eLife Assessment

This important study shows that orientation tuning of V1 neurons is suppressed during a continuous flash suppression paradigm, especially when the neurons have a binocular receptive field. However, the evidence presented is incomplete and, in particular, does not distinguish whether this suppression is due to reduced contrast or due to masking.

Reviewer #1 (Public review):

Disclaimer: While I am familiar with the CFS method and the CFS literature, I am not familiar with primate research or two-photon calcium imaging. Additionally, I may be biased regarding unconscious processing under CFS, as I have extensively investigated this area but have found no compelling evidence in favor of unconscious processing under CFS.

This manuscript reports the results of a nonhuman-primate study (N=2 behaving macaque monkeys) investigating V1 responses under continuous flash suppression (CFS). The results show that CFS substantially suppressed V1 orientation responses, albeit slightly differently in the two monkeys. The authors conclude that CFS-suppressed orientation information "may not suffice for high-level visual and cognitive processing" (abstract).

The manuscript is clearly written and well-organized. The conclusions are supported by the data and analyses presented (but see disclaimer). However, I believe that the manuscript would benefit from a more detailed discussion of the different results observed for monkeys A and B (i.e., inter-individual differences), and how exactly the observed results are related to findings of higher-order cognitive processing under CFS, on the one hand, and the "dorsal-ventral CFS hypothesis", on the other hand.

Major Comments:

(1) Some references are imprecise. For example, l.53: "Nevertheless, two fMRI studies reported that V1 activity is either unaffected or only weakly affected (Watanabe et al., 2011; Yuval-Greenberg & Heeger, 2013)". "To the best of my understanding, the second study reaches a conclusion that is entirely opposite to that of the first, specifically that for low-contrast, invisible stimuli, stimulus-evoked fMRI BOLD activity in the early visual cortex (V1-V3) is statistically indistinguishable from activity observed during stimulus-absent (mask-only) trials. Therefore, high-level unconscious processing under CFS should not be possible if Yuval-Greenberg & Heeger are correct. The two studies contradict each other; they do not imply the same thing.

(2) Line 354: "The flashing masker was a circular white noise pattern with a diameter of 1.89{degree sign}{degree sign}, a contrast of 0.5, and a flickering rate of 10 Hz. The white noise consisted of randomly generated black and white blocks (0.07 × 0.07 each)." Why did the authors choose a white noise stimulus as the CFS mask? It has previously been shown that the depth of suppression engendered by CFS depends jointly on the spatiotemporal composition of the CFS and the stimulus it is competing with (Yang & Blake, 2012). For example, Hesselmann et al. (2016) compared Mondrian versus random dot masks using the probe detection technique (see Supplementary Figure S4 in the reference below) and found only a poor masking performance of the random dot masks.

Yang, E., & Blake, R. (2012). Deconstructing continuous flash suppression. Journal of Vision, 12(3), 8. https://doi.org/10.1167/12.3.8

Hesselmann, G., Darcy, N., Ludwig, K., & Sterzer, P. (2016). Priming in a shape task but not in a category task under continuous flash suppression. Journal of Vision, 16, 1-17.

(3) Related to my previous point: I guess we do not know whether the monkeys saw the CF-suppressed grating stimuli or not? Therefore, could it be that the differences between monkey A and B are due to a different individual visibility of the suppressed stimuli? Interocular suppression has been shown to be extremely variable between participants (see reference below). This inter-individual variability may, in fact, be one of the reasons why the CFS literature is so heterogeneous in terms of unconscious cognitive processing: due to the variability in interocular suppression, a significant amount of data is often excluded prior to analysis, leading to statistical inconsistencies. Moreover, the authors' main conclusion (lines 305-307) builds on the assumption that the stimuli were rendered invisible, but isn't this speculation without a measure of awareness?

Yamashiro, H., Yamamoto, H., Mano, H., Umeda, M., Higuchi, T., & Saiki, J. (2014). Activity in early visual areas predicts interindividual differences in binocular rivalry dynamics. Journal of Neurophysiology, 111(6), 1190-1202. https://doi.org/10.1152/jn.00509.2013

(4) The authors refer to the "tool priming" CFS studies by Almeida et al. (l.33, l.280, and elsewhere) and Sakuraba et al. (l.284). A thorough critique of this line of research can be found here:

Hesselmann, G., Darcy, N., Rothkirch, M., & Sterzer, P. (2018). Investigating Masked Priming Along the "Vision-for-Perception" and "Vision-for-Action" Dimensions of Unconscious Processing. Journal of Experimental Psychology. General. https://doi.org/10.1037/xge0000420

This line of research ("dorsal-ventral CFS hypothesis") has inspired a significant body of behavioral and fMRI/EEG studies (see reference for a review below). The manuscript would benefit from a brief paragraph in the discussion section that addresses how the observed results contribute to this area of research.

Ludwig, K., & Hesselmann, G. (2015). Weighing the evidence for a dorsal processing bias under continuous flash suppression. Consciousness and Cognition, 35, 251-259. https://doi.org/10.1016/j.concog.2014.12.010

Reviewer #2 (Public review):

Summary:

The goal of this study was to investigate the degree to which low-level stimulus features (i.e., grating orientation) are processed in V1 when stimuli are not consciously perceived under conditions of continuous flash suppression (CFS). The authors measured the activity of a population of V1 neurons at single neuron resolution in awake fixating monkeys while they viewed dichoptic stimuli that consisted of an oriented grating presented to one eye and a noise stimulus to the other eye. Under such conditions, the mask stimulus can prevent conscious perception of the grating stimulus. By measuring the activity of neurons (with Ca2+ imaging) that preferred one or the other eye, the authors tested the degree of orientation processing that occurs during CFS.

Strengths:

The greatest strength of this study is the spatial resolution of the measurement and the ability to quantify stimulus representations during CSF in populations of neurons, preferring the eye stimulated by either the grating or the mask. There have been a number of prominent fMRI studies of CFS, but all of them have had the limitation of pooling responses across neurons preferring either eye, effectively measuring the summed response across ocular dominance columns. The ability to isolate separate populations offers an exciting opportunity to study the precise neural mechanisms that give rise to CFS, and potentially provide insights into nonconscious stimulus processing.

Weaknesses:

While this is an impressive experimental setup, the major weakness of this study is that the experiments don't advance any theoretical account of why CFS occurs or what CFS implies for conscious visual perception. There are two broad camps of thinking with regard to CFS. On the one hand, Watanabe et al. (2011) reported that V1 activity remained intact during CFS, implying that CFS interrupts stimulus processing downstream of V1. On the other hand, Yuval-Greenberg and Heeger (2013) showed that V1 activity is, in fact, reduced during CFS. By using a parametric experimental design, they measured the impact of the mask on the stimulus response as a function of contrast and concluded that the mask reduces the gain of neural responses to the grating stimulus. They presented a theoretical model in which the mask effectively reduced the SNR of the grating, making it invisible in the same way that reducing contrast makes a stimulus invisible.

An important discussion point of Yuval-Greenberg and Heeger is that null results (such as those presented by Watanabe et al.) are difficult to interpret, as the lack of an effect may be simply due to insufficient data. I am afraid that this critique also applies to the present study. Here, the authors report that CFS effectively 'abolishes' tuning for stimuli in neurons preferring the eye with the grating stimulus. The authors would have been in a much stronger position to make this claim if they had varied the contrast of the stimulus to show that the loss of tuning was not simply due to masking. So, while this is an incredibly impressive set of measurements that in many ways raises the bar for in vivo Ca2+ imaging in behaving macaques, there isn't anything in the results that constitutes a real theoretical advance.

Reviewer #3 (Public review):

Summary:

In this study, Tang, Yu & colleagues investigate the impact of continuous flash suppression (CFS) on the responses of V1 neurons using 2-photon calcium imaging. The report that CFS substantially suppressed V1 orientation responses. This suppression happens in a graded fashion depending on the binocular preference of the neuron: neurons preferring the eye that was presented with the marker stimuli were most suppressed, while the neurons preferring the eye to which the grating stimuli were presented were least suppressed. The binocular neuron exhibited an intermediate level of suppression.

Strengths:

The imaging techniques are cutting-edge, and the imaging results are convincing and consistent across animals.

Weaknesses:

I am not totally convinced by the conclusions that the authors draw based on their machine learning models.

Author response:

Reviewer #2

We respectfully disagree with Reviewer 2’s critiques, upon which the eLife assessment of “incomplete evidence” is primarily based. We believe these critiques do not accurately reflect our study and are rooted in a misinterpretation of the evidence. Consequently, we suggest that the conclusion of “incomplete evidence” is not warranted.

On the basis of Reviewer 2’s critiques, the eLife assessment states: “However, the evidence presented is incomplete and, in particular, does not distinguish whether this suppression is due to reduced contrast or due to masking.” We emphasize that the suppression we observed is a consequence of interocular masking, not contrast reduction. Reviewer 2 cites Yuval-Greenberg and Heeger (2013), which proposes that during CFS, the mask reduces the gain of neural responses in V1 in a manner analogous to reducing stimulus contrast. We agree that both CFS masking and contrast reduction can decrease signal-to-noise ratio and thereby reduce visibility. However, in our paradigm, the physical stimulus contrast was held constant, while suppression was induced by interocular competition under CFS. This is a fundamentally different mechanism from lowering stimulus contrast. Our results therefore reflect genuine masking-induced suppression, rather than the effect of physical contrast reduction.

Furthermore, Reviewer 2 cited Yuval-Greenberg and Heeger’s discussion that null results can arise from insufficient data, and suggested that this applies to our study. This main critique from Reviewer 2 is misplaced for two reasons: First, our main result is not a null effect. A null effect would mean that CFS masking had no impact on population orientation responses. Instead, we observed significant suppression, including abolished tuning in some conditions, which clearly indicates a strong effect of masking. Second, our findings are based on large neural populations recorded using two-photon calcium imaging, providing extensive sampling and high statistical power. Thus, concerns about “insufficient data” do not apply to our study.

Finally, we used machine learning approaches to examine the effects of CFS masking on orientation discrimination and recognition, providing new insight into the long-standing debate over whether the brain can perform high-level cognitive processing under CFS. Although it is, to some extent, a matter of personal judgment whether our work represents a theoretical advance, Reviewer 2 made no comment, positive or negative, on this major component of our study while forming his/her judgment. (In response to Reviewer 3’s main concern about the suitability of SVMs, we now performed a multi-way classification analysis, which yielded results largely consistent with those obtained using the SVM approach in the original manuscript, confirming the robustness of our mechine learning results.

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eLife Assessment

In this important paper, Garcia et al seek to determine whether the superior frontal sulcus (SFS), an area previously implicated in evidence accumulation for perceptual decisions, plays a causal role in perceptual and/or value-based decisions. Through a combination of careful paradigm design, computational modelling, transcranial magnetic stimulation and fMRI analyses, the authors provide convincing evidence that the SFS supports perceptual but not value-based decisions and that its disruption leads to a lowering of decision boundaries.

Reviewer #1 (Public review):

Summary:

In this study, participants completed two different tasks. A perceptual choice task in which they compared the sizes of pairs of items and a value-different task in which they identified the higher value option among pairs of items with the two tasks involving the same stimuli. Based on previous fMRI research, the authors sought to determine whether the superior frontal sulcus (SFS) is involved in both perceptual and value-based decisions or just one or the other. Initial fMRI analyses were devised to isolate brain regions that were activated for both types of choices and also regions that were unique to each. Transcranial magnetic stimulation was applied to the SFS in between fMRI sessions and it was found to lead to a significant decrease in accuracy and RT on the perceptual choice task but only a decrease in RT on the value-different task. Hierarchical drift diffusion modelling of the data indicated that the TMS had led to a lowering of decision boundaries in the perceptual task and a lowering of non-decision times on the value-based task. Additional analyses show that SFS covaries with model derived estimates of cumulative evidence, that this relationship is weakened by TMS.

The paper has many strengths including the rigorous multi-pronged approach of causal manipulation, fMRI and computational modelling which offers a fresh perspective on the neural drivers of decision making. Some additional strengths include the careful paradigm design which ensured that the two types of tasks were matched for their perceptual content while orthogonalizing trial-to-trial variations in choice difficulty. The paper also lays out a number of specific hypotheses at the outset regarding the behavioural outcomes that are tied to decision model parameters and well justified.

Reviewer #2 (Public review):

Summary:

The authors set out to test whether a TMS-induced reduction in excitability of the left Superior Frontal Sulcus influenced evidence integration in perceptual and value-based decisions. They directly compared behaviour-including fits to a computational decision process model---and fMRI pre and post TMS in one of each type of decision-making task. Their goal was to test domain-specific theories of the prefrontal cortex by examining whether the proposed role of the SFS in evidence integration was selective for perceptual but not value-based evidence.

Strengths:

The paper presents multiple credible sources of evidence for the role of the left SFS in perceptual decision making, finding similar mechanisms to prior literature and a nuanced discussion of where they diverge from prior findings. The value-based and perceptual decision making tasks were carefully matched in terms of stimulus display and motor response, making their comparison credible.

Author response:

The following is the authors’ response to the previous reviews

Public Reviews:

Reviewer #1 (Public review):

Summary:

In this study, participants completed two different tasks. A perceptual choice task in which they compared the sizes of pairs of items and a value-different task in which they identified the higher value option among pairs of items with the two tasks involving the same stimuli. Based on previous fMRI research, the authors sought to determine whether the superior frontal sulcus (SFS) is involved in both perceptual and value-based decisions or just one or the other. Initial fMRI analyses were devised to isolate brain regions that were activated for both types of choices and also regions that were unique to each. Transcranial magnetic stimulation was applied to the SFS in between fMRI sessions and it was found to lead to a significant decrease in accuracy and RT on the perceptual choice task but only a decrease in RT on the value-different task. Hierarchical drift diffusion modelling of the data indicated that the TMS had led to a lowering of decision boundaries in the perceptual task and a lower of nondecision times on the value-based task. Additional analyses show that SFS covaries with model derived estimates of cumulative evidence, that this relationship is weakened by TMS.

Strengths:

The paper has many strengths, including the rigorous multi-pronged approach of causal manipulation, fMRI and computational modelling, which offers a fresh perspective on the neural drivers of decision making. Some additional strengths include the careful paradigm design, which ensured that the two types of tasks were matched for their perceptual content while orthogonalizing trial-to-trial variations in choice difficulty. The paper also lays out a number of specific hypotheses at the outset regarding the behavioural outcomes that are tied to decision model parameters and well justified.

We thank the reviewer for their thoughtful summary of the study and for highlighting these strengths. We are pleased that the multi-pronged approach combining causal manipulation, fMRI, and hierarchical drift–diffusion modelling, as well as the careful matching of perceptual content across the two tasks, came across clearly. We also appreciate the reviewer’s positive remarks on the specificity of our a priori hypotheses and their links to decision-model parameters. In revising the manuscript, we have aimed to further streamline the presentation of these hypotheses and to more explicitly connect the behavioural predictions, model parameters, and neural readouts throughout the Results and Discussion sections.

