Not sure if we have staging Event Reference docs, but this one points to the 13.0.0 version directly.

1st attempt

This is the most abused phrase in all of science. It is sometimes known as "waving your hands," and it is usually a subsitute for doing a big pile of calculus, in this case, three-dimensional calculus. So for PHY2054, we will let discretion be the proverbial better part of valor and accept this important result. Why is it important? It is important because we now have an oscillator system!

Over time, families who have money and time can sue the district and move their kids to private schools, while families without resources have to stay in under-resourced schools. This makes the gap between public schools and social classes even bigger.

This quote illustrates a critical gap between evaluation and implementation. Even when professionals correctly identify needs, the services often never reach the child. I agree with the author that missed interventions in early childhood can have lifelong consequences. It also shows how the responsibility gets pushed around between agencies, leaving families confused and unsupported.

This quote is powerful because it reflects a rare moment of accountability within the article. T.J.’s case exposes failures in evaluations, services, placements, and follow-through. I think this line perfectly summarizes the emotional impact: T.J.’s struggles were preventable. It reinforces the idea that educational inequality is not accidental—it is the result of systemic dysfunction that continues to harm vulnerable students.

This opening immediately shows the urgency of T.J.’s situation. It is heartbreaking because his lack of progress is not due to a lack of effort, but a failure of the system around him. The quote highlights how early delays compound over time when appropriate support is not provided. It connects to class themes about how institutional neglect—rather than individual deficits—creates long-term educational harm.

Out of the 3 test, which is the most reliable in detecting NTD?

If folic acid is recommended, would taking more than the recommended amount be helpful?

If the occipital lobe is damaged, can the person still be able to see but just not interpret what they are seeing?

What is the importance of the ventricles being continuous with the spinal cord?

so is the major arteries the carotid arteries? since they supply blood to the whole brain

is the pituitary gland considered an independent part of the brain or part of the hypothalamus? if independent, then why is the hypothalamus considered the controller of pituitary endocrine function?

eLife Assessment

This important study reveals that connexin43 (Cx43) hemichannels are directly activated by CO₂ through a conserved carbamylation motif, extending a mechanism previously described for β-connexins to α-connexins. The evidence is convincing, supported by complementary biochemical and electrophysiological analyses showing CO₂-induced hemichannel opening and ATP release in cultured cells and hippocampal slices. These findings advance our understanding of connexin regulation by metabolic gases and will be of broad interest to researchers studying cell communication, neural signaling, and gasotransmitter biology.

Reviewer #1 (Public review):

Summary:

This study builds on previous work demonstrating that several beta connexins (Cx26, Cx30 and Cx32) have a carbamylation motif which renders them sensitive to CO2. In response to CO2, hemichannels composed of these connexins open, enabling diffusion of small molecules (such as ATP) between the cytosol and extracellular environment. Here, the authors have identified that an alpha connexin, Cx43, also contains a carbamylation motif, and they demonstrate that CO2 opens Cx43 hemichannels. Most of the study involves using transfected cells expressing wild-type and mutant Cx43 to define amino acids required for CO2 sensitivity. Hippocampal tissue slices in culture were used to show that CO2-induced synaptic transmission was affected by Cx43 hemichannels, providing a physiological context. The authors point out that the Cx43 gene significantly diverges from the beta connexins that are CO2 sensitive, suggesting that the conserved carbamylation motif was present before the alpha and beta connexin genes diverged.

Strengths:

The molecular analysis defining the amino acids which contribute to the CO2 sensitivity of Cx43 is a major strength of the study. The rigor of analysis was strengthened by using three independent assays for hemichannel opening: dye uptake, patch clamp channel measurements and ATP secretion. The resulting analysis identified key lysines in Cx43 that were required for CO2-mediated hemichannel opening. A double K to E Cx43 mutant produced a construct that produced hemichannels that were constitutively open, which further strengthened the analysis.

Using hippocampal tissue sections to demonstrate that CO2 can influence field excitatory postsynaptic potentials (fEPSPs) provides a native context for CO2 regulation of Cx43 hemichannels. Cx43 mutations associated with Oculodentodigital Dysplasia (ODDD) inhibited CO2-induced hemichannel opening, although the mechanism by which this occurs was not elucidated.

Cytosolic pH was measured and it was further demonstrated that Cx43 hemichannels composed of untagged Cx43 are sensitive to CO2.

A molecular phylogenetic survey was performed which identified several other non-beta connexins that have a putative carbamylation motif. How this relates to connexin evolution was added to the discussion.

Weaknesses:

Cultured cells are typically grown in incubators containing 5% CO2 which is ~40 mmHg. Determining compensatory mechanisms that enable the cells to be viable if Cx43 hemichannels are open at this PCO2 would strengthen the study.

Experiments using Gap26 to inhibit Cx43 hemichannels in fEPSP measurements used a scrambled peptide as a control. Including gap peptides specifically targeting Cx26, Cx30 and Cx32 as additional controls would strengthen the study, since the tissue sections have a complex pattern of connexin expression.

Reviewer #2 (Public review):

Summary:

This paper examines the CO2 sensitivity of Cx43 hemichannels and gap junctional channels in transiently transfected Hela cells using several different assays including ethidium dye uptake, ATP release, whole cell patch clamp recordings and an imaging assay of gap junctional dye transfer. The results show that raising pCO2 from 20 to 70 mmHg (at a constant pH of 7.3) cause an increase in opening of Cx43 hemichannels but did not block Cx43 gap junctions. This study also showed that raising pCO2 from 20 to 35 mm Hg resulted in an increase in synaptic strength in hippocampal rat brain slices, presumably due to downstream ATP release, suggesting that the CO2 sensitivity of Cx43 may be physiologically relevant. As a further test of the physiological relevance of the CO2 sensitivity of Cx43, it was shown that two pathological mutations of Cx43 that are associated with ODDD caused loss of Cx43 CO2-sensitivity. Cx43 has a potential carbamylation motif that is homologous to the motif in Cx26. To understand the structural changes involved in CO2 sensitivity, a number of mutations were made in Cx43 sites thought to be the equivalent of those known to be involved in the CO2 sensitivity of Cx26 and the CO2 sensitivity of these mutants was investigated.

Strengths:

This study shows that the apparent lack of functional Cx43 hemichannels observed in a number of previous in vitro function studies may be due to the use of HEPES to buffer the external pH. When Cx43 hemichannels were studied in external solutions in which CO2/bicarbonate was used to buffer pH instead of HEPES, Cx43 hemichannels showed significantly higher levels of dye uptake, ATP release, and ionic conductance. These findings may have major physiological implications since Cx43 hemichannels are found in many organs throughout the body including the brain, heart and immune system.

Weaknesses:

Interpretation of the site-directed mutation studies is complicated. Although Cx43 has a potential carbamylation motif that is homologous to the motif in Cx26, the results of site-directed mutation studies were inconsistent with a simple model in which K144 and K105 interact following carbamylation to cause the opening of Cx43 hemichannels.

Secondly, although it is shown that two Cx43 ODDD associated mutations show a loss of CO2 sensitivity, there is no evidence that the absence of CO2 sensitivity is involved in the pathology of ODDD.

Reviewer #3 (Public review):

In this paper, authors aimed to investigate carbamylation effects on the function of Cx43-based hemichannels. Such effects have previously been characterized for other connexins, e.g. for Cx26, which display increased hemichannel (HC) opening and closure of gap junction channels upon exposure to increased CO2 partial pressure (accompanied by increased bicarbonate to keep pH constant). The authors used HeLa cells transiently transfected with Cx43 to investigate CO2-dependent carbamylation effects on Cx43 HC function. In contrast to Cx43-based gap junction channels that are here reported to be insensitive to PCO2 alterations, they provide evidence that Cx43 HC opening is highly dependent on the PCO2 pressure in the bath solution, over a range of 20 up to 70 mmHg encompassing the physiologically normal resting level of around 40 mmHg. They furthermore identified several Cx43 residues involved in Cx43 HC sensitivity to PCO2: K105, K109, K144 & K234; mutation of 2 or more of these AAs is necessary to abolish CO2 sensitivity. The subject is interesting and the results indicate that a fraction of HCs is open at a physiological 40 mmHg PCO2, which differs from the situation under HEPES buffered solutions where HCs are mostly closed under resting conditions. The mechanism of HC opening with CO2 gassing is linked to carbamylation and authors pinpointed several Lys residues involved in this process. Overall, the work is interesting as it shows that Cx43 HCs have a significant open probability under resting conditions of physiological levels of CO2 gassing, probably applicable to/relevant for brain, heart and other Cx43 expressing organs. The paper gives a detailed account on various experiments performed (dye uptake, electrophysiology, ATP release to assess HC function) and results concluded from those. They further consider many candidate carbamylation sites by mutating them to negatively charged Glu residues. The paper finalizes with hippocampal slice work showing evidence for connexin-dependent increases of the EPSP amplitude that could be inhibited by HC inhibition with Gap26 (Fig. 10). Another line of evidence comes from the Cx43-linked ODDD genetic disease whereby L90V as well as the A44V mutations of Cx43 prevented the CO2 induced hemichannel opening response (Fig. 11). Although the paper is interesting, in its present state it suffers from (i) a problematic Fig. 3, precluding interpretation of the data shown, and (ii) the poor use of hemichannel inhibitors that are necessary to strengthen the evidence in the crucial experiment of Fig. 2 and others.

Comments on revisions:

The traces in Fig.2B show that the HC current is inward at 20 mmHg PCO2, while it switches to an outward current at 55mmHg PCO2. HCs are non-selective channels, so their current should switch direction around 0 mV but not around -50 mV. As such, the -50 mV switching point indicates involvement of another channel distinct from non-selective Cx43 hemichannels. In the revised version, this problem has not been solved nor addressed. Additionally, I identified another problem in that the experimental traces shown lack a trace at the baseline condition of PCO2 35mmHg, while the summary graph depicts a data point. Not showing a trace at baseline PCO2 35mmHg renders data interpretation in the summary graph questionable.

Author response:

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

Reviewer #1 (Public review): 

Summary: 

This study builds on previous work demonstrating that several beta connexins (Cx26, Cx30, and Cx32) have a carbamylation motif which renders them sensitive to CO₂. In response to CO₂, hemichannels composed of these connexins open, enabling diffusion of small molecules (such as ATP) between the cytosol and extracellular environment. Here, the authors have identified that an alpha connexin, Cx43, also contains a carbamylation motif, and they demonstrate that CO₂ opens Cx43 hemichannels. Most of the study involves using transfected cells expressing wildtype and mutant Cx43 to define amino acids required for CO₂ sensitivity. Hippocampal tissue slices in culture were used to show that CO₂-induced synaptic transmission was affected by Cx43 hemichannels, providing a physiological context. The authors point out that the Cx43 gene significantly diverges from the beta connexins that are CO₂ sensitive, suggesting that the conserved carbamylation motif was present before the alpha and beta connexin genes diverged. 

Strengths: 

(1) The molecular analysis defining the amino acids that contribute to the CO₂ sensitivity of Cx43 is a major strength of the study. The rigor of analysis was strengthened by using three independent assays for hemichannel opening: dye uptake, patch clamp channel measurements, and ATP secretion. The resulting analysis identified key lysines in Cx43 that were required for CO₂-mediated hemichannel opening. A double K to E Cx43 mutant produced a construct that produced hemichannels that were constitutively open, which further strengthened the analysis. 

(2) Using hippocampal tissue sections to demonstrate that CO₂ can influence field excitatory postsynaptic potentials (fEPSPs) provides a native context for CO₂ regulation of Cx43 hemichannels. Cx43 mutations associated with Oculodentodigital Dysplasia (ODDD) inhibited CO₂-induced hemichannel opening, although the mechanism by which this occurs was not elucidated. 

Weaknesses: 

(1) Cx43 channels are sensitive to cytosolic pH, which will be affected by CO₂. Cytosolic pH was not measured, and how this affects CO₂-induced Cx43 hemichannel activity was not addressed. 

We have now addressed this with intracellular pH measurements and removal of the C-terminal pH sensor from Cx43 -the hemichannel remains CO₂ sensitive.

(2) Cultured cells are typically grown in incubators containing 5% CO₂, which is ~40 mmHg. It is unclear how cells would be viable if Cx43 hemichannels are open at this PCO2. 

The cells look completely healthy with normal morphology and no sign of excessive cell death in the cultures. Presumably they have ways of compensating for the effects of partially open Cx43 hemichannels.

(3) Experiments using Gap26 to inhibit Cx43 hemichannels in fEPSP measurements used a scrambled peptide as a control. Analysis should also include Gap peptides specifically targeting Cx26, Cx30, and Cx32 as additional controls. 

We don’t feel this is necessary given the extensive prior literature in hippocampus showing the effect of ATP release via open Cx43 hemichannels on fEPSP amplitude that used astrocytic specific knockout of Cx43 and Gap26 (doi: 10.1523/jneurosci.0015-14.2014).

(4) The mechanism by which ODDD mutations impair CO2-mediated hemichannel opening was not addressed. Also, the potential roles for inhibiting Cx43 hemichannels in the pathology of ODDD are unclear. 

These pathological mutations that alter CO<SUB>2</SUB> sensitivity are similar to pathological mutation in Cx26 and Cx32, which also remove CO<SUB>2</SUB> sensitivity. Our cryo-EM studies on Cx26 give clues as to why these mutations have this effect -they alter conformational mobility of the channel (Brotherton et al 2022 doi: 10.1016/j.str.2022.02.010 and Brotherton et al 2024 doi: 10.7554/eLife.93686). We assume that similar considerations apply to Cx43, but this requires improved cryoEM structures of Cx43 hemichannels at differing levels of PCO<SUB>2</SUB>.

We agree that the link between loss of CO<SUB>2</SUB> sensitivity of Cx43 and ODDD is not established and have revised the text to make this clear.

(5) CO2 has no effect on Cx43-mediated gap junctional communication as opposed to Cx26 gap junctions, which are inhibited by CO2. The molecular basis for this difference was not determined. 

Cx26 gap junction channels are so far unique amongst CO<SUB>2</SUB> sensitive connexins in being closed by CO<SUB>2</SUB>. We have addressed the mechanism by which this occurs in Nijjar et al 2025 DOI: 10.1113/JP285885 -the requirement of carbamylation of K108 in Cx26 (in addition to K125) for GJC closure.

(6) Whether there are other non-beta connexins that have a putative carbamylation motif was not addressed. Additional discussion/analysis of how the evolutionary trajectory for Cx43 maintaining a carbamylation motif is unique for non-beta connexins would strengthen the study. 

We have performed a molecular phylogenetic survey to show that the carbamylation motif occurs across the alpha connexin clade and have shown that Cx50 is indeed CO<SUB>2</SUB> sensitive (doi: 10.1101/2025.01.23.634273). This is now in Fig 12.

Reviewer #2 (Public review): 

Summary: 

This paper examines the CO<SUB>2</SUB>  sensitivity of Cx43 hemichannels and gap junctional channels in transiently transfected Hela cells using several different assays, including ethidium dye uptake, ATP release, whole cell patch clamp recordings, and an imaging assay of gap junctional dye transfer. The results show that raising pCO₂ from 20 to 70 mmHg (at a constant pH of 7.3) causes an increase in opening of Cx43 hemichannels but does not block Cx43 gap junctions. This study also showed that raising pCO<SUB>2</SUB> from 20 to 35 mm Hg resulted in an increase in synaptic strength in hippocampal rat brain slices, presumably due to downstream ATP release, suggesting that the CO<SUB>2</SUB> sensitivity of Cx43 may be physiologically relevant. As a further test of the physiological relevance of the CO₂ sensitivity of Cx43, it was shown that two pathological mutations of Cx43 that are associated with ODDD caused loss of Cx43 CO₂-sensitivity. Cx43 has a potential carbamylation motif that is homologous to the motif in Cx26. To understand the structural changes involved in CO<SUB>2</SUB> sensitivity, a number of mutations were made in Cx43 sites thought to be the equivalent of those known to be involved in the CO<SUB>2</SUB> sensitivity of Cx26, and the CO<SUB>2</SUB> sensitivity of these mutants was investigated. 

Strengths: 

This study shows that the apparent lack of functional Cx43 hemichannels observed in a number of previous in vitro function studies may be due to the use of HEPES to buffer the external pH. When Cx43 hemichannels were studied in external solutions in which CO<SUB>2</SUB>/bicarbonate was used to buffer pH instead of HEPES, Cx43 hemichannels showed significantly higher levels of dye uptake, ATP release, and ionic conductance. These findings may have major physiological implications since Cx43 hemichannels are found in many organs throughout the body, including the brain, heart, and immune system. 

Weaknesses: 

(1) Interpretation of the site-directed mutation studies is complicated. Although Cx43 has a potential carbamylation motif that is homologous to the motif in Cx26, the results of site-directed mutation studies were inconsistent with a simple model in which K144 and K105 interact following carbamylation to cause the opening of Cx43 hemichannels. 

The mechanism of opening of Cx43 is more complex than that of Cx26, Cx32 and Cx50 and involves more Lys residues. The 4 Lys residues in Cx43 that are involved in opening the hemichannel have their equivalents in Cx26, but in Cx26 these additional residues seem to be involved in the closing of the GJC rather than opening of the hemichannel (see above). Cx50 is simpler and involves only two Lys residues (doi: 10.1101/2025.01.23.634273), which are equivalent to those in Cx26.

(2) Secondly, although it is shown that two Cx43 ODDD-associated mutations show a loss of CO₂ sensitivity, there is no evidence that the absence of CO2 sensitivity is involved in the pathology of ODD

We agree, but this is probably because this has not been directly tested by experiment, as the CO<Sub>2</sub> sensitivity of Cx43 was not previously known. As mentioned above we have revised the text to ensure that this is clear.

Reviewer #3 (Public review): 

In this paper, the authors aimed to investigate carbamylation effects on the function of Cx43-based hemichannels. Such effects have previously been characterized for other connexins, e.g., for Cx26, which display increased hemichannel (HC) opening and closure of gap junction channels upon exposure to increased CO₂ partial pressure (accompanied by increased bicarbonate to keep pH constant). 

