IL without distinction between glutamatergic and GABAergic populations. Without knowing the relative contribution of these cell types or the percentage of neurons affected, the circuit-level interpretation of the results is unclear.

(2) Extinction retrieval test conflates processes

The retrieval test included 8 tones. Averaging across this many tone presentations conflate extinction retrieval/expression (early tones) with further extinction learning (later tones). A more appropriate analysis would focus on the first 2-4 tones to capture retrieval only. As currently presented, the data do not isolate extinction retrieval.

(3) Under-sampling and poor group matching.

Sample sizes appear small, which may explain why groups are not well matched in several figures (e.g., 2b, 3b, 6b, 6c) and why there are several instances of unexpected interactions (protocol, virus, and period). This baseline mismatch raises concerns about the reliability of group differences.

(4) Incomplete presentation of conditioning data.

Figure 3 only shows a single conditioning session despite five days of training. Without the full dataset, it is difficult to evaluate learning dynamics or whether groups were equivalent before testing.

(5) Interpretation stronger than evidence.

The authors conclude that IL activation facilitates extinction retrieval only when an inhibitory memory has been formed. However, given the caveats above, the data are insufficient to support such a strong mechanistic claim. The results could reflect non-specific facilitation or disruption of behavior by broad prefrontal activation. Moreover, there is compelling evidence that optogenetic activation of IL during fear extinction does facilitate subsequent extinction retrieval without prior extinction training (Do-Monte et al 2015, Chen et al 2021), which the authors do not directly test in this study.

Impact:

The role of IL in extinction retrieval remains a central question in the fear learning literature. However, because the test used conflates extinction retrieval with new learning and the manipulations lack cell-type specificity, the evidence presented here does not convincingly support the main claims. The study highlights the need for more precise manipulations and more rigorous behavioral testing to resolve this issue.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors examine the mechanisms by which stimulation of the infralimbic cortex (IL) facilitates the retention and retrieval of inhibitory memories. Previous work has shown that optogenetic stimulation of the IL suppresses freezing during extinction but does not improve extinction recall when extinction memory is probed one day later. When stimulation occurs during a second extinction session (following a prior stimulation-free extinction session), freezing is suppressed during the second extinction as well as during the tone test the following day. The current study was designed to further explore the facilitatory role of the IL in inhibitory learning and memory recall. The authors conducted a series of experiments to determine whether recruitment of IL extends to other forms of inhibitory learning (e.g., backward conditioning) and to inhibitory learning involving appetitive conditioning. Further, they assessed whether their effects could be explained by stimulus familiarity. The results of their experiments show that backward conditioning, another form of inhibitory learning, also enabled IL stimulation to enhance fear extinction. This phenomenon was not specific to aversive learning, as backward appetitive conditioning similarly allowed IL stimulation to facilitate extinction of aversive memories. Finally, the authors ruled out the possibility that IL facilitated extinction merely because of prior experience with the stimulus (e.g., reducing the novelty of the stimulus). These findings significantly advance our understanding of the contribution of IL to inhibitory learning. Namely, they show that the IL is recruited during various forms of inhibitory learning, and its involvement is independent of the motivational value associated with the unconditioned stimulus.

Strengths:

(1) Transparency about the inclusion of both sexes and the representation of data from both sexes in figures.

(2) Very clear representation of groups and experimental design for each figure.

(3) The authors were very rigorous in determining the neurobehavioral basis for the effects of IL stimulation on extinction. They considered multiple interpretations and designed experiments to address these possible accounts of their data.

(4) The rationale for and the design of the experiments in this manuscript are clearly based on a wealth of knowledge about learning theory. The authors leveraged this expertise to narrow down how the IL encodes and retrieves inhibitory memories.

Weaknesses:

(1) In Experiment 1, although not statistically significant, it does appear as though the stimulation groups (OFF and ON) differ during Extinction 1. It seems like this may be due to a difference between these groups after the first forward conditioning. Could the authors have prevented this potential group difference in Extinction 1 by re-balancing group assignment after the first forward conditioning session to minimize the differences in fear acquisition (the authors do report a marginally significant effect between the groups that would undergo one vs. two extinction sessions in their freezing during the first conditioning session)?

(2) Across all experiments (except for Experiment 1), the authors state that freezing during the initial conditioning increased across "days". The figures that correspond to this text, however, show that freezing changes across trials. In the methods, the authors report that backward conditioning occurred over 5 days. It would be helpful to understand how these data were analyzed and collated to create the final figures. Was the freezing averaged across the five days for each trial for analyses and figures?

(3) In Experiment 3, the authors report a significant Protocol X Virus interaction. It would be useful if the authors could conduct post-hoc analyses to determine the source of this interaction. Inspection of Figure 4B suggests that freezing during the two different variants of backward conditioning differs between the virus groups. Did the authors expect to see a difference in backward conditioning depending on the stimulus used in the conditioning procedure (light vs. tone)? The authors don't really address this confounding interaction, but I do think a discussion is warranted.

(4) In this same experiment, the authors state that freezing decreased during extinction; however, freezing in the Diff-EYFP group at the start of extinction (first bin of trials) doesn't look appreciably different than their freezing at the end of the session. Did this group actually extinguish their fear? Freezing on the tone test day also does not look too different from freezing during the last block of extinction trials.

(5) The Discussion explored the outcomes of the experiments in detail, but it would be useful for the authors to discuss the implications of their findings for our understanding of circuits in which the IL is embedded that are involved in inhibitory learning and memory. It would also be useful for the authors to acknowledge in the Discussion that although they did not have the statistical power to detect sex differences, future work is needed to explore whether IL functions similarly in both sexes.

Reviewer #3 (Public review):

Summary:

This is a really nice manuscript with different lines of evidence to show that the IL encodes inhibitory memories that can then be manipulated by optogenetic stimulation of these neurons during extinction. The behavioral designs are excellent, with converging evidence using extinction/re-extinction, backwards/forwards aversive conditioning, and backwards appetitive/forwards aversive conditioning. Additional factors, such as nonassociative effects of the CS or US, are also considered, and the authors evaluate the inhibitory properties of the CS with tests of conditioned inhibition.

Strengths:

The experimental designs are very rigorous with an unusual level of behavioral sophistication.

Weaknesses:

(1) More justification for parametric choices (number of days of backwards vs forwards conditioning) could be provided.

(2) The current discussion could be condensed and could focus on broader implications for the literature.

Author response:

Reviewer #1 (Public review):

Summary:

The manuscript reports a series of experiments designed to test whether optogenetic activation of infralimbic (IL) neurons facilitates extinction retrieval and whether this depends on animals' prior experience. In Experiment 1, rats underwent fear conditioning followed by either one or two extinction sessions, with IL stimulation given during the second extinction; stimulation facilitated extinction retrieval only in rats with prior extinction experience. Experiments 2 and 3 examined whether backward conditioning (CS presented after the US) could establish inhibitory properties that allowed IL stimulation to enhance extinction, and whether this effect was specific to the same stimulus or generalized to different stimuli. Experiments 5 - 7 extended this approach to appetitive learning: rats received backward or forward appetitive conditioning followed by extinction, and then fear conditioning, to determine whether IL stimulation could enhance extinction in contexts beyond aversive learning and across conditioning sequences. Across studies, the key claim is that IL activation facilitates extinction retrieval only when animals possess a prior inhibitory memory, and that this effect generalizes across aversive and appetitive paradigms.

Strengths:

(1) The design attempts to dissect the role of IL activity as a function of prior learning, which is conceptually valuable.

We thank the Reviewer for their positive assessment.

(2) The experimental design of probing different inhibitory learning approaches to probe how IL activation facilitates extinction learning was creative and innovative.

We thank the Reviewer for their positive assessment.

Weaknesses:

(1) Non-specific manipulation.

ChR2 was expressed in IL without distinction between glutamatergic and GABAergic populations. Without knowing the relative contribution of these cell types or the percentage of neurons affected, the circuit-level interpretation of the results is unclear.

ChR2 was intentionally expressed in the infralimbic cortex (IL) without distinction between local neuronal populations for two reasons. First, this manuscript aimed to uncover some of the features characterizing the encoding of inhibitory memories in the IL, and this encoding likely engages interactions among various neuronal populations within the IL. Second, the hypotheses tested in the manuscript derived from findings that indiscriminately stimulated the IL using the GABA_A receptor antagonist picrotoxin, which is best mimicked by the approach taken. We agree that it is also important to determine the respective contributions of distinct IL neuronal populations to inhibitory encoding; however, the global approach implemented in the present experiments represents a necessary initial step. This rationale will be incorporated into the revised manuscript, which will also make reference to the need to identify the relative contributions of the various neuronal populations within the IL. 

(2) Extinction retrieval test conflates processes

The retrieval test included 8 tones. Averaging across this many tone presentations conflate extinction retrieval/expression (early tones) with further extinction learning (later tones). A more appropriate analysis would focus on the first 2-4 tones to capture retrieval only. As currently presented, the data do not isolate extinction retrieval.

It is unclear when retrieval of what has been learned across extinction ceases and additional extinction learning occurs. In fact, it is only the first stimulus presentation that unequivocally permits a distinction between retrieval and additional extinction learning, as the conditions for this additional learning have not been fulfilled at that presentation. However, confining evidence for retrieval to the first stimulus presentation introduces concerns that other factors could influence performance. For instance, processing of the stimulus present at the start of the session may differ from that present at the end of the previous session, thereby affecting what is retrieved. Such differences between the stimuli present at the start and end of an extinction session have been long recognized as a potential explanation for spontaneous recovery (Estes, 1955). More importantly, whether the test data presented confound retrieval and additional extinction learning or not, the interpretation remains the same with respect to the effects of a prior history of inhibitory learning on enabling the facilitative effects of IL stimulation. Finally, it is unclear how these facilitative effects could occur in the absence of the subjects retrieving the extinction memory formed under the stimulation. Nevertheless, the revised manuscript will provide the trial-by-trial performance during the post-extinction retrieval tests and discuss this issue.

(3) Under-sampling and poor group matching.

Sample sizes appear small, which may explain why groups are not well matched in several figures (e.g., 2b, 3b, 6b, 6c) and why there are several instances of unexpected interactions (protocol, virus, and period). This baseline mismatch raises concerns about the reliability of group differences.

Efforts were made to match group performance upon completion of each training stage and before IL stimulation. Unfortunately, these efforts were not completely successful due to exclusions following post-mortem analyses. However, we acknowledge that the unexpected interactions deserve further discussion, and this will be incorporated into the revised manuscript (see also comment from Reviewer 2). Although we cannot exclude that sample sizes may have contributed to some of these interactions, we remain confident about the reliability of the main findings reported, especially given their replication across the various protocols. Overall, the manuscript provides evidence that IL stimulation does not facilitate brief extinction in the absence of prior inhibitory experience in five different experiments, replicating previous findings (Lingawi et al., 2018; Lingawi et al., 2017). It also replicates these previous findings by showing that prior experience with either fear or appetitive extinction enables IL stimulation to facilitate subsequent fear extinction. Furthermore, the facilitative effects of such stimulation following fear or appetitive backward conditioning are replicated in the present manuscript.  

(4) Incomplete presentation of conditioning data.

Figure 3 only shows a single conditioning session despite five days of training. Without the full dataset, it is difficult to evaluate learning dynamics or whether groups were equivalent before testing.

We apologize, as we incorrectly labeled the X axis for the backward conditioning data set in Figures 3B, 4B, 4D and 5B. It should have indicated “Days” instead of “Trials”. This error will be corrected in the revised manuscript.

(5) Interpretation stronger than evidence.

The authors conclude that IL activation facilitates extinction retrieval only when an inhibitory memory has been formed. However, given the caveats above, the data are insufficient to support such a strong mechanistic claim. The results could reflect non-specific facilitation or disruption of behavior by broad prefrontal activation. Moreover, there is compelling evidence that optogenetic activation of IL during fear extinction does facilitate subsequent extinction retrieval without prior extinction training (Do-Monte et al 2015, Chen et al 2021), which the authors do not directly test in this study.

