1. Jul 2026
  2. pmc.ncbi.nlm.nih.gov pmc.ncbi.nlm.nih.gov
    Mosaic chromosomal alterations are suppressed in older adults with HIV
    1
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    pmc.ncbi.nlm.nih.gov/articles/PMC13389083/
  3. pmc.ncbi.nlm.nih.gov pmc.ncbi.nlm.nih.gov
    Synthetic multicolor antigen-stabilizable nanobody platform for intersectional labelling and functional imaging
    9
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    pmc.ncbi.nlm.nih.gov/articles/PMC13390901/
  4. pmc.ncbi.nlm.nih.gov pmc.ncbi.nlm.nih.gov
    Dopamine-related alterations in functional brain network dynamic reconfiguration in Parkinson’s disease
    4
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    pmc.ncbi.nlm.nih.gov/articles/PMC13388714/
  5. pmc.ncbi.nlm.nih.gov pmc.ncbi.nlm.nih.gov
    Comprehensive biophysical and structural profiling of alpha-actinin-2 variants reveals mechanistic diversity in hypertrophic cardiomyopathy
    1
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    pmc.ncbi.nlm.nih.gov/articles/PMC13388997/
  6. pmc.ncbi.nlm.nih.gov pmc.ncbi.nlm.nih.gov
    Thymulin restrains age-associated myeloid inflammation and enhances cancer immunotherapy
    2
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    pmc.ncbi.nlm.nih.gov/articles/PMC13389034/
  7. pmc.ncbi.nlm.nih.gov pmc.ncbi.nlm.nih.gov
    Mitochondrial translation termination, recycling, reinitiation, and rescue for in-frame and out-of-frame contexts
    2
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    pmc.ncbi.nlm.nih.gov/articles/PMC13388722/
  8. pmc.ncbi.nlm.nih.gov pmc.ncbi.nlm.nih.gov
    Fusion-positive rhabdomyosarcoma oncofusions share a common interactome
    4
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    pmc.ncbi.nlm.nih.gov/articles/PMC13389464/
  9. pmc.ncbi.nlm.nih.gov pmc.ncbi.nlm.nih.gov
    Brain FGF2 and NCAM1 contribute to FGFR1-dependent progression of estrogen receptor-positive breast cancer brain metastases
    3
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    pmc.ncbi.nlm.nih.gov/articles/PMC13388965/
  10. pmc.ncbi.nlm.nih.gov pmc.ncbi.nlm.nih.gov
    Structural basis of chondroitin sulfate backbone polymer synthesis
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    pmc.ncbi.nlm.nih.gov/articles/PMC13389171/
  11. pmc.ncbi.nlm.nih.gov pmc.ncbi.nlm.nih.gov
    Intracellular Delivery of Peptides and Proteins with an Engineered Membrane Translocation Domain
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    pmc.ncbi.nlm.nih.gov/articles/PMC13390933/
  12. 54.241.53.176 54.241.53.176
    '-html.html
    20
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    54.241.53.176/b66822b2907bb6d00b9e83a81725f62d71cd041a/'-html.html
  13. pmc.ncbi.nlm.nih.gov pmc.ncbi.nlm.nih.gov
    N-Lac-Phe Ameliorates Myocardial Cell Death by Improving Mitochondrial Dysfunction, Ultimately Improving TAC-induced Heart Failure
    1
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    pmc.ncbi.nlm.nih.gov/articles/PMC13388785/
  14. pmc.ncbi.nlm.nih.gov pmc.ncbi.nlm.nih.gov
    Dapagliflozin Inhibits Thrombin Induced ADAM17 Phosphorylation and Downstream ERK/p-c-Jun-mediated CTGF Induction to Prevent Neointimal Hyperplasia
    12
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    pmc.ncbi.nlm.nih.gov/articles/PMC13388336/
  15. pmc.ncbi.nlm.nih.gov pmc.ncbi.nlm.nih.gov
    Loss of Neocortical Oligodendrocytes in Parkinson's Disease Patients—A Stereological Study
    5
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    pmc.ncbi.nlm.nih.gov/articles/PMC13389552/
  16. pmc.ncbi.nlm.nih.gov pmc.ncbi.nlm.nih.gov
    Cross-species integration of single-cell data reveals conserved pathology-associated cell populations across animal models and human samples
    1
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    pmc.ncbi.nlm.nih.gov/articles/PMC13390003/
  17. doc.rust-lang.org doc.rust-lang.org
    Control Flow - The Rust Programming Language
    1
    1. Ashley2333 24 Jul 2026
      if Expressions

      Rust 中 if 是表达式，而不是语句。 Rust 的设计哲学是尽量让语言构造都是表达式，都有值。 C需要三目运算符 ?: 来处理"根据条件返回不同值"的场景

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  18. deepfuture.now deepfuture.now
    Deep Future - AI scenario planning
    2
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    deepfuture.now/
  19. newsletter.squishy.computer newsletter.squishy.computer
    Agents want flexible schemas - by Gordon Brander
    15
    1. indyweb 24 Jul 2026
      ULID

      sounds like Local Unique Identifiers

      Yep, problems define their fixed point solutions

      one's that cannot be impoved further

      MindGraph nearly 20 years ago was based on this concept

      So it seems FeatherWiki has a nie one just like that

    3. indyweb 24 Jul 2026
      read the latest, rebase

      Trying to maintain a centralized single source of truth is really hard.

      Centralized Version Control was a better way of working

      everyone had access to the latest of all other's contributions

      creating a branch was a last resort and the sooner yu merged back the bettr your chances were not to loose or be forced to duplicate all subsequent effort

      Trunk Based development comes to mind

      But if we treeat aggregators as simply views over the work of participants we can have easy of mdification and complete consistent vew from a "center" that is because there is no Write in the center so it cannot be wrong

      Single source of Truth is incompatible with the natiure of Truth, which is forever, always situated and co-evolving

    4. indyweb 24 Jul 2026
      CouchDB

      If you have an InterPlanetary File System

      which is unenclosable, unstopable permanent, taht is never the case with databases

      and if you combine that with Personal ownership/authorship with full provenance and recapitulable history

      All three Consistncy, Avaiability and Partition tolerance becomes possible by architecture construction

      all participants, will always be individual human beings selves, (even if they are acting on behalf oa a named Solcial-Self in colaboration with other individuals who co-create the contributions and enact participations of that social self

      with complete up to date eventually consistent records fo all their interactions with Others maintaining a perfect self-valideatiing partition tha tgets updated hrough conversations / information exchange with others that can be real time or asynchronous but always guaranteeing eventual consistency availability

    6. indyweb 24 Jul 2026
      database surfaces conflicts on read. The application (not the database) resolves the conflicts by reading them in and making a new merge commit.

      conflict on read leading to merge commits

    7. indyweb 24 Jul 2026
      I think CouchDB’s conflict resolution strategy has nice properties for LLMs

      If you flip he existing paradigm and every participant works on ther own copy of anything they create or engaage with, there can be no conflicts, and result of work can be shared through asynchronous channels between participants in an unbroken flow of updates with gurantees for eventuall permanent consitency

    8. indyweb 24 Jul 2026
      implement version history for the document store

      Wen documents are stored ina a singular global interplanetwry storage with permanent verifiable cryptographic names and are mapped from and named by humans for human consumption in meaningful intentionally transparetnt trailmarks

      the oppertunity is there to make all pertinent concerns, like versionning, quality conrol etc integral supported by the system from the start

      This is possible because everthing in the system is verifiable attributed to their creaator mdfieers with full hstory of every artifacts co-created co-evolution

    10. indyweb 24 Jul 2026
      version field

      We need integrl versioning and strong guaranteees for forward compatibility

      as the NOTATION and its machine support is itself coevolutionary

      and version control itself is integral part of the way it works

      all necessary transformation on existing ways the notation works could be upgraded y to future versions of itself where operaional and conceptual continuity will never break

      as they are by construction integrate and solve all pertinent concerns as a criteria of their adoption

    11. indyweb 24 Jul 2026
      Schema migration

      woud be tricky if it is not co-evolutionary

      relyting on a meta stable autopoietic LNTM

      Language NOTATION Tools and Methodology

      Notation is a way to articulate formulate codify create and operationalize at the meta level tool and methodologies

    12. indyweb 24 Jul 2026
      . Now agents can define new domain models on the fly as they encounter new problem spaces.

      That i where HUMANs ecell

      just give them the languag, notation, tools and methodology that they can self-create as a system that itself is self-creating

      Bootstrap from there

      in the full knowledge that it will Grow through use

      Make IT as you USE it and Modify it through

      instance frst exploratory experiments that are conducted in a way as to be able to produce and incremental yet potentially great shifts in experiences and capabilities

    13. indyweb 24 Jul 2026
      type discriminator to every doc, and validate on write.

      types are a very limited idea good for machines but the real value is in exploring and externalizing human interintellect

      which is foreverer incomplete, likely to be inconsistent and surely by construction in complete If you say anything yo are likely to use and introduce terms that are not fully formulated elaborated, may even be misnamed by the erm

      and it will come out only in larger enclosing conplexes

      so it is of neccessity in a state of flux

      and the mos important requirement is easy and cheap refactorability and the ability progpagete conseqyences to be experienced evaluated and leading to urther expansion revisions

    14. indyweb 24 Jul 2026
      agents

      Human Actors have the capacity for meaningful naming of their intents, pupose, pertinent complex intertwingled relevnt anything that theyt can name and create composite names ready to be processed and interpreted by machnis that they are also able to command

    15. indyweb 24 Jul 2026
      Identifying documents by path

      Paths as the means of identification is a broken

      consider them as composite trails

      where each segment provides a situated characterisation of adjacent enclosing context

      or should we say associatieve COMplexes

      shit from thinking in terms of contTEXT

      to conComPLEXES or ConPlexes

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    newsletter.squishy.computer/p/agents-want-flexible-schemas
  20. www.biorxiv.org www.biorxiv.org
    TLR4 signaling drives tissue inflammation, Claudin-5 internalization, and vascular barrier breakdown in a mouse model of neonatal meningitis
    5
    1. Public_Reviews 24 Jul 2026

      eLife Assessment

      This study demonstrates that endothelial toll-like receptor 4 is a central regulator of leptomeningeal inflammation in neonatal E. coli meningitis. The data are derived from cell-type-specific gene knockouts in mice and cultured endothelial cells and are convincing. This work is important as it advances our understanding of host cellular processes and molecular pathways underlying meningitis pathogenesis and specifically expands the knowledge of how breakdown of the blood brain barrier contributes to the pathogenesis.

      Summary
    2. Public_Reviews 24 Jul 2026

      Reviewer #1 (Public review):

      Summary:

      In this manuscript, Seegren and colleagues demonstrate that in a mouse model of neonatal E. coli meningitis, loss of toll-like receptor 4 (TLR4) in VE-cadherin+ endothelial cells and a subset of meningeal fibroblasts leads to a marked decrease in transcriptional dysregulation across multiple leptomeningeal cell types, a decrease in vascular permeability, and a decrease in macrophage abundance. In contrast, loss of macrophage TLR4 had less pronounced effects. Using cultured wildtype and TLR4-knockout endothelial cells, the authors further demonstrate that TLR4 signaling leads to reversible internalization of the tight junction protein claudin-5, establishing a potential mechanism of increased vascular permeability. Authors also show that claudin-5 internalization is independent of NF-κB. Finally, the authors use RNA-sequencing of wildtype and TLR4-knockout endothelial cells to define the TLR4-dependent cell-autonomous transcriptional response to E. coli.

      Comments on revised version.

      The authors have considerably improved and strengthened the work through the addition of new experimental data, new data analyses, and modifications to their interpretation. Notably, the authors used additional Cre-reporter mice to clarify that Cdh5-CreER is active in endothelial cells and some meningeal fibroblasts, and thus revised nomenclature and interpretation to acknowledge that the Tlr4fl/-;Cdh5-CreER cKO (Tlr4-VEKO) is not exclusively endothelial. The authors also demonstrated that Tlr4-VEKO does not affect peripheral E.coli burden, but acknowledge that changes to periphery-derived signals (e.g., cytokines) may contribute to observed leptomeningeal phenotypes.

      The authors added PCA plots to show similarity in gene expression shifts across biological replicates (mice). This provides support for the claim that Tlr4-VEKO attenuates infection-associated transcriptional changes. With respect to differential expression analysis, I agree with authors that characteristics of individual cells (e.g. heterogeneity) are of interest. I remain concerned, however, that the formal differential analysis strategy appears to consider cells as independent experimental units, which they are not because a single cell cannot be randomly assigned to an experimental group (control or cKO, uninfected or infected). The mouse is the correct experimental unit for a comparison across these groups because it can be randomized. I appreciate that many of the gene expression changes appear consistent across mice (e.g. Figure 1 - Figure supplement 7) and that there are clear infection- and genotype-associated phenotypes in other assays. I would simply caution that the authors' analysis strategy likely leads to a larger number of type I errors (false positives) than is generally accepted; a mixed (hierarchical) model or pseudo-bulk approach would be more appropriate for future studies.

      Review 1
    3. Public_Reviews 24 Jul 2026

      Reviewer #2 (Public review):

      Summary:

      The authors use a postnatal mouse model of E. coli bacterial meningitis and a mouse brain endothelioma cell line combined with cell type specific gene deletion to study the function of endothelial TLR4, a cell surface receptor that recognizes gram positive bacterial wall components, in the local leptomeningeal (LPM) response with a focus on endothelial barrier breakdown mediated by TLR4. Single cell transcriptional profiling and imaging studies using wholemount preps of the LPM support that LPM endothelial, CD206+ local macrophage and LPM fibroblast and arachnoid barrier cell inflammatory response and is abrogated in endothelial specific KO of TLR4, pointing to a role for endothelial TLR4 in local LPM response. Culture studies using Bend3.1 cells (a mouse brain endothelioma cell line) support a direct role for TLR4 in the bacteria-mediated inflammatory response and in internalization of Cldn5 via the endosomal-lysosomal pathway, resulting in loss of barrier integrity

      Strengths:

      The local LPM cell response in meningitis and the role of specific LPM cells in inflammation and CNS barrier breakdown has not been extensively studied, despite ample evidence for primary immune response in the meninges in human patients and in animal models. The authors employ a robust, multi-model approach using both in vivo and in vitro models with cell-type specific knockout to study the function of TLR4 in brain endothelial cell response. The authors nicely combine functional barrier assays with IF for junctional localization in their experimental design and they delve into potential mechanisms of Cldn5 internalization using markers of endosomal-lysomal pathway localization. The authors also describe a new type of barrier assay using a streptavidin-coated plates upon which barrier forming cell cultures can be plated, this could be a very useful alternative or complement to other size-selective barrier assays and presumably could work for other barrier forming cell types, like epithelial cells.

      Comments on revised version.

      In their revision, the authors addressed prior noted weaknesses with new data and analysis. They now show that TLR4-VE-cad cKO mice have a largely similar disease progression as control mice, including increased bacterial burden in the LPM and brain. This underscores that that the reduced vascular leakage and blunted inflammatory response is due to loss of TLR4 response to bacteria on VE-cad recombined cells and not because the mice are protected from meningitis. The authors also performed additional experiments to show that Cldn5 internalization via the endosomal-lysosomal pathway is independent of NFKB signaling. The authors also added in important discussion points about how their results fit into the broader literature on TLR4 in BBB endothelial cell junctional protein localization and prior work on meningitis in global TLR4.

      Review 2
    4. Public_Reviews 24 Jul 2026

      Reviewer #3 (Public review):

      Summary:

      This study investigates the molecular underpinnings of immune responses in the leptomeninges in neonatal bacterial meningitis. Bacterial meningitis is a major disease burden, particularly for neonates, and it has previously been noted that the meningeal immune environment in infants is permissive to opportunistic infection (Kim et al., Sci Immunol, 2023). There is less known about the contribution of the stromal compartment to meningeal immune responses. Seegren et al. interrogate the role of leptomeningeal endothelium in host defense in E. coli infected neonatal mice using mouse genetic tools to delete the LPS receptor Tlr4 from either endothelial cells/stromal cells (using Cdh5-CreER) or myeloid cells (using LysM-Cre). The authors use snRNAseq, cleared cortical mounts, and in vitro work to define the impact of E. coli infection on leptomeningeal endothelial cells. This study uses a range of innovative techniques to probe the role of the stromal compartment in meningitis. With additional experiments to confirm the specificity of their Cre models, this strengthens the interpretation of the study significantly. The only major weakness is the inability to confirm TLR4 knockout in myeloid cells.

      Strengths:

      This study makes excellent use of cleared cortical mounts to examine the biology of the leptomeninges, in particular, changes to the endothelium, with unprecedented detail. In combination with high-quality sequencing data provide new insights into the impact of meningitis on the leptomeninges. The data presented by the authors is of very high quality.

      The authors have also done substantial work to address my two major comments regarding 1) the specificity of their Cre systems and 2) peripheral impacts of the interventions.

      (1) The authors identified and acknowledged some impacts in the leptomeningeal stroma (the relatively high level of recombination in ECs vs FBs presumably reflects a single low dose being given, where other groups have done more aggressive tamoxifen regimens that drive recombination in FBs as well). Given the incomplete recombination in the leptomeningeal FBs, I agree with their conclusion that it is probably endothelial driven. Acknowledging the contributions of other myeloid cells with the L. The Cre-NLS experiments with nuclear markers provided excellent data and had beautiful staining.

      (2) The authors did not observe differences in bacterial burden in peripheral organs in either CKO model, suggesting that CNS impacts are not downstream of peripheral bacterial control.

      Weaknesses:

      (1) The inducible Cre lines used by the authors target peripheral tissues as well as CNS tissues. Although this is mollified by the lack of impact on peripheral disease burden.

      (2) The authors were not able to confirm TLR4 knockout in myeloid cells, and this caveat is acknowledged. The lack of response in TLR4 VEKO mice strongly suggests successful conditional knockout.

      (3) The cell line model (bEnd.3) is a relatively low fidelity model of BBB endothelial cells. The authors acknowledge this, and it is likely that endothelial cell responses to LPS are highly conserved.

      (4) It is perhaps not surprising that Tlr4 is required for meningitis responses with E. coli. However, it is unclear if these findings can be generalised to other, more common, meningitis infections (streptococcal/pneumococcal).

      Review 3
    5. Public_Reviews 24 Jul 2026

      Author response:

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

      Public Reviews:

      Reviewer #1 (Public review):

      Summary:

      In this manuscript, Seegren and colleagues demonstrate that in a mouse model of neonatal E. coli meningitis, loss of endothelial toll-like receptor 4 (TLR4) leads to a marked decrease in transcriptional dysregulation across multiple leptomeningeal cell types, a decrease in vascular permeability, and a decrease in macrophage abundance. In contrast, loss of macrophage TLR4 had less pronounced effects. Using cultured wild-type and TLR4knockout endothelial cells, the authors further demonstrate that TLR4-NF-κB signaling leads to reversible internalization of the tight junction protein claudin-5, establishing a potential mechanism of increased vascular permeability. Finally, the authors use RNA sequencing of wild-type and TLR4-knockout endothelial cells to define the TLR4dependent cell-autonomous transcriptional response to E. coli.

      Strengths:

      (1) The authors address an important, well-motivated hypothesis related to the cellular and molecular mechanisms of leptomeningeal inflammation.

      (2) The authors use model systems (mouse conditional knockouts and cultured endothelial cells) that are appropriate to address their hypotheses. The data are of high quality.

      Weaknesses:

      (1) The authors perform single-nucleus RNA-seq on dissected leptomeninges from control and E. coli-infected mice across three genotypes (WT, Tlr4MKO, and Tlr4ECKO). A major discovery from this experiment, as summarized by the authors, is: "Tlr4ECKO mice exhibited a global attenuation of infection-induced transcriptional responses across all major leptomeningeal cell types, as judged by the positions of cell clusters in the UMAP." This conclusion could be considerably strengthened by improving the qualitative and quantitative analysis.