The authors used HeLa cells transiently transfected with Cx43 to investigate CO₂ dependent carbamylation effects on Cx43 HC function. In contrast to Cx43-based gap junction channels that are reported here to be insensitive to PCO₂ alterations, they provide evidence that Cx43 HC opening is highly dependent on the PCO2 pressure in the bath solution, over a range of 20 up to 70 mmHg encompassing the physiologically normal resting level of around 40 mmHg. They furthermore identified several Cx43 residues involved in Cx43 HC sensitivity to PCO2: K105, K109, K144 & K234; mutation of 2 or more of these AAs is necessary to abolish CO₂ sensitivity. The subject is interesting and the results indicate that a fraction of HCs is open at a physiological 40 mmHg PCO₂, which differs from the situation under HEPES buffered solutions where HCs are mostly closed under resting conditions. The mechanism of HC opening with CO₂ gassing is linked to carbamylation, and the authors pinpointed several Lys residues involved in this process. 

Overall, the work is interesting as it shows that Cx43 HCs have a significant open probability under resting conditions of physiological levels of CO₂ gassing, probably applicable to the brain, heart, and other Cx43 expressing organs. The paper gives a detailed account of various experiments performed (dye uptake, electrophysiology, ATP release to assess HC function) and results concluded from those. They further consider many candidate carbamylation sites by mutating them to negatively charged Glu residues. The paper ends with hippocampal slice work showing evidence for connexin-dependent increases of the EPSP amplitude that could be inhibited by HC inhibition with Gap26 (Figure 10). Another line of evidence comes from the Cx43-linked ODDD genetic disease, whereby L90V as well as the A44V mutations of Cx43 prevented the CO₂-induced hemichannel opening response (Figure 11). Although the paper is interesting, in its present state, it suffers from (i) a problematic Figure 3, precluding interpretation of the data shown, and (ii) the poor use of hemichannel inhibitors that are necessary to strengthen the evidence in the crucial experiment of Figure 2 and others. 

The panels in Figure 3 were mislabelled in the accompanying legend possibly leading to some confusion. This has now been corrected.

We disagree that hemichannel blockers are needed to strengthen the evidence in Figure 2 and other figures. Our controls show that the CO₂-sensitive responses absolutely requires expression of Cx43 and was modified by mutations of Cx43. It is hard to see how this evidence would be strengthened by use of peptide inhibitors or other blockers of hemichannels that may not be completely selective.

Reviewing Editor Comments:

(1) Improve electrophysiological evidence, addressing concerns about the initial experiment and including peptide inhibitor data where applicable. 

We think the concerns about the electrophysiological evidence arise from a misunderstanding because we gave insufficient information about how we conducted the experiments. We have now provided a much more complete legend, added explanations in the text and given more detail in the Methods. We further respond to the reviewer below.

We do not agree on the necessity of the peptide inhibitor to demonstrate dependence on Cx43.  We have shown that parental HeLa cells do not release ATP to changes in PCO₂ or voltage (Fig 2D; Butler & Dale 2023, 10.3389/fncel.2023.1330983; Lovatt et al 2025, 10.1101/2025.03.12.642803, 10.1101/2025.01.23.634273). Our previous papers have shown many times that parental HeLa cells do not load with dye to CO₂ or zero Ca²⁺ (e.g. Huckstepp et al 2010, 10.1113/jphysiol.2010.192096; Meigh et al 2013, 10.7554/eLife.01213; Meigh et al 2014, 10.7554/eLife.04249), and we have shown that parental HeLa cells do not exhibit the same CO₂ dependent change in whole cell conductance that the Cx43-expressing cells do (Fig 2B). In addition, we shown that mutating key residues in Cx43 alters both CO₂-sensitive release of ATP and the CO₂-dependent dye loading without affecting the respective positive control. To bolster this, we have included data for the K144R mutation as a supplement to Fig 3. Given the expense of Gap26 it is impractical to include this as a standard control and unnecessary given the comprehensive controls outlined.

Collectively, these data show that the responses to CO₂ require expression of Cx43 and can be modified by mutation of Cx43.

(2) Strengthen the manuscript by measuring the effects of CO on cytosolic pH and Cx43 hemichannel opening. Consider using tail truncation mutants to assess the role of the C-terminal pH sensor in CO-mediated channel opening.

We agree and have performed the suggested experiments to address this issue.

(3) Investigate the effect of expressing the K105E/K109E Cx43 double mutant on cell viability.

In our experiments the cells look completely healthy based on their morphology in brightfield microscopy and growth rates. 

(4) Discuss and analyze the uniqueness of Cx43 among alpha connexins in maintaining the carbamylation motif.

now discuss this -Cx43 is not unique. We have added a molecular phylogenetic survey of the alpha connexin clade in Fig 12. Apart from Cx37, the carbamylation motif appears in all the other members of the clade (but not necessarily in the human orthologue). In a different MS, currently posted on bioRxiv, we have documented the CO₂ sensitivity of Cx50 and its dependence on the motif.

(5) Consider omitting data on ODDD-associated mutations unless there is evidence linking CO₂ sensitivity to disease pathology.

This experiment is observational, and we are not making claims that there is a direct causal link. Removing the ODDD mutant findings would lose potentially useful information for anyone studying how these mutations alter channel function. We have reworded the text to ensure that we say that the link between loss of CO₂ sensitivity and ODDD remains unproven.

(6) Justify the choice of high K^⁺ and low external calcium as a positive control in ATP release experiments.

These two manipulations can open the hemichannel independently of the CO₂ stimulus. Extracellular Ca²⁺ is well known to block all connexin hemichannels, and Cx43 is known to be voltage sensitive. The depolarisation from high K⁺ is effective at opening the hemichannel and we preferred this as a more physiological way of opening the Cx43 hemichannel. We have added some explanatory text.

(7) Clarify whether Cx43A44V or Cx43L90V mutations block gap junctional coupling.

This is an interesting point. Since Cx43 GJCs are not CO₂ sensitive we feel this is beyond the scope of our paper. 

(8) Discuss the potential implications of pCO₂ changes on myocardial function through alterations in intracellular pH.

We have modified the discussion to consider this point.

Reviewer #1 (Recommendations for the authors):

(1) Measurements of the effects of CO₂ on cytosolic pH/Cx43 hemichannel opening would strengthen the manuscript. Since the pH sensor of Cx43 is on the C terminus, the authors could consider making tail truncation mutants to see how this affects CO₂-mediated Cx43 channel opening.

We have done this (truncating after residue 256) -the channel remains highly CO₂ and voltage sensitive. We have also documented the effect of the  hypercapnic solutions on intracellular pH measured with BCECF. These new data are now included as figure supplements to Figure 2.

(2) What is the impact of expressing the K105E / K109E Cx43 double mutant on cell viability?

There was no obvious observed impact, cell density was as expected (no evidence of increased cell death), brightfield and fluorescence visualisation indicated normal healthy cells. We have added a movie (Fig 9, movie supplement 1) to show the effect of La³⁺ on the GRAB_ATP signal in cells expressing Cx43^{K105E, K109E} so readers can appreciate the morphology and its stability during the recording.

(3) A quick look at other alpha connexins suggested that Cx43 was unique among alpha connexins in maintaining the carbamylation motif. This merits additional discussion/ analysis.

This is an interesting point. Cx43 is not unique in the alpha clade in having the carbamylation motif as a number of other human alpha connexins also possess: Cx50, Cx59 and Cx62, and non-human alpha connexins (Cx40, Cx59, Cx46) also possess the motif. We have shown that Cx50 is CO₂ sensitive. We have performed a brief molecular phylogenetic analysis of the alpha connexon clade to highlight the occurrence of the carbamylation motif. This is now presented as Fig 12 to go with the accompanying discussion.

(4) There were some minor writing issues that should be addressed. For instance, fEPSP is not defined. Also, insets showing positive controls in some experiments were not described in the figure legends.

We have corrected these issues.

Reviewer #2 (Recommendations for the authors):

(1) I would omit the data on the ODDD-associated mutations since there is no evidence that loss of CO₂ sensitivity plays an important role in the underlying disease pathology.

We are not making the claim CO₂ loss leads to the underlying pathology and have reviewed the text to ensure that we clearly express that this is a correlation not a cause. We think this is worth retaining as many pathological mutations in other CO₂ sensitive connexins (Cx26, Cx32 and Cx50) cause loss of CO₂ sensitivity, and this information may be helpful to other researchers.

(2) Why is high K+ rather than low external calcium used as a positive control in ATP release experiments?

We used of high K⁺ and depolarisation as a positive control as regard this as a more physiological stimulus than the low external Ca²⁺.

(3) Does Cx43A44V or Cx43L90V block gap junctional coupling?

An interesting question but we have not examined this.

(4) Provide references for biophysical recordings of Cx43 hemichannels performed in HEPES-buffered salines, which document Cx43 hemichannels as being shut.

have added the original and some later references which examine Cx43 hemichannel gating in HEPES buffer and shows the need for substantial depolarisation to induce channel opening.

(5) In the heart muscle, changes in PCO₂ have long been hypothesized to cause changes in myocardial function by changing pHi.

This is true and we now add some discussion of this point. Now that we know that Cx43 is directly sensitive to CO₂ a direct action of CO₂ cannot be ruled out and careful experimentation is required to test this possibility. 

Reviewer #3 (Recommendations for the authors):

(1) Page 3: "... homologs of K125 and R104 ... ": the context is linked to Cx26, so Cx26 needs to be added here.

Done

(2) Page 4 text and related Figure 2:

(a) Figure 2A&B: PCO2-dependent Cx43 HC opening is clearly present in the carboxy-fluorescein dye uptake experiments (Figure 2A) as well as in the electrophysiological experiments (Figure 2B). The curves look quite different between these two distinct readouts: dye uptake doubles from 20 to 70 mmHg in Figure 2A while the electrophysiological data double from 45 to 70 mmHg in Figure 2B. These responses look quite distinct and may be linked to a non-linearity of the dye uptake assay or a problem in the electrophysiological measurements of Figure 2B discussed in the next point.

Different molecules/ions may have different permeabilities through the channel, which could explain the observed difference. Also, there is some contamination of the whole cell conductance change with another conductance (evident in recordings from parental HeLa cells). This is evident particularly at 70 mmHg. If this contaminating conductance were subtracted from the total conductance in the Cx43 expressing cells, then the dose response relations would be more similar. However, we are reluctant to add this additional data processing step to the paper.

(b) The traces in Figure 2B show that the HC current is inward at 20 mmHg PCO2, while it switches to an outward current at 55mmHg PCO2. HCs are non-selective channels, so their current should switch direction around 0 mV but not at -50 mV. As such, the -50 mV switching point indicates involvement of another channel distinct from non-selective Cx43 hemichannels.

We think that our incomplete description in the legend led to this misunderstanding. We used a baseline of 35 mmHg (where the channels will be slightly open) and changed to 20 mmHg to close them (or to higher PCO₂ to open them from this baseline), hence a decrease in conductance and loss of outward current for 20 mmHg. The holding potential for the recordings and voltage steps were the same in all recordings. We have now edited the legend and added more information into the methods to clarify this and how we constructed the dose response curve.

We agree that Cx43 hemichannels are relatively nonselective and would normally be expected to have a reversal potential around 0 mV, but we are using K-Gluconate and the lowered reversal potential (~-65 mV) is likely due to poor permeation of this anion via Cx43.

(c) A Hill slope of 6 is reported for this curve, which is extremely steep. The paper does not provide any further consideration, making this an isolated statement without any theoretical framework to understand the present finding in such context (i.e., in relation to the PCO2 dependency of Cx channels).

Yes, we agree -it seems to be the case with all CO₂ sensitive connexins that we have looked at that the Hill coefficient versus CO₂ is >4. Hemichannels are of course hexameric so there is potential for 6 CO₂ molecules to be bound and extensive cooperativity. We have modified the text to give greater context.

(d) A further remark to Figure 2 is that it does not contain any experiment showing the effect of Cx43 hemichannel inhibition with a reliable HC inhibitor such as Gap26, which is only used in the penultimate illustration of Figure 10. Gap26 should be used in Figure 2 and most of the other figures to show evidence of HC contribution. The lanthanum ions used in Figure 9 are a very non-specific hemichannel blocker and should be replaced by experiments with Gap26.

We have addressed the first part of this comment above.

We agree that La³⁺ blocks all hemichannels, but in the context of our experiments and the controls we have performed it is entirely adequate and supports our conclusions. Our controls show (mentioned above and below) show that the expression of Cx43 is absolutely required for CO₂-dependent ATP release (and dye loading). In Figure 9 our use of La³⁺ was to show the presence of a constitutively open Cx43 mutant hemichannel. Gap26 would add little to this. Our further controls show that with expression of Cx43^WT La³⁺ did nothing to the ATP signal under baseline conditions (20 mmHg) supporting our conclusion that the mutant channels are constitutively open.

(e) As the experiments of Figure 2 form the basis of what is to follow, the above remarks cast doubt on the robustness of the experiments and the data produced.

We disagree, our results are extremely robust: 1) we have used three independent assays confirm the presence of the response; 2) parental HeLa cells do not release ATP, dye load or show large conductance changes to CO₂ showing the absolute requirement for expression of Cx43; 3) mutations of Cx43 (in the carbamylation motif) alter the CO₂ evoked ATP release and dye loading giving further confirmation of Cx43 as the conduit for ATP release and dye loading; and 4) we use standard positive controls (0 Ca^², high K) to confirm cells still have functional channels for those mutations that modified CO₂ sensitivity.

(f) The sentence "Cells transfected with GRAB-ATP only, showed ... " should be

modified to "In contrast, cells not expressing Cx43 showed no responses to any applied CO2 concentration as concluded from GRAB-ATP experiments"

We have modified the text.

(3) Page 5 and Figures 3 & 4:

(a) Figure 3 illustrates results obtained with mutations of 4 distinct Lys residues. However, the corresponding legend indicates mutations that are different from the ones shown in the corresponding illustrations, making it impossible to reliably understand and interpret the results shown in panels A-E.

Thanks for pointing this out. Our apologies, we modified the figure so that the order of the images matched the order of the graph (and the legend) but then forgot to put the new version of the figure in the text. We have now corrected this so that Figure and legend match.

(b) Figure 4 lacks control WT traces!

The controls for this (showing that parental HeLa cells do not release ATP in response to CO₂ or depolarisation) are shown in Figure 2.

(c) Figure 4, Supplement 1: High Hill coefficients of 10 are shown here, but they are not discussed anywhere, as is also the case for the remark on p.4. A Hill steepness of 10 is huge and points to many processes potentially involved. As reported above, these data are floating around in the manuscript without any connection.

Yes, we agree this is very high and surprising. It may reflect as mentioned above the hexameric nature of the channel and that 4 Lys residues seem to be involved. We have used this equation to give some quantitative understanding of the effect of the mutations on CO₂ sensitivity and still think this is useful. We have no further evidence to interpret these values one way or the other.

(4) Page 6: Carbamate bridges are proposed to be formed between K105 and K144, and between K109 and K234. The first three of these Lysine residues are located in the 55aa long cytoplasmic loop of Cx43, while K234 is in the juxta membrane region involved in tubulin interactions. Both K144 and and K234 are involved in Cx43 HC inhibition: K144 is the last aa of the L2 peptide (D119-K144 sequence) that inhibits Cx43 hemichannels while K234 is the first aa of the TM2 peptide that reduces hemichannel presence in the membrane (sequence just after TM4, at the start of the C-tail). This context should be added to increase insight and understanding of the CO2 carbamylation effects on Cx43 hemichannel opening.

Thanks for suggesting this. We have added some discussion of CT to CL interactions in the context of regulation by pH and [Ca²⁺].

(5) Page 7: The Cx43 ODDD A44V and L90V mutations lead to loss of pCO2 sensitivity in dye loading and ATP assays. However, A44V located in EL1 is reportedly associated with Cx43 HC activation, while L90V in TM2 is associated with HC inhibition. Remarkably, these mutations are focused on non-Lys residues, which brings up the question of how to link this to the paper's main thread.

This follows the pattern that we have seen for other mutations such as A40V, A88V in Cx26 and several CMTX mutations of Cx32. Our cryoEM structures of Cx26 suggest that these mutations alter the flexibility of the molecule and hence abolish CO₂ sensitivity. We have reworded the text to avoid giving the impression that there is a demonstrated link between loss of CO₂ sensitivity of Cx43 and pathology.

(6) Page 8: HCs constitutively open - 'constutively' perhaps does not have the best connotation as it is not related to HC constitution but CO2 partial pressure.

Yes, we agree and have reworded this.

(7) Page 9: "in all subtypes" -> not clear what is meant - do you mean "in all cell types"?

We agree this is unclear -it refers to all astrocytic subtypes. We have amended the text.

(8) Page 10: Composition of hypocapnic recording solution: bubbling description is incomplete "95%O2/5%" and should be "95%O2/5%CO2".

Changed.

(9) Page 11: Composition of zero Ca^²⁺ hypocapnic recording solution: perhaps better to call this "nominally Ca^²⁺-free hypocapnic recording solution" as no Ca^²⁺ buffer is included in this solution

Thanks for pointing this out. We did in fact add 1 mM EGTA to the solutions but omitted this from the recipe, this has now been corrected.

(10) Page 11: in M&M I found that the NaHCO3- is lowered to 10 mM in the zero Ca^²⁺condition, while the control experimental condition has 26 mM NaHCO3-. The zero Ca condition should be kept at a physiologically normal 26 mM NaHCO3- concentration, so why was this done? Lowering NaHCO3- during hemichannel stimulation may result in smaller responses and introduce non-linearities.

For the dye loading we used 20 mmHg as the baseline condition and increased PCO₂ from this. Hence for the zero Ca²⁺ positive control we modified the 20 mmHg hypocapnic solution by substituting Mg²⁺ for Ca²⁺ and adding EGTA. We have modified the text in the Methods to clarify this.

Further remarks on the figures:

(1) Figure 2A: Add 20 & 70 mmHg to the images, to improve the readability of this illustration.

Done

(2) Figure 3: WT responses are shown in panel F, but experimental data (images and curves) are lacking and should be included in a revised version.

The wild type data is shown in Fig 2A. We have some sympathy for the comment, but we felt that Fig 2 should document CO₂ sensitivity, and then the subsequent Figs should analyse its basis. Hence the separation of Cx43^WT data from the mutant data. In panel F, we state that we have recalculated the WT data from Fig 2A to allow the comparison.

(3) Figures 4, 6, 8: Color codes for mmHg CO₂ pressure make reading these figures difficult; perhaps better to add mmHg values directly in relation to the traces.

We have considered this suggestion but feel that the figures would become very cluttered with the additional labelling.

(4) I wouldn't use colored lines when not necessary, e.g., Figure 9 100 µM La3+; Figure 10 (add 20->35 mmHg PCO2 switch; add scrGap26 above blue bars); Figure 11C & D.