Weaknesses:

In my previous comments (1.3.1 and 1.3.2) I noted that key results could be potentially explained by cTBS leading to faster perceptual decision making in both the perceptual and value-based tasks. The authors responded that if this were the case then we would expect either a reduction in NDT in both tasks or a reduction in decision boundaries in both tasks (whereas they observed a lowering of boundaries in the perceptual task and a shortening of NDT in the value task). I disagree with this statement. First, it is important to note that the perceptual decision that must be completed before the value-based choice process can even be initiated (i.e. the identification of the two stimuli) is no less trivial than that involved in the perceptual choice task (comparison of stimulus size). Given that the perceptual choice must be completed before the value comparison can begin, it would be expected that the model would capture any variations in RT due to the perceptual choice in the NDT parameter and not as the authors suggest in the bound or drift rate parameters since they are designed to account for the strength and final quantity of value evidence specifically. If, in fact, cTBS causes a general lowering of decision boundaries for perceptual decisions (and hence speeding of RTs) then it would be predicted that this would manifest as a short NDT in the value task model, which is what the authors see.

We thank the reviewer for raising these points and for the helpful clarification. We agree that, in principle, the architecture of the value-based task can be conceived as involving an upstream perceptual process that must be completed, to some degree, before value comparison can proceed. Under such a multistage framework, it is indeed possible that cTBS-induced changes in a perceptual decision stage could manifest as a reduction in boundary separation in the pure perceptual task, while the same perturbation appears as a shortening of non-decision time (NDT) when fitting a single-stage DDM to the value task. In this sense, our earlier statement that a “general speeding effect” would necessarily produce identical parameter changes (either NDT or boundaries) in both tasks was too strong, and we are grateful to the reviewer for pointing this out.

At the same time, this alternative explanation remains fully compatible with our central claim that the left SFS plays a perceptual rather than value-based role. We agree with the reviewer that there must be a stimulus-related circuit (in visual and parietal regions) that encodes the physical attributes of the options, and that this upstream processing can influence both tasks. However, a large body of work suggests that left SFS is not part of this primary identification circuitry, but rather contributes specifically to the accumulation and comparison of sensory evidence (e.g., Heekeren et al., 2004, 2006), downstream from areas such as FFA, PPA, or MT/V5 that encode stimulus identity. In other words, stimulus identification (forming a representation of “what is where”) is anatomically and functionally distinct from the accumulation of evidence toward a perceptual decision. Within this framework, the reviewer’s proposal that cTBS speeds “perceptual decisions” across tasks can be understood as targeting precisely the evidence-accumulation stage we ascribe to SFS, with the value-comparison stage proper likely implemented in other regions (e.g., vmPFC and connected valuation circuitry).

We therefore do not rely solely on the dissociation between boundary changes in the perceptual task and NDT changes in the value task as decisive evidence against a “general speeding” account. Instead, our interpretation is based on the convergence of behavioural, model-based, and neural results. First, in the perceptual task, cTBS to left SFS leads to a selective reduction in decision boundary and a concomitant change in trialwise BOLD activity within the stimulated region that covaries with perceptual choice behaviour and with the latent decision variable inferred from the HDDM. Second, in the value task, cTBS does not affect value sensitivity or accuracy, nor does it alter value-related drift or boundary parameters; the only robust HDDM effect is a modest shortening of NDT. Third, critically, left SFS BOLD activity is modulated by perceptual evidence and by cTBS in the perceptual task, but we observe no evidence that SFS activity encodes value evidence or shows value-related cTBS neuronal effects in the value task.

Taken together, these findings indicate that the left SFS serves a causal role in the accumulation of perceptual evidence and in the setting of the choice criterion for perceptual decisions. The reviewer’s suggestion that cTBS may induce a general speeding of perceptual processes that also influences the value task is compatible with this conclusion, in the sense that any contribution of SFS to the value task is best understood as acting via a perceptual component that is upstream of value comparison, rather than via the value accumulation process itself. We have clarified this point in the Discussion of the revised manuscript and now explicitly acknowledge that our DDM dissociation alone does not exclude a general perceptual speeding account, but that the combination of task-specific neural effects in SFS, preserved value-based choice behaviour, and the absence of value-related BOLD changes in SFS strongly support a primarily perceptual role for this region.

Reviewer #2 (Public review):

Summary:

The authors set out to test whether a TMS-induced reduction in excitability of the left Superior Frontal Sulcus influenced evidence integration in perceptual and value-based decisions. They directly compared behaviour-including fits to a computational decision process model---and fMRI pre and post TMS in one of each type of decision-making task. Their goal was to test domain-specific theories of the prefrontal cortex by examining whether the proposed role of the SFS in evidence integration was selective for perceptual but not value-based evidence.

Strengths:

The paper presents multiple credible sources of evidence for the role of the left SFS in perceptual decision making, finding similar mechanisms to prior literature and a nuanced discussion of where they diverge from prior findings. The value-based and perceptual decision-making tasks were carefully matched in terms of stimulus display and motor response, making their comparison credible.

We thank the reviewer for their clear summary of our aims and approach, and for highlighting these strengths. We are pleased that the convergence between causal TMS, fMRI, and hierarchical modelling comes across as providing credible evidence for the role of left SFS in perceptual decision-making, and that our attempt to link these results to the existing literature is seen as appropriately nuanced. We also appreciate the reviewer’s positive assessment of the task design, in particular the close matching of perceptual content and motor output across perceptual and value-based decisions, which was central to our goal of testing domain-specific theories of prefrontal function. In revising the manuscript, we have further clarified these design choices and their rationale, and we have streamlined the exposition of how the hypotheses, model parameters, and neural readouts are connected across the two decision domains.

Weaknesses:

I was confused about the model specification in terms of the relationship between evidence level and drift rate. While the methods (and e.g. supplementary figure 3) specify a linear relationship between evidence level and drift rate, suggesting, unless I misunderstood, that only a single drift rate parameter (kappa) is fit. However, the drift rate parameter estimates in the supplementary tables (and response to reviewers) do not scale linearly with evidence level.

We thank the reviewer for raising this point and appreciate the opportunity to clarify the model specification. In our hierarchical DDM, we did not fit separate, free drift parameters for each evidence level. As shown in Supplementary Fig. 3, the drift on each trial is specified as

where 𝐸_{𝑐,𝑠,𝑖} the trial-wise evidence (difference in size or value) and κ_𝑐,𝑠 is a single drift-scaling parameter per condition and session. Thus, the linear dependence of drift on evidence is implemented at the trial level via 𝜅; we do not estimate independent 𝛿 parameters for each evidence level.

In Supplementary Tables 8 and 9 we report, for descriptive purposes, the posterior means of 𝛿 conditional on each evidence bin (levels 1–4), alongside the corresponding decision boundary and nondecision time summaries. These values are therefore derived quantities that reflect the combination of (i) the single κ_𝑐,𝑠 parameter, (ii) the empirical distribution of continuous evidence values 𝐸 within each bin, and (iii) hierarchical pooling across subjects and sessions. Consequently, they are expected to increase monotonically with evidence level—as they do in our data—but not to lie exactly on a straight line in the discrete level index, because the underlying evidence bins are not equally spaced in physical units and because of between-subject variability and posterior uncertainty.

We will revise the text and table captions to make clear that the evidence-level entries are descriptive summaries of 𝛿 implied by the 𝜅×𝐸 formulation, rather than independently estimated drift parameters, in order to avoid this confusion.

-The fit quality for the value-based decision task is not as good as that for the PDM, and this would be worth commenting on in the paper.

We agree that the HDDM fit for the value-based task is somewhat weaker than for the perceptual task. This is reflected in the somewhat higher DIC values for VDM compared with PDM and in slightly broader posterior-predictive distributions (Supplementary Tables 8–11 and Supplementary Figs. 11–16). We believe this difference primarily reflects the greater intrinsic variability of subjective value-based choices (e.g. trial-to-trial fluctuations in preferences, satiety, or attention), coupled with our decision to use the same relatively simple DDM architecture for both tasks to allow a principled cross-task comparison. Importantly, posterior-predictive checks show that, for VDM as well, the model adequately reproduces both accuracy and full RT distributions at the group and subject level (Supplementary Figs. 11–16), indicating that the fit quality is sufficient for our purposes. In the revised manuscript we now explicitly note that the model captures PDM behaviour more tightly than VDM and that this may reduce sensitivity to very small cTBS effects on value-based decision parameters, even though no systematic effects are evident in our data. Crucially, our central conclusion—that left SFS plays a domain-specific role in setting the decision boundary for perceptual evidence—relies on the robust behavioural, computational, and neural effects observed in PDM and does not depend on assuming a perfect model fit for VDM.

- Supplementary Figure 3 specifies the distribution for kappa hyper-parameter twice.

We thank the reviewer for spotting this typo. We have revised Supplementary Figure 3 legend.

eLife Assessment

Combining state-of-the-art in-situ cell-surface proteomics, functional genetic screening, and single-nucleus RNA sequencing, this fundamental work substantially advances our understanding of glial contributions to organismal lifespan. The evidence supporting the conclusions is compelling, although additional clarification, control experiments, and analysis would further strengthen the study. The work will be of broad interest to researchers studying aging biology, glia-neuron communication, and in vivo proteomic profiling.

Reviewer #1 (Public review):

Summary:

Age-related synaptic dysfunction can have detrimental effects on cognitive and locomotor function. Additionally, aging makes the nervous system vulnerable to late-onset neurodegenerative diseases. This manuscript by Marques et al. seeks to profile the cell surface proteomes of glia to uncover signaling pathways that are implicated in age-related neurodegeneration. They compared the glial cell-surface proteomes in the central brain of young (day 5) and old (day 50) flies, and identified the most up- and down-regulated proteins during the aging process. 48 genes were selected for analysis in a lifespan screen, and interestingly, most sex-specific phenotypes. Among these, adult-specific pan-glial DIP-β overexpression (OE) significantly increased the lifespan of both males and females and improved their motor control ability. To investigate the effect of DIP-β in the aging brain, Marques et al. performed snRNA-seq on 50-day-old Drosophila brains with or without DIP-β OE in glia. Cortex and ensheathing glia showed the most differentially expressed genes. Computational analysis revealed that glial DIP-β OE increased cell-cell communication, particularly with neurons and fat cells.

Strengths:

(1) State-of-the-art methodology to reveal the cell surface proteomes of glia in young and old flies.

(2) Rigorous analyses to identify differentially expressed proteins.

(3) Examination of up- and down-regulated candidates and identification of glial-expressed mediators that impact fly lifespan.

(4) Intriguing sex-specific glial genes that regulate life span.

(5) Follow-up RNA-seq analysis to examine cellular transcriptomes upon overexpression of an identified candidate (DIP-β).

(6) A compelling dataset for the community that should generate extensive interest and spawn many projects.

Weaknesses:

(1) DIP-β OE using flySAM:

a) These flies showed a larger increase in lifespan compared to using UAS-DIP-β (Figure 2 C, D). Do the authors think that flySAM is a more efficient way of OE than UAS? Also, the UAS construct would be specific to one DIP-β isoform, while flySAM would likely express all isoforms. Could this also contribute to the phenotypes observed?

b) The Glial-GS>DIP-β flySAM flies without RU-486 have significantly shorter lifespans (Figure 2C) than their UAS-DIP-β counterparts. flySAM is lethal when expressed under the control of tubulin-GAL4 (Jia et al. 2018), likely due tothe toxicity of such high levels of overexpression. Is it possible that a larger increase in lifespan is due to the already reduced viability of these flies?

c) Statistics: It is stated in the Methods that "statistical methods used are described in the figure legend of each relevant panel." However, there is no description of the statistics or sample sizes used in Figure 2.

(2) Figure 3: The authors use a glial GeneSwitch (GS) to knock down and overexpress candidate genes. In Figure 3A, they look at glial-GS>UAS-GFP with and without RU. Without RU, there is no GFP expression, as expected. With RU, there is GFP expression. It is expected that all cell body GFP signal should colocalize with a glial nuclear marker (Repo). However, there is some signal that does not appear to be glia. Also, many glia do not express GFP, suggesting the glial GS driver does not label all glia. This could impact which glia are being targeted in several experiments.

(3) It is interesting that sex-specific lifespan effects were observed in the candidate screen.

a) The authors should provide a discussion about these sex-specific differences and their thoughts about why these were observed.

b) The authors should also provide information regarding the sex of the flies used in the glial cell surface proteome study.

c) Also, beyond the scope of this study, examining sex-specific glial proteomes could reveal additional insights into age-related pathways affecting males and females differentially.

(4) The behavioral assay used in this study (climbing) tests locomotion driven by motor neurons. The proteomic analysis was performed with the central adult brain, which does not include the nerve cord, where motor neurons reside. While likely beyond the scope of this study, it would be informative to test other behaviors, including learning, circadian rhythms, etc.

(5) It is surprising that overexpressing a CAM in glia has such a broad impact on the transcriptomes of so many different cell types. Could this be due to DIP-β OE maintaining the brain in a "younger" state and indirectly influencing the transcriptomes? Instead of DIP-β OE in glia directly influencing cell-cell interactions? Can the authors comment on this?

Reviewer #2 (Public review):

This manuscript presents an ambitious and technically innovative study that combines in situ cell-surface proteomics, functional genetic screening, and single-nucleus RNA sequencing to uncover glial factors that influence aging in Drosophila. The authors identify DIP-β as a glial protein whose overexpression extends lifespan and report intriguing sex-specific differences in lifespan outcomes. Overall, the study is conceptually compelling and offers a valuable dataset that will be of considerable interest to researchers studying glia-neuron communication, aging biology, and proteomic profiling in vivo.

The in-situ proteomic labeling approach represents a notable methodological advance. If validated more extensively, it has the potential to become a widely used resource for probing glial aging mechanisms. The use of an inducible glial GeneSwitch driver is another strength, enabling the authors to carefully separate aging-relevant effects from developmental confounds. These technical choices meaningfully elevate the rigor of the study and support its central conclusions. The discovery of new candidate genes from the proteomics pipeline, including DIP-β, is intriguing and opens new avenues for understanding glial contributions to organismal lifespan. The observation of sex-specific lifespan effects is particularly interesting and warrants further exploration; the study sets the stage for future work in this direction.