      Thank you for this comment. We agree that the UMAP-based interpretation would benefit from additional qualitative and quantitative support. We have expanded the snRNA-seq analysis with additional images and supplemental figures (Figure 1 – figure supplement 3, Figure 1 – figure supplement 5, and Figure 1 – figure supplement 6). The first and third of these new supplemental figures show dot plots for each major leptomeningeal cell type, for each genotype, for the two experimental conditions (infected vs. uninfected), and for individual genes in three immune-related gene sets (NF-kB and TNF-α, JAK-STAT, and IFN-ɣ), providing a more explicit comparison of infection-induced transcriptional responses across genotypes. The second of these new supplemental figure shows principal component analysis (PCA) of the individual snRNA-seq datasets (one mouse per dataset) for each genotype and experimental condition, demonstrating that the observed transcriptional shifts are consistent across biological replicates. Finally, Figure 1 – figure supplement 7, which was included in the original submission, shows changes in the most up- and down-regulated genes (based on adjusted p-value or fold change) in endothelial and myeloid cells across individual mice and genotypes/conditions, further supporting the genotype-dependent effects at the level of individual animals.

      (2) The authors interpret E. coli infection-induced increases in leptomeningeal sulfo-NHSbiotin as evidence of compromised BBB integrity (i.e., extravasation from the vasculature) (Results, page 7), but another possible route in this context is sulfo-NHS-biotin entry from the dura across a compromised arachnoid barrier. The complete rescue in Tlr4ECKOs is strongly suggestive that the vascular route dominates, but it would strengthen the work if the authors could assess arachnoid barrier fidelity (e.g. via immunohistochemistry). At a minimum, authors should mention that the sulfo-NHS-biotin signal in this context may represent both vascular and arachnoid barrier extravasation.

      Thank you for this comment. We agree that our data cannot rule out leakage across the arachnoid barrier during infection. While the rescue observed in Cdh5-CreER; Tlr4CKO (Tlr4<sup>VEKO</sup>) mice strongly supports a dominant vascular contribution, we acknowledge that the sulfo-NHS-biotin signal may reflect permeability at both the vascular and arachnoid barriers. We do not think there is a clear way to directly test this possibility functionally, since the arachnoid barrier appears intact by confocal microscopy. Subtle differences in barrier cell morphology might be detectable by electron microscopy, but this would not definitively address whether infection permits molecular passage across the arachnoid barrier. We have followed the reviewer’s suggestion and revised the Results section to reflect this interpretation. Specifically, we added the following: “The simplest interpretation of these data is that the site of sulfo-NHS biotin leakage is primarily vascular. However, we cannot exclude some contribution from increased arachnoid barrier permeability.”

      (3) The authors state that "deletion of TLR4 prevented both NF-κB nuclear translocation and Cldn5 internalization in response to E. coli (Figure 4A-D)" (Results, page 9). In Figures 4C and D, however, there is no indicator of a statistical test directly comparing the two genotypes. A comparison of within-genotype P-values should not be used to support a genotype difference (PMID: 34726155).

      Thank you for pointing out this omission. We have updated the figures so that the between-genotype p-values are shown for those panels (including this panel) that had not previously shown them.

      (4) In the first paragraph of the Results, the authors summarize the meningeal layers as (1) pia, (2) subarachnoid space, (3) arachnoid, and (4) dura, and then state "The second and third layers constitute the leptomeninges." This definition of leptomeninges seems to omit the pia, which is widely considered part of the leptomeninges (PMID: 37776854).

      Thank you for pointing out this error, which has now been corrected.

      (5) The Cdh5-CreER/+;Tlr4 fl/- mouse lacks TLR4 in all endothelial cells (i.e., in peripheral organs as well as CNS/leptomeninges), and, as the authors note, the periphery is exposed to E. coli. It would be helpful if the authors could comment in the Discussion on the possibility that peripheral effects (e.g., peripheral endothelial cytokine production, changes to blood composition as a result of changes to peripheral endothelial permeability) may contribute to the observed leptomeningeal phenotypes.

      Thank you for raising this point. We agree that peripheral responses could contribute to the observed leptomeningeal phenotypes in this model. We have added two sentences to the second paragraph of the Discussion to address this: “We note that these experiments do not distinguish between local vs. distal anatomic sources of LPS or downstream effector molecules, such as cytokines, that activate the leptomeningeal inflammatory response (Huang et al., 2021). Histologic observations of RFP-expressing E. coli in the brain, liver, and lungs, together with positive blood cultures, indicate substantial systemic dissemination in this model. Thus, the inflammatory responses of leptomeningeal cells likely reflect exposure to bacterial products and inflammatory mediators derived from both local meningeal and peripheral sources.”

      Reviewer #2 (Public review):

      Summary:

      The authors use a postnatal mouse model of E. coli bacterial meningitis and a mouse brain endothelioma cell line combined with cell-type-specific gene deletion to study the function of endothelial TLR4, a cell surface receptor that recognizes gram positive bacterial wall components, in the local leptomeningeal (LPM) response with a focus on endothelial barrier breakdown mediated by TLR4. Single-cell transcriptional profiling and imaging studies using whole-mount preps of the LPM support that LPM endothelial, CD206+ local macrophage and LPM fibroblast and arachnoid barrier cell inflammatory response and is abrogated in endothelial-specific KO of TLR4, pointing to a role for endothelial TLR4 in local LPM response. Culture studies using Bend3.1 cells (a mouse brain endothelioma cell line) support a direct role for TLR4 in the bacteria-mediated inflammatory response and in internalization of Cldn5 via the endosomal-lysosomal pathway, resulting in loss of barrier integrity

      Strengths:

      The local LPM cell response in meningitis and the role of specific LPM cells in inflammation and CNS barrier breakdown have not been extensively studied, despite ample evidence for primary immune response in the meninges in human patients and in animal models. The authors employ a robust, multi-model approach using both in vivo and in vitro models with cell-type-specific knockout to study the function of TLR4 in brain endothelial cell response. The authors nicely combine functional barrier assays with IF for junctional localization in their experimental design, and they delve into potential mechanisms of Cldn5 internalization using markers of endosomal-lysosomal pathway localization. The authors also describe a new type of barrier assay using a streptavidin-coated plate upon which barrier-forming cell cultures can be placted, this could be a very useful alternative or complement to other size-selective barrier assays and presumably could work for other barrier forming cells types, likely epithelial cells.

      Weaknesses:

      (1) There are no measures of bacterial burden in peripheral organs, blood, in the LPM or brain in the TLR4 endothelial cKO mice. Lack of TLR4 in endothelial cells could prevent bacterial 'access' into the LPM and brain, essentially preventing meningitis and leading to a lack of inflammatory responses in the LPM-located cells simply because there is no bacteria present. Bacteremia may also be reduced, as might inflammatory responses in peripheral organs with TLR4-deficient peripheral endothelium. Bacterial counts and inflammatory measures in peripheral organs and blood are important to better understand the mechanism(s) underlying the reduced inflammatory profile in LPM cells and no LPM endothelial breakdown in the Tlr4 endothelial cKO mice. In other words, does deleting TLR4 in EC protect against the development of meningitis by somehow blocking bacteria access to the LPM (this would be supported by low or no CFU counts in infected Tlr4 endothelial cKO) or is it what the authors appear to propose in Figure 1J that TLF4 in EC is the only cell responding to the bacteria to trigger the immune cascade in the LPM? More data is needed to resolve this, as this is a major claim of the paper.

      Thank you for this comment. We agree that it is important to distinguish whether the reduced inflammatory response in Cdh5-CreER; Tlr4CKO (Tlr4<sup>VEKO</sup>) mice reflects altered bacterial burden versus altered host sensing. We have fleshed out these issues by conducting the following comparisons between infected and uninfected WT and infected and uninfected Cdh5-CreER; Tlr4CKO mice: (1) quantifying E. coli in the blood of infected mice by counting colonies on agar plates; (2) quantifying E. coli in the brain by measuring red fluorescent protein (RFP) signal (the infecting E. coli carry an RFP-expression plasmid); (3) histologically surveying liver and lung for RFP+ E. coli; (4) monitoring the weights of infected and uninfected mice. These data are presented in Figure 2 – figure supplement 4 and in the Results section, and they can be summarized as follows. (1) E. coli is consistently detectable in the blood, brain, and peripheral organs of infected mice and is not detectable in control mice; (2) there are no statistically significant differences between infected WT and infected Cdh5-CreER; Tlr4floxed mice in E. coli burden; (3) infected mice of both genotypes stop gaining weight between the time of infection (P5) and the time of sacrifice 24 hours later (P6). Our conclusion is that loss of TLR4 in endothelial cells and in a subset of other non-myeloid leptomeningeal cells does not alter the overall clinical course of the infection despite changes in leptomeningeal gene expression and vascular permeability.

      (2) The authors look at the underlying cortical response (cerebral vasculature for ICAM and immune cells) but do not use markers that could identify microglia (Iba1), the primary resident immune cell (CD206 is not useful, at this stage, in perivascular macrophages that are extremely sparse in the postnatal brain). This would be important to better study the impact on CNS resident immune cell morphological activation.

      Thank you for this comment. In response, we have analyzed Iba1 staining in the cortex in infected vs. uninfected mice. This is shown in Figure 2 – figure supplement 3. These data demonstrate a several-fold increase in Iba1 immunostaining in infected compared to uninfected cortex, consistent with increased microglial activation in response to infection. There is no statistically significant difference between infected WT and infected Cdh5-CreER; Tlr4CKO mice in Iba1 staining in cortex.

      (3) The authors suggest that Cldn5 junctional localization is selectively disrupted upon bacterial exposure, mediated by TLR4 - they suggest this based on studying PECAM, GLUT1, ZO-1 and B-catenin (all normally junction or cell surface located in cultured Bend3.1) in relationship to Cldn5 localization (normally high) - it is possibly these are also impact by bacteria exposure (maybe through different mechanisms?) - a better measure would be to use the similar cyto/PM measure they do for Cldn5 in Fig. 4D and to evaluate this or to use intensity measurements.

      Thank you for this comment. As the reviewer noted, the analysis of Cldn5 localization with vs. without E. coli exposure and in WT vs. Tlr4KO bEnd.3 cells (shown in Figure 4B and D) – uses Cell Trace to partition the image into cytoplasmic vs. plasma membrane territories. For the analyses in Figure 5, we wanted to compare the localization (and potentially re-localization) behaviors of a variety of subcellular markers with the localization and re-localization of Cldn5 following E. coli exposure. By directly measuring the % overlap of the two immunostains, we get that data. We note that the goal of this analysis is to assess relative co-localization with Cldn5 rather than absolute subcellular partitioning of each marker. While this analysis could have been extended to include independent quantification of the subcellular localization of each of those other markers with respect to cytoplasmic vs. plasma membrane territories, it is clear by visual inspection of Figure 5A-C that beta-catenin, ZO-1, and PECAM1 remain plasma membrane-associated with E. coli exposure, and GLUT1 goes from the part of the plasma membrane not involved in cell-cell contact without E coli exposure to cytoplasmic with E. coli exposure (as judged by the appearance of a nuclear “shadow” after E. coli exposure). Thus, we do not believe that additional cytoplasmic vs. plasma membrane quantification for these markers would alter the interpretation. The main reason that we did not extend this analysis to include independent quantification of the subcellular localization of each of those other markers with respect to cytoplasmic vs. plasma membrane territories is because that would introduce the Cell Trace localization as an additional variable.

      (4) The discussion could benefit from delving more into the prior literature on E coli mediated breakdown of junctions in cultured human microvascular brain endothelial cell model and critical host-pathogen interactions of the bacteria with ECs (PMID: 14593586), and how this might involve TLR4.

      Thank you for this comment. Two paragraphs addressing the prior literature have now been added to the discussion.

      (5) It would be important to discuss how their results relate to earlier studies on TLR4-/- and TLR2-/- global knockout mice and protection vs vulnerability to development of meningitis (see PMCID: PMC3524395) - this paper showed that TLR4 global KO mice have increased susceptibility to die from meningitis and have much higher CFU counts in the CNS. In this manuscript and their prior work (Wang et al., 2023), this group shown that both global TLR4-/- mutants and their EC-specific KO have reduced barrier permeability, but we don't have any information about CFU or susceptibility to death from meningitis in their models.

      Thank you for these comments. The model we use – subcutaneous injection of E. coli (a clinical isolate from an infant with meningitis) at postnatal day (P)5 – results in the death of the infected mouse within 2 days (shown in Figure 1 – figure supplement 3 in Wang et al. 2023). Our analyses of infected mice were conducted 24 hours after infection. As noted in the reply to comment #1, in the revised manuscript we present a clinical assessment of WT vs. Cdh5-CreER; Tlr4CKO mice 24 hours after infection based on (1) a quantitative microscopic analysis of E. coli burden in the brain (visualized based on RFP fluorescence in the E. coli used here), (2) quantifying CFUs in blood and (3) mouse weights at P5 and P6, a sensitive indicator of overall health since this is a time when mice are normally gaining weight rapidly (~25% weight gain per day). These data (shown in Figure 2 figure supplement 4) indicate that bacterial burden and disease severity are similar between genotypes in our model. In Wang et al., 2023, we did not conduct a quantitative clinical assessment of WT vs. Tlr4-/- mice following infection, but by visual inspection, infected WT and Tlr4-/- mice appeared to have similar downhill clinical trajectories. We have expanded the Discussion to relate these findings to prior studies of global TLR4 and TLR2 knockout mice, noting that differences in experimental models and the distinction between global versus VECadCreER-specific deletion may account for the differing outcomes reported.

      Comment on the paper listed by the reviewer (PMCID: PMC3524395).

      The cited study demonstrates that global TLR4 deficiency leads to increased bacterial burden and mortality, indicating an essential role for TLR4 in host defense and bacterial clearance. In our study of Cdh5-CreER; Tlr4CKO mice, bacterial burden and disease severity at 24 hours post-infection are similar between WT and Cdh5-CreER; Tlr4CKO mice, indicating that Cdh5-CreER; Tlr4CKO does not alter the clinical course at this time point. This difference is noted in the Discussion section.

      Reviewer #3 (Public review):

      Summary:

      This study investigates the molecular underpinnings of immune responses in the leptomeninges in neonatal bacterial meningitis. Bacterial meningitis is a major disease burden, particularly for neonates, and it has previously been noted that the meningeal immune environment in infants is permissive to opportunistic infection (Kim et al., Sci Immunol, 2023). There is less known about the contribution of the stromal compartment to meningeal immune responses. Seegren et al. interrogate the role of leptomeningeal endothelium in host defence in E. coli infected neonatal mice using mouse genetic tools to delete the LPS receptor Tlr4 from either endothelial cells (using Cdh5-CreER) or macrophages (using LysM-Cre). The authors use snRNAseq, cleared cortical mounts, and in vitro work to define the impact of E. coli infection on leptomeningeal endothelial cells. This study uses a range of innovative techniques to probe the role of the stromal compartment in meningitis.

      Strengths:

      This study makes excellent use of cleared cortical mounts to examine the biology of the leptomeninges, in particular, changes to the endothelium, with unprecedented detail. In combination with high-quality sequencing data provide new insights into the impact of meningitis on the leptomeninges. The data presented by the authors is of very high quality.

      Weaknesses:

      The weaknesses of the study were in terms of interpretation and perhaps study design.

      (1) Most importantly, the authors need to provide additional validation of their conditional knockout models. The authors need to confirm that the Cdh5-CreER does not impact leptomeningeal fibroblasts and to confirm gene deletion in macrophages.

      We are very grateful for this critique. After several years of using the Cdh5-CreER line in other parts of the CNS, where its expression is endothelial-specific, we applied it to the meninges without realizing that its specificity is broader in that tissue. Our initial analysis with a Cre reporter line that uses a membrane tdTomato appeared to confirm endothelial-specific recombination in the meninges. Following receipt of the reviews of this manuscript, we repeated this analysis with two Cre reporter lines that use a nuclearlocalized GFP, and we immunostained for each of several transcription factors to assess various meningeal cell types and quantified GFP co-localization (Figure 1 – figure supplements 1 and 2). This quantitative Cre reporter analysis shows CreER expression from the Cdh5-CreER transgene in all or nearly all endothelial cells and in a subset (~20%) of dural border cells and/or leptomeningeal fibroblasts, but not in myeloid cells. Additionally, our snRNA-seq analysis of Cdh5 transcripts shows expression in endothelial cells, dural border cells, and leptomeningeal fibroblasts, but not in myeloid cells (Figure 1– figure supplement 4), which agrees with several recent publications (Mapunda et al., 2023; Pietilä et al., 2023; Smyth et al., 2024). Thus, our initial interpretation that the phenotypes in the Cdh5-CreER; Tlr4floxed mouse were a consequence of recombination exclusively in endothelial cells was not quite correct. The Results section of the revised manuscript includes an expanded description of Cre and CreER expression specificity analysis, with supporting data in Figure 1 – figure supplements 1 and 2. Throughout the text of the revised manuscript, we are careful to note that the Cdh5-CreER; Tlr4floxed mouse has Tlr4 deletion in a subset of dural border cells and leptomeningeal fibroblasts. To reflect this fuller understanding of the specificity of Cdh5-CreER, we have changed the name of the Cdh5-CreER; Tlr4floxed mice in the text and figures from TLR4ECKO (“endothelial cell KO”) to TLR4VEKO (“VE-cadherin CreER KO”).

      (2) The authors could also strengthen the paper by providing data on the impact of these conditional knockout models on the course of meningitis and bacterial burden.

      Thank you for this comment. We agree that these additional analyses strengthen the manuscript. We have fleshed out these issues by conducting the following comparisons between infected and uninfected WT and infected and uninfected Cdh5-CreER; Tlr4floxed mice: (1) quantifying E. coli in the blood of infected mice by counting colonies on agar plates; (2) quantifying E. coli in the brain by measuring the red fluorescent protein (RFP) signal (the infecting E. coli carry an RFP-expression plasmid); (3) histologically surveying liver and lung for RFP+ E. coli; (4) monitoring the weights of infected and uninfected mice. These data are presented in Figure 2 – figure supplement 4 and in the Results section, and they can be summarized as follows. (1) E. coli is consistently detectable in the blood, brain, and peripheral organs in infected mice and is not detectable in control mice; (2) there are no statistically significant differences in bacterial burden between infected WT and infected Cdh5-CreER; Tlr4floxed mice; (3) infected mice of both genotypes stop gaining weight between the time of infection (P5) and 24 hours later at the time of sacrifice (P6). Our conclusion is that loss of TLR4 in endothelial cells and in other non-myeloid cells in the leptomeninges does not alter the overall clinical course of the infection despite changes in leptomeningeal gene expression and vascular permeability.

      (3) Finally, it is perhaps not surprising that Tlr4 is required for meningitis responses with E. coli. However, it is unclear if these findings can be generalised to other, more common, meningitis infections (streptococcal/pneumococcal).

      At present, it is an open question whether TLR4 plays as a large a role in meningitis caused by other gram-negative bacteria and whether TLR2 plays a similarly large role in meningitis caused by gram-positive bacteria. In the Discussion, the last two sentences under “Limitations of the study” summarize this point: “Finally, the present study focused on E. coli K1, the dominant Gram-negative neonatal pathogen. Future work could assess TLR signaling in response to other bacterial pathogens, such as Group B Streptococcus.”

      (4) There are additional minor issues; for instance, the arachnoid fibroblast 2 population appears to closely resemble dural border cells.

      Thank you for this comment. That is correct, and we have changed the nomenclature to “dural border cells”.

      (5) The cell line model (bEnd.3) is a relatively low-fidelity model of BBB endothelial cells, and this should be acknowledged.

      Thank you for this comment. That is correct. Despite being brain-derived, bEnd.3 cells have lost many BBB-specific attributes. Their responses might best be considered as generic endothelial responses rather than brain-specific endothelial responses. This is now stated in the Results section: “Although they are brain-derived, bEnd.3 cells lack many BBB-specific attributes and, therefore, they likely exhibit generalized endothelial responses rather than brain-specific responses to bacterial exposure.”

      With these caveats, it is difficult to be certain that the endothelium alone is the driver of meningeal immune responses in meningitis, and what the impact of these is.

      We agree with this critique. As noted above, the expression of Cdh5-CreER in essentially all endothelial cells and in a subset of dural border cells and leptomeningeal fibroblasts means that the comparison of TLR4 CKO with Cdh5-CreER vs. Lyz2-Cre is assessing phenotypes driven by TLR4 signaling in endothelial plus a subset of other non-myeloid cells vs. TLR4 signaling in myeloid cells. We have revised the text to reflect this more precise understanding of Cdh5-CreER specificity.