We agree and can see that in Figs 9 and 10 this muddles our colour scheme in other figures so have modified these figures. There was not space to put the suggested labels.

(5) The mechanism of increased HC opening is not clear.

We agree and have discussed various options and the analogy with what we know about Cx26. Ultimately new cryo-EM data is required.

(6) Figure 10: 35G/35S are weird abbreviations for 35 mmHg Gap26 and scrambled Gap26.

Yes, but we used these to fit into the available space.

(7) Figure 11, legend: '20 mmHg PCO2 for each transfection for 70 mmHg PCO2'. It is not clear what is meant here.

Thanks for pointing this out, we have reworded this to ensure clarity.

From a social point of view, I think this issue goes beyond just individual teachers. Our whole society has long-standing stereotypes about race, behavior, and ability, and these ideas quietly shape how people see students before they even get to know them. So when a teacher misidentifies a student of color or sets lower expectations, it’s not always coming from personal intention but from the social messages they’ve absorbed.

This reinforces research we’ve seen in previous readings about representation and belonging. I agree with the author that diversifying the teacher workforce is essential. Students of color benefit academically and emotionally when they feel understood. This quote also connects with the data showing that Black students are less likely to drop out when they have a same-race teacher at least once

This point is significant because it reveals how much power teachers have in the referral process. When bias shapes this first step, disproportionality becomes almost inevitable. I found this compelling because it shows that inequity isn’t only about policy—it begins with everyday interpersonal judgments. It raises questions about how teacher expectations shape students’ academic identities.

This statistic is alarming because it shows a clear pattern of racial disproportionality. It suggests that Black students are being interpreted through a deficit lens, often due to cultural misunderstandings or implicit bias. The quote supports the argument that special education placement is not just about need—it’s about how teachers and systems perceive certain groups. This reinforces the need for culturally responsive training.

This quote highlights how poverty and disability labeling intersect to create inequity. What stood out to me is how subjective categories—like emotional disturbance—leave room for bias. I agree with the author that these categories, combined with segregation into separate classrooms, reflect systemic problems rather than student deficits. It connects to class themes about how institutions reproduce inequality through supposedly “neutral” practices.

Students of color are easy to identify when they make "trouble" but they’re actually not identified when they really need support. THe real issue is how often they get pushed into lower quality, segregated programs that don’t help them grow. That is just another form of discrimination. How can people in power make fair choices about kids when they don’t understand their backgrounds?

Black children are not troublemakers and the problems that teachers have with them are often misunderstandings. It’s kinda wild how something like valuing harmony or being more cooperative can get labeled as a weakness, just because the teacher doesn’t get the cultural background. This honestly makes it clear that cultural training isn’t optional, it’s necessary if we don’t want kids getting pushed into special ed for the wrong reasons.

I think there are so many consequences such as higher vision impairment, which on paper, does not have any correlation to lower-income, but is actually caused by it. This is the reason that students from low-income families are often mistreated as a result because people does not know the cause.

Interesting to note how an undergrad can interact so freely with their faculty advisor. This would be unlikely in an Asian culture with a rigid hierarchy.

This is counterintuitive to me. I thought exposure to allergens should reduce allergies over time right?

I think the best way is actually not to tell other students or even most teachers that she has any special needs. Instead, the school can assign one coordinator who quietly supports her in the classes where she might need extra help. I’ve always believed that people learn best from people, not from systems or labels. Even a child with special needs still has to learn how to interact with others, and the more normal the environment feels, the easier it is for her to build those skills.

One example that happened around me was when I was coaching after-school sports for elementary and kindergarten students. There was one kid who got really discouraged whenever he didn’t perform well. I didn’t think too much about it and just kept encouraging him like I would with any other student. But later, when I was putting the equipment away, another coach told me that the kid had special needs and was on the autism spectrum. I didn’t ask for this information, but once I heard it, I couldn’t unhear it. After that, even if I tried to act the same, the thought that “he is a special-needs student” would always pop up in my mind. And then I started worrying—if I spend too much extra time helping him, what about the other kids?

This closing statement is emotional and powerful. It captures the author’s plea for humanity, insisting that Lydia’s identity is far richer than her diagnosis. I think this quote reinforces the central message of the article: that true inclusion requires seeing every child as a full person with strengths, dreams, and dignity—rather than as a category to manage. It also reminds me how important it is for educators to acknowledge students as individuals, not labels.

This quote brings attention to the emotional consequences of being “Othered.” It shows how disability is not just a medical category but a social and psychological experience shaped by exclusion. I appreciate how the author frames Lydia’s pain not as an inevitable part of disability but as the result of how schools treat her. It challenges educators to recognize the harm caused by marginalization.

This statement captures the core critique of labeling: it collapses a whole person into a single description. I strongly agree with the author’s point because labels in education often function as limits rather than supports. This connects to class themes about how systems position students—whether due to disability, race, or gender—in ways that restrict their identities and opportunities.

This quote highlights how labels are context-dependent and often imposed by institutions rather than chosen by the child or family. I find it striking that Lydia’s identity shifts depending on the school setting, showing how educational systems construct and reinforce categories. It raises the question of how much of “special needs” is truly about the child, and how much is created by inflexible structures that cannot accommodate difference.

Identity is not simple. Lydia doesnt just sit there and accept the label people give her. The fact that she straight up says “I hate it!” when asked about being labeled shows how schools sometimes act like these terms are neutral or helpful, when for the kid it actually feels hurtful. It also made me think about how often adults talk about children with disabilities instead of actually asking them how they feel, which happens way too much.

I really like how the author is essentially calling out how labels in schools can turn into whole identities, even when they don’t describe the kid fully at all. The way she talks about Lydia being positioned as “one of those children” shows how people act like disability is the only thing that matters. It kind of makes me think about how schools pretend to be supportive but sometimes they just push students into categories because it’s easier for them.

The author challenges the idea that a child’s primary identity in school should be their label, such as “child with special needs.” This point is powerful because it shows how labels, even when intended to provide support, can end up defining and restricting a child’s possibilities. By sharing her own experience as both a teacher educator and a mother, the author demonstrates how deeply personal and interconnected these issues are. Her argument suggests that labeling students based on their differences reinforces narrow expectations and prevents teachers from seeing their full potential. The passage pushes educators to rethink how institutional language shapes children’s identities and to create environments that focus on inclusion rather than categorization.

of two unkown

It feels like many arguments about what is age-appropriate are not really about protecting children. They often come from adults who are uncomfortable with topics they do not understand. Schools can freely talk about male and female bodies in sex education and even show condoms in class, but the moment anything related to LGBTQ+ comes up, it suddenly becomes inappropriate.

For me, my parents always felt that anything I did other than studying was a waste of time. Even when I dyed my hair in high school, I didn’t dare tell my dad because I knew he would think it was meaningless and distracting. When I think about this now, I honestly can’t imagine how he would react if my identity or orientation were LGBTQ+. And the crazy thing is, my parents are already considered pretty open-minded compared to a lot of Asian parents.

From my own experience, a lot of things only start to feel “different” after other people point them out. When you’re just interacting with someone who has bipolar disorder or autism, you might notice that they act in a way that’s not typical, but you usually just adjust how you get along with them and it’s fine. There isn’t anything “wrong.” But once other classmates start saying that this person has a certain disorder or that their orientation is “weird,” you slowly get influenced. Something that was originally just unfamiliar becomes something you start to judge, and without even noticing it, you also begin to look at that person through a tinted lens.

This quote helped me understand why “Don’t Say Gay” policies are so harmful. Erasing LGBTQ+ topics from classrooms doesn’t just ignore queer youth—it opens the door for more restrictive and discriminatory policies. I agree with Ngo that acknowledgment is the foundation for support. Without visibility, there can be no advocacy, protection, or meaningful change.

I found this line very honest and emotionally direct. It captures the psychological harm caused not only by overt harassment, but by erasure. When teachers “know” LGBTQ+ students are present but never talk about them, the silence itself becomes a message of exclusion. This reflects course themes about how institutional neutrality is not neutral—it reinforces heteronormativity and makes students feel unseen.

This stood out to me because it captures how intersectional identities can conflict with family or cultural expectations. The pressure Ngo faced in an Asian American household reflects broader issues of collectivism, academic pressure, and heteronormativity. It shows how racial/cultural identity can intensify the difficulty of exploring gender or sexuality. The quote highlights that LGBTQ+ youth of color often navigate multiple layers of invisibility.

A great way to see the most important knowledge distilled from the Internet!

eLife Assessment

The authors develop an important microfluidic microvascular model called "Vessel-on-Chip", which they use to study Neisseria meningitidis interactions within this in vitro vascular system. Compelling evidence shows that the fabricated channels are lined by endothelial cells, and these can be colonized by N. meningitidis that in turn triggers neutrophil recruitment. This model has advantages over the human skin xenograft mouse model, which requires complex surgical techniques, however, it also carries limitations in that only endothelial cells and supplied specific immune cells in the microfluidics are present, while true vasculature contains a number of other cell types including smooth muscle cells, pericytes, and components of the immune system.

[Editors' note: this paper was reviewed by Review Commons.]

Reviewer #1 (Public review):

Summary:

The work by Pinon et al describes the generation of a microvascular model to study Neisseria meningitidis interactions with blood vessels. The model uses a novel and relatively high throughput fabrication method that allows full control over the geometry of the vessels. The model is well characterized from the vascular standpoint and shows improvements when exposed to flow. The authors show that Neisseria binds to the 3D model in a similar geometry that in the animal xenograft model, induces an increase in permeability short after bacterial perfusion, and endothelial cytoskeleton rearrangements including a honeycomb actin structure. Finally, the authors show neutrophil recruitment to bacterial microcolonies and phagocytosis of Neisseria.

Strengths:

The article is overall well written, and it is a great advancement in the bioengineering and sepsis infection field. The authors achieved their aim at establishing a good model for Neisseria vascular pathogenesis and the results support the conclusions. I support the publication of the manuscript. I include below some clarifications that I consider would be good for readers.

One of the most novel things of the manuscript is the use of a relatively quick photoablation system. Could this technique be applied in other laboratories? While the revised manuscript includes more technical details as requested, the description remains difficult to follow for readers from a biology background. I recommend revising this section to improve clarity and accessibility for a broader scientific audience.

The authors suggest that in the animal model, early 3h infection with Neisseria do not show increase in vascular permeability, contrary to their findings in the 3D in vitro model. However, they show a non-significant increase in permeability of 70 KDa Dextran in the animal xenograft early infection. As a bioengineer this seems to point that if the experiment would have been done with a lower molecular weight tracer, significant increases in permeability could have been detected. I would suggest to do this experiment that could capture early events in vascular disruption.

One of the great advantages of the system is the possibility of visualizing infection-related events at high resolution. The authors show the formation of actin of a honeycomb structure beneath the bacterial microcolonies. This only occurred in 65% of the microcolonies. Is this result similar to in vitro 2D endothelial cultures in static and under flow? Also, the group has shown in the past positive staining of other cytoskeletal proteins, such as ezrin in the ERM complex. Does this also occur in the 3D system?

Significance:

The manuscript is comprehensive, complete and represents the first bioengineered model of sepsis. One of the major strengths is the carful characterization and benchmarking against the animal xenograft model. Beyond the technical achievement, the manuscript is also highly quantitative and includes advanced image analysis that could benefit many scientists. The authors show a quick photoablation method that would be useful for the bioengineering community and improved the state-of-the-art providing a new experimental model for sepsis.

My expertise is on infection bioengineered models.

Comments on revised version:

The authors have addressed all my concerns.

Reviewer #2 (Public review):

Pinon and colleagues have developed a Vessel-on-Chip model showcasing geometrical and physical properties similar to the murine vessels used in the study of systemic infections. The authors succeed on their aim of developing an complex, humanized, in vitro model that can faithfully recapitulate the hallmarks of systemic infections.

The vessel was created via highly controllable laser photoablation in a collagen matrix, subsequent seeding of human endothelial cells, and flow perfusion to induce mechanical cues. This model could be infected with Neisseria meningitidis as a model of systemic infection. In this model, microcolony formation and dynamics, and effects on the host were very similar to those described for the human skin xenograft mouse model (the current gold standard for systemic studies) and were consistent with observations made in patients. The model could also recapitulate the neutrophil response upon N. meningitidis systemic infection.

The claims and the conclusions are supported by the data, the methods are properly presented, and the data is analyzed adequately. The most important strength of this manuscript is the technology developed to build this model, which is impressive and very innovative. The Vessel-on-Chip can be tuned to acquire complex shapes and, according to the authors, the process has been optimized to produce models very quickly. This is a great advancement compared with the technologies used to produce other equivalent models. This model proves to be equivalent to the most advanced model used to date (skin xenograft mouse model). The human skin xenograft mouse model requires complex surgical techniques and has the practical and ethical limitations associated with the use of animals. However, the Vessel-on-chip model is free of ethical concerns, can be produced quickly, and allows to precisely tune the vessel's geometry and to perform higher resolution microscopy. Both models were comparable in terms of the hallmarks defining the disease, suggesting that the presented model can be an effective replacement of the animal use in this area. In addition, the Vessel-on-Chip allows to perform microscopy with higher resolution and ease, which can in turn allow more complex and precise image-based analysis. The authors leverage the image-based analysis to obtain further insights into the infection, highlighting the capabilities of the model in this aspect.

A limitation of this model is that it lacks the multicellularity that characterizes other similar models, which could be useful to research disease more extensively. However, the authors discuss the possibilities of adding other cells to the model, for example, fibroblasts. The methodology would allow for integrating many different types of cells into the model, which would increase the scope of scientific questions that can be addressed. In addition, the technology presented in the current paper is also difficult to adapt for standard biology labs. The methodology is complex and requires specialized equipment and personnel, which might hinder its widespread utilization of this model by researchers in the field.

This manuscript will be of interest for a specialized audience focusing on the development of microphysiological models. The technology presented here can be of great interest to researchers whose main area of interest is the endothelium and the blood vessels, for example, researchers on the study of systemic infections, atherosclerosis, angiogenesis, etc. This manuscript can have great applications for a broad audience focusing on vasculature research. Due to the high degree of expertise required to produce these models, this paper can present an interesting opportunity to begin collaborations with researchers dealing with a wide range of diseases, including atherosclerosis, cancer (metastasis), and systemic infections of all kinds.

Reviewer #3 (Public review):

Summary:

In this manuscript Pinon et al. describe the development of a 3D model of human vasculature within a microchip to study Neisseria meningitidis (Nm)- host interactions and validate it through its comparison to the current gold-standard model consisting of human skin engrafted onto a mouse. There is a pressing need for robust biomimetic models with which to study Nm-host interactions because Nm is a human-specific pathogen for which research has been primarily limited to simple 2D human cell culture assays. Their investigation relies primarily on data derived from microscopy and its quantitative analysis, which support the authors' goal of validating their Vessel-on-Chip (VOC) as a useful tool for studying vascular infections by Nm, and by extension, other pathogens associated with blood vessels.

Strengths:

• Introduces a novel human in vitro system that promotes control of experimental variables and permits greater quantitative analysis than previous models<br /> • The VOC model is validated by direct comparison to the state-of-the-art human skin graft on mouse model<br /> • The authors make significant efforts to quantify, model, and statistically analyze their data<br /> • The laser ablation approach permits defining custom vascular architecture<br /> • The VOC model permits the addition and/or alteration of cell types and microbes added to the model<br /> • The VOC model permits the establishment of an endothelium developed by shear stress and active infusion of reagents into the system

Weaknesses:

• The VOC model contains one cell type, human umbilical cord vascular endothelial cells (HUVECs), while true vasculature contains a number of other cell types that associate with and affect the endothelium, such as smooth muscle cells, pericytes, and components of the immune system. However, adding such complexity may be a future goal of this VOC model.

Impact:

The VOC model presented by Pinon et al. is an exciting advancement in the set of tools available to study human pathogens interacting with the vasculature. This manuscript focuses on validating the model, and as such sets the foundation for impactful research in the future. Of particular value is the photoablation technique that permits the custom design of vascular architecture without the use of artificial scaffolding structures described in previously published works.

Comments on revised version:

The authors have nicely addressed my (and other reviewers') comments.

Author response:

The following is the authors’ response to the original reviews

Public Reviews:

Reviewer #1 (Public review):

One of the most novel things of the manuscript is the use of a relatively quick photoablation system. Could this technique be applied in other laboratories? While the revised manuscript includes more technical details as requested, the description remains difficult to follow for readers from a biology background. I recommend revising this section to improve clarity and accessibility for a broader scientific audience.

As suggested, we have adapted the paragraph related to the photoablation technique in the Material & Method section, starting line 1147. We believe it is now easier to follow.

The authors suggest that in the animal model, early 3h infection with Neisseria do not show increase in vascular permeability, contrary to their findings in the 3D in vitro model. However, they show a non-significant increase in permeability of 70 KDa Dextran in the animal xenograft early infection. As a bioengineer this seems to point that if the experiment would have been done with a lower molecular weight tracer, significant increases in permeability could have been detected. I would suggest to do this experiment that could capture early events in vascular disruption.

Comparing permeability under healthy and infected conditions using Dextran smaller than 70 kDa is challenging. Previous research (1) has shown that molecules below 70 kDa already diffuse freely in healthy tissue. Given this high baseline diffusion, we believe that no significant difference would be observed before and after N. meningitidis infection, and these experiments were not carried out. As discussed in the manuscript, bacteria-induced permeability in mice occurs at later time points, 16h post-infection, as shown previously (2). As discussed in the manuscript, this difference between the xenograft model and the chip could reflect the absence of various cell types present in the tissue parenchyma or simply vessel maturation time.

One of the great advantages of the system is the possibility of visualizing infection-related events at high resolution. The authors show the formation of actin in a honeycomb structure beneath the bacterial microcolonies. This only occurred in 65% of the microcolonies. Is this result similar to in vitro 2D endothelial cultures in static and under flow? Also, the group has shown in the past positive staining of other cytoskeletal proteins, such as ezrin, in the ERM complex. Does this also occur in the 3D system?

We imaged monolayers of endothelial cells in the flat regions of the chip (the two lateral channels) using the same microscopy conditions (i.e., Obj. 40X N.A. 1.05) that have been used to detect honeycomb structures in the 3D vessels in vitro. We showed that more than 56% of infected cells present these honeycomb structures in 2D, which is 13% less than in 3D, and is not significant due to the distributions of both populations. Thus, we conclude that under both in vitro conditions, 2D and 3D, the amount of infected cells exhibiting cortical plaques is similar. These results are in Figure 4E and S4B.