At the same time, several areas would benefit from clarification or additional analysis to fully support the manuscript's claims:

(1) The manuscript frequently refers to "improved" or "increased" cell-cell communication following DIP-β overexpression, but the meaning of this term remains somewhat vague. Because the current analysis relies largely on transcriptomic predictions, it would be helpful to define precisely what metric is being used, e.g., increased numbers of predicted ligand-receptor interactions, enrichment of specific signaling pathways, or altered expression of communication-related components. Strengthening the mechanistic link between DIP-β, cell-cell communication, and lifespan extension, potentially through targeted validation of specific glial interactions, would substantially reinforce the interpretation.

(2) The lifespan screen is central to the paper, and clearer visualization and contextualization of these results would significantly improve the manuscript's impact. For example, Figure 3D is challenging to interpret in its current form. More explicit presentation of which manipulations extend lifespan in each sex, along with effect sizes and significance values, would provide clarity. Including positive controls for lifespan extension would also help contextualize the magnitude of the observed effects. The reported effects of DIP-β, while promising, are modest relative to baseline effects of RU feeding, and a discussion of this would help appropriately calibrate the conclusions.

(3) Several figures would benefit from improved labeling or more detailed legends. For instance, the meaning of "N" and "C" in Figure 1D is unclear; Figure 3A should clarify that Repo is a glial marker; and Figure 5C appears to have truncated labels. Reordering certain panels (e.g., moving control data in Figure 4A-B) may also improve narrative flow. These refinements would greatly aid reader comprehension.

(4) A few claims would be strengthened by more specific references or acknowledgment of alternative interpretations. Examples include the phenoxy-radical labeling radius, the impact of H₂O₂ exposure, and the specificity of neutravidin. Additionally, downregulation of synapse-related GO terms may reflect age-related transcriptional changes rather than impaired glia-neuron communication per se, and this possibility should be recognized. The term "unbiased" to describe the screen may also be reconsidered, given the preselection of candidate genes.

(5) Clarifying the rationale for focusing on central brain glia over optic-lobe glia would be useful.

Reviewer #3 (Public review):

Summary:

Razlan and colleagues provide a detailed anatomical characterization of lamina I projection neurons in the mouse spinal cord that are densely innervated by primary afferents activated by cooling of the skin. The authors, building on their previous anatomical work, validate a Trpm8-Flp mouse line, show synaptic contacts between Trpm8⁺ boutons and projection neurons at the ultrastructural level, and demonstrate at the physiological level that these neurons specifically respond to cooling stimuli. Next, by taking advantage of their previous transcriptomic analysis of ALS neurons, they identify calbindin as a marker for cold-activated lamina I projection neurons and map their ascending projections to the rostral lateral parabrachial area, caudal periaqueductal gray, and ventral posterolateral thalamus, well-known thermosensory and thermoregulatory centers. Altogether, these findings provide strong anatomical and functional evidence for a direct line of transmission from Trpm8⁺ sensory afferents through Calb1⁺ lamina I neurons to key supraspinal centers controlling perception of cold and thermoregulatory responses.

Strengths:

The combination of mouse genetics, electron microscopy, ex vivo physiology, and viral tracing provides convincing evidence for a direct cold pathway. The work validates the Trpm8-Flp line by extensive anatomical and molecular characterization. Integration with previous transcriptomic and anatomical data neatly links the cold-selective lamina I neurons to a molecularly defined cluster of ALS neurons, strengthening the bridge between molecular identity, anatomy, and physiological function.

Weaknesses:

While anatomical evidence for direct synaptic connectivity between Trpm8+ afferents and lamina I projection neurons is compelling, a physiological demonstration of strict monosynaptic transmission is not shown. The conclusion that these inputs are exclusively monosynaptic should be toned down. Similarly, the statement that "Lamina I ALS neurons that are surrounded by Trpm8 afferents are cold-selective" should also be toned down as only a few neurons have been tested and it cannot be excluded that other neurons with similar characteristics may be polymodal.

DOI: 10.21203/rs.3.rs-7483320/v1

Resource: Mutant Mouse Regional Resource Center (RRID:SCR_002953)

Curator: @AleksanderDrozdz

SciCrunch record: RRID:SCR_002953

What is this?

DOI: 10.1016/j.celrep.2025.116658

Resource: RRID:IMSR_JAX:008863

Curator: @areedewitt04

SciCrunch record: RRID:IMSR_JAX:008863

What is this?

DOI: 10.1016/j.celrep.2025.116658

Resource: (IMSR Cat# JAX_006331,RRID:IMSR_JAX:006331)

Curator: @areedewitt04

SciCrunch record: RRID:IMSR_JAX:006331

What is this?

DOI: 10.1016/j.celrep.2025.116658

Resource: (IMSR Cat# JAX_021160,RRID:IMSR_JAX:021160)

Curator: @areedewitt04

SciCrunch record: RRID:IMSR_JAX:021160

What is this?

DOI: 10.1016/j.celrep.2025.116658

Resource: (IMSR Cat# JAX_017586,RRID:IMSR_JAX:017586)

Curator: @areedewitt04

SciCrunch record: RRID:IMSR_JAX:017586

What is this?

DOI: 10.1016/j.celrep.2025.116658

Resource: (IMSR Cat# JAX_007914,RRID:IMSR_JAX:007914)

Curator: @areedewitt04

SciCrunch record: RRID:IMSR_JAX:007914

What is this?

for - James Hansen - youtube - The truth about global warming

Transcript

2:47 We do not have to wait 10 years to conclude that we have reached 1.5 Degrees of warming. Satelllite data shows that earth is strongly out of energy balance.

3:09 An important factor is that IPCC's best estimate of climate sensitivity is a substantial underestimate. I will show that tomorrow in several independent ways.

3:28 Climate sensitivity is probably between 4 and 5 degrees Celcius for doubled CO2 rather than 3 degrees

4:28 What we witness now is scientific reticence on steroids, perhaps because IPCC was granted the position of supreme authority

4:43 But in science, supreme authority is not granted to anyone. Galileo proved that.

4:55 An example of expert herd mentality is the response to our global warming acceleration paper which Annie was coauthor on. The next day, these experts condemned our paper in the media.

5:26 Not one of them discussed the physics in our paper or explained what was wrong. Instead there were ad hominem remarks.

5:51 What could the media do They dropped the paper.

use the same content as in Octa Go

use the same content as in Octa GO

Pokud uvažujete o své prezentaci bez kompromisů, máme pro Vás inovativní řešení! Konstrukce s LED osvětlením zabudovaným přímo do střešních profilů, kterou nikdo jiný nenabízí. +CZ text in the video

use the same content as in Octa Go

use the banner "Grafický návrh zdarma"

use the same content as in Octa GO

Nejvyšší řada nůžkových stanů Nejodolnější hliníková konstrukce s profilem nohy o průměru 54 mm! Bez volných dílů- žádné montování a spojovaní, žádné nářadí. Stan stačí rozložit během 60 s! next paragraphNepromokavá látka a podlepené švy pro absolutní ochranu před nepříznivým počasím. Tuto řadu zvolte pro využití v nejnáročnějších podmínkách. +change the text in video for "Nůžkové stany

pictogram + info about the only octagonal profile on CZ/SK market

for - comparison - energy - China will become the world's first electrostate - The US is doubling down as a leading petrostate

for - planetary tipping points - social tipping points - positive tipping points

SRG Comment - 2025 summary of current state of tipping points - good summary of current state of planetary and social and positive tipping points - crossed our first tipping point - positive one - renewable energy - but it's still too slow, carbon emissions are still too high - comparison - irony - China will become world's first electrostate while the US doubles down as a leading petrostate

for - climate crisis - planetary tipping point - slowing AMOC - Inter-tropical Convergence zone responsible for monsoons - shifts it south

for - quote - illusion of economics - David Suzuki - We live in a human created environment where - it's easy to adopt the illusion that we're different from the rest of life on Earth - we're so smart we create our own habitat - Who needs nature? and - I think this is where we get to where we think we're so clever that what dominates our lives today is economics

for - stats - population - 1900 - 1.5 billion people on the planet

for - ecological civilization - economy should be embed into nature

• SRG comment - David Suzuki - teaching ECOnomy is embedded in ECOlogy

• ECO comes from the Greek root Oikos - for managing the home

• to - David Suzuki talk on interconnectedness - air example

  • https://hyp.is/go?url=https%3A%2F%2Fwww.youtube.com%2Fwatch%3Fv%3Dg2Nw6Yv3S1A&group=world

for - ecological civilization - chinese leaders advocated for it - because many are scientists and engineers

dweb.link: setup-indy0wiki.pad.html

Using the latest indy0pad that produces HTML documents that on Peergos at the point of creation of an index,htmlk file in the right path do - embed hypothesis social annotation tool - set encoding to UTF8 - set title based on the as the last three elements of the path - has a folly operational counterpart in the Inter Planetary Constellation wich is now ready for prime time as IPFS Desktop is now able to share because it is using the new sweep algorithm

The current verison as of 2025-12-09

take this published version as the basis and will be edited with the InterPlanetary Indy0Pad

will use the annotation margin to save updated dweb.links and add them to the document using the Indy0Pad editor @ IPFS which actually save changes into local Storage

and will be able to save to IPFS in the next round of development

I think it's important to note that a lot of the time, for minors, the adults in their lives can often be the ones who oppress them the most. We can see this in how many queer children have to hide their identities in order to be safe, or how in some households, adults may have starkly different views than their children, which may cause conflict.

I also agree with Reagon's criticism. I think the biggest changes come from stepping out of your comfort zone to make a difference in the public. It isn't easy, but once you start creating awareness towards the injustice, it is almost like a domino effect of more people standing up for what they believe in.

I also agree with the authors, it is definitely important to practice allyship among the people in your family and friend group. I think a good mix of the two approaches could be good.

Often in life, we focus on how we can be inclusive to different types of minorities. We see that in the way that companies say they want to hire more diverse people, but in reality, we still see discrimination emerge in places such as the workforce. I think the authors make a great argument of how instead of focusing on including people, we should be holding people accountable for their actions. A mix of the two would be ideal, but in society, we must hold more accountability as it is needed for change. Audre Lorde talks about how anger can be used in creative ways, as a tool for change (Lorde 1997, 283). I agree with her in the sense that anger is not solely destructive. Showing anger is a good way to let people know that there is an inherent problem with the system that must be changed.

This passage really made me think. A lot of the time, when we learn about Indigenous people, we often hear about how they were oppressed, and it focuses on their suffering, but it hardly ever mentions their strength to stand tall despite the oppression they face every single day. I think it's important for both facts to coexist.

As the text mentions, things like social justice aren't always upheld. This is because, inherently, the structures in Western society benefit White colonizers the most. This raises the question of, "What can we do to change the structure that oppresses Indigenous people and people of colour?"

While I do think some White settlers try to challenge racism and colonialism, I ultimately agree with what the authors are saying. A lot of white settlers still hold racist views towards Indigenous people and people of colour because there is hardly backlash for their discriminatory actions. Over the course of the years, racism has become more passive-aggressive rather than "in your face". For example, a lot of people tend to hide their racist views behind jokes, thus trying to get rid of their accountability. This can even happen in friend groups and romantic relationships, whether it is intentional or not.

I resonate a lot with Cindy. I think trying to unlearn colonial ways can be difficult, but once you're aware of what they look like, it makes it a bit easier. This reminds me of when bell hooks stated, "The enemy within must be transformed before we can confront the enemy out." when referring to internalized sexism (hooks 2014, 12). Broadly speaking, when we talk about internalized sexism, it is all these internalized thoughts that we carry with us about gender norms that may be harmful to us. I think this is quite similar to colonial ways of thinking because, just like internalized sexism, colonial thinking is built into us because it played a role in every step of our lives when growing up in a Western society.

Often, we have a lot of behaviour that is deeply rooted in colonialism and patriarchy, yet we don't even realize it. So, it's important to understand this can happen, analyze what we need to change, and try to break free from these norms so that we can work towards decolonization.

The authors focus this article on talking about interpersonal relationships and its impact on decolonization (Hunt and Holmes 2015, 156). Personally, I think this kind of paper is important to write about because it's a good way to show how raising awareness among the people that you're close to can bring the biggest changes. When you are close to people, it's easier to influence their views and behaviours in comparison to trying to persuade a stranger's views. This way, we can show them strategies of decolonization and perhaps bring awareness to problems that they never used to view as problems.

This is such a fantastic way to educate your children. A lot of times, topics like colonialism and its impacts are not things that show up in their education, at least not until later on. By introducing these concepts early on, especially by trying to foster critical thinking, it can benefit them throughout life. Critical thinking is a needed skill in everyone's life as it allows us to foster a deeper understanding and question structures that may be oppressive. By teaching your children critical thinking, you can allow them to be creative and question things on their own, fostering a deeper understanding of the world.

This section kind of made me question myself, and I realized that I don't really take part in any meaningful actions that contribute to decolonization. I think a lot of the time, taking the first step in trying to make a change can be intimidating because a lot of people don't know where to start, myself included. So I really appreciate the authors listing the ways that can support Indigenous people and decolonization

I feel like the authors capture the idea of this perfectly. In colonialism, we see that there is a need for everything to be categorized and defined. For example, in Western culture, a lot of clothes are put into categories of "masculine" and "feminine." This reminded me of the idea of Western epistemology vs Indigenous epistemology that we learned about in class. During week 3, we learned that often times Western knowledge is fixed, whereas Indigenous epistemology is alive and always changing (Bos 2025). I think categorizing things is deeply rooted in colonialism, and it adds to the dichotomous view of gender and sexuality that we see in everyday life through norms, societal views, etc.

This is such an important topic to touch upon because sometimes you have to stop and think about what truly makes you an "ally". There's often a lot of discourse surrounding what it means to be an ally. Are you an ally to a cause simply because you say you are, or does being an ally mean that you take opportunities to involve yourself in those communities and try to make a difference? Maybe the real answer is not so black and white.

As I read the highlighted passage, I thought about how nice it was to see Sarah's mom going to queer events with and without Sarah being there. To me, this feels like genuine allyship. This passage reminded me of enemy feminism and how Sophie Lewis talks about how oftentimes people say they are feminists, but their actions don't always match what they say they stand for (Lewis 2025). Normally, when people say they are allies, they do not do much to showcase this other than verbal support, so it is nice to see Sarah's mom taking part in actions that are meaningful towards the queer community.

In this section, the authors talk about the term "Two-Spirit" and its importance to Indigenous people (Hunt and Holmes 2015, 160). I grew up my whole life not ever hearing this term, which really goes to show how much schools fail to educate their students on the culture of Indigenous people. I later learned this term when I entered university and as someone who is a queer person of colour, I immediately thought the concept of Two-Spirit was something that more people should learn about. In today's society, we have so many gender norms of what people should look like, act like, etc. So it's so cool to see a culture that doesn't follow the norms of colonialism.