As noted above, the revised manuscript will show that the interpretations of the main findings stand whether ore the test data confounds retrieval with additional extinction learning. The revised manuscript will also clarify the plotting of the data for the backward conditioning stages. We do agree that further discussion of the unexpected interactions is necessary, and this will also be incorporated into the revised manuscript. However, the various replications of the core findings provide strong evidence for their reliability and the interpretations advanced in the original manuscript. The proposal that the results reflect non-specific facilitation or disruption of behavior seems highly unlikely. Indeed, the present experiments and previous findings (Lingawi et al., 2018; Lingawi et al., 2017) provide multiple demonstrations that IL stimulation fails to produce any facilitation in the absence of prior inhibitory experience with the target stimulus. Although these demonstrations appear inconsistent with previous studies (Do-Monte et al., 2015; Chen et al., 2021), this inconsistency is likely explained by the fact that these studies manipulated activity in specific IL neuronal populations. Previous work has already revealed differences between manipulations targeting discrete IL neuronal populations as opposed to general IL activity (Kim et al., 2016). Importantly, as previously noted, the present manuscript aimed to generally explore inhibitory encoding in the IL that, as we will acknowledge, is likely to engage several neuronal populations within the IL. Adequate statements on these matters will be included in the revised manuscript.

Impact:

The role of IL in extinction retrieval remains a central question in the fear learning literature. However, because the test used conflates extinction retrieval with new learning and the manipulations lack cell-type specificity, the evidence presented here does not convincingly support the main claims. The study highlights the need for more precise manipulations and more rigorous behavioral testing to resolve this issue.

As noted in our responses, the interpretations of the data presented remain identical whether the test data conflate extinction retrieval with additional extinction learning or not. Although we agree that it is important to establish the role of specific IL neuronal populations in extinction learning, this was beyond the scope of the manuscript and the findings reported remain valuable to our understanding of inhibitory encoding within the IL.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors examine the mechanisms by which stimulation of the infralimbic cortex (IL) facilitates the retention and retrieval of inhibitory memories. Previous work has shown that optogenetic stimulation of the IL suppresses freezing during extinction but does not improve extinction recall when extinction memory is probed one day later. When stimulation occurs during a second extinction session (following a prior stimulation-free extinction session), freezing is suppressed during the second extinction as well as during the tone test the following day. The current study was designed to further explore the facilitatory role of the IL in inhibitory learning and memory recall. The authors conducted a series of experiments to determine whether recruitment of IL extends to other forms of inhibitory learning (e.g., backward conditioning) and to inhibitory learning involving appetitive conditioning. Further, they assessed whether their effects could be explained by stimulus familiarity. The results of their experiments show that backward conditioning, another form of inhibitory learning, also enabled IL stimulation to enhance fear extinction. This phenomenon was not specific to aversive learning, as backward appetitive conditioning similarly allowed IL stimulation to facilitate extinction of aversive memories. Finally, the authors ruled out the possibility that IL facilitated extinction merely because of prior experience with the stimulus (e.g., reducing the novelty of the stimulus). These findings significantly advance our understanding of the contribution of IL to inhibitory learning. Namely, they show that the IL is recruited during various forms of inhibitory learning, and its involvement is independent of the motivational value associated with the unconditioned stimulus.

Strengths:

(1) Transparency about the inclusion of both sexes and the representation of data from both sexes in figures.

We thank the Reviewer for their positive assessment.

(2) Very clear representation of groups and experimental design for each figure.

We thank the Reviewer for their positive assessment.

(3) The authors were very rigorous in determining the neurobehavioral basis for the effects of IL stimulation on extinction. They considered multiple interpretations and designed experiments to address these possible accounts of their data.

We thank the Reviewer for their positive assessment.

(4) The rationale for and the design of the experiments in this manuscript are clearly based on a wealth of knowledge about learning theory. The authors leveraged this expertise to narrow down how the IL encodes and retrieves inhibitory memories.

We thank the Reviewer for their positive assessment.

Weaknesses:

(1) In Experiment 1, although not statistically significant, it does appear as though the stimulation groups (OFF and ON) differ during Extinction 1. It seems like this may be due to a difference between these groups after the first forward conditioning. Could the authors have prevented this potential group difference in Extinction 1 by re-balancing group assignment after the first forward conditioning session to minimize the differences in fear acquisition (the authors do report a marginally significant effect between the groups that would undergo one vs. two extinction sessions in their freezing during the first conditioning session)?

As noted (see response to Reviewer 1), efforts were made daily to match group performance across the training stages, but these efforts were ultimately hampered by the necessary exclusions following post-mortem analyses. This will be made explicit in the revised manuscript. Regarding freezing during Extinction 1, as noted by the Reviewer, the difference, which was not statistically significant, was absent across trials during the subsequent forward fear conditioning stage. Likewise, the protocol difference observed during the initial forward fear conditioning was absent in subsequent stages. We are therefore confident that these initial differences (significant or not) did not impact the main findings at test. Importantly, these findings replicate previous work using identical protocols in which no differences were present during the training stages. These considerations will be addressed in the revised manuscript.

(2) Across all experiments (except for Experiment 1), the authors state that freezing during the initial conditioning increased across "days". The figures that correspond to this text, however, show that freezing changes across trials. In the methods, the authors report that backward conditioning occurred over 5 days. It would be helpful to understand how these data were analyzed and collated to create the final figures. Was the freezing averaged across the five days for each trial for analyses and figures?

We apologize, as noted above, we incorrectly labeled the X axis for the backward conditioning data sets in Figures 3B, 4B, 4D and 5B. It should have indicated “Days” instead of “Trials”. The data shown in these Figures use the average of all trials on a given day. This will be clarified in the methods section of the revised manuscript. The labeling errors on the Figures will be corrected.

(3) In Experiment 3, the authors report a significant Protocol X Virus interaction. It would be useful if the authors could conduct post-hoc analyses to determine the source of this interaction. Inspection of Figure 4B suggests that freezing during the two different variants of backward conditioning differs between the virus groups. Did the authors expect to see a difference in backward conditioning depending on the stimulus used in the conditioning procedure (light vs. tone)? The authors don't really address this confounding interaction, but I do think a discussion is warranted.

We agree with the Reviewer that further discussion of the Protocol x Virus interaction that emerged during the backward conditioning and forward conditioning stages of Experiment 3 is warranted. This will be provided in the revised manuscript. Briefly, during both stages, follow-up analyses did not reveal any differences (main effects or interactions) between the two groups trained with the light stimulus (Diff-EYFP and Diff-ChR2). By contrast, the ChR2 group trained with the tone (Back-ChR2) froze more overall than the EYFP group (Back-EYFP), but there were no other significant differences between the two groups. Based on these analyses, the Protocol x Virus interaction appears to be driven by greater freezing in the ChR2 group trained with the tone rather than a difference in the backward conditioning performance based on stimulus identity. Consistent with this, the statistical analyses did not reveal a main effect of Protocol during either the backward conditioning stage or the stimulus trials during the forward conditioning stage. Nevertheless, during this latter stage, a main effect of Protocol emerged during baseline performance, but once again, this seems to be driven by the Back-ChR2 group. Critically, it is unclear how greater stimulus freezing in the Back-ChR2 group during forward conditioning would lead to lower freezing during the post-extinction retrieval test.  

(4) In this same experiment, the authors state that freezing decreased during extinction; however, freezing in the Diff-EYFP group at the start of extinction (first bin of trials) doesn't look appreciably different than their freezing at the end of the session. Did this group actually extinguish their fear? Freezing on the tone test day also does not look too different from freezing during the last block of extinction trials.

We confirm that overall, there was a significant decline in freezing across the extinction session shown in Figure 4B. The Reviewer is correct to point out that this decline was modest (if not negligible) in the Diff-EYFP group, which was receiving its first inhibitory training with the target tone stimulus. It is worth noting that across all experiments, most groups that did not receive infralimbic stimulation displayed a modest decline in freezing during the extinction session since it was relatively brief, involving only 6 or 8 tone alone presentations. This was intentional, as we aimed for the brief extinction session to generate minimal inhibitory learning and thereby to detect any facilitatory effect of infralimbic stimulation. This issue will be clarified and explained in the revised version of the manuscript.

(5) The Discussion explored the outcomes of the experiments in detail, but it would be useful for the authors to discuss the implications of their findings for our understanding of circuits in which the IL is embedded that are involved in inhibitory learning and memory. It would also be useful for the authors to acknowledge in the Discussion that although they did not have the statistical power to detect sex differences, future work is needed to explore whether IL functions similarly in both sexes.

In line with the Reviewer’s suggestion (see also Reviewer 3), the revised manuscript will include a discussion of the broader implications of the findings regarding inhibitory brain circuitry and will acknowledge the need to further explore sex differences and IL functions.

Reviewer #3 (Public review):

Summary:

This is a really nice manuscript with different lines of evidence to show that the IL encodes inhibitory memories that can then be manipulated by optogenetic stimulation of these neurons during extinction. The behavioral designs are excellent, with converging evidence using extinction/re-extinction, backwards/forwards aversive conditioning, and backwards appetitive/forwards aversive conditioning. Additional factors, such as nonassociative effects of the CS or US, are also considered, and the authors evaluate the inhibitory properties of the CS with tests of conditioned inhibition.

Strengths:

The experimental designs are very rigorous with an unusual level of behavioral sophistication.

We thank the Reviewer for their positive assessment.

Weaknesses:

(1) More justification for parametric choices (number of days of backwards vs forwards conditioning) could be provided.

All experimental parameters were based on previously published experiments showing the capacity of the backward conditioning protocols to generate inhibitory learning and the forward conditioning protocols to produce excitatory learning. Although this was mentioned in the methods section, we acknowledge that further explanation is required to justify the need for multiple days of backward training. This will be provided in the revised manuscript.

(2) The current discussion could be condensed and could focus on broader implications for the literature.

The revised manuscript will make an effort to condense the discussion and focus on broader implications for the literature.

References

Chen, Y.-H., Wu, J.-L., Hu, N.-Y., Zhuang, J.-P., Li, W.-P., Zhang, S.-R., Li, X.-W., Yang, J.-M., & Gao, T.-M. (2021). Distinct projections from the infralimbic cortex exert opposing effects in modulating anxiety and fear. J Clin Invest, 131(14), e145692. https://doi.org/10.1172/JCI145692

Do-Monte, F. H., Manzano-Nieves, G., Quiñones-Laracuente, K., Ramos-Medina, L., & Quirk, G. J. (2015). Revisiting the role of infralimbic cortex in fear extinction with optogenetics. J Neurosci, 35(8), 3607-3615. https://doi.org/10.1523/JNEUROSCI.3137-14.2015

Estes, W. K. (1955). Statistical theory of spontaneous recovery and regression. Psychol Rev, 62(3), 145-154. https://doi.org/10.1037/h0048509

Kim, H.-S., Cho, H.-Y., Augustine, G. J., & Han, J.-H. (2016). Selective Control of Fear Expression by Optogenetic Manipulation of Infralimbic Cortex after Extinction. Neuropsychopharmacology, 41(5), 1261-1273. https://doi.org/10.1038/npp.2015.276

Lingawi, N. W., Holmes, N. M., Westbrook, R. F., & Laurent, V. (2018). The infralimbic cortex encodes inhibition irrespective of motivational significance. Neurobiol Learn Mem, 150, 64-74. https://doi.org/10.1016/j.nlm.2018.03.001

Lingawi, N. W., Westbrook, R. F., & Laurent, V. (2017). Extinction and Latent Inhibition Involve a Similar Form of Inhibitory Learning that is Stored in and Retrieved from the Infralimbic Cortex. Cereb Cortex, 27(12), 5547-5556. https://doi.org/10.1093/cercor/bhw322

eLife Assessment

This important study presents convincing findings on creating an exhaustive library of new enhancer-AAVs targeting astrocytes and oligodendrocytes with high potential for both basic and translational work, which will be of value to a large and growing community. However, the outdated description of glial biology in the Introduction, the overstated claims of utility in the Conclusion, and the loose stringency in the criteria used to assemble the library diminish the strengths of the claims. The work will be of interest to neuroscientists working on glial cell biology.

Reviewer #1 (Public review):

The goal of this study was to generate a library of new enhancer-driven AAVs in order to selectively and efficiently target astrocytes and oligodendrocytes in rodents. The implied criteria are that such viral vectors should have high specificity for the intended cell type and effectively express in all astrocytes/oligos in the brain or, alternatively, be specific for defined brain regions, layers, or subtypes of astrocytes/oligos. In addition, they should be compatible with intravenous retro-orbital delivery to facilitate experimentation and brain-wide targeting (i.e., show organ specificity and high efficiency in the brain). Ideally, these new AAVs would also maintain their characteristics across disease contexts and show applicability in non-human primates. Tools with such characteristics are generally lacking in studying glial cells and would be extremely useful to scale up and accelerate glial research, allowing targeting of astrocytes/oligos with distinct molecular identity and intersectional strategies.