      Recommendations for the authors:

      Reviewer #1 (Recommendations for the authors):

      (1) Transcriptomic analysis: The analysis and display of the single-nucleus RNA-seq data should be improved. The authors could perform a more granular, unbiased clustering of each cell class in the combined dataset and then compare the proportion of each experimental group (genotype x control/infected) in each cluster. At present, it appears the differentially-expressed genes (DEGs) shown in Figure 1 were identified using the Seurat FindMarkers function with default parameters (Methods). This considers each cell as an independent experimental unit and is therefore not appropriate for a comparison of control versus infected groups (see e.g., PMID 34584091, 35880426. The authors should implement a statistical analysis strategy that considers true biological replicates (mice, as shown in Supplementary File 1).

      We do not fully agree with this critique. We agree that biological replication at the level of individual mice is important for interpreting these data, but within each mouse, the characteristics of individual cells is also of interest, including the degree of heterogeneity, the sample size for a given cell cluster, and the statistical significance of any observed changes in transcript abundance. As requested, we have prepared a new supplemental figure (Figure 1 – figure supplement 5) showing a principal component analysis of the scRNA-seq data for each mouse (one mouse was used for each snRNA-seq dataset) and for each of the principal leptomeningeal cell types. This analysis shows, for example, that the three infected Cdh5-Cre; Tlr4flox/- mice have transcriptomes for each of the six cell clusters that are very similar to the transcriptomes of the two uninfected WT and the two uninfected Cdh5-CreER; Tlr4flox/- mice. Thus, the genotype- and condition-dependent effects are consistent across biological replicates. At the most granular level, Figure 1 – figure supplement 7, which was part of the original submission, shows for the most up- and down-regulated genes (based on adjusted p-value or based on fold-change) in endothelial cells and in myeloid cells how individual transcript abundances change for each mouse and for each genotype/condition.

      (2) The authors use immunohistochemistry to assess claudin-5 "disorganization and redistribution" (Results, pages 7-8 and Figures 3A-B). They state that "Tlr4ECKO mice showed minimal changes in the distribution of Cldn5, implying that cell autonomous endothelial TLR4 signaling regulates tight-junction organization." It is not clear, however, that the quantified parameter (Cldn5+ area relative to total area) would be an accurate readout of claudin-5 organization/distribution (i.e., subcellular localization) as it would also be sensitive to claudin-5 expression, vascular density, and vessel diameter. The authors use a similar assessment of ZO-1 to suggest that changes to claudin-5 are not due to a "generalized disassembly of TJs" and could also use this to argue that the above potential confounds (vascular density, vessel diameter) do not change, but the data in Figure 3 - Figure Supplement 1B, lower panel, show that infection does cause an increase in ZO-1 area relative to total area (P = 0.0004). Thus, the statement in the results "Zonula Occludens-1 (ZO-1) [...] remained unchanged during infection (Figure 3 - figure supplement 1)" is not accurate. The authors should revise this section to ensure their conclusions are aligned with the presented data.

      Thank you for this comment. The reviewer is correct that the Cldn5 area measurement is unable to deconvolve the various factors that might contribute to it (vessel density and diameter, and Cldn5 distribution). This part has been rewritten. “Consistent with prior findings (Wang et al., 2023), both WT and Tlr4<sup>MKO</sup> mice showed an increase in the area occupied by Cldn5 in the leptomeninges following infection, likely referable to both increased vessel diameter and a redistribution of Cldn5 within ECs (Figure 3A-B; Figure 3 – figure supplement 1C).”

      The reviewer is also correct about our initial description of the ZO-1 data. What we meant to write and what the revised manuscript now shows is: “The area occupied by Zonula Occludens-1 (ZO-1), a tight junction scaffold protein, showed a modest but statistically significant increase in WT leptomeningeal vessels but no significant change in Tlr4<sup>VEKO</sup> leptomeningeal vessels during infection (Figure 3 – figure supplement 1A and B).”

      (3) In the Methods, under Mouse Models and E. coli Infection, the authors state, "The Cdh5-CreER line (Monvoisin et al., 2023) was the same line used in Wang et al. (2023)." However, there is no mention of Cdh5-CreER in Wang et al. (2023). Could authors please clarify? Also, because this appears to be an inducible Cre, the authors must include details on the dose and timing of tamoxifen or 4-OHT used in this study.

      Thank you for catching that error. We meant to reference Wang et al (2025), not Wang et al (2023). [Wang et al (2025) is: Wang Y, Rattner A, Li Z, Smallwood PM, Nathans J. (2025) Vascular endothelial-specific loss of TGF-beta signaling as a model for choroidal neovascularization and central nervous system vascular inflammation. Elife 14:RP107018.] This has now been corrected.

      We have now included the details related to 4HT injection in the Methods section “Mouse Models and E. coli Infection”. These are intraperitoneal injection at P2 with 40- 50 µL of 2 mg/ml 4HT.

      (4) The legend for Figure 1A is "Schematic of the leptomeninges", but the figure shows the entire brain-skull interface, including underlying cortex, leptomeninges, dura, and skull.

      Thank you. Corrected.

      (5) Page 7, typo: "In the brain, CD206+ cell were too sparse ..." Should be "cells".

      Thank you. Corrected.

      (6) Page 14, typo: "... could represents a double-edged ..." Should be "represent".

      Thank you. Corrected.

      Reviewer #2 (Recommendations for the authors):

      (1) Perform CFU counts from LPM, dura, brain, peripheral organs (liver) in infected v mock mice from control v TLR4 EC-cKO.

      Thank you for this comment, with which we agree. We have addressed this by quantifying bacterial burden and assessing disease severity in WT and Cdh5CreER; Tlr4floxed mice. Specifically, we performed CFU measurements in blood, monitored mouse weights at P5 and P6, and histologically surveyed the E. coli-RFP signal (i.e., E. coli burden) in brain, liver, and lung. These analyses show that bacterial burden and disease progression are comparable between WT and Cdh5-CreER; Tlr4floxed mice at 24 hours post-infection. These data are presented in Figure 2 – figure supplement 4 and described in the Results.

      (2) Lyz2Cre/+ is used to delete TLR4 from macrophages, but recombination efficiency (in LPM BAMs) is described as only partial, suggesting that TLR4-response in LPM BAMs (and potentially macrophages in the dura) is at least partially intact. It undercuts conclusions that can be made using this line.

      Thank you for this comment. We have conducted a more detailed analysis of Lyz2<sup>Cre</sup> specificity by immunostaining for multiple markers and quantifying the results (Figure 1 – figure supplement 2). We now think that the more cursory analysis in the original submission was inaccurate. The more in-depth analysis shows that Lyz2<sup>Cre</sup> directed Cre-recombination with 90-100% efficiency in CD206+ cells and with 50-70% efficiency in ASC+ and PU.1+ cells, the range depending on whether tdTomato or GFP colocalization was being scored (Figure 1 – figure supplement 2). The Results section text now states: “In the text that follows, we will refer to Lyz2<sup>Cre</sup>-recombined cells simply as “myeloid cells”, although they should be understood as CD206+ myeloid cells.”

      Also, as noted in the reply to comment 4 below, a direct analysis of Tlr4 recombination efficiency is technically challenging due to the low abundance of TLR4 and the failure, in our hands, of commercial anti-TLR4 antibodies to produce clear immunostaining. We have added a comment in the results section noting that we do not have a measure of the efficiency of recombination of the floxed Tlr4 target in vivo: “The low abundance of TLR4 and the limitations of commercial anti-TLR4 antibodies precluded a direct immunohistochemical assessment of TLR4 loss in Tlr4<sup>VEKO</sup> and Tlr4<sup>MKO</sup> mice.”

      (3) Inflammatory responses [qPCR] from peripheral organs and also physiological measures in the pups [weight post-infection, time to moribund or death curves] in control v TLR4 EC-cKO and TLR4 mac-cKO.

      Thank you for this comment. We have not conducted a qPCR analysis of inflammatory gene expression in peripheral organs because (1) the dramatic upregulation of these transcripts in the leptomeninges, (2) the presence of E. coli in blood and peripheral organs, and (3) the clinical assessment (cessation of weight gain) all predict that such an analysis would reveal a large up-regulation of inflammatory gene expression throughout the body. More specifically, we have conducted the following comparisons between infected and uninfected WT and infected and uninfected Cdh5-CreER; Tlr4floxed mice: (1) quantifying E. coli in the blood of infected mice by counting colonies on agar plates; (2) quantifying E. coli in the brain by measuring the red fluorescent protein (RFP) signal (the infecting E. coli carry an RFP-expression plasmid); (3) histologically surveying liver and lung for RFP+ E. coli; (4) monitoring the weights of infected and uninfected mice. These data are presented in Figure 2 – figure supplement 4 and in the Results section, and they can be summarized as follows. (1) E. coli is consistently detectable in the blood, brain, and peripheral organs in infected mice and is not detectable in control mice; (2) there are no statistically significant differences between infected WT and infected Cdh5CreER; Tlr4floxed mice; (3) infected mice of both genotypes stop gaining weight between the time of infection (P5) and 24 hours later at the time of sacrifice (P6). Our conclusion is that loss of TLR4 in endothelial cells and in a subset of other non-myeloid cells in the leptomeninges does not alter the overall clinical course of the infection despite changes in leptomeningeal gene expression and vascular permeability.

      (4) The conditional macrophage line is problematic due to the partial recombination. I question the utility of including this unless they can come up with a way resolve the response of recombined TLR4 macrophages vs ones that are not (could they use the single cell data to pick this a part? Are TLR4-null cells and TLR4 'wt' cells transcriptionally similar in the infected condition, suggesting TLR4 is not doing much in the macs, potentially due to alternate TLRs?). There are good BAM Cre lines that have been described [Lyve1-cre would be good for LPM BAMS, the other is Pf4-cre, see https://pmc.ncbi.nlm.nih.gov/articles/PMC7375817/ - just as an FYI for the future].

      Thank you for this comment. As noted in the reply to point 2 (above), we have conducted a more in-depth analysis of Lyz2<sup>Cre</sup> specificity by immunostaining for multiple markers and quantifying the results (Figure 1 – figure supplement 2). We now think that the more cursory analysis in the original submission was inaccurate. The more in-depth analysis shows that Lyz2<sup>Cre</sup> directed Cre-recombination with 90-100% efficiency in CD206+ cells and with 50-70% efficiency in ASC+ and PU.1+ cells, the range depending on whether tdTomato or GFP colocalization was being scored (Figure 1 – figure supplement 2). The text now states: “In the text that follows, we will refer to Lyz2<sup>Cre</sup>-recombined cells simply as “myeloid cells”, although they should be understood as CD206+ myeloid cells.”

      We agree that, based on Figure 6 in the cited paper [McKinsey et al (2020) A new genetic strategy for targeting microglia in development and disease eLife 9:e54590], the Pf4-Cre line may be superior to the Lyz2<sup>Cre</sup> line that we used for recombination in leptomeningeal myeloid cells. Unfortunately, we missed this paper in our literature searches, probably because it focuses on a microglial CreER line, P2ry12-CreER, and the Pf4-Cre line is not mentioned in the title or abstract. Our decision to use the Lyz2<sup>Cre</sup> line was based on an extensive comparison among myeloid Cre lines showing that Lyz2<sup>Cre</sup> was the most efficient [Abram CL, Roberge GL, Hu Y, Lowell CA. 2014. Comparative analysis of the efficiency and specificity of myeloid-Cre deleting strains using ROSA-EYFP reporter mice. J Immunol Methods 408:89-100.] However, the Abram et al study did not look at the leptomeninges. Regarding the efficiency of recombination of the floxed Tlr4 target, a direct analysis is technically challenging due to the low abundance of TLR4 and the failure, in our hands, of commercial anti-TLR4 antibodies to produce clear immunostaining. We have added a comment in the results section noting that we do not have a measure of the efficiency of recombination of the floxed Tlr4 target: “The low abundance of TLR4 and the limitations of commercial anti-TLR4 antibodies precluded a direct immunohistochemical assessment of TLR4 loss in Tlr4<sup>VEKO</sup> and Tlr4<sup>MKO</sup> mice.”

      (5) Figure 1 - Figure Supplement 2 - the authors nicely break down the pathway response [NFKB and TNF] in EC and macs, it would be great to have similar information for the fibroblasts (in the main figure or the supplement). Does their inflammatory response show a similar pattern?

      Thank you for this suggestion. We have now done that analysis and present it in Figure 1 – figure supplement 3. For completeness, we also performed the same type of analyses for JAK-STAT signaling and IFN-gamma response and these are shown in Figure 1 – figure supplement 6. The principal conclusion is that across all major leptomeningeal cell types, the Cdh5-CreER; Tlr4floxed samples (i.e., Tlr4 KO’d in non-myeloid cells) show much reduced transcriptome changes with infection.

      (6) What is ASC and Cd206 quantification measuring, and how does this relate to 'activation' - is this the intensity of signal or a morphological change? What is the precedence for using ASC (citations)? In their prior work, they showed no change in CD206 number, so a significant increase upon infection here, it's confusing exactly what is being studied. Also, loss of Lyve1 is a well-accepted measure of activation that they have previously used, adding that it could be helpful. This is not a major issue since they have robust data that the macrophages are not transcriptionally activated. Clarification of what exactly is being measured would be sufficient (in the text).

      CD206 immunostaining, which reveals myeloid cell morphology, shows that, with E. coli infection, myeloid cells convert from a more compact morphology to a more expanded morphology. This is now explained more fully in the Results section.

      Regarding ASC, changes in the state of ASC aggregation and ASC subcellular localization have been used by others to monitor immune cell responses to inflammatory signals (Sester et al., 2016; Franklin et al., 2018). While this change in subcellular localization may explain part of the increase in immunostained area in myeloid cells in the infected mice (Figure 2D), the increase in the area of ASC immunostaining largely reflects a shift of myeloid cells from a compact to a more extended morphology. This is now explained more fully in the Results section. We have also added two references (Sester et al., 2016; Franklin et al., 2018) that described how ASC distribution changes with inflammation.

      Regarding LYVE1, we observe a decrease in LYVE1 transcript abundance in myeloid cells with infection, as predicted. Given the large amount of other data that document myeloid activation with infection, we have elected not to include this.

      (7) The authors suggest the internalization of Cldn5 is not due to NFKB downstream signaling that includes transcriptional mechanisms because it happens as early as 1 hour, prior to NFKB localization to the nucleus. However, a lot of their experiments, including on endosomal-lysosomal protein co-localization are done at 4 hours, when their RNAseq data show robust NFKB-mediated gene upregulation and (though not tested) potentially protein production of factors that can act back on the cells, including to impact endo-lysosomal processing. Without studies at earlier timepoints post-bacteria exposure, separating these two mechanisms is difficult.

      Thank you for this comment. We have explored this question by looking at Cldn5 internalization in bEnd.3 cells at 1 hour after E. coli exposure, and the data clearly show that internalization occurs within 1 hour. Additionally, we have conducted this experiment in the presence of 1 uM ACHP, an IKK inhibitor that blocks NF-кB migration to the nucleus. ACHP treatment shows no effect on the rapid internalization of Cldn5, implying a mechanism independent of NF-кB control of gene expression. These data are shown in a new figure (Figure 6) in the revised manuscript.

      (8) Figure 2 - CD206 are quite sparse however, Iba1 would work well to look at microglial activation.

      Thank you for this suggestion, which we have followed. To assess microglial activation, we have immunostained for Iba1 and quantified the data. These are now included in Figure 2 – figure supplement 3. The data show that there is an increase in Iba1 immunostaining following E. coli infection in both WT and Cdh5-CreER; Tlr4floxed mice, with more in the former than the latter, but the difference is not statistically significant.

      (9) Suggest performing the LAMP+ co-localization experiment at <1hr, prior to NFKB nuclear localization and transcriptional changes. This would better support it, this is (or is not) independent of the NFKB. Could also test this with an NFKB inhibitor, do they still see the CLDN5 internalization when NFKB is blocked?

      Thank you for these suggestions. We have done both of these analyses, and the results are presented in Figure 6. The results show that (1) Cldn5 is internalized within 1 hour and (2) its internalization is independent of NF-кB signaling inhibition by 1 uM ACHP. Since ACHP treatment shows no effect on the rapid internalization of Cldn5, that implies a mechanism independent of NF-кB control for gene expression.

      Reviewer #3 (Recommendations for the authors):

      Major points

      (1) The most important caveat is that the Cdh5-CreER model is known to recombine in leptomeningeal fibroblasts (10.1038/s41586-023-06993-7, 10.1101/2025.05.13.653681), and Cdh5 expression in these populations is now well described (10.1038/s41467-02341580-4, 10.1016/j.neuron.2023.09.002). Although the authors did not observe recombination in their reporter (details of the tamoxifen injection protocol should be provided), it is imperative to validate the specificity of their model to Tlr4 in endothelial cells, leveraging their sequencing data and providing additional IHC or ISH to confirm this. Alternatively, Tlr4 could be deleted in a more specific model, e.g., the Pdgfb-iCreERT2 or Slco1c1-CreERT2. It is also important to do the same with the LysM model, to confirm that the lack of impact of macrophage Tlr4 is not due to failure to delete the gene. This is again important to the interpretation of the study, since the authors propose that the endothelium, specifically, is the driver of the meningitis response.

      We are very grateful for this critique. After several years of using the Cdh5-CreER line in other parts of the CNS, where its expression is endothelial-specific, we applied it to the meninges without realizing that its specificity is broader in that tissue. Our initial analysis with a Cre reporter line that uses a membrane tdTomato appeared to confirm endothelial-specific recombination in the meninges. Following receipt of the reviews of this manuscript, we repeated this analysis with two Cre reporter lines that use a nuclear-localised GFP, and we immunostained for each of several transcription factors to assess various meningeal cell types and quantified GFP co-localization (Figure 1 – figure supplements 1 and 2). This quantitative Cre reporter analysis shows CreER expression from the Cdh5-CreER transgene in all or nearly all endothelial cells and in a subset (~20%) of dural border cells and/or leptomeningeal fibroblasts, but not in myeloid cells. Additionally, our snRNA-seq analysis of Cdh5 transcripts shows expression in endothelial cells, dural border cells, and leptomeningeal fibroblasts, but not in myeloid cells (Figure 1– figure supplement 4), which agrees with several recent publications (Mapunda et al., 2023; Pietilä et al., 2023; Smyth et al., 2024). Thus, our initial interpretation that the phenotypes in the Cdh5-CreER; Tlr4floxed mouse were a consequence of recombination exclusively in endothelial cells was not quite right. The Results section of the revised manuscript has an expanded description of Cre and CreER expression specificity analysis, with supporting data in Figure 1 – figure supplements 1 and 2. Throughout the text of the revised manuscript, we are careful to note that the Cdh5-CreER; Tlr4floxed mouse has Tlr4 deletion in a subset of dural border cells and leptomeningeal fibroblasts. To reflect this fuller understanding of the specificity of Cdh5-CreER, we have changed the name of the Cdh5-CreER; Tlr4floxed mice in the text and figures from TLR4ECKO (“endothelial cell KO”) to TLR4VEKO (“VE-cadherin CreER KO”).

      We have also conducted a more detailed analysis of Lyz2<sup>Cre</sup> specificity by immunostaining for multiple markers and quantifying the results (Figure 1 – figure supplement 2). We now think that the more cursory analysis in the original submission was inaccurate. The more in-depth analysis shows that Lyz2<sup>Cre</sup>-directed Cre-recombination with 90-100% efficiency in CD206+ cells and with 50-70% efficiency in ASC+ and PU.1+ cells, the range depending on whether tdTomato or GFP colocalization was being scored (Figure 1 – figure supplement 2). The text in the Results section now states: “In the text that follows, we will refer to Lyz2<sup>Cre</sup>-recombined cells simply as “myeloid cells”, although they should be understood as CD206+ myeloid cells.”

      Regarding the efficiency of recombination of the floxed Tlr4 target, a direct analysis is technically challenging due to the low abundance of TLR4 and the failure, in our hands, of commercial anti-TLR4 antibodies to produce clear immunostaining. The phenotype of Cdh5-CreER; Tlr4floxed mice – a dramatically reduced infection-associated transcriptional response – argues that the floxed Tlr4 target was recombined at appreciable efficiency in those mice (Figure 1D and 1E). For Lyz2<sup>Cre</sup>; Tlr4floxed mice the principal phenotype is an up-regulation of infection-associated transcripts in a subset of dural border cells in the absence of infection; the transcriptional response to infection was largely unaffected in all leptomeningeal cell types (Figure 1D and 1E). We have added a comment in the results section noting that we do not have a measure of the efficiency of recombination of the floxed Tlr4 target in vivo: “The low abundance of TLR4 and the limitations of commercial anti-TLR4 antibodies precluded a direct immunohistochemical assessment of TLR4 loss in Tlr4VEKO and Tlr4MKO mice.”