We also performed staining of ezrin in the chip and imaged both the 3D and 2D regions. Although ezrin staining was visible in 3D (Author response image 1), it was not as obvious as other markers under these infected conditions, and we did not include it in the main text. Interpretation of this result is not straightforward, as the substrate of the cells is different, and it would require further studies on the behavior of ERM proteins in these different contexts.

Author response image 1.

F-actin (red) and ezrin (yellow) staining after 3h of infection with N. meningitidis (green) in 2D (top) and 3D (bottom) vessel-on-chip models.

Recommendation to the authors:

Reviewer #1 (Recommendation to the authors):

I appreciate that the authors addressed most of my comments, of special relevance are the change of the title and references to infection-on-chip. I think that the current choice of words better acknowledges the incipient but strong bioengineering infection community. I also appreciate the inclusion of a limitation paragraph that better frames the current work and proposes future advancements.

The addition of more methodological details has improved the manuscript. Although as mentioned earlier the wording needs to be accessible for the biology community. I also appreciated the addition of the quantification of binding under the WSS gradient in the different geometries and shown in Fig 3H. However, the description of the figure and the legend is not clear. What does "vessel" mean on the graph and "normalized histograms ...(blue)" in the figure legend. Could the authors rephrase it?

In Figure 3F, we investigated whether Neisseria meningitidis exhibits preferential sites of infection. We hypothesized that, if bacteria preferentially adhered to specific regions, the local shear stress at these sites would differ from the overall distribution. To test this, we compared the shear stress at bacterial adhesion sites in the VoC (orange dots and curve) with the shear stress along the entire vascular edges (blue dots and curve). The high Spearman correlation indicates that there is no distinct shear stress value associated with bacterial adhesion. This suggests that bacteria can adhere across all regions, independently of local shear stress. To enhance clarity, the legend of Figure 3 and the related text have been rephrased in the revised manuscript (L289-314).

Line 415. Should reference to Fig S5B, not Fig 5B. Also, the titles in Supplementary Figure 4 and 5 are duplicated, and the description of the legend inf Fig S5 seems a bit off. A and B seem to be swapped.

Indeed, the reference to the right figure has been corrected. Also, the title of Figure S4 has been adapted to its contents, and the legend of Figure S5 has been corrected.

Reviewer #2 (Recommendation to the authors):

Minor comments to the authors:

Line 163 "they formed" instead of "formed".

Line 212 "two days" instead of "two day"

Line 269 a space between two words is missing.

These three comments have been addressed in the revised manuscript.

In addition, I appreciate answering the comments, especially those requiring hypothesizing about including further cells. However, when discussing which other cells could be relevant for the model (lines 631 to 632) it would be beneficial to discuss not only the role of those cells but also how could they be included in the model. I think for the reader, inclusion of further cells could be seen as a challenge or limitation, and addressing these technical points in the discussion could be helpful.

We thank Reviewer #2 for the insightful suggestion. Indeed, the method of introducing cells into the VoC depends on their type. Fibroblasts and dendritic cells, which are resident tissue cells, should be embedded in the collagen gel before polymerization and UV carving. This requires careful optimization to preserve chip integrity, as these cells exert pulling forces while migrating within the collagen matrix. In contrast, T cells and macrophages should be introduced through the vessel lumen to mimic their circulation in vivo. Pericytes can be co-seeded with endothelial cells, as they have been shown to self-organize within a few hours post-seeding. These important informations are now included in the manuscript (L577-587).

Reviewer #3 (Recommendation to the authors):

Suggestions and Recommendations

Some suggestions related to the VOC itself:

Figure 1, Fig S1, paragraph starting line 1071: More information would be helpful for the laser photoablation. For instance, is a non-standard UV laser needed? Which form of UV light is used? What is the frequency of laser pulsing? How many pulses/how long is needed to ablate the region of interest?

The photoablation process requires a focused UV-laser, with high frequency (10 kHz) to lower the carving time while providing the required intensity to degrade collagen gel. To carve a reproducible number of 30 µm-large vessels, we used a 2 µm-large laser beam at an energy of 10 mW and moved the stage (i.e., sample) at a maximum speed of 1 mm/s. This information has been added to the related paragraph starting on line 1147 of the revised manuscript.

It is difficult to understand the geometry of the VOC. In Figure 1C, is the light coloration representing open space through which medium can flow, and the dark section the collagen? On a single chip, how many vessels are cut through the collagen? It looks as if at least two are cut in Figure 1C in the righthand photo.

In Figure 1C, the light coloration is the Factin staining. The horizontal upper and lower parts are the 2D lateral channels that also contain endothelial cells, and are connected to inlets and outlets, respectively. In the middle, two vertically carved 3D vessels are shown in the confocal image.

Technically, we designed the PDMS structures to allow carving of 1 to 3 channels, maximizing the number of vessels that can be imaged while minimizing any loss of permeability at the PDMS/collagen/cells interface. This information has been added in the revised manuscript (L. 1147).

If multiple vessels are cut in the center channel between the lateral channels, how do you ensure that medium flow is even between all vessels? A single chip with multiple different vessel architectures through the center channel would be expected to have different hydrostatic resistance with different architectures, thereby causing differences in flow rates in each vessel.

To ensure a consistent flow rate regardless of the number of carved vessels, we opted to control the flow rate directly across the chip with a syringe pump. During experiments, one inlet and one outlet were closed, and a syringe pump was used. Because the carved vessels are arranged in parallel (derivation), the flow rate remains the same in each vessel. If a pressure controller had been used instead, the flow would have been distributed evenly across the different channels. This has been added to the revised manuscript in the paragraph starting on line 1210.

The figures imply that the laser ablation can be performed at depth within the collagen gel, rather than just etching the surface. If this is the case, it should be stated explicitly. If not, this needs to be clarified.

One of the main advantages of the photoablation technique is carving the collagen gel in volume, and not only etching the surface. Thanks to the 3D UV degradation, we can form the 3D architecture surrounded by the bulk collagen. This has been added to the revised manuscript, lines 154-155.

Is the in-vivo-like vessel architecture connected to the lateral channel at an oblique angle, or is the image turned to fit the entire structure? (Figure 1F and 3E). Is that why there is high shear stress at its junction with the lateral channel depicted in Figure 3E?

All structures require connection to the lateral channels to ensure media circulation and nutrient supply. The in vivo-like design must be rotated to allow the upper and lower branches of the complex structure to pass between the fixed PDMS pillars. To remain consistent with the image and the flow direction, we have kept the same orientation as in the COMSOL simulation. This leads to a locally higher shear stress at the top of the architecture. This has been added in the revised manuscript, in the paragraph starting on line 1474.

Figure S1F,G: In the legend, shapes are circles, not squares. On the graphs, what do the numbers in parentheses mean?

Indeed, the terms "squares" have been replaced by "circles" in Figure 1. (1) and (2) refer to the providers of the collagen, FujiFilm and Corning, respectively. We have added this mention in the legend in Figure S1.

Figure 3B: how do the images on the left and right differ? Each of the 4 images needs to be explained.

The four images represent the infected VoC from different viewing angles, illustrating the three-dimensional spread of infection throughout the vessel. A more detailed description has been added in the legend of Figure 3.

Figure S3C is not referenced but should be, likely before sentence starting on line 299.

Indeed, the reference to Figure S3C has been added line 301 of the revised manuscript.

Results in Figure 3 with the pilD mutant are very interesting. It is worth commenting in the Discussion about how T4P functionality in addition to the presence of T4P contributes to Nm infection, and how in the future this could be probed with pilT mutants.

We thank Reviewer #3 for this relevant insight. Following adhesion, a key functionality of Neisseria meningitidis for colony formation and enhanced infection is twitching motility. As suggested, we have added in the Discussion the idea of using a PilT mutant, which can adhere but cannot retract its pili, in the VoC model to investigate the role of motility in colonization in vitro under flow conditions (L611–623).

Which vessel design was used for the data presented in Figures 4, 5, and 6 and associated supplemental figures?

Straight channels have been mostly used in figures 4, 5, and 6. Rarely, we used the branched in vivo-like designs to observe potential similar infection patterns to in vivo, and related neutrophil activity. This has been added in the revised manuscript, lines 1435-1439.

Figure 4B-D: the images presented in Figure 4C are not representative of the averages presented in Figures 4B,D. For instance, the aggregates appear much larger and more elongated in the animal model in Figure 4C, but the animal model and VOC have the colony doubling time (implying same size) in Figure 4B, and same average aggregate elongation in Figure 4D.

The images in Figure 4C were selected to illustrate the elongation of colonies quantified in Figure 4D. The elongation angles are consistent between both images and align with the channel orientation. Representative images of colony expansion over time, corresponding to Figure 4A and 4B, are provided in Figure S4A.

Figures 4E-F: dextran does not appear to diffuse in the VOC in response to histamine in these images, yet there is a significant increase in histamine-induced permeability in Figure 4F. Dotted lines should be used to indicate vessel walls for histamine, and/or a more representative image should be selected. A control set of images should also be included for comparison.

We thank Reviewer #3 for the insightful comment. We confirm that we have carefully selected representative images for the histamine condition and adjusted them to display the same range of gray levels. The apparent increase in permeability with histamine is explained by a slight rise in background fluorescence, combined with the smaller channel size shown in Figure 4E.

Figure S4 title is a duplicate of Figure S5 and is unrelated to the content of Figure S4. Suggest rewording to mention changes in permeability induced by Nm infection in the VOC and animal model.

Indeed, the title of Figure S4 did not correspond to its content. We have, thus, changed it in the revised manuscript.

Line 489 "...our Vessel-on-Chip model has the potential to fully capture the human neutrophil response during vascular infections, in a species-matched microenvironment", is an overstatement. As presented, the VOC model only contains endothelial cells and neutrophils. Many other cell types and structures can affect neutrophil activity. Thus, it is an overstatement to claim that the model can fully capture the human neutrophil response.

We agree with the Reviewer #3, that neutrophil activity is fully recapitulated with other cell types, such as platelets, pericytes, macrophages, dendritic cells, and fibroblasts, that secrete important molecules such as cytokines, chemokines, TNF-α, and histamine. In our simplified model we were able to reconstitute the complex interaction of neutrophils with endothelial cells and with bacteria. The text was modified accordingly.

Supplemental Figure 6 - Does CD62E staining overlap with sites of Nm attachment

E-selectin staining does not systematically colocalize with Neisseria meningitidis colonies although bacterial adhesion is required. Its overall induced expression is heterogeneous across the tissue and shows heterogeneity from cell to cell as seen in vivo.

Line 475, Figure 6E- Phagocytosis of Nm is described, but it is difficult to see. An arrow should be added to make this clear. Perhaps the reference should have been to Figure 6G? Consider changing the colors in Figure 6G away from red/green to be more color-blind friendly.

Indeed, the reference to the right figure is Figure 6G, where the phagocytosis event is zoomed in. We have changed it in the text. Adapting the color of this figure 6G would imply to also change all the color codes of the manuscript, as red has been used for actin and green for Neisseria meningitidis.

Lines 621-632 - This important discussion point should be reworked. Some suggested references to cite and discuss include PMID: 7913984, 15186399, 17991045, 18640287, 19880493.

We have introduced in the discussion parts the following references as suggested (3–7), and discussed more the importance of introducting of immune cells to study immune cell-bacteria interaction and related immune response (L659-678).

Minor corrections:

•  Line 8 - suggest "photoablation-generated" instead of "photoablation-based"

•  Line 57- remove the word "either", or modify the sentence

•  Sentence on lines 162-165 needs rewording

•  Lines 204-205- "loss of vascular permeability" should read "increase in vascular permeability"

•  Line 293- "Measured" shear stress, should be "computed", since it was not directly measured (according to the Materials & Methods)

•  Line 304- "consistently" should be "consistent"

•  Fig. 3 legend, second line: replace "our" with "the VoC"

•  Line 371, change "our" to "the"

•  Line 415- Figure 5B doesn’t appear to show 2-D data. Is this in Figure S5B? Some clarification is needed. The quantification of Nm vessel association in both the VOC and the animal model should be shown in Figure 5, for direct comparison.

•  Supplementary Figure 5C: correlation coefficient with statistical significance should be calculated.

•  Figure 6 title, rephrase to "The infected VOC model"

•  Line 450, replace "important" with "statistically significant"

•  Line 459, suggest rephrasing to "bacterial pilus-mediated adhesion"

•  Line 533- grammar needs correction

•  Line 589- should be "sheds"

•  Line 1106- should be "pellet"

•  Lines 1223-1224 - is the antibody solution introduced into the inlet of the VOC for staining? Please clarify.

•  Line 1295-unclear why Figure 2B is being referenced here

All the suggested minor corrections have been taken into account in the revised manuscript.

References

(1) Gyohei Egawa, Satoshi Nakamizo, Yohei Natsuaki, Hiromi Doi, Yoshiki Miyachi, and Kenji Kabashima. Intravital analysis of vascular permeability in mice using two-photon microscopy. Scientific Reports, 3(1):1932, Jun 2013. ISSN 2045-2322. doi: 10.1038/srep01932.

(2) Valeria Manriquez, Pierre Nivoit, Tomas Urbina, Hebert Echenique-Rivera, Keira Melican, Marie-Paule Fernandez-Gerlinger, Patricia Flamant, Taliah Schmitt, Patrick Bruneval, Dorian Obino, and Guillaume Duménil. Colonization of dermal arterioles by neisseria meningitidis provides a safe haven from neutrophils. Nature Communications, 12(1):4547, Jul 2021. ISSN 2041-1723. doi: 10.1038/s41467-021-24797-z.

(3) Katherine A. Rhodes, Man Cheong Ma, María A. Rendón, and Magdalene So. Neisseria genes required for persistence identified via in vivo screening of a transposon mutant library. PLOS Pathogens, 18(5):1–30, 05 2022. doi: 10.1371/journal.ppat.1010497.

(4) Heli Uronen-Hansson, Liana Steeghs, Jennifer Allen, Garth L. J. Dixon, Mohamed Osman, Peter Van Der Ley, Simon Y. C. Wong, Robin Callard, and Nigel Klein. Human dendritic cell activation by neisseria meningitidis: phagocytosis depends on expression of lipooligosaccharide (los) by the bacteria and is required for optimal cytokine production. Cellular Microbiology, 6(7):625–637, 2004. doi: https://doi.org/10.1111/j.1462-5822.2004.00387.x.

(5) M. C. Jacobsen, P. J. Dusart, K. Kotowicz, M. Bajaj-Elliott, S. L. Hart, N. J. Klein, and G. L. Dixon. A critical role for atf2 transcription factor in the regulation of e-selectin expression in response to non-endotoxin components of neisseria meningitidis. Cellular Microbiology, 18(1):66–79, 2016. doi: https://doi.org/10.1111/cmi.12483.

(6) Andrea Villwock, Corinna Schmitt, Stephanie Schielke, Matthias Frosch, and Oliver Kurzai. Recognition via the class a scavenger receptor modulates cytokine secretion by human dendritic cells after contact with neisseria meningitidis. Microbes and Infection, 10(10):1158–1165, 2008. ISSN 1286-4579. doi: https://doi.org/10.1016/j.micinf.2008.06.009.

(7) Audrey Varin, Subhankar Mukhopadhyay, Georges Herbein, and Siamon Gordon. Alternative activation of macrophages by il-4 impairs phagocytosis of pathogens but potentiates microbial-induced signalling and cytokine secretion. Blood, 115(2):353–362, Jan 2010. ISSN 0006-4971. doi: 10.1182/blood-2009-08-236711.

: Düşük frekanslı akustik enerjinin eklenmesi, dinamik sıvı hareketi oluşturur ve kılların uçlarından biraz uzakta temizlik sağlar.

① : Elektrikli diş fırçaları, plak biyofilmini uzaklaştırmak için öncelikle kıllar ile diş arasındaki mekanik temasa dayanır.

Sert kıllı diş fırçalarının kullanımı, daha fazla diş eti çekilmesi ile ilişkilidir. Ancak, fırçanın kullanım şekli ve diş macununun aşındırıcı özelliği, aşınmayı kılların sertliğinden daha fazla etkiler.

: Daha yumuşak kıllar daha esnektir, sulkus fırçalama tekniği ile kullanıldığında diş eti kenarının biraz altını temizler ve komşu yüzeylere daha iyi ulaşır.

Kıl sertliği, çapın karesi ile doğru orantılı ve kıl uzunluğunun karesi ile ters orantılıdır.

① : Bristles should be 0.2 mm in diameter, 10 mm in length and have rounded tips. ① : Kıllar 0,2 mm çapında, 10 mm uzunluğunda ve yuvarlatılmış uçlu olmalıdır.

② : Handle of the brush should be straight and long enough for the palm to grasp. ② : Fırçanın sapı düz ve avucun kavrayabileceği kadar uzun olmalıdır.

③ : There should be 3-4 rows, each consisting of 5-12 bristle clusters. ③ : Her biri 5-12 kıl demetinden oluşan 3-4 sıra bulunmalıdır.

Her ikisi de mikrobiyal plak biyofilmlerini temizler, ancak piyasada naylon kıllı fırçalar büyük ölçüde baskındır.

Farklı manuel diş fırçalarındaki aşınma, fırça kıllarının kendisinden ziyade, fırça tasarımının fırçalama sırasında daha fazla diş macunu taşımasına izin vermesinin aşınmaya daha fazla katkıda bulunduğunu göstermektedir.

Farklı fırça tiplerinin etkinliği ve potansiyel yaralanmalar büyük ölçüde fırçaların nasıl kullanıldığına bağlıdır.

① : Belirli bir diş fırçası önerilirken, hastanın kullanım kolaylığı ve fırçanın iyi çalıştığı algısı önemli faktörlerdir.

① : Manual Toothbrushes ① : Manuel Diş Fırçaları

② : Size ② : Boyut

③ : Design ③ : Tasarım

④ : Length of bristles ④ : Kıl uzunluğu

⑤ : Hardness of the bristles ⑤ : Kıl sertliği

⑥ : Arrangement of the bristles ⑥ : Kıl düzeni

① : It is a critical component of dental practice, permitting long-term success of periodontal and dental care. ① : Diş hekimliği uygulamasının kritik bir bileşenidir ve periodontal ile diş bakımının uzun vadeli başarısını sağlar. ② : Plaque control is the only practical method for maintaining oral hygiene. ② : Diş plağı kontrolü, ağız hijyenini sürdürmenin tek pratik yöntemidir. ③ : Mechanical debridement with toothbrush and dental floss is the most important part of plaque control. ③ : Diş fırçası ve diş ipi ile yapılan mekanik temizlik, diş plağı kontrolünün en önemli parçasıdır.