At present, however, none of the enhancer-AAVs presented in the study seems to meet this combination of criteria, at least not with the level of stringency typically expected in the field. The main reason is that, in its current form, the study does not present one candidate AAV iteratively improved to meet all these criteria; instead, it presents a catalogue of new AAVs with various degrees of specificity, completeness, and mixed characteristics. Therefore, their utility should be interpreted cautiously. Moreover, the way specificity and completeness are intermixed in the analysis makes it difficult to evaluate the actual utility of any given AAV. The study might have been strengthened by focusing on a small set of the most promising candidates (i.e., AiE0890m_3x2C) and validating them thoroughly for expression specificity, completeness, effective cargo expression, ability to allow specific pan-astrocyte or astrocyte-subtype targeting in vivo, and preserved properties in NHPs and in disease, as this would encourage their adoption by the community. Currently, too many AAVs are assessed inconsistently against the desired criteria, with none being evaluated through and through.

The impact of the catalogue is also greatly diminished by the fact that a suite of AAVs with outstanding specificity and efficiency is already available for the study of astrocytes (e.g., 4x6T AAVs) and was not utilized as a standard to benchmark the new library, making it difficult to appreciate the relative benefits of the new AAVs. The inclusion of expression data in NHPs is very significant, but benchmarking against established AAVs would also be needed to fully appreciate their value.

Importantly, readers should also be aware that the study seems noticeably limited in its literacy with glial biology. The introduction and discussion frame the field in a way that seems outdated, creating the impression that the diverse roles of glia in health and disease have not yet been studied, which may inadvertently be perceived as dismissive and stigmatizing.

In summary, the paper introduces potentially useful viral tools and lays the foundations for future multiplexed targeting of distinct glial cell subpopulations in rodents and in non-human primates, which are extremely important directions. Some of the regionally restricted or even sparsely expressed AAVs may prove valuable in enabling subpopulation-specific targeting or molecular profiling strategies, but currently lack full benchmarking. At present, the promises over the utility of the new tools seem overstated, and the library may not yet represent an actionable resource for targeting astrocytes and oligodendrocytes.

Reviewer #2 (Public review):

Enhancer elements are regulatory DNA sequences that are capable of driving specific expression patterns. As these elements are generally short and context-independent, enhancers can be used in expression vectors (e.g., packaged in an adeno-associated virus, AAV) to limit expression to target cell populations. This approach was identified as a major strategy for cell-type-specific manipulation in the brain and has been pursued by both standard research studies as well as large-scale efforts led by the BRAIN Initiative. This manuscript describes a major effort to generate enhancer-AAVs targeting astrocytes and oligodendrocytes orchestrated by a large research team led by the Allen Institute for Brain Science. This manuscript parallels other recent publications describing sets of enhancer-AAVs, following rigorous, similar methods with relatively broad testing and application.

To identify and screen candidate enhancers, the scientists prioritized candidates via analysis of single-nucleus accessibility and methylation datasets (i.e., snATAC-seq) and tested them in mice. The scientists prioritized candidate enhancers that exhibited specificity of accessibility in the target cell type. Following selection, the scientists cloned the candidate sequences into AAV vectors with a minimal promoter and reporter gene, packaged the virus, delivered it to the mouse brain, and screened for activity based on reporter expression. Candidates that passed initial screening were further characterized via imaging and sorting, followed by single-cell RNA-seq. This process had around a 50% success rate and yielded 25 astrocyte and 21 oligodendrocyte enhancer-AAVs with the targeted cell-type-specific expression patterns.

The scientists went on to test for subtype-specific activity patterns, finding wide diversity in astrocyte activities across sub-populations and conversely, homogenous oligodendrocyte activation. They optimized a few of these via concatenating the enhancer core sequence to increase expression levels of the reporter gene and showed strong specificity and completeness of cell targeting for a set of these enhancer-AAVs. Following characterization and validation, they then deployed these enhancer-AAVs in a number of demonstration applications to show the utility for basic and translational science. All the constructs developed here are available for public use via Addgene, ensuring that these new tools can be used by other researchers.

There really are no obvious weaknesses in the work presented here, from the generation of the enhancer-AAVs to use in sophisticated validation studies. The enhancer-AAV testing is rigorous and provides critical information necessary for other scientists to select and use these constructs. The applications demonstrate the power of enhancer-AAV approaches. The toolbox presented here may not enable specific targeting of all relevant cellular subtypes or activity states for astrocytes and oligodendrocytes, and future work will be needed to fully understand the activity of the enhancers, identity of the target cell types, and context-dependent utility of these constructs. However, the set of enhancer-AAVs developed here should be transformative for researchers working on accessing and manipulating these cell types and have a major impact on the field.

HEAT BITES 1. Itchy private area from the heat 2. Coffee - news reporter 3. Isolating due to hot weather 4. Mailman Moe 5. Ally Gator 6. Libby & Nibbles 7. Daisy 8. Jeff Tracy 9. Walking With Dinosaurs toys 10. Officers Brogan and Haldane

eLife Assessment

This paper presents a collection of analyses relating structure and function in the whole-brain Drosophila EM connectome and whole-brain calcium imaging data. The linkage of detailed anatomical structure with population activity is of broad interest in circuit neuroscience in light of increasingly detailed brain maps, but the analysis methods used made the evidence incomplete. The conclusions are useful for specific network observations, but a more thorough analysis of the anatomical and functional data is needed to support the overall claims.

Reviewer #1 (Public review):

Summary:

In this paper, the authors analyze connectome data from Drosophila and compare the physical wiring with functional connectivity estimated from calcium imaging data. They quantify structure-function relationships as a correlation of the two connectivity modalities. They report correlations roughly comparable to what has been described in the literature on sc/fc relationships in mammalian connectome data at the meso-scale. They then repeat their analysis, focusing on segregated versus unsegregated synapses. They derive separate connectomes using one or the other class of synapse. They show differential contributions to the sc/fc relationships by segregated versus unsegregated synapses.

Strengths:

There is nice synthesis of multimodal imaging data (Ca and EM data from flies and meso-scale data from human and marmoset).

Weaknesses:

(1) The paper is written in an unusual way. The introduction intermingles results with background, making it hard to figure out what precisely is being tested.

(2) There are also major methodological gaps. Though the mammalian connectomes are used as a point of reference, no descriptions of their origins or processing are included.

(3) A major weakness stems from the actual calculation of the sc/fc correlation. In general, SC is sparse. In the case of the EM connectomes, it is *exceptionally* sparse (most neural elements are not connected to one another). The authors calculated sc/fc coupling by correlating the off-diagonal elements of sc (the logarithm of its edge weights) and fc matrices with one another. The logarithmic transformation yields a value of infinity for all zero entries. The authors simply impute these elements with 0. This makes no sense and, depending on whether these zero elements are distributed systematically versus uniformly random, could either inflate or deflate the sc/fc correlations. Care must be taken here.

(4) Further, in constructing the segregated versus unsegregated connectomes, they use absolute thresholds for collecting synapses. It is unclear, however, whether similar numbers of synapses were included in both matrices. If the number is different, that might explain the differential relationship with fc; one matrix has more non-zero entries (and as noted earlier, those zero entries are problematic).

(5) There was also considerable text (in the results) describing the processing of the Ca data. In this section, the authors frequently refer to some pipelines as "better" or "worse" (more or less effective). But it is not clear what measures they adopted to assess the effectiveness of a pipeline.

Reviewer #2 (Public review):

Summary:

Okuno et al. investigate the structure-function relationship in the fruit fly Drosophila melanogaster. To do so, they combine published data from two recent synapse-level connectomes ("hemibrain" and "FlyWire") with a dataset comprising functional whole-brain calcium imaging and behavioural data. First, they investigate the applicability of fMRI pre-processing techniques on data from calcium imaging. They then cross-correlate this pre-processed functional data with structural data extracted from the connectomes, including a comparison to humans. The authors proceed to compare the two connectomes and find significant differences, which they attribute to differences in the accuracy of the synapse detections. Next, they present a novel algorithm to quantify whether neurons are segregated (pre- and postsynapses are spatially separate) or unsegregated (pre- and postsynapses are mixed). Using this approach, they find that unsegregated neurons may contribute more to function than segregated neurons. Applying a general linear model to the functional dataset suggests that activity in two brain areas (Wedge and AVLP) is suppressed during walking. The authors identify a GABAergic neuron in the connectome that could be responsible for this effect and suggest it may provide feedback to the fly's "compass" in the central complex.

Strengths:

The study tackles a relevant question in connectomics by exploring the relationship between structural and functional connectivity in the Drosophila brain. The authors apply a range of established and adapted analytical methods, including fMRI-style preprocessing and a novel synaptic segregation index. The effort to integrate multiple datasets and to compare across species reflects a broad and methodical approach.

Weaknesses:

The manuscript would benefit from a clearer overarching narrative to unify the various analyses, which currently appear somewhat disjointed. While the technical methods are extensive, the writing is often convoluted and lacks crucial details, making it difficult to follow the logic and interpret key findings. Additionally, the conclusions are relatively incremental and lack a compelling conceptual advance, limiting the overall impact of the work.

(1) The introduction currently contains a number of findings and conclusions that would be better placed in the results and discussion to clearly delineate past findings from new results and speculations.

(2) The narrative would benefit greatly from some clear statements along the lines of "we wanted to find out X, therefore we did Y".

(3) More concise terminology would be helpful. For example, the connectomes are currently referred to as either "hemibrain", "FlyEM", "whole-brain", or "FlyWire".

(4) The abstract claims "a new, more robust method to quantify the degree of pre- and post-synaptic segregation". However, the study fails to provide evidence that this method is indeed more robust than existing methods.

(5) The authors define unsegregated neurons as having mixed pre- and postsynapses in the same space. However, this ignores the neurons' topology: a neuron can exhibit a clearly defined dendrite with (mostly) postsynapses and a clearly defined axon with (mostly) presynapses, which then occupy the same space. This is different from genuinely unsegregated neurons with no distinct dendritic and axonal compartments, such as CT1.

(6) It is not entirely clear where the marmoset dataset originates from. Was it generated for this study? If not, why is there a note in the Ethics Declaration?

(7) On the differences between hemibrain and FlyWire: What is the "18.8 million post-synapses" for FlyWire referring to? The (thresholded) FlyWire synapse table has 130M connections (=postsynapses). Subsetting that synapse cloud to the hemibrain volume still gives ~47M synapses. Further subsetting to only connections between proofread neurons inside the hemibrain volume gives 19.4M - perhaps the authors did something like that? Similarly, the hemibrain synapse table contains 64M postsynapses. Do the 21M "FlyEM" post-synapses refer to proofread neurons only? If the authors indeed used only (post-)synapses from proofread neurons, they need to make that explicit in results and methods, and account for differences in reconstruction status when making any comparisons. For example, the mushroom body in the hemibrain got a lot more attention than in FlyWire, which would explain the differences reported here. For that reason, connection weights are often expressed as, e.g., a fraction of the target's inputs instead of the total number of synapses when comparing connectivity across connectomic datasets. Furthermore, in Figure 3b, it looks like the FlyWire synapse cloud was not trimmed to the exact hemibrain boundaries: for example, the trimmed FlyWire synapse cloud seems to extend further into the optic lobes than the hemibrain volume does.

Reviewer #3 (Public review):

Summary:

In this manuscript, Okuno et al. re-analyze whole-brain imaging data collected in another paper (Brezovec et al., 2024) in the context of the two currently available Drosophila connectome datasets: the partial "FlyEM" (hemibrain) dataset (Scheffer et al., 2020) and the whole-brain "FlyWire" dataset (Dorkenwald et al., 2024). They apply existing fMRI signal processing algorithms to the fly imaging data and compute function-structure correlations across a variety of post-processing parameters (noise reduction methods, ROI size), demonstrating an inverse relationship between ROI size and FC-SC correlation. The authors go on to look at structural connectivity amongst more polarized or less polarized neurons, and suggest that stronger FC-SC correlations are driven by more polarized neurons.

Strengths:

(1) The result that larger mesoscale ROIs have a higher correlation with structural data is interesting. This has been previously discussed in Drosophila in Turner et al., 2021, but here it is quantified more extensively.