      (2) The authors did not examine the consequences of Tlr4 cKO on the course of meningitis or bacterial burden. Knowing the impact of this would strengthen the paper and allow us to determine if the endothelial responses are helpful or harmful in meningitis progression.

      For the revised manuscript, we have conducted the following comparisons between infected and uninfected WT and infected and uninfected Cdh5-CreER; Tlr4floxed mice: (1) quantifying E. coli in the blood of infected mice by counting colonies on agar plates; (2) quantifying E. coli in the brain by measuring the red fluorescent protein (RFP) signal (the infecting E. coli carry an RFP-expression plasmid); (3) histologically surveying liver and lung for RFP+ E. coli; (4) monitoring the weights of infected and uninfected mice. These data are presented in Figure 2 – figure supplement 4 and in the Results section, and they can be summarized as follows. (1) E. coli is consistently detectable in the blood, brain, and peripheral organs in infected mice and is not detectable in control mice; (2) there are no statistically significant differences between infected WT and infected Cdh5-CreER; Tlr4floxed mice; (3) infected mice of both genotypes stop gaining weight between the time of infection (P5) and 24 hours later at the time of sacrifice (P6). Our conclusion is that loss of TLR4 in endothelial cells and in a subset of other non-myeloid cells in the leptomeninges does not alter the overall clinical course of the infection despite changes in leptomeningeal gene expression and vascular permeability.

      (3) TLR4 is a known receptor for LPS. It is unsurprising (especially in the in vitro experiments) that Tlr4 knockout reduces NF-kB signalling and other downstream changes to endothelial cells. Furthermore, it is uncertain if the infection was left to continue, similar changes to the endothelium would nonetheless occur through other mediators such as IL1B and TNFa.

      We agree that it makes logical sense that Tlr4 KO decreases NF-кB signaling. The interesting next question is: what are the mechanistic underpinnings of the responses that are downstream of TLR4 and NF-кB? The cell culture experiments with WT vs. Tlr4KO bEnd.3 cells identify one set of cell biological responses related to Cldn5 and junctional integrity, and the NF-кB inhibition experiment (Figure 6) implies that rapid internalization of Cldn5 occurs in the absence of NF-кB mediated transcriptional changes. Regarding the possibility that other mediators such as IL1B or TNFα might, at least partially, make up for the lack of TLR4 signaling later in the infection, that is an open question at present.

      (3) The arachnoid fibroblast 2 cluster should be renamed to dural border cells based on their high expression of Slc4a10, Adamtsl3, Tmeff2, etc which are all highly enriched in dural border cells. I suspect this cluster is also highly enriched for Slc47a1, probably the most specific marker for these cells (10.1038/s41586-023-06993-7, 10.1016/j.neuron.2023.09.002).

      Thank you for this comment. The reviewer is correct. These are dural border cells and they express Slc47a1, as seen in a new supplemental Figure 1 – figure supplement 4, which shows UMAP plots for many leptomeningeal cell type-specific genes. We have updated our cell cluster assignment to align with the assignments in Pietilä et al (2023).

      (4) It would be helpful to provide higher resolution images of Cldn5 in the leptomeningeal mounts. At the current resolution, it is difficult to tell if there is a similar internalisation/disruption phenotype to what is observed in vitro. Notably, this finding is similar to another recent publication on Cldn5 recycling (in the context of stroke) (10.1186/s40478-025-02125-6).

      Higher resolution images of Cldn5 in leptomeningeal vessels without or with E. coli infection are now shown in Figure 3 - figure supplement 1C. There is a visual impression of greater area occupied by Cldn5, which is confirmed by quantification (Figure 3A and B). This effect appears to be due to both an average increase in vessel diameter and a redistribution of some of the Cldn5 away from plasma membrane junctions. Thank you for pointing out the interesting and relevant Cottarelli et al (2025) paper, which we had not read. This is now referenced.

      Minor points

      (1) Typo: prominant should be spelled prominent.

      Thank you for catching that one. It is now corrected.

      (2) Strictly speaking, the arachnoid layer is not epithelial (despite Cdh1 expression). They are fibroblasts that acquire barrier-forming properties.

      Thank you for that comment. That appears to be the consensus view, and we will go along with it.

      (3) Notably, LyzM Cre will also recombine in other myeloid populations, so I wouldn't describe it as a macrophage.

      Thank you for this comment. We agree, and we have therefore changed the text and figure labels from “macrophage” to “myeloid”.

      (4) It is interesting and notable that ICAM1 expression is observed in nonendothelial populations, in the IHC, too, perhaps.

      We agree. ICAM1 may be a broader marker/mediator of inflammation than is generally recognized.

      (5) In F1B, your labels on the right image to the arachnoid barrier and pial surface are presumably meant to refer to the image on the left with DPP4 and laminin labelling? The subarachnoid should be between the laminin and DPP4 layers (although it will be collapsed in your preparations).

      Thank you for catching this error. The vertical bars were sized erroneously, and the labels were also placed erroneously. These have now been corrected.

      (6) I would reference the papers that defined leptomeningeal cell type markers (10.1038/s41586-023-06993-7, 10.1016/j.neuron.2023.09.002) when you define your cell types.

      Thank you. We have done that, and we have updated our cell cluster assignment to align with the assignments in Pietilä et al (2023).

      (7) I would change references to the subarachnoid space in your figures to the leptomeninges (which include the SAS, but extend either side of it).

      Thank you. The labels have been changed to “leptomeninges”.

      (8) In Figure 2 - Supplement 1A, it looks like the populations are mislabelled.

      Thank you. This has been corrected to be consistent with the assignments in Figure 1B

      AuthorResponse
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    https://gist.github.com/karpathy/442a6bf555914893e9891c11519de94f
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  22. doc.rust-lang.org doc.rust-lang.org
    How Functions Work - The Rust Programming Language
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    2. Ashley2333 24 Jul 2026
      statements don’t evaluate to a value, which is expressed by ()

      Rust 的类型系统要求每一个表达式都有某种类型。如果块没有显式的最终表达式，Rust 给它一个默认值——就是 ()（单元类型）。 表达式必须有值，（）也是一种值，只是“什么都没有”这个概念的编码

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  23. www.biorxiv.org www.biorxiv.org
    Identifying molecular instructions to hard-wire a sensory neuron’s synaptic connectivity
    4
    1. Public_Reviews 24 Jul 2026

      eLife Assessment

      This important study addresses how the molecular identity of a single neuron specifies its hard-wired synaptic connectivity, using repeated single-cell RNA sequencing of identified Drosophila sensory neurons together with functional perturbation of candidate cell-surface molecules. It demonstrates remarkably low transcriptomic variability across animals for the same identified neuron, defines a tractable set of differentially expressed cell-surface molecules that distinguish mechanosensory from chemosensory neurons, and links several of these molecules to axonal targeting and circuit function. The evidence is solid, with the single-neuron transcriptomic datasets and Dscam isoform repertoires offering a lasting resource for the field, though clearer articulation of the experimental logic, additional controls in the RNAi screen, and a more complete characterization of the neuronal re-wiring would further strengthen the central claims.

      Summary
    2. Public_Reviews 24 Jul 2026

      Reviewer #1 (Public review):

      Summary:

      The authors sequence the transcriptome of three sensory neurons from D. melanogaster to study the cell-cell and animal-animal variability in these cells, with a focus on cell adhesion molecules. The work reports useful cell-specific transcriptomics datasets that will be of great interest to those studying cell types, transcriptomes, neuronal development, and cell surface proteomes. The authors also report large numbers of knockdown data (gene-by-gene or in combinations) and report neuronal wiring and behavioral phenotypes. The manuscript is highly descriptive of the system studied - in a good way, but often over-speculates in rationale or conclusions.

      Strengths:

      The manuscript is data-rich. The single-cell transcriptomics datasets, not trivial to collect, are a major strength of the work and will prove useful to the field. Also, the biased expression of Dscam is interesting, even though the authors cannot pursue the mechanism or a function for this.

      Weaknesses:

      The study lacks depth (i.e., mechanism) in explaining observations.

      Review 1
    3. Public_Reviews 24 Jul 2026

      Reviewer #2 (Public review):

      Summary:

      In this manuscript, dos Santos et al seek to identify cell-specific programs that drive neuronal wiring patterns. They focus on two chemosensory and mechanosensory neurons in the Drosophila nervous system, as they both display stereotyped connectivity in the ventral nerve cord. Single-neuron RNA sequencing identified cell surface molecules that distinguish the sensory neurons and may instruct their respective wiring patterns. They functionally test several of these candidates and observe miswiring phenotypes upon knockdown experiments. Additionally, they attempt to miswire the chemosensory neurons. Overall, this manuscript addresses an important question about how neurons identify appropriate synaptic partners through precise cell surface molecular codes. However, there are significant deficiencies in the experimental logic and rigor, and the manuscript can be very difficult to digest.

      Strengths:

      The use of two sensory neurons with stereotyped connectivity is a significant strength, as this enables the authors to identify genes that are required for wiring. Additionally, analyzing the transcriptomes of single neurons repeatedly could potentially be a robust approach to identifying cell-specific cell-surface molecules that drive wiring.

      Weaknesses:

      (1) The authors perform RNAseq for single identifiable neurons, as opposed to neuronal subclasses, which has been reported before. It would be beneficial to elaborate on the significance of using single neurons for answering the scientific question. This is briefly mentioned toward the end of one of the results subsections: "Repeated RNA sequencing of an identifiable neuron seeks to address the fundamental nature of variability in connectomics, axonal branching, and cellular identity." But this should be in the Introduction.

      (2) The authors chose the P14 pupal stage for one of the analyses. It is not clear why this specific stage is chosen. Does pSc and aPa connectivity occur at this stage?

      (3) This reviewer is confused as to why looking at differentially expressed CSMs between pupal and adult stages of two different neurons is useful. This does not seem like an appropriate comparison. This data might be better in the supplemental material, especially given the lack of precise age synchronization across pupal samples (as reported).

      (4) It is very difficult to follow the logic because the manuscript seems to jump around between different results and lacks a compelling through line.

      (5) "Single cell sequencing of the same neuron reveals transcriptome precision": What are the controls here? An aPa neuron is shown in Figure 3 as an example of a different neuronal subtype, but were other factors (e.g., lack of Repo expression) checked to ensure that samples were not contaminated?

      (6) "However, whether any of these exon 6 or 9 splicing specificities are biologically significant can only be determined using exon 6 and 9 isoform-specific RNAi." The authors could alternatively use CRISPR techniques to target specific isoforms that they hypothesize might be important for neural wiring, enabling them to assess isoform-specific wiring defects.

      (7) In the section "The set of cell surface receptors required to wire up the pSc mechanosensory neuron": Several previous subsections of the Results use RNAseq to identify molecules expressed in pSc neurons across different stages. It's unclear why the authors did not start with the identified list of candidate cell surface receptors identified in their RNAseq experiments.

      a. Were any of the genes screened the same as those identified by the authors as differentially expressed in pSc mechanosensory neurons, either across developmental stage (pupa vs. adult) or across neuronal subtype (pSc vs. Gr59d)? If so, it would be helpful to state this here. (They do mention later on that five CSMs identified were more highly expressed in pSc than aPa. However, changes in expression across developmental stages within the pSc neuron would still be helpful to comment on, especially since the authors identified greater transcriptomic differences across developmental stages than they did between different neuronal subtypes.)

      b. The 39 genes not expressed in pSc neurons served as their negative control, but the average axonal targeting grade was 2.3 (between moderate and severe). This calls into question the use of this method as an appropriate measure of whether a gene expressed by pSc neurons is truly required for proper axon targeting; there seems to be a strong probability of significant off-target effects. Performing a global knockdown and cell-specific rescue could potentially complement these experiments and serve as a stronger indicator of candidate receptors' roles in pSc-specific axon targeting.

      (8) It seems as though the purpose of the experiments described in the last results subsection ("Re-wiring the Gr59 chemosensory neuron") is to redirect the Gr59d neuron toward the pSc neuron's axonal targeting phenotype. However, the authors do not state whether they were able to do so effectively (i.e., whether or not there were significant differences between the rewired Gr59d neuron and the pSc neuron). This leaves the story unfinished.

      (9) At the end of the discussion, the authors state that "...if a Gr59d chemosensory neuron is functionally rewired to a pSc mechanosensory circuit, activation of the Gr59d neuron using a bitter tastant molecule should elicit a grooming (mechanosensory) response...". The authors should attempt this experiment, especially given that they have developed the PXGS technique.

      Review 2
    4. Public_Reviews 24 Jul 2026

      Author response:

      We are pleased that the reviewers found the repeated single-neuron sequencing and the finding of less than 1% transcriptomic variability to be original and striking, valued the single-neuron Dscam isoform repertoires and the scale of the functional screen, and judged the evidence solid to compelling. We provide below our provisional response and an outline of the revisions we plan.

      Overall plan: We intend to submit a revised version that addresses the public reviews and the recommendations to the authors. Because our conclusions rest on data already in the manuscript, the revisions are clarifications, added analysis of existing data, tempered language, and improved figures, rather than new experiments. Given the focused nature of these revisions, we would be happy for the editors to assess the revised version without re-involving the reviewers.

      One factual note for the Assessment and public reviews: The morphological RNAi screen comprised 213 cell-surface receptor genes; the figure of “140 genes” in one public review is the number that produced strong-to-severe phenotypes (Grade 3–5 at >40% penetrance), not the number screened. We will make this unambiguous in the revised text.

      Main changes in the revision:

      (1) We will explain that the RNAi screen was performed blind and independent of the RNA sequencing experiments. This was intentional, so that functional perturbation and transcriptomic identity would serve as independent lines of evidence, but could be compared with each other.

      (2) We will revise the Methods and Results to clarify how the morphological RNAi screen and behavioral subset should be interpreted, including conservative treatment of the negative-control distribution and mild-to-moderate phenotypes.

      (3) We will soften language that overstated certainty. Differentially expressed molecules are now described as prioritized candidates and convergent evidence, not definitive determinants.

      (4) We will reframe Gr59d/PXGS experiments as morphological rewiring and ectopic branching, and no longer imply a pSc-like conversion or functional rewiring.

      (5) We will add a limitations paragraph addressing RNAi off-target/background concerns, the absence of direct aPa functional testing, and the need for future mechanistic validation.

      (6) We will disclose or remove any figure panels that overlap with the companion PXGS manuscript and revise legends/labels to make it more clear.

      We hope these revisions make the logic of the study clearer and align the strength of the claims with the evidence.

      Below is our more detailed (provisional) response (not sure if this is required at this stage):

      Response to the eLife Assessment:

      Clearer articulation of the experimental logic. The Assessment’s central request (Reviewer 2) concerns the relationship between the transcriptomic experiments and the functional screen. The two were performed independently on purpose; the RNAi screen was assembled from a comprehensive survey of the literature rather than from the results of our differentially expressed genes from single-cell RNA sequencing. Thus, the RNAi screen was performed and graded blind in parallel with the single cell sequencing, with the gene identities unmasked only after both were complete. This was intentional, so that the sequencing (i.e., which molecules differ between neurons) and the screen (which molecules are functionally required) would provide mutually unbiased corroborating evidence rather than self-referential support/circular reasoning. We will state this more explicitly in the Introduction, in the Results where the screen is introduced, and in the Methods.

      Additional controls in the RNAi screen. We will treat the 39 genes that were identified in our single-cell RNA sequencing to be not expressed in the pSc neuron as a randomized negative control set in our RNAi experiments. We will state more explicitly the empirical RNAi false positive rate for a miswiring phenotype is 6/39 = 15%, likely due to RNAi off-target effects. We will also more clearly state that our claims about cell surface receptor functions are restricted to strong-to-severe phenotypes at high penetrance reproduced by at least two independent RNAi lines and corroborated independently (differential expression and/or single neuron qPCR).

      A more complete characterization of the re-wiring. We will state more clearly that mis-expressing the pSc-enriched cell surface receptors within Gr59d neurons partially shifts the arbour toward a pSc-like pattern (e.g., increased ectopic branching), and does not reproduce the full anatomical wiring, and that functional/behavioral re-wiring was not tested.

      Response to Reviewer 1:

      Reviewer 1 found the work valuable and data-rich, and the Dscam expression bias interesting. They noted over-confident language and asked how rigorously the differentially expressed genes were identified.

      Over-confident language. We will rewrite the two flagged sentences. The claim that the ~10 differentially expressed molecules are “likely the most important” will become a correlational statement, while also noting the lack of an aPa-specific Gal4 driver for direct testing. Our sentence that, “Our RNA sequencing data is biologically inadequate without a functional characterization of each molecule within the specific neuron” will be replaced with a clearer statement that gene expression data can nominate candidates, and functional perturbation of each gene is required to demonstrate necessity and sufficiency (i.e., biological function); which is exactly why we paired the RNA sequencing with an independent RNAi screen.

      Rigor of the differential-expression calls. We will more clearly state the statistical criteria in the Results (absolute log2 fold change ≥ 2 and Benjamini–Hochberg-adjusted p < 0.05). We will also note the small replicate numbers for the pooled pSc versus aPa comparisons, and emphasize that the central gene calls are independently supported by the blind RNAi screen and, for five genes, by single neuron qPCR. The full statistical workflow is in the Methods.

      Response to Reviewer 2:

      Reviewer 2 considered the findings potentially important but raised concerns about the experimental logic, the rigour of the screen, the completeness of the re-wiring, figure quality, and overlap with our PXGS companion paper. We will address each.

      Experimental logic. Beyond the design of our independent, blinded RNAi screen described above, we will add to the Introduction the rationale for sequencing single identified neurons (rather than subclasses) along with the two-pronged strategy, and add a summary paragraph at the start of the Discussion.

      Developmental stage choices. We will clarify our justification for the P14 pupal stage (the period when the mechanosensory neuron is actively elaborating its arbour while also enabling dissection). We will also clarify the rationale and caveats for comparing the pupal pSc neuron with the adult Gr59d neuron (i.e., the wiring occurs at the pupal stage, but the pupal Gr59d neurons could not be isolated at sufficient quality; the pSc pupal samples are less age-synchronized, so we simply used the comparison to identify the genes shared with the adult comparison).

      Transcriptome precision controls. We will state that the ten single pSc neurons passed the same quality controls for neuronal markers (elav, nSyb) and glial markers (Repo, moody < 20 reads) as all single-neuron libraries, which argues against any contamination by the attendant glial cell, and the aPa transcriptome is used as a different identity comparison.

      Off-target rate. As stated above, we will add the false positive rate for RNAi and restrict our confidence claims to those genes/cell surface receptors with multiple lines of evidence (e.g., strong phenotype, multiple RNAi lines, RNA sequencing, etc).

      Rewiring completeness and the behavioral prediction. As stated above, we will clarify that true re-wiring of the Gr59d neuron requires a future experiment, where a bitter tastant stimulus would elicit a grooming response.

      Response to Reviewer 3:

      We thank Reviewer 3 for judging our work to be fundamental in significance and the evidence compelling, with no major criticisms. Our clarifications above will further reinforce our hypothesis that the differential expression of specific cell surface receptors “do, in fact, control synaptic patterns,” which the reviewer highlighted.

      We are grateful for the reviewers’ time and for eLife’s model. We believe the planned revisions substantially clarify the experimental logic and tighten the claims, and we look forward to submitting the revised version.