In this section, the authors discuss the importance of friendship and how it can foster allyship (Hunt and Holmes 2015, 161). I agree with this, as it is so easy to make friends by sharing similar interests and things you are passionate about. This is how you make some of the best friends, because when you find people who want to advocate for the same things as you, it can be a huge motivator to continue pro-social behaviours, knowing someone is there to back you up. As the authors mentioned, friends are also a good way to hold each other accountable. For example, perhaps you see a friend who is subconsciously reinforcing something like heteropatriarchal or heteronormative views. In this scenario, you can call them out on their behaviour and show them the error of their ways, holding them accountable. Sometimes we don't even realize that we are upholding certain norms, so it's always good to have a friend or someone you're close to who will point it out.

I feel like not a lot of people talk about how much of a powerful medium writing is. Not only is it a place to write down your thoughts, feelings, etc. But it is also a powerful medium to inform others so they can challenge their views and reflect on their values and norms.

I think here the author defines a perfect definition of what the word "queer" means and a brief history around it. The term "queer" is such an important term to the 2SLGBTQIA+ community for different reasons. For some people, it can be less restrictive compared to labels, allowing for a less categorical approach to gender and sexual identity.

I think a lot of the time, things like this are overlooked. For me, when I read this, I thought about how the world is so white centered and how the aftereffects of colonialism basically shapes everything in Canada, like the gender and sexual norms the author was talking about. For example, gender norms are enforced in the way people expect you to dress, act and behave. Sometimes this even carries over to queer relationships like when you hear someone say, "Okay, but who's the man in the relationship?" to non-men couples. It's even further reinforced by media like shows and movies that continue to showcase colonial gender and sexuality ideals.

Right off the bat, I wanted to say how meaningful all of these involvements are. I feel like a lot of the time, there isn't a lot of awareness for topics such as colonial violence, sex worker rights, etc. In particular, I feel like sex work is something that is often stigmatized. People who do sex work as a living experience a ton of violence, discrimination, and there can even be unfair power dynamics in play that hinder them. There are definitely better laws that need to be put in place to protect them to create a safe and comfortable environment.

This is a broad overview of the main idea of the article. This annotation is explored more in the written reflection.

building onto the major idea

connects to main idea

Although this article clearly mentions that it's both men and women experiencing inequalities in their own ways, male-dominated fields tend to make women feel like they aren't capable enough to fit in the role of the job. For example, surgeons -- a high-paid and difficult job, is used. However, if it were a job such as a server, with an equal amount of men and women working in this field, the results could be different. With "research [showing] that belonging is linked to an increase in job performance, a 50 percent drop in turnover risk, and 75 percent reduction in sick days, resulting in annual savings of more than $52M for a 10,000 person company" (Hutto, n.d.), the title's 'male-dominated' and 'female-dominated' should be unlearned.

connects to the main idea

Yeah you might end up being capable of doing it, but what if you're genuinely risking your life?

Could this connect to the concept of men being perceived 'stronger' and more 'powerful' than women, who are seen as more sensitive and shy?

Why has the 'one size fits all' approach become so normalized?

connects to main idea

In other words, we need to learn to unlearn certain societal norms that have been put in place. Life changes, people change, ideas change. What may have been considered normal back in the last century may be seen as abnormal now. With the help of standing up and raising awareness, whether that's through online petitions, reposting videos related to the issue, or protesting in person, we need to collectively work together for gender equality to be seen within workforces.

on mobile

javascript:(function(){window.hypothesisConfig=function(){return{showHighlights:true,appType:'bookmarklet'};};var d=document,s=d.createElement('script');s.setAttribute('src','https://hypothes.is/embed.js');d.body.appendChild(s)})();

sunset via

this, suggested by the team should work.

Via proxy deprecated by Feb 2026

The Via proxy service can be run on ones own domain as it is open source.

The hypothes.is Via proxy is being closed by Feb 2026. So the option I found to annotate on mobile using 'Share' to Via won't work anymore.

and we can annotate the presentation

e cannot

Hartman's dilemma reminds me of Audre Lorde's famous quote published in This Bridge Called my Back about not using the master's tools to dismantle the master's house (Lorde, 2022). The author wants to help share marginalized stories but is afraid of committing further violence. In my opinion, this "further violence" is likely continuing to keep some voices/perspectives hidden or misinterpreting them to suit their own ideals (which Western epistemologies and archives have done).

This passage speaks directly to what we studied under theories of the flesh. When Hartman writes about the “imperative to respect black noise—the shrieks, the moans, the non-sense, and the opacity,” she invokes the idea that the flesh carries forms of knowledge that cannot be translated into the language of the archive or the law (Moraga and Anzaldúa, 2022). Hartman’s insistence on “noise” directly challenges the logic of capitalism as the author states. In class, we discussed Silvia Federici’s work on capital accumulation where, broadly, capitalism emerges through the transformation of people into laboring bodies, units of value, and property (Federici, 2018). However, respecting the “noise” wouldn't allow for that. The shrieks, moans, and non-sense that Hartman describes cannot be translated into value or productivity. Moreover, respecting the pain experienced and humanizing the affected would make it more difficult to justify that exploitation.

Hartman describes her method as an effort to “topple the hierarchy of discourse” a hierarchy built by colonial, patriarchal, and Western epistemologies. This method "...which weaves past, present, and future..." is tied to the Indigenous epistemologies we learned about in class, regarding conceptions of time as non-linear and relational, something lived with, not simply looked back upon. By "...narrating the time of slavery as our present" Hartman engages in a decolonial act, another concept we learned about in class, with this nonlinear and interconnected approach with time.

The archive of slavery exemplifies epistemic and colonial power as we talked about in class in that it is both produced by and exercises control over people and knowledge. As Hartman emphasizes, the archive does not preserve the full humanity of the enslaved. Instead, it records only what the colonial and slave-owning systems deem legible or valuable. The “founding violence” (as Hartman says) of slavery structures knowledge itself, determining which statements are authorized, and which bodies and which lives are treated as dispensable. In this way, the archive produces authority, and reproduces social hierarchies under the guise of neutrality or documentation. The life of Venus cannot be reconstructed through these records alone, because the archive records only the statements that justify her suffering and death. Feminist methods, such as Hartman’s work against this grain, attempting to recover knowledge and to expose the epistemic violence embedded in the archives (Stoler, 2009).

Hartman is again speaking from the margins, trying to restore Venus’s lost voice as they write "...if I could have detailed the small memories banished from the ledger..." Hartman highlights that the ledger (Western epistemology) only preserves names, indictments, and death, not feelings, relationships, or other lived experiences that align with theories of the flesh (Moraga and Anzaldúa, 2022). Through imagining Venus comforting her friend, Hartman values the grief, intimacy, and care denied by the slave ship and official records.

Hartman’s attention to sensory and bodily effects of enslaved life, such as cries, groans, and melodies, is a type of knowledge derived from embodied experience. Theories of the flesh, as described in This Bridge Called My Back argue that lived bodily experience is a vaild form of knowledge (Moraga and Anzaldúa, 2022). Hartman emphasises the physical, emotional, and other bodily sourced information as a way of knowledge and insight that is otherwise absent from the official archives.

In this passage, Hartman highlights the “daily record of abuses” that documents slavery but simultaneously erases the individuality and humanity of these women. Hartman’s struggle to “liberate them from the obscene descriptions” connects directly to the concept of speaking from the margins, as discussed in class with Ann Laura Stoler's book (Stoler, 2009). By focusing on what is missing or suppressed in these records (the margins), Hartman works against the archival grain, and demonstrates how feminist research methods seek to recover histories that exist at the margins.

I interpret this passage as the author trying to respect the humanity and dignity of those who not only could not speak for themselves but also the ones were spoken for (such as the Venus figure she describes and their dehumanization), while also recognizing their own standpoint. Standpoint theory as described by Harding states that everyone's knowledge is a result of their standpoint (the intersection of the many social positions like race or gender someone may have) (Harding, 1987). "What cannot be known" can mean the limits of the author's standpoint.

This passage illustrates exactly what Ann Laura Stoler calls “working against the archival grain” (Stoler, 2009). The historical records are produced by oppressors and reflect the logic, priorities, and worldview of slave owners and colonialists. They erase the individuality and humanity of the enslaved. Feminist methods, as Hartman tries to follow, aim to recover what these archives conceal despite the challenges therefore "working against the archival grain." Hartman raises the critical question of "How do we center the humanity of those whose existence was reduced to subhuman (e.g. property)?"

Here, Hartman reminds me of Sara Ahmed's description of a "feminist killjoy," which is a feminist that is not complicit or silent to injustices even when others may prioritize agreeability (Ahmed, 2017). Like a killjoy, she refuses to allow readers to enjoy a sanitized version of history. By highlighting structural violence, she “kills the joy” of comfortable narratives and exposes what the archive hides.

This passage draws on speculative fiction as a tool for engaging with history, showing how Dana’s time travel in Kindred parallels our engagement with the archive of slavery. Dana's inability to "rescue" her ancestors highlights the interconnection of violence from the past and present as well as limitations when dealing with trauma from the past. Hartman makes a connection between this and Venus, arguing that we too must "bear what cannot be borne" that is, acknowledge the structural constraints and suffering of the past without erasing or simplifying them. This resonates with decolonial approaches from class, which emphasize bearing witness to historical violence and its ongoing effects rather than imposing closure or resolution.

This passage emphasizes the continuing effects of slavery and dispossession in the present, what Hartman calls the “afterlife of property.” They frame history not as something behind us, but as ongoing: the violence that produced Venus’s life and death continues to shape Black life today. However, I wonder what Hartman means by "a past that has yet to be done." I assume she is talking about time as cyclical, but how can we then "do" the past?

I found this passage very striking as it refers to the feeling of wanting closure or to find comfort in something tragic, even though the situation is very bleak. Throughout learning about colonial and world history, there is plenty of upsetting, disturbing, and violent subject matter. Wanting to make yourself feel better by changing the narrative or minimizing the event is something I and many others have experienced, just like how Hartman wanted the two girls to be friends despite their horrible circumstances and demise.

Hartman questions the ethics and methods of historical writing. To me, it sounds like they are critiquing Western epistemologies and advocating for something different when they wrote "...how might it be possible to generate a different set of descriptions from this archive?" Traditional Western epistemology as we talked about in class prioritizes “objective” records like legal documents, ship logs, and other sources as legitimate knowledge. These often record only the perspectives of the colonialists and reproduce exploitative depictions of enslaved women. Hartman challenges this by asking whether it is possible to write about Venus ethically, centering her humanity rather than the archive’s focus on violence. Hartman, suggesting that ethical historical writing must go beyond documents to center the humanity, experiences, and agency of those rendered voiceless in official accounts, may benefit from Indigenous epistemologies which validate those lived experiences.

commitment to traditional values and ideas with opposition to change or innovation.

What if the frustration (or even shame) of school challenges could be a thing of the past?

Before you get into your about section, I would list out the 3 options you want to steer people towards and specifically call out (one 1:1 offer, your best-selling low-ticket resource, yoru membership, and your checklist freebie). Then have a button underneath to 'Check out all my offers' (linking to your work with me page).

Move this right underneath the 'How my coaching packages...' section.

I would move this logo bar up right underneath the header/above the fold section. As for the podcast features, could you move them into the about page?

source

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I found it be refreshingly pedantic and accurate to refer to it as just a "recommendation" rather than a "requirement" or something... though I'm not actually sure why we call such standard things in the web APIs merely a "recommendation". Can't we just call it a standard at this point?

Stating that dorsal means toward the spine side may help to differentiate "dorsal" from "posterior."

"Away from the midline" might be clearer here?

I like to point out that palms forward allows radius and ulna to be seen distinctly instead of being partially superimposed as a way to help them remember which direction the palms should be facing.

• Highly Processed Foods & Inflammation: Up to one-third of all cancers are linked to poor nutrition, particularly consuming highly processed foods [00:00:00]. These products (full of artificial additives, hardened fats, processed meats, and sweet snacks) act as "fuel" that drives chronic inflammation, creating an environment conducive to cancer development and growth [00:00:24].

• Sugary Drinks (Napoje Słodzone):

  • Regular consumption of sugary drinks (not natural juices) is linked to a higher risk of several cancers [00:00:57].
  • Studies show an increased risk of breast cancer (14%) in women and prostate cancer (18%) in men [00:01:16].
  • Daily consumption of just one serving of soda (like cola) was linked to an up to 55% higher risk of pancreatic cancer [00:01:35].
  • They are also associated with an average 15% greater risk of colon cancer [00:02:15]. Lab studies suggest the sugar in these drinks can increase the mobility and metastatic ability of cancer cells [00:02:42].

• Drinks with Artificial Sweeteners (Słodziki):

  • While many studies suggest no link to cancer, some research indicates a higher risk of leukemia (15% increase per serving), particularly from drinks containing aspartame [00:04:12].
  • The concern is that aspartame can break down into methanol and then formaldehyde (a recognized carcinogen) [00:04:32], which can damage the genetic material in bone marrow stem cells [00:04:49].

• Fried Foods (Żywność Smażona):

  • Regular consumption of fried foods (especially deep-fried meats and plant products) is linked to a 52% higher risk of stomach cancer [00:05:55] and may increase the risk of prostate cancer by 35% [00:06:04]. This is due to the formation of toxic compounds during frying [00:06:13].

• High-Salt Diet (Dieta Bogata w Sól):

  • A long-term diet high in salt, especially from low-quality processed foods, is strongly associated with an increased risk of stomach cancer (up to 68% higher) [00:07:17].
  • Excess salt directly damages the stomach lining, makes it susceptible to toxins [00:07:42], and promotes the growth of the Helicobacter pylori bacteria, which causes chronic inflammation and ulcers that can lead to cancer [00:08:15].

• BPA and BPS (Canned Foods):

  • Bisfenol A (BPA), used in metal can linings [00:09:40], can migrate into food and is a potential pro-carcinogenic agent because it disrupts hormonal balance [00:09:56]. High exposure may promote hormone-dependent cancers like breast and prostate cancer [00:10:06].
  • Bisfenol S (BPS) is a similar chemical still permitted in food-contact materials and is suggested to be more toxic and equally linked to breast cancer [00:11:24]. The speaker recommends avoiding most canned foods unless marked "BPA Free" [00:11:47].

• Phthalates (Ftalany):

  • These chemicals, which increase plastic flexibility [00:12:19], can migrate from plastic packaging into highly processed foods, especially fast food (e.g., in plastic trays) [00:12:37].
  • Phthalates are suggested to promote cancers of the thyroid, breast, and prostate [00:13:19].

• Alcohol:

  • Alcohol consumption is a confirmed risk factor for cancers of the oral cavity, throat, esophagus, and larynx [00:13:44].
  • Regular consumption also increases the risk of colon cancer by an average of 56% [00:13:53]. Even small, frequent amounts are considered harmful [00:14:02].