(2) The quantification of neuron polarization (PPSSI) as applied to these structural data is a promising approach for quantifying differences in spatial synapse distribution.

Weaknesses:

One should not score noise/nuisance removal methods solely by their impact on FC-SC correlation values, because we do not know a priori that direct structural connections correspond with strong functional correlations. In fact, work in C. elegans, where we have access to both a connectome and neuron-resolution functional data, suggests that this relationship is weak (Yemini et al., 2021; Randi et al., 2023). Similarly, I don't think it's appropriate to tune the confidence scores on the EM datasets using FC-SC correlations as an output metric.

Any discussion of FC-SC comparisons should include an analysis of excitatory/inhibitory neurotransmitters, which are available in the fly connectome dataset. However, here the authors do not perform any analyses with neurotransmitter information.<br /> Comparisons between fly and human MRI data are also premature here. Firstly, the fly connectomes, which are derived from neuron-scale EM reconstructions, are a qualitatively different kind of data from human connectomes, which are derived from DSI imaging of large-scale tracts. Likewise, calcium data and fMRI data are very different functional data acquisition methods-the fact that similar processing steps can be used on time-series data does not make them surprisingly similar, and does not in my view, constitute evidence of "similar design concepts."

The comparison of FlyEM/FlyWire connectomes concludes that differences are more likely a result of data processing than of inter-individual variability. If this is the case, the title should not claim that the manuscript covers individual variability.<br /> The analysis of the wedge-AVLP neuron strikes me as highly speculative, given that the alignment precision between the connectome and the functional data is around 5 microns (Brezovec* et al, PNAS 2024).

eLife Assessment

This study by Lapao et al. uncovers a novel role for the Rab27A effector SYTL5 in regulating mitochondrial function and mitophagy under hypoxic conditions. Using a range of imaging and functional assays, the authors demonstrate that SYTL5 localizes to mitochondria in a Rab27A-dependent manner and impacts mitochondrial respiration and metabolic reprogramming. While the findings are solid and valuable in the area of cancer biology, further mechanistic clarity and improved imaging would strengthen the conclusions.

Reviewer #1 (Public review):

Summary:

In this study, Ana Lapao et al. investigated the roles of Rab27 effector SYTL5 in cellular membrane trafficking pathways. The authors found that SYTL5 localizes to mitochondria in a Rab27A-dependent manner. They demonstrated that SYTL5-Rab27A positive vesicles containing mitochondrial material are formed under hypoxic conditions, thus they speculate that SYTL5 and Rab27A play roles in mitophagy. They also found that both SYTL5 and Rab27A are important for normal mitochondrial respiration. Cells lacking SYTL5 undergo a shift from mitochondrial oxygen consumption to glycolysis which is a common process known as the Warburg effect in cancer cells. Based on cancer patient database, the author noticed that low SYTL5 expression is related to reduced survival for adrenocortical carcinoma patients, indicating SYTL5 could be a negative regulator of the Warburg effect and potentially tumorigenesis.

Strengths:

The authors take advantages of multiple techniques and novel methods to perform the experiments.

(1) Live-cell imaging revealed that stably inducible expression of SYTL5 co-localized with filamentous structures positive for mitochondria. This result was further confirmed by using correlative light and EM (CLEM) analysis and western blotting from purified mitochondrial fraction.

(2) In order to investigate whether SYTL5 and RAB27A are required for mitophagy in hypoxic conditions, two established mitophagy reporter U2OS cell lines were used to analyze the autophagic flux.

Weaknesses:

This study revealed a potential function of SYTL5 in mitophagy and mitochondrial metabolism. However, the mechanistic evidence that establishes the relationship between SYTL5/Rab27A and mitophagy is insufficient. The involvement of SYTL5 in ACC needs more investigation. Furthermore, images and results supporting the major conclusions need to be improved.

Comments on revisions: The authors did not revise the paper as suggested.

Reviewer #2 (Public review):

Summary:

The authors provide convincing evidence that Rab27 and STYL5 work together to regulate mitochondrial activity and homeostasis.

Strengths:

The development of models which allow the function to be dissected, and the rigous approach and testing of mitochondrial activity.

This work is carefully done, and supports the importance of the roles of Rab27A and STYL5.

Reviewer #3 (Public review):

In the manuscript by Lapao et al., the authors uncover a role for the RAB27A effector protein SYTL5 in regulating mitochondrial function and apparent selective turnover of mitochondrial components. The authors find that SYTL5 localizes to mitochondria in a RAB27A dependent way and that loss of SYTL5 (or RAB27A) impairs lysosomal turnover of MTCO1 (but not a matrix-based reporter/other mitochondrial proteins). The authors go on to show that loss of SYTL5 impacts mitochondrial respiration and ECAR and as such may influence the Warburg effect and tumorigenesis. Of relevance here, the authors go on to show that SYTL5 expression is reduced in adrenocortical carcinomas and this correlates with reduced survival rates.

As previously reviewed, this is a very intriguing body of work and reveals a new role for SYTL5/RAB27A at the mitochondria. Unfortunately, it appears that SYTL5 is challenging protein to detect endogenously and the authors' cell lines "comprise a heterogenous pool with high variability", which means that a lot of my original concerns remain. It is still also not clear if the conventional autophagy machinery is required for this pathway, especially if SYTL5/RAB27A mitochondrial recruitment is upstream of this. Hopefully, in future work, the authors (and/or others) will be able to address this and build on the mechanisms of this interesting and potentially important pathway.

DOI: 10.1016/j.isci.2025.113584

Resource: (IMSR Cat# JAX_034860,RRID:IMSR_JAX:034860)

Curator: @areedewitt04

SciCrunch record: RRID:IMSR_JAX:034860

What is this?

This sentence highlights a hidden way schools increase inequality. Pay-to-play policies mean that students have to pay to join sports or extracurriculars, which can exclude low-income students. I think this is unfair because it limits opportunities for those who can’t afford it, reinforcing gaps between wealthy and poor students.

This point really stands out because it shows how deeply housing and education are connected. Where a family lives often determines the quality of schooling a child receives. As neighborhoods become more divided by income, schools mirror that inequality, giving wealthier students more advantages while leaving low-income students with fewer opportunities.

I can totally relate to that. In some schools, the competition is so intense that even getting a B feels like you’re falling behind. It creates a lot of pressure, and even though people might seem friendly, there’s still that hidden stress of trying to stay at the top.

This quote highlights how schools in the same area can have completely different student demographics. Factors like poverty, ethnicity, and language ability create unequal learning environments. It shows that educational inequality isn’t just about funding—it’s also about the diverse challenges students face based on their backgrounds.

This passage shows the deep economic and racial divide within Orange County. The contrast between cities like Laguna Beach and Santa Ana highlights how inequality exists even within close distances. It’s striking to see how income and race shape opportunities so differently, emphasizing the need to address these structural disparities.

you ask me, I saw more growth in the Asian and Middle Eastern groups when I went to school near this area at the time. The Latino people who live in Fullerton mostly live near downtown.

There have been big changes in the lives of many people who used to live in Los Angeles and San Diego. Many of them have moved to Orange County.

Large-scale changes in population changed the picture of a lot of people living in Los Angeles and San Diego, with many moving to Orange County.

Wealthier students are more likely to graduate, while poorer students are more likely to drop out. However, the true root causes often form much earlier, at the family and community stages. In other words, college is merely the “final manifestation” of inequality, not its origin. Family structure, early educational resources, parental cultural capital, and social support networks are the deeper causes behind the ultimate disparities in college graduation rates. This passage embodies Putnam's core argument: without addressing the foundational social structures, merely lowering tuition or expanding enrollment cannot truly eliminate educational stratification.

The role of teachers has long since devolved from “educators” to “order-maintaining guards.” Violence and disorder not only disrupt the learning environment for students but also severely undermine teachers' professional passion and instructional quality. Through this description, Putnam conveys a deeper social reality: educational challenges in impoverished communities often stem not merely from shortages of teachers or funding, but reflect structural societal breakdown—broken families, community violence, and economic anxiety all erupt collectively within classrooms. Teachers in such environments often succumb to a sense of powerlessness, while students lose the security and trust essential for learning. This passage thus transcends a mere description of schools; it becomes an indictment of the failure of education at the bottom of society.

Social class has gradually replaced race as the key dividing line determining educational opportunities and academic achievement. Following the civil rights movement, American society made some progress in narrowing racial disparities, yet simultaneously, economic and socioeconomic inequalities deepened. Through this comparison, Putnam highlights that the core issue has shifted from “racial injustice” to “class stratification”—while nominally egalitarian, the education system has in practice reinforced the intergenerational transmission of family wealth and resources. This trend signifies that poverty is no longer primarily a matter of skin color, but a structural socioeconomic issue; the function of education in helping children achieve upward mobility is being progressively eroded.

This stark contrast most vividly reveals the reality that “though both are public schools, they seem worlds apart.” Putnam uses concrete data to illustrate structural inequality—on the surface, the two schools appear similar in “hard metrics” like faculty and funding, yet student backgrounds (social class and family resources) ultimately determine educational outcomes. This demonstrates that disparities in educational quality stem not merely from institutional design, but are deeply rooted in family environments and community ecosystems. Education thus becomes a mechanism for reproducing social stratification.

The American Dream that emphasizes ability above all else is being shattered by the reality where family background prevails. Although high-achieving students from poor families possess academic potential, they may find it difficult to complete their college education due to factors such as economic pressure, lack of family support, and psychological stress. On the other hand, low-achieving students from wealthy families, even with mediocre academic abilities, can make up for the gap through family resources and graduate smoothly. This phenomenon where ability gives way to background means that the American education system has not only failed to narrow the class gap but has instead become a tool for class solidification, completely deviating from the core value of equality of opportunity.

After-school activities are originally an important way to cultivate soft skills such as teamwork and leadership. However, due to the policy of charging fees for participation, they have become the exclusive resources of affluent families. This gap not only affects students' comprehensive qualities, but also creates a hidden disadvantage in further education and employment. The experiences and skills accumulated by affluent students through after-school activities will become bonus points favored by universities during admissions and by employers, further widening the gap with poor students.

esidential spatial differentiation is an invisible driver of educational class segregation. This model of selecting schools through housing prices essentially links educational resources to economic capacity: affluent families secure quality education for their children by purchasing school district housing, while low-income families are excluded. This creates a vicious cycle where the wealthier one is, the more access they have to good education, and the better they can maintain their wealth through education — further solidifying class stratification.

Educational outcomes depend not only on what schools provide, but also on what students bring with them to school. The two schools are barely different in hardware conditions such as per-student funding and teacher experience, yet their students' outcomes vary drastically — the core gap lies in the socioeconomic background of their student bodies.

This section highlights the need to value the strengths of low-income students and build a mentoring culture that promotes activism and confidence. I agree that teachers should work with parents and communities to support students’ growth. It’s a reminder that educators must stay true to their purpose—challenging inequality and helping all students find hope and opportunity.

I really like this story because it shows how a small act of encouragement from a teacher can make a big difference. Sometimes students just need someone to believe in them and guide them through the unspoken rules of school, like participating in discussions. Ms. Hill’s support helped build confidence and showed how positive relationships between teachers and students can truly change a learning experience.

I can really relate to this reflection. Many of us are taught to recognize and resist certain forms of discrimination, like racism, but not others such as classism, which can be more subtle yet equally harmful. It’s eye-opening to realize how deeply class affects educational experiences — from access to resources to the way students are perceived. This shows how important it is for schools to address both race and class openly, so all students can feel equally supported and empowered.

This passage highlights how the education system often overlooks the strengths that poor students bring, such as resilience and creativity, and instead rewards traits that reflect middle- or upper-class norms. It reveals how schools unintentionally reinforce inequality by valuing conformity over diversity in skills and experiences. I think this shows the need for schools to recognize and celebrate different forms of intelligence and success, not just those defined by dominant cultural standards.

The more a teacher engages with the class, the more the students are going to be willing to participate

It’s no secret the treatment disparity amongst blacks and whites

Racism and classism cannot be ignored. They are huge issues and affect people daily

Respect starts whitin the household. Teaching basic fundamentals can further accelerate a student

There cannot be room for participation trophies. Need to start breeding excellence.

Many teachers initially enter the education field to change the status quo and promote fairness, but within institutionalized rules and administrative demands, ideals are often eroded and original aspirations forgotten. True courage lies in upholding the educational principles of fairness and justice within a system that perpetuates inequality.