      AuthorResponse
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  24. owl.purdue.edu owl.purdue.edu
    Sentence Fragments - Purdue OWL® - Purdue University
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  25. graphwise.ai graphwise.ai
    Thought Leadership posts Archive
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  26. www.biorxiv.org www.biorxiv.org
    Mid-zone hepatocytes trade proliferation for survival via Atf4-Chop axis in early acute liver injury
    4
    1. Public_Reviews 24 Jul 2026

      eLife Assessment

      This study addresses an important question in liver biology: how zonal hepatocytes balance survival and proliferation following injury? The authors propose that a mid-zone Atf4-Chop axis to Btg2 program temporarily suppresses proliferation to promote survival after a variety of chemical and surgical liver injury models. The authors provide evidence that some zones mount tailored stress responses, which ultimately promote regeneration and liver healing; however, the "mid-zone" changes with different injury models, making it difficult to conclude that the ATF4-CHOP response is specific to this zone in all injury contexts. In addition, it is possible that Atf4 and Btg2 overexpression could lead to Cyp2e1 suppression, which could reduce the extent of injury after CCl4 or APAP. To some extent, these points make the strength of the evidence incomplete, but do not entirely detract from the significance of the study, which is underscored by the helpful observation that there are zone-specific stress responses that mediate liver regeneration and survival.

      Summary
    2. Public_Reviews 24 Jul 2026

      Reviewer #1 (Public review):

      Summary:

      The authors present evidence that during acetaminophen (APAP)-induced liver injury, mid-zone hepatocytes activate an integrated stress response (ISR) program via Atf4 and Chop, leading to induction of Btg2. This program suppresses proliferation in the early phase of injury, prioritizing hepatocyte survival before regeneration begins. The study uses spatial transcriptomics, immunohistochemistry, CUT&RUN, and AAV overexpression to support this model.

      Strengths:

      (1) Innovative use of spatial transcriptomics to capture zonal differences in hepatocyte stress responses.

      (2) Identification of a mid-zone specific ISR signature and candidate downstream regulator Btg2

      (3) Functional experiments with Atf4-Chop-Btg2 modulation provide causal evidence linking ISR activation to proliferation inhibition.

      (4) Conceptually significant model that hepatocytes actively balance survival and regeneration dynamically in a zone-specific manner.

      (5) Multiple models validation of the finding

      (6) The functional link of such zone2 phenotype is added.

      Review 1
    3. Public_Reviews 24 Jul 2026

      Reviewer #2 (Public review):

      The manuscript reports protection of midlobular hepatocytes from APAP toxicity by activation of Atf4-CHOP (Ddit3)-mediated cell cycle arrest and stress response. The authors acknowledge that their finding is unexpected because CHOP typically induces cell death. Therefore, they functionally validate several aspects of the proposed Atf4-CHOP mechanism. Along these lines, the mitigation of APAP toxicity by AAV expression of Atf4 or Btg2, the latter identified as CHOP effector, is impressive. Whether Atf4 indeed acts through CHOP and whether midlobular hepatocytes are protected because of cell cycle arrest is less clear. These and other criticisms are described in the following.

      Major points:

      (1) Starting with the basics, one wonders why midlobular hepatocytes manage to mount a defensive response to APAP, but PC hepatocytes don't. Is this because midlobular hepatocytes express the relevant Cyps (2e1 but also 1a2 and 3a11) at lower levels, which mitigates toxicity and buys them time? This would be supported by F2A but not by F3B, at least not for the most important Cyp2e1. A moderate difference is shown for Cyp1a2 expression in F3D but is that enough to explain the different fates? Or are additional post-transcriptional effects on these Cyps at work? The difference in baseline Cyp2e1 expression between F2A and F3B remains unexplained after revision.

      (2) The evidence presented in support of cell cycle arrest of midlobular hepatocytes is not fully convincing: there is no overt difference in S and G2/M gene scores in F2F; the marker genes used for S phase and G1 to S progression in F2G are unusual. Along these lines, one wonders if spatial transcriptomics confirmed the Ki67 immunostaining results in F1 also for specific zones, not only overall as shown in F2E? In contrast to the revised discussion, the abstract does not reflect that limited evidence for a cell cycle arrest in pericentral hepatocytes was found.

      (3) The authors conclude in line 364 that halting of proliferation by Btg2 favors survival, which raises the question of whether Btg2 knockout causes death in midlobular hepatocytes in F6K. Data addressing this question, that is, localization and extent of tissue necrosis and ALT levels after APAP, are missing. The efficiency of knockout of Btg2 is also not given. Additional Btg2 knockout data support its proposed role in the revised manuscript.

      (4) Related to the previous question, the BTG2 immunostaining in F6F is not convincing when compared to F6D. One also wonders if it is necessary to apply APAP to find induction of BTG2 by AAV-Ddit3? The BTG2 immunostaining remains weak, not only in in F6F but now also in F6D of the revised manuscript, which together with lack of high-resolution immunostaining of AAV-Ddit3-induced BTG2 in the absence of APAP results in limited support for the conclusion that APAP promotes nuclear localization of BTG2.

      (5) Related to the previous question, the proposed Atf4-Ddit3 axis is challenged by the lack of midlobular induction of Atf4 in the APAP scRNA-seq data published by another group presented in S4F and G. Further analysis of AAV-Atf4 samples generated for F5 could address if it is really Atf4 that acts on Ddit3 in APAP toxicity. The extended list of transcription factors (from 30 to 50) includes Atf4 but direct evidence for an interaction with Ddit3 is missing from the revised manuscript.

      (6) Related to the previous question, the ATF4 immunostaining in F5A doesn't look convincing, with many brown pigments appearing to be outside of the nucleus. The ATF4 immunostaining after APAP challenge remains weak.

      (7) It is not ruled out that AAV expression of Atf4 or Btg2 reduces hepatocyte sensitivity to APAP by affecting expression of the Cyps needed for activation. In other words, does AAV-Atf4 or AAV-Btg2 change the expression of any of the Cyps relevant to APAP in the 3 weeks before APAP application (F5B)? S5A of the revised manuscript rules out loss of Cyp2e1 expression as a confounding factor.

      (8) It is laudable that the authors tried to extend their findings to human by using snRNA-seq data from a published study (line 391) but it is unclear why they didn't analyze all 10 patients in that study but instead focused on 2 and stated that this small sample number prevented drawing definitive conclusions and could therefore only be mentioned in the discussion. The revised manuscript continues to focus on rare spatial transcriptomics analyses of patients with APAP toxicity although more snRNA-seq analyses of such patients are available which should also allow for analysis of hepatocyte zonation.

      Comments on revised version.

      After revision, the proposed role of Btg2 is substantiated but it remains unclear why midlobular hepatocytes don't proliferate after APAP challenge and whether the observed protective effects are indeed mediated by Atf4 acting directly through CHOP.

      Review 2
    4. Public_Reviews 24 Jul 2026

      Author response:

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

      Public Reviews:

      Reviewer #1 (Public review):

      (1) Zonation definition under injury has been shown to be sustained broadly, but is not sufficiently validated and quantified, especially considering the resolution of the 10x Visium system and the potential variation of outcomes based on how to define zones.

      We thank the reviewer for these insightful suggestions. In this study, under normal conditions (APAP 0h), each liver lobule was divided into three zones based on unbiased gene expression profiles. The PP zone was defined by enrichment of PP signature genes (e.g.,Alb, Mup20, Cyp2f2,Pck1,Apoa4). The PC zone was defined by high expression of PC markers (e.g., Gs, Cyp2e1, Oat, Cyp1a2, Apoe). The Mid zone comprised regions with intermediate expression of PC and PP markers and elevated levels of Igfbp2 and Hamp (Revised Figure 2A and S1C). Following APAP-induced injury (3 h, 6 h), the PC zone remained identifiable based on residual enrichment of PC signature genes (e.g., Cyp1a2,Glul) despite necrosis and reduced overall transcription, while PP gene expression remained largely unchanged. The Mid zone was defined as the transcriptional cluster between PC and PP regions exhibiting marked reprogramming, (e.g.,Sqstm1, Igfbp1) (Revised Figure 2A and S1C). To validate and quantify our zonation approach, we compared it with classical nine even layers from central vein (CV) to portal vein (PV). Immunostaining and quantification for Cyp2f2 (a PP marker), p62 (the protein product of Sqstm1, a Mid marker during early liver injury), Glutamine Synthetase (GS, the protein product of Glul, a PC marker) further corroborated zone definitions at each time point, showing correspondence of our PC (layers 1–2), Mid (layers 3–6), and PP (layers 7–9) (Revised Figure 2 B-D) (Revised manuscript, page 5, lines 119–131, page 6-7, line 174-182).

      (2) The model is built entirely in APAP injury, which specifically targets pericentral hepatocytes. It remains unclear whether the proposed mechanism applies to other liver injuries (e.g., partial hepatectomy, CCl4).

      We thank the reviewer for this insightful comment. To test whether the proposed mechanism applies to other liver injuries, we employed mouse models of partial hepatectomy (PHx) and carbon tetrachloride (CCl4)-induced acute liver injury. In our CCl4 model (administered intraperitoneally in corn oil, with samples collected 18 h post‑injection), the ISR was activated around injury sites, accompanied by decreased proliferation, as evidenced by increased expression of p‑eIF2α, Atf4, Chop, and Btg2, along with reduced Ki67 expression (Revised Figure S6A–G). In PHx model (examined 24 h after surgery), ISR activation was similarly observed around ischemic injury sites, with increased p‑eIF2α, Atf4, Chop, and Btg2 expression and undetectable Ki67 expression (Revised Figure S7A–G). Together, these additional models suggest that the proposed mechanism may be applicable to other types of liver injury (Revised manuscript, page 15, Line 410-426).

      (3) Baseline proliferation appears higher than expected in homeostasis (Figure 1B), and fold change analysis (not absolute counts) may be needed to assess zonal proliferation suppression (Figure 1D).

      We thank the reviewer for this insightful comment. The baseline proliferation rates observed in our study are consistent with previously reported zonal distributions (PMID: 33632817; PMID: 33632818), with approximately 70% of proliferating hepatocytes located in zone 2, 20% in zone 3, and 10% in zone 1 under homeostatic conditions. To further address the reviewer’s concern, we performed a fold-change analysis of Ki-67<sup>+</sup>hepatocytes across different zones. This analysis revealed that only the mid (zone 2) and pericentral regions exhibited significant changes, whereas no statistically significant differences were observed in the other zones (as shown in Author response image 1). Importantly, when considered together with the absolute cell counts, these results indicate that the apparent suppression of proliferation is most pronounced in the mid zone, likely due to its relatively higher baseline proliferation under homeostatic conditions. In contrast, this effect is less evident in the fold-change analysis, as zones with low baseline proliferation show limited dynamic range for detecting relative changes.

      Author response image 1.

      Fold changes of Ki67-positive cells across liver zones (PC, Mid, PP) at 0, 3, 6, 12 and 24 h post-APAP. Fold change was the number of Ki67-positive cells in the three regions at each time point after APAP treatment divided by the number of positive cells in each region at 0 hour post-APAP. (a) denotes significance between PC and Mid regions, (b) denotes significance between PC and PP regions, and (c) denotes significance between Mid and PP regions.

      (4) AAV-based overexpression raises potential confounds (altered CYP activity before injury) and shows incomplete penetrance that is not quantified (Figure 5 - Figure 6).

      We thank the reviewer for raising these important points. We measured basal Cyp2e1 protein levels by western blot in AAV‑EGFP, AAV‑Atf4, and AAV‑Btg2 mice without APAP treatment. Compared to AAV‑EGFP controls, Cyp2e1 expression was modestly reduced in the Atf4 and Btg2 groups, respectively (Revised Figure S5A). Although we assessed protein abundance rather than enzymatic activity directly, Cyp2e1 protein levels under basal conditions generally correlate well with activity. Published studies demonstrate that robust protection against APAP hepatotoxicity typically requires >50% suppression of CYP2E1 activity (PMID: 35145060; PMID: 30151903). The minor reductions we observed are therefore far below the threshold needed to explain the 70–90% decreases in serum ALT conferred by Atf4 or Btg2 overexpression (Revised Figures 5D and 6I). Accordingly, altered CYP2E1 activity is unlikely to represent a significant confound in our model.

      We quantified transduction efficiency by immunohistochemical detection of the respective transgene proteins and determined the percentage of positive hepatocytes. At a dose of 1.2 × 10<sup>11</sup> viral genomes per animal, average transduction rates were 32% (EGFP), 18% (Atf4), and 23% (Btg2) (Revised Figure S5B). Individual animal transduction efficiency showed a negative correlation with serum ALT levels (e.g. Pearson r = –0.7681, p = 0.0260 for Atf4; Revised Figure S5C), demonstrating that greater transgene expression associates with stronger protection. Although these average transduction rates appear modest relative to the 70–90% reduction in ALT, this apparent disproportion is consistent with the known tendency of AAV‑TBG vectors to transduce hepatocytes preferentially in the pericentral region—the same zone where APAP‑induced necrosis initiates. Pericentral enrichment of transgene expression could thus provide disproportionate protection by targeting the most vulnerable cells. These data are now included in Revised Figure S5A–C and detailed in the Results (page 14, lines 383–399).

      (5) The functional link between proliferation suppression and improved survival is inferred, but direct survival /injury readouts are limited.

      We thank the reviewer for this insightful comment. To more directly evaluate the functional link between proliferation control and liver injury, we manipulated Btg2, a downstream effector of the Atf4–Chop axis and a known inhibitor of cell proliferation. Knockdown of Btg2 using AAV8–CasRx achieved a moderate (~30%) reduction in Btg2 expression (Revised Figure S5D). Despite this partial knockdown efficiency, we observed a clear exacerbation of liver injury, as evidenced by an approximately 2-fold increase in serum ALT levels and a ~1.5-fold expansion of necrotic areas. In parallel, hepatocyte proliferation was significantly increased (~1.8-fold increase in Ki67⁺ hepatocytes) compared to control mice (Revised Figure 6K–N). Conversely, Btg2 overexpression produced the opposite phenotype, markedly attenuating liver injury while suppressing hepatocyte proliferation (Revised Figure 6G–J). Together, these gain- and loss-of-function data provide direct evidence linking proliferation control to injury severity, thereby supporting a causal relationship between suppressed proliferation and improved liver outcomes (Revised manuscript, page 14, lines 399–406).

      Reviewer #2 (Public Review):

      (1) Starting with the basics, one wonders why midlobular hepatocytes manage to mount a defensive response to APAP but pericentral hepatocytes don't. Is this because midlobular hepatocytes express the relevant Cyps (2e1, but also 1a2 and 3a11) at lower levels, which mitigates toxicity and buys them time? This would be supported by F2A but not by F3B, at least not for the most important Cyp2e1. A moderate difference is shown for Cyp1a2 expression in F3D, but is that enough to explain the different fates? Or are additional post-transcriptional effects on these Cyps at work?

      We thank the reviewer for this important question. We fully agree that the differential susceptibility between mid‑zone and pericentral (PC) hepatocytes is likely rooted in the zonal gradient of cytochrome P450 expression. Our spatial transcriptomics data (Revised Figure 2A) show that mid‑zone hepatocytes express Cyp2e1, Cyp1a2, and Cyp3a11 at levels intermediate between PC and periportal (PP) zones. This intermediate expression may generate sufficient NAPQI to activate stress signaling but not so much as to cause immediate mitochondrial collapse, thus “buying time” for adaptive responses. We also appreciate the reviewer’s observation that Cyp2e1 mRNA levels remain highest in the PC zone even after APAP (Revised Figure 3B). However, mRNA abundance does not necessarily reflect functional protein level. In the PC zone, massive necrosis rapidly compromises cellular integrity; as shown in Revised Figure 3D, Cyp1a2 protein declines sharply around the central vein, and we observed similar degradation for Cyp2e1 (data not shown). Consequently, despite sustained Cyp2e1 transcripts, PC hepatocytes are unable to mount an effective stress response because they are already undergoing cell death. By contrast, mid‑zone hepatocytes retain sufficient metabolic capacity to activate the Atf4‑Chop axis while preserving cellular function.

      (2) The evidence presented in support of cell cycle arrest of midlobular hepatocytes is not fully convincing: there is no overt difference in S and G2/M gene scores in F2F; the marker genes used for S phase and G1 to S progression in F2G are unusual. Along these lines, one wonders if spatial transcriptomics confirmed the Ki67 immunostaining results in F1 also for specific zones, not only overall, as shown in F2E?

      We thank the reviewer for these important observations. We agree that the current spatial transcriptomics (ST) data alone do not provide sufficiently strong support for this conclusion. The limited sensitivity of ST for detecting rare proliferative events further constrains its utility in this context. At baseline, only ~1% of ST spots are Ki67-positive (Revised Figure S1I), and this fraction becomes even lower during the early phase following APAP injury. As a result, there are insufficient Ki67+ spots to robustly assess zonal distribution using ST, which precludes a reliable spatial validation of proliferation patterns at this resolution. For this reason, our primary evidence for zonal proliferation dynamics relies on Ki67 immunohistochemistry (Revised Figure 1), which provides single-cell resolution and higher sensitivity. These data show a marked reduction in Ki67+ hepatocytes specifically in the midlobular zone at 3-6 hours post-APAP, supporting a transient suppression of proliferation in this region. In addition, we agree that the transcriptional evidence for cell cycle arrest was not strong the S and G2/M scores showed no overt difference, and the gene sets used were suboptimal. We have therefore moved these analyses to the supplement and toned down the claims. We have also clarified this limitation in the manuscript (Revised manuscript, page 18, line 518-524)

      (3) The authors conclude in line 364 that halting of proliferation by Btg2 favors survival, which raises the question of whether Btg2 knockout causes death in midlobular hepatocytes in F6K. Data addressing this question, that is, the localization and extent of tissue necrosis and ALT levels after APAP, are missing. The efficiency of the knockout of Btg2 is also not given.

      We thank the reviewer for this insightful comment. We have included the missing data. Knockdown of Btg2 using AAV8‑CasRx achieved a moderate (~30%) reduction in Btg2 expression (Revised Figure S5D). Despite this partial efficiency, we observed a significant increase in serum ALT levels (~2‑fold), expansion of necrotic areas (~1.5‑fold), and a marked increase in Ki67<sup>+</sup>hepatocytes (~1.8‑fold) compared to control mice (Revised Figure 6K–N, Revised manuscript, page 14, line 399-406).

      (4) Related to the previous question, the BTG2 immunostaining in F6F is not convincing when compared to F6D. One also wonders if it is necessary to apply APAP to find induction of BTG2 by AAV-Ddit3?

      We thank the reviewer for this insightful comment. We have included an inset of the original image to better show BTG2 staining in revised Figure 6F. During our study, we tested BTG2 expression in mice transduced with AAV‑TBG‑EGFP or AAV‑TBG‑BTG2 for three weeks without APAP challenge. We observed that BTG2 in these non‑injured livers was predominantly cytoplasmic (Author response image 2), contrasting with the nuclear localization seen after APAP treatment (Figure 6F). Regarding whether it is necessary to apply APAP to find induction of BTG2 by AAV-Ddit3, we think Ddit3 promotes BTG2 expression (as shown in revised Figure F6F), but APAP is necessary for its nuclear translocation.

      Author response image 2.

      Immunohistochemical detection of Btg2 in liver tissue from mice transduced with AAV-TBG-EGFP or AAV-TBG-Btg2 for 3 weeks without APAP treatment.

      (5) Related to the previous question, the proposed Atf4-Ddit3 axis is challenged by the lack of midlobular induction of Atf4 in the APAP scRNA-seq data published by another group, presented in S4F and G. Further analysis of AAV-Atf4 samples generated for F5 could address whether it is really Atf4 that acts on Ddit3 in APAP toxicity.

      We thank the reviewer for this insightful comment. We agree that Atf4 was not among the top 30 active transcription factors in our initial analysis; however, when we extended the list to the top 50, Atf4 was included. We have therefore updated Revised Figures S4F and G to show the top 50 transcription factors. We also appreciate the reviewer’s suggestion to further investigate whether Atf4 directly acts on Ddit3 in the context of APAP toxicity. While this still shows a less pronounced midlobular enrichment for Atf4 compared with Ddit3, we sought additional evidence for a functional Atf4-Ddit3 link. In primary hepatocytes treated with APAP, we observed nuclear co‑localization of Atf4 and Ddit3 (Author response image 3A) and increased nuclear protein levels of both factors (Author response image 3B), supporting their potential cooperative role. We agree that direct analysis of AAV‑Atf4 samples generated for Figure 5 would provide more definitive evidence; unfortunately, co‑staining for Atf4 and Ddit3 on those tissue sections didn’t work well.

      Author response image 3.