① : The initiation, development and progression of diseases are prevented. ① : Hastalıkların başlatılması, gelişimi ve ilerlemesi önlenir.

① : Wound healing becomes easier during periodontal surgical treatment. ① : Periodontal cerrahi tedavi sırasında yara iyileşmesi kolaylaşır.

① : Otherwise, recurrence of the disease after surgery cannot be prevented. ① : Aksi takdirde, cerrahi sonrası hastalığın tekrarı önlenemez.

① : It is the most important factor in the long-term continuity of periodontal health after treatment. ① : Tedavi sonrası periodontal sağlığın uzun vadeli sürekliliğinde en önemli faktördür.

① : Diş plağı kontrolü, diş plağının düzenli olarak uzaklaştırılması ve dişler ile komşu dişeti yüzeylerinde birikmesinin önlenmesidir.

Türkçe: Faz I tedavisi ile inflamatuar değişiklikler kontrol altına alınamaz veya azaltılamazsa, genel prognoz olumsuzdur.

(①) When few teeth remain, the prosthodontic needs become more important, and sometimes periodontally treatable teeth may have to be extracted if they are not compatible with the design of the prosthesis. (①) Az sayıda diş kaldığında, protez ihtiyaçları daha önemli hale gelir ve bazen periodontal olarak tedavi edilebilecek dişler, protez tasarımı ile uyumlu değillerse çekilmek zorunda kalabilir.

①) For example, saving or losing a key tooth may determine whether other teeth are saved or extracted or whether the prosthesis is fixed or removable. (①) Örneğin, kritik bir dişin korunup korunmaması, diğer dişlerin korunup çıkarılacağını veya protezin sabit mi yoksa hareketli mi olacağını belirleyebilir.

(①) At this point, the overall prognosis and individual tooth prognosis overlap because the prognosis for key individual teeth may affect the overall prognosis for prosthetic rehabilitation. (①) Bu noktada, genel prognoz ve bireysel diş prognozu örtüşür çünkü bazı kritik dişlerin prognozu, protez rehabilitasyonu için genel prognozu etkileyebilir.

①) The overall prognosis requires a general consideration of bone levels (evaluated radiographically) and attachment levels (determined clinically) to establish whether enough teeth can be saved either to provide functional and aesthetic dentition or to serve as abutments for a useful prosthetic replacement of the missing teeth. (①) Genel prognoz, yeterli dişin korunup korunamayacağını belirlemek için kemik seviyelerinin (radyografik olarak değerlendirilen) ve tutunma seviyelerinin (klinik olarak belirlenen) genel olarak göz önünde bulundurulmasını gerektirir; bu, işlevsel ve estetik bir dentisyon sağlamak veya eksik dişlerin faydalı bir protez ile yerine konması için destek dişler olarak hizmet etmek amacıyla yapılır.

①) The periodontal prognosis of treated nonvital teeth does not differ from that of vital teeth. New attachment can occur to the cementum of both nonvital and vital teeth. (①) Tedavi edilmiş vital olmayan dişlerin periodontal prognozu, vital dişlerin prognozundan farklı değildir. Yeni tutunma, hem vital hem de vital olmayan dişlerin sementumuna gerçekleşebilir.

(①) Extensive idiopathic root resorption or root resorption resulting from orthodontic therapy jeopardizes the stability of teeth and adversely affects the response to periodontal treatment. (①) Geniş kapsamlı idiyopatik kök rezorpsiyonu veya ortodontik tedaviye bağlı kök rezorpsiyonu, dişlerin stabilitesini tehlikeye atar ve periodontal tedaviye yanıtı olumsuz etkiler

①) For teeth mutilated by extensive caries, the feasibility of adequate restoration and endodontic therapy should be considered before undertaking periodontal treatment. (①) Geniş çürüklerle tahrip olmuş dişlerde, periodontal tedaviye başlamadan önce yeterli restorasyon ve endodontik tedavinin mümkün olup olmadığı değerlendirilmelidir

(①) Principal causes: (①) Başlıca nedenler:

(②) Loss of alveolar bone (②) Alveoler kemik kaybı

(③) Inflammatory changes in the periodontal ligament (③) Periodontal ligamentte inflamatuvar değişiklikler

(④) Trauma from occlusion (④) Oklüzyon travması

①) Meanwhile, if there is no furcation involvement, the tooth with the long trunk has a better prognosis. However, if there is furcation involvement, the tooth with the long trunk will have a poor prognosis because this indicates a large amount of bone loss. (①) Diğer yandan, furkasyon tutulumu yoksa uzun kök gövdeli diş daha iyi prognoza sahiptir. Ancak furkasyon tutulumu varsa, uzun kök gövdeli dişin prognozu kötüdür çünkü bu, büyük miktarda kemik kaybı olduğunu gösterir.

①) Root with short trunks has a better prognosis if there is furcation involvement because this means that the amount of bone destruction is less (①) Kısa kök gövdeli diş, furkasyon tutulumu varsa daha iyi prognoza sahiptir çünkü bu, kemik yıkım miktarının daha az olduğu anlamına gelir

(①) İki dişten biri kısa kök gövdesine, diğeri uzun kök gövdesine sahip olduğunda, grade 2 furkasyon tutulumu açısından hangisinin prognozu daha iyidir?

(①) It's better for the prognosis to have long, wide roots over short, narrow ones, and to have multi-rooted over single-rooted teeth. (①) Prognoz için kısa, dar kökler yerine uzun, geniş köklere sahip olmak ve tek köklü dişler yerine çok köklü dişlere sahip olmak daha iyidir

(①) Disproportionate crown-to-root ratio and the reduced root surface available for periodontal support, the periodontium may be more susceptible to injury by occlusal forces (①) Orantısız taç-kök oranı ve periodontal destek için azalmış kök yüzeyi, periodonsiyumu oklüzal kuvvetlerden kaynaklanan yaralanmalara daha duyarlı hale getirebilir

③) The furcation entrance is narrower than a standard curette in 58% of first molars. (③) İlk büyük azı dişlerinin %58’inde furkasyon girişi standart küretlerden daha dardır

(④) This significantly decreases accessibility, ultimately compromising prognosis. (④) Bu durum, ulaşılabilirliği ciddi şekilde azaltır ve sonuçta prognozu olumsuz etkiler

②) Bu işlemin etkinliğini azaltan anatomik faktörler, prognozu olumsuz etkileyebilir

(①) The microbial challenge in bacterial plaque and calculus is the most important local factor in periodontal diseases. (①) Bakteriyel plak ve diş taşındaki mikrobiyal etki, periodontal hastalıklarda en önemli lokal faktördür

(①) Genetic factors also influence serum IgG2 antibody titers and the expression of Fc-γRII receptors on the neutrophil - aggressive periodontitis. (①) Genetik faktörler, serum IgG2 antikor düzeylerini ve nötrofiller üzerindeki Fc-γRII reseptörlerinin ekspresyonunu da etkiler – agresif periodontitis

🦷 Açıklama:

Genetik faktörler, bağışıklık sisteminin bazı özelliklerini belirler:

IgG2 antikor düzeyleri:

Bakterilere karşı spesifik yanıtı etkiler.

Düşük IgG2 → yetersiz bağışıklık yanıtı → agresif periodontitis riski artar.

Fc-γRII reseptörleri (nötrofillerde):

Nötrofillerin bakteri tanıma ve fagositoz yeteneğini düzenler.

Genetik varyasyonlar, bu reseptörlerin etkinliğini azaltabilir → periodontal yıkım hızlanır.

Özetle: Genetik yapımız, bağışıklık yanıtımızı etkileyerek agresif periodontitis gelişiminde kritik rol oynar.

(①) Smoking (①) Sigara

(②) Risk factor (②) Risk faktörü

(③) Direct relationship - smoking and the prevalence and incidence of periodontitis (③) Doğrudan ilişki – sigara kullanımı ile periodontitisin prevalansı ve insidansı arasında

(④) Affects severity (④) Hastalığın şiddetini etkiler

(⑤) Affects healing (⑤) İyileşmeyi olumsuz etkiler

(⑥) Slight to moderate periodontitis - fair to poor (⑥) Hafif ila orta düzey periodontitis – orta ila kötü prognoz

(⑦) Severe periodontitis - poor to hopeless (⑦) Şiddetli periodontitis – kötü ila umutsuz prognoz

(①) The patient's attitude (①) Hastanın tutumu

(②) Desire to retain the natural teeth, (②) Doğal dişlerini koruma isteği,

(③) Willingness and ability to maintain good oral hygiene (③) İyi ağız hijyenini sürdürme isteği ve yeteneği

(④) Timely periodic maintenance checkups (④) Zamanında ve düzenli periyodik bakım kontrolleri

(②) Prognosis for horizontal bone loss depends on the height of the existing bone. (②) Horizontal kemik kaybının prognozu, mevcut kemik yüksekliğine bağlıdır.

(③) Angular defects; if the contour of the existing bone & the number of osseous walls are favorable, there is an excellent chance that therapy could regenerate bone to approximately the level of the alveolar crest = good prognosis. (③) Açısal (angular) defektlerde; mevcut kemiğin konturu ve kemik duvarlarının sayısı uygun ise, tedavi kemiği alveoler tepe seviyesine yaklaşık olarak yeniden oluşturabilir → iyi prognoz.

(④) When greater bone loss has occurred on one surface of a tooth, the bone height on the less involved surfaces should be taken into consideration when determining the prognosis. (④) Bir dişin bir yüzeyinde daha fazla kemik kaybı olmuşsa, prognozu belirlerken daha az etkilenen yüzeylerdeki kemik yüksekliği dikkate alınmalıdır.

(⑤) Less bone loss = better prognosis. (⑤) Daha az kemik kaybı = daha iyi prognoz.

①) The determination of the level of clinical attachment (CAL) reveals the approximate extent of root surface that is devoid of periodontal ligament. (①) Klinik tutunma seviyesinin (CAL) belirlenmesi, periodontal ligamentten yoksun olan kök yüzeyinin yaklaşık boyutunu ortaya koyar.

(②) Deep pockets with little attachment and bone loss has a better prognosis than one with shallow pockets and severe attachment and bone loss. (②) Az tutunma ve kemik kaybı olan derin cepler, ciddi tutunma ve kemik kaybı olan sığ ceplerden daha iyi prognoza sahiptir.

(③) If CAL is less good to fair prognosis. (③) Eğer CAL seviyesi az ise, prognoz iyi ila orta düzeydedir.

(①) Gingivitis:

Inflammation of the gingiva without loss of connective tissue or bone.

Bağ dokusu veya kemik kaybı olmadan diş etlerinin iltihaplanmasıdır.

(②) Periodontitis:

Inflammation of the supporting structures of the teeth, including alveolar bone, leading to attachment loss.

Dişleri destekleyen yapılar (alveoler kemik, periodontal ligament) etkilenir ve dişin bağ dokusu kaybına yol açar.

(①) Önerilen tedavi planına hastanın uyumu da belirlenebilir.

①) The provisional prognosis allows the clinician to initiate treatment of teeth that have a doubtful outlook in the hope that a favorable response may tip the balance and allow teeth to be retained. (①) Geçici prognoz, klinisyene şüpheli bir geleceğe sahip dişlerin tedavisine başlamasına olanak tanır; amaç, olumlu bir yanıtın durumu lehine çevirebileceği ve dişlerin ağızda korunmasını sağlayabileceği umududur

①) Tedavi sürecindeki yeniden değerlendirme aşaması, klinisyene kazıma (scaling), ağız hijyeni ve kök yüzeyi düzleştirmesine (root planing) karşı dokuların yanıtını, ayrıca gerekli görüldüğü durumlarda kemoterapötik ajanların olası kullanımını inceleme olanağı sağlar.

Hastalığa yakalanma olasılığı

① The prediction of a present disease (done after the disease is there) (Mevcut bir hastalığın tahmini — hastalık ortaya çıktıktan sonra yapılır.)

② Prognostic factors are the factors that affect the prognosis... (Prognozu etkileyen faktörler “prognostik faktörler” olarak adlandırılır...)

③ The likelihood to get the disease (the possibility to get the disease) (Hastalığa yakalanma olasılığı veya riski)

④ Risk factors are the factors which make the patient at risk to get the disease.. (Risk faktörleri, hastanın o hastalığa yakalanma riskini artıran faktörlerdir.)

🦷 Kısaca fark:

Risk faktörleri: Hastalığın oluşmasından önce etkilidir.

Prognostik faktörler: Hastalık oluştuktan sonra seyrini etkiler.

Were the prices low until now only due to VC money in an unsustainable or short term way?

line manager(product or service manager)=is responsible for the production, marketing, and profitability A staff manager, in contrast, leads a function that creates indirect inputs. A project manager has the responsibility for the planning, execution, and closing of any project. A general manager is someone who is responsible for managing a clearly identifiable revenue-producing unit, such as a store, business unit, or product line.

One time when I was filling out my Social Security application, I noticed something that felt a bit off. The form asked for “Parent” information, and then gave two boxes—one labeled “Father” and the other “Mother.” I didn’t think much of it at first, but later I started to wonder. What if someone has two moms? Or what if they don’t know who their biological parents are? How would they fill that out?

To be honest, I keep wondering if she wasn’t a lesbian, would the school have acted differently? Would they have held a memorial for her right away or shown more sympathy from the start? Part of me feels like they probably would have. It’s sad to say, but sometimes it feels like people only show respect when the victim fits into what they see as “normal.” That double standard is exactly what makes LGBTQ+ students feel invisible or unimportant.

I think one of the reasons homophobia persists today is not necessarily because people are against LGBTQ+ identities, but because of how identity politics sometimes shapes public discourse. For example, in the film industry, when a highly anticipated project is handled by LGBTQ+ directors, writers, or cinematographers and the final result doesn't meet public expectations, any criticism toward the work is sometimes labeled as homophobic. However, people rarely ask whether the criticism is about the quality of the work rather than the creator’s identity. In many other cases, straight directors also receive harsh critiques without their identity being part of the conversation. As a result, some neutral audiences feel silenced or unfairly accused, which creates resentment and eventually contributes to homophobia—not out of hate, but out of frustration with not being able to express honest opinions freely. I believe this is a misunderstanding rooted in overprotectiveness and a lack of space for dialogue.

In 2019, I was still in middle school in China. That year, I saw how hard it was for classmates who didn’t fit the “normal” expectations of gender and sexuality. I remember one boy who performed a Blackpink dance during an event—he danced with so much emotion and confidence, but a lot of people laughed at him or called him names. At the time, I didn’t really understand him either. But as I grew older and met more people from the LGBTQ+ community, I started to understand their experiences and slowly began to accept them.

I strongly agree with this point. Calling these incidents “bullying” minimizes the structural nature of the problem. It makes it sound like a behavior issue between individuals instead of a systemic failure. The quote helped me understand why focusing solely on “anti-bullying” policies is not enough—schools must address discrimination, Title IX responsibilities, and broader cultural norms.

This stands out because it shows that rigid masculinity harms everyone. Boys who step outside of what is considered “acceptable” are punished with homophobic taunts, even if they’re not LGBTQ. It demonstrates how gender policing maintains a culture where difference is punished. The intersection with race and ethnicity also adds another layer of vulnerability.

This quote shocked me because it shows that bias is not only coming from peers but from adults who are supposed to model respectful behavior. When teachers or staff use biased language, students receive the message that harassment is normal or acceptable. It makes me think about how important professional training is—not just for protecting LGBTQ students but for shifting the entire school climate.

This line clearly shows how deeply embedded heterosexism is in education. It’s not just about individual attitudes—schools themselves are built around assumptions that erase LGBTQ identities. I think this explains why so many students feel invisible in the curriculum and unsupported by staff. When the system assumes heterosexuality, LGBTQ youth must constantly navigate an environment not designed for them.

This issue is so prevalent with the rise of social media that it's become a normalized joke to call people gay. The stigma and cultural pressure to conform to social norms are harmful to youth.

DRF for the HIA

Depending on the are and situation

Making sure to know the area of where you’re transporting patients can be the extra mile to stay prepared in intense traumas.

Some people may refuse the care against medical advice but will still need glucose or maybe some oxygen.

In some scenarios there is no need for transportation and it is important to restock supplies and take account

Many traumas may be different but making these decision is vital for saving a patients life to receive the proper treatment

Staying within that golden hour but making an educated decision

determine wether the patient is ready to push and if the baby can move through the canal

The three stages are Dilation, expulsion, placental delivery

Making sure we’re constantly prepared inside and outside of the ambulance is crucial to provide the best care and leave little room for error

eLife Assessment

This important study characterises the morphogenesis of cortical folding in the ferret and human cerebral cortex using complementary physical and computational modelling. Notably, these approaches are applied to charting, in the ferret model, known abnormalities of cortical folding in humans. The study finds convincing evidence that variation in cortical thickness and expansion account for deviations in morphology, and supports these findings using cutting-edge approaches from both physical gel models and numerical simulations. The study will be of broad interest to the field of developmental neuroscience.

Reviewer #1 (Public review):

The manuscript by Choi and colleagues investigates the impact of variation in cortical geometry and growth on cortical surface morphology. Specifically, the study uses physical gel models and computational models to evaluate the impact of varying specific features/parameters of the cortical surface. The study makes use of this approach to address the topic of malformations of cortical development and finds that cortical thickness and cortical expansion rate are the drivers of differences in morphogenesis.

The study is composed of two main sections. First, the authors validate numerical simulation and gel model approaches against real cortical postnatal development in the ferret. Next, the study turns to modelling malformations in cortical development using modified tangential growth rate and cortical thickness parameters in numerical simulations. The findings investigate three genetically linked cortical malformations observed in the human brain to demonstrate the impact of the two physical parameters on folding in the ferret brain.

This is a tightly presented study that demonstrates a key insight into cortical morphogenesis and the impact of deviations from normal development. The dual physical and computational modeling approach offers the potential for unique insights into mechanisms driving malformations. This study establishes a strong foundation for further work directly probing the development of cortical folding in the ferret brain.

Reviewer #2 (Public review):

Summary:

Based on MRI data of the ferret (a gyrencephalic non-primate animal, in whom folding happens postnatally), the authors create in vitro physical gel models and in silico numerical simulations of typical cortical gyrification. They then use genetic manipulations of animal models to demonstrate that cortical thickness and expansion rate are primary drivers of atypical morphogenesis. These observations are then used to explain cortical malformations in humans.

Strengths:

The paper is very interesting and original, and combines physical gel experiments, numerical simulations, as well as observations in MCD. The figures are informative, and the results appear to have good overall face validity.