This passage is profoundly powerful because it not only critiques the injustices within educational structures but also reminds educators to embrace their civic responsibility—education is not merely the transmission of knowledge but the practice of social justice. The author encourages educators to proactively connect with students' communities, cultures, and realities, identifying strengths within their lived experiences rather than defining their “shortcomings” by societal standards. This educational philosophy embodies both a professional mission and a moral commitment: ensuring every student has the opportunity to envision and pursue a future filled with hope.

In China's public education system, the opposite may actually be true. In primary school and kindergarten, teachers may prefer you to be an obedient child rather than a bright or curious one. Because China's public education is rigid, it seeks to produce identical, well-calibrated machines rather than nurturing distinct souls.

Students' social circles and social class are thus restricted. If they lack opportunities to meet people from higher social classes and actively engage with them, their chances of securing good job opportunities and advancing socially are greatly diminished. Humans are social creatures, and one's social circle largely determines their class. If students fail to actively network and break out of their circles during college—a time when class divisions among students are less pronounced—they will find it much harder to seize such opportunities in their future careers and lives.

in the Web 2 world

👉 Annotation: This highlights how peer culture also shapes students’ school experiences. I think this is important because even if teachers try to support every student, peer influence can strongly affect motivation, belonging, and academic identity—especially in diverse schools.

This shows how early math placement can create long-term inequality. I think it’s unfair that race and class often determine access to advanced courses. Students who start behind have to work much harder to catch up, even though the system itself set them back.

This sentence really shows how much a single decision can shape a student’s future. I feel bad for Chantelle because it’s not her fault—she just didn’t have the same guidance as others. It makes me realize how important mentorship and support are, especially for students who don’t have that help at home.

The gap in diversity and achievement gap at Berkeley high is not aligning. The efforts are not working and are very contradictory.

Chantelle's choice shows that she gave it some thought and honestly evaluated her academic skills. By thinking about how she learned and how her brother learned, she made a decision that might be better for her future school career. This kind of thinking about the future and making plans is a very useful skill for learning.

I experienced something very similar in my high school Spanish classes. It was common knowledge amongst students at the school that the teaching in the Spanish classes were mediocre, less than satisfactory, and undesirable. Additionally the expectations were very low and I learned very little despite taking 3 years of courses.

I would argue that this type of ranking of language contributes to a perpetuation and reproduction of a racial hierarchy within the school system.

The relationship between students and their school is not solely established through individual interactions between students and adults such as teachers or parents; it is significantly influenced by “peer culture.” In other words, the interactions, habits, values, and behavioral patterns students develop with their peers both inside and outside of school profoundly shape their relationship with the institution. Peer culture can either foster learning—such as by creating a positive study atmosphere and mutual encouragement—or have negative effects, like excluding high-achieving students or encouraging avoidance of academic responsibilities. Therefore, while schools focus on teacher-student relationships, they should also prioritize the social dynamics within student groups. Creating a more positive and inclusive peer culture environment helps students better adapt to school life and achieve their developmental goals.

While the education system nominally grants students “freedom of choice,” it is profoundly unfair to students from different backgrounds. Students with greater social and cultural capital often receive guidance from parents or teachers, enabling them to make decisions that benefit their academic advancement. Students like Chantelle, however, lacking such guidance, are forced to make far-reaching, misguided choices amid information asymmetry. “Freedom of choice” in education is, in fact, a form of structural bias—it appears equitable on the surface but actually widens the class divide.

The author points out that cultural capital encompasses linguistic style, demeanor, behavioral habits, and appearance—characteristics seemingly unrelated to academic performance that nonetheless influence students' school achievements and recognition. For instance, students from upper-middle-class families are more likely to master expressions and behavioral norms aligned with mainstream school culture, making them more easily perceived by teachers as “polite,” “intelligent,” or “promising.” Conversely, students from lower socioeconomic backgrounds may be underestimated despite equal effort, due to differences in their manner of expression or conduct. This demonstrates how the education system subtly favors specific social groups, making cultural capital a key mechanism for reproducing social inequality.

However, such adjustments may be perceived as discriminatory from certain perspectives. If class assignments are made to balance students' family backgrounds, it inevitably creates so-called “good classes” and “less desirable classes.” Educational resources will consequently be skewed toward the good classes, while the less desirable classes are more likely to be neglected. Parents in the better-off classes, benefiting from greater economic means, may initiate more engaging social activities to broaden students' horizons and enhance their resumes. This is something parents in the disadvantaged classes often cannot match. This dynamic subtly widens the class divide between children from different socioeconomic backgrounds.

It is evident that this is a direct manifestation of the disparity in educational resources and foundational advantages stemming from differing family socioeconomic backgrounds. Students from higher socioeconomic backgrounds can supplement and expand upon school knowledge through resources like private tutoring, ensuring they achieve outstanding academic results. In reality, numerous extracurricular opportunities—such as competitions and internships—require access to parental networks and information to secure. These information gaps lead to significant disparities in application essays and background qualifications during university admissions, leaving children from impoverished families without the chance to attend prestigious universities.

Élections au Conseil de la Vie Lycéenne 2025 : Analyse des Programmes

Résumé

Le présent document synthétise les professions de foi des candidats aux élections du Conseil de la Vie Lycéenne (CVL) du lycée Louis Vincent pour le mandat 2025.

Les élections se dérouleront du 30 septembre au 2 octobre via la plateforme Pronote, où cinq binômes seront élus pour un mandat de deux ans.

L'objectif commun déclaré par l'ensemble des candidats est d'améliorer les conditions et la qualité de vie au sein de l'établissement.

L'analyse des programmes révèle plusieurs thèmes transversaux et récurrents :

1. Convivialité et Événements : La quasi-totalité des candidats propose l'organisation de journées à thème (pyjama, sans sac, carnaval, maillot de foot), de ventes de nourriture et de boissons chaudes (notamment en hiver), et de grands événements fédérateurs comme un festival de fin d'année ouvert à tous les niveaux, des tournois sportifs interclasses ou un bal des terminales.

2. Solidarité et Actions Caritatives : Un fort accent est mis sur la solidarité, avec des propositions de collectes (alimentaires, produits d'hygiène, fournitures scolaires) et de levées de fonds dont les bénéfices seraient reversés à des associations caritatives (Restos du cœur, un cahier un crayon) ou à des lycéens dans le besoin de manière anonyme.

3. Culture et Pédagogie : De nombreux programmes ambitionnent de dynamiser la vie culturelle du lycée.

Les idées incluent une meilleure utilisation de la salle de cinéma pour des projections pédagogiques ou des séances gratuites, l'organisation de sorties culturelles, la création de clubs (lecture, débat), et la mise en place de systèmes de tutorat et de parrainage entre élèves.

4. Bien-être et Cadre de Vie : L'amélioration du bien-être des élèves est une priorité centrale.

Les propositions visent à créer des espaces de détente et de travail plus calmes et accueillants, à aménager des espaces verts (potager, jardin partagé), à gérer le stress via des ateliers, et à améliorer les infrastructures pratiques comme l'ajout de casiers.

5. Communication et Représentation : Plusieurs binômes souhaitent renforcer le lien entre le CVL et les élèves par la gestion active d'un compte Instagram, la mise en place d'une boîte à idées, la publication de comptes-rendus mensuels et une promesse générale d'écoute.

Un candidat, Baptiste, fonde même l'intégralité de sa démarche sur l'absence de programme prédéfini afin de représenter directement les demandes des élèves.

En somme, les candidatures de 2025 témoignent d'une volonté partagée de rendre le lycée Louis Vincent plus dynamique, solidaire, culturellement riche et attentif au bien-être de chaque élève.

Présentation des Candidats et de Leurs Plateformes

Le tableau suivant récapitule les différents binômes candidats, leur niveau de classe et les points saillants de leur programme.

Candidats (Titulaires et Suppléants)

Classe(s)

Qualités Revendiquées

Points Clés du Programme

VM & CR

Terminale 6 & Première 2

Créatives, motivées, sérieuses, expérience et énergie

Projet "H24" (camping au lycée), journées à thème (maillot de foot, sans sac, pyjama).

SP et OP

Seconde 12 & Seconde 13

Bienveillants, honnêtes, altruistes, intègres, ouverts au dialogue

Ventes de snacks/boissons chaudes, 2ème service à la cantine, création d'un lieu de travail/repos, tournois sportifs interclasses.

MV et KM

Non spécifié

-

4 axes : écologie (coin nature, potager), vie scolaire (journées à thème, menus spéciaux), culture (concours de talents, tutorat, sorties), bien-être (coin calme).

MO et LO

Première STL & Première 11

Engagée, sympathique, responsable, réfléchi, à l'écoute, sérieux, ambitieux

Récolte caritative, grande journée conviviale de fin d'année, vente de gourdes écoresponsables, communication améliorée (compte-rendu, boîte à idées).

AI et IP

Terminale 2 & Première 7

Inspirés par les anciens CVL

Vente de fournitures scolaires, vente de thé/chocolat chaud pour des associations, festival de fin d'année pour tous les niveaux.

AP et GL

Première 2 & Première 4

Expérience (délégué, ancien CVC)

Ventes de pâtisseries/boissons, gestion active du compte Instagram du CVL (sondages, menus), ajout de casiers pour tous, décorations du lycée.

RS et LH

Seconde 4 & Seconde 1

À l'écoute

Utilisation de la salle de cinéma à but pédagogique, organisation de goûters thématiques pour financer des associations ou des sorties, décorations festives.

HS et JPM

Terminale & Première 6

-

Optimisation des TV de l'accueil (horaires de bus, menus), dynamisation de la web TV, séances de cinéma gratuites, collecte de fournitures, vente de pulls du lycée, changement de la sonnerie via vote.

RB et NL

Seconde 4 & Seconde 6

-

Grand repas mensuel à la cantine, collecte de produits d'hygiène/vêtements pour les SDF, semaines à thème (cinéma), tutorat entre 15h et 18h.

B

Seconde

-

Absence de programme défini. Volonté de représenter directement les idées et besoins des 1600 élèves. Souhaite renouveler les initiatives type "Journées du Patrimoine".

SV et LL

Terminale 4 & Première 9

Souriantes, à l'écoute, motivées

Ateliers de gestion du stress, espaces de détente, tutorat Terminales/Secondes, création d'espaces verts (potager), sorties culturelles, amélioration des salles de travail.

Analyse Détaillée des Thèmes de Campagne

1. Convivialité, Événements et Vie Sociale

Ce thème est le plus largement partagé par les candidats, qui souhaitent briser la routine et renforcer les liens entre les élèves.

• Journées à Thème : Une proposition quasi unanime.

◦ Journée pyjama : Proposée par Victorine & Capucine, et Mabine & Victoria.   

◦ Journée sans sac : Proposée par Victorine & Capucine, Mabine & Victoria, Henry & Maxime, et Sacha & Lili.  

◦ Journée en maillot de foot : Proposée par Victorine & Capucine. 

◦ Journée carnaval : Proposée par Mabine & Victoria, et Sacha & Lili.  

◦ Journée de l'élégance : Proposée par Sacha & Lili.

• Événements de Fin d'Année :

◦ Festival pour tous les niveaux : Avazov & Isaac proposent un événement avec stands de nourriture, concours de talents et concerts pour inclure les Secondes et Premières, souvent exclus du bal des terminales.  

◦ Grande journée conviviale : Méina & Lilian suggèrent une journée avec jeux, sport et musique pour élèves, professeurs et personnel.  

◦ Bal des terminales et "Perçant" : Arnaud & Grégoire s'engagent à organiser ces événements traditionnels.

• Ventes de Nourriture et Boissons :

◦ Ventes de snacks et boissons chaudes (hiver) : Proposées par Sacha & Oscar, Avazov & Isaac.    ◦ Ventes de pâtisseries : Proposées par Arnaud & Grégoire.  

◦ Goûters thématiques (Noël, Halloween) : Proposés par Roman & Louison.

• Événements Sportifs et Sociaux :

◦ Tournois sportifs interclasses : Proposés par Sacha & Oscar.  

◦ Grand repas mensuel à la cantine : Suggéré par Romain & Noa pour favoriser les rencontres.

2. Solidarité, Écologie et Actions Caritatives

De nombreux candidats placent l'engagement solidaire et écologique au cœur de leur projet.

• Collectes et Dons :

◦ Récoltes caritatives générales : Proposées par Méina & Lilian, avec une redistribution anonyme possible aux lycéens dans le besoin.  