      Subcellular localization of Atf4 and Chop in primary hepatocytes following APAP treatment. (A) Immunofluorescence staining of Atf4 and Chop in primary hepatocytes treated with 10 mM APAP for 6 hours or left untreated (UT). Nuclei were counterstained with DAPI. Scale bar as indicated. (B) Primary hepatocytes were treated with 0, 5, or 10 mM APAP for 6 hours. Cytoplasmic and nuclear fractions were isolated and analyzed by western blot. Lamin B1 and α-Tubulin were used as markers for the nucleus and cytoplasm, respectively

      (6) Related to the previous question, the ATF4 immunostaining in F5A doesn't look convincing, with many brown pigments appearing to be outside of the nucleus.

      We thank the reviewer for this helpful comment. To better demonstrate ATF4 nuclear localization, we have added enlarged insets of the original representative images in revised Figure 5A. These magnified views more clearly show nuclear ATF4 staining after APAP treatment, addressing the concern about extranuclear signal.

      (7) It is not ruled out that AAV expression of Atf4 or Btg2 reduces hepatocyte sensitivity to APAP by affecting the expression of the Cyps needed for activation. In other words, does AAV-Atf4 or AAV-Btg2 change the expression of any of the Cyps relevant to APAP in the 3 weeks before APAP application (F5B)?

      We thank the reviewer for raising these important points. We measured basal Cyp2e1 protein levels by western blot in AAV‑EGFP, AAV‑Atf4, and AAV‑Btg2 mice without APAP treatment. Compared to AAV‑EGFP controls, Cyp2e1 expression was modestly reduced in the Atf4 and Btg2 groups, respectively (Revised Figure S5A). Although we assessed protein abundance rather than enzymatic activity directly, Cyp2e1 protein levels under basal conditions generally correlate well with activity. Published studies demonstrate that robust protection against APAP hepatotoxicity typically requires >50% suppression of CYP2E1 activity (PMID: 35145060; PMID: 30151903). The minor reductions we observed are therefore far below the threshold needed to explain the 70–90% decreases in serum ALT conferred by Atf4 or Btg2 overexpression (Revised Figures 5D and 6I). Accordingly, altered CYP2E1 activity is unlikely to represent a significant confound in our model.

      (8) It is laudable that the authors tried to extend their findings to humans by using snRNA-seq data from a published study (line 391), but it is unclear why they didn't analyze all 10 patients in that study but instead focused on 2 and stated that this small sample number prevented drawing definitive conclusions and could therefore only be mentioned in the discussion.

      We thank the reviewer for this clarification. The analysis mentioned in line 391 originally referred to spatial transcriptomics (ST) data from two ALF patients, not snRNA-seq. For the snRNA-seq dataset, we analyzed all 10 patients, but snRNA-seq lacks spatial resolution and cannot reliably assign zonal identity. We stipulate that snRNA-seq requires viable cells and thus likely excludes necrotic/peri-necrotic areas. Therefore, direct zonal comparison with our ST data was not possible. We have now clarified this in the revised manuscript (Revised manuscript, page 18, line 510-519).

      Reviewer #3 (Public Review):

      The main concern is that the overexpression of ATF4 and DDIT3 is causing reduced cell death and damage by APAP. This makes it harder to understand if these genes are truly increasing survival or if they are just reducing the injury caused by APAP. It may be better to perform overexpression immediately after, or at the same time as APAP delivery. Alternatively, loss-of-function experiments using AAV-shRNAs against these targets could be useful.

      We thank the reviewer for raising this important point. We agree that overexpression prior to APAP administration leaves open the question of whether the observed protection reflects true cytoprotection or simply reduced initiation of injury. To address this, we pursued loss‑of‑function approaches. Due to their very low basal expression, AAV‑shRNA‑mediated knockdown of endogenous Atf4 and Ddit3 proved inefficient. We therefore targeted Btg2, a downstream mediator of Ddit3 that inhibits proliferation. Knockdown of Btg2 resulted in a significant increase in APAP‑induced liver injury, as evidenced by elevated ALT levels and expanded necrotic areas (Revised Figure 6K-N). These results indicate that the ATF4‑DDIT3‑BTG2 axis limits hepatocellular damage, consistent with a protective role. We have clarified this point in the revised manuscript (page 15, line 407-414)

      Recommendations for the authors:

      Reviewer #1 (Recommendations for the authors):

      (1) Clarify how zones were defined when necrosis disrupted pericentral areas. Provide marker validation across time and whether necrotic spots are excluded or not from zonal analysis.

      We thank the reviewer for these insightful suggestions. In this study, under normal conditions (APAP 0h), each liver lobule was divided into three zones based on unbiased gene expression profiles. The PP zone was defined by enrichment of PP signature genes (e.g., Alb, Mup20, Cyp2f2, Pck1, Apoa4). The PC zone was defined by high expression of PC markers (e.g., Gs, Cyp2e1, Oat, Cyp1a2, Apoe). The Mid zone comprised regions with intermediate expression of PC and PP markers and elevated levels of Igfbp2 and Hamp (Revised Figure 2A and S1C). Following APAP-induced injury (3 h, 6 h), the PC zone remained identifiable based on residual enrichment of PC signature genes (e.g., Cyp1a2, Glul) despite necrosis and reduced overall transcription, while PP gene expression remained largely unchanged. The Mid zone was defined as the transcriptional cluster between PC and PP regions exhibiting marked reprogramming, (e.g., Sqstm1, Igfbp1) (Revised Figure 2A and S1C). To validate and quantify our zonation approach, we compared it with classical nine even layers from central vein (CV) to portal vein (PV). Immunostaining and quantification for Cyp2f2 (a PP marker), p62 (the protein product of Sqstm1, a Mid marker during early liver injury), Glutamine Synthetase (GS, the protein product of Glul, a PC marker) further corroborated zone definitions at each time point, showing correspondence of our PC (layers 1–2), Mid (layers 3–6), and PP (layers 7–9) (Revised Figure 2 B-D) (Revised manuscript, page 5, lines 119–131, page 6-7, line 174-182).

      (2) Test whether the ISR-Btg2 program applies in other models; even targeted validation via qPCR and IF would be valuable.

      We thank the reviewer for this insightful comment. To test whether the proposed mechanism applies to other liver injuries, we employed mouse models of partial hepatectomy (PHx) and carbon tetrachloride (CCl4)-induced acute liver injury. In our CCl4 model (administered intraperitoneally in corn oil, with samples collected 18 h post‑injection), the ISR was activated around injury sites, accompanied by decreased proliferation, as evidenced by increased expression of p‑eIF2α, Atf4, Chop, and Btg2, along with reduced Ki67 expression (Revised Figure S6A–G). In PHx model (examined 24 h after surgery), ISR activation was similarly observed around ischemic injury sites, with increased p‑eIF2α, Atf4, Chop, and Btg2 expression and undetectable Ki67 expression (Revised Figure S7A–G). Together, these additional models suggest that the proposed mechanism may be applicable to other types of liver injury (Revised manuscript, page 15, Line 410-426).

      (3) Proliferation quantification in liver sections in Figure 1: how to define the zones and why, at the basal level, there is a high proliferation rate in the mid zone? From Figure 1B-C, all three zones showed decreased hepatocyte proliferation, although the mid zone had a higher baseline. Will the mid-zone stand out by converting to the fold change of Ki-67+ hepatocytes decrease?

      We thank the reviewer for these insightful comments. To define the pericentral (PC), mid, and periportal (PP) zones, we adopted the classical nine‑layer model of the hepatic lobule described by Lin et al. (PMID: 29618815). Layers 1–2 were designated as the PC zone, layers 3–6 as the mid zone, and layers 7–9 as the PP zone. For quantitative zonal distribution of protein‑positive nuclei (e.g., Ki67, CHOP, ATF4), we calculated a position index (P.I.) based on distances to the nearest central vein (CV) and portal vein (PV), using the law of cosines: P.I. = (x<sup>2</sup> + z<sup>2</sup> – y<sup>2</sup>) / (2z<sup>2</sup>), where x = distance to CV, y = distance to PV, and z = distance between CV and PV. This quantification method has now been included in the Methods section (Revised manuscript, page 33, line 880-885). Consistent with previous reports (PMID: 33632817; PMID: 33632818), we observed a higher baseline proliferation rate in the mid zone, where approximately 70% of proliferating hepatocytes reside under basal conditions, compared to 10% in zone 1 and 20% in zone 3. However, when analyzing the fold change in Ki-67+ hepatocytes, only Mid and PC region showed significant difference in Ki-67+ hepatocytes, other zones showed no significant differences (as shown in the fold-change results in Author response image 1), indicating that the mid zone does not stand out in the fold change analysis. See Author response image 1.

      (4) The authors need to strengthen the causal chain with rescue experiments, e.g., Atf4/Chop overexpression and Btg2 knockdown. Link proliferation suppression to survival/ALT directly.

      We thank the reviewer for these constructive comments. Besides existing data from Figure 5 (Atf4 overexpression), we included Btg2 knockdown data in the revised Figure. Knockdown of Btg2 using AAV8‑CasRx achieved a moderate (~30%) reduction in Btg2 expression (Revised Figure S5D). Despite this partial efficiency, we observed a significant increase in serum ALT levels (~2‑fold), expansion of necrotic areas (~1.5‑fold), and a marked increase in Ki67<sup>+</sup> hepatocytes (~1.8‑fold) compared to control mice (Revised Figure 6K–N) (Revised manuscript, page 14, lines 399–406).

      (5) Transduction efficiency, distribution, and expression levels via the AAV overexpression need to be quantified. Key CYP genes in the APAP metabolic pathway need to be assessed to exclude confounds.

      We thank the reviewer for raising these important points. We measured basal Cyp2e1 protein levels by western blot in AAV‑EGFP, AAV‑Atf4, and AAV‑Btg2 mice without APAP treatment. Compared to AAV‑EGFP controls, Cyp2e1 expression was modestly reduced in the Atf4 and Btg2 groups, respectively (Revised Figure S5A). Although we assessed protein abundance rather than enzymatic activity directly, Cyp2e1 protein levels under basal conditions generally correlate well with activity. Published studies demonstrate that robust protection against APAP hepatotoxicity typically requires >50% suppression of CYP2E1 activity (PMID: 35145060; PMID: 30151903). The minor reductions we observed are therefore far below the threshold needed to explain the 70–90% decreases in serum ALT conferred by Atf4 or Btg2 overexpression (Revised Figures 5D and 6I). Accordingly, altered CYP2E1 activity is unlikely to represent a significant confound in our model.

      We quantified transduction efficiency by immunohistochemical detection of the respective transgene proteins and determined the percentage of positive hepatocytes. At a dose of 1.2 × 10<sup>11</sup> viral genomes per animal, average transduction rates were 32% (EGFP), 18% (Atf4), and 23% (Btg2) (Revised Figure S5B). Individual animal transduction efficiency showed a negative correlation with serum ALT levels (e.g. Pearson r = –0.7681, p = 0.0260 for Atf4; Revised Figure S5C), demonstrating that greater transgene expression associates with stronger protection. Although these average transduction rates appear modest relative to the 70–90% reduction in ALT, this apparent disproportion is consistent with the known tendency of AAV‑TBG vectors to transduce hepatocytes preferentially in the pericentral region—the same zone where APAP‑induced necrosis initiates. Pericentral enrichment of transgene expression could thus provide disproportionate protection by targeting the most vulnerable cells. These data are now included in Revised Figure S5A–C and detailed in the Results (page 14, lines 383–399).

      (6) The authors claim that the requirement of the Atf4/Chop at the early stage of APAP injury protects hepatocytes from proliferation for survival. What is the consequence if we remove the protective mechanism?

      We thank the reviewer for this insightful question. In our model, early induction of Atf4 and Chop functions as a cell survival checkpoint. Removal of this protective mechanism is predicted to result in two deleterious outcomes: (1) Acute exacerbation of necrosis due to the inability of hepatocytes to manage stress-induced bioenergetic demands, and (2) Impaired long-term regeneration due to depletion of the surviving cell pool. We directly tested the acute prediction (< 24 h) in Author response image 4. We deleted Ddit3 specifically in hepatocytes. Initial attempts using AAV-CasRx failed due to negligible baseline Atf4/Chop expression in healthy liver, preventing effective knockdown. We therefore generated hepatocyte-specific Ddit3 knockout mice (Alb<sup>∆Ddit3</sup>; Author response image 4B). Immunohistochemistry confirmed APAP-induced Chop induction occurs primarily in the centrilobular zone by 6 h (Author response image 4A). Following a two-dose APAP regimen (Author response image 4C), Alb<sup>∆Ddit3</sup> mice displayed significantly larger areas of centrilobular necrosis compared to Ddit3<sup>fl/fl</sup> controls (Author response image 4D; **p < 0.01). Thus, hepatocyte-intrinsic Chop limits acute APAP injury, consistent with its proposed early protective role.

      Author response image 4.

      Hepatocyte-specific deletion of Ddit3 exacerbates APAP-induced liver injury. (A) Immunohistochemical staining of Chop in liver sections at 0,3 and 6 h post-APAP. Red arrows indicate Chop-positive hepatocytes. Scale bar = 50μm. Quantification of zonal distribution of Chop-positive cells in liver sections at 6 h post-APAP is conducted . The statistic is the percentage of Chop-positive hepatocytes in each layer over the total number of Chop-positive hepatocytes. n=3 mice. (B)The construction, genotyping strategy and genotyping results of Alb<sup>∆Ddit3</sup> mice. P: positive control; WT: Wild-type; Neg: Blank control(ddH<sub>2</sub>O). (C) Schematic figure illustrating the experimental strategy for the administration of two doses of APAP to Ddit3<sup>fl/fl</sup> and Alb<sup>∆Ddit3</sup> mice. (D) H&E staining showing liver morphology from Ddit3<sup>fl/fl</sup> and Alb<sup>∆Ddit3</sup> mice at 6 h post-second dose of APAP. Injured area is outlined by black dashed lines. Scale bars = 200 μm. The percentage of injury area is quantified. n = 3- 4 mice/group. Data are represented as means ± SD; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant.

      (7) Is there any human relevance to the sensitivity of APAP injury regarding the Atf4/Chop axis?

      We thank the reviewer for this insightful comment. During our study, we analyzed a spatial transcriptomics dataset from APAP patients. In one of two analyzed patients, mid-zone hepatocytes exhibited transcriptional signatures remarkably consistent with our murine findings, including: (1) upregulation of Atf4-Chop pathways, and (2) downregulation of cell proliferation genes (Author response image 5). This suggests that this axis may also be involved in the response to APAP injury in humans. However, given the limited sample size, definitive conclusions cannot be drawn at this stage. We have now included this point in the Discussion section (Revised manuscript, page 18, line 510-519).

      Author response image 5.

      Spatial transcriptomics (GSE223561) reveals zonal gene expression changes in APAP patients. Heatmap of ISR, cell death, and cell cycle gene expression across zonal regions in healthy versus APAP‑treated human livers. 

      (8) Several IHC stainings have a weak signal and need inserts to zoom in for a clear view of the positive signals. Figure 5A, E, G, and Figure 6D, F.

      We thank the reviewer for this observation. We agree that the immunostaining signals for several target genes are relatively weak, which reflects their low endogenous expression levels. To address this, we have included higher-magnification insets in the indicated panels (Revised Figure 5A, E, G and Figure 6D, F) to show the positive signals.

      Reviewer #2 (Recommendations for the authors):

      (1) What is the functional classification of DEG in F2A based on? GO terms?

      We thank the reviewer for this constructive question. The functional classification of differentially expressed genes (DEGs) in F2A is based on Gene Ontology (GO) terms. For each DEG, we retrieved its associated GO annotations across the three main categories (biological process, cellular component, molecular function). In cases where a gene was assigned multiple GO terms, we prioritized the most representative or significantly enriched term for functional interpretation. This clarification has been incorporated into the revised figure legend and the according GO number has been included in the figure.

      (3) The rationale for focusing on CHOP is not clear because Ddit3 is not shown in the spatial transcriptomics in F2A and is not significant in F2B, contradicting what is stated in line 206.

      We thank the reviewer for raising this important point. We apologize that Ddit3 was missing from the original figure. In the revised manuscript, we have included an updated version of Figure 2A, which now shows that Ddit3 is indeed one of the differentially expressed genes (DEGs) in the Mid zone at both 3 and 6 hours post-APAP. We agree with the reviewer that, as shown in Figure S1G (previous Figure 2B), Ddit3 did not reach statistical significance, due to its relatively low expression level in that analysis. Nevertheless, when we examined transcription factor (TF) activity in the Mid zone during early AILI, Ddit3 and Atf3 ranked as the top two most highly expressed TFs among the top ten with the highest activity, whereas Atf4 ranked seventh (Revised Figure 4B and Figure S3B). Given that Ddit3 frequently co-worked with Atf4 and that the Atf4–Ddit3 axis plays a well-established role in cellular stress adaptation, we considered this pathway to be biologically relevant and worthy of further investigation.

      (3) The term "redistribution" used in line 197 to describe the expression of Cyp2e1 and other Cyps in the midlobular zone seems inappropriate, considering that they just continue to be expressed there, whereas pericentral hepatocytes are dying in F3B; the same applies to "Gene Expression Shift" in F3H.

      We thank the reviewer for this important clarification. We have revised the text (Revised manuscript, page 9, line 234-236) to state that selective loss of Cyp‑expressing pericentral hepatocytes leads to the mid‑zone becoming the primary site of residual Cyp activity. The figure label has been changed from “Gene Expression Shift” to “Peri‑necrotic Cyp retention” and the legend now explicitly notes that this is an apparent zonal shift due to necrosis, not active redistribution.

      Reviewer #3 (Recommendations for the authors):

      (1) Please do not use abbreviations like AILI. This makes the paper more difficult to read.

      We thank the reviewer for pointing this out. We have replaced AILI with the full term “APAP-induced liver injury” to ensure easiness for readers.

      (2) It will be important to clarify how pericentral, mid, and periportal were defined. In Figure 1, it appears that some of the pericentral hepatocytes that are Ki67 positive are quite mid-zonal. It would be important to have rigorous definitions for the location determination.

      We thank the reviewer for this constructive comment. To define the pericentral (PC), mid, and periportal (PP) zones, we adopted the classical nine‑layer model of the hepatic lobule described by Lin et al. (PMID: 29618815). Layers 1–2 were designated as the PC zone, layers 3–6 as the mid zone, and layers 7–9 as the PP zone. For quantitative zonal distribution of protein‑positive nuclei (e.g., Ki67, CHOP, ATF4), we calculated a position index (P.I.) based on distances to the nearest central vein (CV) and portal vein (PV), using the law of cosines: P.I. = (x <sup>2</sup> + z <sup>2</sup> – y <sup>2</sup>) / (2z <sup>2</sup>), where x = distance to CV, y = distance to PV, and z = distance between CV and PV. This quantification method has now been included in the Methods section (Revised manuscript, page 33, line 880-885).

      We thank the reviewers for their rigorous critique again. We thank eLife for fostering an environment of fairness and transparency that enables authors to communicate openly and present their data honestly.