While I understand that it's a logical extension to represent the tree as a vector/matrix format as you've done here to obtain summary statistics, it should on principle be feasilble to do something similar using graph neural networks, which can learn tree/graph representations directly from the original phylogeny/graph. Have you considered taking such an approach?

It looks like there's at least one putative UPRE upstream of FMP52 (425 bases upstream of the ATG, 5'-TACGTGT-3')! I'm curious if you tried looking at endogenous Fmp52-GFP with t-2-hex or other ER stress-inducing treatment, as it may get upregulated and be more visible than in unstressed conditions? This would be consistent with your pFMP52-luciferase results.

In Decolonizing Feminism: Challenging Connections between Settler Colonialism and Hetero-patriarchy, the authors argue that colonialism, and specifically settler-colonialism where the settlers come to stay, happen through both systemic public acts of violence (e.g., control, labour exploitation), but also more intimate ways (assimilation, cultural erasure, sexual identity policing, linguistic oppression). I think this idea connects to the "everyday-ness of decolonization" mentioned earlier, and how resistance must be grounded in local activities and connections with queer Indigenous people, as much on a larger scale. Otherwise, we risk Indigenous erasure and the enforcement of colonial ideas of "progress."

This is an interesting point because it reminds me of the romanticization of social movements. This romanticization often views the process of liberatory struggles through an idealized and oversimplified lens, and overlooks internal conflicts and flaws. This means that progress is only measured through public-facing spectacles/displays of justice, rather than something that happens simultaneously behind closed doors and isn't easily televisual. In regards to this text, a romanticization of these struggles can often dismiss intellectual labour and progress that takes place in unconventional spaces such as within families, homes, and community spaces, or even within ourselves. The authors are trying to make the point that struggling for liberation and decolonialization isn't a one-size-fits-all-approach.

This is a central theme: the reframing of "queer" beyond its initial identity label. Most of my peers think that queer describes a particular sexual orientation or identity. But to be queer is defined through practices and actions that are inherently anti-oppressive.

This reminds me of a quote from bell hooks, where hook's describe queer as "the self that is at odds with everything around it and has to invent and create and find a place to speak and to thrive and to live." In this way, queerness is not a sexual identity but a method of moving through the world, a method that involves self-creation in opposition to hetero-normativity and patriarchy.

This is the central theme of this piece of work; of how decolonization can take place within intimate spaces such as our communities, families, within friendships, and our everyday work. As emphasized by the authors, decolonial practices must first take place within our private, personal experiences, before they can take shape through our laws, policies, and systems. The latter can be a slow process, but the former can start today.

It is interesting to me that even at a specialized school for top performing students, that the teachers were not more educated that these might be common traits for the students they teach.

I can think of many examples of students like these in my own schooling experience and it makes me wonder if they were really an unruly, disrespectful kid or if something else was going on. It is hard as a teacher when you have so many students to dive in deep like this but it SO important for the student if you do.

I am curious to know what the science is behind people being able to communicate through music before speech. I think there must be something very primal about it.

I think this is the case for a lot of people. It is a long process to get any intervention or type of assistance when kids are this young. I wonder why this is.

I found this interesting because how can you have it both ways by separating the gifted kids but including average to below-average students? Who do you pick?

I think this is great point that teachers should recognize more. One of my biggest questions when it comes to teaching students like this who need this differentiation is how do we deal with the social issue that might arise? If other students see that Johnny is getting different work and make a fuss about it, how do we redirect it without making it a big deal?

This disparity shocked me at first but then when I reflected on my own experiences, I couldn't recall a time where I saw a gifted or talented student receive services.

This unfortunately does not surprise me. In all of my studies thus far, it is evident that socioeconomic class plays a HUGE role in what services people receive.

I don't know how to feel about this. I guess there is not one standard so each child can get the services they need. But what happens to a child who moves and who no longer qualifies for services because they don't meet the new score requirement?

I think it is interesting that it took this long to coin the term gifted. Along with other differences, it still fascinates me all of this research and academic stride forward is so recent.

Is there a way that the conductor could’ve had a little more grace with David?

I’ve seen some choirs do this but maybe having a gifted student conduct a piece and rehearse the piece with their peers. This can really challenge their creativity by controlling how the ensemble plays. This will also keep them engaged longer with them thinking about conducting.

Maybe planning more for the gifted students might help. For example, if their task is to learn the Eb concert scale maybe have the gifted students play it in more rhythms or starting on different scale degrees. To try and get them to be occupied for longer.

I think this is a good idea not just for gifted students. Especially when teaching general music and the students sometimes say the most random things.

This surprised me. The fact that so much time is wasted for gifted students.

What is the best way to challenge students while not leaving other students behind?

I haven’t actually thought about the range of being gifted and how some students will show it differently.

• The author describes a comprehensive setup to block virtually all online advertising across devices and services.
• They focus on network-level filtering instead of per-device ad blockers, so that phones, TVs, and other clients benefit automatically.
• The core of the solution is running a self-hosted DNS-based blocker (like Pi-hole or AdGuard Home) to sinkhole common ad and tracker domains.
• Additional blocklists are layered on top to handle more aggressive tracking and region-specific ad domains, trading a bit of breakage for increased privacy.
• For services that hardcode ad endpoints or use techniques that bypass DNS blocking, the author uses more advanced tools such as proxying or firewall rules.
• Some apps and sites break when ads are blocked; in those cases, the author selectively whitelists domains or uses per-device exceptions rather than relaxing global rules.
• On mobile, encrypted DNS and VPN-like tunneling are configured so that all traffic still flows through the home-level blocking setup even on the go.
• The author argues that this configuration significantly improves page load times, reduces bandwidth usage, and makes devices feel faster and less cluttered.
• They acknowledge an ethical gray area with ad blocking but conclude that user safety, privacy, and mental comfort outweigh the downsides of depriving low-quality ad networks of revenue.
• The piece emphasizes that the goal is not absolute perfection but a sustainable setup that requires minimal maintenance once deployed.

Hacker News Discussion

• Commenters discuss additional tools like SponsorBlock for skipping in-video sponsorships on platforms such as YouTube, highlighting that traditional ad blockers do not remove creator-embedded promos.
• Several users point out that DNS-level blocking does not stop ads injected directly by streaming services, noting that such platforms often use certificate pinning or app-level tricks that make proxying and MITM approaches difficult or impossible.
• A highly upvoted comment recommends using a user-agent switcher to bypass sites that block non-Chrome browsers, with examples where services claim to be incompatible with Firefox but run better once the browser “pretends” to be Chrome.
• Participants criticize websites that enforce brittle user-agent checks instead of feature detection, arguing that this needlessly breaks otherwise compatible browsers and punishes privacy-conscious users.
• Some users express skepticism about privacy-focused browsers that are built on or dependent on codebases controlled by ad-driven companies, calling out an inherent tension between privacy promises and ad-based business models.

unclear where on the website this list can be found

This breaks my heart

Though you already report high accuracy with your method, do you think the result would improve or change if you also included the CH stretch region (~2700-2900 cm-1 or so)?

Thanks for sharing this work! Did you notice any alteration in Raman signal of cells due to laser exposure? How did you select the acquisition parameters?

Because these mutants were generated via random mutagenesis, it's difficult to interpret phenotypic variability of each mutant's lipid metabolism in the context of their varied genetic backgrounds. It may be worth showing lipid-productivity data in mutants that have similar levels of starch content to wild-type to show a clear baseline for interpretation.

It would help to show more of the screening process that led to the five chosen lines. For example, reporting how many colonies were screened, the growth conditions used during screening (e.g., BBM vs BBM−N, duration, light), how starch phenotypes were called from iodine staining, and how many candidates fell into each class (low starch / high starch). A semi-quantitative summary of iodine phenotypes would let readers estimate the probability of generating starch phenotypes via this mutagenesis protocol (an informative result on its own).

Here biomass productivity is calculated as u (growth rate) x B_end, but the more common way to calculate BP is B_end - B_start / time. u x B could overstate productivity and/or make comparisons phase-sensitive. Sometimes those early timepoints may be harder to collect/quantify, but is it known that the cultures are in exponential phase for the duration of the experiment?

How many mutants did you have to screen to find five starch high/low phenotypes? Did you also consider mutants that had spatially abnormal starch distribution?

eLife Assessment

This important study presents novel data on temporal variation in sperm whale communication, contributing to a richer understanding of the social transmission of vocal styles across neighbouring clans. The evidence is solid, although some terminology limits comparisons to other taxa. This research will be of interest to bioacoustics and cetacean communication specialists, particularly those working on social learning and culture.

Reviewer #2 (Public review):

Summary:

The current article adapts standard rhythmic measures to describe the temporal organisation of whale song units.

Strengths:

The detailed description of the internal temporal structure of whale songs is something that has thus far been lacking.

Weaknesses:

Conceptual and terminological bases of the paper are problematical and hamper comparison with other taxa, including humans. According to signal theory, codas are indexical rather than symbolic. They signal an individual's group identity. Borrowing from humans and linguistics, coda inter-group variation represents a case of accents -- phonologically different varieties of the same call -- not dialects, confirming they are an index. Moreover, symbolism is not a feature detectable or confirmed through rhythmic analyses or temporal characterisation. This raises serious doubt whether alleged "dialects," "symbolism" and similarity between whales and humans is factual. The comparative scope and relevance of this paper for the broader field is limited and evolutionary claims are potentially misleading and perilous.

Author response:

The following is the authors’ response to the previous reviews

Public Reviews:

Reviewer #2 (Public review):

Summary:

The current article presents a new type of analytical approach to the sequential organisation of whale song units.

Strengths:

The detailed description of the internal temporal structure of whale songs is something that has been thus far lacking.

Weaknesses:

The conceptual and terminological bases of the paper are problematical and hamper comparison with other taxa, including humans. According to signal theory, codas are indexical rather than symbolic. They signal an individual's group identity. Borrowing from humans and linguistics, coda inter-group variation represents a case of accents - phonologically different varieties of the same call - not dialects, confirming they are an index. This raises serious doubt about whether alleged "symbolism" and similarity between whale and human vocal behaviour is factual.

We respect that the reviewer does not agree with describing codas as symbolic markers of cultural identity in sperm whales, but ultimately we find the quantitative evidence presented in Hersh et al. (2022) compelling, and stand by the framing of our manuscript, which builds on this foundation.

The same applies to the difference between ICIs (inter-click interval) and IOIs (inter-onset interval). If the two are equivalent, variation in click duration needs to be shown so small that can be considered negligible. This raises serious doubt about whether the alleged variation in whale codas is indeed rhythmic in nature and prevents future efforts for comparison with the vocal capacities of other species. The scope and relevance of this paper for the broader field is limited.

We believe there has been a miscommunication. Coda inter-click intervals are calculated as the time between the onsets of sequential clicks within a coda. This is identical to definitions of inter-onset intervals in many publications, including:

• Burchardt and Knörnschild (2020): “the duration between the beginning of one element and the next”

• Friberg and Battel (2002): “the time interval between the onset of the tone and the onset of the immediately following tone”

• De Gregorio et al. (2021): “the time between the onset of a note and the next one”

In response to a comment from this reviewer in the first round of revisions, we made the point that we do not believe rhythm analyses need be restricted to inter-onset intervals alone. Regardless of that stance, we did analyze inter-onset intervals in this manuscript and accordingly are capturing aspects of rhythm in our analyses. We have removed a poorly worded sentence in our introduction and apologize for any confusion it caused. We have also made this explicit in lines 30–35: “This classification is based on the total number of clicks and their rhythm and tempo extrapolated from the time interval between the onsets of consecutive clicks: the inter-click interval (ICI) [15, 16] (Fig. 1A). This measure is equivalent to the inter-onset intervals (IOIs) often used in rhythm analyses [17, 18, 19] but for the sake of compatibility with studies on sperm whale acoustics, we use ICI terminology throughout this paper.”

In our analyses, inter-click intervals and inter-onset intervals are equivalent measures.

Recommendations for the authors:

Reviewer #2 (Recommendations for the authors):

My concerns regarding interdisciplinary terminology and methods remain unaddressed. The study's inaccurate terminology hinders reliable comparison with other taxa, including humans. Being "symbolic" bears no weight on the new method that the authors present, thus, the unwillingness for compatibility is limiting and perplexing. The authors state that codas have been previously described as being symbolic, but just because poor terminology has been used before doesn't justify perpetuating it, especially when it confounds and conflicts with broader comparative efforts.

We agree that being symbolic bears no weight on the new method we present, but we believe it does bear weight on our interpretation of what our method reveals about patterns in sperm whale communication. For that reason, we have opted to maintain the current framing of our manuscript.

The same applies to the difference between ICIs and IOIs. The authors resist amending terminology, even though they state the two represent the same measure. If so, want prevents the correct use of IOIs?

We have opted to use ICI throughout the paper because it is standard terminology in sperm whale acoustics, but we have now made the ICI/IOI equivalence explicitly clear in the introduction.

References:

Burchardt LS, Knörnschild M. 2020. Comparison of methods for rhythm analysis of complex animals’ acoustic signals. PLoS Computational Biology 16. doi:10.1371/journal.pcbi.1007755

De Gregorio C, Valente D, Raimondi T, Torti V, Miaretsoa L, Friard O, Giacoma C, Ravignani A, Gamba M. 2021. Categorical rhythms in a singing primate. Current Biology 31:R1379–R1380. doi:10.1016/j.cub.2021.09.032

Friberg A, Battel GU. 2002. Structural communication In: Parncutt R, McPherson G, editors. The Science & Psychology of Music Performance: Creative Strategies for Teaching and Learning. Oxford University Press. doi:10.1093/acprof:oso/9780195138108.001.0001

Hersh TA, Gero S, Rendell L, Cantor M, Weilgart L, Amano M, Dawson SM, Slooten E, Johnson CM, Kerr I, Payne R, Rogan A, Andrews O, Ferguson EL, Hom-Weaver CA, Norris TF, Barkley YM, Merkens KP, Oleson EM, Doniol-Valcroze T, Pilkington J, Gordon J, Fernandes M, Guerra M, Hickmott L, Whitehead H. 2022. Evidence from sperm whale clans of symbolic marking in non-human cultures. Proceedings of the National Academy of Sciences 119:e2201692119. doi:10.1073/pnas.2201692119

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

Learn more at Review Commons

Reply to the reviewers

Reviewer #1

1. First, the authors have not convincingly shown that skin cells, or more specifically skin ECs, are a major source of circulating G-CSF in the psoriasis model as stated in the title and abstract. The data in Figure 4 show selective upregulation of Csf3 gene in skin ECs and their ability to secrete G-CSF upon IMQ treatment in vitro. However, the provided data do not address to what degree the skin EC-derived G-CSF contributes to the elevated level of circulating G-CSF. Additional experiments to selectively deplete G-CSF in skin ECs, or at least in skin cells of the affected site, are warranted to support the authors' claim. Does intradermal injection of G-CSF neutralizing antibody into the psoriatic skin reduce circulating levels of G-CSF?