◦ Collectes pour des associations (SDF, etc.) : Romain & Noa proposent de récolter des produits d'hygiène, de la nourriture et des vêtements chauds. Mabine & Victoria souhaitent organiser des collectes solidaires régulières.  

◦ Collecte de fournitures scolaires : Henry & Maxime suggèrent que les terminales fassent don de leur matériel (ex: calculatrices) aux nouveaux élèves.

• Financement d'Associations :

◦ Plusieurs binômes (Avazov & Isaac, Roman & Louison) proposent que les bénéfices des ventes de nourriture soient reversés à des associations comme les Restos du cœur ou Un cahier, un crayon.

• Écologie et Cadre de Vie :

◦ Espaces verts : Mabine & Victoria et Sacha & Lili veulent créer un "coin nature", un jardin partagé ou un potager pédagogique.  

◦ Gourdes écoresponsables : Méina & Lilian proposent de créer et vendre une gourde en matériaux recyclés.

3. Culture, Éducation et Entraide

L'accès à la culture et le soutien scolaire sont des axes de développement majeurs.

• Utilisation de la Salle de Cinéma : Un projet récurrent.

◦ Usage pédagogique : Roman & Louison souhaitent encourager les professeurs à utiliser la salle pour des projections en lien avec les cours.  

◦ Accès gratuit à la culture : Henry & Maxime et Mabine & Victoria veulent organiser des séances de cinéma gratuites et régulières pour tous.

• Tutorat et Parrainage :

◦ Aide aux devoirs : Romain & Noa suggèrent un système de tutorat entre 15h et 18h pour les élèves en difficulté.   

◦ Parrainage Secondes/Terminales : Sacha & Lili proposent un parrainage pour faciliter l'intégration des Secondes, une idée également mentionnée par Mabine & Victoria.

• Enrichissement Culturel :

◦ Sorties culturelles et sportives : Mabine & Victoria et Sacha & Lili souhaitent en organiser davantage.  

◦ Rencontres avec d'anciens élèves : Mabine & Victoria proposent d'inviter des anciens pour partager leur parcours.   

◦ Concours de talents : Mabine & Victoria veulent lancer un concours annuel (photo, écriture, musique, sport).  

◦ Clubs : L'idée de clubs (lecture, débat) est avancée par Mabine & Victoria.

4. Améliorations Pratiques et Bien-être

Les candidats sont attentifs aux aspects concrets de la vie lycéenne et à la santé mentale des élèves.

• Espaces de Vie et de Travail :

◦ Création d'un lieu de repos/travail : Sacha & Oscar constatent que la Maison des Lycéens est souvent encombrée et bruyante et proposent un nouvel espace.  

◦ Aménagement d'un coin calme et détente : Une priorité pour Mabine & Victoria et Sacha & Lili, qui souhaitent aussi mettre en place des ateliers de gestion du stress.

• Infrastructures et Services :

◦ Ajout de casiers : Arnaud & Grégoire insistent sur le manque de casiers, notamment pour les demi-pensionnaires aux journées longues.  

◦ Deuxième service à la cantine : Proposé par Sacha & Oscar.    ◦ Vente de fournitures scolaires : Avazov & Isaac suggèrent une vente au foyer pour les oublis avant une évaluation.

• Ambiance Sonore et Visuelle :

◦ Changement de la sonnerie : Henry & Maxime proposent un vote via Instagram pour changer la sonnerie plusieurs fois par an.  

◦ Décorations du lycée : Arnaud & Grégoire et Roman & Louison veulent décorer l'établissement pour les fêtes (Noël, Halloween).

5. Communication et Démocratie Lycéenne

Améliorer la transmission de l'information et l'écoute des élèves est un enjeu clé.

• Outils de Communication Numérique :

◦ Gestion du compte Instagram du CVL : Arnaud & Grégoire veulent le rendre plus actif avec des sondages et des informations pratiques (menus de la cantine).   

◦ Utilisation des télévisions de l'accueil : Henry & Maxime veulent y diffuser des informations utiles (horaires de bus, actualités du CVL).

• Mécanismes de Participation :

◦ Boîte à idées : Proposée par Méina & Lilian pour que chaque élève puisse soumettre des propositions.  

◦ Comptes-rendus mensuels : Également une idée de Méina & Lilian pour plus de transparence sur les actions du CVL.

• Philosophie de Représentation :

◦ La quasi-totalité des candidats se disent "à l'écoute".  

◦ Baptiste se distingue par une approche radicale, refusant un programme pour se faire le porte-parole direct et exclusif des demandes formulées par les élèves.

I think that some social media platforms allow certain bots to operate for example a robot that produces positive reaffirmations in an Orc like dialect. On the contrary a spam bot will most likely be banned from a platform because it offers no creativity and doesn't get a lot of likes therefor not offering profit for the social media platform.

I think this example of the gender selection form not only shows how much they care about their users but also how much time they have put into their website. Overall, it is very bad for the company and encourages users to take their time elsewhere.

• local-first,
• person-first,
• autonomous,
• evergreen, -verifiably attributed,
• interpersonal,
• named networked
• co-evolving ,
• emergent,
• massive multiplayer,
• collaborative
• mutual learning by doing spaces

Weaving the Mutual Indy Learning Commons

The source by Joshua Habgood-Cootedescribes how a lot of the racial and sexual injustice produced by search engines is a product of white ignorance. He provides the idea that search engines should be used as information classification systems.

This book really made me think about how decisions in urban planning and design—like gentrification in neighborhoods such as Fremont—can unintentionally harm some communities while benefiting others. It connects to what I’m researching because it highlights the importance of including local residents in decisions that affect their daily lives. Personally, it made me wonder: how often do city planners truly consider the voices of long-term residents when approving new developments, and could applying design justice principles help reduce negative population health outcomes like stress and displacement?

The article explains why 2+2=4 isn't always true in real life, like when mixing vinegar and baking soda. It shows how context can change math facts. Sometimes, 2+2 can seem to equal 5.

I find this article interesting because while Elon Musk claimed he wanted to back out of the Twitter deal because of the plethora of bots, it seems to be an excuse to allow him to back out. Additionally, while this seems to be an excuse to allow him to back out of the deal, legal experts determined it would be an "uphill battle," and may not be grounds to back out of the deal.

I believe that this is an incredibly annoying and aggressive feature that has been added to ads. The marketing industry would be a lot better without it and I feel as though it makes users more frustrated than interested in purchasing whatever the ad is selling.

I appreciated the tension you raised around friction vs. frictionless design — it really made me think, on one hand, low friction feels like “good usability,” but as you note, design can deliberately add friction to push users toward more thoughtful behavior. I wonder: is there a risk that too much deliberate friction becomes paternalistic or manipulative (assuming users can’t be trusted)?

User interfaces, may seem like such a small aspect of an app's design, but in reality contribute tremendously to it's success. For example, TikTok's scrolling algorithm is what makes us addicted to it. The basically endless push of new content is what has personally made me "doom scroll" for hours. Same with tinder, as the swipe right on a person's profile to show interest, or swipe left to indicate you are not interested, dramatically simplifies it's usability to where you could be swiping non stop, compared to if it were a menu with a "next" button. So in total, under interfaces are not only good for creating those annoying little "x"s on ads, but also making an app become successful in the long run.

The text says the user interface is what you view and interact with. My question is what do we call the part of the system that the user doesn't see? For example, the algorithm that decides what order posts appear in user's feed is a huge part of user experience. But it's not part of the UI. Is there a specific name for that "behind-the-scenes" part?

The Tom Knowles source describes how he helped create the online feature known as the "infinite scroll" and how he heavily regrets creating it based on the effect it has had on society. The "infinite scroll" has increased depression rates in teens and has created even more issues beyond what he is describing i.e. doomscrolling.

While I have never used 4chan before or even been on the website, I have always known it as a social media platform with very little moderation where anything goes. I find it interesting how, despite how long it's been since its inception and the reputation it has gotten for being a website that breeds toxicity and hate, it is still very active. I also find it interesting how active it is considering how aged the site looks and how much friction it has.

A detail from the source that I found interesting is that the first radio signals were being sent in the 1970s at the University of Hawaii, but it took until 20 years later in 1992, in Germany for the first actual text message to be sent.

What’s newer to me is that Standage defines social media as part of a long tradition of humans sharing news, gossip, and opinions through networks such as coffeehouses, pamphlets, and letters, rather than a sudden invention of the Internet. The emergence of the Internet has greatly increased the speed of information dissemination, from a quantitative change to a qualitative change. Perhaps the problems we face today, such as misinformation and fake news, are not actually new problems.

For me, My Little Pony always relates back to a place of nostalgia, but it's fandom of grown men called "Bronies" display a great example of strange internet subcultures within social media history. Social media has always been a place for people to find their niches, even if they are "weird" or socially unacceptable. That's why sites like 4chan have existed to allow for the posting of unregulated content. However, one thing I will say about subcultures within social media is they usually always find a way to get political at some point. From furries being tied with leftist views, Taylor Swift fans now being called MAGA with the release of her new album, and even bronies having a Nazi problem, it seems the internet and politics have always been intertwined within history.

It’s cool to see Tom Standage’s Writing on the Wall: Social Media — The First 2,000 Years cited here. His historical framing helps us see that social media isn’t entirely new, only evolved. I’d also suggest adding Shoshana Zuboff’s The Age of Surveillance Capitalism as a counterpoint source: it connects design history with power, data extraction, and economic incentives, deepening the discussion of how “design” choices embed commercial values.

4chan has undeniably been one of those social media sites I had herd about, but never really used. When I recently decided to visit the site, I was shocked by it's retro user interface, and confusing layout. People on 4chan seem to post in their own niche subcategories, and reply in threads like reddit. The whole site itself feels like lost media, but people post regularly, and even talk about current events. Overall, I think the site is a great example of how despite the site looking rather out dated, and confusing to use, it still has a loyal fan base which values un-regulated speech over flashy and newer designs.

With web 1.0 mainly being content created by independent companies, 2.0 being content created by individual people, and web 3.0 being the integration of AI which we are rapidly approaching that possibility, it makes me wonder about the possibilities of web 4.0. I assume it would be content created by a community, with individual ideas seamlessly integrated into a cohesive media, able to be easily understood.

This wikipedia entry caught my eye because I have a personal interest in scrolling, specifically scrolling on social media. This wikipedia page starts by explaining what scrolling is. I think everyone in this day and age knows what scrolling is, but it was interesting to read about it in the most basic, kindergarten level way of thinking. That scrolling is sliding text, images, or video across a screen vertically or horizontally. It gives the example of movie credits, which is interesting to me because all I think about when I hear the word scrolling is physically scrolling on social media. There was also section dedicated to infinite scrolling which is what I really wanted to read about. I learned that Aza Raskin is the person who created this feature in 2006. And that he regrets creating it because he thinks it contributes to smartphone and social media addictions. I agree with him on that.

User friction keeps users from achieving goals that they desire to do online. There are three types of user friction, emotional friction, interaction friction, and cognitive friction. Emotional friction is when something online is hard to use which create negative feeling for users and prevent users from doing what they willing to do. Interaction friction is when aa website is too confusing and hard to navigate. The website is complicated to use which may cause user giving up on using the website. Lastly, cognitive friction occurs when a website doesn't achieve users expectation. This article also talks about three types of clicking which often occurs when users loose patience on doing something and start clicking whatever they see one the website.

In this article it talks about conspiracy theories weren’t just a modern phenomenon and how throughout the 19th century, American political figures accused other country's of secret deals, collusion, and hidden plots. These accusations were especially common during the rise of political parties, and they helped shape distrust in goverment institutions.

The cited Wikipedia Bibliography for "Web 2.0" provides a solid definition of the Web 2.0. It described as the version of the internet we use today. Which mean you can actively interact with websites instead of just reading them. It's often called the "participative social web" because it lets you create your own content and share it or connect with others on platforms like Facebook, YouTube, and Wikipedia. This evolution turned the web from a one-way information source into a collaborative space.

Some people consider this problem to be merely a matter of semantics, but it is not.

I think it's very intriguing that this was an explanation of death as a solution for the overpopulation of their world. I never would have thought that they would take turns dying and coming back to life, and it's such a unique and interesting thing to think about.

Why do they still think of animals as doing good for their people even though they believe animals like Coyote are responsible for death?