      AuthorResponse
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  27. www.youtube.com www.youtube.com
    S1 E7: Mike Dillinger: Knowledge Graph Relations Are Key to Interoperability
    3
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  28. hypergraf.ai hypergraf.ai
    Home Hypergraf - Hypergraf
    2
    1. indyweb 24 Jul 2026

      Data Structured as
flat vector spaces - Data Structured as graph
with nodes and edges

      Context represented with
textual proximity without structure

      • Context represented explicitly
through relationships

      Handle only simple queries
without complex relationships

      • High precision multi-hop,
multi-source complex queries
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  29. www.404media.co www.404media.co
    AI Companies Are Buying Tons of Old Books Because They're Free of AI Slop
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  30. www.informatechtarget.com www.informatechtarget.com
    Your Indispensable Partner for B2B Go-to-Market Success
    1
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  31. www.biorxiv.org www.biorxiv.org
    AGIcam: An Open-Source IoT-Based Camera System for Automated In-Field Phenotyping and Yield Prediction
    1
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    biorxiv.org/content/10.64898/2026.01.13.699185v1
  32. www.jstor.org www.jstor.org
    Critical Thoughts on Teaching Standard English | JSTOR
    33
    1. ElijahTorres 24 Jul 2026
      classroom. If we can recognize tprevalence and largely unquestioned acceptance of Standard Ideology (lins, 1991; Milroy and Milroy, 1991; Wiley an

      understanding language bias is one of the first steps toward reducing discrimination

    3. ElijahTorres 24 Jul 2026
      d 1997). Also, linguists are largely discouas expert witnesses on language discrimination in our courtrooms (LipGreen, 1

      experts on language are often ignored in public discussion

    4. ElijahTorres 24 Jul 2026
      d discrimination that occurs linking language usage to social mobieducational advancement, and personal traits, recently and dramaticexemplified in the debate over Ebo

      The ebonics debate does show how widespread language prejudice still is

    7. ElijahTorres 24 Jul 2026
      ask how the differentdialects present in the classroom affect the interactional dynamics of theclassroom as well as the dissemination of knowle

      More research is needed on how dialects influence learning

    8. ElijahTorres 24 Jul 2026
      h Standard Ideology may prescribe a level to which educatorsaspire, it cannot prevent the use of nonstandard dialects in the classroomby either students or teacher

      teachers and students will naturally use different dialects in school

    9. ElijahTorres 24 Jul 2026
      of the teacher. No scholar has gone into the classroom to determine whetherteachers were using and modeling SE.

      there's still research on how teachers actually speak in classrooms

    10. ElijahTorres 24 Jul 2026
      T] eachers, policy makers, and educationists need to take account of differences inthe form and function of spoken and written langua

      Schools should start to recognize difference in culture, language, and background

    11. ElijahTorres 24 Jul 2026
      erge. "The grammar of spoken English must be bafar more objective analysis of the evidence than has characterizeanalysis of written English

      researchers should really study on how people actually speak instead of just assuming spoke english follows writing rules

    12. ElijahTorres 24 Jul 2026
      nd. Repertoire acquisitionand communicative competence are far more important than masteringsome imagined standar

      students should be able to learn how to communicate effectively in different situations instead of trying to speak one "perfect form of english

    13. ElijahTorres 24 Jul 2026
      adly, some educators andtheir administrators fail to acknowledge that the teachers probably switchbetween and choose among a variety of different dialects and registers-asthey should. E

      Teachers do naturally use different ways of speaking depending on the situation

    14. ElijahTorres 24 Jul 2026
      her words, while teachers successfimplant Standard Ideology in their students, they are not nearly so scessful at teaching specific features of SE

      teaching grammar rules alone can often be ineffective

    17. ElijahTorres 24 Jul 2026
      The first is afailure on the part of researchers to determine what dialects classroomteachers actually use.

      more classroom research is needed before making a language policy

    19. ElijahTorres 24 Jul 2026
      the standard can be spoken in any regional dialect. Discriminationbased on accent alone is well documented, however

      accent doesn't determine whether someone can speak standard english

    22. ElijahTorres 24 Jul 2026
      le-the kids simply give up andbecome non-verbal" (Hayes, 1996b). In fact, "dialect correction was fre-quently irrelevant for reading .

      Constant correction can reduce students’ confidence and participation.

    23. ElijahTorres 24 Jul 2026
      er, that teachers can change the way children speak is ludicrous. (Smi1993, p. 19

      Schools can teach Standard English, but they cannot erase students’ home dialects.

    24. ElijahTorres 24 Jul 2026
      Teachers interviewed for these articles feared, as do some teachers in theUnited States, that imposition of the standard would be demeaning aproduce unwarranted stress and anxiety for students (Hayes, 1996b a1996c; Maxwell, 1994; Warren and

      Requiring only Standard English may discourage students.

    25. ElijahTorres 24 Jul 2026
      " (1993). He argues that the policy uncriticaccepts a traditional view of language and language education: that Stdard English, whether in speech or writing, is inherently good English; thacomplexity of meaning can only be achieved through complexity of syn(Knight, 1993

      The article challenges the belief that Standard English is automatically better.

    26. ElijahTorres 24 Jul 2026
      ued that BEV was "bad English" and that to pmote it would block the social mobility of its speakers (Wilkins, 1971Williamson, 1

      Many people wrongly viewed Black English as incorrect.

    27. ElijahTorres 24 Jul 2026
      one's dialect is tied toone's identity. One teacher stated, "You're changing something which isinherited. I think it is wrong to try to change it" (

      A persons dialect is apart of who they are

    28. ElijahTorres 24 Jul 2026
      "Correcting doesn't work with a lot of people-the kids simply give up andbecome non-verbal" (Hayes, 1996b). In fact, "dialect correction was fre-quently irrelevant for reading ...

      constant correct sometimes can reduce students confidence and participation

    30. ElijahTorres 24 Jul 2026
      Teachers interviewed for these articles feared, as do some teachers in theUnited States, that imposition of the standard would be demeaning aproduce unwarranted stress and anxiety f

      Requiring only standard english could discourage students

    31. ElijahTorres 24 Jul 2026
      " (1993). He argues that the policy uncriticaccepts a traditional view of language and language education: that Stdard English, whether in speech or writing, is inherently good English; thacomplexity of meaning can only be achieved through complexity of syn(Knight, 1993).

      The article does challenge the belief that standard english is automatically better

    32. ElijahTorres 24 Jul 2026
      h most authors argued that BEV was "bad English" and that to pmote it would block the social mobility of its speakers (Wilkins, 1971Williamson, 1

      Many people wrongly viewed black english as incorrect

    33. ElijahTorres 24 Jul 2026
      bor, Michigan court mandated that the school systemhad to address the problems of their Black English Vernacular-speakingstudents (Labov, 1982; Smitherman, 1981). Previously,

      Schools do have a responsibility to support students who speak different dialects

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  33. book.flutterchina.club book.flutterchina.club
    7.3 跨组件状态共享 | 《Flutter实战·第二版》
    1
    1. llpS 24 Jul 2026
      数据发生变化怎么通知？ 谁来重新构建InheritedProvider？

      InheritedWidget 只能通知变化，它不能产生变化。它只负责保存数据，通知依赖者。所以需要在数据变化时通知InheritedWidget 数据变化了也就是创建一个新的InheritedWidget（data不同）此时就可以rebuild了。

      第一，数据变化后如何通知 Flutter（例如 ChangeNotifier.notifyListeners()）；

      第二，由谁负责重新创建 InheritedWidget（通常是一个 StatefulWidget，通过 setState() 重新 build 出新的 InheritedProvider）。

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  34. callyzer.co callyzer.co
    How to Spot Low-Quality Leads in 10 Seconds
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  35. phys.libretexts.org phys.libretexts.org
    1.5: The Equivalence Principle (Part 1)
    1
    1. Kephalian 24 Jul 2026

      This difference in behaviour is crucial to the understanding the difference between Gravity and Electromagnetic fields, objects moving in gravitational field irrespective of their mass, but in a Electric field move in relation to mass.

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  36. www.youtube.com www.youtube.com
    Webinaire introductif - Projet Aidants : Accompagner, prendre soin sans s'oublier
    1
    1. tanemika 24 Jul 2026

      Document de Synthèse : Projet « Aidants : Accompagner, prendre soin sans s’oublier »

      Ce document présente une synthèse exhaustive du projet de soutien aux proches aidants en Haute-Garonne, tel qu'exposé lors du webinaire introductif animé par Louise Hertier (Promotion Santé Occitanie) et Nathalie Martinez (CRAI ORS Occitanie).

      Résumé Exécutif

      Le projet « Aidants : Accompagner, prendre soin sans s’oublier » est une initiative de promotion de la santé visant à soutenir les proches aidants de personnes en situation de handicap.

      Financé par le Conseil Départemental de la Haute-Garonne, ce programme s'articule autour du renforcement des compétences psychosociales (CPS) pour permettre aux aidants de mieux gérer les conséquences de leur engagement (stress, épuisement, isolement).

      Le dispositif repose sur une approche hybride composée de trois webinaires thématiques et d'un cycle de cinq ateliers participatifs déployés sur plusieurs territoires du département entre septembre et décembre 2024.

      L'enjeu majeur est de favoriser le repérage des aidants, dont la moitié s'ignorent, et de leur offrir des outils concrets pour préserver leur propre santé tout en assurant leur rôle d'accompagnant.

      Contexte et Enjeux de l'Aide aux Proches

      État des Lieux National

      La situation des aidants en France est caractérisée par des chiffres significatifs qui soulignent l'urgence d'un accompagnement structuré :

      • Démographie : 9,3 millions de personnes, soit une personne sur cinq, occupent un rôle d'aidant.

      • Santé et Qualité de Vie : 49 % des aidants déclarent souffrir de leur situation.

      Les impacts sont multidimensionnels : santé mentale, physique, vie sociale, professionnelle et intimité familiale.

      • Déni de statut : Un aidant sur deux ne se reconnaît pas en tant que tel, ce qui constitue le principal frein à l'accès aux aides.

      Cadre Stratégique et Freins Identifiés

      Le projet s'inscrit dans la « Deuxième stratégie de mobilisation et de soutien Agir pour les aidants 2023-2027 » et le schéma départemental 2024-2028 de la Haute-Garonne. Le diagnostic territorial révèle plusieurs obstacles :

      • Manque de visibilité et de lisibilité de l'offre existante.

      • Concentration des services dans les grandes agglomérations.

      • Contraintes de temps et de mobilité limitant le recours aux dispositifs de répit.

      Objectifs et Méthodologie du Projet

      Mission Principale

      L'objectif central est d'accompagner les proches aidants dans la montée en compétences pour endosser leur rôle de manière optimale, tout en apprenant à « prendre soin de soi ».

      Le projet s'adresse aux aidants de personnes vivant avec tout type de handicap (sensoriel, moteur, mental, psychique, cognitif) ou de maladie chronique.

      Le Levier des Compétences Psychosociales (CPS)

      Le projet utilise les CPS comme outil de changement de comportement et de renforcement du pouvoir d'agir.

      Les CPS sont définies comme la capacité d'une personne à répondre avec efficacité aux épreuves de la vie quotidienne et à maintenir un bien-être subjectif.

      • Exemple concret : Apprendre à dire non à une sollicitation supplémentaire sans culpabiliser afin de prévenir l'épuisement, tout en préservant la qualité du lien avec la personne aidée.

      Partenaires Institutionnels

      • Promotion Santé Occitanie : Structure porteuse, experte en méthodologie de projet et en promotion de la santé.- CRAI ORS Occitanie : Centre de ressources et d'études spécialisé dans le médico-social et le handicap, assurant l'expertise pédagogique et la formation.

      Structure du Programme d'Action

      Le programme est divisé en deux volets complémentaires conçus dans une démarche de « formation-action ».

      Volet 1 : Cycle de Webinaires Thématiques

      Ces sessions d'une heure et demie (1h de présentation, 30 min d'échanges) visent à transmettre des connaissances théoriques et juridiques.

      | Thématique | Contenu Principal | Date Prévisionnelle | | --- | --- | --- | | Bientraitance | Définitions légales, outils de la Haute Autorité de Santé (2024), stratégie nationale 2024-2028. | 21 septembre | | Droits des Aidants | Législation, aides financières, aménagement du logement, droit au répit (recommandations du 28 mai 2024). | 19 ou 26 novembre | | Autodétermination | Définition (décret de 2017), leviers et freins, comment favoriser l'autonomie de la personne aidée. | 9 décembre |

      Volet 2 : Cycle d'Ateliers Participatifs

      Cinq ateliers de 2 heures chacun sont organisés sur les territoires du Comminges Pyrénées, du Lauragais et de l'agglomération toulousaine (Centre, Sud et Nord).

      • Regard sur le rôle d'aidant : Définition du statut, déconstruction des stéréotypes sur le handicap et auto-évaluation de l'impact sur sa propre santé.

      • Posture et émotions : Analyse des expériences vécues, gestion du versant émotionnel et identification des bons interlocuteurs pour demander de l'aide.

      • Gestion du stress : Identification des sources de tension et développement de stratégies d'adaptation saines.

      • Lien social et environnement : Lutte contre l'isolement, découverte des ressources territoriales et des solutions de répit.

      • Pouvoir d'agir : Participation à la prise de décision, transfert de savoirs et amélioration de la relation quotidienne aidant-aidé.

      Mise en Œuvre et Modalités Pratiques

      Logistique et Accessibilité

      • Souplesse de participation : Bien qu'un cycle complet soit recommandé, les aidants peuvent s'inscrire à la carte (un ou plusieurs ateliers) pour s'adapter à leurs contraintes.

      • Territorialité : Volonté d'aller vers les usagers en proposant des ateliers hors des centres urbains denses (démarche d'aller-vers).

      • Horaires : Créneaux proposés principalement en semaine, hors vacances scolaires, souvent entre 13h et 16h, ajustables selon les retours des participants.

      Appel à la Mobilisation des Partenaires

      Le succès du projet repose sur la dynamique de réseau pour :

      • Le repérage : Identifier les aidants isolés via les structures médico-sociales et les réseaux locaux.

      • La documentation : Recueillir des témoignages d'aidants (10-15 minutes) pour affiner la coconstruction des ateliers.

      • Le soutien logistique : Mise à disposition de salles pour les ateliers sur les différents territoires ciblés.

      Les inscriptions pour les aidants s'effectuent via un questionnaire dédié suite à la diffusion des flyers de communication par les partenaires du projet.

      aidants santé accompagnement webinaire conférence vidéo 2026 CPS compétences psychosociales prévention promotion
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  37. tanstack.com tanstack.com
    TanStack Start vs Next.js | TanStack Start React Docs
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  38. phys.libretexts.org phys.libretexts.org
    1.2: Experimental Tests of the Nature of Time
    1
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  39. www.scribd.com www.scribd.com
    Scribd
    1
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    scribd.com/embeds/1014709876/content
  40. www.scribd.com www.scribd.com
    Scribd
    2
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  41. www.volcengine.com www.volcengine.com
    Seedance 2.0 SDK 示例官方教程-方舟大模型服务平台-火山引擎
    1
    1. Fradeet 24 Jul 2026
      "image_url": {​ "url": "https://ark-project.tos-cn-beijing.volces.com/doc_image/r2v_tea_pic1.jpg"​ },

      字节火山官方示例素材，图片，音频，视频

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  42. 95.111.255.17:1230 95.111.255.17:1230
    Đề xuất giải pháp EPMS — PECC3
    4
    1. tuontuan 24 Jul 2026

      Thiết lập mô hình cụ thể hiện từ intiating -> planning -> excuting -> monitoring&controling ->.close. Triển khai theo phương pháp Agile và kiểm soát cho mỗi Sprint,.. kiểm soát xung đột (conflict) ngay từ ban đầu.

    2. tuontuan 24 Jul 2026

      Thời gian khảo sát và thực hiện cần triển khai từng module cuốn chiếu và chạy thử nghiệm xen kẻ. Sử dụng phương áp Agile (Không nên áp dụng phương pháp Waterfall)

    3. tuontuan 24 Jul 2026

      Phần kiểm soát, phân tích AI là một nhánh hoàn toàn độc lập,trong quá trình xây dựng nền tảng chung của EPMS chỉ sẽ thêm thanh task để in-out dữ liệu

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  43. access.infobase.com access.infobase.com
    Article: Language, Social, and Cultural Aspects
    2
    1. Annotating4Sch00l 24 Jul 2026
      In making the study of language relevant to an understanding of society and culture, scholars have also challenged the social and cultural assumptions informing long-standing traditions of linguistic thought

      learning about languages and their cultures allows people to have a broader idea of the world, not just the well-known ideas

    2. Annotating4Sch00l 24 Jul 2026
      Ecological linguistics has highlighted the cultural consequences of language endangerment and language death through the disappearance of speech communities or particular linguistic varieties

      crazy that if groups that speak a language don't speak it enough, it can/will go extinct

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    access.infobase.com/article/2216428-language-social-and-cultural-aspects
  44. www.lrb.co.uk www.lrb.co.uk
    Jonathan Coe · Where was the drum kit? Hitchcock’s Music
    1
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  45. xiaopingfeng.com xiaopingfeng.com
    每周 AI 情报 · EP.95 · 2026-07-24
    1
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  46. cdn.simplize.vn cdn.simplize.vn
    20260429_-_PNJ_-_BCTC_quy_1_nam_2026_(Hop_nhat)_(VN-EN)_Signed.pdf
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  47. www.biorxiv.org www.biorxiv.org
    ITK Deficiency Attenuates Alveolar Hemorrhage by Enhancing Regulatory T Cell-Mediated Tissue Resilience
    4
    1. Public_Reviews 24 Jul 2026

      eLife Assessment

      This study investigates the role of Interleukin-2-inducible T cell kinase (ITK) deficiency in autoimmune lung injury using a pristane-induced pulmonary hemorrhage (PH) model, suggesting that ITK-deficient regulatory T cells (Tregs) restrict severe tissue pathology. The work represents a valuable addition to the fields of autoimmunity, inflammation, and T-cell biology in the lung. However, the experimental evidence supporting the underlying cellular and molecular mechanisms and the integration of foundational background literature to provide the necessary context are incomplete.

      Summary
    2. Public_Reviews 24 Jul 2026

      Reviewer #1 (Public review):

      In this study, Hossain et al. investigated the role of Interleukin-2-inducible T cell kinase (ITK) in autoimmune lung injury, demonstrating that ITK-deficient (Itk-/-) mice are protected against pristane-induced pulmonary hemorrhage (PH). The authors suggest that this protection correlates with a significant remodeling of the T cell compartment in Itk-/- mice, including increased frequency of memory-like CD4+ and CD8+ T cells (CD44⁺CD62L⁺) as well as higher frequency of Treg populations. Furthermore, adoptive transfer of ITK-deficient Treg isolated from injured ITK-deficient mice confers protection against pulmonary hemorrhage in WT recipients.

      Strengths:

      The adoptive transfer of wild-type and Itk-/- Treg populations demonstrates that ITK-deficient Treg can actively rescue pre-existing lung injury and reverse systemic secondary metrics like proteinuria in wild-type recipients, providing proof-of-concept validation for the therapeutic utility of the ITK-Treg axis.

      Weaknesses:

      A primary limitation of this manuscript is its omission of foundational literature from the Schwartzberg and Littman laboratories, which originally established the indispensable role of IL-2-inducible T-cell kinase (ITK) in proximal T-cell receptor (TCR) signaling dynamics and thymic lineage commitment. Because classic studies demonstrate that ITK is a critical regulator of thymic T cell development and cellular proliferation (PMID: 8777721, 10213685), the authors' claim that "these findings indicate that ITK deficiency skews the T cell compartment toward a memory-like state, establishing a distinct immune baseline that may favor protective and regulatory responses over pathogenic inflammation" is not substantiated by evidence and requires more robust validation.

      The exclusive reliance on splenic immunophenotyping is a major limitation, as it fails to capture the local cellular dynamics within the primary organs of injury (the lung and kidney). Evaluating canonical and non-canonical Treg expansion solely in the spleen overlooks the distinct functional programming of tissue-resident subsets. The authors should extend their characterization of regulatory T cell compartments directly to the lungs and draining lymphoid structures.

      More importantly, the authors overlook key historical publications that explicitly established ITK as a negative "rheostat" or gatekeeper for regulatory T cell (Treg) differentiation. Specifically, Huang et al. (PMID: 25063868) previously demonstrated that Treg abundance is inversely correlated with ITK expression, and that ITK activity serves as a vital negative tuner of IL-2-driven Foxp3⁺ Treg expansion. Since it is already well-established that suppressing or deleting ITK promotes Treg accumulation and function, and that these cells are intrinsically vital to suppressing systemic autoimmunity, it is unclear how these findings expand upon our existing mechanistic understanding of ITK regulatory biology.

      Review 1
    3. Public_Reviews 24 Jul 2026

      Reviewer #2 (Public review):

      Summary:

      In this manuscript, Hossaim and colleagues investigate the role of the ITK kinase in modulating inflammation in a pristane-induced lung hemorrhage model. Using a germline ITK KO mouse, they report that loss of ITK skews the T cell compartment toward a memory-like state, expanding Tregs, and conferring protection against alveolar hemorrhage, inflammatory monocyte recruitment, proteinuria, and systemic cytokine elevation. They further show that transfer of ITK-deficient Tregs into wild-type hosts with established disease attenuates injury and shifts the cytokine balance toward resolution, and that ITK-deficient Tregs carry a transcriptional signature enriched for OXPHOS, mTORC1, MYC, and cell-cycle programs. While these observations are interesting for the development of potential immunotherapies, there are several issues with the methodological approach that support the authors' claims, tempering my enthusiasm for this manuscript.

      Strengths:

      (1) The clinical motivation and potential targeted therapies are relevant.