Comment on the revised version from the Reviewing Editor:

The reviewers are happy with the authors replies and the eLife Assessment has been amended accordingly.

Author response:

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

Reviewer #1 (Public review):

The manuscript by Choi and colleagues investigates the impact of variation in cortical geometry and growth on cortical surface morphology. Specifically, the study uses physical gel models and computational models to evaluate the impact of varying specific features/parameters of the cortical surface. The study makes use of this approach to address the topic of malformations of cortical development and finds that cortical thickness and cortical expansion rate are the drivers of differences in morphogenesis.

The study is composed of two main sections. First, the authors validate numerical simulation and gel model approaches against real cortical postnatal development in the ferret. Next, the study turns to modelling malformations in cortical development using modified tangential growth rate and cortical thickness parameters in numerical simulations. The findings investigate three genetically linked cortical malformations observed in the human brain to demonstrate the impact of the two physical parameters on folding in the ferret brain.

This is a tightly presented study that demonstrates a key insight into cortical morphogenesis and the impact of deviations from normal development. The dual physical and computational modeling approach offers the potential for unique insights into mechanisms driving malformations. This study establishes a strong foundation for further work directly probing the development of cortical folding in the ferret brain. One weakness of the current study is that the interpretation of the results in the context of human cortical development is at present indirect, as the modelling results are solely derived from the ferret. However, these modelling approaches demonstrate proof of concept for investigating related alterations more directly in future work through similar approaches to models of the human cerebral cortex.

We thank the reviewer for the very positive comments. While the current gel and organismal experiments focus on the ferret only, we want to emphasize that our analysis does consider previous observations of human brains and morphologies therein (Tallinen et al., Proc. Natl. Acad. Sci. 2014; Tallinen et al., Nat. Phys. 2016), which we compare and explain. This allows us to analyze the implications of our study broadly to understand the explanations of cortical malformations in humans using the ferret to motivate our study. Further analysis of normal human brain growth using computational and physical gel models can be found in our companion paper (Yin et al., 2025), now also published to eLife: S. Yin, C. Liu, G. P. T. Choi, Y. Jung, K. Heuer, R. Toro, L. Mahadevan, Morphogenesis and morphometry of brain folding patterns across species. eLife, 14, RP107138, 2025. doi:10.7554/eLife.107138

In future work, we plan to obtain malformed human cortical surface data, which would allow us to further investigate related alterations more directly. We have added a remark on this in the revised manuscript (please see page 8–9).

Reviewer 2 (Public review):

Summary:

Based on MRI data of the ferret (a gyrencephalic non-primate animal, in whom folding happens postnatally), the authors create in vitro physical gel models and in silico numerical simulations of typical cortical gyrification. They then use genetic manipulations of animal models to demonstrate that cortical thickness and expansion rate are primary drivers of atypical morphogenesis. These observations are then used to explain cortical malformations in humans.

Strengths:

The paper is very interesting and original, and combines physical gel experiments, numerical simulations, as well as observations in MCD. The figures are informative, and the results appear to have good overall face validity.

We thank the reviewer for the very positive comments.

Weaknesses:

On the other hand, I perceived some lack of quantitative analyses in the different experiments, and currently, there seems to be rather a visual/qualitative interpretation of the different processes and their similarities/differences. Ideally, the authors also quantify local/pointwise surface expansion in the physical and simulation experiments, to more directly compare these processes. Time courses of eg, cortical curvature changes, could also be plotted and compared for those experiments. I had a similar impression about the comparisons between simulation results and human MRI data. Again, face validity appears high, but the comparison appeared mainly qualitative.

We thank the reviewer for the comments. Besides the visual and qualitative comparisons between the models, we would like to point out that we have included the quantification of the shape difference between the real and simulated ferret brain models via spherical parameterization and the curvature-based shape index as detailed in main text Fig. 4 and SI Section 3. We have also utilized spherical harmonics representations for the comparison between the real and simulated ferret brains at different maximum order N. In our revision, we have included more calculations for the comparison between the real and simulated ferret brains at more time points in the SI (please see SI page 6). As for the comparison between the malformation simulation results and human MRI data in the current work, since the human MRI data are two-dimensional while our computational models are threedimensional, we focus on the qualitative comparison between them. In future work, we plan to obtain malformed human cortical surface data, from which we can then perform the parameterization-based and curvature-based shape analysis for a more quantitative assessment.

I felt that MCDs could have been better contextualized in the introduction.

We thank the reviewer for the comment. In our revision, we have revised the description of MCDs in the introduction (please see page 2).

Reviewer #1 (Recommendations for the authors):

The study is beautifully presented and offers an excellent complement to the work presented by Yin et al. In its current form, the malformation portion of the study appears predominantly reliant on the numerical simulations rather than the gel model. It might be helpful, therefore, to further incorporate the results presented in Figure S5 into the main text, as this seems to be a clear application of the physical gel model to modelling malformations. Any additional use of the gel models in the malformation portion of the study would help to further justify the necessity and complementarity of the dual methodological approaches.

We thank the reviewer for the suggestion. We have moved Fig. S5 and the associated description to the main text in the revised manuscript (please see the newly added Figure 5 on page 6 and the description on page 5–7). In particular, we have included a new section on the physical gel and computational models for ferret cortical malformations right before the section on the neurology of ferret and human cortical malformations.

One additional consideration is that the analyses in the current study focus entirely on the ferret cortex. Given the emphasis in the title on the human brain, it may be worthwhile to either consider adding additional modelling of the human cortex or to consider modifying the title to more accurately align with the focus of the methods/results.

We thank the reviewer for the suggestion. While the current gel and organismal experiments focus on the ferret only, we want to emphasize that our analysis does consider previous observations of human brains and morphologies therein (Tallinen et al., Proc. Natl. Acad. Sci. 2014; Tallinen et al., Nat. Phys. 2016), which we compare and explain. This allows us to analyze the implications of our study broadly to understand the explanations of cortical malformations in humans using the ferret to motivate our study. Therefore, we think that the title of the paper seems reasonable. To further highlight the connection between the ferret brain simulations and human brain growth, we have included an additional comparison between human brain surface reconstructions adapted from a prior study and the ferret simulation results in the SI (please see SI Section S4 and SI Fig. S5 on page 9–10).

Two additional minor points:

Table S1 seems sufficiently critical to the motivation for the study and organization of the results section to justify inclusion in the main text. Of course, I would leave any such minor changes to the discretion of the authors.

We thank the reviewer for the suggestion. We have moved Table S1 and the associated description to the main text in the revised manuscript (please see Table 1 on page 7).

Page 7, Column 1: “macacques” → “macaques”.

We thank the reviewer for pointing out the typo. We have fixed it in the revised manuscript (please see page 8).

Reviewer #2 (Recommendations for the authors):

The methods lack details on the human MRI data and patients.

We thank the reviewer for the comment. Note that the human MRI data and patients were from prior works (Smith et al., Neuron 2018; Johnson et al., Nature 2018; Akula et al., Proc. Natl. Acad. Sci. 2023) and were used for the discussion on cortical malformations in Fig. 6. In the revision, we have included a new subsection in the Methods section and provided more details and references of the MRI data and patients (please see page 9–10).

With the fast growth of the internet, online communities easily form their own circles and often define themselves by opposing other circles. This makes the narratives around gender even more amplified. Biological sex is something we are born with, but identity and self-understanding come from personal experience and practice. Society usually does not care about the reasons behind someone’s identity; it only reacts to the result. When people do not accept the way someone understands themselves, they often respond with judgment or hostility, which creates even more division.

I think it is important to remember that religion and culture are not fixed. They have changed across history and will continue to change as society develops. Many ideas that were once seen as absolute were later reinterpreted or replaced. So when some people use tradition to justify strict beliefs about gender or sexuality, they may be holding on to only one version of that tradition. If we look at the past, we can see that many cultures and even some religious communities once accepted more diverse gender roles.

I am curious about how norms will change in the future. For a long time society has created fixed expectations for men and women and these ideas became so common that people often forget they are learned. As transgender people become more visible and more accepted I wonder if new expectations will slowly form around them too. It is possible that society will start creating its own image of what a transgender person should look like act like or live like even though the whole point of acceptance is to allow people to live freely. I think this shows how important it is to stay aware of how norms form so we do not turn one kind of freedom into another kind of pressure.

This social consensus was formed over a long period of time. Early societies viewed heterosexual marriage as the basis for survival and continuation. Later, religion, law, and education reinforced this idea, shaping clear expectations for how men and women should behave. Media and cultural messages kept repeating these images, making people believe this is the norm. Although modern society is becoming more open and diverse, these frameworks still exist, only in more subtle ways within daily communication and thinking. New habits, ideas, values, and even technologies all take time to move from being questioned to being accepted and finally recognized. The world keeps moving forward, but it always takes time for people to truly adapt and accept change.

This quote highlights how school policies—dress codes, bathroom rules, activity assignments—can unintentionally reproduce the very biases they claim to avoid. I agree with the author that when schools enforce traditional gender roles, they strengthen exclusion rather than inclusion. This line also connects to broader course themes about how institutions reproduce inequality through “neutral” rules that are not actually neutral.

I found this especially meaningful. LGBTQ youth often survive hostile environments by building small networks of safety and affirmation. While this shows resilience, it also reveals how school systems fail to provide institutional safety. Students shouldn’t have to hide in “micro-sanctuaries” to feel accepted. It made me think about the emotional labor LGBTQ youth are forced to do just to navigate school.

I find this idea powerful because it explains why homophobia and transphobia are connected: both depend on maintaining the belief that only two genders exist and that heterosexuality is the default. I agree with the author that challenging gender binaries is necessary if schools want to reduce anti-LGBTQ hostility. Without breaking the binary, heteronormativity continues to be reinforced.

This line stood out to me because it reveals how schools reinforce gender binaries through small, everyday routines. Something as common as lining up by “boys” and “girls” teaches children early on that gender is the first and most important way to categorize themselves. It makes me think about how many biases come not from explicit discrimination but from “normal” institutional habits that go unquestioned.

Environmental is important to not only ensure the safety of your patient but yourself as well

eLife Assessment

This study thoroughly assesses tactile acuity on women's breasts, for which no dependable data currently exists. The study provides two important contributions, by convincingly showing that tactile acuity on the breast is poor in comparison to other body parts, and that acuity is worst in larger breasts, indicating that the number of tactile sensors is fixed. However, further arguments concerning the role of the nipple in spatial localisation are not well supported by the current evidence, therefore diluting the overall contribution of the study. This study will be of interest to the broader community of touch, as well as those interested in breast reconstruction and sexual function.

Reviewer #1 (Public review):

The authors investigated tactile spatial perception on the breast using discrimination, categorization, and direct localization tasks. They reach four main conclusions:

(1) The breast has poor tactile spatial resolution.

This conclusion is based on comparing just noticeable differences, a marker of tactile spatial resolution, across four body regions, two on the breast. The data compellingly support the conclusion; the study outshines other studies on tactile spatial resolution that tend to use problematic measures of tactile resolution, such as two-point-discrimination thresholds. The result will interest researchers in the field and possibly in other fields due to the intriguing tension between the finding and the sexually arousing function of touching the breast.

The manuscript incorrectly describes the result as poor spatial acuity. Acuity measures the average absolute error, and acuity is good when response biases are absent. Precision relates to the error variance. It is common to see high precision with low acuity or vice versa. Just noticeable differences assess precision or spatial resolution, while points of subjective equality evaluate acuity or bias. Similar confusions between these terms appear throughout the manuscript.<br /> A paragraph within the next section seems to follow up on this insight by examining the across-participant consistency of the differences in tactile spatial resolution between body parts. To this aim, pairwise rank correlations between body sites are conducted. This analysis raises red flags from a statistical point of view. 1) An ANOVA and its follow-up tests assume no variation in the size of the tested effect but varying base values across participants. Thus, if significant differences between conditions are confirmed by the original statistical analysis, most participants will have better spatial resolution in one condition than the other condition, and the difference between body sites will be similar across participants. 2) Correlations are power-hungry, and non-parametric tests are power-hungry. Thus, the number of participants needed for a reliable rank correlation analysis far exceeds that of the study. In sum, a correlation should emerge between body sites associated with significantly different tactile JNDs; however, these correlations might only be significant for body sites with pronounced differences due to the sample size.

(2) Larger breasts are associated with lower tactile spatial resolution

This conclusion is based on a strong correlation between participants' JNDs and the size of their breasts. The depicted correlation convincingly supports the conclusion. The sample size is below that recommended for correlations based on power analyses, but simulations show that spurious correlations of the reported size are extremely unlikely at N=18. Moreover, visual inspection rules out that outliers drive these correlations. Thus, they are convincing. This result is of interest to the field, as it aligns with the hypothesis that nerve fibers are more sparsely distributed across larger body parts.

(3) The nipple is a unit

The data do not support this conclusion. The conclusion that the nipple is perceived as a unit is based on poor tactile localization performance for touches on the nipple compared to the areola. The problem is that the localization task is a quadrant identification task with the center being at the nipple. Quadrants for the areola could be significantly larger due to the relative size of the areola and the nipple; the results section seems to suggest this was accounted for when placing the tactile stimuli within the quadrants, but the methods section suggests otherwise. Additionally, the areola has an advantage because of its distance from the nipple, which leads to larger Euclidean distances between the centers of the quadrants than for the nipple. Thus, participants should do better for the areola than for the nipple even if both sites have the same tactile resolution.

To justify the conclusion that the nipple is a unit, additional data would be required. 1) One could compare psychometric curves with the nipple as the center and psychometric curves with a nearby point on the areola as the center. 2) Performance in the quadrant task could be compared for the nipple and an equally sized portion of the areola and tactile locations that have the same distance to the border between quadrants in skin coordinates. 3) Tactile resolution could be directly measured for both body sites using a tactile orientation task with either a two-dot probe or a haptic grating.

Categorization accuracy in each area was tested against chance using a Monte Carlo test, which is fine, though the calculation of the test statistic, Z, should be reported in the Methods section, as there are several options. Localization accuracies are then compared between areas using a paired t-test. It is a bit confusing that once a distribution-approximating test is used, and once a test that assumes Gaussian distributions when the data is Bernoulli/Binomial distributed. Sampling-based and t-tests are very robust, so these surprising choices should have hardly any effect on the results.

A correlation based on N=4 participants is dangerously underpowered. A quick simulation shows that correlation coefficients of randomly sampled numbers are uniformly distributed at such a low sample size. This likely spurious correlation is not analyzed, but quite prominently featured in a figure and discussed in the text, which is worrisome.

(4) Localization of tactile events on the breast is biased towards the nipple

The conclusion that tactile percepts are drawn toward the nipple is based on localization biases for tactile stimuli on the breast compared to the back. Unfortunately, the way participants reported the tactile locations introduces a major confound. Participants indicated the perceived locations of the tactile stimulus on 3D models of these body parts. The nipple is a highly distinctive and cognitively represented landmark, far more so than the scapula, making it very likely that responses were biased toward the nipple regardless of the actual percepts. One imperfect but better alternative would have been to ask participants to identify locations on a neutral grey patch and help them relate this patch to their skin by repeatedly tracing its outline on the skin.

Participants also saw their localization responses for the previously touched locations. This is unlikely to induce bias towards the nipple, but it renders any estimate of the size and variance of the errors unreliable. Participants will always make sure that the marked locations are sufficiently distant from each other.

The statistical analysis is again a homebrew solution and hard to follow. It remains unclear why standard and straightforward measures of bias, such as regressing reported against actual locations, were not used.

Null-hypothesis significance testing only lets scientists either reject the null hypothesis or not. The latter does NOT mean the Null hypothesis is true, i.e., it can never be concluded that there is no effect. This rule applies to every NHST test. However, it raises particular concerns with distribution tests. The only conclusion possible is that the data are unlikely from a population with the tested distribution; these tests do not provide insight into the actual distribution of the data, regardless of whether the result is significant or not.

Reviewer #2 (Public review):

Summary:

The authors tested tactile acuity on the breast of females using several tasks.

Results:

Tactile acuity, assessed by just-noticeable differences in judging whether a touch was above or below a comparison stimulus, was lower on both the lateral and medial breast than on the hand and back. Acuity also scaled inversely with breast size, echoing earlier findings that larger hands exhibit lower acuity, presumably because a similar number of tactile receptors must be distributed over larger or smaller body surfaces. Observing this principle in the breast as on the hand strengthens the view that fixed innervation is a general organizing principle of the tactile system. Both methodology and analysis appear sound.

Most participants were unable to localize touch to a specific quadrant of the nipple, suggesting it is perceived as a single tactile unit. However, the study does not address whether touches to the nipple and areola are confused; conceptualizing the nipple as a perceptual (landmark) unit would suggest that such confusion should not take place. Aside from this limitation, the methodology and analysis appear sound.

Absolute touch localization, assessed by asking participants to indicate locations on a 3D rendering of their own torso, revealed a bias toward the nipple. The authors interpret this as evidence that the nipple serves as a landmark attracting perceived touch. However, as reviewers noted during review, alternative explanations cannot be fully ruled out: because the stimulus array was centered on the nipple, the observed bias may stem from stimulus distribution rather than landmark status. Aside from this caveat, the methodology and analysis appear sound.

Overall assessment:

The study offers a welcome exception to the prevailing bias in tactile research that limits investigation to the hand and arm. Its support for the fixed innervation hypothesis and its suggestion that the nipple may serve as a potential landmark-though requiring further scrutiny-illustrate the value of extending research to other body regions. By employing multiple tasks, the authors address several key aspects of tactile perception and create links to earlier findings.

Author response:

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

Reviewer #1 (Public review):

The statistically adequate way of testing the biases is a hierarchical regression model (LMM) with a distance of the physical location from the nipple as a predictor, and a distance of the reported location from the nipple as a dependent variable. Either variable can be unsigned or signed for greater power, for example, coding the lateral breast as negative and the medial breast as positive. The bias will show in regression coefficients smaller than 1.

Thank you for this suggestion. We have subsequently replaced the relevant ANOVA analyses with LMM analyses. Specifically, we use an LMM for breast and back separately to show the different effects of distance, then use a combined LMM to compare the interaction. Finally, we use an LMM to assess the differences between precision and bias on the back and breast. The new analysis confirms earlier statements and do not change the results/interpretation of the data.

Moreover, any bias towards the nipple could simply be another instance of regression to the mean of the stimulus distribution, given that the tested locations were centered on the nipple. This confound can only be experimentally solved by shifting the distribution of the tested locations. Finally, given that participants indicated the locations on a 3D model of the body part, further experimentation would be required to determine whether there is a perceptual bias towards the nipple or whether the authors merely find a response bias.