Author's response:

Thank you for reviewer's comment. We agree with the Reviewer#1 that it is important to directly block G-CSF to the skin via intradermal injection and measure the G-CSF level in the serum afterwards. Therefore, we will perform intradermal injection of IgG-isotype or anti-G-CSF antibody into the IMQ-induced psoriatic mice.

Another concern is insufficient demonstration of G-CSF-mediated emergency granulopoiesis in the psoriasis model. All data in Figure 5 were obtained from experiments with only n=3, and adding more replicates, in particular to those in Figure 5B, which show quite some variation in MPP numbers, is recommended. The relatively small reduction of BM granulocyte numbers (Figure 5C) compared to greater depletion of circulating granulocytes (Figure S5A) raises the possibility that it is the mobilization effect rather than granulopoiesis-stimulating effect that skin-derived G-CSF exerts to promote supply of circulating neutrophils that eventually infiltrate into the affected skin. This could also explain the negligible effect of IL-1blockade (Figure S4), which selectively shut off myelopoiesis-stimulating effect of IL-1 (Pietras et al. Nat Cell Biol 2016, PMID: 27111842). Are the HSPCs in the psoriasis model more cycling? Do they show myeloid-skewed differentiation when cultured ex vivo or upon transplantation?

Author's response: Thank you for these critical comments. We agree to do the following experiments to address them:

1) HSPCs quantification in Figure 5 especially the MPPs will be added with more replicates.

2) We will assess cycling status of HSPCs by flow cytometric analysis of Ki67and Propidium Iodide to characterize G0, G1 and G2/M cell cycle phase.

3) To test myeloid-skewed differentiation, Lin- c-Kit+ Sca-1+ cells containing HSPCs will be isolated from bone marrow of Vas/IMQ-treated mice and transplanted into lethally irradiated syngeneic mice.

The authors' claim that skin-derived G-CSF "induces" neutrophil infiltration warrants further clarification. Alternative explanation is that the upregulated neutrophil-attracting chemokines (Figure S1D) could induce infiltration, whereas G-CSF increase the number of neutrophils to circulate in the vessels near the psoriatic skin. This notion seems supported elsewhere (Moos et al. J Invest Dermatol. 2019, PMID: 30684554). Can the infiltration be inhibited by systemically injecting neutralizing antibody of their receptor, CXCR2?

Author's response: The manuscript focuses on the skin-derived G-CSF function as a long-distance signal for emergency granulopoiesis in the bone marrow upon psoriasis, not the chemoattractant property of it. The sentence of interest is "We found that upon psoriasis induction, skin-resident endothelial cells are activated to produce G-CSF which activates emergency granulopoiesis in bone marrow and induces cutaneous infiltration and accumulation of neutrophil that are functionally inflammatory." in line 28-30. In agreement with point #2 from Reviewer#2, the fact that neutrophil recruitment factors (CXCL1, CXCL2, and CXCL5) were upregulated in psoriatic skin (Figure S1D), suggesting a CXCL-mediated neutrophil recruitment. The sentence of concern need to be changed to "We found that upon psoriasis induction, skin-resident endothelial cells are activated to produce G-CSF which activates emergency granulopoiesis in bone marrow, leading to cutaneous accumulation of neutrophil that are functionally inflammatory.". This revised sentence has omitted the proposal that G-CSF directly dictates neutrophils mobilization to the skin, which is not the key message of the study. Therefore, we found that the CXCR2 (CXCLs receptor) blockade experiment may be of the benefit of future studies.

It remains unclear how skin-derived G-CSF accumulates pathogenic neutrophils. The authors state "pathogenic granulopoiesis," but are the circulating neutrophils in the psoriatic mice already "pathogenic" or do they acquire pathogenic phenotype after cutaneous infiltration? Additional RNA-seq to compare circulating and infiltrated neutrophils would answer this question.

Author's response: We appreciate this valuable comment. We will perform RNA-seq with the peripheral blood-circulating neutrophils (CD45+ CD11b+ Ly6G+ Ly6Cmid) versus skin-infiltrating neutrophils from both Vas/IMQ mice.

In addition, how the accumulated pathogenic neutrophils exacerbate the psoriatic changes remains obscure. Although the authors have attempted to correlate Il17a gene expression in infiltrated neutrophils with psoriatic skin changes, the data do not address to what degree it contributes to cutaneous IL-17A protein levels. The data that cutaneous neutrophil depletion leads to subtle decrease in skin IL-17A expression (Figure 2H) rather supports alternative possibilities. For instance, as indicated elsewhere, IL-17A cutaneous tone could be enhanced by neutrophil-mediated augmentation of Th17 or gamma/delta T cell function (Lambert et al. J Invest Dermatol. 2019, PMID: 30528823). Does neutrophil depletion or G-CSF neutralization alter cell numbers or function of cutaneous Th17 and gamma/delta T cells?

Author's response: Thank you for this insightful comment. To better understand the relative contribution of neutrophils to the cutaneous IL-17A tone in the psoriatic skin, we will perform flowcytometric analysis of Th17 and gamma/delta T cells which are widely known as the major source of IL-17 in psoriatic skin of IMQ-induced mice following injection of isotype-matched or anti-Ly6G antibody.

Finally, as the above conclusions rely solely on the IMQ-induced acute psoriasis model, it would be informative if they could be derived from another psoriasis model. IMQ is known to induce unintended systemic inflammation due to grooming-associated ingestion (Gangwar et al. J Invest Dermatol. 2022, PMID: 34953514), and "pathological crosstalk between skin and BM in psoriatic inflammation" could be strengthened by an intradermal injection model.

Author's response: We appreciate the reviewer for bringing this important point. Regarding the systemic inflammation upon psoriasis, the above-cited study reported increased IFN-B expression in the intestines of IMQ-ingested animal (Grine L et al. Sci Rep. 2016, PMID: 26818707 in Gangwar et al. J Invest Dermatol. 2022, PMID: 34953514). We examined several pro-inflammatory cytokines including IFN-b, IFN-g, and IL-6 and in contrast, found no systemic increase in all these cytokines, except for IFN-g downregulation (Explanation Figure 1), which suggests no evidence of grooming-associated ingestion.

We also examined the Csf3 expression across several distinctively located tissues which showed a selective upregulation in the skin (Figure 4C), suggesting a skin-restricted perturbation. In addition, one study showed that IMQ-ingestion didn't alter number of gut injury-associated CXCR3+ macrophages nor did it aggravate skin inflammation (Pinget et al. Cell Reports. 2022, PMID: 35977500). Together, these findings support that IMQ-induced psoriasis by topical cutaneous application used in our study elicit a local inflammation but not systemic inflammation.

The authors, however, realize that testing alternative psoriasis model such as intradermal injection of IL-23 (Chan et al. J Exp Med. 2006, PMID: 17074928) will strengthen the skin-local insults within the psoriasis model employed, and should be tested in the future.

Minor comments

Figure 1E shows multiple elongated Ly6G+ structures in d0-2 control and d0 IMQ skins that do not appear to be neutrophils.

Author's response: We appreciate the Reviewer#1 pointing this issue. As mentioned by the Reviewer#1, the elongated structures detected in the intravital microscopy are not neutrophils, but autofluorescence from the skin bulge regions (Wun et al. J Invest Dermatol. 2005, PMID: 15816847). We have eliminated these unspecific signals from the transformation and quantification (Figure 1F, S1G, and S1H). We will also add an explanatory sentence in Materials and Methods section "Of note, the fluorescent signal with elongated structures resembling hair bulge were autofluorescence and thus removed from further analysis." to be more precise about our methods.

In Figure 2C, the bottom GSEA seems to be showing type II IFN response, not type I IFN, according to the text.

Author's response: Thank you for the comment, we will correct this misspelling.

Author's response: We appreciate that Reviewer#1 bring up this point. We examined the kinetics of the bone marrow cellularity and GMPs across 4 days of psoriasis induction in mice. The bone marrow cell number was lowered along that span with lowermost count at 2 days. Consistent to the BM-cellularity, the GMP number was also lowered about one-third in the first 2 days of psoriasis. This kinetic is consistent with the previous report showing a rapid reduction of GMPs in the bone marrow within 2 days following systemic G-CSF administration driven emergency granulopoiesis (Hirai et al. Nat. Immunol. 2006, PMID: 16751774). From 2 days to 4 days, the GMP number rapidly increased to slightly above basal number (Explanation Figure 2). This timely coordinated expansion suggests a significant supply of GMPs from the differentiating upstream myeloid progenitors (Figure 3B).

When the psoriatic mice with elevated G-CSF is injected with anti-G-CSF or IgG-isotype antibody, the bone marrow cellularity and GMP numbers at 4 days were (Explanation Figure 3). Firstly, as psoriasis reduced bone marrow cellularity (Explanation Figure 2), the unchanged number after anti-G-CSF injection indicates that administration of 10µg/day for 4 days does not significantly affect mobilization of psoriatic bone marrow cells. Secondly, the similar GMP numbers at 4 days psoriasis is plausibly due to snapshot analysis when it has already in the numerical recovery period (Explanation Figure 2). Importantly, the notion that anti-G-CSF injection to psoriatic mice reduced granulocytes in the bone marrow, peripheral blood, and skin suggesting G-CSF as a key mediator in psoriatic driven emergency granulopoiesis on top of unlikely case of ineffective anti-G-CSF treatment.

Taken together, these data suggest a G-CSF mediated emergency granulopoiesis occurrence in the IMQ-induced psoriasis. We will put these data into a revised Figure.

In Figures 6B, in which cluster of human skin cells IL-17A expression would be enriched?

Author's response: Thank you for this important point. The IL-17A expression is found in the T-cell cluster (Explanation Figure 4). We also expected to see IL-17A contribution from other cell subset(s), in particular neutrophil. However, due to the fragile nature of neutrophils and thereby, technical difficulty to get their sequencing reads, this dataset (GSE173706) doesn't contain neutrophils, but rather monocytes, macrophages, and dendritic cells among the myeloid subset (Explanation Figure 5). With this, it leaves open the question on what potential contribution of IL-17A produced by neutrophils is in human psoriasis (Reich et al. Exp. Dermatol. 2015, PMID: 25828362).

Figure 1E shows multiple elongated Ly6G+ structures in d0-2 control and d0 IMQ skins that do not appear to be neutrophils.

Author's response: We appreciate the Reviewer#1 pointing this issue. As mentioned by the Reviewer#1, the elongated structures detected in the intravital microscopy are not neutrophils, but autofluorescence from the skin bulge regions (Wun et al. J Invest Dermatol. 2005, PMID: 15816847). We have eliminated these unspecific signals from the transformation and quantification (Figure 1F, S1G, and S1H). We will also add an explanatory sentence in Materials and Methods section "Of note, the fluorescent signal with elongated structures resembling hair bulge were autofluorescence and thus removed from further analysis." to be more precise about our methods.

In Figure 2C, the bottom GSEA seems to be showing type II IFN response, not type I IFN, according to the text.

Author's response: Thank you for the comment, we will correct this misspelling.

Reviewer#2

1. Interpretation of neutrophil transcriptomic changes (Figure 2)

The RNA-seq analysis reveals substantial downregulation of several canonical pro inflammatory pathways in neutrophils from psoriatic skin, including IL-6, IL-1, and type II interferon signaling. The authors should discuss the functional relevance of this unexpected transcriptional repression. For example, does this indicate a shift toward specialized effector functions rather than classical cytokine responsiveness? More importantly, the most striking transcriptional change is the upregulation of NADPH oxidase-related genes (e.g., Nox1, Nox3, Nox4, Enox2). This suggests an oxidative stress-driven pathogenic mechanism, potentially more relevant than IL-17A production. Yet this aspect is not explored in the manuscript. Assessing ROS levels or oxidative neutrophil effector functions in this model would considerably strengthen the mechanistic link. Conversely, although IL-17A is upregulated in neutrophils, neutrophil depletion reduces total Il17a expression in skin only partially. This indicates that neutrophils are unlikely to be the dominant IL-17A source in the lesion. The authors' focus on neutrophil-derived IL 17A therefore seems overstated. A more rigorous assessment-e.g., conditional deletion of Il17a specifically in neutrophils-would be required to establish its true contribution. Taken together, the data suggest that oxidative programs, rather than IL-17A production, may represent the principal pathogenic axis downstream of neutrophils, and this deserves deeper discussion.

Author's response: Thank you for raising this valuable views. We have agreed to address these critical points by the following approaches:

1) To address the changes in NADPH oxidase-related gene signature, we will measure ROS production in the neutrophils from skin and peripheral blood with DHR123.

2) Responding to the IL17A contribution by neutrophils, we will flow cytometrically assess the Th17 and gamma/delta T cell population in the skin of psoriatic mice treated with anti-Ly6G or isotype-matched antibody as was suggested by Reviewer#1.

3) We will discuss downregulation of the canonical pro inflammatory and IL-17 pathways in the psoriatic neutrophils in the discussion.

Human data reanalysis (Figure 6):

The re-analysis of bulk and single-cell RNA-seq datasets is valuable but incomplete. Several mechanistically relevant questions could be addressed with the available data:

2.1. GM-CSF (CSF2) is also strongly upregulated in psoriatic lesions (bulk RNA-seq). It would be informative to determine whether endothelial cells also express CSF2 in the scRNA-seq dataset, as this would suggest coordinated regulation of myeloid-supporting cytokines.

2.2. Myeloid cell subsets should be examined more closely. A comparison of human myeloid transcriptomes with the mouse neutrophil RNA-seq would clarify whether similar IL-17A-related or NADPH oxidase-related signatures occur in human disease. In particular, which cell types express IL17A in human lesions?

2.3. Chemokine production should be attributed to specific cell types. Bulk RNA-seq confirms strong induction of CXCL1, CXCL2, CXCL5, but the scRNA-seq dataset allows determining whether these chemokines originate from endothelial cells or other stromal/immune populations. This information is important for defining whether endothelial cells coordinate both neutrophil recruitment and granulopoiesis.

Addressing these points would make the human-mouse comparison substantially stronger.

Author's response: Thank you for pointing these important issues. By reanalyzing the dataset, we found several points regarding the comments, as follows:

2.1) CSF2 is expressed by T-cell cluster in the human skin dataset (Explanation Figure 4), in agreement with previous murine study (Hartwig et al. Cell Reports. 2018, PMID: 30590032). We will add this data in the revised manuscript.