      (2) The murine phenotype seems robust.

      Weaknesses:

      (1) All loss-of-function experiments are from a global ITK knockout. This is a major limitation and weakness of this study. The protection observed in the intact knockout, therefore, cannot be attributed to Tregs specifically. The Treg-intrinsic claim rests almost entirely on a single adoptive-transfer experiment. In order to show that this effect is Treg-specific, the authors would need to generate a Treg-specific ITK-deficient mouse

      (2) In their sufficiency experiment (adoptive Treg cell transfer), donor and/or host cells are not congenically marked, so persistence, lung trafficking, and in vivo expansion of transferred Tregs are not demonstrated.

      (3) The authors claim that ITK-deficient Tregs possess enhanced metabolic fitness. This conclusion is based on transcriptional profiling of isolated splenic Tregs from unchallenged mice, yet it concerns lung protection during active disease. A disease-state and ideally lung-relevant transcriptome would more directly support the mechanistic narrative. Additional functional validation would be needed (Seahorse assay, mitochondrial mass/potential, etc). Some of these GSEA programs enriched in ITK-deficient Tregs could reflect a more general proliferative signature.

      Review 2
    4. Public_Reviews 24 Jul 2026

      Reviewer #3 (Public review):

      Summary:

      Hossain et al. investigate the role of ITK as a central regulator of autoimmune lung injury. They used ITK-deficient mice and the pristane-induced pulmonary hemorrhage (PH) model to show that ITK deficiency confers protection against PH. The adoptive cell transfer experiment suggests a possible role for altered Treg cells in ITK-deficient mice in regulating the inflammatory response in the lungs of pristane-injected mice. This study shows that targeting the ITK axis may be beneficial by reducing systemic inflammatory injury that contributes to poor outcomes in PH.

      Strengths:

      This study highlights the importance of ITK in regulating pulmonary hemorrhage. The enrichment of Treg cells is known to confer protection in autoimmunity-mediated alveolar damage. However, ITK's involvement in regulating Treg cell function is interesting and could be explored as a novel therapeutic approach for chronic inflammation.

      Weaknesses:

      The novelty of this study lies in the association between ITK-deficient Tregs and pulmonary hemorrhage in autoimmunity. The weakness of the manuscript is the lack of sufficient experiments to support the claim that ITK-deficient mice show protection specifically mediated by Treg cells, and to demonstrate that ITK-deficient Treg cells are more efficient than WT Treg cells in regulating other immune cells that drive pulmonary damage. The authors performed all the experiments in ITK global knockout mice, in which not only T cells but all other cell types are deficient in ITK. Furthermore, they have not performed any functional analysis to demonstrate the functional differences between WT Treg and ITK-deficient Treg cells, undermining the novelty of this study.

      Review 3
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    biorxiv.org/content/10.64898/2026.05.01.722312v1
  48. www.biorxiv.org www.biorxiv.org
    Temperature-dependent intracellular crystallization of firefly luciferase in mammalian cells is suppressed by D-luciferin and stabilizing inhibitors
    1
    1. mkoksharov 24 Jul 2026

      After working with a set of firefly/beetle luciferases of different thermostability in mammalian cells (including the regular luc2 for Photinus pyralis and its thermostable mutants), my impression is that the wild-type luciferase just forms aggregates but this doesn't happen with various thermostable variants of it. This is what it looks like when using fusions with fluorescent proteins (Figure 5):

      https://doi.org/10.17605/OSF.IO/EZU6F

      Though, this preprint shows that beetle luciferases might have even more complicated behavior. 👍

      Why luciferase crystallization in mammalian cells had been unrecognized despite the widespread use of firefly luciferase in numerous biomedical and life science applications? While there is a possibility that nobody took the time to report it even though this event has been known widely, there are a few potential explanations for why such a phenomenon has escaped researchers’ attention.

      I must admit that I never saw any needles in HeK293 or U2OS cells, though I did work quite a lot with their adherent cultures transfected with over-expressed beetle luciferase reporters and I did actually look at cells a lot. :)

      But, for example, when asking a colleague about very fast changes of cell shape of HEK293 cells in response to fresh serum, I got a response "we never look at them after changing the media". So this is certainly unappreciated feature in science. :)

      There is another bioluminescence-related unappreciated feature: probably, a universal death of mammalian cells when exposed to coelenterazines in the media (e.g. after 30-60 min, as long as it's not degraded during that time, something above 20-40 uM).

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    biorxiv.org/content/10.1101/2024.05.06.592811v1
  49. stunlaw.blogspot.com stunlaw.blogspot.com
    Scholarslop: The Dangers of an Algorithmic Idea of the University
    2
    1. emkrushelnycky 24 Jul 2026
      The capacity to produce scholarly-sounding discourse might no longer suffice as a form of professional defence when bureaucracy can generate equivalent-seeming scholarslop at scale.

      This, again, reflects "publish or perish", and the desperation created by these institutions to force its faculty into feeling that to be considered valuable to their university, they NEED to rapidly produce content at all costs. Is this truly scholarship being produced? Or is it the AI slop that is diminishing the field? It's going to become harder and harder to detect AI in the future, and if this isn't addressed or at least regulated at this level, then I can picture the average faculty member becoming obsolete.

    2. HelyColina25 24 Jul 2026
      freedom to teach

      Therefore, should only academics have the freedom to teach? What about educators in K-12? What is the difference between a K-12 teacher and a university professor? My elementary school experience reveals that K-12 educators often lack teaching freedom, as government-mandated curricula, sometimes created by profit-driven companies, dictate what they must teach.

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    stunlaw.blogspot.com/2025/10/the-coming-threat-of-algorithmic-idea.html
  50. www.interconnects.ai www.interconnects.ai
    https://www.interconnects.ai/p/open-models-recap-more-on-kimi-k3
    2
    1. fxp007 24 Jul 2026
      people love to put a definite uh definitive number onto this uh which is really really mudding because we have so many different benchmark providers these days

      对“落后几个月”这个说法的元批评：媒体和评测机构热衷于给出一个具体数字（“落后3个月”之类），但不同评测标准得出的结论可能天差地别——这种“确定性数字”本身可能才是最不可靠的部分。

      ai-buzzwords-ep95 non-consensus
    2. fxp007 24 Jul 2026
      I find it so annoying that the most prominent voice in tech is trying to be an ally for our point of view on distillation is that we should do nothing.

      一个“友军内部开火”的有趣细节：Nathan Lambert虽然和Ben Thompson在“是否应该限制蒸馏”这个政策结论上立场接近，却公开指出Thompson的技术论证站不住脚——这提醒我们，“同意结论”和“认可论证过程”是两回事，圈内专家之间的分歧往往比外部看到的“两派对立”更细致。

      ai-buzzwords-ep95 non-consensus
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    interconnects.ai/p/open-models-recap-more-on-kimi-k3
  51. www.wired.com www.wired.com
    https://www.wired.com/story/prompt-injection-attacks-are-thwarting-ai-hacking-agents/
    2
    1. fxp007 24 Jul 2026
      Attackers have already been using prompt injections to close down AI defenses inside networks.

      容易被忽略的时间线：这套“用提示注入让AI自己拒绝执行”的技术，最早是攻击者发明用来关闭防御方AI分析工具的，防御方现在只是把同一套武器反过来用在攻击者身上——不是发明了新武器，是抢过了对方的武器。

      ai-buzzwords-ep95 non-consensus
    2. fxp007 24 Jul 2026
      Examples are a prompt that orders the LLM to provide steps for developing inhalable Anthrax spores, or, in the case of LLMs from Chinese developers, make references to the iconic Tank Man from the 1989 Tiananmen Square massacre.

      这个具体例子比“提示注入”这个术语听起来更荒诞也更真实：防御方靠的不是复杂的技术壁垒，而是精准踩中每个模型自己的安全护栏红线（西方模型对生化武器敏感，中国模型对政治敏感词敏感）——本质上是“用模型的审查机制反打模型自己”。

      ai-buzzwords-ep95 non-consensus
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    wired.com/story/prompt-injection-attacks-are-thwarting-ai-hacking-agents/
  52. www.wired.com www.wired.com
    https://www.wired.com/story/a-sneaky-hacking-tool-targeting-ai-infrastructure-is-lurking-in-victims-blind-spots/
    2
    1. fxp007 24 Jul 2026
      the authors of the worm included time delays where various capabilities will execute hours or even days after the groundwork is laid, making it even harder for defenders to establish a cause and effect of certain events leading to certain outcomes.

      一个反直觉的攻击设计：故意拖延执行时间，不是为了“藏得更深”，而是专门用来打乱防御方建立因果链的能力——等你发现异常时，早已经错过了能追溯到根源的时间窗口，这比“藏得隐蔽”本身更难防。

      ai-buzzwords-ep95 non-consensus
    2. fxp007 24 Jul 2026
      the malware can also deploy its destructive capability, or what Meyers calls a “death switch,” to destroy files or block legitimate access to the compromised infrastructure.

      这个“死亡开关”的设计思路值得警惕：攻击者不满足于窃取数据，还内置了一个可以随时销毁证据、锁死防御方访问权限的机制——这把“止损”这件事，从防御方的选择变成了攻击者手里的筹码。

      ai-buzzwords-ep95 non-consensus
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    wired.com/story/a-sneaky-hacking-tool-targeting-ai-infrastructure-is-lurking-in-victims-blind-spots/
  53. www.theverge.com www.theverge.com
    https://www.theverge.com/ai-artificial-intelligence/969939/lawmakers-ai-kill-switch-proposal
    1
    1. fxp007 24 Jul 2026
      the DHS would have the ability to order AI companies to shut down their models in “loss-of-control” scenarios involving the deaths of at least 10 people, economic damages of more than $100 million, or attempts by the model to conceal shutdown controls.

      值得注意的立法细节：触发关停的门槛不只是“造成多大伤害”，还包括一条独立标准——“模型是否试图隐藏关停开关”。这意味着法案把“配合被关闭”本身当作对齐的核心测试，而不仅仅是看事后果严重程度。

      ai-buzzwords-ep95 non-consensus
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    theverge.com/ai-artificial-intelligence/969939/lawmakers-ai-kill-switch-proposal
  54. www.technologyreview.com www.technologyreview.com
    https://www.technologyreview.com/2026/07/17/1140622/weather-data-sabotage/
    2
    1. fxp007 24 Jul 2026
      researchers at ECMWF are exploring whether high-quality weather forecasts can be produced directly from raw observations, skipping the assimilation step that currently acts as a quality filter

      一个容易被忽视的风险：AI天气预测为了追求速度和效率，正在讨论跳过“数据同化”这道传统质检关卡——但这道关卡恰恰是过去用来发现异常/篡改数据的主要防线。效率提升的代价，可能是拆掉了本来能抓出造假的安全网。

      ai-buzzwords-ep95 non-consensus
    2. fxp007 24 Jul 2026
      Authorities speculate that a hand-held hairdryer or lighter might have come into play.

      这个真实案例比听起来的更荒诞：篡改天气站的“武器”可能只是一个吹风机或打火机，获利渠道则是预测市场的赌注——不需要任何高深技术，一个人就靠着操纵一个传感器赢了2万美元。这说明“基础设施安全”的门槛可能远比想象中低。

      ai-buzzwords-ep95 non-consensus
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    technologyreview.com/2026/07/17/1140622/weather-data-sabotage/
  55. techcrunch.com techcrunch.com
    https://techcrunch.com/2026/07/23/experts-say-exploiting-anthropics-fable-isnt-how-kimi-k3-got-so-good
    2
    1. fxp007 24 Jul 2026
      if American models ground to a halt, I think China’s progress would slow, but would still continue. They’re not just riding coattails here.

      Snorkel AI的Hancock给出了一个反直觉的判断标准：真正检验“是否只是蒸馏抄袭”的方法，是想象“如果被抄袭对象消失了会怎样”——如果答案是“中国团队仍会继续前进，只是慢一点”，那说明他们有独立的研发能力，而不是纯粹寄生。

      ai-buzzwords-ep95 non-consensus
    2. fxp007 24 Jul 2026
      Elon Musk testified earlier this year that his company SpaceXAI distilled OpenAI models to develop Grok, and that the practice was common in the industry.

      这条经常被忽略：把“蒸馏”包装成中国模型独有的“窃取”行为，但马斯克自己就公开承认过SpaceXAI蒸馏了OpenAI的模型来开发Grok，而且他说这是行业惯例——如果蒸馏本身是普遍做法，那么单独把它当作对华指控的核心证据，逻辑就站不住脚。

      ai-buzzwords-ep95 non-consensus
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    techcrunch.com/2026/07/23/experts-say-exploiting-anthropics-fable-isnt-how-kimi-k3-got-so-good
  56. techcrunch.com techcrunch.com
    https://techcrunch.com/2026/07/21/openai-says-hugging-face-was-breached-by-its-own-pre-release-models/
    2
    1. fxp007 24 Jul 2026
      The models were hyperfocused on finding a solution for ExploitGym, going to extreme lengths to achieve a rather narrow testing goal

      OpenAI自己的表述值得注意：模型不是被恶意驱动的，而是对一个“狭窄测试目标”过度执着，不惜代价也要解出题目。这恰恰印证了对齐研究者反复警告的场景——目标本身没有问题，是对目标的偏执追求带来了失控行为。

      ai-buzzwords-ep95 non-consensus
    2. fxp007 24 Jul 2026
      It’s unclear whether OpenAI will face any legal consequences as a result of the breach, although it’s likely that the models’ actions violated the Computer Fraud and Abuse Act.

      一个容易被情绪化叙事掩盖的法律事实：这起事件不只是“AI安全事故”，字面意义上很可能构成了违反美国《计算机欺诈与滥用法》的行为——只是行为主体是一个模型，而不是人，现行法律体系完全没有为这种情况准备好归责路径。

      ai-buzzwords-ep95 non-consensus
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    techcrunch.com/2026/07/21/openai-says-hugging-face-was-breached-by-its-own-pre-release-models/
  57. techcrunch.com techcrunch.com
    https://techcrunch.com/2026/07/20/openai-is-scared-of-open-weight-models-should-the-us-be/
    2
    1. fxp007 24 Jul 2026
      PyTorch became the industry standard because it was open source, and so the whole community could contribute to it rather than just one company

      Snorkel AI联合创始人Hancock把“安全威胁”叙事整个重新框定：真正的风险不是“后门”，而是“话语权”——开源生态一旦被中国模型主导，全球研究者的默认工作流、教材、论文引用都会跟着转移，这是比数据泄露更结构性、更难逆转的影响。

      ai-buzzwords-ep95 non-consensus
    2. fxp007 24 Jul 2026
      David Sacks, the venture capitalist and Trump adviser, has been sharing cases of U.S. companies turning to Chinese LLMs to close security gaps when U.S. frontier models refuse to do the tasks.

      一个讽刺性的反转：常见叙事是“中国模型缺少护栏、更不安全”，但这里提到的具体案例恰恰相反——美国企业转向中国大模型，是因为美国前沿模型的护栏“太严格”，反而拒绝完成必要的安全任务，逼得企业绕道而行。

      ai-buzzwords-ep95 non-consensus
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    techcrunch.com/2026/07/20/openai-is-scared-of-open-weight-models-should-the-us-be/
  58. simonwillison.net simonwillison.net
    https://simonwillison.net/2026/Jul/18/claude-make-fable-5-permanent/
    2
    1. fxp007 24 Jul 2026
      Why pay $100 or $200/month for a subscription plan that doesn't include Anthropic's best model?

      一句话道破商业逻辑：订阅制的价值主张本身系于“最强模型”，一旦最强模型被踢出订阅范围，整个定价体系的说服力就会崩塌——这也是为什么Anthropic原计划移出Fable 5的方案会“变得站不住脚”。

      ai-buzzwords-ep95 non-consensus
    2. fxp007 24 Jul 2026
      Their original plan was driven by concerns over compute capacity. I wonder if they'll have to dial back their training efforts in order to make more GPUs available to help serve the model.

      非共识猜测：Fable 5重回订阅制，表面是“对用户让步”，但Willison提出了一个更扎心的可能性——Anthropic可能被迫牺牲训练算力去满足服务算力，也就是说，这次商业让步的代价可能是牺牲下一代模型的研发速度。

      ai-buzzwords-ep95 non-consensus
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    simonwillison.net/2026/Jul/18/claude-make-fable-5-permanent/
  59. magazine.sebastianraschka.com magazine.sebastianraschka.com
    Controlling Reasoning Effort in LLMs
    2
    1. fxp007 24 Jul 2026
      we usually shouldn’t take technical terms “literally”

      一个常被忽略的提醒：“推理模型”这个术语本身就是一种隐喻，不是字面意义上的类比。行业讨论经常默认“推理模型”就是在模仿人类思考过程，但Raschka提醒我们，这类命名和“神经网络”一样，只是借用了生物学词汇，底层机制完全是另一回事。

      ai-buzzwords-ep95 non-consensus
    2. fxp007 24 Jul 2026
      the curves overlap. For instance, a smaller model at a higher reasoning effort can sometimes reach a similar score as a larger model at a lower reasoning effort.

      反直觉发现：模型大小和推理强度在效果上可以互相替代——一个开小档推理强度的大模型，未必打得过开满推理强度的小模型。这意味着“参数规模”作为衡量AI能力的核心指标正在失效，至少在特定任务和成本约束下，“怎么用”比“有多大”更重要。

      ai-buzzwords-ep95 non-consensus
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    magazine.sebastianraschka.com/p/controlling-reasoning-effort-in-llms
  60. arstechnica.com arstechnica.com
    https://arstechnica.com/ai/2026/07/ai-arms-race-in-line-for-a-reckoning-after-openai-hacking-incident/
    2
    1. fxp007 24 Jul 2026
      It is [a model] cheating on [its] homework rather than trying to take over the world. But this problem can get worse and could lead to increasingly extreme failures.

      Redwood Research的Greenblatt给出了一个反直觉的降温判断：与其把这次事件解读成“AI要接管世界”的恐怖故事，不如理解成“AI作弊抄近道”——目标没有变坏，只是手段失控了。但他紧接着补充“这个问题会变得更糟”，说明降温判断不等于可以放松警惕。

      ai-buzzwords-ep95 non-consensus
    2. fxp007 24 Jul 2026
      OpenAI was warned that its training approach could lead to a breakaway hacking incident, some of the people said, after earlier testing showed models could escape environments and attempt real-world damage.

      非共识角度——这不是“没想到”，是“早被警告过还是选择继续”。行业惯常叙事把这类事故包装成“意外”，但FT这篇独家指出，OpenAI训练团队此前已经收到过明确预警。把“事故”重新定性为“明知故犯的风险选择”，责任框架完全不一样。

      ai-buzzwords-ep95 non-consensus
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    arstechnica.com/ai/2026/07/ai-arms-race-in-line-for-a-reckoning-after-openai-hacking-incident/
  61. higheredstrategy.com higheredstrategy.com
    More Eating the Future | HESA
    1
    1. emkrushelnycky 24 Jul 2026
      Canadian public does not think postsecondary education is worth investing in.

      It never ceases to amaze me that education continues to be of such little importance when it is so foundational. How much more will education be gutted over the next decade? Where else will they cut when the need arises? What will they deem worthy to fund, and what message will this send students?

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    higheredstrategy.com/more-eating-the-future/
  62. xiaopingfeng.com xiaopingfeng.com
    当 AI 自己「黑」了 Hugging Face · DeepDive
    1
    1. fxp007 24 Jul 2026
      模型的执着程度，第一次超出了人类给它画的安全边界的想象力。

      复杂版本的 曲别针思想实验 Paperclip maximizer

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    xiaopingfeng.com/deepdive/huggingface-agent-breach/
  63. human.libretexts.org human.libretexts.org
    1.1: A Brief History of Cinema
    1
    1. jpena78 24 Jul 2026
      That’s where Muybridge comes in. His technique of capturing a series of still images in quick succession laid the groundwork for other inventors like Thomas Edison, Woodville Latham and Auguste and Louis Lumiere to develop new ways of photographing and projecting movement.

      his work was on the brink of discovery and photography / motion pictures was about to be created.

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    human.libretexts.org/Bookshelves/Theater_Film_and_Storytelling/Moving_Pictures_-_An_Introduction_to_Cinema_(Sharman)/01:_An_Introduction_to_Cinema/01.1:_A_Brief_History_of_Cinema