A localization bias toward the nipple in this context does not show that the nipple is the anchor of the breast's tactile coordinate system. The result might simply be an instance of regression to the mean of the stimulus distribution (also known as experimental prior). To convincingly show localization biases towards the nipple, the tested locations should be centered at another location on the breast.

Another problem is the visual salience of the nipple, even though Blender models were uniformly grey. With this type of direct localization, it is very difficult to distinguish perceptual from response biases even if the regression to the mean problem is solved. There are two solutions to this problem: 1) Varying the uncertainty of the tactile spatial information, for example, by using a pen that exerts lighter pressure. A perceptual bias should be stronger for more uncertain sensory information; a response bias should be the same across conditions. 2) Measure bias with a 2IFC procedure by taking advantage of the fact that sensory information is noisier if the test is presented before the standard.

We believe that the fact that we explicitly tested two locations with equally distributed test locations, both of which had landmarks, makes this unlikely. Indeed, testing on the back is exactly what the reviewer suggests. It would also be impossible to test this “on another location on the breast” as we are sampling across the whole breast. Moreover, as markers persisted on the model within each block, the participants were generating additional landmarks on each trial. Thus, if there were any regression to the mean, this would be observed for both locations. Nevertheless, we recognize that this test cannot distinguish between a sensory bias towards the nipple and consistent response bias that is always in the direction of the nipple, though to what extent these are the same thing is difficult to disentangle. That said, if we had restricted testing to half of the breast such that the distribution of points was asymmetrical this would allow us to test the hypothesis put forward by the reviewer. We recognize that this is a limitation of the data and have downplayed statements and added caveats accordingly.

We have changed the appropriate heading and text in the discussion to downplay the finding:

“Reports are biased towards the nipple”

“suggesting that the nipple plays a pivotal role in the mental representation of the breast.”

it might be harder to learn the range of locations on the back given that stimulation is not restricted to an anatomically defined region as it is the case for the breast.

We apologize for any confusion but the point distribution is identical between tasks, as described in the methods.

The stability of the JND differences between body parts across subjects is already captured in the analysis of the JNDs; the ANOVA and the post-hoc testing would not be significant if the order were not relatively stable across participants. Thus, it is unclear why this is being evaluated again with reduced power due to improper statistics.

We apologize for any confusion here. Only one ANOVA with post-hoc testing was performed on the data. The second parenthetical describing the test was perhaps redundant and confusing, so I have removed it.

“(Error! Reference source not found.A, B, 1-way ANOVA with Tukey’s HSD post-hoc t-test: p = 0.0284)”

The null hypothesis of an ANOVA is that at least one of the mean values is different from the others; adding participants as a factor does not provide evidence for similarity.

We agree with this statement and have removed the appropriate text.

The pairwise correlations between body parts seem to be exploratory in nature. Like all exploratory analyses, the question arises of how much potential extra insights outweigh the risk of false positives. It would be hard to generate data with significant differences between several conditions and not find any correlations between pairs of conditions. Thus, the a priori chance of finding a significant correlation is much higher than what a correction accounts for.

We broadly agree with this statement. However, we believe that the analyses were important to determine if participants were systematically more or less acute across body parts. Moreover, both the fact that we actually did not observe any other significant relationships and that we performed post-hoc correction imply that no false positives were observed. Indeed, in the one relationship that was observed, we would need to have an assumed FDR over 10x higher than the existing post hoc correction required implying a true relationship.

If the JND at mid breast (measured with locations centered at the nipple) is roughly the same size as the nipple, it is not surprising that participants have difficulty with the categorical localization task on the nipple but perform better than chance on the significantly larger areola.

We agree that it is not surprising given the previously shown data, however, the initial finding is surprising to many and this experiment serves to reinforce the previous finding.

Neither signed nor absolute localization error can be compared to the results of the previous experiments. The JND should be roughly proportional to the variance of the errors.

We apologize for any confusion, however we are not comparing the values, merely observing that the results are consistent.

Reviewer #2 (Public review):

I had a hard time understanding some parts of the report. What is meant by "broadly no relationship" in line 137?

We have removed the qualifier to simplify the text.

It is suggested that spatial expansion (which is correlated with body part size) is related between medial breast and hand - is this to say that women with large hands have large medial breast size? Nipple size was measured, but hand size was not measured, is this correct?

Correct. We have added text to state as such.

It is furthermore unclear how the authors differentiate medial breast and NAC. The sentence in lines 140-141 seems to imply the two terms are considered the same, as a conclusion about NAC is drawn from a result about the medial breast. This requires clarification.

Thank you for catching this, we have corrected it in the text.

Finally, given that the authors suspect that overall localization ability (or attention) may be overshadowed by a size effect, would not an analysis be adequate that integrates both, e.g. a regression with multiple predictors?

If the reviewer means that participants would be consistently “acute” then we believe that SF1 would have stronger correlations. Consequently, we see no reason to add “overall tactile acuity” as a predictor.

In the paragraph about testing quadrants of the nipple, it is stated that only 3 of 10 participants barely outperformed chance with a p < 0.01. It is unclear how a significant ttest is an indication of "barely above chance".

We have adjusted the text to clarify our meaning.

“On the nipple, however, participants were consistently worse at locating stimuli on the nipple than the breast (paired t-test, t = 3.42, p < 0.01) where only 3 of the 10 participants outperformed chance, though the group as a whole outperformed chance (Error! Reference source not found.B, 36% ± 13%; Z = 5.5, p < 0.01).”

The final part of the paragraph on nipple quadrants (starting line 176) explains that there was a trend (4 of 10 participants) for lower tactile acuity being related to the inability to differentiate quadrants. It seems to me that such a result would not be expected: The stated hypothesis is that all participants have the same number of tactile sensors in their nipple and areola, independent of NAC size. In this section, participants determine the quadrant of a single touch. Theoretically, all participants should be equally able to perform this task, because they all have the same number of receptors in each quadrant of nipple and areola. Thus, the result in Figure 2C is curious.

We agree that this result seemingly contradicts observations from the previous experiment, however we believe that it relates to the distinction between the ability to perform relative distinctions and absolute localizations. In the first experiment, the presentation of two sequential points provides an implicit reference whereas in the quadrant task there is no reference. With the results of the third experiment in mind, biases towards the nipple would effectively reduce the ability of participants to identify the quadrant. What this result may imply is that the degree of bias is greater for women with greater expansion. We have added text to the discussion to lay this out.

“This negative trend implicitly contradicts the previous result where one might expect equal performance regardless of size as the location of the stimuli was scaled to the size of the nipple and areola. However, given the absence of a reference point, systematic biases are more likely to occur and thus may reflect a relationship between localization bias and breast size.”

This section reports an Anova (line 193/194) with a factor "participant". This doesn't appear sensible. Please clarify. The factor distance is also unclear; is this a categorical or a continuous variable? Line 400 implies a 6-level factor, but Anovas and their factors, respectively, are not described in methods (nor are any of the other statistical approaches).

We believe this comment has been addressed above with our replacement of the ANOVA with an LMM. We have also added descriptions of the analysis throughout the methods.

The analysis on imprecision using mean pairwise error (line 199) is unclear: does pairwise refer to x/y or to touch vs. center of the nipple?

We have clarified this to now read:

“To measure the imprecision, we computed the mean pairwise distance between each of the reported locations for a given stimulus location and the mean reported location.”

p8, upper text, what is meant by "relative over-representation of the depth axis"? Does this refer to the breast having depth but the equivalent area on the back not having depth? What are the horizontal planes (probably meant to be singular?) - do you simply mean that depth was ignored for the calculation of errors? This seems to be implied in Figure 3AB.

This is indeed what we meant. We have attempted to clarify in the text.

“Importantly, given the relative over-representation of the depth axis for the breast, we only considered angles in the horizontal planes such that the shape of the breast did not influence the results.” Became:

“Importantly, because the back is a relatively flat surface in comparison to the breast, errors were only computed in the horizontal plane and depth was excluded when computing the angular error.”

Lines 232-241, I cannot follow the conclusions drawn here. First, it is not clear to a reader what the aim of the presented analyses is: what are you looking for when you analyze the vectors? Second, "vector strength" should be briefly explained in the main text. Third, it is not clear how the final conclusion is drawn. If there is a bias of all locations towards the nipple, then a point closer to the nipple cannot exhibit a large bias, because the nipple is close-by. Therefore, one would expect that points close to the nipple exhibit smaller errors, but this would not imply higher acuity - just less space for localizing anything. The higher acuity conclusion is at odds with the remaining results, isn't it: acuity is low on the outer breast, but even lower at the NAC, so why would it be high in between the two?

Thank you for pointing out the circular logic. We have replaced this sentence with a more accurate statement.

“Given these findings, we conclude that the breast has lower tactile acuity than the hand and is instead comparable to the back. Moreover, localization of tactile events to both the back and breast are inaccurate but localizations to the breast are consistently biased towards the nipple.”

The discussion makes some concrete suggestions for sensors in implants (line 283). It is not clear how the stated numbers were computed. Also, why should 4 sensors nipple quadrants receive individual sensors if the result here was that participants cannot distinguish these quadrants?

Thank you for catching this, it should have been 4 sensors for the NAC, not just the nipple. We have fixed this in the text.

I would find it interesting to know whether participants with small breast measurement delta had breast acuity comparable to the back. Alternatively, it would be interesting to know whether breast and back acuity are comparable in men. Such a result would imply that the torso has uniform acuity overall, but any spatial extension of the breast is unaccounted for. The lowest single participant data points in Figure 1B appear similar, which might support this idea.

We agree that this is an interesting question and as you point out, the data does indicate that in cases of minimal expansion acuity may be constant on the torso. However, in the comparison of the JNDs, post-hoc testing revealed no significant difference between the back and either breast region. Consequently, subsampling the group would result in the same result. We have added a sentence to the discussion stating this.

“Consequently, the acuity of the breast is likely determined initially by torso acuity and then any expansion.”

To keep up with a growing society and to continue to thrive in the society.

POLC for Goodwill needs to establish a comprehensive POLC to support people with various backgrounds. The core strategy is doing good for people and the planet. While starbuks forcuses on how to provide good products and services.

DOI: 10.1371/journal.pcbi.1010962

Resource: RRID:IMSR_JAX:000664

Curator: @areedewitt04

SciCrunch record: RRID:IMSR_JAX:000664

What is this?

DOI: 10.1016/j.ymthe.2025.10.050

Resource: (IMSR Cat# JAX_007676,RRID:IMSR_JAX:007676)

Curator: @areedewitt04

SciCrunch record: RRID:IMSR_JAX:007676

What is this?

DOI: 10.1016/j.celrep.2025.116556

Resource: RRID:IMSR_JAX:012899

Curator: @areedewitt04

SciCrunch record: RRID:IMSR_JAX:012899

What is this?

DOI: 10.1016/j.celrep.2025.116516

Resource: (IMSR Cat# JAX_002019,RRID:IMSR_JAX:002019)

Curator: @areedewitt04

SciCrunch record: RRID:IMSR_JAX:002019

What is this?

DOI: 10.1101/2025.11.06.687049

Resource: None

Curator: @dhovakimyan1

SciCrunch record: RRID:IMSR_JAX:000664

What is this?

Version 3 of this preprint has been peer-reviewed and recommended by Peer Community in Evolutionary Biology.<br /> See the peer reviews and the recommendation.

Is this the fault of social media or the people who use it? Or is it the fault of the audience who choose to attend to the fake news and therefore boost it in the algorithm?

Again, this is a major complaint about AI (it steals out data, tells us only what we want to hear so that we continue to utilize the AI), yet there is way more debate about using AI than using social media even though they seem to have similar habits/ downsides

I feel like there are some negative implications to this considering that people will curate content based on what they think people want to hear (it kind of sounds like AI), instead of the truth. How can we be prepared audiences for this golden age of speech?

save content to local filesystem

for indy0pad.save

For the ribbon vibrator portion, they had to choose at least one key to check, and likely choose one of the repair person's favorite default alignment keys.

It's used in alignment because the capital H is both wide and tall and the lower case h goes above the midline which neither m nor n do. On serifed faces (especially), the HHHhhhHHH combination creates a pretty nice visual baseline to ensure the the type has the proper "motion" and is "on feet". These Hs at both ends of the platen and in the center help to check print evenness when doing the ring and cylinder adjustment. They're also useful when adjusting the level of the line indicator though other letters like m, n, z, and k aren't bad either. Letters like v and i are thinner or almost non-existent on the baseline in comparison.

They also frequently use the / character which extends both above and below most other characters to ensure proper alignment with respect to both a bichrome ribbon and the strike against the platen. You want a nice even imprint from top to bottom. % is also good for this as well.

Some of the repair manuals at https://site.xavier.edu/polt/typewriters/tw-manuals.html as well as some of Ted Munk's manuals available through the typewriter database describe many of these adjustments and suggest specific letters for easier visual inspections.

I'd be curious to hear other repair people's favorite letters and characters.

Incidentally, for installing ribbon, many but not all manuals will suggest putting the bichrome setting to red and then simultaneously pressing the G and H keys so that their typebars gently jam together just in front of the typing point. This raises the ribbon vibrator to its highest point and makes it easier to thread the ribbon into it.

reply to https://old.reddit.com/r/typewriters/comments/1ovt8ry/but_why_the_h_key/

the idea that value comes from only those tangible things. This is similar to Epicurius idea of sensation?

I like this acknowledgement of the ultimate art being life itself rather than some specific discipline done within it. The acknowledgement of how difficult that is, and how you don't actually learn it till it's just about over, is correct.

Only under certain criteria is it ok to merely think of joy rather than pursue it.?

This reminds me of the common idea of college students to make a lot of money in the ten years after college and then do what they're passionate about. I agree with this point on a larger scale, that tossing away years as a number is foolish.

Seneca frames this as a bad thing, but once again, it is important to remember that the mind cannot handle "really living" at all times. Additionally, it is part of really living to be unaware at times. To be focussed, to be doing rather than observing.

Although this is a wise statement, and encourages the generally well thought of principal of appreciation, it is important to realize how this can be overdone. If we put so much pressure on every aspect of our life to be packed with meaning, it will lose that very meaning.

Very helpful

eLife Assessment

This valuable study reports that EEG recordings of the earliest stage of information processing in the human visual cortex can be used to predict subsequent choice responses. The findings provide novel, solid evidence for integrative processing in low-level sensory cortices, though the exact nature of the neural signals measured here requires some clarification. While some conceptual issues need to be addressed, the paper is likely to be of interest to neuroscientists interested in the contribution of early sensory signals to decision-making.

Reviewer #1 (Public review):

General assessment of the work:

In this manuscript, Mohr and Kelly show that the C1 component of the human VEP is correlated with binary choices in a contrast discrimination task, even when the stimulus is kept constant and confounding variables are considered in the analysis. They interpret this as evidence for the role V1 plays during perceptual decision formation. Choice-related signals in single sensory cells are enlightening because they speak to the spatial (and temporal) scale of the brain computations underlying perceptual decision-making. However, similar signals in aggregate measures of neural activity offer a less direct window and thus less insight into these computations. For example, although I am not a VEP specialist, it seems doubtful that the measurements are exclusively picking up (an unbiased selection of) V1 spikes. Moreover, although this is not widely known, there is in fact a long history to this line of work. In 1972, Campbell and Kulikowski ("The Visual Evoked Potential as a function of contrast of a grating pattern" - Journal of Physiology) already showed a similar effect in a contrast detection task (this finding inspired the original Choice Probability analyses in the monkey physiology studies conducted in the early 1990's). Finally, it is not clear to me that there is an interesting alternative hypothesis that is somehow ruled out by these results. Should we really consider that simple visual signals such as spatial contrast are *not* mediated by V1? This seems to fly in the face of well-established anatomy and function of visual circuits. Or should we be open to the idea that VEP measurements are almost completely divorced from task-relevant neural signals? Why would this be an interesting technique then? In sum, while this work reports results in line with several single-cell and VEP studies and perhaps is technically superior in its domain, I find it hard to see how these findings would meaningfully impact our thinking about the neural and computational basis of spatial contrast discrimination.

Summary of substantive concerns:

(1) The study of choice probability in V1 cells is more extensive than portrayed in the paper's introduction. In recent years, choice-related activity in V1 has also been studied by Nienborg & Cumming (2014), Goris et al (2017), Jasper et al (2019), Lange et al (2023), and Boundy-Singer et al (2025). These studies paint a complex picture (a mixture of positive, absent, and negative results), but should be mentioned in the paper's introduction.

(2) The very first study to conduct an analysis of stimulus-conditioned neural activity during a perceptual decision-making task was, in fact, a VEP study: Campbell and Kulikowski (1972). This study never gained the fame it perhaps deserves. But it would be appropriate to weave it into the introduction and motivation of this paper.

(3) What are interesting alternative hypotheses to be considered here? I don't understand the (somewhat implicit) suggestion here that contrast representations late in the system can somehow be divorced from early representations. If they were, they would not be correlated with stimulus contrast.

(4) I find the arguments about the timing of the VEP signals somewhat complex and not very compelling, to be honest. It might help if you added a simulation of a process model that illustrated the temporal flow of the neural computations involved in the task. When are sensory signals manifested in V1 activity informing the decision-making process, in your view? And how is your measure of neural activity related to this latent variable? Can you show in a simulation that the combination of this process and linking hypothesis gives rise to inverted U-shaped relationships, as is the case for your data?

Reviewer #2 (Public review):

Summary:

Mohr and Kelly report a high-density EEG study in healthy human volunteers in which they test whether correlations between neural activity in the primary visual cortex and choice behavior can be measured non-invasively. Participants performed a contrast discrimination task on large arrays of Gabor gratings presented in the upper left and lower right quadrants of the visual field. The results indicate that single-trial amplitudes of C1, the earliest cortical component of the visual evoked potential in humans, predict forced-choice behavior over and beyond other behavioral and electrophysiological choice-related signals. These results constitute an important advance for our understanding of the nature and flexibility of early visual processing.

Strengths:

(1) The findings suggest a previously unsuspected role for aggregate early visual cortex activity in shaping behavioral choices.

(2) The authors extend well-established methods for assessing covariation between neural signals and behavioral output to non-invasive EEG recordings.

(3) The effects of initial afferent information in the primary visual cortex on choice behavior are carefully assessed by accounting for a wide range of potential behavioral and electrophysiological confounds.