2.2) In line with point#10 from Reviewer#1, the dataset clearly shows T-cell cluster as the main IL17A source (Explanation Figure 4 above). The dataset, however, doesn't contain phenotypic neutrophils (CEACAM (CD66b) and PGLYRP1) but monocytes, macrophages, and dendritic cells (Explanation Figure 5 above). This loss was probably due to a technical limitation given the difficulty in capturing sequencing reads from fragile neutrophils. Therefore, it is no longer possible to reanalyze IL-17 expression in the absence of neutrophils in the datapool.

2.3) Reanalysis of CXCLs in the human scRNAseq dataset (GSE173706) clarified their secretion dynamics and cellular sources under normal and psoriatic condition. In normal skin, all examined cell subsets show only low CXCLs expression. In contrast, psoriatic skin exhibits significant CXCLs upregulation with distinct cell subsets clearly showing dramatic upregulation, potentially being the major CXCLs source. CXCL1 is markedly upregulated in fibroblasts, myeloid cells, and melanocyte and nerve cells. CXCL2 is strikingly upregulated to myeloid cells, while CXCL5 is hugely increased in fibroblasts, myeloid cells, and mast cells (Explanation Figure 7). Taken together, these results suggest that CXCLs upregulation in the psoriatic skin is coordinatively executed by both stromal and immune compartments. Of note, the endothelial cells show minimal changes in CXCLs expression, even downregulate CXCL2 in psoriasis, indicating that they are unlikely to be the major contributor to CXCL-mediated neutrophil recruitment.

**Referees cross-commenting**

I agree with Reviewer 1 that the contribution of EC-derived G-CSF to circulating G-CSF levels and to emergency myelopoiesis requires additional genetic or neutralization experiments to be fully established.

Author's response: We appreciate that Reviewer#2 raised this key point. In addition to examining the serum G-CSF upon intradermal anti-G-CSF administration in point#1 from Reviewer#1 above, we will also examine the emergency myelopoiesis signs in vivo.

Minor points

1. Line 319: the text likely refers to Figure S4, not S3.

Author's response: Thank you, we will correct the nomenclature.

Line 338: "psoriatic" is misspelled.

Author's response: Thank you, we will change this to "psoriatic".

Reviewer #3

• Place the work in the context of the existing literature (provide references, where appropriate).

Psoriasis is extensively studied, a good recent reference- https://doi.org/10.1016/j.mam.2024.101306

Author's response: Thank you for Reviewer#3's suggestion. The referenced study highlights the current paradigm that largely focus on skin-restricted mechanism and overlook potential cross-organ interaction in the psoriasis inflammation. Our findings provide a new insight into the skin-bone marrow crosstalk in the disease context. In addition, the suggested reference underscores the key roles of diverse innate immune cells including neutrophils, eosinophils, dendritic cells, etc. which is fundamental for our study and might also guide future exploration of additional innate cell subsets beyond neutrophils. We will therefore include the mentioned reference to our revised manuscript.

• Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

It is all good. May add graphical-abstract.

Author's response: Thank you for the reviewer's input, we agree that a graphical-abstract will help the readers more clearly grasp the key messages of our manuscript. We will include it in the revised manuscript.

Major comments:

• Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

No. It is very solid.

Author's response: We appreciate the reviewer's view that the claims in our paper are solid.

• Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

Such a discovery clearly opens many options, and it is fascinating to suggest additional experiments for future studies. It is a complete study, best to publish as-is and let many to read and proceed with this new concept.

Author's response: We thank the reviewer for noting that the current experimental evidence is complete that no additional experiments are necessary at this stage. We agree that the discovery opens prospective directions for future studies.

• Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

N/A - I suggest no additional experiments at this point. Get it published and see how many will follow this new direction!

Author's response: We thank the reviewer for recognizing that the experimental data has been sufficient to be a foundation for the future research.

• Are the data and the methods presented in such a way that they can be reproduced?

Yes.

Author's response: We thank the reviewer for recognizing that our methods are reproducible.

• Are the experiments adequately replicated, and is the statistical analysis adequate?

Yes. The data are of very high quality.

Author's response: We are grateful that the reviewer view our replication strategy and statistical analysis are of a high quality.

Minor comments:

• Specific experimental issues that are easily addressable.

None. It is good as-is. One may always suggest minor things- but this one is better published so many laboratories may rush for this new direction. I think it will be interesting studying some long-term impacts, and changes not only of neutrophils but also of other innate cells, such as DCs, Macrophages, and Eosinophils - so it is best to let laboratories that focus on these cells know of the discovery and pursue independent studies.

Author's response: We appreciate the reviewer's assessment that our paper is already well set for the community to explore the newly proposed direction.

• Are the text and figures clear and accurate?

Yes.

Author's response: We thank the reviewer's evaluation. We have ensured that the text and figures in our manuscript are clear and accurate. Once again, we thank the reviewer for the encouraging and constructive appraisal. We are pleased that the reviewer find the manuscript has already been strong and suitable for publication.

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Referee #3

Evidence, reproducibility and clarity

Summary:

Provide a short summary of the findings and key conclusions (including methodology and model system(s) where appropriate).

Study titled: "Skin-derived G-CSF activates pathological granulopoiesis upon psoriasis" by Kosasih and Takizawa. Paper show establishment of psoriasis model in C57BL/6 mice. They focus on neutrophils infiltration following the Imiquimod cream induction. Importantly, authors show that the induction of psoriasis in the skin cause a robust enhancement of granulopoiesis in the bone marrow. Mechanistically, G-CSF is produced in the skin, especially by endothelial cells. Blocking of G-CSF gained clear inhibition of psoriatic pathology. They further add human data showing that patient with psoriasis have more neutrophils and more G-CSF in their skin endothelial cells.

Parts of the study are simply in line with previous knowledge (e.g.- neutrophils infiltration into psoriatic skin, IL17a). authors show some data that largely confirm the model used. Major discovery: skin endothelial cells are secreting G-CSF that induce granulopoiesis in the bone-marrow. This is a conceptual advancement of this study: psoriatic skin not only recruit neutrophils from the blood, but also enhance the generation of new neutrophils in the bone-marrow. That a major- psoriasis at the level of the model used must not be considered as a confined-pathology. It affect systematically, and might also benefit new systemic treatments. There are plenty of follow-up experiments to pursue now, so it is critical to publish this finding and let many laboratories to know of this new direction. I expect this study to attract high interest and many citations.

Major comments:

• Are the key conclusions convincing?

Yes. The study has excellent data, with good quantification, and very solid support for the discovery and interpretations. - Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

No. It is very solid. - Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

Such a discovery clearly opens many options, and it is fascinating to suggest additional experiments for future studies. It is a complete study, best to publish as-is and let many to read and proceed with this new concept. - Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

N/A - I suggest no additional experiments at this point. Get it published and see how many will follow this new direction! - Are the data and the methods presented in such a way that they can be reproduced?

Yes. - Are the experiments adequately replicated, and is the statistical analysis adequate?

Yes. The data are of very high quality.

Minor comments:

• Specific experimental issues that are easily addressable.

None. It is good as-is. One may always suggest minor things- but this one is better published so many laboratories may rush for this new direction. I think it will be interesting studying some long-term impacts, and changes not only of neutrophils but also of other innate cells, such as DCs, Macrophages, and Eosinophils - so it is best to let laboratories that focus on these cells know of the discovery and pursue independent studies. - Are prior studies referenced appropriately?

Yes. I may suggest adding a recent review by Park and Jung, 2024, https://doi.org/10.1016/j.mam.2024.101306 to cover current concepts of innate immunity in psoriasis. - Are the text and figures clear and accurate?

Yes. - Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

It is all good. May add graphical-abstract.

Significance

• Describe the nature and significance of the advance (e.g., conceptual, technical, clinical) for the field.

Conceptual advancement - discovery of a major impact of psoriasis on bone-marrow granulopoiesis. Explicit finding of endothelial-cells G-CSF as a major communication moiety.

• Place the work in the context of the existing literature (provide references, where appropriate). Psoriasis is extensively studied, a good recent reference- https://doi.org/10.1016/j.mam.2024.101306

Neutrophil recruitment and IL17A are well established. G-CSF of endothelial cells brings the conceptual advancement- psoriasis at the level induced by IMQ develops local pathology, but is tightly linked to systemic changes. The impact on bone-marrow granulopoiesis may have many implications. So far, it was largely considered that chronic inflammation may affect hematopoiesis, but this study reveals an acute and specific communication between skin and bone marrow. The neutrophils are not only recruited from blood- they are made anew, so the disease is enhanced significantly! This discovery led to a novel basic understanding and suggests novel therapeutic options. - State what audience might be interested in and influenced by the reported findings.

Dermatologist, immunologist, haematologist - this one goes for a broad audience. - Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

Immunology and hematology. I am not an expert of dermatology.

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Referee #2

Evidence, reproducibility and clarity

General assessment

This is a well-written and carefully executed study that identifies skin-derived G-CSF as a key driver of pathological emergency granulopoiesis in an imiquimod-induced mouse model of psoriasis. The authors convincingly show that endothelial cells are the dominant source of G-CSF in inflamed skin, and that this cytokine mediates systemic hematopoietic skewing and neutrophil accumulation, ultimately aggravating psoriatic pathology. The eanalysis of human transcriptomic datasets strengthens the translational relevance of the findings. Overall, the conclusions are well supported by the data. However, several mechanistically relevant aspects remain underexplored, particularly regarding the functional state of psoriatic neutrophils and the human data integration. Addressing these points would substantially enhance the impact of the study.

Major points

1. Interpretation of neutrophil transcriptomic changes (Figure 2)

The RNA-seq analysis reveals substantial downregulation of several canonical proinflammatory pathways in neutrophils from psoriatic skin, including IL-6, IL-1, and type II interferon signaling. The authors should discuss the functional relevance of this unexpected transcriptional repression. For example, does this indicate a shift toward specialized effector functions rather than classical cytokine responsiveness? More importantly, the most striking transcriptional change is the upregulation of NADPH oxidase-related genes (e.g., Nox1, Nox3, Nox4, Enox2). This suggests an oxidativestress-driven pathogenic mechanism, potentially more relevant than IL-17A production. Yet this aspect is not explored in the manuscript. Assessing ROS levels or oxidative neutrophil effector functions in this model would considerably strengthen the mechanistic link.

Conversely, although IL-17A is upregulated in neutrophils, neutrophil depletion reduces total Il17a expression in skin only partially. This indicates that neutrophils are unlikely to be the dominant IL-17A source in the lesion. The authors' focus on neutrophil-derived IL17A therefore seems overstated. A more rigorous assessment-e.g., conditional deletion of Il17a specifically in neutrophils-would be required to establish its true contribution. Taken together, the data suggest that oxidative programs, rather than IL-17A production, may represent the principal pathogenic axis downstream of neutrophils, and this deserves deeper discussion. 2. Human data reanalysis (Figure 6):

The re-analysis of bulk and single-cell RNA-seq datasets is valuable but incomplete.

Several mechanistically relevant questions could be addressed with the available data:

2.1. GM-CSF (CSF2) is also strongly upregulated in psoriatic lesions (bulk RNA-seq). It would be informative to determine whether endothelial cells also express CSF2 in the scRNA-seq dataset, as this would suggest coordinated regulation of myeloid-supporting cytokines.

2.2. Myeloid cell subsets should be examined more closely. A comparison of human myeloid transcriptomes with the mouse neutrophil RNA-seq would clarify whether similar IL-17A-related or NADPH oxidase-related signatures occur in human disease. In particular, which cell types express IL17A in human lesions?

2.3. Chemokine production should be attributed to specific cell types. Bulk RNA-seq confirms strong induction of CXCL1, CXCL2, CXCL5, but the scRNA-seq dataset allows determining whether these chemokines originate from endothelial cells or other stromal/immune populations. This information is important for defining whether endothelial cells coordinate both neutrophil recruitment and granulopoiesis. Addressing these points would make the human-mouse comparison substantially stronger.

Minor points

1. Line 319: the text likely refers to Figure S4, not S3.
2. Line 338: "psoriatic" is misspelled.

Referees cross-commenting

I agree with Reviewer 1 that the contribution of EC-derived G-CSF to circulating G-CSF levels and to emergency myelopoiesis requires additional genetic or neutralization experiments to be fully established.

Significance

The study is solid and potentially impactful, particularly for audiences working in inflammation and hematopoiesis, as it uncovers a cross-organ mechanism linking skinderived G-CSF to emergency granulopoiesis in psoriasis. My expertise lies in inflammation and hematopoiesis, and from this perspective several essential mechanistic issues remain insufficiently addressed. In particular, the neutrophil transcriptomic data highlight strong induction of NADPH oxidase-related pathways, which appears more biologically meaningful than the modest Il17a upregulation emphasized by the authors. Likewise, the human RNA-seq reanalyses leave open key questions regarding CSF2 expression, myeloid heterogeneity, and chemokine cellular sources. These issues affect the strength and interpretation of the central claims. For these reasons, I recommend major revision before the manuscript can be considered further.

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Referee #1

Evidence, reproducibility and clarity

Summary:

A role of neutrophils in psoriasis pathogenesis has been highlighted by several past studies; however, how the neutrophils are recruited to the affected skin has not been fully understood. The work by Kosasih et al. tackles a relevant question and has investigated the effect of psoriatic skin inflammation on BM myelopoiesis. Using an IMQ-induced acute psoriasis mouse model, the authors derive 3 major conclusions: (1) skin ECs secrete G-CSF into circulation in response to psoriatic stress, (2) skin EC-derived G-CSF stimulates emergency granulopoiesis, and (3) skin EC-derived G-CSF induces infiltration and accumulation of pathogenic neutrophils in the affected skin. The authors provide many pieces of interesting data, but most of them remain correlative and insufficient to support the conclusions. Many of the experiments were performed in a small number of samples or mice (mostly with n=3), leaving the story still preliminary.