I think everyone, including myself, can relate to this statement. Definitely not to the same extent as Coyote, who is fearing dangerous consequences, maybe even death. However, I think everyone has been in a scenario where they wanted to help out, everyone told them no, and then they went through with it anyway. After actually doing what was advised against, everyone gets angry, and you fear punishment when really you had no ill intent to begin with.

Addressing question three, which I will be answering, is the emotions this text makes me feel. This idea makes me feel annoyed with the people who have become unhappy. At first, they complained that there was overcrowding, and now they complain about the solution. Assuming that temporary death means more people were born in addition to those who came back to life, it seems selfish that people would rather keep coming back to life rather than letting new people who haven't experienced life experience it to the fullest with less overcrowding. Their feeling sad for those who have passed on, I have no problem with, but the overall discontent this text expresses annoys me with its demonstration of humans constantly not being satisfied.

This piece of the text confused me because it seemed to contradict itself. Coyote is recommending that people die forever because temporary death leads to overcrowding, thus creating a lack of food. My question is, why is food needed if death is temporary? If death is a meaningless concept, it just means temporarily being a spirit. Is there really a problem with having too little food?

I wonder why Coyote decided to close the door. Did he truly believe this would help the world, or was it pure jealousy and anger because his idea was rejected? This makes me think about how tricksters often act out of emotion, not reason.

This study chose to use FEES as the "gold standard" of swallow studies to compare with cervical auscultation. However, in my experience, the Videofluoroscopic Swallow Study (VFSS) is typically seen as the gold standard for swallow studies. Also, I have personally found FEES to be a less common procedure completed compared to VFSS. While I understand why the researchers chose to use FEES to compare to CA (to complete multiple swallows at bedside and without radiation exposure), they do a poor job of explaining their reasoning in the passage. They list FEES and VFSS as the gold standards, but do not explain why they chose FEES over VFSS for the study. Additionally, the wide range of experience in SLPs who completed the FEES procedure in this research study introduces a potential variability that could affect the reliability of the test results. This gap in experience provides room for additional factors, such as less experience and less comfort level with FEES, to affect the outcome of the study. To increase the validity of the study, the experience levels should be narrowed.

This section really highlights some of the issues in America with schooling. These children are not getting the same level of education as their peers due to reasons that have nothing to do with them. America needs to do a better job at allocating funds and resources to these places with lower education rates to allow children equal opportunity.

I think that this is an eye opener to many people who do not realize what education is like in other countries. One thing we do see in the graph due to people barely attending higher education in India is their Illiteracy rates is some of the highest in the world between 20% and 49.9%.

This has become another thing that has been lost. Now, more than ever we see kids and parents spending less time together. The parents have to work so many children are put in daycare and other programs where it is less of the parents raising them. Many times some of these kids will be spending less time with their actual parents than at daycare. Do you guys think that this has a drastic change on those kids growing up?

I truly believe this has been one of the biggest reasons people are having less and less kids. Today, women are forced to decide between a career and having more than one child. Many people these days are already struggling to make ends meet in the US and with the estimated cost of raising a kid for 18 years being north of $250,000 dollars many are being forced to work dead end jobs with crazy hours. What do you guys think should be done to make America more affordable for families these days?

• How does culture affect schooling in these countries, and how have these changed over the years?

When reading this, the only thing I somewhat disagree with is that I don't really think most of the time parents don't pass down their social identity. I think a lot of the time, families give a burden for the offspring to carry themselves in their own kids.

The final part about the naps caught my eye. I knew about the sleeping during school but never that this also shows if a kid is working hard. Being the highest school is just a great representation of the culture Japan shows about making your elders proud. Do you think you can clearly see the difference between U.S and Japan in present day?

This quote is quite eye opening to me. Because it shows the much our world of culture and tradition has changed. Don’t you guys also think that the reason the present dropped is because women are now trying to get their own degrees as well and not us finish high school and then try to marry? This text shows the progress women have made.

To me, this passage means a lot. It reminds me everyday to be grateful for what I have and for the people who got us here. I can’t imagine it was easy to live during a time where you could only raise your own food and had to constantly move. To me now, I constantly eat out and live in Phoenix which is a big city. This has always been normal life to me, and seeing how others started making “cities” by using shelters is insane to me. I wonder how many shelters would be in one area back then? How often did they have to move? What kind of food did they eat regularly? Did they know how much of an impact they’d make in the future?

Hearing that 8% of children never go to school and 25% never reach high school is gut wrenching. It made me realize how truly lucky and blessed I am to have a good family, live in a nice area, and a nice country. How do people manage to live life without being able to read or write? Do these people struggle to read labels on food? COVID also affected my own schooling as I was in 7th grade when it hit. School didn’t go back to normal until my sophomore year. Even then, rules that changed after COVID are still in place. For example, retakes used to not be allowed in high school but after COVID, it was district rule we got to retake anything as much times as possible. Sometimes I still wonder what college was like before COVID? How has it changed since then? Are professors more lenient now than they used to be?

This genuinely shocked me. For my entire life, I believed that around 75-80% of people went to religious services weekly. Seeing only 34% say they attended religious services shocked my system. I grew up in an area where almost everyone went to religious services and it was difficult to find someone who didn’t. Are certain parts of the country more religious than others? Are there more religious services in higher populated areas compared to lower populated areas? Do more people attend monthly rather than weekly?

This passage definitely opened my eyes. My whole life, I’ve always thought that getting older meant I had to get married and find my “true love.” This made me realize that our social norm has made our life revolve around love and marriage. It made me start thinking, what would life be without love? What would like be without marriage? Would this make life easier or harder or would not much change?

Reading about gentrification and changing demographics in Fremont made me think about consequentialism, specifically utilitarianism. Cities often justify redevelopment projects by claiming they create the greatest good for the most people—new businesses, higher tax revenue, and improved infrastructure. But from a utilitarian perspective, is the net happiness really maximized if long-term residents are displaced or priced out? Personally, it makes me question how we measure “well-being” in these situations and whether city planners are truly considering the outcomes for all community members, not just newcomers or investors.

Cases of reincarnation are more prominent in younger children the older ones

Their are many cases around the world of reincarnation each one more rare than the last.

Their is no correlation to a wether a persons religious determines them to be reincarnated further explains anyone can be reincarnated even if they don’t believe in it

This proves reincarnation is possible in a way but it could also be a pure coincidence.

This statement highlights the high prevalence of depression and cognitive decline among elderly individuals living in institutional settings, such as nursing homes. It emphasizes that these symptoms are not only common but also crucial considerations when assessing the overall health and functioning of this population. The authors are suggesting that mental health and cognitive status should be integrated into evaluations of institutionalized older adults, as these factors may interact with nutrition, medication use, and functional ability

I think this is so vital to recognize, although it is vague compared to some other statements. Nutrition as we all know is essential to physical performance of everyday life but now, we are realizing it can affect a person's emotional needs. By finding the basics of this, we can recognize and prevent issues such as mental decline and instead promote diets that allow for a person's mind to stay stable.

The author shows how Harold, who comes from a poor family, has big differences with Garrett because they had very different childhoods. Garrett's family has a lot of money, so they can do fun things and get a lot of useful things. But Harold's family doesn't have enough money for food and other basic needs. Harold's mother loves her kids very much, but she can only give them the basics because the family doesn't make enough money, which forever stunts Harold's growth. Kids' minds and feelings get hurt when they are exposed to violence and don't get enough food or medical care. This makes it harder for them to deal with problems that occur in poverty.

The parents play crucial roles in setting up their kids for educational success. Personality traits also determine one’s trajectory

This gap has to close down. Income cannot be the determining factor of ones education

Childhood experiences lead to trauma that affects people for the rest of their lives

By providing stable economic and social security for low-income families, “New Hope” not only alleviates parents' financial burdens but also indirectly improves children's growth environments. Parents' financial stability and psychological relief enable them to devote more time and emotional resources to their children's education and companionship. This demonstrates that educational equity cannot be achieved solely through school-based reforms; social welfare policies also play a crucial role in promoting students' academic and mental well-being. This research reminds policymakers: investing in families is investing in the very foundation of education.

From an economic perspective, high-income families can provide more growth-enhancing “inputs” for their children, such as nutritionally balanced diets, richer home learning environments, higher-quality childcare or extracurricular educational resources, and safe, stimulating community settings. Collectively, these factors give children an early advantage in cognitive, language, and social skills. Psychological and sociological research indicates that economic hardship can undermine the quality of family relationships—such as increased parental stress, insufficient emotional support, and reduced family interaction—thereby indirectly affecting children's mental health and motivation to learn. The roots of educational inequality lie not solely within schools but form much earlier at the family level, even during infancy. This underscores that educational equity cannot be achieved by schools alone; it requires social policy interventions such as improving living conditions for low-income families, providing early education support, and strengthening community resources. Otherwise, the wealth gap will manifest not only in income disparities but also be passed down through generations as a “gap in knowledge and opportunity.”

Educational inequality exists from the very start of schooling, not just in secondary or higher education. Family economic circumstances directly influence children's cognitive development through early educational investments—such as books, language environments, and extracurricular resources—thereby creating “structural inequity” at the starting line of academic achievement. Moreover, this gap widens over time, generating a “cumulative advantage” effect. In other words, the education system often inadvertently replicates society's economic stratification rather than dismantling it.

Researchers are fueled by wanting to solve personal,professional and societal problems .

AI history

In 1963, the Department of Defense funded an AI program at MIT (here’s a fantastic deep dive into that history)

Creating a research plan outline can help by identifying main points in your research.

Make your thoughts understandable and use known facts in your paper.

I was shocked at how astroturfing makes something fake look like real public support. I didn’t realize how often it's used in politics and marketing to manipulate opinions, especially online with bots and fake accounts. It really makes you question what’s real on the internet.

It makes it natural, it is how the Nazis got Germans to be okay with the genocide of the jews. It's how any genocide is justified

one forgets the power of symbols

In my own experience, this is the first time I have ever heard of Metadata, not in school, and I don't recall learning about it, either, due to my poor memory. Is Metadata only for Social Media or does it apply to games, websites, etc...?

so what happens when a child loses points for complaining during the soup test? does the child know theyve lost points? do they ever find out? what are the consequences for that? because if the child knows then there is definitely a punishment. perhaps not an intentional one, but there is a feeling that the child has done something wrong.

Okay so then you're sheltering them from reality and introducing challenges that are not nearly as tough as the challenges of life. How is that preparing them for anything

hmm but these situations are entirely contrived. the only reason the children have any relationship with pains are because theyre decided by the authority. so theyll experience aches and pains in the real world, fine, but those can and will have so many different reasons. if they experience a famine, they won't have the school authority to turn to as the reason behind it. their learning experiences are artificial in nature.

HOW do u know this to be the case, frazier,

yeah this is definitely potentially traumatizing to a child. will it equip the child to be able to handle self control in the future? sure, maybe. it could also teach them to fear authority and break down in a time of need. it could become a trigger. every person reacts differently to this sort of thing. what works for one child may break another. and hard times don't make strong people, they only identify which people make it through alright and which people don't. plenty of people who have experienced trauma are able to snap into action when a high-stakes situation happens. my girlfriend is one of them. she can react to an emergency with great clarity. but what of her ability to perform under low stakes, during times of stability? she's not trained to operate in those situations. its very difficult for her. also this type of lesson can have unintended consequences. the fact that any groaning or complaining results in a wrong answer teaches children to suppress themselves. what if, in the future, the child needs to speak up in a situation where they're being mistreated, but can't because a part of themselves forbids them to?

I like this way of instruction honestly. It's a way I think that can hit the way that each student can learn. It's also not just straight up throwing these kids into the deep end with the lessons and the units.

This is something I really like, outlining the highlights and important parts of our units and lessons really ensures that we will be able to fill in around it some of maybe the lesser stuff or the easier stuff we can scaffold up to the harder stuff and the main points.

This is game-changing for Salem: using shared identities to link parish records, land deeds, family trees, and trial transcripts. The end outcome would be a Linked Salem Dataset, allowing users to move from a confession to a property border to a minister's sermon. It is an ethical rebuilding of context, reuniting the social fabric that panic once ripped apart.

This is precisely how we should approach Salem: begin with the ultimate goal, an accurate, ethical, and transparent record, and then construct the digital record accordingly. The way we encode witness depositions, confessions, and execution maps impacts how future audiences "learn" Salem. Designing from the endpoint involves envisioning the reader or researcher we want to empower, rather than merely the dataset we aim to complete.