(4) Caveats and limitations are transparently addressed and discussed.

Weaknesses:

(1) It is not clear whether integration of contrast information across relatively large arrays is a good test case for decision-related information in C1. The authors raise this issue in the Discussion, and I agree that it is all the more striking that they do find C1 choice probability. Nevertheless, I think the choice of task and stimuli should be explained in more detail.

(2) In a similar vein, while C1 has canonical topographical properties at the grand-average level, these may differ substantially depending on individual anatomy (which the authors did not assess). This means that task-relevant information will be represented to different degrees in individuals' single-trial data. My guess is that this confound was mitigated precisely by choosing relatively extended stimulus arrays. But given the authors' impressive track record on C1 mapping and modeling, I was surprised that the underlying rationale is only roughly outlined. For example, given the topographies shown and the electrode selection procedure employed, I assume that the differences between upper and lower targets are mainly driven by stimulus arms on the main diagonal. Did the authors run pilot experiments with more restricted stimulus arrays? I do not mean to imply that such additional information needs to be detailed in the main article, but it would be worth mentioning.

(3) Also, the stimulus arrangement disregards known differences in conduction velocity between the upper and lower visual fields. While no such differences are evident from the maximal-electrode averages shown in Figure 1B, it is difficult to assess this issue without single-stimulus VEPs and/or a dedicated latency analysis. The authors touch upon this issue when discussing potential pre-C1 signals emanating from the magnocellular pathway.

(4) I suspect that most of these issues are at least partly related to a lack of clarity regarding levels of description: the authors often refer to 'information' contained in C1 or, apparently interchangeably, to 'visual representations' before, during, or following C1. However, if I understand correctly, the signal predicting (or predicted by) behavioral choice is much cruder than what an RSA-primed readership may expect, and also cruder than the other choice-predictive signals entered as control variables: namely, a univariate difference score on single-trial data integrated over a 10 ms window determined on the basis of grand-averaged data. I think it is worth clarifying and emphasizing the nature of this signal as the difference of aggregate contrast responses that *can* only be read out at higher levels of the visual system due to the limited extent of horizontal connectivity in V1. I do not think that this diminishes the importance of the findings - if anything, it makes them more remarkable.

(5) Arguably even more remarkable is the finding that C1 amplitudes themselves appear to be influenced by choice history. The authors address this issue in the Discussion; however, I'm afraid I could not follow their argument regarding preparatory (and differential?) weighting of read-outs across the visual hierarchy. I believe this point is worth developing further, as it bears on the issue of whether C1 modulations are present and ecologically relevant when looking (before and) beyond stimulus-locked averages.

I don't think I particularly understand the difference between what something is and its "is-ness" but would this be liike subgenres for music? Like okay say a wind band is covering pop songs. The "genre" would be classical probably, or at least instrumental, and then the sub genre could be popular music?? Idk am I understanding this?

Exactly what we want to focus on.

Good working definition here.

When the chapter talks about unpaid Reddit moderators, I honestly feel the trade-off is kinda messed up. Reddit is worth billions, but the people actually keeping the communities clean and usable are working for free, and sometimes they even get yelled at by users. I get that mods have some “power” and some of them enjoy shaping the culture of a subreddit, but this power is very fragile, they can also get burned out super fast. For me it feels like Reddit is outsourcing a huge part of its responsibility to volunteers. I think Reddit should at least offer more concrete support: better tools, mental health resources, maybe some small financial compensation or revenue sharing for big subreddits. If the company is making so much money from user content, saying “thanks” in words only is really not enough.

We still use some Old English today. But eventually changed to Middle English

English has come a long way and eventually was promoted to Middle English

Some people might have issues using Standard English. They may come from different backgrounds

In the work setting we use Standard English

Standard English is what we use today and throughout our school years.

Standard English took over.

We use different types of English when it comes to friends or the workplace.

I looked at source [o11], the Vice article “Facebook Is Ignoring Moderators’ Trauma: ‘They Suggest Karaoke and Painting’”. The thing that really stuck in my head is how the company’s response sounds so shallow. These moderators are watching horrible stuff every day, like violence or hate, and then the solution is basically “do some hobbies, try karaoke.” It feel like treating serious psychological damage as just stress from a normal office job. For me this connects to the question in 15.3 about what support moderators should have. After reading this source, I think real support has to include proper therapy, higher pay, and maybe limits on how long someone can do this job. If a platform can afford global expansion and fancy offices, it should also afford not to ignore the people cleaning up its worst content.

eLife Assessment

This study shows that excitatory cholecystokinin (CCK)-expressing neurons in hippocampal area CA3 influence hippocampal-dependent memory using multiple methods to manipulate excitatory CCK-expressing CA3 neurons selectively. The work is valuable because most past studies of CCK-expressing neurons have focused on those neurons that co-express CCK and GABA. Currently, the strength of evidence is incomplete; however, if additional evidence were to be presented that the methods were selective, the evaluation would potentially be higher.

Reviewer #1 (Public review):

Summary:

CCK is the most abundant neuropeptide in the brain, and many studies have investigated the role of CCK and inhibitory CCK interneurons in modulating neural circuits, especially in the hippocampus. The manuscript presents interesting questions regarding the role of excitatory CCK+ neurons in the hippocampus, which has been much less studied compared to the well-known roles of inhibitory CCK neurons in regulating network function. The authors adopt several methods, including transgenic mice and viruses, optogenetics, chemogenetics, RNAi, and behavioral tasks to explore these less-studied roles of excitatory CCK neurons in CA3. They find that the excitatory CCK neurons are involved in hippocampal-dependent tasks such as spatial learning and memory formation, and that CCK-knockdown impairs these tasks.

However, these questions are very dependent on ensuring that the study is properly targeting excitatory CCK neurons (and thus their specific contributions to behavior).

There needs to be much more characterization of the CCK transgenic mice and viruses to confirm the targeting. Without this, it is unclear whether the study is looking at excitatory CCK neurons or a more general heterogeneous CCK neuron population.

Strengths:

This field has focused mainly on inhibitory CCK+ interneurons and their role in network function and activity, and thus, this manuscript raises interesting questions regarding the role of excitatory CCK+ neurons, which have been much less studied.

Weaknesses:

(1a) This manuscript is dependent on ensuring that the study is indeed investigating the role of excitatory CCK-expressing neurons themselves and their specific contribution to behavior. There needs to be much more characterization of the CCK-expressing mice (crossed with Ai14 or transduced with various viruses) to confirm the excitatory-cell targeting. Without this, it is unclear whether the study is looking at excitatory CCK neurons or a more general heterogeneous CCK neuron population.

(1b) For the experiments that use a virus with the CCK-IRES-Cre mouse, there is no information or characterization on how well the virus targets excitatory CCK-expressing neurons. (Additionally, it has been reported that with CaMKIIa-driven protein expression, using viruses, can be seen in both pyramidal and inhibitory cells.)

(2) The methods and figure legends are extremely sparse, leading to many questions regarding methodology and accuracy. More details would be useful in evaluating the tools and data. More details would be useful in evaluating the tools and data. Additionally, further quantification would be useful-e.g. in some places, only % values are noted, or only images are presented.

(3) It is unclear whether the reduced CCK expression is correlated, or directly causing the impairments in hippocampal function. Does the CCK-shRNA have any additional detrimental effects besides affecting CCK-expression (e.g., is the CCK-shRNA also affecting some other essential (but not CCK-related) aspect of the neuron itself?)? Is there any histology comparison between the shRNA and the scrambled shRNA?

Reviewer #2 (Public review):

Summary:

In this study, the authors have demonstrated, through a comprehensive approach combining electrophysiology, chemogenetics, fiber photometry, RNA interference, and multiple behavioral tasks, the necessity of projections from CCK+ CAMKIIergic neurons in the hippocampal CA3 region to the CA1 region for regulating spatial memory in mice. Specifically, authors have shown that CA3-CCK CAMKIIergic neurons are selectively activated by novel locations during a spatial memory task. Furthermore, authors have identified the CA3-CA1 pathway as crucial for this spatial working memory function, thereby suggesting a pivotal role for CA3 excitatory CCK neurons in influencing CA1 LTP. The data presented appear to be well-organized and comprehensive.

Strengths:

(1) This work combined various methods to validate the excitatory CCK neurons in the CA3 area; these data are convincing and solid.

(2) This study demonstrated that the CA3-CCK CAMKIIergic neurons are involved in the spatial memory tasks; these are interesting findings, which suggest that these neurons are important targets for manipulating the memory-related diseases.

(3) This manuscript also measured the endogenous CCK from the CA3-CCK CAMKIIergic neurons; this means that CCK can be released under certain conditions.

Weaknesses:

(1) The authors do not mention which receptors of the CCK modulate these processes.

(2) This author does not test the CCK gene knockout mice or the CCK receptor knockout mice in these neural processes.

(3) The author does not test the source of CCK release during the behavioral tasks.

Reviewer #3 (Public review):

Summary:

Fengwen Huang et al. used multiple neuroscience techniques (transgenetic mouse, immunochemistry, bulk calcium recording, neural sensor, hippocampal-dependent task, optogenetics, chemogenetics, and interfer RNA technique) to elucidate the role of the excitatory cholecystokinin-positive pyramidal neurons in the hippocampus in regulating the hippocampal functions, including navigation and neuroplasticity.

Strengths:

(1) The authors provided the distribution profiles of excitatory cholecystokinin in the dorsal hippocampus via the transgenetic mice (Ai14::CCK Cre mice), immunochemistry, and retrograde AAV.

(2) The authors used the neural sensor and light stimulation to monitor the CCK release from the CA3 area, indicating that CCK can be secreted by activation of the excitatory CCK neurons.

(3) The authors showed that the activity of the excitatory CCK neurons in CA3 is necessary for navigation learning.

(4) The authors demonstrated that inhibition of the excitatory CCK neurons and knockdown of the CCK gene expression in CA3 impaired the navigation learning and the neuroplasticity of CA3-CA1 projections.

Weaknesses:

(1) The causal relationship between navigation learning and CCK secretion?

(2) The effect of overexpression of the CCK gene on hippocampal functions?

(3) What are the functional differences between the excitatory and inhibitory CCK neurons in the hippocampus?

(4) Do CCK sources come from the local CA3 or entorhinal cortex (EC) during the high-frequency electrical stimulation?

eLife Assessment

This study integrates large-scale behavioral, genetic, and molecular analyses in animal models to investigate morphine response. Utilizing high-quality, time-series Quantitative Trait Loci (QTL) mapping, the work provides compelling evidential support for novel, time-dependent genetic interactions (epistasis). A fundamental result of this rigorous analysis is the discovery of a novel Oprm1-Fgf12-MAPK signaling pathway, which offers new insights into the mechanisms of opioid sensitivity.

Reviewer #1 (Public review):

Summary:

The study by Lemen et al. represents a comprehensive and unique analysis of gene networks in rat models of opioid use disorder, using multiple strains and both sexes. It provides a time-series analysis of Quantitative Trait Loci (QTLs) in response to morphine exposure.

Strengths:

A key finding is the identification of a previously unknown morphine-sensitive pathway involving Oprm1 and Fgf12, which activates a cascade through MAPK kinases in D1 medium spiny neurons (MSNs). Strengths include the large-scale, multi-strain, sex-inclusive design, the time-series QTL mapping provides dynamic insights, and the discovery of an Oprm1-Fgf12-MAPK signaling pathway in D1 MSNs, which is novel and relevant.

Weaknesses:

(1) The proposed involvement of Nav1.2 (SCN2A) as a downstream target of the Oprm1-Fgf12 pathway requires further analysis/evidence. Is Nav1.2 (SCN2A) expressed in D1 neurons?

The authors mentioned that SCN8A (Nav1.6) was tested as a candidate mediator of Oprm1-Fgf12 loci and variation in locomotor activity. However, the proposed model supports SCN2A as a target rather than SCN8A. This is somewhat unexpected since SCN8A is highly abundant in MSN.

Can the authors provide expression data for SCN2A, Oprm1, and Fgf12 in D1 vs. D2 MSNs?

(2) The authors should consider adding a reference to FGF12 in Schizophrenia (PMC8027596) in the Introduction.

(3) There is recent evidence supporting the druggability of other intracellular FGFs, such as FGF14 (PMC11696184) and FGF13 (PMC12259270), through their interactions with Nav channels. What are the implications of these findings for drug discovery in the context of the present study? Could FGF12 be considered a potential druggable therapeutic target for opioid use disorder (OUD)?

Reviewer #2 (Public review):

Summary:

This highly novel and significant manuscript re-analyzes behavioral QTL data derived from morphine locomotor activity in the BXD recombinant inbred panel. The combination of interacting behavioral-pharmacology (morphine and naltrexone) time course data, high-resolution mouse genetic analyses, genetic analysis of gene expression (eQTLs), cross-species analysis with human gene expression and genetic data, and molecular modeling approaches with Bayesian network analysis produces new information on loci modulating morphine locomotor activity.

Furthermore, the identification of time-wise epistatic interactions between the Oprm1 and Fgf12 loci is highly novel and points to methodological approaches for identifying other epistatic interactions using animal model genetic studies.

Strengths:

(1) Use of state-of-the art genetic tools for mapping behavioral phenotypes in mouse models.

(2) Adequately powered analysis incorporating both sexes and time course analyses.

(3) Detection of time and sex-dependent interactions of two QTL loci modulating morphine locomotor activity.

(4) Identification of putative candidate genes by combined expression and behavioral genetic analyses.

(5) Use of Bayesian analysis to model causal interactions between multiple genes and behavioral time points.

Weaknesses:

(1) There is a need for careful editing of the text and figures to eliminate multiple typographical and other compositional errors.

(2) There are multiple examples of overstating the possible significance of results that should be corrected or at least directly pointed out as weaknesses in the Discussion. These include:

a) Assumption that the Oprm1 gene is the causal candidate gene for the major morphine locomotor Chr10 QTL at the early time epochs. Oprm1 is 400,000 bp away from the support interval of the Mor10a QTL locus, and there is no mention as to whether the Oprm1 mRNA eQTL overlaps with Mor10a.

b) Although the Bayesian analysis of possible complex interactions between Oprm1, Fgf12, other interacting genes, and behaviors is very innovative and produces testable hypotheses, a more straightforward mediation analysis of causal relationships between genotype, gene expression, and phenotype would have added strength to the arguments for the causal role of these individual genes.

c) The GWAS data analysis for Oprm1 and Fgf12 is incomplete in not mentioning actual significance levels for Oprm1 and perhaps overstating the nominal significance findings for Fgf12.

Appraisal:

The authors largely succeeded in reaching goals with novel findings and methodology.

Significance of Findings:

This study will likely spur future direct experimental studies to test hypotheses generated by this complex analysis. Additionally, the broad methodological approach incorporating time course genetic analyses may encourage other studies to identify epistatic interactions in mouse genetic studies.

Reviewer #3 (Public review):

Summary:

This is a clearly written paper that describes the reanalysis of data from a BXD study of the locomotor response to morphine and naloxone. The authors detect significant loci and an epistatic interaction between two of those loci. Single-cell data from outbred rats is used to investigate the interaction. The authors also use network methods and incorporate human data into their analysis.

Strengths:

One major strength of this work is the use of granular time-series data, enabling the identification of time-point-specific QTL. This allowed for the identification of an additional, distinct QTL (the Fgf12 locus) in this work compared to previously published analysis of these data, as well as the identification of an epistatic effect between Oprm1 (driving early stages of locomotor activation) and Fgf12 (driving later stages).

Weaknesses:

(1) What criteria were used to determine whether the epistatic interaction was significant? How many possible interactions were explored?

(2) Results are presented for males and females separately, but the decision to examine the two sexes separately was never explained or justified. Since it is not standard to perform GWAS broken down by sex, some initial explanation of this decision is needed. Perhaps the discussion could also discuss what (if anything) was learned as a result of the sex-specific analysis. In the end, was it useful?

(3) The confidence intervals for the results were not well described, although I do see them in one of the tables. The authors used a 1.5 support interval, but didn't offer any justification for this decision. Is that a 95% confidence interval? If not, should more consideration have been given to genes outside that interval? For some of the QTLs that are not the focus of this paper, the confidence intervals were very large (>10 Mb). Is that typical for BXDs?

i am an annotation

Gathering your knowledge refers to the process of actively collecting and organizing information from your professor.

I love that they all understood the value of education and highlighted its importance. They wanted the truth behind American facade to be exposed and taught to our people. They wanted to understand their place in history, be represented, and make it be embraced. Unfortunately, to this day the education system hasn't really done that which is sad. It took me until college to be taught deeper into our history. Usually, they just give us a lot of surface level things and show us our oppression but limit showing us how we continously fought for ourselves.

This is a reminded that research should come from curiosity and problems-solving, not from trying to prove what we already believe. It changes how i think about starting any project.

This connects to my experience in school. Most papers focused on sources and citation rules, not personal engagement. I like how Witte points out that this method silence the students own thinking.

This shows how research is often seen as just finding information instead of creating knowledge. It makes me think about how many of my past research papers were just about collecting quotes and not about forming my own ideas.

Children learn more effectively with the areas they like and when they need to learn it they learn effectively by stimulating these areas they learn a lot in a short amount of time.

The use of recurring patterns can be used to educate more effectively that can also show the pile of problems that build up that need to be corrected.

Students can be educators as well and the learning process goes both ways from students to teachers, traditional learning is not wrong but there is a lot more to learning than the teacher just telling the students.

Patterns will always give rise to facts that can back up theories the diverse history of education is vast there can be patterns.

Newer media technologies can help educate people more efficiently but, there are downsides if they don't get executed properly.

The new worlds students inhabit also have to be addressed and when these are utilized the educational process gets better.

Momentum has gathered especially when it comes to change and it will continue to change as mire disappointing programs occur.

They can be exceptional fields if they are properly offered and nurtured for all that want to attend.

The creative programs can create some bright minded individuals in various fields when properly executed.

Change is always inevitable and when no change is done it becomes a growing pain in the figurative sense.

That general crisis of authority is always around especially when authority goes unchecked and uncorrected.

The programs created will always have short comings and don't meet expectations but, they had to try.

The configurations that are created and organized are ones that can help in tremendous waves if done right and ones that can continue to help.

Schools and educational programs are important for students and the more the better.

The connection between student, staff, and teacher is just as important as the material being taught.

Increased rate of heart attacks for survivors

He is a good source for this because his legacy and wealth are at the level of the man he is talking about.

He uses a little bit of ethos here with the credibility of this chief.

In the introduction/first sentence he shows his problem that he is addressing, and his views on it.