Major comments:

1. First, the authors have not convincingly shown that skin cells, or more specifically skin ECs, are a major source of circulating G-CSF in the psoriasis model as stated in the title and abstract. The data in Figure 4 show selective upregulation of Csf3 gene in skin ECs and their ability to secrete G-CSF upon IMQ treatment in vitro. However, the provided data do not address to what degree the skin EC-derived G-CSF contributes to the elevated level of circulating G-CSF. Additional experiments to selectively deplete G-CSF in skin ECs, or at least in skin cells of the affected site, are warranted to support the authors' claim. Does intradermal injection of G-CSF neutralizing antibody into the psoriatic skin reduce circulating levels of G-CSF?
2. Another concern is insufficient demonstration of G-CSF-mediated emergency granulopoiesis in the psoriasis model. All data in Figure 5 were obtained from experiments with only n=3, and adding more replicates, in particular to those in Figure 5B, which show quite some variation in MPP numbers, is recommended. The relatively small reduction of BM granulocyte numbers (Figure 5C) compared to greater depletion of circulating granulocytes (Figure S5A) raises the possibility that it is the mobilization effect rather than granulopoiesis-stimulating effect that skin-derived G-CSF exerts to promote supply of circulating neutrophils that eventually infiltrate into the affected skin. This could also explain the negligible effect of IL-1blockade (Figure S4), which selectively shut off myelopoiesis-stimulating effect of IL-1 (Pietras et al. Nat Cell Biol 2016, PMID: 27111842). Are the HSPCs in the psoriasis model more cycling? Do they show myeloid-skewed differentiation when cultured ex vivo or upon transplantation?
3. The authors' claim that skin-derived G-CSF "induces" neutrophil infiltration warrants further clarification. Alternative explanation is that the upregulated neutrophil-attracting chemokines (Figure S1D) could induce infiltration, whereas G-CSF increase the number of neutrophils to circulate in the vessels near the psoriatic skin. This notion seems supported elsewhere (Moos et al. J Invest Dermatol. 2019, PMID: 30684554). Can the infiltration be inhibited by systemically injecting neutralizing antibody of their receptor, CXCR2?
4. It remains unclear how skin-derived G-CSF accumulates pathogenic neutrophils. The authors state "pathogenic granulopoiesis," but are the circulating neutrophils in the psoriatic mice already "pathogenic" or do they acquire pathogenic phenotype after cutaneous infiltration? Additional RNA-seq to compare circulating and infiltrated neutrophils would answer this question.
5. In addition, how the accumulated pathogenic neutrophils exacerbate the psoriatic changes remains obscure. Although the authors have attempted to correlate Il17a gene expression in infiltrated neutrophils with psoriatic skin changes, the data do not address to what degree it contributes to cutaneous IL-17A protein levels. The data that cutaneous neutrophil depletion leads to subtle decrease in skin IL-17A expression (Figure 2H) rather supports alternative possibilities. For instance, as indicated elsewhere, IL-17A cutaneous tone could be enhanced by neutrophil-mediated augmentation of Th17 or gamma/delta T cell function (Lambert et al. J Invest Dermatol. 2019, PMID: 30528823). Does neutrophil depletion or G-CSF neutralization alter cell numbers or function of cutaneous Th17 and gamma/delta T cells?
6. Finally, as the above conclusions rely solely on the IMQ-induced acute psoriasis model, it would be informative if they could be derived from another psoriasis model. IMQ is known to induce unintended systemic inflammation due to grooming-associated ingestion (Gangwar et al. J Invest Dermatol. 2022, PMID: 34953514), and "pathological crosstalk between skin and BM in psoriatic inflammation" could be strengthened by an intradermal injection model.

Minor comments:

1. Figure 1E shows multiple elongated Ly6G+ structures in d0-2 control and d0 IMQ skins that do not appear to be neutrophils.
2. In Figure 2C, the bottom GSEA seems to be showing type II IFN response, not type I IFN, according to the text.
3. For the BM analysis in Figures 3, 5, S3, and S5, it would be informative if BM cellularity and numbers of committed myeloid progenitors (e.g., GMPs) are shown.
4. In Figures 6B, in which cluster of human skin cells IL-17A expression would be enriched?

Significance

Although quite a few studies have reported various examples of emergency myelopoiesis (Swann et al. Nat Rev Immunol. 2024, PMID: 38467802), there is limited evidence on its occurrence and involvement in locally restricted disease, such as periodontitis (Li et al. Cell 2022, PMID: 35483374; 35483374). As an HSC biologist, I see this study is conceptually interesting as it could extend the above concept to psoriasis, a non-infectious, local inflammatory disease in the skin, and describes a potential causal link between skin-derived G-CSF and emergency myelopoiesis. That said, as detailed in the first section, the conclusions, especially that related to emergency myelopoiesis driven by skin-derived G-CSF, need to be more convincingly supported before taking its value. The findings offer additional understanding of how psoriasis is developed in concert with aberrant hematopoiesis and will be relevant to those working in the field of dermatology, immunology, and hematology.

Archives leave us with fragments and silences. This line acknowledges that engaging with those silences doesn’t just reveal incompleteness in the past it produces incompleteness in us, the ones who encounter it.

Feminist theory values storytelling, embodied knowledge, and reflexivity. This line reflects the feminist method of embracing impossibility by working with fragments, silences, and erasures as sources of insight rather than treating them as dead ends.

Colonialism transformed people into commodities, corpses, or “waste.” The “afterlife of property” refers to how enslaved people were treated as property, and how that logic continues to shape Black life today. The “detritus of lives” is the residue of that commodification, lives reduced, erased, or unattended.

Official archives privilege dominant voices while silencing marginalized ones. Black counter-historical projects attempts to tell history from the perspective of the oppressed are described here as “insurgent” and “disruptive,” but they fail to become recognized as history because the archive itself is structured to exclude them.

Feminist rage and refusal as a feminist practice. This line embodies refusal by rejecting the archive’s version of events and insisting on imagining beyond it.

Feminist epistemology values oral traditions, embodied knowledge, and creative methodologies. Lowe’s “productive attention to the scene of loss” is exactly that, using imagination and alternative modes of storytelling to recover what archives cannot.

Knowledge comes from bodies, trauma, survival, and intimacy. The possibility of communication between the girls is part of that embodied knowledge; truths carried in relationships, even if undocumented.

Epistemic power highlights how power shapes what counts as legitimate knowledge. This line shows that violence itself structures the discourse, determining what can be said about slavery, and who gets to speak.

Feminist memoryscapes contrast patriarchal, linear archives with feminist memory that is emotional, embodied, and collective. This line embodies that feminist memory practice, mourning as a political act, witnessing as a way of keeping the dead present in the now.

Knowledge comes from bodies, trauma, and survival. This line recognizes that representing the girl risks reducing her body to a metaphor or lesson, rather than attending to her lived reality.

Feminist epistemology values oral traditions, embodied knowledge, and storytelling. This line underscores the impossibility of fully recovering erased lives through archival reading alone, pointing to the need for imaginative, disruptive methods.

Knowledge comes from bodies, trauma, and survival. Venus’s body is central to the story, but colonialism reduced it to an object. Representing her means confronting that violence while acknowledging its limits.

Colonialism and slavery highlight how bodies were commodified and dehumanized. “Social death” refers to being stripped of identity, kinship, and recognition; “corporeal death” is literal death. This line situates enslaved lives in the precarious space between those two forms of violence.

The archival grain emphasizes how archives erase marginalized voices. Here, the writer is grappling with how to render lives that the archive has silenced, experimenting with aesthetics to break through those erasures.

The “slave hold” represents the commodification of human beings under colonialism. The line shows how even attempts to narrate history are haunted by the violence of turning lives into cargo, bodies into waste.

The ledger symbolizes colonial record-keeping, which reduced people to property, numbers, or commodities. The absence of friendship in those records shows how colonialism stripped away humanity and relationality.

This is a really interesting point. The difference between 'preventing storage accumulation' and 'rescuing the impacts of already having storage accumulation' are definitely worth considering, and I appreciate that you chose a point that is most in line with what happens clinically.

Nice result! Do you have any idea why the sulfatase acitivity wouldn't be rescued but you can see this dramatic improvement in the downstream product levels? Is this a matter of assay sensitivity, or is something else going on?

This result is really striking! I wonder if the single condition which was not rescued (panel D) suggests maybe a specific sulfatase that still might be underperforming/underactivated?

It's intriguing that some of the sulfatase activities plateau below the point of WT, though. I'd be curious to see the different levels of the sulfatases at each expression level of the FGE as well. Perhaps there are fewer sulfatases for some reason?

this type of thing sounds like what I thought wrt annotation of [[AI agents als virtueel team]]. The example prompts of questions make me think of [[Filosofische stromingen als gereedschap 20030212105451]] die al per stroming een vraagstramien bevat. Making persona's of diff thinking styles, lines of questioning. Idem for reviews, or starting a project etc.

u/capnrefsmmat reply to

u/lodger238 at https://reddit.com/r/typewriters/comments/1hslx56/huh_i_thought_wd40_was_horrible_as_a_lubricant/<br /> Agree completely and to further the point; typewriters were manufactured with low tolerance in many areas just so they wouldn't need lubricating.

• Children at a young age must take proper measures and time to understand their own language or whatever language is being taught to them.
• It establishes a groundwork to help students find their place in the educational field and society,

Instructions for students Purpose: Annotating the syllabus allows you to identify questions and clarify course expectations, in addition to practicing using the annotation tool. You can review a quick-start guide for how to add annotations.

Instructions: As you review the syllabus, add at least 2 annotations. Here’s some guidance for what you might include in your annotations:

Ask a question about something that is unclear or confusing to you Paraphrase instructions in your own words (this can help your instructor understand how you’re interpreting expectations) Comment on a topic or assignment you’re looking forward to and explain why Comment on a topic or assignment that is concerning to you and explain why Comment on a topic or assignment using an outside resource, image or video that might be helpful as supplemental material (review instructions on how to add images, links or videos to annotations) Reply to a classmate with an additive annotation (add to the conversation by answering their question or extending their response). Here are some ideas on how to start an additive response to a classmate: What did you mean by … Did you consider …/ You might consider … I think you should … I connect with … It made me think … Important notes about annotating:

Make sure you hit “post” after you complete your annotation, or else your annotation will not be saved. Make sure it says “post to [this class]” and not “post to only me,” or else people won’t be able to review your annotations. If someone replies to your annotation, you will not receive a notification. Check back periodically to continue the conversation!

Oceans have mroe postive net radiation than land in the mid lats as they asorb more radiation Land masses during summer have low net, due to high albedo of light coloured deserts Polar regions have neggy even in summer as they're ice covered

Earths energy budget is balanced apart from outgoing radiation due to theincreate in greenhhouse gas levels But it's not for everywhere on earth, large scale variation exisits and one fundamental source of this is latitude

Net radiation is the different between incoming SR and outgoing TR

Evapotransipiration is a similar value of incoming SR directly absorbed by the atmopheres, nearly 5x that transferred by sesnisble heat and 22% ofrom surface emitted LW radiation

Global average of SR at the top of the atmosphere is 1/4 solar constant

Energy budget is determined at steady state across one or more years to account for seasonal affects

Greenhouse effect acts as a trap for energy near the surface SE which is emitted as infrared is mostly reabsorbed by the atmosphere, which radiates IR itself This is reabsorbed by other levels in the atmosphere many times over, depedning how transparent it is Radiation can be absorbed and reemitted serval times by gases at dif temps and through the atmsphereic column before escaping into space

Low altitudes the atmospehre is most dense andw arm, which intercepts and remits more radiation Higher altitudes the upward radiation escpates to space as the atmosphere is more transparentlyand colder so emits less radidation in generar The net results is that more radiation returns by being emitted downwards than escapes This recyling of radiation in the lower atmosphere means the surface receives the same energy from the sun more than wonce - first directly as solar radiation and then re-emitted infrared radiation from the atmosphere, raising its temp

-20 is the average torposphere temp, from the surface to tropoause, where LW radiation is emitted to space But gases absorb and remit radation (water/co2) meaning the surface is actually 35 waremer than blackboduy temp of earth which is the greenhosue effect. Without the atmopshere the earhts surface tempw ould be -18.

The earth isn't a single system so its hard to use the law

We can use Stefan-Boltzmann law to estiamte the temp of the Earth (steady state)

The outgoing power of Infrared radiation = the power of absorbed SR, so R = value of absrobed Radiation

Energy coming in is mostly absorbed by SW RD Energy out is infrared LW TR 1/3 SR is refelceted back, and 1/3 of whats left is absrobed by the atmospehre, so half of total radiation incident is absrobed at hte top of the atmophere in the visible region

Based on my own experiences I can positively say that AI is the ruins of this earth. I used to use AI to help me rephrase a prompt or question I didn't understand on its own. However with time I realized this only allowed my comprehension skills to worsen or more so rely on AI to further explain it to me. In the beginning I said hm maybe this is okay because it's like the teacher explaining the prompt to me in a more detailed manner, which is what I needed. But no, I became overly dependent on AI. So I stopped. It was genuinely scary.

Touching on this, I don't understand why this happens. The level of severeness is at an ultimate level, and the developers know this every time. I get that the excitement of trying to see of they can create this new thing is what blinds them but why is there no job position that specifically focuses on making sure they don't create something that will not be ethical in the long run or near future. It should be made so that it is preventable by a committee who isn't so blinded by the project and is focused on the ethics.

Actual solution missing:

• from spacy.util import filter_spans
• Before assigning to doc.ents, filter the spans to resolve overlaps with filtered_ents = filter_spans(original_ents). By default, filter_spans prioritizes longer spans.

It's recursion:

https://hypothes.is/a/nxL8dNRmEfCKiQNmvuH9VQ

Note that in addition to the followup that the author has added to this article (in Feb 2025), he has written a subsequent Tutorial on CWEB article.

Does it defeat the purpose, or is it a form of redundancy?

Same.

I'm amazed by the number of programmers who, having already written down how the problem gets solved, attempt to document/comment their code and don't realize that this is what should be their fundamental concern—what problem does this solve?

Reading John Smith, I realized how times have not changed. How they stated, "If he have nothing but his hands, he may...by industries quickly grow rich" People in corporate now are still the same-- they are able to grow rich through the industries. And everyone else stays beneath even if working twice as hard. It seemed like a genuine talk he had but it has become so toxic

It is important to always ask this question when choosing resources because it helps indicate if the source is credible or not.

It is important to verify that scholarly journals are credible. It is important to do that using the databases in order to verify reputation. In doing this it helps you provide stronger evidence when creating your paper.

It is important that the introduction and conclusion are tied back to the thesis. In doing this it helps to create a cohesive paper.

@chrisaldrich uses the Via proxy on mobile. Or did back in 2020. This will be deprecated by Feb 2026 however.

Earth surfaces also scatter light in different amounts, snow and ice for example have a higher albedo and back scatter much more than forests. All these impact how much SR is absorbed by Earth. Clouds scatter outgoing TR which can 'close' the atmospheirc IR window and keep the surface warmer.

This net effect is complex and the role of clouds remains a big uncertainty in climate model proejctions

Lights also scattered by larger WL, clouds which are white or grey scatter SR in all directions so have a big impact on the atmosphere

SW blue light is a clear day, but as sunrise/set the light has to pass through more atmospehre and rayleigh scattering increases, so more LW red and orange reaches us

When SR encounted agents much smaller than the WL (1/10 size) radiation is dispersed in all directions - this is called Rayleigh scattering In the atmosphere it is caused by indiviudal gas molecules and pirmarily affects SW and is effective for visible light

Another important window is at infrared WL ~8-15um, where there's low air absorption, expect just below 10 um due to ozon. This is where the radiation emitted by Earth is high and transparency in this region allows land to cool on cloudless nights as energy emited by earth is lost to space, the window is closed if clouds are present as they absrob and scatter radiation