This is critical for redigitizing Salem documents. Do we track gender? Occupation? Relation to the accusers? Each field approaches causation differently. If we exclude "social ties," we may overlook the neighbour networks that fueled accusation chains. Data fields equal interpretive power.

I was struck by the biblical reference that occurs with the line “Gentile or Jew.” This line is referencing 1 Corinthians 12: “For by one Spirit we were all baptized into one body – Jews or Greeks, slaves or free – and all were made to drink of one Spirit. For the body does not consist of one member but many.” (1 Corinthians 12: 13-14) The first thing that jumped out to me was the transition from “Greeks” in the translation of the bible given as our source to “Gentile” in The Waste Land. While in the context of the Bible they mean effectively the same thing (non-Jewish people in the early stages of Christianity), Eliot’s choice to use “Gentiles” seems contradictory to the rest of the imagery in the passage. The other sources referenced and “Phlebas the Phoenician” are explicit references to Greek mythology and figures, so Eliot’s choice in using “Gentile” rather than Greek is clearly an intentional one. This choice expands the view of the passage beyond just the Greek myths, which is reflected in the rest of the passage from 1 Corinthians. In the context of “Death by Water,” the language of “baptized” and “drink” in 1 Corinthians has an intentional impact, connecting the Gentiles and Jews to water, and connecting the process of baptism with the drowning of the sailor in The Waste Land. The insertion of “Gentile or Jew” directly after “entering the whirlpool” connects the drowned man to the process of baptism, connecting him to a process of new Christian life in his death. In this context, “Gentile or Jew” serves to unify him with all others, taking his individual story and blurring it together with multiple. The other sources that are blended into this passage fit that pattern, with multiple examples of death by water meshed together in this passage. In a similar sense, the distillation of the previously long “Death by Water” into such a short passage reflects this, combining detailed stories into one small, cryptic moment of the poem.

The passages from 1 Corinthians about humility and connectedness directly parallel Eliot's "Death by Water." When Paul warns "let any one who thinks that he stands take heed lest he fall" and declares that "by one Spirit we were all baptized into one body Jews or Greeks, slaves or free," he establishes universal connectedness and equality. Eliot echoes this precisely with "Gentile or Jew / O you who turn the wheel and look to windward". Eliot directly welcomes all readers regardless of identity to recognize themselves in Phlebas's fate. The warning to "Consider Phlebas, who was once handsome and tall as you" mirrors Paul's warning to those who think they stand. Just as Paul insists "the body does not consist of one member but of many," Eliot reminds us that everyone will rise and fall and pass through the stages of age just like the Phoenician sailor"rose and fell / He passed the stages of his age and youth" captures both the physical motion in the currents and the cycle of life which humans endure. The “wheel” reference suggests a constant spinning cycle, reinforcing that everyone will pass through youth and death. Eliot conveys the same message as Paul. Those who think they are above will fall, as we are all connected and destined for the same fate.

The Salem trials can be viewed as an agent-based model of hysteria. Each villager followed minor contextual principles such as fear, piety, conformity, and suspicion, but they all contributed to widespread dread. Agent-based thinking explains how little individual decisions ("I will name one more person") led to systematic violence. The emergent phenomenon was unplanned; it resulted from repeated micro-actions under specific social conditions.

This shows the information about how covid-19 started and expanded. It said that the virus is founded on 2019.12 in China and WHO announced it is a public health emergency of international concern on 2020.1.30. Until 2023.11, there are 778523540 people had covid and 7100783 were died because of covid-19. However, there might be more excess mortality during this period. Many countries provided vaccines to their people, and quarantine patients. Here is a detail in this website is that many companies and shutdown during covid-19 periods, and caused supply shortage and unemployment rate increased.

Well this is new! Never used bumble before, i fact, a little frightened by swiping. At least this dimistify the risk of cyber security. That said, it is still important to keep everything under control and be aware what information and who you are giving in to.

Now in the social media there are many influencer sustain their fake persona. For example, they might be fake couple, fake job. Or sometimes when they want to sell their product, they will make fake video to shows how good the products are, but the true thing is the products aren't that good, they all want is for money. However, some influencer will make short drama video online, I think it is ok, because if they told everyone first, then people will just watch it as a drama. But if you want to sell some products and make fake video, I think that is pretty bad. So when facing the video online, we should be careful.

Inauthenticity can be both a mask and a mirror — something we wear, and something that reveals how complex we are. Sometimes, by reversal assumption, we get what others are trying to achieve, and thus understand their true motives. It's like psychology game. Reminds me of Hannibal.

in dire need

inti

Explanation: In this passage, Taney describes a procedural moment where Dred Scott’s lawyers asked the judge to instruct the jury to rule in Scott’s favor, as both sides had already agreed on the facts. The judge refused to do so, and Scott’s team officially objected to preserve their right to appeal. This moment illustrates how the case shifted its focus from factual disagreement to legal interpretation.

Connection to Originalist Thinking: Taney’s approach throughout the decision relies on originalist reasoning — he interprets the Constitution according to what he believes the framers’ intent was at the time it was written. By refusing to consider modern moral or social developments, Taney limits the case to what he thinks the founders would have recognized — meaning that, under the Constitution as originally written, enslaved people and their descendants could not be citizens. This is an example of originalism because it looks backward to 18th-century legal definitions instead of evolving ones.

Agree or Disagree: I disagree with Taney’s use of originalism here. His interpretation ignores the moral progress and human rights understanding that had developed since the Constitution was written. By anchoring his reasoning in the framers’ original context — a time when slavery was accepted — he denies Dred Scott’s humanity and right to citizenship. This shows the danger of relying too rigidly on historical intent without considering justice or equality in the present day.

Testing

Absolutely not

I find that using the dication feature helps when pre writing

Example of how structural racism impacts climate change

Just as economic factors, ecological factors have an effect that can affect people's health .

people who are not expected to fall under social norms are often targeted. or more prone to fall under the criminal justice system .

I guess this is why they randomly listed differences between ai and us in the introduction

values quality than fitting into an unjust system

My question is, why did Facebook put all of its competitors out of business so quickly? What did they do so much better that the others could not compete? Social media platforms like Myspace were dominating the industry, and for one social media platform to just completely dominate over the ones that were also leading in the industry.

In the online world today the presence of both 4-chan and 8-chan has become so much more prevalent. It feels like I see it mentioned almost everywhere, and especially when it comes to violent acts. In most instances of mass shootings or political attacks the perpetrator has had some presence on message boards like this.

Although it is important that people have free speech to express themselves on the internet. It is also very important to make the community inclusive and comfortable for all the users. In most of social medias nowadays; there's people who's working for the company to filter what content is okay to be spread on the internet. Internet should not be a place where people go crazy and doesn't care about what they say just become it is anonymous and other people don't know who they are.

This is something that everyone always says when you are growing up. Now, it really resonates with me as a student because there are many failures and mistakes that I have made where I have learned from it.

It is encouraging to hear about struggles from other people. It brings people together knowing that we as people have struggled with the same topics. It encourages us to keep going because we see a role model who also struggled and is now very successful.

This spoke to me because almost every time a scientific discovery was made it was through trial and error. There is no improvement if there is no error.

It is important to know the difference between these two because it is good to know what is there but has not been uncovered yet and what is there naturally.

Labs are meant to learn from your mistakes. When you make a mistake you learn to grow from your past. Its the same as learning from history. History makes you acquire wisdom.

Its the same as when you go to a doctor. You get multiple opinions in order to be sure that you are diagnosed correctly.

Project based learning that can actually allow the student to get creative with the assignment have usually been the assignments I have both learned from and enjoyed the most. the key part about project based learning in my opinion is giving students plenty of choices

I've found that the best teachers in my memory are the teachers that have tried to see how other students perceive the subject, not just how they themselves perceive it. This helps them relate to their students a lot more and thus helps them teach better

You use critical thinking everyday. When talking with others, you have to critically think sometimes just to make a good impression on someone. You also have use critical thinking when decision making as one bad decision can make immense impacts on your life.

Critical thinking makes one actually reflect and think about what they did or they are currently studying. I feel like just critically thinking about a topic could be a studying technique.

I think it it helpful because it makes the mind actually think instead of just coasting through easy assignments.

I think critical thinking is very much useable in everyday life, even outside of just school. It can be a very helpful skill to use later on in life if you're able to develop and use it.

It is especially important to take advantage of any resources you can in order to make the most out of the material you are learning. For example, if I am stuck on a problem some resources I can use are textbooks, the help of teachers and outside tutoring to use as a reference for the problem that I am having trouble solving.

I relate to this a lot because I feel that having an open mind really helps in understanding complicated problems from different perspectives. Especially when working in groups and listening to the different ideas that your peers have to share.

I often find this essential to get a deeper understanding on a concept I do not understand because helps me understand why a certain answer is wrong and to avoid it the next time I make a mistake.

Stoneditch pointed out an unusual period of media concentration in the United States in the 1900s. This centralization creates a more unified national narrative that feels very different from today’s fragmented social media environment. This “solidarity” can silence minority voices because, as the passage points out, those mainstream newsrooms are dominated by white people, resulting in limited diversity in the news media, further increasing the promotion of a single group narrative.

As the work media entails, and form of interaction with the physical world can count as a form of media. And as such, any interaction with the physical world can also count as a form of social media. Mediums can also become social through the exposure to other people, but this also brings up the question of is a media only social if shared or perceived by some body else.

I found this interesting because I have never considered how graffiti and books would be social media, but it makes sense. In the world of the internet, so much more media types can be spread and go "viral," so it's interesting to think about how much more work it was for a piece of media to go viral before the internet was created.

It is important to use rhetoric to create designs that appeal to the customers. Using rhetoric you can build upon your original designs and add more ways to benefit the user, which helps make sure more people continue using the product.

I think when she says it's easy to forget what she came for, it;s because there's so much to take in down there ; not just the wounds and broken things, but also strange , hauntingly beautiful details that make you want to stay and wander.Tat's why there seem to be people who have 'always lived here' - they've settles among the ruins, maybe trapped by it or maybe finding a kind of comfort in it.

I think this is powerful because it shows that we are all part of this search. Even if our names weren't written in the old stories, we can still go down there and see the wreck for ourselves. I think the wreck here might be our traumas or things we try to forgetting. this line is really deep

I love how she describes the wreck so vividly that we can picture it. it makes me think of the dark times in our own lives when we wanted to reach the sunlight so badly. some things we believed would last forever ended up drifting into the 'ocean of lost memories' I especially like the part about the ribs, it feels like those old hurts we try to ignore, but every now and then they poke us again. we can't really erase them ;we just learn to move around them, like tentative haunters of our own past.

the ladder is a symbol of a way into depths of history/memory, but here she wrote 'we who have used it' implying that not everybody chooses descend..Only the ones who choose to climb down can really start to face themselves and hidden truths.

unlike heroic male explorers, the speaker dives alone leads to feminist emphasis on personal, solitary quest

Reading this reminds me a lot of modern day Discord, so you could defiantly say that IRC was ancestor of modern multiple room based chats like Discord and other similar things. Even the layout as shown in this image is almost exactly like Discord and how it is laid out now, with a series of "channels" with different conversations to switch between on the left, the main conversation for that room in the middle (complete with the handle of whoever said something with when they said it), and the list of users on the right. If it ain't broke don't fix it I guess.

Removing political oppenents from the arena

tort liability

I think that this section and the following one would benefit from some actual outputs illustrated - e.g., stand and stock tables. I show students (in excel) how to generate stand and stock tables from plot and point sampling data in Baker. Seeing the outputs would make the illustrations more clear. But maybe the code spits those out?

When is that? I would say that we can sometimes eliminate the need to measure DBH, as in VBAR, and only count logs, instead of measuring both. I don't think there is any situation where measuring height is easier than DBH. Choice 1. Measure DBH and height on in trees. Choice 2. Measure height only and use VBAR as a ratio estimator.

Sig figs in the table for volume and TPA?

Is it worth showing an illustration of the mirage or walkthrough with point sampling?

As above, three decimal places is likely overkill and the measurements aren't that precise.

Maybe round the numbers in the data to realistic values for precision?

I'd put double asterisks or a different mark on the walkthrough trees counted twice in the figure. Makes it easier to read and understand quickly.

At this point, it looks as though Iago's manipulative behavior is no longer needed because Othello is internalizing the lies and believes them at this point. He has fully submerged himself in the lies that come out of Iago's mouth that he deems true. He doesn't even sound like himself, calling Desdemona a "cunning whore of Venice".